Potentiation of the anticancer effects of everolimus using a dual mTORC1/2 inhibitor in hepatocellular carcinoma cells

Abstract

There is lots of evidence to support the critical involvement of mTOR signaling in the carcinogenesis of hepatocellular carcinoma (HCC). However, it has not been determined how the roles of individual mTORC1 and mTORC2 inhibitors played in the HCC therapeutics. We thus compared the effects of everolimus, Ku0063794, and a combination of the two therapies on HCC cells, using various in vitro studies (HepG2, Hep3B, and Huh7 cells), ex vivo culturing of HCC tissues obtained from patients, and the in vivo mouse xenograft model of HCC cells. Our in vitro, ex vivo, and in vivo experiments consistently demonstrated that everolimus and Ku0063794 combination therapy was superior to individual monotherapies, as manifested by higher reduction of proliferation, migration, and invasion of HCC cells, and the higher inhibition of EMT process as well. Although individual monotherapies could not inhibit SIRT1 (positive regulator of EMT) expression, the combination therapy significantly inhibited SIRT1 expression. However, overexpression of SIRT1 mitigated the EMT-inhibiting effect of the combination therapy, suggesting that the combination therapy inhibits the EMT by way of suppressing SIRT1 expression. Therefore, when considering everolimus as an anti-HCC agent, the improved anticancer effects provided by combining it with an inhibitor of both mTORC1 and mTORC2 should be recognized.

mTOR inhibition reduces growth and adhesion of hepatocellular carcinoma cells in vitro

“…In addition, the half maximal inhibitory concentration values of everolimus or temsirolimus in HepG2 cells have been reported to range between 0.9 nM (16) and 9 µM (17). Similar sensitivity to mTOR inhibition has been reported for Hep3B and HuH7 cells (18,19), indicating that mTOR inhibition may be of clinical relevance in treating HCC. When discussing the behavior of the cell lines used in the present study, it must be emphasized that HepG2 cells were isolated from a liver biopsy with primary hepatoblastoma and HCC characteristics (20).…”

“…Whether the moderate elevation in cell migration, based on absolute cell number count, is clinically relevant or unspecific remains to be elucidated. Based on a wound-healing assay, the motility of HepG2, HuH7 and Hep3B cells has recently been reported to be significantly reduced by everolimus, whereas a Transwell migration assay, which was conducted in parallel, demonstrated a slight elevation in HepG2 and HuH7 migration (18). These data suggested a partial assay-dependent effect.…”

“…Possibly, identification of a predictive biomarker may aid in selecting patients most likely to respond to everolimus or temsirolimus. In addition to optimizing the mTOR inhibitor-based protocol, which includes dual targeting of the mTOR complexes (mTORCs), mTORC1 and mTORC2 (18,33), further studies should focus on combination strategies that interfere not only with the mTOR signaling cascade, but with different tumor targets.…”

The Effect of Immunosuppression on Coagulation After Liver Transplantation

“…EVR in combination with a reduced dose of TAC results in better renal function than that seen with standard immunosuppression (IS) with TAC alone . Furthermore, EVR had been shown to exert antiproliferative effects and antitumor activity on hepatoma cells in vitro . However, in either clinical or in vivo trials, clear evidence is still missing confirming that the application of EVR could reduce recurrence rates of hepatocellular carcinoma (HCC) after LT .…”

“…(1)(2)(3) Furthermore, EVR had been shown to exert antiproliferative effects and antitumor activity on hepatoma cells in vitro. (4,5) However, in either clinical or in vivo trials, clear evidence is still missing confirming that the application of EVR could reduce recurrence rates of hepatocellular carcinoma (HCC) after LT. (6)(7)(8) New evidence suggests that EVR protects LT patients from the development of donor-specific antibodies, which may complicate longterm graft viability. (9,10) Moreover, the administration of EVR is related to a reduction in the risk of cytomegalovirus infections among immunosuppressed patients (11)(12)(13) and has been shown to reduce the viral load of LT patients infected with hepatitis C virus genotypes 2a and 3.…”