br Correlation of Bach with Slug and HMGA in
3.5. Correlation of Bach1 with Slug and HMGA2 in human ovarian cancers
To further confirm the relationship among Bach1, HMGA2 and Slug, we analyzed the expression of these proteins in normal ovarian tissues and EOC samples. The protein levels of Bach1 and the EMT markers Vimentin, Slug, Snail and Twist were significantly increased in human ovarian cancer samples compared with normal tissues (Fig. 5A–B); moreover, the expression of Slug and HMGA2 was significantly in-creased in the early stages (stage I and II) of EOC and further increased in the advanced stages (stage III and IV) of EOC (Fig. 5B–C). Combined with the expression of Bach1 in these samples (Fig. 1A), the results of the correlation analysis revealed that Bach1 expression was positively correlated with Slug and HMGA2 expression in human ovarian cancers (Fig. 5D). Furthermore, Fig. 5D shows a positive correlation between the expression of HMGA2 and Slug.
3.6. Bach1 enhances EOC cell growth in vitro and in vivo
We evaluated the role of Bach1 in ovarian cancer cell growth in vitro and in vivo. The numbers of Ad-Bach1 POM 1 and colonies were significantly greater than those of Ad-GFP cells; those of Bach1-siRNA cells were significantly lower than those of Con-siRNA cells (Fig. 6A–C). HMGA2 downregulation did not impair the cell growth promoted by Bach1 (Fig. 6A), although HMGA2 knockdown alone slightly reduced Cancer Letters 445 (2019) 45–56
cell growth, indicating that Bach1-mediated tumor cell growth was independent of HMGA2. The overexpression of Bach1 significantly upregulated the levels of p-AKT, p-p70S6K and Cyclin D1, and the op-posite results were observed after silencing Bach1 expression by siRNA in A2780 cells (Fig. 6D). These results demonstrate that the AKT/ p70S6K pathway is activated after Bach1 overexpression.
To further confirm the growth-promoting eﬀects of Bach1, we per-formed an in vivo xenograft tumor experiment in nude mice. The tumor volumes, growth rates, and tumor weights were significantly increased in the tumors derived from Bach1-overexpressing cells and markedly decreased in the tumors derived from Bach1-KO cells (Fig. 7A–C). Tu-mors derived from Bach1-overexpressing cells exhibited an increase in Ki67, p-AKT and Cyclin D1 expression by immunohistochemistry or western blotting, whereas tumors derived from the Bach1-KO cells showed a reduction in the expression of these proteins (Fig. 7D–F).
Ovarian cancer patients with metastasis have low survival rates . The investigation of genes involved in ovarian cancer migration and metastasis is critical for understanding their therapeutic potential. However, the mechanisms of ovarian cancer metastasis have not been thoroughly elucidated. Although Bach1 functions as a regulator of metastasis, the role of Bach1 in ovarian cancer metastasis is unknown. Here, our data demonstrated that high levels of Bach1 were associated with metastasis, tumor growth, and poor prognosis in EOC. Bach1 promoted EMT and ovarian cancer metastasis by recruiting HMGA2. Moreover, Bach1 promoted ovarian cancer cell growth and tumor-igenesis. Thus, our findings suggest that Bach1 is a key regulator of ovarian cancer metastasis and growth.
There is increasing evidence linking Bach1 to tumor metastasis. Bach1 has been shown to promote the metastasis of breast cancer , colon cancer , prostate cancer  and neuroendocrine tumors . Bach1 forms a complex with MAFG and the DNA methyl-transferase DNMT3B, leading to the hypermethylation and transcrip-tional silencing of tumor suppressor genes [29,30]. Furthermore, Bach1 both suppresses and is suppressed by the metastasis suppressor Raf kinase inhibitory protein (RKIP) and is a let-7-regulated transcription factor that promotes the metastasis of breast cancer by upregulating metastatic genes such as CXCR4 and MMP1 [13,31]. In the present study, we found that Bach1 was highly expressed in human ovarian cancers. The upregulation of Bach1 in ovarian cancer may be associated with decreases in the expression of metastasis-suppressors such as RKIP and let-7, but the specific mechanisms of the upregulation of Bach1 in ovarian cancers have yet to be determined.
Furthermore, Bach1 expression was positively correlated with EMT-related gene expression in human ovarian cancers, and the prometa-static activity of Bach1 was confirmed in the model of metastatic ovarian cancer in mice. Bach1 enhanced the expression of the EMT-related genes Vimentin, Slug, Snail, Twist, MMP1 and CXCR4, sig-nificantly reduced the expression of E-cadherin and promoted cell mi-gration in the EOC cell line. EMT, a process by which epithelial cells lose their polarity and acquire a migratory mesenchymal phenotype, has been widely regarded as a process that initiates cancer metastasis . Several EMT-associated genes have been identified, and their functions have been characterized in ovarian cancers . Thus, the prometastatic properties of Bach1 in ovarian cancer may be mediated by its regulation of the genes involved in EMT.