A 5:2 intermittent fasting regimen ameliorates NASH and fibrosis and blunts HCC development via hepatic PPARα and PCK1.

The role and molecular mechanisms of intermittent fasting (IF) in non-alcoholic steatohepatitis (NASH) and its transition to hepatocellular carcinoma (HCC) are unknown. Here, we identified that an IF 5:2 regimen prevents NASH development as well as ameliorates established NASH and fibrosis without affecting total calorie intake. Furthermore, the IF 5:2 regimen blunted NASH-HCC transition when applied therapeutically. The timing, length, and number of fasting cycles as well as the type of NASH diet were critical parameters determining the benefits of fasting. Combined proteome, transcriptome, and metabolome analyses identified that peroxisome-proliferator-activated receptor alpha (PPARα) and glucocorticoid-signaling-induced PCK1 act co-operatively as hepatic executors of the fasting response. In line with this, PPARα targets and PCK1 were reduced in human NASH. Notably, only fasting initiated during the active phase of mice robustly induced glucocorticoid signaling and free-fatty-acid-induced PPARα signaling. However, hepatocyte-specific glucocorticoid receptor deletion only partially abrogated the hepatic fasting response. In contrast, the combined knockdown of Ppara and Pck1 in vivo abolished the beneficial outcomes of fasting against inflammation and fibrosis. Moreover, overexpression of Pck1 alone or together with Ppara in vivo lowered hepatic triglycerides and steatosis. Our data support the notion that the IF 5:2 regimen is a promising intervention against NASH and subsequent liver cancer.

Spatially Resolved Multi-Omics Single-Cell Analyses Inform Mechanisms of Immune Dysfunction in Pancreatic Cancer.

BACKGROUND & AIMS: As pancreatic ductal adenocarcinoma (PDAC) continues to be recalcitrant to therapeutic interventions, including poor response to immunotherapy, albeit effective in other solid malignancies, a more nuanced understanding of the immune microenvironment in PDAC is urgently needed. We aimed to unveil a detailed view of the immune micromilieu in PDAC using a spatially resolved multimodal single-cell approach. METHODS: We applied single-cell RNA sequencing, spatial transcriptomics, multiplex immunohistochemistry, and mass cytometry to profile the immune compartment in treatment-naïve PDAC tumors and matched adjacent normal pancreatic tissue, as well as in the systemic circulation. We determined prognostic associations of immune signatures and performed a meta-analysis of the immune microenvironment in PDAC and lung adenocarcinoma on single-cell level. RESULTS: We provided a spatially resolved fine map of the immune landscape in PDAC. We substantiated the exhausted phenotype of CD8 T cells and immunosuppressive features of myeloid cells, and highlighted immune subsets with potentially underappreciated roles in PDAC that diverged from immune populations within adjacent normal areas, particularly CD4 T cell subsets and natural killer T cells that are terminally exhausted and acquire a regulatory phenotype. Differential analysis of immune phenotypes in PDAC and lung adenocarcinoma revealed the presence of extraordinarily immunosuppressive subtypes in PDAC, along with a distinctive immune checkpoint composition. CONCLUSIONS: Our study sheds light on the multilayered immune dysfunction in PDAC and presents a holistic view of the immune landscape in PDAC and lung adenocarcinoma, providing a comprehensive resource for functional studies and the exploration of therapeutically actionable targets in PDAC.

Anoikis resistant gastric cancer cells promote angiogenesis and peritoneal metastasis through C/EBPß-mediated PDGFB autocrine and paracrine signaling.

Anoikis is a type of programmed cell death induced by loss of anchorage to the extracellular matrix (ECM). Anoikis resistance (AR) is crucial for the survival of metastatic cancer cells in blood, lymphatic circulation and distant organs. Compared to ordinary cancer cells, anoikis resistant cancer cells undergo various cellular and molecular alterations, probably characterizing the cells with unique features not limited to anoikis resistance. However, the molecular mechanisms connecting anoikis resistance to other metastatic properties are still poorly understood. Here, the biological interaction between anoikis resistance and angiogenesis as well as their involvement into peritoneal metastasis of gastric cancer (GC) were investigated in vitro and in vivo. The prognostic value of key components involved in this interaction was evaluated in the GC cohort. Compared to ordinary GC cells, GCAR cells exhibited stronger metastatic and pro-angiogenic traits corresponding to elevated PDGFB secretion. Mechanistically, transcription factor C/EBPß facilitated PDGFB transcription by directly binding to and interacting with PDGFB promoter elements, subsequently increasing PDGFB secretion. Secreted PDGFB promoted the survival of detached GC cells through a C/EBPß-dependent self-feedback loop. Moreover, secreted PDGFB promoted angiogenesis in metastases via activation of the MAPK/ERK signaling pathway in vascular endothelial cells. Both C/EBPß activation level and PDGFB expression were significantly elevated in GC and correlated with metastatic progression and poor prognosis of patients with GC. Overall, interaction between GCAR cells and vascular endothelial cells promotes angiogenesis and peritoneal metastasis of GC based on C/EBPß-mediated PDGFB autocrine and paracrine signaling. C/EBPß-PDGFB-PDGFRß-MAPK axis promises to be potential prognostic biomarkers and therapeutic targets for peritoneal metastasis of GC.

NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes1-5. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need6,7. Here we report the progressive accumulation of exhausted, unconventionally activated CD8+PD1+ T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8+PD1+ T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8+PD1+CXCR6+, TOX+, and TNF+ T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8+ T cells or TNF neutralization, suggesting that CD8+ T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8+PD1+ T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.

Class I transactivator, NLRC5: a central player in the MHC class I pathway and cancer immune surveillance.

Major histocompatibility complex (MHC) class I and class II molecules play critical roles in the activation of the adaptive immune system by presenting antigens to CD8+ and CD4+ T cells, respectively. Although it has been well known that CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, as a master regulator of MHC class II gene expression, the mechanism of MHC class I gene transactivation was unclear. Recently, another NLR protein, NLRC5 (NLR family, CARD domain-containing 5), was identified as an MHC class I transactivator (CITA). NLRC5 is a critical regulator for the transcriptional activation of MHC class I genes and other genes involved in the MHC class I antigen presentation pathway. CITA/NLRC5 plays a crucial role in human cancer immunity through the recruitment and activation of tumor killing CD8+ T cells. Here, we discuss the molecular function and mechanism of CITA/NLRC5 in the MHC class I pathway and its role in cancer.

Iron-regulated protein HupB of Mycobacterium tuberculosis positively regulates siderophore biosynthesis and is essential for growth in macrophages.

Mycobacterium tuberculosis expresses the 28-kDa protein HupB (Rv2986c) and the Fe(3+)-specific high-affinity siderophores mycobactin and carboxymycobactin upon iron limitation. The objective of this study was to understand the functional role of HupB in iron acquisition. A hupB mutant strain of M. tuberculosis, subjected to growth in low-iron medium (0.02 µg Fe ml(-1)), showed a marked reduction of both siderophores with low transcript levels of the mbt genes encoding the MB biosynthetic machinery. Complementation of the mutant strain with hupB restored siderophore production to levels comparable to that of the wild type. We demonstrated the binding of HupB to the mbtB promoter by both electrophoretic mobility shift assays and DNA footprinting. The latter revealed the HupB binding site to be a 10-bp AT-rich region. While negative regulation of the mbt machinery by IdeR is known, this is the first report of positive regulation of the mbt operon by HupB. Interestingly, the mutant strain failed to survive inside macrophages, suggesting that HupB plays an important role in vivo.