Influence of mTOR-inhibitors and mycophenolic acid on human cholangiocellular carcinoma and cancer associated fibroblasts

Abstract

BackgroundThe incidence of Cholangiocellular Carcinoma (CCA) is increasing in the western world. The tumour has a high proportion of desmoplastic stroma and is correlated with a worse prognosis when cancer associated myofibroblasts (CAFs) are present. Recent studies showed promising results after liver transplantation (LTx) in non-resectable early stage CCA. Mycophenolic acid (MPA) and the mTor inhibitor Everolimus are used to prevent organ rejection but recently were shown to exhibit an antiproliferative effect on CCA-cells. Little is known about the influence of immunosuppressive drugs on tumour cell proliferation and migration after paracrine stimulation by CAFs. Moreover, it is still unknown, which signaling pathways are activated following these specific cell-cell interactions.MethodsCCA cell lines HuCCT1 and TFK1 were utilized for the study. CAFs were derived from resected CCA cancer tissue. Cell viability was measured by the crystal violet assay and tumour cell invasion was quantified using a modified co-culture transmigration assay. Semiquantitative cytokine-expression was measured using a cytokine-array. Protein expression and phosphorylation of ERK, STAT3 and AKT was determined by Western-blot analysis.ResultsCCA cells treated with MPA exhibited a dose related decrease in cell viability in contrast to Cyclosporine A (CSA) treatment which had no effect on cell viability. Everolimus significantly inhibited proliferation at very low concentrations. The pro-invasive effect of CAFs in co-culture transmigration assay was significantly reduced by Everolimus at a concentration of 1nM (p = 0.047). In contrast, MPA and CSA showed no effect on tumour cell invasion. Treatment of CAFs with 1nM Everolimus showed a significant reduction in the expression of IL 8, IL 13, MCP1, MIF and Serpin E1. CCA-cells showed significant increases in phosphorylation of ERK, STAT3 and AKT under the influence of conditioned CAF-media. This effect was suppressed by Everolimus.ConclusionsThe secretion of proinflammatory cytokines by CAFs may lead to increased activation of JAK/STAT3-, ERK- and AKT-signaling and increased migration of CCA-cells. Everolimus abrogates this effect and inhibits proliferation of CCA-cells even at low concentrations.LTx for non-resectable early stage CCA is currently performed in several clinical studies. Consistent with a role for common immunosuppressants in inhibiting tumour cell-proliferation and -invasion, our study indicates that a combination of standard therapies with Everolimus and MPA is a promising therapy option to treat CCA following LTx.

Up‐regulation of miR‐497 confers resistance to temozolomide in human glioma cells by targeting mTOR/Bcl‐2

“…Numerous studies have demonstrated that the IGF1R/IRS1 pathway was involved in cell proliferation and metastasis control of several solid tumors . mTOR is associated with various biological processes including cell proliferation, differentiation, migration and invasion, angiogenesis, survival as well as tumorigenesis . Recent studies demonstrated that mTOR also plays an important role in chemotherapeutic resistance .…”

Molecular Mechanisms Driving Cholangiocarcinoma Invasiveness: An Overview

“…Several studies showed that co-culture of CCA cells with either CAFs or HSCs resulted in increased cancer cell migration and/or invasiveness, in vitro 28–31 . In addition, Claperon et al demonstrated that subcutaneous co-injection of CCA cells with liver myofibroblasts in immunodeficient mice enhanced both tumor growth and the incidence of intrahepatic micrometastases, compared with mice inoculated with cancer cells alone 32 .…”

Tumor reactive stroma in cholangiocarcinoma: The fuel behind cancer aggressiveness

“…Indeed, high stromal expression of α-SMA was reported as an independent prognostic factor for overall and disease-free survival[27,29]. In line with these findings, both incubation of CCA cells with CAF conditioned medium and co-culture of CCA cells with CAFs resulted in increased cancer cell proliferation and migration, in vitro [27,30]. On the contrary, slighter pro-tumorigenic effects were elicited by liver fibroblasts isolated from the peritumoral areas, arguing for a deep biological gap between CAFs and their naïve counterpart (see below)[27].…”

“…By translating these findings in an in vivo , orthotopic syngeneic rat model of CCA, navitoclax markedly reduced tumor growth and metastasis, and significantly improved survival, an effect related to a quantitative depletion of CAFs from the stroma. Taking a different approach, the mammalian target of rapamycin inhibitor everolimus, in addition to directly reduce CCA cell proliferation and invasion[100], was reported to hamper the cross-talk between CAFs and CCA cells, by both impairing the activation of CAF-induced motogenic pathways in cancer cells, and inhibiting the secretion of tumor-promoting cyto/chemokines by CAFs[30]. Interestingly, everolimus is already an FDA-approved drug for the treatment of breast, neuroendocrine and renal cell carcinomas[101].…”