Suppression Of Cyclin D1 By Hypoxia-Inducible Factor-1 Via Direct Mechanism Mediates Chemoresistance In Lung Cancer Cells

Part I: Suppression of Cyclin D1 by Hypoxia-Inducible Factor-1 via Direct Mechanism Mediates Chemoresistance in Lung Cancer CellsHypoxia-inducible factor (HIF) and cyclin D1 are key mediators of cell growth and proliferation in both normal and cancer cells. However, the interrelation between HIF and cyclin D1 remains unclear. Although genetic studies from the mouse HIF-1αnull cells had strongly indicated that HIF-1αis required for hypoxia-induced cell cycle arrest, the mechanism underlying hypoxia-induced cell cycle arrest remains undetermined so far. Sequence analysis of the cyclin D1 promoter revealed the putative binding sites for the HIF-1. Therefore, it is important to investigate whether HIF-1 regulates cyclin D1 expression or not, and if it does, what will be the detailed mechanism and subsequent outcome.In current study, arsenite which is regarded as a heavy metal and can mimic hypoxia condition by enhancing the stability of HIF-1αprotein was used to study the correlation between HIF and cyclin D1. To test whether HIF-1 was able to regulate cyclin D1 expression, dominant negative mutant of HIF-1α(DN-HIF) stable transfectants were established. Cyclin D1 expression in DN-HIF stable transfectant was significantly increased, indicating the suppression of cyclin D1 by HIF-1 in Beas-2B cells exposed to arsenite. To further confirm this finding, HIF-1αknockout MEF cells(HIF-1α-/-) was used in current study and the results showed that cyclin D1 induction by arsenite in HIF-1αknockout cells was notably enhanced compared with that in WT cells. In addition, we found that impairment of HIF1-αincreased cyclin D1 expression in A549 pulmonary cancer cells, and which in turn promoted G1/S cell cycle transition and cell proliferation. Furthermore, cyclin D1 expression was increased in subcutaneous xenograft of DN-HIF stably-transfected A549 cells in nude mice compared with that of control cells. ChIP assay revealed that HIF was able to directly bind to the promoter region of cyclin D1, which indicates the negative regulation of cyclin D1 by HIF was through a direct mechanism. A significant increase of cylin D1 promoter activation by arsenite was observed inΔHRE cyclin D1 reporter transiently transfected A549 cells compared with wide-type cyclin D1 reporter transfected cells, which indicates HIF negatively regulates cyclin D1 transcription via the interaction with cyclin D1 promoter. It has been reported that HDAC7 was able to interact with HIF-1αand mediated the expression of HIF downstream target gene. Not only TSA pretreatment but also introduction of shRNA of HDAC7 significantly antagonized the suppression of cyclin D1 by HIF revealing that HDAC7 was required in HIF-mediated cyclin D1 down-regulation. Moreover, we found the increased HIF-1αexpression or decreased cyclin D1 expression both led to the chemoresistance of A549 cells upon 5-FU treatment, suggesting HIF-1 over-expression associated chemoresistance might be due to, at least partially, the negative regulation of cyclin D1.Base on the data achieved in present study, we conclude that the negative regulation of HIF-1αon cyclin D1 is through its interaction with HRE sequence in cyclin D1 proximal promoter, which also requires HDAC7 involvement. HIF-1 induced G0/G1 phase arrest may account for the resistance of cancer cells to some certain chemotherapeutic agents that target DNA synthesis in S phase.Elevated expression of HIF-1 is usually detected in various solid tumors including colon, breast, prostate, and lung cancers, and is associated with resistance to therapies and poor prognosis in head and neck cancer, ovarian cancer and oesophageal cancer. The maximal therapeutic effects are likely to be achieved by treating patients with several different types of anticancer drugs, just as we use"cock-tail therapy"for infections and hypertension. Given that HIF-1 is likely to be one of the most valuable therapeutic targets, the possible clinical implications of targeting HIF-1 in cancer still need to be fully defined. Part II: Cyclin G1 Facilitates Liver Cancer Metastasis via Promotion of Epithelia Mesenchymal TransitionLiver cancer is the sixth most common cancer worldwide in terms of number of cases (626,000 or 5.7% of new cancer cases) but because of the rather poor prognosis, the number of deaths is almost the same (598,000). It is therefore the third most common cause of death from cancer. Survival rates are 3% to 5% in cancer registries for the United States and developing countries. Distant metastasis is one of the main reasons for the death of liver cancer. It is very important to find out the patients who belong to the high-risk group of recurrence and metastasis, and give them sufficient adjuvant therapy to improve the survival. Traditional clinical prognostic indexes fail to reflect the biological feature of liver cancer completely. With the development of molecular biology, it has become a hotspot in the field of liver cancer research that seeking new indexes to direct the diagnosis or treatment and predict the prognosis.Cyclin G1 belongs to a subgroup of cyclins, which also includes cyclin G2 and cyclin I. Functional cyclin-dependent kinase partners for these cyclins have not been described, and the biologic function(s) of this subgroup remain to be firmly established. In fact, cyclin G1 deregulation is associated with genomic instability and increased cyclin G1 levels have been described in colorectal cancer, breast cancer, and leiomyoma. Moreover, experimental evidences achieved inform cancer cell lines and tumor xenografts have shown that suppression of cyclin G1 results in the inhibition of tumor growth through the reduction of proliferation and induction of apoptosis. In experimental hepatocarcinogenesis, loss of cyclin G1 is associated with a significantly lower tumor incidence after carcinogenic challenge and cyclin G1–null hepatocytes enter S phase at a lower rate. Cyclin G1 is transcriptionally activated by p53 and p73, and, in turn, it negatively regulates p53 family proteins via a feedback regulation. Taken together, these data suggests a link between cyclin G1 and liver carcinogenesis and progression.At present, the relationship between cyclin G1 and prognosis of liver cancer has been poorly reported and no functional study on cyclin G1 in late stage of liver cancer was reported. Therefore, in this study we investigated the expression of cyclin G1 in liver cancer, as well as the possible functions of cyclin G1 in liver cancer metastasis and the underlying molecular mechanism.In the results of this study, we showed that cyclin G1 mRNA level in liver cancer cell lines is higher than that in normal liver cells, and could be induced by carcinogens such as DEN. By immunohistochemical analysis, the high expression of cyclin G1 protein was detected in all of the paraffin embedded specimens of liver cancer and mainly located in tumor cell nuclei. Western blot assay revealed 62.5% cases with high expression of cyclin G1 in protein extracts of liver cancer tissue versus the peri-tumor tissue. To investigate the effect of cyclin G1 on behavior of tumor cells, lenti-virus was utilized to establish cyclin G1 overexpression cell lines. There was no significant difference in proliferation index of cyclin G1 stable transfectants and the control cells. However, results from cell invasion assay and wound healing assay demonstrated that over-expression of cyclin G1 enhances the migration and invasion abilities of SMMC-7721 and HepG2 cells. Importantly, more intrahepatic metastatic nodes were observed in the nude mice inoculated with SMMC-7721 cyclin G1 mass cells after 6 weeks than those inoculated with control cells via spleen. To clarify the underlying molecular mechanism we tested several signaling pathways which are closely related to tumor metastasis. We found that cyclin G1 could promote EMT through PI-3K/AKT cascade and further triggered the dedifferentiation of liver cancer cells which in turn facilitates the invasion and metastases of tumor cells.Taken together, in present study, we observed cyclin G1 is over-expressed in liver cancer tissues and Demonstrated that cyclin G1 regulates EMT by activation of AKT pathway, which is responsible, at least partially, for the invasion and metastasis of liver cancer. We suggest that cyclin G1 may be a valuable marker for assessing the prognosis of liver cancer, and can be used as therapeutic target for preventing liver cancer from metastasis.