Mind the GAP: A Novel Tumor-Promoting Mechanism

RAS switches between active and inactive conformations. Inappropriate RAS signaling can lead to excessive proliferation and the formation of cancer. One newly identified mechanism used to promote RAS activation is the loss of GAP proteins, which help to turn RAS off. Reactivating these GAPs may be therapeutically beneficial in tumors with reduced GAP expression.

RAS switches between active and inactive conformations. Inappropriate RAS signaling can lead to excessive proliferation and the formation of cancer. One newly identified mechanism used to promote RAS activation is the loss of GAP proteins, which help to turn RAS off. Reactivating these GAPs may be therapeutically beneficial in tumors with reduced GAP expression.

RAS proteins, like light switches, toggle between an “on” conformation where they promote cell growth, survival, and/or the formation of blood vessels (known as angiogenesis) and an “off” conformation in which they are unable to stimulate their target effector proteins. Nearly one-third of human tumors express a mutated RAS gene, which encodes a protein locked permanently in the active state. Other tumors, including liver hepatocellular carcinomas (HCCs), display aberrant RAS pathway signaling but lack RAS gene mutations, suggesting alternative mechanisms for this excessive RAS activity.

Two classes of regulatory proteins, GEFs and GAPs, help to turn RAS on and off, respectively, and are critical for appropriately balancing normal RAS activity. While mutations in upstream activators or downstream effectors of RAS have been identified, the role of altered GAP activity in tumor formation was unknown until Diego Calvisi, M.D., Ph.D. working with Snorri Thorgeirsson, M.D., Ph.D. of the Laboratory of Experimental Carcinogenesis and their colleagues examined GAP expression in HCC. These results were recently published in the Journal of Hepatology.

To begin investigating GAP proteins in HCC, the researchers assembled a collection of 10 normal liver and 88 HCC tissue samples. Sequencing of DNA from the HCCs revealed no mutations in RAS or in several activator or effector genes. In a subset of HCC samples from patients with a poor prognosis, the scientists observed slightly elevated RAS protein levels. However, in all HCC tissue samples examined, they saw significantly increased RAS activity, indicating that this higher activity was not a result of higher RAS protein levels or the presence of RAS mutations.

The authors next examined the message levels of several known RAS GAPs and discovered that DAB2IP and RASAL1 were almost ubiquitously repressed in the HCC samples while NF1 was decreased in a small subset. One method of silencing gene expression is to modify DNA with methyl groups. The researchers found that the regulatory regions or promoters of the RASAL1 and DAB2IP genes were excessively methylated in a large percentage of the HCC tissues and that the NF1 promoter showed increased methylation in a small subset, analogous to their message levels. Intriguingly, all the HCC samples displayed methylation of at least one of these three GAPs, suggesting their loss is critical for the development of HCC. Unlike DAB2IP and NF1, approximately 1 in 5 HCCs with decreased RASAL1 expression had no promoter methylation or other alterations to the RASAL1 gene, implicating another regulatory mechanism for its loss. Previous studies showed that the protein PITX1 could control RASAL1 expression levels leading the scientists to examine PITX1 in the HCC samples. They found reduced PITX1 message and protein levels, especially in HCC tissues from patients with a poor prognosis and those with no RASAL1 methylation. The reduction in PITX1 was also traced to excessive methylation of its promoter.

The researchers wanted to verify their observations from the HCC tissue samples by investigating the effects of changing GAP levels in HCC cell lines. RASAL1, DAB2IP, and PITX1 silencing increased cell viability and RAS GEF activity. Conversely, over-expression of each reduced RAS pathway signaling, HCC cell survival, and GEF activity. These studies support the scientists’ hypothesis that RAS GAP proteins play an important role in HCC tumor induction.

Since activation of the RAS pathway plays a role in numerous cancer types, the scientists examined tissues from breast, lung, colon and pancreatic cancer for mutations in the RAS gene and for RAS GAP promoter methylation. With the exception of one lung tumor sample, the two types of alterations are mutually exclusive, suggesting that mutational activation of RAS and hypermethylation of GAP genes are independent mechanisms used by tumors to promote their growth. The work by Drs. Calvisi and Thorgeirsson, and their colleagues supports the idea that reactivating DAB2IP, PITX1, or RASAL1 may be an effective way to inhibit wild type Ras activity and block tumor growth in HCC and other cancers with reduced GAP protein expression.

Summary Posted: 09/2010

Reference

J. Hepatol. 2010 Sept 7 [Epub ahead of print] PubMed Link