In our previous study, we observed that miR-29a is globally repressed in tumors as well as in PSC- and epithelial cell- specific manner in PDAC . We further demonstrated that TGF-β1 via SMAD3 signaling negatively regulates miR-29a expression in PSCs and upregulates several ECM proteins including collagens, laminin and fibronectin . In the current study, using RNAseq, we characterize the mechanism and pathway interactions by which miR-29a contributes to PSC-mediated regulation of ECM and tumor-stromal crosstalk. This will allow for a comprehensive understanding of the therapeutic applicability of the molecule in the context of PDAC stroma.
RNAseq analysis with miR-29a overexpressing PSCs and controls identified a number of DEGs, which included predicted direct and indirect targets of the molecule. Because miRNAs primarily regulate genes either by mRNA decay or translational repression, we focused on the direct targets that were downregulated with miR-29a overexpression. We validated the translational repression of the targets namely IGF-1, COL5A3, CLDN1, E2F7, MYBL2, which exhibited the highest fold changes in the RNAseq dataset, along with ITGA6 and ADAMTS2, which had functional relevance in stromal regulation. Our western blot analysis indicated the highest repression of ITGA6, ADAMTS2 and IGF-1 protein levels with miR-29a overexpression in the PSCs (Fig. 2b). Among these identified direct targets, association of IGF-1 and COL5A3 with PSCs in PDAC has been reported previously [32, 51]. Network analysis with the targets identified three overlapping pathways related to IGF, RAS/MAPK signaling and laminin interactions. IGF-1 secreted by activated PSCs and CAFs via sonic hedgehog pathway activates IGF-1R in cancer cells triggering phosphorylation of insulin-receptor or Src substrates to promote PDAC metastasis via intracellular pathways such as RAS/MAPK [36, 52]. In addition, high IGF-1 with low IGFBP3 expressions associated with enhanced risks for PDAC . Expectedly, patients with advance clinical stages (II and III) of PDAC had higher levels of IGF-1R and low IGFBP3 and exhibited poor prognosis . Interestingly, the IGF-1R expressions in these patients associated with high stromal abundance, suggesting the regulation of tumor-stromal crosstalk via IGF/IGF-1R signaling . Another identified miR-29a target CLDN1 is a tight junction protein that facilitates cell-ECM communication and EMT in various cancer types [54–56]. The gene is shown to be a contributor in tumor-stroma crosstalk in pancreatic cancer . Although the regulation of CLDN1 in PSCs has not been reported previously, studies have shown the gene to be under the regulation of IGF-1 signaling [58, 59]. Upregulation of collagens, including COL5A3, is a salient feature of fibrosis and malignant tumor stroma, including that in PDAC [51, 60, 61]. Collagens are abundantly expressed in PDAC ECM; and collagen V, by binding with α2β1 integrin receptors, stimulate migration, proliferation and metastasis in PDAC . Interestingly, ADAMTS2, another identified miR-29a downregulated target, primarily functions to process collagens I, II, III and V precursors into mature molecules [26, 27]. The gene promotes fibrosis via activation of TGF-β signaling . Evidently, miR-29a plays an anti-fibrotic role in PDAC by influencing ECM deposition via modulation of multiple targets in the collagen pathway. In addition to these genes that directly regulate tumor-microenvironment and desmoplasia, the top targets identified from our dataset consisted of the two additional genes E2F7 and MYBL2, which play essential roles in cell cycle regulation. E2F7 associate with poor patient outcome in several types of cancer including PDAC [64–66] and has been shown essential for mouse embryonic survival . Inhibition of E2F7 enhanced G1 phase percentage in prostate cancer reducing cellular proliferation . Similarly, MYBL2 is a transcription factor which promotes cell proliferation and differentiation by fostering cell cycle entry into S and M phases, and is dysregulated in types of cancer [38, 68, 69]. A recent study demonstrated the regulatory role of MYBL2 in promoting PDAC desmoplasia and PSCs’ growth through sonic hedgehog and adrenomedullin via paracrine and autocrine signaling , however the role of the gene in PSCs has not been reported. A negative feedback regulatory mechanism between miR-29a and MYBL2 influencing the activation of PSCs is possible, but this requires future validation. Nonetheless, the identified set of miR-29a target genes exhibit a pro-fibrotic and tumorigenic function in PDAC desmoplasia and progression via multiple targeted pathways, although PSC-specific function of some of the identified target genes such as E2F7, CLDN1, MYBL2 and ADAMTS2 has not been studied previously. Together, the observations in the current study signify that overexpression of miR-29a may lead to inhibition of PSC-induced pro-fibrotic and desmoplastic effects by targeting these genes to impair signaling such as sonic hedgehog, IGF, RAS/MAPK, collagen metabolism and laminin pathways to perturb their normal cellular responses promoting PDAC progression.
As mentioned above, IGF-1 signaling axis is a key mechanism in promoting PDAC tumor-stromal crosstalk and drug resistance. In our RNAseq dataset, we observed the most robust downregulation of the IGF-1 gene among all miR-29a targets. It is possible that in addition to IGF-1 alone, miR-29a regulates IGF-signaling via modulation multiple components of its pathway in PSCs, such as indirect regulation of genes including IGF-1R, INSR and direct targeting of some others. It is worthy to note that MYBL2 and E2F7 are miR-29a targets that are at the functional convergence of p53-IGF-1 pathways. Stromal p53 has been implicated as a key component that reprograms activated pancreatic and hepatic stellate cells to transform them into quiescence states [71, 72]. Depletion of p53 in stromal cells caused faster and more aggressive tumor development with enhanced invasion and metastasis of cancer cells, suggesting a paracrine mechanism of p53 in tumor progression [73, 74]. In addition, studies have reported inactivating p53 mutations in fibroblastic stromal cells and their association in promoting tumor progression and cancer cell metastasis in types of carcinogenesis , although the molecular mechanisms are still unclear. MYBL2 is a downstream effector of the p53 pathway . With p53 mutations, MYBL2 repression is uncoupled allowing enhanced binding of the molecule with MuvB and FOXM1 leading to activation of mitotic genes [68, 75]. FOXM1 is an essential component of Akt signaling, which functions both in the context of tumor stroma and cancer cells to promote tumorigenesis [76–79]. Interestingly, Akt pathway is under inverse regulation of IGF-1 signaling [78, 80, 81]. Similarly, E2F7 is a crucial transcription factor, which promotes E2F1-p53 dependent apoptosis and cell-cycle arrest [67, 82]. In our RNAseq data with miR-29a overexpressing PSCs, we found E2F1 to be one of the indirect downregulated targets. In addition, E2F7 has also been shown to be activated by Akt signaling in carcinomas [82–84]. Although the exact mechanisms of MYBL2 and E2F7 in PSCs is still to be understood, our results suggest that dysregulation of miR-29a in PSCs derepresses genes such as IGF-1, MYBL2 and E2F7, which may in turn disrupt stromal p53 regulation promoting PSC-mediated tumor proliferation.
GO analysis showed that the direct and indirect miR-29a downregulated targets were enriched in crucial cellular and molecular functions associated with PDAC stromal remodeling and proliferation. The biological processes consisted of those related to cell cycle regulation, collagen formation, ECM organization and immune signaling (Table 2). Our study further identified inter-connected networks comprising of essential pathways in PDAC stromal regulation and desmoplasia (Table 3). Although a single miRNA is known to target hundreds of genes to result in their post- transcriptional repression, based on the functional network of the differentially expressed targets, the predominant phenotypic effect of a miRNA can be systematically analyzed in a context-specific manner. Our analysis using PSCs identifies miR-29a target genes that are some of the crucial players of PDAC stromal remodeling and tumor-stromal crosstalk, suggesting the importance of the molecule in their pathway regulations to modulate PDAC microenvironment and tumor progression.