Unmasking the role of KRAS and BRAF pathways in MSI colorectal tumors

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1 Unmasking the role of KRAS and BRAF pathways in MSI colorectal tumors Expert Rev. Gastroenterol. Hepatol. 3(1), 5 9 (2009) Raquel Seruca, MD, PhD Author for correspondence Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Dr Roberto Frias, Porto, Portugal Tel.: Fax: rseruca@ipatimup.pt Sérgia Velho Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Dr Roberto Frias, Porto, Portugal svelho@ipatimup.pt Carla Oliveira, PhD Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal carlaol@ipatimup.pt Marina Leite, PhD Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal mleite@ipatimup.pt Paulo Matos, PhD Centro de Genética Humana, Instituto Nacional de Saúde Dr Ricardo Jorge, Lisboa, Portugal Tel.: Fax: paulo.matos@insa.min-saude.pt Peter Jordan, PhD Centro de Genética Humana Instituto Nacional de Saúde Dr Ricardo Jorge, Lisboa, Portugal peter.jordan@insa.min-saude.pt We found that mutations in KRAS or BRAF in these premalignant lesions occur in a mutually exclusive manner, as earlier verified for microsatellite instability colorectal cancer. Colorectal cancer (CRC) represents one of the leading causes of cancer mortality in the Western world. Approximately 5% of cases are attributable to familial cancer syndromes, while the remaining cases arise as sporadic forms. The vast majority of these sporadic CRC cases present as an adenoma carcinoma sequence, involving somatic mutations in the APC tumor suppressor gene and in the KRAS oncogene. In this article, we will discuss new insights gained into the molecular pathways involved in a specific subset of tumors, those CRCs harboring hundreds of thousands of mutations throughout the genome: microsatellite instability (MSI) CRC. Within this setting, we will emphasize the role of KRAS and BRAF activation in MSI colorectal tumori genesis, mainly drawing upon our own experience in the field. Approximately 15% of sporadic and 90% of hereditary nonpolyposis colon carcin oma (HNPCC) show MSI in multiple repeat sequences due to defects in mismatch repair (MMR) genes. HNPCC is an autosomal, dominantly inherited form of cancer that is caused mainly by mutations and rearrangements of MMR genes, and is characterized by the development of CRC and extracolonic malignancies, particularly cancers of the endometrium, stomach, renal pelvis and/or ureter, small bowel, ovary, biliary tract and brain, as well as sebaceous tumors. The diagnosis of HNPCC can be made on molecular grounds, by finding an alteration in one of the MMR genes, or on clinical grounds, according to the so-called Amsterdam criteria. Patients with incomplete Amsterdam criteria or fulfilment of the Bethesda criteria are also suspected of having HNPCC. Detection of a clearly pathogenic germline MMR gene mutation has helped to identify presympto matic individuals at risk for HNPCC. hmsh2, hmlh1 and hmsh6 are the most frequently mutated MMR genes in HNPCC families [1]. In the majority of sporadic forms of MSI CRC, the ablation of MMR is achieved by the promotion of hypermethylation of hmlh1. Nevertheless, rare somatic mutations in either hmlh1 or other MMR genes have also been reported in sporadic cases [2]. Although sporadic and hereditary forms of MSI CRC show distinct molecular mechanisms for MMR silencing, both subsets show a very similar molecular profile of mutations in noncoding and coding repetitive tracts. The genes that accumulate mutations in their coding sequences are called target genes and, among these, TGFBRII, IGFIIR, BAX and TCF-4 show the highest rate of mutations, with their alterations demonstrated to be functionally important [3]. As a consequence, signaling pathways that are pivo tal for colorectal differentiation, such as the Wnt/APC/β catenin pathway and the TGF-β pathway, are aberrantly switched on or off, leading to abnormal cell prolif eration, differentiation, cell cycle regulation and apoptosis, inducing rapid progression of these tumors. Moreover, MMR defects, besides giving rise to instability at microsatellite sequences (both coding and noncoding), also induce an increased frequency of point mutations, namely in proto-oncogenes / Expert Reviews Ltd ISSN

2 Seruca, Velho, Oliveira, Leite, Matos & Jordan KRAS & BRAF alterations in MSI CRCs Unstable microsatellite CRCs have recently been demonstrated to harbor activating missense mutations in genes coding for protein members of the Ras Raf MAPK pathway, another crucial pathway in colorectal tumorigenesis. The BRAF V600E hotspot has been reported in approximately 40% of sporadic MSI CRC cases. Several hotspot point mutations in the KRAS sequence have been identified in approximately 20% of sporadic MSI CRCs, clustering in cases negative for BRAF mutations [4]. The alternative occurrence of oncogenic activation in these two genes, of the same pathway, emphasizes the importance of the activation of the MAPK pathway in MSI CRC tumorigenesis. Furthermore, and importantly in clinical terms, other researchers as well as ourselves have found that the BRAF V600E mutation occurs exclusively in sporadic forms of MSI CRC. Thus, the combined ana lysis of MSI and the BRAF V600E mutation is now included in the current protocols for screening HNPCC, since it is a reliable, fast and low-cost strategy that helps to identify sporadic cases and, thus, avoids the time-consuming screening of MMR germline mutation analyses [4,5]. In contrast to the scenario observed for the BRAF V600E mutation, HNPCC cases show a high level of KRAS mutations (40%) in comparison with sporadic MSI CRC (20%). Moreover, the frequency of KRAS mutations varies among HNPCC tumors depending on the MMR gene affected. Tumors from patients carrying either hmsh2 or hmsh6 germline mutations showed a higher frequency of KRAS mutations when compared with CRCs from patients with an hmlh1 germline alteration [6,7]. It remains to be clarified whether this finding can explain the apparently different clinical phenotypes seen in HNPCC families carrying mutations in specific MMR genes. Not only is the frequency of KRAS mutations different between HNPCC and sporadic forms of MSI CRC but, most interestingly, the type of KRAS mutations also differ between these two settings. While sporadic MSI CRC bears 80% of KRAS mutations at codon 12, HNPCC CRC harbors 50% of KRAS mutations at codon 13. Although more frequent in HNPCC tumors, the presence of KRAS codon 13 mutations was not associated with a specific MMR gene affected in the germline of HNPCC patients [7]. In clinical terms, and in contrast to the BRAF V600E mutation, these results are unlikely to benefit HNPCC screening protocols, since KRAS mutations occur in both forms of MSI CRC (sporadic and hereditary), as well as in approximately 35% of microsatellite stable CRCs. KRAS & BRAF alterations in colorectal premalignant lesions Little was known regarding the frequency of KRAS and BRAF mutations in premalignant lesions and in CRCs at different stages of progression. In order to determine the oncogenic importance and the timing of the occurrence of KRAS and BRAF mutations in the process of colorectal tumorigenesis, we recently analyzed a series of mixed hyperplastic and adenomatous colorectal polyps. We found that mutations in KRAS or BRAF in these premalignant lesions occur in a mutually exclusive manner, as earlier verified for MSI CRC. Moreover, KRAS and BRAF V600E mutations occur in 35 and 30% of the polyps, respectively. When comparing the frequency of KRAS and BRAF mutations found in our series of colorectal polyps with that observed in MSI CRC, no difference was observed. These observations highlight the role of both KRAS and BRAF activation as primary genetic events and their role in the initiation of this subset of sporadic CRC [8]....the combined ana lysis of microsatellite instability and the BRAF V600E mutation is now included in the current protocols for screening hereditary nonpolyposis colon carcinoma... These polyps were also studied for molecular phenotypes previously associated with sporadic MSI CRC. In sporadic MSI CRC, BRAF mutations were preferentially found in CRC associated with hmlh1 promoter hypermethylation. In our series of polyps, none of the cases, including those with BRAF mutations, harbored methylation at the hmlh1 promoter and all were microsatellite stable. These results demonstrate that KRAS or BRAF mutations precede hmlh1 promoter methylation and the MSI phenotype [8]. These data challenge the current assumption that MMR deficiency in sporadic CRC induces an accumulation of mutations in noncoding and coding repetitive tracts and an increased rate of missense mutations, namely in proto-oncogenes. Instead, it suggests that, in sporadic MSI CRC, KRAS and BRAF oncogenic mutations are triggers of malignancy rather than gene targets of MMR deficiency. Moreover, it is likely that, in the sporadic MSI CRC pathway, the late acquisition of hmlh1 methylation leading to loss of MMR occurs mostly in the transition from a premalignant to a malignant stage. Interestingly, in the same study, we verified that the CpGisland methylation phenotype (CIMP) occurred in 25% of the polyps and all were mutated for BRAF. These results are in accordance with previous findings in CRC regarding the association of BRAF mutations with the CIMP. Furthermore, both results (in polyps and in CRCs) suggest that BRAF mutant CRC cells need to accumulate extra epigenetic/genetic alterations in order to acquire full transformation. Functional relevance of BRAF V600E in MSI CRCs In 2002, Davies et al. showed that the BRAF V600E mutation increases the BRAF kinase activity by tenfold and promotes activation of the MEK/ERK kinase cascade independent of Ras, resulting in the activation of transcription factors that stimulate cell proliferation. However, the BRAF V600E mutation alone has a 50-fold lower transforming activity, in a fibroblast focus-formation assay, compared with oncogenic KRAS. We studied the functional importance of the BRAF V600E mutation in MSI CRCs using RNAi by selectively knocking down BRAF in CRC cell lines that harbored KRAS- or BRAF-activating mutations. Inhibition of BRAF in MSI CRCs with BRAF V600E or KRAS G13D mutations significantly inhibited cell cycle progression (through BrdU incorporation). Inhibition of BRAF induces a significant increase 6 Expert Rev. Gastroenterol. Hepatol. 3(1), (2009)

3 Unmasking the role of KRAS & BRAF pathways in MSI colorectal tumors Editorial in apoptosis (by terminal transferase dutp nick-end labeling assay) in cell lines with the BRAF V600E mutation only, and no significant difference was seen in apoptosis in the cell line with a KRAS G13D mutation. We further analyzed several signaling mediators that could be implicated in the proliferative (e.g., perk1/2, cyclin D1 and p27) and apoptotic (e.g., B-cell lymphoma-2, Bax, mantle cell lymphoma-1, pakt, pbad and X-linked inhibitor of apoptosis protein) pathways that were altered in these CRC cell lines. We found a decrease in phosphorylation of ERK1 and -2 and in cyclin D1 levels in all cell lines, independent of the genetic background. Moreover, we found an increase in p27 levels and a decrease in levels of the antiapoptotic protein B-cell lymphoma-2 in cell lines with mutated BRAF. Overall, these results show that MSI KRAS and BRAF mutant cancer cell lines respond differently to BRAF knockdown. We further demonstrate that the BRAF V600E mutation, in MSI CRCs, provides cell survival signals that involve B-cell lymphoma-2 [9]. In addition, we verified that knockdown of BRAF in BRAF V600E mutant cancer cell lines increased apoptosis by fourfold, in comparison with the control sirna. Together, these data provided evidence, using in vitro assays, that BRAF is likely to be a good therapeutic target in MSI CRCs carrying BRAF mutations but not in those with KRAS mutations. BRAF V600E & Rac1b overexpression cooperate to induce malignant progression What other genetic alterations do BRAF mutant CRC cells require for malignant progression? Oncogenic KRAS also requires Rac1 signaling for transformation, most likely via the NF-κB-mediated stimulation of cell cycle progression and cell survival activated by Rac1. Very recently, we reported that approximately 80% of BRAF V600E-positive tumors also overexpress Rac1b, a highly activated splice variant of the signaling GTPase Rac1 [10]. In accordance with these data and taking into account the results obtained by the analyses of the colorectal polyps, we proposed a working model in which BRAF V600E mutant cells would cooperate with Rac1b to acquire similar KRAS oncogenic potential (Fi g u r e 1). To test our hypothesis, we made use of a classical fibroblast focus formation assay to understand the importance of BRAF V600E and Rac1b in cellular transformation. We verified that coexpression of wild-type Rac1b, but not of wild-type Rac1, produces a synergistic increase in the focus-forming ability of mutant BRAF V600E, similar to the coexpression of the constitutively active form of Rac1 (Rac1-L61), raising efficiency to levels that approximate those of oncogenic KRAS. Considering these results and taking advantage of colon cancer cell lines, we further studied in vitro the cellular effects associated with suppression of mutant BRAF and Rac1b. The specific suppression of the mutant BRAF V600E allele and Rac1b revealed that both proteins provide an independent, but additive, contribution to cell cycle progression in colorectal cells, with their simultaneous suppression producing a 3.3-fold reduction in BrdU-positive cells, which correlated with decreasing cyclin D1 levels. However, when apoptosis was assessed, under the same double-knockdown conditions, a striking 7.8-fold, synergistic increase in terminal transferase dutp nick-end labeling assay-positive cells was observed. A BRAF WT MEK ERK Mutant KRAS Cell cycle progression Cell survival PI3K Rac1 NF-κB Mutant BRAF MEK ERK Cell cycle progression Rac1 KRAS WT PI3K RAC1B NF-κB Cell survival Figure 1. A model summarizing the role of RAC1B in cooperation with BRAF V600E to sustain colorectal cell survival and cell cycle progression. (A) In colorectal tumors with oncogenic mutations in the KRAS gene, cell proliferation is promoted via the ERK pathway and cell survival is stimulated via PI3K and Rac1 signaling. (B) In another subgroup of colorectal tumors, an oncogenic BRAF mutation stimulates the ERK pathway; however, BRAF lies downstream of KRAS and by itself cannot activate Rac1. In these tumors, the survival stimulus usually provided by mutant KRAS is achieved by overexpression of hyperactive Rac1b. Terminal transferase dutp nick-end labeling assay results were confirmed by western blot, showing an increase in the 89-kDa fragment of poly(adp-ribose) polymerase-1, a well-defined, caspase 3/7-cleaved apoptosis marker. The simultaneous suppression of both proteins dramatically decreased CRC cell viability through impaired cell cycle progression and increased apoptosis. These results demonstrate that Rac1b and BRAF V600E functionally cooperate to sustain colorectal cell viability and suggest they constitute an alternative survival pathway to oncogenic KRAS [10]. An intriguing issue is now to clarify what genetic alterations cause the change in alternative splicing of the Rac1 pre-mrna. At least in CRC cell lines with different levels of Rac1b expression, we found no evidence that direct splice-site mutations in the Rac1 gene were responsible for the differences in alternative splicing. Therefore, alternative splicing is most likely deregulated downstream of genetic changes in key colorectal cell signaling pathways, such as the PI3K/Akt or the β-catenin/tcf4 pathways. These pathways have recently been described to affect the level or activity of RNA splicing factors and, thus, change splicing decisions in cells [11,12]. A major challenge will now be to identify the upstream pathways responsible for Rac1b generation. We also expect that a just-right amount of pathway activation will exist, which will promote selection for Rac1b overexpression without causing cells to undergo apoptosis, cell cycle arrest or senescence. This knowledge can be expected to unearth novel therapeutic targets for the BRAF V600E/Rac1b subgroup of colorectal tumors. B 7

4 Seruca, Velho, Oliveira, Leite, Matos & Jordan A further unresolved issue concerns how the initiating BRAF V600E mutation and/or Rac1b overexpression are related to the occurrence of CIMP; is CIMP a consequence or prerequisite of tumor progression? Altogether, the discussed results reveal novel molecular charac teristics of colon tumors containing BRAF V600E mutations and should help in defining novel targets for individual tumor therapy. Implications of KRAS & BRAF mutations in the treatment of metastatic CRCs Besides the role of KRAS and BRAF in the initiation and progression of colorectal tumors, the mutational status of these genes has also been implicated in the response to therapy of CRC patients. Anti-EGF receptor (EGFR) therapies are being applied to treat patients with metastatic CRCs (mcrcs). Cetuximab and panitumumab are anti-egfr monoclonal antibodies that, alone or in combination with chemotherapy agents, have been shown to be helpful in the treatment of patients with mcrc [13,14]. Currently, KRAS gene mutations are being pointed out not only as a molecular prognostic marker in CRC, but also as a marker to predict the sensitivity of tumors to anti-egfr therapeutics in mcrc. KRAS mutant mcrc patients show a lower response rate to therapy when compared with patients with KRAS wild-type mcrc [15 23]. This finding led to the exclusion of KRAS-mutated mcrc patients from being eligible for EGFR-blocking therapies. Nevertheless, among the group of mcrc patients with tumors harboring the wild-type KRAS gene, the response to anti-egfr monoclonal antibodies is limited [15 23]. The gene make-up that contributes to this therapeutical resistance remains to be determined. As previously mentioned, CRCs frequently harbor alterations in other genes involved in EGFR-associated signaling cascades, such as mutations in BRAF and PIK3CA, as well as loss of PTEN expression or overexpression of Rac1b. Can BRAF, PIK3CA and Rac1b activation render CRC patients refractory to EGFR-blocking therapies? Accordingly, it was reported that the presence of a BRAF mutation in mcrc was associated with a lack of response to cetuximab treatment [24]. Recently, in vitro and in vivo studies have shown that the presence of PIK3CA mutations or loss of PTEN expression predicts the response of colon cancer cells to cetuximab [25,26]. Furthermore, in CRC cell lines, constitutive and simultaneous activation of the KRAS and PIK3CA pathways lead to an increased resistance to cetuximab treatment [26]. The present data indicate a possible synergistic effect of EGFR, Ras/Raf/MAPK and PIK3CA/Rac1b pathways in the acquisition of resistance to anti-egfr therapies. In fact, we verified that, in CRC, most oncogenic mutations do not occur as single events but rather preferentially occur together, as illustrated by the presence of PIK3CA mutations in cases displaying KRAS or BRAF mutations [27]. In our view, the use of KRAS mutation detection in primary CRC is an oversimplified method to select patients for anti-egfr monoclonal antibodies [28]. Thus, we consider that a cautionary note should be added to this strategy of selecting mcrc patients for therapy with anti-egfr agents. Furthermore, it remains to be seen if different KRAS mutations may influence the response to cetuximab and panitumumab in mcrc patients, since it has been previously demonstrated that the type of KRAS amino acid substitution has a clinical impact on the outcome of colon cancer patients [29]. In conclusion, there is still much to unravel in MSI CRC biology so as to forge new routes of therapy for this disease, not only for economic reasons but, more importantly, for the success of therapy in the outcome of each individual patient. Financial & competing interests disclosure The Portuguese Foundation for Science and Technology (POCTI/SAU- OBD/68310/2006) and the Sixth Framework Program from EU- FP6 (LSHC-CT ) provided funding. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. References 1 Lynch HT, de la Chapelle A. Hereditary colorectal cancer. N. Engl. J. Med. 348(10), (2003). 2 Kane MF, Loda M, Gaida GM et al. 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