Vasodilator-stimulated Phosphoprotein Expression Research Articles

Clinical Significance of Detection of Peripheral Blood VASP Level in Lung Cancer Patients.

This paper explores the relationship between the clinical value of vasodilator-stimulated phosphoprotein (VASP) in lung cancer tissue and its diagnosis and severity. Totally 100 patients who were clinically diagnosed with lung cancer from January 2018 to December 2020 were enrolled in our study. They were assigned into two groups according to the presence of lymph node metastasis. The VASP levels were measured by flow cytometry. The correlation between the expression of VASP in tumor tissue and the clinical characteristics and prognosis in patients was analyzed. The diagnostic efficacy of plasma VASP with squamous cell carcinoma antigen (SCC), neuron-specific enolase (NSE), cytokeratin-19 fragment (CYFRA21-1), prosecretin-releasing peptide (proGRP), and lung cancer was analyzed. The results were compared with APACHE III score to evaluate the accuracy of VASP in determining the severity of patients. This paper finds that the value of VASP in the non-lymph node metastasis group was significantly higher than that in the healthy control group, and the VASP level in the lymph node metastasis group was significantly higher than that in the non-lymph node metastasis group and the healthy control group (all p values <0.05). The APACHE III score of the lymph node metastasis group was higher than that of the non-lymph node metastasis group (p value <0.05). The diagnostic efficacy of VASP is similar to that of SCC, NSE, CYFRA21-1, and proGRP. The plasma VASP value was statistically different in the survival group and the death group, with higher level observed in the death group compared to survival group (all p values <0.05). The value of plasma VASP alone and acute physiology and chronic health evaluations III (APACHE III) score for lung cancer mortality was similar (47.06% vs. 52.94%, p value >0.05). Similar accuracy was observed in VASP and APACHE III score in predicting mortality of lung cancer (84.37% vs. 85.77%, p value>0.05). This paper concludes that the level of VASP correlates to the severity of the lung cancer and survival of the patients.

Role of vasodilator-stimulated phosphoprotein in RANKL-differentiated murine macrophage RAW264.7 cells: Modulation of NF-κB, c-Fos and NFATc1 transcription factors.

Vasodilator-stimulated phosphoprotein (VASP) is essential for osteoclast differentiation, and reduced VASP expression results in depressed osteoclast differentiation. Previously, we demonstrated the importance of VASP and Ras-related C3 botulinum toxin substrate 1 interactions in osteosarcoma cell migration and metastasis using Mg-63 and Saos2 cells. However, the molecular details of the functional role of VASP in cell motility and migration remain to be elucidated. The present study demonstrated that VASP affects the expression of αV-integrin, tartrate-resistant acid phosphatase (TRAP) and lamellipodia protrusion in RAW 264.7 murine macrophage cells. The RAW 264.7 mouse monocyte macrophage cell line was used as an osteoclast precursor. RAW 264.7 cells were treated with 50 ng/ml of receptor activator of nuclear factor κ-Β ligand (RANKL) in order to induce cell differentiation (osteoclastogenesis). Small interfering RNA (siRNA) was used to silence VASP, and RT-PCR and western blotting were used to determine the expression for genes and proteins, respectively. TRAP staining as a histochemical marker for osteoclast and fluorescent microscopy for lamellipodia protrusion was performed. RANKL treatment significantly increased the gene and protein expression of VASP, αV-integrin and TRAP in RAW 264.7 cells. Silencing of VASP significantly reduced the RANKL-induced expression of αV-integrin, TRAP and lamellipodia protrusion. In addition, knockdown of VASP attenuated RANKL-stimulated activation of NF-κB, c-Fos and nuclear factor of activated T cells cytoplasmic 1 transcription factors, and the phosphorylation of the p65 and IκBα. These results suggest the critical role of VASP in regulating osteoclast differentiation, which should be further explored in osteosarcoma research.

LncRNA ATXN8OS enhances tamoxifen resistance in breast cancer.

BackgroundTamoxifen (TAMR) resistance remains a massive obstacle for breast cancer (BC) management. The precise parts of long non-coding RNA ataxin 8 opposite strand (ATXN8OS) in BC TAMR resistance have not been defined.MethodsThe levels of ATXN8OS, vasodilator-stimulated phosphoprotein (VASP), and miR-16-5p were assessed by quantitative real-time polymerase chain reaction or western blot. Colony formation and cell viability were analyzed by MTT and colony formation assays, respectively. Targeted interactions among miR-16-5p, ATXN8OS, and VASP were confirmed by dual-luciferase reporter assay. Animal studies were performed to observe the role of ATXN8OS in TAMR sensitivity in vivo.ResultsATXN8OS expression was increased in BC tissues and cells. ATXN8OS depletion promoted BC cell sensitivity to TAMR. ATXN8OS sequestered miR-16-5p by directly binding to miR-16-5p. The promotional effect of ATXN8OS knockdown on BC cell TAMR sensitivity was mediated by miR-16-5p. VASP was a direct target of miR-16-5p, and miR-16-5p overexpression enhanced TAMR sensitivity by VASP. Moreover, ATXN8OS regulated VASP expression by acting as a miR-16-5p sponge. In addition, ATXN8OS knockdown augmented BC TAMR sensitivity in vivo.ConclusionATXN8OS knockdown enhanced BC TAMR sensitivity partially through the miR-16-5p/VASP axis, highlighting a potential therapeutic target for improving the clinical benefits of TAMR treatment in BC patients.

Betulinic acid inhibits cell proliferation and migration in gastric cancer by targeting the NF-κB/VASP pathway

Gastric cancer (GC) is one of the most common malignant neoplasms of the digestive system, with China leading in terms of morbidity and mortality rates. Betulinic acid (BA) is a widely-occurring pentacyclic triterpenoid that has been reported to exhibit potent anti-inflammatory, antioxidant, and antitumor activities. BA can combat tumors by inducing apoptosis, regulating cell cycle, and inhibiting autophagy, but its mechanism of action in the context of GC is unclear. A preliminary study found that higher expression of vasodilator-stimulated phosphoprotein (VASP) was correlated with migration in the GC cell line. In this study, BGC-823 cells and MNK45 cells were treated with BA for investigating its effect on the proliferation and migration of cells. Moreover, the expression of VASP and upstream signal molecules were also investigated in this background. The results showed BA could inhibit the proliferation and migration the GC cells. Furthermore, NF-κB acted as a transcription factor to upregulate VASP expression. Moreover, BA could downregulate the expression of VASP at the protein and mRNA level by inhibiting NF-κB activity. In conclusion, these results suggest that BA could inhibit the expression of VASP by negatively regulating NF-κB, thereby inhibiting the proliferation and migration of the GC cells. Our study provides a theoretical basis for exploring the molecular mechanism underlying BA-induced inhibition of proliferation and migration in GC cells.

Up-regulation of SNHG15 facilitates cell proliferation, migration, invasion and suppresses cell apoptosis in breast cancer by regulating miR-411-5p/VASP axis.

Breast cancer (BC) is an intractable cancer with a rising incidence. Small nucleolar RNA host gene 15 (SNHG15) is a novel biomarker of multiple cancers. However, the molecular mechanism of SNHG15 during oncogenesis of BC is still poorly understood. Expression of SNHG15, microRNA (miR)-411-5p and vasodilator stimulated phosphoprotein (VASP) was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was evaluated by colony formation and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. Cell apoptosis was determined by flow cytometry and caspase-3 activity assay. Cell migration and invasion were examined by transwell assay. The interaction between miR-411-5p and SNHG15 or VASP was validated by dual-luciferase reporter assay. Protein expression of VASP, B cell lymphoma (Bcl-2), Bcl-2 associated X (Bax), vascular endothelial growth factor (VEGF), and matrix metalloproteinases (MMP-9, MMP-14) was measured by Western blot. Xenograft mice were established by subcutaneously injecting SKBR-3 cells transfected with sh-SNHG15 and sh-NC. SNHG15 and VASP were over-expressed whereas miR-411-5p was low-expressed in BC tumors and cells compared with the normal counterparts. Next, SNHG15 knockdown attenuated cell proliferation, migration, invasion and stimulated cell apoptosis in BC. In addition, SNHG15 acted as a sponge while VASP acted as a target of miR-411-5p. Rescue experiment revealed that miR-411-5p inhibitor could alleviate SNHG15 silencing-induced inhibitive effects on cell proliferation, migration, invasion and promotive effects on cell apoptosis. Similarly, VASP attenuated the regulatory effects of SNHG15 silencing on BC cell progression. Furthermore, SNHG15 elimination hindered tumor growth in vivo. SNHG15 contributes to BC cell progression by sponging miR-411-5p and enhancing VASP expression, providing essential biomarkers for BC therapy.

Interleukin 1β-mediated HOXC10 Overexpression Promotes Hepatocellular Carcinoma Metastasis by Upregulating PDPK1 and VASP.

Rationale: Metastasis and recurrence are the primary reasons for the high mortality rate of human hepatocellular carcinoma (HCC) patients. However, the exact mechanism underlying HCC metastasis remains unclear. The Homeobox (HOX) family proteins, which are a highly conserved transcription factor superfamily, play important roles in cancer metastasis. Here, we report a novel role of HOXC10, one of the most upregulated HOX genes in human HCC tissues, in promoting HCC metastasis.Methods: The expression of HOXC10 and its functional targets was detected by immunohistochemistry in two independent human HCC cohorts. Luciferase reporter and chromatin immunoprecipitation assays were used to measure the transcriptional regulation of target genes by HOXC10. The effect of HOXC10-mediated invasion and metastasis were analyzed by Transwell assays and by an orthotopic metastasis model.Results: Elevated expression of HOXC10 was positively correlated with the loss of tumor encapsulation and with higher tumor-nodule-metastasis (TNM) stage and poor prognosis in human HCC. Overexpression of HOXC10 promoted HCC metastasis by upregulating metastasis-related genes, including 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and vasodilator-stimulated phosphoprotein (VASP). Knockdown of PDPK1 and VASP inhibited HOXC10-enhanced HCC metastasis, whereas upregulation of PDPK1 and VASP rescued the decreased metastasis induced by HOXC10 knockdown. Interleukin-1β (IL-1β), which is the ligand of IL-1R1, upregulated HOXC10 expression through the c-Jun NH2-terminal kinase (JNK)/c-Jun pathway. HOXC10 knockdown significantly reduced IL-1β-mediated HCC metastasis. Furthermore, Anakinra, a specific antagonist of IL-1R1, inhibited IL-1β-induced HOXC10 upregulation and HCC metastasis. In human HCC tissues, HOXC10 expression was positively correlated with PDPK1, VASP and IL-1R1 expression, and patients with positive coexpression of HOXC10/PDPK1, HOXC10/VASP or IL-1R1/HOXC10 exhibited the poorest prognosis.Conclusions: Upregulated HOXC10 induced by IL-1β promotes HCC metastasis by transactivating PDPK1 and VASP expression. Thus, our study implicates HOXC10 as a prognostic biomarker, and targeting this pathway may be a promising therapeutic option for the clinical prevention of HCC metastasis.

Antitumor effects of saikosaponin b2 on breast cancer cell proliferation and migration.

Saikosaponin b2 (SSb2) can be extracted from Bupleurum spp. roots (Radix Bupleuri), which belongs to the Umbelliferae family. The current study aimed to explore the effects of SSb2 on proliferation of breast cancer cells and to identify the mechanism by which SSb2 affects breast cancer cell migration. mRNA expression levels of STAT3 and vasodilator‑stimulated phosphoprotein (VASP) were determined and increased expression was observed in 16 breast cancer tissues compared with the paracancerous tissues. MTT, wound healing, colony formation assays and western blot suggested that SSb2 inhibited MCF‑7 proliferation and migration. It was further identified by western blot analysis that SSb2 treatment reduced levels of phosphorylated STAT3, VASP, matrix metallopeptidase (MMP) 2 and MMP9 in MCF‑7 compared with the untreated cells. In addition, it was demonstrated that inhibition of STAT3 phosphorylation decreased VASP expression levels and induction of STAT3 phosphorylation increased VASP levels. Furthermore, it was observed that the treatment of Kunming mice with SSb2 at 30mg/kg/day for 30days induced no obvious changes in the liver or kidney tissues, as determined by haematoxylin and eosin staining. In conclusion, these results indicated that SSb2 may be a potential antitumor drug for the treatment of breast cancer, which acts by suppressing proliferation and migration by downregulating the STAT3 signalling pathway and inhibiting the expression of VASP, MMP2 and MMP9 expression.

The Increase of pFAK and THBS1 Protein and Gene Expression Levels in Vascular Smooth Muscle Cells by Histamine-treated M1 Macrophages

Atherosclerosis is developed due to the formation of atheroma plaques in the coronary arteries. In this process, M1 macrophages and vascular smooth muscle cells (VSMCs) are the main functional cells. Inflammatory mediators such as histamine may inflame M1 macrophages. The aim of this study was to determine the effect of M1 macrophage secretion contents on the gene and protein expression levels of focal adhesion kinase (FAK), vasodilator-stimulated phosphoprotein (VASP), and thrombospondin1 (THBS1). Whole blood samples from the six healthy subjects (stenosis<5%), and six patients (stenosis>70%) were prepared and peripheral blood mononuclear cells (PBMCs) were isolated. Then monocytes were differentiated into M1 macrophages using 100 ng/mL granulocyte-macrophage colony stimulating factor (GM-CSF). The differentiated M1 macrophages were treated with histamine (10-6 M), and their secretion contents were harvested and added to the culture medium of VSMCs. The FAK, VASP, and THBS1 gene expression and protein levels were measured using RT-qPCR and western blot techniques in VSMCs, respectively. The FAK and THBS1 gene expression levels significantly increased in VSMCs after adding secretion contents obtained from histamine-treated M1 macrophages (p=0.023 and 0.05, respectively), while significant results were not observed for VASP gene (p=0.45). In converse with the phosphorylated VASP (pVASP) (p<0.34), the phosphorylated FAK (pFAK) and THBS1 protein levels increased in VSMCs (p<0.001). We concluded that in inflammatory conditions, the immune events could affect the macrophages by histamine. The activated macrophages could locally activate signaling pathways via FAK and THBS1 genes that are effective in the proliferation and migration of VSMCs.

TNF‑α inhibits xenograft tumor formation by A549 lung cancer cells in nude mice via the HIF‑1α/VASP signaling pathway.

Lung cancer is the leading cause of cancer‑associated mortality worldwide. Tumor necrosis factor α (TNF‑α) is an important cytokine in the tumor microenvironment that serves a function in the balance of cell survival and cell death pathways. Our previous studies indicated that hypoxia‑inducible factor 1α (HIF‑1α) acts downstream of TNF‑α in MCF‑7 luminal breast cancer cells. However, whether vasodilator‑stimulated phosphoprotein (VASP) is implicated in the direct regulation of HIF‑1α in response to TNF‑α in lung cancer remains unknown. In vitro studies were performed using A549 and H226 lung carcinoma cells and in vivo studies of tumor xenograft models were performed to investigate the effects of TNF‑α. The results demonstrated that TNF‑α decreased VASP expression by upregulating the expression of HIF‑1α to inhibit A549 cell proliferation and adhesion. Inhibition of transplanted tumor growth was associated with downregulation of VASP expression in nude mice. Bioinformatics analysis indicated that expression levels of VASP or HIF‑1α lead to differential outcomes of overall survival in lung carcinoma. These results suggest that the HIF‑1α/VASP signaling pathway serves an important function in the regulation of TNF‑α‑induced suppression of A549 cell proliferation and xenograft growth. This may improve our understanding of the antitumor effect of TNF‑α.

Vasodilator-stimulated phosphoprotein (VASP), a novel target of miR-4455, promotes gastric cancer cell proliferation, migration, and invasion, through activating the PI3K/AKT signaling pathway

BackgroundMicroRNAs (miRNAs) are small non-coding RNAs which play important roles in the carcinogenesis of gastric cancer (GC). Expression profiling of miRNAs in paired gastric cancer and adjacent normal gastric tissues has demonstrated that miR-4455 is down-regulated in gastric cancer tissues, but its functional role in the carcinogenesis of GC had not previously been investigated.AimsThe purpose of this study was to investigate the functional and biological mechanisms of miR-4455 in the progression of GC, in vitro.MethodsExpression of miR-4455 was compared in human GC tissue samples and paired adjacent normal tissue samples. The in vitro effects of miR-4455 expression in MGC-803 cells on their proliferation, invasion, and migration were assessed by MTT assays and 5-bromo-2′-deoxyuridine staining, matrigel-invasion analysis and wound healing assays. Bioinformatics analysis (using PicTar, target scan and miRBase target) was used to identify potential targets for miR-4455, and the luciferase reporter assay, qRT-PCR and Western-blotting analyses were used to confirm VASP as the target of miR-4455. In addition, the effects of downregulation of VASP on the activation of PI3K/AKT signaling pathway were measured using Western-blot analysis.ResultsThe expression of miR-4455 was markedly down-regulated in gastric cancer tissues vs. adjacent normal tissues, and miR-4455 expression inhibited the proliferation, invasion and migration of MGC-803 GC cells in vitro. Luciferase reporter assays revealed that miR-4455 inhibited VASP expression by targeting the 3′-UTR sequence of VASP. Furthermore, silencing of VASP markedly inhibited the activation of the PI3K/AKT signaling pathway.ConclusionOur results suggest that miR-4455 functions as a tumor suppressor in gastric cancer, by targeting VASP leading to activation of the PI3K/AKT signaling pathway and the inhibition of VASP mediated proliferation, migration and invasion of gastric cancer cells.

Role of vasodilator-stimulated phosphoprotein in human cytomegalovirus-induced hyperpermeability of human endothelial cells.

Atherosclerosis (AS) is a common chronic vascular disease and epidemiological evidence demonstrates that infection is closely associated with the occurrence of AS, including infection by human cytomegalovirus (HCMV) and Chlamydophila pneumoniae. The aim of the present study was to investigate the effect of HCMV AD169 infection on the barrier function of human umbilical vein endothelial cells (HUVECs) and to understand the role of vasodilator-stimulated phosphoprotein (VASP) during this process. In cultured HUVEC-CRL-1730 cells, knockdown of VASP expression with small interfering (si)RNA-VASP resulted in impaired cellular barrier function. Furthermore, knockdown of Ras-related C3 botulinum toxin substrate 1 (Rac1) using siRNA-Rac1 could induce downregulation of VASP expression in HUVEC-CRL-1730 cells. Additionally, following the infection of the cells by HCMV, cellular morphological alterations could be observed under an inverted microscope, the mRNA and protein levels of Rac1 and VASP were transiently reduced, and what appeared to be a time-dependent impairment of the barrier function was observed. Finally, transfection of siRNA-VASP or siRNA-Rac1 into HCMV-infected HUVEC-CRL-1730 cells resulted in increased impairment of the cellular barrier function. Taken together, these data demonstrated that HCMV infection could induce impairment of the barrier function in monolayer HUVEC-CRL-1730 cells via interference with Rac1/VASP expression.

Knockdown of RAC1 and VASP gene expression inhibits breast cancer cell migration.

The ability of tumor cells to migrate is biologically fundamental for tumorigenesis, growth, metastasis and invasion. The present study examined the role of Ras-related C3 botulinum toxin substrate (RAC1) and vasodilator-stimulated phosphoprotein (VASP) in breast cancer cell migration. According to data in Kaplan, Oncomine and The Cancer Genome Atlas, increased expression levels of RAC1 and VASP in breast cancer are associated with decreased cancer cell differentiation, advanced pathological stage and more aggressive tumor subtypes, while increased VASP mRNA expression levels are positively correlated with a poor prognosis in patients with breast cancer. The short hairpin (sh)RNA technique was employed to knock down the expression of RAC1 or VASP. Stable interference with the expression of RAC1 or VASP using RAC1-shRNA or VASP-shRNA, respectively, was established in MCF-7 breast cancer cells. In RAC1-shRNA or VASP-shRNA cells, the protein expression levels of RAC1 or VASP were significantly downregulated compared with control cells. The proliferation and migration rates of the RAC1-shRNA or VASP-shRNA cells were significantly lower compared with control cells. It was observed that the protein expression levels of VASP also decreased in RAC1-shRNA cells compared with control cells. The results revealed that RAC1 and VASP may serve important roles in promoting the migration of MCF-7 breast cancer cells, and that VASP may among the downstream signaling molecules associated with RAC1.

Label-Free Quantitative Phosphoproteomics Reveals Regulation of Vasodilator-Stimulated Phosphoprotein upon Stathmin-1 Silencing in a Pair of Isogenic Colorectal Cancer Cell Lines.

In this communication, we present the phosphoproteome changes in an isogenic pair of colorectal cancer cell lines, viz., the poorly metastatic HCT-116 and the highly metastatic derivative E1, upon stathmin-1 (STMN1) knockdown. The aim was to better understand how the alterations of the phosphoproteins in these cells are involved in cancer metastasis. After the phosphopeptides were enriched using the TiO2 HAMMOC approach, comparative proteomics analysis was carried out using sequential window acquisition of all theoretical mass spectra-MS. Following bioinformatics analysis using MarkerView and OneOmics platforms, we identified a list of regulated phosphoproteins that may play a potential role in signaling, maintenance of cytoskeletal structure, and focal adhesion. Among these phosphoproteins, was the actin cytoskeleton regulator protein, vasodilator-stimulated phosphoprotein (VASP), where its change in phosphorylation status was found to be concomitant with STMN1-associated roles in metastasis. We further showed that silencing of stathmin-1 altered the expression, subcellular localization and phosphorylation status of VASP, which suggested that it might be associated with remodeling of the cell cytoskeleton in colorectal cancer metastasis.

Hypoxia-induced up-regulation of VASP promotes invasiveness and metastasis of hepatocellular carcinoma.

Rational: Patients with hepatocellular carcinoma (HCC) have a poor prognosis mostly due to intrahepatic as well as distal metastasis. Vasodilator-stimulated phosphoprotein (VASP), a regulator of actin cytoskeleton and cell migration, is overexpressed in HCC and correlated with its malignant features and poor prognosis. Very little is known about its function in HCC.Methods: qRT-PCR, Western blot and IHC were used to detect the VASP expression in tissues and cells. Transwell and wound healing assays were used to measure the migration and invasion of HCC cells. Immunoblotting and immunofluorescence were used for detection of epithelial-to-mesenchymal transition (EMT) progression in HCC cells. A lung metastasis mouse model was used to evaluate metastasis of HCC in vivo. The putative targets of miR-204 were disclosed by public databases and a dual-luciferase reporter assay. IP was used to show the interaction between VASP and CRKL. ChIP was used to analyze the binding of HIF-1α to VASP promoter region.Results: Our data involving both gain- and loss-of-function studies revealed that VASP activated AKT and ERK signaling and promoted HCC migration and invasion in vitro and in vivo by altering the EMT phenotype and expression of MMPs. We investigated the positive correlation between VASP and an adapter protein, CRKL. VASP dynamically co-localized at the SH3N domain of CRKL and mediated its function. Mechanistically, VASP overexpression at the transcriptional level was mediated by HIF-1α through direct binding to two hypoxia response elements (HRE) in the VASP promoter region. Furthermore, we identified hypoxia-induced down-regulation of miR-204, which functioned as the regulator of VASP overexpression at the post-transcriptional level. Also, hypoxia-activated p-Smad3 dependent TGF-β signaling indirectly promoted VASP expression.Conclusion: A variety of hypoxia-induced molecular mechanisms contributed to the upregulation of VASP at transcriptional and post-transcriptional levels. These mechanisms involved CRKL, HIF-1α, miR-204, and TGF-β activating the AKT and ERK signaling to promote EMT and expression of MMPs. Taken together, our results defined VASP as an oncogene of HCC pathogenesis and metastasis with the potential to serve as a prognostic biomarker.

Vasodilator-Stimulated Phosphoprotein Biomarkers Are Associated with Invasion and Metastasis in Colorectal Cancer.

Background and Aims:The benefit of adjuvant chemotherapy for stage II colorectal cancer (CRC) patients remains unclear, emphasizing the need for improved prognostic biomarkers to identify patients at risk of metastatic recurrence. To address this unmet clinical need, we examined the expression and phosphorylation status of the vasodilator-stimulated phosphoprotein (VASP) in CRC tumor progression. VASP, a processive actin polymerase, promotes the formation of invasive membrane structures leading to extracellular matrix remodeling and tumor invasion. Phosphorylation of VASP serine (Ser) residues 157 and 239 regulate VASP function, directing subcellular localization and inhibiting actin polymerization, respectively.Methods:The expression levels of VASP protein, pSer157-VASP, and pSer239-VASP were determined by immunohistochemistry in tumors and matched normal adjacent tissue from 141 CRC patients, divided into 2 cohorts, and the association of VASP biomarker expression with clinicopathologic features and disease recurrence was examined.Results:We report that changes in VASP expression and phosphorylation were significantly associated with tumor invasion and disease recurrence. Furthermore, we disclose a novel 2-tiered methodology to maximize VASP positive and negative predictive value performance for prognostication.Conclusion:VASP biomarkers may serve as prognostic biomarkers in CRC and should be evaluated in a larger clinical study.

Interleukin-17 augments tumor necrosis factor α-mediated increase of hypoxia-inducible factor-1α and inhibits vasodilator-stimulated phosphoprotein expression to reduce the adhesion of breast cancer cells.

Interleukin-17 (IL-17) and tumor necrosis factor (TNF)-α are able to cooperatively alter the expression levels of a number of genes. In the present study, the mRNA expression levels of hypoxia-inducible factor (HIF)-1α were analyzed in MDA-MB-231 breast cancer cells following treatment with IL-17, TNF-α or the combination of IL-17 and TNF-α. The protein expression levels of HIF-1α and vasodilator-stimulated phosphoprotein (VASP) were evaluated using western blot analysis. The adhesive ability of the cells was determined using an MTT assay following treatment with HIF-1α-small interfering RNA and short hairpin RNA-VASP that were used to suppress the expression levels of HIF-1α and VASP protein, respectively. These results demonstrated that IL-17 augmented TNF-α-induced gene expression of HIF-1α. The combination of IL-17 and TNF-α promoted an increase in HIF-1α expression and a decrease in VASP expression and a reduction in the adhesive ability of cells. These results demonstrated that IL-17 effectively enhanced the TNF-α-induced increase in HIF-1α and inhibited VASP expression, thus reducing the adhesion of MDA-MB-231 cells.

Vasodilator-Stimulated Phosphoprotein (VASP) depletion from breast cancer MDA-MB-231 cells inhibits tumor spheroid invasion through downregulation of Migfilin, β-catenin and urokinase-plasminogen activator (uPA)

A hallmark of cancer cells is their ability to invade surrounding tissues and form metastases. Cell-extracellular matrix (ECM)-adhesion proteins are crucial in metastasis, connecting tumor ECM with actin cytoskeleton thus enabling cells to respond to mechanical cues. Vasodilator-stimulated phosphoprotein (VASP) is an actin-polymerization regulator which interacts with cell-ECM adhesion protein Migfilin, and regulates cell migration. We compared VASP expression in MCF-7 and MDA-MB-231 breast cancer (BC) cells and found that more invasive MDA-MB-231 cells overexpress VASP. We then utilized a 3-dimensional (3D) approach to study metastasis in MDA-MB-231 cells using a system that considers mechanical forces exerted by the ECM. We prepared 3D collagen I gels of increasing concentration, imaged them by atomic force microscopy, and used them to either embed cells or tumor spheroids, in the presence or absence of VASP. We show, for the first time, that VASP silencing downregulated Migfilin, β-catenin and urokinase plasminogen activator both in 2D and 3D, suggesting a matrix-independent mechanism. Tumor spheroids lacking VASP demonstrated impaired invasion, indicating VASP’s involvement in metastasis, which was corroborated by Kaplan-Meier plotter showing high VASP expression to be associated with poor remission-free survival in lymph node-positive BC patients. Hence, VASP may be a novel BC metastasis biomarker.

Genome-Wide Prioritization and Transcriptomics Reveal Novel Signatures Associated With Thiazide Diuretics Blood Pressure Response.

Thiazide diuretics are among the most commonly prescribed antihypertensives. However, <50% of thiazide-treated patients achieve blood pressure (BP) control. Herein, we used different omics (genomics and transcriptomics) to identify novel biomarkers of thiazide diuretics BP response. Genome-wide analysis included 228 white hypertensives with BP determined at baseline and after 9 weeks of hydrochlorothiazide. Single-nucleotide polymorphisms with P <5×10-5 were prioritized according to their biological function, using RegulomeDB, haploreg, and Genome-Wide Annotation of Variants. The results from the prioritization approach revealed rs10995 as the most likely functional single-nucleotide polymorphism, among single-nucleotide polymorphisms tested, that has been associated with hydrochlorothiazide BP response. The rs10995 G-allele was associated with better BP response to hydrochlorothiazide versus noncarriers (Δ systolic BP/Δ diastolic BP: -12.3/-8.2 versus -6.8/-3.5 mm Hg, respectively, Δ systolic BP P=3×10-4, Δ diastolic BP P=5×10-5). This association was replicated in independent participants treated with chlorthalidone. In addition, rs10995 G-allele was associated with increased mRNA expression of VASP (vasodilator-stimulated phosphoprotein). Moreover, baseline expression of the VASP mRNA was significantly higher in 25 good responders to hydrochlorothiazide compared with 25 poor responders (P=0.01). This finding was replicated in independent participants treated with chlorthalidone (P=0.04). Last, allelic-specific expression analysis revealed a significant but modest imbalance with rs10995 and rs10156, a single-nucleotide polymorphism in high linkage disequilibrium (r2=0.7) with rs10995, which both could contribute to the observed genetic effects by affecting VASP mRNA expression. This study highlights the strength of using different omics to identify novel biomarkers of drug response and suggests VASP as a potential determinant of thiazide diuretics BP response. URL: http://www.clinicaltrials.gov. Unique identifiers: NCT00246519 and NCT01203852.

Vasodilator-Stimulated Phosphoprotein Activity Is Required for Coxiella burnetii Growth in Human Macrophages.

Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute flu-like illness that can progress to chronic endocarditis and liver and bone infections. Humans are typically infected by aerosol-mediated transmission, and C. burnetii initially targets alveolar macrophages wherein the pathogen replicates in a phagolysosome-like niche known as the parasitophorous vacuole (PV). C. burnetii manipulates host cAMP-dependent protein kinase (PKA) signaling to promote PV formation, cell survival, and bacterial replication. In this study, we identified the actin regulatory protein vasodilator-stimulated phosphoprotein (VASP) as a PKA substrate that is increasingly phosphorylated at S157 and S239 during C. burnetii infection. Avirulent and virulent C. burnetii triggered increased levels of phosphorylated VASP in macrophage-like THP-1 cells and primary human alveolar macrophages, and this event required the Cα subunit of PKA. VASP phosphorylation also required bacterial protein synthesis and secretion of effector proteins via a type IV secretion system, indicating the pathogen actively triggers prolonged VASP phosphorylation. Optimal PV formation and intracellular bacterial replication required VASP activity, as siRNA-mediated depletion of VASP reduced PV size and bacterial growth. Interestingly, ectopic expression of a phospho-mimetic VASP (S239E) mutant protein prevented optimal PV formation, whereas VASP (S157E) mutant expression had no effect. VASP (S239E) expression also prevented trafficking of bead-containing phagosomes to the PV, indicating proper VASP activity is critical for heterotypic fusion events that control PV expansion in macrophages. Finally, expression of dominant negative VASP (S157A) in C. burnetii-infected cells impaired PV formation, confirming importance of the protein for proper infection. This study provides the first evidence of VASP manipulation by an intravacuolar bacterial pathogen via activation of PKA in human macrophages.

Expression of miRNA-26a in platelets is associated with clopidogrel resistance following coronary stenting.

The present study aimed to evaluate the association between platelet microRNA (miRNA)-26a expression and clopidogrel resistance in patients who underwent coronary stenting. Between September 2013 and August 2014, 43 patients with coronary heart disease underwent percutaneous coronary intervention at Heibei General Hospital (Shijiazhuang, China). In the same period, 20 healthy volunteers without any history of cardiovascular disease were enrolled in the present study as the control group. Flow cytometry was used to measure the phosphorylation levels of vasodilator-stimulated phosphoprotein (VASP), and to calculate the platelet reactivity index (PRI). Low response to clopidogrel was defined as PRI ≥50% on day 7 following clopidogrel administration. Western blotting was used to measure protein expression of VASP and reverse transcription-quantitative polymerase chain reaction analysis was performed to determine the expression levels of mRNA and miRNAs. Bioinformatics tools were employed to predict that miR-26a, miR-199 and miR-23a may target VASP mRNA. The results of the present study demonstrated that the activity of platelets in patients with low or high clopidogrel response was increased, as compared with healthy subjects. No differences in platelet VASP protein expression levels were detected between patients with high clopidogrel response and healthy subjects; whereas VASP protein expression was elevated in patients with low clopidogrel response. Furthermore VASP gene transcription was maintained at low levels in healthy subjects and patients with high clopidogrel response, whereas patients with low clopidogrel response exhibited increased VASP mRNA expression levels. Platelet expression of miRNA-26a, but not miRNA-199 or miRNA-23a, was associated with high platelet reactivity. Serum miRNA-26a, miRNA-199 and miRNA-23a were not demonstrated to be involved in clopidogrel resistance. Therefore, the present study demonstrated that platelet miRNA-26a has an important role in clopidogrel resistance. Combined miRNA and VASP PRI tests may aid the early diagnosis and prediction of clopidogrel resistance.