RasGAP Research Articles

Runx1: a new driver in neurofibromagenesis.

The Runt-related transcription factor-1 (RUNX1 or AML1) encodes a transcription factor that serves as a master developmental regulator. It is important for hematopoiesis, angiogenesis, maturation of megakaryocytes, and differentiation of T and B cells [1]. Runx1 is also important for neuronal development and glial cell differentiation [2]. Runx1 is integrated into a complex regulatory network which acts both at the transcriptional and post-transcriptional levels. Runx1 activity can be regulated by several posttranslational modifications, including phosphorylation, de-phosphorylation, SUMOylation, acetylation, methylation and ubiquitination. These modifications control various aspects of transcriptional factors' activities such as auto inhibition, dimerization and ubiquitin-mediated degradation [3]. Besides its developmental determination role, RUNX1 is involved in malignant tumor formation. Reports have shown that RUNX1 is frequently de-regulated and has paradoxical effects in human cancers, in which it can function either as a tumor suppressor or oncogene [3, 4]. RUNX1 has been implicated as a tumor suppressor in several solid tumors including breast cancer, esophageal adenocarcinoma, colon cancer and possibly prostate cancer but acts as an oncogene in head/neck squamous cell carcinomas, endometrial cancer, and epithelial cancer [3, 4]. Because Runx1 is a sequence specific DNA-binding transcription factor, whether it functions as oncogene or tumor suppressor is dependent on its interaction with specific co-regulatory proteins. We recently showed that RUNX1 acts as an oncogene in the context of loss of neurofibromatosis type 1 (Nf1). Instead of chromosomal translocation and mutation frequently detected in other cancers, Runx1 is overexpressed in human and mouse neurofibroma-initiating cells, both at the messenger RNA and protein levels. Specifically, loss of Nf1 increases number of embryonic day 12.5 Runx1+/Blbp+ Schwann cell progenitors that enable neurofibroma formation in a mouse model (Figure ​(Figure1).1). Targeted genetic deletion of RUNX1 in Schwann cells and Schwann cell progenitors delays mouse neurofibroma formation in vivo (5). Figure 1 Model of neurofibromagenesis It is not clear how loss of Nf1 induces Runx1 overexpression and serves as an oncogene. There are several potential possibilities: 1) NF1 is known to encode a Ras-GTPase activating protein (Ras-GAP) and the Ras-MEK-ERK pathway is important for Nf1 neurofibroma formation [6]. Runx1 may be phosphorylated by the elevated MEK signaling to initiate the tumor formation process. 2) Elevated Wnt or Notch signaling can directly or indirectly activate Runx1, which can accelerate G1-S transition and stimulates cell proliferation. Consistently, our results show that loss of Runx1 in Schwann cells decreased cell proliferation by activating Trp53-p21 or increased cell apoptosis by inhibiting anti-apoptotic gene Bcl-2 in the context of Nf1−/− Schwann cell environment. It is possible that the elevated Runx1 within the neurofibroma cell alters cell fate (i.e. proliferation or differentiation) through Trp53-p21. Further experiments are needed to determine how p21 and Trp53 were activated or Bcl-2 was inactivated in the Runx1fl/fl;Nf1fl/fl; Dhhre tumors. 3) Runx1 might interact with epigenetic regulators such as the chromatin remodeling complexes, SWI/SNF, or polycomb repressive complexes to affect Runx1 activity by post-translational modification [7]. Overall, our study supports an oncogenic role of Runx1 in neurofibroma initiation and/or maintenance, but the underlying mechanism(s) are not clear. With increasing knowledge of RUNX1 biology, targeting the transcription factor RUNX1 or RUNX1 pathway might provide a novel therapy for neurofibroma as well as other tumors which overexpress RUNX1.

Abstract B1-58: Computational modeling provides new insights into KRAS mutant-specific responses to targeted therapy

Abstract Emerging data suggest different activating Ras mutants may have different biological behaviors. The most striking example may be in colon cancer, where activating KRAS mutations generally indicate a lack of benefit to treatment with cetuximab, with the activating KRAS G13D mutation an apparent exception. It is unclear why patients with KRAS G13D would respond to cetuximab; KRAS G13D shares the same general biochemical defects as the other oncogenic KRAS mutants. Here, a previously developed mathematical model of the biochemical reactions that regulate Ras signaling is used to investigate this problem. The purpose of the analysis is to determine whether known mechanisms of Ras signaling allow similar, but different, Ras mutants to respond differently to the same inhibitor, and to determine if the known biochemical properties of the G13D Ras mutant are consistent with increased sensitivity to upstream inhibition. Computational analysis of the mathematical model finds that differential response to upstream inhibition between different Ras mutants is indeed consistent with known mechanisms of Ras biology. Moreover, modeling demonstrates that the subtle biochemical differences between the responsive G13D mutant and the non-responsive G12D and G12V mutants are sufficient to explain the different responses to cetuximab. Simulations further identify differences in wild-type Ras signaling as the key difference between the mutants. The analysis suggests the elevated Km for the Ras GAP reaction on the G13D mutant is the biochemical property that most contributes to differences in wild-type Ras signaling. The identification of a single parameter that influences sensitivity to a targeted therapy is significant in that it suggests an approach to evaluate other, less common, Ras mutations for their anticipated response to cetuximab. Note: This abstract was not presented at the conference. Citation Format: Edward C. Stites. Computational modeling provides new insights into KRAS mutant-specific responses to targeted therapy. [abstract]. In: Proceedings of the AACR Special Conference on Computational and Systems Biology of Cancer; Feb 8-11 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 2):Abstract nr B1-58.

Nuclear import mechanism of neurofibromin for localization on the spindle and function in chromosome congression.

Neurofibromatosis type-1 (NF-1) is caused by mutations in the tumor suppressor gene NF1; its protein product neurofibromin is a RasGTPase-activating protein, a property that has yet to explain aneuploidy, most often observed in astrocytes in NF-1. Here, we provide a mechanistic model for the regulated nuclear import of neurofibromin during the cell cycle and for a role in chromosome congression. Specifically, we demonstrate that neurofibromin, phosphorylated on Ser2808, a residue adjacent to a nuclear localization signal in the C-terminal domain (CTD), by Protein Kinase C-epsilon (PKC-ε), accumulates in a Ran-dependent manner and through binding to lamin in the nucleus at G2 in glioblastoma cells. Furthermore, we identify CTD as a tubulin-binding domain and show that a phosphomimetic substitution of its Ser2808 results in a predominantly nuclear localization. Confocal analysis shows that endogenous neurofibromin localizes on the centrosomes at interphase, as well as on the mitotic spindle, through direct associations with tubulins, in glioblastoma cells and primary astrocytes. More importantly, analysis of mitotic phenotypes after siRNA-mediated depletion shows that acute loss of this tumor suppressor protein leads to aberrant chromosome congression at the metaphase plate. Therefore, neurofibromin protein abundance and nuclear import are mechanistically linked to an error-free chromosome congression. Concerned with neurofibromin's, a tumor suppressor, mechanism of action, we demonstrate in astrocytic cells that its synthesis, phosphorylation by Protein Kinase C-ε on Ser2808 (a residue adjacent to a nuclear localization sequence), and nuclear import are cell cycle-dependent, being maximal at G2. During mitosis, neurofibromin is an integral part of the spindle, while its depletion leads to aberrant chromosome congression, possibly explaining the development of chromosomal instability in Neurofibromatosis type-1. Read the Editorial Highlight for this article on page 11. Cover Image for this issue: doi: 10.1111/jnc.13300.

Loss of neurofibromin Ras-GAP activity enhances the formation of cardiac blood islands in murine embryos.

Type I neurofibromatosis (NF1) is caused by mutations in the NF1 gene encoding neurofibromin. Neurofibromin exhibits Ras GTPase activating protein (Ras-GAP) activity that is thought to mediate cellular functions relevant to disease phenotypes. Loss of murine Nf1 results in embryonic lethality due to heart defects, while mice with monoallelic loss of function mutations or with tissue-specific inactivation have been used to model NF1. Here, we characterize previously unappreciated phenotypes in Nf1-/- embryos, which are inhibition of hemogenic endothelial specification in the dorsal aorta, enhanced yolk sac hematopoiesis, and exuberant cardiac blood island formation. We show that a missense mutation engineered into the active site of the Ras-GAP domain is sufficient to reproduce ectopic blood island formation, cardiac defects, and overgrowth of neural crest-derived structures seen in Nf1-/-embryos. These findings demonstrate a role for Ras-GAP activity in suppressing the hemogenic potential of the heart and restricting growth of neural crest-derived tissues.

Abstract IA14: Preclinical models for targeting oncogenic Ras signaling in cancer.

Abstract Somatic RAS mutations occur in 30% of human cancers, and are common in both melanoma and hematopoietic cancers. Importantly, whereas KRAS mutations predominate in epithelial cancers, NRAS is mutated much more often in melanoma and hematopoietic cancers. The most obvious therapeutic strategy for cancers with RAS mutations – developing specific inhibitors of the mutant protein – is extremely challenging due to the very high affinity of Ras for GTP, structural considerations of the Ras/GTPase activating protein (Ras/GAP) molecular switch, and the loss of normal enzymatic activity as a consequence of oncogenic RAS mutations. Given these issues, most recent drug discovery efforts have focused on inhibiting downstream biochemical targets of mutant Ras such as Raf, MEK, and Akt with many compounds undergoing clinical evaluation. We have exploited strains of mice that recapitulate endogenous expression of cancer-associated RAS alleles or NF1 inactivation to model human hematologic cancers and to conduct biologic and preclinical studies. In particular, we have deployed the MOL4070LTR retrovirus as an insertional mutagen to generate genetically heterogeneous transplantable myeloid and lymphoid leukemias characterized by hyperactive Ras signaling. We utilize cohorts of mice that are engrafted with the same primary leukemias for preclinical testing. In this presentation, I will focus on three biologic and therapeutic questions related to NRAS in tumorigenesis: (1) loss of the normal NRAS allele in cancers with oncogenic NRAS mutations; (2) responses of primary Nras mutant leukemias to MEK and PI3 kinase inhibitors; and, (3) potential strategies for selectively targeting oncogenic N-Ras in cancer. Note: This abstract was presented as part of the Joint Session: Clinical Science Interactions between Melanoma and Hematologic Malignancies with the AACR Special Conference on Advances in Melanoma: From Biology to Therapy held September 20-23, 2014 in Philadelphia, PA. Citation Format: Kevin Shannon. Preclinical models for targeting oncogenic Ras signaling in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA14.

Abstract 2730: Additional mutations in genes relevant to the Ras signaling pathway promote the malignant characteristics of NF1-associated malignant peripheral nerve sheath tumor (NF1-MPNST) cells

Abstract Malignant peripheral nerve sheath tumors (MPNST) are soft tissue sarcomas surrounding the peripheral nerve and occur sporadically or more often in association with neurofibromatosis type 1 (NF1, von Recklinghausen disease), which is an inherited disease caused by NF1 gene germline mutations. The NF1 tumor suppressor gene encodes neurofibromin and is a functional Ras GTPase-activating protein (RasGAP) that regulates the Ras signal negatively by accelerating the conversion of activated Ras-GTP to inactive Ras-GDP. We hypothesized that additional genetic alterations promote malignancy of NF1-associated tumors. Hence, we inoculated a GFP-labeled human NF1-MPNST cell line, sNF96.2-GFP, which has a frame-shift mutation (c.3683delC, p.Asn1229MetfsTer11) in the NF1 gene, into the renal sub-capsules of immunodeficient mice. A subclonal cell line, the A-1 cell, was established from the developed tumor. We found that A-1 cells show much higher tumorigenic activity and Akt activation than parental sNF96.2-GFP cells. We analyzed the genomic DNA of both the sNF96.2 and the A-1 cells by using the next-generation sequencing and the medical exome panel including 4813 genes, which are known to be responsible for most human genetic disorders. We identified 18 heterozygous variants in 17 genes that were present in the A-1 cells, but not in the original sNF96.2 cells. Two of these genes are associated with the regulation of the Ras signaling pathway. Our data suggests that the additional gene mutations that regulate Ras pathways do in fact promote the malignant characteristics of NF1-associated tumors through the Akt activation. Citation Format: Yoshimi Arima, Kenjiro Kosaki, Chikako Hirose, Hideyuki Saya. Additional mutations in genes relevant to the Ras signaling pathway promote the malignant characteristics of NF1-associated malignant peripheral nerve sheath tumor (NF1-MPNST) cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2730. doi:10.1158/1538-7445.AM2015-2730

A Novel Monoclonal Antibody Against Human DAB2IP.

DAB2 interactive protein (DAB2IP), also known as ASK1-interacting protein-1 (AIP1), a novel member of the RasGTPase-activating protein family, plays a key role in tumor suppression during cancer progression and is highly expressed in vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). To further explore its function as a cancer suppressor, in this study, we immunized BALB/c mice with synthesized human DAB2IP polypeptide and obtained a novel monoclonal antibody (MAb) against human DAB2IP. A stable strain of hybridoma was screened and successfully established by the hybridoma technique. The immunohistochemistry, immunocytochemistry, and Western blot analysis revealed that the MAb was directed against human DAB2IP with high specificity. Therefore, this MAb may be a useful tool and facilitate studies on tumorigenesis associated with DAB2IP.

Neurofibromatosis type 1: Its association with the Ras/MAPK pathway syndromes

Neurofibromatosis type 1 (NF1) is a common (affects ∼1 in 4000 individuals worldwide) autosomal dominant neurocutaneous syndrome, resulting from functional inactivation of the NF1 gene. The main clinical features include dermal pigmentary changes (cafe-au-lait spots and skin fold freckling), development of benign peripheral nerve sheath-derived tumors (neurofibromas) and hamartomas of the iris (Lisch nodules). Benign tumor development, involving peripheral nerves and the central nervous system, are hallmark features of NF1. Associated clinical manifestations can include abnormalities of the cardiovascular, gastrointestinal, renal and endocrine systems, orthopedic problems, macrocephaly, small stature, facial and body dysmorphism, learning disabilities, and an increased malignancy risk. Neurofibromin, the NF1 gene product, is a mammalian Ras GTPase-activating protein expressed in most tissues, with functional inactivation leading to increased cellular Ras signaling, resulting in cell growth, proliferation and tumorigenesis, underlining its role as a tumor suppressor gene. At least 1050 different disease-causing NF1 mutations are known, although correlating different mutations with specific clinical features has proved largely unsuccessful, with only two such associations identified. One relating to severely affected NF1 patients with large genomic deletions that involve the entire NF1 gene, and the other, relating to some mildly affected NF1 patients without any cutaneous neurofibromas, who have the same specific 3bp NF1 gene deletion. Several animal NF1 models have been developed that have allowed analysis of NF1 gene inactivation and proved crucial in unravelling the complexity of the Ras signaling pathway and its role in regulating cell proliferation, differentiation, motility, apoptosis and tumorigenesis. Such models have also better defined the probable causes of NF1-related learning difficulties, skeletal abnormalities, and also identified neurofibromin as a cellular cyclic AMP regulator. It is now recognized that mutations of a number of the genes in the complex RAS-MAPK signaling pathway are associated with several neuro-cardio-facial-cutaneous syndromes, a group of pheno- and genotypically similar developmental disorders, that includes NF1. The recent identification of SPRED1 gene mutations in individuals with mild NF1-like features extends this disease association with spred proteins known to be directly involved in RAS-MAPK pathway. No effective treatment for NF1 is currently available, with many proposed therapies, often aimed at preventing tumor growth, showing little success, although a statin-based treatment has proved useful in treating a mouse NF1 model with learning disability and tibial dysplasia. Several proposed therapies targeting mast cell function, different Ras signaling pathway components, as well as growth factor receptors, may hopefully prove more successful.

Abstract IA12: Preclinical models for targeting oncogenic Ras signaling in cancer

Abstract Somatic RAS mutations occur in 30% of human cancers, and are common in both melanoma and hematopoietic cancers. Importantly, whereas KRAS mutations predominate in epithelial cancers, NRAS is mutated much more often in melanoma and hematopoietic cancers. The most obvious therapeutic strategy for cancers with RAS mutations – developing specific inhibitors of the mutant protein – is extremely challenging due to the very high affinity of Ras for GTP, structural considerations of the Ras/GTPase activating protein (Ras/GAP) molecular switch, and the loss of normal enzymatic activity as a consequence of oncogenic RAS mutations. Given these issues, most recent drug discovery efforts have focused on inhibiting downstream biochemical targets of mutant Ras such as Raf, MEK, and Akt with many compounds undergoing clinical evaluation. We have exploited strains of mice that recapitulate endogenous expression of cancer-associated RAS alleles or NF1 inactivation to model human hematologic cancers and to conduct biologic and preclinical studies. In particular, we have deployed the MOL4070LTR retrovirus as an insertional mutagen to generate genetically heterogeneous transplantable myeloid and lymphoid leukemias characterized by hyperactive Ras signaling. We utilize cohorts of mice that are engrafted with the same primary leukemias for preclinical testing. In this presentation, I will focus on three biologic and therapeutic questions related to NRAS in tumorigenesis: (1) loss of the normal NRAS allele in cancers with oncogenic NRAS mutations; (2) responses of primary Nras mutant leukemias to MEK and PI3 kinase inhibitors; and, (3) potential strategies for selectively targeting oncogenic N-Ras in cancer. Note: This abstract was presented as part of the Joint Session: Clinical Science Interactions between Melanoma and Hematologic Malignancies with the AACR Special Conference: Hematologic Malignancies: Translating Discoveries to Novel Therapies held September 20-23, 2014 in Philadelphia, PA. Citation Format: Kevin Shannon. Preclinical models for targeting oncogenic Ras signaling in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr IA12.

MYCNOS functions as an antisense RNA regulating MYCN

Amplification or overexpression of neuronal MYC (MYCN) is associated with poor prognosis of human neuroblastoma. Three isoforms of the MYCN protein have been described as well as a protein encoded by an antisense transcript (MYCNOS) that originates from the opposite strand at the MYCN locus. Recent findings suggest that some antisense long non-coding RNAs (lncRNAs) can play a role in epigenetically regulating gene expression. Here we report that MYCNOS transcripts function as a modulator of the MYCN locus, affecting MYCN promoter usage and recruiting various proteins, including the Ras GTPase-activating protein-binding protein G3BP1, to the upstream MYCN promoter. Overexpression of MYCNOS results in a reduction of upstream MYCN promoter usage and increased MYCN expression, suggesting that the protein-coding MYCNOS also functions as a regulator of MYCN ultimately controlling MYCN transcriptional variants. The observations presented here demonstrate that protein-coding transcripts can regulate gene transcription and can tether regulatory proteins to target loci.

Cutting Edge: Codeletion of the Ras GTPase-Activating Proteins (RasGAPs) Neurofibromin 1 and p120 RasGAP in T Cells Results in the Development of T Cell Acute Lymphoblastic Leukemia.

Ras GTPase-activating proteins (RasGAPs) inhibit signal transduction initiated through the Ras small GTP-binding protein. However, which members of the RasGAP family act as negative regulators of T cell responses is not completely understood. In this study, we investigated potential roles for the RasGAPs RASA1 and neurofibromin 1 (NF1) in T cells through the generation and analysis of T cell-specific RASA1 and NF1 double-deficient mice. In contrast to mice lacking either RasGAP alone in T cells, double-deficient mice developed T cell acute lymphoblastic leukemia/lymphoma, which originated at an early point in T cell development and was dependent on activating mutations in the Notch1 gene. These findings highlight RASA1 and NF1 as cotumor suppressors in the T cell lineage.

Wnt-related SynGAP1 is a neuroprotective factor of glutamatergic synapses against Aβ oligomers.

Wnt-5a is a synaptogenic factor that modulates glutamatergic synapses and generates neuroprotection against Aβ oligomers. It is known that Wnt-5a plays a key role in the adult nervous system and synaptic plasticity. Emerging evidence indicates that miRNAs are actively involved in the regulation of synaptic plasticity. Recently, we showed that Wnt-5a is able to control the expression of several miRNAs including miR-101b, which has been extensively studied in carcinogenesis. However, its role in brain is just beginning to be explored. That is why we aim to study the relationship between Wnt-5a and miRNAs in glutamatergic synapses. We performed in silico analysis which predicted that miR-101b may inhibit the expression of synaptic GTPase-Activating Protein (SynGAP1), a Ras GTPase-activating protein critical for the development of cognition and proper synaptic function. Through overexpression of miR-101b, we showed that miR-101b is able to regulate the expression of SynGAP1 in an hippocampal cell line. Moreover and consistent with a decrease of miR-101b, Wnt-5a enhances SynGAP expression in cultured hippocampal neurons. Additionally, Wnt-5a increases the activity of SynGAP in a time-dependent manner, with a similar kinetic to CaMKII phosphorylation. This also, correlates with a modulation in the SynGAP clusters density. On the other hand, Aβ oligomers permanently decrease the number of SynGAP clusters. Interestingly, when neurons are co-incubated with Wnt-5a and Aβ oligomers, we do not observe the detrimental effect of Aβ oligomers, indicating that, Wnt-5a protects neurons from the synaptic failure triggered by Aβ oligomers. Overall, our findings suggest that SynGAP1 is part of the signaling pathways induced by Wnt-5a. Therefore, possibility exists that SynGAP is involved in the synaptic protection against Aβ oligomers.

EBP1 suppresses growth, migration, and invasion of thyroid cancer cells through upregulating RASAL expression

Ebp1, a protein identified by its interactions with the ErbB3 receptor, has been characterized as a negative regulator of cancers. RAS GTPase-activating protein (RasGAP), RASAL1, was recently identified as a major tumor suppressor in thyroid cancer. In this study, we examined EBP1 expression in papillary and follicular thyroid cancer cells. We found that compared with normal thyroid cells, TPC1, WRO, and FTC133 thyroid tumor cells exhibited lower EBP1 expression at messenger RNA (mRNA) and protein levels. We then investigated the effects of forced EBP1 expression on growth, migration, and invasiveness of thyroid tumor cells. By using MTT and Boyden chamber assays, we showed that EBP1 overexpression dramatically reduced growth rate, migration, and invasiveness of K1 and FTC133 thyroid tumor cells. Furthermore, we explored the molecular mechanism underlying the effects of EBP1 on the cells by disclosing the correlation of EBP1 and RASAL1 expression. RASAL expression was elevated in thyroid tumor cells overexpressing EBP1. Knockdown RASAL by transduction of RASAL1 shRNA lentiviral particles markedly reduced RASAL levels with restoration of EBP1, and RASAL1 knockdown abrogated the effects of forced EBP1 expression on cell growth, migration, and invasiveness of thyroid tumor cells. These findings suggest that Ebp1 suppressed thyroid cancer cell lines by upregulating RASRAL expression.

Involvement of Ras GTPase-activating protein SH3 domain-binding protein 1 in the epithelial-to-mesenchymal transition-induced metastasis of breast cancer cells via the Smad signaling pathway.

In situ models of epithelial-to-mesenchymal transition (EMT)-induced carcinoma develop into metastatic carcinoma, which is associated with drug resistance and disease recurrence in human breast cancer. Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1), an essential Ras mediator, has been implicated in cancer development, including cell growth, motility, invasion and apoptosis. Here, we demonstrated that the upregulation of G3BP1 activates the EMT in breast cancer cells. Silencing Smads almost completely blocked this G3BP1-induced EMT, suggesting that this process depends on the Smad signaling pathway. We also found that G3BP1 interacted with the Smad complex. Based on these results, we proposed that G3BP1 might act as a novel co-factor of Smads by regulating their phosphorylation status. Moreover, knockdown of G3BP1 suppressed the mesenchymal phenotype of MDA-MB-231 cells in vitro and suppressed tumor growth and lung metastasis of 4T1 cells in vivo. Our findings identified a novel function of G3BP1 in the progression of breast cancer via activation of the EMT, indicating that G3BP1 might represent a potential therapeutic target for metastatic human breast cancer.

A RASopathy gene commonly mutated in cancer: the neurofibromatosis type 1 tumour suppressor.

Neurofibromatosis type 1 (NF1) is a common genetic disorder that predisposes affected individuals to tumours. The NF1 gene encodes a RAS GTPase-activating protein called neurofibromin and is one of several genes that (when mutant) affect RAS-MAPK signalling, causing related diseases collectively known as RASopathies. Several RASopathies, beyond NF1, are cancer predisposition syndromes. Somatic NF1 mutations also occur in 5-10% of human sporadic cancers and may contribute to resistance to therapy. To highlight areas for investigation in RASopathies and sporadic tumours with NF1 mutations, we summarize current knowledge of NF1 disease, the NF1 gene and neurofibromin, neurofibromin signalling pathways and recent developments in NF1 therapeutics.

C/EBP-β-activated microRNA-223 promotes tumour growth through targeting RASA1 in human colorectal cancer.

Background:Evidences have shown that the RAS signalling pathway plays an important role in colorectal cancer (CRC). Moreover, RAS-GTPase-activating proteins (RASGAPs) as RAS signalling terminators are associated with tumourigenicity and tumour progression. In this study, we used bioinformatics analysis to predict and study important miRNAs that could target RAS p21 GTPase-activating protein 1 (RASA1), an important member of RASGAPs.Methods:The levels of RASA1 and miR-223 were analysed by real-time PCR, western blotting or in situ immunofluorescence analyses. The functional effects of miR-223 and the effects of miR-223-targeted inhibitors were examined in vivo using established assays.Results:Upregulation of miR-223 was detected in CRC tissues (P<0.01) and was involved in downregulation of RASA1 in CRC tissues. Furthermore, the direct inhibition of RASA1 translation by miR-223 and the activation of miR-223 by CCAAT/enhancer binding protein-β (C/EBP-β) were evaluated in CRC cells. An in vivo xenograft model of CRC suggested that the upregulation of miR-223 could promote tumour growth and that the inhibition of miR-223 might prevent solid tumour growth.Conclusions:These results identify that C/EBP-β-activated miR-223 contributes to tumour growth by targeting RASA1 in CRC and miR-223-targeted inhibitors may have clinical promise for CRC treatment via suppression of miR-223.

The Ras GTPase-activating protein Rasal3 supports survival of naive T cells.

The Ras-mitogen-activated protein kinase (MAPK) pathway is crucial for T cell receptor (TCR) signaling in the development and function of T cells. The significance of various modulators of the Ras-MAPK pathway in T cells, however, remains to be fully understood. Ras-activating protein-like 3 (Rasal3) is an uncharacterized member of the SynGAP family that contains a conserved Ras GTPase-activating protein (GAP) domain, and is predominantly expressed in the T cell lineage. In the current study, we investigated the function and physiological roles of Rasal3. Our results showed that Rasal3 possesses RasGAP activity, but not Rap1GAP activity, and represses TCR-stimulated ERK phosphorylation in a T cell line. In systemic Rasal3-deficient mice, T cell development in the thymus including positive selection, negative selection, and β-selection was unaffected. However, the number of naive, but not effector memory CD4 and CD8 T cell in the periphery was significantly reduced in Rasal3-deficient mice, and associated with a marked increase in apoptosis of these cells. Indeed, survival of Rasal3 deficient naive CD4 T cells in vivo by adoptive transfer was significantly impaired, whereas IL-7-dependent survival of naive CD4 T cells in vitro was unaltered. Collectively, Rasal3 is required for in vivo survival of peripheral naive T cells, contributing to the maintenance of optimal T cell numbers.

RASAL3, a novel hematopoietic RasGAP protein, regulates the number and functions of NKT cells.

Ras GTPase-activating proteins negatively regulate the Ras/Erk signaling pathway, thereby playing crucial roles in the proliferation, function, and development of various types of cells. In this study, we identified a novel Ras GTPase-activating proteins protein, RASAL3, which is predominantly expressed in cells of hematopoietic lineages, including NKT, B, and Tcells. We established systemic RASAL3-deficient mice, and the mice exhibited a severe decrease in NKT cells in the liver at 8 weeks of age. The treatment of RASAL3-deficient mice with α-GalCer, a specific agonist for NKT cells, induced liver damage, but the level was less severe than that in RASAL3-competent mice, and the attenuated liver damage was accompanied by a reduced production of interleukin-4 and interferon-γ from NKT cells. RASAL3-deficient NKT cells treated with α-GalCer in vitro presented augmented Erk phosphorylation, suggesting that there is dysregulated Ras signaling in the NKT cells of RASAL3-deficient mice. Taken together, these results suggest that RASAL3 plays an important role in the expansion and functions of NKT cells in the liver by negatively regulating Ras/Erk signaling, and might be a therapeutic target for NKT-associated diseases.

Extracellular superoxide dismutase regulates the expression of small gtpase regulatory proteins GEFs, GAPs, and GDI.

Extracellular superoxide dismutase (SOD3), which catalyzes the dismutation of superoxide anions to hydrogen peroxide at the cell membranes, regulates the cellular growth in a dose-dependent manner. This enzyme induces primary cell proliferation and immortalization at low expression levels whereas it activates cancer barrier signaling through the p53-p21 pathway at high expression levels, causing growth arrest, senescence, and apoptosis. Because previous reports suggested that the SOD3–induced reduction in the rates of cellular growth and migration also occurred in the absence of functional p53 signaling, in the current study we investigated the SOD3-induced growth-suppressive mechanisms in anaplastic thyroid cancer cells. Based on our data, the robust over-expression of SOD3 increased the level of phosphorylation of the EGFR, ERBB2, RYK, ALK, FLT3, and EPHA10 receptor tyrosine kinases with the consequent downstream activation of the SRC, FYN, YES, HCK, and LYN kinases. However, pull-down experiments focusing on the small GTPase RAS, RAC, CDC42, and RHO revealed a reduced level of growth and migration signal transduction, such as the lack of stimulation of the mitogen pathway, in the SOD3 over-expressing cells, which was confirmed by MEK1/2 and ERK1/2 Western blotting analysis. Interestingly, the mRNA expression analyses indicated that SOD3 regulated the expression of guanine nucleotide-exchange factors (RHO GEF16, RAL GEF RGL1), GTPase-activating proteins (ARFGAP ADAP2, RAS GAP RASAL1, RGS4), and a Rho guanine nucleotide-disassociation inhibitor (RHO GDI 2) in a dose dependent manner, thus controlling signaling through the small G protein GTPases. Therefore, our current data may suggest the occurrence of dose-dependent SOD3–driven control of the GTP loading of small G proteins indicating a novel growth regulatory mechanism of this enzyme.

Abstract A36: Unveiling biomarker and mechanism of drug resistance to molecular targeted therapeutics in renal cell carcinoma

Abstract Introduction and Objective: Renal cell carcinoma (RCC) is the most lethal of the urologic malignancies and it is largely incurable when it becomes metastatic. Recent development of molecular targeted agents such as tyrosine kinase inhibitors (TKIs) and mammalian target of rapamycin (mTOR) inhibitors have provided some survival benefit for a subset of RCC patients. Overall, only 30% patients exhibit a response to these agents, however, almost all patients eventually develop drug resistance. Currently, there is no reliable biomarker(s) for predicting drug response for individual patients and the mechanism(s) of drug resistance is largely unknown. It is known that DAB2IP, a novel member of the Ras GTPase-activating protein gene family, is associated with cell proliferation, apoptosis, metastasis and chemo- or radio-resistance in several cancer types. In the present study, we decided to investigate the association between DAB2IP expression and therapeutic response to molecular targeted agents in RCC patients and explored the possible mechanism of DAB2IP in regulating drug response of RCC cells. Methods: DAB2IP expression was evaluated by immunohistochemistry (IHC) in a cohort of RCC patients who had received targeted therapies. Correlation between DAB2IP expression and patients' survival was analyzed. The effect of DAB2IP on RCC cells was examined using a panel of knockdown (KD) or overexpression cells generated by gene transfection and also determined for their in vitro and in vivo (xenograft model) responses to targeted agents treatment. The mechanism of drug resistance was delineated by signaling cascade profiling with a variety of molecular biologic techniques such as cDNA array, ChIP, reporter gene assay, real-time PCR and Western blot. Results: The levels of DAB2IP expression in RCC specimens determined by IHC positively correlate with the survival of patients receiving targeted therapies. DAB2IP KD RCC cells exhibit drug resistance to mTOR inhibitor Temsirolimus (Tem) treatment compared with the control from both in vitro and in vivo experiments. In contrast, increased DAB2IP in RCC cells by gene transfection can sensitize their response to Tem. Mechanistically, loss of DAB2IP was able to activate both the PI3K/mTOR pathway and the ERK/RSK1 pathway that further led to increased expression of hypoxia-inducible factor (HIF)-2α protein. Inhibitor(s) of either the mTOR pathway or the ERK pathway only partially suppressed growth of DAB2IP KD cells. However, the combination of both inhibitors achieved a synergistic growth inhibition in resistant cells. In this event, HIF-2α, but not HIF-1α, appeared to be a key converging factor and we further demonstrated that silencing of HIF-2α expression with specific shRNA was able to reverse the resistant phenotype in DAB2IP KD cells. Conclusions: DAB2IP represents a new predictive biomarker for the therapeutic response of RCC patients to targeted therapy. Most importantly, its functional role in RCC provides new therapeutic strategies in combating this lethal disease. Citation Format: Jiancheng Zhou, Kaijie Wu, Eunjin Yun, Payal Kapur, Rey-Chen Pong, Dalin He, Jer-Tsong Hsieh. Unveiling biomarker and mechanism of drug resistance to molecular targeted therapeutics in renal cell carcinoma. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A36.