Immune checkpoint inhibitors in HCC: Cellular, molecular and systemic data.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related deaths in the world, and for patients with advanced disease there are few therapeutic options available. The complex immunological microenvironment of HCC and the success of immunotherapy in several types of tumours, has raised the prospect of potential benefit for immune based therapies, such as immune checkpoint inhibitors (ICIs), in HCC. This has led to significant breakthrough research, numerous clinical trials and the rapid approval of multiple systemic drugs for HCC by regulatory bodies worldwide. Although some patients responded well to ICIs, many have failed to achieve significant benefit, while others showed unexpected and paradoxical deterioration. The aim of this review is to discuss the pathophysiology of HCC, the tumour microenvironment, key clinical trials evaluating ICIs in HCC, various resistance mechanisms to ICIs, and possible ways to overcome these impediments to improve patient outcomes.

Adiponectin confers protection from acute colitis and restricts a B cell immune response.

Adiponectin demonstrates beneficial effects in various metabolic diseases, including diabetes, and in bowel cancer. Recent data also suggest a protective role in colitis. However, the precise molecular mechanisms by which adiponectin and its receptors modulate colitis and the nature of the adaptive immune response in murine models are yet to be elucidated. Adiponectin knock-out mice were orally administered dextran sulfate sodium for 7 days and were compared with wild-type mice. The severity of disease was analyzed histopathologically and through cytokine profiling. HCT116 colonic epithelial cells were employed to analyze the in vitro effects of adiponectin and AdipoR1 interactions in colonic injury following dextran sulfate sodium treatment. Adiponectin knock-out mice receiving dextran sulfate sodium exhibited severe colitis, had greater inflammatory cell infiltration, and an increased presence of activated B cells compared with controls. This was accompanied by an exaggerated proinflammatory cytokine profile and increased STAT3 signaling. Adiponectin knock-out mouse colons had markedly reduced proliferation and increased epithelial apoptosis and cellular stress. In vitro, adiponectin reduced apoptotic, anti-proliferative, and stress signals and restored STAT3 signaling. Following the abrogation of AdipoR1 in vitro, these protective effects of adiponectin were abolished. In summary, adiponectin maintains intestinal homeostasis and protects against murine colitis through interactions with its receptor AdipoR1 and by modulating adaptive immunity.

The role of AdipoR1 and AdipoR2 in liver fibrosis.

Activation of the adiponectin (APN) signaling axis retards liver fibrosis. However, understanding of the role of AdipoR1 and AdipoR2 in mediating this response is still rudimentary. Here, we sought to elucidate the APN receptor responsible for limiting liver fibrosis by employing AdipoR1 and AdipoR2 knock-out mice in the carbon tetrachloride (CCl4) model of liver fibrosis. In addition, we knocked down receptor function in primary hepatic stellate cells (HSCs) in vitro. Following the development of fibrosis, AdipoR1 and AdipoR2 KO mice had no quantitative difference in fibrosis by Sirius red staining. However, AdipoR2 KO mice had an enhanced fibrotic signature with increased Col1-α1, TGFß-1, TIMP-1, IL-10, MMP-2 and MMP-9. Knockdown of AdipoR1 or AdipoR2 in HSCs followed by APN treatment demonstrated that AdipoR1 and AdipoR2 did not affect proliferation or TIMP-1 gene expression, while AdipoR2 modulated Col1-α1 and α-SMA gene expression, HSC migration, and AMPK activity. These finding suggest that AdipoR2 is the major APN receptor on HSCs responsible for mediating its anti-fibrotic effects.

Testing Cell Migration, Invasion, Proliferation, and Apoptosis in Hepatic Stellate Cells.

The hepatic wound repair process involves cell types including healthy and injured hepatocytes, Kupffer and inflammatory cells, sinusoidal endothelial cells (SECs), and hepatic stellate cells (HSCs). Normally, in their quiescent state, HSCs are a reservoir for vitamin A, but in response to hepatic injury, they become activated myofibroblasts that play a key role in the hepatic fibrotic response. Activated HSCs express extracellular matrix (ECM) proteins, elicit anti-apoptotic responses, and proliferate, migrate, and invade hepatic tissues to protect hepatic lobules from damage. Extended liver injury can lead to fibrosis and cirrhosis, the deposition of ECM that is driven by HSCs. Here we describe in vitro assays that quantify activated HSC responses in the presence of inhibitors targeting hepatic fibrosis.

Ets2 regulates colonic stem cells and sensitivity to tumorigenesis.

Ets2 has both tumor repressive and supportive functions for different types of cancer. We have investigated the role of Ets2 within intestinal epithelial cells in postnatal mouse colon development and tumorigenesis. Conditional inactivation of Ets2 within intestinal epithelial cells results in over representation of Ets2-deficient colon crypts within young and adult animals. This preferential representation is associated with an increased number of proliferative cells within the stem cell region and an increased rate of crypt fission in young mice that result in larger patches of Ets2-deficient crypts. These effects are consistent with a selective advantage of Ets2-deficient intestinal stem cells in colonizing colonic crypts and driving crypt fission. Ets2-deficient colon crypts have an increased mucosal thickness, an increased number of goblet cells, and an increased density. Mice with Ets2-deficient intestinal cells develop more colon tumors in response to treatment with azoxymethane and dextran sulfate sodium. The selective population of colon crypts, the altered differentiation state and increased sensitivity to carcinogen-induced tumors all indicate that Ets2 deficiency alters colon stem cell number or behavior. Ets2-dependent, epithelial cell-autonomous repression of intestinal tumors may contribute to protection from colon cancer of persons with increased dosage of chromosome 21.

Cell adhesion an important determinant of myogenesis and satellite cell activity.

Skeletal muscles represent a complex and highly organised tissue responsible for all voluntary body movements. Developed through an intricate and tightly controlled process known as myogenesis, muscles form early in development and are maintained throughout life. Due to the constant stresses that muscles are subjected to, skeletal muscles maintain a complex course of regeneration to both replace and repair damaged myofibers and to form new functional myofibers. This process, made possible by a pool of resident muscle stem cells, termed satellite cells, and controlled by an array of transcription factors, is additionally reliant on a diverse range of cell adhesion molecules and the numerous signaling cascades that they initiate. This article will review the literature surrounding adhesion molecules and their roles in skeletal muscle myogenesis and repair.

Anoikis effector Bit1 negatively regulates Erk activity.

Bcl-2 inhibitor of transcription (Bit1) is a mitochondrial protein that functions as a peptidyl-tRNA hydrolase, but, when released into the cytoplasm, it elicits apoptosis. The proapoptotic function is uniquely counteracted by integrin-mediated cell attachment. We generated a conditional KO mouse of the Bit1 gene by using the Cre-LoxP recombination system. Bit1-null mice were born alive but with some developmental abnormalities. They developed a runting syndrome after birth and died within the first 2 weeks. Cultured fibroblasts from the Bit1-null embryos [mouse embryo fibroblasts (MEFs)] were more resistant to cell death induced by loss of attachment to extracellular matrix (anoikis) than cells from the wild-type or heterozygous littermates. MEFs and tissues from Bit1 KO mice displayed a marked increase in Erk phosphorylation. Knocking down Bit1 expression in cultured cells resulted in increased Erk activation, and partially knocking down Erk reversed the increased anoikis resistance of Bit1 knockdown. The enhanced Erk activation was associated with decreased Erk phosphatase activity. These studies establish the physiological significance of Bit1 activity and begin to delineate a Bit1 signaling pathway that acts through Erk regulation.

The role of DNA damage and repair in liver cancer.

Hepatocellular carcinoma is rapidly becoming a major cause of global mortality due to the ever-increasing prevalence of obesity. DNA damage is known to play an important role in cancer initiation, however DNA repair systems are also vital for the survival of cancer cells. Given the function of the liver and its exposure to the gut, it is likely that DNA damage and repair would be of particular importance in hepatocellular carcinoma. However, many contemporary reports have neglected the role of individual pathways of DNA damage and repair in their hypotheses. This review, therefore, aims to provide a concise overview for researchers in the field of liver cancer to understand the pathways of DNA damage and repair and their individual roles in hepatocellular carcinoma.

Targeting the PI3K/Akt/mTOR Pathway in Hepatocellular Carcinoma.

Despite advances in the treatment of cancers through surgical procedures and new pharmaceuticals, the treatment of hepatocellular carcinoma (HCC) remains challenging as reflected by low survival rates. The PI3K/Akt/mTOR pathway is an important signaling mechanism that regulates the cell cycle, proliferation, apoptosis, and metabolism. Importantly, deregulation of the PI3K/Akt/mTOR pathway leading to activation is common in HCC and is hence the subject of intense investigation and the focus of current therapeutics. In this review article, we consider the role of this pathway in the pathogenesis of HCC, focusing on its downstream effectors such as glycogen synthase kinase-3 (GSK-3), cAMP-response element-binding protein (CREB), forkhead box O protein (FOXO), murine double minute 2 (MDM2), p53, and nuclear factor-κB (NF-κB), and the cellular processes of lipogenesis and autophagy. In addition, we provide an update on the current ongoing clinical development of agents targeting this pathway for HCC treatments.

The effects of fructose and metabolic inhibition on hepatocellular carcinoma.

Hepatocellular carcinoma is rapidly becoming one of the leading causes of cancer-related deaths, largely due to the increasing incidence of non-alcoholic fatty liver disease. This in part may be attributed to Westernised diets high in fructose sugar. While many studies have shown the effects of fructose on inducing metabolic-related liver diseases, little research has investigated the effects of fructose sugar on liver cancer metabolism. The present study aimed to examine the metabolic effects of fructose on hepatocellular carcinoma growth in vitro and in vivo. Fructose sugar was found to reduce cell growth in vitro, and caused alterations in the expression of enzymes involved in the serine-glycine synthesis and pentose phosphate pathways. These biosynthesis pathways are highly active in cancer cells and they utilise glycolytic by-products to produce energy and nucleotides for growth. Hence, the study further investigated the efficacy of two novel drugs that inhibit these pathways, namely NCT-503 and Physcion. The study is the first to show that the combination treatment of NCT-503 and Physcion substantially inhibited hepatocellular carcinoma growth in vitro and in vivo. The combination of fructose diet and metabolism-inhibiting drugs may provide a unique metabolic environment that warrants further investigation in targeting hepatocellular carcinoma.

Keratin 8 protection of placental barrier function.

The intermediate filament protein keratin 8 (K8) is critical for the development of most mouse embryos beyond midgestation. We find that 68% of K8-/- embryos, in a sensitive genetic background, are rescued from placental bleeding and subsequent death by cellular complementation with wild-type tetraploid extraembryonic cells. This indicates that the primary defect responsible for K8-/- lethality is trophoblast giant cell layer failure. Furthermore, the genetic absence of maternal but not paternal TNF doubles the number of viable K8-/- embryos. Finally, we show that K8-/- concepti are more sensitive to a TNF-dependent epithelial apoptosis induced by the administration of concanavalin A (ConA) to pregnant mothers. The ConA-induced failure of the trophoblast giant cell barrier results in hematoma formation between the trophoblast giant cell layer and the embryonic yolk sac in a phenocopy of dying K8-deficient concepti in a sensitive genetic background. We conclude the lethality of K8-/- embryos is due to a TNF-sensitive failure of trophoblast giant cell barrier function. The keratin-dependent protection of trophoblast giant cells from a maternal TNF-dependent apoptotic challenge may be a key function of simple epithelial keratins.

The role of obesity in inflammatory bowel disease.

In just over a generation overweight and obesity has become a worldwide health concern. The ramifications for this on future health care costs and longevity are consequent, whilst increased adiposity is a harbinger for diabetes, kidney and bone failure, and cancer. An area of intense interest where the role of adiposity is avidly discussed is in inflammatory bowel disease (IBD), which presents mainly as Crohn's disease (CD) and ulcerative colitis (UC). Studies in patients associating IBD with a western diet are divergent. Nevertheless, elegant studies have found gene polymorphisms in humans that in murine models parallel the inflammatory and gut microbiome changes seen in IBD patients. However, an area not to be ignored are the alterations in adipocyte function with ensuing adiposity, in particular and a focus of this review, the dysregulation of the levels of adipocytokines such as leptin and adiponectin. Herein, we present and discuss the known influences of a western diet on IBD in patients and rodent models and how adipocytokines could influence the IBD disease process.

Targeting mTOR and Src restricts hepatocellular carcinoma growth in a novel murine liver cancer model.

Liver cancer is a poor prognosis cancer with limited treatment options. To develop a new therapeutic approach, we derived HCC cells from a known model of murine hepatocellular carcinoma (HCC). We treated adiponectin (APN) knock-out mice with the carcinogen diethylnitrosamine, and the resulting tumors were 7-fold larger than wild-type controls. Tumors were disassociated from both genotypes and their growth characteristics evaluated. A52 cells from APN KO mice had the most robust growth in vitro and in vivo, and presented with pathology similar to the parental tumor. All primary tumors and cell lines exhibited activity of the mammalian target of Rapamycin (mTOR) and Src pathways. Subsequent combinatorial treatment, with the mTOR inhibitor Rapamycin and the Src inhibitor Dasatinib reduced A52 HCC growth 29-fold in vivo. Through protein and histological analyzes we observed activation of these pathways in human HCC, suggesting that targeting both mTOR and Src may be a novel approach for the treatment of HCC.

Langerhans cells are strongly reduced in the skin of transgenic mice overexpressing follistatin in the epidermis.

Activins are members of the transforming growth factor-beta (TGF-beta) family and are important for skin morphogenesis and wound healing. TGF-beta1 is necessary for the population of the epidermis with Langerhans cells (LC). However, a role for activin in LC biology is not known. To address this question, we analyzed skin from transgenic mice overexpressing the activin antagonist follistatin in the epidermis. Using immunofluorescence, we observed a striking decrease in the number of LC in the epidermis of transgenic mice in comparison to wild-type mice. Nevertheless, these LC expressed normal levels of major histocompatibility complex (MHC)-class II and Langerin/ CD207 in situ. In explant cultures of whole ear skin the number of dendritic cells (DC), which migrated into the culture medium, was reduced. This reduction was even more pronounced in cultures of epidermal sheets. Virtually all emigrated cutaneous DC displayed typical morphology with cytoplasmic "veils", showed translocation of MHC-class II to the surface membrane, and expressed the maturation marker 2A1. Thus, cutaneous DC from transgenic mice seemed to mature normally. These results demonstrate that overexpression of follistatin in the epidermis affects LC trafficking but not maturation and suggest a novel role of the follistatin-binding partner activin in LC biology.

Activin: an important regulator of wound repair, fibrosis, and neuroprotection.

We recently identified the gene encoding the activin betaA chain as a novel injury-regulated gene. We showed that activin over-expression in the skin of transgenic mice enhances the speed of wound healing but also the scarring response. By contrast, inhibition of activin action by over-expression of the activin antagonist follistatin caused a severe delay in wound repair, but the quality of the healed wound was improved. In a search for activin-regulated genes in keratinocytes we identified the Mad1 transcription factor as a direct target of activin in these cells. Since Mad1 inhibits proliferation and induces differentiation of various cell types, our results suggest that activin regulates these processes in keratinocytes via induction of mad1. In addition to its role in the skin, we recently identified activin as a novel neuroprotective factor in vivo. Together with results from other laboratories, these findings suggest that activin is an important player in inflammation, repair and cytoprotection in various organs.

The roles of activin in cytoprotection and tissue repair.

Activin is a member of the transforming growth factor beta family of growth and differentiation factors. Initially discovered as a protein that stimulates release of follicle-stimulating hormone, it is now well accepted as an important regulator of cell growth and differentiation. Most interestingly, a series of previous studies have revealed novel roles of activin in inflammation and repair. Our own results have provided evidence for an important function of activin in cutaneous wound repair as well as in neuroprotection, and these data will be summarized and discussed in this chapter.

Modulation of activin/bone morphogenetic protein signaling by follistatin is required for the morphogenesis of mouse molar teeth.

Teeth form as ectodermal appendages, and their morphogenesis is regulated by conserved signaling pathways. The shape of the tooth crown results from growth and folding of inner dental epithelium, and the cusp patterning is regulated by transient signaling centers, the enamel knots. Several signal proteins in the transforming growth factor-beta (TGF beta) superfamily are required for tooth development. Follistatin is an extracellular inhibitor of TGF beta signaling. To investigate the roles of follistatin during tooth development, we analyzed in detail the expression patterns of follistatin, activin beta A, as well as Bmp2, Bmp4, and Bmp7 during tooth morphogenesis. We also examined the tooth phenotypes of follistatin knockout mice and of transgenic mice overexpressing follistatin in the epithelium under the keratin 14 (K14) promoter. The folding of the dental epithelium was aberrant in the molars of follistatin knockout mice, and the cusps were shallow with reduced cell proliferation and lack of anteroposterior polarization. The functions of both primary and secondary enamel knots were apparently disturbed. In K14-follistatin transgenic mice, the molar cusp pattern was also seriously affected (although different from the follistatin knockouts) and the occlusal surfaces of the molars were whorled. Their enamel was prematurely worn. In addition, all of the third molars were missing. Our results indicate that follistatin regulates morphogenesis and shaping of the tooth crown. We propose that finely tuned antagonistic effects between follistatin and TGF beta superfamily signals are critical for enamel knot formation and function, as well as for patterning of tooth cusps.