Biology subjects

Guda, C.

Publications and source records attributed to Guda, C..

9 recordsLinked to original sources

bioRxiv2026

Integrative Proteomic Analysis Implicates Inhibition of Intracellular Protein Trafficking in Therapy-Induced Migrastasis in Prostate Cancer

BackgroundDysregulated cell migration leading to metastasis remains the primary cause of cancer-related mortality. It has been challenging to understand how cells regulate migration. We have previously created the first selective inhibitor of cell migration, KBU2046. Here, we use it as a probe to identify regulatory processes. MethodsMetastatic and primary human prostate cancer cells were treated for different times and at different concentrations with KBU2046. Immunofluorescent microscopy examined protein localization in cells. Label-free mass spectrometry (MS) was performed on total cell proteins, Tandem Mass Tag (TMT) labeling MS was used on membrane fractions, and temporal phosphoproteomic profiling. Results were analyzed with a suite of bioinformatic tools. ResultsKBU2046-induced migrastasis is associated with the accumulation of activated integrin {beta}1 into focal adhesions. Whole-cell proteomics demonstrated suppression of processes that mediate intracellular protein trafficking and increases in mitochondrial energy-generation signatures. Evaluation of the membrane fraction identified increases in membrane repair and maintenance processes and decreases in those that drive motility. Temporal- and concentration-dependent phosphoproteomic profiling revealed that KBU2046 initiates a dynamic, cascading sequence of transient signaling waves rather than a static block. ConclusionsKBU2046-induced migrastasis appears to operate through spatial decoupling rather than structural degradation. By restricting the intracellular trafficking machinery required for receptor recycling, KBU2046 limits focal adhesion turnover, providing a correlative framework to inhibit metastatic dissemination independent of direct cytotoxicity. O_FIG O_LINKSMALLFIG WIDTH=122 HEIGHT=200 SRC="FIGDIR/small/736165v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1efd74dorg.highwire.dtl.DTLVardef@1202106org.highwire.dtl.DTLVardef@581ed3org.highwire.dtl.DTLVardef@14fdcfb_HPS_FORMAT_FIGEXP M_FIG Graphic Abstract C_FIG

cancer biology↗

bioRxiv2026

PRMT5 as an Epigenetic Target for Group 3 (MYC-driven) Medulloblastoma

BackgroundGroup 3 (MYC-driven) medulloblastoma (MB) is a highly aggressive brain tumor with poor-prognosis and limited treatment options. We previously identified protein-arginine methyltransferase-5 (PRMT5) as a promising target in Group 3 MB with its control on MYC protein stability. In this follow up study, we further mechanistically investigated PRMT5 control on MYC transcription and targeted it pharmacologically for therapeutic proof-of-concept. MethodsUsing pharmacogenetic inhibition approaches against PRMT5 in MYC-amplified (Group 3) MB cell line and neurosphere models in vitro and in vivo, we investigated molecular mechanism(s) and anti-cancer efficacy of PRMT5 inhibition. ResultsOur experiments demonstrated that PRMT5 epigenetically regulates MYC transcription in MYC-amplified MB cells by binding to the proximal-promoter region of the MYC gene and contributing to the enriched symmetric-dimethylation of histone H4R3 in the same region. We further showed that PRMT5 is recruited to the MYC promoter by its interaction with BRD4, the major BET-protein responsible for MYC transcription. PRMT5 inhibition caused the suppression of MYC-induced transcriptional programs and target genes, with widespread disruption of splicing across the transcriptome, particularly affecting metabolism-related gene products. Pharmacologic inhibition of PRMT5 using a panel of selective small-molecule inhibitors demonstrates suppression of cell growth/survival in a MYC-dependent manner in MB cells. Moreover, our in vivo analyses of PRMT5 inhibition, in mice treated with one of the potent pharmacologic inhibitors, particularly a lipid-decorated form of it, demonstrated reduced cerebellar tumor growth with suppressed MYC expression and prolonged survival of mice with MYC-amplified MB xenografts. ConclusionsOur findings establish a functional link between PRMT5 and MYC-mediated transcriptional regulation, suggesting a promising therapeutic approach targeting the PRMT5-MYC axis for MYC-driven MB. Key PointsO_LIPRMT5 acts as an epigenetic regulator of MYC transcription, RNA splicing and associated energy metabolism in MYC-driven MB. C_LIO_LIPRMT5 inhibition selectively suppresses cell growth/survival in MYC-driven MB. C_LIO_LIPRMT5 inhibition reduces tumor burden and prolongs survival in a MYC-driven MB mouse model. C_LI Importance of the StudyGroup 3 medulloblastoma is a highly aggressive pediatric brain tumor marked by MYC amplification, malignant clinical behavior, and poor survival outcomes despite intensive multimodal therapy. Because MYC remains largely undruggable, there is an urgent need for effective and less toxic treatment options for affected children. This study identifies protein arginine methyltransferase 5 (PRMT5) as a key epigenetic regulator of MYC transcription and MYC-dependent oncogenic programs in Group 3 MB. We show that PRMT5 is recruited to the MYC promoter via BRD4, sustains MYC-driven transcription and RNA splicing networks associated with metabolism, and supports MB tumor growth. Importantly, pharmacologic inhibition of PRMT5 using a selective brain-penetrant inhibitor suppresses MYC expression, reduces cerebellar tumor burden, and prolongs survival in MYC-amplified MB models. These findings provide a strong translational rationale for PRMT5 inhibition as a targeted therapeutic strategy for high-risk MB, with the potential to improve outcomes while reducing treatment-related toxicity.

cancer biology↗

bioRxiv2022

Morphine Suppresses Peripheral Responses and Transforms Brain Myeloid Gene Expression to Favor Neuropathogenesis in SIV Infection

The twin pandemics of opioid abuse and HIV infection can have devastating effects on physiological systems, including on the brain. Our previous work found that morphine increased the viral reservoir in the brains of treated SIV-infected macaques. In this study, we investigated the interaction of morphine and SIV to identify novel host-specific targets using a multimodal approach. We probed systemic parameters and performed single-cell examination of the targets for infection in the brain, microglia and macrophages. Morphine treatment created an immunosuppressive environment, blunting initial responses to infection, which persisted during antiretroviral treatment. Antiretroviral drug concentrations and penetration into the cerebrospinal fluid and brain were unchanged by morphine treatment. Interestingly, the transcriptional signature of both microglia and brain macrophages was transformed to one of a neurodegenerative phenotype. Notably, the expression of osteopontin, a pleiotropic cytokine, was significantly elevated in microglia. This was especially notable in the white matter, which is also dually affected by HIV and opioids. Increased osteopontin expression was linked to numerous HIV neuropathogenic mechanisms, including those that can maintain a viral reservoir. The opioid morphine is detrimental to SIV/HIV infection, especially in the brain.

neuroscience↗

bioRxiv2022

Post-transcriptional control of SRSF9 promotes the epithelial-to-mesenchymal transition (EMT) in colorectal cancer cells

In human colorectal cancer (CRC) cells the Raf/MEK/ERK scaffold Kinase Suppressor of Ras 1 (KSR1)-dependent signaling is required for the epithelial-to-mesenchymal transition (EMT)-like phenotype. Here we show that KSR1 promotes the association of differentially spliced mRNA bearing recognition sites for the Serine/Arginine-Rich (SR) splicing factor SRSF9. CRISPR/Cas9 disruption of KSR1 destabilizes SRSF9 protein, which interacts preferentially with mRNA encoding Epithelial Stromal Interaction 1 (EPSTI1). EPSTI1 protein mediates Ras and KSR1-dependent induction of EMT. Analysis of EPSTI1 splice variants reveals that inclusion of exon 8 is critical to the ability of EPSTI1 to promote the E-to N-cadherin switch and CRC cell motile and invasive behavior. These data reveal a mechanism in CRC cells in which Ras-induced and KSR1-dependent signaling affects pre-mRNA splicing to control behaviors critical to cancer cell dissemination and metastasis.

cell biology↗

bioRxiv2021

KSR1-and ERK-dependent Translational Regulation of the Epithelial-to-Mesenchymal Transition

The epithelial-to-mesenchymal transition (EMT) is considered a transcriptional process that induces a switch in cells from a polarized state to a migratory phenotype. Here we show that KSR1 and ERK promote EMT through the preferential translation of Epithelial-Stromal Interaction 1 (EPSTI1), which is required to induce the switch from E-to N-cadherin and coordinate migratory and invasive behavior. EPSTI1 is overexpressed in human colorectal cancer (CRC) cells. Disruption of KSR1 or EPSTI1 significantly impairs cell migration and invasion in vitro, and reverses EMT, in part, by decreasing the expression of N-cadherin and the transcriptional repressors of E-cadherin expression, ZEB1 and Slug. In CRC cells lacking KSR1, ectopic EPSTI1 expression restored the E-to N-cadherin switch, migration, invasion, and anchorage-independent growth. KSR1-dependent induction of EMT via selective translation of mRNAs reveals its underappreciated role in remodeling the translational landscape of CRC cells to promote their migratory and invasive behavior.

cancer biology↗

bioRxiv2020

High-Throughput Analysis of Lung Immune Cells in a Murine Model of Rheumatoid Arthritis-Associated Lung Disease

Rheumatoid arthritis (RA)-associated lung disease is a leading cause of mortality in RA, yet the mechanisms linking lung disease and RA remain unknown. Using an established murine model of RA-associated lung disease combining collagen-induced arthritis (CIA) with organic dust extract (ODE)-induced airway inflammation, differences among lung immune cell populations were analyzed by single cell RNA-sequencing. Additionally, four lung myeloid-derived immune cell populations including macrophages, monocytes/macrophages, monocytes, and neutrophils were isolated by fluorescence cell sorting and gene expression was determined by NanoString analysis. Unsupervised clustering revealed 14 discrete clusters among Sham, CIA, ODE, and CIA+ODE treatment groups: 3 neutrophils (inflammatory, resident/transitional, autoreactive/suppressor), 5 macrophages (airspace, differentiating/recruited, recruited, resident/interstitial, and proliferative airspace), 2 T-cells (differentiating and effector), and a single cluster each of inflammatory monocytes, dendritic cells, B-cells and natural killer cells. Inflammatory monocytes, autoreactive/suppressor neutrophils, and recruited/differentiating macrophages were predominant with arthritis induction (CIA and CIA+ODE). By specific lung cell isolation, several interferon-related and autoimmune genes were disproportionately expressed among CIA and CIA+ODE (e.g. Oasl1, Oas2, Ifit3, Gbp2, Ifi44, and Zbp1), corresponding to RA and RA-associated lung disease. Monocytic myeloid-derived suppressor cells were reduced, while complement genes (e.g. C1s1 and Cfb) were uniquely increased in CIA+ODE mice across cell populations. Recruited and inflammatory macrophages/monocytes and neutrophils expressing interferon-, autoimmune-, and complement-related genes might contribute towards pro-fibrotic inflammatory lung responses following airborne biohazard exposures in setting of autoimmune arthritis and could be predictive and/or targeted to reduce disease burden.

immunology↗

bioRxiv2020

Bromelain Inhibits SARS-CoV-2 Infection in VeroE6 Cells

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The initial interaction between Transmembrane Serine Protease 2 (TMPRSS2) primed SARS-CoV-2 spike (S) protein and host cell receptor angiotensin-converting enzyme 2 (ACE-2) is a pre-requisite step for this novel coronavirus pathogenesis. Here, we expressed a GFP-tagged SARS-CoV-2 S-Ectodomain in Tni insect cells. That contained sialic acid-enriched N- and O-glycans. Surface resonance plasmon (SPR) and Luminex assay showed that the purified S-Ectodomain binding to human ACE-2 and immunoreactivity with COVID-19 positive samples. We demonstrate that bromelain (isolated from pineapple stem and used as a dietary supplement) treatment diminishes the expression of ACE-2 and TMPRSS2 in VeroE6 cells and dramatically lowers the expression of S-Ectodomain. Importantly, bromelain treatment reduced the interaction between S-Ectodomain and VeroE6 cells. Most importantly, bromelain treatment significantly diminished the SARS-CoV-2 infection in VeroE6 cells. Altogether, our results suggest that bromelain or bromelain rich pineapple stem may be used as an antiviral against COVID-19. Highlights O_FIG O_LINKSMALLFIG WIDTH=178 HEIGHT=200 SRC="FIGDIR/small/297366v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@b7afb0org.highwire.dtl.DTLVardef@16f8185org.highwire.dtl.DTLVardef@1a07df8org.highwire.dtl.DTLVardef@1ae33fa_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIBromelain inhibits / cleaves the expression of ACE-2 and TMPRSS2 C_LIO_LIBromelain cleaves / degrades SARS-CoV-2 spike protein C_LIO_LIBromelain inhibits S-Ectodomain binding and SARS-CoV-2 infection C_LI

microbiology↗

bioRxiv2020

Red Panda: A novel method for detecting variants in single-cell RNA sequencing

Single-cell sequencing enables us to better understand genetic diseases, such as cancer or autoimmune disorders, which are often affected by changes in rare cells. Currently, no existing software is aimed at identifying single nucleotide variations or micro (1-50bp) insertions and deletions in single-cell RNA sequencing (scRNA-seq) data. Generating high-quality variant data is vital to the study of the aforementioned diseases, among others. In this study, we report the design and implementation of Red Panda, a novel method to accurately identify variants in scRNA-seq data. Variants were called on scRNA-seq data from human articular chondrocytes, mouse embryonic fibroblasts (MEFs), and simulated data stemming from the MEF alignments. Red Panda had the highest Positive Predictive Value at 45.0%, while other tools--FreeBayes, GATK HaplotypeCaller, GATK UnifiedGenotyper, Monovar, and Platypus--ranged from 5.8%-41.53%. From the simulated data, Red Panda had the highest sensitivity at 72.44%. We show that our method provides a novel and improved mechanism to identify variants in scRNA-seq as compared to currently-existing software. AvailabilitySource code freely available under the MIT License at https://github.com/adambioi/red_panda, and is supported on Linux

bioinformatics↗

bioRxiv2018

Placental epigenetics for evaluation of fetal congenital heart defects: Ventricular septal defect (VSD)

Ventricular Septal Defect (VSD), the most common congenital heart defect, is characterized by a hole in the septum between the right and left ventricles. The pathogenesis of VSD is unknown in most clinical cases. There is a paucity of data relevant to epigenetic changes in VSD. The placenta is a fetal tissue and is a potentially useful surrogate for the evaluation of fetal organ development. To understand epigenetic mechanisms that may play a role in the development of VSD, a genome-wide DNA methylation assay of the placentas of 8 term subjects with isolated VSD and no known or suspected genetic syndromes and 10 normal controls was performed using the Illumina HumanMethylation450 BeadChip assay. The study identified a total of 80 highly accurate potential epigenomic markers in 80 genes for the detection of VSD; area under the receiver operating characteristic curve (AUC ROC) = 1.0 with significant 95% CI (FDR) p-values < 0.05. The biological processes and functions for these differentially methylated genes are known to be associated with heart development or heart disease, including cardiac ventricle development (HEY2, ISL1), heart looping (SRF), cardiac muscle cell differentiation (ACTC1, HEY2), cardiac septum development (ISL1), heart morphogenesis (SRF, HEY2, ISL1, HEYL), Notch signaling pathway (HEY2, HEYL), cardiac chamber development (ISL1), and cardiac muscle tissue development (ACTC1, ISL1). The study also identified eight microRNA genes that have the potential to be biomarkers for the early detection of VSD including miR-191, miR-548F1, miR-148A, miR-423, miR-92B, miR-611, miR-2110, and miR-548H4. To our knowledge this is the first report in which placental analysis has been used for determining the pathogenesis of and predicting CHD.

genomics↗