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The let-7 family of microRNAs are important regulatory molecules in lung cancer. One downstream target of let-7 is the RAS gene family, including KRAS, an important oncogene in the etiology and clinical outcome of lung adenocarcinoma. Recently, a SNP in the let-7 binding region of the KRAS 3’ UTR was identified (termed LCS6). This functional polymorphism alters let-7 binding, resulting in both increased KRAS expression and decreased let-7 exposure. Further, this SNP has been reported as a risk trait for lung cancer risk among low-moderate smokers. Given the functionality of LCS6, we tested the hypothesis that this SNP is associated with the occurrence of KRAS mutation as well as patient survival. Here, we report there is no association between the LCS6 KRAS polymorphism and KRAS mutation. Further, we find no association between the LCS6 polymorphism and lung cancer survival. These unexpected findings imply that this newly reported KRAS-LCS6 polymorphism will have limited clinical utility for NSCLC.
Altered expression of the let-7 family of microRNAs (miRNA) is implicated in many human cancers, including non-small cell lung cancer [1–3]. miRNAs hybridize to the 3’UTR of target mRNA altering the stability and expression of target sequences. The human RAS genes contain let-7 complementary sites in their 3’UTR, and let-7 has been show to repress RAS expression . Further, let-7 is down-regulated in lung cancer . Mechanistically, this would lead to enhanced expression of the lung oncogene KRAS, also successfully demonstrated by Johnson et al .
We, and others, have reported a strong association between KRAS mutation and survival in non-small cell lung cancer . Consistent with these findings, reduced let-7 expression is also associated with poor prognosis in lung cancer [3, 6]. Mitigating factors (i.e. exposures or polymorphisms) that alter expression of let-7 or KRAS might be expected to have a significant impact on patient survival.
Recently, a report by Chin et al  described a novel SNP in the 3’UTR of the KRAS gene that alters binding of let-7. This variant, the 6th let-7 complementary site in the KRAS 3’UTR (LCS6), results in up-regulation of the KRAS gene and concomitant down-regulation of let-7. In addition, Chin et al  report that this polymorphism is associated with a modest increase in lung cancer risk, particularly among low-dose smokers, suggesting that this variant is a biomarker of susceptibility to the carcinogenic effects of tobacco smoke. Presumably, the enhanced risk is related to the combined effects of up-regulated expression of KRAS, and down-regulation of the let-7 miRNA among those with the LCS6 variant genotype.
To further understand this model of cancer susceptibility, we have evaluated whether the LCS6 polymorphism is associated with KRAS mutation in tumors (previously described for this population ). Given the up-regulation of KRAS expression associated with the variant allele, our a priori hypothesis was that KRAS mutations would be preferentially selected for among those who were constitutively wild type for the LCS6 SNP. Further, given prior evidence for lower let-7 levels to correlate with poor prognosis , we hypothesize that the LCS6 variant (with accompanying low level let-7), is associated with reduced survival time.
Details on the study population have been previously described . Briefly, cases consisted of all newly diagnosed patients with resectable lung cancer who received treatment at the Massachusetts General Hospital (MGH) Thoracic Surgery, Oncology, and Pulmonary Services from November 1992 through December 1996. Data on case demographics and exposures were derived from an administered questionnaire, clinical data were obtained from pathology reports and clinical record, and patient outcome data were obtained from the Massachusettes General Hospital cancer registry supplemented with SSDI query. KRAS codon 12 mutation was determined by RFLP (n=365) .
From this surgical case series, a subset of cases were evaluated for the LCS6 genotype (n=218); inclusion criteria was determined by availability of blood-derived DNA. Compared to those without available DNA, the subset of cases studied were slightly younger (65.9 years vs 66.8 years), had smoked more (60.3 vs 52.3 packyears), tended to be male (57% vs 47%), and had a lower proportion of adenocarcinomas (62% vs 69%). None of these differences were statistically significant. DNA was extracted from whole blood with the QIAmp DNA mini kit according to the manufacturer’s protocol (Qiagen, Valencia, CA). Genotyping of the KRAS-LCS6 SNP was done using Taqman® allelic discrimination (Applied Biosystems, Foster City, CA) with a custom designed primer probe set. Genotyping was performed in a blinded fashion, appropriate controls were included in each run, and approximately 10% of samples were embedded duplicates. There was over >95% concordance observed between replicates.
Statistical analysis of LCS6 polymorphism and mutation status included χ2 and unconditional logistic regression. Survival time was defined as the time from surgery to the patient’s death, or the last time that the patient was known to be alive. There were 200 deaths, and the mean follow-up time for survivors was 4.94 years. Kaplan-Meier survival probability curves were constructed, and differences between groups were tested with the use of the logrank method. In addition, a Cox proportional hazards model was used to evaluate the association between genotype and outcome controlling for age, sex, and stage. All P values represent two-sided statistical tests.
The overall allele prevelance was 6.6%, consistent with the report by Chin et al . In this case series, there was no significant difference in the variant allele frequency comparing adenocarcinoma (14.7%) and squamous cell carcinoma (11.8%). Further, there was no significant association of the LCS6 polymorphism and KRAS mutation overall, nor in the adenocarcinoma subgroup (Table 1). Given the modifying effects of smoking reported by Chin , we further examined the data stratified by 40 packyears smoked, and there remained no evidence of an association between KRAS mutation and the polymorphism (data not shown). Finally, as Chin et al suggested that the LCS6 polymorphism may impact patient outcome, we evaluated survival time as a function of genotype, and found no association between the LCS6 polymorphism and survival among all histologies (figure 1), or restricted to histologic subgroups (data not shown).
We tested the hypothesis i) that there is negative selection pressure in lung cancer for KRAS mutation to occur in individuals who do not have the KRAS-LCS6 polymorphism and, ii), that the LCS6 variant would be associated with poor survival. In both instances, we observed no evidence of any association. These findings are unexpected given the recent report by Chin et al  suggesting that the polymorphism is associated with susceptibility to the carcinogenic effects of tobacco. As the KRAS mutation prevalence was the same in both genotype groups, it seems clear that, despite the upregulation in KRAS that occurs with the LCS6 variant, this does not result in any selective pressure for, or against, KRAS mutations.
Let-7 expression levels are notably decreased in lung tumors [3, 4], and this has been associated with a worse clinical outcome [3, 6]. However, in our case series, we find that the LCS6 polymorphism does not correlate with outcome, despite its reported association with reduced let-7 . The reason for this is unclear, although it is possible, for example, that the variant is not associated with a sufficient decrease in let-7 expression to alter outcome. Other possibilities for our null observations may lie with study design. Our analysis was restricted to a subset of cases from a larger case-series study from a single institution. However, we have demonstrated that the subset available for study was similar to those cases not studied. Further, while our statistical power is limited, within this subset of cases there remains a clear association between k-ras mutation and outcome.
In the case-control risk context, there is marked lung cancer risk estimates attributed to this polymorphism (2.5-fold) and this was most evident among those who were low to moderate smokers. Restricting our case-only analysis in this way did not alter any of our observations; LCS6 polymorphism and KRAS mutation status remained independent events, and there was no impact of LCS6 polymorphism on survival. The LCS6 variant genotype may predispose individuals to a somatic event that is ubiquitous in tumors, allowing an observation of risk in the case-control framework, but undetectable in the tumor-series. For instance, let-7 is known to down-regulate Dicer, a critical component of the miRNA processing machinery; and reduced let-7 levels lead to increased levels of Dicer and other mature miRNAs . Although an altered balance of miRNAs associated with the LCS6 variant appears to not be associated with prognosis of NSCLC, the same alterations in miRNA expression levels may have important clinical implications for other tumor types. In fact, a recent paper demonstrated that the LCS6 variant is associated with poor prognosis in head and neck cancer . Hence, while our data suggest that this newly reported KRAS-LCS6 polymorphism will have limited (if any) clinical utility for NSCLC, additional studies in other cancer sites remain of interest.
This work was supported by NIH grant P30 CA-077598.
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Conflict of Interest Statement: None declared.