Biology subjects

Ivaska, J.

Publications and source records attributed to Ivaska, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2022

AXL-TBK1 driven nuclear AKT3 promotes metastasis

Epithelial-to-mesenchymal transition (EMT) contributes to tumor cell survival, immune evasion, migration, invasion, and therapy resistance. Across human cancer, tumors that are high grade, poorly differentiated, and have undergone EMT carry a worse prognosis with a higher likelihood of metastasis. AXL, a receptor tyrosine kinase, drives EMT and is implicated in tumor progression, metastasis, and therapy resistance in multiple cancer types including pancreatic cancer and breast cancer. TANK-binding kinase 1 (TBK1) is central to AXL-driven EMT yet, the mechanism of how TBK1 induces EMT remains unclear. Here, we report that AXL activation stimulates TBK1 binding and phosphorylation of AKT3. TBK1 activation of AKT3 drives binding and phosphorylation of slug/snail resulting in protection from proteasomal degradation and translocation of the complex into the nucleus. We show that nuclear translocation of AKT3 is required for AXL-driven EMT and metastasis. Congruently, nuclear AKT3 expression correlates with worse outcome in aggressive breast cancer. To advance AKT3 as a therapeutic target, an AKT3-isoform selective allosteric small molecule inhibitor, BGB214, was developed. BGB214 inhibits AKT3 nuclear translocation, EMT-TF stability, AKT3-mediated invasion of breast cancer cells and reduces tumor initiation in vivo. Our results suggest that AKT3 nuclear activity is an important feature of AXL-driven epithelial plasticity and that selective AKT3 inhibition represents a novel therapeutic avenue for treating aggressive cancer. SignificanceNuclear AKT3 activity is an important feature of AXL-TBK1 driven EMT and metastasis, thus selective AKT3 targeting represents a novel approach to treat aggressive cancer.

cancer biology↗

bioRxiv2021

Myosin-X-dependent assembly of the extracellular matrix limits breast cancer invasion

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. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/464987v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d90a4eorg.highwire.dtl.DTLVardef@133ba9borg.highwire.dtl.DTLVardef@1204a05org.highwire.dtl.DTLVardef@dba254_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Filopodia sculpt the tumor-proximal stroma in pre-invasive ductal carcinoma in situ (DCIS). - Filopodia-dependent basement membrane (BM) assembly limits invasive transition of DCIS-like tumors in vivo. - Loss of MYO10-dependent filopodia impairs BM assembly and induces an EMT-like phenotype at the tumor-stroma interface in vivo. - MYO10 filopodia are anti-invasive in DCIS but facilitate dissemination in invasive breast cancer.

cell biology↗

bioRxiv2021

Sortilin-related receptor is a druggable therapeutic target in breast cancer

In breast cancer, the currently approved anti-receptor tyrosine-protein kinase erbB-2 (HER2) therapies do not fully meet the expected clinical goals due to therapy resistance. Identifying alternative HER2-related therapeutic targets could offer means to overcome these resistance mechanisms. We have previously demonstrated that an endosomal sorting protein, sortilin-related receptor (SorLA), regulates the traffic and signaling of HER2 and HER3, thus promoting resistance to HER2-targeted therapy in breast cancer. This study aims to assess the feasibility of targeting SorLA using a monoclonal antibody. Our results demonstrate that anti-SorLA antibody (SorLA ab) alters the resistance of breast cancer cells to HER2 monoclonal antibody trastuzumab in vitro and in ovo. We found that SorLA ab and trastuzumab combination therapy also inhibits tumor cell proliferation and tumor cellularity in a mouse xenograft model of HER2-positive breast cancer. In addition, SorLA ab inhibits the proliferation of breast cancer patient-derived explant three-dimensional cultures. These results provide for the first time proof-of-principle that SorLA is a druggable target in breast cancer.

cancer biology↗

bioRxiv2020

Conformational dynamics regulate SHANK3 actin and Rap1 binding

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.

cell biology↗

bioRxiv2020

Negative durotaxis: cell movement toward softer environments

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 cells contractile and adhesive machinery dictating its capacity to exert traction on mechanically distinct substrates, directing cell migration.

biophysics↗

bioRxiv2020

Cargo-specific recruitment in clathrin and dynamin-independent endocytosis

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis, and cancer invasion and is often hijacked by viral infections 1. Unlike clathrin-mediated endocytosis, which exploits cargo-specific adaptors for selective protein internalization, the clathrin and dynamin-independent endocytic pathway (CLIC-GEEC, CG-pathway) has until now been considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids 2,3. Although the core molecular players of CG endocytosis have been recently defined, no cargo-specific adaptors are known and evidence of selective protein uptake into the pathway is lacking 3. Here, we identify the first cargo-specific adaptor for CG-endocytosis and demonstrate its clinical relevance in breast cancer progression. By combining unbiased molecular characterization and super-resolution imaging, we identified the actin-binding protein swiprosin-1 (EFHD2) as a cargo-specific adaptor regulating integrin internalization via the CG-pathway. Swiprosin-1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery, IRSp53 and actin. Swiprosin-1 is critical for integrin endocytosis, but not for other CG-cargo and supports integrin-dependent cancer cell migration and invasion, with clinically relevant implications for breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG-pathway and opens the possibility to discover more adaptors regulating it.

cell biology↗

bioRxiv2020

A feed-forward loop between SorLA and HER3 determines heregulin response and neratinib resistance

Current evidence indicates that resistance to HER2-targeted therapies is frequently associated with HER3 and active signaling via HER2-HER3 dimers, particularly in the context of breast cancer. Thus, understanding the response to HER2-HER3 signaling and the regulation of the dimer per se remains essential to decipher therapy relapse mechanisms. Here, we demonstrate that signaling by HER3 growth factor ligands, heregulins, support the transcription of a type-1 transmembrane sorting receptor, sortilin-related receptor (SorLA; SORL1) downstream of the mitogen-activated protein kinase pathway. In addition, we demonstrate that SorLA interacts directly with HER3, forming a trimeric complex with HER2 and HER3 to attenuate lysosomal degradation of the dimer through a Rab4-dependent manner. In line with a role for SorLA in supporting the stability of the HER2 and HER3 receptors, loss of SorLA compromised heregulin-induced cell proliferation and sensitized metastatic anti-HER2 therapy-resistant breast cancer cells to neratinib in cancer spheroids in vitro and in vivo in a zebrafish brain xenograft model. Collectively, our results demonstrate a novel feed-forward loop consisting of heregulin, HER2-HER3 and SorLA, which controls breast cancer growth and anti-HER2 therapy resistance in vitro and in vivo. SignificanceHER3 signaling, through ERK/MAPK, upregulates SorLA and SorLA controls the trafficking and stability of HER3 to support cancer proliferation and neratinib resistance.

cancer biology↗

bioRxiv2020

Talin Rod Domain Containing Protein 1 (TLNRD1) is a novel actin- bundling protein which promotes filopodia formation

Talin is a mechanosensitive adapter protein which couples integrins to the cytoskeleton and regulates integrin-mediated adhesion. Talin rod domain-containing protein-1 (TLNRD1) shares 22% homology with the R7R8 domains of talin, and is highly conserved throughout vertebrate evolution, however little is known about its function. Here we show that TLNRD1 is an -helical protein which shares the same atypical topology as talin R7R8, but forms a novel antiparallel dimer arrangement. Actin co-sedimentation assays and electron microscopy reveal TLNRD1 is an actin-bundling protein that forms tight actin bundles. In addition, TLNRD1 binds to the same LD-motif containing proteins, RIAM and KANK, as talin, and thus may act in competition with talin. Filopodia are cell protrusions supported by tightly bundled actin filaments and TLNRD1 localises to filopodia tips, increases filopodia number and promotes cell migration in 2D. Together our results suggest that TLNRD1 has similar functionality to talin R7R8, serving as a nexus between the actin and microtubule cytoskeletons independent of adhesion complexes.

cell biology↗

bioRxiv2020

Myosin-X is required for integrin activation at filopodia tips

Filopodia assemble unique integrin-adhesion complexes to sense the extracellular matrix. However, the mechanisms of integrin localization and regulation in filopodia are poorly defined. Here, we observed that active integrins accumulate at the tip of myosin-X(MYO10)-positive filopodia while inactive integrins are uniformly distributed. RNAi depletion of 10 integrin activity modulators identified talin as the principal integrin activator in filopodia. Deletion of the MYO10-FERM domain, or mutation of the {beta}1-integrin-binding residues within, revealed MYO10 as facilitating integrin activation but not transport in filopodia. However, MYO10-FERM alone could not activate integrins, potentially due to dual binding to both a- and {beta}-integrin tails. As swapping MYO10-FERM with talin-FERM enabled integrin activation in filopodia, our data indicate that an integrin-binding FERM domain coupled to a myosin motor is a core requirement for integrin activation in filopodia. Therefore, we propose a two-step integrin activation model in filopodia: receptor tethering by MYO10 followed by talin-mediated integrin activation.

cell biology↗

bioRxiv2019

Laminin alpha 5 is Necessary for Mammary Epithelial Growth and Function by Maintaining Luminal Epithelial Cell Identity

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we demonstrate that expression of distinct laminin -isoforms by luminal and basal mammary epithelial cells enforces lineage identity that is necessary for normal mammary gland growth and function. Laminin 5 (LM5) is mainly expressed by the luminal epithelial cells, and it is necessary for pubertal mammary gland growth, pregnancy induced gland remodeling, and for alveolar function. Adhesion to LM5 containing laminin promotes luminal traits in both luminal and basal epithelial cells, and reduces progenitor activity of basal epithelial cells. Mechanistically, we show that Lama5 loss interferes with differentiation of hormone receptor positive luminal cells, which results in reduced Wnt4 expression and defective crosstalk between luminal and basal epithelial cells during gland remodeling. Our results reveal a novel BM-mediated mechanism, which regulates mammary gland remodeling and function via specification of luminal epithelial cells.

cell biology↗

bioRxiv2019

Mechano-responsiveness of fibrillar adhesions on stiffness-gradient gels

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 5{beta}1-integrin exhibits the most definitive fibrillar adhesion localisation compared to other proteins, such as tensin-1, 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 extracellular matrix-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.

cell biology↗

bioRxiv2019

Fluctuation-based Super-Resolution Traction Force Microscopy

Cellular mechanics play a crucial role in tissue morphogenesis and homeostasis and are often misregulated in disease. Traction force microscopy (TFM) is one of the key methods that has enabled researchers to study fundamental aspects of mechanobiology; however, the power of TFM is limited by poor resolution and low throughput. Here, we propose a simplified protocol and imaging strategy, relying on super-resolution microscopy enabled by fluorophore fluctuation analysis, to enhance the output of TFM, by increasing both bead density as well as the accuracy of bead tracking in TFM gels. Our analysis pipeline can be used on either camera-based confocal or widefield microscopes and is fully compatible with available TFM analysis software. In addition, we demonstrate that our workflow can be used to gain biologically relevant information and is suitable for long-term live measurement of traction forces even in light-sensitive cells. Finally, we propose that our strategy could be used to considerably simplify the implementation of TFM screens. Our streamlined protocol can be performed with minimal hardware and software investment, and has the potential to standardize high-resolution TFM.

cell biology↗

bioRxiv2019

PP2A inhibitor PME-1 suppresses anoikis, and is associated with therapy relapse of PTEN-deficient prostate cancers

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.

cancer biology↗

bioRxiv2019

Integrin binding dynamics modulate ligand-specific mechanosensing in mammary gland fibroblasts

The link between the modulation of integrin activity and cellular mechanosensing of tissue rigidity, especially on different extracellular matrix ligands, remains poorly understood. Here, we find that primary mouse mammary gland stromal fibroblasts (MSFs) are able to spread efficiently on soft collagen-coated substrates, resembling the soft mammary gland tissue. In addition, MSFs generate high forces and display nuclear YAP at a low matrix stiffness, supported by mature focal adhesions, prominent actin stress fibers, and myosin phosphorylation. We describe that loss of the cytosolic integrin inhibitor, SHARPIN, impedes MSF spreading specifically on soft type I collagen but not on fibronectin. Through quantitative experiments and computational modelling, we find that SHARPIN-deficient MSFs display faster force-induced unbinding of adhesions from collagen-coated beads. Faster unbinding, in turn, impairs force transmission in these cells, particularly, at the stiffness optimum observed for wild-type cells, and increases actin retrograde flow. Mechanistically, we link the impaired mechanotransduction of SHARPIN-deficient cells on collagen to reduced levels of the collagen-binding integrin 11{beta}1. Our results unveil a collagen-specific mechanosensing mechanism and suggest a key function for integrin activity regulation and integrin 11{beta}1 in MSF mechanotransduction.

cell biology↗

bioRxiv2018

Superresolution architecture of pluripotency guarding adhesions

Human pluripotent stem cells (hPSC) can generate almost all adult cell lineages. While it is clear that key transcriptional programmes are important elements for maintaining pluripotency, the equally essential requirement for cell adhesion to specific extracellular matrix components remains poorly defined. Our recent observation that hPSC colonies form unusually large \"cornerstone\" focal adhesions (FA), distinct from parental somatic cells, that are lost following differentiation, emphasises the potential of these atypical FA as gatekeepers of pluripotency. Here, using nanopatterns, we further demonstrate that physical restriction of adhesion size, in hPSC colonies, is sufficient to trigger differentiation. Using superresolution two-colour interfero-metric photo-activated localization microscopy (iPALM), we examined the three-dimensional architecture of these cornerstone adhesions and report vertical lamination of FA proteins with three main structural peculiarities: 1) integrin {beta}5 and talin are present at high density, at the edges of cornerstone FA, adjacent to a vertical kank-rich protein wall. 2) Vinculin localises higher than expected with respect to the substrata and displays a head-above-tail orientation, and 3) surprisingly, actin and -actinin are present in two discrete layers, a previously undescribed localisation for these proteins. Finally, we report that depletion of kanks diminishes FA patterning, and actin organisation within the colony, indicating a key role for kanks in hPSC colony architecture.

cell biology↗

bioRxiv2018

Filopodome mapping identifies p130Cas as a mechanosensitive regulator of filopodia stability

Filopodia are adhesive cellular protrusions specialised in the detection of extracellular matrix (ECM)-derived cues. While ECM engagement at focal adhesions is known to trigger the recruitment of hundreds of proteins (\"adhesome\") to fine-tune cellular behaviour, the components of the filopodia adhesions remain undefined. Here, we performed a structured illumination microscopy-based screen to map the localisation of 80 target proteins, linked to cell adhesion and migration, within filopodia. We demonstrate preferential enrichment of several adhesion proteins to either filopodia tips, filopodia shafts, or shaft subdomains suggesting divergent, spatially restricted functions for these proteins. Moreover, proteins with phospho-inositide (PI) binding sites are particularly enriched in filopodia. This, together with the strong localisation of PI(3,4)P2 in filopodia tips, predicts critical roles for PIs in regulating filopodia ultra-structure and function. Our mapping further reveals that filopodia adhesions consist of a unique set of proteins, the filopodome, that are distinct from classical nascent adhesions, focal adhesions and fibrillar adhesions. Using live imaging, we observe that filopodia adhesions can give rise to nascent adhesions, which, in turn, form focal adhesions. Finally, we demonstrate that p130Cas (BCAR1) is recruited to filopodia tips via its CCHD domain and acts as a mechanosensitive regulator of filopodia stability.

cell biology↗

bioRxiv2018

GGA2 and RAB13 regulate activity-dependent β1-integrin recycling

{beta}1-integrins mediate cell-matrix interactions and their trafficking is important in the dynamic regulation of cell adhesion, migration and malignant processes like cancer cell invasion. Here we employ an RNAi screen to characterize regulators of integrin traffic and identify the association of Golgi-localized gamma ear-containing Arf-binding protein 2 (GGA2) with {beta}1-integrin and its role in recycling of the active but not inactive {beta}1-integrin receptors. Silencing of GGA2 limits active {beta}1-integrin levels in focal adhesions and decreases cancer cell migration and invasion congruent with its ability to regulate the dynamics of active integrins. Using the proximity-dependent biotin identification (BioID) method, we identify two RAB family small GTPases, RAB13 and RAB10, associating with GGA2 and {beta}1-integrin. Functionally, RAB13 silencing triggers the intracellular accumulation of active {beta}1-integrin, reduces integrin activity, in focal adhesions, and cell migration, similarly to GGA2 depletion, indicating that both facilitate active {beta}1-integrin recycling the plasma membrane. Thus, GGA2 and RAB13 are important specificity determinants for integrin activity-dependent traffic.

cell biology↗

bioRxiv2018

SORLA-driven endosomal trafficking regulates the oncogenic fitness of HER2

Human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. Endosomal trafficking of many other receptor tyrosine kinases regulates their oncogenic signaling, but the prevailing view is that HER2 is retained on the cell surface. Here we reveal that in cancer cells Sortilin related receptor 1 (SORLA; SORL1) forms a complex with HER2 and regulates its subcellular distribution by promoting recycling of endosomal HER2 back to plasma membrane. Expression of SORLA in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA targets HER2 to late endosomal/lysosomal compartments, impairs HER2-driven signaling and in vivo tumor growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.

cell biology↗