Biology subjects

Urisman, A.

Publications and source records attributed to Urisman, A..

11 recordsLinked to original sources

bioRxiv2025

A Fibroblast State Choreographs an Epithelial YAP-dependent Regenerative Program Essential to (Pre)malignancy via ECM-mediated Mechanotransduction

Chronic lung injury generates metaplasia which occasionally, but ominously, progresses to squamous dysplasia and squamous lung cancer. To identify mechanisms through which disrupted tissue homeostasis contributes to malignant initiation and progression, we used in vivo and in vitro heterotypic recombinant models of human bronchial epithelial cells (hBECs) and fibroblasts. We demonstrate that injury-associated TGF-{beta} signaling creates a fibroblast state dependent upon HSP47 upregulation. These fibroblasts accumulated collagen, thus elevating tissue stiffness and activating mechanosignaling that sustained YAP-dependent embryonic-like, pro-malignant activities in adjacent hBECs. This Stress/Tension-Instructive Fibroblast (STIF) state, exhibited by stressed fibroblasts in premalignant and malignant lesions across multiple cancer types, was sufficient to reprogram disease-free hBECs to metaplasia and to drive hBECs with compromised tumor suppressor function to dysplasia, yet could be inhibited and reversed. STIFs suffice to activate epithelial phenotypes reminiscent of oncogene-mediated cell transformation and induce (pre)malignancy via increased force transmission, providing novel targets for prevention. Statement of significanceTissue injury creates a regenerative pro-tumorigenic Stress/Tension-Instructive Fibroblast (STIF) state which is sufficient to activate a YAP-dependent, pre-malignant program to induce or unmask pre-cancerous phenotypes in epithelial cells through mechanotransduction. Inhibition of STIF activity or mechanosignaling prevents metaplasia and progression to dysplasia. HighlightsO_LITissue injury creates a pro-tumorigenic Stress/Tension-Instructive Fibroblast (STIF) state in multiple organs that precedes and persists through cancer C_LIO_LISTIF signaling alone, working through fibroblasts and not epithelial cells, is sufficient to activate embryonic-like plasticity and induce epithelial pre-cancerous metaplastic lesions C_LIO_LISTIFs program (pre)malignant phenotypes in adjacent epithelial cells through mechanosignaling by activating YAP prior to tumor formation C_LIO_LIInhibiting STIFs or mechanosignaling prevents/reverts metaplasia and prevents progression to dysplasia C_LI

cancer biology↗

bioRxiv2023

The deubiquitinase USP9X regulates RIT1 protein abundance and oncogenic phenotypes

RIT1 is a rare and understudied oncogene in lung cancer. Despite structural similarity to other RAS GTPase proteins such as KRAS, oncogenic RIT1 activity does not appear to be tightly regulated by nucleotide exchange or hydrolysis. Instead, there is a growing understanding that the protein abundance of RIT1 is important for its regulation and function. We previously identified the deubiquitinase USP9X as a RIT1 dependency in RIT1-mutant cells. Here, we demonstrate that both wild-type and mutant forms of RIT1 are substrates of USP9X. Depletion of USP9X leads to decreased RIT1 protein stability and abundance and resensitizes cells to EGFR tyrosine kinase inhibitors. Our work expands upon the current understanding of RIT1 protein regulation and presents USP9X as a key regulator of RIT1-driven oncogenic phenotypes.

cancer biology↗

bioRxiv2022

Data-Independent Acquisition and Quantification of Extracellular Matrix from Human Lung in Chronic Inflammation-Associated Carcinomas

Early events associated with chronic inflammation and cancer involve significant remodeling of the extracellular matrix (ECM), which greatly affects its composition and functional properties. Using lung squamous cell carcinoma (LSCC), a chronic inflammation-associated cancer (CIAC), we optimized a robust proteomic pipeline to discover potential biomarker signatures and protein changes specifically in the stroma. We combined ECM enrichment from fresh human tissues, data-independent acquisition strategies, and stringent statistical processing to analyze Tumor and matched adjacent histologically normal ( Matched Normal) tissues from patients with LSCC. Overall, 1,802 protein groups were quantified with at least two unique peptides, and 56% of those proteins were annotated as extracellular. Confirming dramatic ECM remodeling during CIAC progression, 529 proteins were significantly altered in the Tumor compared to Matched Normal tissues. The signature was typified by a coordinated loss of basement membrane proteins and small leucine-rich proteins. The dramatic increase in the stromal levels of SERPINH1/heat shock protein 47, that was discovered using our ECM proteomic pipeline, was validated by immunohistochemistry (IHC) of Tumor and Matched Normal tissues, obtained from an independent cohort of LSCC patients. This integrated workflow provided novel insights into ECM remodeling during CIAC progression, and identified potential biomarker signatures and future therapeutic targets. Statement of significance of the studyThe extracellular matrix (ECM) is a complex scaffolding network composed of glycoproteins, proteoglycans and collagens, which binds soluble factors and, most importantly, significantly impacts cell fate and function. Alterations of ECM homeostasis create a microenvironment promoting tumor formation and progression, therefore deciphering molecular details of aberrant ECM remodeling is essential. Here, we present a multi-laboratory and refined proteomic workflow, featuring i) the prospective collection of tumor and matched histologically normal tissues from patients with lung squamous cell carcinoma, ii) the enrichment for ECM proteins, and iii) subsequent label-free data-independent acquisition (DIA)-based quantification. DIA is a powerful strategy to comprehensively profile and quantify all detectable precursor ions contained in the biological samples, with high quantification accuracy and reproducibility. When combined with very stringent statistical cutoffs, this unbiased strategy succeeded in capturing robust and highly confident proteins changes associated with cancer, despite biological variability between individuals. This label-free quantification workflow provided the flexibility required for ongoing prospective studies. Discussions with clinicians, surgeons, pathologists, and cancer biologists represent an opportunity to interrogate the DIA digitalized maps of the samples for newly formulated questions and hypotheses, thus gaining insights into the continuum of the disease and opening the path to novel ECM-targeted therapies.

cancer biology↗

bioRxiv2022

PKC-alpha regulates the phosphorylation of KRAS that suppresses its oncogenic properties

Oncogenic KRAS-driven cancers have long been considered as "undruggable" due to limited therapeutic options. While the recent success of KRAS-G12C inhibitors argues against the "undruggability" of KRAS, this treatment only benefits a small proportion of patients with KRAS mutant cancers, leaving an urgent need for modalities to target other KRAS mutants. KRAS-calmodulin (CaM) signaling axis reportedly regulates the oncogenic properties of KRAS through its C-terminal hypervariable region. Phosphorylation of KRAS by activated protein kinase C (PKC) uncouples KRAS-CaM, resulting in growth inhibition effective against the entire spectrum of KRAS hotspot mutations. However, broadly activating PKC could mediate tumor promoting signaling nodes and cause systemic toxicity, undermining its applicability as an anti-KRAS therapy. Here, we found that prostratin induces KRAS phosphorylation, resulting in an elevated level of active CaM in the cytosol of KRAS mutant cells, and consequentially suppresses their malignancies. A whole-genome wide CRISPR/Cas9 knockout screening, further confirmed by biochemical analysis, revealed that prostratin acts through activating PKC. Functional studies confirmed PKC as the sole kinase to phosphorylate KRAS and, therefore, a KRAS suppressor. Activation of PKC induces senescence in KRAS mutant tumor cells through PTPN14, accompanied by a secretory phenotype contributing to the growth inhibition, and parallelly mediates a nuclear translocation of a CaM-dependent transcription activator, CAMTA-1, which can be a biomarker to indicate the activity of PKC-KRAS-CaM axis. Our findings reveal a previously understudied regulation of KRAS-CaM axis by PKC, which can be an actionable target for developing anti-KRAS therapeutics. One Sentence SummaryThis study deciphers a PKC-led tumor suppressive effect specific to the "undruggable" KRAS-mutant tumor cells through the phosphorylation of KRAS and a consequently altered KRAS-CaM signaling axis.

cancer biology↗

bioRxiv2022

Deep tissue infection by an invasive human fungal pathogen requires novel lipid-based suppression of the IL-17 response

Candida albicans is the most common cause of human fungal infection, but the mechanisms of invasive pathogenesis remain poorly defined. Here we identify an unexpected mechanism: lipid-mediated immunosuppression. Through forward genetics, we found that C. albicans secretes a lipase, Lip2, that is critical for invasive disease. Murine infection with C. albicans strains that lack Lip2 display an exaggerated host IL-17 response that leads to fungal clearance from solid organs and host survival. IL-17 signaling is required for Lip2 action. The lipase activity of Lip2 inhibits IL-17 production indirectly through suppression of IL-23 production by tissue resident dendritic cells. We conclude that C. albicans suppresses antifungal IL-17 defense in solid organs by altering the tissue lipid milieu.

microbiology↗

bioRxiv2022

Cross-species analysis of LZTR1 loss-of-function mutants demonstrates dependency to RIT1 orthologs

RAS GTPases are highly conserved proteins involved in the regulation of mitogenic signaling. We have previously described a novel Cullin 3 RING E3 ubiquitin ligase complex formed by the substrate adaptor protein LZTR1 that binds, ubiquitinates, and promotes proteasomal degradation of the RAS GTPase RIT1. In addition, others have described that this complex is also responsible for the ubiquitination of canonical RAS GTPases. Here, we have analyzed the phenotypes of LZTR1 loss-of-function mutants in both fruit flies and mice and have demonstrated biochemical dependency on their RIT1 orthologs. Moreover, we show that LZTR1 is haplosufficient in mice and that embryonic lethality of the homozygous null allele can be rescued by deletion of RIT1.

genetics↗

bioRxiv2020

The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression

The spindle assembly checkpoint (SAC) is an evolutionarily conserved safety mechanism that maintains genomic stability. However, despite the understanding of the fundamental mechanisms that control the SAC, it remains unknown how signaling pathways directly interact with and regulate the mitotic checkpoint activity. In response to extracellular stimuli, a diverse network of signaling pathways involved in cell growth, survival, and differentiation are activated and this process is prominently regulated by the Ras family of GTPases. Here we show that RIT1, a Ras-related GTPase, is essential for timely progression through mitosis and proper chromosome segregation. Furthermore, pathogenic levels of RIT1 silence the SAC, accelerate transit through mitosis, and promote chromosome segregation errors through direct association with SAC proteins MAD2 and p31comet. Our results highlight a unique function of RIT1 compared to other Ras GTPases and elucidate a direct link between a signaling pathway and the SAC through a novel regulatory mechanism. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/386177v1_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@fd6655org.highwire.dtl.DTLVardef@4974b4org.highwire.dtl.DTLVardef@8cffa0org.highwire.dtl.DTLVardef@e28cbd_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2020

Deficiency of the splicing factor RBM10 limits EGFR inhibitor response in EGFR mutant lung cancer

Molecularly targeted cancer therapy has improved outcomes for cancer patients with targetable oncoproteins, such as mutant epidermal growth factor receptor (EGFR) in lung cancer. Yet, long-term patient survival remains limited because treatment responses are typically incomplete. One potential explanation for the lack of complete and durable responses is that oncogene-driven cancers with activating mutations in the EGFR often harbor additional co-occurring genetic alterations. This hypothesis remains untested for most genetic alterations that co-occur with mutant EGFR. Here, we report the functional impact of inactivating genetic alteration of the mRNA splicing factor RBM10 that co-occur with mutant EGFR. RBM10 deficiency decreased EGFR inhibitor efficacy in patient-derived EGFR mutant tumor models. RBM10 modulated mRNA alternative splicing of the mitochondrial apoptotic regulator Bcl-x to regulate tumor cell apoptosis during treatment. Genetic inactivation of RBM10 diminished EGFR inhibitor-mediated apoptosis by decreasing the ratio of Bcl-xS-(pro-apoptotic)-to-Bcl-xL(anti-apoptotic) Bcl-x isoforms. RBM10 deficiency was a biomarker of poor response to EGFR inhibitor treatment in clinical samples. Co-inhibition of Bcl-xL and mutant EGFR overcame resistance induced by RBM10 deficiency. This study sheds light on the role of co-occurring genetic alterations, and on the impact of splicing factor deficiency in the modulation of sensitivity to targeted kinase inhibitor cancer therapy.

cancer biology↗

bioRxiv2020

Intra-axonal translation of Khsrp mRNA slows axon regeneration by destabilizing localized mRNAs

Proteins generated by localized mRNA translation in axons support nerve regeneration through retrograde injury signaling and localized axon growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional control of localized mRNAs, but only a limited number of axonal RBPs have been reported. We used a targeted mass spectrometry approach to profile the axonal RBPs in naive, injured and regenerating PNS axons. We detected 76 axonal proteins that are reported to have RNA binding activity, with the levels of several of these axonal RBPs changing with axonal injury and regeneration. These axonal RBPs with altered axoplasm levels include KHSRP that we previously reported decreases neurite outgrowth in developing CNS neurons. We show that KHSRP levels rapidly increase in sciatic nerve axons after crush injury and remain elevated increasing in levels out to 28 days post-sciatic nerve crush injury. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP after axotomy is mediated by the local translation of its mRNA. KHSRP binds to mRNAs with a 3UTR AU-rich element and targets those mRNAs to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of defined KHSRP target mRNAs, Gap43 and Snap25 mRNAs, following sciatic nerve injury and accelerated nerve regeneration in vivo. These data indicate that axonal translation of Khsrp mRNA following nerve injury serves to destabilize other axonal mRNAs and slow axon regeneration.

neuroscience↗

bioRxiv2019

Heterogeneity and targeted therapy-induced adaptations in lung cancer revealed by longitudinal single-cell RNA sequencing

Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNAseq) of metastatic lung cancer was performed using 44 tumor biopsies obtained longitudinally from 27 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNAseq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNAseq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes.

cancer biology↗

bioRxiv2019

Targeted phosphoproteomics of the Ras signaling network reveal regulatory mechanisms mediated by oncogenic KRAS

BackgroundKRAS mutations are present in up to 30% of lung adenocarcinoma cases and are associated with poor survival. No effective targeted therapy against KRAS is currently available, and novel strategies to counteract oncogenic KRAS signaling are needed.\n\nResultsWe used targeted proteomics to monitor abundance and site-specific phosphorylation in a network of over 150 upstream and downstream effectors of KRAS signaling in H358 cells (KRAS G12C). We compared patterns of protein regulation following sustained signaling blockade in the RAS/ERK module at two different levels, KRAS and MEK. Network-based analysis demonstrated complex non-linear patterns of regulation with wide-spread crosstalk among diverse subnetworks. Among 85 most regulated proteins in the network, only 12 proteins showed concordant regulation in response to signaling blockade at both KRAS and MEK levels, while the remainder were either specifically regulated in response to KRAS knockdown or MEK inhibition or showed orthogonal regulation in both conditions. Dephosphorylation of DNA methyltransferase 1 (DNMT1) at S714 was identified among the changes unique to KRAS knockdown, and here we elucidate the role of this phosphorylation in KRAS-dependent transcriptional silencing of tumor suppressor genes.\n\nConclusionsNetwork-based analysis of the Ras signaling has shown complex non-linear patterns of regulation with wide-spread crosstalk among diverse subnetworks. Our work illustrates a targeted proteomics approach to functional interrogation of complex signaling networks focused on identification of readily testable hypotheses. These methods are widely applicable to diverse questions in tumor biology and other signaling paradigms.

cancer biology↗