Mutant Kras- and p16-regulated NOX4 activation overcomes metabolic checkpoints in development of pancreatic ductal adenocarcinoma

Kras activation and p16 inactivation are required to develop pancreatic ductal adenocarcinoma (PDAC). However, the biochemical mechanisms underlying these double alterations remain unclear. Here we discover that NAD(P)H oxidase 4 (NOX4), an enzyme known to catalyse the oxidation of NAD(P)H, is upregulated when p16 is inactivated by looking at gene expression profiling studies. Activation of NOX4 requires catalytic subunit p22phox, which is upregulated following Kras activation. Both alterations are also detectable in PDAC cell lines and patient specimens. Furthermore, we show that elevated NOX4 activity accelerates oxidation of NADH and supports increased glycolysis by generating NAD+, a substrate for GAPDH-mediated glycolytic reaction, promoting PDAC cell growth. Mechanistically, NOX4 was induced through p16-Rb-regulated E2F and p22phox was induced by KrasG12V-activated NF-κB. In conclusion, we provide a biochemical explanation for the cooperation between p16 inactivation and Kras activation in PDAC development and suggest that NOX4 is a potential therapeutic target for PDAC

Cellular Location of HNF4α is Linked With Terminal Liver Failure in Humans

Hepatocyte nuclear factor 4 alpha (HNF4α) is a transcription factor that plays a critical role in hepatocyte function, and HNF4α-based reprogramming corrects terminal liver failure in rats with chronic liver disease. In the livers of patients with advanced cirrhosis, HNF4α RNA expression levels decrease as hepatic function deteriorates, and protein expression is found in the cytoplasm. These findings could explain impaired hepatic function in patients with degenerative liver disease. In this study, we analyzed HNF4α localization and the pathways involved in post-translational modification of HNF4α in human hepatocytes from patients with decompensated liver function. RNA-sequencing analysis revealed that AKT-related pathways, specifically phospho-AKT, is down-regulated in cirrhotic hepatocytes from patients with terminal failure, in whom nuclear levels of HNF4α were significantly reduced, and cytoplasmic expression of HNF4α was increased. cMET was also significantly reduced in failing hepatocytes. Moreover, metabolic profiling showed a glycolytic phenotype in failing human hepatocytes. The contribution of cMET and phospho-AKT to nuclear localization of HNF4α was confirmed using Spearman's rank correlation test and pathway analysis, and further correlated with hepatic dysfunction by principal component analysis. HNF4α acetylation, a posttranslational modification important for nuclear retention, was also significantly reduced in failing human hepatocytes when compared with normal controls. Conclusion: These results suggest that the alterations in the cMET-AKT pathway directly correlate with HNF4α localization and level of hepatocyte dysfunction. This study suggests that manipulation of HNF4α and pathways involved in HNF4α posttranslational modification may restore hepatocyte function in patients with terminal liver failure.Fil: Florentino, Rodrigo M.. Univeristy of Pittsburgh. School of Medicine; Estados Unidos. Universidade Federal de Minas Gerais; BrasilFil: Fraunhoffer Navarro, Nicolas Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Morita, Kazutoyo. University of Pittsburgh at Johnstown; Estados UnidosFil: Takeishi, Kazuki. University of Pittsburgh at Johnstown; Estados UnidosFil: Ostrowska, Alina. University of Pittsburgh at Johnstown; Estados UnidosFil: Achreja, Abhinav. Michigan State University; Estados UnidosFil: Animasahun, Olamide. Michigan State University; Estados UnidosFil: Haep, Nils. University of Pittsburgh at Johnstown; Estados UnidosFil: Arazov, Shohrat. University of Pittsburgh at Johnstown; Estados UnidosFil: Agarwal, Nandini. University of Pittsburgh at Johnstown; Estados UnidosFil: Collin de lHortet, Alexandra. University of Pittsburgh at Johnstown; Estados UnidosFil: Guzman Lepe, Jorge. University of Pittsburgh at Johnstown; Estados UnidosFil: Tafaleng, Edgar N.. University of Pittsburgh at Johnstown; Estados UnidosFil: Mukherjee, Amitava. University of Pittsburgh at Johnstown; Estados UnidosFil: Troy, Kris. University of Pittsburgh at Johnstown; Estados UnidosFil: Banerjee, Swati. University of Pittsburgh at Johnstown; Estados UnidosFil: Paranjpe, Shirish. University of Pittsburgh at Johnstown; Estados UnidosFil: Michalopoulos, George K.. University of Pittsburgh at Johnstown; Estados UnidosFil: Bell, Aaron. University of Pittsburgh at Johnstown; Estados UnidosFil: Nagrath, Deepak. Michigan State University; Estados UnidosFil: Hainer, Sarah J.. University of Pittsburgh at Johnstown; Estados UnidosFil: Fox, Ira J.. University of Pittsburgh at Johnstown; Estados UnidosFil: Soto Gutierrez, Alejandro. University of Pittsburgh at Johnstown; Estados Unido

Lactate-Mediated Epigenetic Reprogramming Regulates Formation of Human Pancreatic Cancer-Associated Fibroblasts

Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation

Comprehensive sampling of the lung microbiome in early-stage non–small cell lung cancerCentral MessagePerspective

Objectives: Data are scarce on whether the composition of the lung microbiome (extending from the nasopharynx to the peripheral lung tissue) varies according to histology or grade of non–small cell lung cancer. We hypothesized that the composition of the lung microbiome would vary according to the histology and the grade of non–small cell lung cancer. Methods: We collected naso-oral and central lobar (cancer affected, ipsilateral unaffected, and contralateral unaffected) bronchoalveolar lavage fluid and brushing samples from patients with clinical early-stage lung cancer between July 2018 and February 2020 at a single academic center. We performed bacterial 16S rRNA sequencing and then compared clinical and pathologic findings with microbiome signatures. Results: Samples were collected from 28 patients. Microbial composition in affected lobes displayed unique enrichment of oropharyngeal bacterial species that was significantly different compared with that from the unaffected contralateral lobes; patients with chronic obstructive pulmonary disease had similar diversity to those without chronic obstructive pulmonary disease (P = .1312). The lung microbiome diversity in patients with adenocarcinoma was similar to those with squamous cell cancer (P = .27). There were no differences in diversity or composition in the unaffected lobes of patients with adenocarcinoma versus squamous cell cancer. There was a trend toward lower lung microbial diversity in poorly differentiated adenocarcinomas compared with well-differentiated adenocarcinomas (P = .08). Conclusions: The lung microbiota differs between cancer affected and unaffected lobes in the same patient. Furthermore, poorly differentiated lung cancers were associated with lower microbial diversity. Larger studies will be required to confirm these findings

ITLN1 modulates invasive potential and metabolic reprogramming of ovarian cancer cells in omental microenvironment

Advanced ovarian cancer usually spreads to the omentum. Here, the authors show that circulating intelectin-1 (ITLN1) has prognostic significance in patients with advanced ovarian cancer, and that mesothelial cell-derived ITLN1 in the omental tumor microenvironment suppresses ovarian cancer progression

Genomic deletion of malic enzyme 2 confers collateral lethality in pancreatic cancer

The genome of pancreatic ductal adenocarcinoma (PDAC) frequently contains deletions of tumour suppressor gene loci, most notably SMAD4, which is homozygously deleted in nearly one-third of cases. As loss of neighbouring housekeeping genes can confer collateral lethality, we sought to determine whether loss of the metabolic gene malic enzyme 2 (ME2) in the SMAD4 locus would create cancer-specific metabolic vulnerability upon targeting of its paralogous isoform ME3. The mitochondrial malic enzymes (ME2 and ME3) are oxidative decarboxylases that catalyse the conversion of malate to pyruvate and are essential for NADPH regeneration and reactive oxygen species homeostasis. Here we show that ME3 depletion selectively kills ME2-null PDAC cells in a manner consistent with an essential function for ME3 in ME2-null cancer cells. Mechanistically, integrated metabolomic and molecular investigation of cells deficient in mitochondrial malic enzymes revealed diminished NADPH production and consequent high levels of reactive oxygen species. These changes activate AMP activated protein kinase (AMPK), which in turn directly suppresses sterol regulatory element-binding protein 1 (SREBP1)-directed transcription of its direct targets including the BCAT2 branched-chain amino acid transaminase 2) gene. BCAT2 catalyses the transfer of the amino group from branched-chain amino acids to α-ketoglutarate (α-KG) thereby regenerating glutamate, which functions in part to support de novo nucleotide synthesis. Thus, mitochondrial malic enzyme deficiency, which results in impaired NADPH production, provides a prime 'collateral lethality' therapeutic strategy for the treatment of a substantial fraction of patients diagnosed with this intractable disease

Metabolic shifts toward glutamine regulate tumor growth, invasion and bioenergetics in ovarian cancer

Glutamine can play a critical role in cellular growth in multiple cancers. Glutamine‐addicted cancer cells are dependent on glutamine for viability, and their metabolism is reprogrammed for glutamine utilization through the tricarboxylic acid (TCA) cycle. Here, we have uncovered a missing link between cancer invasiveness and glutamine dependence. Using isotope tracer and bioenergetic analysis, we found that low‐invasive ovarian cancer (OVCA) cells are glutamine independent, whereas high‐invasive OVCA cells are markedly glutamine dependent. Consistent with our findings, OVCA patients’ microarray data suggest that glutaminolysis correlates with poor survival. Notably, the ratio of gene expression associated with glutamine anabolism versus catabolism has emerged as a novel biomarker for patient prognosis. Significantly, we found that glutamine regulates the activation of STAT3, a mediator of signaling pathways which regulates cancer hallmarks in invasive OVCA cells. Our findings suggest that a combined approach of targeting high‐invasive OVCA cells by blocking glutamine's entry into the TCA cycle, along with targeting low‐invasive OVCA cells by inhibiting glutamine synthesis and STAT3 may lead to potential therapeutic approaches for treating OVCAs

Targeting integrated epigenetic and metabolic pathways in lethal childhood PFA ependymomas

Childhood posterior fossa group A ependymomas (PFAs) have limited treatment options and bear dismal prognoses compared to group B ependymomas (PFBs). PFAs overexpress the oncohistone-like protein EZHIP (enhancer of Zeste homologs inhibitory protein), causing global reduction of repressive histone H3 lysine 27 trimethylation (H3K27me3), similar to the oncohistone H3K27M. Integrated metabolic analyses in patient-derived cells and tumors, single-cell RNA sequencing of tumors, and noninvasive metabolic imaging in patients demonstrated enhanced glycolysis and tricarboxylic acid (TCA) cycle metabolism in PFAs. Furthermore, high glycolytic gene expression in PFAs was associated with a poor outcome. PFAs demonstrated high EZHIP expression associated with poor prognosis and elevated activating mark histone H3 lysine 27 acetylation (H3K27ac). Genomic H3K27ac was enriched in PFAs at key glycolytic and TCA cycle–related genes including hexokinase-2 and pyruvate dehydrogenase. Similarly, mouse neuronal stem cells (NSCs) expressing wild-type EZHIP (EZHIP-WT) versus catalytically attenuated EZHIP-M406K demonstrated H3K27ac enrichment at hexokinase-2 and pyruvate dehydrogenase, accompanied by enhanced glycolysis and TCA cycle metabolism. AMPKα-2, a key component of the metabolic regulator AMP-activated protein kinase (AMPK), also showed H3K27ac enrichment in PFAs and EZHIP-WT NSCs. The AMPK activator metformin lowered EZHIP protein concentrations, increased H3K27me3, suppressed TCA cycle metabolism, and showed therapeutic efficacy in vitro and in vivo in patient-derived PFA xenografts in mice. Our data indicate that PFAs and EZHIP-WT–expressing NSCs are characterized by enhanced glycolysis and TCA cycle metabolism. Repurposing the antidiabetic drug metformin lowered pathogenic EZHIP, increased H3K27me3, and suppressed tumor growth, suggesting that targeting integrated metabolic/epigenetic pathways is a potential therapeutic strategy for treating childhood ependymomas