Int Urol Nephrol DOI 10.1007/s11255-015-0915-2

UROLOGY – ORIGINAL PAPER

Relationship between LSD1 expression and E‑cadherin expression in prostate cancer Min Wang · Xiuheng Liu · Guanjun Jiang · Hui Chen · Jia Guo · Xiaodong Weng 

Received: 22 November 2014 / Accepted: 13 January 2015 © Springer Science+Business Media Dordrecht 2015

Abstract  Purpose  To investigate the relationship between the expression of LSD1 and E-cadherin in prostate cancer and their prognostic significance. Methods  The expression of LSD1 and E-cadherin in prostate cancer was detected using immunohistochemistry, and the relationship between the expressions of these two molecules was analyzed by correlation analysis. Furthermore, LNCap cell line was treated with Pargyline (an inhibitor of LSD1), and Western blot was used to analyze LSD1 and E-cadherin expression. Results  LSD1 expression increased significantly in prostate cancer specimens compared with benign prostatic hyperplasia (P < 0.05). Further analysis testified that LSD1 expression was positively correlated with higher Gleason Score, distant metastases, and poor prognosis (P < 0.05). Nevertheless, E-cadherin expression decreased significantly in prostate cancer specimens compared with benign prostatic hyperplasia (P < 0.05) and was negatively correlated with higher Gleason Score, distant metastases (P < 0.05). Correlation analysis revealed that LSD1 expression was negatively correlated with E-cadherin expression in prostate cancer (rs  =  −0.486, P  = 0.001). Positive LSD1 expression and negative E-cadherin expression were significantly correlated with high 2-year progression (occurrence of castration-resistant prostate cancer) rate and low 5-year survival rate (P < 0.05). Moreover, Pargyline

M. Wang · X. Liu (*) · G. Jiang · H. Chen · J. Guo · X. Weng  Department of Urology, Renmin Hospital of Wuhan University, Wuhan University, Jiefang Road 238, Wuhan 430060, Hubei, People’s Republic of China e-mail: [email protected] M. Wang e-mail: [email protected]

inhibited activity of LSD1 and up-regulated E-cadherin expression. Conclusion  High LSD1 expression combined with low E-cadherin expression might be predictors of prostate cancer progression and metastasis. Inhibition of LSD1 may be a potential therapeutic target for prevention of prostate cancer. Keywords  LSD1 · E-cadherin · Prostate cancer · Progression · Metastases

Introduction Prostate cancer (PCa) was the most common noncutaneous malignancy in men. In USA, it was estimated that nearly 217,730 new incidences of PCa was diagnosed in 2010, and it was the second leading cause of cancer-related deaths among US men [1]. The incidence of PCa had consistently risen and might become the leading cause of cancer-related deaths in men living in Western developed countries [2]. The standard treatments for localized PCa and metastatic PCa were radical prostatectomy and androgen deprivation therapy (ADT), respectively. Unfortunately, after an initial response in the majority of cases, most patients would ultimately relapse and progress to more aggressive castrationresistant prostate cancer (CRPC) [3]. In recent years, the phenomenon of epithelial–mesenchymal transition (EMT) had been considered to play an important role in tumorigenesis and metastasis. EMT endowed epithelial cells with fibroblast-like characteristics and accelerated cancer cells to escape from the rigid structural constraints and to spread to distal sites. During EMT, carcinoma cells acquired a malignant phenotype, and benign tumors were allowed to progress to invasive and

13



metastatic cancers showing increased resistance to traditional anticancer therapies [4–6]. E-cadherin was a critical caretaker of the epithelial state, and the loss of E-cadherin was regarded as a hallmark of EMT. Lysine-specific demethylase 1 (LSD1) was the first discovered histone demethylase [7]. It had been analyzed in kinds of human tumors and been showed to be overproduced in neuroblastoma [8], lung, colorectal, and bladder cancer [9, 10], as well as PCa [11, 12]. Snail could facilitate the process of EMT by strongly repressing transcription of E-cadherin gene [13]. Furthermore, LSD1 was essential for Snai1-mediated transcriptional repression, and Snai1 was unable to repress E-cadherin in the absence of LSD1, as the formation of a Snail1-LSD1-CoREST ternary complex played a key role in keeping the stability and function of these proteins [14, 15]. In colon cancer, it was proved that the expression of LSD1 was negatively correlated to the expression of E-cadherin (rs = −0.138, P = 0.001) [16]. However, no studies on relationship between LSD1 expression and E-cadherin expression had been reported in PCa. Our study investigated the expression of LSD1 and E-cadherin in PCa specimen using immunohistochemical methods and correlated their expression levels with clinicopathological data from a patient cohort. Meanwhile, the change of E-cadherin expression was investigated by using a inhibitor of LSD1 in vitro.

Int Urol Nephrol

1:100, cell signal technology). Serial sections (thickness 5 µm) were cut from the tissue blocks, deparaffinized in xylene, and hydrated in a graded series of alcohol. Staining was then performed using the DAB chromogenic agent (Dako Corp, Carpinteria, CA). Negative control experiments were routinely performed. The slides were scored independently by two experienced pathologists who were unaware of the origin of the slides. The semiquantitative scoring system suggested by Remmele and Stegner [17] considering staining intensity and percentage of positive cell nuclei was used for analysis of the immunohistochemical staining results. The staining intensity was described by scores between 0 and 3 (0 = no reaction, 1 = low, 2 = moderate, 3 = strong). Accordingly, the number of positive cell nuclei was counted and scored between 0 and 4 (0 = no positive cell nuclei, 1  ≤ 25 % positive cell nuclei, 2 = 26–50 % positive cell nuclei, 3 = 51–75 % positive cell nuclei, 4 ≥ 75 % positive cell nuclei). The product of staining intensity and percentage of positive cell nuclei resulted in a overall score (IRS) between 0 and 12. Each sample was categorized by this rating score in which an overall score of 0–1 was taken to be negative (−), 2–3 as weak (+), 4–6 as moderate (++), and >6 as strong (+++). Cell culture

Materials and methods

LNCaP cells were cultured in RPMI 1640 medium supplemented with 10 % FBS. Cells were treated with 0, 1, 3 mM Pargyline for 48 h, respectively.

Patients and specimens

Western blot analyses

Archived formalin-fixed and paraffin wax-embedded tissue blocks of 46 PCa and 25 benign prostatic hyperplasia removed by surgery from 2006 to 2008 were retrieved from the Department of Pathology, Renmin Hospital of Wuhan University, China. There were no differences between the two groups in age. Among these 46 cases of PCa, 15 cases were classified in low Gleason Score (4–7) and the other 31 cases were classified in high Gleason Score (8–10); 18 cases were diagnosed with no metastasis, and the other 28 cases were diagnosed with metastasis; 13 cases were treated with radical prostatectomy (RP) with 5-year disease-free survival rate being estimated, and the other 33 cases were treated with ADT with 2-year progression (defines as occurrence of CRPC) rate being estimated.

The protein expression levels of LSD1 and E-cadherin were examined by Western blotting. Briefly, proteins were from LNCaP cells treated with or without Pargyline, separated on 10 % SDS-PAGE gels (50 μg/lane) and then transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were blocked with 5 % nonfat milk in TBST buffer (10 mmol/L Tris–HCl, 0.15 mol/L NaCl, and 0.05 %Tween 20, pH 7.2) for 2 h and incubated with primary antibodies overnight at 4 °C. Primary antibodies used here were monoclonal mouse antibodies against LSD1 (1:50 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and E-cadherin (1:50 dilution; Santa Cruz Biotechnology). After extensive washing with TBST buffer, the membranes were incubated with HRP-conjugated anti-mouse secondary antibodies (1:2,000 dilution; Santa Cruz Biotechnology). The proteins were detected using an enhanced chemiluminescence system (ECL kit, Pierce Biotechnology, Beijing, China) and captured on light-sensitive X-ray film (Kodak, Shanghai, China). Optical densities were detected using ImageJ software.

Immunohistochemical analysis and evaluation Tissue sections were stained by immunohistochemistry (IHC) using specific antibodies for LSD1 (mouse monoclonal, 1:50, Santacruz), E-cadherin (mouse monoclonal,

13

Int Urol Nephrol

Fig. 1  Immunohistochemical expression of LSD1 and E-cadherin in BPH and PCa (magnification, 400). H&E staining showed standard morphology (magnification, 400). BPH showed weak LSD1 expression and strong E-cadherin expression; Low Gleason score PCa showed moderate LSD1 expression and moderate E-cadherin expres-

sion; High Gleason score PCa showed strong LSD1 expression and weak E-cadherin expression; PCa metastasis showed stronger LSD1 expression and weaker E-cadherin expression than PCa without metastasis

Statistical analysis

progression rate after ADT and lower 5-year disease-free survival rate after RP. However, E-cadherin expression significantly down-regulated in cases of PCa and was negatively correlated with Gleason score and metastasis, and lower E-cadherin expression significantly indicated higher 2-year progression rate after ADT, but was not significantly correlated with 5-year disease-free survival rate after RP.

All data were presented as mean ± SEM. Differences were considered statistically significant when P values were <0.05. The means of the different groups were compared using Student’s t test. Spearman’s rank correlation test was used to analyze the correlation between the expression of LSD1 and that of E-cadherin, as assessed by immunohistochemistry.

Correlation of LSD1 expression with E‑cadherin expression Results Expression levels of LSD1, E‑cadherin, and clinicopathological characteristics in patients of BPH and PCa Immunohistochemical staining showed that LSD1 protein was mainly localized in nuclei of luminal cells of normal prostate glands and of prostate carcinoma cells, while E-cadherin protein was localized in the cell membrane and cytoplasm of luminal cells of normal prostate glands and of prostate carcinoma cells (Fig. 1). As shown in Table 1, LSD1 expression significantly up-regulated in cases of PCa and was positively correlated with Gleason score and metastasis, and higher LSD1 expression significantly indicated a poorer prognosis, including higher 2-year

Spearman’s rank correlation test was used to analyze the relationship between LSD1 and E-cadherin expression levels based on the overall staining score. As shown in Table 2, the results showed that LSD1 expression in the PCa specimens was negatively correlated to E-cadherin expression (rs = −0.486, P = 0.001). Pargyline inhibited activity of LSD1 and up‑regulated E‑cadherin expression in vitro To investigate the different levels of protein expression, we measured LSD1 and E-cadherin by Western blot. The expression of LSD1 was not changed in LNCaP cells treated with Pargyline, but the expression of E-cadherin was significantly up-regulated (Fig. 2).

13

Table 1  Clinicopathological and immunohistochemical parameters in relation to LSD1 and E-cadherin immunoreactivity in BPH and PCa

Int Urol Nephrol Item

Disease  BPH 25  PCa 46 Gleason score  4–7 15  8–10 31 Metastasis  No 18  Yes 28 Progression (years)  ≤2 9  >2 24 Survival (years)  ≤5 3  >5

Table 2  Correlation analysis between LSD1 and E-cadherin immunoreactivity in PCa specimens

An overall score of 0–1 was taken to be negative (−), 2–3 as weak (+), 4–6 as moderate (++), and >6 as strong (+++)

n

10

LSD1 expression

+++ ++ + − Total

LSD1 expression X ± SEM

t

P

X ± SEM

t

P

3.72 ± 0.69 7.74 ± 0.50

4.73

<0.01

6.64 ± 0.60 3.44 ± 0.31

5.23

<0.01

5.33 ± 0.73 8.90 ± 0.55

3.79

<0.01

2.45 ± 0.161 5.47 ± 0.67

6.02

<0.01

6.22 ± 0.74 8.71 ± 0.61

2.57

4.33 ± 0.60 2.86 ± 0.30

2.42

0.02

6.44 ± 1.24 9.58 ± 0.49

2.89

<0.01

3.78 ± 072 7.74 ± 0.50

2.51

0.02

9.00 ± 1.73

3.53

<0.01

5.30 ± 0.88

0.37

0.72

0.0138

4.10 ± 0.59

4.67 ± 0.67

E-Cadherin expression +++

++

+

−

Total

1 0 3 1

3 4 1 1

22 6 1 0

2 1 0 0

28 11 5 2

5

9

29

3

46

Discussion Histone methylation played an important role in epigenetic modification and was once considered to be an irreversible process. LSD1 was the first discovered histone demethylase [7], which could demethylate only mono- and dimethylated lysine residues but not trimethylated lysine residues. LSD1 had been analyzed in numerous human tumors, including PCa. Importantly, it was involved in many pathological processes of cancer, including carcinogenesis, proliferation, metastasis, and apoptosis [8, 9, 18–20]. The LSD1 expression had been found to be evidently higher in multiple cancers, such as neuroblastoma, lung, colorectal, and bladder cancer [8–10]. Consistent with precious study [12], this study revealed that LSD1 level significantly increased in PCa specimens compared with BPH (P < 0.05). Furthermore, LSD1 expression positively correlated with higher Gleason Score, distant metastases, and progression (P < 0.05). Consequently, it was speculated that LSD1 might play a vital role in PCa metastases and progression.

13

E-cadherin expression

rs

P

−0.486

0.001

In one hand, it was speculated that LSD1 might mediate PCa progression via interacting with AR. The standard treatment for metastatic PCa was ADT, but a number of patients relapsed and developed more aggressive CRPC after a median of only 2–3 years [5]. Multiple studies had shown that not only AR protein but also AR mRNA was expressed at high levels in CRPC compared with levels in untreated tumors [21–26]. So, reactivation of AR was believed to be one of the mechanisms for CRPC. In accordance with previous study [12], this study showed that higher expression level of LSD1 was significantly positively correlated with earlier occurence of CRPC after ADT (P < 0.05). It was demonstrated that LSD1 co-localizes with AR in nucleus of normal human prostate and PCa [27]. By forming chromatin-associated complexes with AR in a ligand-dependent manner, LSD1 demethylated the repressing histone marks mono- and dimethyl H3-K9 and thereby leaded to de-repression of AR target genes, including PSA [27], which was considered as a marker of occurrence and progression. In the other hand, LSD1 might promote process of EMT by down-regulating E-cadherin expression.

Int Urol Nephrol

study, inhibiting LSD1 function by Pargyline significantly up-regulated the expression level of E-cadherin in vitro (P < 0.05). Then, inhibiting LSD1 function or down-regulating LSD1 expression might serve as a potential therapeutic intervention to delay or suppress PCa progression and metastasis, via repressing AR target genes and up-regulating E-cadherin. In conclusion, LSD1 expression was significantly upregulated, while E-cadherin expression was significantly down-regulated in PCa specimens at high Gleason score and in distant metastasis. Specimens which expressed higher levels of LSD1 accompanied with lower levels of E-cadherin might indicate a worse prognosis. Meanwhile, inhibition of LSD1 might offer a promising strategy for treating CRPC and PCa metastasis. However, these hypotheses were limited, as they were mainly based on a retrospective study. Better designed prospective studies and further experiments in vitro and animal models were required to confirm this study.

Fig. 2  LNCaP cells were treated with 0, 1, or 3 mM Pargyline for 48 h and LSD1, E-cadherin or β-actin were immunoblotted. (*P < 0.05 versus 0 mM, **P < 0.05 versus 1 mM)

Epithelial cells could be reprogrammed into mesenchymal cells, a process defined as EMT [28]. During cancer progression, the process of EMT had been associated with the acquisition of stemness properties, treatment resistance, and metastatic progression, hallmarks of malignancy [29, 30]. EMT was regarded as an important step in PCa metastasis [31] and was involved in deregulation of the androgen axis [32], which might be correlated with CRPC. This study revealed that loss of E-cadherin was significantly correlated with metastasis and progression to CRPC (P < 0.05). Loss of E-cadherin was considered as a hallmark of EMT and a prerequisite for tumor cell invasion and metastasis formation [33, 34]. Previous studies [14, 15] had demonstrated that LSD1 was necessary for Snai1-mediated transcriptional repression of E-cadherin. This study also revealed that LSD1 expression was negatively correlated with E-cadherin expression in PCa specimen by correlation analysis (rs = −0.486, P = 0.001). LSD1 was composed of three domains, including a C-terminal Tower domain, an N-terminal SWIRM domain, and an amino oxidase damain (AOD). As AOD of LSD1 shared high structural and mechanistic similarities with amine oxidases, monoamine oxidase (MAO) covalent inhibitors including Pargyline, tranylcypromine, and polymine analogues had been shown to inhibit LSD1 enzymatic activity. The process of EMT was reversible which might be reverted by re-expression of E-cadherin. In our

Acknowledgments  This study is supported by the Grants from the National Natural Science Foundation of China (No. 2013RMFH012), the Province Natural Science Foundation of Hubei (No. 2012FFA096), and supported by the Fundamental Research Funds for the Central Universities (No. 302-274231). We thank all the authors whose work was included in this study. Conflict of interest None.

References 1. Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60:277–300 2. Brooke GN, Bevan CL (2009) The role of androgen receptor mutations in prostate cancer progression. Curr Genomics 10:18–25 3. Taplin ME, Balk SP (2004) Androgen receptor: a key molecule in the progression of prostate cancer to hormone independence. J Cell Biochem 91:483–490 4. Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelialmesenchymal transitions in development and disease. Cell 139:871–890 5. Iwatsuki M, Mimori K, Yokobori T, Ishi H, Beppu T, Nakamori S, Baba H, Mori M (2010) Epithelial-mesenchymal transition in cancer development and its clinical significance. Cancer Sci 101:293–299 6. Voulgari A, Pintzas A (2009) Epithelial-mesenchymal transition in cancer metastasis: mechanisms, markers and strategies to overcome drug resistance in the clinic. Biochim Biophys Acta 1796:75–90 7. Shi Y, Lan F, Matson C, Mulligan P, Whetstine JR, Cole PA, Casero RA (2004) Histone demethylation mediated by the nuclear amine oxidase homolog LSD1. Cell 119:941–953 8. Schulte JH, Lim S, Schramm A, Friedrichs N, Koster J, Versteeg R, Ora I, Pajtler K, Klein-Hitpass L, Kuhfittig-Kulle S, Metzger E, Schule R, Eggert A, Buettner R, Kirfel J (2009) Lysine-specific demethylase 1 is strongly expressed in poorly differentiated neuroblastoma: implications for therapy. Cancer Res 69:2065–2071

13

9. Hayami S, Kelly JD, Cho HS, Yoshimatsu M, Unoki M, Tsunoda T, Field HI, Neal DE, Yamaue H, Ponder BA, Nakamura Y, Hamamoto R (2011) Overexpression of LSD1 contributes to human carcinogenesis through chromatin regulation in various cancers. Int J Cancer 128:574–586 10. Kauffman EC, Robinson BD, Downes MJ, Powell LG, Lee MM, Scherr DS, Gudas LJ, Mongan NP (2011) Role of androgen receptor and associated lysine-demethylase coregulators, LSD1 and JMJD2A, in localized and advanced human bladder cancer. Mol Carcinog 50:931–944 11. Metzger E, Imhof A, Patel D, Kahl P, Hoffmeyer K, Friedrichs N, Muller JM, Greschik H, Kirfel J, Ji S, Kunowska N, BeisenherzHuss C, Gunther T, Buettner R, Schule R (2010) Phosphorylation of histone H3T6 by PKCbeta(I) controls demethylation at histone H3K4. Nature 464:792–796 12. Kahl P, Gullotti L, Heukamp LC, Wolf S, Friedrichs N, Vorreuther R, Solleder G, Bastian PJ, Ellinger J, Metzger E, Schule R, Buettner R (2006) Androgen receptor coactivators lysinespecific histone demethylase 1 and four and a half LIM domain protein 2 predict risk of prostate cancer recurrence. Cancer Res 66:11341–11347 13. Batlle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J, Garcia De Herreros A (2000) The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2:84–89 14. Lin T, Ponn A, Hu X, Law BK, Lu J (2010) Requirement of the histone demethylase LSD1 in Snai1-mediated transcriptional repression during epithelial-mesenchymal transition. Oncogene 29:4896–4904 15. Lin Y, Wu Y, Li J, Dong C, Ye X, Chi YI, Evers BM, Zhou BP (2010) The SNAG domain of Snail1 functions as a molecular hook for recruiting lysine-specific demethylase 1. EMBO J 29:1803–1816 16. Jie D, Zhongmin Z, Guoqing L, Sheng L, Yi Z, Jing W, Liang Z (2013) Positive expression of LSD1 and negative expression of E-cadherin correlate with metastasis and poor prognosis of colon cancer. Dig Dis Sci 58:1581–1589 17. Remmele W, Stegner HE (1987) Recommendation for uniform definition of an immunoreactive score (IRS) for immunohistochemical estrogen receptor detection (ER-ICA) in breast cancer tissue. Pathologe 8:138–140 18. Huang J, Sengupta R, Espejo AB, Lee MG, Dorsey JA, Richter M, Opravil S, Shiekhattar R, Bedford MT, Jenuwein T, Berger SL (2007) p53 is regulated by the lysine demethylase LSD1. Nature 449:105–108 19. Scoumanne A, Chen X (2007) The lysine-specific demethylase 1 is required for cell proliferation in both p53-dependent and -independent manners. J Biol Chem 282:15471–15475 20. Bennani-Baiti IM, Machado I, Llombart-Bosch A, Kovar H (2012) Lysine-specific demethylase 1 (LSD1/KDM1A/AOF2/ BHC110) is expressed and is an epigenetic drug target in chondrosarcoma, Ewing’s sarcoma, osteosarcoma, and rhabdomyosarcoma. Hum Pathol 43:1300–1307

13

Int Urol Nephrol 21. van der Kwast TH, Schalken J, Ruizeveld de Winter JA, van Vroonhoven CC, Mulder E, Boersma W, Trapman J (1991) Androgen receptors in endocrine-therapy-resistant human prostate cancer. Int J Cancer 48:189–193 22. Ruizeveld de Winter JA, Janssen PJ, Sleddens HM, Verleun-Mooijman MC, Trapman J, Brinkmann AO, Santerse AB, Schroder FH, van der Kwast TH (1994) Androgen receptor status in localized and locally progressive hormone refractory human prostate cancer. Am J Pathol 144:735–746 23. Mohler JL, Gregory CW, Ford OH 3rd, Kim D, Weaver CM, Petrusz P, Wilson EM, French FS (2004) The androgen axis in recurrent prostate cancer. Clin Cancer Res 10:440–448 24. Taplin ME, Bubley GJ, Shuster TD, Frantz ME, Spooner AE, Ogata GK, Keer HN, Balk SP (1995) Mutation of the androgenreceptor gene in metastatic androgen-independent prostate cancer. N Engl J Med 332:1393–1398 25. Holzbeierlein J, Lal P, LaTulippe E, Smith A, Satagopan J, Zhang L, Ryan C, Smith S, Scher H, Scardino P, Reuter V, Gerald WL (2004) Gene expression analysis of human prostate carcinoma during hormonal therapy identifies androgen-responsive genes and mechanisms of therapy resistance. Am J Pathol 164:217–227 26. Stanbrough M, Bubley GJ, Ross K, Golub TR, Rubin MA, Penning TM, Febbo PG, Balk SP (2006) Increased expression of genes converting adrenal androgens to testosterone in androgenindependent prostate cancer. Cancer Res 66:2815–2825 27. Metzger E, Wissmann M, Yin N, Muller JM, Schneider R, Peters AH, Gunther T, Buettner R, Schule R (2005) LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription. Nature 437:436–439 28. Hay ED (1995) An overview of epithelio-mesenchymal transformation. Acta Anat (Basel) 154:8–20 29. Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES (2009) Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138:645–659 30. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA (2008) The epithelialmesenchymal transition generates cells with properties of stem cells. Cell 133:704–715 31. Emadi Baygi M, Soheili ZS, Schmitz I, Sameie S, Schulz WA (2010) Snail regulates cell survival and inhibits cellular senescence in human metastatic prostate cancer cell lines. Cell Biol Toxicol 26:553–567 32. Zhu ML, Kyprianou N (2010) Role of androgens and the androgen receptor in epithelial-mesenchymal transition and invasion of prostate cancer cells. FASEB J 24:769–777 33. Friedl P, Alexander S (2011) Cancer invasion and the microenvironment: plasticity and reciprocity. Cell 147:992–1009 34. Yip WK, Seow HF (2012) Activation of phosphatidylinositol 3-kinase/Akt signaling by EGF downregulates membranous E-cadherin and beta-catenin and enhances invasion in nasopharyngeal carcinoma cells. Cancer Lett 318:162–172