Pancreatic cancer (PC) survival rates are dismally on track to make it the second most lethal malignancy by the end of the decade. Over half of all patients are diagnosed at stage 4 disease limiting the availability and success of therapeutic intervention. In PC, presentation with advanced disease is theresult of both asymptomatic disease initiation and early metastasis. While surgical resection currently yields the best outcome, less than one quarter of patients are eligible at presentation, frequently limited by appreciable metastasis or vascular involvement. Compounding the issue of late stage diagnosis is the innate therapeutic resistance observed in PC tumors, either directly or resulting from reduced treatment delivery to tumors. PC tumors demonstrate a range of aberrant findings, including a high rate of KRAS mutations, CDKN2A deletion, and anomalous early expression of mucins. Mucins are large glycoproteins that have been demonstrated to significantly contribute to disease progression, metastasis, and drug resistance. A number of alternatively spliced mucin species have been reported, but their role in PC pathology remains elusive. Using RNA-seq data from TCGA, I discovered that select MUC1, MUC4, and MUC16 transcripts are significantly associated with survival outcome in PC patients. Of these 5 significant transcripts, a single MUC4 isoform (MUC4?6) contains an in-frame deletion of exon 6 and has previously been found by our lab in patient tumors. Exon 6 in MUC4 corresponds to the NIDO domain and may alter interactions between the expressing cell and tumor stroma. Because of the high rate of expression, I hypothesize that this transcript serves as a prognostic molecule, detectable in patient biofluids, and contributes to metastatic behavior of PC cells. To facilitate addressing this hypothesis, I propose two independent specific aims.
Aim 1 will employ a custom multiplexed assay for the detection of PC-specific RNA transcripts using gold nanoparticle-bound fluorescent oligonucleotide probes. We will demonstrate that this assay can detect the presence MUC4?6, KRAS mutant, and CDKN2A transcripts in patient plasma with high sensitivity. Detection and quantification of target RNA transcripts using our nanoprobe-based approach requires perfect hybridization of both probe and RNA followed by subsequent enzymatic cleavage. The ultility in this novel assay technology is that it can detect down to single nucleotide mutations, permits quantification, and requires a minimal amount of blood for testing. This will be validated in orthotopic animals and PC cell lines.
Aim 2 involves the generation of MUC4?6-sparing knockdown of MUC4 using constitutively expression of shRNA in PC cell lines. These cell lines will be used for in vitro functional studies of MUC4?6 to elucidate its role in tumor cell mobility, stromal interactions, drug resistance, and proliferative effects. Subsequently, these features will be tested in vivo using orthotopic implantation in a mouse model. At the conclusion of my study, I expect to conclusively demonstrate that MUC4?6 plays a unique role in PC biology and serves as an important diagnostic/prognostic molecule detectable in PC patient blood plasma.
Expression of mucins in pancreatic cancer (PC) and their association with PC pathology have been clearly demonstrated; however, the role of alternatively spliced species have not been thoroughly documented. We propose the use of a novel technology to detect mucin splice variants with significant correlation to survival outcome in pancreatic cancer patients from blood plasma. Further, we propose extending this technology to screen for the presence of disease-associated mutant gene messenger RNA from biofluid samples. Based on in silico analysis, this proposal will also explore the functional contribution of MUC4?6 isoform to PC disease pathology.
