Biology subjects

Campbell, M. J.

Publications and source records attributed to Campbell, M. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Transporter-Mediated Uptake of Microcystin-LR in Human Trophoblasts: Regulation By Oxygen Concentration and Cell Fusion

BackgroundRising global temperatures and eutrophication are increasing the intensity and frequency of cyanobacterial harmful algal blooms that release toxins including microcystin-LR (MC-LR). MC-LR inhibits protein phosphatases in the human liver and brain, but its accumulation in the placenta is unclear. Placental transporter expression varies across pregnancy and is influenced by physiological cues, such as low oxygen concentrations which activate HIF1A, and trophoblast cell fusion forming syncytiotrophoblasts that engage CREB-driven transcription. This study examined whether MC-LR accumulates in placental cells, which transporters mediate uptake, and how these transporters are regulated by HIF1A and CREB. MethodsIntracellular accumulation of MC-LR (0.1-10 {micro}M, 3 hour) was measured in human cytotrophoblasts (JAR, BeWo) and extravillous trophoblasts (HTR-8/SVneo) by western blotting for MC-LR-adducted proteins. Organic anion transporting polypeptide (OATP) involvement was tested using cyclosporin A (10 {micro}M), an OATP inhibitor, before exposure to the OATP substrate or MC-LR. Cells were also cultured under 3%, 8%, or 20% O2 to induce hypoxic responses or treated with forskolin (a potent intracellular cAMP inducer) to stimulate cell fusion before MC-LR exposure. ResultsMC-LR accumulated in all three placenta cell lines in a concentration-dependent manner. Cyclosporin A reduced MC-LR uptake by 57% in JAR cells, confirming OATP-mediated transport. Low O2 increased OATP4A1 expression and function but reduced protein phosphatase expression, decreasing MC-LR-bound proteins by 52-72%. Forskolin increased OATP4A1 expression and enhanced MC-LR uptake >2.5-fold. ConclusionMC-LR enters placental trophoblasts via active OATP transport, likely OATP4A1, and uptake increases under hypoxia and trophoblast fusion.

pharmacology and toxicology↗

bioRxiv2025

Crowdsourced Protein Design: Lessons From the Adaptyv EGFR Binder Competition

In this report, we summarize and analyze the 2024 Adaptyv protein design competition. Participants used computational and Machine Learning (ML) methods of their choice to design proteins that bind the Epidermal Growth Factor Receptor (EGFR), a key drug target involved in cell growth, differentiation, and cancer development. Over 1,800 designs were submitted across two rounds. Of these, 601 proteins were selected and characterized for expression and binding affinity to EGFR, with competitors both optimizing existing binders (KD = 1.21 nM) and creating de novo binders (KD = 82 nM). All selected designs were experimentally validated using Adaptyvs automated Bio-Layer Interferometry (BLI) pipeline. This competition illustrates the potential of crowdsourcing to drive creativity and innovation in protein design. However, it also exposed key challenges, such as the lack of standardized benchmarks, experimental design targets, and robust computational metrics for method comparison. We anticipate that future competitions will address these gaps and further motivate progress in computational protein design.

bioengineering↗

bioRxiv2024

Aminolipid biosynthesis in response to phosphate limitation promotes colistin tolerance in Acinetobacter baumannii

The Gram-negative outer membrane protects bacterial cells from environmental toxins such as antibiotics. The outer membrane lipid bilayer is asymmetric; while glycerophospholipids compose the periplasmic facing leaflet, the surface layer is enriched with phosphate-containing lipopolysaccharides. The anionic phosphates that decorate the cell surface promote electrostatic interactions with cationic antimicrobial peptides such as colistin, allowing them to penetrate the bilayer, form pores, and lyse the cell. Colistin is prescribed as a last-line therapy to treat multidrug-resistant Gram-negative infections. Acinetobacter baumannii is an ESKAPE pathogen that rapidly develops resistance to antibiotics and persists for extended periods in the host or on abiotic surfaces. Survival in environmental stress such as phosphate scarcity, represents a clinically significant challenge for nosocomial pathogens. In the face of phosphate starvation, certain bacteria encode adaptive strategies, including the substitution of glycerophospholipids with phosphorus-free lipids. In bacteria, phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin are conserved glycerophospholipids that can form lipid bilayers, particularly in the presence of other lipids. Here, we demonstrate that in response to phosphate limitation, conserved regulatory mechanisms induce alternative lipid production in A. baumannii. Specifically, phosphate limitation induces formation of three lipids, including amine-containing ornithine and lysine aminolipids. Mutations that inactivate aminolipid biosynthesis exhibit fitness defects relative to wild type in colistin growth and killing assays. Furthermore, we show that other Gram-negative ESKAPE pathogens accumulate aminolipids under phosphate limiting growth conditions, suggesting aminolipid biosynthesis may represent a broad strategy to overcome cationic antimicrobial peptide-mediated killing. Author SummaryGram-negative ESKAPE pathogens, including Acinetobacter baumannii, are responsible for a dramatic increase in the morbidity and mortality of patients in healthcare settings over the past two decades. Infections are difficult to treat due to antibiotic resistance and tolerance; however, broadly conserved mechanisms that promote antibiotic treatment failure have not been extensively studied. Herein, we identify an alternative lipid biosynthesis pathway that is induced in phosphate starvation that enables Gram-negative ESKAPE pathogens, including A. baumannii, Klebsiella pneumoniae, and Enterobacter cloacae to build lipid bilayers in the absence of glycerophospholipids, which are the canonical bilayers lipid. Replacement of the anionic phosphate in the lipid headgroup with zwitterionic ornithine and lysine promote survival against colistin, a last resort antimicrobial used against Gram-negative infections. These studies suggest that ESKAPE pathogens can remodel their bilayers with phosphate free lipids to overcome colistin treatment and that aminolipid biosynthesis could be targeted to improve antimicrobial treatment.

microbiology↗

bioRxiv2024

Interactive effects of temperature acclimation and dietary fatty acids on metabolic rate and body composition of zebra finches (Taeniopygia guttata)

Climate change is contributing to geographic range shifts in many bird species, with possible exposure to novel diets. How individuals respond physiologically across chronic time frames to the interacting effects of diet and environmental temperature has been little explored. We acclimated zebra finches to either cool (20-24{degrees}C) or thermoneutral (35{degrees}C) temperatures over 6-months and provided them with diets enriched in either unsaturated or saturated fatty acids. We measured body mass throughout the study, and basal metabolic rate (BMR) and body composition at 3- and 6-months, respectively. Individuals held in cool conditions and fed a diet enriched with unsaturated fatty acids lost mass initially relative to the other groups, however, effects were reversible, and all individuals had a similar mass at 6- months. Chronic exposure to cool conditions increased BMR and the mass of the pectoral muscle and visceral organs. However, we could detect no long-term effect of diet on any physiological parameter. Our results contrast with those of birds studied over acute time frames, in which diet and temperature interact to determine energy expenditure. Over chronic time frames individuals appear to reach a new steady-state, with long-term physiological responses driven primarily by thermoregulatory responses to environmental temperature. Research HighlightsO_LIWith climate change, birds may encounter novel diets and temperatures C_LIO_LIIn zebra finches we show that chronic acclimation to cool temperatures increased energy expenditure and changed body composition. C_LIO_LIDietary fatty acid content had little long-term impact on the physiological parameters we measured. C_LI

zoology↗

bioRxiv2024

VIPER: A General Model for Prediction of Enzyme Substrates

Enzymes, natures catalysts, possess remarkable properties such as high stereo-, regio-, and chemo-specificity. These properties allow enzymes to greatly simplify complex synthetic processes, resulting in improved yields and reduced manufacturing costs compared to traditional chemical methods. However, the lack of experimental characterization of enzyme substrates, with only a few thousand out of tens of millions of known enzymes in Uniprot having annotated substrates, severely limits the ability of chemists to repurpose enzymes for industrial applications. Previous machine learning models aimed at predicting enzyme substrates have been hampered by poor generalization to new substrates. Here, we introduce VIPER (Virtual Interaction Predictor for Enzyme Reactivity), a model that achieves an average 30% improvement over the previous state-of-the-art model (ProSmith) in reaction prediction for unseen substrates. Furthermore, we reveal flaws in previous enzyme-substrate reaction datasets, and introduce a novel high-quality enzyme-substrate reaction dataset to alleviate these issues.

bioinformatics↗

bioRxiv2024

Dual agonism of sodium iodide symporter function in vivo

New approaches are urgently needed to enhance the radioiodide (RAI) ablation of aggressive and metastatic thyroid cancer. We recently discovered that valosin-containing protein inhibitors (VCPi) such as clotrimazole and disulfiram transiently block sodium iodide symporter (NIS) proteasomal degradation, hence promoting RAI uptake. However, poor bioavailability diminishes their potential impact in vivo. Following 3D modelling and iterative drug design we appraised 26 novel analogues of clotrimazole, as well as albumin nano-encapsulated copper-diethyldithiocarbamate [Cu(DDC)2-alb] - a stabilised reformulation of a disulfiram metabolite. While several clotrimazole analogues specifically increased RAI uptake, the greatest impact was observed with Cu(DDC)2-alb in thyroid cancer cells as well as human primary thyrocytes from patients with thyroid hyperplasia. NanoBRET assays revealed that Cu(DDC)2 enhanced the plasma membrane accumulation of NIS in living cells. In BALB/c mice, both intraperitoneal and intravenous administration of Cu(DDC)2-alb significantly enhanced thyroidal 99mTc-uptake. RNA-Seq revealed the surprising observation that Cu(DDC)2-alb induced key thyroid transcription factors. Accordingly, expression of PAX8 and NKX2.1 was upregulated in thyroid glands from drug treated mice, with NIS levels correlating closely to 99mTc-uptake. As Cu(DDC)2 inhibits the VCP cofactor NPL4, with VCP being critical to the proteostatic processing of NIS protein, we identify a new dual agonist of RAI uptake in vivo, with the potential to directly impact RAI therapy for patients with aggressive thyroid cancer.

cancer biology↗

bioRxiv2023

Epigenetic disruption of the RARγ complex impairs its function to bookmark AR enhancer interactions required for enzalutamide sensitivity in prostate cancer

The current study in prostate cancer (PCa) focused on the genomic mechanisms at the cross-roads of pro-differentiation signals and the emergence of lineage plasticity. We explored an understudied cistromic mechanism involving RAR{gamma}s ability to govern AR cistrome-transcriptome relationships, including those associated with more aggressive PCa features. The RAR{gamma} complex in PCa cell models was enriched for canonical cofactors, as well as proteins involved in RNA processing and bookmarking. Identifying the repertoire of miR-96 bound and regulated gene targets, including those recognition elements marked by m6A, revealed their significant enrichment in the RAR{gamma} complex. RAR{gamma} significantly enhanced the AR cistrome, particularly in active enhancers and super-enhancers, and overlapped with the binding of bookmarking factors. Furthermore, RAR{gamma} expression led to nucleosome-free chromatin enriched with H3K27ac, and significantly enhanced the AR cistrome in G2/M cells. RAR{gamma} functions also antagonized the transcriptional actions of the lineage master regulator ONECUT2. Similarly, gene programs regulated by either miR-96 or antagonized by RAR{gamma} were enriched in alternative lineages and more aggressive PCa phenotypes. Together these findings reveal an under-investigated role for RAR{gamma}, modulated by miR-96, to bookmark enhancer sites during mitosis. These sites are required by the AR to promote transcriptional competence, and emphasize luminal differentiation, while antagonizing ONECUT2.

cancer biology↗

bioRxiv2023

Tumor microenvironmental determinants of high-risk DCIS progression

ABSTRACT/SUMMARYDuctal carcinoma in situ (DCIS) constitutes an array of morphologically recognized intraductal neoplasms in the mammary ductal tree defined by an increased risk for subsequent invasive carcinomas at or near the site of biopsy detection. However, only 15-45% of untreated DCIS cases progress to invasive cancer, so understanding mechanisms that prevent progression is key to avoid overtreatment and provides a basis for alternative therapies and prevention. This study was designed to characterize the tumor microenvironment and molecular profile of high-risk DCIS that grew to a large size but remained as DCIS. All patients had DCIS lesions >5cm in size with at least one additional high-risk feature: young age (<45 years), high nuclear grade, hormone receptor negativity, HER2 positivity, the presence of comedonecrosis, or a palpable mass. The tumor immune microenvironment was characterized using multiplex immunofluorescence to identify immune cells and their spatial relationships within the ducts and stroma. Gene copy number analysis and whole exome DNA sequencing identified the mutational burden and driver mutations, and quantitative whole-transcriptome/gene expression analyses were performed. There was no association between the percent of the DCIS genome characterized by copy number variants (CNAs) and recurrence events (DCIS or invasive). Mutations, especially missense mutations, in the breast cancer driver genes PIK3CA and TP53 were common in this high-risk DCIS cohort (47% of evaluated lesions). Tumor infiltrating lymphocyte (TIL) density was higher in DCIS lesions with TP53 mutations (p=0.0079) compared to wildtype lesions, but not in lesions with PIK3CA mutations (p=0.44). Immune infiltrates were negatively associated with hormone receptor status and positively associated with HER2 expression. High levels of CD3+CD8-T cells were associated with good outcomes with respect to any subsequent recurrence (DCIS or invasive cancer), whereas high levels of CD3+Foxp3+ Treg cells were associated with poor outcomes. Spatial proximity analyses of immune cells and tumor cells demonstrated that close proximity of T cells with tumor cells was associated with good outcomes with respect to any recurrence as well as invasive recurrences. Interestingly, we found that myoepithelial continuity (distance between myoepithelial cells surrounding the involved ducts) was significantly lower in DCIS lesions compared to normal tissue (p=0.0002) or to atypical ductal hyperplasia (p=0.011). Gene set enrichment analysis identified several immune pathways associated with low myoepithelial continuity and a low myoepithelial continuity score was associated with better outcomes, suggesting that gaps in the myoepithelial layer may allow access/interactions between immune infiltrates and tumor cells. Our study demonstrates the immune microenvironment of DCIS, in particular the spatial proximity of tumor cells and T cells, and myoepithelial continuity are important determinants for progression of disease.

cancer biology↗

bioRxiv2023

The MYC axis in advanced prostate cancer is impacted through concurrent targeting of ERβ and AR using a novel ERβ-selective ligand alongside Enzalutamide

We have dissected the role of Estrogen receptor beta (ER{beta}) in prostate cancer (PCa) with a novel ER{beta} ligand, OSU-ERb-12. Drug screens revealed additive interactions between OSU-ERB-12 and either epigenetic inhibitors or the androgen receptor antagonist, Enzalutamide (Enza). Clonogenic and cell biolody studies supported the potent additive effects of OSU-ERB-12 (100nM) and Enza (1{micro}M). The cooperative behavior was in PCa cell lines treated with either OSU- ERB-12 plus Enza or combinations involving 17{beta}-estradiol (E2). OSU-ERb-12 plus Enza uniquely impacted the transcriptiome, accessible chromatin, and the AR, MYC and H3K27ac cistromes. This included skewed transcriptional responses including suppression of the androgen and MYC transcriptomes, and repressed MYC protein. OSU-ERb-12 plus Enza uniquely impacted chromatin accessibility at approximately 3000 nucleosome-free sites, enriched at enhancers, enriched for basic Helix-Loop-Helix motifs. CUT&RUN experiments revealed combination treatment targeting of MYC, AR, and H3K27ac again shaping enhancer accessibility. Specifically, it repressed MYC interactions at enhancer regions enriched for bHLH motifs, and overlapped with publicly-available bHLH cistromes. Finally, cistrome-transcriptome analyses identified [~]200 genes that distinguished advanced PCa tumors in the SU2C cohort with high androgen and low neuroendocrine scores. Statement of ImplicationTargeting ER{beta} has potentially to augment AR antagonism to restrain MYC signaling and limit growth of advanced prostate cancer.

cancer biology↗

bioRxiv2023

ONECUT2 Activates Diverse Resistance Drivers of Androgen Receptor-Independent Heterogeneity in Prostate Cancer

Significance StatementONECUT2 (OC2) is a master transcription factor that alters lineage identity by activating gene networks associated with both neuroendocrine prostate cancer and prostate adenocarcinoma. A small molecule inhibitor of OC2 represses the lineage plasticity program activated by enzalutamide, suggesting OC2 inhibition as a novel therapeutic strategy to prevent emergence of treatment-resistant variants. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/560025v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@13278a4org.highwire.dtl.DTLVardef@8e82aeorg.highwire.dtl.DTLVardef@25a115org.highwire.dtl.DTLVardef@728eb4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphic AbstractC_FLOATNO C_FIG Androgen receptor-(AR-) indifference is a mechanism of resistance to hormonal therapy in prostate cancer (PC). Here we demonstrate that the HOX/CUT transcription factor ONECUT2 (OC2) activates resistance through multiple drivers associated with adenocarcinoma, stem-like and neuroendocrine (NE) variants. Direct OC2 targets include the glucocorticoid receptor and the NE splicing factor SRRM4, among others. OC2 regulates gene expression by promoter binding, enhancement of chromatin accessibility, and formation of novel super-enhancers. OC2 also activates glucuronidation genes that irreversibly disable androgen, thereby evoking phenotypic heterogeneity indirectly by hormone depletion. Pharmacologic inhibition of OC2 suppresses lineage plasticity reprogramming induced by the AR signaling inhibitor enzalutamide. These results demonstrate that OC2 activation promotes a range of drug resistance mechanisms associated with treatment-emergent lineage variation in PC. Our findings support enhanced efforts to therapeutically target this protein as a means of suppressing treatment-resistant disease.

cancer biology↗

bioRxiv2023

Dissecting endocytic mechanisms reveals new molecular targets to enhance sodium iodide symporter activity with clinical relevance to radioiodide therapy

The sodium/iodide symporter (NIS) frequently shows diminished plasma membrane (PM) targeting in differentiated thyroid cancer (DTC), resulting in suboptimal radioiodide (RAI) treatment and poor prognosis. The mechanisms which govern the endocytosis of NIS away from the PM are ill-defined. Here, we challenged the hypothesis that new mechanistic understanding of NIS endocytosis would facilitate prediction of patient outcomes and enable specific drug modulation of RAI uptake in vivo. Through mutagenesis, NanoBiT interaction assays, cell surface biotinylation assays, RAI uptake and NanoBRET, we identify an acidic dipeptide within the NIS C-terminus which mediates binding to the {sigma}2 subunit of the Adaptor Protein 2 (AP2) heterotetramer. We discovered that the FDA-approved drug chloroquine modulates NIS accumulation at the PM in a functional manner that is AP2 dependent. In vivo, chloroquine treatment of BALB/c mice significantly enhanced thyroidal uptake of 99mTc pertechnetate in combination with the histone deacetylase (HDAC) inhibitor SAHA, accompanied by increased thyroidal NIS mRNA. Bioinformatic analyses validated the clinical relevance of AP2 genes with disease-free survival in RAI-treated DTC, enabling construction of an AP2 gene-related risk score classifier for predicting recurrence. We propose that NIS internalisation is orchestrated by the interaction of a C-terminal diacidic motif with AP2{sigma}2, together with the proto-oncogene PBF acting via AP22. Given that NIS internalisation was specifically druggable in vivo, our data provide new translatable potential for improving RAI therapy using FDA-approved drugs in patients with aggressive thyroid cancer. SummaryWe delineate the role of endocytic genes in regulating NIS activity at the plasma membrane and highlight the potential for systemic targeting of endocytosis to enhance radioiodine effectiveness in radioiodine-refractory cancer cells.

cancer biology↗

bioRxiv2022

The major role of sarA in limiting Staphylococcus aureus extracellular protease production is correlated with decreased virulence in diverse clinical isolates in osteomyelitis

We previously demonstrated that MgrA, SarA, SarR, SarS, SarZ, and Rot bind at least three of the four promoters associated with genes encoding primary extracellular proteases in Staphylococcus aureus. We also showed that mutation of sarA results in a greater increase in protease production, and decrease in biofilm formation, than mutation of the loci encoding any of these other proteins. However, these conclusions were based on in vitro studies. Thus, the goal of the experiments reported here was to determine the relative impact of the regulatory loci encoding these proteins in vivo. To this end, we compared the virulence of mgrA, sarA, sarR, sarS, sarZ, and rot mutants in a murine osteomyelitis model. Mutants were generated in the methicillin-resistant USA300 strain LAC and the methicillin-sensitive USA200 strain UAMS-1. As assessed based on an overall osteomyelitis pathology score derived from the incidence of bone fracture, bacterial burdens in the bone, cortical bone destruction, and reactive bone formation, mutation of mgrA and rot limited virulence to a statistically significant extent in UAMS-1, but not in LAC. In contrast, the sarA mutant exhibited reduced virulence in both strains. This illustrates the importance of considering diverse clinical isolates when evaluating the impact of regulatory mutations on virulence. The reduced virulence of the sarA mutant was correlated with reduced cytotoxicity for osteoblasts and osteoclasts, reduced biofilm formation, and reduced sensitivity to the antimicrobial peptide indolicidin, all of which were directly attributable to increased protease production in both LAC and UAMS-1. This suggests that these in vitro phenotypes, either alone or in combination with each other, may be useful in prioritizing additional mutants for in vivo evaluation. Most importantly, they illustrate the significance of limiting protease production in vivo in S. aureus, and confirm that SarA plays the primary role in this regard. Author SummaryStaphylococcus aureus causes a diverse array of infections due to its ability to produce an arsenal of virulence factors. Among these are extracellular proteases, which serve several purposes on behalf of the bacterium. However, it has become increasingly apparent that it is also critical to limit the production of these proteases to prevent them from compromising the S. aureus virulence factor repertoire. Many regulatory loci have been implicated in this respect, but it is difficult to draw relative conclusions because few reports have made direct comparisons, and fewer still have done so in vivo. We addressed this by assessing the impact on virulence of six regulatory loci previously implicated in protease production. We did this in the clinical context of osteomyelitis using mutants generated in two divergent clinical isolates. Our results confirm significant strain-dependent differences, reinforcing the importance of considering such diverse clinical isolates when evaluating targets for potential therapeutic intervention. In this respect, only mutation of sarA attenuated virulence in both strains. This illustrates the importance of limiting protease production as a means of post-translational regulatory control in S. aureus and confirms that sarA plays a predominant role in this regard.

microbiology↗

bioRxiv2022

Vitamin D receptor cistrome-transcriptome analyses establishes quantitatively distinct receptor genomic interactions in African American prostate cancer regulated by BAZ1A

BackgroundAfrican American (AA) prostate cancer (PCa) appears uniquely sensitive to 1,25(OH)2D3 signaling, compared to European American (EA) PCa, but the extent and impact of vitamin D receptor genomic functions remain poorly defined. ResultsA panel of EA and AA prostate epithelial cells (EA: HPr1-AR, LNCaP, AA: RC43N, RC43T, RC77N, RC77T) were analyzed with RIME to reveal the cell-specific composition of the VDR- complex. 1,25(OH)2D3-dependent ATAC-Seq revealed the greatest impact on nucleosome positioning in RC43N and RC43T, with gain of nucleosome-free at enhancer regions. VDR ChIP-Seq identified stronger and more frequent VDR binding in RC43N and RC43T that was enriched for a larger and distinct motif repertoire, than EA cells. VDR binding significantly overlapped with core circadian rhythm transcription factors in AA cell line models. RNA-Seq also revealed significantly stronger 1,25(OH)2D3 dependent VDR transcriptional responses enriched for circadian rhythm and inflammation networks in AA cells. Whilst RC43N was most responsive, RC43T displayed distorted responses. Significantly reduced BAZ1A/SMARCA5 in AA PCa samples was identified, and restored BAZ1A expression uniquely and significantly increased 1,25(OH)2D3-regulated VDR targets in AA cells. These VDR- dependent cistrome-annotated genes were also uniquely and most significantly identified in three cohorts of AA PCa patients. ConclusionThese data suggest VDR transcriptional control in the prostate is more potent and dynamic in AA men, and primed to govern inflammatory and circadian pathways. Reduced BAZ1A/SMARCA5 expression and/or reduced environmentally-regulated serum vitamin D3 levels suppress these actions. Therefore, the VDR axis lies at the cross-roads of biopsychosocial processes including stress responses, access to quality early detection and treatment, social determinants and that collectively contribute to PCa health disparities.

cancer biology↗

bioRxiv2020

Cytoprotective effects of combination of gypenosides and Costus pictus D.Don extract in BRIN-BD11 β-cells

Ethnopharmacological RelevanceGypenosides and Costus pictus D.Don are used as an anti-diabetic herbal remedy in China and India respectively. However, the synergistic effect of these two extracts on {beta}-cell protection is not yet elucidated. IntroductionIn Type 2 diabetes mellitus (T2DM), pro-inflammatory cytokines and lipotoxicity are known causes of pancreatic {beta}-cell dysfunction and impaired insulin secretion and eventually {beta}-cell death. Thus, any cytoprotective drug supplements can protect the {beta}-cell and may help in T2DM treatment. Gypenosides, extracted from the Chinese medicinal herb Gynostemma pentaphyllum and the leaf extract from an Indian medicinal herb Costus pictus D. Don are used in traditional medicine due to their insulin secretory properties. In our previous studies, both extracts have shown significant cytoprotective effects in insulin-secreting BRIN-BD11 cells. In the present study, we aim to investigate the synergistic effects of a combination of these extracts on BRIN-BD11 {beta}-cell protection. MethodsCombination of extracts was prepared by adding Gypenosides with Costus pictus at 2:1 to a concentration of 18.75mg/ml. Cell viability was determined by MTT assay following treatment with combination and/or palmitate and cytokine cocktail for 24-48h. Following 24h treatment, proliferation was measured by Ki67 staining and cytoprotective gene expression was quantified by qPCR. ResultsCombination treatment of 25{micro}g/ml enhanced cell viability both at 24h (n=8; P<0.05) and 48h (n=8; P<0.0001) treatment. Over 24h, combination treatment (25&12.5 {micro}g/ml) showed a significant protective effect against 125{micro}M and 250{micro}M palmitate induced (P<0.0001) and cytokine cocktail-(TNF 1000U, IL-1{beta} 50U & IFN{gamma} 1000U) (P<0.0001 & P<0.01 respectively) induced toxicity. Combination treatment over 24h increased expression of antioxidant genes Nrf2 (P<0.001), Cat (P<0.001) and Sod1 (P<0.05) along with pro-proliferative Erk1 (P<0.01) while pro-inflammatory Nfkb1 expression was reduced(P<0.001). ConclusionThe results suggest that a combination of gypenosides and costus pictus may protect {beta}-cells against inflammatory cytokines and lipotoxicity caused by saturated free fatty acids associated with obesity and diabetes.

pharmacology and toxicology↗

bioRxiv2020

Reduced NCOR2 expression accelerates androgen deprivation therapy failure in prostate cancer

NCOR2 is frequently and significantly mutated in late stage androgen deprivation therapy resistant prostate cancer (ADT-RPCa). NCOR2 has been characterized as a transcriptional corepressor and has mechanistic links to DNA methylation, but its global functions and overall contributions to PCa progression remain enigmatic. In the current study, we utilize immunohistochemical staining of samples from over 700 PCa patients and reveal associations of reduced NCOR2 expression with correlates of aggressive primary PCa and recurrence in patients who received adjuvant androgen deprivation therapy. We mapped the dihydrotestosterone (DHT) dependent and independent effects of NCOR2 on the transcriptome, cistrome and DNA methylome in androgen sensitive (AS) and ADT-RPCa cells using the isogenic LNCaP and LNCaP-C4-2 (C4-2) cell models. Transcriptional profiling identified androgen dependent and independent regulatory roles of NCOR2, the latter of which was enhanced in the ADT-RPCa state and included neuronal differentiation. Interestingly, reduced expression of NCOR2 resulted in a striking global DNA hypermethylation pattern that significantly enriched at enhancer regions. ChIP-seq revealed that NCOR2 was more clearly associated with promoters in AS LNCaP cells, which was modestly enhanced by DHT treatment. However, in ADT-RPCa C4-2 cells, the NCOR2 cistrome was larger and more distal. Motif analyses and integration of large-scale public cistrome data revealed strong enrichment for FOXA1 in mediating NCOR2 binding, and included additional factors such as AR, E2F, TET2, MED1 and MBD2. Utilizing the CWR22 xenograft model, we demonstrate a direct role for NCOR2 in PCa progression as reduced NCOR2 expression attenuated the impact of ADT, and significantly accelerated recurrence of disease. Transcriptomic analyses in recurrent CWR22 tumors indicated NCOR2-dependent gene expression profiles during ADT that were enriched for neuroendocrine associated genes and also associated with worse survival in human patients with ADT-RPCa. DNA methylation profiles in CWR22 tumors with reduced NCOR2 expression recapitulated the hypermethylation observed in vitro, and further revealed that hypermethylation patterns are commonly associated with ADT-RPCa disease, which was also confirmed in human samples. These studies reveal robust roles for NCOR2 in regulating the PCa transcriptome and epigenome and underscore recent mutational studies linking NCOR2 loss of function to PCa disease progression.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cancer biology↗

bioRxiv2020

Genome-wide analyses of transcription factors and co-regulators across seven cohorts identified reduced PPARGC1A expression as a driver of prostate cancer progression

Defining altered transcription factor (TF) and coregulators that are oncogenic drivers remains a challenge, in part because of the multitude of TFs and coregulators. We addressed this challenge by using bootstrap approaches to test how expression, copy number alterations or mutation of TFs (n = 2662), coactivators (COA; n= 766); corepressor (COR; n = 599); mixed function coregulators (MIXED; n = 511) varied across seven prostate cancer (PCa) cohorts (three of localized and four advanced disease). COAS, CORS, MIXED and TFs all displayed significant down-regulated expression (q.value < 0.1) and correlated with protein expression ({rho} 0.4 to 0.55). Stringent expression filtering identified commonly altered TFs and coregulators including well-established (e.g. ERG) and underexplored (e.g. PPARGC1A, encodes PGC1) in localized PCa. Reduced PPARGC1A expression significantly associated with worse disease-free survival in two cohorts of localized PCa. Stable PGC1 knockdown in LNCaP cells increased growth rates and invasiveness and RNA-Seq revealed a profound basal impact on gene expression (~2300 genes; FDR < 0.05, logFC > 1.5), but only modestly impacted PPAR{gamma} responses. GSEA analyses of the PGC1 transcriptome revealed that it significantly altered the AR-dependent transcriptome, and was enriched for epigenetic modifiers. PGC1-dependent genes were overlapped with PGC1-ChIP-Seq genes and significantly associated in TCGA with higher grade tumors and worse disease-free survival. Together these data demonstrate an approach to identify cancer-driver coregulators in cancer and that PGC1 expression is clinically significant yet underexplored coregulator in aggressive early stage PCa.

cancer biology↗

bioRxiv2020

Germline genetic contribution to the immune landscape of cancer

The role of germline genetics in shaping the tumor immune landscape is largely unknown. Using genotypes from >9,000 individuals in The Cancer Genome Atlas, we investigated the association of common and rare variants with 139 well-defined immune traits. Our analysis of common variants identified 10 immune traits with significant heritability estimates, and an additional 23 with suggestive heritability, including estimates of T-cell subset abundance and interferon signaling. We performed genome-wide association on the 33 heritable traits and identified 23 genome-wide significant loci associated with at least one immune trait, including SNPs in the IFIH1 locus previously associated with several autoimmune diseases. We also found significant associations between immune traits and pathogenic or likely-pathogenic rare variants in BRCA1 and in genes functionally linked to telomere stabilization, and Wnt/Beta-catenin signaling. We conclude that germline genetic variants significantly impact the composition and functional orientation of the tumor immune microenvironment.

immunology↗

bioRxiv2019

Pharmacologically targeting a novel pathway of sodium iodide symporter trafficking to enhance radioiodine uptake

Radioiodine treatment fails [≥]25% of patients with thyroid cancer and has been proposed as a potential treatment for breast cancer. Cellular iodide uptake is governed by the sodium iodide symporter (NIS), which is frequently mislocalized in thyroid and breast tumours. However, the trafficking of NIS to the plasma membrane (PM) is ill-defined. Through mass spectrometry, co-immunoprecipitation, cell surface biotinylation and proximity ligation assays we identify two proteins which control NIS subcellular trafficking: ADP-ribosylation factor 4 (ARF4) and valosin-containing protein (VCP). HiLo microscopy revealed ARF4 enhanced NIS trafficking in co-incident PM vesicles, governed by a C-terminal VXPX motif, whilst papillary thyroid cancers (PTC) demonstrate repressed ARF4 expression. In contrast, VCP, the central protein in ER-associated degradation, specifically bound NIS and decreased its PM localization. Five chemically distinct allosteric VCP inhibitors all overcame VCP-mediated repression of NIS function. In mice, two re-purposed FDA-approved VCP inhibitors significantly enhanced radioiodine uptake into thyrocytes, whilst human primary thyrocytes showed similar increases. Critically, PTC patients with high tumoural VCP expression who received radioiodine had strikingly worse disease-free survival. These studies now delineate the mechanisms of NIS trafficking, and for the first time open the therapeutic possibility of systemically enhancing radioiodine uptake in patients via FDA-approved drugs.\n\nOne Sentence SummaryNovel NIS interactors ARF4 and VCP alter NIS trafficking in vitro, and FDA-approved VCP inhibitors can significantly enhance radioiodine uptake.

cancer biology↗