| Abstract: | ![]()
The small GTPase KRAS is frequently mutated in human cancer and currently there are no targeted therapies for KRAS mutant tumors. Here, we show that the small ubiquitin-like modifier (SUMO) pathway is required for KRAS-driven transformation. RNAi depletion of the SUMO E2 ligase Ubc9 suppresses 3D growth of KRAS mutant colorectal cancer cells in vitro and attenuates tumor growth in vivo. In KRAS mutant cells, a subset of proteins exhibit elevated levels of SUMOylation. Among these proteins, KAP1, CHD1, and EIF3L collectively support anchorage-independent growth, and the SUMOylation of KAP1 is necessary for its activity in this context. Thus, the SUMO pathway critically contributes to the transformed phenotype of KRAS mutant cells and Ubc9 presents a potential target for the treatment of KRAS mutant colorectal cancer.The Ras family of small GTPases are signal transduction molecules downstream of growth factor receptors. Ras activates a number of downstream effector pathways to regulate cell proliferation, survival and motility, these effectors include the MAP kinase (MAPK) pathway, the PI3-kinase (PI3K) pathway, the small GTPases RalA, RalB, and Rho, and phospholipase-Cε (1). Activating mutations in Ras are frequently found in human malignancies, with mutations in the KRAS gene being particularly prevalent. KRAS mutations occur in ∼60% of pancreatic ductal carcinomas, 26% of lung adenocarcinomas, and 45% of colorectal carcinomas, as well as a significant fraction of ovarian, endometrial, and biliary track cancers (2, 3). A salient hallmark of the Ras oncogene is its ability to transform cells to enable anchorage-independent 3D colony growth in vitro and tumor growth in vivo. Consequently, Ras mutant cancer cells often exhibit oncogene addiction to Ras such that extinction of the Ras oncogene leads to either a reversion of the transformed phenotype or loss of viability (4, 5). Therapeutically, the Ras oncoprotein has proven pharmacologically intractable thus far: intensive drug screening efforts have not yielded high-affinity, selective Ras inhibitors. Farnesyltransferase inhibitors that aimed to block Ras membrane localization are ineffective against KRAS because of its alternative geranylgeranylation. Inhibitors targeting Ras effector kinases, including MEK, PI3K, and Akt, are currently undergoing clinical evaluations, but they have yet to demonstrate clear clinical benefits (6). Thus, KRAS mutant tumors represent a class of “recalcitrant cancer” with urgent, unmet therapeutic needs.To gain new insight into the genetic dependencies of Ras mutant cancers and discover new therapeutic targets, we and others have previously carried out genome-wide synthetic lethal screens in KRAS mutant and WT cells to identify genes whose depletion leads to greater toxicity in KRAS mutant cells. In our screen we found a wide array of genes, many of which are involved in cellular stress response, that are required to maintain the viability of KRAS mutant cells (7). We proposed the concept of “nononcogene addiction” to explain the heightened dependency of cancer cells on stress response and other indirect cellular pathways for survival, and we suggested that this form of addiction could be exploited for therapeutic gain (8).In our primary screen we identified the small ubiquitin-like modifier (SUMO) E2 ligase Ubc9 (encoded by the UBE2I gene) and the E1 ligase subunit SAE1 as candidate KRAS synthetic lethal partners. Similar to the ubiquitin pathway, the SUMO pathway modulates the function and stability of cellular proteins through the reversible conjugation of SUMO on their lysine residues, often in a “ΨKxE” motif (9). In human, the SUMO pathway consists of three SUMO proteins (SUMO1, SUMO2, and SUMO3), a single heterodimeric E1 ligase SAE1/UBA2, a single E2 ligase Ubc9, and several E3 ligases. SUMO proteins are conjugated onto target proteins either directly by Ubc9 or through a family of E3s, and removed by the sentrin/SUMO-specific protease (SENP) family of SUMO peptidases. SUMOylation occurs in a highly dynamic manner in the cell and substrate proteins can be modified with either mono- or poly-SUMOylation. The SUMO pathway plays a critical role in cellular stress response, such as DNA damage, genomic stability, and heat shock (10–12), and it has also been recently implicated in prostate and breast cancer (13–16). However, the role of this pathway in KRAS mutant cancers is not clear.In this study we provide evidence for the requirement for the SUMO pathway in the transformation growth of KRAS mutant colorectal cancer (CRC) cells. We found that these cells are highly dependent on Ubc9 for their clonogenic growth under both anchorage-dependent (AD) and anchorage-independent (AI) conditions. Quantitative proteomics analysis revealed that the SUMOylation patterns of a subset of cellular proteins are altered by the KRAS oncogene, and these SUMO target proteins functionally support the 3D growth of KRAS mutant cells. Our findings thus provide evidence that the SUMO pathway is critical for the transformation growth of KRAS mutant cancer cells, and suggests Ubc9 could be a potential drug target. |
