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Background/Aim: Aggressive breast cancer (BC) cells show high expression of Rho GTPase activating protein 29 (ARHGAP29), a negative regulator of RhoA. In breast cancer cells in which mesenchymal transformation was induced, ARHGAP29 was the only one of 32 GTPaseactivating enzymes whose expression increased significantly. Therefore, we investigated whether there is a correlation between expression of ARHGAP29 and tumor progression in BC. Since tamoxifen-resistant BC cells exhibit increased mesenchymal properties and invasiveness, we additionally investigated the relationship between ARHGAP29 and increased invasion rate in tamoxifen resistance. The question arises as to whether ARHGAP29 is a suitable prognostic marker for the progression of BC. Materials and Methods: Tissue microarrays were used to investigate expression of ARHGAP29 in BC and adjacent normal breast tissues. Knockdown experiments using siRNA were performed to investigate the influence of ARHGAP29 and the possible downstream actors RhoC and pAKT1 on invasive growth of tamoxifen-resistant BC spheroids in vitro. Results: Expression of ARHGAP29 was frequently increased in BC tissues compared to adjacent normal breast tissues. In addition, there was evidence of a correlation between high ARHGAP29 expression and advanced clinical tumor stage. Tamoxifenresistant BC cells show a significantly higher expression of ARHGAP29 compared to their parental wild-type cells. After knockdown of ARHGAP29 in tamoxifen-resistant BC cells, expression of RhoC was significantly reduced. Further, expression of pAKT1 decreased significantly. Invasive growth of three-dimensional tamoxifen-resistant BC spheroids was reduced after knockdown of ARHGAP29. This could be partially reversed by AKT1 activator SC79. Conclusion: Expression of ARHGAP29 correlates with the clinical tumor parameters of BC patients. In addition, ARHGAP29 is involved in increased invasiveness of tamoxifen-resistant BC cells. ARHGAP29 alone or in combination with its downstream partners RhoC and pAKT1 could be suitable prognostic markers for BC progression. [ABSTRACT FROM AUTHOR]

Constitutive activation of GNAQ/11 is the initiative oncogenic event in uveal melanoma (UM). Direct targeting GNAQ/11 has yet to be proven feasible as they are vital for a plethora of cellular functions. In search of genetic vulnerability for UM, we found that inhibition of euchromatic histone lysine methyltransferase 2 (EHMT2) expression or activity significantly reduced the proliferation and migration capacity of cancer cells. Notably, elevated expression of EHMT2 had been validated in UM samples. Furthermore, Kaplan–Meier survival analysis indicated high EHMT2 protein level was related to poor recurrence-free survival and a more advanced T stage. Chromatin immunoprecipitation sequencing analysis and the following mechanistic investigation showed that ARHGAP29 was a downstream target of EHMT2. Its transcription was suppressed by EHMT2 in a methyltransferase-dependent pattern in GNAQ/11-mutant UM cells, leading to elevated RhoA activity. Rescuing constitutively active RhoA in UM cells lacking EHMT2 restored oncogenic phenotypes. Simultaneously blocking EHMT2 and GNAQ/11 signaling in vitro and in vivo showed a synergistic effect on UM growth, suggesting the driver role of these two key molecules. In summary, our study shows evidence for an epigenetic program of EHMT2 regulation that influences UM progression and indicates inhibiting EHMT2 and MEK/ERK simultaneously as a therapeutic strategy in GNAQ/11-mutant UM.

Constitutive activation of GNAQ/11 is the initiative oncogenic event in uveal melanoma (UM). Direct targeting GNAQ/11 has yet to be proven feasible as they are vital for a plethora of cellular functions. In search of genetic vulnerability for UM, we found that inhibition of euchromatic histone lysine methyltransferase 2 (EHMT2) expression or activity significantly reduced the proliferation and migration capacity of cancer cells. Notably, elevated expression of EHMT2 had been validated in UM samples. Furthermore, Kaplan–Meier survival analysis indicated high EHMT2 protein level was related to poor recurrence-free survival and a more advanced T stage. Chromatin immunoprecipitation sequencing analysis and the following mechanistic investigation showed that ARHGAP29 was a downstream target of EHMT2. Its transcription was suppressed by EHMT2 in a methyltransferase-dependent pattern in GNAQ/11- mutant UM cells, leading to elevated RhoA activity. Rescuing constitutively active RhoA in UM cells lacking EHMT2 restored oncogenic phenotypes. Simultaneously blocking EHMT2 and GNAQ/11 signaling in vitro and in vivo showed a synergistic effect on UM growth, suggesting the driver role of these two key molecules. In summary, our study shows evidence for an epigenetic program of EHMT2 regulation that influences UM progression and indicates inhibiting EHMT2 and MEK/ERK simultaneously as a therapeutic strategy in GNAQ/11- mutant UM. EHMT2-dependent epigenetic program promotes RhoA signaling in GNAQ/11 -mutant uveal melanoma and targeting EHMT2 and MEK/ERK simultaneously is a therapeutic strategy to overcome resistance. [Display omitted] [ABSTRACT FROM AUTHOR]

Purpose: Previous studies have shown that TBX21 (T-Box Transcription Factor 21) plays a vital role in coordinating multiple aspects of the immune response especially type 1 immune response as well as tumor progression. However, the function of TBX21 in colorectal cancer (CRC) remains unclear. Methods: IHC to investigate TBX21 expression in CRC tissues. Cell proliferation and apoptosis assays to validate TBX21 function in vitro and in vivo. RNA-seq assay to explore target genes of TBX21. Human phospho-kinase array assay to explore down-stream signaling of TBX21. Results: We disclosed that the expression of TBX21 was marked decreased in CRC versus normal tissue, and negatively correlated with CRC TNM stages. Surprisingly, we found that the CRC and normal cell lines show no TBX21 expression levels. Ectopic expression of TBX21 inhibited cell proliferation and promoted cell apoptosis in vitro. Moreover, RNA-sequence data first time showed that ARHGAP29 acts as the target gene of TBX21 to mediate down-stream signaling activation. Human phospho-kinase array data first time displayed that ectopic expression of TBX21 reduced kinase RSK and GSK3β activation. In contrast, knocked down the expression of TBX21 or ARHGAP29 alternatively abolished TBX21 mediated cell proliferation suppression, cell apoptosis enhancement and RSK/GSK3β activation. In addition, xenograft model studies demonstrated that TBX21 inhibits colorectal tumor progression via ARHGAP29/ RSK/ GSK3β signaling in vivo. Conclusions: In summary, the aforementioned findings suggest a model of TBX21 in suppressing CRC progression. This may provide a promising target for CRC therapy. [ABSTRACT FROM AUTHOR]

Glomerular disease due to podocyte malfunction is a major factor in the pathogenesis of chronic kidney disease. Identification of podocyte-specific signaling pathways is therefore a prerequisite to characterizing relevant disease pathways and developing novel treatment approaches. Here, we employed loss of function studies for EPB41L5 (Yurt) as a central podocyte gene to generate a cell type-specific disease model. Loss of Yurt in fly nephrocytes caused protein uptake and slit diaphragm defects. Transcriptomic and proteomic analysis of human EPB41L5 knockout podocytes demonstrated impaired mechanotransduction via the YAP/TAZ signaling pathway. Further analysis of specific inhibition of the YAP/TAZ-TEAD transcription factor complex by TEADi led to the identification of ARGHAP29 as an EPB41L5 and YAP/TAZ-dependently expressed podocyte RhoGAP. Knockdown of ARHGAP29 caused increased RhoA activation, defective lamellipodia formation, and increased maturation of integrin adhesion complexes, explaining similar phenotypes caused by loss of EPB41L5 and TEADi expression in podocytes. Detection of increased levels of ARHGAP29 in early disease stages of human glomerular disease implies a novel negative feedback loop for mechanotransductive RhoA—YAP/TAZ signaling in podocyte physiology and disease. [ABSTRACT FROM AUTHOR]

Aim: Circular RNAs (circRNAs) are a novel class of noncoding RNAs and are conserved in various species. Although numerous circRNAs have been identified, their role in cancer remains unclear. Methods: The expression of circTMEM181 in 90 paired human hepatocellular carcinoma (HCC) and adjacent nontumor tissues were detected using quantitative reverse transcription–polymerase chain reaction. Transwell assay was performed for functional analysis of HCC cell migration and invasion. Luciferase reporter assay was used to verify the combination of circTMEM181 and miR‐519a‐5p. Results: In this study, we identified a novel circRNA, named circTMEM181, was downregulated in HCC tissues. Decreased expression of circTMEM181 was associated with shorter overall survival of patients with HCC. CircTMEM181 overexpression reduced HCC cell migration and invasion abilities, while circTMEM181 knockdown increased cell motility. Mechanically, circTMEM181 could directly bind to miR‐519a‐5p and subsequently upregulate ARHGAP29 protein expression. Conclusion: These data provide the first evidence of clinical significance and function of circTMEM181, and suggest the circTMEM181/miR‐519a‐5p/ARHGAP29 axis in HCC development. [ABSTRACT FROM AUTHOR]

Glomerular disease due to podocyte malfunction is a major factor in the pathogenesis of chronic kidney disease. Identification of podocyte-specific signaling pathways is therefore a prerequisite to characterizing relevant disease pathways and developing novel treatment approaches. Here, we employed loss of function studies for EPB41L5 (Yurt) as a central podocyte gene to generate a cell type-specific disease model. Loss of Yurt in fly nephrocytes caused protein uptake and slit diaphragm defects. Transcriptomic and proteomic analysis of human EPB41L5 knockout podocytes demonstrated impaired mechanotransduction via the YAP/TAZ signaling pathway. Further analysis of specific inhibition of the YAP/TAZ-TEAD transcription factor complex by TEADi led to the identification of ARGHAP29 as an EPB41L5 and YAP/TAZ-dependently expressed podocyte RhoGAP. Knockdown of ARHGAP29 caused increased RhoA activation, defective lamellipodia formation, and increased maturation of integrin adhesion complexes, explaining similar phenotypes caused by loss of EPB41L5 and TEADi expression in podocytes. Detection of increased levels of ARHGAP29 in early disease stages of human glomerular disease implies a novel negative feedback loop for mechanotransductive RhoA—YAP/TAZ signaling in podocyte physiology and disease.

Yes-associated protein (YAP) is regulated by mechanical cues via the interaction of the Hippo pathway with cytoskeleton. Previous studies showed that YAP plays a role in regulating the actomyosin network by suppressing Rho GTPase in medaka fish. Here, we identify Rho GTPase activating protein 29 (ARHGAP29) as a transcriptional target of YAP in a human gastric cancer cell line. YAP promotes the expression of ARHGAP29 to suppress the RhoA-LIMK-cofilin pathway, destabilizing F-actin. The overexpression of YAP causes cytoskeletal rearrangement by altering the dynamics of F-actin/G-actin turnover, thus promoting migration. In a mouse model, circulating tumor cells (CTCs) exhibit an increased ARHGAP29 RNA level compared with cells at primary tumor sites, and the metastatic potential of CTCs is positively correlated with ARHGAP29 expression. Moreover, increased ARHGAP29 expression is correlated with shortened survival of human gastric cancer patients. Our study provides a model to understand YAP’s contribution to cancer metastasis via regulation of actin dynamics.

Fibroblasts show a high range of phenotypic plasticity, including transdifferentiation into myofibroblasts. Myofibroblasts are responsible for generation of the contraction forces that are important for wound healing and scar formation. Overactive myofibroblasts, by contrast, are involved in abnormal scarring. Cell stretching and extracellular signals such as transforming growth factor β can induce the myofibroblastic program, whereas microenvironmental conditions such as reduced tissue oxygenation have an inhibitory effect. We investigated the effects of hypoxia on myofibroblastic properties and linked this to RhoA activity. Hypoxia reversed the myofibroblastic phenotype of primary fibroblasts. This was accompanied by decreased αSMA (ACTA2) expression, alterations in cell contractility, actin reorganization and RhoA activity. We identified a hypoxia-inducible induction of ARHGAP29, which is critically involved in myocardin-related transcription factor-A (MRTF-A) signaling, the differentiation state of myofibroblasts and modulates RhoA activity. This novel link between hypoxia and MRTF-A signaling is likely to be important for ischemiainduced tissue remodeling and the fibrotic response. This article has an associated First Person interview with the first author of the paper [ABSTRACT FROM AUTHOR]

Aggressive and mesenchymal-transformed breast cancer cells show high expression levels of Rho GTPase activating protein 29 (ARHGAP29), a negative regulator of RhoA. ARHGAP29 was the only one of 32 GTPase-activating enzymes whose expression significantly increased after the induction of mesenchymal transformation in breast cancer cells. Therefore, we investigated the influence of ARHGAP29 on the invasiveness of aggressive and mesenchymal-transformed breast cancer cells. After knock-down of ARHGAP29 using siRNA, invasion of HCC1806, MCF-7-EMT, and T-47D-EMT breast cancer cells was significantly reduced. This could be explained by reduced inhibition of RhoA and a consequent increase in stress fiber formation. Proliferation of the breast cancer cell line T-47D-EMT was slightly increased by reduced expression of ARHGAP29, whereas that of HCC1806 and MCF-7-EMT significantly increased. Using interaction analyses we found that AKT1 is a possible interaction partner of ARHGAP29. Therefore, the expression of AKT1 after siRNA knock-down of ARHGAP29 was tested. Reduced ARHGAP29 expression was accompanied by significantly reduced AKT1 expression. However, the ratio of active pAKT1 to total AKT1 remained unchanged or was significantly increased after ARHGAP29 knock-down. Our results show that ARHGAP29 could be an important factor in the invasion of aggressive and mesenchymal-transformed breast cancer cells. Further research is required to fully understand the underlying mechanisms.

Lipomyelomeningocele is a type of neural tube defect characterized by lipomatous tissue causing a defect in the vertebrae, infiltrating the dura, and tethering the spinal cord. Despite significant neurologic consequences, the underlying etiology remains poorly understood. We present a father and son with remarkably similar presentations of lipomyelomeningocele. Genetic testing did not reveal an underlying cause but whole exome sequencing identified variants in the ARHGAP29 and RADIL genes in the proband and his affected father. Genetic analyses of asymptomatic family members revealed several carriers of the ARHGAP29 or RADIL variants, but only the proband and his father carried both variants, suggesting a possible shared genetic mechanism. Rare cases of siblings affected with lipomyelomeningocele have suggested the possibility of autosomal recessive or germline mosaicism. We present the first documented cases of transgenerational lipomyelomeningocele with important implications for family counseling about the recurrence of lipomyelomeningocele. [ABSTRACT FROM AUTHOR]

Summary Yes-associated protein (YAP) is regulated by mechanical cues via the interaction of the Hippo pathway with cytoskeleton. Previous studies showed that YAP plays a role in regulating the actomyosin network by suppressing Rho GTPase in medaka fish. Here, we identify Rho GTPase activating protein 29 (ARHGAP29) as a transcriptional target of YAP in a human gastric cancer cell line. YAP promotes the expression of ARHGAP29 to suppress the RhoA-LIMK-cofilin pathway, destabilizing F-actin. The overexpression of YAP causes cytoskeletal rearrangement by altering the dynamics of F-actin/G-actin turnover, thus promoting migration. In a mouse model, circulating tumor cells (CTCs) exhibit an increased ARHGAP29 RNA level compared with cells at primary tumor sites, and the metastatic potential of CTCs is positively correlated with ARHGAP29 expression. Moreover, increased ARHGAP29 expression is correlated with shortened survival of human gastric cancer patients. Our study provides a model to understand YAP’s contribution to cancer metastasis via regulation of actin dynamics. [ABSTRACT FROM AUTHOR]

Background: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect of complex etiology. Several genes have been implicated in the etiology of NSCL/P, although only a few have been replicated across datasets. Methods: ARHGAP29 was suggested as a candidate gene for NSCL/ P as it is located in close proximity to ABCA4 (1p22), a gene previously identified in a genome-wide association study of NSCL/P. Results: Rare, potentially damaging, coding variants in ARHGAP29 were found in NSCL/P cases, and its expression was detected during murine craniofacial development. In this study, we investigated whether variations in ARHGAP29 were associated with NSCL/P in our family based dataset. Five singlenucleotide polymorphisms (SNPs) flanking and within ARHGAP29 were genotyped in our NSCL/P datasets consisting of simplex and multiplex families of non-Hispanic white (NHW, primarily European) and Hispanic ethnicities. Results showed strong association of three ARHGAP29 SNPs with NSCL/P in the NHW families. Two intronic SNPs (rs1541098 and rs3789688) showed strong association with NSCL/P in all NHW families (p=0.0005 and p=0.0002, respectively), and simplex NHW families (p=0.003 for both SNPs). A SNP in the 3' untranslated region (rs1576593) also showed strong association with NSCL/P in all NHW families (p=0.002), and the multiplex subset (p=0.002). ARHGAP29 SNP haplotypes were also associated with NSCL/P. Evidence of gene--gene interaction was found between ARHGAP29 and additional cleft susceptibility genes. Conclusion: This study further supports ARHGAP29 as a candidate gene for human NSCL/P in families of Caucasian descent. [ABSTRACT FROM AUTHOR]

Background: Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common birth defects, and occurs in approximately 1/700 live births worldwide. The correlation between the ABCA4-ARHGAP29 region and NSCL/P was first identified by genome-wide association studies (GWAS), but few reports have examined NSCL/P caused by ARHGAP29 mutations in the Chinese population., Methods: We performed chromosome microarray analysis (CMA) for two consecutive abnormal fetuses and whole exome sequencing (WES) for the family, including 3 patients and 2 normal family members, Sanger sequencing and RT-PCR were used to confirm the mutation., Results: We identified a novel splice donor mutation ( ARHGAP29 c.1920 + 1G > A) in two consecutive NSCL/P fetuses, and the variant was inherited from the mother and grandfather. The mutation caused abnormal skipping of exon 17, and the mRNA level of ARHGAP29 was significantly decreased compared to the wild type., Conclusions: In this study, we successfully diagnosed the genetic cause of NSCL/P in a family and first report that the c.1920 + 1G > A mutation in ARHGAP29 is associated with NSCL/P. Our study enriches the genetic landscape of NSCL/P, extends the mutation spectrum of ARHGAP29 , and provides a new direction for the diagnosis of NSCL/P in patients and its prenatal diagnosis in fetuses.

BACKGROUND Nonsyndromic cleft lip with or without cleft palate (NSCL/P) is a common birth defect with complex etiology reflecting the action of multiple genetic and environmental factors. Genome-wide association studies have successfully identified five novel loci associated with NSCL/P, including a locus on 1p22.1 near the ABCA4 gene. Because neither expression analysis nor mutation screening support a role for ABCA4 in NSCL/P, we investigated the adjacent gene ARHGAP29. METHODS Mutation screening for ARHGAP29 protein coding exons was conducted in 180 individuals with NSCL/P and controls from the United States and the Philippines. Nine exons with variants in ARHGAP29 were then screened in an independent set of 872 cases and 802 controls. Arhgap29 expression was evaluated using in situ hybridization in murine embryos. RESULTS Sequencing of ARHGAP29 revealed eight potentially deleterious variants in cases including a frameshift and a nonsense variant. Arhgap29 showed craniofacial expression and was reduced in a mouse deficient for Irf6, a gene previously shown to have a critical role in craniofacial development. CONCLUSION The combination of genome-wide association, rare coding sequence variants, craniofacial specific expression, and interactions with IRF6 support a role for ARHGAP29 in NSCL/P and as the etiologic gene at the 1p22 genome-wide association study locus for NSCL/P. This work suggests a novel pathway in which the IRF6 gene regulatory network interacts with the Rho pathway via ARHGAP29. Birth Defects Research (Part A) 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The ability to form and maintain a functional system of contiguous hollow tubes is a critical feature of vascular endothelial cells (ECs). Lumen formation, or tubulogenesis, occurs in blood vessels during both vasculogenesis and angiogenesis in the embryo. Formation of vascular lumens takes place prior to the establishment of blood flow and to vascular remodeling which results in a characteristic hierarchical vessel organization. While epithelial lumen formation has received intense attention in past decades, more recent work has only just begun to elucidate the mechanisms controlling the initiation and morphogenesis of endothelial lumens. Studies using in vitro and in vivo models, including zebrafish and mammals, are beginning to paint an emerging picture of how blood vessels establish their characteristic morphology and become patent. In this article, we review and discuss the molecular and cellular mechanisms driving the formation of vascular tubes, primarily in vivo, and we compare and contrast proposed models for blood vessel lumen formation. [Copyright &y& Elsevier]

Aggressive and mesenchymal-transformed breast cancer cells show high expression levels of Rho GTPase activating protein 29 (ARHGAP29), a negative regulator of RhoA. ARHGAP29 was the only one of 32 GTPase-activating enzymes whose expression significantly increased after the induction of mesenchymal transformation in breast cancer cells. Therefore, we investigated the influence of ARHGAP29 on the invasiveness of aggressive and mesenchymal-transformed breast cancer cells. After knock-down of ARHGAP29 using siRNA, invasion of HCC1806, MCF-7-EMT, and T-47D-EMT breast cancer cells was significantly reduced. This could be explained by reduced inhibition of RhoA and a consequent increase in stress fiber formation. Proliferation of the breast cancer cell line T-47D-EMT was slightly increased by reduced expression of ARHGAP29, whereas that of HCC1806 and MCF-7-EMT significantly increased. Using interaction analyses we found that AKT1 is a possible interaction partner of ARHGAP29. Therefore, the expression of AKT1 after siRNA knock-down of ARHGAP29 was tested. Reduced ARHGAP29 expression was accompanied by significantly reduced AKT1 expression. However, the ratio of active pAKT1 to total AKT1 remained unchanged or was significantly increased after ARHGAP29 knock-down. Our results show that ARHGAP29 could be an important factor in the invasion of aggressive and mesenchymal-transformed breast cancer cells. Further research is required to fully understand the underlying mechanisms. [ABSTRACT FROM AUTHOR]

Yes‑associated protein (YAP) is a transcription‑coupling factor that plays a central role in the Hippo pathway, and its activation regulates cell proliferation and carcinogenesis. YAP activation has been reported in various malignancies, conferring tumors with migratory and invasive abilities. Several studies have suggested that YAP expression is closely associated with prostate cancer. Furthermore, YAP has been revealed to regulate destabilization of F‑actin associated with the cytoskeleton via Rho GTPase‑activating protein 29 (ARHGAP29), suggesting that ARHGAP29 is associated with cancer metastasis. In the present study, the functions of ARHGAP29 were examined in four prostate cancer cell lines (22Rv1, LNCaP, DU145 and PC‑3) and it was revealed that upregulation of ARHGAP29 in LNCaP and DU145 cells with the lowest expression of ARHGAP29 promoted cell proliferation and invasion. Conversely, ARHGAP29 knockdown in PC‑3 cells with its highest expression level significantly reduced cell proliferation and invasion. In addition, immunohistochemistry of specimens from 133 patients who underwent radical prostatectomy was performed to investigate the clinical association between ARHGAP29 expression and prognosis in prostate cancer patients. Multivariate analysis demonstrated that ARHGAP29 was an independent prognostic factor for biochemical progression‑free survival (P=0.0123). These findings indicated that ARHGAP29 in prostate cancer may be a potential prognostic biomarker and therapeutic target.

Objective: Mutations in exon 1 of ARHGAP29, a RhoA specific GTPase have been identified in North American and Filipino subjects with nonsyndromic cleft palate and cleft lip with or without cleft palate. Since the genetic status of ARHGAP29 in Indian subjects with nonsyndromic oral clefts is not known, we designed the present study to investigate the occurrence of the above mutations in them., Materials and Methods: Total genomic DNA extracted from peripheral blood of 60 subjects with nonsyndromic cleft palate and cleft lip with or without cleft palate, and equal number of control healthy subjects were amplified with primers flanking exon 1 of ARHGAP29 gene and subjected to direct sequencing., Results: Sequencing analysis identified a nonsense mutation in exon 1 of ARHGAP29 that caused substitution of lysine to stop codon at codon position 32 in a subject with nonsyndromic cleft lip with cleft palate. The mutation, however, occurred in heterozygous condition. None of the other subjects carried mutation in this region., Conclusion: The study has thus identified a rare but novel truncation mutation in ARHGAP29 gene for the first time in nonsyndromic oral clefts.