Targeting HIF-2α Enhances Sorafenib Antitumor Activity Via Wnt/β-catenin Dependent Pathways In Hepatocellular Carcinoma

Background:Hepatocellular carcinoma (HCC) is the second most frequent cause of cancer death in men worldwide. Surgical resection, liver transplantation and local ablation are curative treatments. However, most HCCs arise in cirrhotic livers and most of HCC patients were found at an advanced stage, only 30%-40% of patients are suitable for the curative treatment and tumor recurrence is expected in about 70% at five years. Tanscatheter arterial chemoembolization could ameliorate the clinical symptoms and shows no significant improvement on the prognosis of HCC patients. Sorafenib, a multikinase inhibitor with antiangiogenic and antiproliferative effects, is the unique first-line drug recommended for advanced HCC. But it has not been widely used because of its actually quite low response rate and sorafenib resistance. Thus it is essential to investigate the underlying mechanisms for the resistance to sorafenib and seek potential strategy to enhance its efficacy in the therapy of HCC.Chronic infection with HBV is the major risk factor for HCC in China, it stimulates portal fibroblast and stellate and eventually leads to liver cirrhosis. Cirrhosis induces the impairment of blood supply system and results in the development of hypoxia. HCC might induce hypoxia due to accumulative increase in tumor mass from OAH, AAH, and early HCC in the progress of HCC. To make it worse, administration of sorafenib resulted in a decrease of vasculature, leading to elevated level of tumor hypoxia. The effects of hypoxia on cancer biology include not only promotion of tumor progression but also the resistance to radiotherapy or chemotherapy.The hypoxic response is governed by hypoxia-inducible factors (HIFs). HIFs are heterodimers composed of an α-and β-subunit and belong to bHLH/PAS protein family. There are two major HIF-a subunits, HIF-laand HIF-2a. Although the two subunits share 48% amino acid identity, the difference between them is being explored. While HIF-la is ubiquitously expressed, HIF-2a is only expressed by certain types of cells including hepatocytes. The hepatic erythropoietin is preferentially regulatedby HIF-2a and the expression of angiogenic genes in livers is predominantly regulated by HIF-2a, indicating that HIF-2α may be a therapeutic target for hepatocellular carcinoma.Preliminary study on the role of HIF-2α in the progression of HCC has been made. Most studies conclude that HIF-2a promotes the progression of HCC, whereas others argue that HIF-2a inhibits hepatocellular carcinoma growth. Hence, it’s necessary to elucidate the exact role of HIF-2a in the development of hepatocellular carcinoma. It is widely accepted that hypoxia in solid tumors is associated with chemotherapy failure and is a major cause of tumor resistance to drug therapy. Recent studies found that sustained sorafenib therapy could lead to increased intratumor hypoxia, which was associated with decreased sensitivity of sorafenib. But the exact molecular mechanisms of hypoxia involved in sorafenib resistance remains unknown. Therefore, we designed the present study to investigate the possible relationship between HIF-2a and sorafenib resistance under hypoxic conditions, providing an experimental evidence for the pathogenesis research of tumors.Methods:1. The protein expressions of HIF-2a in hepatocellular carcinoma tissue, paired peritumoral tissue samples and Human HCC cell lines were investigated by immunohistochemical staining and Western blot assay, respectively.2. Western blot was used to detect the protein expressions of HIF-laand HIF-2a in Human HCC cell lines in the presence or absense of sorafenib under normoxia or hypoxia. The effect of HIF-2a on the proliferation in vivo and in vitro was assessed.3. The correlation between HIF-2a and Wnt/β-catenin signaling pathway in HCC cell lines was explored using cell immunochemistry and TOP-Flash Luciferase reporter assay.4. The key protein of Wnt/β-catenin signaling pathway was evaluated by Western blot assay knockdown HIF-2a or c-Myc in vivo and in vitro, and the relationship between HIF-2a and sorafenib resistance was further explored.Results:1.The expression level of HIF-2α in the hepatocellular carcinoma tissues was higher than that in the paired peritumoral tissues (P<0.01).The immunohistochemical staining showed that HIF-2α was mainly expressed in the cytoplasm of the cells. The HIF-2α protein was localized mainly in the cytoplasm of the cells in the cancerous tissue as well as in the surrounding noncancerous tissue. High expression of HIF-2α was found in 65.2%(43/66) of tumor specimens, compared with (21.2%,15/66) in peritumoral tissues. the positive expression of HIF-2α was significantly correlated with TNM stage (P=0.033).2. The expression of HIF-1α and HIF-2α was undetectable under normoxia in the presence or absence of sorafenib, while incubation of cells under hypoxia resulted in significantly higher levels of both subunits. Compared with the expression of HIF-1α reduced, the expression of HIF-2α increased significantly incubation with sorafenib. The cells were transfected with HIF-2a shRNA or control shRNA and then treated with sorafenib, and then the cell viability was mesaured. Compared with the control shRNA, HIF-2a shRNA further enhanced the anti-proliferation activity of sorafenib in all of the four cell lines under hypoxic conditins. Tumors treated with either sorafenib or HIF-2a siRNA were significantly smaller than the tumors in the control group (P<0.001). The combination group further suppressed tumor growth and significantly smaller than the tumors treated with sorafenib (P<0.05) or HIF-2a siRNA (P<0.001) monotherapy. There were fewer Ki-67 positive cells in tumors treated with either sorafenib or HIF-2a siRNA compared with the control. Tumors treated with sorafenib and HIF-2a siRNA had a significantly lower proliferation index than the control group and the groups treated with monotherapy (P< 0.001).3. The results of immunocytochemistry indicated that the protein expression of β-catenin was low under normoxic conditions, while incubation of cells under hypoxia resulted in significantly higher levels of both the control and the cells treated with HIF-2a shRNA. However, the knockdown of HIF-2a significantly decreased the the protein expression of β-catenin. To verify the results, the TOP-Flash reporter assay was introduced. We found that the luciferase activity of the cells of control and HIF-1a knockdown significantly increased under normoxia compared with the same cells under hypoxia, while the luciferase activity of HIF-2a knockdown cells were significantly decreased compared with the control or HIF-la knockdown cells (P<0.05). These results indicated that it was HIF-2a rather than HIF-la could enhanced the activity of Wnt/β-catenin signaling pathway under hypoxia.4. Transfection of HIF-2a shRNA significantly reduced the expression of HIF-2a, β-catenin, c-Myc and PCNA in HepG2 cells. As c-Myc is a key downstream protein in β-catenin mediated cell proliferation, and then c-Myc shRNA was further transfented into HepG2 cells. c-Myc shRNA significantly decreased c-Myc and PCNA expression with no effect on the expression of HIF-2a and β-catenin. These results indicated that it was HIF-2a that regulated HCC cells proliferation in Wnt/β-catenin signaling pathway under hypoxic conditions. The results of xenograft experiments in nude mice were consistent with the results obtained in vitro. HIF-2 siRNA significantly downregulated the expression of HIF-2a, P-catenin,c-Myc and PCNA, whereas sorafenib only downregulated the expression of PCNA and increased the expressions of HIF-2a, β-catenin and c-Myc Combination treatment with HIF-2a siRNA plus sorafenib not only downregulated the expression of HIF-2a, P-catenin and c-Myc but also resulted in significantly lower levels of PCNA expression compared with the monotheraphy.Conclusions:1. There was high expression of HIF-2a protein in hepatocellular carcinoma tissues. And it was significantly correlated with tumor TNM Stage.2 Sorafenib inhibits the synthesis of HIF-la protein, making the hypoxic response switch from HIF-1α to HIF-2a-dependent pathways, and upregulates the expression of HIF-2a protein in hypoxic HCC cells. Upregulation of HIF-2a contributes to the sorafenib resistance by promoting proliferation, and targeting HIF-2a could significantly enhance sorafenib antitumor sensitivity under hypoxic conditions.3. It was HIF-2a rather than HIF-1a could enhanced the activity of Wnt/β-catenin signaling pathway under hypoxia.4. HIF-2a contributes to the sorafenib resistance by activating the canonical Wnt/β-catenin signaling pathway in hepatocellular carcinoma cells.Significance:1. The study revealed the relationship between the expressions of HIF-2a with the clinicopathologic characteristics of hepatocellular carcinoma tissues, and provided some clues for further study of the progression of HCC.2.We verified that HIF-2a contributes to the sorafenib resistance by activating the canonical Wnt/β-catenin signaling pathway in hepatocellular carcinoma cells, but targeting HIF-2a could significantly enhance sorafenib antitumor sensitivity under hypoxic conditions.3. The results indicated that HIF-2a was closely related to the resistance to sorafenib under hypoxia and may be a new target in the therapy of hepatocellular carcinoma.