Biology subjects

Clarke, C. J.

Publications and source records attributed to Clarke, C. J..

11 recordsLinked to original sources

bioRxiv2026

Cholesterol and p53 promote senescence and systemic fibrosis in metabolic dysfunction-associated steatohepatitis

Background & aims: TP53 (p53) coordinates diverse cellular stress response programmes including pro-survival activities, senescence, and cell death. During tissue damage, p53 can shape both the local cellular response to injury, including the fibrotic response, and influence distal organ biology. Fibrosis in the liver is a major driver of hepatocellular carcinoma (HCC) risk within metabolic dysfunction-associated steatohepatitis (MASH). It is also an important determinant of dysfunction in multiple distal tissues including the kidneys, lungs, and heart. Despite significant clinical burden, our understanding of the molecular determinants of fibrotic MASH and its relationship to multiorgan fibrosis remain incomplete. Here, we investigate local and systemic effects of hepatocellular p53 activity and cholesterol during MASH development, with implications for disease prevention. Methods: This study utilised a genetic model of stabilised p53, diet-induced MASH models with varying cholesterol compositions, and an in vitro obesogenic system to investigate p53 activity during liver disease development. Non-invasive imaging and histopathological analyses were employed to monitor p53 activity, MASH, and multiorgan fibrosis in vivo. Complementary approaches, including in vitro human multicomponent liver spheroids, cytokine arrays, and analyses of human MASH transcriptomic and proteomic datasets, were used to examine molecular drivers and patient relevance. Results: Using an inducible mouse model of MDM2 E3 ubiquitin ligase deficiency to stabilise p53, we report that hepatocellular MDM2 E3 loss results in progressive fibrotic damage, robust hepatocellular expression of the p53 target gene CDKN1A/p21 (p21),and induces p21 and fibrosis in the kidneys of male mice in a sex-specific manner. In diet-induced MASH, we observe cholesterol and p53-dependent development of liver fibrosis, high expression of hepatocellular p21, and induction of p21 and fibrosis in the kidneys of male mice-reminiscent of features observed in MDM2 E3-deficient mice. We also observe fibrosis in the lungs and heart of male MASH mice. Both a cholesterol-free obesogenic diet and liver-specific loss of p53 mitigate hepatic fibrosis and systemic induction of p21 and fibrosis. Mechanistically, p53 induces hepatic expression of senescence-associated secretory phenotype (SASP) factors, including GDF15, in vivo. A human multicomponent LiverACE spheroid model showed a concordant trend towards increased GDF15 protein abundance under steatotic stress, while in humans, elevated circulating GDF15 levels in advanced MASH correlate with increased TNFRSF1A and EPHA2, circulating markers linked to kidney injury. Conclusions: Our work identifies undue p53 activity within the liver as a driver of multiorgan fibrosis in a sex-specific manner, affecting male but not female mice. We implicate cholesterol in promoting this pro-fibrotic environment in vivo and highlight circulating factors that could identify at-risk patients for multiorgan fibrosis in MASH.

cancer biology↗

bioRxiv2026

Neutral Sphingomyelinase-2 Restrains TAZ to Suppress Breast Tumor Growth

Loss of tumor suppressor gene (TSG) activity is pervasive across cancers and linked to worse clinical outcomes, yet therapeutic efforts aimed at restoring TSGs have remained elusive. One underexplored avenue to address this problem is the targeting of metabolic signaling pathways that actively enforce tumor suppressive programs. Ceramide (Cer), the central hub of the sphingolipid (SL) metabolic network, has long been thought to have tumor suppressive functions, though its mechanistic roles remain incompletely defined. Here, we identify neutral sphingomyelinase-2 (nSMase2) as a critical mediator of Cer-dependent tumor suppression. We show that nSMase2 is frequently suppressed in breast cancer (BC) and its restoration inhibits tumorigenesis. Biologically, this was linked to the suppression of anchorage-independent growth (AIG) and to restraint of the HIPPO pathway effector TAZ, but not its paralog YAP. Taken together, these findings define a previously unrecognized metabolic tumor suppressor pathway, clarify ambiguities in both SL and HIPPO signaling networks, and highlight reactivation of nSMase2-Cer signaling as a potential therapeutic strategy in BC.

cancer biology↗

bioRxiv2026

Metabolic plasticity of sphingolipids governs cancer cell fitness in acidic tumor ecosystems

Cell state plasticity enables cancer cells to rapidly adapt to fluctuating microenvironments without requiring genetic alteration, shaping tumor evolution under stress. Extracellular acidosis is a persistent feature of solid tumors that impose strong selective pressure, yet how cancer cells maintain fitness under acute and chronic acidic conditions remains unclear. Here, we show that adaptation to acidosis is mediated by plastic rewiring of sphingolipid metabolism centered on ceramide turnover. Spatial multi-omics analysis of three-dimensional tumor models revealed enrichment of ceramides within acidic niches, consistent with a stress-induced phenotype. While acute acidosis promoted ceramide accumulation and reduced fitness, chronic exposure selected for cells capable of dynamically redistributing sphingolipid flux across multiple clearance pathways. Functional perturbation demonstrated that inhibition of individual pathways was insufficient to compromise survival, whereas simultaneous disruption of all ceramide clearance routes resulted in cell death, revealing a degenerate metabolic architecture. This network-level flexibility enables cancer cells to maintain fitness by switching between alternative metabolic states under acidic stress. Together, our findings identify sphingolipid metabolic plasticity as an adaptive strategy that supports tumor persistence in acidic ecosystems and suggest that targeting metabolic flexibility, rather than individual pathways, may provide a more effective therapeutic approach.

cancer biology↗

bioRxiv2025

A Critical Role for Neutral Sphingomyelinase-2 in Doxorubicin-induced Cardiotoxicity

Cardiotoxicity is a major side effect of Doxorubicin (Dox) that has hampered its clinical utility, and strategies to mitigate this cardiotoxicity are limited. Sphingolipids (SL) are central to the chemotherapy response in cancer but their role in normal tissue is less clear. Here, we identified the SL enzyme neutral sphingomyelinase-2 (nSMase2) as a critical mediator of chronic Dox-induced cardiotoxicity, establishing nSMase2 as a key downstream effector of Dox in cardiomyocytes (CM) and showing that in vivo loss of nSMase2 activity is protecting against chronic Dox-induced cardiac damage and dysfunction. Biologically, these studies link nSMase2 with Dox-induced CM senescence both in vitro and in vivo and identify the dual specificity phosphatase DUSP4 as a novel effector of nSMase2 in the Dox response. In addition to cementing a role for SL metabolism in Dox effects in normal tissue, this study advances nSMase2 as a target of interest for cardioprotection.

cell biology↗

bioRxiv2025

An optimized SP3 sample processing workflow for in-depth and re-producible phosphoproteomics

Protein phosphorylation is a ubiquitous post-translational modification (PTM) found across the kingdoms of life, and is critical for the regulation of protein function in health & disease. Advances in high-throughput mass spectrometry have transformed our ability to interrogate the phosphoproteome. However, sample preparation methodologies optimized for phosphoproteomics have not kept pace, compromising the ability to fully exploit these technological advances. In this study, we present an optimized phosphoproteomics workflow using carboxylated SP3 magnetic beads which have simplified proteomics sample preparation. By employing a washing step with 8 M urea and omitting the conventional C18 SPE clean-up, we demonstrate a significant improvement in phosphopeptide identifications, with application of this refined protocol to HEK-293T cell extracts increasing the number nearly 2-fold compared to standard SP3 techniques (7908 cf. 4129). We also observed a substantial improvement in the detection of multiply phosphorylated peptides. Our findings suggest that the complexity of PTM crosstalk using current peptide-based proteomics workflows is currently under-represented and underscores the necessity of methodological innovations to better capture the intricacies of the phosphoproteome landscape.

biochemistry↗

bioRxiv2025

Muscle stem cell activation in response to acute injury is promoted by transient exposure to neutrophil elastase

The molecular mechanisms underlying the activation of muscle stem cells in response to skeletal muscle injury are complex and only beginning to be elucidated since new tools were developed in the last decade. The immune response to tissue injury, often referred to as sterile inflammation, is a known key contributor to the mechanisms supporting muscle stem cell proliferation and differentiation in the muscle stem cell niche, but it is unclear whether muscle stem cell activation is also affected by the immune response. Here we show that neutrophil elastase, released in abundance during the first two days after muscle injury by infiltrating neutrophils, contributes to muscle stem cell re-entry into the cell cycle and commitment to the myogenic program. When neutrophil elastase is genetically ablated in mice, muscle stem cell activation is impaired, and muscle regeneration is delayed. Additionally, we identified cMet/mTOR and FGFR/ERK1/2 as two pathways activated by elastase in two different in vitro systems, via elastase-mediated cleavage and activation of pro-HGF and via elastase-mediated cleavage of extracellular matrix and release of bioactive FGF, respectively. While chronic exposure to neutrophil elastase impairs myoblast survival and differentiation, ultimately causing muscle regeneration defects, its transient presence in injured muscle during the first 24-48 hours post injury is beneficial and necessary. Thus, our results emphasize the importance of a tight regulation of the immune response for successful muscle regeneration, and provide strong evidence in support of a role for neutrophil elastase that is mainly to promote muscle healing in response to injury. Mechanistically, we highlight a key role for the cross-talk between neutrophils and muscle stem cells in muscle stem cell activation that involves neutrophil-derived elastase-mediated activation of mTOR and ERK1/2 via cMet and FGFR in muscle stem cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=180 SRC="FIGDIR/small/640469v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@1d4fc1corg.highwire.dtl.DTLVardef@1f3a6b2org.highwire.dtl.DTLVardef@144f84corg.highwire.dtl.DTLVardef@109691b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

SERUM AND PLASMA SPHINGOLIPIDS AS BIOMARKERS OFANTHRACYCLINE-INDUCED CARDIOTOXICITY

Although effective as a chemotherapy, the utility of Doxorubicin (Dox) is hampered by cardiotoxicity. Despite this, the ability to predict and guide monitoring of patients receiving Dox or other anthracyclines is hampered by a lack of effective biomarkers to identify susceptible patients, and to detect early signs of subclinical cardiotoxicity. Based on their well-established roles in the response to Dox and other chemotherapies, we performed a retrospective analysis of serum and plasma sphingolipids (SLs) from patients undergoing anthracycline-containing therapy, correlating with cardiac parameters assessed by echocardiography. Results showed there were substantial changes in both plasma and serum SL species during therapy including ceramide (Cer), deoxydihydroCer, and dihydrosphingosine with reversion towards baseline following treatment. Linear mixed-effects model analysis revealed that at baseline, a number of SLs correlated with adverse cardiac outcomes with serum sphingosine-1-phosphate (S1P) and dihydroS1P, and plasma Cer performing comparably to the prognostic value of pro-NT-BNP, an established biomarker of cardiotoxicity. Intriguingly, while pro-NT-BNP had no predictive value at mid- and post-therapy timepoints, serum S1P and dhS1P and plasma Cer levels showed correlation with adverse outcomes, particularly at the post-therapy timepoint. Finally, analysis of plasma and serum C16:C24- Cer ratios - previously reported as predictive of adverse cardiac outcomes - showed no correlation in the context of anthracycline treatment. Taken together, this pilot study provides supporting evidence that plasma and serum SLs may have benefit as both prognostic and diagnostic biomarkers for patients undergoing anthracycline-containing therapy. This suggests that diagnostic SL measurements - recently implemented for metabolic-associated cardiac disorders - could have wider utility.

physiology↗

bioRxiv2024

Remote organ cancer adversely alters renal function and induces kidney injury, inflammation, and fibrosis.

Approximately 30% of cancer patients experience kidney complications, which hinder optimal cancer management, imposing a burden on patients quality of life and the healthcare system. The etiology of kidney complications in cancer patients is often attributed to nephrotoxic oncological therapies. However, the direct impact of cancer on kidney health is underestimated, as most nephrotoxic oncological therapies have been studied in animal models that do not have cancer. Our previous study demonstrated that metastatic lung cancer adversely alters kidney physiology and function, and exacerbates chemotherapy-induced nephrotoxicity, indicating lung cancer-kidney crosstalk. The current study examines whether this phenomenon is specific to the employed cancer model. Female and male mice of various strains were injected with different cell lines representing human and mouse lung cancer, breast cancer, and melanoma, and their kidney tissues were analyzed for toxicity and fibrosis. The impact of cancer on the kidney varied by cancer type. Breast cancer and specific subtypes of lung cancer, including KRAS- and EGFR-mutant cancer, pathologically altered kidney physiology and function in a manner dependent on the metastatic potential of the cell line. This was independent of mouse strain, sex, and cancer cell line origin. Moreover, tumor DNA was not detected in the renal tissue, excluding metastases to the kidney as a causative factor for the observed pathological alterations. Lewis lung carcinoma and B16 melanoma did not cause nephrotoxicity, regardless of the tumor size. Our results confirm cancer-kidney crosstalk in specific cancer types and highlight gaps in understanding the risk of renal complications in cancer patients. In the era of precision medicine, further research is essential to identify at-risk oncology populations, enabling early detection and management of renal complications.

physiology↗

bioRxiv2024

Uridine Phosphorylase-1 supports metastasis of mammary cancer by altering immune and extracellular matrix landscapes of the lung

Understanding the mechanisms that facilitate early events in metastatic seeding is key to developing therapeutic approaches to reduce metastasis - the leading cause of cancer-related death. Using whole animal screens in genetically engineered mouse models of cancer we have identified circulating metabolites associated with metastasis. Specifically, we highlight the pyrimidine uracil as a prominent metastasis-associated metabolite. Uracil is generated by neutrophils expressing the enzyme uridine phosphorylase-1 (UPP1), and neutrophil specific Upp1 expression is increased in cancer. Altered UPP1 activity influences expression of adhesion molecules on the surface of neutrophils, leading to decreased neutrophil motility in the pre-metastatic lung. Furthermore, we find that UPP1-expressing neutrophils suppress T-cell proliferation, and the UPP1 product uracil can increase fibronectin deposition in the extracellular microenvironment. Consistently, knockout or inhibition of UPP1 in mice with mammary tumours increases the number of T-cells and reduces fibronectin content in the lung and decreases the proportion of mice that develop lung metastasis. These data indicate that UPP1 influences neutrophil behaviour and extracellular matrix deposition in the lung and suggest that pharmacological targeting of this pathway could be an effective strategy to reduce metastasis.

cancer biology↗

bioRxiv2021

Ca2+-activated sphingomyelin scrambling and turnover mediate ESCRT-independent lysosomal repair

Lysosomes are vital organelles vulnerable to injuries from diverse materials. Failure to repair or sequester damaged lysosomes poses a threat to cell viability. Here we report that cells exploit a sphingomyelin-based lysosomal repair pathway that operates independently of ESCRT to reverse potentially lethal membrane damage. Various conditions perturbing organelle integrity trigger a rapid calcium-activated scrambling and cytosolic exposure of sphingomyelin. Subsequent metabolic conversion of sphingomyelin by neutral sphingomyelinases on the cytosolic surface of injured lysosomes promotes their repair, also when ESCRT function is compromised. Conversely, blocking turnover of cytosolic sphingomyelin renders cells more sensitive to lysosome-damaging drugs. Our data indicate that calcium-activated scramblases, sphingomyelin, and neutral sphingomyelinases are core components of a previously unrecognized membrane restoration pathway by which cells preserve the functional integrity of lysosomes.

cell biology↗

bioRxiv2020

Use of the Polo-like kinase 4 (PLK4) inhibitor centrinone to investigate intracellular signaling networks using SILAC-based phosphoproteomics

Polo-like kinase 4 (PLK4) is the master regulator of centriole duplication in metazoan organisms. Catalytic activity and protein turnover of PLK4 are tightly coupled in human cells, since changes in PLK4 concentration and catalysis have profound effects on centriole duplication and supernumerary centrosomes, which are associated with aneuploidy and cancer. Recently, PLK4 has been targeted with a variety of small molecule kinase inhibitors exemplified by centrinone, which rapidly induces inhibitory effects on PLK4 and leads to on-target centrosome depletion. Despite this, relatively few PLK4 substrates have been identified unequivocally in human cells, and PLK4 signaling outside centriolar networks remains poorly characterised. We report an unbiased mass spectrometry (MS)-based quantitative analysis of cellular protein phosphorylation in stable PLK4-expressing U2OS human cells exposed to centrinone. PLK4 phosphorylation was itself sensitive to brief exposure to the compound, resulting in PLK4 stabilization. Analysing asynchronous cell populations, we report hundreds of centrinone-regulated cellular phosphoproteins, including centrosomal and cell cycle proteins and a variety of likely non-canonical substrates. Surprisingly, sequence interrogation of ~300 significantly down-regulated phosphoproteins reveals an extensive network of centrinone-sensitive [Ser/Thr]Pro phosphorylation sequence motifs, which based on our analysis might be either direct or indirect targets of PLK4. In addition, we confirm that NMYC and PTPN12 are PLK4 substrates, both in vitro and in human cells. Our findings suggest that PLK4 catalytic output directly controls the phosphorylation of a diverse set of cellular proteins, including Pro-directed targets that are likely to be important in PLK4-mediated cell signaling.

biochemistry↗