Biology subjects

Cox, T. R.

Publications and source records attributed to Cox, T. R..

10 recordsLinked to original sources

bioRxiv2026

Cholesterol remodels the endoplasmic reticulum to control myofibroblastic CAF function

Chronic exposure of local fibroblasts to the reactive tumour niche results in the emergence of cancer-associated fibroblasts (CAFs), which can be classed into multiple subtypes with distinct functional and molecular characteristics. Of these subtypes, myofibroblastic CAFs (myCAFs), contribute to tumour progression primarily by depositing and remodelling extracellular matrix (ECM) within the tumour microenvironment (TME). ECM production is known to be a metabolically demanding process, but specific metabolic pathways that are reprogrammed during myCAF activation are poorly understood. Here, we show that cholesterol biosynthesis is elevated in myCAFs relative to normal fibroblasts. We find that cholesterol is specifically enriched in the myCAF endoplasmic reticulum (ER) membrane and is associated with augmented ER mass and enhanced ER secretory function. Importantly, we demonstrate that pharmacological or genetic inhibition of cholesterol biosynthesis plays a critical role in regulating the ECM-secreting and ECM remodelling function of myCAFs. Moreover, we show that inhibition of cholesterol biosynthesis in myCAFs markedly constrains mammary tumour metastasis in vivo. These studies suggest that cholesterol biosynthesis represents a metabolic vulnerability that can be exploited to normalise myCAF-mediated ECM dysfunction in the TME.

cell biology↗

bioRxiv2025

ROCK2 inhibition has a dual role in reducing ECM remodelling and cell growth, while impairing migration and invasion

The Rho-associated coiled-coil containing protein kinases 1/2 (ROCK1/2) are key signalling proteins involved in the regulation of the actin cytoskeleton and control a variety of cellular processes. This includes cell proliferation, stemness, cell migration and invasion as well as actomyosin contraction and extracellular matrix (ECM) remodelling. Due to a lack of ROCK2 specific inhibitors, previous pharmacological studies of ROCK function have relied on pan-ROCK1/2 inhibition. Here, we use the ROCK2 specific inhibitor, GV101, in the context of breast cancer (BC) models to uncouple the effects of ROCK2 pharmacological inhibition on both epithelial and stromal fibroblast cell populations. We demonstrate that ROCK2 inhibition with GV101 reduces fibroblast-mediated remodelling of pre-existing and de novo synthesised ECM resulting in a reduction in biomechanical stiffness, while also limiting epithelial cell growth in 2D and 3D settings. We also demonstrate that ROCK2 inhibition exposes epithelial cell vulnerability to fluid flow-induced shear stress. Furthermore, using 3D co-cultures we reveal that GV101 reduces single-cell migratory capacity, while the disruption of stromal ECM architecture is sufficient to impede collective cell migration and invasion. Finally, we assessed ROCK2 expression in human BC patient tumours revealing that ROCK2 expression is up-regulated during BC progression and that high ROCK2 expression correlates with poor patient outcome in the triple-negative subtype of BC. Together, these results demonstrate that ROCK2 specific inhibition disrupts key functions in both epithelial cell and stromal fibroblast compartments warranting further assessment of ROCK2 in highly proliferative, fibrotic or metastatic diseases, such as BC. HighlightsO_LIFibroblast-mediated remodelling of pre-existing and de novo synthesised extracellular matrix is reduced upon ROCK2 specific targeting. C_LIO_LIROCK2 inhibition impairs epithelial cancer cell growth and colony formation in 2D and 3D contexts, while also limiting single-cell migration and cell survival upon fluid flow-induced shear stress. C_LIO_LIROCK2 targeting during fibroblast-mediated ECM remodelling is sufficient to disrupt collective cell migration as well as 3D invasion into organotypic fibroblast-contracted collagen matrices. C_LIO_LIROCK2 expression is up-regulated during breast cancer progression and high expression correlates with poor patient outcome in triple-negative breast cancer. C_LI

cell biology↗

bioRxiv2025

A selective inhibitor of oncogenic JNK signalling perturbs metastatic outgrowth of triple-negative breast cancer through metabolic blockade

Although c-Jun N-terminal Kinase (JNK) represents an attractive anti-cancer target, its pleiotropic functionality limits the use of direct JNK inhibitors. Here, we identify a distinct subcellular pattern of JNK activity as a therapeutic vulnerability in breast cancer, where cytoplasmic JNK activity predicts poor survival outcomes, is elevated in triple-negative breast cancers (TNBC) and is essential for metastatic outgrowth. Mechanistic analyses reveal cytoplasmic JNK acts through multiple mechanisms, with downstream targets involved in cellular metabolism and cytoskeletal regulation. On this basis, we leveraged actin-based phenotypic drug-screening and identified K12, an indirect but selective inhibitor of cytoplasmic JNK that blocks TNBC metastatic outgrowth in vivo. We reveal that K12 inhibits glutaminase-1 and the pyruvate dehydrogenase complex, and that this poly-pharmacology overcomes pyruvate anaplerosis, a known resistance mechanism of existing glutaminase inhibitors. These findings demonstrate the potential of selectively targeting the oncogenic function of JNK, offering new treatment options for early-stage metastatic TNBC.

cancer biology↗

bioRxiv2025

Dynamic dysregulation of Tenascin-X/Tenascin-C balance controlled by Transforming Growth Factor-beta leads to tumor cell proliferation during pancreatic carcinogenesis

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers due to late diagnosis and poor therapeutic efficiency. Throughout PDAC progression, a dense extracellular matrix (ECM) is deposited around neoplastic cells and accompanies tumor development and aggressiveness. This significant stroma, both in terms of quantity and through its impact on tumor cells, is now considered as a target for innovative therapies to improve patient survival. Among ECM proteins, Tenascins (TNs) are a family of four glycoproteins (TNC, TNW, TNR and TNX) sharing a common modular structure, but exhibiting different expression patterns and functions depending on the physiological and physio-pathological contexts. In PDAC, TNC is up-regulated and is considered as a pro-tumorigenic actor, whereas TNXs role remains to be elucidated. Herein, we demonstrated that unlike TNC, TNX is drastically decreased in PDAC, and that this loss is correlated with reduced patient survival. Dysregulation of the TNX/TNC balance is attributable to Transforming Growth Factor-beta (TGF-{beta}) upregulation during pancreatic carcinogenesis. Interestingly, we found that TNX is first highly deposited in low grade lesions before being clearly decreased in later stages suggesting an elaborate stromal remodelling during PDAC development. Finally, low TNX and high TNC deposition around precursor lesions are correlated with increased preneoplastic cell proliferation. Altogether, our results (i) demonstrate the importance of Tenascin ratios during pancreatic carcinogenesis, (ii) suggest an anti-proliferative role for TNX unlike its pro-tumoral counterpart, TNC and (iii) underscore TNX and TNC as valuable targets for the development of new drugs for pancreatic cancer and/or solid tumor treatment. Statement of significanceThe tumor microenvironment plays a prominent but contradictory role in pancreatic cancer. Our study highlights the role of a TGF-{beta}-controlled TNX/TNC balance and designates TNX as an inhibitor of tumor cell proliferation.

cancer biology↗

bioRxiv2024

Cleavage of the vascular matrix attracts glioblastoma cells to infiltrate the brain parenchyma

BackgroundGlioblastoma is a highly aggressive brain cancer and, unlike many other cancers types, the median survival for patients after treatment (14.6 months) has barely improved in the last 20 years. Infiltrative growth into the surrounding brain parenchyma facilitates tumor recurrence and ultimately the death of the patient - novel therapies targeting this process are desperately needed. Lysyl oxidase inhibition has been shown to decrease invasive growth in a variety of solid tumours and is a potential therapy for glioblastoma patients. MethodsGenes highly expressed in the mesenchymal subtype of glioblastoma were analyzed in a data set from the Cancer Genome Atlas and tissue microarrays. Two patient-derived human glioblastoma stem cell lines were used to assess the involvement of lysyl oxidase (LOX). The effect of LOX on infiltration was examined in an organotypic brain slice assay and in an orthotopic mouse model. Chemotactic assays, protease and cleavage arrays were used to assess the underlying mechanism behind LOX-mediated infiltration. The orthotopic model was used to evaluate potential clinical utility of targeting LOX in glioblastoma. ResultsLOX is overexpressed in the mesenchymal glioblastoma subtype and strongly associated with poor patient survival. LOX expression upregulates MMP7 expression, which subsequently cleaves the vascular matrix resulting in increased chemotaxis of glioblastoma cells. ConclusionsWe have uncovered a novel mechanism of glioblastoma infiltration and suggest that targeting LOX represent an effective therapeutic approach blocking glioblastoma infiltration. Importance of the studyThe ability of glioblastoma cells to infiltrate the surrounding normal brain tissue facilitates their evasion of current therapies, leading to tumor recurrence and ultimately the death of the patient. To improve targeted therapies for glioblastoma patients we need to understand the molecular mechanisms of glioblastoma cell infiltration and how cells interact with the unique microenvironment of the brain. We have identified a novel mechanism whereby tumor-derived LOX mediates chemotaxis of glioblastoma cells to the laminin rich perivascular niche, enabling infiltrative growth. Inhibiting this infiltrative pathway is a potential anti-invasive therapy that is desperately needed for glioblastoma patients.

cancer biology↗

bioRxiv2022

Spatial analysis of the metastatic brain tumor immune and extracellular matrix microenvironment

Metastatic cancer is responsible for the overwhelming majority of cancer-related deaths with metastatic tumors being the most common neoplasms affecting the central nervous system. One of the major factors regulating tumor biology is the tumor microenvironment. However, little is known about the cellular and non-cellular composition of metastatic brain tumors and how tumor cell ontogeny influences the metastatic brain tumor microenvironment. By integrating multiplex immunohistochemistry and histopathological analysis to investigate composition and the spatial relationship between neoplastic cells, infiltrating and brain resident immune cells and the extracellular matrix, we demonstrate that metastatic brain tumors exhibit differences in ECM deposition, compared with the most common primary brain tumor type, glioblastoma, and that the dominant immune cell types in metastatic brain tumors are immunosuppressive macrophages, which preferentially localize to ECM-rich stromal regions.

cancer biology↗

bioRxiv2021

Micromechanical Characterisation of 3D Bioprinted neural cell models using Brillouin Microscopy

Biofabrication of artificial 3D in vitro neural cell models that closely mimic the central nervous system (CNS) is an emerging field of research with applications from fundamental biology to regenerative medicine, and far reaching benefits for the economy, healthcare and the ethical use of animals. The micromechanical properties of such models are an important factor dictating the success of modelling outcomes in relation to accurate reproduction of the processes in native tissues. Characterising the micromechanical properties of such models non-destructively and over a prolonged span of time, however, are key challenges. Brillouin microscopy (BM) could provide a solution to this problem since this technology is non-invasive, label-free and is capable of microscale 3D imaging. In this work, the viscoelasticity of 3D bioprinted neural cell models consisting of NG 108-15 neuronal cells and GelMA hydrogels of various concentrations were investigated using BM. We demonstrate changes in the micro- and macro-scale mechanical properties of these models over a 7 day period, in which the hydrogel component of the model are found to soften as the cells grow, multiply and form stiffer spheroid-type structures. These findings signify the necessity to resolve in microscopic detail the mechanics of in vitro 3D tissue models and suggest Brillouin microscopy to be a suitable technology to bridge this gap.

bioengineering↗

bioRxiv2020

Single-cell analysis reveals diverse stromal subsets associated with immune evasion in triple-negative breast cancer

The tumour stroma regulates nearly all stages of carcinogenesis. Stromal heterogeneity in human triple-negative breast cancers (TNBCs) remains poorly understood, limiting the development of stromal-targeted therapies. Single cell RNA-sequencing of five TNBCs revealed two cancer-associated fibroblast (CAF) and two perivascular-like (PVL) subpopulations. CAFs clustered into two states, the first with features of myofibroblasts and the second characterised by high expression of growth factors and immunomodulatory molecules. PVL cells clustered into two states consistent with a differentiated and immature phenotype. We showed that these stromal states have distinct morphologies, spatial relationships and functional properties in regulating the extracellular matrix. Using cell-signalling predictions, we provide evidence that stromal-immune crosstalk acts via a diverse array of immunoregulatory molecules. Importantly, the investigation of gene signatures from inflammatory-CAFs and differentiated-PVL cells in independent TNBC patient cohorts revealed strong associations with cytotoxic T-cell dysfunction and exclusion, respectively. Such insights present promising candidates to further investigate for new therapeutic strategies in the treatment of TNBCs.

cancer biology↗

bioRxiv2020

ID4 represses myoepithelial differentiation of mammary stem cells through its interaction with HEB

Differentiation of stem cells embedded within the mammary epithelium is orchestrated by lineage-specifying transcription factors. Unlike the well-defined luminal hierarchy, dissection of the basal lineage has been hindered by a lack of specific markers. Inhibitor of Differentiation 4 (ID4) is a basally-restricted helix-loop-helix (HLH) transcription factor essential for mammary development. Here we show that ID4 is highly expressed in basal stem cells and decreases during myoepithelial differentiation. By integrating transcriptomic, proteomic, and ChIP-sequencing data, we reveal that ID4 is required to suppress myoepithelial gene expression and cell fate. We identify the bHLH protein HEB as a direct binding partner of ID4, and describe a previously-unknown role for this regulator in mammary development. HEB binds to E-boxes in regulatory elements of developmental genes, negatively regulated by ID4, involved in extracellular matrix synthesis and cytoskeletal contraction. Together our findings support a model whereby ID4 binds to HEB and blocks it from promoting myoepithelial specialisation. These new insights expand our current understanding into control of myoepithelial differentiation and mammary gland morphogenesis.

developmental biology↗

bioRxiv2017

Targeting stromal remodeling and cancer stem cell plasticity to overcome chemoresistance in triple negative breast cancer

The cellular and molecular basis of stromal cell recruitment, activation and crosstalk in carcinomas is poorly understood, limiting the development of targeted anti-stromal therapies. In mouse models of triple negative breast cancer (TNBC), Hh ligand produced by neoplastic cells reprogrammed cancer-associated fibroblast (CAF) gene expression, driving tumor growth and metastasis. Hh-activated CAFs upregulated expression of FGF5 and production of fibrillar collagen, leading to FGFR and FAK activation in adjacent neoplastic cells, which then acquired a stem-like, drug-resistant phenotype. Treatment with smoothened inhibitors (SMOi) reversed these phenotypes. Stromal treatment of TNBC patient-derived xenograft (PDX) models with SMOi downregulated the expression of cancer stem cell markers and sensitized tumors to docetaxel, leading to markedly improved survival and reduced metastatic burden. In the phase I clinical trial EDALINE, 3 of 12 patients with metastatic TNBC derived clinical benefit from combination therapy with the SMOi Sonidegib and docetaxel chemotherapy, with one patient experiencing a complete response. Markers of pathway activity correlated with response. These studies identify Hh signaling to CAFs as a novel mediator of cancer stem cell plasticity and an exciting new therapeutic target in TNBC.\n\nSIGNIFICANCECompared to other breast cancer subtypes, TNBCs are associated with significantly worse patient outcomes. Standard of care systemic treatment for patients with non-BRCA1/2 positive TNBC is cytotoxic chemotherapy. However, the failure of 70% of treated TNBCs to attain complete pathological response reflects the relative chemoresistance of these tumors. New therapeutic strategies are needed to improve patient survival and quality of life. Here, we provide new insights into the dynamic interactions between heterotypic cells within a tumor. Specifically, we establish the mechanisms by which CAFs define cancer cell phenotype and demonstrate that the bidirectional CAF-cancer cell crosstalk can be successfully targeted in mice and humans using anti-stromal therapy.

cancer biology↗