Homeodomain transcription factors play important roles in directing cellular proliferation and differentiation, and their dysregulation has been implicated in various human cancers. In some ovarian cancers, HOXB7 is expressed at high levels, and overexpression of HOXB7 in normal HOSE cells enhances cell proliferation (15
). Other homeobox proteins such as MEIS, PBX, and PAX8 are also frequently up regulated in ovarian carcinomas (16
). Moreover, up regulation of DLX4 has been observed in high grade ovarian cancers, and overexpression of DLX4 promotes ovarian cancer cell proliferation and increases clonogenicity in vitro
, and it induces vascular endothelial growth factor transcription and enhances tumor vascularization in vivo
Our findings suggest that overexpression of DLX5 promotes the proliferation of ovarian cancer cells and that knockdown of DLX5 causes cell cycle arrest in vitro and diminished tumor size in xenografts of ovarian cancer cells in SCID mice. The arrest appears to occur in both G1 and G2/M. Moreover, down regulation of cyclin proteins was shown to underlie the reduced cell proliferation rate seen in tumor cells following knockdown of DLX5. Thus. the primary consequence of DLX5 knockdown may be the inhibition of DNA synthesis and S phase entry, which may be related to the down regulation of Cyclin A and Cyclin E that we observed. At the same time, down regulation of Cyclin D2 as well as Cyclin B may cause the arrest of cells at G1 phase and G2/M phase, respectively.
We also found that overexpression of DLX5 increases HRAS-induced colony formation in HOSE cells. It has been reported that activated HRAS can transform SV40-imortalized HOSE in vitro
). Our study implies that DLX5 is not only important in maintaining ovarian cancer cell proliferation but also participates in the transformation of SV40-imortalized HOSE cells by cooperating with activated HRAS, similar to the oncogenic cooperativity we observed between Dlx5 and activated Akt2 in Rat-1 fibroblasts (20
The receptor kinases EGFR, HER2, MET and IGF1R are often overexpressed in ovarian cancers and may contribute to tumor progression (21
). Inhibition of IGF1R function induces apoptosis in ovarian cancer cells (23
). Its adaptor proteins IRS-1 and IRS-2 have also been shown to be oncogenic. IRS proteins have been shown to be overexpressed in hepatic and pancreatic cancers and to possess constitutive activity in breast cancer, myosarcoma, and multiple endocrine neoplasia, among others (26
). Furthermore, transgenic mice overexpressing IRS-1 or IRS-2 develop breast cancer (27
). Interestingly, IRS-2 has been implicated in positive feedback regulation of IGFR specifically through the mTOR pathway (28
). The elevated IRS levels observed in certain cancers are related to activation of the AKT pathway, and dephosphorylation of IRS protein has been shown to inhibit AKT signaling (29
). Similarly, inhibition of MET represses AKT signaling and abolishes tumor cell invasion (22
). Importantly, our findings indicate that knockdown of DLX5 reduces the expression of IRS-2 and MET in ovarian cancer cells overexpressing DLX5, which in turn resulted in decreased AKT activity.
The basal activity of IRS-2 promoter is maintained by the transcription factor AP1 but is susceptible to certain oncogenic stimuli. In breast cancer cells, IRS-2 transcription can be augmented by EGFR signaling through the JNK-AP1 pathway (32
). Moreover, in some prostate cancer cells, the steroid receptor coactivator-3 binds to the IRS-2 promoter and enhances IRS-2 transcription (33
). Our studies have revealed that IRS-2 protein levels are up regulated in ovarian cancer cells overexpressing DLX5 and that DLX5 can bind to the IRS-2 promoter and augment its activity and downstream AKT signaling. Interestingly, the truncated DLX5 forms also retained partial activity on IRS-2 promoter, possibly because shorter mRNAs having a higher translation rate than the full-length mRNA and/or that truncated DLX5 proteins are more stable. Identification of other potential targets of DLX5 participating in oncogenic signaling is worthy of investigation.