Androgen receptor, neovascularization and liver cancer metastasis

The mechanisms underlying the recurrence and metastasis of hepatocellular carcinoma (HCC) are poorly understood. In 2015, Zhuang and her colleagues identified a unique histopathological structure in HCC tissues, which they dubbed VETC, standing for vessels that encapsulate tumor clusters.¹ In the VETC structure, sinusoid-like vessels form a cobweb-like network that encapsulates individual tumor cell clusters. These endothelium-wrapped tumor cell clusters were released into the blood stream and formed metastatic tumors in a manner independent of epithelial-mesenchymal transition (EMT), another well-known tumor metastatic process.² Mechanistically, the authors found that angiopoietin-2 (Angpt2) secreted by tumor cells played a critical role in the induction of VETC formation and intrahepatic tumor metastasis and recurrence.

In this issue, Zhuang’s group report a very interesting functional relationship between androgen receptor (AR) and VETC formation in intrahepatic and extrahepatic metastasis of liver cancer.³ AR is a member of the nuclear hormone receptor superfamily whose elevated expression has been implicated in HCC development.⁴ Paradoxically, genetic deletion of AR in hepatocytes delayed mouse HCC initiation driven by diethylnitrosamine, but exacerbated later tumor growth and metastasis to the lung, demonstrating complex mechanisms of AR activity in HCC.⁵ Consistently, an AR antagonist did not show significant therapeutic benefit for patients with HCC in clinical trials.⁶

To further elucidate the roles of AR in HCC, the investigators examined nuclear AR contents in 241 HCC samples collected in the Cancer Center of Sun Yat-sen University. Interestingly, lower levels of nuclear AR and VETC formation significantly correlated with higher risk of HCC recurrence and poor survival. This data was validated in another cohort of patients with HCC using the TCGA dataset. Reduced AR expression was also associated with HCC cases with portal vein tumor thrombus. To explore a causative role of AR in VETC-dependent metastasis, the authors expressed mouse AR in Hepa1–6 hepatoma cells, which form VETC+ tumors. Overexpression of AR suppressed VETC formation in xenografts and also intrahepatic metastasis, while increasing the numbers and sizes of metastasized foci in the lung. Next, the authors interrogated the molecular mechanisms underlying the effects of AR. Their previous experiments showed that high expression of Angpt2 was required for VETC formation.¹ In the present study, the authors detected much reduced levels of Angpt2 expression in xenografts derived from AR-overexpressing Hepa cells, and high levels of nuclear AR staining in HCC tissues in association with low Angpt2 expression.

A potent AR agonist 5α-DHT was shown to suppress Angpt2 expression and secretion from MHCC-97H cells. This effect was mediated by AR, as AR knockdown abolished the effect of 5α-DHT, but ectopic expression of AR reduced both cellular and secreted Angpt2 in 2 different HCC cell lines, indicating an inhibitory role of AR on Angpt2 production. Expressing AR in VETC-2 cells indeed suppressed intrahepatic metastasis but increased lung metastasis in tumor xenografts. Restoring Angpt2 expression in AR-expressing VETC cells abrogated the negative effect of AR in VETC formation and intrahepatic metastasis without impacting pulmonary metastasis. These data suggest that AR inhibited VETC-dependent intrahepatic metastasis by suppressing Angpt2 expression. The authors identified 3 putative AR response elements in the Angpt2 promoter region. Using a luciferase reporter assay, they observed a robust inhibitory effect of 5α-DHT on Angpt2 promoter activity. A direct interaction of AR with the Angpt2 promoter was validated in a chromatin immunoprecipitation assay, identifying Angpt2 as a direct transcriptional target of AR.

The authors then interrogated how AR played an opposite role in promoting lung metastasis. In cell culture, AR overexpression increased cell migration and invasiveness independently of Angpt2 expression. Transcriptomic data analysis of the Cancer Cell Line Encyclopedia (CCLE) database revealed higher expression of genes involved in actin cytoskeleton and EMT in AR-high cells. They focused on Rac1, which is known to regulate actin cytoskeleton and lamellipodia formation. Rac1 expression was upregulated in AR-overexpressing cells and also in primary and metastasized tumors derived from Hepa-AR cells. A Rac1 inhibitor (NSC23766) attenuated the AR effect in promoting cell migration and invasion in vitro, without influencing VETC formation and intrahepatic metastasis. However, Rac1 inhibition significantly suppressed AR-promoted lung metastasis and improved the survival of tumor-bearing mice if administered in combination with AR restoration. A clinically approved AR inhibitor, enzalutamide, suppressed both intrahepatic and pulmonary metastasis, which was abrogated by re-introduction of Rac1.

In conclusion, the authors proposed an interesting model that AR inhibits intra-hepatic metastasis of VETC+ tumors by repressing Angpt2 expression, while promoting pulmonary metastasis of both VETC+ and VETC− tumors by upregulating Rac1 expression. Although it may represent a somewhat simplified view, this model provides important information regarding the complexity and multiple pathways involved in HCC metastasis, and helps explain the disappointing outcomes achieved with an AR inhibitor in clinical trials. Stratifying patients with HCC into VETC+ and VETC− groups may lead to better therapeutic results with AR and Rac1 inhibitors in the era of personalized/precision medicine. Comprehensive searches and the identification of molecules that drive intrahepatic and extrahepatic metastasis of HCC will lead to the design of novel therapeutic strategies for advanced liver cancer.