title: 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.
abstract: 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.
authors: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan. teku@koto.kpu-m.ac.jp.
authors: Takuro
authors: T
authors: Yamamoto
authors: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan. moriman@koto.kpu-m.ac.jp.
authors: Taisuke
authors: T
authors: Mori
authors: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan. mosawada@koto.kpu-m.ac.jp.
authors: Morio
authors: M
authors: Sawada
authors: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan. m-hiro@koto.kup-m.ac.jp.
authors: Hiroshi
authors: H
authors: Matsushima
authors: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan. fitoh@koto.kpu-m.ac.jp.
authors: Fumitake
authors: F
authors: Ito
authors: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan. mt-fall@koto.kpu-m.ac.jp.
authors: Makoto
authors: M
authors: Akiyama
authors: Department of Obstetrics and Gynecology, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan. kitawaki@koto.kpu-m.ac.jp.
authors: Jo
authors: J
authors: Kitawaki
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D002945:Cisplatin / Q000008:administration & dosage; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D016889:Endometrial Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D006801:Humans; D016547:Kinesins / Q000096:biosynthesis* / Q000235:genetics; D020935:MAP Kinase Signaling System / Q000187:drug effects; D008875:Middle Aged; D009218:Myosins / Q000096:biosynthesis* / Q000235:genetics; D009361:Neoplasm Invasiveness / Q000235:genetics; D017239:Paclitaxel / Q000008:administration & dosage
chemical_list: C095516:AFDN protein, human; D009218:Myosins; D016547:Kinesins; D017239:Paclitaxel; D002945:Cisplatin