In this study, we have shown that systemic delivery of TSG-miRs using a nanovector delivery platform can inhibit the growth of pancreatic cancer xenografts in both subcutaneous and orthotopic milieus. Most molecularly targeted therapeutic approaches are directed towards blocking aberrant hyperfunction of oncogenic components; however, the current miRNA delivery strategy is geared towards regaining function lost specifically in cancer cells.
We selected two miRNA candidates for systemic delivery in pancreatic cancer models: miR-34a and the miR-143/145 cluster. As data from our group and others have established (8
), these miRs are downregulated (or completely absent) in the majority of pancreatic cancers. Functionally, miR-34a is a component of the p53 transcriptional network and its loss in cancer cells is associated with resistance to apoptosis induced by p53 activating agents (27
). In cancer cells with wild type p53, ectopic expression of miR-34a levels can restore p53 function by repressing the deacetylase SIRT1, thereby enhancing levels of active (acetylated) p53 (42
). The repertoire of miR-34a targets are expectedly quite diverse, and include molecules involved in promoting cellular proliferation (cyclin D1, cyclin-dependent kinases CDK4 and 6) and blocking apoptosis (Bcl-2) (43
). Thus, restoring miR-34a function in cancer cells is expected to have both pro-apoptotic and anti-proliferative effects, as was observed in the miR-34a nanovector-treated xenografts by TUNEL and Ki-67 labeling, respectively. Recently, miR-34a has also been implicated in regulating the number and function of tumor-initiating cells (i.e. "cancer stem cells" or CSCs) in solid cancers (25
). Specifically, studies in pancreatic and prostate cancer models have demonstrated that CSCs in these cancer types harbor low miR-34a levels, while re-expression of this miRNA significantly decreases CSC clonogenicity and survival, and tumor engraftment capacity in vivo
. In pancreatic cancer, these profound deleterious effects on CSCs are observed irrespective of p53 functional status (32
), underscoring the applicability of therapeutic miR-34a restitution to a disease that harbors TP53
mutations in approximately 70% of cases (2
). Indeed, using two credentialed surrogate measures of pancreatic cancer CSCs, ALDH
), we demonstrated a significant downregulation in expression of both transcripts in miR-34a nanovector-treated xenografts. Finally, given the widespread loss of expression of miR-34a in many other solid cancers (39
), there is also compelling rationale to test the efficacy of the nanovector platform in corresponding preclinical disease models.
The second candidate we tested using nanovector delivery is a cluster of two co-transcribed miRNAs, miR-143/145, whose expression is also frequently lost in many solid and hematological malignancies, including colorectal cancers where a consistent downregulation was first identified (47
). Our recent work has identified the existence of a feed-forward loop in pancreatic cancer cells, wherein the Ras effector protein RREB1 directly represses the expression of miR-143/145, thereby relieving the miRNA-mediated repression of KRAS2
). Not unexpectedly, the robust tumor growth inhibition observed in vivo
with miR-143/145 nanovector therapy is accompanied by significant downregulation in KRAS2
transcripts, as well as decreased RREB1 protein expression by immunohistochemistry. The seminal importance of the KRAS2
oncogene to pancreatic cancer cannot be overstated - somatic activating mutations are found in greater than 90% of cases, and Ras is implicated in both tumor initiation and tumor maintenance (1
). Nonetheless, pharmacological blockade of this small GTPase protein has been challenging, as small molecule inhibitors of Ras farnesylation have failed to improve median survival in clinical trials (49
). The miR-143/145 nanovector represents a tangible genetic approach towards direct inhibition of KRAS2
in pancreatic cancer, and future studies in the Ras-driven genetically engineered models of pancreatic cancer (50
) will provide additional insights on the therapeutic potential of this modality in an autochthonous setting.
We performed two independent experiments using TSG-miRs (i.e. "miRNA monotherapy"), each of which demonstrated significant and comparable tumor growth inhibition in vivo. We are currently developing delivery methods for concurrent restitution of two or more diverse TSG miRs targeting non-overlapping coding genes (i.e., "miRNA combination therapy"), with the intent of achieving therapeutic synergy. This approach is based on either the concurrent administration of two independent nanovectors (for example, miR-34a or miR-143/145 nanovectors illustrated in this study), or the generation of a single nanovector capable of delivering dual therapeutic cargo (such as a bi-cistronic vector expressing two miRNAs simultaneously). Additionally, combination therapy with other traditional chemotherapy agents (e.g., gemcitabine) may yield improved effects in pancreatic cancer. The lack of demonstrable adverse effects at the histological or biochemical level is encouraging and likely because saturation of endogenous levels of miR-34a and miR-143/145 are already achieved in normal cells. Thus, to conclude, the nanovector platform we have designed can be used for systemic delivery of TSG miRNAs to cancer cells. Although the proof-of-principle studies presented here utilize pancreatic cancer as a disease model, it is conceivable that this approach will be broadly applicable across other tumor types, delivering potentially any TSG miR that is a candidate for restitution in cancer cells.