Evaluation of the Glypican-3 promoter for transcriptional targeting of Hepatocellular Carcinoma

Background: Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and represents the third most common cause of death from cancer globally. Limited therapeutic options, especially in an advanced stage, combined with the presence of underlying liver dysfunction in most of the patients limit its treatment. Targeted gene therapy may be a promising treatment in this setting. Transcriptional targeting of cancer can be achieved using promoters preferentially active in tumor cells (tumor specific promoters (TSPs)). Glypican 3 (GPC3) is an oncofetal protein belonging to the proteoglycan family and is highly expressed in HCC and not in normal or cirrhotic liver. Given the HCC-specific nature of GPC3 expression, we hypothesized that the promoter for this gene should be preferentially active in HCC

Spotlight on Bortezomib: Potential in the treatment of hepatocellular carcinoma.

This study reviews the evidence for the use of Bortezomib (BZB), a first-in-class proteasome inhibitor in advanced Hepatocellular carcinoma (HCC). This review aims to delineate the role of BZB within the management of non-surgical and metastatic HCC, either as an alternative or as an adjunct to the current treatment paradigm. Areas covered: In addition to BZB pharmacology and mechanism of action, safety and tolerance profiles of the drug obtained from clinical trials are explored. The utility of BZB as a therapeutic agent either alone or in combination with other therapies against HCC, including its application in both preclinical and clinical settings has been reviewed. In particular, we highlight the importance of preclinical evaluation of BZB as a combinatorial agent in synergism with other therapies for the use in the management of HCC. Expert opinion: There has been much interest surrounding the use of BZB, a first-in-class proteasome inhibitor for HCC therapy. The discernment of outcomes of BZB clinical trials for HCC need to take into consideration the disease-specific factors that can affect survival outcomes including patient selection and aetiological differences. Further preclinical testing of BZB in combination with other therapeutic modalities can be important for eliciting enhanced anti-HCC effects

Evaluation of the Glypican 3 promoter for transcriptional targeting of hepatocellular carcinoma

© 2018 Macmillan Publishers Ltd., part of Springer Nature. Hepatocellular carcinoma (HCC) is a major health problem as evidenced by its increasing incidence and high morbidity and mortality rates. Most patients with HCC have underlying liver disease and dysfunction which limits the current therapeutic options. Treatments that spare the liver and destroy the HCC are needed. Targeting transcriptional differences between HCC and liver cells may provide this therapeutic window. In this study, we examine the potential of the Glypican 3 (GPC3) promoter as a targeting strategy. GPC3 is an oncofetal protein belonging to the proteoglycan family which is normally only expressed during fetal development. However, in HCC, the expression of this protein is reactivated. Here, we show that GPC3 is expressed primarily in HCC and not in normal liver lines. We show that the GPC3 promoter can be used to drive expression of significantly more luciferase and eYFP in HCC cell lines compared to normal liver cells. Further, we show that vectors containing cytosine deaminase (CD) under GPC3 promotor control induced significantly more killing of HCC cell lines after treatment with 5-FC compared to normal liver cell lines. These data suggest that transcriptionally targeted delivery of transgene in HCC cells can be achieved using the GPC3 promoter and this targeting strategy produces limited toxicity to normal liver cells

Characterizing and combating the immune modulatory and epithelial-to-mesenchymal plasticity properties of hepatocellular carcinoma-derived cancer stem cells

Background: Hepatocellular carcinoma (HCC) is the fastest increasing cause of cancer mortality in Australia despite the development of various therapeutic strategies. The development of an effective immunotherapy for HCC has proven difficult, with the induction of anticancer-directed immune responses seldom resulting in complete tumor eradication. Understanding the mechanisms by which cancer cells are able to escape host immune responses is critical for the development of successful immunotherapeutic treatments. Cancer stem cells (CSCs) represent a specialized population of transformed cells distinct from the majority of “differentiated” tumor cells. These cells are responsible for tumor initiation, organization and maintenance and play a major role in the resistance to radiation and chemotherapy. Recently, epithelial-to-mesenchymal plasticity (EMP) that enables tumor metastasis has emerged as an important regulator of CSC immune modulation. Despite the importance of CSCs, very little is known about the sensitivity of these cells to immune surveillance and immune killing in HCC

Monitoring immune checkpoint regulators as predictive biomarkers in hepatocellular carcinoma

The global burden of hepatocellular carcinoma (HCC), one of the frequent causes of cancer-related deaths worldwide, is rapidly increasing partly due to the limited treatment options available for this disease and recurrence due to therapy resistance. Immune checkpoint inhibitors that are proved to be beneficial in the treatment of advanced melanoma and other cancer types are currently in clinical trials in HCC. These ongoing trials are testing the efficacy and safety of a few select checkpoints in HCC. Similar to observations in other cancers, these immune checkpoint blockade treatments as monotherapy may benefit only a fraction of HCC patients. Studies that assess the prevalence and distribution of other immune checkpoints/modulatory molecules in HCC have been limited. Moreover, robust predictors to identify which HCC patients will respond to immunotherapy are currently lacking. The objective of this study is to perform a comprehensive evaluation on different immune modulators as predictive biomarkers to monitor HCC patients at high risk for poor prognosis. We screened publically available HCC patient databases for the expression of previously well described immune checkpoint regulators and evaluated the usefulness of these immune modulators to predict high risk, patient overall survival and recurrence. We also identified the immune modulators that synergized with known immune evasion molecules programmed death receptor ligand-1 (PD-L1), programmed cell death protein-1 (PD-1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and correlated with worse patient outcomes. We evaluated the association between the expression of epithelial-to-mesenchymal transition (EMT) markers and PD-L1 in HCC patient tumors. We also examined the relationship of tumor mutational burden with HCC patient survival. Notably, expression of immune modulators B7-H4, PD-L2, TIM-3, and VISTA were independently associated with worse prognosis, while B7-H4, CD73, and VISTA predicted low recurrence-free survival. Moreover, the prognosis of patients expressing high PD-L1 with high B7-H4, TIM-3, VISTA, CD73, and PD-L2 expression was significantly worse. Interestingly, PD-L1 expression in HCC patients in the high-risk group was closely associated with EMT marker expression and prognosticates poor survival. In HCC patients, high tumor mutational burden (TMB) predicted worse patient outcomes than those with low TMB. © 2018 Shrestha, Prithviraj, Anaka, Bridle, Crawford, Dhungel, Steel and Jayachandran

Murine hepatocellular carcinoma derived stem cells reveal epithelial-to-mesenchymal plasticity

AIM To establish a model to enrich and characterize stemlike cells from murine normal liver and hepatocellular carcinoma (HCC) cell lines and to further investigate stem-like cell association with epithelial-to-mesenchymal transition (EMT). METHODS In this study, we utilized a stem cell conditioned serumfree medium to enrich stem-like cells from mouse HCC and normal liver cell lines, Hepa 1-6 and AML12, respectively. We isolated the 3-dimensional spheres and assessed their stemness characteristics by evaluating the RNA levels of stemness genes and a cell surface stem cell marker by quantitative reverse transcriptase-PCR (qRTPCR). Next, we examined the relationship between stem cells and EMT using qRT-PCR. RESULTS Three-dimensional spheres were enriched by culturing murine HCC and normal hepatocyte cell lines in stem cell conditioned serum-free medium supplemented with epidermal growth factor, basic fibroblast growth factor and heparin sulfate. The 3-dimensional spheres had enhanced stemness markers such as Klf4 and Bmi1 and hepatic cancer stem cell (CSC) marker Cd44 compared to parental cells grown as adherent cultures. We report that epithelial markers E-cadherin and ZO-1 were downregulated, while mesenchymal markers Vimentin and Fibronectin were upregulated in 3-dimensional spheres. The 3-dimensional spheres also exhibited changes in expression of Snai , Zeb and Twist family of EMT transcription factors. CONCLUSION Our novel method successfully enriched stem-like cells which possessed an EMT phenotype. The isolation and characterization of murine hepatic CSCs could establish a precise target for the development of more effective therapies for HCC