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While organ‐confined prostate cancer (PCa) is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumours with concomitant inhibition of two tumour suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having < 50% 5‐year secondary‐therapy‐free patient survival. Functionally, overexpression of PME‐1, a methylesterase for the catalytic PP2A‐C subunit, inhibits anoikis in PTEN‐deficient PCa cells. In vivo, PME‐1 inhibition increased apoptosis in in ovo PCa tumour xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME‐1‐deficient PC3 cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype known to correlate with increased apoptosis sensitivity. In summary, our results demonstrate that PME‐1 supports anoikis resistance in PTEN‐deficient PCa cells. Clinically, these results identify PME‐1 as a candidate biomarker for a subset of particularly aggressive PTEN‐deficient PCa. A subset of prostate cancer (PCa) tumours present simultaneous inactivation of two tumour suppressor phosphatases; phosphatase and tensin homolog (PTEN) and protein phosphatase 2A (PP2A). PP2A is inhibited via overexpression of PME‐1. Such cancers are particularly aggressive and often relapse from standard therapy, indicating PME‐1 as a potential clinically applicable biomarker for PCa. Mechanistically, PME‐1 expression protects cancer cells from anoikis, promoting their survival outside the primary tumour.

How cells sense tissue stiffness to guide cell migration is a fundamental question in development, fibrosis and cancer. Although durotaxis-cell migration towards increasing substrate stiffness-is well established, it remains unknown whether individual cells can migrate towards softer environments. Here, using microfabricated stiffness gradients, we describe the directed migration of U-251MG glioma cells towards less stiff regions. This 'negative durotaxis' does not coincide with changes in canonical mechanosensitive signalling or actomyosin contractility. Instead, as predicted by the motor-clutch-based model, migration occurs towards areas of 'optimal stiffness', where cells can generate maximal traction. In agreement with this model, negative durotaxis is selectively disrupted and even reversed by the partial inhibition of actomyosin contractility. Conversely, positive durotaxis can be switched to negative by lowering the optimal stiffness by the downregulation of talin-a key clutch component. Our results identify the molecular mechanism driving context-dependent positive or negative durotaxis, determined by a cell's contractile and adhesive machinery.

How cells sense tissue stiffness to guide cell migration is a fundamental question in development, fibrosis and cancer. Although durotaxis – traditionally defined as cell migration toward increasing substrate stiffness – is well established, it remains unknown whether individual cells can migrate toward softer environments. Using microfabricated stiffness gradients, we observed directed migration of U-251MG glioma cells toward lower stiffness. This ‘negative durotaxis’ did not coincide with changes in canonical mechanosensitive signaling or actomyosin contractility. Instead, motor-clutch-based modeling predicted migration toward cell-intrinsic ‘optimal stiffness’, where cells generate maximal traction. As predicted by the model, negative durotaxis was selectively disrupted and even reversed by partial inhibition of actomyosin contractility. Conversely, positive durotaxis was switched to negative experimentally by lowering the optimal stiffness via downregulation of a key clutch component, talin. Our results identify the molecular mechanism driving context-dependent positive or negative durotaxis, determined by a cell’s contractile and adhesive machinery.

Fibrillar adhesions are important structural and adhesive components in fibroblasts that are critical for fibronectin fibrillogenesis. While nascent and focal adhesions are known to respond to mechanical cues, the mechanoresponsive nature of fibrillar adhesions remains unclear. Here, we used ratiometric analysis of paired adhesion components to determine an appropriate fibrillar adhesion marker. We found that active α5β1-integrin exhibits the most definitive fibrillar adhesion localisation compared to other proteins, such as tensin1, reported to be in fibrillar adhesions. To elucidate the mechanoresponsiveness of fibrillar adhesions, we designed and fabricated thin polyacrylamide (PA) hydrogels, embedded with fluorescently labelled beads, with physiologically relevant stiffness gradients using a cost-effective and reproducible technique. We generated a correlation curve between bead density and hydrogel stiffness, thus allowing the use of bead density as a readout of stiffness, eliminating the need for specialised knowhow including atomic force microscopy (AFM). We find that stiffness promotes the growth of fibrillar adhesions in a tensin-dependent manner. Thus, the formation of these ECM depositing structures is coupled to the mechanical parameters of the cell environment and may enable cells to fine-tune their matrix environment in response to alternating physical conditions.

While organ-confined PCa is mostly therapeutically manageable, metastatic progression of PCa remains an unmet clinical challenge. Resistance to anoikis, a form of cell death initiated by cell detachment from the surrounding extracellular matrix, is one of the cellular processes critical for PCa progression towards aggressive disease. Therefore, further understanding of anoikis regulation in PCa might provide therapeutic opportunities. Here, we discover that PCa tumors with concomitantly compromised function of two tumor suppressor phosphatases, PP2A and PTEN, are particularly aggressive, having less than 50% 5-year secondary-therapy free patient survival. Functionally, overexpression of PME-1, a PP2A inhibitor protein, inhibits anoikis in PTEN-deficient PCa cells. In vivo, PME-1 inhibition increased apoptosis in in ovo PCa tumor xenografts, and attenuated PCa cell survival in zebrafish circulation. Molecularly, PME-1 deficient PCa cells display increased trimethylation at lysines 9 and 27 of histone H3 (H3K9me3 and H3K27me3), a phenotype corresponding to increased apoptosis sensitivity. In summary, we discover that PME-1 overexpression supports anoikis resistance in PTEN-deficient PCa cells. Clinically, the results identify PME-1 as a candidate biomarker for a subset of particularly aggressive PTEN-deficient PCa. Competing Interest Statement The authors have declared no competing interest.

Ductal carcinoma in situ (DCIS) is a pre-invasive stage of breast cancer, where the tumor is encapsulated by a basement membrane (BM). At the invasive phase, the BM barrier is compromised enabling tumor cells to escape into the surrounding stroma. The molecular mechanisms that establish and maintain an epithelial BM barrier in vivo are poorly understood. Myosin-X (MYO10) is a filopodia-inducing motor protein implicated in metastasis and poor clinical outcome in patients with invasive breast cancer (IBC). We compared MYO10 expression in patient-matched normal breast tissue and DCIS lesions and found elevated MYO10 expression in DCIS samples, suggesting that MYO10 might facilitate the transition from DCIS to IBC. Indeed, MYO10 promoted the formation of filopodia and cell invasion in vitro and positively regulated the dissemination of individual cancer cells from IBC lesions in vivo. However, MYO10-depleted DCIS xenografts were, unexpectedly, more invasive. In these xenografts, MYO10 depletion compromised BM formation around the lesions resulting in poorly defined tumor borders and increased cancer cell dispersal into the surrounding stroma. Moreover, MYO10-depleted tumors showed increased EMT-marker-positive cells, specifically at the tumor periphery. We also observed cancer spheroids undergoing rotational motion and recruiting BM components in a filopodia-dependent manner to generate a near-continuous extracellular matrix boundary. Taken together, our data identify a protective role for MYO10 in early-stage breast cancer, where MYO10-dependent tumor cell protrusions support BM assembly at the tumor-stroma interface to limit cancer progression, and a pro-invasive role that facilitates cancer cell dissemination at later stages. Competing Interest Statement The authors have declared no competing interest.

Abstract Actin-rich cellular protrusions direct versatile biological processes from cancer cell invasion to dendritic spine development. The stability, morphology and specific biological function of these protrusions are regulated by crosstalk between three main signaling axes: integrins, actin regulators and small GTPases. SHANK3 is a multifunctional scaffold protein, interacting with several actin-binding proteins, and a well-established autism risk gene. Recently, SHANK3 was demonstrated to sequester integrin-activating small GTPases Rap1 and R-Ras to inhibit integrin activity via its N-terminal SPN domain. Here, we demonstrate that SHANK3 interacts directly with actin using its SPN domain. Actin binding can be inhibited by an intramolecular closed conformation of SHANK3, where the adjacent ARR domain covers the actin-binding interface of the SPN domain. Actin and Rap1 compete with each other for binding to SHANK3 and loss of SHANK3-actin binding augments inhibition of Rap1-mediated integrin activity. This dynamic crosstalk has functional implications for filopodia formation in cancer cells, dendritic spine morphology in neurons and autism-linked phenotypes in vivo. Competing Interest Statement The authors have declared no competing interest.

Abstract Durotaxis – the ability of cells to sense and migrate along stiffness gradients – is important for embryonic development and has been implicated in pathologies including fibrosis and cancer. Although cellular processes can sometimes turn toward softer environments, durotaxis at the level of cells has thus far been observed exclusively as migration from soft to stiff regions. The molecular basis of durotaxis, especially the factors that contribute to different durotactic behaviors in various cell types, are still inadequately understood. With the recent discovery of ‘optimal stiffness’, where cells generate maximal traction forces on substrates in an intermediate stiffness range, we hypothesized that some migratory cells may be capable of moving away from stiff environments and toward matrix on which they can generate more traction. Combining hydrogel-based stiffness gradients, live-cell imaging, genetic manipulations, and computational modeling, we found that cells move preferentially toward their stiffness optimum for maximal force transmission. Importantly, we directly observed biased migration toward softer environments, i.e. ‘negative durotaxis’, in human glioblastoma cells. This directional migration did not coincide with changes in FAK, ERK or YAP signaling, or with altered actomyosin contractility. Instead, integrin-mediated adhesion and motor-clutch dynamics alone are sufficient to generate asymmetric traction to drive both positive and negative durotaxis. We verified this mechanistically by applying a motor-clutch-based model to explain negative durotaxis in the glioblastoma cells and in neurites, and experimentally by switching breast cancer cells from positive to negative durotaxis via talin downregulation. Our results identify the likely molecular mechanisms of durotaxis, with a cell’s contractile and adhesive machinery dictating its capacity to exert traction on mechanically distinct substrates, directing cell migration. Competing Interest Statement The authors have declared no competing interest.