SUPPLEMENTAL INFORMATION

The neuronal repellent SLIT2 is a target for repression

by EZH2 in prostate cancer
Jindan Yu#,1,2,3, Qi Cao1,2, Jianjun Yu1, Longtao Wu#, Ashraf Dallol6, James Li1, Guoan Chen1,
Catherine Grasso1,2, Xuhong Cao1,2, Robert J. Lonigro1,3, Sooryanarayana Varambally1, 2, Rohit
Mehra1,2, Nallasivam Palanisamy1,2, Jane Y. Wu#, Farida Latif6, Arul M. Chinnaiyan1,2,3,4,5

Division of Hematology/Oncology, Northwestern University, Robert H. Lurie Comprehensive

Cancer Center, Chicago, IL 60611, United States. 1Michigan Center for Translational
Pathology,2Department of Pathology, 3Comprehensive Cancer Center, 4Howard Hughes Medical
Institute, 5Department of Urology, University of Michigan, Ann Arbor, Michigan, USA.
6
Department of Medical and Molecular Genetics, Institute of Biomedical Research, University of
Birmingham, Edgbaston, B15 2TT, UK

Content:
Supplemental Figures S1: ChIP-on-chip shows PRC2 binding to the SLIT2 promoter.
Supplemental Figures S2: HDAC inhibitor SAHA reduces H3K27me3 and increases H3K4me3
mark at the promoters of EZH2 target genes.
Supplemental Figures S3: PRC1 proteins bind to the SLIT2 promoter.
Supplemental Figures S4: Knockdown of EZH2 increases SLIT2 expression
Supplemental Figures S5: SLIT2 upregulation by SAHA is not due to changes in cell cycle.
Supplemental Figures S6: Negative correlation between SLIT2 and EZH2 expression in cancer
microarray datasets.
Supplemental Figures S7: Kaplan-Meier Analysis of PSA Recurrence-Free Survival by SLIT2
status.
Supplementary Table S1: Univariate associations between PSA recurrence and clinical variables.
Supplementary Table S2: Multivariate analysis of SLIT2 as a predictor of PSA recurrence.
Supplementary Table S3: Primers used for RT-PCR, and ChIP-PCR analysis in this study.

Supplementary Figures:

Figure S1. ChIP-on-chip shows PRC2 binding to the SLIT2 promoter. (a) Images of hybridized
arrays showing five probes representing the proximal promoter region of the SLIT2 gene in 2
replicate ChIP-on-chip analyses of 3mH3K27 and one ChIP-on-chip analysis of SUZ12. Red
signal indicates strong enrichment. (b) Chromosomal view of 3mH3K27 signal near SLIT2
promoter revealed by ChIP-on-chip experiment. The plots show unprocessed enrichment ratios
for all probes near SLIT2 genomic region (ChIP-enriched vs. input DNA). Chromosome
positions are from NCBI build 35 of the human genome. Refseq genes are shown above the
chromosomal view with exons represented by boxes. The position and ChIP signal of SLIT2 are
denoted by arrows.

Figure S2. HDAC inhibitor SAHA reduces H3K27me3 and increases H3K4me3 mark at the
promoters of EZH2 target genes. By contrast, DNA methylation inhibitor 5-Aza has marginal
effects. LNCaP cells were treated with DMSO, 5 M 5-Aza or 5 M SAHA for 36 hours before
ChIP using anti-H3K4me3 (Millipore) and anti-H3K27me3 (Millipore) antibodies. CNR1
promoter was used as a positive control, and KIAA0066 introgenic region as a negative control.

Figure S3. PRC1 proteins bind to the SLIT2 promoter. ChIP was performed in LNCaP cells
using antibodies against PRC1 proteins BMI1, RING1, RING2, and IgG control. ChIP-PCR was
performed using primers specific to the promoter regions of SLIT2 and of known EZH2 target
genes CNR1 and ADRB2.

Figure S4. Knockdown of EZH2 increases SLIT2 expression. DU145 and PC-3 cells were
subjected to RNA interference against EZH2 for 48 hours. Cells were then harvested for qRTPCR analysis of EZH2 (a) and SLIT2 (b) transcript levels. GAPDH was used as a control.

Figure S5. SLIT2 upregulation by SAHA is not due to changes in cell cycle. (a) SAHA
treatments results in slightly increased G1 cell cycle arrest. LNCaP cells were treated with
SAHA or DMSO control for 48hrs before flow cytometry analysis. (b) SLIT2 is upregulated by
SAHA regardless of increases G1 cell cycle arrest. qRT-PCR analysis was used to determine the
expression levels of SLIT2 and control cell cycle genes CCND1 and CCNE1 that are not
sensitive to SAHA.

Figure S6. Negative correlation between SLIT2 and EZH2 expression in cancer microarray
datasets. EZH2 and SLIT2 expression in normalized unites are shown from all profiled samples
in two large-scale gene expression studies: (a) our in-house dataset and (b) Yu et al. dataset.
Sample classes are indicated according to the color scale. Scatter plots of EZH2 and SLIT2
expression across all of the samples in each dataset are shown (right).

Figure S7. Kaplan-Meier Analysis of PSA Recurrence-Free Survival by SLIT2 status (Product
Score >= 270 vs. Product Score < 270). A Kaplan-Meier plot of recurrence-free survival
illustrates the magnitude of the differences between outcomes for the sets of patients with high
and low product scores (staining intensity multiplied by percentage of staining). Product Score
270 was chosen as a cutoff because it represents the median product score for the entire cohort.

Supplementary Table S1. Univariate associations between PSA recurrence and clinical
variables. Univariate Cox regression models were used to investigate associations between each
clinical variable (including SLIT2 product score) and PSA recurrence. SLIT2 product score is
significantly associated with hazard of PSA recurrence (p = 0.04).
Variable
Gland Weight
Multifocal
Race
Age
Tumor Size
Surgical Margin
PSA
PSA
SVI
DRE
EPE
Gleason Score
Path Stage
Median Product Score

Comparison
1-unit increase
Yes vs. No
Caucasian vs. African-American

> 60 vs. <= 60

>= 1.5 vs. < 1.5
Positive vs. Negative
4-10 vs. <= 4
> 10 vs. <= 4
Present vs. Absent
T2 or higher vs. T1 or lower
Positive vs. Negative
>= 7 vs. < 7
T3 or Higher vs. T2 or Lower
>= 270 vs. < 270

Hazard Ratio
1
1.16
0.79
2.03
1.75
2.67
4.56
8.41
4.25
1.11
3.06
2.98
3.06
0.49

CI
(0.98,1.02)
(0.55,2.43)
(0.3,2.08)
(0.99,4.14)
(0.84,3.66)
(1.33,5.33)
(1.06,19.52)
(1.86,38)
(1.59,11.35)
(0.56,2.21)
(1.53,6.14)
(1.23,7.24)
(1.53,6.14)
(0.25,0.98)

P-Value
P=0.69
P=0.7
P=0.64
P=0.05
P=0.14
P=0.006
P=0.04
P=0.006
P=0.004
P=0.76
P=0.002
P=0.02
P=0.002
P=0.04

Supplementary Table S2. Multivariate analysis of SLIT2 as a predictor of PSA recurrence.

Multivariate model incorporating age, PSA, Gleason score, path stage, and SLIT2 product score
was used as predictors of PSA recurrence. After adjusting for the other variables, SLIT2 was not
a statistically significant predictor of risk of PSA recurrence (P=0.13). Taken together with
univariate analysis, this data suggests that SLIT2 has some association with clinical outcome but
not at an independent level.
Variable
Age
PSA
Gleason Sum
Path Stage
SLIT2 Product Score

Comparison
> 60 vs. <= 60
<= 10 vs. > 10
>= 7 vs. < 7
T3 or Higher vs. T2 or Lower
>= 270 vs. < 270

Hazard Ratio
1.47
0.62
2.5
2.14
0.57

95% CI
(0.71,3.05)
(0.29,1.33)
(1.01,6.21)
(1.04,4.39)
(0.28,1.17)

P-value
P=0.29
P=0.23
P=0.03
P=0.04
P=0.13

Supplementary Table S3: Primers use for RT-PCR, and ChIP-PCR analysis in this study.
WNT1_pF1
WNT1_pR1
NUP214_E34
NUP214_E34
B-ACTIN_Q pF1
B-ACTIN_Q pR1
SLIT2_promt F2
SLIT2_promt R2
SLIT2_cDNA F1
SLIT2_cDNA R1
EZH2 F1
EZH2 R1
CCNE1 F2
CCNE1 R2
CCND1 F1
CCND1 R1
IL-3 pF1
IL-3 pR1
KIAA0079_E23 F1
KIAA0079_E23 R1
CNR1 pF1
CNR1 pR1
ADRB2pQ_p F1
ADRB2pQ_p R1
KIAA0066_pF2
KIAA0066_pR2

ACCCGTCAGCTCTCGGCTCA
TGCAGTTGCGGCGACTTTGG
CAGTGAGGTCTCAGCATCAGCA
CTGGAGGCTATGGGGGTACTTG
AGTGTGGTCCTGCGACTTCTAAG
CCTGGGCTTGAGAGGTAGAGTGT
GAGAGGCCGCGTGAGTGAGC
CAGCGGGCCCGTAGCAGA
GCGGCGGGGAAAGATGC
AGCGCCAGCCCGTGACAG
TGCAGTTGCTTCAGTACCCATAAT
ATCCCCGTGTACTTTCCCATCATAAT
ATCCTCCAAAGTTGCACCAG
AGGGGACTTAAACGCCACTT
CCCTCGGTGTCCTACTTCAA
AGGAAGCGGTCCAGGTAGTT
ACTGATCTTGAGTACTAGAAAGTCATGGA
GGAAGGATCTTTATCTGACATGGAA
TCTGTCATGTCCTGCTGATGGA
GCCCAAGAAGGACTGACCACTT
GCAGAGCTCTCCGTAGTCAG
AACAGGCTGGGGCCATACAG
TCGGGTGAGGCAAGTTCGGAGTA
GAAACGCGGCCAGAAACAAACAC
CTAGGAGGGTGGAGGTAGGG
GCCCCAAACAGGAGTAATGA

ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR
RT-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR
ChIP-PCR