GTPase-activating Protein Family Research Articles

Integrated analysis identified the role of three family members of ARHGAP in pancreatic adenocarcinoma

The Rho GTPase activating protein family (ARHGAPs) is expressed in pancreatic adenocarcinoma (PAAD) but its function is unclear. The aim of this study was to explore the role and potential clinical value of ARHGAPs in PAAD. Using TCGA and GEO databases to analyze expression of ARHGAPs in PAAD and normal tissues. Survival curve was drawn by Kaplan–Meier. ARHGAPs were integrated analyzed by GEPIA2, TIMER, UCLCAN, cBioPortal and R language. Protein level and prognostic value were evaluated via IHC staining or survival analysis. We totally identify 18 differentially expressed (DE) ARHGAPs in PAAD. Among the 18 DE genes, 8 were positively correlated with tumor grade; abnorrmal expression of 5 was positively correlated with copy number variation; expression of 4 was positively correlated with promoter hypomethylation. Multivariate Cox regression identified ARHGAP5, ARHGAP11A, and ARHGAP12 as independent prognostic factors of PAAD. The function of ARHGAPs was mainly related to GTPase activity and signaling, axon guidance, proteoglycans in cancer and focal adhesion. Expression of 7 ARHGAPs was strongly correlated with immune infiltration. Immunohistochemistry showed increased protein levels of ARHGAP5, ARHGAP11A, and ARHGAP12 in PAAD tissues. Survival analysis confirmed a negative correlation between ARHGAP5, ARHGAP11A, and ARHGAP12 expression and patient prognosis. Multivariate Cox regression proved ARHGAP5, ARHGAP11A, and ARHGAP12 could serve as independent prognostic indicators for PAAD. Finally, this study verified ARHGAP5, ARHGAP11A, and ARHGAP12 as independent prognostic factors in PAAD, suggesting their significance for the diagnosis and treatment of PAAD.

ARHGAP44-mediated regulation of the p53/C-myc/Cyclin D1 pathway in modulating the malignant biological behavior of osteosarcoma cells

ObjectiveOsteosarcoma is a rare primary malignant tumor of the bone characterized by poor survival rates, owing to its unclear pathogenesis. Rho GTPase-activating protein 44 (ARHGAP44), which belongs to the Rho GTPase-activating protein family, has promising applications in the targeted therapy of tumors. Therefore, this study aimed to investigate the biological function of ARHGAP44 in osteosarcoma and its possible application as a therapeutic target.MethodsThe expression level of ARHGAP44 in osteosarcoma and its relationship with tumor prognosis were detected using Gene Expression Omnibus database analysis and immunohistochemical staining of clinical specimens. The cell model of ARHGAP44 knockdown was constructed, and the effects of this gene on the malignant biological behavior of osteosarcoma cells were investigated using CCK-8, clone formation, transwell invasion, wound healing, and flow cytometry assays. Western blotting was performed to detect the expression of ARHGAP44, p53, C-myc, and Cyclin D1 in osteosarcoma.ResultsBiogenic analysis showed that ARHGAP44 was highly expressed in osteosarcoma. This result was associated with poor tumor prognosis and negatively correlated with the expression of the tumor suppressor gene p53. Immunohistochemistry and western blotting revealed significantly upregulated expression of ARHGAP44 in osteosarcoma tissues. Additionally, Kaplan–Meier analysis of clinical specimens suggested that ARHGAP44 was negatively correlated with tumor prognosis. CCK-8, clone formation, transwell invasion, wound healing, and flow cytometry assays showed that downregulation of ARHGAP44 expression significantly reduced the malignant biological behavior of osteosarcoma cells. Furthermore, western blotting showed that the expression level of p53 in osteosarcoma cells was significantly increased after the downregulation of ARHGAP44 expression, whereas the expression of C-myc and Cyclin D1 was significantly decreased compared with that in the control group.ConclusionARHGAP44 was highly expressed in osteosarcoma and was negatively correlated with its prognosis. The downregulation of ARHGAP44 expression reduced the malignant biological behavior of osteosarcoma cells. These findings suggest that the downregulation of ARHGAP44 expression inhibits the malignant progression of osteosarcoma by regulating the p53/C-myc/Cyclin D1 pathway, demonstrating the potential of ARHGAP44 as a therapeutic target for osteosarcoma.

Diverse p120RasGAP interactions with doubly phosphorylated partners EphB4, p190RhoGAP, and Dok1

RasGAP (p120RasGAP), the founding member of the GTPase-activating protein (GAP) family, is one of only nine human proteins to contain two SH2 domains and is essential for proper vascular development. Despite its importance, its interactions with key binding partners remains unclear. In this study we provide a detailed viewpoint of RasGAP recruitment to various binding partners and assess their impact on RasGAP activity. We reveal the RasGAP SH2 domains generate distinct binding interactions with three well-known doubly phosphorylated binding partners: p190RhoGAP, Dok1, and EphB4. Affinity measurements demonstrate a 100-fold weakened affinity for RasGAP-EphB4 binding compared to RasGAP-p190RhoGAP or RasGAP-Dok1 binding, possibly driven by single versus dual SH2 domain engagement with a dominant N-terminal SH2 interaction. Small-angle X-ray scattering reveals conformational differences between RasGAP-EphB4 binding and RasGAP-p190RhoGAP binding. Importantly, these interactions do not impact catalytic activity, implying RasGAP utilizes its SH2 domains to achieve diverse spatial-temporal regulation of Ras signaling in a previously unrecognized fashion.

ARHGAP4 promotes leukemogenesis in acute myeloid leukemia by inhibiting DRAM1 signaling.

Rho GTPase-activating protein 4 (ARHGAP4) is an important Rho family GTPase-activating protein that is strongly associated with the onset and progression of some tumors. We found that ARHGAP4 mRNA and protein are overexpressed in human acute myeloid leukemia (AML) patients and are associated with a poor prognosis. ARHGAP4 knockdown significantly impairs viability and colony formation capacity and induces apoptosis in AML cells. Further results demonstrate that ARHGAP4 deletion impairs AML progression in vivo. Interestingly, DRAM1 signaling is significantly activated in AML cells with ARHGAP4 knockdown. Our results also indicated that ARHGAP4 might function in AML cells by binding with p53 to inhibit DRAM1. Moreover, knockdown of DRAM1 rescues the defects of ARHGAP4 in AML cells. This newly described role of the ARHGAP4/DRAM1 axis in regulating AML progression may have important therapeutic implications.

ELMOD3-Rab1A-Flotillin2 cascade regulates lumen formation via vesicle trafficking in Ciona notochord.

Lumen development is a crucial phase in tubulogenesis, although its molecular mechanisms are largely unknown. In this study, we discovered an ELMO domain-containing 3 (ELMOD3), which belongs to ADP-ribosylation factor GTPase-activating protein family, was necessary to form the notochord lumen in Ciona larvae. We demonstrated that ELMOD3 interacted with lipid raft protein Flotillin2 and regulated its subcellular localization. The loss-of-function of Flotillin2 prevented notochord lumen formation. Furthermore, we found that ELMOD3 also interacted with Rab1A, which is the regulatory GTPase for vesicle trafficking and located at the notochord cell surface. Rab1A mutations arrested the lumen formation, phenocopying the loss-of-function of ELMOD3 and Flotillin2. Our findings further suggested that Rab1A interactions influenced Flotillin2 localization. We thus identified a unique pathway in which ELMOD3 interacted with Rab1A, which controlled the Flotillin2-mediated vesicle trafficking from cytoplasm to apical membrane, required for Ciona notochord lumen formation.

The Antithetic Roles of IQGAP2 and IQGAP3 in Cancers

The scaffold protein family of IQ motif-containing GTPase-activating proteins (IQGAP1, 2, and 3) share a high degree of homology and comprise six functional domains. IQGAPs bind and regulate the cytoskeleton, interact with MAP kinases and calmodulin, and have GTPase-related activity, as well as a RasGAP domain. Thus, IQGAPs regulate multiple cellular processes and pathways, affecting cell division, growth, cell-cell interactions, migration, and invasion. In the past decade, significant evidence on the function of IQGAPs in signal transduction during carcinogenesis has emerged. Compared with IQGAP1, IQGAP2 and IQGAP3 were less analyzed. In this review, we summarize the different signaling pathways affected by IQGAP2 and IQGAP3, and the antithetic roles of IQGAP2 and IQGAP3 in different types of cancer. IQGAP2 expression is reduced and plays a tumor suppressor role in most solid cancer types, while IQGAP3 is overexpressed and acts as an oncogene. In lymphoma, for example, IQGAPs have partially opposite functions. There is considerable evidence that IQGAPs regulate a multitude of pathways to modulate cancer processes and chemoresistance, but some questions, such as how they trigger this signaling, through which domains, and why they play opposite roles on the same pathways, are still unanswered.

Expression Analyses of Rich2/Arhgap44, a Rho Family GTPase-Activating Protein, during Mouse Brain Development

Rho family small GTPases, such as Rho, Rac, and Cdc42, play essential roles during brain development, by regulating cellular signaling and actin cytoskeletal reorganization. Rich2/Arhgap44, a Rac- and Cdc42-specific GTPase-activating protein, has been reported to be a key regulator for dendritic spine morphology and synaptic function. Given the essential roles of Rac and Cdc42 in brain development, Rich2 is supposed to take part in brain development. However, not only the molecular mechanism involved but also the expression profile of Rich2 during neurodevelopment has not yet been elucidated. In this study, we carried out expression analyses of Rich2 by focusing on mouse brain development. In immunoblotting, Rich2 exhibited a tissue-dependent expression profile in the young adult mouse, and the expression was increased during brain development. In immunohistochemical analyses, Rich2 was observed in the cytoplasm of cortical neurons at postnatal day (P) 0 and then came to be enriched in the nucleus with moderate distribution in neuropils at P7. Later at P30, a complex immunostaining pattern of Rich2 was observed; Rich2 was distributed in the nucleus, cytoplasm, and neuropils in many cortical neurons, whereas other neurons frequently displayed little expression. In the hippocampus at P7, Rich2 was distributed mainly in the cytoplasm of excitatory neurons in the cornu ammonis regions, while it was moderately detected in the nucleus in the dentate granule cells. Notably, Rich2 was distributed in excitatory synapses of the cornu ammonis 1 region at P30. Biochemical fractionation analyses also detected Rich2 in the postsynaptic density. Taken together, Rich2 is found to be expressed in the central nervous system in a developmental stage-dependent manner and may be involved in synapse formation/maintenance in cortical neurons.

CDC42-IQGAP Interactions Scrutinized: New Insights into the Binding Properties of the GAP-Related Domain.

The IQ motif-containing GTPase-activating protein (IQGAP) family composes of three highly-related and evolutionarily conserved paralogs (IQGAP1, IQGAP2 and IQGAP3), which fine tune as scaffolding proteins numerous fundamental cellular processes. IQGAP1 is described as an effector of CDC42, although its effector function yet re-mains unclear. Biophysical, biochemical and molecular dynamic simulation studies have proposed that IQGAP RASGAP-related domains (GRDs) bind to the switch regions and the insert helix of CDC42 in a GTP-dependent manner. Our kinetic and equilibrium studies have shown that IQGAP1 GRD binds, in contrast to its C-terminal 794 amino acids (called C794), CDC42 in a nucleotide-independent manner indicating a binding outside the switch regions. To resolve this discrepancy and move beyond the one-sided view of GRD, we carried out affinity measurements and a systematic mutational analysis of the interfacing residues between GRD and CDC42 based on the crystal structure of the IQGAP2 GRD-CDC42Q61L GTP complex. We determined a 100-fold lower affinity of the GRD1 of IQGAP1 and of GRD2 of IQGAP2 for CDC42 mGppNHp in comparison to C794/C795 proteins. Moreover, partial and major mutation of CDC42 switch regions substantially affected C794/C795 binding but only a little GRD1 and remarkably not at all the GRD2 binding. However, we clearly showed that GRD2 contributes to the overall affinity of C795 by using a 11 amino acid mutated GRD variant. Furthermore, the GRD1 binding to the CDC42 was abolished using specific point mutations within the insert helix of CDC42 clearly supporting the notion that CDC42 binding site(s) of IQGAP GRD lies outside the switch regions among others in the insert helix. Collectively, this study provides further evidence for a mechanistic framework model that is based on a multi-step binding process, in which IQGAP GRD might act as a ‘scaffolding domain’ by binding CDC42 irrespective of its nucleotide-bound forms, followed by other IQGAP domains downstream of GRD that act as an effector domain and is in charge for a GTP-dependent interaction with CDC42.

ARHGAP11A Promotes the Malignant Progression of Gastric Cancer by Regulating the Stability of Actin Filaments through TPM1.

The mechanism underlying the poor prognosis of gastric cancer, including its high degree of malignancy, invasion, and metastasis, is extremely complicated. Rho GTPases are involved in the occurrence and development of a variety of malignant tumors. ARHGAP11A, in the Rho GTPase activating protein family, is highly expressed in gastric cancer, but its function and mechanism have not yet been explored. In this study, the effect of ARHGAP11A on the occurrence and development of gastric cancer and the mechanism related to this effect were studied. The expression of ARHGAP11A was increased in gastric cancer cells and tissues, and high ARHGAP11A expression in tissues was related to the degree of tumor differentiation and poor prognosis. Moreover, ARHGAP11A knockout significantly inhibited cell proliferation, cell migration, and invasion in vitro and significantly inhibited the tumorigenic ability of gastric cancer cells in nude mice in vivo. Further studies revealed that ARHGAP11A promotes the malignant progression of gastric cancer cells by interacting with TPM1 to affect cell migration and invasion and the stability of actin filaments. These results suggest that ARHGAP11A plays an important role in gastric cancer and may become a useful prognostic biomarker and therapeutic target for gastric cancer patients.

Structure-activity mapping of ARHGAP36 reveals regulatory roles for its GAP homology and C-terminal domains.

ARHGAP36 is an atypical Rho GTPase-activating protein (GAP) family member that drives both spinal cord development and tumorigenesis, acting in part through an N-terminal motif that suppresses protein kinase A and activates Gli transcription factors. ARHGAP36 also contains isoform-specific N-terminal sequences, a central GAP-like module, and a unique C-terminal domain, and the functions of these regions remain unknown. Here we have mapped the ARHGAP36 structure-activity landscape using a deep sequencing-based mutagenesis screen and truncation mutant analyses. Using this approach, we have discovered several residues in the GAP homology domain that are essential for Gli activation and a role for the C-terminal domain in counteracting an N-terminal autoinhibitory motif that is present in certain ARHGAP36 isoforms. In addition, each of these sites modulates ARHGAP36 recruitment to the plasma membrane or primary cilium. Through comparative proteomics, we also have identified proteins that preferentially interact with active ARHGAP36, and we demonstrate that one binding partner, prolyl oligopeptidase-like protein, is a novel ARHGAP36 antagonist. Our work reveals multiple modes of ARHGAP36 regulation and establishes an experimental framework that can be applied towards other signaling proteins.

Abstract 6178: Loss of DAB2IP promotes resistance to radiation in rectal cancer by activating HIF-1α

Abstract Rectal cancer is one of the most common cancers in the United State. It shows that approximately 72.20% of the diagnosed rectal cancers are T3-4N0 or TxN+ from the SEER database. Preoperative radiotherapy has been performed as a prevalent therapy to decrease the local recurrence after total mesorectal excision. However, some patients could not get benefits from the local radiotherapy because of the resistance to radiation. While different expressions of numerous proteins have been found in radiation resistant cancer, the mechanism has been still veiled. Previously, we reported that DAB2IP, a novel member of the Ras GTPase-activating protein family, was down-regulated in various human cancers. DAB2IP functions as a scaffold protein to balance proliferation and apoptosis, and also inhibits metastasis in prostate cancer cells. In CRC, loss of DAB2IP increases the properties of cancer stem cells by regulating the NF-kB signal pathway, which also indicates the poor survival outcome. However, the role and mechanism of DAB2IP involving the resistance to radiation has not been elucidated. In this study, firstly, we compared the expression of DAB2IP in rectal cancers sensitive (42 patients) and non-sensitive (47 patients) to radiation by immunohistochemistry. Our data showed that non- or low-expression of DAB2IP was found in patients resistant to radiation (P<0.001). And these patients had worse disease free survival than high DAB2IP expression patients (p<0.01). Multivariate analyses indicated that DAB2IP expression level was an independently prognostic marker for survival of patients with rectal cancer (p<0.05). Then, we explored the biological function of DAB2IP by knocking down endogenous DAB2IP in cultured rectal cancer cell line SW837. Our results showed that knocking down DAB2IP in SW837 increased the survival rate when exposed to 2Gy dose radiation demonstrated by MTT assay and Anexin V/PI assay in flow cytometery. In xenograft model, SW837 with DAB2IP knocking down grew faster by recording the growth curve even if given total 10Gy dose radiation. Moreover, we found the amount of cleaved PARP and phosphorylated γ-H2AX decreased in DAB2IP knocking down cells exposed to 2Gy dose radiation for 6h. In addition, loss of DAB2IP could promote HIF-1α expression in nucleus and activate the signal pathway by HIF-1α luciferase analysis. In SW837 with DAB2IP knocking down, knocking down the HIF-1a by siRNA could almost rescue DAB2IP loss inducing the resistance to radiation. Taken together, our data provides strong evidence that loss of DAB2IP may contribute to the resistance to radiation in rectal cancer by activating the HIF-1α pathway. Citation Format: Shenglan Yang, Jiang Min, Liang liu, Daxing Xie. Loss of DAB2IP promotes resistance to radiation in rectal cancer by activating HIF-1α [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6178.

Integrative Analysis of the IQ Motif-Containing GTPase-Activating Protein Family Indicates That the IQGAP3-PIK3C2B Axis Promotes Invasion in Colon Cancer.

BackgroundColon cancer (CRC) is a common type of tumour, and IQGAP family proteins play an important role in many tumours. However, their roles in CRC remain unclear.MethodsFirst, we searched many public databases to comprehensively analyze expression of IQGAPs in CRC. Next, real-time PCR, immunohistochemical (IHC), transwell, siRNA transfection and Western blot assays were used to evaluate relationships among IQGAP3 expression, clinical pathological parameters and CRC prognosis, and the underlying molecular mechanism was investigated.ResultsIQGAP3 was elevated in CRC tissues, whereas there was no significant change in expression of IQGAP1 or IQGAP2. Additionally, IQGAP3 expression in CRC tissues was associated with tumour progression, invasion and poor prognosis. In mechanistic studies, we found that IQGAP3 was positively coexpressed with PIK3C2B. In an in vitro assay, the PIK3C2B expression level was increased after exogenous overexpression of IQGAP3, resulting in the promotion of cell invasion, which was blocked by pretransfecting cells with PIK3C2B siRNA. Furthermore, we found that high expression of IQGAP3 and PIK3C2B correlated with tumour stage and vessel invasion in human CRC, whereby patients with high expression of both in tumours had a worse prognosis compared with patients with single-positive or double-negative tumours.ConclusionThe results of our current study and corresponding previous studies provide evidence that IQGAP3 is elevated in CRC and promotes colon cancer growth and metastasis by regulating PIK3C2B activation.

P190A inactivating mutations cause aberrant RhoA activation and promote malignant transformation via the Hippo-YAP pathway in endometrial cancer

The Rho family of GTPases is strictly regulated by a large family of GTPase-activating proteins (GAPs) that stimulate the relatively weak intrinsic GTP-hydrolyzing activity of Rho GTPases. p190A is a potent and widely expressed GAP that acts on RhoA GTPases. p190A is frequently mutated in endometrial cancer, but the contribution of p190A mutations to endometrial tumorigenesis remains unclear. Here we identified that p190A is an upstream regulator of the Hippo-YAP signaling pathway, which is a critical regulator of cell proliferation, apoptosis, and cell fate. p190A knockout in endometrial cancer cells promoted cell proliferation, migration, and epithelial–mesenchymal transition (EMT), which were partially dependent on YAP activation. Wild-type p190A, but not endometrial cancer-associated mutants, suppressed the nuclear localization, transcriptional activity, and malignant transformation function of YAP. Moreover, the nuclear localization of YAP was enhanced in p190A-mutated endometrial cancer. These findings reveal novel molecular mechanisms underlying Hippo-YAP pathway-driven endometrial tumorigenesis and elucidate the potential for therapy targeting the Hippo-YAP pathway in p190A-mutated endometrial cancer.

Identification of a truncated β1-chimaerin variant that inactivates nuclear Rac1.

β1-chimaerin belongs to the chimaerin family of GTPase-activating proteins (GAPs) and is encoded by the CHN2 gene, which also encodes the β2- and β3-chimaerin isoforms. All chimaerin isoforms have a C1 domain that binds diacylglycerol as well as tumor-promoting phorbol esters and a catalytic GAP domain that inactivates the small GTPase Rac. Nuclear Rac has emerged as a key regulator of various cell functions, including cell division, and has a pathological role by promoting tumorigenesis and metastasis. However, how nuclear Rac is regulated has not been fully addressed. Here, using several approaches, including siRNA-mediated gene silencing, confocal microscopy, and subcellular fractionation, we identified a nuclear variant of β1-chimaerin, β1-Δ7p-chimaerin, that participates in the regulation of nuclear Rac1. We show that β1-Δ7p-chimaerin is a truncated variant generated by alternative splicing at a cryptic splice site in exon 7. We found that, unlike other chimaerin isoforms, β1-Δ7p-chimaerin lacks a functional C1 domain and is not regulated by diacylglycerol. We found that β1-Δ7p-chimaerin localizes to the nucleus via a nuclear localization signal in its N terminus. We also identified a key nuclear export signal in β1-chimaerin that is absent in β1-Δ7p-chimaerin, causing nuclear retention of this truncated variant. Functionally analyses revealed that β1-Δ7p-chimaerin inactivates nuclear Rac and negatively regulates the cell cycle. Our results provide important insights into the diversity of chimaerin Rac-GAP regulation and function and highlight a potential mechanism of nuclear Rac inactivation that may play significant roles in pathologies such as cancer.

RASAL2 Plays Inconsistent Roles in Different Cancers.

RAS protein activator like 2 (RASAL2) belongs to the RAS GTPase-activating protein family and plays an important role in several cancers, including ovarian cancer, nasopharyngeal carcinoma, malignant astrocytoma, renal cell carcinoma, bladder cancer, colorectal cancer, liver cancer, triple-negative breast cancer, lung adenocarcinoma, and pancreatic ductal adenocarcinoma. Traditionally, RASAL2 has been regarded as a tumor suppressor but recent studies have found that it is an oncogene in specific types of cancer, such as colorectal cancer, liver cancer, triple-negative breast cancer, triple-negative/estrogen receptor-negative breast cancer. In this review, we summarize the latest findings regarding RASAL2 in cancers, which may be important and useful in clinical practice. We discussed the specific functions and mechanisms of RASAL2 in different kinds of cancer cells (including its inhibition of invasion, metastasis and angiogenesis and its opposite effects), which may provide new directions for cancer research and treatments. RASAL2 exhibits different relationship with clinical cancer stage, histological grade, prognosis and overall survival in different kinds of tumor. RASAL2 is a potential prognostic factor and a new therapeutic target for diagnosis and treatment.

Clinical value of detecting IQGAP3, B7-H4 and cyclooxygenase-2 in the diagnosis and prognostic evaluation of colorectal cancer

BackgroundThe IQ-motif-containing GTPase-activating protein (IQGAP) family comprises three members, IQGAP1, IQGAP2 and IQGAP3. IQGAP3 is the latest addition to the family. This study mainly investigated the novel marker IQGAP3 at serum and tumor tissue levels compared with the markers B7-H4 and cyclooxygenase-2 (COX-2) in patients with colorectal cancer (CRC) and in healthy individuals, aiming to evaluate the diagnostic and prognostic value of IQGAP3 for CRC.Materials and methodsSerum samples were collected prior to any therapy in 118 CRC patients and as part of a routine examination in 85 healthy individuals. Serum IQGAP3, B7-H4 and COX-2 levels were measured using commercially available ELISA kits. Immunohistochemistry was performed to detect the IQGAP3, B7-H4 and COX-2 in tumor tissues and normal para-carcinoma tissues. The receiver operating characteristics (ROC) curve and the area under the curve (AUC) were used to evaluate and compare the diagnostic value of different serum tumor markers. Univariate and multivariate analyses were performed to identify the prognostic risk factors for CRC.ResultsIQGAP3, B7-H4 and COX-2 showed low or high expression in tumor tissues while no expression in normal para-carcinoma tissues. Serum levels of IQGAP3 in CRC group were significantly higher than those in healthy control group (P < 0.001). The IQGAP3 AUC was 0.799, while the B7-H4 AUC was 0.795 and the COX-2 AUC was 0.796. IQGAP3 seemed to be superior to B7-H4 and COX-2 in detecting CRC, with the highest sensitivity among the three markers. Multivariate analysis showed that T stage, N stage, differentiation degree, TNM stage and both serum and tissue IQGAP3, B7-H4 and COX-2 levels were significant prognostic factors for CRC.ConclusionsIQGAP3 has a better diagnostic efficacy than B7-H4 and COX-2 in detecting CRC and it has value in predicting the prognosis of patients with CRC.

IN SILICO STUDY OF MIRNA-REGULATED IQ MOTIF-CONTAINING GTPASE-ACTIVATING PROTEIN FAMILY IN LIVER CANCER

Objective: The aim of this paper is to identify the list of microRNA (miRNA) which can regulate the aberrant expression of IQGAP in liver cancer formation. The aberrant expression of IQ motif-containing GTPase-activating protein (IQGAP) family which consists of IQGAP1, IQGAP2, and IQGAP3 has been linked to carcinogenesis in human cancers. The reciprocal expression of IQGAP family in human cancer has been studied to act as oncogenes or tumor suppressor genes. A growing number of studies suggest that upregulated or downregulated expression of IQGAP family triggers cancer development.Methods: A correlation study was performed to construct a pathway to inhibit or activate IQGAP family between miRNAs and IQGAPs. A pre-processing step was conducted to download, filter and process the dataset from TCGA. It yields miRNA and IQGAP gene expression matrix. Then, correlation computation was computed using MATLAB. Moreover, this study linked the results to the MiRTarBase to validate the prediction result with the wet lab experimental result.Results: This study identified significantly inversely correlation in 51 miRNAs-IQGAP1, 169 miRNAs-IQGAP2, and 33 miRNAs-IQGAP3, respectively, which may potentially play a role in a liver cancer formation. Some of the results also can be found in miRTarBase. It supports the precision of those miRNA and IQGAP interaction between dry lab and wet lab study. IQGAP1 and IQGAP2 mostly has been identified as an oncogene in cancer but IQGAP2 has been discovered as tumor suppressor gene. The list of miRNA in the result of this study can become a potential therapy to target the aberrant expression of IQGAP family.Conclusion: miRNA function is known as an oncogene or tumor suppressor gene in cancer development. Therefore, it can be one of the important molecular biology which may target the aberrant expression of IQGAP in liver cancer.

ARHGAP30 suppressed lung cancer cell proliferation, migration, and invasion through inhibition of the Wnt/β-catenin signaling pathway.

ObjectiveRho GTPase-activating protein 30 (ARHGAP30), a member of the Rho GTPase-activating proteins (Rho GAPs) family, plays an important role in the regulation of cytoskeleton organization and cell adhesion.Materials and methodsmRNA and protein expression was assessed by quantitative real-time PCR and Western blotting, respectively. Cell Counting Kit-8 (CCK-8) and Transwell assays were conducted to detect cell proliferation, migration, and invasion.ResultsARHGAP30 expression was downregulated in specimens and cell lines of lung cancer in comparison to non-cancerous specimens and normal bronchial epithelial cell lines, respectively. Moreover, in vitro experiments demonstrated that ARHGAP30 overexpression impeded the proliferative, migratory, and invasive abilities of lung cancer cells. Moreover, bioinformatics analysis with The Cancer Genome Atlas (TCGA) lung cancer dataset showed a negative association between ARHGAP30 expression and the Wnt signaling pathway. Enforced expression of ARHGAP30 decreased the mRNA and protein levels of β-catenin, c-Myc, matrix metalloproteinase-2 (MMP-2) and MMP-9. Besides, the β-catenin inhibitor XAV939 blocked the enhanced cell growth, migration, and invasion caused by ARHGAP30 knockdown. Thus, the Wnt/β-catenin pathway mediated the functions of ARHGAP30 in lung cancer cells.ConclusionARHGAP30 acts as a tumor suppressor in lung cancer by suppressing Wnt/β-catenin signaling.

Abstract B07: Targeting DAB2IP in ovarian cancer stem cells

Abstract Ovarian cancer is the leading cause of death from gynecologic malignancies. Failure to eliminate ovarian cancer stem cells (OCSCs) by the end of conventional chemotherapy contributes to tumor relapse and metastasis. Understanding the molecular features of OCSCs may allow for effective targeting and eradicating these cells and tumor remission. We reported tumor-initiation and expansion capacity by ALDH+ OCSCs after platinum-based treatment of ovarian cancer xenografts. In addition, loss of DAB2IP in prostate and colon cancer promoted CSC-like features, suggesting a critical role in modulating CSC maintenance. DAB2IP, a novel member of Ras GTPase-activating (GAP) protein family, was identified as a potent tumor suppressor that interfered with many aspects of cancer progression and was frequently downregulated in tumors. In the current study, we aimed to elucidate the tumor-suppressor role of DAB2IP in ovarian cancer in the context of OCSCs and explore epigenetic approaches to upregulate its expression. OCSCs and non-CSCs subpopulations were isolated from multiple high-grade serous ovarian cancer cell lines (Kuramochi, OVCAR3, and COV362). Reduced (p&amp;lt;0.05) DAB2IP expression in OCSCs compared to non-CSC counterparts was observed, suggesting that DAB2IP suppresses OCSC maintenance. Overexpression of DAB2IP in OCSCs derived from Kuramochi decreased (p&amp;lt;0.05) OCSC population, and DAB2IP overexpression inhibited stemness-related phenotypes, including reduced (P&amp;lt;0.05) spheroid formation ability after 14-day incubation under stem cell conditions and decreased (P&amp;lt;0.05) colony formation. Decreased expression of stem cell markers (Oct4, Nanog, and Twist) in DAB2IP-overexpressing cells was also observed, indicating potential key effectors downstream of DAB2IP. Furthermore, elevated DAB2IP expression in OCSCs decreased (p&amp;lt;0.05) cisplatin IC50 and cell migration capacity in transwell assay, suggesting DAB2IP plays a role in OCSC function. Aberrant DNA promoter methylation and histone modifications were previously shown to be two major mechanisms of DAB2IP inactivation in cancer. We treated OCSCs with next-generation DNMT inhibitor guadecitabine and EZH2 inhibitor GSK-126, either alone or in combination. Single-agent guadecitabine or GSK-126 decreased ALDH expression with greater (p&amp;lt;0.05) effect in combination. Single GSK-126 was sufficient to increase DAB2IP expression (p&amp;lt;0.05) with additional increase when combined with guadecitabine, correlating with decreased ALDH expression. Collectively, our data suggested a possible combination epigenetic therapy to eliminate OCSCs, potentially through targeting DAB2IP, an important OCSC regulator. Citation Format: Xingyue Zong, Ali R. Ozes, Kenneth P. Nephew. Targeting DAB2IP in ovarian cancer stem cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B07.

CYK-4 functions independently of its centralspindlin partner ZEN-4 to cellularize oocytes in germline syncytia.

Throughout metazoans, germ cells undergo incomplete cytokinesis to form syncytia connected by intercellular bridges. Gamete formation ultimately requires bridge closure, yet how bridges are reactivated to close is not known. The most conserved bridge component is centralspindlin, a complex of the Rho family GTPase-activating protein (GAP) CYK-4/MgcRacGAP and the microtubule motor ZEN-4/kinesin-6. Here, we show that oocyte production by the syncytial Caenorhabditis elegans germline requires CYK-4 but not ZEN-4, which contrasts with cytokinesis, where both are essential. Longitudinal imaging after conditional inactivation revealed that CYK-4 activity is important for oocyte cellularization, but not for the cytokinesis-like events that generate syncytial compartments. CYK-4's lipid-binding C1 domain and the GTPase-binding interface of its GAP domain were both required to target CYK-4 to intercellular bridges and to cellularize oocytes. These results suggest that the conserved C1-GAP region of CYK-4 constitutes a targeting module required for closure of intercellular bridges in germline syncytia.