Combined inhibition of TGF-beta 1-induced EMT and PD-L1 silencing re-sensitizes hepatocellular carcinoma to sorafenib treatment

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic malignancy. HCC is one of the leading causes of cancer deaths worldwide. The oral multi-tyrosine kinase inhibitor Sorafenib is the standard first-line therapy in patients with advanced unresectable HCC. Despite the significant survival benefit in HCC patients post treatment with Sorafenib, many patients had progressive disease as a result of acquiring drug resistance. Circumventing resistance to Sorafenib by exploring and targeting possible molecular mechanisms and pathways is an area of active investigation worldwide. Epithelial-to-mesenchymal transition (EMT) is a cellular process allowing epithelial cells to assume mesenchymal traits. HCC tumour cells undergo EMT to become immune evasive and develop resistance to Sorafenib treatment. Immune checkpoint molecules control immune escape in many tumours, including HCC. The aim of this study is to investigate whether combined inhibition of EMT and immune checkpoints can re-sensitise HCC to Sorafenib treatment. Post treatment with Sorafenib, HCC cells PLC/PRF/5 and Hep3B were monitored for induction of EMT and immune checkpoint molecules using quantitative reverse transcriptase (qRT)- PCR, western blot, immunofluorescence, and motility assays. The effect of combination treatment with SB431542, a specific inhibitor of the transforming growth factor (TGF)-β receptor kinase, and siRNA mediated knockdown of programmed cell death protein ligand-1 (PD-L1) on Sorafenib resistance was examined using a cell viability assay. We found that three days of Sorafenib treatment activated EMT with overexpression of TGF-β1 in both HCC cell lines. Following Sorafenib exposure, increase in the expression of PD-L1 and other immune checkpoints was observed. SB431542 blocked the TGF-β1-mediated EMT in HCC cells and also repressed PD-L1 expression. Likewise, knockdown of PD-L1 inhibited EMT. Moreover, the sensitivity of HCC cells to Sorafenib was enhanced by combining a blockade of EMT with SB431542 and knockdown of PD-L1 expression. Sorafenib-induced motility was attenuated with the combined treatment of SB431542 and PD-L1 knockdown. Our findings indicate that treatment with Sorafenib induces EMT and expression of immune checkpoint molecules, which contributes to Sorafenib resistance in HCC cells. Thus, the combination treatment strategy of inhibiting EMT and immune checkpoint molecules can re-sensitise HCC cells to Sorafenib

Promoter hypomethylation of NY-ESO-1, association with clinicopathological features and PD-L1 expression in non-small cell lung cancer.

Cancer-Testis antigens (CTA) are immunogenic molecules with normal tissue expression restricted to testes but with aberrant expression in up to 30% of non-small cell lung cancers (NSCLCs). Regulation of CTA expression is mediated in part through promoter DNA methylation. Recently, immunotherapy has altered treatment paradigms in NSCLC. Given its immunogenicity and ability to be re-expressed through demethylation, NY-ESO-1 promoter methylation, protein expression and its association with programmed death receptor ligand-1 (PD-L1) expression and clinicopathological features were investigated. Lung cancer cell line demethylation resulting from 5-Aza-2'-deoxycytidine treatment was associated with both NY-ESO-1 and PD-L1 re-expression in vitro but not increased chemosensitivity. NY-ESO-1 hypomethylation was observed in 15/94 (16%) of patient samples and associated with positive protein expression (P < 0.0001). In contrast, PD-L1 expression was observed in 50/91 (55%) but strong expression in only 12/91 (13%) cases. There was no association between NY-ESO-1 and PD-L1 expression, despite resultant re-expression of both by 5-Aza-2'-deoxycytidine. Importantly, NY-ESO-1 hypomethylation was found to be an independent marker of poor prognosis in patients not treated with chemotherapy (HR 3.59, P = 0.003) in multivariate analysis. In patients treated with chemotherapy there were no differences in survival associated with NY-ESO-1 hypomethylation. Collectively, these results provided supporting evidence for the potential use of NY-ESO-1 hypomethylation as a prognostic biomarker in stage 3 NSCLCs. In addition, these data highlight the potential to incorporate demethylating agents to enhance immune activation, in tumours currently devoid of immune infiltrates and expression of immune checkpoint genes

Epithelial-to-mesenchymal plasticity of cancer stem cells: therapeutic targets in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide despite the development of various therapeutic strategies. A better understanding of the mechanisms responsible for HCC initiation and progression is essential for the development of more effective therapies. The cancer stem cell (CSC) model has provided new insights into the development and progression of HCC. CSCs are specialized tumor cells that are capable of self-renewal and have long-term repopulation potential. As they are important mediators of tumor proliferation, invasion, metastasis, therapy resistance, and cancer relapse, the selective targeting of this crucial population of cells has the potential to improve HCC patient outcomes and survival. In recent years, the role of epithelial-to-mesenchymal transition (EMT) in the advancement of HCC has gained increasing attention. This multi-step reprograming process resulting in a phenotype switch from an epithelial to a mesenchymal cellular state has been closely associated with the acquisition of stem cell-like attributes in tumors. Moreover, CSC mediates tumor metastasis by maintaining plasticity to transition between epithelial or mesenchymal states. Therefore, understanding the molecular mechanisms of the reprograming switches that determine the progression through EMT and generation of CSC is essential for developing clinically relevant drug targets. This review provides an overview of the proposed roles of CSC in HCC and discusses recent results supporting the emerging role of EMT in facilitating hepatic CSC plasticity. In particular, we discuss how these important new insights may facilitate rational development of combining CSC- and EMT-targeted therapies in the future

Association between hereditary hemochromatosis and hepatocellular carcinoma: a comprehensive review

Hepatocellular carcinoma (HCC) is a significant global health problem with high morbidity and mortality. Its incidence is increasing exponentially worldwide with a close overlap between annual incidence and death rates. Even though significant advances have been made in HCC treatment, fewer than 20% of patients with HCC are suitable for potentially curative treatment. Hereditary hemochromatosis (HH) is an important genetic risk factor for HCC. HH is an autosomal recessive disorder of iron metabolism, characterised by elevated iron deposition in most organs including the liver, leading to progressive organ dysfunction. HCC is a complication of HH, nearly always occurring in patients with cirrhosis and contributes to increased mortality rates. Identifying the susceptibility of development of HCC in HH patients has gained much traction. This review summarises the current knowledge with regard to the association of HH and HCC in order to encourage further research. In this review, we focus particularly on HFE gene-related HH. Herein, we highlight and discuss emerging clinical research which addresses the prevalence of HCC in HH patients and the coincidence of HH with other risk factors for HCC development. We also focus on the therapeutic tools in the management of HCC associated with HH

Crosstalk between Immune Checkpoint Modulators, Metabolic Reprogramming and Cellular Plasticity in Triple-Negative Breast Cancer

Breast cancer is one of the major causes of mortality in women worldwide. Accounting for 15–20% of all breast cancer diagnoses, the triple-negative breast cancer (TNBC) subtype presents with an aggressive clinical course, heightened metastatic potential and the poorest short-term prognosis. TNBC does not respond to hormonal therapy, only partially responds to radio- and chemotherapy, and has limited targeted therapy options, thus underlining the critical need for better therapeutic treatments. Although immunotherapy based on immune checkpoint inhibition is emerging as a promising treatment option for TNBC patients, activation of cellular plasticity programs such as metabolic reprogramming (MR) and epithelial-to-mesenchymal transition (EMT) causes immunotherapy to fail. In this report, we review the role of MR and EMT in immune checkpoint dysregulation in TNBCs and specifically shed light on development of novel combination treatment modalities for this challenging disease. We highlight the clinical relevance of crosstalk between MR, EMT, and immune checkpoints in TNBCs

Effects of Epithelial to Mesenchymal Transition on T Cell Targeting of Melanoma Cells

Melanoma cells can switch phenotype in a manner similar to epithelial to mesenchymal transition (EMT). In this perspective article, we address the effects of such phenotype switching on T cell targeting of tumor cells. During the EMT-like switch in phenotype, a concomitant change in expression of multiple tumor antigens occurs. Melanoma cells undergoing EMT escape from killing by T cells specific for antigens whose expression is downregulated by this process. We discuss melanoma antigens whose expression is influenced by EMT. We assess the effect of changes in the expressed tumor antigen repertoire on T-cell mediated tumor recognition and killing. In addition to escape from T cell immunity via changes in antigen expression, mesenchymal-like melanoma cells are generally more resistant to classical chemotherapy and radiotherapy. However, we demonstrate that when targeting antigens whose expression is unaltered during EMT, the capacity of T cells to kill melanoma cell lines in vitro is not influenced by their phenotype. When considering immune therapies such as cancer vaccination, these data suggest escape from T cell killing due to phenotype switching in melanoma could potentially be avoided by careful selection of target antigen