| dc.description.abstracteng | Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival
rate of less than 8 %. Hallmarks of pancreatic cancer are extensive desmoplasia and strong
resistance to standard chemotherapeutic agents, e.g. gemcitabine. In this context, impaired
drug delivery and drug metabolism pathways might play a crucial role in mediating this
pronounced chemoresistance.
In this study, I investigated tumor cell intrinsic and extrinsic mechanisms of
chemotherapeutic resistance in PDAC.
Pharmacokinetic characteristics of gemcitabine (dFdC) were analyzed in the widely used LSL-Kras<sup>G12D/+</sup>;LSL-Trp53<sup>R172H/+</sup>;Pdx-1-Cre
(KPC) mouse model by liquid chromatography
tandem mass-spectrometry (LC-MS/MS). Surprisingly, the levels of gemcitabine were
elevated in the primary, stroma-rich and hypovascular tumor samples compared with
matched normal liver samples and samples from liver metastases. A more detailed analysis
by our group revealed an increased ratio of cancer-associated fibroblasts (CAFs) in primary
tumors compared to liver metastases. Notably, gemcitabine metabolizing enzymes were
highly expressed in epithelial but not stromal cells. The inactivating enzymes cytidine
deaminase (CDA), deoxycytidylate deaminase (DCTD), and cytosolic 5’-nucleotidase 1A
(NT5C1A) were hardly expressed in the stromal compartment of murine and human PDAC
samples in vivo. In contrast, these enzymes were robustly expressed in the epithelial
compartment. Consequently, the cytotoxic gemcitabine metabolite dFdCTP accumulated in
murine CAFs, as the phosphorylated metabolites are unable to cross the cell membrane.
Drug scavenging of CAFs was confirmed in conditioned medium (CM) assays. Incubation
of tumor cells with CM of gemcitabine treated CAFs reduced the available amount of
gemcitabine for tumor cells in vitro. Due to the low proliferation rate of CAFs in vivo, these
cells might have exhibited intrinsic resistance to the increased amounts of dFdCTP.
Further experiments were aimed to characterize NT5C1A, a previously unrecognized
gemcitabine inactivating enzyme in pancreatic cancer that reverses the initial
phosphorylation step of gemcitabine. Immunohistochemical staining of tissue microarrays
(TMAs) with more than 400 tumor samples, from two independent cohorts of resected
PDAC patients, were used to study the expression pattern of NT5C1A in PDAC. We found
robust protein expression in the epithelial compartment of 64-70 % of PDAC patients,
whereas robust stromal expression of NT5C1A was detectable in less than 20 % of these
patients. A prognostic role of NT5C1A was not observed in both patient cohorts. Recombinant expression of this enzyme was used to elucidate its impact on
chemotherapeutic resistance. Re-expression of NT5C1A in pancreatic stellate cells (PSCs)
reduced the intracellular levels of the active gemcitabine metabolite dFdCTP, suggesting
NT5C1A as novel target for stromal reprogramming.
Gemcitabine response in tumor cells overexpressing NT5C1A was investigated using
standard biochemical assays and orthotopic transplantation of the modified tumor cells into
mice. Indeed, cells overexpressing NT5C1A showed higher resistance towards gemcitabine
and had decreased levels of intracellular dFdCTP and of cleaved caspase 3 (CC3) levels
following treatment with gemcitabine. Tumor weights were increased in mice that were
transplanted with NT5C1A expressing cells compared to control cells upon gemcitabine
treatment, showing the relevance of this enzyme in therapeutic effectiveness. Given its role
in dephosphorylation of nucleoside monophosphates, NT5C1A overexpression in
pancreatic cancer cells did not reduce chemosensitivity towards paclitaxel, a standard
chemotherapeutic agent that acts independently of intracellular phosphorylation.
In conclusion, our study gave new insight into the impact of drug metabolizing enzymes on
chemotherapeutic resistance in PDAC. We demonstrated that alterations in drug
metabolism and not impaired drug delivery mainly determine the response to gemcitabine
in PDAC. Our results further demonstrated NT5C1A as target for stromal reprogramming.
Most importantly, our findings pave the way for a more detailed stratification of patients for
treatments and suggest NT5C1A to be considered as a possible predictor of treatment
response to gemcitabine in PDAC patients. | de |