Inactivation of Ras GTPase-activating proteins promotes unrestrained activity of wild-type Ras in human liver cancer

Background & Aims

Aberrant activation of the RAS pathway is ubiquitous in human hepatocarcinogenesis, but the molecular mechanisms leading to RAS induction in the absence of RAS mutations remain under-investigated. We defined the role of Ras GTPase activating proteins (GAPs) in the constitutive activity of Ras signaling during human hepatocarcinogenesis.

Methods

The mutation status of RAS genes and RAS effectors was assessed in a collection of human hepatocellular carcinomas (HCC). Levels of RAS GAPs (RASA1-4, RASAL1, nGAP, SYNGAP1, DAB2IP, and NF1) and the RASAL1 upstream inducer PITX1 were determined by real-time RT-PCR and immunoblotting. The promoter and genomic status of RASAL1, DAB2IP, NF1, and PITX1 were assessed by methylation assays and microsatellite analysis. Effects of RASAL1, DAB2IP, and PITX1 on HCC growth were evaluated by transfection and siRNA analyses of HCC cell lines.

Results

In the absence of Ras mutations, downregulation of at least one RAS GAP (RASAL1, DAB2IP, or NF1) was found in all HCC samples. Low levels of DAB2IP and PITX1 were detected mostly in a HCC subclass from patients with poor survival, indicating that these proteins control tumor aggressiveness. In HCC cells, reactivation of RASAL1, DAB2IP, and PITX1 inhibited proliferation and induced apoptosis, whereas their silencing increased proliferation and resistance to apoptosis.

Conclusions

Selective suppression of RASAL1, DAB2IP, or NF1 RAS GAPs results in unrestrained activation of Ras signaling in the presence of wild-type RAS in HCC.

Introduction

RAS proteins are molecular switches for signalling cascades modulating many aspects of cell biology [1], [2]. Two distinct conformations characterize RAS proteins: an inactive GDP-bound and an active GTP-bound state, which are controlled by RAS guanine nucleotide exchange factors (GEFs) and RAS GTPase-activating proteins (GAPs) [3]. RAS GEFs trigger the activation of RAS by increasing the exchange of GDP for GTP, whereas RAS GAPs enhance the intrinsic RAS GTPase activity, leading to RAS inactivation through conversion of GTP into GDP [3]. Approximately 30% of human tumors express an oncogenic form of RAS genes (Ha-, K-, and N-RAS), which is locked in the active conformation as a result of being insensitive to RAS GAPs [1], [2]. Besides oncogenic mutations, an aberrant activation of the RAS cascade may also occur in the presence of wild-type RAS genes in cancer. Indeed, the RAS pathway is often deregulated in tumors through mutations in upstream inducers and downstream effectors, or via inactivation of RAS inhibitors, including DAB2, RASSF1A, and SPRY2 [4]. Suppression of RAS GAPs may constitute an additional mechanism whereby aberrant Ras activation promotes tumorigenesis [5]. Various RAS GAPs have been identified, including p120GAP or RASA1, the SynGAPs (Disabled homolog 2 interacting protein or DAB2IP, nGAP, and SynGAP1), neurofibromin (NF1), and the GAP1 proteins (GAP1m or RASA2, GAP1^IP4BP or RASA3, Ca²⁺-promoted RAS inactivator or RASA4, and RAS GTPase-activating-like protein 1 or RASAL1) [3], [5]. The role of RAS GAPs in carcinogenesis remains unexplored. Only NF1 was shown to be a bona fide oncosuppressor gene [5], whereas few studies were conducted on other RAS GAPs. Recently, DAB2IP downregulation was detected in human prostate cancer [6], and two genome-wide studies identified DAB2IP as a putative oncosuppressor gene in aggressive prostate adenocarcinomas [7]. In human hepatocellular carcinoma (HCC), DAB2IP is often epigenetically silenced [8], while RASAL1 is downregulated in HCC cells [9]. However, RAS GAPs status has not been comprehensively examined, and no functional studies on the role of RAS GAPs in liver cancer cells have been performed.

Here, we investigated the expression levels of RAS GAPs in a large collection of human HCC, determined the molecular mechanisms responsible for the inactivation of RAS GAPs, and studied their biologic role in HCC cell lines. Our findings indicate that inactivation of RASAL1, DAB2IP, or pituitary homeobox 1 (PITX1; a RASAL1 upstream inducer) is a major oncogenic event leading to unconstrained activation of wild-type RAS in human HCC.