Lin, Qingxiang

Abstract Pancreatic cancer (PaCA) is highly refractory to treatment, with a median survival of 6 months. Currently, gemcitabine (Gem), a difluoro analog of deoxycytidine, is the standard care for PaCA, but it renders only a modest survival benefit. Combination therapy, integrating novel agents into standard-of-care regimens, may maximize efficacy of Gem-based therapy. Fibroblast growth factor receptor (FGFR) function is aberrant in pancreatic cancer, and suppression of FGFRs inhibits pancreatic cancer growth and invasion. However, the mechanisms of FGFR action and their therapeutic utility in pancreatic cancer remain to be elucidated. Here we tested the hypothesis that FGFRs can impact Gem sensitivity of PaCA cells by employing pan-FGFR inhibitors and shRNAs against FGFRs. NVP-BGJ398 and AZD4547 are novel FGFR inhibitors (FGFRIs) specific to FGFR1, FGFR2 and FGFR3, and are in various stages of clinical trial. The FGFRIs modestly but significantly inhibited proliferation, migration, and invasion of PANC-1, MIAPaCa-2 cells in wound-healing and trans-well assays. Treatment with low-concentration Gem (7.5nM) enhanced MIAPaCa-2 cell migration at 24hrs, but migration was reduced subsequently, suggesting a sequence of delayed effects of Gem upon cell migration. After 72hrs of exposure, Gem combined with FGFRIs reduced MIAPaCa-2 cell proliferation significantly compared to controls or Gem- and FGFRI alone. Pharmacodynamic modeling was employed to evaluate drug interaction, and the interaction coefficient Psi was estimated by simultaneous modeling of 16 different drug combinations over a period of 0-96 hrs. The analysis estimated Psi=0.78, which indicates synergism. Compared to BGJ398 or Gem alone, the combination also increased the percentage of cells arrested at S phase for a longer period of time. Western blots revealed significant downregulation of ribonucleoside-diphosphate reductase large subunit (RRM1), DNA excision repair protein (ERCC1), and Epithelial-to-Mesenchymal transition biomarkers (vimentin and snail) with FGFRI/Gem treatment compared to Gem-treated cells, indicating that FGFRIs inhibit numerous factors that may contribute to Gem resistance. Loss-of-function by shRNA-mediated knockdown of FGFR1 in MIAPaCa-2 cells showed decreased expression of RRM1, whereas knockdown of FGFR2 and FGFR3 mediated an increase in RRM1 expression, implicating FGFR1 suppression in improving Gem-sensitivity. These results suggest that inhibition of FGFR1 could potentially enhance sensitivity of PaCA to Gem therapy, and that combining FGFRs with gemcitabine is a promising treatment strategy in pancreatic cancer. Citation Format: Qingxiang Lin, Tista Roy Chaudhuri, Ninfa L. Straubinger, Wen Wee Ma, Robert M. Straubinger. FGFR inhibitors enhance gemcitabine sensitivity in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4072. doi:10.1158/1538-7445.AM2017-4072

Abstract The majority of Pancreatic cancer (PaCA) patients present with metastatic disease and receive little benefit from chemotherapy. Although nanoparticulate carriers can potentially deposit sustained and high doses of chemotherapeutics in tumors, PaCA tumors are poorly perfused, which limits drug deposition. Ten days of oral dosing with NVP-LDE225 (40 mg/kg), an inhibitor of the sonic hedgehog (SHH) pathway (sHHI), increased tumor deposition of blood-borne, fluorescently labeled, sterically-stabilized lipidic (SSL) nanoparticles by enhancing the perfusion and permeability of the otherwise-impermeable microvasculature of PaCA patient-derived xenograft (PDX) tumors. However, literature indicates sHH ablation is associated with increased Epithelial-to-Mesenchymal (EMT) transition and invasiveness in PaCA. Here we investigated the compensatory role of FGFR1 in sHHI-induced tumor progression. Four to 8 daily treatments with the sHHI resulted in no significant reduction of tumor volume, but markedly increased the ratio of tumor cells (human mitochondria+) to Collagen1 in the stroma, in two s.c. low-passage PDX models of varying stromal content. Immunostaining of sHHI-treated tumors revealed no alteration in total FGFR1 expression, but showed a significant elevation in the (1) percent of FGFR1+ pixels correlating with DAPI+ nuclei in tumor (human mitochondria+) cells and stromal cells, (2) number of vimentin+ (EMT) cells, and (3) proliferative index marked by Ki67+ nuclei, compared to controls, suggesting accelerated tumor progression. Nuclear FGFR1 was associated with vimentin+ cells, but was excluded from the nuclei of Ki67+ cells in all groups, implicating nuclear translocation of FGFR1 in EMT. NVP-BGJ398, a small-molecule pan-FGFR inhibitor (FGFRI), administered p.o. for 3 days (15 mg/kg/day) after 7d of sHHI-pretreatment (1) significantly reduced nuclear localization of FGFR1 compared to sHHI-treated animals, (2) reversed the ratio of human mitochondria+ tumor cells to Collagen 1+ stroma, (3) depleted vimentin+ structures by >2 fold, and (4) reduced Ki67+ cells to control levels. FGFRI without sHHI-pretreatment resulted in decreased nuclear FGFR1 relative to controls, but did not impact proliferation, EMT, or tumor cell-to-stroma ratio. Finally, the sHHI/FGFRI sequence increased functional (FITC-L. esculentum lectin+) tumor vessel density and area the perfused by vascular permeability probes (fluorescent SSL) compared to both controls and sHHI-treated animals. FGFRI administration in vehicle-treated controls had no impact on vascular permeability or perfusion. We conclude that the combination of sHHI in sequence with FGFRI may reverse potentially deleterious effects of sHHI and mediate a transient enhancement of tumor permeability, and increase deposition and penetration of nanoparticulate SSL drug formulations into pancreatic cancers. Citation Format: Tista Roy Chaudhuri, Qingxiang Lin, Ninfa L. Straubinger, Wen Wee Ma, Robert M. Straubinger. FGFR1 inhibitor reverses Smoothened inhibition-induced epithelial-to-mesenchymal transition in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4071. doi:10.1158/1538-7445.AM2017-4071