Loss of AF-6/afadin induces cell invasion, suppresses the formation of glandular structures and might be a predictive marker of resistance to chemotherapy in endometrial cancer

BMC Cancer

Jo Kitawaki¹  Makoto Akiyama¹  Fumitake Ito¹  Hiroshi Matsushima¹  Morio Sawada¹  Taisuke Mori¹  Takuro Yamamoto¹ 

[1]Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku 602-8566, Kyoto, Japan

关键词: Src;    ERK;    Morphology;    Invasion;    Endometrial cancer;    AF-6/afadin;   

Others  :  1171661
DOI  :  10.1186/s12885-015-1286-x

 received in 2014-12-25, accepted in 2015-03-30,  发布年份 2015

【 摘 要 】

Background

AF-6/afadin plays an important role in the formation of adherence junctions. In breast and colon cancer, loss of AF-6/afadin induces cell migration and cell invasion. We aimed to elucidate the role of AF-6/afadin in human endometrial cancer.

Methods

Morphology and AF-6/afadin expression in endometrial cancer cell lines was investigated by 3-dimensional culture. We used Matrigel invasion assay to demonstrate AF-6/afadin knockdown induced invasive capability. Cell proliferation assay was performed to estimate chemoresistance to doxorubicin, paclitaxel and cisplatin induced by AF-6/afadin knockdown. The associations between AF-6/afadin expression and clinicopathological status were determined by immunohistochemical analysis in endometrial cancer tissues. Informed consent was obtained from all patients before the study.

Results

The majority of cell clumps in 3-dimensional cultures of Ishikawa cells that strongly expressed AF-6/afadin showed round gland-like structures. In contrast, the cell clumps in 3-dimensional cultures of HEC1A and AN3CA cells—both weakly expressing AF-6/afadin—showed irregular gland-like structures and disorganized colonies with no gland-like structures, respectively. AF-6/afadin knockdown resulted in reduced number of gland-like structures in 3-dimensional cultures and enhancement of cell invasion and phosphorylation of ERK1/2 and Src in the highly AF-6/afadin-expressing endometrial cancer cell line. Inhibitors of MAPK/ERK kinase (MEK) (U0126) and Src (SU6656) suppressed the AF-6/afadin knockdown-induced invasive capability. AF-6/afadin knockdown induced chemoresistance to doxorubicin, paclitaxel and cisplatin in Ishikawa cells, not in HEC1A. Immunohistochemical analysis showed that AF-6/afadin expression was significantly associated with myometrial invasion and high histological grade.

Conclusions

AF-6/afadin regulates cell morphology and invasiveness. Invasive capability is partly regulated through the ERK and Src pathway. The inhibitors to these pathways might be molecular-targeted drugs which suppress myometrial invasion in endometrial cancer. AF-6/afadin could be a useful selection marker for fertility-sparing therapy for patients with atypical hyperplasia or grade 1 endometrioid adenocarcinoma with no myometrial invasion. AF-6/afadin knockdown induced chemoresistance especially to cisplatin. Therefore, loss of AF-6/afadin might be a predictive marker of chemoresistance to cisplatin.

【 授权许可】

   
2015 Yamamoto et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Aoki D: Annual report of gynecologic oncology committee, Japan society of obstetrics and gynecology, 2013. J Obstet Gynaecol Res 2014, 40(2):338-48.
• [2]Ushijima K: Current status of gynecologic cancer in Japan. J Gynecol Oncol 2009, 20(2):67-71.
• [3]Sorosky JI: Endometrial cancer. Obstet Gynecol 2008, 111(2 Pt 1):436-47.
• [4]Yilmaz M, Christofori G: EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev 2009, 28(1–2):15-33.
• [5]Amant F, Moerman P, Neven P, Timmerman D, Van Limbergen E, Vergote I: Endometrial cancer. Lancet 2005, 366(9484):491-505.
• [6]Saha V, Lillington DM, Shelling AN, Chaplin T, Yaspo ML, Ganesan TS, et al.: AF6 gene on chromosome band 6q27 maps distal to the minimal region of deletion in epithelial ovarian cancer. Genes Chromosomes Cancer 1995, 14(3):220-2.
• [7]Takahashi K, Nakanishi H, Miyahara M, Mandai K, Satoh K, Satoh A, et al.: Nectin/PRR: an immunoglobulin-like cell adhesion molecule recruited to cadherin-based adherens junctions through interaction with Afadin, a PDZ domain-containing protein. J Cell Biol 1999, 145(3):539-49.
• [8]Asakura T, Nakanishi H, Sakisaka T, Takahashi K, Mandai K, Nishimura M, et al.: Similar and differential behaviour between the nectin-afadin-ponsin and cadherin-catenin systems during the formation and disruption of the polarized junctional alignment in epithelial cells. Genes Cells 1999, 4(10):573-81.
• [9]Takai Y, Nakanishi H: Nectin and afadin: novel organizers of intercellular junctions. J Cell Sci 2003, 116(Pt 1):17-27.
• [10]Miyata M, Ogita H, Komura H, Nakata S, Okamoto R, Ozaki M, et al.: Localization of nectin-free afadin at the leading edge and its involvement in directional cell movement induced by platelet-derived growth factor. J Cell Sci 2009, 122(Pt 23):4319-29.
• [11]Elloul S, Kedrin D, Knoblauch NW, Beck AH, Toker A. The Adherens Junction Protein Afadin Is an AKT Substrate that Regulates Breast Cancer Cell Migration. Mol Cancer Res. 2014.
• [12]Fournier G, Cabaud O, Josselin E, Chaix A, Adelaide J, Isnardon D, et al.: Loss of AF6/afadin, a marker of poor outcome in breast cancer, induces cell migration, invasiveness and tumor growth. Oncogene 2011, 30(36):3862-74.
• [13]Sun TT, Wang Y, Cheng H, Zhang XH, Xiang JJ, Zhang JT, et al.: Disrupted interaction between CFTR and AF-6/afadin aggravates malignant phenotypes of colon cancer. Biochim Biophys Acta 2013, 1843(3):618-28.
• [14]Letessier A, Garrido-Urbani S, Ginestier C, Fournier G, Esterni B, Monville F, et al.: Correlated break at PARK2/FRA6E and loss of AF-6/Afadin protein expression are associated with poor outcome in breast cancer. Oncogene 2007, 26(2):298-307.
• [15]Koshiba H, Hosokawa K, Kubo A, Tokumitsu N, Watanabe A, Honjo H: Junctional adhesion molecule A [corrected] expression in human endometrial carcinoma. Int J Gynecol Cancer 2009, 19(2):208-13.
• [16]Yamamoto T, Mori T, Sawada M, Kuroboshi H, Tatsumi H, Yoshioka T, et al.: Estrogen-related receptor-gamma regulates estrogen receptor-alpha responsiveness in uterine endometrial cancer. Int J Gynecol Cancer 2012, 22(9):1509-16.
• [17]Mandai K, Nakanishi H, Satoh A, Obaishi H, Wada M, Nishioka H, et al.: Afadin: A novel actin filament-binding protein with one PDZ domain localized at cadherin-based cell-to-cell adherens junction. J Cell Biol 1997, 139(2):517-28.
• [18]Ooshio T, Irie K, Morimoto K, Fukuhara A, Imai T, Takai Y: Involvement of LMO7 in the association of two cell-cell adhesion molecules, nectin and E-cadherin, through afadin and alpha-actinin in epithelial cells. J Biol Chem 2004, 279(30):31365-73.
• [19]Sato T, Fujita N, Yamada A, Ooshio T, Okamoto R, Irie K, et al.: Regulation of the assembly and adhesion activity of E-cadherin by nectin and afadin for the formation of adherens junctions in Madin-Darby canine kidney cells. J Biol Chem 2006, 281(8):5288-99.
• [20]Mell LK, Meyer JJ, Tretiakova M, Khramtsov A, Gong C, Yamada SD, et al.: Prognostic significance of E-cadherin protein expression in pathological stage I-III endometrial cancer. Clin Cancer Res 2004, 10(16):5546-53.
• [21]Moreno-Bueno G, Hardisson D, Sarrio D, Sanchez C, Cassia R, Prat J, et al.: Abnormalities of E- and P-cadherin and catenin (beta-, gamma-catenin, and p120ctn) expression in endometrial cancer and endometrial atypical hyperplasia. J Pathol 2003, 199(4):471-8.
• [22]Gonzalez-Rodilla I, Aller L, Llorca J, Munoz AB, Verna V: The E-Cadherin expression vs. tumor cell proliferation paradox in endometrial cancer. Anticancer Res 2013, 33(11):5091-5.
• [23]O’Brien LE, Zegers MM, Mostov KE: Opinion: Building epithelial architecture: insights from three-dimensional culture models. Nat Rev Mol Cell Biol 2002, 3(7):531-7.
• [24]Mariani A, Dowdy SC, Podratz KC: New surgical staging of endometrial cancer: 20 years later. Int J Gynaecol Obstet 2009, 105(2):110-1.
• [25]Thomas SM, Brugge JS: Cellular functions regulated by Src family kinases. Annu Rev Cell Dev Biol 1997, 13:513-609.
• [26]Yeatman TJ: A renaissance for SRC. Nat Rev Cancer 2004, 4(6):470-80.
• [27]Sugimura M, Kobayashi K, Sagae S, Nishioka Y, Ishioka S, Terasawa K, et al.: Mutation of the SRC gene in endometrial carcinoma. Jpn J Cancer Res 2000, 91(4):395-8.
• [28]Boucher MJ, Morisset J, Vachon PH, Reed JC, Laine J, Rivard N: MEK/ERK signaling pathway regulates the expression of Bcl-2, Bcl-X(L), and Mcl-1 and promotes survival of human pancreatic cancer cells. J Cell Biochem 2000, 79(3):355-69.
• [29]Li C, Ahlborn TE, Kraemer FB, Liu J: Oncostatin M-induced growth inhibition and morphological changes of MDA-MB231 breast cancer cells are abolished by blocking the MEK/ERK signaling pathway. Breast Cancer Res Treat 2001, 66(2):111-21.
• [30]Mitsui H, Takuwa N, Maruyama T, Maekawa H, Hirayama M, Sawatari T, et al.: The MEK1-ERK map kinase pathway and the PI 3-kinase-Akt pathway independently mediate anti-apoptotic signals in HepG2 liver cancer cells. Int J Cancer 2001, 92(1):55-62.
• [31]Kanai M, Konda Y, Nakajima T, Izumi Y, Kanda N, Nanakin A, et al.: Differentiation-inducing factor-1 (DIF-1) inhibits STAT3 activity involved in gastric cancer cell proliferation via MEK-ERK-dependent pathway. Oncogene 2003, 22(4):548-54.
• [32]Koochekpour S, Sartor O, Lee TJ, Zieske A, Patten DY, Hiraiwa M, et al.: Prosaptide TX14A stimulates growth, migration, and invasion and activates the Raf-MEK-ERK-RSK-Elk-1 signaling pathway in prostate cancer cells. Prostate 2004, 61(2):114-23.
• [33]Nguyen TT, Tran E, Nguyen TH, Do PT, Huynh TH, Huynh H: The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cells. Carcinogenesis 2004, 25(5):647-59.
• [34]Radziwill G, Erdmann RA, Margelisch U, Moelling K: The Bcr kinase downregulates Ras signaling by phosphorylating AF-6 and binding to its PDZ domain. Mol Cell Biol 2003, 23(13):4663-72.
• [35]Chen T, Wang C, Liu Q, Meng Q, Sun H, Huo X, et al.: Dasatinib reverses the multidrug resistance of breast cancer MCF-7 cells to doxorubicin by downregulating P-gp expression via inhibiting the activation of ERK signaling pathway. Cancer Biol Ther 2015, 16(1):106-14.
• [36]Wu G, Qin XQ, Guo JJ, Li TY, Chen JH: AKT/ERK activation is associated with gastric cancer cell resistance to paclitaxel. Int J Clin Exp Pathol 2014, 7(4):1449-58.
• [37]Fu X, Feng J, Zeng D, Ding Y, Yu C, Yang B: PAK4 confers cisplatin resistance in gastric cancer cells via PI3K/Akt- and MEK/Erk-dependent pathways. Biosci Rep 2014, 34(2):art:e00094.
• [38]Wang J, Zhou JY, Wu GS: ERK-dependent MKP-1-mediated cisplatin resistance in human ovarian cancer cells. Cancer Res 2007, 67(24):11933-41.
• [39]Wu DW, Wu TC, Wu JY, Cheng YW, Chen YC, Lee MC, et al.: Phosphorylation of paxillin confers cisplatin resistance in non-small cell lung cancer via activating ERK-mediated Bcl-2 expression. Oncogene 2014, 33(35):4385-95.