The potent modulator of cholesterol biosynthesis insulin-induced gene 1 (INSIG1) is a novel hypoxia-regulated tumor suppressor in hepatocellular carcinoma

Introduction:

Excessive lipid biosynthesis is considered to be a hallmark of metabolic deregulation in cancer cells. In line with this, increased cellular membrane cholesterol levels are thought to induce intracellular hypoxia by generating membrane resistance and reducing oxygen permeability. Insulin-induced genes including insulin-induced gene 1 (INSIG1) are major modulators of cholesterol and fatty acid biosynthesis in hepatocytes. However, little is known about the molecular function of INSIG1 in hepatocellular carcinoma (HCC). The aim of this study was to characterize the regulation of INSIG1 expression and function under hypoxic and normoxic conditions in HCC in vitro and in vivo.

Methods:

Human HCC cell lines (PLC, Hep3B, HepG2, and Huh-7) and primary human hepatocytes were used for expression and functional analyses. Patient-derived samples of HCC and corresponding non-tumorous liver tissues (CNTLT) were used for in vivo validation of gene expression patterns. Gene expression levels were assessed by qRT-PCR and Western blot analyses. Hypoxic conditions were established by treating HCC cells with the hypoxia-mimicking agent desferrioxamine (DFX) or by culturing cells under low (< 1%) oxygen levels. Functional in vitro assays were performed after siRNA-mediated knockdown of target genes. Functional experiments included Boyden chamber migration assays, clonogenicity assays, and real-time cell proliferation assays.

Results:

INSIG1 mRNA levels were significantly downregulated in HCC cell lines and patient-derived HCC tissue samples as compared to matched non-cancerous controls. Intracellular hypoxia resulted in strong repression of INSIG1 mRNA levels in HCC cell lines. Si-RNA-mediated knockdown of INSIG1 expression strongly enhanced migration of HCC cells under hypoxic conditions. However, this effect on HCC cell migration was not observed under normoxic conditions. In silico survival analyses revealed that low INSIG1 expression levels were associated with a poor prognosis for HCC patients.

Conclusions:

INSIG1 was found to be strongly downregulated in HCC in vivo and in vitro. Moreover, hypoxia even further suppressed INSIG1 expression and inhibition of INSIG1 induced migration of HCC cells. Our findings suggest that INSIG1 is a potential novel tumor suppressor in HCC.