The role of WWOX tumor suppressor gene in the regulation of EMT process via regulation of CDH1-ZEB1-VIM expression in endometrial cancer

Płuciennik,Elżbieta; Nowakowska,Magdalena; Pospiech,Karolina; Stępień,Anna; Wołkowicz,Mateusz; Gałdyszyńska,Małgorzata; Popęda,Marta; Wójcik-Krowiranda,Katarzyna; Bieńkiewicz,Andrzej; Bednarek,Andrzej  K.

doi:10.3892/ijo.2015.2964

The role of WWOX tumor suppressor gene in the regulation of EMT process via regulation of CDH1-ZEB1-VIM expression in endometrial cancer

Authors:
- Elżbieta Płuciennik
- Magdalena Nowakowska
- Karolina Pospiech
- Anna Stępień
- Mateusz Wołkowicz
- Małgorzata Gałdyszyńska
- Marta Popęda
- Katarzyna Wójcik-Krowiranda
- Andrzej Bieńkiewicz
- Andrzej K. Bednarek
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Affiliations: Department of Molecular Carcinogenesis, Medical University of Lodz, PL 90-752 Lodz, Poland, Laboratory of Clinical and Transplant Immunology and Genetics, Copernicus Memorial Hospital in Lodz, PL 90-752 Lodz, Poland, Bio-Tech Consulting Sp. z o.o, Faculty of Biomedical Science and Postgraduate Education, Medical University of Lodz, PL 90-752 Lodz, Poland, Faculty of Biomedical Sciences and Postgraduate Education, Medical University of Lodz, PL 90-752 Lodz, Poland, Clinical Division of Gynecological Oncology, Medical University of Lodz, PL 90-752 Lodz, Poland
Published online on: April 16, 2015 https://doi.org/10.3892/ijo.2015.2964
Pages: 2639-2648

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Abstract

This study defines the role of WWOX in the regulation of epithelial to mesenchymal transition. A group of 164 endometrial adenocarcinoma patients was studied as well as an ECC1 well-differentiated steroid-responsive endometrial cell line, which was transducted with WWOX cDNA by a retroviral system. The relationship between WWOX gene and EMT marker (CDH1, VIM, ZEB1, SNAI1) expression on mRNA (RT-qPCR) and protein levels (western blotting) was evaluated. The EMT processes were also analysed in vitro by adhesion of cells to extracellular matrix proteins, migration through a basement membrane, anchorage-independent growth and MMP activity assay. DNA microarrays (HumanOneArray™) were used to determine WWOX-dependent pathways in an ECC1 cell line. A positive correlation was observed between WWOX and ZEB1, and a negative correlation between CDH1 and VIM. WWOX expression was found to inversely correlate with the risk of recurrence of tumors in patients. However, in the WWOX-expressing ECC1 cell line, WWOX expression was found to be inversely related with VIM and positively with CDH1. The ECC1/WWOX cell line variant demonstrated increased migratory capacity, with increased expression of metalloproteinases MMP2/MMP9. However, these cells were not able to form colonies in suspension and revealed decreased adhesion to fibronectin and fibrinogen. Microarray analysis demonstrated that WWOX has an impact on the variety of cellular pathways including the cadherin and integrin signalling pathways. Our results suggest that the WWOX gene plays a role in the regulation of EMT processes in endometrial cancer by controlling the expression of proteins associated with cell motility, thus influencing tissue remodeling, with the suppression of mesenchymal markers.

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1	Amant F, Moerman P, Neven P, Timmerman D, Van LE and Vergote I: Endometrial cancer. Lancet. 366:491–505. 2005. View Article : Google Scholar : PubMed/NCBI
2	Leslie KK, Thiel KW, Goodheart MJ, De Geest K, Jia Y and Yang S: Endometrial cancer. Obstet Gynecol Clin North Am. 39:255–268. 2012. View Article : Google Scholar : PubMed/NCBI
3	Merritt MA and Cramer DW: Molecular pathogenesis of endometrial and ovarian cancer. Cancer Biomark. 9:287–305. 2010.
4	Troisi R, Potischman N, Hoover RN, Siiteri P and Brinton LA: Insulin and endometrial cancer. Am J Epidemiol. 146:476–482. 1997. View Article : Google Scholar : PubMed/NCBI
5	Bednarek AK, Laflin KJ, Daniel RL, Liao Q, Hawkins KA and Aldaz CM: WWOX, a novel WW domain-containing protein mapping to human chromosome 16q23.3–24.1, a region frequently affected in breast cancer. Cancer Res. 60:2140–2145. 2000.PubMed/NCBI
6	Thiery JP, Acloque H, Huang RY and Nieto MA: Epithelial-mesenchymal transitions in development and disease. Cell. 139:871–890. 2009. View Article : Google Scholar : PubMed/NCBI
7	Garg M: Epithelial-mesenchymal transition - activating transcription factors - multifunctional regulators in cancer. World J Stem Cells. 5:188–195. 2013. View Article : Google Scholar : PubMed/NCBI
8	Kalluri R and Weinberg RA: The basics of epithelial-mesenchymal transition. J Clin Invest. 119:1420–1428. 2009. View Article : Google Scholar : PubMed/NCBI
9	Tanaka Y, Terai Y, Kawaguchi H, Fujiwara S, Yoo S, Tsunetoh S, Takai M, Kanemura M, Tanabe A and Ohmichi M: Prognostic impact of EMT (epithelial-mesenchymal-transition)-related protein expression in endometrial cancer. Cancer Biol Ther. 14:13–19. 2013. View Article : Google Scholar :
10	Huber MA, Kraut N and Beug H: Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol. 17:548–558. 2005. View Article : Google Scholar : PubMed/NCBI
11	Thiery JP: Epithelial-mesenchymal transitions in development and pathologies. Curr Opin Cell Biol. 15:740–746. 2003. View Article : Google Scholar : PubMed/NCBI
12	Grünert S, Jechlinger M and Beug H: Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis. Nat Rev Mol Cell Biol. 4:657–665. 2003. View Article : Google Scholar : PubMed/NCBI
13	Montserrat N, Mozos A, Llobet D, Dolcet X, Pons C, de Herreros AG, Matias-Guiu X and Prat J: Epithelial to mesenchymal transition in early stage endometrioid endometrial carcinoma. Hum Pathol. 43:632–643. 2012. View Article : Google Scholar
14	Abal M, Llauradó M, Doll A, Monge M, Colas E, González M, Rigau M, Alazzouzi H, Demajo S, Castellví J, et al: Molecular determinants of invasion in endometrial cancer. Clin Transl Oncol. 9:272–277. 2007. View Article : Google Scholar : PubMed/NCBI
15	Aqeilan RI, Donati V, Palamarchuk A, Trapasso F, Kaou M, Pekarsky Y, Sudol M and Croce CM: WW domain-containing proteins, WWOX and YAP, compete for interaction with ErbB-4 and modulate its transcriptional function. Cancer Res. 65:6764–6772. 2005. View Article : Google Scholar : PubMed/NCBI
16	Aqeilan RI, Donati V, Gaudio E, Nicoloso MS, Sundvall M, Korhonen A, Lundin J, Isola J, Sudol M, Joensuu H, et al: Association of Wwox with ErbB4 in breast cancer. Cancer Res. 67:9330–9336. 2007. View Article : Google Scholar : PubMed/NCBI
17	Aqeilan RI, Palamarchuk A, Weigel RJ, Herrero JJ, Pekarsky Y and Croce CM: Physical and functional interactions between the Wwox tumor suppressor protein and the AP-2gamma transcription factor. Cancer Res. 64:8256–8261. 2004. View Article : Google Scholar : PubMed/NCBI
18	Guler G, Huebner K, Himmetoglu C, Jimenez RE, Costinean S, Volinia S, Pilarski RT, Hayran M and Shapiro CL: Fragile histidine triad protein, WW domain-containing oxidoreductase protein Wwox, and activator protein 2gamma expression levels correlate with basal phenotype In breast cancer. Cancer. 115:899–908. 2009. View Article : Google Scholar : PubMed/NCBI
19	Gaudio E, Palamarchuk A, Palumbo T, Trapasso F, Pekarsky Y, Croce CM and Aqeilan RI: Physical association with WWOX suppresses c-Jun transcriptional activity. Cancer Res. 66:11585–11589. 2006. View Article : Google Scholar : PubMed/NCBI
20	Abu-Remaileh M and Aqeilan RI: Tumor suppressor WWOX regulates glucose metabolism via HIF1α modulation. Cell Death Differ. 21:1805–1814. 2014. View Article : Google Scholar : PubMed/NCBI
21	Abu-Odeh M, Bar-Mag T, Huang H, Kim T, Salah Z, Abdeen SK, Sudol M, Reichmann D, Sidhu S, Kim PM, et al: Characterizing WW domain interactions of tumor suppressor WWOX reveals its association with multiprotein networks. J Biol Chem. 289:8865–8880. 2014. View Article : Google Scholar : PubMed/NCBI
22	Sałuda-Gorgul A, Seta K, Nowakowska M and Bednarek AK: WWOX oxidoreductase - substrate and enzymatic characterization. Z Naturforsch C. 66:73–82. 2011. View Article : Google Scholar
23	Aqeilan RI, Hagan JP, de Bruin A, Rawahneh M, Salah Z, Gaudio E, Siddiqui H, Volinia S, Alder H, Lian JB, et al: Targeted ablation of the WW domain-containing oxidoreductase tumor suppressor leads to impaired steroidogenesis. Endocrinology. 150:1530–1535. 2009. View Article : Google Scholar :
24	Aqeilan RI and Croce CM: WWOX in biological control and tumorigenesis. J Cell Physiol. 212:307–310. 2007. View Article : Google Scholar : PubMed/NCBI
25	Qin HR, Iliopoulos D, Semba S, Fabbri M, Druck T, Volinia S, Croce CM, Morrison CD, Klein RD and Huebner K: A role for the WWOX gene in prostate cancer. Cancer Res. 66:6477–6481. 2006. View Article : Google Scholar : PubMed/NCBI
26	Nunez MI, Rosen DG, Ludes-Meyers JH, Abba MC, Kil H, Page R, Klein-Szanto AJ, Godwin AK, Liu J, Mills GB, et al: WWOX protein expression varies among ovarian carcinoma histotypes and correlates with less favorable outcome. BMC Cancer. 5:642005. View Article : Google Scholar : PubMed/NCBI
27	Kosla K, Pluciennik E, Kurzyk A, Jesionek-Kupnicka D, Kordek R, Potemski P and Bednarek AK: Molecular analysis of WWOX expression correlation with proliferation and apoptosis in glioblastoma multiforme. J Neurooncol. 101:207–213. 2011. View Article : Google Scholar :
28	Kuroki T, Yendamuri S, Trapasso F, Matsuyama A, Aqeilan RI, Alder H, Rattan S, Cesari R, Nolli ML, Williams NN, et al: The tumor suppressor gene WWOX at FRA16D is involved in pancreatic carcinogenesis. Clin Cancer Res. 10:2459–2465. 2004. View Article : Google Scholar : PubMed/NCBI
29	Płuciennik E, Nowakowska M, Wujcicka WI, Sitkiewicz A, Kazanowska B, Zielińska E and Bednarek AK: Genetic alterations of WWOX in Wilms’ tumor are involved in its carcinogenesis. Oncol Rep. 28:1417–1422. 2012.
30	Żelazowski MJ, Płuciennik E, Pasz-Walczak G, Potemski P, Kordek R and Bednarek AK: WWOX expression in colorectal cancer - a real-time quantitative RT-PCR study. Tumor Biol. 32:551–560. 2011. View Article : Google Scholar
31	Li YP, Wu CC, Chen WT, Huang YC and Chai CY: The expression and significance of WWOX and β-catenin in hepatocellular carcinoma. APMIS. 121:120–126. 2013. View Article : Google Scholar
32	Xiong Z, Hu S and Wang Z: Cloning of WWOX gene and its growth-inhibiting effects on ovarian cancer cells. J Huazhong Univ Sci Technolog Med Sci. 30:365–369. 2010. View Article : Google Scholar : PubMed/NCBI
33	Qu J, Lu W, Li B, Lu C and Wan X: WWOX induces apoptosis and inhibits proliferation in cervical cancer and cell lines. Int J Mol Med. 31:1139–1147. 2013.PubMed/NCBI
34	Bednarek AK, Keck-Waggoner CL, Daniel RL, Laflin KJ, Bergsagel PL, Kiguchi K, Brenner AJ and Aldaz CM: WWOX, the FRA16D gene, behaves as a suppressor of tumor growth. Cancer Res. 61:8068–8073. 2001.PubMed/NCBI
35	Gourley C, Paige AJ, Taylor KJ, Ward C, Kuske B, Zhang J, Sun M, Janczar S, Harrison DJ, Muir M, et al: WWOX gene expression abolishes ovarian cancer tumorigenicity in vivo and decreases attachment to fibronectin via integrin alpha3. Cancer Res. 69:4835–4842. 2009. View Article : Google Scholar : PubMed/NCBI
36	Nowakowska M, Pospiech K, Lewandowska U, Piastowska-Ciesielska AW and Bednarek AK: Diverse effect of WWOX overexpression in HT29 and SW480 colon cancer cell lines. Tumor Biol. 35:9291–9301. 2014. View Article : Google Scholar
37	Lewandowska U, Zelazowski M, Seta K, Byczewska M, Pluciennik E and Bednarek AK: WWOX, the tumor suppressor gene affected in multiple cancers. J Physiol Pharmacol. 60(Suppl 1): 47–56. 2009.
38	Płuciennik E, Kośla K, Wójcik-Krowiranda K, Bieńkiewicz A and Bednarek AK: The WWOX tumor suppressor gene in endometrial adenocarcinoma. Int J Mol Med. 32:1458–1464. 2013.
39	Pecorelli S: Revised FIGO staging for carcinoma of the vulva, cervix, and endometrium. Int J Gynaecol Obstet. 105:103–104. 2009. View Article : Google Scholar : PubMed/NCBI
40	Mo B, Vendrov AE, Palomino WA, DuPont BR, Apparao KB and Lessey BA: ECC-1 cells: A well-differentiated steroid-responsive endometrial cell line with characteristics of luminal epithelium. Biol Reprod. 75:387–394. 2006. View Article : Google Scholar : PubMed/NCBI
41	Pfaffl MW, Horgan GW and Dempfle L: Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30:e362002. View Article : Google Scholar : PubMed/NCBI
42	Sakuragi N, Nishiya M, Ikeda K, Ohkouch T, Furth EE, Hareyama H, Satoh C and Fujimoto S: Decreased E-cadherin expression in endometrial carcinoma is associated with tumor dedifferentiation and deep myometrial invasion. Gynecol Oncol. 53:183–189. 1994. View Article : Google Scholar : PubMed/NCBI
43	Płuciennik E, Kusińska R, Potemski P, Kubiak R, Kordek R and Bednarek AK: WWOX - the FRA16D cancer gene: Expression correlation with breast cancer progression and prognosis. Eur J Surg Oncol. 32:153–157. 2006. View Article : Google Scholar
44	Wang X, Chao L, Ma G, Chen L, Zang Y and Sun J: The prognostic significance of WWOX expression in patients with breast cancer and its association with the basal-like phenotype. J Cancer Res Clin Oncol. 137:271–278. 2011. View Article : Google Scholar
45	Spoelstra NS, Manning NG, Higashi Y, Darling D, Singh M, Shroyer KR, Broaddus RR, Horwitz KB and Richer JK: The transcription factor ZEB1 is aberrantly expressed in aggressive uterine cancers. Cancer Res. 66:3893–3902. 2006. View Article : Google Scholar : PubMed/NCBI
46	Singh M, Spoelstra NS, Jean A, Howe E, Torkko KC, Clark HR, Darling DS, Shroyer KR, Horwitz KB, Broaddus RR, et al: ZEB1 expression in type I vs type II endometrial cancers: A marker of aggressive disease. Mod Pathol. 21:912–923. 2008. View Article : Google Scholar : PubMed/NCBI
47	Yan H and Sun Y: Evaluation of the mechanism of epithelial-mesenchymal transition in human ovarian cancer stem cells transfected with a WW domain-containing oxidoreductase gene. Oncol Lett. 8:426–430. 2014.PubMed/NCBI
48	Palumbo JS, Kombrinck KW, Drew AF, Grimes TS, Kiser JH, Degen JL and Bugge TH: Fibrinogen is an important determinant of the metastatic potential of circulating tumor cells. Blood. 96:3302–3309. 2000.PubMed/NCBI
49	Sahni A, Arévalo MT, Sahni SK and Simpson-Haidaris PJ: The VE-cadherin binding domain of fibrinogen induces endothelial barrier permeability and enhances transendothelial migration of malignant breast epithelial cells. Int J Cancer. 125:577–584. 2009. View Article : Google Scholar : PubMed/NCBI