Aldehydes alter TGF-ß signaling and induce obesity and cancer.

Obesity and fatty liver diseases-metabolic dysfunction-associated steatotic liver disease (MASLD) and metabolic dysfunction-associated steatohepatitis (MASH)-affect over one-third of the global population and are exacerbated in individuals with reduced functional aldehyde dehydrogenase 2 (ALDH2), observed in approximately 560 million people. Current treatment to prevent disease progression to cancer remains inadequate, requiring innovative approaches. We observe that Aldh2-/- and Aldh2-/-Sptbn1+/- mice develop phenotypes of human metabolic syndrome (MetS) and MASH with accumulation of endogenous aldehydes such as 4-hydroxynonenal (4-HNE). Mechanistic studies demonstrate aberrant transforming growth factor ß (TGF-ß) signaling through 4-HNE modification of the SMAD3 adaptor SPTBN1 (ß2-spectrin) to pro-fibrotic and pro-oncogenic phenotypes, which is restored to normal SMAD3 signaling by targeting SPTBN1 with small interfering RNA (siRNA). Significantly, therapeutic inhibition of SPTBN1 blocks MASH and fibrosis in a human model and, additionally, improves glucose handling in Aldh2-/- and Aldh2-/-Sptbn1+/- mice. This study identifies SPTBN1 as a critical regulator of the functional phenotype of toxic aldehyde-induced MASH and a potential therapeutic target.

Detection of gamma radiation processed onion during storage using propidium iodide based fluorescence microscopy.

The determination of absorbed dose in gamma radiation processed onion (treated with 20-100 Gy for sprout inhibition) during storage is an important regulatory requirement to control unfair practices. To address this problem, a microscopy based method was developed using propidium iodide (PI) staining of onion adaxial epidermis. A proportional radiation dose dependent increase in nuclei count was observed during ambient (26 ± 2 °C) and low (2 ± 1 °C) temperature storage. The method was validated and dose of radiation could be determined accurately in stored onions using blind tests. During mechanism studies, boron-dipyrromethene (BODIPY) dye staining and malondialdehyde (MDA) estimation showed dose dependent increase in peroxidation of membrane lipids. The fluorescein diacetate (FDA) stained onion adaxial epidermis showed decrease in fluorescence indicating lowering of physiological activity. Enzyme peroxidase (POD), phenylalanine ammonia lyase (PAL) activities and phytochemicals (phenolics, flavonoids, ascorbic acid and pyruvic acid) did not change significantly with increasing dose of gamma radiation.

Impaired reciprocal regulation between SIRT6 and TGF-ß signaling in fatty liver.

Dysregulated transforming growth factor-beta (TGF-ß) signaling contributes to fibrotic liver disease and hepatocellular cancer (HCC), both of which are associated with fatty liver disease. SIRT6 limits fibrosis by inhibiting TGF-ß signaling through deacetylating SMAD2 and SMAD3 and limits lipogenesis by inhibiting SREBP1 and SREBP2 activity. Here, we showed that, compared to wild-type mice, high-fat diet-induced fatty liver is worse in TGF-ß signaling-deficient mice (SPTBN1+/- ) and the mutant mice had reduced SIRT6 abundance in the liver. Therefore, we hypothesized that altered reciprocal regulation between TGF-ß signaling and SIRT6 contributes to these liver pathologies. We found that deficiency in SMAD3 or SPTBN1 reduced SIRT6 mRNA and protein abundance and impaired TGF-ß induction of SIRT6 transcripts, and that SMAD3 bound to the SIRT6 promoter, suggesting that an SMAD3-SPTBN1 pathway mediated the induction of SIRT6 in response to TGF-ß. Overexpression of SIRT6 in HCC cells reduced the expression of TGF-ß-induced genes, consistent with the suppressive role of SIRT6 on TGF-ß signaling. Manipulation of SIRT6 abundance in HCC cells altered sterol regulatory element-binding protein (SREBP) activity and overexpression of SIRT6 reduced the amount of acetylated SPTBN1 and the abundance of both SMAD3 and SPTBN1. Furthermore, induction of SREBP target genes in response to SIRT6 overexpression was impaired in SPTBN1 heterozygous cells. Thus, we identified a regulatory loop between SIRT6 and SPTBN1 that represents a potential mechanism for susceptibility to fatty liver in the presence of dysfunctional TGF-ß signaling.

Mycobacterium tuberculosis Acetyltransferase Suppresses Oxidative Stress by Inducing Peroxisome Formation in Macrophages.

Mycobacterium tuberculosis (Mtb) inhibits host oxidative stress responses facilitating its survival in macrophages; however, the underlying molecular mechanisms are poorly understood. Here, we identified a Mtb acetyltransferase (Rv3034c) as a novel counter actor of macrophage oxidative stress responses by inducing peroxisome formation. An inducible Rv3034c deletion mutant of Mtb failed to induce peroxisome biogenesis, expression of the peroxisomal ß-oxidation pathway intermediates (ACOX1, ACAA1, MFP2) in macrophages, resulting in reduced intracellular survival compared to the parental strain. This reduced virulence phenotype was rescued by repletion of Rv3034c. Peroxisome induction depended on the interaction between Rv3034c and the macrophage mannose receptor (MR). Interaction between Rv3034c and MR induced expression of the peroxisomal biogenesis proteins PEX5p, PEX13p, PEX14p, PEX11ß, PEX19p, the peroxisomal membrane lipid transporter ABCD3, and catalase. Expression of PEX14p and ABCD3 was also enhanced in lungs from Mtb aerosol-infected mice. This is the first report that peroxisome-mediated control of ROS balance is essential for innate immune responses to Mtb but can be counteracted by the mycobacterial acetyltransferase Rv3034c. Thus, peroxisomes represent interesting targets for host-directed therapeutics to tuberculosis.

ß2-spectrin (SPTBN1) as a therapeutic target for diet-induced liver disease and preventing cancer development.

The prevalence of nonalcoholic steatohepatitis (NASH) and liver cancer is increasing. De novo lipogenesis and fibrosis contribute to disease progression and cancerous transformation. Here, we found that ß2-spectrin (SPTBN1) promotes sterol regulatory element (SRE)­binding protein (SREBP)­stimulated lipogenesis and development of liver cancer in mice fed a high-fat diet (HFD) or a western diet (WD). Either hepatocyte-specific knockout of SPTBN1 or siRNA-mediated therapy protected mice from HFD/WD-induced obesity and fibrosis, lipid accumulation, and tissue damage in the liver. Biochemical analysis suggested that HFD/WD induces SPTBN1 and SREBP1 cleavage by CASPASE-3 and that the cleaved products interact to promote expression of genes with sterol response elements. Analysis of human NASH tissue revealed increased SPTBN1 and CASPASE-3 expression. Thus, our data indicate that SPTBN1 represents a potential target for therapeutic intervention in NASH and liver cancer.

Evaluation of Berberine as an Adjunct to TB Treatment.

Tuberculosis (TB) is the global health problem with the second highest number of deaths from a communicable disease after COVID-19. Although TB is curable, poor health infrastructure, long and grueling TB treatments have led to the spread of TB pandemic with alarmingly increasing multidrug-resistant (MDR)-TB prevalence. Alternative host modulating therapies can be employed to improve TB drug efficacies or dampen the exaggerated inflammatory responses to improve lung function. Here, we investigated the adjunct therapy of natural immune-modulatory compound berberine in C57BL/6 mouse model of pulmonary TB. Berberine treatment did not affect Mtb growth in axenic cultures; however, it showed increased bacterial killing in primary murine bone marrow-derived macrophages and human monocyte-derived macrophages. Ad libitum berberine administration was beneficial to the host in combination with rifampicin and isoniazid. Berberine adjunctive treatment resulted in decreased lung pathology with no additive or synergistic effects on bacterial burdens in mice. Lung immune cell flow cytometry analysis showed that adjunctive berberine treatment decreased neutrophil, CD11b+ dendritic cell and recruited interstitial macrophage numbers. Late onset of adjunctive berberine treatment resulted in a similar phenotype with consistently reduced numbers of neutrophils both in lungs and the spleen. Together, our results suggest that berberine can be supplemented as an immunomodulatory agent depending on the disease stage and inflammatory status of the host.

Evaluation of IL-1 Blockade as an Adjunct to Linezolid Therapy for Tuberculosis in Mice and Macaques.

In 2017 over 550,000 estimated new cases of multi-drug/rifampicin resistant tuberculosis (MDR/RR-TB) occurred, emphasizing a need for new treatment strategies. Linezolid (LZD) is a potent antibiotic for drug-resistant Gram-positive infections and is an effective treatment for TB. However, extended LZD use can lead to LZD-associated host toxicities, most commonly bone marrow suppression. LZD toxicities may be mediated by IL-1, an inflammatory pathway important for early immunity during M. tuberculosis infection. However, IL-1 can contribute to pathology and disease severity late in TB progression. Since IL-1 may contribute to LZD toxicity and does influence TB pathology, we targeted this pathway with a potential host-directed therapy (HDT). We hypothesized LZD efficacy could be enhanced by modulation of IL-1 pathway to reduce bone marrow toxicity and TB associated-inflammation. We used two animal models of TB to test our hypothesis, a TB-susceptible mouse model and clinically relevant cynomolgus macaques. Antagonizing IL-1 in mice with established infection reduced lung neutrophil numbers and partially restored the erythroid progenitor populations that are depleted by LZD. In macaques, we found no conclusive evidence of bone marrow suppression associated with LZD, indicating our treatment time may have been short enough to avoid the toxicities observed in humans. Though treatment was only 4 weeks (the FDA approved regimen at the time of study), we observed sterilization of the majority of granulomas regardless of co-administration of the FDA-approved IL-1 receptor antagonist (IL-1Rn), also known as Anakinra. However, total lung inflammation was significantly reduced in macaques treated with IL-1Rn and LZD compared to LZD alone. Importantly, IL-1Rn administration did not impair the host response against Mtb or LZD efficacy in either animal model. Together, our data support that inhibition of IL-1 in combination with LZD has potential to be an effective HDT for TB and the need for further research in this area.

Alterations in TGF-ß signaling leads to high HMGA2 levels potentially through modulation of PJA1/SMAD3 in HCC cells.

Recently, we observed that the TGF-ß pathway is altered in 39% of HCCs. The alterations are correlated with a raised HMGA2 level. Therefore, we compared genetic alterations of HMGA2 and 43 TGF-ß pathway core genes in HCC patients from TCGA database. Genetic alterations of 15 genes, including INHBE, INHBC, GDF11, ACVRL and TGFB2 out of 43 core genes, highly-moderately matched that of HMGA2. Co-occurrences of mutation amplification, gains, deletions and high/low mRNA of HMGA2 with those of the core genes were highly significant in INHBE, INHBC, ACVR1B, ACVRL and GDF11. Mass spectrometry studies revealed that HMGA2 interacted with an E3 ligase, PJA1, and that this interaction is enhanced by TGF-ß treatment in the nuclear of HCC cells. Co-localization of nuclear PJA1 and HMGA2 in HCC cells increased upon TGF-ß treatment. Raised HMGA2 levels that occur with alterations in the TGF-ß signaling pathway may reflect an altered activity of E3 ligases, such as PJA1, and potentially contribute to the tumor-promoting roles of TGF-ß signaling. Here, we report that the co-occurrence of genetic alterations in HMGA2 and TGF-ß pathway core genes is implicated in HCC progression, and propose that HMGA2 and PJA1 may be potential novel targets in dysfunctional TGF-ß signaling in HCC.

Targeting the E3 Ubiquitin Ligase PJA1 Enhances Tumor-Suppressing TGFß Signaling.

RING-finger E3 ligases are instrumental in the regulation of inflammatory cascades, apoptosis, and cancer. However, their roles are relatively unknown in TGFß/SMAD signaling. SMAD3 and its adaptors, such as ß2SP, are important mediators of TGFß signaling and regulate gene expression to suppress stem cell-like phenotypes in diverse cancers, including hepatocellular carcinoma (HCC). Here, PJA1, an E3 ligase, promoted ubiquitination and degradation of phosphorylated SMAD3 and impaired a SMAD3/ß2SP-dependent tumor-suppressing pathway in multiple HCC cell lines. In mice deficient for SMAD3 (Smad3 +/-), PJA1 overexpression promoted the transformation of liver stem cells. Analysis of genes regulated by PJA1 knockdown and TGFß1 signaling revealed 1,584 co-upregulated genes and 1,280 co-downregulated genes, including many implicated in cancer. The E3 ligase inhibitor RTA405 enhanced SMAD3-regulated gene expression and reduced growth of HCC cells in culture and xenografts of HCC tumors, suggesting that inhibition of PJA1 may be beneficial in treating HCC or preventing HCC development in at-risk patients.Significance: These findings provide a novel mechanism regulating the tumor suppressor function of TGFß in liver carcinogenesis.

Mutated CEACAMs Disrupt Transforming Growth Factor Beta Signaling and Alter the Intestinal Microbiome to Promote Colorectal Carcinogenesis.

BACKGROUND & AIMS: We studied interactions among proteins of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, which interact with microbes, and transforming growth factor beta (TGFB) signaling pathway, which is often altered in colorectal cancer cells. We investigated mechanisms by which CEACAM proteins inhibit TGFB signaling and alter the intestinal microbiome to promote colorectal carcinogenesis. METHODS: We collected data on DNA sequences, messenger RNA expression levels, and patient survival times from 456 colorectal adenocarcinoma cases, and a separate set of 594 samples of colorectal adenocarcinomas, in The Cancer Genome Atlas. We performed shotgun metagenomic sequencing analyses of feces from wild-type mice and mice with defects in TGFB signaling (Sptbn1+/- and Smad4+/-/Sptbn1+/-) to identify changes in microbiota composition before development of colon tumors. CEACAM protein and its mutants were overexpressed in SW480 and HCT116 colorectal cancer cell lines, which were analyzed by immunoblotting and proliferation and colony formation assays. RESULTS: In colorectal adenocarcinomas, high expression levels of genes encoding CEACAM proteins, especially CEACAM5, were associated with reduced survival times of patients. There was an inverse correlation between expression of CEACAM genes and expression of TGFB pathway genes (TGFBR1, TGFBR2, and SMAD3). In colorectal adenocarcinomas, we also found an inverse correlation between expression of genes in the TGFB signaling pathway and genes that regulate stem cell features of cells. We found mutations encoding L640I and A643T in the B3 domain of human CEACAM5 in colorectal adenocarcinomas; structural studies indicated that these mutations would alter the interaction between CEACAM5 and TGFBR1. Overexpression of these mutants in SW480 and HCT116 colorectal cancer cell lines increased their anchorage-independent growth and inhibited TGFB signaling to a greater extent than overexpression of wild-type CEACAM5, indicating that they are gain-of-function mutations. Compared with feces from wild-type mice, feces from mice with defects in TGFB signaling had increased abundance of bacterial species that have been associated with the development of colon tumors, including Clostridium septicum, and decreased amounts of beneficial bacteria, such as Bacteroides vulgatus and Parabacteroides distasonis. CONCLUSION: We found expression of CEACAMs and genes that regulate stem cell features of cells to be increased in colorectal adenocarcinomas and inversely correlated with expression of TGFB pathway genes. We found colorectal adenocarcinomas to express mutant forms of CEACAM5 that inhibit TGFB signaling and increase proliferation and colony formation. We propose that CEACAM proteins disrupt TGFB signaling, which alters the composition of the intestinal microbiome to promote colorectal carcinogenesis.

Klebsiella pneumoniae infection of murine neutrophils impairs their efferocytic clearance by modulating cell death machinery.

Neutrophils are the first infiltrating cell type essential for combating pneumoseptic infections by bacterial pathogens including Klebsiella pneumoniae (KPn). Following an infection or injury, removal of apoptotic infiltrates via a highly regulated process called efferocytosis is required for restoration of homeostasis, but little is known regarding the effect of bacterial infection on this process. Here we demonstrate that KPn infection impedes the efferocytic uptake of neutrophils in-vitro and in-vivo in lungs by macrophages. This impaired efferocytosis of infected neutrophils coincides with drastic reduction in the neutrophil surface exposure of apoptosis signature phospholipid phosphatidyserine (PS); and increased activity of phospholipid transporter flippases, which maintain PS in the inner leaflet of plasma membrane. Concomitantly, pharmacological inhibition of flippase activity enhanced PS externalization and restored the efferocytosis of KPn infected neutrophils. We further show that KPn infection interferes with apoptosis activation and instead activates non-apoptotic programmed cell death via activation of necroptosis machinery in neutrophils. Accordingly, pharmacological inhibition of necroptosis by RIPK1 and RIPK3 inhibitors restored the efferocytic uptake of KPn infected neutrophils in-vitro. Importantly, treatment of KPn infected mice with necroptosis inhibitor improved the disease outcome in-vivo in preclinical mouse model of KPn pneumonia. To our knowledge, this is the first report of neutrophil efferocytosis impairment by KPn via modulation of cell death pathway, which may provide novel targets for therapeutic intervention of this infection.

Galectin-3 in M2 Macrophages Plays a Protective Role in Resolution of Neuropathology in Brain Parasitic Infection by Regulating Neutrophil Turnover.

Macrophages/microglia with M2-activation phenotype are thought to play important anti-inflammatory and tissue reparative functions in the brain, yet the molecular bases of their functions in the CNS remain to be clearly defined. In a preclinical model of neurocysticercosis using brain infection with a parasite Mesocestoides corti, we previously reported the presence of large numbers of M2 cells in the CNS. In this study using female mice, we report that M2 macrophages in the parasite-infected brain display abundant galectin-3 expression. Disease severity was increased in Galectin-3-/- mice correlating with increased neurological defects, augmented cell death and, importantly, massive accumulation of neutrophils and M2 macrophages in the CNS of these mice. Because neutrophil clearance by efferocytosis is an important function of M2 macrophages, we investigated a possible role of galectin-3 in this process. Indeed, galectin-3-deficient M2 macrophages exhibited a defect in efferocytic clearance of neutrophils in vitro Furthermore, adoptive transfer of M2 macrophages from galectin-3-sufficient WT mice reduced neutrophilia in the CNS and ameliorated disease severity in parasite-infected Galectin-3-/- mice. Together, these results demonstrate, for the first time, a novel role of galectin-3 in M2 macrophage function in neutrophil turnover and resolution of inflammatory pathology in the CNS. This likely will have implications in neurocysticercosis and neuroinflammatory diseases.SIGNIFICANCE STATEMENT Macrophages/microglia with M1-activation phenotype are thought to promote CNS pathology, whereas M2-anti-inflammatory phenotype promote CNS repair. However, the mechanisms regulating M2 cell-protective functions in the CNS microenvironment are undefined. The current study reports that helminth infection of the brain induces an increased expression of galectin-3 in M2 macrophages accumulated in the CNS. Using multiple experimental models in vivo and in vitro, they show that galectin-3 in M2 macrophages functions to clear neutrophils accumulated in the CNS. Importantly, galectin-3 in M2 macrophages plays a central role in the containment of neuropathology and disease severity. These results provide a direct mechanistic evidence of the protective function of M2 macrophages in the CNS.

Precision Medicine for CRC Patients in the Veteran Population: State-of-the-Art, Challenges and Research Directions.

Colorectal cancer (CRC) accounts for ~9% of all cancers in the Veteran population, a fact which has focused a great deal of the attention of the VA's research and development efforts. A field-based meeting of CRC experts was convened to discuss both challenges and opportunities in precision medicine for CRC. This group, designated as the VA Colorectal Cancer Cell-genomics Consortium (VA4C), discussed advances in CRC biology, biomarkers, and imaging for early detection and prevention. There was also a discussion of precision treatment involving fluorescence-guided surgery, targeted chemotherapies and immunotherapies, and personalized cancer treatment approaches. The overarching goal was to identify modalities that might ultimately lead to personalized cancer diagnosis and treatment. This review summarizes the findings of this VA field-based meeting, in which much of the current knowledge on CRC prescreening and treatment was discussed. It was concluded that there is a need and an opportunity to identify new targets for both the prevention of CRC and the development of effective therapies for advanced disease. Also, developing methods integrating genomic testing with tumoroid-based clinical drug response might lead to more accurate diagnosis and prognostication and more effective personalized treatment of CRC.

Analysis of Genomes and Transcriptomes of Hepatocellular Carcinomas Identifies Mutations and Gene Expression Changes in the Transforming Growth Factor-ß Pathway.

BACKGROUND & AIMS: Development of hepatocellular carcinoma (HCC) is associated with alterations in the transforming growth factor-beta (TGF-ß) signaling pathway, which regulates liver inflammation and can have tumor suppressor or promoter activities. Little is known about the roles of specific members of this pathway at specific of HCC development. We took an integrated approach to identify and validate the effects of changes in this pathway in HCC and identify therapeutic targets. METHODS: We performed transcriptome analyses for a total of 488 HCCs that include data from The Cancer Genome Atlas. We also screened 301 HCCs reported in the Catalogue of Somatic Mutations in Cancer and 202 from Cancer Genome Atlas for mutations in genome sequences. We expressed mutant forms of spectrin beta, non-erythrocytic 1 (SPTBN1) in HepG2, SNU398, and SNU475 cells and measured phosphorylation, nuclear translocation, and transcriptional activity of SMAD family member 3 (SMAD3). RESULTS: We found somatic mutations in at least 1 gene whose product is a member of TGF-ß signaling pathway in 38% of HCC samples. SPTBN1 was mutated in the largest proportion of samples (12 of 202, 6%). Unsupervised clustering of transcriptome data identified a group of HCCs with activation of the TGF-ß signaling pathway (increased transcription of genes in the pathway) and a group of HCCs with inactivation of TGF-ß signaling (reduced expression of genes in this pathway). Patients with tumors with inactivation of TGF-ß signaling had shorter survival times than patients with tumors with activation of TGF-ß signaling (P = .0129). Patterns of TGF-ß signaling correlated with activation of the DNA damage response and sirtuin signaling pathways. HepG2, SNU398, and SNU475 cells that expressed the D1089Y mutant or with knockdown of SPTBN1 had increased sensitivity to DNA crosslinking agents and reduced survival compared with cells that expressed normal SPTBN1 (controls). CONCLUSIONS: In genome and transcriptome analyses of HCC samples, we found mutations in genes in the TGF-ß signaling pathway in almost 40% of samples. These correlated with changes in expression of genes in the pathways; up-regulation of genes in this pathway would contribute to inflammation and fibrosis, whereas down-regulation would indicate loss of TGF-ß tumor suppressor activity. Our findings indicate that therapeutic agents for HCCs can be effective, based on genetic features of the TGF-ß pathway; agents that block TGF-ß should be used only in patients with specific types of HCCs.

Alcohol, stem cells and cancer.

Dosage, gender, and genetic susceptibility to the effects of alcohol remained only partially elucidated. In this review, we summarize the current knowledge of the mechanisms underlying the role of alcohol in liver and gastrointestinal cancers. In addition, two recent pathways- DNA repair and TGF-ß signaling which provide new insights into alcohol in the regulation of cancers and stem cells are also discussed here.

Erratum: PRAJA is overexpressed in glioblastoma and contributes to neural precursor development.

[This corrects the article DOI: 10.18632/genesandcancer.151.].

PRAJA is overexpressed in glioblastoma and contributes to neural precursor development.

PRAJA, a RING-H2 E3 ligase, is abundantly expressed in brain tissues such as the cerebellum and frontal cortex, amongst others, and more specifically in neural progenitor cells as well as in multiple cancers that include glioblastomas. However, the specific role that Praja plays in neural development and gliomas remains unclear. In this investigation, we performed bioinformatic analyses to examine Praja1 and Praja2 expression across 29 cancer types, and observed raised levels of Praja1 and Praja2 in gliomas with an inverse relationship between Praja1 and apoptotic genes and Praja substrates such as Smad3. We analyzed the role of Praja in the developing brain through loss of function studies, using morpholinos targeting Praja1 in embryonic zebrafish, and observed that Praja1 is expressed prominently in regions enriched with neural precursor cell subtypes. Antisense Praja morpholinos resulted in multiple embryonic defects including delayed neural development likely through increased apoptosis. Further studies revealed high levels of Cdk1 with loss of Praja1 in TGF-ß or insulin treated cells, supporting the link between Praja1 and cell cycle regulation. In summary, these studies underscore Praja's role in mammalian brain development and Praja1 deregulation may lead to gliomas possibly through the regulation of cell cycle and/or apoptosis.