DOG1 regulates growth and IGFBP5 in gastrointestinal stromal tumors

Abstract

Gastrointestinal stromal tumors (GIST) are characterized by activating mutations of KIT or platelet-derived growth factor receptor α(PDGFRA), which can be therapeutically targeted by tyrosine kinase inhibitors (TKI) such as imatinib. Despite long-lasting responses, most patients eventually progress after TKI therapy. The calcium-dependent chloride channel DOG1 (ANO1/TMEM16A), which is strongly and specifically expressed in GIST, is used as a diagnostic marker to differentiate GIST from other sarcomas. Here, we report that loss of DOG1 expression occurs together with loss of KIT expression in a subset of GIST resistant to KIT inhibitors, and we illustrate the functional role of DOG1 in tumor growth, KIT expression, and imatinib response. Although DOG1 is a crucial regulator of chloride balance in GIST cells, we found that RNAi-mediated silencing or pharmacologic inhibition of DOG1 did not alter cell growth or KIT signaling in vitro. In contrast, DOG1 silencing delayed the growth of GIST xenografts in vivo. Expression profiling of explanted tumors after DOG1 blockade revealed a strong upregulation in the expression of insulin-like growth factor-binding protein 5 (IGFBP5), a potent antiangiogenic factor implicated in tumor suppression. Similar results were obtained after selection of imatinib-resistant DOG1- and KIT-negative cells derived from parental DOG1 and KIT-positive GIST cells, where a 5,000-fold increase in IGFBP5 mRNA transcripts were documented. In summary, our findings establish the oncogenic activity of DOG1 in GIST involving modulation of IGF/IGF receptor signaling in the tumor microenvironment through the antiangiogenic factor IGFBP5.

Figure 1

Coexpression of KIT and DOG1 in different GIST cell lines. A, Whole transcriptome sequencing data for KIT and DOG1 in different GIST cell lines. B, Western blot analyses of KIT, pKIT and DOG1 expression in KIT-positive (GIST882, GIST430, GIST48) and KIT-negative (GIST882B, GIST430B, GIST48B, GIST5, GIST474, GIST62) GIST cell lines. C, Western blot analyses of GIST-T1 and GIST882 after 24h of incubation with imatinib (1µM) and 17-AAG (500nM).

Figure 2

Effects of DOG1 knockdown. A, Western blot analyses of DOG1 expression in GIST-T1 and GIST882 cells. B, Whole-cell patch-clamp measurements of scrambled and DOG1 knockdown cells. C, BrdU cell proliferation assay. D, Cells were treated for 72h with increasing doses of IM (1nm – 10µM) and cytotoxicity was measured using SRB assays. Results represent the mean ± S.D. of quadruplicate values of a representative experiment.

Figure 3

Cytotoxicity studies with the CaCC inhibitors A01, NAC and NPPB. GIST-T1 and GIST882 cells with or without DOG1 knockdown were treated with increasing doses of inhibitors for 72h and the relative amount of remaining cells was measured using the SRB assay. Results represent the mean ± S.D. of quadruplicate values of a representative experiment.

Figure 4

In vivo growth of DOG1 knockdown xenografts. A, Western blot analyses of DOG1 and KIT expression in GIST-T1 and GIST882 xenografts. B, Immunohistochemical analysis of GIST-T1 and GIST882 xenografts. Samples were stained with H&E and with antibodies against DOG1, KIT and Ki-67.B. C, Tumor volume over time in nude mice implanted with GIST-T1, GIST882 and GIST430 cells after shRNA-mediated DOG1 suppression compared to scrambled shRNA controls.

Figure 5

Heat map representing color-coded expression levels of differentially expressed genes (up-regulated (red) or down-regulated (green)) in GIST-T1 xenograft and cell line.

Figure 6

Analysis of gene expression data. A, Quantitative Real Time RT-PCR evaluation of KIT, DOG1, MNK1 (MAP kinase interacting serine/threonine kinase 1), LTN1 (listerin E3 ubiquitin protein ligase 1), CDC14A (CDC14 cell division cycle 14 homolog A), DDX17 (DEAD (Asp-Glu-Ala-Asp) box polypeptide 17), EPHA4 (EPH receptor A4), CDKN1C (cyclin-dependent kinase inhibitor 1C) and IGFBP5 mRNA in GIST-T1 xenografts.. B; Quantitative Real Time RT-PCR evaluation of IGFBP5 in GIST-T1 and GIST882. Values were normalized to the scrambled control. C, Whole transcriptome sequencing data for IGFBP5 in GIST882 (KIT- and DOG1-positive), GIST882B (KIT- and DOG1-negative), GIST430 (KIT- and DOG1-positive) and GIST430B (KIT- and DOG1-negative) cells.