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.

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.

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.

Non-canonical HIF-1 stabilization contributes to intestinal tumorigenesis.

The hypoxia-inducible transcription factor HIF-1 is appreciated as a promising target for cancer therapy. However, conditional deletion of HIF-1 and HIF-1 target genes in cells of the tumor microenvironment can result in accelerated tumor growth, calling for a detailed characterization of the cellular context to fully comprehend HIF-1's role in tumorigenesis. We dissected cell type-specific functions of HIF-1 for intestinal tumorigenesis by lineage-restricted deletion of the Hif1a locus. Intestinal epithelial cell-specific Hif1a loss reduced activation of Wnt/ß-catenin, tumor-specific metabolism and inflammation, significantly inhibiting tumor growth. Deletion of Hif1a in myeloid cells reduced the expression of fibroblast-activating factors in tumor-associated macrophages resulting in decreased abundance of tumor-associated fibroblasts (TAF) and robustly reduced tumor formation. Interestingly, hypoxia was detectable only sparsely and without spatial association with HIF-1α, arguing for an importance of hypoxia-independent, i.e., non-canonical, HIF-1 stabilization for intestinal tumorigenesis that has not been previously appreciated. This adds a further layer of complexity to the regulation of HIF-1 and suggests that hypoxia and HIF-1α stabilization can be uncoupled in cancer. Collectively, our data show that HIF-1 is a pivotal pro-tumorigenic factor for intestinal tumor formation, controlling key oncogenic programs in both the epithelial tumor compartment and the tumor microenvironment.

PHD3 Acts as Tumor Suppressor in Mouse Osteosarcoma and Influences Tumor Vascularization via PDGF-C Signaling.

Cancer cell proliferation and insufficient blood supply can lead to the development of hypoxic areas in the tumor tissue. The adaptation to the hypoxic environment is mediated by a transcriptional complex called hypoxia-inducible factor (HIF). HIF protein levels are tightly controlled by oxygen-dependent prolyl hydroxylase domain proteins (PHDs). However, the precise roles of these enzymes in tumor progression and their downstream signaling pathways are not fully characterized. Here, we study PHD3 function in murine experimental osteosarcoma. Unexpectedly, PHD3 silencing in LM8 cells affects neither HIF-1α protein levels, nor the expression of various HIF-1 target genes. Subcutaneous injection of PHD3-silenced tumor cells accelerated tumor progression and was accompanied by dramatic phenotypic changes in the tumor vasculature. Blood vessels in advanced PHD3-silenced tumors were enlarged whereas their density was greatly reduced. Examination of the molecular pathways underlying these alterations revealed that platelet-derived growth factor (PDGF)-C signaling is activated in the vasculature of PHD3-deficient tumors. Silencing of PDGF-C depleted tumor growth, increased vessel density and reduced vessel size. Our data show that PHD3 controls tumor growth and vessel architecture in LM8 osteosarcoma by regulating the PDGF-C pathway, and support the hypothesis that different members of the PHD family exert unique functions in tumors.

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.