The Ras Inhibitor Farnesylthiosalicylic Acid (Salirasib) Disrupts The Spatiotemporal Localization Of Active Ras: A Potential Treatment For Cancer

Abstract

Chronic activation of Ras proteins by mutational activation or by growth factor stimulation is a common occurrence in many human cancers and was shown to induce and be required for tumor growth. Even if additional genetic defects are present, “correction” of the Ras defect has been shown to reverse Ras‐dependent tumorigenesis. One way to block Ras protein activity is by interfering with their spatiotemporal localization in cellular membranes or in membrane microdomains, a prerequisite for Ras signaling and biological activity. Detailed reports describe the use of this method in studies employing farnesylthiosalicylic acid (FTS, Salirasib), a Ras farnesylcysteine mimetic, which selectively disrupts the association of chronically active Ras proteins with the plasma membrane. FTS competes with Ras for binding to Ras‐escort proteins, which possess putative farnesyl‐binding domains and interact only with the activated form of Ras proteins, thereby promoting Ras nanoclusterization in the plasma membrane and robust signals. This chapter presents three‐dimensional time‐lapse images that track the FTS‐induced inhibition of membrane‐activated Ras in live cells on a real‐time scale. It also describes a mechanistic model that explains FTS selectivity toward activated Ras. Selective blocking of activated Ras proteins results in the inhibition of Ras transformation in vitro and in animal models, with no accompanying toxicity. Phase I clinical trials have demonstrated a safe profile for oral FTS, with minimal side effects and promising activity in hematological malignancies. Salirasib is currently undergoing trials in patients with pancreatic cancer and with nonsmall cell lung cancer, with or without identified K‐Ras mutations. The findings might indicate whether with the disruption of the spatiotemporal localization of oncogenic Ras proteins and the targeting of prenyl‐binding domains by anticancer drugs is worth developing as a means of cancer treatment.

Introduction

Mutations in ras genes occur in 30% of all human cancers, with the highest incidence of mutational activation of Ras being detected in pancreatic (90%) and colon (50%) cancers (Baines 2005, Chin 1999, Cox 2002, Downward 2003b, Hahn 1999, Hanahan 2000). K‐ras and N‐ras are the most frequently mutated genes (in most cases displaying a point mutation at codon 12), yet all three of the highly homologous Ras isoforms (H‐Ras, K‐Ras, and N‐Ras) promote malignant transformation (Baines 2005, Chin 1999, Cox 2002, Downward 2003b, Hahn 1999, Hanahan 2000). Oncogenic Ras isoforms (e.g., G12V mutants) are constitutively active (bound to GTP) and are critically required for both initiation and maintenance of the transformed phenotype of cancer cells that harbor the mutated Ras (Baines 2005, Chin 1999, Cox 2002, Hahn 1999, Hanahan 2000). Thus, for example, constitutively active Ras causes growth transformation of primary human cells, but other genetic alterations are required to facilitate the Ras transformation (Hahn et al., 1999). Ras activation induces and is required for tumor growth, and even if many genes are defective, “correction” of the Ras defect alone is sufficient to reverse the process (Chin et al., 1999). Blockage of capan‐1 (a human pancreatic cell line that harbors K‐RasG12V tumor growth in mice) by siRNA for K‐RasG12V provides additional strong support for the notion that blockage of Ras will be of clinical benefit (Baines et al., 2005). The aforementioned experimental findings highlight the importance of Ras proteins as a target for cancer drugs. One way to block the functions of Ras proteins is by interfering with their trafficking to and from cellular membranes, as well as with their proper localizations in different cellular localities and microdomains (Kloog 2000, Philips 2007)—all of which are required for Ras signaling and biological activities (Cox 2002, Dong 2003, Hancock 2005, Kloog 2000, Philips 2007, Silvius 2006). This chapter describes the methods employed to study the effects of farnesylthiosalicylic acid (FTS; Salirasib), a Ras farnesylcysteine mimetic shown to selectively disrupt the association of chronically active Ras proteins with the plasma membrane, on Ras membrane anchorage and function (Elad 1999, Goldberg 2006, Haklai 1998, Yaari 2005).