Individual Palmitoyl Residues Serve Distinct Roles in H-ras Trafficking, Microlocalization, and Signaling

H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopallmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cbolesterol-dependent and cholesterol-independent microdomains. In contrast, monopallmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-pahnitate is weaker. Thus, membrane affinity of a pallmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopahnitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

RAS mutations and cetuximab in locally advanced rectal cancer: results of the EXPERT-C trial.

Background: RAS mutations predict resistance to anti-epidermal growthfactor receptor (EGFR) monoclonal antibodies in metastatic colorectal cancer. We analysed RAS mutations in 30 non-metastatic rectal cancer patients treated with or without cetuximab within the 31 EXPERT-C trial.

Methods: Ninety of 149 patients with tumours available for analysis were KRAS/BRAF wild-type, and randomly assigned to capecitabine plus oxaliplatin (CAPOX) followed by chemoradiotherapy, surgery and adjuvant CAPOX or the same regimen plus cetuximab (CAPOX-C). Of these, four had a mutation of NRAS exon 3, and 84 were retrospectively analysed for additional KRAS (exon 4) and NRAS (exons 2/4) mutations by using bi-directional Sanger sequencing. The effect of cetuximab on study end-points in the RAS wild-type population was analysed.

Results: Eleven (13%) of 84 patients initially classified as KRAS/BRAF wild-type were found to have a mutation in KRAS exon 4 (11%) or NRAS exons 2/4 (2%). Overall, 78/149 (52%) assessable patients were RAS wild-type (CAPOX, n = 40; CAPOX-C, n = 38). In this population, after a median follow-up of 63.8 months, in line with the initial analysis, the addition of cetuximab was associated with numerically higher, but not statistically significant, rates of complete response (15.8% versus 7.5%, p = 0.31), 5-year progression-free survival (75.5% versus 67.5%, hazard ratio (HR) 0.61, p = 0.25) and 5-year overall survival (83.8% versus 70%, HR 0.54, p = 0.20).

Conclusions: RAS mutations beyond KRAS exon 2 and 3 were identified in 17% of locally advanced rectal cancer patients. Given the small sample size, no definitive conclusions on the effect of additional RAS mutations on cetuximab treatment in this setting can be drawn and further investigation of RAS in larger studies is warranted.

A genome-based strategy uncovers frequent BRAF mutations in melanoma

Using a genome-scanning approach to search for oncogenes, a recent report identifies somatic mutations in the signaling gene BRAF that are particularly prevalent in melanoma.

Microarray expression profiling in melanoma reveals a BRAF mutation signature

We have used microarray gene expression profiling and machine learning to predict the presence of BRAF mutations in a panel of 61 melanoma cell lines. The BRAF gene was found to be mutated in 42 samples (69%) and intragenic mutations of the NRAS gene were detected in seven samples (11%). No cell line carried mutations of both genes. Using support vector machines, we have built a classifier that differentiates between melanoma cell lines based on BRAF mutation status. As few as 83 genes are able to discriminate between BRAF mutant and BRAF wild-type samples with clear separation observed using hierarchical clustering. Multidimensional scaling was used to visualize the relationship between a BRAF mutation signature and that of a generalized mitogen-activated protein kinase (MAPK) activation (either BRAF or NRAS mutation) in the context of the discriminating gene list. We observed that samples carrying NRAS mutations lie somewhere between those with or without BRAF mutations. These observations suggest that there are gene-specific mutation signals in addition to a common MAPK activation that result from the pleiotropic effects of either BRAF or NRAS on other signaling pathways, leading to measurably different transcriptional changes.

Notch and NOXA-related pathways in melanoma cells

Notch receptor-mediated intracellular events represent an ancient cell signaling system, and alterations in Notch expression are associated with various malignancies in which Notch may function as an oncogene or less commonly as a tumor suppressor. Notch signaling regulates cell fate decisions in the epidermis, including influencing stem cell dynamics and growth/differentiation control of cells in skin. Because of increasing evidence that the Notch signaling network is deregulated in human malignancies, Notch receptors have become attractive targets for selective killing of malignant cells. Compared with proliferating normal human melanocytes, melanoma cell lines are characterized by markedly enhanced levels of activated Notch-1 receptor. By using a small molecule gamma-secretase inhibitor (GSI) consisting of a tripeptide aldehyde, N-benzyloxycarbonyl-Leu-Leu-Nle-CHO, which can block processing and activation of all four different Notch receptors, we identified a specific apoptotic vulnerability in melanoma cells. GSI triggers apoptosis in melanoma cells, but only G2/M growth arrest in melanocytes without subsequent cell death. Moreover, GSI treatment induced a pro-apoptotic BH3-only protein, NOXA, in melanoma cells but not in normal melanocytes. The use of GSI to induce NOXA induction overcomes the apoptotic resistance of melanoma cells, which commonly express numerous cell survival proteins such as Mcl-1, Bcl-2, and survivin. Taken together, these results highlight the concept of synthetic lethality in which exposure to GSI, in combination with melanoma cells overexpressing activated Notch receptors, has lethal consequences, producing selective killing of melanoma cells, while sparing normal melanocytes. By identifying signaling pathways that contribute to the transformation of melanoma cells (e.g. Notch signaling), and anti-cancer agents that achieve tumor selectivity (e.g., GSI-induced NOXA), this experimental approach provides a useful framework for future therapeutic strategies in cutaneous oncology.

Molecular biomarkers in non-small-cell lung cancer : a retrospective analysis of data from the phase 3 FLEX study

Background: Findings from the phase 3 FLEX study showed that the addition of cetuximab to cisplatin and vinorelbine significantly improved overall survival, compared with cisplatin and vinorelbine alone, in the first-line treatment of EGFR-expressing, advanced non-small-cell lung cancer (NSCLC). We investigated whether candidate biomarkers were predictive for the efficacy of chemotherapy plus cetuximab in this setting. Methods: Genomic DNA extracted from formalin-fixed paraffin-embedded (FFPE) tumour tissue of patients enrolled in the FLEX study was screened for KRAS codon 12 and 13 and EGFR kinase domain mutations with PCR-based assays. In FFPE tissue sections, EGFR copy number was assessed by dual-colour fluorescence in-situ hybridisation and PTEN expression by immunohistochemistry. Treatment outcome was investigated according to biomarker status in all available samples from patients in the intention-to-treat population. The primary endpoint in the FLEX study was overall survival. The FLEX study, which is ongoing but not recruiting participants, is registered with ClinicalTrials.gov, number NCT00148798. Findings: KRAS mutations were detected in 75 of 395 (19%) tumours and activating EGFR mutations in 64 of 436 (15%). EGFR copy number was scored as increased in 102 of 279 (37%) tumours and PTEN expression as negative in 107 of 303 (35%). Comparisons of treatment outcome between the two groups (chemotherapy plus cetuximab vs chemotherapy alone) according to biomarker status provided no indication that these biomarkers were of predictive value. Activating EGFR mutations were identified as indicators of good prognosis, with patients in both treatment groups whose tumours carried such mutations having improved survival compared with those whose tumours did not (chemotherapy plus cetuximab: median 17·5 months [95% CI 11·7-23·4] vs 8·5 months [7·1-10·8], hazard ratio [HR] 0·52 [0·32-0·84], p=0·0063; chemotherapy alone: 23·8 months [15·2-not reached] vs 10·0 months [8·7-11·0], HR 0·35 [0·21-0·59], p<0·0001). Expression of PTEN seemed to be a potential indicator of good prognosis, with patients whose tumours expressed PTEN having improved survival compared with those whose tumours did not, although this finding was not significant (chemotherapy plus cetuximab: median 11·4 months [8·6-13·6] vs 6·8 months [5·9-12·7], HR 0·80 [0·55-1·16], p=0·24; chemotherapy alone: 11·0 months [9·2-12·6] vs 9·3 months [7·6-11·9], HR 0·77 [0·54-1·10], p=0·16). Interpretation: The efficacy of chemotherapy plus cetuximab in the first-line treatment of advanced NSCLC seems to be independent of each of the biomarkers assessed. Funding: Merck KGaA. © 2011 Elsevier Ltd.

Prognostic value of TP53, KRAS and EGFR mutations in nonsmall cell lung cancer : the EUELC cohort

Nonsmall cell lung cancer samples from the European Early Lung Cancer biobank were analysed to assess the prognostic significance of mutations in the TP53, KRAS and EGFR genes. The series included 11 never-smokers, 86 former smokers, 152 current smokers and one patient without informed smoking status. There were 110 squamous cell carcinomas (SCCs), 133 adenocarcinomas (ADCs) and seven large cell carcinomas or mixed histologies. Expression of p53 was analysed by immunohistochemistry. DNA was extracted from frozen tumour tissues. TP53 mutations were detected in 48.8% of cases and were more frequent among SCCs than ADCs (p<0.0001). TP53 mutation status was not associated with prognosis. G to T transversions, known to be associated with smoking, were marginally more common among patients who developed a second primary lung cancer or recurrence/metastasis (progressive disease). EGFR mutations were almost exclusively found in never-smoking females (p=0.0067). KRAS mutations were detected in 18.5% of cases, mainly ADC (p<0.0001), and showed a tendency toward association with progressive disease status. These results suggest that mutations are good markers of different aetiologies and histopathological forms of lung cancers but have little prognostic value, with the exception of KRAS mutation, which may have a prognostic value in ADC. Copyright©ERS 2012.

No evidence for genetic association with the let-7 microRNA-binding site or other common KRAS variants in risk of endometriosis

STUDY QUESTION Is there a contribution of the minor allele at the KRAS single nucleotide polymorphism (SNP) rs61764370 in the let-7 microRNA-binding site to endometriosis risk? SUMMARY ANSWER We found no evidence for association between endometriosis risk and rs61764370 or any other SNPs in KRAS. WHAT IS KNOWN ALREADY The rs61764370 SNP in the 3' untranslated region of the KRAS gene is predicted to disrupt a complementary binding site (LCS6) for the let-7 microRNA, and was recently reported to be at a high frequency (31%) in 132 women of varying ancestry with endometriosis compared with frequencies in a database of population controls (up to 7.6% depending on ancestry), suggesting a strong effect of this KRAS SNP in the aetiology of endometriosis. STUDY DESIGN, SIZE AND DURATION This was a case-control study with a total of 11 206 subjects. The study was performed between February 2012 and July 2012. PARTICIPANTS/MATERIALS, SETTINGAND METHODS We first investigated a possible association between common markers in KRAS and endometriosis risk from our genome-wide association (GWA) data in 3194 surgically confirmed endometriosis cases and 7060 controls of European ancestry. Although rs61764370 was not genotyped on the GWA arrays, five SNPs typed in the study were highly correlated with this variant. The rs61764370 and two SNPs highly correlated with rs61764370 were then genotyped in 933 endometriosis cases and 952 controls using the Sequenom MassARRAY platform. MAIN RESULTS AND THE ROLE OF CHANCE There was no evidence for an association between rs61764370 and endometriosis risk P = 0.411 and odds ratio = 1.10 (95% confidence intervals: 0.88-1.36). We also found no evidence for an association between the highly correlated SNP rs17387019 and endometriosis. Their minor allele frequencies in cases and controls were of 0.087-0.091 similar to the population frequency reported previously for this variant in controls. Analyses of endometriosis cases with revised American Fertility Society stage III/IV disease also showed no evidence for an association between these SNPs and endometriosis risk. LIMITATIONS AND REASONS FOR CAUTION The GWA and genotyped data sets were not independent since individuals and cases from some families overlap. Controls in our GWA study were not screened for endometriosis. WIDER IMPLICATIONS OF THE FINDINGS The key SNP, rs61764370, was genotyped in a subset of samples. Our results do not support the suggestion that carrying the minor allele at rs61764370 contributes to a significant number of endometriosis cases and rs61764370 is, therefore, unlikely to be a useful marker of endometriosis risk. STUDY FUNDING/COMPETING INTEREST(S) The research was funded by grants from the Australian National Health and Medical Research Council and Wellcome Trust. None of the authors has competing interests for the study.

RUNX3 downregulation in human lung adenocarcinoma is independent of p53, EGFR or KRAS status

RUNX3 aberrations play a pivotal role in the oncogenesis of breast, gastric, colon, skin and lung tissues. The aim of this study was to characterize further the expression of RUNX3 in lung cancers. To achieve this, a lung cancer tissue microarray (TMA), frozen lung cancer tissues and lung cell lines were examined for RUNX3 expression by immunohistochemistry, while the TMA was also examined for EGFR and p53 expression. RUNX3 promoter methylation status, and EGFR and KRAS mutation status were also investigated. Inactivation of RUNX3 was observed in 70% of the adenocarcinoma samples, and this was associated with promoter hypermethylation but not biased to EGFR/KRAS mutations. Our results suggest a central role of RUNX3 downregulation in pulmonary adenocarcinoma, which may not be dependent of other established cancer-causing pathways and may have important diagnostic and screening implications.

Overexpression of neurone glial-related cell adhesion molecule is an independent predictor of poor prognosis in advanced colorectal cancer

A downstream target of the Wnt pathway, neurone glial-related cell adhesion molecule (Nr-CAM) has recently been implicated in human cancer development. However, its role in colorectal cancer (CRC) pathobiology and clinical relevance remains unknown. In this study, we examined the clinical significance of Nr-CAM protein expression in a retrospective series of 428 CRCs using immunohistochemistry and tissue microarrays. Cox proportional hazards regression was used to calculate hazard ratios (HR) of mortality according to various clinicopathological features and molecular markers. All CRC samples were immunoreactive for Nr-CAM protein expression, compared to 10 / 245 (4%) matched normal tissue (P <0.0001). Of 428 CRC samples, 97 (23%) showed Nr-CAM overexpression, which was significantly associated with nodal (P = 0.012) and distant (P = 0.039) metastasis, but not with extent of local invasion or tumor size. Additionally, Nr-CAM overexpression was associated with vascular invasion (P = 0.0029), p53 expression (P = 0.036), and peritoneal metastasis at diagnosis (P = 0.013). In a multivariate model adjusted for other clinicopathological predictors of survival, Nr-CAM overexpression correlated with a significant increase in disease-specific (HR 1.66; 95% confidence interval 1.11-2.47; P = 0.014) and overall mortality (HR 1.57; 95% confidence interval 1.07-2.30; P = 0.023) in advanced but not early stage disease. Notably, 5-fluorouracil-based chemotherapy conferred significant survival benefit to patients with tumors negative for Nr-CAM overexpression but not to those with Nr-CAM overexpressed tumors. In conclusion, Nr-CAM protein expression is upregulated in CRC tissues. Nr-CAM overexpression is an independent marker of poor prognosis among advanced CRC patients, and is a possible predictive marker for non-beneficence to 5-fluorouracil- based chemotherapy.

Validation of Next Generation Sequencing Technologies in Comparison to Current Diagnostic Gold Standards for BRAF, EGFR and KRAS Mutational Analysis

Next Generation Sequencing (NGS) has the potential of becoming an important tool in clinical diagnosis and therapeutic decision-making in oncology owing to its enhanced sensitivity in DNA mutation detection, fast-turnaround of samples in comparison to current gold standard methods and the potential to sequence a large number of cancer-driving genes at the one time. We aim to test the diagnostic accuracy of current NGS technology in the analysis of mutations that represent current standard-of-care, and its reliability to generate concomitant information on other key genes in human oncogenesis. Thirteen clinical samples (8 lung adenocarcinomas, 3 colon carcinomas and 2 malignant melanomas) already genotyped for EGFR, KRAS and BRAF mutations by current standard-of-care methods (Sanger Sequencing and q-PCR), were analysed for detection of mutations in the same three genes using two NGS platforms and an additional 43 genes with one of these platforms. The results were analysed using closed platform-specific proprietary bioinformatics software as well as open third party applications. Our results indicate that the existing format of the NGS technology performed well in detecting the clinically relevant mutations stated above but may not be reliable for a broader unsupervised analysis of the wider genome in its current design. Our study represents a diagnostically lead validation of the major strengths and weaknesses of this technology before consideration for diagnostic use.

DNA methylation subgroups and the CpG island methylator phenotype in gastric cancer: a comprehensive profiling approach

BACKGROUND: Methylation-induced silencing of promoter CpG islands in tumor suppressor genes plays an important role in human carcinogenesis. In colorectal cancer, the CpG island methylator phenotype (CIMP) is defined as widespread and elevated levels of DNA methylation and CIMP+ tumors have distinctive clinicopathological and molecular features. In contrast, the existence of a comparable CIMP subtype in gastric cancer (GC) has not been clearly established. To further investigate this issue, in the present study we performed comprehensive DNA methylation profiling of a well-characterised series of primary GC.

METHODS: The methylation status of 1,421 autosomal CpG sites located within 768 cancer-related genes was investigated using the Illumina GoldenGate Methylation Panel I assay on DNA extracted from 60 gastric tumors and matched tumor-adjacent gastric tissue pairs. Methylation data was analysed using a recursively partitioned mixture model and investigated for associations with clinicopathological and molecular features including age, Helicobacter pylori status, tumor site, patient survival, microsatellite instability and BRAF and KRAS mutations.

RESULTS: A total of 147 genes were differentially methylated between tumor and matched tumor-adjacent gastric tissue, with HOXA5 and hedgehog signalling being the top-ranked gene and signalling pathway, respectively. Unsupervised clustering of methylation data revealed the existence of 6 subgroups under two main clusters, referred to as L (low methylation; 28% of cases) and H (high methylation; 72%). Female patients were over-represented in the H tumor group compared to L group (36% vs 6%; P = 0.024), however no other significant differences in clinicopathological or molecular features were apparent. CpG sites that were hypermethylated in group H were more frequently located in CpG islands and marked for polycomb occupancy.

CONCLUSIONS: High-throughput methylation analysis implicates genes involved in embryonic development and hedgehog signaling in gastric tumorigenesis. GC is comprised of two major methylation subtypes, with the highly methylated group showing some features consistent with a CpG island methylator phenotype.

Selecting the best targeted agent in first-line treatment of unresectable liver metastases from colorectal cancer:does the bench have the answers?

For physicians facing patients with organ-limited metastases from colorectal cancer, tumor shrinkage and sterilization of micrometastatic disease is the main goal, giving the opportunity for secondary surgical resection. At the same time, for the majority of patients who will not achieve a sufficient tumor response, disease control remains the predominant objective. Since FOLFOX or FOLFIRI have similar efficacies, the challenge is to define which could be the most effective targeted agent (anti-EGFR or anti-VEGF) to reach these goals. Therefore, a priori molecular identification of patients that could benefit from anti-EGFR or anti-VEGF monoclonal antibodies (i.e. the currently approved targeted therapies for metastatic colorectal cancer) is of critical importance. In this setting, the KRAS mutation status was the first identified predictive marker of response to anti-EGFR therapy. Since it has been demonstrated that tumors with KRAS mutation do not respond to anti-EGFR therapy, KRAS status must be determined prior to treatment. Thus, for KRAS wild-type patients, the choices that remain are either anti-VEGF or anti-EGFR. In this review, we present the most updated data from translational research programs dealing with the identification of biomarkers for response to targeted therapies.