Abstract
Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is a lethal malignancy that is tightly associated with chronic hepatitis B virus (HBV) infection. HBV encodes a viral onco-protein, transactivator protein X (HBx), which interacts with proteins of hepatocytes to promote oncogenesis. Our current study focused on the interaction of HBx with a transcription factor, hypoxia-inducible factor-1α (HIF-1α), which is stabilized by low O2 condition (hypoxia) and is found to be frequently overexpressed in HCC intra-tumorally due to poor blood perfusion. Here, we showed that overexpression of HBx by tetracycline-inducible systems further stabilized HIF-1α under hypoxia in HBV-negative HCC cell lines. Reversely, knockdown of HBx reduced HIF-1α protein stabilization under hypoxia in HBV-positive HCC cell lines. More intriguingly, overexpression of HBx elevated the mRNA and protein expression of a family of HIF-1α target genes, the lysyl oxidase (LOX) family in HCC. The LOX family members function to cross-link collagen in the extracellular matrix (ECM) to promote cancer progression and metastasis. By analyzing the collagens under scanning electron microscope, we found that collagen fibers were significantly smaller in size when incubated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells in vitro. Transwell invasion assay further revealed that less cells were able to invade through the matrigel which was pre-treated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells. Orthotopic and subcutaneous HCC models further showed that knockdown of HBx in HCC cells reduced collagen crosslinking and stiffness in vivo and repressed HCC growth and metastasis. Taken together, our in vitro and in vivo studies showed the HBx remodeled the ECM through HIF-1α/LOX pathway to promote HCC metastasis.
Highlights
Hepatocellular carcinoma (HCC), malignancy transformed from hepatocytes, accounts for 90% of primary liver cancer
Establishment of tetracycline-inducible stable expression of HBx cell model and HBx knockdown cell model To study the effect of HBx on hypoxia-inducible factor-1α (HIF-1α) stability, we employed gain-of-function and loss-of-function HBx models in HCC cell lines
polymerase chain reaction (PCR) using primers flanking HBx and western blotting showed that HBx was successfully expressed upon tetracycline withdrawal (Tet-OFF) or tetracycline addition (Tet-ON) (Fig. 1a, b and Supplementary Fig. 1)
Summary
Hepatocellular carcinoma (HCC), malignancy transformed from hepatocytes, accounts for 90% of primary liver cancer. HCC is the fifth most prevalent cancer and the third leading cause of cancer-associated death. The high mortality rate of HCC is mainly associated with late. Regorafenib (TKI) and Nivolumab (immune checkpoint inhibitor targeting programmed death 1 [PD1]) were recently approved by FDA as second-line treatments[3,4]. Regorafenib could only prolong the survival of Sorafenibresistant HCC patients for another 2–3 months[3]. Nivolumab has an unprecedented response rate of. 20% in HCC patients[4]. Through blocking inhibitory T cell receptor PD1, reactivates T cells to suppress HCC. Nivolumab highlighted the immense potential of monoclonal antibodies against other inhibitory T cell receptors, such as TIM3 and LAG3 in HCC treatment
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