DLC1 promotes mechanotransductive feedback for YAP via RhoGAP-mediated focal adhesion turnover.

Angiogenesis is a tightly controlled dynamic process demanding a delicate equilibrium between pro-angiogenic signals and factors that promote vascular stability. The spatiotemporal activation of the transcriptional co-factors YAP (herein referring to YAP1) and TAZ (also known WWTR1), collectively denoted YAP/TAZ, is crucial to allow for efficient collective endothelial migration in angiogenesis. The focal adhesion protein deleted-in-liver-cancer-1 (DLC1) was recently described as a transcriptional downstream target of YAP/TAZ in endothelial cells. In this study, we uncover a negative feedback loop between DLC1 expression and YAP activity during collective migration and sprouting angiogenesis. In particular, our study demonstrates that signaling via the RhoGAP domain of DLC1 reduces nuclear localization of YAP and its transcriptional activity. Moreover, the RhoGAP activity of DLC1 is essential for YAP-mediated cellular processes, including the regulation of focal adhesion turnover, traction forces, and sprouting angiogenesis. We show that DLC1 restricts intracellular cytoskeletal tension by inhibiting Rho signaling at the basal adhesion plane, consequently reducing nuclear YAP localization. Collectively, these findings underscore the significance of DLC1 expression levels and its function in mitigating intracellular tension as a pivotal mechanotransductive feedback mechanism that finely tunes YAP activity throughout the process of sprouting angiogenesis.

ITIH5 as a multifaceted player in pancreatic cancer suppression, impairing tyrosine kinase signaling, cell adhesion and migration.

Inter-alpha-trypsin inhibitor heavy chain 5 (ITIH5) has been identified as a metastasis suppressor gene in pancreatic cancer. Here, we analyzed ITIH5 promoter methylation and protein expression in The Cancer Genome Atlas (TCGA) dataset and three tissue microarray cohorts (n = 618), respectively. Cellular effects, including cell migration, focal adhesion formation and protein tyrosine kinase activity, induced by forced ITIH5 expression in pancreatic cancer cell lines were studied in stable transfectants. ITIH5 promoter hypermethylation was associated with unfavorable prognosis, while immunohistochemistry demonstrated loss of ITIH5 in the metastatic setting and worsened overall survival. Gain-of-function models showed a significant reduction in migration capacity, but no alteration in proliferation. Focal adhesions in cells re-expressing ITIH5 exhibited a smaller and more rounded phenotype, typical for slow-moving cells. An impressive increase of acetylated alpha-tubulin was observed in ITIH5-positive cells, indicating more stable microtubules. In addition, we found significantly decreased activities of kinases related to focal adhesion. Our results indicate that loss of ITIH5 in pancreatic cancer profoundly affects its molecular profile: ITIH5 potentially interferes with a variety of oncogenic signaling pathways, including the PI3K/AKT pathway. This may lead to altered cell migration and focal adhesion formation. These cellular alterations may contribute to the metastasis-inhibiting properties of ITIH5 in pancreatic cancer.

HPIP and RUFY3 are noncanonical guanine nucleotide exchange factors of Rab5 to regulate endocytosis-coupled focal adhesion turnover.

While the role of endocytosis in focal adhesion turnover-coupled cell migration has been established in addition to its conventional role in cellular functions, the molecular regulators and precise molecular mechanisms that underlie this process remain largely unknown. In this study, we report that proto-oncoprotein hematopoietic PBX-interacting protein (HPIP) localizes to focal adhesions as well as endosomal compartments along with RUN FYVE domain-containing protein 3 (RUFY3) and Rab5, an early endosomal protein. HPIP contains two coiled-coil domains (CC1 and CC2) that are necessary for its association with Rab5 and RUFY3 as CC domain double mutant, that is, mtHPIPΔCC1-2 failed to support it. Furthermore, we show that HPIP and RUFY3 activate Rab5 by serving as noncanonical guanine nucleotide exchange factors of Rab5. In support of this, either deletion of coiled-coil domains or silencing of HPIP or RUFY3 impairs Rab5 activation and Rab5-dependent cell migration. Mechanistic studies further revealed that loss of HPIP or RUFY3 expression severely impairs Rab5-mediated focal adhesion disassembly, FAK activation, fibronectin-associated-ß1 integrin trafficking, and thus cell migration. Together, this study underscores the importance of HPIP and RUFY3 as noncanonical guanine nucleotide exchange factors of Rab5 and in integrin trafficking and focal adhesion turnover, which implicates in cell migration.

Vinculin Y822 is an important determinant of ligand binding.

Vinculin is an actin-binding protein present at cell-matrix and cell-cell adhesions, which plays a critical role in bearing force experienced by cells and dissipating it onto the cytoskeleton. Recently, we identified a key tyrosine residue, Y822, whose phosphorylation plays a critical role in force transmission at cell-cell adhesions. The role of Y822 in human cancer remains unknown, even though Y822 is mutated to Y822C in uterine cancers. Here, we investigated the effect of this amino acid substitution and that of a phosphodeficient Y822F vinculin in cancer cells. We observed that the presence of the Y822C mutation led to cells that proliferate and migrate more rapidly and contained smaller focal adhesions when compared to cells with wild-type vinculin. In contrast, the presence of the Y822F mutation led to highly spread cells with larger focal adhesions and increased contractility. Furthermore, we provide evidence that Y822C vinculin forms a disulfide bond with paxillin, accounting for some of the elevated phosphorylated paxillin recruitment. Taken together, these data suggest that vinculin Y822 modulates the recruitment of ligands.

A novel focal adhesion-related risk model predicts prognosis of bladder cancer -- a bioinformatic study based on TCGA and GEO database.

BACKGROUND: Bladder cancer (BLCA) is the ninth most common cancer globally, as well as the fourth most common cancer in men, with an incidence of 7%. However, few effective prognostic biomarkers or models of BLCA are available at present. METHODS: The prognostic genes of BLCA were screened from one cohort of The Cancer Genome Atlas (TCGA) database through univariate Cox regression analysis and functionally annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The intersecting genes of the BLCA gene set and focal adhesion-related gene were obtained and subjected to the least absolute shrinkage and selection operator regression (LASSO) to construct a prognostic model. Gene set enrichment analysis (GSEA) of high- and low-risk patients was performed to explore further the biological process related to focal adhesion genes. Univariate and multivariate Cox analysis, receiver operating characteristic (ROC) curve analysis, and Kaplan-Meier survival analysis (KM) were used to evaluate the prognostic model. DNA methylation analysis was presented to explore the relationship between prognosis and gene methylation. Furthermore, immune cell infiltration was assessed by CIBERSORT, ESTIMATE, and TIMER. The model was verified in an external GSE32894 cohort of the Gene Expression Omnibus (GEO) database, and the Prognoscan database presented further validation of genes. The HPA database validated the related protein level, and functional experiments verified significant risk factors in the model. RESULTS: VCL, COL6A1, RAC3, PDGFD, JUN, LAMA2, and ITGB6 were used to construct a prognostic model in the TCGA-BLCA cohort and validated in the GSE32894 cohort. The 7-gene model successfully stratified the patients into both cohorts' high- and low-risk groups. The higher risk score was associated with a worse prognosis. CONCLUSIONS: The 7-gene prognostic model can classify BLCA patients into high- and low-risk groups based on the risk score and predict the overall survival, which may aid clinical decision-making.

Focal adhesion proteins in hepatocellular carcinoma: RSU1 a novel tumour suppressor with prognostic significance.

AIM: Hepatocellular carcinoma (HCC) is a common cause a cancer-related death. Focal adhesions (FAs) represent multiprotein complexes at integrin-mediated cell-extracellular matrix adhesion sites that orchestrate vital cellular functions. The heterotrimeric ILK-PINCH-PARVB (IPP) complex, RSU1, a PINCH binding protein and CTEN, a member of the tensin family of proteins exert a critical role in FAs, where they regulate important cancer related functions such as cell adhesion, migration, proliferation and survival. Previous studies implicate these FA proteins in liver pathophysiology but their detailed role in human HCC is not fully understood. Here in we investigated expression and function of IPP, RSU1 and CTEN in human HCC. METHODS: The expression of focal adhesion proteins was studied in human HCC by immunohistochemistry in relation to clinicopathological parameters, previous studied genomic instability markers and patient's survival. Effects on cell proliferation and FA proteins expression upon ILK inhibition and RSU1 silencing were also investigated in HCC in vitro. RESULTS: IPP complex and CTEN proteins are overexpressed while RSU1 expression is decreased in human HCC. CTEN expression correlates with reduced patients' survival while RSU1 represents an independent favorable prognostic indicator in human HCC. Nuclear ILK expression correlates with markers of genomic instability. Pharmacological targeting of ILK suppresses, while RSU1 silencing promotes cell growth of HCC cells in vitro, while in both experimental conditions expression and/or localization of focal adhesion proteins is deregulated. CONCLUSION: Our results suggest that FA signaling is implicated in hepatocellular carcinogenesis with prognostic significance. RSU1 seems to exert tumor suppressive functions in HCC and represents a novel favorable prognostic indicator.

Integrin ß-like 1 protein (ITGBL1) promotes cell migration by preferentially inhibiting integrin-ECM binding at the trailing edge.

BACKGROUND: Cell migration is a basic cellular behavior involved in multiple phenomena in the human body such as embryonic development, wound healing, immune reactions, and cancer metastasis. For proper cell migration, integrin and the ECM binding complex must be disassembled for the retraction of trailing edges. OBJECTIVE: Integrin must be differentially regulated at leading edges or trailing edges during cell migration. Previously, we showed that ITGBL1 was a secreted protein and inhibits integrin activity. Therefore, we examined the function of ITGBL1 on the retraction of trailing edges during cell migration. METHODS: To examined the function of ITGBL1 on cell migration, we knocked-down or overexpressed ITGBL1 by using ITGBL1 siRNA or ITGBL1 plasmid DNA in human chondrocytes or ATDC5 cells. We then characterized cellular migration and directionality by performing wound healing assays. Also, to analyze leading-edge formation and trailing-edge retraction, we labeled cell membranes with membrane-GFP and performed live imaging of migrating cells and. Finally, we specifically detected active forms of integrin, FAK and Vinculin using specific antibodies upon ITGBL1 depletion or overexpression. RESULT: In this study, ITGBL1 preferentially inhibited integrin activity at the trailing edges to promote cell migration. ITGBL1-depleted cells showed increased focal adhesions at the membranous traces of trailing edges to prevent the retraction of trailing edges. In contrast, overexpression of ITGBL1 upregulated directional cell migration by promoting focal adhesion disassembly at the trailing edges. CONCLUSION: ITGBL1 facilitates directional cell migration by promoting disassembly of the trailing edge focal adhesion complex.

lncRNA ITGB8-AS1 functions as a ceRNA to promote colorectal cancer growth and migration through integrin-mediated focal adhesion signaling.

Long non-coding RNAs (lncRNAs) play critical roles in tumorigenesis and progression of colorectal cancer (CRC). However, functions of most lncRNAs in CRC and their molecular mechanisms remain uncharacterized. Here we found that lncRNA ITGB8-AS1 was highly expressed in CRC. Knockdown of ITGB8-AS1 suppressed cell proliferation, colony formation, and tumor growth in CRC, suggesting oncogenic roles of ITGB8-AS1. Transcriptomic analysis followed by KEGG analysis revealed that focal adhesion signaling was the most significantly enriched pathway for genes positively regulated by ITGB8-AS1. Consistently, knockdown of ITGB8-AS1 attenuated the phosphorylation of SRC, ERK, and p38 MAPK. Mechanistically, ITGB8-AS1 could sponge miR-33b-5p and let-7c-5p/let-7d-5p to regulate the expression of integrin family genes ITGA3 and ITGB3, respectively, in the cytosol of cells. Targeting ITGB8-AS1 using antisense oligonucleotide (ASO) markedly reduced cell proliferation and tumor growth in CRC, indicating the therapeutic potential of ITGB8-AS1 in CRC. Furthermore, ITGB8-AS1 was easily detected in plasma of CRC patients, which was positively correlated with differentiation and TNM stage, as well as plasma levels of ITGA3 and ITGB3. In conclusion, ITGB8-AS1 functions as a competing endogenous RNA (ceRNA) to regulate cell proliferation and tumor growth of CRC via regulating focal adhesion signaling. Targeting ITGB8-AS1 is effective in suppressing CRC cell growth and tumor growth. Elevated plasma levels of ITGB8-AS1 were detected in advanced-stage CRC. Thus, ITGB8-AS1 could serve as a potential therapeutic target and circulating biomarker in CRC.

YAP facilitates melanoma migration through regulation of actin-related protein 2/3 complex subunit 5 (ARPC5).

Yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are transcriptional coactivators that have been implicated in driving metastasis and progression in many cancers, mainly through their transcriptional regulation of downstream targets. Although YAP and TAZ have shown redundancy in many contexts, it is still unknown whether or not this is true in melanoma. Here, we show that while both YAP and TAZ are expressed in a panel of melanoma cell lines, depletion of YAP results in decreased cell numbers, focal adhesions, and the ability to invade matrigel. Using non-biased RNA-sequencing analysis, we find that melanoma cells depleted of YAP, TAZ, or YAP/TAZ exhibit drastically different transcriptomes. We further uncover the ARP2/3 subunit ARPC5 as a specific target of YAP but not TAZ and that ARPC5 is essential for YAP-dependent maintenance of melanoma cell focal adhesion numbers. Our findings suggest that in melanoma, YAP drives melanoma progression, survival, and invasion.

Nuclear pore protein NUP210 depletion suppresses metastasis through heterochromatin-mediated disruption of tumor cell mechanical response.

Mechanical signals from the extracellular microenvironment have been implicated in tumor and metastatic progression. Here, we identify nucleoporin NUP210 as a metastasis susceptibility gene for human estrogen receptor positive (ER+) breast cancer and a cellular mechanosensor. Nup210 depletion suppresses lung metastasis in mouse models of breast cancer. Mechanistically, NUP210 interacts with LINC complex protein SUN2 which connects the nucleus to the cytoskeleton. In addition, the NUP210/SUN2 complex interacts with chromatin via the short isoform of BRD4 and histone H3.1/H3.2 at the nuclear periphery. In Nup210 knockout cells, mechanosensitive genes accumulate H3K27me3 heterochromatin modification, mediated by the polycomb repressive complex 2 and differentially reposition within the nucleus. Transcriptional repression in Nup210 knockout cells results in defective mechanotransduction and focal adhesion necessary for their metastatic capacity. Our study provides an important role of nuclear pore protein in cellular mechanosensation and metastasis.

The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids.

Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds.

ARHGAP4-SEPT2-SEPT9 complex enables both up- and down-modulation of integrin-mediated focal adhesions, cell migration, and invasion.

The Rho family of GTPases are inactivated in a cell context-dependent manner by Rho-GTPase-activating proteins (Rho-GAPs), but their signaling mechanisms are poorly understood. Here we demonstrate that ARHGAP4, one of the Rho-GAPs, forms a complex with SEPT2 and SEPT9 via its Rho-GAP domain and SH3 domain to enable both up- and down-modulation of integrin-mediated focal adhesions (FAs). We show that silencing ARHGAP4 and overexpressing its two mutually independent upstream regulators, SEPT2 and SEPT9, all induce reorganization of FAs to newly express Integrin Beta 1 and also enhance both cell migration and invasion. Interestingly, even if these cell migration/invasion-associated phenotypic changes are induced upon perturbations to the complex, it does not necessarily cause enhanced clustering of FAs. Instead, its extent depends on whether the microenvironment contains ligands suitable for the up-regulated Integrin Beta 1. These results provide novel insights into cell migration, invasion, and microenvironment-dependent phenotypic changes regulated by the newly identified complex.

Campylobacter jejuni Triggers Signaling through Host Cell Focal Adhesions To Inhibit Cell Motility.

Campylobacter jejuni is a major foodborne pathogen that exploits the focal adhesions of intestinal cells to promote invasion and cause severe gastritis. Focal adhesions are multiprotein complexes involved in bidirectional signaling between the actin cytoskeleton and the extracellular matrix. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected cells using a comprehensive set of approaches, including confocal microscopy of live and fixed cells, immunoblotting, and superresolution interferometric photoactivated localization microscopy (iPALM). We found that C. jejuni infection of epithelial cells results in increased focal adhesion size and altered topology. These changes resulted in a persistent modulatory effect on the host cell focal adhesion, evidenced by an increase in cell adhesion strength, a decrease in individual cell motility, and a reduction in collective cell migration. We discovered that C. jejuni infection causes an increase in phosphorylation of paxillin and an alteration of paxillin turnover at the focal adhesion, which together represent a potential mechanistic basis for altered cell motility. Finally, we observed that infection of epithelial cells with the C. jejuni wild-type strain in the presence of a protein synthesis inhibitor, a C. jejuni CadF and FlpA fibronectin-binding protein mutant, or a C. jejuni flagellar export mutant blunts paxillin phosphorylation and partially reestablishes individual host cell motility and collective cell migration. These findings provide a potential mechanism for the restricted intestinal repair observed in C. jejuni-infected animals and raise the possibility that bacteria targeting extracellular matrix components can alter cell behavior after binding and internalization by manipulating focal adhesions. IMPORTANCE Campylobacter jejuni is a major foodborne pathogen that causes severe gastritis. We investigated the dynamics of focal adhesion structure and function in C. jejuni-infected epithelial cells. Focal adhesions act as signaling complexes that connect the extracellular matrix to the intracellular cytoskeleton. The key findings of this study show that C. jejuni changes the structure (size and position), composition, and function of cellular focal adhesions using a combination of virulence factors. Mechanistically, we found that the changes in focal adhesion dynamics are dependent upon the activation of host cell signaling pathways, which affect the assembly and disassembly of cellular proteins from the focal adhesion. To summarize, we have identified a new cellular phenotype in C. jejuni-infected cells that may be responsible for the restricted intestinal repair observed in C. jejuni-infected animals.

An alternate covalent form of platelet αIIbß3 integrin that resides in focal adhesions and has altered function.

The αIIbß3 integrin receptor coordinates platelet adhesion, activation, and mechanosensing in thrombosis and hemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in ∼1 in 3 integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbß3 is predetermined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts transfected with recombinant integrin. From coimmunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in baby hamster kidney fibroblast cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signaling but extended residency time in focal adhesions due to a reduced rate of clathrin-mediated integrin internalization and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein 2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbß3 integrin receptor is produced in various covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits, and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that the alternate integrin form and function are also conserved.

CCM3 is a gatekeeper in focal adhesions regulating mechanotransduction and YAP/TAZ signalling.

The YAP/TAZ transcriptional programme is not only a well-established driver of cancer progression and metastasis but also an important stimulator of tissue regeneration. Here we identified Cerebral cavernous malformations 3 (CCM3) as a regulator of mechanical cue-driven YAP/TAZ signalling, controlling both tumour progression and stem cell differentiation. We demonstrate that CCM3 localizes to focal adhesion sites in cancer-associated fibroblasts, where it regulates mechanotransduction and YAP/TAZ activation. Mechanistically, CCM3 and focal adhesion kinase (FAK) mutually compete for binding to paxillin to fine-tune FAK/Src/paxillin-driven mechanotransduction and YAP/TAZ activation. In mouse models of breast cancer, specific loss of CCM3 in cancer-associated fibroblasts leads to exacerbated tissue remodelling and force transmission to the matrix, resulting in reciprocal YAP/TAZ activation in the neighbouring tumour cells and dissemination of metastasis to distant organs. Similarly, CCM3 regulates the differentiation of mesenchymal stromal/stem cells. In conclusion, CCM3 is a gatekeeper in focal adhesions that controls mechanotransduction and YAP/TAZ signalling.

MAGI1, a Scaffold Protein with Tumor Suppressive and Vascular Functions.

MAGI1 is a cytoplasmic scaffolding protein initially identified as a component of cell-to-cell contacts stabilizing cadherin-mediated cell-cell adhesion in epithelial and endothelial cells. Clinical-pathological and experimental evidence indicates that MAGI1 expression is decreased in some inflammatory diseases, and also in several cancers, including hepatocellular carcinoma, colorectal, cervical, breast, brain, and gastric cancers and appears to act as a tumor suppressor, modulating the activity of oncogenic pathways such as the PI3K/AKT and the Wnt/ß-catenin pathways. Genomic mutations and other mechanisms such as mechanical stress or inflammation have been described to regulate MAGI1 expression. Intriguingly, in breast and colorectal cancers, MAGI1 expression is induced by non-steroidal anti-inflammatory drugs (NSAIDs), suggesting a role in mediating the tumor suppressive activity of NSAIDs. More recently, MAGI1 was found to localize at mature focal adhesion and to regulate integrin-mediated adhesion and signaling in endothelial cells. Here, we review MAGI1's role as scaffolding protein, recent developments in the understanding of MAGI1 function as tumor suppressor gene, its role in endothelial cells and its implication in cancer and vascular biology. We also discuss outstanding questions about its regulation and potential translational implications in oncology.

Exploration of Alternative Splicing Events in Mesenchymal Stem Cells from Human Induced Pluripotent Stem Cells.

Although comparative genome-wide transcriptomic analysis has provided insight into the biology of human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs), the distinct alternative splicing (AS) signatures of iMSCs remain elusive. Here, we performed Illumina RNA sequencing analysis to characterize AS events in iMSCs compared with tissue-derived MSCs. A total of 4586 differentially expressed genes (|FC| > 2) were identified between iMSCs and umbilical cord blood-derived MSCs (UCB-MSCs), including 2169 upregulated and 2417 downregulated genes. Of these, 164 differentially spliced events (BF > 20) in 112 genes were identified between iMSCs and UCB-MSCs. The predominant type of AS found in iMSCs was skipped exons (43.3%), followed by retained introns (19.5%), alternative 3' (15.2%) and 5' (12.8%) splice sites, and mutually exclusive exons (9.1%). Functional enrichment analysis showed that the differentially spliced genes (|FC| > 2 and BF > 20) were mainly enriched in functions associated with focal adhesion, extracellular exosomes, extracellular matrix organization, cell adhesion, and actin binding. Splice isoforms of selected genes including TRPT1, CNN2, and AP1G2, identified in sashimi plots, were further validated by RT-PCR analysis. This study provides valuable insight into the biology of iMSCs and the translation of mechanistic understanding of iMSCs into therapeutic applications.

A genetic variant conferred high expression of CAV2 promotes pancreatic cancer progression and associates with poor prognosis.

AIM: This study aimed to identify the functional genes and genetic variants associated with the prognosis of pancreatic ductal adenocarcinoma (PDAC) and reveal the mechanism underlying their prognostic roles. METHODS: First, we implement a two-stage exome-wide association study in a total of 1070 patients to identify the genetic variant correlated with PDAC prognosis. Then we performed fine mapping through bioinformatics analysis and dual-luciferase reporter assays to reveal the causal functional variant and prognostic gene. Next, we established the gene knockdown, knockout, and overexpression cell lines with small interfering RNA, CRISPR/Cas9, and lentivirus, respectively, and investigated the gene function on cell proliferation and migration in vivo and in vitro. Finally, we performed the RNA-seq to elucidate downstream genes and mechanisms altering PDAC prognosis. RESULTS: We identified the CAV1-CAV2 locus tagged by rs8940 was significantly associated with PDAC prognosis, and rs10249656 in the 3'untranslated region of CAV2 was the real functional variant, which upregulated CAV2 expression through abolishing miR-548s binding. We observed upregulated CAV2 in PDAC and the higher expression correlated with worse prognosis. Transient knockdown of CAV2 inhibited PDAC migration without affecting proliferation rate. Knockout of CAV2 suppressed PDAC progression and metastasis, whereas stable overexpression of CAV2 promoted. Overexpressed CAV2 promoted the PDAC progression and metastasis via perturbing genes in the focal adhesion (CCND1, IGTA1, and ZYX) and extracellular matrix organisation (PLOD2, CAST, and ITGA1) pathways mechanically. CONCLUSION: These findings shed light on an important role of CAV2 on PDAC progression and the prognostic impact of its genetic variation.

A novel focal adhesion related gene signature for prognostic prediction in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly heterogeneous disease. Reduced expression of focal adhesion is considered as an important prerequisite for tumor cell invasion and metastasis. However, the prognostic value of focal adhesion related genes in HCC remains to be further determined. In this study, RNA expression profiles were downloaded from public databases. A five focal adhesion related gene signature model was established by the least absolute shrinkage and selection operator Cox regression analysis, which categorized patients into high- and low-risk groups. Multivariate Cox regression analysis showed that the risk score was an independent predictor for overall survival. Single-sample gene set enrichment analysis revealed that immune status was different between the two risk groups, and tumor-related pathways were enriched in high-risk group. The risk score was significantly associated with tumor grade, tumor stage, immune scores, and immune infiltrate types. Pearson correlation showed that the expression level of prognostic genes was associated with anti-tumor drug sensitivity. Besides, the mRNA and protein expression of prognostic genes was significantly different between HCC tissues and adjacent non-tumorous tissues in our separate cohort. Taken together, a novel focal adhesion related gene signature can be used for prognostic prediction in HCC, which may be a therapeutic alternative.

Cell shape-based chemical screening reveals an epigenetic network mediated by focal adhesions.

Adapter proteins CRK and CRKL participate in a variety of signaling pathways, including cell adhesion, and fate regulation of mammalian cells. However, the molecular functions of CRK/CRKL in epigenetic regulation remain largely unknown. Here, we developed a pipeline to evaluate cell morphology using high-content image analysis combined with chemical screening of kinase and epigenetic modulators. We found that CRK/CRKL modulates gene regulatory networks associated with cell morphology through epigenetic alteration in mouse embryonic fibroblasts. Integrated epigenome and transcriptome analyses revealed that CRK/CRKL is involved in super-enhancer activity and upregulation of Cdt1, Rin1, and Spp1 expression for the regulation of cell morphology. Screening of a library of 80 epigenetic inhibitors showed that histone H3 modifiers, euchromatic histone methyltransferase 2 and mitogen- and stress-activated kinase 1, may be important for CRK/CRKL-mediated morphological changes. Taken together, our results indicate that CRK/CRKL plays a critical role in gene regulatory networks through epigenetic modification. DATABASES: Chromatin immunoprecipitation sequencing and RNA sequencing data were deposited in the DNA Data Bank of Japan under DRA011080 and DRA011081 accession numbers, respectively.