FOXP4 inhibits squamous differentiation of atypical cells in cervical intraepithelial neoplasia via an ELF3-dependent pathway.

Although the human papillomavirus (HPV) vaccine is effective for preventing cervical cancers, this vaccine does not eliminate pre-existing infections, and alternative strategies have been warranted. Here, we report that FOXP4 is a new target molecule for differentiation therapy of cervical intraepithelial neoplasia (CIN). An immunohistochemical study showed that FOXP4 was expressed in columnar epithelial, reserve, and immature squamous cells, but not in mature squamous cells of the normal uterine cervix. In contrast with normal mature squamous cells, FOXP4 was expressed in atypical squamous cells in CIN and squamous cell carcinoma lesions. The FOXP4-positive areas significantly increased according to the CIN stages from CIN1 to CIN3. In monolayer cultures, downregulation of FOXP4 attenuated proliferation and induced squamous differentiation in CIN1-derived HPV 16-positive W12 cells via an ELF3-dependent pathway. In organotypic raft cultures, FOXP4-downregulated W12 cells showed mature squamous phenotypes of CIN lesions. In human keratinocyte-derived HaCaT cells, FOXP4 downregulation also induced squamous differentiation via an ELF3-dependent pathway. These findings suggest that downregulation of FOXP4 inhibits cell proliferation and promotes the differentiation of atypical cells in CIN lesions. Based on these results, we propose that FOXP4 is a novel target molecule for nonsurgical CIN treatment that inhibits CIN progression by inducing squamous differentiation.

Spheroid formation induces chemokine production in trophoblast-derived Swan71 cells.

PROBLEM: In the cell column of anchoring villi, the cytotrophoblast differentiates into extravillous trophoblast (EVT) and invades the endometrium in contact with maternal immune cells. Recently, chemokines were proposed to regulate the decidual immune response. To investigate the roles of chemokines around the anchoring villi, we examined the expression profiles of chemokines in the first-trimester trophoblast-derived Swan71 cells using a three-dimensional culture model. METHOD OF STUDY: The gene expressions in the spheroid-formed Swan71 cells were examined by microarray and qPCR analyses. The protein expressions were examined by immunochemical staining. The chemoattractant effects of spheroid-formed Swan71 cells were examined by migration assay using monocyte-derived THP-1 cells. RESULTS: The expressions of an EVT marker, laeverin, and matrix metalloproteases, MMP2 and MMP9, were increased in the spheroid-cultured Swan71 cells. Microarray and qPCR analysis revealed that mRNA expressions of various chemokines, CCL2, CCL7, CCL20, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, and CXCL10, in the spheroid-cultured Swan71 cells were up-regulated as compared with those in the monolayer-cultured Swan71 cells. These expressions were significantly suppressed by hypoxia. Migration assay showed that culture media derived from the spheroid-formed Swan71 cells promoted THP-1 cell migration. CONCLUSION: This study indicated that chemokine expressions in Swan71 cells increase under a spheroid-forming culture and the culture media have chemoattractant effects. Since three-dimensional cell assembling in the spheroid resembles the structure of the cell column, this study also suggests that chemokines play important roles in the interaction between EVT and immune cells in their early differentiation stage.

Androgen promotes squamous differentiation of atypical cells in cervical intraepithelial neoplasia via an ELF3-dependent pathway.

BACKGROUND: Since the human papillomavirus vaccines do not eliminate preexisting infections, nonsurgical alternative approaches to cervical intraepithelial neoplasia (CIN) have been required. We previously reported that FOXP4 (forkhead box transcription factor P4) promoted proliferation and inhibited squamous differentiation of CIN1-derived W12 cells. Since it was reported that FOXP expressions were regulated by the androgen/androgen receptor (AR) complex and AR was expressed on the CIN lesions, in this study we examined the effects of androgen on CIN progression. METHODS: Since AR expression was negative in W12 cells and HaCaT cells, a human male skin-derived keratinocyte cell line, we transfected AR to these cell lines and investigated the effects of dihydrotestosterone (DHT) on their proliferation and squamous differentiation. We also examined the immunohistochemical expression of AR in CIN lesions. RESULTS: DHT reduced the intranuclear expression of FOXP4, attenuating cell proliferation and promoting squamous differentiation in AR-transfected W12 cells. Si-RNA treatments showed that DHT induced the expression of squamous differentiation-related genes in AR-transfected W12 cells via an ELF3-dependent pathway. DHT also reduced FOXP4 expression in AR-transfected HaCaT cells. An immunohistochemical study showed that AR was expressed in the basal to parabasal layers of the normal cervical epithelium. In CIN1 and 2 lesions, AR was detected in atypical squamous cells, whereas AR expression had almost disappeared in the CIN3 lesion and was not detected in SCC, suggesting that androgens do not act to promote squamous differentiation in the late stages of CIN. CONCLUSION: Androgen is a novel factor that regulates squamous differentiation in the early stage of CIN, providing a new strategy for nonsurgical and hormone-induced differentiation therapy against CIN1 and CIN2.