Use of phosphotyrosine-containing peptides to target SH2 domains: Antagonist peptides of the Crk/CrkL-p130Cas axis.

Protein-protein interactions between SH2 domains and segments of proteins that include a post-translationally phosphorylated tyrosine residue (pY) underpin numerous signal transduction cascades that allow cells to respond to their environment. Dysregulation of the writing, erasing, and reading of these posttranslational modifications is a hallmark of human disease, notably cancer. Elucidating the precise role of the SH2 domain-containing adaptor proteins Crk and CrkL in tumor cell migration and invasion is challenging because there are no specific and potent antagonists available. Crk and CrkL SH2s interact with a region of the docking protein p130Cas containing 15 potential pY-containing tetrapeptide motifs. This chapter summarizes recent efforts toward peptide antagonists for this Crk/CrkL-p130Cas interaction. We describe our protocol for recombinant expression and purification of Crk and CrkL SH2s for functional assays and our procedure to determine the consensus binding motif from the p130Cas sequence. To develop a more potent antagonist, we employ methods often associated with structure-based drug design. Computational docking using Rosetta FlexPepDock, which accounts for peptides having a greater number of conformational degrees of freedom than small organic molecules that typically constitute libraries, provides quantitative docking metrics to prioritize candidate peptides for experimental testing. A battery of biophysical assays, including fluorescence polarization, differential scanning fluorimetry and saturation transfer difference nuclear magnetic resonance spectroscopy, were employed to assess the candidates. In parallel, GST pulldown competition assays characterized protein-protein binding in vitro. Taken together, our methodology yields peptide antagonists of the Crk/CrkL-p130Cas axis that will be used to validate targets, assess druggability, foster in vitro assay development, and potentially serve as lead compounds for therapeutic intervention.

FLOT1 promotes gastric cancer progression and metastasis through BCAR1/ERK signaling.

Flotillin-1 (FLOT1) is a member of the flotillin family and serves as a hallmark of lipid rafts involved in the process of signaling transduction and vesicular trafficking. Here, we find FLOT1 promotes gastric cancer cell progression and metastasis by interacting with BCAR1, through ERK signaling. FLOT1 regulates BCAR1 phosphorylation and translocation. Overexpression of FLOT1 increases, while knockdown of FLOT1 decreases gastric cancer cell proliferation, migration and invasion. BCAR1 knockdown could block FLOT1 induced gastric cancer cell proliferation, migration and invasion. Re-expression of wildtype rather than mutant BCAR1 (Y410F) could partially restore FLOT1 knockdown induced gastric cancer cell migration and invasion, while the restore could be inhibited by ERK inhibitor. Furthermore, FLOT1 and BCAR1 expression is closely related to gastric cancer patients' poor outcome. Thus, our findings confirm that BCAR1 mediates FLOT1 induced gastric cancer progression and metastasis through ERK signaling, which may provide a novel pathway for gastric cancer treatment.

Cas phosphorylation regulates focal adhesion assembly.

Integrin-mediated cell attachment rapidly induces tyrosine kinase signaling. Despite years of research, the role of this signaling in integrin activation and focal adhesion assembly is unclear. We provide evidence that the Src-family kinase (SFK) substrate Cas (Crk-associated substrate, p130Cas, BCAR1) is phosphorylated and associated with its Crk/CrkL effectors in clusters that are precursors of focal adhesions. The initial phospho-Cas clusters contain integrin ß1 in its inactive, bent closed, conformation. Later, phospho-Cas and total Cas levels decrease as integrin ß1 is activated and core focal adhesion proteins including vinculin, talin, kindlin, and paxillin are recruited. Cas is required for cell spreading and focal adhesion assembly in epithelial and fibroblast cells on collagen and fibronectin. Cas cluster formation requires Cas, Crk/CrkL, SFKs, and Rac1 but not vinculin. Rac1 provides positive feedback onto Cas through reactive oxygen, opposed by negative feedback from the ubiquitin proteasome system. The results suggest a two-step model for focal adhesion assembly in which clusters of phospho-Cas, effectors and inactive integrin ß1 grow through positive feedback prior to integrin activation and recruitment of core focal adhesion proteins.

The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells.

Tumor suppressor p53 and its related proteins, p63 and p73, can be synthesized as multiple isoforms lacking part of the N- or C-terminal regions. Specifically, high expression of the ΔNp73α isoform is notoriously associated with various human malignancies characterized by poor prognosis. This isoform is also accumulated by oncogenic viruses, such as Epstein-Barr virus (EBV), as well as genus beta human papillomaviruses (HPV) that appear to be involved in carcinogenesis. To gain additional insight into ΔNp73α mechanisms, we have performed proteomics analyses using human keratinocytes transformed by the E6 and E7 proteins of the beta-HPV type 38 virus as an experimental model (38HK). We find that ΔNp73α associates with the E2F4/p130 repressor complex through a direct interaction with E2F4. This interaction is favored by the N-terminal truncation of p73 characteristic of ΔNp73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of ΔNp73 isoforms (α, ß, γ, δ, ε, ζ, θ, η, and η1). We show that the ΔNp73α-E2F4/p130 complex inhibits the expression of specific genes, including genes encoding for negative regulators of proliferation, both in 38HK and in HPV-negative cancer-derived cell lines. Such genes are not inhibited by E2F4/p130 in primary keratinocytes lacking ΔNp73α, indicating that the interaction with ΔNp73α rewires the E2F4 transcriptional program. In conclusion, we have identified and characterized a novel transcriptional regulatory complex with potential implications in oncogenesis. IMPORTANCE The TP53 gene is mutated in about 50% of human cancers. In contrast, the TP63 and TP73 genes are rarely mutated but rather expressed as ΔNp63 and ΔNp73 isoforms in a wide range of malignancies, where they act as p53 antagonists. Accumulation of ΔNp63 and ΔNp73, which is associated with chemoresistance, can result from infection by oncogenic viruses such as EBV or HPV. Our study focuses on the highly carcinogenic ΔNp73α isoform and uses a viral model of cellular transformation. We unveil a physical interaction between ΔNp73α and the E2F4/p130 complex involved in cell cycle control, which rewires the E2F4/p130 transcriptional program. Our work shows that ΔNp73 isoforms can establish interactions with proteins that do not bind to the TAp73α tumor suppressor. This situation is analogous to the gain-of-function interactions of p53 mutants supporting cellular proliferation.

p130Cas is required for androgen-dependent postnatal development regulation of submandibular glands.

Salivary glands develop through epithelial-mesenchymal interactions and are formed through repeated branching. The Crk-associated substrate protein (p130Cas) serves as an adapter that forms a complex with various proteins via integrin and growth factor signaling, with important regulatory roles in several essential cellular processes. We found that p130Cas is expressed in ductal epithelial cells of the submandibular gland (SMG). We generated epithelial tissue-specific p130Cas-deficient (p130CasΔepi-) mice and aimed to investigate the physiological role of p130Cas in the postnatal development of salivary glands. Histological analysis showed immature development of granular convoluted tubules (GCT) of the SMG in male p130CasΔepi- mice. Immunofluorescence staining showed that nuclear-localized androgen receptors (AR) were specifically decreased in GCT cells in p130CasΔepi- mice. Furthermore, epidermal growth factor-positive secretory granules contained in GCT cells were significantly reduced in p130CasΔepi- mice with downregulated AR signaling. GCTs lacking p130Cas showed reduced numbers and size of secretory granules, disrupted subcellular localization of the cis-Golgi matrix protein GM130, and sparse endoplasmic reticulum membranes in GCT cells. These results suggest that p130Cas plays a crucial role in androgen-dependent GCT development accompanied with ER-Golgi network formation in SMG by regulating the AR signaling.

BTG1 inactivation drives lymphomagenesis and promotes lymphoma dissemination through activation of BCAR1.

Understanding the functional role of mutated genes in cancer is required to translate the findings of cancer genomics into therapeutic improvement. BTG1 is recurrently mutated in the MCD/C5 subtype of diffuse large B-cell lymphoma (DLBCL), which is associated with extranodal dissemination. Here, we provide evidence that Btg1 knock out accelerates the development of a lethal lymphoproliferative disease driven by Bcl2 overexpression. Furthermore, we show that the scaffolding protein BCAR1 is a BTG1 partner. Moreover, after BTG1 deletion or expression of BTG1 mutations observed in patients with DLBCL, the overactivation of the BCAR1-RAC1 pathway confers increased migration ability in vitro and in vivo. These modifications are targetable with the SRC inhibitor dasatinib, which opens novel therapeutic opportunities in BTG1 mutated DLBCL.

Endothelial p130cas confers resistance to anti-angiogenesis therapy.

Anti-angiogenic therapies, such as anti-VEGF antibodies (AVAs), have shown promise in clinical settings. However, adaptive resistance to such therapies occurs frequently. We use orthotopic ovarian cancer models with AVA-adaptive resistance to investigate the underlying mechanisms. Genomic profiling of AVA-resistant tumors guides us to endothelial p130cas. We find that bevacizumab induces cleavage of VEGFR2 in endothelial cells by caspase-10 and that VEGFR2 fragments internalize into the nucleus and autophagosomes. Nuclear VEGFR2 and p130cas fragments, together with TNKS1BP1 (tankyrase-1-binding protein), initiate endothelial cell death. Blockade of autophagy in AVA-resistant endothelial cells retains VEGFR2 at the membrane with bevacizumab treatment. Targeting host p130cas with RGD (Arg-Gly-Asp)-tagged nanoparticles or genomic ablation of vascular p130cas in p130casflox/floxTie2Cre mice significantly extends the survival of mice with AVA-resistant ovarian tumors. Higher vascular p130cas is associated with shorter survival of individuals with ovarian cancer. Our findings identify opportunities for new strategies to overcome adaptive resistance to AVA therapy.

Docking Protein p130Cas Regulates Acinar to Ductal Metaplasia During Pancreatic Adenocarcinoma Development and Pancreatitis.

BACKGROUND & AIMS: Acinar to ductal metaplasia is the prerequisite for the initiation of Kras-driven pancreatic ductal adenocarcinoma (PDAC), and candidate genes regulating this process are emerging from genome-wide association studies. The adaptor protein p130Cas emerged as a potential PDAC susceptibility gene and a Kras-synthetic lethal interactor in pancreatic cell lines; however, its role in PDAC development has remained largely unknown. METHODS: Human PDAC samples and murine KrasG12D-dependent pancreatic cancer models of increasing aggressiveness were used. p130Cas was conditionally ablated in pancreatic cancer models to investigate its role during Kras-induced tumorigenesis. RESULTS: We found that high expression of p130Cas is frequently detected in PDAC and correlates with higher histologic grade and poor prognosis. In a model of Kras-driven PDAC, loss of p130Cas inhibits tumor development and potently extends median survival. Deletion of p130Cas suppresses acinar-derived tumorigenesis and progression by means of repressing PI3K-AKT signaling, even in the presence of a worsening condition like pancreatitis. CONCLUSIONS: Our observations finally demonstrated that p130Cas acts downstream of Kras to boost the PI3K activity required for acinar to ductal metaplasia and subsequent tumor initiation. This demonstrates an unexpected driving role of p130Cas downstream of Kras through PI3K/AKT, thus indicating a rational therapeutic strategy of targeting the PI3K pathway in tumors with high expression of p130Cas.

The unique function of p130Cas in regulating the bone metabolism.

p130 Crk-associated substrate (Cas) functions as an adapter protein and plays important roles in certain cell functions, such as cell proliferation, spreading, migration, and invasion. Furthermore, it has recently been reported to have a new function as a mechanosensor. Since bone is a tissue that is constantly under gravity, it is exposed to mechanical stress. Mechanical unloading, such as in a microgravity environment in space or during bed rest, leads to a decrease in bone mass because of the suppression of bone formation and the stimulation of bone resorption. Osteoclasts are multinucleated bone-resorbing giant cells that recognize bone and then form a ruffled border in the resorption lacuna. p130Cas is a molecule located downstream of c-Src that is important for the formation of a ruffled border in osteoclasts. Indeed, osteoclast-specific p130Cas-deficient mice exhibit osteopetrosis due to osteoclast dysfunction, similar to c-Src-deficient mice. Osteoblasts subjected to mechanical stress induce both the phosphorylation of p130Cas and osteoblast differentiation. In osteocytes, mechanical stress regulates bone mass by shuttling p130Cas between the cytoplasm and nucleus. Oral squamous cell carcinoma (OSCC) cells express p130Cas more strongly than epithelial cells in normal tissues. The knockdown of p130Cas in OSCC cells suppressed the cell growth, the expression of receptor activator of NF-κB ligand, which induces osteoclast formation, and bone invasion by OSCC. Taken together, these findings suggest that p130Cas might be a novel therapeutic target molecule in bone diseases, such as osteoporosis, rheumatoid arthritis, bone loss due to bed rest, and bone invasion and metastasis of cancer.

Deletion of epithelial cell-specific p130Cas impairs the maturation stage of amelogenesis.

Amelogenesis consists of secretory, transition, maturation, and post-maturation stages, and the morphological changes of ameloblasts at each stage are closely related to their function. p130 Crk-associated substrate (Cas) is a scaffold protein that modulates essential cellular processes, including cell adhesion, cytoskeletal changes, and polarization. The expression of p130Cas was observed from the secretory stage to the maturation stage in ameloblasts. Epithelial cell-specific p130Cas-deficient (p130CasΔepi-) mice exhibited enamel hypomineralization with chalk-like white mandibular incisors in young mice and attrition in aged mouse molars. A micro-computed tomography analysis and Vickers micro-hardness testing showed thinner enamel, lower enamel mineral density and hardness in p130CasΔepi- mice in comparison to p130Casflox/flox mice. Scanning electron microscopy, and an energy dispersive X-ray spectroscopy analysis indicated the disturbance of the enamel rod structure and lower Ca and P contents in p130CasΔepi- mice, respectively. The disorganized arrangement of ameloblasts, especially in the maturation stage, was observed in p130CasΔepi- mice. Furthermore, expression levels of enamel matrix proteins, such as amelogenin and ameloblastin in the secretory stage, and functional markers, such as alkaline phosphatase and iron accumulation, and Na+/Ca2++K+-exchanger in the maturation stage were reduced in p130CasΔepi- mice. These findings suggest that p130Cas plays important roles in amelogenesis (197 words).

Protumor Effects of Histone H3-H4 Chaperone Antisilencing Feature 1B Gene on Lung Adenocarcinoma: In Silico and In Vitro Analyses.

BACKGROUND: ASF1B is a member of the histone H3-H4 chaperone antisilencing feature 1 (ASF1). ASF1B reportedly acts as an oncogene in several cancers including, breast cancer and cervical cancer. To date, the role of ASF1B in lung adenocarcinoma (LUAD) is not elucidated. METHODS: The TCGA database, containing data for 33 cancer types, was used to explore the dysregulation and prognostic value of the ASF1B gene in pan-cancer data. R software packages and public databases/webservers were applied for bioinformatics and statistical analyses. Using in vitro models, immunoprecipitation and immunofluorescence were utilized to investigate if BCAR1 interacted with ASF1B in LUAD. Further, transfection experiments were performed to validate the expression pattern of ASF1B in LUAD and examine its regulating role in tumor-associated processes including tumor cell proliferation and migration. RESULTS: ASF1B was found to be significantly elevated in LUAD and the majority of cancer types, except PCPG (pheochromocytoma and paraganglioma). The overexpression of ASF1B was associated with worse prognostic outcomes in most cancer types including LUAD. ASF1B was associated with lymph node metastasis, and in vitro, it promoted the proliferation and migration of LUAD cells. ASF1B knockdown suppressed LUAD cell proliferation and migration and also diminished the expression of cell cycle, metastasis, and EMT signaling-associated proteins. BCAR1 was found positively correlated and interacting with ASF1B, and BCAR1 overexpression reversed the effects of ASF1B knockdown in LUAD cells. CONCLUSION: These findings indicated that ASF1B plays a significant role in the tumor progression of LUAD and BCAR1 mediates the tumor-promotive effects of ASF1B, acting as an intermediate protein. Therefore, the ASF1B/BCAR1 axis might be regarded as a putative therapeutic target for LUAD.

p130Cas Is Correlated with EREG Expression and a Prognostic Factor Depending on Colorectal Cancer Stage and Localization Reducing FOLFIRI Efficacy.

p130 Crk-associated substrate (p130Cas) is associated with poor prognosis and treatment resistance in breast and lung cancers. To elucidate p130Cas functional and clinical role in colorectal cancer (CRC) progression/therapy resistance, we performed cell culture experiments and bioinformatic/statistical analyses of clinical data sets. p130Cas expression was associated with poor survival in the cancer genome atlas (TCGA) data set. Knockdown/reconstitution experiments showed that p130Cas drives migration but, unexpectedly, inhibits proliferation in CRC cells. TCGA data analyses identified the growth factor epiregulin (EREG) as inversely correlated with p130Cas. p130Cas knockdown and simultaneous EREG treatment further enhanced proliferation. RNA interference and EREG treatment experiments suggested that p130Cas/EREG limit each other's expression/activity. Inverse p130Cas/EREG Spearman correlations were prominent in right-sided and earlier stage CRC. p130Cas was inducible by 5-fluorouracil (5-FU) and FOLFIRI (folinic acid, 5-FU, irinotecan), and p130Cas and EREG were upregulated in distant metastases (GSE121418). Positive p130Cas/EREG correlations were observed in metastases, preferentially in post-treatment samples (especially pulmonary metastases). p130Cas knockdown sensitized CRC cells to FOLFIRI independent of EREG treatment. RNA sequencing and gene ontology analyses revealed that p130Cas is involved in cytochrome P450 drug metabolism and epithelial-mesenchymal transition. p130Cas expression was associated with poor survival in right-sided, stage I/II, MSS (microsatellite stable), or BRAF-mutated CRC. In summary, p130Cas represents a prognostic factor and potential therapeutic target in CRC.

BCAR1 plays critical roles in the formation and immunoevasion of invasive circulating tumor cells in lung adenocarcinoma.

Background: We investigated the roles of breast cancer anti-estrogen resistance 1 (BCAR1/p130Cas) in the formation and immunoevasion of invasive circulating tumor cells (CTCs) in lung adenocarcinoma (LUAD). Methods: Biomarkers of CTCs including BCAR1 and CD274, were evaluated by the CanPatrol method. Proteomics analysis of LUAD cells and exosomes after BCAR1 overexpression (BCAR1-OE) was performed by mass spectrometry. Cell functions and relevant signaling pathways were investigated after BCAR1 knockdown (BCAR1-KO) or BCAR1-OE in LUAD cells. Lastly, in vitro and in vivo experiments were performed to confirm the roles of BCAR1 in the formation and immunoevasion of CTCs. Results: High expression of BCAR1 by CTCs correlated with CD274 expression and epithelial-to-mesenchymal transition (EMT). RAC1, together with BCAR1, was found to play an important role in the carcinogenesis of LUAD. RAC1 functioned with BCAR1 to induce EMT and to enhance cell proliferation, colony formation, cell invasion and migration, and anoikis resistance in LUAD cells. BCAR1 up-regulated CD274 expression probably by shuttling the short isoform of BRD4 (BRD4-S) into the nucleus. CTCs, as well as tumor formation, were prohibited in nude mice xenografted with BCAR1-KO cells. The co-expression of BCAR1/RAC1 and BCAR1/CD274 was confirmed in LUAD. BCAR1 expression in LUAD is an indicator of poor prognosis, and it associates with immunoevasion. Conclusion: BCAR1, as a new target for the treatment of LUAD, plays roles in the formation and immunoevasion of invasive CTCs. The mechanism includes triggering EMT via RAC1 signaling and up-regulating CD274 expression by shuttling BRD4-S into the nucleus.

A Cas-BCAR3 co-regulatory circuit controls lamellipodia dynamics.

Integrin adhesion complexes regulate cytoskeletal dynamics during cell migration. Adhesion activates phosphorylation of integrin-associated signaling proteins, including Cas (p130Cas, BCAR1), by Src-family kinases. Cas regulates leading-edge protrusion and migration in cooperation with its binding partner, BCAR3. However, it has been unclear how Cas and BCAR3 cooperate. Here, using normal epithelial cells, we find that BCAR3 localization to integrin adhesions requires Cas. In return, Cas phosphorylation, as well as lamellipodia dynamics and cell migration, requires BCAR3. These functions require the BCAR3 SH2 domain and a specific phosphorylation site, Tyr 117, that is also required for BCAR3 downregulation by the ubiquitin-proteasome system. These findings place BCAR3 in a co-regulatory positive-feedback circuit with Cas, with BCAR3 requiring Cas for localization and Cas requiring BCAR3 for activation and downstream signaling. The use of a single phosphorylation site in BCAR3 for activation and degradation ensures reliable negative feedback by the ubiquitin-proteasome system.

Proteasomal degradation of p130 facilitate cell cycle deregulation and impairment of cellular differentiation in high-risk Human Papillomavirus 16 and 18 E7 transfected cells.

The High-Risk Human Papillomaviruses (HR-HPVs) 16 and 18 are known to cause cervical cancer, which is primarily attributed to E6 and E7 oncoproteins. In addition, recent studies have focused on the vital role of the p130 pocket protein as an oncosuppressor to limit the expression of E2F transcription factors required for cell cycle progression. In view of this, the current study was conducted to investigate the mechanism by which transfection with HPV16/18 E7 leads to the deregulation of the host cell cycle, altering the localisation of p130, and expression of differentiation genes in Human Keratinocytes (HaCaT) cells. Co-immunoprecipitation, Western blot analysis, immunofluorescence microscopy, flow cytometry, quantitative-Polymerase Chain Reaction (qPCR), and the inhibition of p130 by MG132 inhibitor were employed to investigate the loss of p130 and its disruption in HPV 16/18 E7-transfected HaCaT cells. The HPV16- and HPV18-transformed cells, known as CaSki and HeLa, respectively, were also used to complement the ectopic expressions of E7 in HaCaT cells. Normal keratinocytes displayed higher level of p130 expression than HPV-transformed cells. In addition, the immunofluorescence analysis revealed that both HPV 16/18 E7-transfected HaCaT and HPV-transformed cells exhibited higher level of cytoplasmic p130 compared to nuclear p130. A significant increase in the number of S/G2 phase cells in HPV-transformed cells was also recorded since E7 has been shown to stimulate proliferation through the deactivation of Retinoblastoma Protein (pRB)-dependent G1/S checkpoint. Furthermore, the findings recorded the down-regulation of keratinocyte differentiation markers, namely p130, keratin10, and involucrin. The proteasomal degradation of the exported p130 confirmed the cellular localisation pattern of p130, which was commonly observed in cancerous cells. The findings provide strong evidence that the localisation of nuclear p130 nuclear was disrupted by HPV16/18 E7 led to the deregulation of the cell cycle and the impairment of cellular differentiation ultimately lead to cellular transformation.

Targeting p130Cas- and microtubule-dependent MYC regulation sensitizes pancreatic cancer to ERK MAPK inhibition.

To identify therapeutic targets for KRAS mutant pancreatic cancer, we conduct a druggable genome small interfering RNA (siRNA) screen and determine that suppression of BCAR1 sensitizes pancreatic cancer cells to ERK inhibition. Integrative analysis of genome-scale CRISPR-Cas9 screens also identify BCAR1 as a top synthetic lethal interactor with mutant KRAS. BCAR1 encodes the SRC substrate p130Cas. We determine that SRC-inhibitor-mediated suppression of p130Cas phosphorylation impairs MYC transcription through a DOCK1-RAC1-ß-catenin-dependent mechanism. Additionally, genetic suppression of TUBB3, encoding the ßIII-tubulin subunit of microtubules, or pharmacological inhibition of microtubule function decreases levels of MYC protein in a calpain-dependent manner and potently sensitizes pancreatic cancer cells to ERK inhibition. Accordingly, the combination of a dual SRC/tubulin inhibitor with an ERK inhibitor cooperates to reduce MYC protein and synergistically suppress the growth of KRAS mutant pancreatic cancer. Thus, we demonstrate that mechanistically diverse combinations with ERK inhibition suppress MYC to impair pancreatic cancer proliferation.

The role of E26 transformation-specific variant transcription factor 5 in colorectal cancer cell proliferation and cell cycle progression.

E26 transformation-specific variant transcription factor 5 (ETV5) contributes to tumor growth and progression and promotes colorectal cancer (CRC) angiogenesis. Previous studies indicate that ETV5 may regulate the cell cycle, but its detailed mechanism remain unclear. Gene Ontology (GO) analysis of RNA-seq data revealed that ETV5 possibly regulates the cell cycle in CRC. Here, in vitro and in vivo experiments were performed to verify that ETV5 promoted tumor progression and influenced cell cycle G1/S transition. Cell cycle PCR array and co-immunoprecipitation (Co-IP) helped identify the p21-CDKs pathway. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays were performed to determine whether ETV5 binds to the p21 promoter. ETV5 and p21 were detected by immunohistochemistry, and the effects of their expression on CRC patients were evaluated. ETV5 upregulation enhanced tumor proliferative capacity and promoted G1 phase transfer to the S phase. ETV5 knockdown slowed the growth of CRC cells and repressed the G1/S transition. We also found p21 as a downstream target of ETV5. p21 knockdown resulted in faster CRC cell growth and in more cells being driven from the G0/1 phase into the S phase. Co-IP experiments showed that p21 banding to CDK2, CDK4, and CDK6 inhibited p130 phosphorylation. Using the ChIP and luciferase reporter assay, we confirmed that ETV5 bound to the p21 promoter and repressed p21 expression. CRC patients with high ETV5 expression and low p21 expression showed the worst prognosis. Finally, by targeting p21 to regulate CDK function, ETV5 also changed drug-sensitivity to palbociclib and dinaciclib. In conclusion, ETV5 promoted cell cycle G1/S transition through transcriptional inhibition of p21, thereby accelerating tumor growth. Moreover, ETV5 changed drug-sensitivity to palbociclib and dinaciclib. Therefore, therapeutic regimens targeting ETV5 may be promising in improving the efficacy of target-CDK treatment in CRC.

Suppression of macrophage migration by down-regulating Src/FAK/P130Cas activation contributed to the anti-inflammatory activity of sinomenine.

A large number of macrophages in inflamed sites not only amplify the severity of inflammatory responses but also contribute to the deleterious progression of many chronic inflammatory diseases, autoimmune diseases and cancers. Macrophage migration is a prerequisite for their entry into inflammatory sites and their participation of macrophages in the pathologic processes. Inhibition of macrophage migration is therefore a potential anti-inflammatory mechanism. Moreover, alleviation of inflammation also prevents the macrophages infiltration. Sinomenine (SIN) is an alkaloid derived from the Chinese medicinal plant Sinomenium acutum. It has multiple pharmacological effects, including anti-inflammation, immunosuppression, and anti-arthritis. However, its anti-inflammatory molecular mechanisms and effect on macrophage migration are not fully understood. The purpose of this research was to investigate the pharmacological effects and the molecular mechanism of SIN on macrophage migration in vivo and in vitro as well as to elucidate its anti-inflammatory mechanisms associated with macrophage migration. Our results showed that SIN reduced the number of RAW264.7 cells migrating into inflammatory paws and blocked lipopolysaccharide (LPS)-induced RAW264.7 cells and bone marrow-derived macrophages (BMDMs) migration in vitro. Furthermore, SIN attenuated the 3D mesenchymal migration of BMDMs. The absence of macrophage migration after circulatory and periphery macrophages depletion led to a reduction in the severity of inflammatory response. In macrophages depleted (macrophages-/-) mice, as inflammatory severity decreased, RAW264.7 cells migration was suppressed. A non-obvious effect of SIN on the inflammatory response was found in macrophages-/- mice, while the inhibitory effect of SIN on RAW264.7 cells migration was still observed. Furthermore, the migration of RAW264.7 cells pre-treated with SIN was suppressed in normal mice. Finally, Src/focal adhesion kinase (FAK)/P130Cas axis activation, which supports macrophages mesenchymal migration, and iNOS expression, NO production, integrin αV and in integrin ß3 expressions, which promote Src/FAK/P130Cas activation, were down-regulated by SIN. However, SIN had no obvious effect on the expression of the monocyte chemoattractant protein-1 (MCP-1), which is an important chemokine for macrophage migration. These results indicated that SIN significantly inhibited macrophage mesenchymal migration by down-regulating on Src/FAK/P130Cas axis activation. There was a mutual regulatory correlation between the inflammatory response and macrophage migration, and the effects of SIN on macrophage migration were involved in its anti-inflammatory activity.

Mutant TP53 interacts with BCAR1 to contribute to cancer cell invasion.

BACKGROUND: Mutant TP53 interacts with other proteins to produce gain-of-function properties that contribute to cancer metastasis. However, the underlying mechanisms are still not fully understood. METHODS: Using immunoprecipitation and proximity ligation assays, we evaluated breast cancer anti-estrogen resistance 1 (BCAR1) as a novel binding partner of TP53R273H, a TP53 mutant frequently found in human cancers. The biological functions of their binding were examined by the transwell invasion assay. Clinical outcome of patients was analysed based on TP53 status and BCAR1 expression using public database. RESULTS: We discovered a novel interaction between TP53R273H and BCAR1. We found that BCAR1 translocates from the cytoplasm into the nucleus and binds to TP53R273H in a manner dependent on SRC family kinases (SFKs), which are known to enhance metastasis. The expression of full-length TP53R273H, but not the BCAR1 binding-deficient mutant TP53R273HΔ102-207, promoted cancer cell invasion. Furthermore, among the patients with mutant TP53, high BCAR1 expression was associated with a poorer prognosis. CONCLUSIONS: The interaction between TP53R273H and BCAR1 plays an important role in enhancing cancer cell invasion. Thus, our study suggests a disruption of the TP53R273H-BCAR1 binding as a potential therapeutic approach for TP53R273H-harbouring cancer patients.

Src kinase: Key effector in mechanosignalling.

Cells have developed a unique set of molecular mechanisms that allows them to probe mechanical properties of the surrounding environment. These systems are based on deformable primary mechanosensors coupled to tension transmitting proteins and enzymes generating biochemical signals. This modular setup enables to transform a mechanical load into more versatile biochemical information. Src kinase appears to be one of the central components of the mechanotransduction network mediating force-induced signalling across multiple cellular contexts. In tight cooperation with primary sensors and the cytoskeleton, Src functions as an effector molecule necessary for transformation of mechanical stimuli into biochemical outputs executing cellular response and adaptation to mechanical cues.