The Role of Specificity Protein Transcription Factors And Noncoding Rnas In Colorectal And Hepatocellular Carcinomas

Specificity protein transcription factors, Spl, Sp3 and Sp4 are overexpressed in multiple cancer cell lines and tumor types and Sp-regulated genes play an important role in cancer cell proliferation, survival, angiogenesis and inflammation. Noncoding RNAs have been implicated in carcinogenesis and cancer progression, and our studies investigated the role of Sp proteins and noncoding RNAs in colorectal and hepatocellular carcinomas. We investigated the anticancer activity of curcumin and several synthetic analogs in colon cancer cells. Curcumin inhibits growth of several cancer cell lines, and studies in this laboratory in bladder and pancreatic cancer cells show that curcumin downregulates Spl, Sp3 and Sp4 and pro-oncogenic Sp-regulated genes. The IC50 (half- maximal) values for growth inhibition (24 hr) of colon cancer cells by curcumin and synthetic cyclohexanone and piperidine analogs of curcumin varied from 10 /zM for curcumin to 0.7 //M for the most active synthetic piperidine analog RL197, which was used along with curcumin as model agents in this study. Curcumin and RL197 inhibited RKO and SW480 colon cancer cell growth and induced apoptosis, and this was accompanied by downregulation of specificity protein (Sp) transcription factors Spl, Sp3 and Sp4 and Sp-regulated genes including the epidermal growth factor receptor (EGFR), hepatocyte growth factor receptor (c-MET), survivin, bcl-2, cyclin D1 and NFkB (p65 and p50). Curcumin and RL197 also induced reactive oxygen species (ROS), and cotreatment with the antioxidant glutathione significantly attenuated curcumin- and RL197-induced growth inhibition and downregulation of Spl, Sp3, Sp4 and Sp-regulated genes. The mechanism of curcumin-/RL197-induced repression of Sp transcription factors was ROS-dependent and due to induction of the Sp repressors ZBTB10 and ZBTB4 and downregulation of microRNAs (miR)-27a, miR-20a and miR- 17-5p that regulate these repressors. These results identify a new and highly potent curcumin derivative and demonstrate that in cells where curcumin and RL197 induce ROS, an important underlying mechanism of action involves perturbation of miR- ZBTB10/ZBTB4, resulting in the induction of these repressors which downregulate Sp transcription factors and Sp-regulated genes. In our second study, we investigated the effects of Sp proteins and Sp-regulated long noncoding RNAs (IncRNAs) in hepatocellular carcinoma (HCC). LncRNAs have recently garnered attention because of their pivotal role in cancer and other diseases, and IncRNA HULC (Highly Upregulated in Liver Cancer) was identified as a liver cancer- specific noncoding RNA and the most upregulated gene in HCC. Both HULC and Spl are upregulated in HCC, but the relationship between HULC and Spl and the underlying mechanism by which HULC contributes to HCC metastasis have not been investigated. We examined the expression of Sp proteins and IncRNAs in three HCC cell lines and found that HULC is an Spl-regulated IncRNA involved in HCC growth and metastasis. Both Spl and HULC contribute to epithelial to mesenchymal transition (EMT) of HCC cells and downregulation of either resulted in reversal of EMT and acquisition of epithelial characteristics. Our findings identify a novel role for HULC and, as an Spl- regulated IncRNA, make it an attractive target for drug treatment in HCC. Together, these studies provide conclusive evidence regarding the pro-oncogenic role of Sp proteins and Sp-linked ncRNAs, and illustrate the utility of drugs that downregulate Sp proteins as anticancer agents.