Clin Transl Oncol DOI 10.1007/s12094-014-1228-0

RESEARCH ARTICLE

Overexpression of ribonuclease inhibitor defines good prognosis and suppresses proliferation and metastasis in human colorectal cancer cells via PI3K/AKT pathway Y. Tang • P. Liu • Y. Tian • Y. Xu F. Ren • X. Cui • J. Fan

•

Received: 16 May 2014 / Accepted: 12 September 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014

Abstract Purpose We aim to evaluate the diagnostic value of ribonuclease inhibitor (RI) in colorectal cancer (CRC) and investigate the important role of RI in cell growth and metastasis of CRC. Methods/patients In this study, the expression of RI was evaluated in human CRC samples with different histological grade and the association between RI expression and clinicopathological parameters was investigated. Furthermore, the exogenous RI gene was introduced into human HT29 CRC cells and the effects of RI on cell proliferation and metastasis were determined in vitro. The PI3K/Akt signaling pathway and some related protein molecules were detected. Results RI is downregulated in CRC tissues compared to adjacent normal tissues and its expression is inversely associated with histological grade, pathological stage, and venous invasion, respectively. Multivariate analysis showed that RI expression was an independent prognostic factor for overall survival. In addition, the exogenous overexpression of RI reduced the proliferation and migration of HT29 CRC cell line in vitro by inhibiting the PI3K/ Akt signaling pathway and suppressing the expression of Y. Tang Department of Pathology, The Second Affiliated Hospital of Dalian Medical University, Dalian 116023, China P. Liu Regenerative Medicine Centre, The First Affiliated Hospital of Dalian Medical University, Dalian 116011, China Y. Tian  Y. Xu  F. Ren  X. Cui  J. Fan (&) Department of Biochemistry and Molecular Biology, Dalian Medical University, 9 South Lvshun Road Western Section, Dalian 116044, China e-mail: [email protected]

vascular endothelial growth factor (VEGF) and upregulating phosphatase and tensin homolog (PTEN). Conclusions RI represents an important predictor of progression in patients with CRC and suppresses proliferation and metastasis in CRC cells through inhibiting PI3K/ AkT pathway. Keywords Ribonuclease inhibitor  Colorectal cancer  Metastasis  PI3K/Akt signal pathway

Introduction It has been estimated that nearly 102,480 new cases of colorectal cancer (CRC) were diagnosed in the US in 2013, with approximately 50,830 people dying of the disease [1]. Colorectal cancer is the most common malignancy in China, and the incidence of this disease is gradually increasing [2]. Despite considerable progress in its treatment, the prognosis of CRC patients remains poor. Current prognostic parameters cannot predict long-term outcome of CRC with any certainty. Therefore, specific molecular markers that could serve as standard prognostic factors are needed. Human ribonuclease inhibitor (RI), very abundant in cell cytosol, is a ubiquitous acidic protein of about 50 kDa with a high content of cysteine residues consisting of 15 leucine-rich repeats (LRR). The three-dimensional structure, a striking horseshoe shape being characterized by alternating units of a-helix and b-strand, is one of the most captivating properties of RI [3, 4]. Such repeats have been identified in more than 100 proteins that exhibit a wide range of functions, including cell-cycle regulation, DNA repair, extracellular matrix interaction, and enzyme inhibition [5].

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RI has been found in the cytosol of many cell types. Although it inhibits secretory ribonucleases, RI has not been detected in extracellular fluids, such as plasma, saliva, and urine [6, 7]. The expression of RI has been investigated in melanoma and bladder cancer cells [8, 9], with the hope of revealing insight into the biological role of RI. Till today, the relationship between RI expression and underlying tumorigenesis mechanism is still not fully understood. Previous study indicated that the exogenous RI prolonged the survival time of B16 melanoma tumor bearing mice [10] and decreased RI expression has been determined significantly in human breast cancer tissues [11]. In the present study, we examined the expression profile of RI in patients with CRC and investigated its clinicopathological and prognostic significance by immunohistochemical analysis. We also determined the survivals and prognostic value of RI expression for CRC patients by Kaplan–Meier method. Furthermore, exogenous RI gene was introduced into CRC cells, and the important effects of RI on proliferation and metastasis were detected in vitro, the potentially related mechanisms were explored finally.

Hematoxylin and eosin staining and Immunohistochemistry assay

Materials and methods

The samples were cut at a thickness of 4 lm and subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC), respectively, as described before [12]. Briefly, For IHC, slides were incubated overnight at 4 °C with polyclonal anti-RI (Bioss, Woburn, MA, USA) followed by incubation with proper secondary antibody (Abcam, Cambridge, MA, USA). Diaminobenzidine was used as chromogen. The number of positive-staining cells showing immunoreactivity in the cytoplasm in 10 representative microscopic fields was counted and the percentage of positive cells was calculated. Given the homogeneity of the staining of the target proteins, tumor specimens were scored in a semi-quantitative manner based on the percentage of tumor cells that showed immunoreactivity. The criteria for the assessment of RI expression were as following: where 0 (negative, B5 %); 1?(positive,[5 %) of the tumor cells stained. The results of immunostaining were examined by two independent observers without prior knowledge of the clinical status of the patients. The scores of the two pathologists were compared and any discrepant scores were trained through re-examining the staining by both pathologists to achieve a consensus score.

Patients and tissue samples

Cloning and plasmid construction

For detection of RI expression by qRT-PCR and western blot analysis, 27 surgically removed CRC specimens and 18 adjacent normal tissues were collected from patients in the second affiliated hospital of Dalian Medical University in 2013. Tumor histological grade were as follows: well and moderately (n = 18), poorly (n = 9). All samples were obtained immediately after surgical removal, snap frozen in liquid nitrogen, and stored at -80 °C until use. In addition, a total of 108 paraffin-embedded samples of CRC and 34 specimens of adjacent normal tissue were collected from second affiliated hospital of Dalian Medical University between January 2006 and December 2008. The criteria for study enrollment were histopathological diagnosis of CRC, newly diagnosed and untreated, no history of other tumor. This study was approved by the Ethical Committees of Dalian Medical University Hospital, and all patients gave informed consent to have their tissues before collection. To investigate the overall survival (OS), all patients were followed up at intervals of 3 months until December 2013 with an end-time point of 60 months. OS was determined from the date of surgery to the time of the last follow-up or cancer-related death. Patients who died from other diseases or unexpected events were excluded from the case collection.

Wild-type human RI cDNA (accession number: NM 002939) was amplified by RT-PCR from total RNA of human MCF-7 cells and cloned into the pcDNA 3.1 plasmid (Invitrogen, Carlsbad, CA, USA). The recombinant plasmid sequence was verified by DNA sequencing.

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Cell culture and transfections Human CRC cells HT29 were cultured in RPMI 1640 medium supplemented with 10 % fetal bovine serum in a humidified atmosphere containing 5 % CO2 at 37 °C. The cells were seeded into six well plates and were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen). 48 hours after transfection, the selection was performed with 800 lg/ml of G418 for 2 weeks and the individual G418-resistant monocolonies were obtained and expanded to generate stable transfected cell line HT29/RI that overexpress RI, HT-29/Mock cell line which was transfected with empty vector as control. Quantitative RT-PCR assay For the quantification of RI in tissues samples, real-time RTPCR was employed. Total RNA was prepared using Trizol reagent (Invitrogen). cDNAs were synthesized using SYBRÒ

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PrimeScriptTM RT-PCR Kit (Takara, Tokyo, Japan). The primers were as follows: RI forward: 50 -TCAGCGACA ACCTCT TGG G-30 and reverse: 50 -CACAATGCCGCA CAGGTC-30 ; GAPDH forward 50 -AAATGGTGAAGGTC GGTGTG-30 , GAPDH reverse 50 -TGAAGGGGTCGTT GATGG-30 . RI mRNA expression was normalized to GAP DH which was used as the internal control. Western blot assay

bottom layer of medium containing 10 % fetal bovine serum, 1 % penicillin and streptomycin with agar (low melt 0.7 %) was poured first, after solidifying this is followed by a top layer containing the same medium with 1.2 % agar, mixed with 1,000 cells. The plates were placed in a humidified incubator at 37 °C with 5 % CO2 for 2 weeks. The plates were photographed and the numbers of distinct colonies (C50 cells per colony) were counted under a light microscope. The results averaged for each treatment group.

Total cell lysate was prepared and protein concentration was determined by BCA kit (Beyotime, Haimen, Jiangsu, PR China). Equal amounts of protein samples (25 mg) were run in 12 % SDS-PAGE and then transferred to nitrocellulose membrane. The membranes were blocked in Tris-buffered saline (TBS) with 3 % nonfat milk, followed by incubation with proper primary antibody. After washed in TBS-Tween 20 buffer, membranes were incubated with HRP-conjugated secondary antibody. The immunoreactive bands were visualized using ECL plus (GE Healthcare, Piscataway, NJ, USA) and the signal intensity was measured using VersaDocTM Imaging system (Bio-Rad, Hercules, CA, USA).

Statistical analysis

Cell proliferation assay

Expression of RI in CRC samples and its relationship with overall survival of CRC patients

The cell proliferation was determined by Cell Counting Kit-8 (CCK-8) assay. Briefly, each group cells about 3,000–4,000 per well were seeded in 96-well plates with 100 ll medium, the plates were incubated at 37 °C with 5 % CO2 for 12, 24, 48, and 72 h, respectively. CCK-8 solution (10 ll) was added to each well and the absorbance was measured at 450 nm after 3 h incubation. Each point represents the mean of 6 independent samples and assay was repeated 3 times.

The significance of the relationships between RI protein expression and clinicopathological parameters was evaluated using v2 tests and unpaired Student’s t test. Survival rates were estimated by Kaplan–Meier curves. Differences between groups were evaluated by log-rank test. SPSS 16.0 software was used for statistical analysis. A value of P \ 0.05 was considered statistically significant.

Results

The expression status of RI mRNA and protein in CRC tissues are summarized in Fig. 1a–c. mRNA expression of RI

Transwell migration assay To evaluate cell migration capability, the transwell plates (Corning, Tewksbury, MA, USA) with 8.0 lm pore size were used. Transwell chambers were inserted into 24-well plate. 1 9 105 cells were plated in the upper compartment in 200 ll serum-free medium per chamber, and 500 ll of complete medium was added to the lower wells. The chambers were incubated for 24 h to allow cells to migrate from the upper chamber to the lower well. Cells migrating through the pores and adherent on the undersurface of the membrane were stained with crystal violet. The number of cells was counted under a microscope. Data were obtained from three individual experiments performed in triplicate. Soft-agar colony-formation assay To determine the transformation potential of the cells, colony-formation assay was performed in 6 cm dishes. The

Fig. 1 Expression of RI in CRC tissues detected by qRT-PCR and western blot. a mRNA expression of RI in colorectal tumors and adjacent normal tissues. b Expression levels of RI mRNA in different histological grade tumor tissues. c Western blot analysis of RI protein expression in normal and different tumor tissues of CRC. GAPDH expression acts as an internal control. N1–N3, normal samples; T1– T3, well and moderate tumor samples; T4–T6, poorly tumor samples. **P \ 0.01

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Clin Transl Oncol Fig. 2 Expression of RI in CRC samples and its relationship with overall survival of CRC. a H&E staining and IHC detection for RI protein expression in representative CRC tissues. a0 , b0 , and c0 , H&E staining showed normal, well and moderately, poorly differentiate CRC tissues, respectively. d0 , e0 , and f0 , RI immunostaining was mainly localized within the cytoplasm and its expression was observed in cancer cells. d0 , strong positive RI. e0 , weak RI staining. f0 , negative RI staining. b Kaplan–Meier survival curves of 108 CRC patients. The RI high expression group had significantly longer survival than the low expression group (log-rank, P = 0.016). Scale bars = 50 lm

decreased in tumor tissues compared with adjacent normal tissues (Fig. 1a). RI mRNA expression levels were also higher in well and moderately differentiate tissues compared with poorly histological grade CRC tissues (Fig. 1b). Western blot analysis showed that RI protein was highly expressed in the adjacent normal tissues compared with tumor samples (Fig. 1c). This data showed that RI mainly expressed in normal colorectal tissues, while its expression decreased with worse histological differentiation. To further identify the RI expression status, IHC was performed to detect the RI expression in different CRC samples. Among the RI-positive cases, the brown IHC staining was strongly present in normal colorectal tissues

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(in the cytoplasm) (Fig. 2a0 -d0 ), and RI-positive staining was also can be detected in well and moderately histological type of CRC cells with weak staining (Fig. 2a0 -e0 ). On the other hand, the poorly histological cancer cells were negative for RI (Fig. 2a0 -f0 ). The IHC staining showed the same tendency of RI expression which detected by qRTPCR and western blot assay. Furthermore, Kaplan–Meier curves have been figured out according to RI staining. RI expression correlated to overall survival of patients with CRC as an independent prognosis predictor. As shown in Fig. 2b, the high expression group had a significantly better long-term prognosis than the low expression group (P \ 0.05, log-rank test).

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Association between RI expression and clinicopathological parameters of CRC patients We grouped the 108 CRC cases into two groups according to the RI expression level [i.e., the low (absent/weak) expression group (n = 64) and the high (intermediate/ strong) expression group (n = 44)]. We compared these two groups for clinicopathological features in CRC patients. As summarized in Table 1, RI protein expression was inversely correlated with tumor histological grade, pathological stage, and venous invasion significantly (v2 test). However, RI expression showed no significant association with other clinicopathological features such as age, gender, tumor location, and lymph vascular invasion. The effects of RI on proliferation and metastatic capability in vitro

Effects of RI on PI3K/Akt signaling pathway in vitro

0.769

PI3K/Akt signaling pathway is known to play a major role in signaling pathways responsible for the metastasis of various cancers. To further study whether RI participates in the proliferation and metastasis of CRC cells via PI3k/Akt pathway, signal transduction molecules were examined in CRC cells. As shown in Fig. 4, the expression p-PI3K and p-Akt in HT29/RI was significantly lower than that in the control group. Phosphatase and tensin homolog (PTEN) is a tumor suppressor with phosphatase activity that can inhibit tumor metastasis via negative regulation of the PI3K/Akt pathway. In this study, upregulation of PTEN was found compared to that in the control group. Previous study showed that PTEN regulates the expression of vascular endothelial growth factor (VEGF) [13]. Our results showed silencing expression of VEGF with the upregulated RI in CRC cells. These results revealed that RI is able to inhibit the PI3k/Akt pathway whereas upregulate the expression of the PTEN protein.

0.008**

Discussion

To assess the effect of RI on tumor cell proliferation and metastatic capability, the exogenous RI gene was Table 1 Association between RI expression and clinicopathological parameters of CRC patients Parameters

Case no.

RI expression High

Low

108

44

64

69 39

30 14

39 25

Male

58

20

38

Female

50

24

26

Colon

62

26

36

Rectum

46

18

28

Well and moderate

52

28

24

Poor

56

16

40

All cases Age C60 \60

0.154

Location

Histology

Pathological stageb

0.018*

pT1–pT2

44

20

24

pT3–pT4

64

24

40

66 42

26 18

40 24

Lymph vascular invasion Negative Positive

0.721

Venous invasion

a b

P valuea

0.441

Gender

0.042*

Negative

61

30

31

Positive

47

14

33

introduced into HT29 cells. After G418 selection, the upregulation of RI was identified by RT-PCR (Fig. 3a) and western blot (Fig. 3b). Furthermore, as shown in CCK-8 assay, upregulated RI in HT29 cells inhibited cell proliferation apparently (Fig. 3c). In transwell assay, the HT29/ RI cells have a lower potential to migrate than wild-type and mock cells (Fig. 3d). Likewise, soft-agar colony-formation assay in vitro further confirmed the effect of RI on tumor metastatic capability. As shown that the colony numbers and size of HT29/RI cells were apparently less and smaller than those of wide-type and mock cells (Fig. 3e). Taken together, the data suggested that the expression of RI markedly affects cell proliferation and migration in CRC cells.

2

v -test, * P \ 0.05, ** P \ 0.01

According to TNM classification of malignant tumors (AJCC/UICC 2010)

RI is a cytosolic protein that binds to ribonucleases with high affinity and renders them inactive [5, 14]. The distinguished LRRs structure which is present in a large family of proteins endues RI with very important proteinprotein interactions [15]. Initially, RI was known as a laboratory reagent to eliminate ribonucleolytic activity based on the unique structure. While additional roles unrelated to inhibition of RNase A has been explored by researchers. For example, RI inhibited angiogenesis by binding to human angiogenin (ANG), which stimulates neovascularization by activating transcription in the nucleus [16, 17]. These observations have led researchers to summarize multiple biological roles for RI [18, 19].

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Fig. 3 Overexpression of RI inhibits cell proliferation and metastatic capability in vitro. a Analysis of RI mRNA expression in HT29 cells by RT-PCR. b Determination of RI protein expression with western blot in HT29 cells. GAPDH was used as internal control. c Cell growth was determined by CCK-8 for definite time. d Cells that migrated through membrane of transwell chambers were fixed and stained with 0.1 % crystal violet and then examined under a light microscope and photographed at 9200 magnifications. The number of

migrating cells was determined by counting the stained cells. (right panel). e Colony-formation assay was performed in 6 cm dishes. After incubation at 37 °C in 5 % CO2, cell colonies were photographed. Cell colonies were counted under the microscope according to diameter (right panel; small, less than 0.3 mm; middle, 0.3–0.6 mm; large, more than 0.6 mm). 1, HT29; 2, HT29/Mock; 3, HT29/RI; data expressed as the mean ± SD from three independent experiments. *P \ 0.05, **P \ 0.01

Ribonuclease inhibitor expression has previously been investigated in a few studies in different human cancers including melanomas [20], breast cancer [21], and cervix cancer [22]. In the present study, human RI expression was evaluated as an independent prognostic biomarker in CRC. The results indicated that RI was significantly down regulated in CRC patients compared with corresponding normal tissues, and the comparison among well, moderately and poorly differentiate CRC samples also showed that RI was much lower in poor outcome patients. Furthermore, in this study, the

relationship between RI expression and clinicopathological parameters showed the down regulation of RI in tumor samples was significantly associated with tumor histological grade, pathological stage, and venous invasion in 108 CRC patients and the patients with high expression group have significantly longer survival time after surgery. Taken together, adding RI expression to conventional clinicopathological risk factors made it possible to make a better prediction of progression and survival to CRC patients. This influence was especially of importance in long-term follow-up.

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Clin Transl Oncol Fig. 4 Effect of RI on the PI3K/Akt signal pathway in CRC cells. a Proteins were extracted, then VEGF, p-PI3K, PI3K, p-Akt, Akt, PTEN expressions were determined by western blot analysis. The results showed downregulation of VEGF, p-PI3K, and p-Akt, and upregulation of PTEN in the RI upregulated group compared to that in the control group. The expression of PI3K, Akt had no significance difference in any of the groups. b–e Densitometric values of protein bands were normalized to those of control. a0 , HT29 cells; b0 , HT29/Mock cells; c0 , HT29/RI cells. The data represent the mean ± S.D, *P \ 0.05, **P \ 0.01

An important finding in our study was that lower expression of RI was strongly associated with metastasis and venous invasion of CRC. To further elucidate the biological function of RI, exogenous RI gene was cloned into eukaryotic expression plasmids that transfected into HT29 CRC cells. The data showed that overexpressing RI could obviously repress cell proliferation and restrain cell migration and colony-formation capability. In addition, Chen et al. demonstrated that the upregulation of RI inhibited the growth and metastasis of murine melanoma cells and that the downregulation of RI facilitates the metastasis potential of noninvasive bladder cancer cells [23]. Also, a recent study identified that upregulation of RI can inhibit tumorigenesis and metastasis of bladder cancer by regulating epithelial–mesenchymal transition through integrin-linked kinase-mediated signaling pathway [8]. These findings indicate that RI plays a significant role in tumor progression. Tumor invasion and metastasis are complex multistage processes. Angiogenesis is essential for carcinogenesis and tumor growth and metastasis. The most potent tumor angiogenic factor, VEGF, can stimulate the proliferation of endothelial cells in various human cancers. VEGF expression is commonly upregulated in tumors and plays a key role in invasion and migration of tumor cells [24]. As known, RI inhibited angiogenesis by binding to angiogenin. VEGF maybe also involved in RI-mediated metastasis in CRC cells. To test this hypothesis, we investigated the expression of VEGF. The results showed that RI upregulation decreases the expression of VEGF. Xiao et al. demonstrated that VEGF promotes angiogenesis via the

PI3K/Akt signaling pathway [25]. However, the potential mechanisms of interaction between RI and VEGF are poorly understood. The PI3K/Akt signaling pathway has been reported to play an essential role in various cellular processes including proliferation, survival, and metastasis [26]. Activated PI3K initiates the activation of the downstream Akt kinase. Moreover, phosphorylated Akt could facilitate cell survival and proliferation and regulate various signaling pathways [27]. Conversely, the PI3K/Akt signaling pathway also regulates the expression of VEGF [28]. The PI3K/Akt signaling pathway has been closely related to the occurrence of human malignant tumors, such as endometrial cancer and nonsmall cell lung cancer [29]. In this study, RI upregulation reduced phosphorylated PI3K and phosphorylated Akt levels, accompanied by inhibition of the expression of VEGF. The tumor suppressor gene PTEN is one of the most commonly lost or mutated phosphatase genes in CRC [30]. PTEN antagonises PI3K/Akt signaling, thereby negatively regulating aggressive tumor behavior. We also found that RI increased the expression of PTEN in HT29/RI cells. Thus, these results suggested that upregulation of RI leads to inhibition of the PI3K/Akt pathway and VEGF expression. In summary, RI is downregulated in CRC tissues compared to adjacent normal tissues, and lower expression is strongly correlated with tumor grade, stage, and venous invasion. In addition, High RI expression is associated with the good prognosis of CRC and is an independent prognostic factor for overall survival. Upregulation of RI significantly inhibited proliferation and metastasis of CRC

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cells by enhancing the expression of PTEN, thereby suppressing PI3K/Akt signaling pathway. These findings might help us to understand the molecular mechanism of RI underlying its antitumor effects, and RI protein thus might potentially represent an important predictor of progression in patients with CRC. Acknowledgments This work was supported by Grant from the National Science Foundation of China (No. 81302310 and No. 31400687). Conflict of interest interest exists.

The authors have declared that no conflict of

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