Applying Small Molecule Signal Transducer and Activator of Transcription-3 (STAT3) Protein Inhibitors as Pancreatic Cancer Therapeutics

Abstract

Constitutively activated Signal Transducer and Activator of Transcription 3 (STAT3) protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. In this study PG-S3-001, a small molecule derived from the SH-4-54 class of STAT3 inhibitors, was found to inhibit patient-derived pancreatic cancer cell proliferation in vitro and in vivo in the low μM range. PG-S3-001 binds the STAT3 protein potently, Kd = 324 nM by SPR, showed no effect in a kinome screen (> 100 cancer-relevant kinases). In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in pancreatic cancer cells. To better model the tumor and its microenvironment, we utilized 3-Dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAFs). In this co-culture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. Confocal microscopy was used to verify tumor cell death following treatment of 3D co-cultures with PG-S3-001. The 3D model was predictive of in vivo efficacy as significant tumor growth inhibition was observed upon administration of PG-S3-001. These studies showed that the inhibition of STAT3 was able to impact the survival of tumor cells in a relevant 3D model, as well as in a xenograft model using patient-derived cells.

Regulation of HIF1α under Hypoxia by APE1/Ref-1 Impacts CA9 Expression: Dual Targeting in Patient-Derived 3D Pancreatic Cancer Models

“…The cooperative activities of STAT3 and HIF-1 have been demonstrated in a variety of cancers (56, 57); however the finding that APE1/Ref-1 binding to STAT3 is stimulated by exposure to hypoxia in PDAC cells, presented here for the first time, further indicates the importance of both APE1/Ref-1 and STAT3 as potential therapeutic targets in PDAC. These findings will be further pursued with preclinical STAT3 inhibitors that are being developed for eventual clinical trials (42). Furthermore, our results demonstrating that APX3330 treatment decreases hypoxia-induced HIF-1 transcriptional activity and CA9 mRNA levels (21) is exciting since CA9 inhibitors are either entering or are in clinical trials.…”

“…Ultra low attachment 96-well plates (Corning Inc., Life Sciences) were used to generate 3-dimensional tumor spheroids in the presence and absence of CAFs (75 μL/well) as described previously (41, 42). Cells were stably transduced with EGFP (green) or TdTomato (red) as indicated to preserve the genetic characteristics of the low passage patient cells (34, 42).…”

“…Cells were stably transduced with EGFP (green) or TdTomato (red) as indicated to preserve the genetic characteristics of the low passage patient cells (34, 42). Cells were re-suspended in colorless DMEM growth media containing 3% Reduced Growth Factor Matrigel (RGF, BD Biosciences) and 5% FBS.…”

Identification and Characterization of New Chemical Entities Targeting Apurinic/Apyrimidinic Endonuclease 1 for the Prevention of Chemotherapy-Induced Peripheral Neuropathy

“…All cell lines were authenticated via short tandem repeat analysis (IDEXX BioResearch) and checked routinely for mycoplasma contamination. Ultra-low attachment 96-well plates (Corning Life Sciences, Corning, NY) were used to generate threedimensional tumor spheroids in the presence and absence of CAFs as described previously (Sempere et al, 2011;Arpin et al, 2016). TdTomato-labeled pancreatic ductal adenocarcinoma (PDAC) cells and enhanced green fluorescent protein-labeled CAFs were resuspended in colorless Dulbecco's modified Eagle's medium containing 3% growth factor reduced Matrigel (BD Biosciences, San Jose, CA) and 5% fetal bovine serum at a cell ratio of 1:4 (tumor/CAF) and were fed on days 4 and 8 after plating.…”

“…Although the neuroprotective effects of APX2009 are evident, we also wanted to investigate whether these E3330 analogs were capable of tumor cell killing similar to what we have observed with E3330 (Vasko et al, 2011;Kelley et al, 2014;Kim et al, 2015). A three-dimensional coculture model of pancreatic cancer established in our laboratories was used as an ex vivo system that included both low-passage, patient-derived tumor cells and cancer-associated fibroblasts (Arpin et al, 2016). We assessed the effects of APX2009-induced cytotoxicity on the area and intensity of tumor cells both alone and in coculture with CAFs.…”