Utility of Human Relevant Preclinical Animal Models in Navigating NAFLD to MAFLD Paradigm.

Fatty liver disease is an emerging contributor to disease burden worldwide. The past decades of work established the heterogeneous nature of non-alcoholic fatty liver disease (NAFLD) etiology and systemic contributions to the pathogenesis of the disease. This called for the proposal of a redefinition in 2020 to that of metabolic dysfunction-associated fatty liver disease (MAFLD) to better reflect the current understanding of the disease. To date, several clinical cohort studies comparing NAFLD and MAFLD hint at the relevancy of the new nomenclature in enriching for patients with more severe hepatic injury and extrahepatic comorbidities. However, the underlying systemic pathogenesis is still not fully understood. Preclinical animal models have been imperative in elucidating key biological mechanisms in various contexts, including intrahepatic disease progression, interorgan crosstalk and systemic dysregulation. Furthermore, they are integral in developing novel therapeutics against MAFLD. However, substantial contextual variabilities exist across different models due to the lack of standardization in several aspects. As such, it is crucial to understand the strengths and weaknesses of existing models to better align them to the human condition. In this review, we consolidate the implications arising from the change in nomenclature and summarize MAFLD pathogenesis. Subsequently, we provide an updated evaluation of existing MAFLD preclinical models in alignment with the new definitions and perspectives to improve their translational relevance.

Novel therapeutic strategies: targeting epithelial-mesenchymal transition in colorectal cancer.

Epithelial-mesenchymal transition (EMT) is a process during which cells lose their epithelial characteristics, for instance apical-basal cell polarity and cell-cell contact, and gain mesenchymal properties, such as increased motility. In colorectal cancer, EMT has an important role in tumour progression, metastasis, and drug resistance. There has been accumulating evidence from preclinical and early clinical studies that show that EMT markers might serve as outcome predictors and potential therapeutic targets in colorectal cancer. This Review describes the fundamentals of EMT, including biology, newly partial EMT, and associated changes. We also provide a comprehensive summary of therapeutic compounds capable of targeting EMT markers, including drugs in preclinical and clinical trials and those with repurpose potential. Lastly, we explore the obstacles of EMT bench-to-bedside drug development.

The LIDPAD Mouse Model Captures the Multisystem Interactions and Extrahepatic Complications in MASLD.

Metabolic dysfunction-associated steatotic liver disease (MASLD) represents an impending global health challenge. Current management strategies often face setbacks, emphasizing the need for preclinical models that faithfully mimic the human disease and its comorbidities. The liver disease progression aggravation diet (LIDPAD), a diet-induced murine model, extensively characterized under thermoneutral conditions and refined diets is introduced to ensure reproducibility and minimize species differences. LIDPAD recapitulates key phenotypic, genetic, and metabolic hallmarks of human MASLD, including multiorgan communications, and disease progression within 4 to 16 weeks. These findings reveal gut-liver dysregulation as an early event and compensatory pancreatic islet hyperplasia, underscoring the gut-pancreas axis in MASLD pathogenesis. A robust computational pipeline is also detailed for transcriptomic-guided disease staging, validated against multiple harmonized human hepatic transcriptomic datasets, thereby enabling comparative studies between human and mouse models. This approach underscores the remarkable similarity of the LIDPAD model to human MASLD. The LIDPAD model fidelity to human MASLD is further confirmed by its responsiveness to dietary interventions, with improvements in metabolic profiles, liver histopathology, hepatic transcriptomes, and gut microbial diversity. These results, alongside the closely aligned changing disease-associated molecular signatures between the human MASLD and LIDPAD model, affirm the model's relevance and potential for driving therapeutic development.

Commonalities and differences in the mutational signature and somatic driver mutation landscape across solid and hollow viscus organs.

Advances in sequencing have revealed a highly variegated landscape of mutational signatures and somatic driver mutations in a range of normal tissues. Normal tissues accumulate mutations at varying rates ranging from 11 per cell per year in the liver, to 1879 per cell per year in the bladder. In addition, some normal tissues are also comprised of a large proportion of cells which possess driver mutations while appearing phenotypically normal, as in the oesophagus where a majority of cells harbour driver mutations. Individual tissue proliferation and mutation rate, unique mutagenic stimuli, and local tissue architecture contribute to this highly variegated landscape which confounds the functional characterization of driver mutations found in normal tissue. In particular, our understanding of the relationship between normal tissue somatic mutations and tumour initiation or future cancer risk remains poor. Here, we describe the mutational signatures and somatic driver mutations in solid and hollow viscus organs, highlighting unique characteristics in a tissue-specific manner, while simultaneously seeking to describe commonalities which can bring forward a basic unified theory on the role of these driver mutations in tumour initiation. We discuss novel findings which can be used to inform future research in this field.

GTP Cyclohydrolase Drives Breast Cancer Development and Promotes EMT in an Enzyme-Independent Manner.

GTP cyclohydrolase (GCH1) is the rate-limiting enzyme for tetrahydrobiopterin (BH4) biosynthesis. The catalysis of BH4 biosynthesis is tightly regulated for physiological neurotransmission, inflammation, and vascular tone. Paradoxically, BH4 has emerged as an oncometabolite regulating tumor growth, but the effects on tumor development remain controversial. Here, we found that GCH1 potentiated the growth of triple-negative breast cancer (TNBC) and HER2+ breast cancer and transformed nontumor breast epithelial cells. Independent of BH4 production, GCH1 protein induced epithelial-to-mesenchymal transition by binding to vimentin (Vim), which was mediated by HSP90. Conversely, GCH1 ablation impaired tumor growth, suppressed Vim in TNBC, and inhibited EGFR/ERK signaling while activating the p53 pathway in estrogen receptor-positive tumor cells. GCH1 deficiency increases tumor cell sensitivity to HSP90 inhibition and endocrine treatments. In addition, high GCH1 correlated with poor breast cancer survival. Together, this study reveals an enzyme-independent oncogenic role of GCH1, presenting it as a potential target for therapeutic development. SIGNIFICANCE: GTP cyclohydrolase functions as an oncogene in breast cancer and binds vimentin to induce epithelial-to-mesenchymal transition independently of its enzyme activity, which confers targetable vulnerabilities for developing breast cancer treatment strategies.

A "spindle and thread" mechanism unblocks p53 translation by modulating N-terminal disorder.

Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of "life on the edge of solubility." Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular "spindle and thread" mechanism unblocks protein translation in vitro.

Quantification of T-Cell Migratory Phenotypes Using High-Content Analysis.

The exploration screening of phenotypic changes in motile T-cells within a signaling environment has always been an arduous task due to the sheer population of these microscopic cells. In recent years, High-Content Analysis (HCA) has gained epochal momentum and has allowed for a wider range of quantitative multiplexed cell-based assays in the field of lymphocyte signaling. In this chapter, we consolidate our understanding and describe the technical approach and methodology to quantify T-cell migratory phenotypes using HCA. Optimizations to be adopted to generate high-quality cytological images of motile T-cells and subsequent analysis using HCA are detailed as well.

Extracellular K+ Dampens T Cell Functions: Implications for Immune Suppression in the Tumor Microenvironment.

Background: Dying tumor cells release intracellular potassium (K+), raising extracellular K+ ([K+]e) in the tumor microenvironment (TME) to 40-50 mM (high-[K+]e). Here, we investigated the effect of high-[K+]e on T cell functions. Materials and Methods: Functional impacts of high-[K+]e on human T cells were determined by cellular, molecular, and imaging assays. Results: Exposure to high-[K+]e suppressed the proliferation of central memory and effector memory T cells, while T memory stem cells were unaffected. High-[K+]e inhibited T cell cytokine production and dampened antitumor cytotoxicity, by modulating the Akt signaling pathway. High-[K+]e caused significant upregulation of the immune checkpoint protein PD-1 in activated T cells. Although the number of KCa3.1 calcium-activated potassium channels expressed in T cells remained unaffected under high-[K+]e, a novel KCa3.1 activator, SKA-346, rescued T cells from high-[K+]e-mediated suppression. Conclusion: High-[K+]e represents a so far overlooked secondary checkpoint in cancer. KCa3.1 activators could overcome such "ionic-checkpoint"-mediated immunosuppression in the TME, and be administered together with known PD-1 inhibitors and other cancer therapeutics to improve outcomes.