Cancer Cell 20

Supplemental Information

Coordinated Regulation of Polycomb Group Complexes through microRNAs in Cancer

Qi Cao, Ram-Shankar Mani, Bushra Ateeq, Saravana M. Dhanasekaren, Irfan A. Asangani, John R. Prensner, Jung H. Kim, J. Chad Brenner, Xiaojun Jing, Xuhong Cao, Rui Wang, Yong Li, Arun Dahiya, Lei Wang, Mithil Pandhi, Robert J. Lonigro, Yi-Mi

Wu, Scott A.Tomlins, Nallasivam Palanisamy, Zhaohui Qin, Jindan Yu, Christopher A. Maher, Sooryanarayana Varambally, Arul M. Chinnaiyan

Inventory

Supplemental Data

Figure S1, related to Figure 1.

Table S1, related to Figure 1.

Figure S2, related to Figure 2.

Figure S3, related to Figure 3.

Figure S4, related to Figure 4.

Table S2 (provided as excel files), related to Figure 4.

Table S3 (provided as excel files), related to Figure 4.

Table S4 (provided as excel files), related to Figure 4.

Figure S5, related to Figure 5.

Figure S6, related to Figure 6. 

Supplemental Experimental Procedures

Supplemental References

1  

Coordinated Regulation of Polycomb Group Complexes through microRNAs in Cancer

Qi Cao1,2, Ram-Shankar Mani1,2, Bushra Ateeq1,2, Saravana M. Dhanasekaren1,2, Irfan A.

Asangani1,2, John R. Prensner1,2, Jung H. Kim1,2, J. Chad Brenner1,2, Xiaojun Jing1,2, Xuhong Cao1,3, Rui Wang1,2, Yong Li1,2, Arun Dahiya1, Lei Wang1,2, Mithil Pandhi1,

Robert J. Lonigro1,2, Yi-Mi Wu1,2, Scott A.Tomlins1,2, Nallasivam Palanisamy1,2, Zhaohui Qin7, Jindan Yu1,2,#, Christopher A. Maher1,2,4, Sooryanarayana Varambally1,2,6*, Arul M.

Chinnaiyan1,2,3,5,6,*

1Michigan Center for Translational Pathology, Ann Arbor, MI, 48109, USA, 2Department of Pathology, University of Michigan, Ann Arbor, MI, 48109, USA, 3Howard Hughes

Medical Institute, University of Michigan Medical School, Ann Arbor, MI, 48109, USA, 4Center for Computational Medicine and Bioinformatics, Ann Arbor, MI, 48109, USA,

5Department of Urology, University of Michigan, Ann Arbor, MI, 48109, USA, 6Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, MI,

48109, USA 7Department of Biostatistics and Bioinformatics, Center for Comprehensive Informatics,

Emory University, Atlanta, GA 30329 RUNNING TITLE: microRNAs link PRC2 to PRC1 *These authors share senior authorship #Present address: Division of Hematology/Oncology, Northwestern University, Robert H. Lurie Comprehensive Cancer Center, Chicago, IL 60611, United States.

To whom correspondence should be addressed:

Arul M. Chinnaiyan, M.D., Ph.D.

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Figure S1, related to Figure 1; PcG proteins are regulated by miRNAs.

(A) Dicer knockdown increases PRC2 and PRC1 protein levels. PC3 cells were transfected with Dicer specific duplexes (siDicer-d2, siDicer-d3, siDicer-d4), control siRNA (siControl) or mock treated. EZH2, SUZ12, EED, BMI1, RING2 and RING1 protein levels were examined by immunoblot analysis. β-Actin was used as control. (B) EZH2-repressed miRNAs have predicted PRC1 binding sites and 13 of the 14 identified miRNAs fall into several major family clusters.

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Only microRNAs with probe p-values < 0.05 were including in this analysis. Values represent the ratio of average normalized intensity values between two samples. DU145 cells are evaluated with a siRNA targeting EZH2 relative to a control siRNA. The remaining columns represent the comparison of microRNA expression in DU145 cells against PrEC, RWPE, HME and H16N2, or SKBr3 and BT-549 against HME and H16N2.

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BT-

54

9

cont

rol m

iR

miR

-19b

miR

-20a

miR

-93

miR

-106

a

miR

-106

b

miR

-27a

miR

-183

miR

-200

b

BMI1

RING2

-Actin

BMI1

RING2

-Actin

DU

14

5

cont

rol m

iR

miR

-17

miR

-454

miR

-200

cA

B

p16 p21 p19 HOXC13

Fol

d of

Enr

ichm

ent

0

1

2

3

Control miR

miR-200b

miR-203

BMI1

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Figure S2, related to Figure 2; EZH2-repressed miRNAs inhibit expression of BMI1 and RING2.

(A) BT-549 and DU145 cells were transfected with indicated miRNAs. miR-200b and miR-200c were used as positive controls which repress BMI1 and RING2 as shown in main figure 2A. BMI1 and RING2 protein levels were examined by immunoblot analysis. β-Actin was used as a loading control. (B) ChIP-qPCR analysis of BMI1 occupancy at indicated BMI1 target gene promoter regions in BT-549 cells transfected with indicated miRNAs. (C) Predicted binding site of miR-181a and miR-181b to RING2 3’UTR (left panel); mutants of RING2 3’UTR (middle and right panels). (D) Predicted binding site of miR-200b and miR-200c to RING2 3’UTR (left panel); mutants of RING2 3’UTR (middle and right panels). (E) Predicted binding site of miR-200b and miR-200c to BMI1 3’UTR (left panel); mutants of BMI1 3’UTR (middle and right panels). (F) Predicted binding site of miR-203 to BMI1 3’UTR (left panel); mutants of BMI1 3’UTR (middle and right panels). Red letters indicated the mutation sites. All bar graphs are shown with ±SEM.

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8  

miR

-181

a1,b

1

miR

-181

a2

miR

-181

b2

miR

-200

a,b

miR

-200

c

miR

-203

MYT

GAPDH

Fol

d of

Enr

ichm

ent

0

5

10

15

20

25

30

35 Anti-H3K27me3

E

0

1

2

3

4

5

6anti-EZH2anti-BMI1

Fol

d of

Enr

ichm

ent

miR

-181

a1,b

1

miR

-181

a2

miR

-181

b2

miR

-200

a,b

miR

-200

c

miR

-203

MYT

GAPDH

5siControlsiEZH2

0

1

2

3

4

6

Fol

d of

Enr

ichm

ent

miR

-200

b,a

WNT1

F

Figure S3, Continued

Figure S3, related to Figure 2; Depletion of EZH2 restores EZH2-repressed miRNA levels.

(A) Depletion of EZH2 restores EZH2-downregulated miRNA levels. EZH2 was depleted in DU145 (left panel) or SKB3 (right panel) cells by stable knockdown with EZH2 shRNA or overexpression of miR-101. Cells treated with scrambled shRNA or vector alone were used as controls. miR-181a, miR-181b, miR- 200a, miR-200b, miR-200c, miR-203, miR-217 and miR-219 expression levels were examined by Taqman miRNA qPCR, and normalized to U6. EZH2 transcript levels were examined by Sybr green qPCR, and normalized to GAPDH. (B) Deletion of the SET domain abrogates EZH2-mediated repression of miRNAs. H16N2 cells were transfected with wild-type EZH2, EZH2 lacking the SET domain (EZH2Δ SET) or vector adenovirus. miRNA expression levels were measured by Taqman miRNA qPCR and normalized to U6. (C) Taqman miRNA qPCR analysis of indicated miRs in DU145 cells infected by adeno-Vec or adeno-EZH2, and treated with or without 5uM DZNep for 48 hours. (D) As in (C), except SAHA and 5-aza-dC were used in DU145 cells. (E) ChIP-qPCR was performed to examine the occupancy of H3K27me3 (left panel), EZH2 and BMI1 (right panel) on miR-181a, miR-181b, miR-200a, miR- 200b and miR-203 regions. EZH2 targets MYT was used as positive controls and GAPDH served as negative control for ChIP-qPCR. (F) Knockdown of EZH2 decreases H3K27me3 occupancy at EZH2 repressed miRNAs. BT-549 cells were transfected with control siRNA (siControl) or EZH2 siRNA duplex (siEZH2) followed by ChIP with anti-H3K27me3 antibody or control IgG. ChIP-qPCR was performed to examine the occupancy of H3K27me3 on miR-181a, miR-181b, miR-200a, miR-200b and miR-203 regions. EZH2 targets ADRB2, WNT1 and RUNX3 were used as positive controls and GAPDH served as negative control for ChIP-qPCR. All bar graphs are shown with ±SEM.

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Figure S4, related to Figure 4; EZH2-repressed miRNA overexpression attenuates cell proliferation and metastasis and abrogates cancer stem cell phenotype.

(A) EZH2-regulated miRNAs reduce cell proliferation. DU145 cells were transfected with control miR, miR-101, miR-181a, miR-181b, miR-200a, miR-200b, miR-200c, miR-203, miR-217 and miR-219, and were disassociated by trypsin at indicated time points. Cell numbers for each treatment and each time point were determined by cell counter in triplicates.*p<0.005, **p<0.01 (student’s t-test).(B) Baydon chamber invasion assays using RWPE controls, RWPE-UBE2L3-KRAS transfected cells with indicated miRNAs (top panel), or RWPE-SLC45A3-BRAF transfected cells with indicated miRNAs (bottom panel). (C) Proliferation assays by cell counting using RWPE-UBE2L3-KRAS (top panel), or RWPE-SLC45A3-BRAF transfected with indicated miRNAs (bottom panel). (D) EZH2-repressed miRNAs inhibit stem-cell properties. BT-549 cells were transfected with control miR, miR-101, miR-181a, miR-181b, miR-200a, miR-200b, miR-200c, miR-203, miR-217 and miR-219. RNA was extracted and Sybr green and qPCR were performed to examine NANOG, OCT4, KLF4, SOX2 and c-MYC. CD24, CTBP2 and PHF8 were used as negative controls. (E) Relative expression levels of iPS factors NANOG, OCT4, KLF4, SOX2 and c-MYC, and polycomb group genes EZH2, BMI1 and RING2 in hES cell lines E002, H7 and cancer cell lines BT-549 and DU145 cells. For each indicated genes, Ct values were normalized with Ct values of GAPDH. All bar graphs are shown with ±SEM.

Table S2, related to Figure 4; mRNA profiling results intersected with predicted miRNA:mRNA interactions in BT-549 cells. (Attached as supplemental Excel file).

Table S3, related to Figure 4; mRNA profiling results intersected with predicted miRNA:mRNA interactions in DU145 cells. (Attached as supplemental Excel file).

Table S4, related to Figure4; MCM analysis of genes suppressed by miR203 overexpression in DU145 cells. (Attached as supplemental Excel file).

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A

0

2

4

6

8

10

12

14

16

miR-vec miR-101 miR-200b miR-203 parental

miR-101

miR-200b

miR-203 Nom

aliz

ed ta

rget

/U6

Scram

ble

BMI1

-sh1

BMI1

-sh2

BMI1

-sh3

BMI1

-sh4

BMI1

-sh5

RING2-

sh1

Scram

ble

RING2-

sh2

RING2-

sh3

RING2-

sh4

Scram

ble-2

RING2BMI1Actin Actin

B

C

RING2

Actin

miR

-NT

miR

-181

b

0

2

4

6

8

10

12

miR‐NT miR‐181bNor

mal

ized

miR

-181

b/U

6

Figure S5, related to Figure 5; Generating DU145 cells stably overexpressing PRC2-repressed miRNAs or knocking down BMI1 and RING2.

(A) Validation of miRNA overexpression experiments. miR-101, miR-200b and miR- 203 levels were examined by qPCR in DU145 cells stably expressing miR-101, miR-200b, miR-203, or vector alone (miR-vec). (B) Generating BMI1 (left panel), and RING2 (right panel) stably knockdown DU145 cells by infecting with indicated BMI1 or RING2 specific shRNA lentivirus. (C) Generating miR-181b stably overexpressing DU145 cells. qPCR analyses (left panel) or immunoblot analyses were performed using miR-NT (non-targeting) or miR-181b stable cells. All bar graphs are shown with ±SEM.

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EZH2

BMI1

RING2

-Actin

ubiquityl-H2A

Benign n=4

PCA n=5

MET n=5

A

y = 0.1638x + 1.216R = 0.8572

0

2

4

6

8

10

EZH2

BM

I1

B

y = 0.1022x + 1.6182R = 0.7381

0

2

4

6

8

EZH2

y = 1.1076x + 13.631R = 0.6944

0

20

40

60

80

100

EZH2

0 10 20 30 40 50

0 10 20 30 40 50

0 10 20 30 40 50

Fol

d E

nric

hmen

t

PCA

MET

0

1

2

3

4

5

6

KIAA00

66CNR

miR

-181

a1,b

1

miR

-200

c

miR

-203

miR

-181

b2

miR

-200

a,b

22.85

29.18

21.30

C

uH2A

RIN

G2

RING1

Figure S6, related to Figure 6; PRC components are upregulated in prostate cancer.

(A) Expression of EZH2, BMI1, RING2 and ubiquityl-H2A in prostate tissues. Immunoblot analysis was performed with protein lysates from 4 benign prostate tissues, 5 clinical localized prostate cancer (PCA) and 5 metastatic prostate cancer (MET) tissues (samples are unique from those in Fig.4B) with anti-EZH2, BMI1, RING2 or ubiquityl-H2A antibodies. β-Actin was used as control. (B) Protein levels of EZH2, BMI1, RING2, and uH2A, but not RING1, are elevated in prostate cancer. Protein levels of EZH2, BMI1, RING2, and uH2A were measured by immunoblot followed by band intensity quantification with ImageJ in tissue samples derived from Fig.4B. The X-axis displays normalized EZH2 and the Y-axis displays normalized BMI1, RING2, or uH2A protein levels from Fig. 4B. EZH2 levels are positively correlated with all three PRC1-related proteins. The respective R values are displayed. (C) ChIP assay of a metastatic prostate cancer tissue shows increased occupancy of H3K27me3 at EZH2-repressed miRNAs. ChIP-qPCR for H3K27me3 on the regions of miR-181a1,b1, miR-181b2 miR-200a,b, miR-200c, miR- 203 were performed on a localized (PCA) or metastatic (MET) prostate cancer tissues. CNR (an

EZH2 target) was used as positive control and KIAA0066 was used as negative control. All bar graphs are shown with ±SEM.

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Supplemental Experimental Procedures

Immunoblot Analyses

Normal and prostate cancer tissues were homogenized in NP-40 lysis buffer containing

50 mM Tris-HCl, pH 7.4, 1% Nonidet P-40 (Sigma) and complete protease inhibitor

cocktail (Roche, Indianapolis, IN). The breast cancer cell lines BT-549 and prostate

cancer cell DU145 were transfected with miRNAs or controls. The breast cell lines

H16N2 were transfected with antagomiRs or negative controls. Post 72 hours transfection,

cells were homogenized in NP40 lysis buffer, and complete proteinase inhibitor mixture

(Roche, Indianapolis, IN). Ten micrograms of each protein extract were boiled in sample

buffer, separated by SDS-PAGE, and transferred onto Polyvinylidene Difluoride

membrane (GE Healthcare, Piscataway, NJ). The membrane was incubated for one hour

in blocking buffer [Tris-buffered saline, 0.1% Tween (TBS-T), 5% nonfat dry milk] and

incubated overnight at 4ºC with the following: anti-EZH2 mouse monoclonal (1:1000 in

blocking buffer, BD Biosciences Cat # 612667, San Jose, CA), anti-BMI1 mouse

monoclonal (1:500, Millipore, Cat#: 05-637), anti-RING2 rabbit polyclonal (1:1000,

Proteintech Group, Cat#: 16031-1-AP), anti-RING1 mouse monoclonal (1:1000,

Millipore, Cat#: 05-1362), anti-uH2A mouse monoclonal (1:1000, ProteinTech Group,

Chicago, IL, Cat#: 16031-1-AP), anti-trimethyl-H3K27 rabbit polyclonal (1:2000,

Upstate, Cat #: 07-449), anti-total Histone H3 rabbit polyclonal (1:5000, Cell Signaling,

Cat #: 9715), anti-p16INK4A mouse monoclonal (1:500, Biosciences Cat #551154), anti-

p21 rabbit monoclonal (1:500, cell Signaling, Cat #: 2947S) and anti-β-Actin mouse

monoclonal antibody (1:10000, Sigma, Cat #: A5316-500ul). Following a wash with

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TBS-T, the blot was incubated with horseradish peroxidase-conjugated secondary

antibody and the signals visualized by enhanced chemiluminescence system as per

manufacturer’s instructions (GE Healthcare).

Quantitative Real-Time PCR Assays

Total RNA was isolated from BT-549 and DU145 cells that were transfected either with

miRNAs, or control miRNA (Qiagen). Quantitative PCR (QPCR) and miRNA Taqman

qPCR were performed as described  (Varambally et al., 2008). The primer sequences for

the transcript analyzed are provided below.

Gene Name Forward primer Reverse primer EZH2 TGCAGTTGCTTCAGTACCCATAAT ATCCCCGTGTACTTTCCCATCATAAT BMI1 CCAGGGCTTTTCAAAAATGA CCGATCCAATCTGTTCTGGT RING2 ATGGCACACAGAACAATGGA ATGTGGCAACCCAAAATGAT NANOG CTGCCGTCTCTGGCTATAGATAA TACGAATACATCTTCATCACCAA OCT4 GACTATGCACAACGAGAGGATTT AGTGAAGTGAGGGCTCCCATAG KLF4 GTCATCAGCGTCAGCAAAGG CTCCTGCTTGATCTTGGGG SOX2 CCATGCACCGCTACGAC CTGCGAGTAGGACATGCTGTAG CD24 TCTAAATGTGGCTATTCTGATCCA TATTTGGGAAGTGAAGACTGGAA CTBP2 GAGGACTGCTTTGCCTTTTG TGTCCAATCGCTGTCTCTTG PHF8 TTCTGCCCAGCTTTGCTTAT ACTACCCTCTGCTGCCTTGA  

Chromatin Immunoprecipitation (ChIP) Assays

The ChIP assays were performed as described  (Varambally et al., 2008; Yu et al., 2007).

BMI1 antibody was obtained from Millipore (cat#: 05-637), H3K27me3 antibody was

obtained from Abcam (cat#: ab6002) and EZH2 antibody was obtained from Cell

Signaling (cat#: 5246). The primer sequences for the promoters analyzed are provided

below

 

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Gene Name Forward primer Reverse primer ADBR2 GTGACTTTATGCCCCTTTAGAGACAA GAAGGGCTACAACTGGAACTGGAATA WNT1 GTTTCTGCTACGCTGCTGCT CACCAGCTCACTTACCACCA RUNX3 TGTCCCGGGATCCTCTTCT TAGAGACGTTGGTGCGGAAAT miR-181a,b-1 TCATGCTTCTTATTTGTCTTCTTTTG GGTTAGCCATAGGGTACAATCAAC miR-181a-2 ACCATTCAAAGACATTTTCTCAGAC CTGATAGCCCTTCTTCATTCTCC miR-181b-2 CCAATGTCAATGTTATGGACACCT CATTGATCAGTGAGTTGATTCAGA miR-200b,a CGTCTGGCCAGGACACTT AATGCTGCCCAGTAAGATGG miR-200c AGGGCTCACCAGGAAGTGT CCATCATTACCCGGCAGTAT miR-203 GAGGACTGCTTTGCCTTTTG TGTCCAATCGCTGTCTCTTG KIAA0066 CTAGGAGGGTGGAGGTAGGG GCCCCAAACAGGAGTAATGA CNR GCAGAGCTCTCCGTAGTCAG AACAGGCTGGGGCCATACAG P16 GCACTCAAACACGCCTTTGC AGAGCCAGCGTTGGCAAGGA P19 GCAGGCTCAGAGCAGACC CGGGTCTCCCCCTAACTC P21 GGGGCGGTTGTATATCAGG GGCTCCACAAGGAACTGACT HoxC13 GAGGGAACCCCAGGAGAC CGCTCTCCACCTCTCAGC GAPDH TACTAGCGGTTTTACGGGCG TCGAACAGGAGGAGCAGAGAGCGA

LMNA Obtained from Diagenode pp-1011-500 MB Obtained from Diagenode pp-1006-500