Acta Pharmacologica Sinica (2015) 36: 291–297 npg © 2015 CPS and SIMM All rights reserved 1671-4083/15 $32.00 www.nature.com/aps Review The RAS-RAL axis in cancer: evidence for mutation- specific selectivity in non-small cell lung cancer Sunny GUIN1, 2, Dan THEODORESCU1, 2, 3, * 1Department of Surgery, University of Colorado, Aurora, CO 80045, USA; 2Department of Pharmacology, University of Colorado, Aurora, CO 80045, USA; 3University of Colorado Comprehensive Cancer Center, Aurora, CO 80045, USA Activating RAS mutations are common in human tumors. These mutations are often markers for resistance to therapy and subsequent poor prognosis. So far, targeting the RAF-MEK-ERK and PI3K-AKT signaling pathways downstream of RAS is the only promising approach in the treatment of cancer patients harboring RAS mutations. RAL GTPase, another downstream effector of RAS, is also considered as a therapeutic option for the treatment of RAS-mutant cancers. The RAL GTPase family comprises RALA and RALB, which can have either divergent or similar functions in different tumor models. Recent studies on non-small cell lung cancer (NSCLC) have showed that different RAS mutations selectively activate specific effector pathways. This observation requires broader validation in other tumor tissue types, but if true, will provide a new approach to the treatment of RAS-mutant cancer patients by targeting specific downstream RAS effectors according to the type of RAS mutation. It also suggests that RAL GTPase inhibition will be an important treatment strategy for tumors harboring RAS glycine to cysteine (G12C) or glycien to valine (G12V) mutations, which are commonly found in NSCLC and pancreatic cancer. Keywords: RAS; RAL; RAS mutation; RALBP1; non-small cell lung cancer Acta Pharmacologica Sinica (2015) 36: 291–297; doi: 10.1038/aps.2014.129; published online 5 Jan 2015 RAS GTPase overview RAS is the most extensively studied GTPase[1]. It is known to regulate various cellular functions, including proliferation, survival, growth, migration, differentiation and cytoskeletal dynamics[2–4] (Figure 1). Not coincidentally, increased activi- ties of all of these processes are required by tumor cells to promote tumor growth. Activating oncogenic RAS mutations lead to treatment resistance in various tumor models and poor patient outcomes[3, 5–9]. Three human RAS genes have been identified: HRAS, KRAS, and NRAS[1, 2]. These encode the related proteins HRAS, KRAS and NRAS, respectively, of 189 amino acids in length[1, 4]. KRAS exists as two isoforms, 4A and 4B, which are generated by alternative exon splicing[10]. These different RAS genes are well known to have differential cellular specificities and intrinsic transforming potentials[3, 4, 10]. In normal cells, RAS proteins act as molecular switches for critical cellular functions. RAS proteins are activated Figure 1. by upstream receptors such as receptor tyrosine kinases RAS GTPase activation and downstream signaling. The cartoon shows the mechanism of RAS GTPase activation by upstream stimuli and numerous downstream effectors stimulated by activated RAS. RAS * To whom correspondence should be addressed. regulates critical cellular functions through these effectors. Constitutive E-mail [email protected] RAS activation due to mutations leads to protumorigenic signaling through Received 2014-08-07 Accepted 2014-10-30 these effectors. npg www.nature.com/aps Guin S et al 292 (RTKs), G-protein coupled receptors (GPCRs) and serpen- naling pathways. For example, mutant KRAS with either a tine and cytokine receptors[1, 4, 11]. RAS protein activity is glycine to cysteine (G12C) or glycine to valine (G12V) muta- tightly regulated by Guanine Nucleotide Exchange Factors tion at codon 12 preferentially binds to RAL guanine nucleo- (GEFs) and GTPase Activating Proteins (GAPs). Constitu- tide dissociation stimulator (RALGDS), a RAL GTPase-specific tive activation of the RAS protein, in which RAS is unable to GEF, whereas KRAS harboring a glycine to aspartate mutation hydrolyze GTP, leads to cancer and other diseases[1, 9, 12]. Acti- at codon 12 (G12D) has higher affinity for phosphatidylino- vating RAS mutations have been observed in 30% of human sitol 3-kinase (PI3K)[20]. These recent studies have brought to tumors[2, 13]; KRAS is the most commonly mutated isoform, light the need to clarify the impact of such KRAS mutations on mutated in 25%–30% of human cancers[3, 13]. KRAS mutations the RAL GTPase signaling pathway. are dominant in colorectal cancer (40%–45%), non-small cell lung cancer (NSCLC) (15%–40%) and pancreatic carcinoma The RAL GTPase family and effectors (69%–95%)[2, 3] and are associated with poor patient outcomes RAL GTPase falls under the RAS family of GTPases. RAL and treatment responses in these malignancies[2, 3]. NRAS and shares a high degree of sequence similarity with the three RAS HRAS mutations are less common (8% and 3% of all tumors, genes, hence the name RAL (RAS-like)[1]. The RAL GTPase respec tively)[2, 13], with melanoma harboring the most NRAS sub-family comprises the two isoforms RALA and RALB, mutations at 20%–30%[2, 13]. which share high sequence homology[21]. Approximately 85% Activating mutations occur at codons 12, 13, and 61, which of the amino acid sequences of these two isoforms are identi- are within the GTPase catalytic domain, in all three RAS iso- cal[21]. RAL GTPase can be activated by six GEFs (RALGEFs), forms[14]. Approximately 80% of KRAS mutations are found RALGDS, RGL, RGL2/Rlf, RGL3, RALGPS1, and RALGPS2, in codon 12, whereas approximately 60% of NRAS mutations and inactivated by two GAPs, RALGAP1, and RALGAP2[22, 23]. are found in codon 61, with 35% in codon 12[2, 14]. HRAS muta- Four RALGEFs (RALGDS, RGL, RGL2, and RGL3) are known tions are equally divided between codons 12 and 61[2, 14]. All to directly interact with the effector binding region of GTP- of these activating mutations inhibit RAS GTPase activity by bound RAS and are thus important for RAS-mediated tumori- preventing GAP-stimulated GTP hydrolysis of GTP-bound genesis[22]. RALGEFs and RAL play a dominant role in the activated RAS. RAS-mediated transformation of several different immor- Approximately 20 downstream effectors bind to RAS-GTP talized human cell lines, as well as in a RAS-driven tumor and trigger signaling. RAS drives tumor growth via a number model[24, 25]. of prominent pathways, including the following: RAF-MEK- RAL proteins mediate various cellular activities, including ERK[15, 16]; p110 catalytic subunits (p110α, β, γ, and δ) of class I filopodia formation/membrane ruffling, glycolysis, autoph- PI3K; TIAM1, a small RAC GTPase-specific GEF; RAL-specific agy, secretion, the maintenance of polarity, apoptosis and GEFs (RALGDS, RGL, RGL2, and RGL3); and phospholipase transcription[21, 26] (Figure 2). Alterations to these activities can C epsilon[1, 2, 16]. lead to tumor invasion, metastasis, altered cellular energy lev- els, proliferative signaling and resistance to cell death. These RAS mutation and cancer therapeutics activities are mediated by effectors that interact with activated Devising an effective treatment strategy for patients with RAS (GTP-bound) RAL[22]. Of all the RAL effectors, the most exten- mutations has been a major challenge[2, 17]. However, recent sively studied are RALBP1 and the members of the exocyst attempts have been promising[17, 18]. RNA interference is an complex Sec5 and Exo84[26]. Other RAL effectors include Fila- interesting approach but has many technical hurdles, includ- min, PLD1 and ZONAB[26]. ing the lack of an efficient delivery system, poor uptake and RALBP1 has GAP activity for RAC/CDC42 proteins. In low gene silencing efficiency[2]. The inhibition of RAS mem- this way, RALBP1 is important for actin dynamics, filopodia brane localization via the inhibition of RAS farnesylation has formation and membrane ruffling[1, 26, 27], thereby contributing been investigated, but this approach has failed to materialize to cancer cell adhesion, invasion and migration. RALBP1 is into a therapeutic strategy due to several limitations, including critical for the migration of bladder and prostate cancer cells, toxicity, and mainly the appearance of a compensatory mecha- which is partly independent of its interaction with RAL[28]. nism via geranylgeranylation[2, 8, 19]. RALBP1 is also important for endocytosis, including receptor- So far, the only approach that has shown promise in treat- mediated endocytosis of proteins such as epidermal growth ing cancer patients with RAS mutations is the targeting of its factor receptor, insulin receptor and transferrin receptor, downstream signaling cascades such as RAF-MEK-ERK and through its interactions with POB1, Reps1 and AP2[29–31]. The PI3K-AKT[2, 8, 16, 19]. Targeting these two pathways either sepa- involvement of RALBP1 in receptor endocytosis points to the rately or together is beneficial in preventing in vitro and in vivo prominence of RAL in signal transduction by these receptors, progression of tumors harboring a RAS mutation[2]. Currently, which regulate various cellular processes such as survival clinical trials are being conducted to study the therapeutic and proliferation[32, 33]. RALBP1 has also been identified as a effects of MEK and PI3K inhibitors in cancer patients harbor- non-ABC transporter that causes resistance to chemothera- ing RAS mutations[2]. However, a wrinkle in this approach peutic drugs[34]. RALBP1 is known to cause resistance to has appeared. Recent studies have shown that different KRAS anthracycline derivatives such as doxorubicin and glutathione mutations preferentially activate different downstream sig- conjugates by increasing their efflux from cells in an ATP- Acta Pharmacologica Sinica www.chinaphar.com npg Guin S et al 293 Figure 2. RAL effectors and their functions. Upon activation, RAL GTPase regulates numerous biological processes through its effectors. Abnormal regulation of these biological processes by activated RAL leads to protumorigenic biological outcomes. The various dotted lines show the cellular processes regulated by each RAL effector and the corresponding biological outcomes.