Significance of KRAS/PAK1/Crk pathway in non-small cell lung cancer oncogenesis.

BackgroundKey effector(s) of mutated KRAS in lung cancer progression and metastasis are unknown. Here we investigated the role of PAK1/Crk axis in transduction of the oncogenic KRAS signal in non-small cell lung cancer (NSCLC).MethodsWe used NSCLC clinical specimens to examine the correlation among KRAS mutations (codon 12, 13 and 61); PAK1/Crk axis activation [p-PAK1(Thr423), p-Crk(Ser41)]; and adhesion molecules expression by immunohistochemistry. For assessing the role of proto-oncogene c-Crk as a KRAS effector, we inhibited KRAS in NSCLC cells by a combination of farnesyltransferase inhibitor (FTI) and geranylgeranyltransferase inhibitor (GGTI) and measured p-Crk-II(Ser41) by western blotting. Finally, we disrupted the signaling network downstream of KRAS by blocking KRAS/PAK1/Crk axis with PAK1 inhibitors (i.e., IPA-3, FRAX597 or FRAX1036) along with partial inhibition of all other KRAS effectors by prenylation inhibitors (FTI + GGTI) and examined the motility, morphology and proliferation of the NSCLC cells.ResultsImmunohistochemical analysis demonstrated an inverse correlation between PAK1/Crk phosphorylation and E-cadherin/p120-catenin expression. Furthermore, KRAS mutant tumors expressed higher p-PAK1(Thr423) compared to KRAS wild type. KRAS prenylation inhibition by (FTI + GGTI) completely dephosphorylated proto-oncogene c-Crk on Serine 41 while Crk phosphorylation did not change by individual prenylation inhibitors or diluent. Combination of PAK1 inhibition and partial inhibition of all other KRAS effectors by (FTI + GGTI) dramatically altered morphology, motility and proliferation of H157 and A549 cells.ConclusionsOur data provide evidence that proto-oncogene c-Crk is operative downstream of KRAS in NSCLC. Previously we demonstrated that Crk receives oncogenic signals from PAK1. These data in conjunction with the work of others that have specified the role of PAK1 in transduction of KRAS signal bring forward the importance of KRAS/PAK1/Crk axis as a prominent pathway in the oncogenesis of KRAS mutant lung cancer

An integrative approach unveils FOSL1 as an oncogene vulnerability in KRAS-driven lung and pancreatic cancer

KRAS mutated tumours represent a large fraction of human cancers, but the vast majority remains refractory to current clinical therapies. Thus, a deeper understanding of the molecular mechanisms triggered by KRAS oncogene may yield alternative therapeutic strategies. Here we report the identification of a common transcriptional signature across mutant KRAS cancers of distinct tissue origin that includes the transcription factor FOSL1. High FOSL1 expression identifies mutant KRAS lung and pancreatic cancer patients with the worst survival outcome. Furthermore, FOSL1 genetic inhibition is detrimental to both KRAS-driven tumour types. Mechanistically, FOSL1 links the KRAS oncogene to components of the mitotic machinery, a pathway previously postulated to function orthogonally to oncogenic KRAS. FOSL1 targets include AURKA, whose inhibition impairs viability of mutant KRAS cells. Lastly, combination of AURKA and MEK inhibitors induces a deleterious effect on mutant KRAS cells. Our findings unveil KRAS downstream effectors that provide opportunities to treat KRAS-driven cancers

A polymorphism in a let-7 microRNA binding site of KRAS in women with endometriosis.

Endometriosis is found in 5-15% of women of reproductive age and is more frequent in relatives of women with the disease. Activation of KRAS results in de novo endometriosis in mice, however, activating KRAS mutations have not been identified in women. We screened 150 women with endometriosis for a polymorphism in a let-7 microRNA (miRNA) binding site in the 3'-UTR of KRAS and detected a KRAS variant allele in 31% of women with endometriosis as opposed to 5% of a large diverse control population. KRAS mRNA and protein expression were increased in cultured endometrial stromal cells of women with the KRAS variant. Increased KRAS protein was due to altered miRNA binding as demonstrated in reporter assays. Endometrial stromal cells from women with the KRAS variant showed increased proliferation and invasion. In a murine model, endometrial xenografts containing the KRAS variant demonstrated increased proliferation and decreased progesterone receptor levels. These findings suggest that an inherited polymorphism of a let-7 miRNA binding site in KRAS leads to abnormal endometrial growth and endometriosis. The LCS6 polymorphism is the first described genetic marker of endometriosis risk

Impact of the Specific Mutation in KRAS Codon 12 Mutated Tumors on Treatment Efficacy in Patients with Metastatic Colorectal Cancer Receiving Cetuximab-Based First-Line Therapy: A Pooled Analysis of Three Trials

Purpose: This study investigated the impact of specific mutations in codon 12 of the Kirsten-ras (KRAS) gene on treatment efficacy in patients with metastatic colorectal cancer (mCRC). Patients: Overall, 119 patients bearing a KRAS mutation in codon 12 were evaluated. All patients received cetuximab-based first-line chemotherapy within the Central European Cooperative Oncology Group (CECOG), AIO KRK-0104 or AIO KRK-0306 trials. Results: Patients with KRAS codon 12 mutant mCRC showed a broad range of outcome when treated with cetuximab-based first-line regimens. Patients with tumors bearing a KRAS p.G12D mutation showed a strong trend to a more favorable outcome compared to other mutations (overall survival 23.3 vs. 14-18 months; hazard ratio 0.66, range 0.43-1.03). An interaction model illustrated that KRAS p.G12C was associated with unfavorable outcome when treated with oxaliplatin plus cetuximab. Conclusion: The present analysis suggests that KRAS codon 12 mutation may not represent a homogeneous entity in mCRC when treated with cetuximab-based first-line therapy. Copyright (C) 2012 S. Karger AG, Base

Outcomes of patients with advanced cancer and KRAS mutations in phase I clinical trials.

BackgroundKRAS mutation is common in human cancer. We assessed the clinical factors, including type of KRAS mutation and treatment, of patients with advanced cancer and tumor KRAS mutations and their association with treatment outcomes.MethodsPatients referred to the Phase I Clinic for treatment who underwent testing for KRAS mutations were analyzed.ResultsOf 1,781 patients, 365 (21%) had a KRAS mutation. The G12D mutation was the most common mutation (29%). PIK3CA mutations were found in 24% and 10% of patients with and without KRAS mutations (p<0.0001). Of 223 patients with a KRAS mutation who were evaluable for response, 56 were treated with a MEK inhibitor-containing therapy and 167 with other therapies. The clinical benefit (partial response and stable disease lasting ≥6 months) rates were 23% and 9%, respectively, for the MEK inhibitor versus other therapies (p=0.005). The median progression-free survival (PFS) was 3.3 and 2.2 months, respectively (p=0.09). The respective median overall survival was 8.4 and 7.0 months (p=0.38). Of 66 patients with a KRAS mutation and additional alterations, higher rates of clinical benefit (p=0.04), PFS (p=0.045), and overall survival (p=0.02) were noted in patients treated with MEK inhibitor-containing therapy (n=9) compared to those treated with targeted therapy matched to the additional alterations (n=24) or other therapy (n=33).ConclusionsMEK inhibitors in patients with KRAS-mutated advanced cancer were associated with higher clinical benefit rates compared to other therapies. Therapeutic strategies that include MEK inhibitors or novel agents combined with other targeted therapies or chemotherapy need further investigation

KRAS early testing. Consensus initiative and cost-effectiveness evaluation for metastatic colorectal patients in an italian setting

KRAS testing is relevant for the choice of the most appropriate first-line therapy of metastatic colorectal cancer (CRC). Strategies for preventing unequal access to the test should be implemented, but their relevance in the practice is related to economic sustainability. The study adopted the Delphi technique to reach a consensus on several topics. Issues related to execution of KRAS testing were identified by an expert's board and proposed to 108 Italian oncologists and pathologists through two subsequent questionnaires. The emerging proposal was evaluated by decision analyses models employed by technology assessment agencies in order to assess cost-effectiveness. Alternative therapeutic strategies included most commonly used chemotherapy regimens alone or in combination with cetuximab or bevacizumab. The survey indicated that time interval for obtaining KRAS test should not exceed 15 days, 10 days being an optimal interval. To assure the access to proper treatment, a useful strategy should be to anticipate the test after radical resection in patients at high risk of relapse. Early KRAS testing in high risk CRC patients generates incremental cost-effectiveness ratios between 6,000 and 13,000 Euro per quality adjusted life year (QALY) gained. In extensive sensitivity analyses ICER's were always below 15,000 Euro per QALY gained, far within the threshold of 60,000 Euro/QALY gained accepted by regulatory institutions in Italy. In metastatic CRC a time interval higher than 15 days for result of KRAS testing limits access to therapeutic choices. Anticipating KRAS testing before the onset of metastatic disease in patients at high risk does not affect the sustainability and cost-effectiveness profile of cetuximab in first-line mCRC. Early KRAS testing may prevent this inequality in high-risk patients, whether they develop metastases, and is a cost-effective strategy. Based on these results, present joined recommendations of Italian societies of Oncology and Pathology should be updated including early KRAS testing

High-Resolution Three-Dimensional NMR Structure Of The KRAS Proto-Oncogene Promoter Reveals Key Features Of A G-Quadruplex Involved In Transcriptional Regulation

Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5′-AGGGCGGTGTGGGAATAGGGAA-3′) and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression