The dysadherin/FAK axis promotes individual cell migration in colon cancer.

Dysregulation of cancer cell motility is a key driver of invasion and metastasis. High dysadherin expression in cancer cells is correlated with invasion and metastasis. Here, we found the molecular mechanism by which dysadherin regulates the migration and invasion of colon cancer (CC). Comprehensive analysis using single-cell RNA sequencing data from CC patients revealed that high dysadherin expression in cells is linked to cell migration-related gene signatures. We confirmed that the deletion of dysadherin in tumor cells hindered local invasion and distant migration using in vivo tumor models. In this context, by performing cell morphological analysis, we found that aberrant cell migration resulted from impaired actin dynamics, focal adhesion turnover and protrusive structure formation upon dysadherin expression. Mechanistically, the activation of focal adhesion kinase (FAK) was observed in dysadherin-enriched cells. The dysadherin/FAK axis enhanced cell migration and invasion by activating the FAK downstream cascade, which includes the Rho family of small GTPases. Overall, this study illuminates the role of dysadherin in modulating cancer cell migration by forcing actin dynamics and protrusive structure formation via FAK signaling, indicating that targeting dysadherin may be a potential therapeutic strategy for CC patients.

Tumor microenvironment-induced FOXM1 regulates ovarian cancer stemness.

In ovarian tumors, the omental microenvironment profoundly influences the behavior of cancer cells and sustains the acquisition of stem-like traits, with major impacts on tumor aggressiveness and relapse. Here, we leverage a patient-derived platform of organotypic cultures to study the crosstalk between the tumor microenvironment and ovarian cancer stem cells. We discovered that the pro-tumorigenic transcription factor FOXM1 is specifically induced by the microenvironment in ovarian cancer stem cells, through activation of FAK/YAP signaling. The microenvironment-induced FOXM1 sustains stemness, and its inactivation reduces cancer stem cells survival in the omental niche and enhances their response to the PARP inhibitor Olaparib. By unveiling the novel role of FOXM1 in ovarian cancer stemness, our findings highlight patient-derived organotypic co-cultures as a powerful tool to capture clinically relevant mechanisms of the microenvironment/cancer stem cells crosstalk, contributing to the identification of tumor vulnerabilities.

Cancer-associated fibroblast-derived periostin promotes papillary thyroid tumor growth through integrin-FAK-STAT3 signaling.

Background: Periostin (POSTN) is a critical extracellular matrix protein in various tumor microenvironments. However, the function of POSTN in thyroid cancer progression remains largely unknown. Methods: Postn and Rag1 knock-out mice and orthotopic mouse models were used to determine the role of POSTN on papillary thyroid tumor progression. Immunofluorescence, cell co-culture, fluorescence in situ hybridization, chromatin immunoprecipitation assay, recombinant protein and inhibitor treatment were performed to explore the underlying mechanisms of POSTN-promoted papillary thyroid tumor growth. Results: POSTN is up-regulated in papillary thyroid tumors and negatively correlates with the overall survival of patients with thyroid cancer. Cancer-associated fibroblast (CAF)-derived POSTN promotes papillary thyroid tumor growth in vivo and in vitro. POSTN deficiency in CAFs significantly impairs CAF-promoted papillary thyroid tumor growth. POSTN promotes papillary thyroid tumor cell proliferation and IL-4 expression through integrin-FAK-STAT3 signaling. In turn, tumor cell-derived IL-4 induces the activation of CAFs and stimulates POSTN expression by activating STAT6. We reveal the crucial role of CAF-derived POSTN and tumor cell-derived IL-4 in driving the development of papillary thyroid tumors through the POSTN-integrin-FAK-STAT3-IL-4 pathway in tumor cells and IL-4-STAT6-POSTN signaling in CAFs. Conclusion: Our findings underscore the significance of POSTN and IL-4 as critical molecular mediators in the dynamic interplay between CAFs and tumor cells, ultimately supporting the growth of papillary thyroid tumors.

FOXS1 acts as an oncogene and induces EMT through FAK/PI3K/AKT pathway by upregulating HILPDA in prostate cancer.

Prostate cancer (PCa) is a widespread global health concern characterized by elevated rates of occurrence, and there is a need for novel therapeutic targets to enhance patient outcomes. FOXS1 is closely linked to different cancers, but its function in PCa is still unknown. The expression of FOXS1, its prognostic role, clinical significance in PCa, and the potential mechanism by which FOXS1 affects PCa progression were investigated through bioinformatics analysis utilizing public data. The levels of FOXS1 and HILPDA were evaluated in clinical PCa samples using various methods, such as western blotting, immunohistochemistry, and qRT-PCR. To examine the function and molecular mechanisms of FOXS1 in PCa, a combination of experimental techniques including CCK-8 assay, flow cytometry, wound-healing assay, Transwell assay, and Co-IP assay were employed. The FOXS1 expression levels were significantly raised in PCa, correlating strongly with tumor aggressiveness and an unfavorable prognosis. Regulating FOXS1 expression, whether upregulating or downregulating it, correspondingly enhanced or inhibited the growth, migration, and invasion capabilities of PCa cells. Mechanistically, we detected a direct interaction between FOXS1 and HILPDA, resulting in the pathway activation of FAK/PI3K/AKT and facilitation EMT in PCa cells. FOXS1 collaborates with HILPDA to initiate EMT, thereby facilitating the PCa progression through the FAK/PI3K/AKT pathway activation.

Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer.

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

Muscone restores anoikis sensitivity in TMZ-resistant glioblastoma cells by suppressing TOP2A via the EGFR/Integrin ß1/FAK signaling pathway.

BACKGROUND: Temozolomide (TMZ) resistance is the main obstacle faced by glioblastoma multiforme (GBM) treatment. Muscone, one of the primary active pharmacological ingredients of Shexiang (Moschus), can cross the blood-brain barrier (BBB) and is being investigated as an antineoplastic medication. However, muscone treatment for GBM has received little research, and its possible mechanisms are still unclear. PURPOSE: This study aims to evaluate the effect and the potential molecular mechanism of muscone on TMZ-resistant GBM cells. METHODS: The differentially expressed genes (DEGs) between TMZ-resistant GBM cells and TMZ-sensitive GBM cells were screened using GEO2R. By progressively raising the TMZ concentration, a relatively stable TMZ-resistant human GBM cell line was established. The drug-resistance traits of U251-TR cells were assessed via the CCK-8 assay and Western Blot analysis of MGMT and TOP2A expression. Cell viability, cell proliferation, cell migration ability, and drug synergism were detected by the CCK-8 assay, colony formation assay, wound healing assay, and drug interaction relationship test, respectively. Anoikis was quantified by Calcein-AM/EthD-1 staining, MTT assay, and flow cytometry. Measurements of cell cycle arrest, apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) were performed using cell cycle staining, Annexin V-FITC/PI labeling, JC-1 assay, and ROS assay, respectively. DNA damage was measured by TUNEL assay, alkaline comet assay, and γ-H2AX foci assay. GEPIA was used to investigate the link between the anoikis marker (FAK)/drug resistance gene and critical proteins in the EGFR/Integrin ß1 signaling pathway. Molecular docking was used to anticipate the probable targets of muscone. The intracellular co-localization and expression of EGFR and FAK were shown using immunofluorescence. The U251-TR cell line stably overexpressing EGFR was constructed using lentiviral transduction to assess the involvement of EGFR-related signaling in anoikis resistance. Western Blot was employed to detect the expression of migration-related proteins, cyclins, anoikis-related proteins, DNA damage/repair-related proteins, and associated pathway proteins. RESULTS: DEGs analysis identified 97 deregulated chemotherapy-resistant genes and 3779 upregulated genes in TMZ-resistant GBM cells. Subsequent experiments verified TMZ resistance and the hyper-expression of DNA repair-related genes (TOP2A and MGMT) in continuously low-dose TMZ-induced U251-TR cells. Muscone exhibited dose-dependent inhibition of U251-TR cell migration and proliferation, and its co-administration with TMZ showed the potential for enhanced therapeutic efficacy. By downregulating FAK, muscone reduced anoikis resistance in anchorage-independent U251-TR cells. It also caused cell cycle arrest in the G2/M phase by upregulating p21 and downregulating CDK1, CDK2, and Cyclin E1. Muscone-induced anoikis was accompanied by mitochondrial membrane potential collapse, ROS production, an increase in the BAX/Bcl-2 ratio, as well as elevated levels of Cytochrome c (Cyt c), cleaved caspase-9, and cleaved caspase-3. These findings indicated that muscone might trigger mitochondrial-dependent anoikis via ROS generation. Moreover, significant DNA damage, DNA double-strand breaks (DSBs), the formation of γ-H2AX foci, and a reduction in TOP2A expression are also associated with muscone-induced anoikis. Overexpression of EGFR in U251-TR cells boosted the expression of Integrin ß1, FAK, ß-Catenin, and TOP2A, whereas muscone suppressed the expression levels of EGFR, Integrin ß1, ß-Catenin, FAK, and TOP2A. Muscone may influence the expression of the key DNA repair enzyme, TOP2A, by suppressing the EGFR/Integrin ß1/FAK pathway. CONCLUSION: We first demonstrated that muscone suppressed TOP2A expression through the EGFR/Integrin ß1/FAK pathway, hence restoring anoikis sensitivity in TMZ-resistant GBM cells. These data suggest that muscone may be a promising co-therapeutic agent for enhancing GBM treatment, particularly in cases of TMZ-resistant GBM with elevated TOP2A expression.

CD276 enhances sunitinib resistance in clear cell renal cell carcinoma by promoting DNA damage repair and activation of FAK-MAPK signaling pathway.

OBJECTIVE: This study aimed to explore the effect of CD276 expression on the sunitinib sensitivity of clear cell renal cell carcinoma (ccRCC) cell and animal models and the potential mechanisms involved. METHODS: CD276 expression levels of ccRCC and normal samples were analyzed via online databases and real-time quantitative PCR (RT-qPCR). CD276 was knocked down in ccRCC cell models (sunitinib-resistant 786-O/R cells and sunitinib-sensitive 786-O cells) using shRNA transfection, and the cells were exposed to a sunitinib (2 µM) environment. Cells proliferation was then analyzed using MTT assay and colony formation experiment. Alkaline comet assay, immunofluorescent staining, and western blot experiments were conducted to assess the DNA damage repair ability of the cells. Western blot was also used to observe the activation of FAK-MAPK pathway within the cells. Finally, a nude mouse xenograft model was established and the nude mice were orally administered sunitinib (40 mg/kg/d) to evaluate the in vivo effects of CD276 knockdown on the therapeutic efficacy of sunitinib against ccRCC. RESULTS: CD276 was significantly upregulated in both ccRCC clinical tissue samples and cell models. In vitro experiments showed that knocking down CD276 reduced the survival rate, IC50 value, and colony-forming ability of ccRCC cells. Knocking down CD276 increased the comet tail moment (TM) values and γH2AX foci number, and reduced BRCA1 and RAD51 protein levels. Knocking down CD276 also decreased the levels of p-FAK, p-MEK, and p-ERK proteins. CONCLUSION: Knocking down CD276 effectively improved the sensitivity of ccRCC cell and animal models to sunitinib treatment.

A new invertebrate NPY-like polypeptide, ZoaNPY, from the Zoanthus sociatus, as a novel ligand of human NPY Y2 receptor rescues vascular insufficiency via PLC/PKC and Src- FAK-dependent signaling pathways.

Our recent multi-omics studies have revealed rich sources of novel bioactive proteins and polypeptides from marine organisms including cnidarians. In the present study, we initially conducted a transcriptomic analysis to review the composition profile of polypeptides from Zoanthus sociatus. Then, a newly discovered NPY-like polypeptide-ZoaNPY was selected for further in silico structural, binding and virtually pharmacological studies. To evaluate the pro-angiogenic effects of ZoaNPY, we employed an in vitro HUVECs model and an in vivo zebrafish model. Our results indicate that ZoaNPY, at 1-100 pmol, enhances cell survival, migration and tube formation in the endothelial cells. Besides, treatment with ZoaNPY could restore a chemically-induced vascular insufficiency in zebrafish embryos. Western blot results demonstrated the application of ZoaNPY could increase the phosphorylation of proteins related to angiogenesis signaling including PKC, PLC, FAK, Src, Akt, mTOR, MEK, and ERK1/2. Furthermore, through molecular docking and surface plasmon resonance (SPR) verification, ZoaNPY was shown to directly and physically interact with NPY Y2 receptor. In view of this, all evidence showed that the pro-angiogenic effects of ZoaNPY involve the activation of NPY Y2 receptor, thereby activating the Akt/mTOR, PLC/PKC, ERK/MEK and Src- FAK-dependent signaling pathways. Furthermore, in an excision wound model, the treatment with ZoaNPY was shown to accelerate the wound healing process in mice. Our findings provide new insights into the discovery and development of novel pro-angiogenic drugs derived from NPY-like polypeptides in the future.

The c-Abl-RACK1-FAK signaling axis promotes renal fibrosis in mice through regulating fibroblast-myofibroblast transition.

BACKGROUND: Renal fibrosis is a prevalent manifestation of chronic kidney disease (CKD), and effective treatments for this disease are currently lacking. Myofibroblasts, which originate from interstitial fibroblasts, aggregate in the renal interstitium, leading to significant accumulation of extracellular matrix and impairment of renal function. The nonreceptor tyrosine kinase c-Abl (encoded by the Abl1 gene) has been implicated in the development of renal fibrosis. However, the precise role of c-Abl in this process and its involvement in fibroblast-myofibroblast transition (FMT) remain poorly understood. METHODS: To investigate the effect of c-Abl in FMT during renal fibrosis, we investigated the expression of c-Abl in fibrotic renal tissues of patients with CKD and mouse models. We studied the phenotypic changes in fibroblast or myofibroblast-specific c-Abl conditional knockout mice. We explored the potential targets of c-Abl in NRK-49F fibroblasts. RESULTS: In this study, fibrotic mouse and cell models demonstrated that c-Abl deficiency in fibroblasts mitigated fibrosis by suppressing fibroblast activation, fibroblast-myofibroblast transition, and extracellular matrix deposition. Mechanistically, c-Abl maintains the stability of the RACK1 protein, which serves as a scaffold for proteins such as c-Abl and focal adhesion kinase at focal adhesions, driving fibroblast activation and differentiation during renal fibrosis. Moreover, specifically targeting c-Abl deletion in renal myofibroblasts could prove beneficial in established kidney fibrosis by reducing RACK1 expression and diminishing the extent of fibrosis. CONCLUSIONS: Our findings suggest that c-Abl plays a pathogenic role in interstitial fibrosis through the regulation of RACK1 protein stabilization and myofibroblast differentiation, suggesting a promising strategy for the treatment of CKD.

Effect of Focal Adhesion Kinase and Vinculin Expression on Migration Parameters of Normal and Tumor Epitheliocytes.

Focal adhesions (FAs) are mechanosensory structures that transform physical stimuli into chemical signals guiding cell migration. Comprehensive studies postulate correlation between the FA parameters and cell motility metrics for individual migrating cells. However, which properties of the FAs are critical for epithelial cell motility in a monolayer remains poorly elucidated. We used high-throughput microscopy to describe relationship between the FA parameters and cell migration in immortalized epithelial keratinocytes (HaCaT) and lung carcinoma cells (A549) with depleted or inhibited vinculin and focal adhesion kinase (FAK) FA proteins. To evaluate relationship between the FA morphology and cell migration, we used substrates with varying stiffness in the model of wound healing. Cells cultivated on fibronectin had the highest FA area values, migration rate, and upregulated expression of FAK and vinculin mRNAs, while the smallest FA area and slower migration rate to the wound were specific to cells cultivated on glass. Suppression of vinculin expression in both normal and tumor cells caused decrease of the FA size and fluorescence intensity but did not affect cell migration into the wound. In contrast, downregulation or inactivation of FAK did not affect the FA size but significantly slowed down the wound closure rate by both HaCaT and A549 cell lines. We also showed that the FAK knockdown results in the FA lifetime decrease for the cells cultivated both on glass and fibronectin. Our data indicate that the FA lifetime is the most important parameter defining migration of epithelial cells in a monolayer. The observed change in the cell migration rate in a monolayer caused by changes in expression/activation of FAK kinase makes FAK a promising target for anticancer therapy of lung carcinoma.

Biological evaluation of sulfonate and sulfate analogues of lithocholic acid: A bioisosterism-guided approach towards the discovery of potential sialyltransferase inhibitors for antimetastatic study.

The naturally occurring bile acid lithocholic acid (LCA) has been a crucial core structure for many non-sugar-containing sialyltranferase (ST) inhibitors documented in literature. With the aim of elucidating the impact of the terminal carboxyl acid substituent of LCA on its ST inhibition, in this present study, we report the (bio)isosteric replacement-based design and synthesis of sulfonate and sulfate analogues of LCA. Among these compounds, the sulfate analogue SPP-002 was found to selectively inhibit N-glycan sialylation by at least an order of magnitude, indicating a substantial improvement in both potency and selectivity when compared to the unmodified parent bile acid. Molecular docking analysis supported the stronger binding of the synthetic analogue in the enzyme active site. Treatment with SPP-002 also hampered the migration, adhesion, and invasion of MDA-MB-231 cells in vitro by suppressing the expression of signaling proteins involved in the cancer metastasis-associated integrin/FAK/paxillin pathway. In totality, these findings offer not only a novel structural scaffold but also valuable insights for the future development of more potent and selective ST inhibitors with potential therapeutic effects against tumor cancer metastasis.

Direct contact between tumor cells and platelets initiates a FAK-dependent F3/TGF-ß positive feedback loop that promotes tumor progression and EMT in osteosarcoma.

Platelets have received growing attention for their roles in hematogenous tumor metastasis. However, the tumor-platelet interaction in osteosarcoma (OS) remains poorly understood. Here, using platelet-specific focal adhesion kinase (FAK)-deficient mice, we uncover a FAK-dependent F3/TGF-ß positive feedback loop in OS. Disruption of the feedback loop by inhibition of F3, TGF-ß, or FAK significantly suppresses OS progression. We demonstrate that OS F3 initiated the feedback loop by increasing platelet TGF-ß secretion, and platelet-derived TGF-ß promoted OS F3 expression in turn and modulated OS EMT process. Immunofluorescence results indicate platelet infiltration in OS niche and we verified it was mediated by platelet FAK. In addition, platelet FAK was proved to mediate platelet adhesion to OS cells, which was vital for the initiation of F3/TGF-ß feedback loop. Collectively, these findings provide a rationale for novel therapeutic strategies targeting tumor-platelet interplay in metastatic OS.

LAMC1 attenuates neuronal apoptosis via FAK/PI3K/AKT signaling pathway after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The poor prognosis in patients with subarachnoid hemorrhage (SAH) is often attributed to neuronal apoptosis. Recent evidence suggests that Laminin subunit gamma 1 (LAMC1) is essential for cell survival and proliferation. However, the effects of LAMC1 on early brain injury after SAH and the underlying mechanisms are unknown. The current study aimed to reveal the anti-neuronal apoptotic effect and the potential mechanism of LAMC1 in the rat and in the in vitro SAH models. METHODS: The SAH model of Sprague-Dawley rats was established by endovascular perforation. Recombinant LAMC1 (rLAMC1) was administered intranasally 30 min after modeling. LAMC1 small interfering RNA (LAMC1 siRNA), focal adhesion kinase (FAK)-specific inhibitor Y15 and PI3K-specific inhibitor LY294002 were administered before SAH modeling to explore the neuroprotection mechanism of rLAMC1. HT22 cells were cultured and stimulated by oxyhemoglobin to establish an in vitro model of SAH. Subsequently, SAH grades, neurobehavioral tests, brain water content, blood-brain barrier permeability, western blotting, immunofluorescence, TUNEL, and Fluoro-Jade C staining were performed. RESULTS: The expression of endogenous LAMC1 was markedly decreased after SAH, both in vitro and in vivo. rLAMC1 significantly reduced the brain water content and blood-brain barrier permeability, improved short- and long-term neurobehavior, and decreased neuronal apoptosis. Furthermore, rLAMC1 treatment significantly increased the expression of p-FAK, p-PI3K, p-AKT, Bcl-XL, and Bcl-2 and decreased the expression of Bax and cleaved caspase -3. Conversely, knockdown of endogenous LAMC1 aggravated the neurological impairment, suppressed the expression of Bcl-XL and Bcl-2, and upregulated the expression of Bax and cleaved caspase-3. Additionally, the administration of Y15 and LY294002 abolished the protective roles of rLAMC1. In vitro, rLAMC1 significantly reduced neuronal apoptosis, and the protective effects were also abolished by Y15 and LY294002. CONCLUSION: Exogenous LAMC1 treatment improved neurological deficits after SAH in rats, and attenuated neuronal apoptosis in both in vitro and in vivo SAH models, at least partially through the FAK/PI3K/AKT pathway.

Comprehensive pan-cancer analysis of 33 human cancers reveals immunotherapeutic value of focal adhesion tyrosine kinase.

The immune environment in tumors is the key factor affecting the survival and immunotherapeutic response of patients. This research aimed to explore the underlying association between focal adhesion tyrosine kinase (FAK/PTK2) and cancer immunotherapy in 33 human cancers. Gene expression data and clinical features of 33 cancers were retrieved from the Cancer Genome Atlas Database. The immunotherapy cohorts included GSE67501, GSE78220, and IMVIGOR210, which were derived from the comprehensive gene expression database or from previous studies. Clinical parameters including patient age, gender, survival rate, and tumor stage were analyzed to evaluate the prognostic value of FAK/PTK2. FAK/PTK2 activity was detected by single-sample gene set enrichment analysis and used to compare the difference between FAK/PTK2 transcriptome and protein expression levels. To better understand the role of FAK/PTK2 in cancer immunotherapy, we analyzed its correlations with tumor microenvironment and with immune processes/elements (e.g., immune cell infiltration, immunosuppressants, and stimulants) and major histocompatible complexes. Potential pathways associated with FAK/PTK2 signaling in cancers were also explored. Correlations between FAK/PTK2 and 2 immunotherapeutic biomarkers (tumor mutation load and microsatellite instability) were studied. Finally, the 3 independent immunotherapy cohorts were used to study the relationship between FAK/PTK2 and immunotherapeutic response. Although FAK/PTK2 is not closely associated with age (13/33), gender (5/33), or tumor stage (5/33) in any of the studied human cancers, it has potential prognostic value for predicting patient survival. Consistency between FAK/PTK2 activity and expression exists in some cancers (3/33). Generally, FAK/PTK2 is robustly correlated with immune cell infiltration, immune modulators, and immunotherapeutic markers. Moreover, high FAK/PTK2 expression is significantly related to immune-relevant pathways. However, FAK/PTK2 is not significantly correlated with the immunotherapeutic response. Research on the immunotherapeutic value of FAK/PTK2 in 33 human cancers provides evidence regarding the function of FAK/PTK2 and its role in clinical treatment. However, given the use of a bioinformatics approach, our results are preliminary and require further validation.

IQGAP1 and NWASP promote human cancer cell dissemination and metastasis by regulating ß1-integrin via FAK and MRTF/SRF.

Attachment of circulating tumor cells to the endothelial cells (ECs) lining blood vessels is a critical step in cancer metastatic colonization, which leads to metastatic outgrowth. Breast and prostate cancers are common malignancies in women and men, respectively. Here, we observe that ß1-integrin is required for human prostate and breast cancer cell adhesion to ECs under shear-stress conditions in vitro and to lung blood vessel ECs in vivo. We identify IQGAP1 and neural Wiskott-Aldrich syndrome protein (NWASP) as regulators of ß1-integrin transcription and protein expression in prostate and breast cancer cells. IQGAP1 and NWASP depletion in cancer cells decreases adhesion to ECs in vitro and retention in the lung vasculature and metastatic lung nodule formation in vivo. Mechanistically, NWASP and IQGAP1 act downstream of Cdc42 to increase ß1-integrin expression both via extracellular signal-regulated kinase (ERK)/focal adhesion kinase signaling at the protein level and by myocardin-related transcription factor/serum response factor (SRF) transcriptionally. Our results identify IQGAP1 and NWASP as potential therapeutic targets to reduce early metastatic dissemination.

FAK, vinculin, and talin control mechanosensitive YAP nuclear localization.

Focal adhesions (FAs) are nanoscale complexes containing clustered integrin receptors and intracellular structural and signaling proteins that function as principal sites of mechanotransduction in part via promoting the nuclear translocation and activation of the transcriptional coactivator yes-associated protein (YAP). Knockdown of FA proteins such as focal adhesion kinase (FAK), talin, and vinculin can prevent YAP nuclear localization. However, the mechanism(s) of action remain poorly understood. Herein, we investigated the role of different functional domains in vinculin, talin, and FAK in regulating YAP nuclear localization. Using genetic or pharmacological inhibition of fibroblasts and human mesenchymal stem cells (hMSCs) adhering to deformable substrates, we find that disruption of vinculin-talin binding versus talin-FAK binding reduces YAP nuclear localization and transcriptional activity via different mechanisms. Disruption of vinculin-talin binding or knockdown of talin-1 reduces nuclear size, traction forces, and YAP nuclear localization. In contrast, disruption of the talin binding site on FAK or elimination of FAK catalytic activity did not alter nuclear size yet still prevented YAP nuclear localization and activity. These data support both nuclear tension-dependent and independent models for matrix stiffness-regulated YAP nuclear localization. Our results highlight the importance of vinculin-talin-FAK interactions at FAs of adherent cells, controlling YAP nuclear localization and activity.

Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.

Postbiotics of Lacticaseibacillus paracasei CECT 9610 and Lactiplantibacillus plantarum CECT 9608 attenuates store-operated calcium entry and FAK phosphorylation in colorectal cancer cells.

Store-operated Ca2+ entry (SOCE) is a major mechanism for Ca2+ influx in colorectal cancer (CRC) cells. This mechanism, regulated by the filling state of the intracellular Ca2+ stores, is mediated by the endoplasmic reticulum Ca2+ sensors of the stromal interaction molecules (STIM) family [stromal interaction molecule 1 (STIM1) and STIM2] and the Ca2+-release-activated Ca2+ channels constituted by Orai family members, with predominance of calcium release-activated calcium channel protein 1 (Orai1). CRC cells exhibit enhanced SOCE due to remodeling of the expression of the key SOCE molecular components. The enhanced SOCE supports a variety of cancer hallmarks. Here, we show that treatment of the colorectal adenocarcinoma cell lines HT-29 and Caco-2 with inanimate Lacticaseibacillus paracasei (CECT9610) and Lactiplantibacillus plantarum (CECT9608) attenuates SOCE, although no detectable effect is seen on SOCE in normal colon mucosa cells. The effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics was mediated by downregulation of Orai1 and STIM1, while the expression levels of Orai3 and STIM2 remained unaltered. Treatment of HT-29 and Caco-2 cells with inanimate Lacticaseibacillus paracasei and Lactiplantibacillus plantarum impairs in vitro migration by a mechanism likely involving attenuation of focal adhesion kinase (FAK) tyrosine phosphorylation. Cell treatment with the Orai1 inhibitor synta-66 attenuates SOCE and prevents any further effect of Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics. Together, our results indicate for the first time that Lacticaseibacillus paracasei and Lactiplantibacillus plantarum postbiotics selectively exert negative effects on Ca2+ influx through SOCE in colorectal adenocarcinoma cell lines, providing evidence for an attractive strategy against CRC.

The FAK/occludin/ZO-1 complex is critical for cadmium-induced testicular damage by disruption of the integrity of the blood-testis barrier in chickens.

Cadmium (Cd) is a well-known testis toxicant. The blood-testis barrier (BTB) is a crucial component of the testis. Cd can disrupt the integrity of the BTB and reproductive function. However, the mechanism of Cd-induced disruption of BTB and testicular damage has not been fully elucidated. Here, our study investigates the effects of Cd on BTB integrity and testicular dysfunction. 80 (aged 1 day) Hy-Line white variety chickens were randomly designed into 4 groups and treated for 90 days, as follows: control group (essential diet), 35 Cd, 70 Cd and 140 Cd groups (35, 70 and 140 mg/kg Cd). The results found that Cd exposure diminished volume of the testes and induced histopathological lesions in the testes. Exposure to Cd induced an inflammatory response, disrupted the structure and function of the FAK/occludin/ZO-1 protein complex and disrupted the tight junction and adherens junction in the BTB. In addition, Cd exposure reduced the expression of steroid-related proteins and inhibited testosterone synthesis. Taken together, these data elucidate that Cd disrupts the integrity of the BTB and further inhibits spermatogenesis by dissociating the FAK/occludin/ZO-1 complex, which provides a basis for further investigation into the mechanisms of Cd-induced impairment of male reproductive function and pharmacological protection.

FAK mediates hypoxia-induced pulmonary artery smooth muscle cell proliferation by modulating mitochondrial transcription termination factor 1/cyclin D1.

This study aimed to investigate the mechanism of FAK-dependent hypoxia-induced proliferation on human pulmonary artery smooth muscle cells (HPASMCs). Primary HPASMCs were isolated and cultured in vitro under normal and hypoxia conditions to assess cell proliferation with cell counting kit-8. FAK and mitochondrial transcription termination factor 1 (mTERF1) were silenced with siRNA, mRNA, and protein levels of FAK, mTERF1, and cyclin D1 were determined. HPASMC proliferation increased under hypoxia compared to normal conditions. Knocking down FAK or mTERF1 with siRNA led to decreased cell proliferation under both normal and hypoxia conditions. FAK knockdown led to the reduction of both mTERF1 and cyclin D1 expressions under the hypoxia conditions, whereas mTERF1 knockdown led to the downregulation of cyclin D1 expression but not FAK expression under the same condition. However, under normal conditions, knocking down either FAK or mTERF1 had no impact on cyclin D1 expression. These results suggested that FAK may regulate the mTERF1/cyclin D1 signaling pathway to modulate cell proliferation in hypoxia.