| dc.description.abstracteng | Pancreatic cancer is a dismal malignancy with a 5-year survival rate of 7-9%, one of the worst among all cancer types. Patients with pancreatic ductal adenocarcinoma (PDAC) usually present an advanced stage of the disease upon diagnosis and often develop chemotherapy resistance. As the disease progresses, patients are commonly administered a gemcitabine-based therapy, which is known for its clinical benefits, but also low response and concomitant high resistance rates. For this reason, the mechanism driving gemcitabine resistance has been extensively studied in pancreatic cancer. In fact, several gemcitabine metabolizing enzymes have been identified as prognostic, correlating with gemcitabine response rates in patient biopsies. Still, the molecular consequences of gemcitabine resistance in tumors remain elusive. Chemotherapeutic agents are known to not only act on their targets, but to also elicit stress and therefore trigger stress-induced apoptosis. Thus, it is plausible that chemotherapy resistance is not only mediated by a bypass of the pathway directly targeted by the chemotherapeutic agent, but also by an altered response to stress cues.
In this study we investigated the molecular consequences of gemcitabine resistance in PDAC tumors. For this, a gemcitabine resistant cell line was established by treating treatment-naïve PDAC cells with increasing concentrations of gemcitabine. By studying the genomic, epigenomic and transcriptomic changes associated with acquired gemcitabine resistance, we identified a main driver of gemcitabine resistance and unraveled a novel mechanism employed by these tumors to overcome stress and activate alternative pathways. Copy number variation analyses revealed an amplification of a segment of chromosome 11, which included genes previously associated with gemcitabine resistance, such as Ribonucleotide Reductase Catalytic Subunit M1 (RRM1) as well as other genes, like Stromal Interaction Molecule 1 (STIM1). RRM1 is a known target of gemcitabine and proliferation studies confirmed that its amplification and upregulation drove gemcitabine resistance in our system.
In order to elucidate further molecular mechanisms affected by acquired gemcitabine resistance, an epigenetic profiling of the cells was traced. This led to the identification of a dampened ER stress response in gemcitabine resistant compared to parental cells. Gemcitabine resistant cells failed to activate stress responsive transcription factors, such as Activating Transcription Factor 4 (ATF4), while also displaying a drop in active transcription histone marks around ATF4 binding sites and target genes. Interestingly, the stress response is tightly coupled to calcium signaling and an important ER calcium sensor, STIM1, was identified to be co-amplified with RRM1 in gemcitabine resistant cells. In fact, the co-amplification of the neighboring genes, RRM1 and STIM1, was shown to have a high co-occurrence rate in different treatment naïve cancer cell lines as well as several primary tumors, suggesting it may also spontaneously occur in tumors.
STIM1 is an ER calcium sensor, which upon ER calcium depletion interacts with the calcium channel ORAI calcium release-activated calcium modulator 1 (ORAI1). This stimulates the influx of calcium from the extracellular matrix into the cytosol, a process referred to as Store Operated Calcium Entry (SOCE). Interestingly, calcium measurements revealed that STIM1-amplifying cells displayed an increased SOCE, which in turn led to a dampened ER stress response. Moreover, this increase in SOCE elicited an aberrant activation of the Nuclear Factor of Activated T cells (NFAT) family of transcription factors. Finally, analysis of primary tumors as well as treatment-naïve and gemcitabine treated Patient-Derived Xenografts (PDXs) corroborated our findings in vivo.
Taken together, our study characterizes molecular mechanisms driving gemcitabine resistance in PDAC and unravels the role of calcium signaling in these tumors. While the amplification of RRM1 drove gemcitabine resistance, the upregulation of STIM1 elicited a heightened SOCE leading to ER stress resistance and aberrant NFAT activation. Thus, STIM1 was identified as a rheostat balancing between ER stress-responsive and NFAT-driven epigenetic programs upon stress. Finally, we propose STIM1 as a novel therapeutic target for the treatment of gemcitabine resistant as well as STIM1-overexpressing tumors. | de |