M.W. Spinrad1, C. Cai1, L. Gattie1, E. Glazer1 1Univeristy Of Tennessee Health Science Center, Memphis, TN, USA
Introduction:
Pancreatic Adenocarcinoma (PDAC) is one of deadliest cancers, with one of the worst five-year relative survival rates, at only 12%. Incidence is increasing and PDAC is projected to become the second leading cause of cancer related deaths within the next decade. There is an urgent need to develop new and effective therapies. Microtubule inhibition is a promising therapeutic target as microtubule dynamics play a critical role in cell growth and division. In PDAC, several β-tubulin subtypes are highly upregulated compared to normal pancreas cells, specifically βIII- and βIVb-tubulin subtypes. Previous studies have shown that decreased expression of βIII- and βIVb-tubulin decreases PDAC cell growth. Another potential therapeutic target is Bromodomain and extra-terminal domain (BET) proteins, transcription factors involved in multiple cell processes. Our lab has previously shown that inhibition of BET proteins can inhibit mitochondrial function and prevent proliferation. In this study, we hypothesize that SB-216, a novel colchicine binding site β-tubulin inhibitor, which has also been shown to bind to the BD2 domain of BRD4, a BET protein, will inhibit cell growth and reduce expression of βIII- and βIVb-tubulin subtypes.
Methods:
Cell Growth was analyzed on the IncuCyte Live-Cell Analysis system for 5 days. PDCL110, a PDAC patient-derived cell line, and Panc-1 a commercially available PDAC cell line were incubated with .1% DMSO (Control) 2nM, 5nM, 10 nM, and 15 nM concentrations of SB-216. mRNA expression of βIII- and βIVb-tubulin was evaluated with quantitative real time PCR with samples of PDCL110 and PANC1 treated with 5nM of SB-216 for 48 hours. Western blot analysis was performed for βIII- and βIVb-tubulin, as well as for autophagy and mitophagy markers LC3B, and p62/SQSTM1,
Results:
Cell growth was greatly inhibited across all doses in both cell lines (p < .0001). mRNA expression of TUBB3 (βIII subtypes) and TUBB4 (βIVb subtypes) fold change in expression was significantly decreased (p < .05) in both cell lines. Western blot analysis of TUBB3 proteins demonstrated reduced protein quantification in Panc-1 cells (p < .05). Western blot analysis also showed decreased levels of LC3B in Panc-1 cells treated with SB-216.
Conclusion:
Our preliminary data demonstrates that SB-216 inhibits cell growth and expression of oncogenic β-tubulin subtypes in commercial and patient derived PDAC cell clines, and may impact key cellular functions such as autophagy. Further studies will elucidate the effect of SB-216 on murine and organoid models, as well as characterize the effect of SB-216 on mitochondrial function in PDAC cells with mitochondrial function assays.