Impact of dietary carbohydrate restriction on the pathobiology of Hepatocellular Carcinoma: The gut-liver axis and beyond.

Despite decades of fiercely competitive research and colossal financial investments, the majority of patients with advanced solid cancers cannot be treated with curative intent. To improve this situation, conceptually novel treatment approaches are urgently needed. Cancer is increasingly appreciated as a systemic disease and numerous organismal factors are functionally linked to neoplastic growth, e.g. systemic metabolic dysregulation, chronic inflammation, intestinal dysbiosis and disrupted circadian rhythms. It is tempting to hypothesize that interventions targeting these processes could be of significant account for cancer patients. One important driver of tumor-supporting systemic derangements is inordinate consumption of simple and highly processed carbohydrates. This dietary pattern is causally linked to hyperinsulinemia, insulin resistance, chronic inflammation and intestinal dysbiosis, begging the pertinent question whether the adoption of dietary carbohydrate restriction can be beneficial for patients with cancer. This review summarizes the published data on the role of dietary carbohydrate restriction in the pathogenesis of Hepatocellular Carcinoma (HCC), the most frequent type of primary liver cancer. In addition to outlining the functional interplay between diet, the intestinal microbiome and immunity, the review underscores the importance of bile acids as interconnectors between the intestinal microbiota and immune cells.

Stabilization but No Functional Influence of HIF-1α Expression in the Intestinal Epithelium during Salmonella Typhimurium Infection.

Hypoxia-inducible transcription factor 1 (HIF-1) has been shown to enhance microbial killing and ameliorate the course of bacterial infections. While the impact of HIF-1 on inflammatory diseases of the gut has been studied intensively, its function in bacterial infections of the gastrointestinal tract remains largely elusive. With the help of a publicly available gene expression data set, we inferred significant activation of HIF-1 after oral infection of mice with Salmonella enterica serovar Typhimurium. Immunohistochemistry and Western blot analyses confirmed marked HIF-1α protein stabilization, especially in the intestinal epithelium. This prompted us to analyze conditional Hif1a-deficient mice to examine cell type-specific functions of HIF-1 in this model. Our results demonstrate enhanced noncanonical induction of HIF-1 activity upon Salmonella infection in the intestinal epithelium as well as in macrophages. Surprisingly, Hif1a deletion in intestinal epithelial cells did not impact inflammatory gene expression, bacterial spread, or disease outcomes. In contrast, Hif1a deletion in myeloid cells enhanced intestinal Cxcl2 expression and reduced the cecal Salmonella load. In vitro, HIF-1α-deficient macrophages showed overall impaired transcription of mRNA encoding proinflammatory factors; however, the intracellular survival of Salmonella was not impacted by HIF-1α deficiency.

Severe hypoxia is a typical characteristic of human hepatocellular carcinoma: Scientific fact or fallacy?

Hepatocellular carcinoma (HCC) is characterised by a robust resistance to therapy, resulting in the very poor prognosis usually seen in patients with unresectable HCC. A thorough understanding of the molecular and cellular pathogenesis of HCC is of paramount importance for the identification of more effective treatment options. As hypoxia in tumours is associated with the malignant phenotype, molecules involved in the hypoxic response are being investigated as potential targets for cancer therapy. One key hallmark of human HCC is the hypervascularisation and arterialisation of the tumour's blood supply. Hypoxia being a strong inducer of neo-angiogenesis, it was hypothesised over 20 years ago that reduced oxygen levels in human HCC are a crucial feature of this deadly disease. However, while there is a considerable body of literature espousing the presumed functional relevance of hypoxia in HCC, direct measurements of oxygen partial pressures or O2 concentrations in human HCCs have yet to be performed. This narrative review seeks to demonstrate how overinterpretation of in vitro experiments and incorrect citations have resulted in HCCs being perceived as severely hypoxic tumours.

Deletion of mTOR in liver epithelial cells enhances hepatic metastasis of colon cancer.

Activation of the mechanistic target of rapamycin (mTOR) pathway is frequently found in cancer, but mTOR inhibitors have thus far failed to demonstrate significant antiproliferative efficacy in the majority of cancer types. Besides cancer cell-intrinsic resistance mechanisms, it is conceivable that mTOR inhibitors impact on non-malignant host cells in a manner that ultimately supports resistance of cancer cells. Against this background, we sought to analyze the functional consequences of mTOR inhibition in hepatocytes for the growth of metastatic colon cancer. To this end, we established liver epithelial cell (LEC)-specific knockout (KO) of mTOR (mTORLEC ) mice. We used these mice to characterize the growth of colorectal liver metastases with or without partial hepatectomy to model different clinical settings. Although the LEC-specific loss of mTOR remained without effect on metastasis growth in intact liver, partial liver resection resulted in the formation of larger metastases in mTORLEC mice compared with wildtype controls. This was accompanied by significantly enhanced inflammatory activity in LEC-specific mTOR KO livers after partial liver resection. Analysis of NF-ĸB target gene expression and immunohistochemistry of p65 displayed a significant activation of NF-ĸB in mTORLEC mice, suggesting a functional importance of this pathway for the observed inflammatory phenotype. Taken together, we show an unexpected acceleration of liver metastases upon deletion of mTOR in LECs. Our results support the notion that non-malignant host cells can contribute to resistance against mTOR inhibitors and encourage testing whether anti-inflammatory drugs are able to improve the efficacy of mTOR inhibitors for cancer therapy. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Patient-specific logic models of signaling pathways from screenings on cancer biopsies to prioritize personalized combination therapies.

Mechanistic modeling of signaling pathways mediating patient-specific response to therapy can help to unveil resistance mechanisms and improve therapeutic strategies. Yet, creating such models for patients, in particular for solid malignancies, is challenging. A major hurdle to build these models is the limited material available that precludes the generation of large-scale perturbation data. Here, we present an approach that couples ex vivo high-throughput screenings of cancer biopsies using microfluidics with logic-based modeling to generate patient-specific dynamic models of extrinsic and intrinsic apoptosis signaling pathways. We used the resulting models to investigate heterogeneity in pancreatic cancer patients, showing dissimilarities especially in the PI3K-Akt pathway. Variation in model parameters reflected well the different tumor stages. Finally, we used our dynamic models to efficaciously predict new personalized combinatorial treatments. Our results suggest that our combination of microfluidic experiments and mathematical model can be a novel tool toward cancer precision medicine.

Kinetic modelling of quantitative proteome data predicts metabolic reprogramming of liver cancer.

BACKGROUND: Metabolic alterations can serve as targets for diagnosis and cancer therapy. Due to the highly complex regulation of cellular metabolism, definite identification of metabolic pathway alterations remains challenging and requires sophisticated experimentation. METHODS: We applied a comprehensive kinetic model of the central carbon metabolism (CCM) to characterise metabolic reprogramming in murine liver cancer. RESULTS: We show that relative differences of protein abundances of metabolic enzymes obtained by mass spectrometry can be used to assess their maximal velocity values. Model simulations predicted tumour-specific alterations of various components of the CCM, a selected number of which were subsequently verified by in vitro and in vivo experiments. Furthermore, we demonstrate the ability of the kinetic model to identify metabolic pathways whose inhibition results in selective tumour cell killing. CONCLUSIONS: Our systems biology approach establishes that combining cellular experimentation with computer simulations of physiology-based metabolic models enables a comprehensive understanding of deregulated energetics in cancer. We propose that modelling proteomics data from human HCC with our approach will enable an individualised metabolic profiling of tumours and predictions of the efficacy of drug therapies targeting specific metabolic pathways.

The tumour microenvironment and immune milieu of cholangiocarcinoma.

Tumour microenvironment is a complex, multicellular functional compartment that, particularly when assembled as an abundant desmoplastic reaction, may profoundly affect the proliferative and invasive abilities of epithelial cancer cells. Tumour microenvironment comprises not only stromal cells, mainly cancer-associated fibroblasts, but also immune cells of both the innate and adaptive system (tumour-associated macrophages, neutrophils, natural killer cells, and T and B lymphocytes), and endothelial cells. This results in an intricate web of mutual communications regulated by an extensively remodelled extracellular matrix, where the tumour cells are centrally engaged. In this regard, cholangiocarcinoma, in particular the intrahepatic variant, has become the focus of mounting interest in the last years, largely because of the lack of effective therapies despite its rising incidence and high mortality rates worldwide. On the other hand, recent studies in pancreatic cancer, which similarly to cholangiocarcinoma, is highly desmoplastic, have argued against a tumour-promoting function of the tumour microenvironment. In this review, we will discuss recent developments concerning the role of each cellular population and their multifaceted interplay with the malignant biliary epithelial counterpart. We ultimately hope to provide the working knowledge on how their manipulation may lead to a therapeutic gain in cholangiocarcinoma.

A Newly Established Murine Cell Line as a Model for Hepatocellular Cancer in Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) has become a major risk factor for hepatocellular cancer (HCC) due to the worldwide increasing prevalence of obesity. However, the pathophysiology of NASH and its progression to HCC is incompletely understood. Thus, the aim of this study was to generate a model specific NASH-derived HCC cell line. A murine NASH-HCC model was conducted and the obtained cancer cells (N-HCC25) were investigated towards chromosomal aberrations, the expression of cell type-specific markers, dependency on nutrients, and functional importance of mTOR. N-HCC25 exhibited several chromosomal aberrations as compared to healthy hepatocytes. Hepatocytic (HNF4), EMT (Twist, Snail), and cancer stem cell markers (CD44, EpCAM, CK19, Sox9) were simultaneously expressed in these cells. Proliferation highly depended on the supply of glucose and FBS, but not glutamine. Treatment with a second generation mTOR inhibitor (KU-0063794) resulted in a strong decrease of cell growth in a dose-dependent manner. In contrast, a first generation mTOR inhibitor (Everolimus) only slightly reduced cell proliferation. Cell cycle analyses revealed that the observed growth reduction was most likely due to G1/G0 cell cycle arrest. These results indicate that N-HCC25 is a highly proliferative HCC cell line from a NASH background, which might serve as a suitable in vitro model for future investigations of NASH-derived HCC.

The Response of Macrophages and Neutrophils to Hypoxia in the Context of Cancer and Other Inflammatory Diseases.

Lack of oxygen (hypoxia) is a hallmark of a multitude of acute and chronic diseases and can be either beneficial or detrimental for organ restitution and recovery. In the context of inflammation, hypoxia is particularly important and can significantly influence the course of inflammatory diseases. Macrophages and neutrophils, the chief cellular components of innate immunity, display distinct properties when exposed to hypoxic conditions. Virtually every aspect of macrophage and neutrophil function is affected by hypoxia, amongst others, morphology, migration, chemotaxis, adherence to endothelial cells, bacterial killing, differentiation/polarization, and protumorigenic activity. Prominent arenas of macrophage and neutrophil function, for example, acute/chronic inflammation and the microenvironment of solid tumors, are characterized by low oxygen levels, demonstrating the paramount importance of the hypoxic response for proper function of these cells. Members of the hypoxia-inducible transcription factor (HIF) family emerged as pivotal molecular regulators of macrophages and neutrophils. In this review, we will summarize the molecular responses of macrophages and neutrophils to hypoxia in the context of cancer and other chronic inflammatory diseases and discuss the potential avenues for therapeutic intervention that arise from this knowledge.

HIF1α modulates cell fate reprogramming through early glycolytic shift and upregulation of PDK1-3 and PKM2.

Reprogramming somatic cells to a pluripotent state drastically reconfigures the cellular anabolic requirements, thus potentially inducing cancer-like metabolic transformation. Accordingly, we and others previously showed that somatic mitochondria and bioenergetics are extensively remodeled upon derivation of induced pluripotent stem cells (iPSCs), as the cells transit from oxidative to glycolytic metabolism. In the attempt to identify possible regulatory mechanisms underlying this metabolic restructuring, we investigated the contributing role of hypoxia-inducible factor one alpha (HIF1α), a master regulator of energy metabolism, in the induction and maintenance of pluripotency. We discovered that the ablation of HIF1α function in dermal fibroblasts dramatically hampers reprogramming efficiency, while small molecule-based activation of HIF1α significantly improves cell fate conversion. Transcriptional and bioenergetic analysis during reprogramming initiation indicated that the transduction of the four factors is sufficient to upregulate the HIF1α target pyruvate dehydrogenase kinase (PDK) one and set in motion the glycolytic shift. However, additional HIF1α activation appears critical in the early upregulation of other HIF1α-associated metabolic regulators, including PDK3 and pyruvate kinase (PK) isoform M2 (PKM2), resulting in increased glycolysis and enhanced reprogramming. Accordingly, elevated levels of PDK1, PDK3, and PKM2 and reduced PK activity could be observed in iPSCs and human embryonic stem cells in the undifferentiated state. Overall, the findings suggest that the early induction of HIF1α targets may be instrumental in iPSC derivation via the activation of a glycolytic program. These findings implicate the HIF1α pathway as an enabling regulator of cellular reprogramming.

Kinetic Modeling of Hepatic Metabolism and Simulation of Treatment Effects.

Mathematical modeling is a promising strategy to fill the experimentally unapproachable knowledge gaps about the relative contribution of various molecular processes to cellular metabolic function. To this end, we developed detailed kinetic models of the central metabolism of different cell types, comprising multiple metabolic functionalities. We used the model to simulate metabolic changes in several cell types under different experimental settings in health and disease. In this way, we show that it is possible to decipher and characterize the relative influence of various metabolic pathways and enzymes to overall metabolic performance and phenotype.Quantitative Systems Metabolism (QSM™) allows quantitative assessment of metabolic functionality and metabolic profiling based on proteomic data. Here, we describe the technique, namely, molecular resolved kinetic modeling, underlying QSM™. We explain the necessary steps for the generation of cell-specific models to functionally interpret proteomic data and point out some unresolved challenges and open questions.

Hepatocellular loss of mTOR aggravates tumor burden in nonalcoholic steatohepatitis-related HCC.

Obesity and associated nonalcoholic steatohepatitis (NASH) are on the rise globally. NASH became an important driver of hepatocellular carcinoma (HCC) in recent years. Activation of the central metabolic regulator mTOR (mechanistic target of rapamycin) is frequently observed in HCCs. However, mTOR inhibition failed to improve the outcome of HCC therapies, demonstrating the need for a better understanding of the molecular and functional consequences of mTOR blockade. We established a murine NASH-driven HCC model based on long-term western diet feeding combined with hepatocellular mTOR-inactivation. We evaluated tumor load and whole-body fat percentage via µCT-scans, analyzed metabolic blood parameters and tissue proteome profiles. Additionally, we used a bioinformatic model to access liver and HCC mitochondrial metabolic functions. The tumor burden was massively increased via mTOR-knockout. Several signs argue for extensive metabolic reprogramming of glucose, fatty acid, bile acid and cholesterol metabolism. Kinetic modeling revealed reduced oxygen consumption in KO-tumors. NASH-derived HCC pathogenesis is driven by metabolic disturbances and should be considered separately from those caused by other etiologies. We conclude that mTOR functions as tumor suppressor in hepatocytes especially under long-term western diet feeding. However, some of the detrimental consequences of this diet are attenuated by mTOR blockade.

Combination of AFP vaccine and immune checkpoint inhibitors slows hepatocellular carcinoma progression in preclinical models.

Many patients with hepatocellular carcinoma (HCC) do not respond to the first-line immune checkpoint inhibitor treatment. Immunization with effective cancer vaccines is an attractive alternative approach to immunotherapy. However, its efficacy remains insufficiently evaluated in preclinical studies. Here, we investigated HCC-associated self/tumor antigen, α-fetoprotein-based (AFP-based) vaccine immunization for treating AFP (+) HCC mouse models. We found that AFP immunization effectively induced AFP-specific CD8+ T cells in vivo. However, these CD8+ T cells expressed exhaustion markers, including PD1, LAG3, and Tim3. Furthermore, the AFP vaccine effectively prevented c-MYC/Mcl1 HCC initiation when administered before tumor formation, while it was ineffective against full-blown c-MYC/Mcl1 tumors. Similarly, anti-PD1 and anti-PD-L1 monotherapy showed no efficacy in this murine HCC model. In striking contrast, AFP immunization combined with anti-PD-L1 treatment triggered significant inhibition of HCC progression in most liver tumor nodules, while in combination with anti-PD1, it induced slower tumor progression. Mechanistically, we demonstrated that HCC-intrinsic PD-L1 expression was the primary target of anti-PD-L1 in this combination therapy. Notably, the combination therapy had a similar therapeutic effect in the cMet/ß-catenin mouse HCC model. These findings suggest that combining the AFP vaccine and immune checkpoint inhibitors may be effective for AFP (+) HCC treatment.

Hypoxia-mediated drug resistance: novel insights on the functional interaction of HIFs and cell death pathways.

Resistance towards chemotherapy, either primary or acquired, represents a major obstacle in clinical oncology. Three basic categories underlie most cases of chemotherapy failure: Inadequate pharmacokinetic properties of the drug, tumor cell intrinsic factors such as the expression of drug efflux pumps and tumor cell extrinsic conditions present in the tumor microenvironment, characterized by such hostile conditions as hypoxia, acidosis, nutrient starvation and increased interstitial pressure. Tumor hypoxia has been known to negatively affect therapy outcome for decades. Hypoxia inhibits tumor cell proliferation and induces cell cycle arrest, ultimately conferring chemoresistance since anticancer drugs preferentially target rapidly proliferating cells. However, this knowledge has been largely neglected while screening for anti-proliferative substances in vitro, resulting in hypoxia-mediated failure of most newly identified substances in vivo. To achieve a tangible therapeutic benefit from this knowledge, the mechanisms that drive tumoral responses to hypoxia need to be identified and exploited for their validity as innovative therapy targets. The HIF family of hypoxia-inducible transcription factors represents the main mediator of the hypoxic response and is widely upregulated in human cancers. HIF-1α and to a lesser extent HIF-2α, the oxygen-regulated HIF isoforms, have been associated with chemotherapy failure and interference with HIF function holds great promise to improve future anticancer therapy. In this review we summarize recent findings on the molecular mechanisms that underlie the role of the HIFs in drug resistance. Specifically, we will highlight the multifaceted interaction of HIF with apoptosis, senescence, autophagy, p53 and mitochondrial activity and outline how these are at the heart of HIF-mediated therapy failure.

Impact of microvessel density on lymph node metastasis and survival after curative resection of pancreatic cancer.

PURPOSE: The roles of angiogenesis and the most prominent angiogenic vascular endothelial growth factor (VEGF) in diseases of the pancreas remain controversial. We compared microvessel density (MVD) and VEGF status in normal pancreatic, chronic pancreatic, and pancreatic cancer (PC) tissues to establish their prognostic relevance. METHODS: Eighty samples of PC tissue, 32 samples of normal pancreatic tissue, and 20 samples of chronic pancreatitis (cP) tissue were immunostained with monoclonal anti-CD31 and polyclonal anti-VEGF antibody. The MVD was correlated with clinicopathological features and survival. RESULTS: Microvessel density was higher in PC than in cP (P < 0.001). Residual tumor status was highly predictive for survival (P < 0.001). After stratification for residual tumor status, we identified lymph node metastasis (LNM) in more than two lymph nodes (P < 0.04) and high MVD (P < 0.03) as risk factors for mortality. Multivariate analysis revealed only a high MVD (P = 0.03, odds ratio 0.441, 95% confidence interval 0.211-0.821) as an independent predictor of poor survival. Vascular endothelial growth factor was found over stromal cells in cP and over ductal adenocarcinoma cells in PC. Vascular endothelial growth factor expression status was not predictive of survival (P < 0.07). CONCLUSION: This study confirms the role of angiogenesis in PC and identifies MVD as an independent prognostic factor in patients with curatively resected PC.

Hypoxia-induced neutrophil survival is mediated by HIF-1alpha-dependent NF-kappaB activity.

Neutrophils are key effector cells of the innate immune response and are required to migrate and function within adverse microenvironmental conditions. These inflammatory sites are characterized by low levels of oxygen and glucose and high levels of reductive metabolites. A major regulator of neutrophil functional longevity is the ability of these cells to undergo apoptosis. We examined the mechanism by which hypoxia causes an inhibition of neutrophil apoptosis in human and murine neutrophils. We show that neutrophils possess the hypoxia-inducible factor (HIF)-1alpha and factor inhibiting HIF (FIH) hydroxylase oxygen-sensing pathway and using HIF-1alpha-deficient myeloid cells demonstrate that HIF-1alpha is directly involved in regulating neutrophil survival in hypoxia. Gene array, TaqMan PCR, Western blotting, and oligonucleotide binding assays identify NF-kappaB as a novel hypoxia-regulated and HIF-dependent target, with inhibition of NF-kappaB by gliotoxin or parthenolide resulting in the abrogation of hypoxic survival. In addition, we identify macrophage inflammatory protein-1beta as a novel hypoxia-induced neutrophil survival factor.

Role of mammalian target of rapamycin complex 2 in primary and secondary liver cancer.

The mammalian target of rapamycin (mTOR) acts in two structurally and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Upon deregulation, activated mTOR signaling is associated with multiple processes involved in tumor growth and metastasis. Compared with mTORC1, much less is known about mTORC2 in cancer, mainly because of the unavailability of a selective inhibitor. However, existing data suggest that mTORC2 with its two distinct subunits Rictor and mSin1 might play a more important role than assumed so far. It is one of the key effectors of the PI3K/AKT/mTOR pathway and stimulates cell growth, cell survival, metabolism, and cytoskeletal organization. It is not only implicated in tumor progression, metastasis, and the tumor microenvironment but also in resistance to therapy. Rictor, the central subunit of mTORC2, was found to be upregulated in different kinds of cancers and is associated with advanced tumor stages and a bad prognosis. Moreover, AKT, the main downstream regulator of mTORC2/Rictor, is one of the most highly activated proteins in cancer. Primary and secondary liver cancer are major problems for current cancer therapy due to the lack of specific medical treatment, emphasizing the need for further therapeutic options. This review, therefore, summarizes the role of mTORC2/Rictor in cancer, with special focus on primary liver cancer but also on liver metastases.

Cyclin E1 in Murine and Human Liver Cancer: A Promising Target for Therapeutic Intervention during Tumour Progression.

Cyclin E1 (CCNE1) is a regulatory subunit of Cyclin-dependent kinase 2 (CDK2) and is thought to control the transition of quiescent cells into cell cycle progression. Recently, we identified CCNE1 and CDK2 as key factors for the initiation of hepatocellular carcinoma (HCC). In the present study, we dissected the contributions of CCNE1 and CDK2 for HCC progression in mice and patients. Therefore, we generated genetically modified mice allowing inducible deletion of Ccne1 or Cdk2. After initiation of HCC, using the hepatocarcinogen diethylnitrosamine (DEN), we deleted Ccne1 or Cdk2 and subsequently analysed HCC progression. The relevance of CCNE1 or CDK2 for human HCC progression was investigated by in silico database analysis. Interventional deletion of Ccne1, but not of Cdk2, substantially reduced the HCC burden in mice. Ccne1-deficient HCCs were characterised by attenuated proliferation, impaired DNA damage response and downregulation of markers for stemness and microinvasion. Additionally, the tumour microenvironment of Ccne1-deficient mice showed a reduction in immune mediators, myeloid cells and cancer-associated fibroblasts. In sharp contrast, Cdk2 was dispensable for HCC progression in mice. In agreement with our mouse data, CCNE1 was overexpressed in HCC patients independent of risk factors, and associated with reduced disease-free survival, a common signature for enhanced chromosomal instability, proliferation, dedifferentiation and invasion. However, CDK2 lacked diagnostic or prognostic value in HCC patients. In summary, CCNE1 drives HCC progression in a CDK2-independent manner in mice and man. Therefore, interventional inactivation of CCNE1 represents a promising strategy the treatment of liver cancer.