Bardsley, Michael R.

Ano1 is a Ca 2+ activated Cl − channel expressed in several proliferating cell types including various epithelia, gastrointestinal stromal tumors, interstitial cells of Cajal (ICC) and CFPAC‐1 cells. The aim of this study was to determine if Ano1 regulates proliferation. ICC from Ano1 (−/−) and Ano1 (+/+) mice were cultured with Cl − channel inhibitors or in low Cl − media. CFPAC‐1 cells grown in low Cl − media were pulsed with EdU for 1 h. Incorporation was measured by flow cytometry. For immunoblots on mouse jejunum, antibodies to pRb, phospho(Ser773)‐pRb and GAPDH were used. Fewer proliferating ICC were detected in Ano1 (−/−) cultures (15 ± 3.5% Ki67 + ICC vs 25 ± 4% in Ano1 (+/+) , mean ± SEM, n = 4, p < 0.05). Cl − channel inhibitors DIDS (10 μM), niflumate (10 μM) and tamoxifen (1 μM) decreased ICC proliferation by 53, 53 and 59%, respectively ( p < 0.05). This effect was reduced in cultures from Ano1 (−/−) mice (29% and 19 % decrease, 22% increase, p > 0.05). Fewer proliferating ICC and CFPAC‐1 cells were detected in low Cl − media (% EdU + ICC: 120 mM; 19.8 ± 5.3, 12 mM; 8.5 ± 3.2, n = 4, and % EdU + CFPAC‐1: 120 mM; 29.5, 12 mM; 14.3 p < 0.05). Proportionally less Ser773‐phosphorylated pRb was detected in jejunum from Ano1 (−/−) mice (Ano1 (+/+) ; 5.98 ± 0.78, Ano1 (−/−) ; 3.60 ± 0.49, mean ± SEM, n = 7, p < 0.05). In conclusion, Ano1 regulates proliferation possibly by promoting the G 1 /S transition of the cell cycle. Support: DK57061 and DK68055.

Abstract Background & Aims: Most GIST harbor oncogenic KIT or PDGFRA mutations, which are in most cases heterozygous. However, most GIST co-express both wild-type KIT and PDGFRA with the mutated KIT or PDGFRA. Signaling mediated by heterodimers of the wild-type receptors contribute to GIST growth even after blocking oncogenic signaling by KIT/PDGFRA inhibitors such as imatinib. Previously we found that crenolanib (CP-868596), a PDGFRA/B tyrosine kinase inhibitor with &gt;100-fold selectivity over other tyrosine kinases including KIT and a potent inhibitor of oncogenic PDGFRA signaling in vitro, inhibited the proliferation of KIT+PDGFRA+PDGFRB− GIST-T1 cells with a potency comparable to that of imatinib (IC50: 5.3 nM; imatinib: IC50: 3.5 nM) but had very little effect in the KITlow/−PDGFRAlow/−PDGFRB+ GIST48B cells (maximum inhibition: &lt;50%; IC50&gt;75 nM) and in mouse stem cells for interstitial cells of Cajal (ICC-SC), a presumed cellular source for GIST, which share a phenotype with GIST48B cells (IC50 &gt;600 nM; Hayashi et al., Gastroenterology 2012; 142:S30). Here we investigated whether the antiproliferative effect of crenolanib was dependent on KIT expression, and studied crenolanib's effect on KIT/PDGFRA signaling and on the stability of ETV1, a transcription factor required for GIST growth and survival. Methods: Protein expression and phosphorylation were studied by Western blotting. KITlow/−PDGFRAlow/+PDGFRB+ murine ICC-SC (2xSCS2F10) were retrovirally transduced to express wild-type murine KIT. Cell proliferation was studied by methyl-tetrazolium salt assay. Results: Expression of wild-type KIT in retrovirally transduced ICC-SC dramatically reduced crenolanib's IC50 from 1157 nM to 121 nM. In GIST-T1 cells, crenolanib significantly reduced PDGFRA and KIT protein expression, inhibited PDGFRA phosphorylation (IC50: 5 nM) but had no effect on KIT phosphorylation. Crenolanib applied at 8.3 nM concentration rapidly and profoundly inhibited phosphorylation of ERK1/2 MAP kinases and reduced ETV1 protein levels by &gt;50% in 2 h. The MEK inhibitor PD98059 (50 μM) reproduced crenolanib's effect on ETV1 protein levels with an identical time-course. The inhibitory effects of both crenolanib and PD98059 on ETV1 protein expression could be reversed with the proteasome inhibitor MG132 (10 μM) and accentuated with the protein synthesis inhibitor cycloheximide (10 μg/mL). ETV1 was undetectable in the crenolanib-resistant, KITlow/−PDGFRAlow/−PDGFRB+ GIST48B cells. Conclusions: In KIT mutant, KIT+PDGFRA+PDGFRB− GIST cells, crenolanib induced ETV1 protein degradation by suppressing PDGFRA-dependent MAPK phosphorylation. Crenolanib's antiproliferative effect in these cells may depend on KIT-induced ETV1 expression and reflect the destabilization of ETV1. Grant support: NIH DK58185. Citation Format: Yujiro Hayashi, Michael R. Bardsley, Yoshitaka Toyomasu, Takahiro Taguchi, Brian P. Rubin, Monique Carter, Abhijit Ramachandran, Tamas Ordog. Crenolanib, a highly selective platelet-derived growth factor receptor α/β (PDGFRA/B) tyrosine kinase inhibitor, destabilizes ETV1 protein in KIT-mutant gastrointestinal stromal tumor (GIST) cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1035. doi:10.1158/1538-7445.AM2013-1035

Ano1 is a recently discovered Ca2+-activated Cl− channel expressed on interstitial cells of Cajal (ICC) that has been implicated in slow-wave activity in the gut. However, Ano1 is expressed on all classes of ICC, even those that do not contribute to generation of the slow wave, suggesting that Ano1 may have an alternate function in these cells. Ano1 is also highly expressed in gastrointestinal stromal tumors. Mice lacking Ano1 had fewer proliferating ICC in whole mount preparations and in culture, raising the possibility that Ano1 is involved in proliferation. Cl− channel blockers decreased proliferation in cells expressing Ano1, including primary cultures of ICC and in the pancreatic cancer-derived cell line, CFPAC-1. Cl− channel blockers had a reduced effect on Ano1(−/−) cultures, confirming that the blockers are acting on Ano1. Ki67 immunoreactivity, 5-ethynyl-2′-deoxyuridine incorporation, and cell-cycle analysis of cells grown in low-Cl− media showed fewer proliferating cells than in cultures grown in regular medium. We confirmed that mice lacking Ano1 had less phosphorylated retinoblastoma protein compared with controls. These data led us to conclude that Ano1 regulates proliferation at the G1/S transition of the cell cycle and may play a role in tumorigenesis.

SP/KLF (Specificity protein/Krüppel-like factor) transcription factors comprise an emerging group of proteins that may behave as tumour suppressors. Incidentally, many cancers that display alterations in certain KLF proteins are also associated with a high incidence of KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homologue) mutations. Therefore in the present paper we investigate whether SP/KLF proteins suppress KRAS-mediated cell growth, and more importantly, the potential mechanisms underlying these effects. Using a comprehensive family-wide screening of the 24 SP/KLF members, we discovered that SP5, SP8, KLF2, KLF3, KLF4, KLF11, KLF13, KLF14, KLF15 and KLF16 inhibit cellular growth and suppress transformation mediated by oncogenic KRAS. Each protein in this subset of SP/KLF members individually inhibits BrdU (5-bromo-2-deoxyuridine) incorporation in KRAS oncogenic-mutant cancer cells. SP5, KLF3, KLF11, KLF13, KLF14 and KLF16 also increase apoptosis in these cells. Using KLF11 as a representative model for mechanistic studies, we demonstrate that this protein inhibits the ability of cancer cells to form both colonies in soft agar and tumour growth in vivo. Molecular studies demonstrate that these effects of KLF11 are mediated, at least in part, through silencing cyclin A via binding to its promoter and leading to cell-cycle arrest in S-phase. Interestingly, similar to KLF11, KLF14 and KLF16 mechanistically share the ability to modulate the expression of cyclin A. Collectively, the present study stringently defines a distinct subset of SP/KLF proteins that impairs KRAS-mediated cell growth, and that mechanistically some members of this subset accomplish this, at least in part, through regulation of the cyclin A promoter.