To our knowledge, we have performed the largest study to date that assesses the potential diagnostic and prognostic utility of miRNAs in esophageal cancer. MiRNA expression was evaluated in 170 cancerous and adjacent non-cancerous tissue pairs and we identified miRNAs important for classification of samples into diagnostic and Barrett's categories. Elevated miR-21 levels were observed in both SCC and ADC samples indicating that miR-21 involvement in esophageal carcinogenesis is independent of histological type. Furthermore, increased expression of miR-194, miR-192, miR-223 and reduced expression of miR-203 are observed in ADC patients from two independent clinical centers while decreased expression of miR-375 is detected in SCC patients from three clinical centers. Altered expression of these miRNAs is specific to histological type, which may provide clues as to the different molecular progression undertaken by these cancers and suggests a potential avenue for histology-specific therapy to improve prognosis.
Expression levels of miRNAs mentioned above were validated in samples from the testing and validation cohorts using qRT-PCR. Over-expression of miR-21 is of interest since it is ubiquitously induced in solid tumors, including esophagus, liver, lung, breast, stomach, prostate, colon, pancreas (10,11,13,16–21) and in chronic lymphocytic leukemia (22
). MiR-21 targets tumor and metastasis suppressor genes, including phosphatase and tensin homolog PTEN (31
), tumor suppressor gene tropomyosin 1 TPM1 (32
), programmed cell death 4 PDCD4 (33
), and Sprouty2 (35
), thereby demonstrating its involvement in tumor growth, invasion, and metastasis. Our laboratory has also revealed that elevated miR-21 in tumors are associated with poor prognosis and therapeutic outcome in colon cancer (16
Importantly, we demonstrate here that altered levels of miR-21 in non-cancerous tissue of SCC patients are associated with survival, suggesting that miR-21 may have an effect in SCC tumors via the stroma. Knowing that inflammatory responses are implicated in all stages of tumorigenesis (36
), targeting mir-21 in stromal cells provides a possible avenue for chemoprevention. We have previously established that the combination of cytokine expression in non-cancerous and cancerous tissue of lung ADC patients are predictors of survival, suggesting a possible interaction between the tumor and its surrounding lung environment (37
). A similar association between cytokine expression and survival was observed in esophageal cancer (unpublished results). Furthermore, there is growing evidence for the role of miRNAs in regulating innate and acquired immune response (38–40). Specifically, mir-21 expression has been associated with immune-related diseases, including B-cell lymphoma (41
) and chronic lymphocytic leukemia (22
). Furthermore, a recent study demonstrated the Stat3-dependent effect of interleukin-6 on miR-21 induction, which contributed to the oncogenic potential of Stat3 (42
). Consequently, our finding that increased levels of miR-21 are associated with worse prognosis in non-cancerous tissue is possibly a reflection of an immune response that is associated with tumorigenesis.
In addition, our study shows that mir-375 levels in cancerous tissue are associated with prognosis in Barrett's associated ADC patients. A recent study has demonstrated that mir-375-deficient mice are hyperglycemic and that the pancreatic alpha-cell mass is decreased, a sign of impaired proliferation (43
). Furthermore, the same group performed a combined analysis of putative targets and microarray expression showing that putative mir-375 targets control cellular growth and proliferation (43
). Experimentally validated targets of mir-375, as described in Tarbase (44
), include Mxi1, JAK2, and Ahr. MXi1 is a c-MYC antagonist previously found to be over-expressed in esophageal ADC (45
). A direct association between JAK2 and PI3-kinase upon glycine-extended gastrin stimulation has been shown in Barrett's associated ADC cells (46
). The suppression of Ahr signaling as a chemopreventive approach was supported by a study that showed a reduction of DNA adducts in esophageal SCC cell lines by blocking the transcription factor Ahr (47
). These results combined with our finding that reduced levels of mir-375 are associated with worse prognosis suggest that mir-375 may be a good marker for cell proliferation.
In concordance with our observations, a recent study based on a cohort of 7 patients reported that miR-21 is over-expressed in ADC, miR-143 is under-expressed in ADC, and miR-194 is over-expressed in Barrett's (28
) . The study also reported over-expression of miR-203, miR-205, miR-143, and miR-215 in Barrett's. Another study reported the analysis of 20 cases and 9 normal epithelial tissue and revealed an over-expression of miR-21 and under-expression of miR-203 and miR-205 in cancerous tissue in both histological subtypes (26
), which is concordant with our microarray results. In a previous study evaluating miRNA expression in SCC patients, high expression of miR-103 and miR-107 correlated with poor survival in 30 patients, a finding confirmed in an independent set of 22 SCC patients (27
). These results were not in concordance with our analysis, perhaps due to their use of a different microarray platform and more limited sample size.
The administration of neo-adjuvant chemoradiation therapy (prior to surgery) in 54% of patients used in this study and complete pathologic response in 22% of patients limits our ability to negate the role of therapy on associations between miRNA expression and diagnosis/prognosis. Differential expression in cancerous tissue and non-cancerous tissue was observed in patients that had or had not undergone neo-adjuvant chemoradiation therapy (Supplemental Table 10
) although associations were not consistent across different cohort types. Because therapy was administered prior to tissue collection and no pre-therapy tissue is available, it was not possible to directly predict therapeutic response.
Of note, patients with complete pathologic response are not necessarily cured, perhaps due to remaining systemic processes or the inability to detect small metastatic disease (48
). It is still a debate whether such patients have longer survival than patients without complete pathologic response (49
). This observation further demonstrates the importance of identifying molecular biomarkers, such as miRNAs, that would help refine staging and predict treatment response. Furthermore, while chronic alcohol consumption and smoking may adversely affect survival of esophageal cancer patients (50
), we were unable to adequately assess the influence of those covariates in our multivariate Cox analysis due to missing values (16% and 23% missing values for smoking and alcohol consumption, respectively). Although preliminary analysis of available data did not show an association between smoking, alcohol consumption and survival (data not shown), further studies are warranted to confirm the association of miRNAs with survival, independent of these two factors.
In conclusion, we identified miRNAs whose expression is altered in and between SCC and ADC cancerous tissue, and in cancerous tissue between Barrett's associated and sporadic ADC cancerous tissue. Furthermore, we have established a strong association between elevated miR-21 levels in the non-cancerous tissue of SCC patients with worse prognosis, thereby suggesting a possible association between miR-21 in the stromal environment, immune response, and SCC. Prognostic association of miRNA expression in non-cancerous tissue is of particular interest because altered levels of these miRNAs may be evident prior to advanced disease stage and the occurrence of symptoms. In Barrett's associated ADC patients, high levels of mir-375, which targets genes associated with tumor cell proliferation, in the cancerous tissue was associated with worse prognosis. The ability to block miRNA transcription may open avenues for the possible use of miRNAs in identifying novel drug targets and therapies for esophageal carcinoma.