Survivin Is a Novel Transcription Regulator of KIT and ...

Survivin Is a Novel Transcription Regulator of KIT and Is Downregulated by miRNA494 in Gastrointestinal Stromal Tumors

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SeongJu Yun , Won Kyu Kim , Yujin Kwon , Mi Jang , Sebastian Bauer , and Hoguen Kim

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Department of Pathology and Brain Korea 21 PLUS Projects for Medical Science, Yonsei University 2

College of Medicine, Seoul, Republic of Korea; Sarcoma Center, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany and German Cancer Consortium (DKTK), Heidelberg, Germany SeongJu Yun and Won Kyu Kim contributed equally to this article

Running Title: MiR-494 targets survivin and KIT in GISTs

*Correspondence Hoguen Kim, M.D., Ph.D. Professor of Pathology Yonsei University, College of Medicine CPO Box 8044, Seoul, Korea Tel: +82-2-2228-1761/ Fax: +82-2-363-5263/ Email: [email protected]

Supporting Information (Materials and Methods)

Materials and Methods Cell Lines and Culture Colon cancer cell lines (DLD-1, Colo320DM) and GIST cell lines (GIST882 [K642E], GIST430 [exon 11 in-frame deletion] were used for the analysis. GIST cell lines were developed by Jonathan Fletcher, Brigham & Womens Hospital in Boston, MA. The colon cancer cell lines expressing wildtype KIT (DLD-1 and Colo320DM) and the HeLa cell line were purchased from the Korean Cell Line Bank (Cancer Research Institute, Seoul, Korea). The DLD-1 and Colo320DM cell lines were maintained in RPMI1640 medium supplemented with 10% FBS (Life Technologies) and 1% penicillin/streptomycin. The GIST430 cell line was cultured in Iscove's Modified Dulbecco's Medium supplemented with 15% FCS (Life Technologies) and 1% penicillin/streptomycin. The GIST882 cell line was cultured in RPMI1640 medium supplemented with 15% FBS (Life Technologies) and 1% penicillin/streptomycin. Microscopic examination, qPCR, and western blot analysis were regularly performed to monitor the morphology, growth patterns, and expression of KIT proteins in all cell lines. The cell lines were also regularly screened to monitor for mycoplasma infection on a bi-monthly basis.

Dual Luciferase Assay Dual luciferase assays were performed in HeLa cells by cotransfecting Renilla or Firefly luciferase vectors conjugated with the 3’-UTR of each gene or the KIT promoter region with siNC, miR-494, or the miR-494 inhibitor. Two days after transfection, the luciferase activity was measured according to the manufacturer’s instructions.

Quantitative (q)PCR

cDNA synthesis was performed using the M-MLV reverse transcriptase (Life Technologies). Expression of each transcript (KIT, survivin, and 18S rRNA) was measured using SYBR Premix Ex Taq II (TaKaRa) and the ABI PRISM 7500 Sequence Detector (Applied Biosystems). The amounts of KIT mRNA and survivin mRNA were normalized to that of 18S rRNA. For a TaqMan miRNA qPCR assay, total RNA was extracted from two GIST cells and 35 GIST frozen tissues using TRIZOL (Life Technology). cDNA synthesis and subsequent qPCR analysis were performed using TaqMan miRNA assay kit (Applied Biosystems) and the ABI PRISM 7500 Sequence Detector. The miR-494 level was normalized to the RNU6B level.

Western Blotting and Immunofluorescence Protein extracts were prepared using a passive lysis buffer (Promega) with a protease inhibitor cocktail (Roche). The membranes were incubated with primary antibodies against GAPDH (Trevigen), FLAG (Sigma), KIT (Dako), CKS1B (Thermo), AKT (Cell Signaling Technology), phospho-AKT (Cell Signaling Technology), phospho-KIT (Cell Signaling Technology), phospho-STAT (Cell Signaling Technology), survivin (Cell Signaling Technology), survivin (Novus Biologicals), ERK (Santa Cruz Biotechnology), STAT3 (Santa Cruz Biotechnology), and phospho-ERK (Santa Cruz Biotechnology) overnight at 4°C. Western blot images were recorded using an LAS 4000 imager (Fujifilm). The relative density of each band was quantified using ImageJ software (NIH). GIST882 or GIST430 cells were seeded onto a chambered coverglass. The cells were fixed with 4% paraformaldehyde for 30 minutes and then permeabilized in 0.1% Triton X-100 in PBS. The slides were incubated with primary antibody overnight at 4°C and were incubated for 1 hour with the fluorescent conjugated secondary antibody (Invitrogen). An LSM700 confocal microscope (Carl Zeiss) was used to acquire all images.

Colony Formation Assay

Approximately 40,000 GIST430 and GIST882 cells transfected with siNC, miR-494, or siSurvivin were seeded for colony formation. The colonies were stained with crystal violet and counted using Colony V1.1 software (Fujifilm) after 14 days of incubation.

Cell-cycle and Apoptosis Analysis For flow cytometry analysis, GIST882 and GIST430 cells were transfected with siNC, miR-494, or siSurvivin. For cell cycle analysis, at 5 days after transfection cells were fixed in 95% ethanol at 4°C for 15 min and each sample was stained using propidium iodide (Abcam). For apoptosis analysis, at 5 days after transfection cells were stained using propidium iodide and FITC conjugated Annexin V (BD Biosciences) according to the manufacturer’s protocol. All samples were analyzed using an FACS Calibur cell analyzer (BD Biosciences).

Supporting Information Figure S1

Supporting Information Figure S1. Microarray results were validated using randomly selected cell cycle related genes (n=6) and qPCR. An independent set of GIST430 and GIST882 cells treated with miR-494 was used for this validation.


Supporting Information Figure S2. Survivin expression is regulated by miR-494 independent of changes in KIT expression. (a) Flag-conjugated survivin expression vectors with and without the 3’-UTR and a mutant KIT (K642E) expression vector were constructed. EC (extracellular domain), TM (transmembrane domain), JM (juxtamembrane domain), TK1 (tyrosine kinase 1 domain), KI (kinase insert domain), and TK2 (tyrosine kinase 2 domain). (b) Western blotting analysis of survivin expression in HeLa cells transfected with the survivin expression vector with nontargeting siRNA (siNC) or miR-494. (c) Western blotting analysis of survivin expression in HeLa cells transfected with the survivin expression vector containing the 3’-UTR with siNC or miR-494. (d) Western blotting analysis of miR-494–mediated survivin inhibition in GIST882 cells when KIT expression was rescued by overexpression of mutant KIT after RNAi depletion.


Supporting Information Figure S3. Levels of total KIT, survivin, and miR-494 expression in tissues from 35 GIST cases.


Supporting Information Figure S4. The representative photomicrographs of GIST430 and GIST882 cells. Representative photomicrographs of GIST430 and GIST882 cells at days 3 and 5 after transfection of siNC, miR-494, or miR-494 with the survivin expression vector.


Supporting Information Figure S5. Survivin-mediated transcriptional regulation of KIT was evaulated in two colon cancer cell lines (DLD-1 and Colo320DM) expressing wild-type KIT. (a, b) Wild-type KIT expression and KIT mRNA expression were measured 3 days after transfection of siSurvivin. (c, d) Wild-type KIT expression and KIT mRNA expression were meausred 2 days after transfection of a survivin expression vector.


Supporting Information Figure S6. Kaplan-Meier analysis of 113 GIST tissues according to the survivin expression. (a) Representative immunohistochemical analysis of survivin in GIST tissues. (b) Relationship between survivin expression and survival (disease-free and overall survival) of GIST patients.


Supporting Information Figure S7. Kaplan-Meier analysis of 34 GIST tissues according to the miR-494 expression. Disease-free and overall survival analysis in 34 GIST patients according to the miR-494 expression.


Supporting Information Figure S8. MicroRNA-494 synergistically suppresses the PI3K pathway in GISTs by inhibiting the activation loop between KIT and survivin.

Supporting Information Table 1. Primers used for qPCR, semi-qPCR and construction of expression vectors Primers used for qPCR and semi-qPCR Gene Direction Sequence Forward 5'-GAGGTAGTGACGAAAAATAACAAT 18S ribosomal RNA (18S rRNA) Reverse 5'-TTGCCCTCCAATGGATCCT Forward 5'-AGGACCACCGCATCTCTACAT Survivin Reverse 5'-AAGTCTGGCTCGTTCTCAGTG Forward 5'-GGCGACGAGATTAGGCTGTT KIT Reverse 5'-GCCTTTTCCGTGATCCATTC Forward 5'-GGGAAAACGTGTATGAAAACCT ChiP KIT promoter fragment 1 Reverse 5'-ATTGTTTCTGCTCAATTTCTCCAC Forward 5'-TAACGCTCCCCTCCCCATC ChiP KIT promoter fragment 2 Reverse 5'-TTGTGAAGCTGGGCACCAC Forward 5'-AGAGCTGGAACGTGGACCAGAG ChiP KIT promoter fragment 3 Reverse 5'-AGGCACAGGCTTCGCCGAGTAGT Forward 5'-CTAGGTTGCAGACTTTTTGCTTTC ChiP KIT promoter fragment 4 Reverse 5'-GAGTCAGGGAACGCAGAGTCC Primers used for construction or mutagenesis of luciferase vectors Gene Direction Sequence Forward 5'-GCAAAGGAAACCAACAATAAGAAG Reporter vector conjugated with survivin 3’-UTR Reverse 5'-GACAATATTTTAAAAACCCAGTAGGG Forward 5'-ACTACCCAAGAAACCAAAGAAATGAA Reporter vector conjugated with CKS1B 3’-UTR Reverse 5'-TCCTCTTTAATCAAGGCTTTTAACAT Forward 5'-CAGGAGACCTACCCTCCACA Reporter vector conjugated with DNMT3B 3’-UTR Reverse 5'-AGGCATCCGTCATCTTTCAG Forward 5'-CTGATCTTTGCAGGGTGGTC Reporter vector conjugated with FOXM1 3’-UTR Reverse 5'-TCTCAAGCCTCCACCTGAGT Forward 5'-ACATTTTCAAATTAGATCCGGCAACTGTGCTCTTGTTTTG Reporter vector conjugated with mutated survivin 3’-UTR Reverse 5'-CAAAACAAGAGCACAGTTGCCGGATCTAATTTGAAAATGT Forward 5'-GTGGTGACTTGCGGATTTATCCGGCAGTGTACTGGAAAC Reporter vector conjugated with mutated CKS1B 3’-UTR Reverse 5'-GTTTCCAGTACACTGCCGGATAAATCCGCAAGTCACCAC Forward 5'-GAGCCCTAGCCCTAAAACTGTCGT KIT promoter fragment 1 Reverse 5'-GTTAGGAAATTGAGCCCCGACATT Forward 5'-TCCTAACGCTCCCCTCCCCATC KIT promoter fragment 2 Reverse 5'-GTTCCCGGACCAAGGAGGTGCT Forward 5'-CACATCCCAGGGGTGGAAAGGT KIT promoter fragment 3 Reverse 5'-GCAGTCCTCTCTCCGGATGCAC Forward 5'-GGTGCATCCGGAGAGAGGACTG KIT promoter fragment 4 Reverse 5'-TTGCAGAAAGTTCCACGCCATCT Primers used for construction of expression vectors Gene Direction Sequence Forward 5'-ATGGGTGCCCCGACGTTGCCCCCT Survivin expression vector Reverse 5'-CCAGAGGCCTCAATCCA

Supporting Information Table 2. Pathway analysis using commonly down-regulated genes (n=443) by miR-494 in GIST430 and GIST882 cells Category

Term

P-Value

Benjamini P-value*

GOTERM_BP_FAT

cell division

2.90E-11

4.70E-08

GOTERM_BP_FAT

nuclear division

6.00E-10

4.80E-07

GOTERM_BP_FAT

mitosis

6.00E-10

4.80E-07

GOTERM_BP_FAT

cell cycle

6.30E-10

3.40E-07

GOTERM_BP_FAT

M phase of mitotic cell cycle

8.30E-10

3.40E-07

GOTERM_BP_FAT

organelle fission

1.20E-09

4.00E-07

GOTERM_BP_FAT

M phase

1.60E-09

4.40E-07

GOTERM_BP_FAT

cell cycle phase

4.00E-08

9.20E-06

GOTERM_BP_FAT

mitotic cell cycle

7.50E-08

1.50E-05

GOTERM_BP_FAT

cell cycle process

1.50E-07

2.60E-05

GOTERM_BP_FAT

chromosome segregation

2.10E-06

3.30E-04

GOTERM_BP_FAT

mitotic sister chromatid segregation

3.30E-06

4.80E-04

GOTERM_BP_FAT

sister chromatid segregation

4.00E-06

5.40E-04

GOTERM_BP_FAT

DNA replication

4.60E-05

5.70E-03

GOTERM_BP_FAT

DNA metabolic process

5.40E-05

6.30E-03

GOTERM_BP_FAT

DNA packaging

5.50E-05

5.90E-03

GOTERM_BP_FAT

mitotic chromosome condensation

6.80E-05

6.90E-03

GOTERM_BP_FAT

chromosome condensation

7.70E-05

7.30E-03

GOTERM_BP_FAT

chromosome organization

2.30E-04

2.00E-02

GOTERM_BP_FAT

hemopoiesis

1.30E-03

1.10E-01

* Multiple testing P-value adjusted based on Benjamini-Hochberg correction

Supporting Information Table 3. Clinicopathological features of 113 GISTs according to the survivin overexpression Category

Variables

GISTs with KIT mutation Case Survivin expression no. Strong (%) Moderate (%) Weak (%) (n=93) (n=8) (n=38) (n=47)

GISTs lacking KIT mutation Case Survivin expression P-value no. P-value Moderate (%) Weak (%) (n=20) (n=11) (n=9)

Age (years) 63.1±8.64

56.3±11.7

57.4±12.7

0.371

53.3±10.9

56.5±11.4

0.603

Gender Male Female

47 46

5 (62.5) 3 (37.5)

21 (55.3) 17 (44.7)

21 (44.7) 26 (55.3)

0.577

11 9

8 (72.7) 3 (27.3)

3 (33.3) 6 (66.7)

0.175

Stomach Duodenum

67 2

7 (87.5) 0 (0)

25 (65.8) 0 (0)

35 (74.4) 2 (4.3)

0.145

16 0

9 (81.8) 0 (0.0)

7 (77.8) 0 (0.0)

0.625

16

1 (12.5)

11 (28.9)

4 (8.5)

4

2 (18.2)

2 (22.2)

8

0 (0)

2 (5.3)

6 (12.8)

0

0 (0.0)

0 (0.0)

≤5.0 5.1∼10.0 ＞10.0

53 32

1 (12.5) 4 (50)

21 (55.3) 15 (39.5)

31 (66.0) 13 (27.6)

13 5

7 (63.6) 2 (18.2)

6 (66.7) 3 (33.3)

8

3 (37.5)

2 (5.2)

3 (6.4)

2

2 (18.2)

0 (0)

≤5 6∼10 ＞10

48 17

1 (12.5) 0 (0)

21 (55.3) 6 (15.8)

26 (55.3) 11 (23.4)

15 3

10 (90.9) 0 (0.0)

5 (55.6) 3 (33.3)

28

7 (87.5)

11 (28.9)

10 (21.3)

2

1 (9.1)

1 (11.1)

35

0 (0)

15 (39.5)

20 (42.6)

11

7 (63.6)

4 (44.5)

18 40

1 (12.5) 7 (87.5)

6 (15.8) 17 (44.7)

11 (23.4) 16 (34.0)

4 5

2 (18.2) 2 (18.2)

2 (22.2) 3 (33.3)

Negative Positive

74 19

5 (62.5) 3 (37.5)

29 (76.3) 9 (23.7)

40 (85.1) 7 (14.9)

0.23

19 1

11 (100) 0 (0.0)

8 (88.9) 1 (11.1)

0.45

Alive Expired

77 16

4 (50) 4 (50)

30 (78.9) 8 (21.1)

43 (91.5) 4 (8.5)

0.016

19 1

11 (100) 0 (0)

8 (88.9) 1 (11.1)

0.45

Location

Jejunum/ Ileum Rectum Tumor size (cm)

0.018

0.642

Mitotic index (/50HPFs) 0.013

0.103

Grade (Modified NIH) Very low & Low Intermediate High

0.068

0.835

Recurrence /Metastasis

Survivial

Supporting Information Table 4. Multivariable analysis of 113 GISTs for the impact of variable clinicopathologic factors on recurrence/metastasis and survival Recurrence/metastasis

Category

Variables

Gender

Female

1

Male

1.234(0.415~3.669)

Stomach

1

Location

HR (95% Cl)

Survival P-value

HR (95% Cl)

P-value

1 0.705

1.521(0.333~6.957)

0.14

1

Duodenum

3.483(0.345~35.212)

0.29

68.512(0.803~5846.82)

0.062

Jejunum/Ileum

5.584(1.888~16.516)

0.002

11.571(1.283~104.321)

0.029

1.165(0.236~5.741)

0.851

15.244(0.802~289.738)

0.07

Tumor size

Rectum ≤ 5.0

(cm)

5.1∼10.0

2.307(0.811~6.563)

0.117

0.487(0.077~3.077)

0.445

＞10.0

6.298(1.599~24.805)

0.009

0.331(0.028~3.991)

0.384

Mitotic index

≤ 5

1

(/50HPFs)

6∼10

1.874(0.251~13.991)

0.54

25.139(1.048~602.811)

0.047

＞10

2.710(0.312~23.563)

0.366

14.614(1.037~205.964)

0.047

Grade Very low & Low (Modified NIH) Intermediate High Mutation

1

1

1

1

1

2.864(0.490~16.743)

0.243

7609(0~3.420E+114)

0.945

2.650(0.445~15.791).

0.284

14319(0~6.404E+114)

0.941

Wild

1

KIT

7.079(0.836~59.974)

0.073

0.923(0.059~14.381)

0.995

PDGFRA

0

0.987

0(0~8.729E+283)

0.976

Survivin

Weak

1

expression

Moderate

0.965(0.323~2.885)

0.949

2.041(0.296~14.047)

0.469

(IHC)

Strong

2.134(0.449~10.145)

0.34

16.981(1.434~201.085)

0.025

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Supporting Information Table 5. Clinicopathological features of 34 GISTs according to the miRNA-494 expression Category

Variables

miRNA-494 expression Case no. Strong (%) Moderate (%) Weak (%) (n=34) (n=8) (n=18) (n=8)

P-value

Age (years) 64.0±8.81

53.4±12.4

64.0±9.97

0.350

Gender Male Female

16 18

3 (37.5) 5 (62.5)

7 (38.9) 11 (61.1)

6 (75.0) 2 (25.0)

0.256

Stomach Duodenum

23 2

5 (62.5) 1 (12.5)

11 (61.1) 1 (5.6)

7 (87.5) 0 (0)

0.662

9

2 (25)

6 (33.3)

1 (12.5)

Location

Jejunum/ Ileum Tumor size (cm) ≤5.0

9

7 (87.5)

2 (11.1)

0 (0)

5.1∼10.0

17

1 (12.5)

10 (55.6)

6 (75.0)

＞10.0

8

0 (0)

6 (33.3)

2 (25.0)

≤5

14

6 (75.0)

8 (44.5)

0 (0)

6∼10 ＞10

7

1 (12.5)

4 (22.2)

2 (25.0)

13

1 (12.5)

6 (33.3)

6 (75.0)

Very low & Low

6

5 (62.5)

1 (5.6)

0 (0)

Intermediate High

5 23

1 (12.5) 2 (25.0)

4 (22.2) 13 (72.2)

0 (0) 8 (100)

Negative

25

7 (87.5)

12 (66.7)

6 (75.0)

Positive

9

1 (12.5)

6 (33.3)

2 (25.0)

Alive Expired

29 5

8 (100) 0 (0)

17 (94.4) 1 (5.6)

4 (50.0) 4 (50.0)

0.001

Mitotic index (/50HPFs) 0.024

Grade (Modified NIH) 0.003

Recurrence /Metastasis 0.689

Survivial 0.013

Supporting Information Table 6. Multivariable analysis of 34 GISTs for the impact of variable clinicopathologic factors on recurrence/metastasis and survival Recurrence/metastasis

Survival

Category

Variables

Gender

Female

1

Male

1.859(0.249~13.900)

Location

Stomach

1

Duodenum

0

0.998

1.000(0~3416.533)

1.000

4.063(0.562~29.359)

0.165

1.000(0.076~13.090)

1.000

Tumor size

Jejunum/Ileum ≤ 5.0

(cm)

5.1∼10.0

0.953(0.11~84.041)

0.983

1.000(0.006~178.744)

1.000

＞10.0

1.744(0.025~124.055)

0.798

1.000(0.004~268.421)

1.000

Mitotic index

≤ 5

1

(/50HPFs)

6∼10

6.309(0.180~221.577)

0.310

1.000(0.025~39.508)

1.000

＞10

0.849(0.055~13.215)

0.907

1.000(0.045~22.471)

1.000

Grade Very low & Low (Modified NIH) Intermediate

P-value

HR (95% Cl)

P-value

1 0.546

1.000(0.125~8.005)

1.000

1

1

1

1

1

1

3.962(0~9.57E+145)

0.994

1.000(0.006~162.898)

1.000

0.942

1.000(0.009~116.087)

1.000

Wild

39910(0~2.059E+129). 1

KIT

0

0.984

1.000(0.001~1085.245)

1.000

PDGFRA

0

0.997

0

1.000

High Mutation

HR (95% Cl)

1

MiRNA-494

Weak

1

expression

Moderate

0.587(0.029~11.700)

0.727

1.000(0.057~17.587)

1 1.000

Strong

1.716(0.015~202.499)

0.825

1.000(0.009~106.774)

1.000