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Src Kinase Inhibition Reverses P27/kip1 Mislocalization And Reduces Growth Of Pre-neoplastic Barrett’S Esophagus Cell Lines.
Aaron J. Fowler1, Valerie M. Felton1, Amanda L. Richer1, Kimberly M. Paquette2, Nhan L Tran2, *Ross M. Bremner1, Landon J Inge1
1Heart and Lung Inst., St. Joseph's Hospital and Medical Center, Phoenix, AZ;2Translation Genomics Research Institute, Phoenix, AZ
Local ablation (RFA or Cryo), or esophagectomy, are currently the only therapies available for the treatment of high-grade dysplasia (HGD) in Barrett's esophagus (BE). There are no chemopreventative strategies to offer BE patients with HGD in lieu of more invasive procedures. Our understanding of the molecular changes that occur in HGD offer an opportunity to inhibit the progression of HGD to esophageal adenocarcinoma (EAC).
The tumor suppressor, p27-kip1 functions to regulate cell growth and is commonly deregulated EAC and BE. Deregulation of p27-kip1 occurs through post-translational modification, specifically protein phosphorylation, which induces nuclear export to the cytoplasm, inhibiting its regulatory function. Functionally, targeted inactivation of p27-kip1 by phosphorylation is due to several protein kinases, especially the Src family. Src kinases promote nuclear export of p27-kip1 and Src displays increased activity in HGD-BE. We hypothesize that targeted inhibition of Src may restore p27-kip1’s regulatory function and thus may serve as a potential chemopreventative modality for BE patients with HGD.
To test this hypothesis, we examined the effects of a well-tolerated and FDA approved Src kinase inhibitor, dasatinib upon growth and localization of p27-kip1 in immortalized dysplastic (CP-D) and metaplastic (CP-A) BE cell lines. Localization of p27-kip1 was analyzed using confocal microscopy, while growth of BE cell lines was assessed via CellTiter-Glo following treatment with dasatinib. Dasatinib effects upon mRNA levels of the proliferative markers Cyclin D1 and E1 were also evaluated. Immunoblot analysis of protein markers was also performed upon treated BE cell lines.
Confocal analysis of CP-A and CP-D BE cells revealed cytoplasmic localization of p27-kip1, which relocalized to the nucleus with dasatinib treatment (Figure 1A). Dasatinib treatment reduced cell viability in BE cell lines (Figure 2B), which correlated with reduced mRNA levels of both Cyclins D1 and E1. At lower doses of dasatinib, dysplastic BE cells were more sensitive to the treatment compared to metaplastic BE cells (Figure 2B). p27 mRNA and protein levels were unaffected by dasatinib treatment, however dasatinib treatment resulted in cleavage of the apoptotic marker, PARP, indicating activation of cell death (Figure 2C).
Dasatinib reverses the cytoplasmic mislocalization of p27 by inhibiting src kinase activation in BE and HGD cell lines. After treatment, the expression of the proliferative markers Cyclin D1 and Cyclin E1 decreased correlating with reduced cell growth and activated apoptosis within BE cell lines. Dysplastic BE cells appeared to be more sensitive to lower doses of dasatinib compared to the metaplastic line. Taken together, these findings suggest that dasatinib, a targeted agent with minimal side effects, has potential as a treatment for patients with high-grade Barrett’s esophagus and sub-cellular localization of p27-kip1 has potential as a biomarker for HGD-BE patients who could be treated with dasatinib.
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