Recent clinical trials show that different histological subtypes of NSCLC have different responses to therapy (
31–
34). Yet, current treatment is similar across histologies because histology-specific molecular targets that could be therapeutically exploited are lacking. In the largest study of miR expression in lung cancer to date, we found a miR profile that strongly and consistently differentiated AD from SQ. We confirmed the largest differences in an independent set of lung cancer cases and identified a set of putative target genes whose expression was correlated with the expression of miRs differentiating the two histology groups, providing preliminary evidence of miR-target correlations for further experimental validation. Expression differences by histology were highly significant in the early stage tumors but not significant in the advanced stages, suggesting that in advanced, less differentiated tumors, miR expression loses histology specificity. This finding implies that efforts to exploit these differences for mechanistic insight or therapeutic benefit should focus on early stage tumors. Given the different miR profiles by histology, we conducted survival analyses separately in the two histology groups, with a rigorous statistical approach. Many of the miRs down-regulated in early stage SQ
vs. AD were associated with increased risk of mortality from SQ, suggesting a role for these miRs in the repression of genes involved in lung cancer progression. This finding may also provide insight into why some early stage, apparently surgically cured, patients recur with a fatal outcome. Among these miRs, we identified a five-miR signature that strongly predicted survival from SQ.
All members of the let-7 family highly differentiated AD from SQ and increased mortality risk in SQ, suggesting that this group exerts its influence most profoundly within the early stage SQ histology. Pioneering studies described let-7 miR as a negative regulator of the oncogenic family of RAS, and repressor of cell proliferation pathways and NSCLC growth (
11;
27;
35). As it has been observed for other pathways (e.g., the EGFR pathway in non small cell lung cancer (
36)) or other tumors (e.g., the NRAS/BRAF mutations in melanoma (
37)), it is possible that let-7 expression alterations and KRAS mutations are mutually exclusive in lung carcinogenesis, with let-7 having a more predominant effect in SQ and KRAS mutations in AD. If further confirmed by mutational analyses, this finding may improve our understanding of the carcinogenetic pathways leading to non small cell lung cancer. Among the let-7 miRs, let-7g showed the largest fold-change together with miR 26a, an hypoxia-induced miR known to decrease pro-apoptotic signaling (
38). Interestingly, both let-7g and miR-26a are localized to chromosome 3p21-22. Allele loss and genetic alteration at this locus are the most frequent and earliest genomic abnormalities in NSCLC (
39) and are more common in SQ than AD (
40;
41). We also found histological differences for miRs clustering at chromosome 14q32 and 19q13, both loci involved in lung cancer development and progression (
28;
29). All these loci have been extensively investigated for potential tumor suppressor genes without clear success. However, if the chromosomal alterations involve miRs that target distant genes, the potentially relevant lung cancer genes likely extend beyond these loci.
Other miRs strongly differentiating the two histology groups included miR-29a, which affects apoptosis (
42) and epigenetic normalization of NSCLC (
43), and miR-21, which acts as an oncogene or “oncomiR” in many tumor types and plays an important role in tumor metastasis (
44;
45). Interestingly, in our study miR-21 strongly differentiated the histology groups with high levels in AD in stage II but not in stage I tumors, suggesting that miR-21 may be a marker of tumor progression in AD, identifying tumors on the verge of acquiring metastatic potential.
The large majority of miRs were down-regulated in SQ vs. AD in our study. This pattern suggests that miRs that function as tumor suppressors, like let-7 and miR-29a, may be more relevant for SQ tumorigenesis because they exhibited poor expression in this tumor type. In contrast, miRs with oncogenic potentials in many tissue types, like miR-21 and -26a, may be crucial for AD development since they were over-expressed in AD compared to SQ. If functionally confirmed, these miRs may identify histology-specific therapeutic targets, especially for surgically resectable lesions.
The only previous study showing miR expression differences among lung cancer histologies found 5 mature miRs and 1 miR precursor differentiating 65 AD from 39 SQ (
14) in an unadjusted analysis of Caucasians and African Americans, with different smoking patterns than Southern Europeans. Dissimilarity in sample sizes, analytical approaches, assays and ethnic groups may have contributed to the discrepancies between studies. Moreover, differential expression and variable ratio of mature
vs. immature miRs in specific subpopulations of malignant tissues (
45;
46) may account for some differences.
We examined the impact of tobacco smoking on miR expression and obtained interesting preliminary findings. The comparison between smokers and never smokers did not elicit significant results, possibly because of the small number of never smokers (only 5 in males). In smokers, we found that the expression of each member of the let-7 family, which has tumor suppression potential, was inversely associated with number of cigarettes per day in females but not in males. We confirmed this finding also in the independent samples from the CHTN. Down-regulation of let-7 by tobacco smoking was also observed in rats (
47). The gender discrepancy might be related to hormonal effects, since estrogen levels may modify miR expression (
48). Sex and smoking status may have differential impact on miR expression as has been observed for mutational patterns of EGFR in non small cell lung cancer (
49). Interestingly, expression of miR21, which acts as an oncogene, increased with increasing cigarettes per day in SQ but not AD, suggesting a histology-specific response to tobacco carcinogens. However, small numbers or other unknown factors may have contributed to these results, and larger studies and functional tests are necessary to confirm our findings. If confirmed, they may provide an important piece in the mechanistic puzzle relating tobacco smoking to lung cancer development.
In the survival analysis, we found that low expression of several miRs was associated with up to 4-fold excess mortality from SQ overall and in the subgroup including only male smokers with stage I to IIIA. Interestingly, many of these miRs were down-regulated in SQ vs. AD even in stage I, suggesting an early role for these miRs in regulation of tumor progression. We confirmed the findings by QRT-PCR for selected miRs.
Lower expression of let-7e, miR-34a, miR-34c-5p, miR-25, and miR-191 constituted a “poor survival” signature. Reduced expression of the let-7 family has been previously correlated with poor post-operative survival in lung cancer (
13). MiR-34a and, to a lesser extent, miR-34c are targets of p53 and are involved in p53-dependent apoptosis, cell cycle arrest, senescence, and DNA damage response (
50). MiR34a has been recently found to be associated with poor survival of NSCLC in a study investigating the role of the miR34 family (
51) (). Recently, Mascaux et al. found that miR-34c expression progressively decreased from normal bronchial tissues of nonsmokers to SQ (
52), while Liu et al., (
53) identified miR34c as a growth-suppressive miR in murine lung cancer, suggesting that this miR is involved in bronchial carcinogenesis from the very early steps of this process. MiR-25, which targets important cell cycle regulators, like AURKA (p<1e-05, miRBase database), may contribute to the change from bronchioalveolar stem cell to lung cancer stem cells (
54) with metastatic potential. Finally, expression of miR-191 has been found associated with survival from SQ (
55) and acute myeloid leukemia (
56). Although risk prediction models should be interpreted cautiously (
57) and need confirmation in multiple studies, taken together these findings suggest that targeting these miRs in the treatment of SQ may prove fruitful.
| Table 5MiRs associated with worse survival from lung cancer across studies |
Previously, few studies explored the association of miR expression with survival from lung cancer and found different sets of miRs (). Four of the 5 miRs identified by our risk prediction model were also found to be associated with lung cancer survival in other studies, while we could not confirm associations with some of the previously identified miRs. As shows, there is large heterogeneity across studies with regard to subjects’ and tumors’ characteristics, sample size, assays, analytical approaches, and tissue types, which likely explain the discrepant results. Results may also be affected by the different numbers of miRs tested and different cut-offs used to exclude miRs with low intensity across multiple samples or arrays. As an example, Yu et al (
15) found a group of 5 mature miRs that together predicted survival in 57 SQ and 60 AD stage I-III Asian patients. We could not verify this finding since three of the five miRs identified in this study were excluded from our analyses because they had more than 50% missing data across subjects. However, of the remaining two miRs, one (let-7a) was associated with survival in our study.
In conclusion, in the largest study of miR expression in lung cancer to date, miR expression profiles strongly differed between AD and SQ, suggesting that different sets of miRs contribute to the pathogenesis of different NSCLC histologies and may become targets of histology-specific treatment in the future. Among miRs whose expression was reduced in SQ from the early stages, we identified a profile that predicted survival for SQ. These miRs may have important implications for prognosis and treatment of this histology subgroup of lung cancer.
The publisher's final edited version of this article is available free at 
