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Avoidance of therapy-induced apoptosis is a hallmark of acquired resistance towards radiotherapy. Thus, breaking resistance still challenges modern cancer therapy. The Bcl-2 protein family is known for its regulatory role in apoptosis signaling, making Bcl-2, Mcl-1 and Bcl-x L promising targets. This study evaluates the effects of highly specific inhibitors for Bcl-x L (WEHI-539), Bcl-2 (ABT-199) and Mcl-1 (S63845) as radiosensitizers. Covering a broad spectrum of solid tumors, Non-Small-Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC) and synovial sarcoma cell lines were exposed to fractionated radiation as standard therapy with or without Bcl-2 protein inhibition. Protein expression was detected by Western blot and cell death was assessed by flow cytometry measuring apoptosis. In contrast to NSCLC, a high level of Bcl-x L and its upregulation during radiotherapy indicated radioresistance in HNSCC and synovial sarcoma. Radioresistant cell lines across all entities benefited synergistically from combined therapy with Bcl-x L inhibition and fractionated radiation. In NSCLC cell lines, Mcl-1 inhibition significantly augmented radiotherapy independent of the expression level. Our data suggest that among antiapoptotic Bcl-2 proteins, targeting Bcl-x L may break resistance to radiation in HNSCC, synovial sarcoma and NSCLC in vitro. In NSCLC, Mcl-1 might be a promising target that needs further investigation.

Liver cancers, which are mostly hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are very aggressive tumors with poor prognosis. Therapeutic options with curative intent are largely limited to surgery and available systemic therapies show limited benefit. Signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) are key transcription factors activated by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6 (IL-6). In this study, we combined in vitro cell culture experiments and immunohistochemical analyses of human HCC ( N = 124) and CCA ( N = 138) specimens. We observed that in the absence of STAT3, IL-6 induced the activation of STAT1 and its target genes suggesting that IL-6 derived from the tumor microenvironment may activate both STAT1 and STAT3 target genes in HCC tumor cells. In addition, STAT1 and STAT3 were highly activated in a subset of HCC, which exhibited a high degree of infiltrating CD8- and FOXP3-positive immune cells and PD-L1 expression. Our results demonstrate that STAT1 and STAT3 are expressed and activated in HCC and tumor infiltrating immune cells. In addition, HCC cases with high STAT1 and STAT3 expression also exhibited a high degree of immune cell infiltration, suggesting increased immunological tolerance.

Although knowledge on inflammatory signaling pathways driving cancer initiation and progression has been increasing, molecular mechanisms in hepatocarcinogenesis are still far from being completely understood. Hepatocyte-specific deletion of the MAPKKK Tak1 in mice recapitulates important steps of hepatocellular carcinoma (HCC) development, including the occurrence of cell death, steatohepatitis, dysplastic nodules, and HCCs. However, overactivation of Tak1 in mice upon deletion of its deubiquitinase Cyld also results in steatohepatitis and HCC development. To investigate Tak1 and Cyld in human HCCs, we created a tissue microarray to analyze their expression by immunohistochemistry in a large and well-characterized cohort of 871 HCCs of 561 patients. In the human liver and HCC, Tak1 is predominantly present as its isoform Tak1A and predominantly localizes to cell nuclei. Tak1 is upregulated in diethylnitrosamine-induced mouse HCCs as well as in human HCCs independent of etiology and is further induced in distant metastases. A high nuclear Tak1 expression is associated with short survival and vascular invasion. When we overexpressed Tak1A in Huh7 cells, we observed increased tumor cell migration, whereas overexpression of full-length Tak1 had no significant effect. A combined score of low Cyld and high Tak1 expression was an independent prognostic marker in a multivariate Cox regression model.

Cholangiocarcinoma (CCA) is a heterogeneous malignancy with a dismal prognosis. Therapeutic options are largely limited to surgery and conventional chemotherapy offers limited benefit. As immunotherapy has proven highly effective in various cancer types, we have undertaken a quantitative immunohistopathological assessment of immune cells expressing the immunoinhibitory T cell immune response cDNA 7 receptor (TIRC7), an emerging immunoinhibitory receptor, in a cohort of 135 CCA patients. TIRC7 + immune cells were present in both the tumor epithelia and stroma in the majority of CCA cases with the highest levels found in intrahepatic CCA. While intraepithelial density of TIRC7 + immune cells was decreased compared to matched non-neoplastic bile ducts, stromal quantity was higher in the tumor samples. Tumors exhibiting signet ring cell or adenosquamous morphology were exclusively associated with an intraepithelial TIRC7 + phenotype. Survival analysis showed intraepithelial TIRC7 + immune cell density to be a highly significant favorable prognosticator in intrahepatic but not proximal or distal CCA. Furthermore, intraepithelial TIRC7 + immune cell density correlated with the number of intraepithelial CD8 + immune cells and with the total number of CD4 + immune cells. Our results suggest the presence and prognostic relevance of TIRC7 + immune cells in CCA and warrant further functional studies on its pharmacological modulation.

Autophagy is a catabolic process that enables cells to degrade obsolete content and refuel energy depots. In colorectal cancer (CRC) autophagy has been shown to promote tumorigenesis through energy delivery in the condition of uncontrolled proliferation. With this study, we aimed at evaluating whether autophagy sustains CRC cell viability and if it impacts therapy resistance. Initially, a colorectal cancer tissue micro array, containing mucosa ( n = 10), adenoma ( n = 18) and adenocarcinoma ( n = 49) spots, was stained for expression of essential autophagy proteins LC3b, Atg7, p62 and Beclin-1. Subsequently, central autophagy proteins were downregulated in CRC cells using siRNA technology. Viability assays, flow cytometry and immunoblotting were performed and three-dimensional cell culture was utilized to study autophagy in a tissue mimicking environment. In our study we found an upregulation of Atg7 in CRC. Furthermore, we identified Atg7 as crucial factor within the autophagy network for CRC cell viability. Its disruption induced cell death via triggering apoptosis and in combination with conventional chemotherapy it exerted synergistic effects in inducing CRC cell death. Cell death was strictly dependent on nuclear LC3b, since simultaneous knockdown of Atg7 and LC3b completely restored viability. This study unravels a novel cell death preventing function of Atg7 in interaction with LC3b, thereby unmasking a promising therapeutic target in CRC.

Venetoclax (ABT-199) represents a specific B-cell lymphoma 2 (Bcl-2) inhibitor that is currently under development for the treatment of lymphoid malignancies. So far, there is no published information on its interaction potential with important drug metabolizing enzymes and drug transporters, or its efficacy in multidrug resistant (MDR) cells. We therefore scrutinized its drug-drug interaction potential in vitro. Inhibition of cytochrome P450 enzymes (CYPs) was quantified by commercial kits. Inhibition of drug transporters (P-glycoprotein (P-gp, ABCB1), breast cancer resistance protein (BCRP), and organic anion transporting polypeptides (OATPs)) was evaluated by the use of fluorescent probe substrates. Induction of drug transporters and drug metabolizing enzymes was quantified by real-time RT-PCR. The efficacy of venetoclax in MDR cells lines was evaluated with proliferation assays. Venetoclax moderately inhibited P-gp, BCRP, OATP1B1, OATP1B3, CYP3A4, and CYP2C19, whereas CYP2B6 activity was increased. Venetoclax induced the mRNA expression of CYP1A1, CYP1A2, UGT1A3, and UGT1A9. In contrast, expression of ABCB1 was suppressed, which might revert tumor resistance towards antineoplastic P-gp substrates. P-gp over-expression led to reduced antiproliferative effects of venetoclax. Effective concentrations for inhibition and induction lay in the range of maximum plasma concentrations of venetoclax, indicating that it might act as a perpetrator drug in pharmacokinetic drug-drug interactions.