Epigenetic silencing of a Ca(2+)-regulated Ras GTPase-activating protein RASAL defines a new mechanism of Ras activation in human cancers

Abstract

Ras has achieved notoriety as an oncogene aberrantly activated in multiple human tumors. Approximately 30% of all human tumors express an oncogenic form of this GTPase that is locked in an active conformation as a result of being insensitive to Ras GTPase-activating proteins (GAPs), proteins that normally regulate the inactivation of Ras by enhancing its intrinsic GTPase activity. Besides oncogenic mutations in Ras, signaling by wild-type Ras is also frequently deregulated in tumors through aberrant coupling to activated cell surface receptors. This indicates that alternative mechanisms of aberrant wild-type Ras activation may be involved in tumorigenesis. Here, we describe another mechanism through which aberrant Ras activation is achieved in human cancers. We have established that Ras GTPase-activating-like protein (RASAL), a Ca(2+)-regulated Ras GAP that decodes the frequency of Ca(2+) oscillations, is silenced through CpG methylation in multiple tumors. With the finding that ectopic expression of catalytically active RASAL leads to growth inhibition of these tumor cells by Ras inactivation, we have provided evidence that epigenetically silencing of this Ras GAP represents a mechanism of aberrant Ras activation in certain cancers. Our demonstration that RASAL constitutes a tumor suppressor gene has therefore further emphasized the importance of Ca(2+) in the regulation of Ras signaling and has established that deregulation of this pathway is an important step in Ras-mediated tumorigenesis.

Fig. 1.

CAPRI and RASAL showed broad tissue expression. RASAL and CAPRI expression in a panel of normal adult tissues was examined by semiquantitative RT-PCR, using GAPDH as a control. Normal tissues underlined represent tissues whose corresponding tumors have been studied in this report. Sk, skeletal.

Fig. 2.

In contrast to CAPRI, RASAL expression is frequently down-regulated in tumor cell lines. RASAL (A) and CAPRI (B) expression in a variety of tumor cell lines was examined by semiquantitative RT-PCR, using GAPDH as a control. Ca, carcinoma; NPC, nasopharyngeal carcinoma; BrCa, breast carcinoma; HCC, hepatocellular carcinoma; EsCa, esophageal carcinoma. Normal tissues and normal epithelial cell lines are underlined.

Fig. 3.

Infrequent deletion of 12q24 in tumor cell lines. Representative results of 1-Mb array comparative genomic hybridization show a small hemizygous deletion including the RASAL (RASAL1) locus in some cell lines. Cytoband of 12q is shown in Top. Normalized log₂ signal intensity ratios from −1 to 1 are plotted. Each black dot represents a single BAC clone. Two BAC clones closest to the RASAL locus (dJ363l18 and bA438N16) are indicated with two vertical dashed lines. The RASAL locus is shown in Lower as in Ensemble Human Contigview (www.ensemble.org/).

Fig. 4.

Epigenetic down-regulation of RASAL in tumor cell lines. (A) The structure of the RASAL gene. The CGI, exon 1 and 2, the PITX1 binding site, MSP, and BGS primer regions are labeled. Each short vertical line represents a CpG site. (B) The methylation status of the RASAL CGI was analyzed by MSP. M, methylated; U, unmethylated. NP69 is an immortalized but nontransformed normal nasopharyngeal epithelial cell line, and NE1 and NE3 are immortalized normal esophageal epithelial cell lines. For clarity, some RT-PCR data from Fig. 2A are reproduced in this figure. (C) Methylation of the RASAL CGI was confirmed by BGS in esophageal (EC109) and NPC cell lines (HKM1), but not in normal esophageal epithelial cell lines (NE1, NE3). One row of circles represents an individual allele of the RASAL CGI analyzed. One circle indicates one CpG site. Filled circles represent methylated CpG sites.

Fig. 5.

RASAL CGI is also methylated in different primary tumors (carcinomas and lymphoma) but not in nontumor tissues or normal cells. The methylation status of the RASAL CGI was analyzed by MSP. M, methylated; U, unmethylated. Representative results are shown.

Fig. 6.

Demethylation reactivates RASAL expression. (A) Cell lines were treated with 5-aza-2′-deoxycytidine, with (A+T) or without (A) TSA, and the levels of RASAL expression were determined by semiquantitative RT-PCR. Methylation status of the RASAL CGI was analyzed by MSP (B) and BGS (C). −, not treated.

Fig. 7.

Ectopic RASAL expression inhibits tumor cell growth through Ras inactivation. (A) The effect of ectopic expression of RASAL wild type (RASAL-wt) and its mutants on tumor cell growth as investigated by soft agar assay. Quantitative analyses of colony numbers are shown in Lower as values of mean ± standard deviation. P values were calculated with Student's t test. The asterisk indicates statistical significance (P ≤ 0.01). (B) The active form of Ras was pulled down by Raf-RBD and subjected to Western blot analysis after SDS/PAGE. The expression levels of RASAL and its mutants were determined by RT-PCR. (C) The effect of RASAL expression on cellular growth of CNE2 cells in the presence of wild-type (wt) or mutant (mu) RAS(Q61L) was investigated by soft agar assay. Quantitative analysis of colony numbers is shown as in A. (D) Proposed model that CpG methylation-mediated silencing of RASAL contributes to RAS-mediated tumorigenesis. wt, wild type.