IQGAP1 Expression Research Articles

Structure and function of IQ-domain GTPase-activating protein 1 and its association with tumor progression (Review)

IQ-domain GTPase-activating proteins (IQGAPs) are evolutionary conserved multidomain proteins that are found in numerous organisms, from yeast to mammals. To date, three IQGAP proteins have been identified in humans, of which IQGAP1 is the best characterized. As a scaffold protein, IQGAP1 contains multiple protein-interacting domains, which modulate binding to target proteins. Recent mounting studies demonstrated a role for IQGAP1 in tumor progression, supported by the altered expression and subcellular distribution of IQGAP1 in tumors. The contribution of IQGAP1 to tumor progression appears to involve a complex interplay of cell functions by integrating diverse signal transduction pathways and coordinating activities, such as cell adhesion, migration, invasion, proliferation and angiogenesis.

Bile Acids Activate YAP to Promote Liver Carcinogenesis

Elevated bile acid levels increase hepatocellular carcinoma by unknown mechanisms. Here, we show that mice with a severe defect in bile acid homeostasis due to the loss of the nuclear receptors FXR and SHP have enlarged livers, progenitor cell proliferation, and Yes-associated protein (YAP) activation and develop spontaneous liver tumorigenesis. This phenotype mirrors mice with loss of hippo kinases or overexpression of their downstream target, YAP. Bile acids act as upstream regulators of YAP via a pathway dependent on the induction of the scaffold protein IQGAP1. Patients with diverse biliary dysfunctions exhibit enhanced IQGAP1 and nuclear YAP expression. Our findings reveal an unexpected mechanism for bile acid regulation of liver growth and tumorigenesis via the Hippo pathway.

Overexpression of IQGAP1 in human pancreatic cancer

Pancreatic cancer is a highly aggressive malignant tumor with the lowest survival rate. A better understanding of the molecular mechanisms which contribute to pancreatic cancer occurrence and progression will aid in the development of new approaches to the early diagnosis, prevention, and treatment of this deadly disease. The scaffold protein IQGAP1 shows elevated levels in a variety of cancer types. Currently, we investigated whether or not IQGAP1 is also overexpressed in pancreatic cancer. IQGAP1 expression was examined in pancreatic cancer and normal tissues adjacent to cancerous tissues (adjacent tissues) by Western blotting and real-time RT-PCR as well as in paraffin sections of tissue microarray by immunohistochemistry. The correlations between IQGAP1 expression and various clinicopathological characteristics were analyzed. Western blotting and real-time RT-PCR revealed that the levels of IQGAP1 protein and mRNA expression in pancreatic cancer tissues were significantly increased compared with adjacent tissues. Immunohistochemistry analysis on tissue microarray showed that IQGAP1 protein expression was significantly higher in pancreatic cancer (80.0%, 48/60) compared with adjacent tissues (18.3%, 11/60) (P<0.001). Moreover, overexpression of IQGAP1 was shown to be associated with the grades of tumor differentiation (P<0.05). The overexpression of IQGAP1 may play an important role in pancreatic cancer occurrence and progression, and IQGAP1 may serve as a novel molecular target for the diagnosis and treatment of pancreatic cancer.

IQGAP1 expression in spared CA1 neurons after an excitotoxic lesion in the mouse hippocampus.

Repeated seizures induce permanent alterations in the hippocampal circuits in experimental models with intractable temporal lobe epilepsy. Sprouting and synaptic reorganization induced by seizures has been well-studied in the mossy fiber pathway. However, studies investigating sprouting and synaptic reorganization beyond the mossy fiber pathway are limited. The present study examined the biochemical changes of CA1 pyramidal neurons undergoing morphological changes after excitotoxicity-induced hippocampal CA3 neuronal death. IQ-domain GTPase-activating proteins (IQGAP1), is an effector of Rac1 and Cdc42 and an actin-binding protein, was upregulated in CA1 pyramidal neurons after kainic acid-induced hippocampal CA3 neuronal degeneration. IQGAP1+cells were colocalized with Nestin, but not in astrocytes or mature neurons. Furthermore, IQGAP1 did not originate from newly divided local precursors or NG2+cells. IQGAP1 and adenomatous polyposis coli localized in CA1 pyramidal neurons, and Cdc42 activation was followed by IQGAP1 recruitment. These findings suggest that IQGAP1 is upregulated in pre-existed sparing neurons of the CA1 layer undergoing morphological changes after excitoxicity-induced hippocampal CA3 neuronal death. It demonstrates the utility of IQGAP1 as a possible marker for spared pyramidal neurons, which may contribute to structural and functional alternations responsible for the development of epilepsy.

Increasing our IQ of vascular smooth muscle cell migration with IQGAP1. Focus on “IQGAP1 links PDGF receptor-β signal to focal adhesions involved in vascular smooth muscle cell migration: role in neointimal formation after vascular injury”

<i>Increasing our IQ of vascular smooth muscle cell migration with IQGAP1</i>. Focus on “IQGAP1 links PDGF receptor-β signal to focal adhesions involved in vascular smooth muscle cell migration: role in neointimal formation after vascular injury”

IQGAP1 Functions as a Modulator of Dishevelled Nuclear Localization in Wnt Signaling

Dishevelled (DVL) is a central factor in the Wnt signaling pathway, which is highly conserved among various organisms. DVL plays important roles in transcriptional activation in the nucleus, but the molecular mechanisms underlying their nuclear localization remain unclear. In the present study, we identified IQGAP1 as a regulator of DVL function. In Xenopus embryos, depletion of IQGAP1 reduced Wnt-induced nuclear accumulation of DVL, and expression of Wnt target genes during early embryogenesis. The domains in DVL and IQGAP1 that mediated their interaction are also required for their nuclear localization. Endogenous expression of Wnt target genes was reduced by depletion of IQGAP1 during early embryogenesis, but notably not by depletion of other IQGAP family genes. Moreover, expression of Wnt target genes caused by depletion of endogenous IQGAP1 could be rescued by expression of wild-type IQGAP1, but not IQGAP1 deleting DVL binding region. These results provide the first evidence that IQGAP1 functions as a modulator in the canonical Wnt signaling pathway.

Endothelial cell IQGAP1 is an important regulator of the transendothelial migration of leukocytes

One of the key processes in the inflammatory response is the movement of leukocytes across the endothelium (transendothelial migration, TEM). We have shown that TEM requires a novel endothelial cell compartment, the Lateral Border Recycling Compartment (LBRC), which delivers requisite membrane proteins to surround the migrating leukocyte. In a recent proteomic screen to identify the components of the LBRC we discovered that IQGAP1 is specifically associated with it. Moreover, IQGAP1 is a critical factor in TEM. shRNA mediated knockdown of IQGAP1 expression in endothelial cells significantly impairs their ability to support TEM. In IQGAP1 knockdown cells, the targeted movement of the LBRC to the leukocyte is severely disrupted. This defect is rescued by re‐expression of full length IQGAP1. Using fluorescently tagged IQGAP1 constructs, we can follow IQGAP1 during TEM and have found that it moves with the LBRC to surround the migrating leukocyte. Furthermore, we are performing domain truncations of IQGAP1 to determine which of its many functional interactions are required for TEM. These data suggest that IQGAP1 is an important factor in the regulation of LBRC trafficking during TEM. This research was supported by F32 AI084454 to DPS and R21 HL102519, R01 HL046849, and R37 HL064774 to WAM.

Expression pattern of IQGAP1 in sinonasal inverted papillomas and squamous cell carcinomas

The scaffold protein IQGAP1 is a ubiquitously expressed 190 kDa protein that participates in protein-protein interactions, integrating diverse signaling pathways. In this study, we examined the expression of IQGAP1 in sinonasal inverted papillomas (IP) and sinonasal squamous cell carcinomas (SCC), which include IP with SCC (IPcSCC) and SCC alone. We also tried to elucidate whether the amount of IQGAP1 expression is related with any clinicopathologic features of sinonasal SCCs. This was a retrospective study. The expression of IQGAP1 was analyzed by means of immunohistochemistry. The degrees of IQGAP1 expression and subcellular localization (rim formation) were analyzed with respect to the following clinicopathologic variables: age, gender, T stage, histologic differentiation, local recurrence, and distant metastasis. The expression of IQGAP1 was significantly higher in patients with IPcSCC (3.62 ± 0.14), and SCC (2.93 ± 0.34) compared with IP (1.43 ± 0.26). The rim formation scale was significantly higher in IPcSCC (2.39 ± 0.24) compared with IP (1.68 ± 0.15). Distant metastasis in patients with SCC significantly correlated with an increased IQGAP1 rim formation (p = 0.011). Histologic differentiation was also significantly correlated with a positive staining scale for IQGAP1 in IPcSCCs (p = 0.038). These results indicated that IQGAP1 expression is associated with both the histologic differentiation of IPcSCCs and is present in SCC cases where distant metastasis has occurred.

IQGAP1 Interacts with Components of the Slit Diaphragm Complex in Podocytes and Is Involved in Podocyte Migration and Permeability In Vitro

IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (α-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties.

Endothelial cell IQGAP1 is an important regulator of the transendothelial migration of leukocytes

The movement of leukocytes across the endothelium (transendothelial migration, TEM) into tissue is a complex process that is critical for both beneficial and deleterious inflammation. Our lab recently identified a novel endothelial cell compartment, (the Lateral Border Recycling Compartment, LBRC) that delivers critical membrane proteins to surround the migrating leukocyte and is required for TEM. Despite its importance in inflammation, the protein composition of the LBRC is largely unknown. Using biochemistry and subcellular fractionation we recently identified IQGAP1 as a protein associated with the LBRC. Our data indicate that IQGAP1 is required for TEM and is likely involved with regulating LBRC function during TEM. siRNA mediated knockdown of IQGAP1 expression in endothelial cells significantly impairs their ability to support TEM without altering the structure or size of the LBRC. Furthermore, IQGAP1 appears to be recruited to the site of transmigration along with the LBRC. These data suggest that IQGAP1 is involved in the regulation of LBRC trafficking during TEM. This research was supported by F32 AI084454 to DPS and R01 HL046849, R37 HL064774, and R21 HL102519 to WAM.

Abstract A49: The ERK/MAPK scaffold IQGAP1 is a tumor-selective target

Abstract To advance insight into skin biology and pathophysiology, our lab has focused on understanding how the normal processes of cell division and migration are regulated and how they become dysregulated in pathological states, including cancer. One particular pathway of interest in the lab is the extracellular-regulated kinase/mitogen-activated protein kinase (Erk/MAPK) pathway. This highly conserved pathway is critical to epidermal homeostasis and tumorigenesis, but its mechanisms of action remain unclear. This research explores how the Erk/MAPK-interacting protein IQGAP1 mediates signaling in tumorigenesis. IQGAP1 physically interacts with canonical Erk/MAPK pathway members in order to precisely modulate their signal output and in its absence, signaling through this pathway is severely diminished. We recently found that depleting expression of IQGAP1, a Mek- and Erk-binding MAPK-IP, exerted no effect on normal tissue but effectively blocked invasive neoplasia. We are currently working to define the functional domains of IQGAP1 required for Erk MAPK-driven epidermal neoplasia. These efforts will help identify potentially important and therapeutically relevant neoplasia-selective interactions of IQGAP1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A49.

IQGAP1 involvement in MTOC and granule polarization in NK‐cell cytotoxicity

Natural killer (NK) cells form a region of tight contact called the NK immunological synapse (NKIS) with their target cells. This is a dynamic region serving as a platform for targeted signaling and exocytotic events. We previously identified IQGAP1 as a cytoskeletal component of the NK-like cell line YTS. The present study was undertaken to determine the role of IQGAP1 in the function of NK cells. Silencing of IQGAP1 expression resulted in almost complete loss of the cytotoxic activity of YTS cells. Loss of IQGAP1 did not prevent conjugate formation with target cells but it did result in a failure to reorient the microtubule organizing centre to the immune synapse. Significantly, IQGAP1 expression was required for the perigranular accumulation of an F-actin network. IQGAP1 was shown to undergo marked rearrangements during synapse maturation in effector target conjugates of YTS or primary NK cells. These results suggest previously undescribed role(s) for IQGAP1 in regulating multiple aspects of cytoskeletal organization and granule polarization in NK cells.

Alteration of gene expression of IQGAP1 and Rho-family GTPases in the cadmium-induced ventral body wall defects in the chick model

Cadmium (Cd) induces ventral body wall defects (VBWD) in the chick embryo, with adherens junctions (AJs) breakdown at 4 h post treatment (4H). Signalling by which Cd disrupts AJs in this model remains unclear. IQGAP1 regulates AJs via binding to Cdc42 and Rac1, Rho-family GTPases. Activation of IQGAP1-Cdc42 interaction regulates AJs positively, whereas Rac1 activation inhibits AJs. We hypothesised that IQGAP1 and Cdc42 are downregulated and Rac1 is upregulated during embryogenesis in the Cd chick model. Chick embryos were explanted and treated with saline or Cd after 60 h incubation. Chicks were harvested at 1H, 4H and 8H post treatment and RT-PCR and immunohistochemistry were performed. Gene expression levels of IQGAP1 and Cdc42 were significantly downregulated and Rac1 was upregulated in Cd group compared to controls only at 4H. Immunoreactivity of IQGAP1 and Cdc42 was also markedly decreased, whereas Rac1 was increased in Cd group compared to controls at 4H. Alteration of IQGAP and Rho-family GTPases may cause VBWD in Cd chick model by inducing the dissociation of cadherin-mediated AJs.

RhoC protein stimulates migration of gastric cancer cells through interaction with scaffold protein IQGAP1

The scaffold protein IQGAP1 is closely related to certain RhoGTPases. Research has revealed that IQGAP1 acts as an effector of Cdc42 and Rac1 in the regulation of cell activity such as proliferation and migration. However, whether IQGAP1 is associated with RhoC, another important RhoGTPase, is unclear. Previous results from our laboratory indicated that IQGAP1 and RhoC are highly expressed in gastric cancer tissues and cells. This study was designed to investigate the possible interaction between IQGAP1 and RhoC in the regulation of the migration of cancer cells. The expression of IQGAP1 and RhoC in gastric cancer tissues and cell lines was detected by Western blotting. siRNAs targeting IQGAP1 or RhoC were transfected into gastric cancer cells to knock down the expression of the proteins. Adenoviral constructs encoding full length IQGAP1, the C‑terminal fragment of IQGAP1, and the constitutively active RhoC gene were used to infect gastric cancer cells to increase the expression of the proteins. The migratory activity of a gastric cancer cell line was measured by a transwell migration assay. Westernblotting revealed that the IQGAP1 and RhoC proteins were highly expressed in gastric cancer tissues and cells. Spearman's rank correlation analysis indicated that the increases in the expression of IQGAP1 and RhoC were closely correlated. The transwell migration assay revealed that both IQGAP1 and RhoC stimulated the migration activity of the gastric cancer cell line AGS. The knockdown of IQGAP1 expression by siRNA blocked the migration‑stimulating activity of RhoC, while the knockdown of RhoC expression had no effect on the migration-stimulatory activity of IQGAP1. Co-IP results showed that RhoC and IQGAP1 bound to each other. These results reveal a previously unrecognized interaction between IQGAP1 and RhoC, and demonstrate that IQGAP1 is a downstream effector of RhoC in the regulation of the migration activity of gastric cancer cells.

Genetic variants at the miR-124 binding site on the cytoskeleton-organizing IQGAP1 gene confer differential predisposition to breast cancer

IQGAP1 knockout mice develop gastric cancer, but the IQGAP1 protein is associated with some advanced-stage human cancers. IQGAP1 expression is regulated by a microRNA, miR-124, through a binding site at the 3'-untranslated region, where a single nucleotide polymorphism (SNP) exists in the core binding region. We asked whether IQGAP1 expression is associated with breast cancer development and whether genetic variants at the miR-124 binding site are important. We genotyped the IQGAP1 SNP rs1042538 A/T in 1,541 breast cancer cases and 1,598 controls and analyzed the frequency of the variant and interactions with major risk factors in these populations. We also measured the expression of IQGAP1 at both mRNA and protein levels in different IQGAP1 genotypes. The IQGAP1 TT genotype, compared with the AA genotype, was associated with a significantly lower risk of developing breast cancer [P=0.049, odds ratio (OR), 0.78; 95% confidence interval (CI), 0.61-0.99]. In case-only analyses, the TT, compared with the AA, genotype was associated with progesterone receptor-positive subjects (OR, 1.35; 95% CI, 1.00-1.83). The expression levels of IQGAP1 protein were significantly higher in the TT genotype compated to the AA genotype. The presence of SNPs at the miR-124 binding site may be a marker for predicting breast cancer risk and prognosis.

IQGAP1 and IQGAP2 are Reciprocally Altered in Hepatocellular Carcinoma

BackgroundIQGAP1 and IQGAP2 are homologous members of the IQGAP family of scaffold proteins. Accumulating evidence implicates IQGAPs in tumorigenesis. We recently reported that IQGAP2 deficiency leads to the development of hepatocellular carcinoma (HCC) in mice. In the current study we extend these findings, and investigate IQGAP1 and IQGAP2 expression in human HCC.MethodsIQGAP1 and IQGAP2 protein expression was assessed by Western blotting and immunohistochemistry. IQGAP mRNA was measured by quantitative RT-PCR. The methylation status of the Iqgap2 promoter was determined by pyrosequencing of bisulfite-treated genomic DNA.ResultsIQGAP1 and IQGAP2 expression was reciprocally altered in 6/6 liver cancer cell lines. Similarly, immunohistochemical staining of 82 HCC samples showed that IQGAP2 protein expression was reduced in 64/82 (78.0%), while IQGAP1 was present in 69/82 (84.1%). No IQGAP1 staining was detected in 23/28 (82.1%) normal livers, 4/4 (100.0%) hepatic adenomas and 23/23 (100.0%) cirrhosis cases, while IQGAP2 was increased in 22/28 (78.6%), 4/4 (100.0%) and 23/23 (100.0%), respectively. Although the Iqgap2 promoter was not hypermethylated in HCC at any of the 25 CpG sites studied (N = 17), IQGAP2 mRNA levels were significantly lower in HCC specimens (N = 23) than normal livers (N = 6).ConclusionsWe conclude that increased IQGAP1 and/or decreased IQGAP2 contribute to the pathogenesis of human HCC. Furthermore, downregulation of IQGAP2 in HCC occurs independently of hypermethylation of the Iqgap2 promoter. Immunostaining of IQGAP1 and IQGAP2 may aid in the diagnosis of HCC, and their pharmacologic modulation may represent a novel therapeutic strategy for the treatment of liver cancer.

IQGAP1 Is Involved in Post-Ischemic Neovascularization by Regulating Angiogenesis and Macrophage Infiltration

BackgroundNeovascularization is an important repair mechanism in response to ischemic injury and is dependent on inflammation, angiogenesis and reactive oxygen species (ROS). IQGAP1, an actin-binding scaffold protein, is a key regulator for actin cytoskeleton and motility. We previously demonstrated that IQGAP1 mediates vascular endothelial growth factor (VEGF)-induced ROS production and migration of cultured endothelial cells (ECs); however, its role in post-ischemic neovascularization is unknown.Methodology/Principal FindingsIschemia was induced by left femoral artery ligation, which resulted in increased IQGAP1 expression in Mac3+ macrophages and CD31+ capillary-like ECs in ischemic legs. Mice lacking IQGAP1 exhibited a significant reduction in the post-ischemic neovascularization as evaluated by laser Doppler blood flow, capillary density and α-actin positive arterioles. Furthermore, IQGAP1−/− mice showed a decrease in macrophage infiltration and ROS production in ischemic muscles, leading to impaired muscle regeneration and increased necrosis and fibrosis. The numbers of bone marrow (BM)-derived cells in the peripheral blood were not affected in these knockout mice. BM transplantation revealed that IQGAP1 expressed in both BM-derived cells and tissue resident cells, such as ECs, is required for post-ischemic neovascularization. Moreover, thioglycollate-induced peritoneal macrophage recruitment and ROS production were inhibited in IQGAP1−/− mice. In vitro, IQGAP1−/− BM-derived macrophages showed inhibition of migration and adhesion capacity, which may explain the defective macrophage recruitment into the ischemic tissue in IQGAP1−/− mice.Conclusions/SignificanceIQGAP1 plays a key role in post-ischemic neovascularization by regulating, not only, ECs-mediated angiogenesis but also macrophage infiltration as well as ROS production. Thus, IQGAP1 is a potential therapeutic target for inflammation- and angiogenesis-dependent ischemic cardiovascular diseases.

MiR-124a Is Important for Migratory Cell Fate Transition During Gastrulation of Human Embryonic Stem Cells

Precise control of gene expression is of paramount importance for proper embryonic development. Although a number of microRNAs (miRNAs) has been implicated in fine-tuning mRNA translation during development, their exact roles for gastrulation, particularly in connection with functional targets, have yet to be clarified, with regard to stage-specific cell migration to form three embryonic germ layers. We found that miR-124a is expressed in human embryonic stem cells (hESC), but is gradually downregulated during embryoid body (EB) formation in vitro. We also provide evidence that SLUG and IQGAP1, which modulates rearrangement of the migratory cytoskeleton, are specific targets for miR-124a during EB formation. Furthermore, we show that the beginning of cell migration, a hallmark event in gastrulation, is tightly coupled with downregulation of miR-124a during EB formation and induction of SLUG and IQGAP1. Overexpressed miR-124a in hESC reduced expression of SLUG and IQGAP1 and blocked migratory cell behavior in EB. An expression level of MIXL1, associated with gastulation process, was also inversely correlated with expression of miR-124a. Taken together, our results strongly suggest that miR-124a may play an active role in inhibiting hESCs from differentiation into EB by downregulating expression of SLUG and IQGAP1, thereby maintaining stemness.

Abstract 4996: GTPase-activating protein IQGAP2 is a novel tumor suppressor in hepatocellular carcinoma

Abstract Highly homologous putative GTPase-activating proteins IQGAP1 and IQGAP2 are multidomain scaffolding proteins that juxtapose Rho GTPases Rac1 and Cdc42, Ca2+/calmodulin signals and cytoskeletal reorganization events. Previously, we found that IQGAP2 deficiency in mice leads to the development of hepatocellular carcinoma (HCC), identifying IQGAP2 as a potential tumor suppressor gene (Schmidt VA, et al. MCB 2008; 28:1489-502). Moreover, mice deficient in both Iqgap1 and Iqgap2 genes displayed relative protection against HCC and enhanced long-term survival, indicating opposing roles for IQGAP2 and IQGAP1 in hepatic carcinogenesis. We hypothesized that diminished levels of hepatic IQGAP2 expression, along with subsequent upregulation of IQGAP1, may have a causative effect in the development of HCC in humans. To test this, we first evaluated mRNA transcript and protein expression in an array of liver samples from patients with HCC of different etiologies using qRT-PCR and immunohistochemistry (IHC). qPCR TissueScan Array™ containing 23 individual cDNAs from patients with primary HCC of different stages and non-malignant adjacent tissue controls demonstrated decreased expression of IQGAP2 transcript in HCC tumors compared to normal tissue, and the magnitude of the decrease in expression correlated with progression of disease. The lowest IQGAP2 transcript expression was observed at the most advanced stage IV HCC, a 4-fold decrease compared to the non-malignant controls. At the protein level, lack of IQGAP2 expression was confirmed in 100% (36/36) of formalin-fixed paraffin-embedded human HCC livers studied by IHC. All samples also showed a significant overexpression of IQGAP1. Normal livers (13/13), cirrhotic livers (23/23), and benign hepatic adenomas (4/4) showed the reverse, i.e. a high level of IQGAP2 expression and very low levels of IQGAP1. Next, functional studies using RNAi technology showed that knockdown of IQGAP2 expression in HepG2 cells leads to a 50% increase in their migratory capacity, while IQGAP1 siRNA has the opposite effect. Finally, the gene expression profile of Iqgap2−/− mouse liver using the Mouse Genome 430 2.0 Array chip (Affymetrix) identified 294 genes which were expressed differentially in Iqgap2−/− HCC livers compared to age-matched disease-free wild-type controls (N=3 in each group). The differentially expressed genes (at least 2-fold change, p&amp;lt;0.05) included those encoding proteins associated with Wnt/beta-catenin signaling pathway activation (Igf2r, Sp5, Ccnb2, Ccnd1, Wif1 and Ctnnbip1) and tumor metastasis (Cdh1, Ctsl, Hgf, Fn1, Hpse and Timp2). Collectively, these findings validate the relevance of the Iqgap2−/− mouse model to human disease and identify a novel IQGAP-dependent pathway regulating HCC development. Modulation of both IQGAP1 and IQGAP2 expression represents a new potential therapeutic strategy for liver cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4996.

Abstract 2050: MicroRNA-124a: A master regulator of glioblastoma migration

Abstract For most patients diagnosed with a glioblastoma multiforme (GBM), the invasiveness of this tumor and its resistance to treatment predicts a poor outcome with no realistic possibility of a cure. Although brain tumor cells rarely metastasize outside the brain, they have an extraordinary capacity to migrate long distances and infiltrate normal brain, making complete surgical removal impossible. New therapeutic strategies are needed to target the invading cell population to further reduce tumor cell spread and a better understanding of the mechanisms promoting tumor migration and invasion are urgently needed. MiR-124a, a microRNA, normally abundantly expressed in normal adult brain tissue, shows marked down regulation (&amp;gt;1000 fold) in GBM cell lines (A172, T98G, MO59J, MO59K and U87MG). Conversely, a predicted miR-124a target, IQGAP1, is over-expressed in GBM cell lines and tissue samples. We previously demonstrated IQGAP1 overexpression in 86% of GBM patients and associated high levels with poor prognosis. IQGAP1 expression was prominent in the actively migrating and rapidly dividing palisading neoplastic cells that surround the necrotic foci. We hypothesized a reciprocal interaction between miR-124a and IQGAP1. To show this, GBM cell lines were transfected with a synthetic miR-124a precursor molecule to increase endogenous miR-124a expression. Cell migration was measured by impedence of a current (ECIS 9600) and tumor cell invasion was measured using transwell inserts. The interaction between miR-124a and IQGAP1 was ascertained using quantitative PCR, Western Blot analysis and luciferase reporter assays. Transfected miR-124a-GBM cells did not show any alterations in cell proliferation however significant reductions in tumor cell migration and invasion were observed when compared to control cells. Reducedmigration and invasion was also observed when GBM cells were transiently transfected with siRNA directed at IQGAP1. A direct effect of miR-124a on IQGAP1 expression levels was demonstrated at the mRNA and protein levels via miR-124a targeting the IQGAP1 mRNA 3’-UTR. In summary, we show that miR-124 functions as a tumour suppressor miRNA being downregulated in GBM which results in overexpression of IQGAP1. Additional migratory proteins commonly overexpressed in GBM also possess predicted miR-124a target sites and are currently been evaluated. miR-124a may therefore act as a master regulator of GBM invasiveness. Furthermore, delivery of miR-124a into GBMs may represent as a novel therapeutic approach to limit GBM invasion both alone and in combination with convential chemotherapy treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2050.