MiRNAs regulate various biological functions in normal cells such as growth and differentiation, and they are increasingly recognized as playing critical roles in cancer development and progression. Dysregulation of miRNA expression resulting from amplification or loss of miRNAs in tumors compared to their normal tissue counterparts suggests that miRNAs can function as either oncogenes or tumor suppressor genes . Studies evaluating miRNA expression in spontaneously occurring tumors in dogs demonstrate that similar to human cancers, alteration of miRNAs likely contributes to tumorigenesis and that high-throughput methodologies used for the study of miRNAs in human tissues can also be applied to dogs [26–32].
Cutaneous MCTs are the most common skin tumor in dogs; however, little is known regarding mechanisms underlying malignant transformation of these cells. The biological behavior of canine MCTs ranges from relatively benign disease cured with surgical removal to aggressive, highly metastatic tumors ultimately resulting in the death of affected dogs. While the presence of activating KIT mutations helps to explain the behavior of some canine MCTs, little is known regarding the potential role of miRNAs in both normal and malignant mast cells. The purpose of this study was to begin to investigate the potential role of miRNA dysregulation in canine MCTs that exhibit aggressive biologic behavior.
MiRNA expression profiling of primary canine MCTs identified unique miRNA signatures associated with aggressive MCTs as compared to benign MCTs. The unsupervised hierarchical clustering of primary cutaneous MCTs based on their miRNA expression profiles recapitulated the grouping of the tumors based on their biological behavior, supporting the notion that miRNA dysregulation is associated with the biologic behavior of canine MCTs. Furthermore, we found that miR-9 expression was significantly upregulated in aggressive MCTs compared to benign MCTs. Interestingly, miR-9 was identified as a pro-metastatic miRNA in human breast cancer cell lines through its ability to enhance cell motility and invasiveness in vitro and metastasis formation in vivo. More recently, miR-9 expression was found to be significantly increased in paired primary tumors and distant metastatic sites, suggesting direct involvement of miR-9 in the metastatic process [34, 35]. In concordance with the potential role of miR-9 in malignant mast cell behavior, the BR and C2 canine malignant cell lines expressed high levels of miR-9 compared to normal canine BMMCs. Taken together, these data support the notion that dysregulation of miR-9 may contribute to the aggressive biologic behavior of some canine MCTs.
While activating KIT mutations clearly contribute to the malignant behavior of mast cells, additional cooperating or initiating genetic defects may be required for the malignant transformation and promotion of the metastatic phenotype . Our data demonstrate that overexpression of miR-9 in the C57 and P815 mouse malignant mast cell lines and normal mouse BMMCs significantly enhanced the invasive behavior of mast cells and indicate that miR-9 induces a pattern of gene dysfunction associated with an invasive phenotype regardless of KIT mutation status.
While some studies have shown that miR-9 promotes metastasis formation [33, 36–39] other contrasting studies suggest that increased expression of miR-9 suppresses metastasis formation [40, 41] and that miR-9 inhibits tumor growth . The opposing roles of miR-9 in various tissues may be explained by the expression of different mRNA targets in distinct cellular and developmental contexts. Indeed, miRNA effects do appear to be cell type/tissue specific and contextual in nature. Previous studies have demonstrated that miR-9 is overexpressed in CDX2-negative primary gastric cancers and miR-9 knockdown inhibits proliferation of human gastric cancer cell lines . In contrast, miR-9 is downregulated in human ovarian tumor cells and overexpression of miR-9 suppresses their proliferation, in part by downregulating NFκB1 [40, 42]. Moreover, miRNA dysregulation may affect only certain aspects of cell behavior. In our studies, miR-9 expression in mast cell lines did not provide a survival advantage or prevent apoptosis, but it did alter the invasive phenotype, supporting the contextual nature of miR-9 induced effects.
To gain insight into possible mechanisms underlying the observed miR-9-dependent invasive behavior of mast cells, we evaluated the effects of miR-9 expression on the transcriptional profiles of BMMCs and P815 cells. MiR-9 modulated the expression of a large number of gene transcripts, including down-regulation of several putative miR-9 targets identified by computational prediction programs. Furthermore, down-regulation of peroxisome proliferator-activated receptor δ (PPARG) was observed in BMMCs following enforced miR-9 expression, a finding consistent with recent studies demonstrating that regulation of PPARG expression is mediated by miR-9 through direct targeting of its 3’-UTR . To draw firm conclusions regarding direct regulation of target gene expression by miR-9, a functional approach for each gene would be required to validate whether these genes are true miR-9 targets, which although relevant, was outside the scope of this study.
Overexpression of miR-9 significantly altered gene expression in both BMMCs and P815 cells, however, most gene transcripts affected by miR-9 expression differed between normal and malignant mast cells. These observed differences likely reflect variations in the impact of miR-9 that are dependent on cellular context. In our study, we identified gene transcripts that showed similar changes in expression following miR-9 overexpression in both normal and malignant mast cells and validated several genes demonstrating significant changes in expression (interferon-induced transmembrane protein protein 3, IFITM3; PDZK1 interacting protein 1, PDZK1IP1) or implicated in promoting the metastatic phenotype (mast cell chymase, CMA1). IFITM3 belongs to a family of interferon-induced transmembrane proteins that contribute to diverse biological processes, such as antiviral immunity, germ cell homing and maturation, and bone mineralization. The function of these proteins in mast cells is currently unclear . PDZK1IP1 is a small, non-gycosylated membrane-associated protein that localizes to the plasma membrane and Golgi apparatus. While the function of PDZK1IP1 has not been evaluated in mast cells, overexpression of PDZK1IP1 has been documented in human ovarian, breast, and prostate carcinomas and this strongly correlates with tumor progression [45, 46]. Furthermore, overexpression of PDKZK1IP1 in melanoma cell lines enhances cell proliferation, decreases apoptosis, increases cell migration and is, in part, mediated by an increase in reactive oxygen species (ROS) production .
Chymases are serine proteases possessing chymotrypsin-like activity expressed exclusively by mast cells that promote matrix destruction, tissue remodeling and modulation of immune responses by hydrolyzing chemokines and cytokines . Given the role of chymase in the activation of matrix metalloproteases and extracellular matrix degradation, our findings suggest that miR-9 enhances invasion, in part, through increased expression chymase. Indeed, miR-9 overexpression in normal mast cells resulted in increased expression of CMA1 with a concomitant decrease in the expression of secretory leukocyte peptidase inhibitor (SLPI), a direct inhibitor of chymase . These findings are consistent with the notion that that miR-9 promotes a pattern of gene expression contributing to enhanced invasion and suggests a role for chymase in mediating the biologic functions of miR-9.
Interestingly, miR-9 modulated the expression of other proteases in normal mast cells, including up-regulation of heparinase (HSPE). Heparinase is an endogylocosidase that functions in the degradation and release of heparan sulfate-bound growth factors . Previous studies have shown that enzymatic cleavage of heparin sulfate by heparinase results in disassembly of the extracellular matrix and basement membrane dissolution, inducing structural modifications that loosen the extracellular matrix barrier and enable cell invasion . Heparinase increases tumor invasion in both cell lines and spontaneous tumor models, through both extracellular matrix remodeling and increased peritumoral lymphangiogenesis . Our data show that normal mast cells overexpressing miR-9 exhibit markedly increased HSPE expression, supporting the assertion that miR-9 may promote the metastatic phenotype by enhancing the proteolytic activity of a number of proteases important in physical remodeling of the extracellular matrix and activate mediators responsible for cell dissemination.
The present study investigated alterations in gene transcript expression affected by miR-9; however, these changes were not demonstrated at the protein level. Gene expression does not directly correlate with changes at the protein level and miRNAs may suppress protein expression by post-transcriptional silencing mechanisms that are not reflected in transcriptional profiling analyses. Furthermore, inhibition of miR-9 in canine mast cell lines would provide further convincing evidence of its importance in mast cell invasion. As such, identifying proteins altered by miR-9 that promote cell invasion and validating these targets in canine cell lines/tumors represents an area of ongoing investigation.