In the present study we show that a high expression level of miR-21 measured by quantitation of chromogenic ISH signal is associated with short DFS and OS in stage II CC patients. The relative miR-21 expression estimates were found to be independent of other clinical parameters including age, gender, K-RAS and MSI status. We found an intense and well-defined miR-21 ISH signal in the stromal compartment of the tumors using three distinct high affinity antisense LNA probes against the mature miRNA. This localization was supported by RT-qPCR analysis of LCM tissue compartments identifying a 6-fold higher miR-21 level in the stromal compartment compared to that in the cancer cell compartments. The formation of ISH signal was strongly reduced by mix-incubating excess un-labeled probe with the DIG-labeled miR-21 probe on the tissue sections. In addition, no specific ISH signal was observed with two negative control DIG-labeled oligos, one similar to the full-length miR-21 oligo but containing three mis-matches and one with a scrambled sequence. Based on these specificity tests, we conclude that the ISH signal observed represented the presence of genuine miR-21. Taken together, our findings suggest that miR-21 derived from the stromal cell population contributes to the hostile malignant properties of the primary CCs.
The stromal miR-21 positive cells observed in the CRC were generally fibroblast-like in morphology. Cancer associated fibroblasts constitute a heterogeneous cell population with significant growth-promoting impact on tumor progression [32
]. Most cancer-associated fibroblasts are myo-differentiated so-called myofibroblasts that may derive from one or more local cell populations like pericryptal myofibroblasts, quiescent interstitial fibroblasts, smooth muscle cells, circulating fibrocytes, and/or by epithelial-to-mesenchymal transition (EMT) [34
]. Fibroblasts may act on tumor cells through expression of growth factors, such as TGF-β, and thereby contribute to the survival and proliferation of tumor cells [32
]. In addition, a number of extracellular proteases are expressed in myofibroblasts, facilitating tissue remodeling and growth factor activation during tumor growth, for example, well-established prognostic cancer biomarkers like uPA and PAI-1 as well as TIMP-1 and MMP-2 are primarily expressed in cancer associated myofibroblasts [37
]. Sempere et al.
] included CC samples in their study and also found miR-21 signal in both tumor cells and tumor associated fibroblast using a double fluorescence approach. A prevalent expression of miR-21 in CRC stroma was not reported by Schetter et al. [7
] who found discrete ISH signals in the tumor area and described the miR-21 positive cells as tumor cells. Yamamichi et al. [25
] used an FITC-labeled LNA oligo and TSA-based amplification and found expression of miR-21 ISH signal primarily in cancer cells but also reported stroma cell expression. The reason for these discrepancies is likely explained by cross-reaction of the oligo to “similar sequences” allowed under suboptimal hybridization conditions. Another possible reason for the discrepancy is linked to the different reagents employed to detect the labeled LNA oligo. It should be noted that in the latter two studies [7
] only the scrambled probe was included to support the specificity of the ISH signal observed.
For our study we developed a novel ISH procedure for LNA-based miRNA ISH considering procedures described by others [39
], but suitable for an automated set-up using a Genepaint instrument [42
]. We recently established a manual ISH procedure and confirmed the expression pattern for miR-21 as well as for a number of other miRNAs in various tissues [43
]. For semiquantitative analysis, we employed an unbiased image sampling and analysis approach to replace the manual and more subjective scoring of the histological expression intensities. Using image analysis we obtained quantitative estimates of the ISH signal for miR-21 (TB) as well as the area ratio to the nuclear density (TBR). Both estimates showed that the high levels correlated significantly to short DFS in the CC patient group of which the TBR values provided the best correlation. Adjusting the ISH signal to the cell density provided a lower inter slide variation and may therefore explain the slightly better correlation obtained for TBR. The precision of the TBR and TB estimates for each patient was fairly high compared to the variation among individuals. For both TBR and TB estimates, we obtained high ICC values, 84% for both TBR and TB, confirming that the inter-individual variation was substantially larger than the intra-individual variation and a good indication that the two parameters could be significant biomarkers.
The significant correlation of miR-21 to DFS and OS may prove useful for future development of tests to identify stage II CC patients at risk of disease recurrence. In this study we found that miR-21 correlation to CC DFS was independent of other clinical parameters available for some or all of the patients. Thus, none of the parameters available (including age, gender, total leukocyte count, K-RAS or MSI) for the CC patient group (alone or in combination) correlated with DFS or miR-21 ISH levels. A significant correlation between high miR-21 levels and poor survival in CC patients has also been reported by Schetter et al.
] These authors isolated RNA from normal tissue and CC tissue from 52 stage II patients and obtained cancer/normal ratios of miR-21 by qPCR. Recently, Schetter et al.
found that combining the miR-21 levels with a mRNA expression score for inflammation (Il-6, IL-8, IL10, IL-12a and NOS2a) allowed an even better prognostication of high risk CC patients [6
]. This is an interesting observation, which in addition to enforcing the prognostic value of miR-21, also suggests that the molecular profile of the inflammatory response in CC better reflects the risk than the total leukocyte count available in our patient cohort. Increased miR-21 levels measured by either microarray or qPCR have been shown to correlate with poor prognosis in several other cancer types including NSCLC [10
], breast cancer [11
], and head-and-neck cancer [44
]. It has been demonstrated that the cellular origin of miR-21 is also partly stromal in breast [46
] and lung cancers [43
]. All these data further demonstrate miR-21 as an inherent cancer associated miRNA with a significant impact on tumor growth and dissemination.
Our quantitative measurements of the miR-21 ISH signal identified a significant correlation to DFS in CC patients but not in the RC group. This could be a result of surgical intervention being different or due to different molecular pathways for cancer invasion and dissemination in the two parts of the intestines. The RC patients included in the present study were all operated before the introduction of the TME (Total Mesorectal Excision) operational procedure [47
]. The TME practice is associated with a significantly longer DFS probably due to excision of more malignant tissue and/or lymph nodes [48
]. Thus, the surgical procedure of the RC patients in this study is a likely confounding parameter that may have prevented the detection of relations between biological events (here miR-21) and disease progression. It cannot be excluded, however, that also other molecular pathways being specific for the RC could contribute to the observed lack of a significant association between miR-21 and DFS in the RC patients.
The miR-21 promoter contains putative binding sites for AP-1, Ets/PU.1, C/EBPα, NFI, SRF, p53, and STAT3 [16
]. Activation of the ERK-MAP kinase pathway, induced for example by TGF-β, leads to AP-1 activation and has been found to be augmented by miR-21 in cardiac fibroblasts [19
]. TGF-β is expressed at high levels in CRC and induces fibroblast activation and differentiation into myofibroblasts in model systems [49
] as well as in CRC tissue [50
] where most fibroblasts are myofibroblasts. The mechanism(s) of action of miR-21 in cancer fibroblasts is not known. In various settings, miR-21 has been reported to affect expression of the transcription factor NF1-B in PMA-stimulated HL60 cells [16
], Spry1 in cardiac fibroblasts [19
], the tumor suppressor PTEN in hepatocytes and cardiac fibroblasts [22
], and the tumor suppressor PDCD4 in a variety of cell lines [17
]. The identified mRNA targets vary according to the particular cell line being analyzed.
We have demonstrated that the prevalent expression of miR-21 in the stromal compartment of CC patients is correlated with short DFS and shorter OS. However, it remains unsolved how the miR-21 positive stromal cells contribute to the malignant characteristics of these tumors and whether the sporadic miR-21 positive epithelial cancer cells are also important. If the stromal cell population is directing tumor progression, how do we then explain the recurrent disease after tumor excision? Is the submucosa already primed for promoting local re-growth or are potential metastatic sites already primed for stimulating growth of un-detected micro-metastases? In this context, the miRNAs are truly playing the role as molecular regulators critical to disease progression. Such central roles in cancer have led miRNAs to be considered attractive novel therapeutic cancer targets, both for inhibiting oncogenic miRNAs or by re-introducing miRNAs lost in cancer [52
]. To this end, recently it has been demonstrated that miR-21 is strongly upregulated in cardiac fibroblasts in a murine heart failure model and that silencing the fibroblast-derived miR-21 using miR-21 antagonists can prevent or even cure the functional deterioration [19