Cell Oncol. DOI 10.1007/s13402-017-0345-5

REVIEW

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options Meysam Yousefi 1,2 & Tayyeb Bahrami 3 & Arash Salmaninejad 4 & Rahim Nosrati 5 & Parisa Ghaffari 2 & Seyed H. Ghaffari 2

Accepted: 16 August 2017 # International Society for Cellular Oncology 2017

Abstract Background Lung cancer is the most common cause of cancer-related mortality in humans. There are several reasons for this high rate of mortality, including metastasis to several organs, especially the brain. In fact, lung cancer is responsible for approximately 50% of all brain metastases, which are very difficult to manage. Understanding the cellular and molecular mechanisms underlying lung cancer-associated brain metastasis brings up novel therapeutic promises with the hope to ameliorate the severity of the disease. Here, we provide an overview of the molecular mechanisms underlying the pathogenesis of lung cancer dissemination and metastasis to the brain, as well as promising horizons for impeding lung cancer brain metastasis, including the role of cancer stem cells, the blood-brain barrier, interactions of lung cancer cells with the brain microenvironment and lung cancer-driven systemic processes, as well as the role of growth factor/receptor tyrosine kinases, cell adhesion molecules and non-coding RNAs. In addition, we provide an overview of current and novel

* Seyed H. Ghaffari [email protected] 1

Department of Medical Genetics, Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

2

Hematology, Oncology and Stem Cell Transplantation Research Center, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran

3

Genetic Research Center, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran

4

Drug Applied Research Center, Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran

5

Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

therapeutic approaches, including radiotherapy, surgery and stereotactic radiosurgery, chemotherapy, as also targeted cancer stem cell and epithelial-mesenchymal transition (EMT)based therapies, micro-RNA-based therapies and other small molecule or antibody-based therapies. We will also discuss the daunting potential of some combined therapies. Conclusions The identification of molecular mechanisms underlying lung cancer metastasis has opened up new avenues towards their eradication and provides interesting opportunities for future research aimed at the development of novel targeted therapies. Keywords Lung cancer . Brain metastasis . Molecular mechanisms . Blood-brain barrier

1 Introduction Lung cancer is the most common cause of mortality among all human cancers [1] and in 2017 more than 155,000 lung cancer-related deaths in the USA are expected, accounting for >25% of all cancer-related deaths [2]. Lung cancer is the second most common cancer in both men and women [3]. Since a high lung cancer rate is a reflection of a population’s smoking behavior, recent declines in the prevalence of smoking has resulted in concomitant declines in the incidence of lung cancer [4, 5]. According to the appearance and morphology of malignant cells, lung cancers can be classified into small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) [6]. NSCLC accounts for more than 80% of all lung cancer cases and encompasses squamous cell carcinoma and adenocarcinoma [7]. Most lung cancer-associated deaths result from metastasis [8]. The brain is the most common site of lung cancer metastasis, i.e., up to 50% of all lung cancers develop into brain

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metastasis (BM) during the course of the disease [9–11]. Conversely, more than half of BMs originate from lung cancer, followed by breast cancer and melanoma [12]. The propensity for metastasis to the brain remains a major clinical hurdle that negatively impacts the five-year survival rate of lung cancer patients. In addition, it has been reported that the rate of lung cancer brain metastasis (LCBM) has been increasing in recent years [13], which places a great burden on public health services. Several factors may have contributed to the recent increase in the incidence of BMs. For instance, a longer survival of lung cancer patients and advances that have been made in neuroimaging techniques have resulted in increases in detection rates, and consequently, the incidence of BM [14]. Other factors that may have contributed to this increase include a rise in advanced cancer stages [15] and a growth of the aging population [16]. In spite of many advances that have been made in the treatment of lung cancer patients, the prognosis of patients with BM is still dismal. This is in part due to the blood-brain barrier (BBB), which curbs the accessibility of therapeutic agents to the brain parenchyma. A plethora of studies has attempted to identify high-risk individuals and to delineate prognostic factors for LCBM, and it has been found that in locally advanced NSCLC (LA-NSCLC) factors such as an age of ≤ 60 years, non-squamous cell carcinoma [17] and the presence of clinical bulky mediastinal lymph nodes (˃ 2 cm) [18] are associated with a high BM rate. In addition, it has been found that a Karnofsky performance status (KPS) > 70%, the absence of extracranial metastases and control of the primary tumor are prognostic factors that correlate with a better median survival in patients with BMs (Fig. 1) [19, 20]. Whereas a role of PD-1, LKB1, KRAS and EGFR mutations and ALK rearrangements has been confirmed in the progression of lung cancer [21–23], their putative involvement in the development of BM is still a matter of debate [24–33]. Patients with completely resected pIIIA-N2 nonsquamous cell carcinoma and a lymph node ratio (LNR) ≥ 30% are at the highest risk for developing BM and, as such, are most likely to benefit from prophylactic cranial irradiation (PCI) [34]. These risk factors warrant further studies on prophylactic chemotherapy and PCI, as well as intensive radiologic follow-up studies on high-risk populations. Since BM predicts a poor outcome, with a median survival rate of ˂ 6 months [9, 35], an in-depth understanding of the molecular mechanisms underlying LCBM is considered necessary for the identification of new therapeutic targets.

2 Metastatic cascade and cancer stem cell hypothesis Metastasis is a process by which cancer cells spread to distant organs or to other regions within the same organ [36]. This process contributes to more than 90% of cancer-related deaths

[36, 37], and it encompasses a series of interrelated steps beginning with local cancer cell invasion, followed by intravasation and invasion of the hematogenous and/or lymphatic systems. The resulting circulating tumor cells (CTCs) may subsequently extravasate to the parenchyma of distant tissues and adapt to local supportive niches, thereby forming micro-metastases. These micro-metastases may eventually form macroscopic tumors through a process referred to as Bmetastatic colonization^ [38, 39]. The composition of cell populations within primary tumors is heterogeneous. Most neoplastic tissues harbor rare subpopulations of self-renewing, stem cell-like cells referred to as Bcancer stem cells^ (CSCs) [40]. It is thought that CSCs may emanate from differentiated cancer cells that have undergone dedifferentiation and, by doing so, have adapted stem cell-like properties (Fig. 2a). Epithelial-mesenchymal transition (EMT) is an evolutionary-conserved process that is considered essential for normal embryonic development [41]. Recent evidence has, however, indicated that EMT is also implicated in the processes of cancer progression and metastasis [42]. During EMT cancer cells acquire characteristics of stem cells such as self-renewal and differentiation. Usually, CSCs lose their polarity and cell-cell adhesion structures, rearrange their cytoskeleton and become motile and resistant to apoptosis [42]. These characteristics enable CSCs to seed new tumors and, therefore, they are also called Btumor-initiating cells^ [40, 43]. Several cell surface antigens such as CD44, CD24, CD34, CD133 and CD117, as well as aldehyde dehydrogenase 1A1 (ALDH1A1) expression, are currently used for the identification, isolation and targeting of CSCs [44]. EMT can be induced by various growth factor receptor tyrosine kinases (RTKs) and cellular signaling pathways, including the TGF-β, Wnt and Notch pathways. TGF-β is one of the most extensively studied EMT inducers. After binding to its receptor, TGF-β leads to phosphorylation and activation of Smad2 and Smad3, which enables them to form trimers with Smad4 and to, subsequently, translocate to the nucleus where they regulate the expression of TGF-β target genes (Fig. 2b) [45]. TGF-β may also cooperate with the Wnt and Notch signaling pathways to induce EMT [46–49]. Also, the SNAIL, TWIST and ZEB transcription factors may serve as master inducers of EMT [50]. These transcription factors can downregulate epithelial markers and upregulate mesenchymal markers and can be activated by Ras/Raf/MAPK, PI3K/Akt or other signaling pathways (Fig. 2b) [50, 51]. In addition, it has been found that the Smad complex is dependent on the aforementioned transcription factors to achieve a high affinity and selectivity for TGF-β target genes [51]. Ample evidence supports a role of EMT in the development of LCBM [52]. A well-known alteration during LCBM development is a loss of epithelial attributes. By forming adherent junctions via E-cadherins, epithelial cells adhere to adjacent epithelial cells, thereby allowing them to assemble epithelial

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options

Fig. 1 Schematic picture of LCBM and several factors affecting this process. The identification of factors involved in BM formation may result in a more accurate diagnosis and clinical management, as well as

the design of more effective therapies. The factors marked with question marks are not decisively confirmed

cell sheets and to maintain the organization of cells within these sheets [53]. Recent studies have shown that NSCLC patients with BMs exhibit a lower E-cadherin expression compared to NSCLC patients without BMs [54]. Conversely, it has been found that mesenchymal markers (such as N-cadherin, vimentin, fibronectin and proteolytic enzymes), which contribute to cell migration during embryogenesis and inflammation, are usually upregulated during lung cancer metastasis [55]. Grinberg-Rashi et al. found that N-cadherin was significantly overexpressed in BM-positive NSCLCs, suggesting that this protein may serve as a predictive BM marker [56]. Others found that vimentin was associated with NSCLC spread and suggested that a high vimentin expression may be of prognostic value for the occurrence of LCBM [57]. These reports are, however, inconsistent with previous in vitro studies [58–60]. In addition, it has been found that fibronectin has the ability to induce EMT in NSCLC cells through integrin α9-mediated activation of the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways [61]. Under hypoxic conditions Sox2 and Oct4, which are induced by HIF1 and HIF2, were found to activate the CD133 promoter in NSCLC-derived cell lines, and overexpression of Nanog and Oct4 in the same cell lines was found to induce stem cell-like characteristics, such as self-renewal and invasion [62]. EMT inducers have also been found to degrade the stromal matrix by upregulating matrix metalloproteinases (MMPs) and plasminogen activators (PAs), which are associated with the invasive phenotype of lung cancer cells [63, 64]. EMT may also lead to angiogenesis by the induction of proangiogenic factors, such as the vascular endothelial growth factor (VEGF), leading

to an excessive vascularization within the primary tumor and, thus, a contribution to CSC invasion [65]. In fact, a significant part of tumor cells may intravasate into the blood vessel and lymphatic systems, but given that these tumor cells must subsequently evade immunity and apoptotic signals, only a small proportion of them may survive and extravasate into parenchyma at distant sites to initiate metastases [66]. Although EMT may be involved in cancer dissemination, recent evidence has shown that EMT may also be dispensable for metastasis formation. Specifically, animal models for mammary and pancreatic cancers have challenged the role of EMT as exclusive process for cancer dissemination. [67, 68]. These studies suggested that both epithelial and mesenchymal tumor cells may have the potential to invade and to establish metastases. More interestingly, it was found that EMT prohibition through overexpression of the microRNA miR-200 (which suppresses EMT through targeting ZEB1/2 factors) did not show any negative effect on the capacity of cancer cells to form distant metastases. Fisher et al. [67] found that lung metastases could be derived from non-EMT tumor cells, despite the contribution of EMT to the acquisition of chemo-resistance and aggressiveness of cancer cells. These observations suggest that the mechanisms underlying lung cancer metastasis may be more diverse than previously thought. It has for example been suggested that CSCs may originate from deregulated normal tissue stem cells that have acquired oncogenic mutations (Fig. 2a) [69]. Indeed, CSCs share features with both normal tissue stem cells and cancer cells, suggesting that the accumulation of oncogenic mutations in tissue stem cells may result in stem cell populations exhibiting uncontrolled proliferation [69].

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Fig. 2 A schematic view of CSC formation and the most studied signaling pathways in EMT. (a) CSCs may originate from somatic mutations of normal tissue stem cells or dedifferentiation of cancer cells via EMT. (b) Several signaling pathways may be involved in EMT. TGFβ is the most studied signaling pathway: After the binding of the TGF-β to its receptor, Smad2 and Smad3 are phosphorylated, thus, able to form trimers with Smad4 and translocate to the nucleus where they cooperate with key EMT transcription factors such as Snail, ZEB and Twist. Activation of these transcription factors results in downregulation of epithelial markers and upregulation of mesenchymal markers giving cancer cells their stem-like cell properties. TGF-β cooperates with Wnt and Notch signaling pathways and can also stimulate the NF-кB module to induce EMT. Wnt signaling begins when a Wnt protein binds to the extracellular domain of a Frizzled (Fz) family receptor. Upon activation

of the receptor, a signal is sent to Dishevelled (Dsh), which is located in the cytoplasm. Dsh then inhibits glycogen synthase kinase 3 (GSK3) and keeps it from forming a destruction complex with Axin and APC, thus interrupting its inhibitory effect on the key EMT transcription factors. Notch signaling is another inducer of EMT: When Notch ligands bind to the extracellular domain of the Notch receptor, they induce proteolytic cleavage of the receptor in such a way that the Notch intracellular domain (NICD) is released. The NICD then enters the cell nucleus where it binds to the DNA-binding protein CSL (CBF1/Suppressor of Hairless/LAG-1) and activates the transcription of Notch target genes in cooperation with other auxiliary proteins. Moreover, SNAIL, TWIST and ZEB transcription factors can be activated by Ras/Raf/MAPK, PI3K/AKT or other signaling pathways

Apart from their origin, CSCs may play important roles in organ-specific metastasis of human cancers. In case of breast cancer, for example, CD44 expression on the surface of breast cancer stem cells (BCSCs) has revealed their contribution to formation of brain metastases where common CD44 ligands (hyaluronan and osteopontin) are abundant [70, 71]. Moreover, breast cancer cells highly express the chemokine receptor CXCR4, which may be instrumental in their targeting to bone, lung and brain where the CXCR4 ligands CXCL12/ SDF-1 are abundant [72, 73]. Given the fact that the stem cell marker CD44 and the chemokine receptor CXCR4 are also expressed in NSCLC stem cells [74, 75], it may not be irrational to assume that CSCs may contribute to the organ-specific metastasis of lung cancer.

3 Organ-specific metastasis of lung cancer Primary tumors may metastasize to distinct organs. This process is referred to as Borganotropism^ or Borgan-specific metastasis^ [37]. Two fundamental hypotheses may explain organ-specific metastatic processes. The first hypothesis, Bthe hemodynamic or mechanical hypothesis^, refers to an anatomic distribution system (the spreading of tumor cells into the lymphatic system or a body cavity followed by a remote spread via the venous system). Since organ-draining routes invoke metastatic patterns, this hypothesis proclaims that tumors with diverse draining routes may exhibit diverse metastatic patterns [76]. Even though circulation may affect dissemination patterns, the mechanical hypothesis does not

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options

always explain the organ-specific metastasis of human cancers. The liver and the brain, for instance, receive an equal volume of blood, but these organs exhibit distinct metastatic patterns [12, 77]. Thus, there must exist other mechanisms explaining the organ tropisms of cancer metastases. Accordingly, the second Bseed-and-soil hypothesis^, which is a more widely accepted hypothesis for the formation of metastasis, denotes that metastatic tumor cells can blossom and form secondary tumors only in permissive tissues with organ-specific Bsoils^ [78]. In other words, organ-specific metastasis may not only depend on the distribution pattern of the CTCs, but also on other extrinsic factors such as the architecture of the vascular and/or lymphatic system and intrinsic abilities of CTCs to cross physical barriers and survive at distant sites [77]. Overall, it may be assumed that lung cancer Bseeds^ are conveyed in different directions, but due to factors that are discussed in the following sub-sections, the brain is the preferred site for lung cancer metastasis (up to 50%) [9], followed by bones, liver and adrenal glands, respectively [79]. The concomitant histological breakdown of patients with BMs has been found to be as follows: 31% SCLC, 21% adenocarcinoma, 21% large cell carcinoma and 8% squamous cell carcinoma [80]. 3.1 Transmigration of disseminated tumor cells across the blood-brain barrier Transmigration across the BBB is a rate-limiting step in the development of BMs. In contrast to those in the peripheral system, brain microvascular endothelial cells (BMECs) are joined by tight junctions enabling them to maintain their selective permeability and to prevent substances exchanging freely between the blood and the interstitial fluid of the brain [81]. However, in metastatic brain tumors the integrity of the BBB is compromised, i.e., the BBB at metastatic tumor sites exhibits an increased level of permeability (Fig. 3) [20]. As yet, the molecular mechanisms underlying this hyper-permeability are not fully understood. In SCLC, Rho GTPases are activated during trans-endothelial migration, thereby facilitating the breakdown of intercellular junctions through increasing actomyosin contractility. It has been found that inhibition of the endothelial Rho kinase (ROCK) can prevent the transendothelial migration of NCI-H209 SCLC-derived cells, hence suggesting a role of the Rho/ROCK signaling pathway in the transmigration of disseminated SCLC cells across the BBB [82, 83]. Tumor cells may also release vascular endothelial growth factor (VEGF) and other cytokines that can increase vessel permeability [84, 85]. In the case of breast-to-brain metastasis, several factors have been identified that may enhance the transmigration of tumor cells across the BBB. It has for instance been found that cathepsin S may contribute to the proteolytic digestion of the junctional adhesion molecule JAM-B, while the alpha2,6-sialyltransferase ST6GALNAC5

may confer a co-opted glycosylation pattern to brain-seeking breast cancer cells [86, 87]. In addition, it has been found that the microRNAs miR-105 and miR-181c may target the tight junction protein ZO-1 and induce an abnormal localization of actin via downregulation of its target gene PDPK1, respectively [88, 89]. Overall, compromised tight junctions, increased perivascular spaces, the presence of fenestrations and an increased number of pinocytic vacuoles have been noted in micro-vessels of metastatic brain sites. These characteristics reflect the vasculature in the periphery and suggest that micro-vessels in metastatic sites of the brain are similar to those of the tumor of origin rather than of the CNS [90]. On the other hand, it has been reported that the expression levels of transporters in endothelial cells are significantly decreased at metastatic sites compared to those in the normal brain vasculature. Regina et al. [91] reported, for instance, that in LCBMs the micro-vessels around the metastatic sites express only 40% of the P-glycoprotein found in the normal brain vasculature. Therefore, once tumor cells enter the brain, the BBB plays a protective role against various agents. This characteristic hampers the efficient delivery of chemotherapeutic drugs to the brain (see section 4). Most cancer cells that enter the brain will instantly die [92]. Therefore, after crossing the BBB, disseminated cancer cells must have acquired sophisticated mechanisms that enable them to reside in the brain parenchyma, to evade death signals and to form macro-metastases. These mechanisms include cell-autonomous processes that enable cancer cells to have paracrine interactions with the brain microenvironment and tumor-driven systemic processes that stimulate the growth of disseminated cancer cells by generating a favorable microenvironment [93]. 3.2 Molecular interactions between metastatic tumor cells and the brain microenvironment A favorable microenvironment for metastatic tumor cells, also referred to as a Bpremetastatic microenvironment^, is a sophisticated network that supports the survival and proliferation of cancer cells [94, 95]. In each organ, tissue stem cells reside in niches that provide a balance between stem-cell proliferation and quiescence, as well as self-renewal and differentiation. These niches or microenvironments are usually rich in selfrenewal signals such as hedgehog, chemokine CXCL12, TGF-β and Wnt signals [96, 97]. When CSCs disperse to distant organs, they take advantage of their similarities with tissue stem cells and home in stem cell niches where they have the potential to develop into macro-metastases [98]. Although the exact nature of the bidirectional interactions between metastatic tumor cells and the brain microenvironment remains to be resolved, several molecules involved in these interactions have recently been identified. Astrocytes, the most abundant cell type of the brain, secrete a serine

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Fig. 3 Migration of SCLC cells across a compromised blood-brain barrier (BBB). Rho GTPases have been shown to activate trans-endothelial migration through tight junction (TJ) destabilization and actomyosin contractility

protease, plasminogen activator (PA), which is responsible for converting plasminogen to plasmin. Plasmin, in turn, mobilizes the pro-apoptotic cytokine Fas ligand (FasL) to kill extravasated cancer cells. Also, plasmin inactivates the axon path-finding molecule L1 cell adhesion molecule (L1CAM), which is expressed by metastatic cells to facilitate their spread along perivascular sites (Fig. 4) [99, 100]. Interestingly, it has recently been found that metastatic tumor cells from breast and lung adenocarcinomas produce anti-PA serpins, including neuroserpin and serpin B2. These serpins help tumor cells to avert Fas-dependent programmed cell death and allows them to proliferate along brain capillaries, finally resulting in the engulfment and remodeling of a co-opted capillary network [100]. After

evasion of Fas-dependent apoptosis, breast and lung tumor cells engage astrocytes and microglia to express endothelin1 (ET-1). ET-1 signals through ET-1 receptors on the tumor cells and activates the PI3K/AKT and MAPK/ERK signaling pathways, thereby, leading to the survival and chemoresistance of metastatic cancer cells [101]. Additional tumor cell-astrocyte crosstalk comes from the expression of protocadherin 7 (PCDH7) by the tumor cells, which favors the assembly of connexin 43 (Cx43) gap junctions between the tumor cells and astrocytes. After gap junction formation, the tumor cells transfer cGAMP to the astrocytes resulting in activation of the innate immune response STING pathway and, consequently, the production of the inflammatory cytokines IFNα and TNFα. These inflammatory cytokines act as

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options

Fig. 4 Molecular interactions of metastatic tumor cells with the brain microenvironment. After arriving in the brain parenchyma, tumor cells must avoid apoptotic signals from astrocytes. Astrocytes secrete plasminogen activator (PA) to produce plasmin. Plasmin can mobilize the pro-apoptotic cytokine Fas ligand (FasL) and produce soluble FasL (sFasL), which can induce cell death in cancer cells. In addition, plasmin inactivates L1CAM, which is expressed by metastatic cells to facilitate their spread along perivascular sites. To overcome these inhibitory signals, lung (and breast) tumor cells produce anti-PA serpins, which enable them to evade Fas-dependent programmed cell death and to

proliferate in the brain microenvironment. Tumor cells also recruit astrocytes and microglia to express endothelin-1 (ET-1). ET-1 activates the PI3K/AKT and MAPK/ERK signaling pathways in the tumor cells, which results in tumor cell survival and chemo-resistance. Additional crosstalk comes from the expression of protocadherin 7 (PCDH7) by the tumor cells, which favors the assembly of connexin 43 (Cx43) gap junctions between the tumor cells and astrocytes and, subsequently, transfers the messenger cGAMP to the astrocytes. Finally, this causes activation of the STAT1 and NF-κB pathways in the metastatic tumor cells, resulting in tumor outgrowth and chemo-resistance

paracrine signals and activate the STAT1 and NF-κB pathways in tumor cells which, in turn, facilitate their growth and chemo-resistance (Fig. 4) [102]. A detailed comprehension of the molecular mechanisms underlying the interactions between metastatic tumor cells and the brain microenvironment may provide promising avenues towards the design of new therapeutics to target LCBMs.

3.3 Lung cancer-driven systemic processes It has been shown that different types of systemic mediators may precondition distant organs by creating premetastatic niches before the arrival of metastatic cancer cells [103]. These mediators include extracellular matrix (ECM)-remodeling enzymes, inflammatory cytokines and tumor-derived

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extracellular vesicles. Different ECM-remodeling proteases have been implicated in the process of BM. ADAM9, a member of the Bdisintegrin and metalloprotease^ family, is a membrane-tethered protease that regulates cell-cell and cellmatrix interactions [104]. A role of ADAM9 in the invasion and metastasis of cancer cells has been well established [105]. The expression of ADAM9 has been found to be significantly higher in lung cancer-derived EBC-1 brain metastatic cells compared to parental EBC-1 cells [106]. It has been reported that ADAM9 over-expression may enhance metastatic foci in the brain through upregulation of integrin α3β1 [106, 107]. Matrix metalloproteinases (MMPs) represent another class of proteolytic enzymes that play a special role in the degradation of tight junction proteins, thereby resulting in a compromised BBB [108]. In this regard, MMP-9 has been reported to be specifically overexpressed by BMs from lung adenocarcinomas, suggesting a significant role of this enzyme in the transmigration of these tumor cells through the blood vasculature to the brain parenchyma [109]. Chemokines and chemokine receptors are commonly implicated in tumor progression and metastasis [110]. Several studies have e.g. shown that the expression of CXCL12 and its receptor, CXCR4, in BM-positive NSCLCs is significantly higher than in BM-negative NSCLCs and primary brain tumors [111, 112]. CXCL12 and CXCR4 overexpression in histopathological specimens has also been found to correlate with BMs in a NSCLC cohort study [75]. Although the exact role of the CXCL12/CXCR4 axis in BMs has not been elucidated, it has been suggested that CXCR4 may contribute to the adhesion and chemo-resistance of SCLC cells through a cooperation with integrins [113]. CX3CR1 is a chemokine receptor that has been associated with a site-specific metastasis of lung cancer cells and Mauri et al. [114] reported that CX3CR1 protein expression is significantly elevated in NSCLC compared to SCLC. They also reported that CX3CR1-positive lung adenocarcinomas tend to disseminate to various metastatic sites, whereas CX3CR1-negative carcinomas preferentially metastasize to the brain. The chemokine receptor CXCR6 and its ligand CXCL16 have also been found to be involved in the pathobiology of NSCLC. Mir et al. [115] reported that the expression of CXCR6 and CXCL16 was significantly higher in lung adenocarcinomas and squamous cell carcinomas compared to non-neoplastic tissues. In addition, they found that the serum level of CXCL16 was significantly higher in patients with lung adenocarcinomas than in patients with squamous cell carcinomas. They validated the biological significance of this axis in vitro in lung cancerderived cell lines, and found that CXCR6 expression was relatively higher in adenocarcinoma-derived cell lines, which may explain the higher migratory and invasive potential of these cell lines resulting from CXCL16-induced expression of metalloproteinases [115]. Nearby metastatic lesions, brain sections may present a considerable increase in glial fibrillary

acidic protein (GFAP)-positive astrocytes. Seike et al. [116] found that the astrocytes may be activated by tumor cellderived factors such as interleukin-8 (IL-8), macrophage migration inhibitory factor (MIF) and plasminogen activator inhibitor-1 (PAI-1). When astrocytes are stimulated, they produce tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1b (IL-1b) which, in turn, stimulate tumor cell proliferation. These findings indicate that astrocytes and tumor cells may stimulate each other. An in-depth understanding of these mutual relations is essential to comprehend how lung cancer cells proliferate and colonize in the brain. Exosomes are able to contribute to the formation of premetastatic niches through the ‘education’ of neighboring cells and raising inflammation at distant sites, thus facilitating the outgrowth of metastases [117, 118]. Tumor-derived exosomes that are taken up by organ-specific cells have been found to support premetastatic niche formation [119]. Exosomes may contain diverse integrins, and the exosomal integrins α6β1 and α6β4 have been found to correlate with lung metastasis, whereas α6β4 targeting was found to result in a reduced exosome uptake and a decrease in the occurrence of lung metastases. Hoshino et al. [120, 121] reported that exosome integrin uptake by resident cells may trigger Src phosphorylation and pro-inflammatory S100 gene expression. As a consequence, integrins that are expressed by exosomes may direct premetastatic niche formation. It has also been found that astrocyte-derived exosomes can promote the outgrowth of brain metastatic cancer cells by transferring PTEN-targeting microRNA-19a to these cells [121, 122]. Thus, additional inputs for the formation of LCBMs may come from exosomes. 3.4 Growth factors and their receptor tyrosine kinases Growth factors and/or their corresponding receptor tyrosine kinases (RTKs) have been implicated in BMs. A microarraybased study of 42 breast cancer and NSCLC brain metastatic patients revealed that the expression of the EGFR/ERK protein network is elevated in BMs [123]. Another study of 50 BMs and their matched primary NSCLCs indicated that the EGF and amphiregulin protein expression levels were significantly higher in BMs than in the primary tumors [124]. This latter study also revealed that the EGFR and ERBB receptor tyrosine kinase phosphorylation levels were upregulated in the BM cell membranes. Others, however, failed to find EGFR gene mutations in NSCLC BMs [27, 30]. The hepatocyte growth factor (HGF) and its receptor, c-MET, represent another growth factor/tyrosine kinase receptor combination that has been associated with cancer cell invasion and metastasis [125]. c-MET expression deregulation and gene amplification have been observed in 30% and 10% of adenocarcinomas, respectively [126]. These aberrations contribute to tyrosine kinase inhibitor (TKI) resistance [127–129]. Benedettini et al. [130] reported that in NSCLC

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options

patients c-MET activation is associated with BM development and resistance to EGFR inhibitors. Therefore, the HGF/cMET signaling pathway may hold further promises for therapeutically targeting brain metastases. Vascular endothelial growth factor (VEGF) subfamily members serve as additional growth factors involved in brain metastasis. VEGF induces angiogenesis and vessel permeability and it has been shown that VEGF expression levels are elevated in NSCLC cells that have metastasized to the brain, indicating that VEGF is necessary for BM both in humans and metastatic xenografted mice [131]. Additional experiments in lung adenocarcinoma-derived cell lines have shown that VEGF gene silencing may result in a decrease in the incidence of BMs, thus confirming its role in the establishment of nonsquamous cell carcinoma BMs [131]. Next to the observed higher VEGF expression in BMs compared to their matched NSCLC primaries, Jubb et al. [132] found that the vasculature in these BMs is relatively mature, explaining why these patients poorly respond to the VEGF inhibitor bevacizumab. Placental growth factor (PLGF) is another member of the VEGF subfamily which plays a role in the processes of angiogenesis, cellular cytoskeleton rearrangement and cell migration [78]. Li et al. [133] found that the serum PLGF levels were significantly elevated in BM-positive SCLC patients compared to BM-negative SCLC patients and normal individuals. They also found that SCLC-derived PLGF can stimulate the disassembly of tight junctions between brain endothelial cells through activation of the VEGFR1/Rho/ERK signaling pathway, which may facilitate the trans-endothelial migration of SCLC cells into the brain parenchyma. Therefore, VEGF subfamily members may serve as potential therapeutic targets for either SCLC or NSCLC. 3.5 Cell adhesion molecules CD44 is a cell surface glycoprotein that plays an important role in cell adhesion. Its isoforms are expressed in a range of human cell types, as well as various cancer types [134]. In addition, it has been found that CD44 is expressed at varying degrees during the various stages of NSCLC development, including metastasis. Thus, a regular clinical examination of CD44 expression may be instrumental for evaluating disease progression to metastasis, and for monitoring the treatment of NSCLC patients [74]. The role of tumor cell-derived protocadherin expression in the recruitment of astrocytes has recently extensively been discussed by others [102]. In addition, it has frequently been reported that cadherin deregulation may affect the metastatic process. As aforementioned, it has been reported that during EMT E-cadherin is downregulated while N-cadherin is upregulated [54, 56]. Interestingly, it has been found that Ecadherin is re-expressed in metastatic lesions, resulting in the adhesion and proliferation of tumor cells at the site of metastasis. High levels of E-cadherin expression have been

encountered in BMs, including those originating from lung cancers, thereby facilitating the proliferation and development of brain macro-metastases [135]. Integrins are cell adhesion molecules that can interact with components of the ECM and can trigger the transduction of signals to mediate cell survival [136]. It has been reported that a high expression of α3β1 integrin in lung cancer is associated with its propensity to metastasize to the brain. This propensity may be due to interaction of α3β1 integrin with the ECM component laminin in the brain parenchyma [137]. Endothelial cells express several proinflammatory adhesion molecules, including members of the intercellular adhesion molecules (ICAMs), vascular-cell adhesion molecule (VCAM) and platelet-endothelial-cell adhesion molecule (PECAM), all of which belong to the immunoglobulin superfamily of proteins and are essential in immune responses and inflammation [138]. Ample evidence supports a role of these adhesion molecules in the interaction of vascular endothelial cells with tumor cells, which may result in the extravasation of tumor cells and, consequently, the formation of metastases. In this context, ICAM-1 and VCAM-1 have recently been shown to facilitate polychlorinated biphenyl-mediated induction of brain metastasis [139]. 3.6 The role of non-coding RNAs in lung cancer brain metastasis Aside the abovementioned altered genes and signaling pathways, further LCBM input comes from the deregulation of non-coding RNAs (ncRNAs). MicroRNAs (miRNAs) represent a group of small non-coding endogenous RNAs containing 18–24 nucleotides that are capable of regulating gene expression mostly at the posttranscriptional level [140, 141]. MiRNAs are known to play a crucial role in normal development, proliferation, differentiation and apoptosis [142]. Accordingly, alterations in miRNA expression have been observed in various pathological conditions, including cancer [142]. In humans, almost 50% of the miRNA genes are located in areas of the genome that are known to be associated with carcinogenesis. Loss or inactivation of tumor suppressive miRNAs and/or gain or upregulation of oncogenic miRNAs may stimulate oncogenic pathways and, by doing so, confer cancerous phenotypes [143]. The role of miRNAs in the development of brain metastases has only recently been established [144]. The brain microenvironment may interact with tumor cells through various mechanisms, including miRNAs, to promote the outgrowth of tumors and metastases, and several studies have shown that the brain microenvironment can change miRNA profiles in primary tumor cells [145]. Importantly, it has been found that some miRNAs may confer a Bbrain-seeking^ behavior to lung cancer cells (Table 1). These miRNAs herald the necessity for a more aggressive treatment in order to prevent the development of intracranial metastases.

M. Yousefi et al. Table 1 Comparison of deregulated non-coding RNAs in brain metastases to their matched primary lung tumors

NcRNA

Expression status

Primary tumor

Putative target

Reference

miRNA-768-3p

Downregulated

Lung adenocarcinoma

KRAS

[146]

miR-145

Downregulated

Lung adenocarcinoma

JAM-A and fascin

[147]

miR-95-3p miR-146a

Downregulated Downregulated

Lung adenocarcinoma NSCLC (and breast)

Cyclin D1 B-catenin and hnRNPC

[148] [149, 150]

miR-328

Upregulated

NSCLC

PRKCA

[151]

miR-378 miR-197

Upregulated Upregulated

NSCLC EGFR-mutant NSCLC

MMP-7, MMP-9 and VEGF NOXA, BMF

[152] [153, 154]

miRNA-184

Upregulated

EGFR-mutant NSCLC

c-Myc

[153, 155]

miR-200 MALAT1

Upregulated Upregulated

Lung adenocarcinoma Lung adenocarcinoma

ZEB1/2 transcription factors EMT

[156] [157]

MiRNA deregulation has been reported to have implications for LCBM. MiR-378 has for example been shown to be differentially expressed in matched BM-positive NSCLC cases compared to BM-negative NSCLC cases [152]. MiR378 has been found to promote NSCLC cell migration and metastasis through upregulation of MMP-7, MMP-9 and VEGF and downregulation of Sufu [152]. The MiR-200 family of miRNAs (including miR-141 and miR-200a/b/c) has also been reported to be deregulated in metastatic lung cancers. MiR-200 can suppress EMT mainly through the targeting of ZEB1/2 transcription factors. It has also been reported that miR-200 family member levels may be elevated in cerebrospinal fluid (CSF) of patients with secondary brain tumors that have originated from lung and breast carcinomas compared to those of patients with glioblastoma or non-cancer patients [156]. MiRNA-328 overexpression has been found to promote the migration of NSCLC cells and subsequent BM formation via the deregulation of PRKCA, a member of the VEGF-IL1 family [151]. Cheng et al. [158] have reported that IL1-β can induce the urokinase plasminogen activator (uPA) in NSCLC-derived A549 cells, which results in cell migration and BM formation. Therefore, miR-328 overexpression may confer invasive properties to NSCLC cells resulting in BM. MiRNA-768-3p downregulation has been observed in tumor cells co-cultured with astrocytes, indicating that proliferative astrocytes form main components of the premetastatic niches of the brain parenchyma. MiRNA-768-3p downregulation results in KRAS overexpression, tumor outgrowth and drug resistance [146]. Zhao et al. examined the differential expression of miRNAs in primary lung adenocarcinomas and its BMs and found that miR-145, miR-214, miR-9 and miR1471 were deregulated [147]. They further found that miR145, which was downregulated in BMs, regulates the proliferation of lung cancer-derived A549 and SPC-A1 cells through the targeting of JAM-A and fascin. Interestingly, they also found that miR-145 downregulation was not associated with lymph node invasion and metastasis, suggesting that it is specific for BM [147]. Also miR-146a has been found to be deregulated in brain-seeking metastatic breast and lung cancer

cells. In metastatic breast cancer cells, it has been found that miR-146a overexpression inhibits β-catenin degradation and suppresses heterogeneous nuclear ribonucleoprotein C (HNRNPC) expression, both resulting in inhibition of cell invasion and migration [149]. Moreover, it has been shown that miR-146a can induce apoptosis and suppress the proliferation of NSCLC cells, and can enhance the inhibitory effects of drugs targeting the epidermal growth factor receptor (EGFR) [150]. Therefore, in patients with NSCLC miR146a downregulation may have a brain-seeking effect, similar to what has been seen in breast cancer patients [150]. Remon et al. found that miRNA-197 and miRNA-184 were upregulated in the EGFR-mutant of NSCLC BMs. Consequently, they concluded that these miRNAs may serve as biomarkers for stratifying NSCLC BM risk [153]. Hwang et al. found that miR-95-3p was downregulated in lung adenocarcinomaderived BMs [148]. MiR-95-3p directly targets the expression of cyclin D1. Correspondingly, its downregulation in BMs may result in increased invasiveness and clonogenicity, concomitant with cyclin D1 upregulation. As such, miR-95-3p may serve as a potential therapeutic target for LCBMs [148]. Long non-coding RNAs (lncRNAs) represent another class of non-protein coding transcripts with implications for cancer progression and metastasis [159–161]. Numerous lncRNAs have been reported to be associated with the development of cancer, such as the HOX antisense intergenic RNA (HOTAIR) [162, 163] and the antisense non-coding RNA in the INK4 locus (ANRIL) [164]. The latter lncRNA has been found to promote NSCLC cell proliferation and to inhibit its apoptosis by silencing the expression of KLF2 and P21 [164]. The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), also known as the non-coding nuclear-enriched abundant transcript 2 (NEAT2), is an extremely conserved lncRNA among mammals with implications in lung cancer metastasis [165]. Shen et al. [157] reported that MALAT1 expression may be elevated in invasive subtypes of lung cancer cells and to result in a propensity of these cells to metastasize to the brain. Concordantly, additional functional studies indicated that MALAT1 expression silencing may result in

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options

migration inhibition and BM formation of lung cancer cells through EMT suppression [157]. Therefore, augmentation of lncRNA MALAT1 expression may be a therapeutic avenue towards BM targeting in the near future.

with LCBM. By doing so, Hoffman et al. [181] found that SRS alone (without WBRT) resulted in a high risk of new BMs. Several other studies have subsequently confirmed this observation [182, 183]. Therefore, it seems that SRS combined with WBRT may be a more effective strategy for improving LCBM patient survival than SRS alone.

4 Therapeutic approaches 4.2 Chemotherapy Of the major challenges in medicine, an important one has been experienced in the field of cancer treatment [166]. Brain metastasis represents a substantial complication in the management of cancer, especially lung cancer [167]. The median survival of untreated patients with LCBM is approximately 4–7 weeks [168, 169]. Various strategies have been employed for the treatment of BM, including conventional approaches (surgery and radiotherapy), chemotherapy and targeted therapy, each with its own advantages and drawbacks. Since targeted therapies are expected to provide new opportunities beyond initial conventional therapies, the discovery of new molecular targets may result in more effective and specific treatment modalities. 4.1 Conventional therapies The initial treatment options for BMs include whole brain radiotherapy (WBRT), surgery and stereotactic radiosurgery (SRS) [37]. Although WBRT as first-line treatment for BMs in patients with lung cancer is widely accepted [170, 171], poor survival rates and radiation neurotoxicity are factors of concern [172]. Retrospective studies have shown that surgery may be an effective strategy to delay recurrence in patients with a small number of BMs, thereby improving their median survival rate [173]. There is also a growing body of evidence from prospective studies indicating that surgery followed by WBRT may be used as a standard treatment option for patients with single BMs [174, 175]. Patchell et al. [176] performed a randomized study on patients with single BMs and found that the interval from the time of treatment to recurrence was considerably increased in patients who underwent surgery and WBRT compared to those who underwent WBRT alone. Also, the median survival rate was found to be significantly improved after surgery combined with WBRT. These results have been corroborated by other prospective randomized studies [177]. However, due to the still inherent limitations of WBRT and surgery, stereotactic radiosurgery (SRS) has recently been investigated as an alternative strategy. SRS is a non-invasive technique by which high doses of radiation are delivered to tumor deposits. The major benefits of SRS include a quick treatment, low rates of complications and minimal side effects such as neurotoxicity [178, 179]. The median survival rate of patients following SRS is, however, still only 8–10 months [180]. Therefore, several investigators have set out to evaluate the effectiveness of SRS with or without WBRT in patients

Despite WBRT-based treatment approaches with or without SRS or surgery, BMs may still recur after treatment. Over the last decades, the efficacy of chemotherapeutic agents has extensively been studied and it has been found that metastatic lung cancer is one of the most chemotherapy-sensitive solid tumors [171, 184]. Currently, platinum-based drugs plus agents such as gemcitabine, paclitaxel, docetaxel or vinorelbine are adopted for first-line therapy in patients with metastatic lung cancer, whereas other agents such as docetaxel, erlotinib and pemetrexed have been clinically approved for second-line therapy. Clinical findings indicate that, with these therapies, the 60 month median survival (MS) is currently still <5% [185]. There are several limitations to the use of chemotherapeutics for the treatment of BMs. The BBB has, for instance, been identified as an obstacle blocking the diffusion of chemotherapeutic agents into the brain. Several efforts have been made to overcome this hurdle. One approach is the injection of agents directly into the cerebral arterial circulation to increase the driving gradient across the BBB [186, 187]. Another approach is to specifically use drugs that are known to cross the BBB, such as the alkylating agent temozolomide (Temodal®, Schering Plough; TMZ) [188]. In contrast to gliomas, which exhibit high P-glycoprotein expression levels similar to those in the normal brain vasculature, the Pglycoprotein expression levels in the vasculature at metastatic sites in the brain are similar to those in the vasculature of the periphery [91]. Based on this decreased P-glycoprotein expression, as well as the increased tissue pharmacologic concentrations of paclitaxel in metastatic brain tumors compared with gliomas, it may be concluded that the use of chemotherapeutic agents should be based on the histologic features of the brain tumor rather than the lipophilicity of the drug. As such, metastatic brain tumors with a low P-glycoprotein expression may be more sensitive to chemotherapy than primary brain tumors [189]. Also worth mentioning is that the BBB may be disrupted when the micro-metastatic deposits grow to a certain size, which may result in neo-angiogenesis and, consequently, access to the CNS of systemically administered chemotherapeutic drugs [171, 190]. 4.3 Novel targeted therapies Our increasing understanding of the molecular mechanisms underlying cancer progression and metastasis has led to the

M. Yousefi et al.

emergence of the era of ‘targeted therapy’. Targeted therapies, which are based on the use of miRNAs, anti-sense oligonucleotides, monoclonal antibodies and peptide mimetics to target angiogenesis-inducing factors, signaling molecules, modulators of apoptosis, growth factors and cell cycle regulators, have shown promising results in patients with advanced stage lung cancer and are anticipated to provide new opportunities beyond the above mentioned conventional therapies and chemotherapy [166]. 4.3.1 Targeting CSCs and EMT Given the fact that cancer stem cell-like cells (CSCs) may act as cancer cell progenitors and may confer chemo-resistance to patients, therapeutic targeting of CSCs may be used as a strategy to eradicate human cancers. It has been found that various signaling pathways that are involved in CSC formation may serve as therapeutic targets and, thus, may also be used for the targeting of lung cancer metastases [191]. Surface markers are commonly used for the identification of CSC populations. In case of SCLC, for instance, a CD44High/CD90+ phenotype has been correlated with CSCs, since it confers self-renewal and spheroid forming capacities to these cells [192]. Clinical studies have shown that the presence of CD133+/ABCG2+ cells poses a relapse risk to stage 1 NSCLC cases, whereas CD166+/CD44+ cells represent a population that possesses CSC characteristics such as self-renewal, differentiation and a transcript profile of stem cells [193]. As such, these stem cell-associated markers may unlock new avenues towards targeting CSC populations. Another promising strategy for eradicating CSCs is targeting their niche, i.e., a microenvironment that offers suitable conditions for stem cell growth. As indicated above, the CSC niche is composed of different cell types, molecular networks, soluble factors and extracellular matrix components [194] and supports CSCs to give rise to tumors [195]. Integrin, which is the primary receptor involved in cell–matrix adhesion, is an important player in the CSC microenvironment and is involved in cancer progression and drug resistance. It has been found, for instance, that treatment of NSCLC mouse models with cisplatin may result in the enrichment of CSC populations expressing integrin β4 [196]. Additional clinical studies have shown that upon chemotherapy integrin β1 and integrin α6 may be upregulated in cancers of the head and neck and breast, respectively. This latter observation supports a role of integrins in chemo-resistance, thereby highlighting them as potential targets [197, 198]. A putative role of other components of the lung CSC microenvironment as therapeutic targets remains to be established. EMT targeting may serve as another avenue towards the prevention of CSC formation and the development of metastases. Several lines of evidence suggest, for instance, that Oct4, Sox2 and Nanog can efficiently be used to impede the

growth of cancerous cells through EMT in vitro [199, 200]. Accordingly, the identification of EMT-related pathways involved in the CSC formation has become an appealing approach for the identification of suitable treatment targets [200]. It has already been found that anti-Wnt-2 monoclonal antibodies can be employed to induce cell death in NSCLC cells through the inhibition of Wnt signaling and, subsequently, the suppression of EMT [201]. Additional studies have shown that Wnt-7a may act as a tumor suppressor in a subset of NSCLC-derived cell lines, and that exogenous expression of Wnt-7a in these cell lines may lead to cellular transformation and epithelial differentiation [202]. As of yet, various drugs have been evaluated for the targeting lung CSCs, some of which have shown significant responses. For instance, thioridazine, which is a classical neurological drug, has recently been shown to dramatically inhibit sphere formation and to sensitize lung CSCs to the chemotherapeutic drugs 5-FU and cisplatin [203]. Curcumin, a yellow-pigmented polyphenol derived from turmeric, is another agent that has shown a great potential against CSCs. Currently, curcumin is gradually becoming known as a chemo-preventive and therapeutic agent against many human cancers, and it has been shown that curcumin may have significant effects on various characteristics of cancer cells including proliferation, inflammation, apoptosis, metastasis and angiogenesis [204–206]. Recently, curcumin has been shown to effectively inhibit the ability of NSCLC CSCs to form spheres, to suppress specific markers and to inhibit signaling pathways, such as the Wnt/β-catenin pathway [207], and the ability to sensitize them to cisplatin [208]. Aside from its potential use in medicine, we should consider that there are still many challenges ahead to eradicate CSCs. CSC populations are, for example, heterogeneous and none of the known CSC markers is selective and specific enough to target all CSCs. For instance, it has been shown that CD133 expression does not always mark CSCs, since there are no differences between CD133+ and CD133− with respect to stem cell characteristics such as self-renewal and differentiation [209]. In addition, it has been found that both CD44 + /CD24 low and CD44 + /CD24 + NSCLCderived A549 cells are capable of developing spheroids and forming colonies, indicating that at least for these cells CD44 and CD24 may not serve as exclusive CSC markers [210]. Therefore, cellular biomarkers should be used Bin combination^ to enhance their capacity to identify CSCs. In addition, it should be kept in mind that CSCs may share characteristics with normal tissue stem cells, including self-renewal and differentiation. Since, consequently, normal tissue stem cells may also be affected, new therapeutic procedures should be developed in such a way that they specifically target CSCs and not normal stem cells.

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options

4.3.2 MiRNA-based therapies The discovery of a role of miRNAs in cancer dissemination and metastasis has led to attempts to apply this knowledge to the design of novel diagnostic and therapeutic approaches [211]. It has been found, for example, that altered plasma miRNA levels, such as decreased miRNA-768-3p [212] or increased miRNA-200 [213] levels, may represent early signals for the application of aggressive treatment modalities [214]. Two approaches may be used for miRNA-based therapeutics. The first is a direct strategy, which is based on BmiRNA replacement^. By using (antisense) oligonucleotide mimics that are reminiscent of the original miRNA, a loss of function of a tumor suppressor miRNA can be restored or a gain of function of an oncogenic miRNA can be blocked. Through a second Bindirect strategy^, the expression and/or processing of miRNAs can be modulated using traditional compound-library screens [215]. Currently, the generation of efficient systems to increase the stability of miRNA mimics in the circulation and to adequately deliver them to target tissues are among the major challenges in this field. Several methods, including viral and non-viral delivery systems, have been used to increase the efficiency of miRNA-based treatment protocols [216]. Liposomes have recently emerged as an efficient non-viral delivery system since they are more stable than adenovirus-associated vector (AAV)-based delivery methods. Liposomal phospholipid bilayers have been successfully used to deliver miRNA-133b in mouse lung cancer models. Although liposomes may be superior to AAV-based delivery methods, their application is limited due to their toxicity, which is caused by their strong cationic charge [217]. Therefore, a neutral lipid emulsion has been generated with the purpose to reduce the toxicity of liposomes. The resulting neutral liposomes have successfully been used to deliver let-7 and miRNA-34a in mouse lung cancer models [218]. miRNAs may target several genes and pathways involved in the development of cancer. Consequently, a small number of miRNAs may affect a broad array of genes and pathways in cancer cells. In addition, trivial changes in miRNA expression patterns may induce broad phenotypic alterations. Therefore, the use of miRNAs seems to be more incisive than concoctions of small interfering RNAs (siRNAs) that are already used for treatment [219]. However, the same miRNA can affect a plethora of genes, regulating different processes in different cell types. Therefore, modulation of a specific miRNA may be beneficial in one cell type and harmful in another and, consequently, the question of how to deliver therapeutic miRNAs to target cells without eliciting side effects in other cells needs to be considered [220]. Another challenge for the targeting of BMs with miRNAs is that the BBB can be a major obstacle for the successful delivery of miRNA to the CNS. To overcome this obstacle, a few methods have been considered. The BTrojan Horse Liposome^ (THL) system encapsulates miRNA replacements or antagomirs

as therapeutic agents. In order to protect its cargo against nuclease degradation, this system is covered by liposomes. To stabilize the liposomes and target-specific BBB receptors, polyethyleneglycol (PEG) and peptidomimetic monoclonal antibodies are being used, respectively [221]. An additional polyethylenimine (PEI)-based delivery system, which is widely used in gene therapy, represents another novel method to increase the transmigration of cargo across the BBB. Through the rabies virus glycoprotein (RVG) and binding to the acetylcholine receptor, this system can easily bypass the BBB. Regarding the limitations mentioned for miRNA therapy and its potential solutions, it may be useful to evaluate the possibilities to modulate deregulated miRNAs in LCBM (Table 1). As of yet, the use of miRNAs for treating BMs still has to be explored and tested at pre-clinical levels [215]. 4.3.3 Other targeted therapies Pharmacological studies have indicated that suppression of the epidermal growth factor receptor (EGFR) may be used as a therapeutic approach for metastatic lung cancer. These studies have shown that EGFR inhibitors can increase the median survival time to 12 months [222]. Interestingly, other studies revealed that treatment of EGFR mutation-harboring LCBMs with EGFR-specific tyrosine kinase inhibitors (TKIs) may result in a median survival time of 15–20 months, with a response rate of 60–100% [223]. To date, several EGFR inhibitors have been studied in the context of LCBM treatment, including erlotinib [224], cetuximab [225], gefitinib [226] and murine monoclonal antibodies (mAbs) 225 and 528 [227]. One clinical trial has reported that erlotinib may extend the median survival to approximately 10 months with an overall response rate of 58% [228]. It is worth mentioning that the molecular mechanisms underlying the action of these antagonists have been uncovered. It has, for example, been found that murine mAbs 225 and 528 inhibit EGFR activation by competing with TGF-α or EGF, while cetuximab treatment results in EGFR expression downregulation by blocking EGFR-ligand binding and inducing receptor internalization [166]. Several ALK inhibitors have also been found to exhibit activity in the brain. These inhibitors, including crizotinib [229], ceritinib [230], alectinib [231] and PF-06463922, have successfully been tested in phase II/III clinical trials for the treatment of BM-positive NSCLC patients [232]. Gefitinib and erlotinib are small-molecule inhibitors that can reversibly inhibit the EGFR (without decreasing its protein level) by blocking the intracellular adenosine triphosphate-binding domain. Based on results of phase III clinical trials by the National Cancer Institute of Canada Clinical Trials Group, these antagonists have been approved by the USA food and drug administration (FDA) as a targeted therapy for metastatic NSCLC patients [233]. Dasatinib, a Src kinase inhibitor, is another attractive antagonist and, according to several clinical

M. Yousefi et al.

trials, it can inhibit the growth of cancerous cells through EMT interference in vitro [199]. In addition to the inhibition of the EGFR, ALK and Src, several angiogenesis-targeting agents, including aflibercept and bevacizumab, are currently being tested for their clinical efficacies. Aflibercept is an engineered, fully human and soluble VEGF receptor that has been licensed for phase III clinical trials in metastatic NSCLC as well as in metastatic colorectal cancer (CRC) to the pancreas. Bevacizumab, the first VEGF inhibitor approved by the USA FDA (2004), is the most advanced targeting agent for the treatment of solid tumors. This humanized IgG1 mAb neutralizes all human VEGF-A isoforms, thereby preventing the spread of cancer [166]. Programmed cell death protein 1 (PD-1)-axis inhibitors are currently being evaluated for the treatment of BMs in patients with advanced NSCLC. All available inhibitors, including atezolizumab, pembrolizumab, nivolumab and MEDI4736, have revealed promising results. In addition, it has been found that the PD-1 inhibitor pembrolizumab can enhance the efficacy of breast cancer treatment [234, 235], whereas a phase II trial is ongoing for brain metastatic melanoma and NSCLC patients [236]. Among the NSCLC trial patients, so far only one patient having a systemic response developed disease progression to the brain, suggesting an acceptable efficacy of PD-1 inhibitors for the treatment of BMs. Additional in-depth research is warranted in order to fully understand the PD-1-axis and other immune factors in the pathogenesis of LCBM. Targeted-therapy is highly dependent on the identification of ignite genes and its related signaling pathways. Once known, it can be turned into the most promising and successful molecular therapeutic strategy thus far [237]. Currently, there are several ongoing clinical trials and in vivo studies evaluating the anticancer effects of several targeted therapeutic agents in metastatic lung cancers (Table 2). It is expected that a further understanding of the exact molecular mechanisms involved in LCBMs will bring new prospects for targeted therapies through the design of small molecules and/or mAbs. 4.4 Combination therapy To increase the treatment efficacy of lung cancer patients with BMs, combination therapies may be promising alternatives. One clinical trial (RTOG 0320) studied the combination of EGFR TKI with WBRT plus SRS in patients with advanced NSCLC and found that the cytotoxicity may increase when WBRT is combined with either erlotinib or temozolomide. Others have shown that the combination of EGFR TKI with WBRT is efficient and safe for the treatment of advanced lung cancer [223]. In a retrospective study, upfront use of erlotinib was compared with its combination with SRS or WBRT in EGFR mutation-positive NSCLC patients with BMs. The overall survival for patients treated with SRS or WBRT

compared to erlotinib alone was found to be similar, but the time of intracranial progression in patients treated with SRS or WBRT was longer compared to treatment with erlotinib alone [238]. Sandler et al. [166] conducted a randomized phase III clinical trial to compare chemotherapeutic drugs alone or in co mbina tion w ith mABs dir ecte d ag ainst V EGF (bevacizumab) in patients with advanced NSCLC. In patients receiving additional bevacizumab treatment the median survival (MS) was 12.3 months compared to 10.3 months in the control group. This study also revealed a 35% response rate in patients treated with bevacizumab and a 15% response in the control group. Based on this study, bevacizumab was approved by the FDA in 2006 as a first-line combination therapy for advanced and metastatic NSCLC. Lim et al. [239] reported that treatment with SRS plus chemotherapy resulted in a median survival of 14.6 months in LCBM patients, whereas Liu et al. [240] found that there was no difference in the median survival of LCBM patients treated with WBRT plus EGFR TKIs compared to treatment with WBRT alone. As yet, insufficient reliable data are available on the efficacy of other combination therapies in BM-positive lung cancer patients, including that of miRNA-based therapies combined with other conventional and/or targeted therapies. In the future, additional potent therapeutic HER2 inhibiting agents may provide clinical advantage for the treatment of LCBMs.

5 Concluding remarks and future perspectives LCBMs account for a significant proportion of cancer-related deaths and, therefore, require serious attention. Conventional treatments do not lead to a complete eradication of tumor cells and, therefore, often result in relapse. Another complicating factor is that chemotherapeutic agents fail to cross the BBB. With such dismal implications, novel targeted therapies have gained interest. However, targeted therapies are highly dependent on knowledge of the molecular mechanisms and signaling pathways underlying the formation of BMs. Brain-specific metastasis of lung cancer depends not only on the distribution pattern of CTCs via the peripheral circulation, but also on the architecture of the brain vasculature and intrinsic abilities of CTCs to cross physical barriers and survive in the brain parenchyma. CSC-associated characteristics and surface markers may direct organ-specific metastasis. For instance, the expression of CD44 and the chemokine receptor CXCR4 on the surface of CSCs is thought to contribute to the metastasis of lung cancer cells to the brain, where their ligands are abundant. While transmigration across the BBB is a ratelimiting step in BM formation, it does not seem to be difficult for cancer cells to survive in the brain parenchyma. Endothelial cells in the brain are usually joined by tight junctions and, as such, ensure a selective permeability. However, in metastatic brain tumors the integrity of the BBB is

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options Table 2

Phase 1–3 clinical trials for targeted therapy of LCBM

Agent

Histology

Phase

Purpose

Status/outcome

http://clinicaltrials.gov identifier

GRN1005

NSCLC

2

NSCLC

2

CR or PR: ≥ 30% decrease in the sum of the largest diameter of target lesions Recruiting

NCT01497665

MK-3475

Erlotinib

NSCLC

3

Completed/no study results recorded

NCT01887795

Sunitinib

NSCLC

2

Completed/no study results recorded

NCT00372775

Gefitinib and pemetrexed/ cisplatin

NSCLC

2

Recruiting

NCT01951469

Dasatinib

NSCLC

2

To assess the efficacy, safety, and tolerability of GRN1005 in LCBM patients To assess the activity of pembrolizumab (MK-3475) in untreated melanoma or NSCLC-associated BMs To assess the efficacy of erlotinib concurrent with WBRT as first-line treatment compared to WBRT alone in LCBM patients To assess the safety, tolerability and efficacy of SU011248 in LCBM patients To assess the efficacy of gefitinib alone and in combination with pemetrexed/ cisplatin in LCBM patients harboring EGFR mutations To assess the safety of and response to dasatinib in LCBM patients

NCT00787267

Cetuximab

-

2

CR or PR: at least a 30% decrease in the sum of the largest diameter of target lesions Completed/no study results recorded

BKM120, cisplatin, etoposide

SCLC

1

Completed/no study results recorded

NCT02194049

Crizotinib, AT13387

NSCLC

1/2

Ongoing, not recruiting

NCT01712217

Alectinib, bevacizumab

NSCLC

1/2

Recruiting

NCT02521051

To assess the efficacy of cetuximab in patients with recurrent or stage IIIB or stage IV lung cancer To assess side-effects and dose optimum of the PI3K inhibitor BKM120 when administered together with cisplatin and etoposide to LCBM patients To assess the safety and efficacy of AT13387 alone or in combination with crizotinib in LCBM patients To assess the efficacy of alectinib and bevacizumab as combination therapy in LCBN patients

compromised resulting in an increased permeability. The exact molecular mechanism underlying this hyper-permeability still remains to be resolved. In SCLC, the Rho/ROCK signaling pathway is known to play a pivotal role in the breakdown of intercellular junctions through increasing actomyosin contractility. In addition, it has been found that tumor cells may release VEGF and other cytokines that increase vessel permeability. After transmigration of tumor cells across the BBB, anti-PA serpins have been found to play a significant role in the evasion of cancer cells from apoptosis, whereas endothelin-1 (ET-1) receptors have been found to play important roles in cancer cell growth and chemo-resistance within the brain parenchyma. The expression of PCDH7 by tumor cells favors the assembly of connexin 43 (Cx43) gap junctions and the transfer of cGAMP from tumor cells to astrocytes which, consequently, results in activation of the STAT1 and NF-κB signaling pathways in the tumor cells, resulting in tumor growth and chemo-resistance. Systemic mediators, such as the ECM-remodeling protease ADAM9 and the chemokines CXCL12 and CXCL16, have been found to

NCT02085070

NCT00103207

precondition the brain by creating pre-metastatic niches before the actual arrival of metastatic lung cancer cells. EGF/EGFR, HGF/c-MET, VEGF and PLGF are among the most important growth factors and RTKs to be involved in LCBM formation. According to pharmacological studies, suppression of these modules through TKIs should be a therapeutic focus for metastatic lung cancer. The application of TKIs has already shown to increase the median survival of the patients for up to 20 months. Adhesion molecules represent another class of potential targets for the suppression of LCBMs, but the efficacy of their targeting still remains to be established. Noncoding RNAs offer another avenue towards identification of the molecular mechanisms underlying LCBM formation. Several miRNAs, including miR-378, miR-328 and miR200 have been reported to be deregulated in lung cancers with implications for BMs. Given the fact that simple changes in miRNA expression can have broad phenotypic effects, miRNAs may conceivably serve as potential targets for the suppression of LCBMs. However, miRNA expression modulation may elicit unwanted side effects through expression

M. Yousefi et al.

alteration of off-target genes. Therefore, studies aimed at targeting miRNAs must be designed with ultimate care. Lastly, combination therapies through the simultaneous targeting of multiple arms in LCBM treatment protocols may be an interesting area of study. Clearly, the in-depth elucidation of the molecular mechanisms underlying lung cancer metastasis will open up new avenues towards LCBM targeting and will provide interesting opportunities for research aimed at developing novel efficacious therapies. Acknowledgments The authors would like to thank the faculty members of the Department of Medical Genetics, School of Medicine, Mashhad University of Medical Sciences for their assistance. Compliance with ethical standards Conflict of interest The authors declare no conflict of interest.

References 1.

2.

3. 4.

5.

6. 7. 8.

9. 10.

11.

12.

13.

R. Siegel, E. Ward, O. Brawley, A. Jemal, Cancer statistics, 2011: The impact of eliminating socioeconomic and racial disparities on premature cancer deaths. CA Cancer J. Clin. 61, 212–236 (2011) SEER. Stat fact sheets: lung and bronchus cancer 2016 [cited 2016 September 2016] Available from: http://seer.cancer.gov/statfacts/ html/lungb.html R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2016. CA Cancer J. Clin. 66, 7–30 (2016) SEER. Cancer statistics review 1975-2013 2013 [cited 2016 September 2016] Available from: http://seer.cancer.gov/csr/ 1975_2013/browse_csr.php Centers for Disease Control and Prevention. Smoking & tobacco use. [cited 2016 September 2016]. Available from: http://www. cdc.gov/tobacco/data_statistics/tables/trends/cig_smoking/ K. Sawada. [Lung cancer: classification by cell types]. Nihon rinsho Japanese journal of clinical medicine 38, 2574–80 (1980) W.C. Dempke, T. Suto, M. Reck, Targeted therapies for non-small cell lung cancer. Lung Cancer 67, 257–274 (2010) M. Dragoj, Z. Milosevic, J. Bankovic, N. Tanic, M. Pesic, T. Stankovic, Targeting CXCR4 and FAK reverses doxorubicin resistance and suppresses invasion in non-small cell lung carcinoma. Cell. Oncol. 40, 47–62 (2017) I.T. Gavrilovic, J.B. Posner, Brain metastases: Epidemiology and pathophysiology. J. Neuro-Oncol. 75, 5–14 (2005) J.B. Sorensen, H.H. Hansen, M. Hansen, P. Dombernowsky, Brain metastases in adenocarcinoma of the lung: Frequency, risk groups, and prognosis. J. Clin. Oncol. 6, 1474–1480 (1988) A. Mujoomdar, J.H. Austin, R. Malhotra, C.A. Powell, G.D. Pearson, M.C. Shiau, H. Raftopoulos, Clinical predictors of metastatic disease to the brain from non-small cell lung carcinoma: Primary tumor size, cell type, and lymph node metastases. Radiology 242, 882–888 (2007) J. Budczies, M. von Winterfeld, F. Klauschen, M. Bockmayr, J.K. Lennerz, C. Denkert, T. Wolf, A. Warth, M. Dietel, I. Anagnostopoulos, W. Weichert, D. Wittschieber, A. Stenzinger, The landscape of metastatic progression patterns across major human cancers. Oncotarget 6, 570–583 (2015) B.D. Fox, V.J. Cheung, A.J. Patel, D. Suki, G. Rao. Epidemiology of metastatic brain tumors. Neurosurg. Clin. N. Am. 22, 1–6 (2011)

14.

J.D. Cox, R.A. Yesner, Adenocarcinoma of the lung: Recent results from the veterans administration lung group. Am. Rev. Respir. Dis. 120, 1025–1029 (1979) 15. J.S. Barnholtz-Sloan, A.E. Sloan, F.G. Davis, F.D. Vigneau, P. Lai, R.E. Sawaya, Incidence proportions of brain metastases in patients diagnosed (1973 to 2001) in the metropolitan Detroit cancer surveillance system. J. Clin. Oncol. 22, 2865–2872 (2004) 16. F.G. Davis, T.A. Dolecek, B.J. McCarthy, J.L. Villano, Toward determining the lifetime occurrence of metastatic brain tumors estimated from 2007 United States cancer incidence data. NeuroOncology 14, 1171–1177 (2012) 17. Z. Ji, N. Bi, J. Wang, Z. Hui, Z. Xiao, Q. Feng, Z. Zhou, D. Chen, J. Lv, J. Liang, C. Fan, L. Liu, L. Wang, Risk factors for brain metastases in locally advanced non-small cell lung cancer with definitive chest radiation. Int. J. Radiat. Oncol. Biol. Phys. 89, 330–337 (2014) 18. G.L. Ceresoli, M. Reni, G. Chiesa, A. Carretta, S. Schipani, P. Passoni, A. Bolognesi, P. Zannini, E. Villa, Brain metastases in locally advanced nonsmall cell lung carcinoma after multimodality treatment: Risk factors analysis. Cancer 95, 605– 612 (2002) 19. L. Gaspar, C. Scott, M. Rotman, S. Asbell, T. Phillips, T. Wasserman, W.G. McKenna, R. Byhardt, Recursive partitioning analysis (RPA) of prognostic factors in three radiation therapy oncology group (RTOG) brain metastases trials. Int. J. Radiat. Oncol. Biol. Phys. 37, 745–751 (1997) 20. M. Hanibuchi, S.J. Kim, I.J. Fidler, Y. Nishioka, The molecular biology of lung cancer brain metastasis: An overview of current comprehensions and future perspectives. J. Med. Investig. 61, 241–253 (2014) 21. L. Ding, G. Getz, D.A. Wheeler, E.R. Mardis, M.D. McLellan, K. Cibulskis, C. Sougnez, H. Greulich, D.M. Muzny, M.B. Morgan, L. Fulton, R.S. Fulton, Q. Zhang, M.C. Wendl, M.S. Lawrence, D.E. Larson, K. Chen, D.J. Dooling, A. Sabo, A.C. Hawes, H. Shen, S.N. Jhangiani, L.R. Lewis, O. Hall, Y. Zhu, T. Mathew, Y. Ren, J. Yao, S.E. Scherer, K. Clerc, G.A. Metcalf, B. Ng, A. Milosavljevic, M.L. Gonzalez-Garay, J.R. Osborne, R. Meyer, X. Shi, Y. Tang, D.C. Koboldt, L. Lin, R. Abbott, T.L. Miner, C. Pohl, G. Fewell, C. Haipek, H. Schmidt, B.H. Dunford-Shore, A. Kraja, S.D. Crosby, C.S. Sawyer, T. Vickery, S. Sander, J. Robinson, W. Winckler, J. Baldwin, L.R. Chirieac, A. Dutt, T. Fennell, M. Hanna, B.E. Johnson, R.C. Onofrio, R.K. Thomas, G. Tonon, B.A. Weir, X. Zhao, L. Ziaugra, M.C. Zody, T. Giordano, M.B. Orringer, J.A. Roth, M.R. Spitz, I.I. Wistuba, B. Ozenberger, P.J. Good, A.C. Chang, D.G. Beer, M.A. Watson, M. Ladanyi, S. Broderick, A. Yoshizawa, W.D. Travis, W. Pao, M.A. Province, G.M. Weinstock, H.E. Varmus, S.B. Gabriel, E.S. Lander, R.A. Gibbs, M. Meyerson, R.K. Wilson, Somatic mutations affect key pathways in lung adenocarcinoma. Nature 455, 1069–1075 (2008) 22. S. Matsumoto, R. Iwakawa, K. Takahashi, T. Kohno, Y. Nakanishi, Y. Matsuno, K. Suzuki, M. Nakamoto, E. Shimizu, J.D. Minna, J. Yokota, Prevalence and specificity of LKB1 genetic alterations in lung cancers. Oncogene 26, 5911–5918 (2007) 23. M. Ji, Y. Liu, Q. Li, X.D. Li, W.Q. Zhao, H. Zhang, X. Zhang, J.T. Jiang, C.P. Wu, PD-1/PD-L1 pathway in non-small-cell lung cancer and its relation with EGFR mutation. J. Transl. Med. 13 (2015) 24. W. Shi, A.P. Dicker, CNS metastases in patients with non-smallcell lung cancer and ALK gene rearrangement. J. Clin. Oncol. 34, 107–109 (2016) 25. R.C. Doebele, X. Lu, C. Sumey, D.A. Maxson, A.J. Weickhardt, A.B. Oton, P.A. Bunn Jr., A.E. Baron, W.A. Franklin, D.L. Aisner, M. Varella-Garcia, D.R. Camidge, Oncogene status predicts patterns of metastatic spread in treatment-naive nonsmall cell lung cancer. Cancer 118, 4502–4511 (2012)

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options 26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38. 39. 40. 41.

42.

43. 44.

D.Y. Shin, I.I. Na, C.H. Kim, S. Park, H. Baek, S.H. Yang, EGFR mutation and brain metastasis in pulmonary adenocarcinomas. J. Thorac. Oncol. 9, 195–199 (2014) C. Villalva, V. Duranton-Tanneur, K. Guilloteau, F. BurelVandenbos, M. Wager, J. Doyen, P.M. Levillain, D. Fontaine, H. Blons, F. Pedeutour, L. Karayan-Tapon, EGFR, KRAS, BRAF, and HER-2 molecular status in brain metastases from 77 NSCLC patients. Cancer Med. 2, 296–304 (2013) A.B. Cortot, A. Italiano, F. Burel-Vandenbos, G. Martel-Planche, P. Hainaut, KRAS mutation status in primary nonsmall cell lung cancer and matched metastases. Cancer 116, 2682–2687 (2010) L. Daniele, P. Cassoni, E. Bacillo, S. Cappia, L. Righi, M. Volante, F. Tondat, G. Inghirami, A. Sapino, G.V. Scagliotti, M. Papotti, S. Novello, Epidermal growth factor receptor gene in primary tumor and metastatic sites from non-small cell lung cancer. J. Thorac. Oncol. 4, 684–688 (2009) C. Han, H. Zou, J. Ma, Y. Zhou, J. Zhao. [Comparison of EGFR and KRAS status between primary non-small cell lung cancer and corresponding metastases: a systematic review and meta-analysis]. Zhongguo Fei Ai Za Zhi 13, 882–891 (2010) S.E. Monaco, M.N. Nikiforova, K. Cieply, L.A. Teot, W.E. Khalbuss, S. Dacic, A comparison of EGFR and KRAS status in primary lung carcinoma and matched metastases. Hum. Pathol. 41, 94–102 (2010) D. Munfus-McCray, S. Harada, C. Adams, F. Askin, D. Clark, E. Gabrielson, Q.K. Li, EGFR and KRAS mutations in metastatic lung adenocarcinomas. Hum. Pathol. 42, 1447–1453 (2011) N. Zhao, M.D. Wilkerson, U. Shah, X. Yin, A. Wang, M.C. Hayward, P. Roberts, C.B. Lee, A.M. Parsons, L.B. Thorne, B.E. Haithcock, J.E. Grilley-Olson, T.E. Stinchcombe, W.K. Funkhouser, K.K. Wong, N.E. Sharpless, D.N. Hayes, Alterations of LKB1 and KRAS and risk of brain metastasis: Comprehensive characterization by mutation analysis, copy number, and gene expression in non-small-cell lung carcinoma. Lung Cancer 86, 255–261 (2014) X. Ding, H. Dai, Z. Hui, W. Ji, J. Liang, J. Lv, Z. Zhou, W. Yin, J. He, L. Wang, Risk factors of brain metastases in completely resected pathological stage IIIA-N2 non-small cell lung cancer. Radiat. Oncol. 7, 119 (2012) E.A. Maher, J. Mietz, C.L. Arteaga, R.A. DePinho, S. Mohla, Brain metastasis: Opportunities in basic and translational research. Cancer Res. 69, 6015–6020 (2009) I.J. Fidler, K. Balasubramanian, Q. Lin, S.W. Kim, S.J. Kim, The brain microenvironment and cancer metastasis. Mol. Cell 30, 93– 98 (2010) B. Tayyeb, M. Parvin, Pathogenesis of breast cancer metastasis to brain: A comprehensive approach to the signaling network. Mol. Neurobiol. 53, 446–454 (2016) D. Hanahan, R.A. Weinberg, Hallmarks of cancer: The next generation. Cell 144, 646–674 (2011) R. Paduch, The role of lymphangiogenesis and angiogenesis in tumor metastasis. Cell. Oncol. 39, 397–410 (2016) M. S. Wicha, S. Liu, G. Dontu. Cancer stem cells: an old idea–a paradigm shift. Cancer Res 66, 1883–90; discussion 95–6 (2006) J.P. Thiery, H. Acloque, R.Y. Huang, M.A. Nieto, Epithelialmesenchymal transitions in development and disease. Cell 139, 871–890 (2009) S.A. Mani, W. Guo, M.J. Liao, E.N. Eaton, A. Ayyanan, A.Y. Zhou, M. Brooks, F. Reinhard, C.C. Zhang, M. Shipitsin, L.L. Campbell, K. Polyak, C. Brisken, J. Yang, R.A. Weinberg, The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133, 704–715 (2008) T. Reya, S.J. Morrison, M.F. Clarke, I.L. Weissman, Stem cells, cancer, and cancer stem cells. Nature 414, 105–111 (2001) S. Gottschling, P.A. Schnabel, F.J.F. Herth, E. Herpel, Are we missing the target? – Cancer stem cells and drug resistance in

45.

46.

47.

48.

49. 50. 51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

non-small cell lung cancer. Cancer Genomics Proteomics 9, 275–286 (2012) J. Fuxe, T. Vincent, A. Garcia de Herreros, Transcriptional crosstalk between TGF-beta and stem cell pathways in tumor cell invasion: Role of EMT promoting Smad complexes. Cell Cycle 9, 2363–2374 (2010) S.Y. Shin, O. Rath, A. Zebisch, S.M. Choo, W. Kolch, K.H. Cho, Functional roles of multiple feedback loops in extracellular signalregulated kinase and Wnt signaling pathways that regulate epithelial-mesenchymal transition. Cancer Res. 70, 6715–6724 (2010) A. Eger, A. Stockinger, J. Park, E. Langkopf, M. Mikula, J. Gotzmann, W. Mikulits, H. Beug, R. Foisner, beta-Catenin and TGFbeta signalling cooperate to maintain a mesenchymal phenotype after FosER-induced epithelial to mesenchymal transition. Oncogene 23, 2672–2680 (2004) L.A. Timmerman, J. Grego-Bessa, A. Raya, E. Bertran, J.M. Perez-Pomares, J. Diez, S. Aranda, S. Palomo, F. McCormick, J.C. Izpisua-Belmonte, J.L. de la Pompa, Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation. Genes Dev. 18, 99–115 (2004) D. Xiao, J. He, Epithelial mesenchymal transition and lung cancer. J Thorac Dis 2, 154–159 (2010) B. De Craene, G. Berx, Regulatory networks defining EMT during cancer initiation and progression. Nat. Rev. Cancer 13, 97–110 (2013) U. Valcourt, M. Kowanetz, H. Niimi, C.H. Heldin, A. Moustakas, TGF-beta and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition. Mol. Biol. Cell 16, 1987–2002 (2005) P. Chunhacha, V. Sriuranpong, P. Chanvorachote, Epithelialmesenchymal transition mediates anoikis resistance and enhances invasion in pleural effusion-derived human lung cancer cells. Oncol. Lett. 5, 1043–1047 (2013) G. Berx, F. van Roy, Involvement of members of the cadherin superfamily in cancer. Cold Spring Harb. Perspect. Biol. 1, a003129 (2009) J.Y. Yoo, S.H. Yang, J.E. Lee, D.G. Cho, H.K. Kim, S.H. Kim, I.S. Kim, J.T. Hong, J.H. Sung, B.C. Son, S.W. Lee, E-cadherin as a predictive marker of brain metastasis in non-small-cell lung cancer, and its regulation by pioglitazone in a preclinical model. J. Neuro-Oncol. 109, 219–227 (2012) U. Cavallaro, G. Christofori, Cell adhesion and signalling by cadherins and Ig-CAMs in cancer. Nat. Rev. Cancer 4, 118–132 (2004) H. Grinberg-Rashi, E. Ofek, M. Perelman, J. Skarda, P. Yaron, M. Hajduch, J. Jacob-Hirsch, N. Amariglio, M. Krupsky, D.A. Simansky, Z. Ram, R. Pfeffer, I. Galernter, D.M. Steinberg, I. Ben-Dov, G. Rechavi, S. Izraeli, The expression of three genes in primary non-small cell lung cancer is associated with metastatic spread to the brain. Clin. Cancer Res. 15, 1755–1761 (2009) M. Dauphin, C. Barbe, S. Lemaire, B. Nawrocki-Raby, E. Lagonotte, G. Delepine, P. Birembaut, C. Gilles, M. Polette, Vimentin expression predicts the occurrence of metastases in non small cell lung carcinomas. Lung Cancer 81, 117–122 (2013) M. Polette, C. Gilles, S. de Bentzmann, D. Gruenert, J.M. Tournier, P. Birembaut, Association of fibroblastoid features with the invasive phenotype in human bronchial cancer cell lines. Clin. Exp. Metastasis 16, 105–112 (1998) R.L. Yauch, T. Januario, D.A. Eberhard, G. Cavet, W. Zhu, L. Fu, T.Q. Pham, R. Soriano, J. Stinson, S. Seshagiri, Z. Modrusan, C.Y. Lin, V. O'Neill, L.C. Amler, Epithelial versus mesenchymal phenotype determines in vitro sensitivity and predicts clinical activity of erlotinib in lung cancer patients. Clin. Cancer Res. 11, 8686– 8698 (2005) S. Thomson, E. Buck, F. Petti, G. Griffin, E. Brown, N. Ramnarine, K.K. Iwata, N. Gibson, J.D. Haley, Epithelial to mesenchymal

M. Yousefi et al. transition is a determinant of sensitivity of non-small-cell lung carcinoma cell lines and xenografts to epidermal growth factor receptor inhibition. Cancer Res. 65, 9455–9462 (2005) 61. X. Sun, P. Fa, Z. Cui, Y. Xia, L. Sun, Z. Li, A. Tang, Y. Gui, Z. Cai, The EDA-containing cellular fibronectin induces epithelialmesenchymal transition in lung cancer cells through integrin alpha9beta1-mediated activation of PI3-K/AKT and Erk1/2. Carcinogenesis 35, 184–191 (2014) 62. S. Singh, S. Chellappan, Lung cancer stem cells: Molecular features and therapeutic targets. Mol. Asp. Med. 39, 50–60 (2014) 63. L.S. Orlichenko, D.C. Radisky, Matrix metalloproteinases stimulate epithelial-mesenchymal transition during tumor development. Clin. Exp. Metastasis 25, 593–600 (2008) 64. J.P. Thiery, J.P. Sleeman, Complex networks orchestrate epithelial-mesenchymal transitions. Nat. Rev. Mol. Cell Biol. 7, 131–142 (2006) 65. B. De Craene, B. Gilbert, C. Stove, E. Bruyneel, F. van Roy, G. Berx, The transcription factor snail induces tumor cell invasion through modulation of the epithelial cell differentiation program. Cancer Res. 65, 6237–6244 (2005) 66. J.P. Thiery, Epithelial-mesenchymal transitions in tumour progression. Nat. Rev. Cancer 2, 442–454 (2002) 67. K.R. Fischer, A. Durrans, S. Lee, J. Sheng, F. Li, S.T. Wong, H. Choi, T. El Rayes, S. Ryu, J. Troeger, R.F. Schwabe, L.T. Vahdat, N.K. Altorki, V. Mittal, D. Gao, Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance. Nature 527, 472–476 (2015) 68. X. Zheng, J.L. Carstens, J. Kim, M. Scheible, J. Kaye, H. Sugimoto, C.C. Wu, V.S. LeBleu, R. Kalluri, Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer. Nature 527, 525–530 (2015) 69. J.E. Chu, A.L. Allan, The role of cancer stem cells in the organ tropism of breast cancer metastasis: A mechanistic balance between the "seed" and the "soil"? Intl. J. Breast Cancer 2012, 209748 (2012) 70. L.F. Brown, B. Berse, L. Van de Water, A. Papadopoulos-Sergiou, C.A. Perruzzi, E.J. Manseau, H.F. Dvorak, D.R. Senger, Expression and distribution of osteopontin in human tissues: Widespread association with luminal epithelial surfaces. Mol. Biol. Cell 3, 1169–1180 (1992) 71. J.E. Draffin, S. McFarlane, A. Hill, P.G. Johnston, D.J. Waugh, CD44 potentiates the adherence of metastatic prostate and breast cancer cells to bone marrow endothelial cells. Cancer Res. 64, 5702–5711 (2004) 72. M.Z. Dewan, S. Ahmed, Y. Iwasaki, K. Ohba, M. Toi, N. Yamamoto. Stromal cell-derived factor-1 and CXCR4 receptor interaction in tumor growth and metastasis of breast cancer. Biomed. Pharmacother. 60, 273–276 (2006) 73. B. Furusato, A. Mohamed, M. Uhlen, J.S. Rhim, CXCR4 and cancer. Pathol. Int. 60, 497–505 (2010) 74. H.A. Kargi, M.F. Kuyucuoglu, M. Alakavuklar, O. Akpinar, S. Erk, CD44 expression in metastatic and non-metastatic non-small cell lung cancers. Cancer Lett. 119, 27–30 (1997) 75. A. Salmaggi, E. Maderna, C. Calatozzolo, P. Gaviani, A. Canazza, I. Milanesi, A. Silvani, F. DiMeco, A. Carbone, B. Pollo, CXCL12, CXCR4 and CXCR7 expression in brain metastases. Cancer Biol Ther 8, 1608–1614 (2009) 76. K.R. Hess, G.R. Varadhachary, S.H. Taylor, W. Wei, M.N. Raber, R. Lenzi, J.L. Abbruzzese, Metastatic patterns in adenocarcinoma. Cancer 106, 1624–1633 (2006) 77. A.C. Obenauf, J. Massague, Surviving at a distance: Organ specific metastasis. Trends Cancer 1, 76–91 (2015) 78. R.R. Langley, I.J. Fidler, The seed and soil hypothesis revisited– the role of tumor-stroma interactions in metastasis to different organs. Int. J. Cancer 128, 2527–2535 (2011)

79.

80. 81.

82.

83. 84.

85.

86.

87.

88.

89.

90.

91.

92.

93. 94. 95.

96.

97.

H. Sugiura, K. Yamada, T. Sugiura, T. Hida, T. Mitsudomi, Predictors of survival in patients with bone metastasis of lung cancer. Clin. Orthop. Relat. Res. 466, 729–736 (2008) L.E. Gaspar, Brain metastases in lung cancer. Expert. Rev. Anticancer. Ther. 4, 259–270 (2004) N.J. Abbott, L. Ronnback, E. Hansson, Astrocyte-endothelial interactions at the blood-brain barrier. Nat. Rev. Neurosci. 7, 41–53 (2006) B. Li, W.D. Zhao, Z.M. Tan, W.G. Fang, L. Zhu, Y.H. Chen, Involvement of rho/ROCK signalling in small cell lung cancer migration through human brain microvascular endothelial cells. FEBS Lett. 580, 4252–4260 (2006) B. Wojciak-Stothard, A.J. Ridley, Rho GTPases and the regulation of endothelial permeability. Vasc. Pharmacol. 39, 187–199 (2002) J. JuanYin, K. Tracy, L. Zhang, J. Munasinghe, E. Shapiro, A. Koretsky, K. Kelly, Noninvasive imaging of the functional effects of anti-VEGF therapy on tumor cell extravasation and regional blood volume in an experimental brain metastasis model. Clin. Exp. Metastasis 26, 403–414 (2009) M. Lorger, B. Felding-Habermann, Capturing changes in the brain microenvironment during initial steps of breast cancer brain metastasis. Am. J. Pathol. 176, 2958–2971 (2010) L. Sevenich, R.L. Bowman, S.D. Mason, D.F. Quail, F. Rapaport, B.T. Elie, E. Brogi, P.K. Brastianos, W.C. Hahn, L.J. Holsinger, J. Massague, C.S. Leslie, J.A. Joyce, Analysis of tumour- and stroma-supplied proteolytic networks reveals a brain-metastasispromoting role for cathepsin S. Nat. Cell Biol. 16, 876–888 (2014) P.D. Bos, X.H. Zhang, C. Nadal, W. Shu, R.R. Gomis, D.X. Nguyen, A.J. Minn, M.J. van de Vijver, W.L. Gerald, J.A. Foekens, J. Massague, Genes that mediate breast cancer metastasis to the brain. Nature 459, 1005–1009 (2009) W. Zhou, M.Y. Fong, Y. Min, G. Somlo, L. Liu, M.R. Palomares, Y. Yu, A. Chow, S.T. O'Connor, A.R. Chin, Y. Yen, Y. Wang, E.G. Marcusson, P. Chu, J. Wu, X. Wu, A.X. Li, Z. Li, H. Gao, X. Ren, M.P. Boldin, P.C. Lin, S.E. Wang, Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell 25, 501–515 (2014) N. Tominaga, N. Kosaka, M. Ono, T. Katsuda, Y. Yoshioka, K. Tamura, J. Lotvall, H. Nakagama, T. Ochiya, Brain metastatic cancer cells release microRNA-181c-containing extracellular vesicles capable of destructing blood-brain barrier. Nat. Commun. 6, 6716 (2015) J.F. Deeken, W. Loscher, The blood-brain barrier and cancer: Transporters, treatment, and Trojan horses. Clin. Cancer Res. 13, 1663–1674 (2007) A. Regina, M. Demeule, A. Laplante, J. Jodoin, C. Dagenais, F. Berthelet, A. Moghrabi, R. Beliveau, Multidrug resistance in brain tumors: Roles of the blood-brain barrier. Cancer Metastasis Rev. 20, 13–25 (2001) Y. Kienast, L. von Baumgarten, M. Fuhrmann, W.E. Klinkert, R. Goldbrunner, J. Herms, F. Winkler, Real-time imaging reveals the single steps of brain metastasis formation. Nat. Med. 16, 116–122 (2010) J. Massague, A.C. Obenauf, Metastatic colonization by circulating tumour cells. Nature 529, 298–306 (2016) D. Spano, M. Zollo, Tumor microenvironment: A main actor in the metastasis process. Clin. Exp. Metastasis 29, 381–395 (2012) H. Fazilaty, M. Gardaneh, T. Bahrami, A. Salmaninejad, B. Behnam, Crosstalk between breast cancer stem cells and metastatic niche: Emerging molecular metastasis pathway? Tumour Biol. 34, 2019–2030 (2013) S.J. Morrison, A.C. Spradling, Stem cells and niches: Mechanisms that promote stem cell maintenance throughout life. Cell 132, 598–611 (2008) Y.C. Hsu, L. Li, E. Fuchs, Emerging interactions between skin stem cells and their niches. Nat. Med. 20, 847–856 (2014)

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options 98.

99.

100.

101.

102.

103.

104. 105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

115.

T. Oskarsson, E. Batlle, J. Massague, Metastatic stem cells: Sources, niches, and vital pathways. Cell Stem Cell 14, 306–321 (2014) W.S. Carbonell, O. Ansorge, N. Sibson, R. Muschel, The vascular basement membrane as "soil" in brain metastasis. PLoS One 4, e5857 (2009) M. Valiente, A.C. Obenauf, X. Jin, Q. Chen, X.H. Zhang, D.J. Lee, J.E. Chaft, M.G. Kris, J.T. Huse, E. Brogi, J. Massague, Serpins promote cancer cell survival and vascular co-option in brain metastasis. Cell 156, 1002–1016 (2014) S.W. Kim, H.J. Choi, H.J. Lee, J. He, Q. Wu, R.R. Langley, I.J. Fidler, S.J. Kim, Role of the endothelin axis in astrocyte- and endothelial cell-mediated chemoprotection of cancer cells. Neuro-Oncology 16, 1585–1598 (2014) Q. Chen, A. Boire, X. Jin, M. Valiente, E.E. Er, A. Lopez-Soto, L.S. Jacob, R. Patwa, H. Shah, K. Xu, J.R. Cross, J. Massague, Carcinoma-astrocyte gap junctions promote brain metastasis by cGAMP transfer. Nature 533, 493–498 (2016) S.S. McAllister, R.A. Weinberg, The tumour-induced systemic environment as a critical regulator of cancer progression and metastasis. Nat. Cell Biol. 16, 717–727 (2014) D.R. Edwards, M.M. Handsley, C.J. Pennington, The ADAM metalloproteinases. Mol. Asp. Med. 29, 258–289 (2008) N. Rocks, G. Paulissen, M. El Hour, F. Quesada, C. Crahay, M. Gueders, J.M. Foidart, A. Noel, D. Cataldo, Emerging roles of ADAM and ADAMTS metalloproteinases in cancer. Biochimie 90, 369–379 (2008) Y. Shintani, S. Higashiyama, M. Ohta, H. Hirabayashi, S. Yamamoto, T. Yoshimasu, H. Matsuda, N. Matsuura, Overexpression of ADAM9 in non-small cell lung cancer correlates with brain metastasis. Cancer Res. 64, 4190–4196 (2004) M. Noda, T. Seike, K. Fujita, Y. Yamakawa, M. Kido, H. Iguchi, The role of immune cells in brain metastasis of lung cancer cells and neuron-tumor cell interaction. Ross. Fiziol. Zh. Im. I M Sechenova 95, 1386–1396 (2009) S. Feng, J. Cen, Y. Huang, H. Shen, L. Yao, Y. Wang, Z. Chen, Matrix metalloproteinase-2 and -9 secreted by leukemic cells increase the permeability of blood-brain barrier by disrupting tight junction proteins. PLoS One 6, e20599 (2011) L. Hu, J. Zhang, H. Zhu, J. Min, Y. Feng, H. Zhang, Biological characteristics of a specific brain metastatic cell line derived from human lung adenocarcinoma. Med. Oncol. 27, 708–714 (2010) E.C. Keeley, B. Mehrad, R.M. Strieter, CXC chemokines in cancer angiogenesis and metastases. Adv. Cancer Res. 106, 91–111 (2010) S. Paratore, G.L. Banna, M. D'Arrigo, S. Saita, R. Iemmolo, L. Lucenti, D. Bellia, H. Lipari, C. Buscarino, R. Cunsolo, S. Cavallaro, CXCR4 and CXCL12 immunoreactivities differentiate primary non-small-cell lung cancer with or without brain metastases. Cancer Biomark 10, 79–89 (2011) G. Chen, Z. Wang, X.Y. Liu, F.Y. Liu, High-level CXCR4 expression correlates with brain-specific metastasis of non-small cell lung cancer. World J. Surg. 35, 56–61 (2011) T.N. Hartmann, J.A. Burger, A. Glodek, N. Fujii, M. Burger, CXCR4 chemokine receptor and integrin signaling co-operate in mediating adhesion and chemoresistance in small cell lung cancer (SCLC) cells. Oncogene 24, 4462–4471 (2005) F.A. Mauri, D.J. Pinato, P. Trivedi, R. Sharma, R.J. Shiner, Isogeneic comparison of primary and metastatic lung cancer identifies CX3CR1 as a molecular determinant of site-specific metastatic diffusion. Oncol. Rep. 28, 647–653 (2012) H. Mir, R. Singh, G.H. Kloecker, J.W. Lillard Jr., S. Singh, CXCR6 expression in non-small cell lung carcinoma supports metastatic process via modulating metalloproteinases. Oncotarget 6, 9985–9998 (2015)

116.

117.

118.

119.

120.

121. 122.

123.

124.

125.

126.

T. Seike, K. Fujita, Y. Yamakawa, M.A. Kido, S. Takiguchi, N. Teramoto, H. Iguchi, M. Noda, Interaction between lung cancer cells and astrocytes via specific inflammatory cytokines in the microenvironment of brain metastasis. Clin. Exp. Metastasis 28, 13–25 (2011) H. Peinado, M. Aleckovic, S. Lavotshkin, I. Matei, B. Costa-Silva, G. Moreno-Bueno, M. Hergueta-Redondo, C. Williams, G. Garcia-Santos, C. Ghajar, A. Nitadori-Hoshino, C. Hoffman, K. Badal, B.A. Garcia, M.K. Callahan, J. Yuan, V.R. Martins, J. Skog, R.N. Kaplan, M.S. Brady, J.D. Wolchok, P.B. Chapman, Y. Kang, J. Bromberg, D. Lyden, Melanoma exosomes educate bone marrow progenitor cells toward a pro-metastatic phenotype through MET. Nat. Med. 18, 883–891 (2012) A. Caivano, F. La Rocca, V. Simeon, M. Girasole, S. Dinarelli, I. Laurenzana, A. De Stradis, L. De Luca, S. Trino, A. Traficante, G. D'Arena, G. Mansueto, O. Villani, G. Pietrantuono, L. Laurenti, L. Del Vecchio, P. Musto, MicroRNA-155 in serum-derived extracellular vesicles as a potential biomarker for hematologic malignancies - a short report. Cell. Oncol. 40, 97–103 (2017) B. Costa-Silva, N.M. Aiello, A.J. Ocean, S. Singh, H. Zhang, B.K. Thakur, A. Becker, A. Hoshino, M.T. Mark, H. Molina, J. Xiang, T. Zhang, T.M. Theilen, G. Garcia-Santos, C. Williams, Y. Ararso, Y. Huang, G. Rodrigues, T.L. Shen, K.J. Labori, I.M. Lothe, E.H. Kure, J. Hernandez, A. Doussot, S.H. Ebbesen, P.M. Grandgenett, M.A. Hollingsworth, M. Jain, K. Mallya, S.K. Batra, W.R. Jarnagin, R.E. Schwartz, I. Matei, H. Peinado, B.Z. Stanger, J. Bromberg, D. Lyden, Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat. Cell Biol. 17, 816–826 (2015) A. Hoshino, B. Costa-Silva, T.L. Shen, G. Rodrigues, A. Hashimoto, M. Tesic Mark, H. Molina, S. Kohsaka, A. Di Giannatale, S. Ceder, S. Singh, C. Williams, N. Soplop, K. Uryu, L. Pharmer, T. King, L. Bojmar, A.E. Davies, Y. Ararso, T. Zhang, H. Zhang, J. Hernandez, J.M. Weiss, V.D. DumontCole, K. Kramer, L.H. Wexler, A. Narendran, G.K. Schwartz, J.H. Healey, P. Sandstrom, K.J. Labori, E.H. Kure, P.M. Grandgenett, M.A. Hollingsworth, M. de Sousa, S. Kaur, M. Jain, K. Mallya, S.K. Batra, W.R. Jarnagin, M.S. Brady, O. Fodstad, V. Muller, K. Pantel, A.J. Minn, M.J. Bissell, B.A. Garcia, Y. Kang, V.K. Rajasekhar, C.M. Ghajar, I. Matei, H. Peinado, J. Bromberg, D. Lyden, Tumour exosome integrins determine organotropic metastasis. Nature 527, 329–335 (2015) Y. Liu, X. Cao, Organotropic metastasis: Role of tumor exosomes. Cell Res. 26, 149–150 (2016) L. Zhang, S. Zhang, J. Yao, F.J. Lowery, Q. Zhang, W.C. Huang, P. Li, M. Li, X. Wang, C. Zhang, H. Wang, K. Ellis, M. Cheerathodi, J.H. McCarty, D. Palmieri, J. Saunus, S. Lakhani, S. Huang, A.A. Sahin, K.D. Aldape, P.S. Steeg, D. Yu, Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature 527, 100–104 (2015) G. Improta, A. Zupa, H. Fillmore, J. Deng, M. Aieta, P. Musto, L.A. Liotta, W. Broaddus, E.F. Petricoin 3rd, J.D. Wulfkuhle, Protein pathway activation mapping of brain metastasis from lung and breast cancers reveals organ type specific drug target activation. J. Proteome Res. 10, 3089–3097 (2011) M. Sun, C. Behrens, L. Feng, N. Ozburn, X. Tang, G. Yin, R. Komaki, M. Varella-Garcia, W. K. Hong, K. D. Aldape, Wistuba, II. HER family receptor abnormalities in lung cancer brain metastases and corresponding primary tumors Clin. Cancer Res. 15, 4829–37 (2009) G.M. Stella, S. Benvenuti, P.M. Comoglio, Targeting the MET oncogene in cancer and metastases. Expert Opin. Investig. Drugs 19, 1381–1394 (2010) K. Tsuta, Y. Kozu, T. Mimae, A. Yoshida, T. Kohno, I. Sekine, T. Tamura, H. Asamura, K. Furuta, H. Tsuda. c-MET/phospho-MET protein expression and MET gene copy number in non-small cell lung carcinomas. J Thorac Oncol 7, 331–9 (2012)

M. Yousefi et al. 127.

J.A. Engelman, K. Zejnullahu, T. Mitsudomi, Y. Song, C. Hyland, J.O. Park, N. Lindeman, C.M. Gale, X. Zhao, J. Christensen, T. Kosaka, A.J. Holmes, A.M. Rogers, F. Cappuzzo, T. Mok, C. Lee, B.E. Johnson, L.C. Cantley, P.A. Janne, MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science 316, 1039–1043 (2007) 128. J. Bean, C. Brennan, J.Y. Shih, G. Riely, A. Viale, L. Wang, D. Chitale, N. Motoi, J. Szoke, S. Broderick, M. Balak, W.C. Chang, C.J. Yu, A. Gazdar, H. Pass, V. Rusch, W. Gerald, S.F. Huang, P.C. Yang, V. Miller, M. Ladanyi, C.H. Yang, W. Pao, MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib. Proc. Natl. Acad. Sci. U. S. A. 104, 20932–20937 (2007) 129. S. Yano, W. Wang, Q. Li, K. Matsumoto, H. Sakurama, T. Nakamura, H. Ogino, S. Kakiuchi, M. Hanibuchi, Y. Nishioka, H. Uehara, T. Mitsudomi, Y. Yatabe, T. Nakamura, S. Sone, Hepatocyte growth factor induces gefitinib resistance of lung adenocarcinoma with epidermal growth factor receptor-activating mutations. Cancer Res. 68, 9479–9487 (2008) 130. E. Benedettini, L.M. Sholl, M. Peyton, J. Reilly, C. Ware, L. Davis, N. Vena, D. Bailey, B.Y. Yeap, M. Fiorentino, A.H. Ligon, B.S. Pan, V. Richon, J.D. Minna, A.F. Gazdar, G. Draetta, S. Bosari, L.R. Chirieac, B. Lutterbach, M. Loda, Met activation in non-small cell lung cancer is associated with de novo resistance to EGFR inhibitors and the development of brain metastasis. Am. J. Pathol. 177, 415–423 (2010) 131. S. Yano, H. Shinohara, R.S. Herbst, H. Kuniyasu, C.D. Bucana, L.M. Ellis, D.W. Davis, D.J. McConkey, I.J. Fidler, Expression of vascular endothelial growth factor is necessary but not sufficient for production and growth of brain metastasis. Cancer Res. 60, 4959–4967 (2000) 132. A.M. Jubb, A. Cesario, M. Ferguson, M.T. Congedo, K.C. Gatter, F. Lococo, A. Mule, F. Pezzella, Vascular phenotypes in primary non-small cell lung carcinomas and matched brain metastases. Br. J. Cancer 104, 1877–1881 (2011) 133. B. Li, C. Wang, Y. Zhang, X.Y. Zhao, B. Huang, P.F. Wu, Q. Li, H. Li, Y.S. Liu, L.Y. Cao, W.M. Dai, W.G. Fang, D.S. Shang, L. Cao, W.D. Zhao, Y.H. Chen, Elevated PLGF contributes to small-cell lung cancer brain metastasis. Oncogene 32, 2952–2962 (2013) 134. S. Jothy, CD44 and its partners in metastasis. Clin. Exp. Metastasis 20, 195–201 (2003) 135. H.K. Shabani, G. Kitange, K. Tsunoda, T. Anda, Y. Tokunaga, S. Shibata, M. Kaminogo, T. Hayashi, H. Ayabe, M. Iseki, Immunohistochemical expression of E-cadherin in metastatic brain tumors. Brain Tumor Pathol. 20, 7–12 (2003) 136. J.S. Desgrosellier, D.A. Cheresh, Integrins in cancer: Biological implications and therapeutic opportunities. Nat. Rev. Cancer 10, 9–22 (2010) 137. T. Yoshimasu, T. Sakurai, S. Oura, I. Hirai, H. Tanino, Y. Kokawa, Y. Naito, Y. Okamura, I. Ota, N. Tani, N. Matsuura, Increased expression of integrin alpha3beta1 in highly brain metastatic subclone of a human non-small cell lung cancer cell line. Cancer Sci. 95, 142–148 (2004) 138. I. Wilhelm, J. Molnar, C. Fazakas, J. Hasko, I.A. Krizbai, Role of the blood-brain barrier in the formation of brain metastases. Int. J. Mol. Sci. 14, 1383–1411 (2013) 139. E. Sipos, L. Chen, I.E. Andras, J. Wrobel, B. Zhang, H. Pu, M. Park, S.Y. Eum, M. Toborek, Proinflammatory adhesion molecules facilitate polychlorinated biphenyl-mediated enhancement of brain metastasis formation. Toxicol. Sci. 126, 362–371 (2012) 140. E. Huntzinger, E. Izaurralde, Gene silencing by microRNAs: Contributions of translational repression and mRNA decay. Nat. Rev. Genet. 12, 99–110 (2011) 141. G.S. Markopoulos, E. Roupakia, M. Tokamani, E. Chavdoula, M. Hatziapostolou, C. Polytarchou, K.B. Marcu, A.G. Papavassiliou, R.

Sandaltzopoulos, E. Kolettas, A step-by-step microRNA guide to cancer development and metastasis. Cell. Oncol. 40, 303–339 (2017) 142. S.A. Melo, M. Esteller, Dysregulation of microRNAs in cancer: Playing with fire. FEBS Lett. 585, 2087–2099 (2011) 143. G.A. Calin, C. Sevignani, C.D. Dumitru, T. Hyslop, E. Noch, S. Yendamuri, M. Shimizu, S. Rattan, F. Bullrich, M. Negrini, C.M. Croce, Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. U. S. A. 101, 2999–3004 (2004) 144. S. Alsidawi, E. Malek, J.J. Driscoll, MicroRNAs in brain metastases: Potential role as diagnostics and therapeutics. Int. J. Mol. Sci. 15, 10508–10526 (2014) 145. A.T. Grupenmacher, A.L. Halpern, F. Bonaldo Mde, C.C. Huang, C.A. Hamm, A. de Andrade, T. Tomita, S.T. Sredni, Study of the gene expression and microRNA expression profiles of malignant rhabdoid tumors originated in the brain (AT/RT) and in the kidney (RTK). Childs Nerv. Syst. 29, 1977–1983 (2013) 146. L.T. Chen, S.D. Xu, H. Xu, J.F. Zhang, J.F. Ning, S.F. Wang, MicroRNA-378 is associated with non-small cell lung cancer brain metastasis by promoting cell migration, invasion and tumor angiogenesis. Med. Oncol. 29, 1673–1680 (2012) 147. N.M. Teplyuk, B. Mollenhauer, G. Gabriely, A. Giese, E. Kim, M. Smolsky, R.Y. Kim, M.G. Saria, S. Pastorino, S. Kesari, A.M. Krichevsky, MicroRNAs in cerebrospinal fluid identify glioblastoma and metastatic brain cancers and reflect disease activity. Neuro-Oncology 14, 689–700 (2012) 148. S. Arora, A.R. Ranade, N.L. Tran, S. Nasser, S. Sridhar, R.L. Korn, J.T. Ross, H. Dhruv, K.M. Foss, Z. Sibenaller, T. Ryken, M.B. Gotway, S. Kim, G.J. Weiss, MicroRNA-328 is associated with (non-small) cell lung cancer (NSCLC) brain metastasis and mediates NSCLC migration. Int. J. Cancer 129, 2621–2631 (2011) 149. C.Y. Cheng, H.L. Hsieh, C.C. Sun, C.C. Lin, S.F. Luo, C.M. Yang, IL-1 beta induces urokinase-plasminogen activator expression and cell migration through PKC alpha, JNK1/2, and NF-kappaB in A549 cells. J. Cell. Physiol. 219, 183–193 (2009) 150. A. Subramani, S. Alsidawi, S. Jagannathan, K. Sumita, A.T. Sasaki, B. Aronow, R.E. Warnick, S. Lawler, J.J. Driscoll, The brain microenvironment negatively regulates miRNA-768-3p to promote K-ras expression and lung cancer metastasis. Sci Rep 3, 2392 (2013) 151. C. Zhao, Y. Xu, Y. Zhang, W. Tan, J. Xue, Z. Yang, Y. Lu, X. Hu, Downregulation of miR-145 contributes to lung adenocarcinoma cell growth to form brain metastases. Oncol. Rep. 30, 2027–2034 (2013) 152. S.J. Hwang, H.J. Seol, Y.M. Park, K.H. Kim, M. Gorospe, D.H. Nam, H.H. Kim, MicroRNA-146a suppresses metastatic activity in brain metastasis. Mol. Cell 34, 329–334 (2012) 153. G. Chen, I. A. Umelo, S. Lv, E. Teugels, K. Fostier, P. Kronenberger, A. Dewaele, J. Sadones, C. Geers, J. De Greve. miR-146a inhibits cell growth, cell migration and induces apoptosis in non-small cell lung cancer cells. PLoS One 8, e60317 (2013) 154. J. Remon, D. Alvarez-Berdugo, M. Majem, T. Moran, N. Reguart, P. Lianes. miRNA-197 and miRNA-184 are associated with brain metastasis in EGFR-mutant lung cancers. Clin. Transl. Oncol. 18, 153–9 (2016) 155. S.J. Hwang, H.W. Lee, H.R. Kim, H.J. Song, D.H. Lee, H. Lee, C.H. Shin, J.G. Joung, D.H. Kim, K.M. Joo, H.H. Kim, Overexpression of microRNA-95-3p suppresses brain metastasis of lung adenocarcinoma through downregulation of cyclin D1. Oncotarget 6, 20434–20448 (2015) 156. T. Gutschner, S. Diederichs, The hallmarks of cancer: A long noncoding RNA point of view. RNA Biol. 9, 703–719 (2012) 157. M.C. Tsai, R.C. Spitale, H.Y. Chang, Long intergenic noncoding RNAs: New links in cancer progression. Cancer Res. 71, 3–7 (2011)

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options 158.

159.

160.

161.

162.

163.

164. 165. 166.

167. 168.

169.

170. 171.

172.

173.

174.

175.

M. Vitiello, A. Tuccoli, L. Poliseno, Long non-coding RNAs in cancer: Implications for personalized therapy. Cell. Oncol. 38, 17– 28 (2015) R.A. Gupta, N. Shah, K.C. Wang, J. Kim, H.M. Horlings, D.J. Wong, M.C. Tsai, T. Hung, P. Argani, J.L. Rinn, Y. Wang, P. Brzoska, B. Kong, R. Li, R.B. West, M.J. van de Vijver, S. Sukumar, H.Y. Chang, Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis. Nature 464, 1071–1076 (2010) S. Sharma Saha, R. Roy Chowdhury, N.R. Mondal, B. Chakravarty, T. Chatterjee, S. Roy, S. Sengupta, Identification of genetic variation in the lncRNA HOTAIR associated with HPV16-related cervical cancer pathogenesis. Cell. Oncol. 39, 559–572 (2016) F.Q. Nie, M. Sun, J.S. Yang, M. Xie, T.P. Xu, R. Xia, Y.W. Liu, X.H. Liu, E.B. Zhang, K.H. Lu, Y.Q. Shu, Long noncoding RNA ANRIL promotes non-small cell lung cancer cell proliferation and inhibits apoptosis by silencing KLF2 and P21 expression. Mol. Cancer Ther. 14, 268–277 (2015) T. Gutschner, M. Hammerle, S. Diederichs, MALAT1 – a paradigm for long noncoding RNA function in cancer. J. Mol. Med. 91, 791–801 (2013) L. Shen, L. Chen, Y. Wang, X. Jiang, H. Xia, Z. Zhuang, Long noncoding RNA MALAT1 promotes brain metastasis by inducing epithelial-mesenchymal transition in lung cancer. J. Neuro-Oncol. 121, 101–108 (2015) A. Stoffel, Targeted therapies for solid tumors. BioDrugs 24, 303– 316 (2010) A. Chi, R. Komaki, Treatment of brain metastasis from lung cancer. Cancer 2, 2100–2137 (2010) D.-B. Chang, P.-C. Yang, K.-T. Luh, S.-H. Kuo, R.-L. Hong, L.-N. Lee, Late survival of non-small cell lung cancer patients with brain metastases. Influence of treatment. CHEST J. 101, 1293–1297 (1992) P. Richards, W. McKissock, Intracranial metastases. Br. Med. J. 1, 15 (1963) A. Zabel, J. Debus, Treatment of brain metastases from non-smallcell lung cancer (NSCLC): Radiotherapy. Lung Cancer 45, S247– SS52 (2004) R. Soffietti, A. Costanza, E. Laguzzi, M. Nobile, R. Ruda, Radiotherapy and chemotherapy of brain metastases. J. NeuroOncol. 75, 31–42 (2005) W.A. Castrucci, J.P. Knisely, An update on the treatment of CNS metastases in small cell lung cancer. Cancer J. 14, 138–146 (2008) J.P. Sheehan, M.-H. Sun, D. Kondziolka, J. Flickinger, L.D. Lunsford, Radiosurgery for non-small cell lung carcinoma metastatic to the brain: Long-term outcomes and prognostic factors influencing patient survival time and local tumor control. J. Neurosurg. 97, 1276–1281 (2002) R.A. Patchell, P.A. Tibbs, J.W. Walsh, R.J. Dempsey, Y. Maruyama, R.J. Kryscio, W.R. Markesbery, J.S. Macdonald, B. Young, A randomized trial of surgery in the treatment of single metastases to the brain. N. Engl. J. Med. 322, 494–500 (1990) E. M. Noordijk, C. J. Vecht, H. Haaxma-Reiche, G. W. Padberg, J. H. Voormolen, F. H. Hoekstra, J. T. J. Tans, N. Lambooij, J. A. Metsaars, A. R. Wattendorff. The choice of treatment of single brain metastasis should be based on extracranial tumor activity and age. International Journal of Radiation Oncology* Biology* Physics 29, 711–7 (1994) R.A. Patchell, P.A. Tibbs, W.F. Regine, R.J. Dempsey, M. Mohiuddin, R.J. Kryscio, W.R. Markesbery, K.A. Foon, B. Young, Postoperative radiotherapy in the treatment of single metastases to the brain: A randomized trial. JAMA 280, 1485–1489 (1998) M.A. Chidel, J.H. Suh, J.F. Greskovich, P.A. Kupelian, G.H. Barnett, Treatment outcome for patients with primary nonsmall-

176. 177.

178.

179.

180.

181.

182.

183.

184.

185.

186.

187.

188. 189.

190.

191.

cell lung cancer and synchronous brain metastasis. Radiat. Oncol. Investig. 7, 313–319 (1999) J.H. Suh, Stereotactic radiosurgery for the management of brain metastases. N. Engl. J. Med. 362, 1119–1127 (2010) H. Aoyama, H. Shirato, M. Tago, K. Nakagawa, T. Toyoda, K. Hatano, M. Kenjyo, N. Oya, S. Hirota, H. Shioura, Stereotactic radiosurgery plus whole-brain radiation therapy vs stereotactic radiosurgery alone for treatment of brain metastases: A randomized controlled trial. JAMA 295, 2483–2491 (2006) H. Qin, C. Wang, Y. Jiang, X. Zhang, Y. Zhang, Z. Ruan, Patients with single brain metastasis from non-small cell lung cancer equally benefit from stereotactic radiosurgery and surgery: A systematic review. Med. Sci. Monit Intern. Med. J. Exp. Clin. Res. 21, 144 (2015) R. Hoffman, P. K. Sneed, M. W. McDermott, S. Chang, K. R. Lamborn, E. Park, W. Wara, D. A. Larson. Radiosurgery for brain metastases from primary lung carcinoma. Cancer J. (Sudbury, Mass) 7, 121–31 (2000) W. F. Regine, J. L. Huhn, R. A. Patchell, W. H. S. Clair, J. Strottmann, A. Meigooni, M. Sanders, A. B. Young. Risk of symptomatic brain tumor recurrence and neurologic deficit after radiosurgery alone in patients with newly diagonised brain metastases: results and implications. Int. J. Radiat. Oncol. Biol. Phys. 52, 333–8 (2002) B. Li, J. Yu, M. Suntharalingam, A.S. Kennedy, P.P. Amin, Z. Chen, R. Yin, S. Guo, T. Han, Y. Wang, Comparison of three treatment options for single brain metastasis from lung cancer. Int. J. Cancer 90, 37–45 (2000) Z. Wang, Y. Li, A. Ahmad, A.S. Azmi, D. Kong, S. Banerjee, F.H. Sarkar, Targeting miRNAs involved in cancer stem cell and EMT regulation: An emerging concept in overcoming drug resistance. Drug Resist. Updat. 13, 109–118 (2010) D.S. Ettinger, W. Akerley, H. Borghaei, A.C. Chang, R.T. Cheney, L.R. Chirieac, T.A. D’Amico, T.L. Demmy, A.K.P. Ganti, R. Govindan, Non–small cell lung cancer. J. Natl. Compr. Cancer Netw. 10, 1236–1271 (2012) H.B. Newton, M.A. Slivka, C. Volpi, E.C. Bourekas, G.A. Christoforidis, M.A. Baujan, W. Slone, D.W. Chakeres, Intraarterial carboplatin and intravenous etoposide for the treatment of metastatic brain tumors. J. Neuro-Oncol. 61, 35–44 (2003) D. Fortin, C. Gendron, M. Boudrias, M.P. Garant, Enhanced chemotherapy delivery by intraarterial infusion and blood-brain barrier disruption in the treatment of cerebral metastasis. Cancer 109, 751–760 (2007) L.E. Abrey, J.D. Olson, J.J. Raizer, M. Mack, A. Rodavitch, D.Y. Boutros, M.G. Malkin, A phase II trial of temozolomide for patients with recurrent or progressive brain metastases. J. NeuroOncol. 53, 259–265 (2001) E.R. Gerstner, R.L. Fine, Increased permeability of the bloodbrain barrier to chemotherapy in metastatic brain tumors: Establishing a treatment paradigm. J. Clin. Oncol. 25, 2306– 2312 (2007) W. Schuette, Treatment of brain metastases from lung cancer: Chemotherapy. Lung Cancer 45, S253–S2S7 (2004) N. Zakaria, N.A. Satar, N.H. Abu Halim, S.H. Ngalim, N.M. Yusoff, J. Lin, B.H. Yahaya, Targeting lung cancer stem cells: Research and clinical impacts. Front. Oncol. 7 (2017) H. Yue, D. Huang, L. Qin, Z. Zheng, L. Hua, G. Wang, J. Huang, H. Huang, Targeting lung cancer stem cells with antipsychological drug thioridazine. Biomed Res Int 2016, 1–7 (2016) N. Zakaria, N.M. Yusoff, Z. Zakaria, M.N. Lim, P.J.N. Baharuddin, K.S. Fakiruddin, B. Yahaya, Human non-small cell lung cancer expresses putative cancer stem cell markers and exhibits the transcriptomic profile of multipotent cells. BMC Cancer 15, 84 (2015)

M. Yousefi et al. 192.

T. Borovski, E.M.F. De Sousa, L. Vermeulen, J.P. Medema, Cancer stem cell niche: The place to be. Cancer Res. 71, 634– 639 (2011) 193. C. Calabrese, H. Poppleton, M. Kocak, T.L. Hogg, C. Fuller, B. Hamner, E.Y. Oh, M.W. Gaber, D. Finklestein, M. Allen, A. Frank, I.T. Bayazitov, S.S. Zakharenko, A. Gajjar, A. Davidoff, R.J. Gilbertson, A perivascular niche for brain tumor stem cells. Cancer Cell 11, 69–82 (2007) 194. Y. Zheng, C.C. de la Cruz, L.C. Sayles, C. Alleyne-Chin, D. Vaka, T.D. Knaak, M. Bigos, Y. Xu, C.D. Hoang, J.B. Shrager, H.J. Fehling, D. French, W. Forrest, Z. Jiang, R.A. Carano, K.H. Barck, E.L. Jackson, E.A. Sweet-Cordero, A rare population of CD24(+)ITGB4(+)notch(hi) cells drives tumor propagation in NSCLC and requires Notch3 for self-renewal. Cancer Cell 24, 59–74 (2013) 195. H.L. Goel, T. Gritsko, B. Pursell, C. Chang, L.D. Shultz, D.L. Greiner, J.H. Norum, R. Toftgard, L.M. Shaw, A.M. Mercurio, Regulated splicing of the alpha6 integrin cytoplasmic domain determines the fate of breast cancer stem cells. Cell Rep. 7, 747–761 (2014) 196. E. Dickreuter, I. Eke, M. Krause, K. Borgmann, M.A. van Vugt, N. Cordes, Targeting of beta1 integrins impairs DNA repair for radiosensitization of head and neck cancer cells. Oncogene 35, 1353–1362 (2016) 197. A. Voulgari, A. Pintzas, Epithelial–mesenchymal transition in cancer metastasis: Mechanisms, markers and strategies to overcome drug resistance in the clinic. Biochim Biophys Acta (BBA)-reviews on Cancer 1796, 75–90 (2009) 198. S. Singh, S. Chellappan, Lung cancer stem cells: Molecular features and therapeutic targets. Mol. Asp. Med. 39, 50–60 (2014) 199. L. You, B. He, Z. Xu, K. Uematsu, J. Mazieres, I. Mikami, N. Reguart, T.W. Moody, J. Kitajewski, F. McCormick, Inhibition of Wnt-2-mediated signaling induces programmed cell death in nonsmall-cell lung cancer cells. Oncogene 23, 6170–6174 (2004) 200. R.A. Winn, M. Van Scoyk, M. Hammond, K. Rodriguez, J.T. Crossno, L.E. Heasley, R.A. Nemenoff, Antitumorigenic effect of Wnt 7a and Fzd 9 in non-small cell lung cancer cells is mediated through ERK-5-dependent activation of peroxisome proliferatoractivated receptor γ. J. Biol. Chem. 281, 26943–26950 (2006) 201. H. Yue, D. Huang, L. Qin, Z. Zheng, L. Hua, G. Wang, J. Huang, H. Huang. Targeting Lung Cancer Stem Cells with Antipsychological Drug Thioridazine. 2016, 6709828 (2016) 202. S.S. Lin, K.C. Lai, S.C. Hsu, J.S. Yang, C.L. Kuo, J.P. Lin, Y.S. Ma, C.C. Wu, J.G. Chung, Curcumin inhibits the migration and invasion of human A549 lung cancer cells through the inhibition of matrix metalloproteinase-2 and -9 and vascular endothelial growth factor (VEGF). Cancer Lett. 285, 127–133 (2009) 203. Y. Li, T. Zhang, Targeting cancer stem cells by curcumin and clinical applications. Cancer Lett. 346, 197–205 (2014) 204. E. Robles-Escajeda, U. Das, N.M. Ortega, K. Parra, G. Francia, J.R. Dimmock, A. Varela-Ramirez, R.J. Aguilera, A novel curcumin-like dienone induces apoptosis in triple-negative breast cancer cells. Cell. Oncol. 39, 265–277 (2016) 205. J.Y. Zhu, X. Yang, Y. Chen, Y. Jiang, S.J. Wang, Y. Li, X.Q. Wang, Y. Meng, M.M. Zhu, X. Ma, C. Huang, R. Wu, C.F. Xie, X.T. Li, S.S. Geng, J.S. Wu, C.Y. Zhong. Curcumin Suppresses Lung Cancer Stem Cells via Inhibiting Wnt/beta-catenin and Sonic Hedgehog Pathways. 31, 680–688 (2017) 206. P. Baharuddin, N. Satar, K.S. Fakiruddin, N. Zakaria, M.N. Lim, N.M. Yusoff, Z. Zakaria, B.H. Yahaya, Curcumin improves the efficacy of cisplatin by targeting cancer stem-like cells through p21 and cyclin D1-mediated tumour cell inhibition in non-small cell lung cancer cell lines. Oncol. Rep. 35, 13–25 (2016) 207. X. Meng, M. Li, X. Wang, Y. Wang, D. Ma, Both CD133+ and CD133- subpopulations of A549 and H446 cells contain cancerinitiating cells. Cancer Sci. 100, 1040–1046 (2009)

208.

209.

210.

211.

212.

213.

214.

215.

216.

217. 218.

219.

220.

221.

222.

223. 224.

R. Roudi, Z. Madjd, M. Ebrahimi, F.S. Samani, A. Samadikuchaksaraei, CD44 and CD24 cannot act as cancer stem cell markers in human lung adenocarcinoma cell line A549. Cell. Mol. Biol. Lett. 19, 23–36 (2014) Y. Lu, R. Govindan, L. Wang, P.-y. Liu, B. Goodgame, W. Wen, A. Sezhiyan, J. Pfeifer, Y.-f. Li, X. Hua. MicroRNA profiling and prediction of recurrence/relapse-free survival in stage I lung cancer. Carcinogenesis 33, 1046-1054 (2012) A. Subramani, S. Alsidawi, S. Jagannathan, K. Sumita, A.T. Sasaki, B. Aronow, R.E. Warnick, S. Lawler, J.J. Driscoll, The brain microenvironment negatively regulates miRNA-768-3p to promote K-ras expression and lung cancer metastasis. Sci Rep 3 (2013) N.M. Teplyuk, B. Mollenhauer, G. Gabriely, A. Giese, E. Kim, M. Smolsky, R.Y. Kim, M.G. Saria, S. Pastorino, S. Kesari. MicroRNAs in cerebrospinal fluid identify glioblastoma and metastatic brain cancers and reflect disease activity. Neuro-oncol. 14, 689-700 (2012) P.S. Mitchell, R.K. Parkin, E.M. Kroh, B.R. Fritz, S.K. Wyman, E.L. Pogosova-Agadjanyan, A. Peterson, J. Noteboom, K.C. O'Briant, A. Allen, Circulating microRNAs as stable bloodbased markers for cancer detection. Proc. Natl. Acad. Sci. 105, 10513–10518 (2008) S. Alsidawi, E. Malek, J.J. Driscoll, MicroRNAs in brain metastases: Potential role as diagnostics and therapeutics. Int. J. Mol. Sci. 15, 10508–10526 (2014) J. Kota, R.R. Chivukula, K.A. O'Donnell, E.A. Wentzel, C.L. Montgomery, H.-W. Hwang, T.-C. Chang, P. Vivekanandan, M. Torbenson, K.R. Clark, Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell 137, 1005–1017 (2009) Y. Wu, M. Crawford, B. Yu, Y. Mao, S.P. Nana-Sinkam, L.J. Lee, MicroRNA delivery by cationic lipoplexes for lung cancer therapy. Mol. Pharm. 8, 1381–1389 (2011) H.Y. Lee, K.A. Mohammed, F. Kaye, P. Sharma, B.M. Moudgil, W.L. Clapp, N. Nasreen, Targeted delivery of let-7a microRNA encapsulated ephrin-A1 conjugated liposomal nanoparticles inhibit tumor growth in lung cancer. Int. J. Nanomedicine 8, 4481–4494 (2013) A. Esquela-Kerscher, F.J. Slack, Oncomirs - microRNAs with a role in cancer. Nat. Rev. Cancer 6, 259–269 (2006) O. Fortunato, M. Boeri, C. Verri, M. Moro, G. Sozzi, Therapeutic use of microRNAs in lung cancer. Biomed. Res. Int. 2014, 756975 (2014) Y. Zhang, Y.-f. Zhang, J. Bryant, A. Charles, R.J. Boado, W.M. Pardridge, Intravenous RNA interference gene therapy targeting the human epidermal growth factor receptor prolongs survival in intracranial brain cancer. Clin. Cancer Res. 10, 3667–3677 (2004) T.K. Owonikoko, J. Arbiser, A. Zelnak, H.-K.G. Shu, H. Shim, A.M. Robin, S.N. Kalkanis, T.G. Whitsett, B. Salhia, N.L. Tran, Current approaches to the treatment of metastatic brain tumours. Nat. Rev. Clin. Oncol. 11, 203 (2014) S. Zimmermann, R. Dziadziuszko, S. Peters, Indications and limitations of chemotherapy and targeted agents in non-small cell lung cancer brain metastases. Cancer Treat. Rev. 40, 716–722 (2014) F.A. Shepherd, J. Rodrigues Pereira, T. Ciuleanu, E.H. Tan, V. Hirsh, S. Thongprasert, D. Campos, S. Maoleekoonpiroj, M. Smylie, R. Martins, Erlotinib in previously treated non–small-cell lung cancer. N. Engl. J. Med. 353, 123–132 (2005) J. Baselga, The EGFR as a target for anticancer therapy—Focus on cetuximab. Eur. J. Cancer 37, 16–22 (2001) M. Fukuoka, S. Yano, G. Giaccone, T. Tamura, K. Nakagawa, J.Y. Douillard, Y. Nishiwaki, J. Vansteenkiste, S. Kudoh, D. Rischin, Multi-institutional randomized phase II trial of gefitinib

Lung cancer-associated brain metastasis: Molecular mechanisms and therapeutic options for previously treated patients with advanced non–small-cell lung cancer. J. Clin. Oncol. 21, 2237–2246 (2003) 225. J. Sato, T. Kawamoto, A. Le, J. Mendelsohn, J. Polikoff, G. Sato, Biological effects in vitro of monoclonal antibodies to human epidermal growth factor receptors. Molecular Biol. Med. 1, 511– 529 (1983) 226. Y.-L. Wu, C. Zhou, Y. Cheng, S. Lu, G.-Y. Chen, C. Huang, Y.-S. Huang, H.-H. Yan, S. Ren, Y. Liu, Erlotinib as second-line treatment in patients with advanced non-small-cell lung cancer and asymptomatic brain metastases: A phase II study (CTONG– 0803). Ann. Oncol. 24, 993–999 (2013) 227. D.B. Costa, A.T. Shaw, S.-H. I. Ou, B.J. Solomon, G.J. Riely, M.J. Ahn, C. Zhou, S.M. Shreeve, P. Selaru, A. Polli. Clinical experience with crizotinib in patients with advanced ALK-rearranged non–small-cell lung cancer and brain metastases. J. Clin. Oncol. 33, 1881-1888 (2015) 228. A. T. Shaw, R. Mehra, D. S. Tan, E. Felip, L. Chow, D. R. Camidge, J. Vansteenkiste, S. Sharma, T. De Pas, G. J. Riely. 1293Pevaluation of ceritinib-treated patients (PTS) with anaplastic lymphoma kinase rearranged (ALK+) non-small cell lung cancer (NSCLC) and brain metastases in the ascend-1 study. Ann. Oncol. 25, iv455-iv6 (2014) 229. S.-H. I. Ou, J. S. Ahn, L. De Petris, R. Govindan, J. C.-H. Yang, B. G. M. Hughes, H. Lena, D. Moro-Sibilot, A. Bearz, S. V. Ramirez, editors. Efficacy and safety of the ALK inhibitor alectinib in ALK+ non-small-cell lung cancer (NSCLC) patients who have failed prior crizotinib: An open-label, single-arm, global phase 2 study (NP28673). ASCO Annual Meeting Proceedings; 2015 230. A. T. Shaw, T. M. Bauer, E. Felip, B. Besse, L. P. James, J. S. Clancy, G. Mugundu, J.-F. Martini, A. Abbattista, B. J. Solomon, editors. Clinical activity and safety of PF-06463922 from a dose escalation study in patients with advanced ALK+ or ROS1+ NSCLC. ASCO Annual Meeting Proceedings; 2015 231. M.G. Kris, R.B. Natale, R.S. Herbst, T.J. Lynch Jr., D. Prager, C.P. Belani, J.H. Schiller, K. Kelly, H. Spiridonidis, A. Sandler, Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic patients with non–small cell lung cancer: A randomized trial. JAMA 290, 2149–2158 (2003)

232. 233.

234. 235.

236.

237.

238.

239.

240.

S.B. Goldberg, J.N. Contessa, S.B. Omay, V. Chiang, Lung cancer brain metastases. Cancer J. 21, 398–403 (2015) A. Salmaninejad, V. Khoramshahi, A. Azani, E. Soltaninejad, S. Aslani, M.R. Zamani, M. Zal, A. Nesaei, S.M. Hosseini, PD-1 and cancer: Molecular mechanisms and polymorphisms. Immunogenetics. https://doi.org/10.1007/s00251-017-10155 (2017) J.V. Cohen, H.M. Kluger, Systemic immunotherapy for the treatment of brain metastases. Front. Oncol. 6, 49 (2016) T. Bahrami, S. Mokmeli, H. Hossieni, R. Pourpaknia, Z. Makani, A. Salmaninejad, M.A. Estiar, A. Hossieni, A. Farshbaf, The molecular signature of breast cancer metastasis to bone. Anti-Cancer Drugs 27, 824–831 (2016) N.K. Gerber, Y. Yamada, A. Rimner, W. Shi, G.J. Riely, K. Beal, A.Y. Helena, T.A. Chan, Z. Zhang, A.J. Wu. Erlotinib versus radiation therapy for brain metastases in patients with EGFRmutant lung adenocarcinoma. Int. J. Radiat. Oncol. Biol. Phys. 89, 322–329 (2014) S.H. Lim, J.Y. Lee, M.-Y. Lee, H. Kim, J. Lee, J.-M. Sun, J. Ahn, S.-W. Um, H. Kim, B. Kim. A randomized phase III trial of stereotactic radiosurgery (SRS) versus observation for patients with asymptomatic cerebral oligo-metastases in non-small cell lung cancer. Ann. Oncol. 26, 762-768 (2014) L. Liu, M. Yang, J. Guan, Y. Zhang, Q. Li, Y. Zhang, M. Chen, L. Li, N. Xiao, Y. Dai, editors. Whole Brain Radiotherapy (WBRT) plus EGFR tyrosine kinase inhibitors (TKIs) versus WBRT alone for brain metastases (BMs) in non-small cell lung cancer (NSCLC) patients: A meta-analysis. ASCO Annual Meeting Proceedings; 2015 M.E. Fiori, C. Barbini, T.L. Haas, N. Marroncelli, M. Patrizii, M. Biffoni, R. De Maria, Antitumor effect of miR-197 targeting in p53 wild-type lung cancer. Cell Death Differ. 21, 774–782 (2014) Z. Liu, C. Mai, H. Yang, Y. Zhen, X. Yu, S. Hua, Q. Wu, Q. Jiang, Y. Zhang, X. Song, W. Fang, Candidate tumour suppressor CCDC19 regulates miR-184 direct targeting of C-Myc thereby suppressing cell growth in non-small cell lung cancers. J. Cell. Mol. Med. 18, 1667–1679 (2014)