Biology subjects

Graham, M. K.

Publications and source records attributed to Graham, M. K..

8 recordsLinked to original sources

bioRxiv2025

A transition zone enriched WIF1⁺ basal cell subtype is associated with benign prostatic hyperplasia

ABSTRACT/SUMMARYThe cellular composition and disease susceptibilities of the distinct zones of the human prostate remain incompletely understood. Through extensive single-cell RNA sequencing (scRNA-seq) of benign regions from prostatectomy specimens, we identified a basal cell population expressing WIF1, VCAN, and NRG1, among other genes, that was significantly enriched in the transition zone (TZ). Benign prostatic hyperplasia (BPH) is a common condition that causes widespread morbidity and is nearly exclusively localized to the TZ. Analysis of previously published scRNA-seq datasets further confirmed that WIF1+ basal cells were significantly enriched in BPH compared to normal prostate. Pathway and cell-cell communication analyses revealed that this basal subtype is associated with programs related to cell proliferation, epithelial-mesenchymal transition (EMT), angiogenesis, and hormone response. Together, the molecular signature, zonal distribution, and pathway enrichment suggest that TZ-enriched WIF1+ basal cells may contribute to BPH pathogenesis by promoting epithelial and stromal remodeling.

cell biology↗

bioRxiv2025

Unmasking Pathogen Traits for Chronic Colonization in Neurogenic Bladder Patients

Individuals with neurogenic bladder are particularly susceptible to both chronic bacterial colonization of the bladder and urinary tract infections (UTIs). Neurogenic bladder can arise from a variety of diseases such as diabetes, spinal cord injuries, and spina bifida. To study the ecological and evolutionary dynamics of the microbiome in neurogenic bladder, we developed a longitudinal cohort of 77 children and young adults with spina bifida from two medical centers. We used enhanced urine culture, 16S rRNA sequencing, and whole genome sequencing to characterize the microbial composition of urine and fecal samples. In addition to prospective sample collection, we retrieved prior bacterial isolates from enrolled patients from Vanderbilts clinical microbial biobank, MicroVU. This allowed us to compare bacterial isolates from the same patients over a period of five years. Urine samples were characterized by high abundance of urinary pathogens, such as E. coli and Klebsiella. From longitudinal isolates from individual patients, we identified two common patterns of urinary tract colonization. We observed either the rapid cycling of strains and/or species, often following antibiotic treatment, or we observed the persistence of a single strain across timepoints. Neither persistence of a strain nor colonization with a new strain or species was associated with increased antibiotic resistance. Rather, in paired longitudinally collected strains from the same patients, mutations were identified in genes that code for cell envelope components associated with immune or phage evasion. Experimental testing revealed that O-antigen/LPS biosynthesis mutations confer protection from the immune system while altering susceptibility to phage predation, reflecting a fitness trade-off. We argue that this unparalleled cohort offers the opportunity to identify mechanisms of bacterial adaptation to the urinary tract that can be exploited in future therapeutic approaches.

microbiology↗

bioRxiv2025

Loss of DAXX induces alternative lengthening of telomeres (ALT)-associated hallmarks in prostate cancer cells in a context-specific manner

BackgroundThe histone chaperone complex, consisting of the death domain-associated protein (DAXX) and the alpha-thalassemia/mental retardation X-linked protein (ATRX), plays a pivotal role in maintaining chromatin through the deposition of the histone variant H3.3. Mutations leading to loss of ATRX or DAXX function are linked to the non-telomerase, alternative lengthening of telomeres (ALT) phenotype in certain cancers. Engineered ATRX mutations have previously been found to induce features of ALT in prostate cancer cell lines, notably in LAPC-4, but not in CWR22Rv1. This study determined the impact of DAXX mutations on ALT-associated characteristics in CWR22Rv1 and LAPC-4. MethodologyMutations were induced in CWR22Rv1 and LAPC-4 cells by targeting exon 2 of DAXX using the CRISPR-Cas9 genome editing strategy. The resulting mutant clones were then evaluated for ALT-associated characteristics, including the presence of ALT-associated PML bodies (APBs), C-circles, telomere length heterogeneity, and a lack of telomerase activity. ResultsFour CWR22Rv1 DAXX mutant clones (DAXXMut1-4) and five LAPC-4 clones (DAXXMut1-5) were evaluated. In CWR22Rv1, DAXXMut1, DAXXMut2, and DAXXMut4 were true knockout clones with frameshift mutations in both copies, while CWR22Rv1 DAXXMut3 had a frameshift mutation in one copy and an in-frame mutation in the other. Protein expression was undetectable in all the CWR22Rv1 clones, including CWR22Rv1 DAXXMut3. In LAPC-4, DAXXMut1 was a true knockout, while DAXXMut2, DAXXMut3, DAXXMut4, and DAXXMut5 clones had at least one in-frame mutation. Among these LAPC-4 clones, only DAXXMut1 had undetectable protein by western blotting. ALT-associated characteristics such as APBs, C-circles, and telomere length heterogeneity were observed only in CWR22Rv1 DAXXMut4. All the clones maintained telomerase activity, regardless of whether ALT-associated hallmarks were observed. ImplicationsThe CWR22Rv1 and LAPC-4 DAXX mutant clone models provide useful tools for future studies on telomere maintenance mechanisms and DAXX-related biology, particularly in prostate cancer.

cancer biology↗

bioRxiv2025

Cell Type-Specific Effects of miR-21 Loss Attenuate Tumor Progression in MYC-Driven Prostate Cancer

Aberrant microRNA expression is common in cancer, yet cell-type-specific microRNA activity in the tumor microenvironment (TME) remains poorly understood. Here, we show that germline deletion of miR-21 significantly attenuated the progression of MYC-driven prostate cancer (PCa), reducing prostate weight, tumor burden, and proliferation index in Hi-Myc mice. In situ hybridization revealed elevated miR-21 expression in multiple cell types during disease progression. Inflammatory and premalignant lesions in mouse and human prostate showed increased miR-21 in both stroma and epithelium, with further enrichment in the stroma of invasive adenocarcinoma. In Hi-Myc mice, single cell RNA-sequencing revealed miR-21 gene regulation in neoplastic, stromal, and immune cells in a cell-type-specific manner, impacting both direct and indirect targets. Notably, miR-21 deletion reduced immune infiltration into the prostate TME, particularly Trem2-expressing macrophages and regulatory T cells. The Timp1-fibroblast gene signature in MYC-driven PCa was suppressed in miR-21 knockout prostates. Cell-cell communication analysis showed that miR-21 suppressed TGF-beta signaling in the TME, partially through Ski and Smad7 suppression in cancer-associated fibroblasts. These findings underscore the crucial role of miR-21 in PCa and provide some of the first in situ insights into cell-type-specific miRNA activity in solid tumors.

cancer biology↗

bioRxiv2023

Convergent alterations in the tumor microenvironment of MYC-driven human and murine prostate cancer

The tissue microenvironment in prostate cancer is profoundly altered. While such alterations have been implicated in driving prostate cancer initiation and progression to aggressive disease, how prostate cancer cells and their precursors mediate those changes is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we performed extensive single-cell RNA-sequencing (scRNA-seq) and rigorous molecular pathology of the comparative biology between human prostate cancer and key time points in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues, with validation in a large external data set, revealed that cancer cell-intrinsic activation of MYC signaling was the top up-regulated pathway in human cancers, representing a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Likewise, numerous non-malignant cell states in the tumor microenvironment (TME), including non-cancerous epithelial, immune, and fibroblast cell compartments, were conserved across individuals, raising the possibility that these cell types may be a sequelae of the convergent MYC activation in the cancer cells. To test this hypothesis, we employed a GEMM of prostate epithelial cell-specific MYC activation in two mouse strains. Cell communication network and pathway analyses suggested that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogrammed the TME during carcinogenesis, leading to the emergence of cascading cell state alterations in neighboring epithelial, immune, and fibroblast cell types that paralleled key findings in human prostate cancer. Importantly, among these changes, the progression from a precursor-enriched to invasive-cancer-enriched state was accompanied by a cell-intrinsic switch from pro-immunogenic to immunosuppressive transcriptional programs with coinciding enrichment of immunosuppressive myeloid and Treg cells in the immune microenvironment. These findings implicate activation of MYC signaling in reshaping convergent aspects of the TME of prostate cancer as a common denominator across the otherwise well-documented molecular heterogeneity of human prostate cancer.

cancer biology↗

bioRxiv2022

The TERT promoter is polycomb-repressed in neuroblastoma cells with long telomeres

Acquiring a telomere maintenance mechanism is one of the hallmarks of high-risk neuroblastoma and commonly occurs by expressing telomerase (TERT). Telomerase-negative neuroblastoma has, characteristically, long telomeres and most utilize the telomerase-independent alternative lengthening of telomeres (ALT) mechanism. Conversely, no discernable telomere maintenance mechanism is detected in a fraction of neuroblastoma with long telomeres, representing a phenomenon referred to as ever-shorter telomeres. Here, we show that, unlike most cancers, DNA of the TERT promoter is broadly hypomethylated in neuroblastoma. In telomerase-positive neuroblastoma cells, the hypomethylated DNA promoter is approximately 1.5-kb in length and is bound by hypermethylated TERT gene body and upstream intergenic sequences. The TERT locus shows active chromatin marks including H3K4me3, H3K27Ac, H3K14Ac, RNA PolII and BRD4 with low enrichment for the repressive mark, H3K27me3. Strikingly, in neuroblastoma with long telomeres, the hypomethylated region spans the entire TERT locus, including multiple nearby genes with enrichment for the repressive H3K27me3 chromatin mark. Furthermore, subtelomeric regions showed enrichment of repressive chromatin marks in neuroblastomas with long telomeres relative to those with short telomeres. These repressive marks were even more evident at the genic loci, suggesting a telomere position effect. Inhibiting H3K27 methylation by the EZH2 inhibitor, Tazemetostat, induced the expression of TERT, particularly in cell lines with long telomeres and H3K27me3 marks in the promoter region. Taken together, these data suggest that epigenetic regulation of TERT expression differs in neuroblastoma depending on the telomere maintenance status, and H3K27 methylation is important in repressing TERT expression in neuroblastoma with long telomeres.

cancer biology↗

bioRxiv2022

Single-cell atlas of epithelial and stromal cell heterogeneity by lobe and strain in the mouse prostate

Evaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered mouse models in different mouse strains and prostate lobes within each mouse strain, yet, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood. To address this, we examined the single-cell transcriptomes of individual mouse prostate lobes from two commonly used mouse strains, FVB/NJ and C57BL/6J. Data dimensionality reduction and clustering analysis revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2 and Atp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressing Chga, Chgb, and Syp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate. Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.

cell biology↗

bioRxiv2020

Pervasive promoter hypermethylation of silenced TERT alleles in human cancers

In cancers, maintenance of telomeres often occurs through activation of the catalytic subunit of telomerase, encoded by TERT. Yet, most cancers show only modest levels of telomerase gene expression, even in the context of activating hotspot promoter mutations (C228T and C250T). The role of epigenetic mechanisms, including DNA methylation, in regulating telomerase gene expression in cancer cells is not fully understood. Here, we have carried out the most comprehensive characterization to date of TERT promoter methylation using ultra-deep bisulfite sequencing spanning the CpG island surrounding the core TERT promoter in 96 different human cell lines. In general, we observed that immortalized and cancer cell lines were hypermethylated in a region upstream of the recurrent C228T and C250T TERT promoter mutations, while non-malignant primary cells were comparatively hypomethylated in this region. However, at the allele-level, we generally observe hypermethylation of promoter sequences in cancer cells is associated with repressed expression, and the remaining unmethylated alleles marked with open chromatin are largely responsible for the observed TERT expression in cancer cells. Our findings suggest that hypermethylation of the TERT promoter alleles signals transcriptional repression of those alleles, leading to the attenuation of TERT activation in cancer cells. SIGNIFICANCEHypermethylation of the TERT promoter alleles to attenuate TERT activation in cancer cells may account for the modest activation of TERT expression in most cancers.

cancer biology↗