The Wnt/b-catenin signaling pathway is activated during the malignant transformation of keratinocytes that originate from the human uterine cervix. Dkk1, 2 and 4 have been shown to modulate the Wnt-induced stabilization of the b-catenin signaling pathway. However, the function of Dkk3 in this pathway is unknown. Comparison of the Dkk3 gene expression profiles in cervical cancer and normal cervical tissue by cDNA microarray and subsequent real-time PCR revealed that the Dkk3 gene is frequently downregulated in the cancer. Methylation studies showed that the promoter of Dkk3 was methylated in cervical cancer cell lines and 22 (31.4%) of 70 cervical cancer tissue specimens. This promoter methylation was associated with reduced expression of Dkk3 mRNA in the paired normal and tumor tissue samples. Further, the reintroduction of Dkk3 into HeLa cervical cancer cells resulted in reduced colony formation and retarded cell growth. The forced expression of Dkk3 markedly attenuated b-catenin-responsive luciferase activity in a dose-dependent manner and decreased the b-catenin levels. By utilizing a yeast two-hybrid screen, bTrCP, a negative regulator of b-catenin was identified as a novel Dkk3-interacting partner. Coexpression with bTrCP synergistically enhanced the inhibitory function of Dkk3 on b-catenin. The stable expression of Dkk3 blocks the nuclear translocation of b-catenin, resulting in downregulation of its downstream targets (VEGF and cylcin D), whereas knockdown of Dkk3 abrogates this blocking. We conclude from our finding that Dkk3 is a negative regulator of b-catenin and its downregulation contribute to an activation of the b-catenin signaling pathway.

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Ovarian cancer is a leading cause of death in women. Early detection of ovarian cancer is essential to decrease mortality. However, the early diagnosis of ovarian cancer is difficult due to a lack of clinical symptoms and suitable molecular diagnostic markers. Thus, identification of meaningful tumor biomarkers with potential clinical application is clearly needed. To search for a biomarker for the early detection of ovarian cancer, we identified human anterior gradient 2 (AGR2) from our systematic analysis of paired normal and ovarian tumor tissue cDNA microarray. We noted a marked overexpression of AGR2 mRNA and protein in early stage mucinous ovarian tumors compared to normal ovarian tissues and serous type ovarian tumors by Western blot analysis and immunohistochemistry. To further elucidate the role of AGR2 in ovarian tumorigenesis, stable 2774 human ovarian cancer cell lines overexpressing AGR2 were established. Forced expression of AGR2 in 2774 cells enhanced the growth and migration of ovarian cancer cells. AGR2 protein was detected in the serum of mucinous ovarian cancer patients by Western blot and ELISA analysis. Thus, AGR2 is a potential biomarker for the diagnosis of mucinous ovarian cancer and an ELISA assay may facilitate the early detection of mucinous ovarian cancer using patient serum.

Mammalian target of rapamycin (mTOR) is known to be a major negative regulator of autophagy. Recent studies have shown that mTOR activity is abnormally increased in endometriotic lesions. In endometriosis, abnormal mTOR activity may contribute to the alteration of endometrial cell autophagy, which may affect apoptosis because endometrial cell autophagy is directly involved in the regulation of apoptosis. To test this hypothesis, we investigated whether endometrial cell autophagy is altered by aberrant mTOR activity and is associated with apoptosis in ovarian endometriotic cysts. Our results show that endometrial cell autophagy induction was increased by mTOR inhibition as the menstrual cycle progresses in the normal endometrium, and that it is correlated with apoptosis. However, in endometriotic tissues from ovarian endometriotic cysts, autophagy, mTOR activity and apoptosis were constant throughout the menstrual cycle, suggesting that a constant level of autophagy is maintained by disinhibition of mTOR activity during the menstrual cycle in endometriotic tissues and is related to decreased apoptosis. Indeed, compared with normal endometrium, increased mTOR activity during the secretory phase in endometriotic tissues inhibited autophagy and apoptosis induction. In addition, to determine the direct effect of autophagy induction mediated by mTOR on endometriotic cell apoptosis, endometriotic cells were treated with rapamacin (mTOR inhibitor) with and without 3-methyladenine (3-MA, autophagy inhibitor). Although rapamycin treatment induced autophagy and led to apoptosis promotion, the pro-apoptotic effect of rapamycin was reversed by the addition of 3-MA, suggesting that mTOR inhibition promotes endometriotic cell apoptosis via autophagy induction. In conclusion, our results suggest that aberrant mTOR activity in ovarian endometriotic cysts leads to alteration of endometrial cell autophagy, which is associated with abnormal apoptosis.