Biology subjects

Lajoie, G. A.

Publications and source records attributed to Lajoie, G. A..

11 recordsLinked to original sources

bioRxiv2025

Cell State Chaos Underpins the Evolution of SMARCA4-Deficient Dedifferentiated Endometrial Cancer

Dedifferentiated endometrial carcinoma (DDEC) is a histologically unique cancer type, wherein well-differentiated regions lie adjacent to morphologically distinct, high-grade lesions that are histologically undifferentiated. Previous studies have determined that in nearly half of the cases dedifferentiation is associated with the genomic inactivation of SMARCA4, a catalytic subunit belonging to the SWI/SNF chromatin remodelling complex (SWI/SNF CRC), suggesting that SMARCA4 loss causes dedifferentiation. Herein, using gene editing, we reveal that when serially passaged in mice, SMARCA4-deficient endometrial cancer cells repeatably and predictably generate heterogeneous admixtures of differentiated and undifferentiated cells, resembling human DDEC. Surprisingly, despite this metamorphosis, SMARCA4 loss does not induce lineage plasticity nor reprogramming to a less differentiated fate. Rather, single-cell sequencing combined with barcoding demonstrated that SMARCA4 loss induces a dysregulated epigenome that allows cells to randomly move through cellular states that are otherwise shared with SMARCA4-expressing well-differentiated cancer cells. This finding was validated using a cohort of patient samples, such that epithelial fate markers (E-CADHERIN) can be detected in morphologically undifferentiated cells. Collectively, this work constitutes the first repeatable model of human dedifferentiated cancer and suggests that histological dedifferentiation is not due to the acquisition of a stem cell-like fate. Rather, undifferentiated tissue emerges due to epigenomic dysfunction concomitant with the arbitrary movement of cancer cells between cellular states.

cancer biology↗

bioRxiv2025

Isolation of Extracellular Vesicles from Minimal Volume Ascites Fluid Using Strong Anion Exchange Magnetic Beads

Ovarian cancer (OC) remains a leading cause of gynecologic cancer mortality due to late-stage diagnosis and limited early detection strategies. Ascites fluid, a pathological hallmark of OC, is a rich source of tumor-derived extracellular vesicles (EVs) that reflect the tumor microenvironment and hold promise for biomarker discovery. However, isolating EVs from minimal ascites volumes (<100 {micro}L) poses technical challenges using conventional methods like ultracentrifugation or size-exclusion chromatography (SEC). This study explores the application of strong anion exchange (SAX) magnetic beads (Mag-Net) for efficient EV isolation from as little as 2 {micro}L of ascites fluid from both murine models and a human patient with mucinous borderline tumor. We demonstrate that SAX achieves robust EV capture at 10{micro}l of input volume, enabling comprehensive proteomic profiling and single-EV surface-enhanced Raman spectroscopy (SERS) with a >2-fold increase in proteomic depth compared to raw ascites. Notably, this study was able to identify 1000 proteins not previously annotated in Vesiclepedia for OC-derived EVs, alongside distinct SERS signatures, highlighting the potential for multiomic analysis. Comparative analysis with UC revealed enhanced proteomic depth obtained with SAX beads, albeit we also observed differential detection of canonical markers (e.g., CD9, CD81) between input volumes of ascites fluid. These findings establish SAX as a scalable, low-input platform for EV-based biomarker discovery, paving the way for improved early detection and molecular insights into OC progression.

biochemistry↗

bioRxiv2025

Proteomic Signatures of Podocyte Injury Are Reflected in Urinary Extracellular Vesicles in Pediatric Nephrotic Syndrome.

Idiopathic nephrotic syndrome (NS) is a common glomerulopathy in children and presents with significant proteinuria. There are no reliable clinical or biochemical markers of disease relapse, or prognosis. Extracellular vesicles (EVs) are small, membrane-bound biological effectors released from stressed cells. We previously showed increases in podocyte-specific urinary EVs from children with disease relapse in NS, with numbers returning to near-zero in remission. Herein, we have expanded this work to evaluate puromycin aminonucleoside (PAN) injury by characterizing proteomic signatures of podocytes and their EVs in vitro. In addition, we performed data-independent proteomic analysis (DIA) to characterize changes in signatures of EVs from pediatric patients with active NS versus remission. Our key findings reveal PAN-injured podocytes increase large EV (LEV) secretion in vitro; moreover, DIA uncovered changes in cellular and LEV proteomes that were also observed in urinary LEVs from patients with active disease. Urinary LEV proteomes from children with active NS were significantly different than those in remission, highlighted by 645 and 240 unique proteins associated with disease or remission, respectively. This foundational work provides the impetus for a larger, prospective biomarker study aimed at identifying EV-specific proteins associated with relapse versus remission.

cell biology↗

bioRxiv2024

Mining the Proteome of Human Ovarian Cancer Extracellular Vesicles Using Thermolysin Proteolysis

This study investigates the utility of Thermolysin as a proteolytic enzyme to enhance the depth and coverage of proteomic analysis in ovarian cancer (OC) extracellular vesicles (EVs). EVs were isolated from OC cell lines and ascites fluid samples from women diagnosed with high-grade serous carcinoma. Proteins were digested using Thermolysin and Trypsin/LysC, followed by label-free data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry. The proteolytic efficiency, sequence coverage, peptide complexity, and proteomic depth were compared between Thermolysin and Trypsin/LysC digests. In silico analyses predicted theoretical benchmarks of these parameters using a core set of 22 proteins, and Gene set enrichment analyses (GSEA) highlighted the biological relevance of proteins identified throughout the study. Thermolysin digestion significantly increased the complexity or of peptide pools compared to Trypsin/LysC leading to limited peptide and protein identification, albeit total sequence coverage was increased through complementation to tryptic peptides. In both cell line and ascites EVs, Thermolysin identified unique proteins not detected by Trypsin/LysC that are known drivers of metastatic solid cancers, such as Ly6E. Offline strong cation exchange (SCX) fractionation improved proteomic depth and sequence coverage obtained with Thermolysin to generate a spectral library for DIA. Our DIA analysis of Thermolysin digests revealed the presence of NODAL in OC ascites EVs, a protein associated with poor clinical prognosis, which was not detected in Trypsin/LysC digests. The importance of NODAL was cross-validated in TGCA-OV and AOCS datasets by clinical cohorts by assessing RNA levels in solid tumors or ascites fluid, respectively. Collectively, we demonstrate that Thermolysin complements traditional enzymes like Trypsin/LysC to provide a more comprehensive proteomic landscape for biomarker discovery.

cancer biology↗

bioRxiv2024

Interplay between β-propeller subunits WDR26 and muskelin regulates the CTLH E3 ligase supramolecular complex

The Pro/N-degron recognizing C-terminal to LisH (CTLH) complex is an E3 ligase of emerging interest in the developmental field and for targeted protein degradation (TPD) modalities. The human CTLH complex forms distinct supramolecular ring-shaped structures dependent on the multimerization of WDR26 or muskelin {beta}-propeller proteins. Here, we find that, in human cells, CTLH complex E3 ligase activity is dictated by a dynamic exchange between WDR26 and muskelin in tandem with muskelin autoregulation. Proteomic experiments revealed that complex-associated muskelin protein turnover is a major ubiquitin-mediated degradation event dependent on the CTLH complex in unstimulated HeLa cells. We observed that muskelin and WDR26 binding to the scaffold of the complex is interchangeable, indicative of the formation of separate WDR26 and muskelin complexes, which correlated with distinct proteomes in WDR26 and muskelin knockout cells. We found that mTOR inhibition-induced degradation of Pro/N-degron containing protein HMGCS1 is distinctly regulated by a muskelin-specific CTLH complex. Finally, we found that mTOR inhibition also activated muskelin degradation, likely as an autoregulatory feedback mechanism to regulate CTLH complex activity. Thus, rather than swapping substrate receptors, the CTLH E3 ligase complex controls substrate selectivity and its autoregulation through exchanging its {beta}-propeller oligomeric subunits WDR26 and muskelin.

molecular biology↗

bioRxiv2023

A chemical probe to modulate human GID4 Pro/N-degron interactions

The CTLH complex is a multi-subunit ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via the substrate receptor GID4. Recently, focus has turned to this complex as a potential mediator of targeted protein degradation, but the role GID4-mediated substrate ubiquitylation and proteasomal degradation plays in humans has thus far remained unclear. Here, we report PFI-7, a potent, selective, and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation enabled the identification of dozens of endogenous GID4-interacting proteins that bind via the GID4 substrate binding pocket, only a subset of which possess canonical Pro/N-degron sequences. GID4 interactors are enriched for nuclear and nucleolar proteins including RNA helicases. GID4 antagonism by PFI-7 altered protein levels of several proteins including RNA helicases as measured by label-free quantitative proteomics, defining proteins that are regulated by GID4 and the CTLH complex in humans. Interactions with GID4 via Pro/N-degron pathway did not result in proteasomal degradation, demonstrating that CTLH interactors are regulated through a combination of degradative and non-degradative functions. The lack of degradation of GID4 interactors highlights potential challenges in utilizing GID4-recruiting bifunctional molecules for targeted protein degradation. Going forward, PFI-7 will be a valuable research tool for defining CTLH complex biology and honing targeted protein degradation strategies.

molecular biology↗

bioRxiv2022

Noncannonical functions of Ku may underlie essentiality in human cells

The Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair but has also been involved in other cellular functions like telomere regulation and maintenance, in which Kus role is not fully characterized. It was previously reported that knockout of Ku80 in a human cell line results in lethality, but the underlying cause of Ku essentiality in human cells has yet to be fully explored. Here, we established conditional Ku70 knockout cells to study the essentiality of Ku70 function. Endogenous Ku70 knockout was achieved using CRISPR/Cas9 editing in cells where Ku70 expression was maintained through integration of an HA-tagged Ku70 cDNA under the control of a doxycycline-inducible promoter. Ku70 conditional knockout cell lines were identified via western blotting, and edits were validated by Sanger sequencing. We visually observed cell death in Ku70 knockout cells 8-10 days post Ku70-HA depletion, and loss of viability following Ku depletion was quantified using crystal violet assays. Interestingly, assessment of telomere length in Ku70 knockout cells using telomere restriction fragment analyses did not reveal any changes in average telomere length following Ku70-HA depletion. Immunofluorescence analysis used to assess {gamma}H2AX foci accumulation as a measure of double-stranded DNA breaks following Ku70-HA depletion allowed us to conclude that increased DNA damage is not the driving cause of loss of cell viability. Finally, quantitative proteome analysis of Ku70 knockout cells following Ku70-HA depletion identified a number of pathways and proteins that are significantly dysregulated following the loss of Ku70, including processes which Ku function has been previously associated with such as cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Overall, this conditional Ku70 knockout system reveals that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in other cellular processes beyond DNA repair and telomere maintenance to maintain cell viability. Author SummaryThe Ku70/80 heterodimer is a key player in non-homologous end-joining DNA repair, where it acts as a scaffold for other repair factors needed to process double-stranded DNA breaks. Ku has also been involved in other cellular functions like telomere regulation and maintenance, in which Kus role is not fully characterized. Previous data suggest that while loss of Ku70/80 can be tolerated in other species, Ku is essential to humans. We have established a conditional Ku70 knockout in HEK293 cells to evaluate the basis of Ku essentiality in human cells. While we observed loss of cell viability upon Ku depletion, we did not observe significant changes in telomere length nor did we record lethal levels of DNA damage upon loss of Ku, suggesting that the reasons for the loss of viability is not linked to the functions of Ku in DNA repair or at telomeres. Analysis of global proteome changes following Ku70 depletion revealed dysregulations of several cellular pathways including cell cycle/mitosis, RNA related processes, and translation/ribosome biogenesis. Our study reveals that loss of Ku affects multiple cellular processes and pathways and suggests that Ku plays critical roles in cellular processes beyond DNA repair and telomere maintenance to maintain cell viability.

molecular biology↗

bioRxiv2022

Never in Mitosis Kinase 2 regulation of metabolism is required for neural differentiation

1.Wnt and Hh are known signalling pathways involved in neural differentiation and recent work has shown the cell cycle regulator, Never in Mitosis Kinase 2 (Nek2) is able to regulate both pathways. Despite its known function in pathway regulation, few studies have explored Nek2 within embryonic development. The P19 embryonal carcinoma cell model was used to investigate Nek2 and neural differentiation through CRISPR knockout and overexpression studies. Loss of Nek2 reduced cell proliferation in the undifferentiated state and during directed differentiation, while overexpression increased cell proliferation. Despite these changes in proliferation rates, Nek2 deficient cells maintained pluripotency markers after neural induction while Nek2 overexpressing cells lost these markers in the undifferentiated state. Nek2 deficient cells lost the ability to differentiate into both neurons and astrocytes, although Nek2 overexpressing cells enhanced neuron differentiation at the expense of astrocytes. Hh and Wnt signaling were explored, however there was no clear connection between Nek2 and these pathways causing the observed changes to differentiation phenotypes. Mass spectrometry was also used during wildtype and Nek2 knockout cell differentiation and we identified reduced electron transport chain components in the knockout population. Immunoblotting confirmed the loss of these components and additional studies showed cells lacking Nek2 were exclusively glycolytic. Interestingly, hypoxia inducible factor 1 was stabilized in these Nek2 knockout cells despite culturing them under normoxic conditions. Since neural differentiation requires a metabolic switch from glycolysis to oxidative phosphorylation, we propose a mechanism where Nek2 prevents HIF1 stabilization, thereby allowing cells to use oxidative phosphorylation to facilitate neuron and astrocyte differentiation.

developmental biology↗

bioRxiv2022

Targeted Proteomics and Support Vector Classification Reveal Potential Biomarkers for the Early Detection of High-grade Serous Ovarian Cancer

PurposeThe five-year prognosis for patients with late-stage high-grade serous carcinoma (HGSC) remains dismal, underscoring the critical need for identifying early-stage biomarkers. This study explores the potential of extracellular vesicles (EVs) circulating in blood, which are believed to harbor proteomic cargo reflective of the HGSC microenvironment, as a source for biomarker discovery. Experimental DesignWe conducted a comprehensive proteomic profiling of EVs isolated from blood plasma, ascites, and cell lines of patients, employing both data-dependent (DDA) and data-independent acquisition (DIA) methods to construct a spectral library tailored for targeted proteomics. Our investigation aimed at uncovering novel biomarkers for the early detection of HGSC by comparing the proteomic signatures of EVs from women with HGSC to those with benign gynecological conditions. The initial cohort, comprising 9-10 donors, utilized DDA proteomics for spectral library development. The subsequent cohort, involving 30 HGSC patients and 30 control subjects, employed DIA proteomics for a similar purpose. Support vector machine (SVM) classification was applied in both cohorts to identify combinatorial biomarkers with high specificity and sensitivity (ROC-AUC > 0.90). ResultsNotably, MUC1 emerged as a significant biomarker in both cohorts when used in combination with additional biomarkers. Validation through an ELISA assay on a subset of benign (n=18), Stage I (n=9), and stage II (n=9) plasma samples corroborated the diagnostic utility of MUC1 in the early-stage detection of HGSC. ConclusionsThis study highlights the value of EV-based proteomic analysis in the discovery of combinatorial biomarkers for early ovarian cancer detection.

cancer biology↗

bioRxiv2020

Lactate Accelerates Mouse ES Cell Differentiation Towards the XEN Lineage

Metabolism plays a crucial role for cell survival and function; however, recent evidence has implicated it in regulating embryonic development. The inner cell mass undergoes orchestrated cellular divisions resulting in the formation of embryonic stem cells and extraembryonic endoderm (XEN) cells. Concomitantly, changes in the metabolic profile occurs during development and are well-documented in the embryonic lineages. However, a comprehensive multi-omics analysis of these features in XEN cells remains lacking. We observed that feeder-free XEN cells exhibited high sensitivity to glycolytic inhibition in addition to maintaining elevated intra- and extracellular lactate levels. XEN cells maintain high lactate levels by increased LDHA activity and re-routing pyruvate away from the mitochondria. Importantly, exogenous lactate supplementation or promoting intracellular lactate accumulation enhances XEN differentiation in vitro. Our results highlight how lactate contributes to XEN differentiation in the mammalian embryo and may serve to enhance reprogramming efficiency of cells used for regenerative medicine. HighlightsO_LIFeeder-free XEN cells exhibit high sensitivity to glycolytic inhibition C_LIO_LIDistinct transcriptomic, proteomic and metabolomic profile exists between feeder-free ES and XEN cells C_LIO_LIElevated intracellular and extracellular lactate is observed in feeder-free XEN cells C_LIO_LILactate enhances feeder-free XEN differentiation in vitro C_LI

developmental biology↗

bioRxiv2020

Ultrafiltration Segregates Tissue Regenerative Stimuli Harboured Within and Independent of Extracellular Vesicles

The release of extracellular vesicles (EVs) from human multipotent stromal cells (MSC) has been proposed as a mechanism by which MSC mediate regenerative functions in vivo. Our recent work has characterized MSC derived from human pancreatic tissues (Panc-MSC) that generated a tissue regenerative secretome. Despite these advancements, it remains unknown whether regenerative stimuli are released independent or within extracellular vesicles. Herein, this study demonstrates ultrafiltration is a simple method to enrich for EVs which can be injected in murine models of tissue regeneration. The enrichment of EVs from Panc-MSC conditioned media (CM) was validated using nanoscale flow cytometry and atomic force microscopy; in addition to the exclusive detection of classical EV-markers CD9, CD81, CD63 using label-free mass spectrometry. Additionally, we identified several pro-regenerative stimuli, such as WNT5A or ANGPT1, exclusive to EV-enriched CM. Endothelial cell tubule formation was enhanced in response to both Panc-MSC CM fractions in vitro yet only intramuscular injection of EV-enriched CM demonstrated vascular regenerative functions in NOD/SCID mice with unilateral hind-limb ischemia (*<p<0.05). Furthermore, both EV-depleted and EV-enriched CM reduced hyperglycemia following intrapancreatic injection in hyperglycemic mice (**p<0.01). Collectively, understanding the functional synergy between compartments of the secretome is required to advance cell-free biotherapeutics into applications of regenerative medicine.

cell biology↗