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. 2015 Apr 10;9(7):1421–1433. doi: 10.1016/j.molonc.2015.03.013

miRNA‐221 and miRNA‐222 induce apoptosis via the KIT/AKT signalling pathway in gastrointestinal stromal tumours

Michaela Angelika Ihle 1,✉, Marcel Trautmann 1,†, Helen Kuenstlinger 1, Sebastian Huss 1,†, Carina Heydt 1, Jana Fassunke 1, Eva Wardelmann 1,†, Sebastian Bauer 2, Hans-Ulrich Schildhaus 1,‡, Reinhard Buettner 1, Sabine Merkelbach-Bruse 1
PMCID: PMC5528808  PMID: 25898773

Abstract

Aberrantly expressed microRNAs (miRNAs) are involved in many diseases including cancer. In gastrointestinal stromal tumours (GISTs) expression of miR‐221 and miR‐222 is reduced compared to control tissue and other sarcomas but the functional effects of this downregulation are not fully understood. This study aimed at evaluating the miR‐221 and miR‐222 expression profiles in different GIST subtypes and the functional role of these miRNAs. Expression of miR‐221 and miR‐222 was analysed in six KIT exon 9 and three KIT exon 11 mutated and nine wildtype GISTs by qPCR. Viability and apoptosis were examined in three different, KIT positive GIST cell lines (GIST882, GIST‐T1 and GIST48) after overexpression of these miRNAs. The modulation of KIT and the PI3K/AKT pathways was determined by Western blot. Wildtype and KIT mutated GISTs revealed reduced miRNA expression compared to adequate control tissue. miRNA expression was lower for wildtype compared to mutated GISTs. Transient transfection of miR‐221 and miR‐222 reduced viability and induced apoptosis by inhibition of KIT expression and its phosphorylation and activation of caspases 3 and 7 in all three GIST cell lines. p‐AKT, AKT and BCL2 expression was reduced after miRNA transfection whereas only slight influence on p‐MTOR, MTOR and BCL2L11 (BIM) was detected. Our results demonstrate that miR‐221 and miR‐222 which are downregulated in wildtype and mutated GISTs, induce apoptosis in vitro by a signalling cascade involving KIT, AKT and BCL2. Therefore, overexpression of these miRNAs seems to functionally counteract oncogenic signalling pathways in GIST.

Keywords: miR-221, miR-222, GIST, KIT, Expression, Apoptosis

Highlights

  • miR‐221 and miR‐222 are downregulated in GIST.

  • Overexpression of miR‐221 and miR‐222 reduce cellular proliferation.

  • This antiproliferative effect correlated with an induction of apoptosis.

  • Induction of apoptosis is mediated by a signaling cascade involving KIT, AKT and BCL2.

1. Introduction

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal tumours of the digestive tract. They are characterised by activating mutations in the KIT (stem cell factor receptor) or platelet derived growth factor receptor alpha (PDGFRA) genes encoding two type III receptor tyrosine kinases. KIT activation drives a number of downstream pathways associated with malignant transformation including mitogen‐activated protein kinase (MAPK), phosphatidylinositol 3‐kinase (PI3K) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways (Taylor and Metcalfe, 2000). With the introduction of the selective tyrosine kinase inhibitor imatinib, treatment options for GISTs have been improved. Clinical responses to imatinib are seen in 80% of patients with metastatic GISTs (Bauer et al., 2007). However, treatment with imatinib is not uniformly successful depending on the localisation of the mutation in the KIT or PDGFRA gene. Also, for a minority of cases without a mutation in the hotspots of KIT and PDGFRA, there is limited benefit of imatinib therapy (Debiec‐Rychter et al., 2004; Demetri et al., 2010). Moreover, the acquisition of secondary KIT mutations during imatinib treatment may lead to therapy resistance (Wardelmann et al., 2006). Even the clinical activity of second and third generation selective inhibitors as the multi‐target KIT inhibitor sunitinib is influenced by both primary and secondary mutations in the KIT or PDGFRA gene (Rutkowski et al., 2012; Yang et al., 2011b).

Therefore, the identification of genetic factors regulating KIT and PDGFRA expression, regardless of primary and secondary mutations, may offer a highly relevant therapeutic approach. One such regulating factor might be microRNAs (miRNA). miRNAs are small non‐coding RNAs of approximately 22 nucleotides that regulate the expression of proteins post‐transcriptionally. This regulation is conveyed through an imperfect or perfect base pairing with the 3′‐untranslated region (3′UTR) of targeted mRNA leading to translational inhibition or mRNA degradation depending on the sequence complementarities (Bartel, 2004). A motif referred to as seed‐sequence from nucleotides 2 to 7 at the 5′‐end of the miRNA is required for target recognition (Lewis et al., 2005). Putative target genes contain seed‐matched sites in their 3′UTR. By regulating target genes miRNAs are involved in physiological processes including cell proliferation, apoptosis, differentiation, metabolism and growth (Fan et al., 2014).

Recent studies showed that miRNAs are often deregulated in human malignancies and contribute to the occurrence, development and invasion of cancer. Depending on the specific miRNA expression level, they may act either as oncogenes or tumour suppressors (Suzuki et al., 2013). Dysregulation of miRNAs was also reported for GISTs. These tumours can be clearly distinguished from other sarcomas by their miRNA expression profiles (Subramanian et al., 2004). Furthermore, small bowel and retroperitoneal wildtype have a similar miRNA expression profile compared to adult wildtype gastric GISTs, adult mutant and paediatric wildtype cases (Kelly et al., 2013). MiR‐196a upregulation has been found to be related to high‐risk status, metastasis and poor prognosis in GISTs (Niinuma et al., 2012). Overexpression of miR‐494 leads to KIT downregulation and repression of proliferation in vitro (Kim et al., 2011).

Especially, miR‐221 and miR‐222 were shown to target the 3′UTR of KIT. Both miRNAs are downregulated in GISTs compared to other sarcomas (Subramanian et al., 2004; Gits et al., 2013) and normal gastrointestinal control tissue (Haller et al., 2010). Both miRNAs are associated with KIT overexpression (Choi et al., 2010; Koelz et al., 2011). Whereas Koelz et al. (2011) did not find any correlation of miRNA expression with mutational status, Haller et al. (2010) described a significantly higher expression of miR‐221 and miR–222 in wildtype than in mutated GISTs. Further studies showed that miR‐222 and miR‐17/20a directly target KIT and ETV1 in GISTs and that overexpression of these two miRNAs inhibited significantly cell proliferation and induced apoptosis in the cell line GIST‐T1 (Gits et al., 2013). Although all studies correlated miRNA expression signatures with various parameters including anatomical localisation, mutational status, tumour risk and histomorphological parameters, possible mechanisms by which altered expression of miR‐221 and miR‐222 may contribute to the pathogenesis of GISTs are still unknown.

Therefore, we examined the miRNA expression in different GIST subtypes and analysed the regulatory effects of miR‐221 and miR‐222 on KIT expression and on the signal transduction in GISTs. We detected differential miRNA expression of both miRNAs in GISTs depending on the mutational status. By analysing three different GIST cell lines (two imatinib sensitive and one resistant cell line) we provide first evidence that exogenous miR‐221 and miR‐222 induce apoptosis and lead to a decrease of proliferation via the KIT/AKT signalling pathway in different GIST cell lines. In summary, our results underline the important role of miR‐221 and miR‐222 in GIST tumourigenesis and may represent an approach to inhibit increased KIT activity.

2. Material and methods

2.1. Samples

24 samples (18 tumour tissue samples and six smooth muscle tissue samples as control group) were included in this study. The tumour tissues were comprised of six GISTs with KIT exon 9 mutations, three GISTs with KIT exon 11 mutations and nine wildtype GISTs. Six samples of gastric or intestinal muscularis propria served as control group. These samples consisted predominantly of smooth muscle tissue and were obtained from normal exterior organ wall of intestines and stomachs which were surgically removed because of carcinomas. None of these six patients had a history of GIST. All samples were fixed in neutral‐buffered formalin prior to paraffin embedding (FFPE samples). GISTs were diagnosed and classified by an experienced pathologist (HUS, EW, RB) according to Lasota and Miettinen, 2006. Immunohistochemical staining results for KIT (CD117) are displayed in Table 1.

Table 1.

Clinicopathologic and molecular characteristics of 18 GISTs.

Case no. Age/Gender Localisation Size (cm) Morphological subtype Mitotic count (per 50 HPF) Risk classificationa Mutation status KIT mutational status IHC
KIT exon 9 KIT exon 11 KIT
1 67/F Small bowel, duodenum 13.0 Mixed 0 High +
2 74/F Small bowel 5.5 Spindle 0 High +
3 30/M Small bowel 9.0 Spindle 0 Intermediate +
4 23/NA Small bowel 35.0 Mixed 2 Intermediate +
5 13/M Stomach 5.0 Mixed 1 Low +
6 52/M Stomach 5.0 Epitheloid 1 Low +
7 68/M Stomach 3.5 Mixed 0 Very low +
8 29/F Stomach 2.5 Mixed 1 Very low +
9 44/M Stomach 4.0 Spindle 3 Very low +
10 63/M Stomach 11.0 Spindle 59 High p.V560D +
11 57/M Stomach 25.0 Mixed 29 High p.K550_K558del +
12 68/NA Small bowel 7.5 Mixed 2 Intermediate p.V559A +
13 47/F Small bowel 9.5 Spindle 4 Intermediate p.A502_Y503dup +
14 68/F Small bowel, duodenum >5.0 Mixed 15 High p.A502_Y503dup +
15 60/F Small bowel 4.8 Spindle 2 Low p.A502_Y503dup +
16 29/F Small bowel, duodenum 13.0 Spindle 3 High p.A502_Y503dup +
17 77/F Small bowel 4.0 Spindle 0 Low p.A502_Y503dup +
18 50/F Small bowel 12.0 Spindle 2 High p.A502_Y503dup +
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NA: not available; F: female, M: male, HPF: high power field, IHC: immunohistochemistry,+ indicates that tumour cells express KIT.

a

According to (Miettinen and Lasota, 2006).

Sequence analyses of KIT (exons 8, 9, 11, 13, 14, 15 and 17) and PDGFRA (exons 12, 14 and 18) were carried out as described earlier (Kunstlinger et al., 2013; Lasota et al., 2000). Additionally, wildtype status of the BRAF gene was ascertained in all samples.

2.2. RNA isolation and expression analysis

miRNA was isolated from FFPE tissue using the miRNeasy FFPE Kit (Qiagen, Hilden, DE) according to the manufacturer's instructions. mRNA was isolated using the Dynabeads® mRNA DIRECT™ Micro Kit (Thermo Fisher Scientific Inc., Waltham, US) according to manufacturer's instructions.

From each sample 50 ng total RNA were reversely transcribed into cDNA twice using TaqMan® MicroRNA Reverse Transcription Kit for miRNA expression analyses (Thermo Fisher Scientific Inc.). Both preparations were pooled. Cycling conditions were as recommended.

miRNA quantification was performed in triplicates using TaqMan® MicroRNA Expression assays (Thermo Fisher Scientific Inc.) and a sample dilution of 1:10. The PCR mixture was incubated at 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Results were normalised to hsa‐miR‐26b as reference miRNA according to the application note for TaqMan® MicroRNA Assays (Thermo Fisher Scientific Inc.) and analysed by the 2−ΔΔCT method (Livak and Schmittgen, 2001).

2.3. Cell lines

The cell line GIST882 was kindly provided by Jonathan Fletcher (Brigham and Women's Hospital, Harvard Medical School, Boston, MA, US). It was established from an untreated primary tumour harbouring a homozygous imatinib sensitive mutation in KIT exon 13 (p.K642E) (Tuveson et al., 2001). Cells were maintained in RPMI 1640, supplemented with 15% heat inactivated foetal calf serum (FCS) and 1 mM l‐glutamine (Thermo Fisher Scientific Inc.). GIST‐T1 cell line was established from the primary tumour of a metastasised primary GIST with a heterozygous imatinib sensitive KIT exon 11 deletion (p.V560_Y578del) (Taguchi et al., 2002). Cells were cultured in DMEM + Glutamax supplemented with 10% heat inactivated FCS and 1 mM l‐glutamine. The GIST48 cell line is an imatinib resistant cell line from a patient showing initial response under imatinib therapy but then progressed (Bauer et al., 2006). This cell line is characterised by a homozygous KIT exon 11 mutation (p.V560D) and a heterozygous, secondary KIT exon 17 mutation (p.D820A) and was maintained in IMDM supplemented with 10% heat inactivated FCS and 1 mM l‐glutamine.

All cell lines were cultured at 37 °C in a 5% CO2 atmosphere and were checked for genetic alterations by Sanger sequencing. All cell lines showed positive KIT immunohistochemical staining (Appendices, Figure A1).

2.4. Immunohistochemistry

Immunohistochemical staining was performed with a specific primary antibody against KIT (polyclonal rabbit anti human KIT antibody, 1:100, Agilent Technologies, Santa Clara, US, A4502). 3 μm sections of FFPE tissue samples or FFPE cell pellets were dried overnight at 37 °C. Deparaffinisation, rehydration and heat induced epitope retrieval (30 min in citrate buffer pH 6) were performed semiautomatically on a DAKO‐TechMate™ 500 immunostainer (Agilent Technologies). Immunohistochemical staining was carried out within 2 weeks after cutting the 3 μm sections.

2.5. Cell viability and apoptosis analyses

2 × 104 GIST882, 4 × 103 GIST‐T1 and 7 × 103 GIST48 cells/well were plated in a 96 well plate with five replicates for each condition and cultured in serum containing media. After 24 h, cells were transiently transfected with syn‐hsa‐miR‐221‐3p miScript miRNA Mimic (miR‐221), syn‐hsa‐miR‐222‐3p miScript miRNA Mimic (miR‐222), a combination of both, non‐targeting AllStars Negative Control siRNA (N.C.), AllStars Hs Cell Death siRNA (P.C.) or Hs_KIT_5 FlexiTube siRNA (all purchased from Qiagen) using Lipofectamine® RNAiMAX for the cell lines GIST882 and GIST48 and Lipofectamine® 2000 for the cell line GIST‐T1 (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Lipofectamine® 2000 was chosen for the cell line GIST‐T1 as Lipofectamine® RNAiMAX was too cytotoxic in this cell line. Transfection efficiency was equivalent in all three GIST cell lines with the different transfection reagents. miRNA or siRNA were applied in a final concentration of 100 nM. Cells treated only with transfection reagent were used as Mock control and Camptothecin treated cells as positive control. Viability of transfected cells and corresponding controls was measured using MTT assay. 10 μl of MTT staining solution (5 mg/ml of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide in PBS, sterile filtered) was added to each sample 0, 24, 48 and 72 h after transfection and cells were further incubated for at least 5 h. The reaction was stopped at each time point with 100 μl MTT solvent (10% SDS in 0.01 M HCl) and formed crystals were lysed overnight at 37 °C. Absorption at 550 nm was measured and background absorption at 690 nm was deducted. Data of miRNA transfected samples were normalised to N.C. at timepoint 0 h. To verify and further analyse the results, cell viability, cytotoxicity and apoptosis were measured 24, 48 and 72 h after transfection using the ApoTox‐Glo™ Triplex Assay (Promega, Madison, US) according to the manufacturer's instructions. All experiments were repeated once to confirm the observations.

2.6. Western blot analyses

3 × 105 GIST882 or GIST48 and 2 × 105 GIST‐T1 cells/well were plated in a 12 well plate and cultured in serum containing media. Transfection was performed after 24 h with miR‐221, miR‐222 or a combination of both at final concentration of 100 nM as described in Section 2.5 of this study. Lipofectamine alone was used as Mock control.

After 24, 48 and 72 h proteins were isolated according to standard protocols with RIPA‐puffer. Protein concentrations were measured using the BCA Protein Assay Kit (Thermo Fisher Scientific Inc.). Equal amounts of total protein were applied to SDS‐PAGE. After electrophoresis, proteins were blotted to polyvinylidene fluoride (PVDF) membranes (Carl Roth GmbH & Co.KG, Karlsruhe, DE) and blocked in TBS‐T (Tris Buffered Saline containing 0.1% Tween‐20) supplemented with 5% skimmed milk powder. The membranes were then probed with specific primary antibodies and incubated at 4 °C overnight: p‐AKT (1:500, Ser473, monoclonal rabbit anti p‐AKT), AKT (1:500, polyclonal rabbit anti AKT), p‐MTOR (1:500, Ser2448, polyclonal rabbit anti p‐mTOR), BCL2 (1:1000, polyclonal rabbit anti BCL2) and ACTB (also known as beta‐actin; 1:500, monoclonal mouse anti ACTB, all purchased from Cell Signaling Technology®, Danvers, US), BCL2L11 (1:250, polyclonal rabbit anti BCL2L11) and MTOR (1:500, polyclonal rabbit anti MTOR, both purchased from Acris Antibodies GmbH, Herford, DE). Membranes were washed three times with TBS‐T and probed with the appropriate horseradish‐peroxidase‐conjugated (HRP) secondary antibody at room temperature for 1 h (polyclonal goat anti rabbit IgG HRP, 1:1000, Thermo Fisher Scientific Inc., and polyclonal goat anti mouse IgG HRP, 1:1000, Cell Signaling). The specific protein was detected by SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific Inc.) according to the manufacturer's instructions. Quantification of Western blot images was performed with the ImageJ software (v.1.42q (Schneider et al., 2012)). Target protein expression levels were normalised to those of ACTB as loading control.

2.7. Statistical analyses

Data are depicted as the mean ± standard error (S.E.M) of five replicates. p < 0.05 was considered as statistically significant using one way ANOVA (Dunnett's multiple comparison) test. Graphs were illustrated using GraphPad Prism 4.02 software (GraphPad Software, Inc.).

3. Results

3.1. MiR‐221 and miR‐222 expression in GISTs

Our data show that miR‐221 and miR‐222 were both downregulated in the tumour tissues in general (wildtype GISTs combined with mutated GISTs) compared to control (fold change −5.2 for miR‐221 and ‐6.5 and for miR‐222, Figure 1). Looking more detailed into the different subgroups of the analysed GISTs, wildtype GISTs (n = 9) exhibited lower levels of miR‐221 compared to mutant GISTs (n = 9). Fold changes of miR‐221 in wildtype GISTs were −6.6 compared to CT whereas mutant GISTs exhibited a fold change of −3.6 compared to CT.

Figure 1.

Figure 1

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miR‐221 and miR‐222 expression in GISTs (six GISTs with KIT exon 9 mutations, three GISTs with KIT exon 11 mutations and nine wildtype GISTs) compared to control (six samples of gastric or intestinal muscularis propria, CT). A) Downregulation of miR‐221 and B) miR‐222 in tumour tissue in general as well as in wildtype and mutated GISTs. Displayed is mean with SEM. CT: smooth muscle tissue as control tissue, wt‐GISTs: wildtype GISTs, mut‐GISTs: KIT exon 9 or 11 mutated GISTs, *: p < 0.05.

Similar results were obtained for miR‐222. Significant lower expression could be observed in tumour tissues in general (wildtype combined with mutated GISTs) and in wildtype GISTs alone (fold change −6.5 and −7.7 respectively) compared to CT. The same tendency was observed in mutant GISTs but this did not reach significant levels (fold change −4.9).

3.2. MiR‐221 and miR‐222 reduce cell viability

We analysed the capacity of miR‐221 and miR‐222 to suppress the KIT mediated survival signalling. Three different GIST cell lines (two imatinib sensitive cell lines (GIST882 and GIST‐T1) and one imatinib resistant cell line (GIST48)) were used to evaluate whether antiproliferative effects of the analysed miRNAs are independent of the mutational status. After 48 h overexpression of miR‐221 and miR‐222 induced morphological changes most prominently in the cell line GIST882 (Figure 2A). Strongest effects could be observed after transfection of miR‐221 being similar to those of the specific knock‐down of KIT by siRNA interference. In contrast, the cell line GIST‐T1 was most sensitive towards miR‐222 but also showed morphological changes after transfection of each miRNA compared to a non‐targeting control siRNA (N.C.). Overexpression of each miRNA in the GIST48 cell line resulted in a slight morphological change being similar for each miRNA.

Figure 2.

Figure 2

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miR‐221 and miR‐222 reduce cell viability in GIST. A) Morphological changes in the cell lines GIST882, GIST‐T1 and GIST48 48 h after transfection of miR‐221, miR‐222, non‐targeting AllStars Negative Control siRNA (N.C.) or Hs_KIT_5 FlexiTube siRNA at a final concentration of 100 nM. B) MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromid) assay performed 0, 24, 48 and 72 h after transfection of miR‐221, miR‐222, a combination of both, non‐targeting AllStars Negative Control siRNA (N.C.), AllStars Hs Cell Death siRNA or a specific siRNA targeting KIT (Hs_KIT_5 FlexiTube siRNA) at a final concentration of 100 nM compared to transfection reagent (Mock) or Camptothecin treated controls. Data were normalised to N.C. at time point 0 h.

To verify these observations MTT assays were performed 0, 24, 48 and 72 h after transient transfection of miR‐221, miR‐222 and a combination of both compared to non‐targeting control siRNA (N.C.), a specific siRNA directed against KIT and two positive controls known to induce apoptosis (AllStars Hs Cell Death siRNA and Camptothecin). In the cell line GIST882 miR‐221, miR‐222 and a combination of both reduced cell proliferation by almost a quarter showing the strongest effect among all evaluated GIST cell lines (Figure 2B). In the cell line GIST‐T1 the overexpression of miR‐222 exhibited the most prominent effect. miR‐221 reduced cell proliferation only slightly compared to N.C. In contrast, miR‐222 and a combination of both were not effective in the cell line GIST48. Only the overexpression of miR‐221 reduced cell proliferation compared to the N.C. but in a lesser extent as shown for the other GIST cell lines. The combination of both miRNAs showed no additive effect in all evaluated cell lines.

3.3. MiR‐221 and miR‐222 induce apoptosis

To further analyse the antiproliferative effect ApoTox‐Glo™ Triplex Assay was performed with all three GIST cell lines. Viability and apoptosis were measured 24, 48 and 72 h after transient transfection of miR‐221, miR‐222, a combination of both and adequate controls. In all cell lines, no effect could be observed after 24 h (Figure 3).

Figure 3.

Figure 3

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Cell viability is inversely correlated with apoptosis. GIST cell lines were treated for 24, 48 and 72 h with miR‐221, miR‐222, a combination of both, non‐targeting AllStars Negative Control siRNA (N.C.), AllStars Hs Cell Death siRNA (P.C.) or Hs_KIT_5 FlexiTube siRNA at a final concentration of 100 nM compared to transfection reagent alone (Mock). Significant and time dependent reduction of viability (A) correlating to induction of apoptosis (B) after miRNA‐transfection was detected. Blue indicates viability and apoptosis after 24 h, red after 48 h and green after 72h of miRNA treatment. RFU: relative fluorescence units, RLU: relative luminescence units, *: p < 0.05, **: p < 0.01, ***: p < 0.001, Column bars display mean + SEM.

After 48 and 72 h miR‐221, miR‐222 and a combination of both reduced significantly cell viability in the cell line GIST882. In correlation with the observed reduction of viability a significant induction of apoptosis could be seen 48 and 72 h after transfection of miR‐221, miR‐222 and a combination of both.

In the cell line GIST‐T1 reduction of viability could be confirmed 48 h after transfection of miR‐221, miR‐222 and their combination. After 72 h, only miR‐222 and the combination of both miRNAs caused a significant antiproliferative effect. miR‐221 was also able to reduce viability but this effect did not reach significant levels. Induction of apoptosis was observed 48 and 72 h after transient transfection of miR‐222 and a combination of both miRNAs. miR‐221 showed significant induction of apoptosis 72 h after transfection.

The cell line GIST48 was sensitive to miR‐221, miR‐222 and a combination of both resulting in a significant reduction of viability. Reduction of viability correlated to the induction of apoptosis after 48 and 72 h. Even though an induction of apoptosis could be seen for all transfections those of miR‐222 did not reach significant levels after 72 h. Again, no additive effect of both miRNAs could be observed in all evaluated cell lines.

To sum up, miR‐221, miR‐222 and a combination of both were able to reduce viability and to induce apoptosis in all three GIST cell lines independent of the mutational status.

3.4. MiR‐221 and miR‐222 regulate proliferation and apoptosis via the KIT/AKT signalling cascade

To evaluate whether both miRNAs regulate endogenous KIT in GISTs and whether this is independent of the mutational status Western blot analyses were performed 24, 48 and 72 h after transient transfection of miRNAs and adequate controls. Furthermore, to compare the miRNA dependent effects, RNA interference with a specific siRNA directed against KIT was performed and analysed by Western blot. Given that the PI3K/AKT and MTOR cascade is associated with apoptosis after imatinib treatment (Bauer et al., 2007; Duensing et al., 2004), we evaluated the effect of miR‐221 and miR‐222 on AKT, MTOR, BCL2 and BCL2L11 in all cell lines to further analyse the downstream signalling cascade.

In the cell line GIST882, miR‐221, miR‐222 and a combination of both were able to reduce the phosphorylation of KIT and the expression of total KIT after 24 h (Figure 4A). After 48 and 72 h the effect was more pronounced. RNA interference with a specific siRNA directed against KIT exhibited similar effects (Figure 4B). Analysing the subsequent signalling cascade, we could show that the phosphorylation of AKT was downregulated by miR‐221, miR‐222 and the combination in all evaluated time points (Figure 4C). Here, miR‐221 was most effective reducing the phosphorylation levels in half. The effect on total AKT was most prominent 72 h after transfection of miR.221, miR‐222 and a combination of both. Looking further downstream of the signalling cascade, miR‐221, miR‐222 and the combination of both miRNAs showed almost no effect on p‐MTOR as well as on total MTOR. To evaluate the induction of apoptosis demonstrated under section 4.3 of this study, the protein expression of BCL2 and BCL2L11 was analysed after miRNA transfection. BCL2 expression was downregulated by miR‐221, miR‐222 and a combination of both at all time points. BCL2L11 protein expression did not change under miRNA treatment.

Figure 4.

Figure 4

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miR‐221 and miR‐222 dependent induction of apoptosis is mediated by the KIT signalling cascade in the imatinib sensitive cell line GIST882. Cells were treated for 24, 48 and 72 h with miR‐221, miR‐222, a combination of both (final concentration 100 nM) or transfection reagent alone (Mock). A) MiR‐221 and miR‐222 mediate reduced expression of phosphorylated and total KIT protein in the cell line GIST882. B) Western blot analyses of total KIT and phosphorylated KIT after RNA interference showing that the expression of both proteins markedly decreased after transfection of KIT siRNA compared to non‐targeting AllStars negative control siRNA (N.C.) and Mock control C) Analyses of the downstream signalling cascade of KIT after transfection of miRNAs revealed a decrease in phosphorylated and total AKT and total BCL2. Phosphorylated and total MTOR as well as total BCL2L11 are less affected.

In the cell line GIST‐T1 miR‐221, miR‐222 and a combination of both were also able to reduce the phosphorylation of KIT at all evaluated time points (Figure 5A). Here, miR‐222 showed the most prominent effect. Transfection of the miRNAs showed no effect on KIT protein expression after 24 h. After 48 h a strong reduction of total KIT expression could be observed by transfection of the combination of both miRNAs. After 72 h a reduction could also be observed by transfection of miR‐221 and miR‐222 alone. siRNA directed knock down of KIT reduced both phosphorylated KIT and total KIT protein levels almost completely (Figure 5B). The phosphorylation of AKT was reduced by miR‐221, miR‐222 and a combination of both after 24 h (Figure 5C). Similar results were obtained by each miRNA for AKT showing a stronger downregulation of AKT expression after transfection of miR‐222. The p‐MTOR and MTOR Western blot analyses in the cell line GIST‐T1 resembled those of the GIST882 cell line showing almost no regulation of the protein levels. BCL2 expression was reduced in the cell line GIST‐T1 especially 48 and 72 h after transfection of miR‐221, miR‐222 and a combination of both. BCL2L11 expression was almost unaffected by the transfection of miRNAs.

Figure 5.

Figure 5

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miR‐221 and miR‐222 dependent induction of apoptosis is mediated by the KIT signalling cascade in the imatinib sensitive cell line GIST‐T1. Western blot analyses of GIST‐T1 cells treated with miR‐221, miR‐222, a combination of both (final concentration 100 nM) or transfection reagent alone (Mock) at three different time points (24, 48 and 72 h). A) MiR‐221 and miR‐222 reduced the expression of phosphorylated and to a lesser extend total KIT protein expression in the cell line GIST‐T1 shown by Western blot. B) RNA interference abolished almost completely the expression of phosphorylated and total KIT protein in the cell line GIST‐T1. C) Western blot analyses showing a reduction of phosphorylated AKT, total AKT and to a lesser extent total BCL2 expression after transfection of miR‐221, miR‐222 and a combination of both in the cell line GIST‐T1. miRNA transfection had not much influence on phosphorylated and total MTOR as well as on total BCL2L11.

In the cell line GIST48, miRNAs had no effect on p‐KIT after 24 h. After 48 h miR‐221, miR‐222 and a combination of both reduced p‐KIT protein level (Figure 6A). Similar results were obtained after 72 h. KIT protein expression was already reduced after 24 h by all transfection approaches and lasted up to 72 h. The miRNA dependent downregulation on KIT expression was stronger for miR‐222. RNA interference with a specific siRNA directed against KIT reduced p‐KIT and KIT protein levels stronger than demonstrated for miRNAs but showed similar results as in the cell lines GIST882 and GIST‐T1 abolishing almost completely the protein levels of p‐KIT and KIT (Figure 6B). Concerning the downstream signalling cascade, miR‐221, miR‐222 and a combination of both could reduce the phosphorylation of AKT whereas the effect on total AKT was only weak (Figure 6C). The phosphorylation of MTOR and the expression of total MTOR was almost unaffected by the transfection of both miRNAs being in concordance with the results of the cell lines GIST882 and GIST‐T1. BCL2 expression was downregulated by the transfection of and the combination. In contrast, protein expression of BCL2L11 was reduced only slightly by both miRNAs as well als the combination.

Figure 6.

Figure 6

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miR‐221 and miR‐222 dependent induction of apoptosis is mediated by the KIT signalling cascade even in the imatinib resistant cell line GIST48. Effects of the transfection of miR‐221, miR‐222, a combination of both (final concentration 100 nM) or transfection reagent alone (Mock) was measured by Western blot in the cell line GIST48 at three different time points. A) Reduction of phosphorylated KIT could be observed after 48 h whereas total KIT was already reduced after 24 h of transfection. B) Western blot after RNA interference showing a strong reduction of phosphorylated as well as total KIT protein expression already after 24 h. C) Induction of apoptosis is mediated by a downregulation of phosphorylated and total AKT as well as total BCL2 protein expression but not by regulation of phosphorylated and total MTOR protein or total BCL2L11 in the cell line GIST48.

4. Discussion

In recent years, miRNAs have gained importance as post‐transcriptional regulators of gene expression in mammals. Approximately 50% of miRNAs are located in unstable and tumour associated gene regions (Calin et al., 2004) contributing to the pathogenesis of tumours by abnormal expression profiles (Esau and Monia, 2007; He et al., 2005; Suzuki et al., 2013). Although miRNAs have been extensively studied in different types of cancer, their role in the pathogenesis of GISTs has only been proposed for few miRNAs. miR‐221 and miR‐222, both on the same gene cluster, have been reported to be expressed at lower levels in GISTs compared to other sarcomas (Subramanian et al., 2004), compared to peripheral non‐tumour tissue (Koelz et al., 2011) and to normal intestinal tissue (Gits et al., 2013). In our study, we could confirm the latter observations in our small sample cohort. Reconsidering wildtype GISTs and KIT mutated GISTs separately more detailed, we found the same tendency for both molecular subtypes. However the reduction of miRNA expression was more distinct in wildtype GISTs reaching significant levels for miR‐222. In contrast, Haller et al. (2010) found a significantly higher expression of miR‐221 and miR‐222 in wildtype than in mutated GISTs by analysing a sample cohort including only three KIT exon 9 mutated samples and five wildtype GISTs (Haller et al., 2010). Wildtype GISTs are probably less dependent on miR‐221 and miR‐222 and consequently KIT compared to KIT mutated GISTs as KIT is a direct target of miR‐221 and miR‐222 (Gits et al., 2013). These observations led to the assumption of a mutation dependent miRNA expression profile. However, to verify these results more cases have to be investigated as our study and those of Haller et al., 2010 analysed only a small sample cohort concerning the miRNA expression profile in different GIST subgroups. Furthermore, Koelz et al. (2011) did not find any correlation of miRNA expression with histomorphological parameters. Another recent study with a different focus showed that the miRNA expression of small bowel and retroperitoneal wildtype GISTs clustered together whereas adult wildtype gastric GISTs, adult mutant and paediatric wildtype cases built up a different cluster strengthening the correlation of clinicopathological parameters with miRNA expression in GISTs (Kelly et al., 2013).

As activating mutations in the receptor tyrosine kinases KIT or PDGFRA are initial steps in the tumourigenesis of GISTs, miRNAs targeting these two genes are of particular interest. Reporter gene analyses showed that miR‐221 and miR‐222 target directly the 3′UTR of KIT. Downregulation of these miRNAs led to increased KIT protein expression in haematopoietic progenitor cells and correlated with KIT overexpression in GISTs. These observations suggest a functional role of these miRNAs in the pathogenesis of GISTs (Choi et al., 2010; Felli et al., 2005; Subramanian et al., 2004). In pancreatic adenocarcinoma, the antisense inhibition of miR‐221 arrests cell cycle and induces apoptosis (Park et al., 2009) whereas in thyroid papillary carcinoma, overexpressed miR‐221 regulates cell cycle progression (Visone et al., 2007). In gastric carcinoma, miR‐221 and miR‐222 regulate cell proliferation and radiation resistance (Chun‐Zhi et al., 2010). In prostate cancer miR‐221 downregulation is associated with tumour aggressiveness (Kneitz et al., 2014).

However, effects of the direct interaction of miR‐221 and miR‐222 with KIT on physiological processes and the signalling cascade are not completely understood. We could show in this study that both miRNAs are able to reduce proliferation in three GIST cell lines. The reduction was inversely correlated with a significant induction of apoptosis. Although both miRNA are transcribed from the same gene cluster from chromosome Xp11.3, they exhibited a different extent of regulation in the GIST cell lines. The cell line GIST‐T1 was more sensitive towards miR‐222 and the cell line GIST48 to lesser extends more towards miR‐221. In the cell line GIST882 the effects of miR‐221 and miR‐222 were comparable. In contrast to our results, Gits et al., analysing only miR‐222 in the cell lines GIST882 and GIST‐T1, showed that miR‐222 dependent effects on proliferation and apoptosis were less pronounced in GIST882 cells (Gits et al., 2013). Nevertheless they could also show a miRNA dependent reduction of viability and induction of apoptosis in both cell lines. Interestingly, their KIT mRNA expression analyses revealed a 38% decrease of KIT mRNA in the cell line GIST‐T1 but a higher decrease of 46% in the cell line GIST882.

This differential regulation by miRNAs from the same gene cluster was also observed in other tumour entities (Gan et al., 2014; Yang et al., 2011a; Yu et al., 2012). One explanation might be that the regulation of both miRNAs is different post‐transcriptionally. The regulatory effect of both miRNAs on KIT seems to be different. According to the database microRNA.org (Dec. 2014) miR‐221 and miR‐222 have a different miRSVR value for the KIT receptor (−2.11 and −2.20, respectively). This value predicts the probability of downregulation of the target‐mRNA by the evaluated miRNA based on the structure of the mRNA/miRNA interaction. These different miRSVR values are also predicted for other target genes. Furthermore, a miRNA does not only regulate one target‐mRNA. 5670 target genes are predicted to be regulated by miR‐221 but only 5434 for miR‐222 even though both miRNAs are transcribed from the same gene cluster and belong to the same miRNA family.

GISTs with mutated KIT have a high phosphorylation status of KIT and show activation of downstream pathways associated with malignant transformation including MAPK 3/1, AKT, STAT1 and STAT3 (Duensing et al., 2004). Recent studies reported that downregulation of KIT transcription by Flavopiridol induces apoptosis in GIST cells. Molecular analysis revealed that this induction was based on downregulation of KIT and AKT (Sambol et al., 2006). This is consistent with our findings that exogenous miR‐221 and miR‐222 reduced phosphorylated KIT and total KIT protein, resulting in suppression of phosphorylated AKT and total AKT protein levels with little influence on phosphorylated or total MTOR. It seems that not only the phosphorylation status of AKT but also the expression level of AKT is affected in this signalling cascade as shown by others (Kim et al., 2011; Muhlenberg et al., 2009). Hereby, the extent of protein regulation resembled those of cellular proliferation and apoptosis induction of each miRNA in the evaluated GIST cell lines. Furthermore, the level of KIT inhibition correlated to the inhibition of downstream located protein AKT. These findings suggest that miRNA dependent effects on cellular proliferation and apoptosis are mediated by KIT and AKT but not by MTOR. To further analyse and generalise this effect also wildtype and PDGFRA mutated GISTs need to be investigated. Our cell lines are all KIT dependent exhibiting KIT mutation and expression (Appendices, Figure A1). However miR‐221 is able to induce apoptosis and cisplatin resistance by a mechanism involving the PI3K/AKT pathway (Zhao et al., 2013) in osteosarcoma representing a tumour entity that is not KIT dependent.

To further evaluate the molecular mechanisms inducing apoptosis, we analysed BCL2L11 (also known as BIM: “BCL2‐interacting mediator of apoptosis”) and BCL2 as potential targets. In physiological circumstances AKT enhances the survival of cells by blocking several pro‐apoptotic members of the BCL2 homology domain 3 (BH3)‐ only proteins such as BCL2L11 (Manning and Cantley, 2007). BCL2L11 acts as a direct antagonist of the pro‐survival protein BCL2 that functions as an inhibitor of apoptosis (Strasser et al., 2011). Recent studies have shown that 80% of GISTs express BCL2 and that the synergistic use of BCL2 inhibitor ABT‐737 and imatinib resulted in apoptosis in the cell lines GIST882 and GIST‐T1 (Reynoso et al., 2011). In gastric cancer cells the overexpression of miR‐15 and miR‐16 triggered apoptosis by downregulation of BCL2 (Cimmino et al., 2005). In our study, we could show that the overexpression of miR‐221 and miR‐222 led to the downregulation of the prosurvival protein BCL2.

Moreover, inhibition of constitutively activated KIT by imatinib triggered upregulation of BCL2L11 in GISTs (Gordon and Fisher, 2010). In our study the expression of BCL2L11 was almost unaffected.

Our results show that overexpression of miR‐221 and miR‐222 seems to destroy the prosurvival function of AKT by downregulating BCL2 resulting in an induction of apoptosis in GISTs. According to the databases PicTar (http://pictar.mdc‐berlin.de/) and microRNA.org (http://www.microrna.org/microrna/home.do) AKT and BCL2 are no direct predicted targets of miR‐221 and miR‐222 confirming that KIT itself as only direct target is the mediator of enhanced apoptosis (Betel et al., 2008; Krek et al., 2005). However, as one miRNA is able to target multiple mRNAs and therefore multiple pathways further analyses are needed to understand this regulation mechanism in more detail.

5. Conclusion

Currently, there are increasing treatment options for GISTs. Nevertheless novel therapeutic approaches are needed since complete responses are rare and most patients develop drug resistance (Blanke et al., 2008; Debiec‐Rychter et al., 2004). Alternative strategies aim at inhibiting the oncogenic signal of constitutively activated KIT regardless of mutation status and emerged therapy resistance. miRNAs display such an alternative therapy option. Here, we could show for the first time that not only miR‐222 but also miR‐221 are able to reduce viability and induce apoptosis in vitro in three different GIST cell lines. These cellular effects are mediated by the KIT, AKT and BCL2 signalling cascade. Therefore, miR‐221 and miR‐222 seem to be potential tumour suppressors in some GIST subtypes and further analyses may contribute to the development of new therapeutic options based on the post‐transcriptional regulation of KIT by miR‐221 and miR‐222.

Conflict of interest

There is no conflict of interest and source of funding to declare.

Supporting information

The following is the supplementary data related to this article:

Figure A1 All cell lines exhibited a positive KIT immunohistochemical staining and mutations in the KIT gene. A) Immunohistochemical staining of GIST cell line pellets showing that all three evaluated cell lines are immunohistochemically positive for KIT. B) Confirmation of the described genetic alteration of the different GIST cell lines. The cell line GIST882 harbours a homozygous mutation in KIT exon 13 (p.K642E) (Tuveson et al., 2001). The cell line GIST‐T1 is described with a heterozygous KIT exon 11 deletion (p.V560_Y578del) (Taguchi et al., 2002). The cell line GIST48 is characterised by a homozygous KIT exon 11 mutation (p.V560D) and a heterozygous secondary KIT exon 17 mutation (p.D820A) (Tuveson et al., 2001). K: Lysine, V: Valine, D: aspartic acid, bp: base pair.

Click here for additional data file. (206.2KB, jpg)

Acknowledgements

We appreciate the expert technical assistance of Elke Binot and Theresa Buhl.

Supplementary data 1.

1.1.

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.molonc.2015.03.013.

Ihle Michaela Angelika, Trautmann Marcel, Kuenstlinger Helen, Huss Sebastian, Heydt Carina, Fassunke Jana, Wardelmann Eva, Bauer Sebastian, Schildhaus Hans-Ulrich, Buettner Reinhard, Merkelbach-Bruse Sabine, (2015), miRNA-221 and miRNA-222 induce apoptosis via the KIT/AKT signalling pathway in gastrointestinal stromal tumours, Molecular Oncology, 9, doi: 10.1016/j.molonc.2015.03.013.

References

  1. Bartel, D.P. , 2004. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281–297. [DOI] [PubMed] [Google Scholar]
  2. Bauer, S. , Duensing, A. , Demetri, G.D. , Fletcher, J.A. , 2007. KIT oncogenic signaling mechanisms in imatinib-resistant gastrointestinal stromal tumor: PI3-kinase/AKT is a crucial survival pathway. Oncogene 26, 7560–7568. 10.1038/sj.onc.1210558 [DOI] [PubMed] [Google Scholar]
  3. Bauer, S. , Yu, L.K. , Demetri, G.D. , Fletcher, J.A. , 2006. Heat shock protein 90 inhibition in imatinib-resistant gastrointestinal stromal tumor. Cancer Res. 66, 9153–9161. 10.1158/0008-5472.CAN-06-0165 [DOI] [PubMed] [Google Scholar]
  4. Betel, D. , Wilson, M. , Gabow, A. , Marks, D.S. , Sander, C. , 2008. The microRNA.org resource: targets and expression. Nucleic Acids Res. 36, D149–D153. 10.1093/nar/gkm995 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Blanke, C.D. , Rankin, C. , Demetri, G.D. , Ryan, C.W. , von Mehren, M. , Benjamin, R.S. , Raymond, A.K. , Bramwell, V.H. , Baker, L.H. , Maki, R.G. , Tanaka, M. , Hecht, J.R. , Heinrich, M.C. , Fletcher, C.D. , Crowley, J.J. , Borden, E.C. , 2008. Phase III randomized, intergroup trial assessing imatinib mesylate at two dose levels in patients with unresectable or metastatic gastrointestinal stromal tumors expressing the kit receptor tyrosine kinase: S0033. J. Clin. Oncol. 26, 626–632. 10.1200/JCO.2007.13.4452 [DOI] [PubMed] [Google Scholar]
  6. Calin, G.A. , Sevignani, C. , Dumitru, C.D. , Hyslop, T. , Noch, E. , Yendamuri, S. , Shimizu, M. , Rattan, S. , Bullrich, F. , Negrini, M. , Croce, C.M. , 2004. Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc. Natl. Acad. Sci. USA 101, 2999–3004. 10.1073/pnas.0307323101 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Choi, H.-J. , Lee, H. , Kim, H. , Kwon, J.E. , Kang, H.J. , You, K.T. , Rhee, H. , Noh, S.H. , Paik, Y.-K. , Hyung, W.J. , Kim, H. , 2010. MicroRNA expression profile of gastrointestinal stromal tumors is distinguished by 14q loss and anatomic site. Int. J. Cancer J. Int. Cancer 126, 1640–1650. 10.1002/ijc.24897 [DOI] [PubMed] [Google Scholar]
  8. Chun-Zhi, Z. , Lei, H. , An-Ling, Z. , Yan-Chao, F. , Xiao, Y. , Guang-Xiu, W. , Zhi-Fan, J. , Pei-Yu, P. , Qing-Yu, Z. , Chun-Sheng, K. , 2010. MicroRNA-221 and microRNA-222 regulate gastric carcinoma cell proliferation and radioresistance by targeting PTEN. BMC Cancer 10, 10.1186/1471-2407-10-367 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cimmino, A. , Calin, G.A. , Fabbri, M. , Iorio, M.V. , Ferracin, M. , Shimizu, M. , Wojcik, S.E. , Aqeilan, R.I. , Zupo, S. , Dono, M. , Rassenti, L. , Alder, H. , Volinia, S. , Liu, C.-G. , Kipps, T.J. , Negrini, M. , Croce, C.M. , 2005. miR-15 and miR-16 induce apoptosis by targeting BCL2. Proc. Natl. Acad. Sci. U. S. A 102, 13944–13949. 10.1073/pnas.0506654102 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Debiec-Rychter, M. , Dumez, H. , Judson, I. , Wasag, B. , Verweij, J. , Brown, M. , Dimitrijevic, S. , Sciot, R. , Stul, M. , Vranck, H. , Scurr, M. , Hagemeijer, A. , van Glabbeke, M. , van Oosterom, A.T. , 2004. Use of c-KIT/PDGFRA mutational analysis to predict the clinical response to imatinib in patients with advanced gastrointestinal stromal tumours entered on phase I and II studies of the EORTC Soft Tissue and Bone Sarcoma Group. Eur. J. Cancer Oxf. Engl. 1990 40, 689–695. 10.1016/j.ejca.2003.11.025 [DOI] [PubMed] [Google Scholar]
  11. Demetri, G.D. , von Mehren, M. , Antonescu, C.R. , DeMatteo, R.P. , Ganjoo, K.N. , Maki, R.G. , Pisters, P.W.T. , Raut, C.P. , Riedel, R.F. , Schuetze, S. , Sundar, H.M. , Trent, J.C. , Wayne, J.D. , 2010. NCCN Task Force report: update on the management of patients with gastrointestinal stromal tumors. J. Natl. Compr. Cancer Netw. JNCCN 8, (Suppl 2) S1–S41. quiz S42–44 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Duensing, A. , Medeiros, F. , McConarty, B. , Joseph, N.E. , Panigrahy, D. , Singer, S. , Fletcher, C.D. , Demetri, G.D. , Fletcher, J.A. , 2004. Mechanisms of oncogenic KIT signal transduction in primary gastrointestinal stromal tumors (GISTs). Oncogene 23, 3999–4006. 10.1038/sj.onc.1207525 [DOI] [PubMed] [Google Scholar]
  13. Esau, C.C. , Monia, B.P. , 2007. Therapeutic potential for microRNAs. Adv. Drug Deliv. Rev. 59, 101–114. 10.1016/j.addr.2007.03.007 [DOI] [PubMed] [Google Scholar]
  14. Fan, R. , Zhong, J. , Zheng, S. , Wang, Z. , Xu, Y. , Li, S. , Zhou, J. , Yuan, F. , 2014. MicroRNA-218 inhibits gastrointestinal stromal tumor cell and invasion by targeting KIT. Tumour Biol. J. Int. Soc. Oncodevelopmental Biol. Med. 35, 4209–4217. 10.1007/s13277-013-1551-z [DOI] [PubMed] [Google Scholar]
  15. Felli, N. , Fontana, L. , Pelosi, E. , Botta, R. , Bonci, D. , Facchiano, F. , Liuzzi, F. , Lulli, V. , Morsilli, O. , Santoro, S. , Valtieri, M. , Calin, G.A. , Liu, C.G. , Sorrentino, A. , Croce, C.M. , Peschle, C. , 2005. MicroRNAs 221 and 222 inhibit normal erythropoiesis and erythroleukemic cell growth via kit receptor down-modulation. Proc. Natl. Acad. Sci. USA 102, 18081–18086. 10.1073/pnas.0506216102 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gan, R. , Yang, Y. , Yang, X. , Zhao, L. , Lu, J. , Meng, Q.H. , 2014. Downregulation of miR-221/222 enhances sensitivity of breast cancer cells to tamoxifen through upregulation of TIMP3. Cancer Gene Ther. 21, 290–296. 10.1038/cgt.2014.29 [DOI] [PubMed] [Google Scholar]
  17. Gits, C.M.M. , van Kuijk, P.F. , Jonkers, M.B.E. , Boersma, A.W.M. , van Ijcken, W.F. , Wozniak, A. , Sciot, R. , Rutkowski, P. , Schoffski, P. , Taguchi, T. , Mathijssen, R.H.J. , Verweij, J. , Sleijfer, S. , Debiec-Rychter, M. , Wiemer, E.A.C. , 2013. MiR-17-92 and miR-221/222 cluster members target KIT and ETV1 in human gastrointestinal stromal tumours. Br. J. Cancer 109, 1625–1635. 10.1038/bjc.2013.483 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gordon, P.M. , Fisher, D.E. , 2010. Role for the proapoptotic factor BIM in mediating imatinib-induced apoptosis in a c-KIT-dependent gastrointestinal stromal tumor cell line. J. Biol. Chem. 285, 14109–14114. 10.1074/jbc.M109.078592 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Haller, F. , von Heydebreck, A. , Zhang, J.D. , Gunawan, B. , Langer, C. , Ramadori, G. , Wiemann, S. , Sahin, O. , 2010. Localization- and mutation-dependent microRNA (miRNA) expression signatures in gastrointestinal stromal tumours (GISTs), with a cluster of co-expressed miRNAs located at 14q32.31. J. Pathol. 220, 71–86. 10.1002/path.2610 [DOI] [PubMed] [Google Scholar]
  20. He, H. , Jazdzewski, K. , Li, W. , Liyanarachchi, S. , Nagy, R. , Volinia, S. , Calin, G.A. , Liu, C.G. , Franssila, K. , Suster, S. , Kloos, R.T. , Croce, C.M. , de la Chapelle, A. , 2005. The role of microRNA genes in papillary thyroid carcinoma. Proc. Natl. Acad. Sci. USA 102, 19075–19080. 10.1073/pnas.0509603102 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kelly, L. , Bryan, K. , Kim, S.Y. , Janeway, K.A. , Killian, J.K. , Schildhaus, H.-U. , Miettinen, M. , Helman, L. , Meltzer, P.S. , van de Rijn, M. , Debiec-Rychter, M. , O'Sullivan, M. , 2013. Post-transcriptional dysregulation by miRNAs is implicated in the pathogenesis of gastrointestinal stromal tumor [GIST]. PloS One 8, 10.1371/journal.pone.0064102 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kim, W.K. , Park, M. , Kim, Y.K. , Tae, Y.K. , Yang, H.K. , Lee, J.M. , Kim, H. , 2011. MicroRNA-494 downregulates KIT and inhibits gastrointestinal stromal tumor cell proliferation. Clin. Cancer Res. 17, 7584–7594. 10.1158/1078-0432.CCR-11-0166 [DOI] [PubMed] [Google Scholar]
  23. Kneitz, B. , Krebs, M. , Kalogirou, C. , Schubert, M. , Joniau, S. , van Poppel, H. , Lerut, E. , Kneitz, S. , Scholz, C.J. , Strobel, P. , Gessler, M. , Riedmiller, H. , Spahn, M. , 2014. Survival in patients with high-risk prostate cancer is predicted by miR-221, which regulates proliferation, apoptosis, and invasion of prostate cancer cells by inhibiting IRF2 and SOCS3. Cancer Res. 74, 2591–2603. 10.1158/0008-5472.CAN-13-1606 [DOI] [PubMed] [Google Scholar]
  24. Koelz, M. , Lense, J. , Wrba, F. , Scheffler, M. , Dienes, H.P. , Odenthal, M. , 2011. Down-regulation of miR-221 and miR-222 correlates with pronounced Kit expression in gastrointestinal stromal tumors. Int. J. Oncol. 38, 503–511. 10.3892/ijo.2010.857 [DOI] [PubMed] [Google Scholar]
  25. Krek, A. , Grun, D. , Poy, M.N. , Wolf, R. , Rosenberg, L. , Epstein, E.J. , MacMenamin, P. , da Piedade, I. , Gunsalus, K.C. , Stoffel, M. , Rajewsky, N. , 2005. Combinatorial microRNA target predictions. Nat. Genet. 37, 10.1038/ng1536 [DOI] [PubMed] [Google Scholar]
  26. Kunstlinger, H. , Binot, E. , Merkelbach-Bruse, S. , Huss, S. , Wardelmann, E. , Buettner, R. , Schildhaus, H.U. , 2013. High-resolution melting analysis is a sensitive diagnostic tool to detect imatinib-resistant and imatinib-sensitive PDGFRA exon 18 mutations in gastrointestinal stromal tumors. Hum. Pathol. 45, 573–582. 10.1016/j.humpath.2013.10.025 [DOI] [PubMed] [Google Scholar]
  27. Lasota, J. , Miettinen, M. , 2006. KIT and PDGFRA mutations in gastrointestinal stromal tumors (GISTs). Semin. Diagn. Pathol. 23, 91–102. [DOI] [PubMed] [Google Scholar]
  28. Lasota, J. , Wozniak, A. , Sarlomo-Rikala, M. , Rys, J. , Kordek, R. , Nassar, A. , Sobin, L.H. , Miettinen, M. , 2000. Mutations in exons 9 and 13 of KIT gene are rare events in gastrointestinal stromal tumors. A study of 200 cases. Am. J. Pathol. 157, 1091–1095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lewis, B.P. , Burge, C.B. , Bartel, D.P. , 2005. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20. 10.1016/j.cell.2004.12.035 [DOI] [PubMed] [Google Scholar]
  30. Livak, K.J. , Schmittgen, T.D. , 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25, 402–408. [DOI] [PubMed] [Google Scholar]
  31. Manning, B.D. , Cantley, L.C. , 2007. AKT/PKB signaling: navigating downstream. Cell 129, 1261–1274. 10.1016/j.cell.2007.06.009 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miettinen, M. , Lasota, J. , 2006. Gastrointestinal stromal tumors: pathology and prognosis at different sites. Semin. Diagn. Pathol. 23, 70–83. [DOI] [PubMed] [Google Scholar]
  33. Muhlenberg, T. , Zhang, Y. , Wagner, A.J. , Grabellus, F. , Bradner, J. , Taeger, G. , Lang, H. , Taguchi, T. , Schuler, M. , Fletcher, J.A. , Bauer, S. , 2009. Inhibitors of deacetylases suppress oncogenic KIT signaling, acetylate HSP90, and induce apoptosis in gastrointestinal stromal tumors. Cancer Res. 69, 6941–6950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Niinuma, T. , Suzuki, H. , Nojima, M. , Nosho, K. , Yamamoto, H. , Takamaru, H. , Yamamoto, E. , Maruyama, R. , Nobuoka, T. , Miyazaki, Y. , Nishida, T. , Bamba, T. , Kanda, T. , Ajioka, Y. , Taguchi, T. , Okahara, S. , Takahashi, H. , Nishida, Y. , Hosokawa, M. , Hasegawa, T. , Tokino, T. , Hirata, K. , Imai, K. , Toyota, M. , Shinomura, Y. , 2012. Upregulation of miR-196a and HOTAIR drive malignant character in gastrointestinal stromal tumors. Cancer Res. 72, 1126–1136. 10.1158/0008-5472.CAN-11-1803 [DOI] [PubMed] [Google Scholar]
  35. Park, J.K. , Lee, E.J. , Esau, C. , Schmittgen, T.D. , 2009. Antisense inhibition of microRNA-21 or -221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic adenocarcinoma. Pancreas 38, e190–e199. 10.1097/MPA.0b013e3181ba82e1 [DOI] [PubMed] [Google Scholar]
  36. Reynoso, D. , Nolden, L.K. , Yang, D. , Dumont, S.N. , Conley, A.P. , Dumont, A.G.P. , Zhou, K. , Duensing, A. , Trent, J.C. , 2011. Synergistic induction of apoptosis by the Bcl-2 inhibitor ABT-737 and imatinib mesylate in gastrointestinal stromal tumor cells. Mol. Oncol. 5, 10.1016/j.molonc.2010.10.003 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rutkowski, P. , Bylina, E. , Klimczak, A. , Switaj, T. , Falkowski, S. , Kroc, J. , Lugowska, I. , Brzeskwiniewicz, M. , Melerowicz, W. , Osuch, C. , Mierzejewska, E. , Wasielewski, K. , Wozniak, A. , Grzesiakowska, U. , Nowecki, Z.I. , Siedlecki, J.A. , Limon, J. , 2012. The outcome and predictive factors of sunitinib therapy in advanced gastrointestinal stromal tumors (GIST) after imatinib failure - one institution study. BMC Cancer 12, 10.1186/1471-2407-12-107 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sambol, E.B. , Ambrosini, G. , Geha, R.C. , Kennealey, P.T. , Decarolis, P. , O'connor, R. , Wu, Y.V. , Motwani, M. , Chen, J.-H. , Schwartz, G.K. , Singer, S. , 2006. Flavopiridol targets c-KIT transcription and induces apoptosis in gastrointestinal stromal tumor cells. Cancer Res. 66, 5858–5866. 10.1158/0008-5472.CAN-05-2933 [DOI] [PubMed] [Google Scholar]
  39. Schneider, C.A. , Rasband, W.S. , Eliceiri, K.W. , 2012. NIH image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Strasser, A. , Cory, S. , Adams, J.M. , 2011. Deciphering the rules of programmed cell death to improve therapy of cancer and other diseases. EMBO J. 30, 3667–3683. 10.1038/emboj.2011.307 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Subramanian, S. , West, R.B. , Corless, C.L. , Ou, W. , Rubin, B.P. , Chu, K.M. , Leung, S.Y. , Yuen, S.T. , Zhu, S. , Hernandez-Boussard, T. , Montgomery, K. , Nielsen, T.O. , Patel, R.M. , Goldblum, J.R. , Heinrich, M.C. , Fletcher, J.A. , van de Rijn, M. , 2004. Gastrointestinal stromal tumors (GISTs) with KIT and PDGFRA mutations have distinct gene expression profiles. Oncogene 23, 7780–7790. 10.1038/sj.onc.1208056 [DOI] [PubMed] [Google Scholar]
  42. Suzuki, H. , Maruyama, R. , Yamamoto, E. , Kai, M. , 2013. Epigenetic alteration and microRNA dysregulation in cancer. Front. Genet. 4, 10.3389/fgene.2013.00258 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taguchi, T. , Sonobe, H. , Toyonaga, S. , Yamasaki, I. , Shuin, T. , Takano, A. , Araki, K. , Akimaru, K. , Yuri, K. , 2002. Conventional and molecular cytogenetic characterization of a new human cell line, GIST-T1, established from gastrointestinal stromal tumor. Lab Invest 82, 663–665. [DOI] [PubMed] [Google Scholar]
  44. Taylor, M.L. , Metcalfe, D.D. , 2000. Kit signal transduction. Hematol. Oncol. Clin. North Am. 14, 517–535. [DOI] [PubMed] [Google Scholar]
  45. Tuveson, D.A. , Willis, N.A. , Jacks, T. , Griffin, J.D. , Singer, S. , Fletcher, C.D. , Fletcher, J.A. , Demetri, G.D. , 2001. STI571 inactivation of the gastrointestinal stromal tumor c-KIT oncoprotein: biological and clinical implications. Oncogene 20, 5054–5058. 10.1038/sj.onc.1204704 [DOI] [PubMed] [Google Scholar]
  46. Visone, R. , Russo, L. , Pallante, P. , De Martino, I. , Ferraro, A. , Leone, V. , Borbone, E. , Petrocca, F. , Alder, H. , Croce, C.M. , Fusco, A. , 2007. MicroRNAs (miR)-221 and miR-222, both overexpressed in human thyroid papillary carcinomas, regulate p27Kip1 protein levels and cell cycle. Endocr. Relat. Cancer 14, 791–798. 10.1677/ERC-07-0129 [DOI] [PubMed] [Google Scholar]
  47. Wardelmann, E. , Merkelbach-Bruse, S. , Pauls, K. , Thomas, N. , Schildhaus, H.-U. , Heinicke, T. , Speidel, N. , Pietsch, T. , Buettner, R. , Pink, D. , Reichardt, P. , Hohenberger, P. , 2006. Polyclonal evolution of multiple secondary KIT mutations in gastrointestinal stromal tumors under treatment with imatinib mesylate. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 12, 1743–1749. 10.1158/1078-0432.CCR-05-1211 [DOI] [PubMed] [Google Scholar]
  48. Yang, C.-J. , Shen, W.G. , Liu, C.-J. , Chen, Y.-W. , Lu, H.-H. , Tsai, M.-M. , Lin, S.-C. , 2011. miR-221 and miR-222 expression increased the growth and tumorigenesis of oral carcinoma cells. J. Oral Pathol. Med. Off. Publ. Int. Assoc. Oral Pathol. Am. Acad. Oral Pathol. 40, 560–566. 10.1111/j.1600-0714.2010.01005.x [DOI] [PubMed] [Google Scholar]
  49. Yang, J. , Ikezoe, T. , Nishioka, C. , Takezaki, Y. , Hanazaki, K. , Taguchi, T. , Yokoyama, A. , 2011. Long-term exposure of gastrointestinal stromal tumor cells to sunitinib induces epigenetic silencing of the PTEN gene. Int. J. Cancer 130, 959–966. 10.1002/ijc.26095 [DOI] [PubMed] [Google Scholar]
  50. Yu, B. , Zhou, S. , Wang, Y. , Qian, T. , Ding, G. , Ding, F. , Gu, X. , 2012. miR-221 and miR-222 promote Schwann cell proliferation and migration by targeting LASS2 after sciatic nerve injury. J. Cell Sci. 125, 2675–2683. 10.1242/jcs.098996 [DOI] [PubMed] [Google Scholar]
  51. Zhao, G. , Cai, C. , Yang, T. , Qiu, X. , Liao, B. , Li, W. , Ji, Z. , Zhao, J. , Zhao, H. , Guo, M. , Ma, Q. , Xiao, C. , Fan, Q. , Ma, B. , 2013. MicroRNA-221 induces cell survival and cisplatin resistance through PI3K/Akt pathway in human osteosarcoma. PloS One 8, 10.1371/journal.pone.0053906 [DOI] [PMC free article] [PubMed] [Google Scholar]

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Figure A1 All cell lines exhibited a positive KIT immunohistochemical staining and mutations in the KIT gene. A) Immunohistochemical staining of GIST cell line pellets showing that all three evaluated cell lines are immunohistochemically positive for KIT. B) Confirmation of the described genetic alteration of the different GIST cell lines. The cell line GIST882 harbours a homozygous mutation in KIT exon 13 (p.K642E) (Tuveson et al., 2001). The cell line GIST‐T1 is described with a heterozygous KIT exon 11 deletion (p.V560_Y578del) (Taguchi et al., 2002). The cell line GIST48 is characterised by a homozygous KIT exon 11 mutation (p.V560D) and a heterozygous secondary KIT exon 17 mutation (p.D820A) (Tuveson et al., 2001). K: Lysine, V: Valine, D: aspartic acid, bp: base pair.

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