Modulation of ATP8B1 Gene Expression in Colorectal Cancer Cells Suggest its Role as a Tumor Suppressor.

AIM: The study aims to understand the role of tumor suppressor genes in colorectal cancer initiation and progression. BACKGROUND: Sporadic colorectal cancer (CRC) develops through distinct molecular events. Loss of the 18q chromosome is a conspicuous event in the progression of adenoma to carcinoma. There is limited information regarding the molecular effectors of this event. Earlier, we had reported ATP8B1 as a novel gene associated with CRC. ATP8B1 belongs to the family of P-type ATPases (P4 ATPase) that primarily function to facilitate the translocation of phospholipids. OBJECTIVE: In this study, we attempt to implicate the ATP8B1 gene located on chromosome 18q as a tumor suppressor gene. METHODS: Cells culture, Patient data analysis, Generation of stable ATP8B1 overexpressing SW480 cell line, Preparation of viral particles, Cell Transduction, Generation of stable ATP8B1 knockdown HT29 cell line with CRISPR/Cas9, Generation of stable ATP8B1 knockdown HT29 cell line with shRNA, Quantification of ATP8B1 gene expression, Real-time cell proliferation and migration assays, Cell proliferation assay, Cell migration assay, Protein isolation and western blotting, Endpoint cell viability assay, Uptake and efflux of sphingolipid, Statistical and computational analyses. RESULTS: We studied indigenous patient data and confirmed the reduced expression of ATP8B1 in tumor samples. CRC cell lines were engineered with reduced and enhanced levels of ATP8B1, which provided a tool to study its role in cancer progression. Forced reduction of ATP8B1 expression either by CRISPR/Cas9 or shRNA was associated with increased growth and proliferation of CRC cell line - HT29. In contrast, overexpression of ATP8B1 resulted in reduced growth and proliferation of SW480 cell lines. We generated a network of genes that are downstream of ATP8B1. Further, we provide the predicted effect of modulation of ATP8B1 levels on this network and the possible effect on fatty acid metabolism-related genes. CONCLUSION: Tumor suppressor gene (ATP8B1) located on chromosome 18q could be responsible in the progression of colorectal cancer. Knocking down of this gene causes an increased rate of cell proliferation and reduced cell death, suggesting its role as a tumor suppressor. Increasing the expression of this gene in colorectal cancer cells slowed down their growth and increased cell death. These evidences suggest the role of ATP8B1 as a tumor suppressor gene.

Mental health and well-being during the COVID-19 pandemic in higher education: Evidence from G20 countries.

Background: The spread of coronavirus (COVID-19) has resulted in a drastic alteration to billions of individuals' emotional, physical, mental, social, and financial status. As of July 21st, 2020, there had been 14.35 million confirmed cases of COVID-19, including 0.60 million deaths in 216 countries. Design and Methods: The study explores health and wellbeing in universities within the G20 countries (19 member countries and the European Union) during the COVID-19 pandemic. The sample selection of these countries was considered since it serves around 80% of the world's economic output, two-thirds of the global population (including more than half of the world's poor), and 75% of international trade. Specifically, due to this public health concern, schools' nationwide closures are impacting over 60% of the world's student population to promote their quality of life and well-being. Results: This study investigates the G20 policies and procedures within higher education regarding health and well-being measures during the COVID-19 epidemic. The findings reveal that the lockdown, social distancing, and self-isolation requirements are stressful and detrimental for many individuals and have caused students' health and well-being concerns. Conclusions: Several countries within the G20 have taken significant steps to support health and well-being issues for university students; however, numerous countries are far behind in addressing this issue. Hence, government leaders of G20 countries, policymakers, and health providers should promptly take the necessary measures to regulate the outbreak, improve safety measures to decrease disease transmission, and administer those who demand medical attention.

A Mechanism of Resistance to Antibody-Targeted Immune Attack.

Targeted monoclonal antibody therapy is a promising therapeutic strategy for cancer, and antibody-dependent cell-mediated cytotoxicity (ADCC) represents a crucial mechanism underlying these approaches. The majority of patients have limited responses to monoclonal antibody therapy due to the development of resistance. Models of ADCC provide a system for uncovering immune-resistance mechanisms. We continuously exposed epidermal growth factor receptor (EGFR+) A431 cells to KIR-deficient NK92-CD16V effector cells and the anti-EGFR cetuximab. Persistent ADCC exposure yielded ADCC-resistant cells (ADCCR1) that, compared with control ADCC-sensitive cells (ADCCS1), exhibited reduced EGFR expression, overexpression of histone- and interferon-related genes, and a failure to activate NK cells, without evidence of epithelial-to-mesenchymal transition. These properties gradually reversed following withdrawal of ADCC selection pressure. The development of resistance was associated with lower expression of multiple cell-surface molecules that contribute to cell-cell interactions and immune synapse formation. Classic immune checkpoints did not modulate ADCC in this unique model system of immune resistance. We showed that the induction of ADCC resistance involves genetic and epigenetic changes that lead to a general loss of target cell adhesion properties that are required for the establishment of an immune synapse, killer cell activation, and target cell cytotoxicity.

Enhancing antibody-dependent cell-mediated cytotoxicity: a strategy for improving antibody-based immunotherapy.

The targeting of surface antigens expressed on tumor cells by monoclonal antibodies (mAbs) has revolutionized cancer therapeutics. One mechanism of action of antibody-based immunotherapy is the activation of immune effector cells to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). This review will summarize the process of ADCC, its important role in the efficacy of mAb therapy, how to measure it, and finally future strategies for antibody design that can take advantage of it to improve clinical performance.

Intrahepatic Transcriptional Signature Associated with Response to Interferon-α Treatment in the Woodchuck Model of Chronic Hepatitis B.

Recombinant interferon-alpha (IFN-α) is an approved therapy for chronic hepatitis B (CHB), but the molecular basis of treatment response remains to be determined. The woodchuck model of chronic hepatitis B virus (HBV) infection displays many characteristics of human disease and has been extensively used to evaluate antiviral therapeutics. In this study, woodchucks with chronic woodchuck hepatitis virus (WHV) infection were treated with recombinant woodchuck IFN-α (wIFN-α) or placebo (n = 12/group) for 15 weeks. Treatment with wIFN-α strongly reduced viral markers in the serum and liver in a subset of animals, with viral rebound typically being observed following cessation of treatment. To define the intrahepatic cellular and molecular characteristics of the antiviral response to wIFN-α, we characterized the transcriptional profiles of liver biopsies taken from animals (n = 8-12/group) at various times during the study. Unexpectedly, this revealed that the antiviral response to treatment did not correlate with intrahepatic induction of the majority of IFN-stimulated genes (ISGs) by wIFN-α. Instead, treatment response was associated with the induction of an NK/T cell signature in the liver, as well as an intrahepatic IFN-γ transcriptional response and elevation of liver injury biomarkers. Collectively, these data suggest that NK/T cell cytolytic and non-cytolytic mechanisms mediate the antiviral response to wIFN-α treatment. In summary, by studying recombinant IFN-α in a fully immunocompetent animal model of CHB, we determined that the immunomodulatory effects, but not the direct antiviral activity, of this pleiotropic cytokine are most closely correlated with treatment response. This has important implications for the rational design of new therapeutics for the treatment of CHB.

Beyond peptides and mAbs--current status and future perspectives for biotherapeutics with novel constructs.

Biotherapeutics are attractive anti-cancer agents due to their high specificity and limited toxicity compared to conventional small molecules. Antibodies are widely used in cancer therapy, either directly or conjugated to a cytotoxic payload. Peptide therapies, though not as prevalent, have been utilized in hormonal therapy and imaging. The limitations associated with unmodified forms of both types of biotherapeutics have led to the design and development of novel structures, which incorporate key features and structures that have improved the molecules' abilities to bind to tumor targets, avoid degradation, and exhibit favorable pharmacokinetics. In this review, we highlight the current status of monoclonal antibodies and peptides, and provide a perspective on the future of biotherapeutics using novel constructs.

Sustained efficacy and seroconversion with the Toll-like receptor 7 agonist GS-9620 in the Woodchuck model of chronic hepatitis B.

BACKGROUND & AIMS: New therapies for chronic hepatitis B (CHB) are urgently needed since current treatments rarely lead to cure. We evaluated whether the oral small molecule toll-like receptor (TLR7) agonist GS-9620 could induce durable antiviral efficacy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to human hepatitis B virus (HBV). METHODS: After evaluating the pharmacokinetics, pharmacodynamics and tolerability of oral GS-9620 in uninfected woodchucks, adult woodchucks chronically infected with WHV (n = 7 per group) were dosed with GS-9620 or placebo for 4 or 8 weeks with different treatment schedules. RESULTS: GS-9620 treatment induced rapid, marked and sustained reduction in serum viral DNA (mean maximal 6.2log10 reduction), and hepatic WHV DNA replicative intermediates, WHV cccDNA and WHV RNA, as well as loss of detectable serum WHV surface antigen (WHsAg). GS-9620 treatment also induced a sustained antibody response against WHsAg in a subset of animals. Strikingly, treatment reduced the incidence of hepatocellular carcinoma (HCC) from 71% in the placebo group to 8% in GS-9620-treated woodchucks with sustained viral load reduction. GS-9620 treatment was associated with reversible increases in serum liver enzymes and thrombocytopenia, and induced intrahepatic CD8(+) T cell, NK cell, B cell and interferon response transcriptional signatures. CONCLUSIONS: The data demonstrate that short duration, finite treatment with the oral TLR7 agonist GS-9620 can induce a sustained antiviral response in the woodchuck model of CHB, and support investigation of this compound as a therapeutic approach to attain a functional cure in CHB patients.

The development of immunoconjugates for targeted cancer therapy.

Immunoconjugates are specific, highly effective, minimally toxic anticancer therapies that are beginning to show promise in the clinic. Immunoconjugates consist of three separate components: an antibody that binds to a cancer cell antigen with high specificity, an effector molecule that has a high capacity to kill the cancer cell, and a linker that will ensure the effector does not separate from the antibody during transit and will reliably release the effector to the cancer cell or tumour stroma. The high affinity antibody-antigen interaction allows specific and selective delivery of a range of effectors, including pharmacologic agents, radioisotopes, and toxins, to cancer cells. Some anticancer molecules are not well tolerated when administered systemically owing to unacceptable toxicity to the host. However, this limitation can be overcome through the linking of such cytotoxins to specific antibodies, which mask the toxic effects of the drug until it reaches its target. Conversely, many unconjugated antibodies are highly specific for a cancer target, but have low therapeutic potential and can be repurposed as delivery vehicles for highly potent effectors. In this Review, we summarize the successes and shortcomings of immunoconjugates, and discuss the future potential for the development of these therapies.

c-Abl modulates tumor cell sensitivity to antibody-dependent cellular cytotoxicity.

Monoclonal antibodies (mAb) can modulate cancer cell signal transduction and recruit antitumor immune effector mechanisms-including antibody-dependent cellular cytotoxicity (ADCC). Although several clinically effective antibodies can promote ADCC, therapeutic resistance is common. We hypothesized that oncogenic signaling networks within tumor cells affect their sensitivity to ADCC. We developed a screening platform and targeted 60 genes derived from an EGFR gene network using RNAi in an in vitro ADCC model system. Knockdown of GRB7, PRKCE, and ABL1 enhanced ADCC by primary and secondary screens. ABL1 knockdown also reduced cell proliferation, independent of its ADCC enhancement effects. c-Abl overexpression decreased ADCC sensitivity and rescued the effects of ABL1 knockdown. Imatinib inhibition of c-Abl kinase activity also enhanced ADCC-phenocopying ABL1 knockdown-against several EGFR-expressing head-and-neck squamous cell carcinoma cell lines by ex vivo primary natural killer cells. Our findings suggest that combining c-Abl inhibition with ADCC-promoting antibodies, such as cetuximab, could translate into increased therapeutic efficacy of mAbs.