The Sustained Induction of c-MYC Drives Nab-Paclitaxel Resistance in Primary Pancreatic Ductal Carcinoma Cells

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with limited and very often, ineffective medical and surgical therapeutic options. The treatment of patients with advanced unresectable PDAC is restricted to systemic chemotherapy, a therapeutic intervention to which most eventually develop resistance. Recently, nab-paclitaxel has been added to the arsenal of first line therapies, and the combination of gemcitabine and nab-paclitaxel has modestly prolonged median overall survival. However, patients almost invariably succumb to the disease, and little is known about the mechanisms underlying nab-paclitaxel (n-PTX) resistance. Using the conditionally reprogrammed (CR) cell approach, we established and verified continuously growing cell cultures from treatment-naive PDAC patients. To study the mechanisms of primary drug resistance, nab-paclitaxel-resistant (n-PTX-R) cells were generated from primary cultures and drug resistance was verified in vivo, both in zebrafish and in athymic nude mouse xenograft models. Molecular analyses identified the sustained induction of c-MYC in the nab-paclitaxelresistant cells. Depletion of c-Myc restored nab-paclitaxel sensitivity, as did treatment with either the MEK inhibitor, trametinib, or a small molecule activator of protein phosphatase 2a (SMAP). Implications: The strategies we have devised, including the patient-derived primary cells and the unique, drug resistant isogenic cells, are rapid and easily applied in vitro and in vivo platforms to better understand the mechanisms of drug resistance and for defining effective therapeutic options on a patient by patient basis.

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“…Similarly, the successes achieved with our CR approach to treat lethal diseases is evident by our earlier studies. 37 Our recent studies have further established that the CR culture conditions support long term genomic stability and can validate drug response in vivo and in vitro 18 and, therefore, may be vital resources for personalized drug identification.…”

“…6,8 Briefly, each drug's Z-score (Zi), which represents the final quantification of the drug-PCa patient sample association, was calculated for a ranking using the following equation were used, as previously described. 14,18 3 | RESULTS Figure 1A).…”

“…Following detailed pathological analyses that documented that the tissue sections collected were more than 70% tumor cells, the specimens were processed via protease dissociation as previously described. 11,18 Primary cultures were established at Georgetown University using the CR method, and drug senstivity experiments were performed in conditioned media (CM) as previously described. 19 Genetic profiles, obtained by performing metaphase spreads for the normal and tumor-derived CR cells were performed.…”

“…Chromosomal analyses revealed that both of the tumor-derived CR cells remained diploid, however, increased incidences of chromosomal aberrations were observed in the tumor-derived cells from patient 2, which exhibited numerous chromosomal translocations and markers 14. Gene transcription microarrays were performed by Illumina bead array on the matched normal and tumor cells cultured in CM as previously described 14,18,19. The data were analyzed by IPA based on differential gene expression between the normal CR cells and the matched tumor-derived CR cells as described 14,18.…”

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“…Morphologically normal cells upregulated 7 out of 8 proteins in the chaperonin containing TCP-1 complex (adjusted p value = 7.64e-15; Figure 4E), which is critical for tubulin folding and has been previously associated with paclitaxel resistance in ovarian cancer 39 . Morphologically normal cells also upregulated the transcriptional targets of two pacltiaxel resistance-associated signaling pathways 42,43 : c-Myc (adjusted p value = 1.66e-30) and mTORC1 (adjusted p value = 6.19e-17; Figure 4F). Together, these results suggest that the morphologically normal, paclitaxel-treated cells mounted a biosynthetic and proteostatic response to drug treatment, with remarkable similarities to the gene expression profiles observed in paclitaxel resistant cell lines and cancers.…”