SUPPLEMENTARY INFORMATION

A Chromosome 3q25

LXN RARRES1

B Transmembrane CPA binding domain site RARRES1 TS 1

RARRES1 TS 2

LXN

C

RARRES1 TS 1 RARRES1 TS 2 Transmembrane LXN domain

RARRES1 TS 1 RARRES1 TS 2 LXN CPA binding site

RARRES1 TS 1 RARRES1 TS 2 Key residues LXN needed to interact with CPA4

RARRES1 TS 1 RARRES1 TS 2 LXN

RARRES1 TS 1 RARRES1 TS 2 LXN

RARRES1 TS 1 RARRES1 TS 2 LXN

1 Supplementary Figure S1. RARRES1 and LXN lie adjacent and differ only by the lack of a transmembrane domain in LXN. (a) RARRES1 and LXN lie adjacent on chromosome 3q25.32. (b)

RARRES1 and LXN share 30% amino acid similarity predominantly in the C-terminus, share a CPA binding domain but differ in the N-terminus, where LXN lacks a transmembrane domain. (c) Alignment of transcript 1 (TS1; NP_996846.1) and 2 (TS2; NP_002879.2) of RARRES1 and LXN (NP_064554.3) aligned by the Clustal W method using the Lasergene MegAlign software. Underlined is the N’- terminal transmembrane domain present in RARRES1 (dotted line; 1-61aa), the CPA4 binding domain present in both RARRES1 TS1 and LXN (solid line; 240-249aa; taken from ) and the key amino acids from LXN that are required to bind to CPA4 (240, 242, 247-249aa), which are fully conserved in

RARRES1 (double solid line).

2 A RARRES1

B LATEXIN

Supplementary Figure S2. RARRES1 and LXN possess CpG islands within their first exon. (a)

Diagram depicting the location of the CpG islands relative to exon 1 of the RARRES1 and (b) LXN genes identified by bioinformatics analysis using the EMBOSS CpGPlot software. PCR primers and sequencing primers used for pyrosequencing analysis are shown. Individual CpG sites are depicted by vertical lines.

3 4 A

5 B BPH Stem vs CaP Stem vs Gene Probe BPH Comm CaP Comm Stem vs Comm Stem vs RARRES1 206391_at 0.046 0.122 0.011 Comm RARRES1 206392_s_at 0.010 0.058 0.002 P < 0.05 RARRES1 221872_at 0.002 0.038 0.0005 P < 0.01 LXN 218729_at 0.026 0.119 0.008 P < 0.001

C BPH Stem vs BPH Comm vs Gene Probe BPH vs CaP CaP Stem CaPComm BPH vs RARRES1 206391_at 0.872 0.354 0.512 CaP RARRES1 206392_s_at 0.787 0.365 0.455 RARRES1 221872_at 0.614 0.381 0.259 LXN 218729_at 0.962 0.251 0.544

6 Supplementary Figure S3. RARRES1 and LXN are expressed at low levels in prostate epithelial cultures enriched for stem cells. (a) Affymetrix gene expression data showing the expression of

RARRES1 and LXN in primary prostate epithelial cultures enriched for SC and CB cells derived from human BPH (n=7) and CaP (n=12) tissues containing a minimum Gleason score 7 pathology. (BS: benign SC; MS: malignant SC; BC: benign committed cell; MC: malignant committed cell). (b)

Statistical significance values of stem vs. committed and (c) BPH vs. CaP were measured by the

Student’s T-test (Unpaired, two-tailed). Significance values are highlighted in grey.

7 A RARRES1 100 90 Prostate Enriched Epithelial Cultures 80 70 60 50 40 30 20

D 10 N

A 0

M 0% 100% SC TA CB SC TA CB SC TA CB SC TA CB e

t Controls Y003/10 Y088/09 Y089/09 PE671 h

y BPH Enriched Cultures CaP Enriched Cultures l a t i o n

(

% B

) LXN 100 90 Prostate Enriched Epithelial Cultures 80 70 60 50 40 30 20

D 10 N

A 0

M 0% 100% SC TA CB SC TA CB SC TA CB SC TA CB e

t Y003/10 Y088/09 Y089/09 PE671 h Controls y

l Benign Enriched Cultures Cancer Enriched Cultures a t i o n

( % )

Supplementary Figure S4. RARRES1 and LXN are not hypermethylated in enriched primary prostate epithelial cultures. (a) Pyrosequencing analysis data showing the percentage promoter methylation of RARRES1 and (b) LXN in enriched SC, TA and CB cell subpopulations of primary prostate epithelial cultures derived from BPH (n=2) and CaP (n=2). (Bars = single CpG sites; black line

= average of individual CpG sites; error bars expressed as standard deviation of n=3 technical replicates).

8 A 600 500 DMSO 10nM 80 100nM 500nM 1uM 60

40

R 20 e l a t i v

e 0

E 24 48 72 96 x p r

e Time (Hrs) s s i

o B 200 n 180 DMSO 10nM 160 100nM 500nM 140 1uM 120 100 80 60

R 40 e l

a 20 t i v 0 e

E 24 48 72 96 x p r e s s i Supplementary Figureo S5. The expression of RARRES1 and LXN is induced by retinoic acid in n basal primary prostate epithelial cultures. (a) qRT-PCR expression data quantifying the expression of RARRES1 and (b) LXN after treatment of a primary prostate CaP culture with various concentrations of atRA over a time course. Expression relative to an RPLPO control gene; n=3 technical replicates; error bars expressed as range of the mean.

9 Knockdown Overexpression Overexpression A 1.2 B 100000 C 100000

LNCaP PNT1a PNT1a n n n o o 1.0 o 10000 10000 i i i s s s s s s 1000

0.8 1000 e e e r r r p p p x x 0.6 x 100 100 E E E

e e 0.4 e 10 10 v v v i i i t t t a a a l l 0.2 0.09 l 1 1 e e 0.0 e R R 0.0 R 0 0 Scr RR1 Scr LXN Vect RR1 Vect LXN Vect RR1 Vect LXN

D Scr RR1 E Vect RR1 F Vect RR1

RARRES1 RARRES1 RARRES1

β-actin β-actin β-actin 1.0 0.2

Scr LXN Vect LXN Vect LXN

LXN LXN LXN

β-actin β-actin β-actin 1.0 0.0

Supplementary Figure S6. RARRES1 and LXN are knocked down and over-expressed in prostate epithelial cell lines. qRT-PCR data showing expression of (a) RARRES1 and LXN relative to an 18S endogenous control gene at 48 hr after transfection with 10nM scrambled siRNA (Scr),

Rarres1 siRNA (RR1) or LXN siRNA (LXN) in PNT1a cells. (b) RARRES1 and LXN relative to a

GAPDH endogenous control gene over time in PNT1a and (c) LNCaP cells transfected with empty vector (Vect), Rarres1 (RR1) or LXN (LXN) vectors (all values are normalised to Scr/Vect, which is set at 1 for each time point; UD: undetectable expression after 40 cycles; n=3 technical replicates; error bars expressed as range of the mean). (d) Quantified Western blot data showing protein levels of

RARRES1 (33kDa) and LXN (29kDa) over time in Scr, RR1 and LXN samples from PNT1a cells. (e)

Protein levels of RARRES1 and LXN at 72 hr in PNT1a and (f) LNCaP cells transfected with Vect,

RR1 or LXN vectors. Protein expression was quantified relative to a β-actin (42kDa) loading control and relative to the scr control at each time point (set at 1; values below each blot).

10 A Average cells per field KNOCKDOWN No Matrigel With Matrigel Motility Invasion - PNT1a 77.5 3.3 + MDA-MB-231 48.3 83.0 PNT1a Scr 72.5 5.8 RARRES1 77.5 9.2 LXN 28.3 6.7

B Average cells per field OVEREXPRESSION No Matrigel With Matrigel Motility Invasion - PNT1a 144.0 17.3 + MDA-MB-231 31.7 71.6 LNCaP Vector 3.8 4.8 RARRES1 5.7 5.9 LXN 7.9 6.3

Supplementary Figure S7. RARRES1 regulates invasion but LXN regulates motility of prostate epithelial cell lines. Raw data tables showing the average number of motile and invasive cells per field of view, and the respective relative invasion percentages after transfection (a) of PNT1a cells with

10nM scrambled (Scr), RARRES1 or LXN siRNA or (b) LNCaP cells with empty vector, RARRES1 or

LXN vectors.

11 Knockdown A 1.2 Primary n Scr LXN o 1.0 B Scr RR1 i s

s 0.8 e RARRES1 LXN r p

x 0.6 E β-actin e 0.4 β-actin v i t

a 1.0 0.0 l 0.2 0.03 1.0 0.0 e 0.0

R 0.0 Scr RR1 Scr LXN

Supplementary Figure S8. SiRNA knockdown efficiencies of RARRES1 and LXN in primary cells. qRT-PCR data showing expression of (a) RARRES1 and (b) LXN relative to an 18S endogenous control gene at 48 hr after transfection with 50nM scrambled siRNA (Scr), RARRES1 siRNA (RR1) or LXN siRNA (LXN) in a primary CaP culture (all values are normalised to Scr, which is set at 1 for each time point; n=3 technical replicates; error bars expressed as range of the mean).

Quantified Western blot data showing protein levels of (c) RARRES1 (33kDa) and (d) LXN (29kDa) at

72hrs in primary cultures transfected with Scr, RR1 or LXN. Protein expression was quantified relative to a β-actin (42kDa) loading control and relative to the Scr control at each time point (set at 1; values below each blot).

12 A Number of cells per field B Number of cells per field KNOCKDOWN No Matrigel With Matrigel KNOCKDOWN No Matrigel With Matrigel Motility Invasion Motility Invasion Media Scr 90.2 57.7 99.3 36.0 H135/11 RR1 112.3 102.3 H159/12 Scr 182.8 92.7 Gleason 9 RR1 127.1 100.4 LXN 91.8 79.8 Scr 93.9 51.6 LXN 123.3 85.1 H146/12 Rb Media 88.2 35.4 Gleason 8 RR1 89.0 64.3 Scr 167.6 92.3 LXN 91.4 59.9 H158/12 RR1 168.6 106.3 Media 23.5 12.1 BPH Scr 33.0 20.8 LXN 151.0 101.9 H062/12 RR1 53.1 33.3 Scr 123.7 42.4 Cancer Gleason 7 H169/12 RR1 75.5 75.2 LXN 64.3 39.8 LXN 154.0 118.8 Media 78.1 44.6 Media 1.9 0.7 H144/12 Scr 104.1 51.3 Gleason 7 RR1 106.8 67.3 H189/12 † Scr 1.7 0.8 RR1 2.1 1.5 LXN 104.6 80.8 LXN 2.7 1.4 Media 3.7 2.0 H046/11 † Scr 2.3 1.4 Gleason 8 RR1 2.6 2.0 LXN 6.0 6.6

Supplementary Figure S9. RARRES1 regulates invasion but LXN regulates motility of primary prostate epithelial cultures. Raw data tables showing the average number of motile and invasive cells per field of view from (a) BPH and (b) CaP cultures, and the respective relative invasion percentages. The samples marked with a † indicate experiments where 25 000 cells instead of 250

000 cells were plated per insert.

13 A LNCaP DDK (CPA4) pulldown

DDK (CPA4)-tag 35kDa

HA (RR1)-tag 33kDa

B LNCaP HA (LXN) pulldown

HA (LXN)-tag 26kDa

DDK (CPA4)-tag 35kDa

Supplementary Figure S10. RARRES1 and LXN do not co-immunoprecipitate with CPA4 in transfected epithelial cell lines. (a) Immunoprecipitation carried out using Direct IP kit (Pierce) and

DDK antibody coupled to the resin to pull down the DDK tag (CPA4) and then probed with HA tag

(RARRES1). Transfection of RARRES1 and CPA4 (CoT), CPA4 only (CPA4), RARRES1 only (RR1), untransfected cells (U/T) and reagent only treatment (Mock). (b) Immunoprecipitation using Profound

Mammalian HA-Tag IP/ Co-IP Kit (Pierce) with HA-antibody coupled to the beads to pull-down LXN and probed with the DDK tag (CPA4). Transfection of LXN and CPA4 (CoT), CPA4 only (CPA4), LXN only (RR1), reagent only treatment (Mock) and HA-tag positive control protein.

14 Cell Line Medium Origin PNT1a RPMI (Invitrogen) supplemented with 10% FCS (PAA) Obtained with kind and 2mM L-glutamine (Invitrogen) [R10] permission from Dr. P. Berthon PNT2-C2 R10 Generated and characterised in our own laboratory LNCaP R10 European Collection of Cell Cultures (ECACC) BPH-1 RPMI supplemented with 5% FCS and 2mM L-Glutamine A gift from Dr. S. [R5] Hayward PC3 Ham's F-12 (Lonza) supplemented with 7% FCS and 2 ECACC mM L-glutamine [H7] P4E6 Keratinocyte serum-free medium (KSFM; Invitrogen) Generated and supplemented with 2% FCS, 2 mM L-glutamine, 5 ng/mL characterised in our own epidermal growth factor (EGF; Invitrogen), and 5 μg/mL laboratory bovine pituitary extract (Invitrogen) [K2] RC-165N/ KSFM supplemented with 50µg/ml bovine pituitary Obtained with kind hTERT extract, 5ng/ml human EGF and 2mM L-Glutamine permission from Dr. J. Miki MDA-MB-231 DMEM supplemented with 10% FCS and 2mM L- ATCC Glutamine [D10] Bob KSFM supplemented with 5ng/ml human EGF, 50µg/ml Obtained with kind bovine pituitary extract, 2ng/ml leukaemia inhibitory factor permission from Dr. D. (Chemicon), 100ng/ml cholera toxin (Sigma), 1ng/ml Hudson granulocyte macrophage colony stimulating factor (Miltenyi Biotec), 2ng/ml stem cell factor (First Link UK Ltd) and 2mM L-Glutamine [SCM]

Supplementary Table S1. List of culture conditions and origin of each epithelial cell line used.

15 Patient Sample Diagnosis Patient Sample Diagnosis Gleason grade PE109 BPH PE025 Cancer 4+4 Y082/06 BPH PE107 Cancer 3+3 Y088/09 BPH PE519 Cancer 3+3 Y003/10 BPH PE531 Cancer 4+5 H040/10 BPH PE665 Cancer 3+4 Y048/10 BPH PE671 Cancer 3+4 Y052/10 BPH Y089/09 Cancer CRPC Y059/10 BPH Y091/09 Cancer CRPC Y061/10 BPH H031/11 Cancer 7 H040/11 BPH H043/11 Cancer 7 Y054/11 BPH H046/11 Cancer 3+5 H094/11 BPH H062/11 Cancer - H150/12 BPH H082/11 Cancer - H158/12 BPH H103/11 Cancer - H159/12 BPH H131/11 Cancer - H169/12 BPH H135/11 Cancer 9 H189/12 BPH H144/11 Cancer 7 H146/12 Cancer 7

Supplementary Table S2. Human prostatic tissue was obtained with informed patient consent and ethical approval from the York Research Ethics Committee from patients undergoing trans-urethral resection of the prostate or radical prostatectomy. BPH or CaP diagnosis was confirmed by histological examination of adjacent tissue fragments.

16 Primer Name Primer Sequence (5’ to 3’) Purpose RARRES1 forward AGT TTT AGG ATG TTG GGG TTT RARRES1 reverse biotin TAC CCA AAT ATC ACC TCC CAA RARRES1 sequencing GGA GTT TTA TTT TTT TAA TT LXN nested PCR forward GTT GGT GTT TGA TAA GTA TGT T Pyrosequencing LXN nested PCR reverse CCC CTA CTA AAC TCA CCT CCA T analysis LXN forward GAT GTA GGG AGT TTG GGT TTA AAT AG LXN reverse biotin CCA ATA AAC AAT AAC TTC AAA ACT T LXN sequencing GGG AGT TTG GGT TTA AAT A RARRES1 Hs00161204_m1 LXN Hs00220138_m1 Taqman qRT-PCR GAPDH Hs99999905_m1 assays (Applied RPLPO Hs99999902_m1 Biosystems) 18S Hs03003631_g1

Supplementary Table S3. Sequences of primers for pyrosequencing analysis and product codes of primers for qRT-PCR analysis.

SUPPLEMENTARY REFERENCES

1. Pallares I, Bonet R, Garcia-Castellanos R, Ventura S, Aviles FX, Vendrell J, et al. Structure of human carboxypeptidase A4 with its endogenous protein inhibitor, latexin. Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):3978-83. 2. Berthon P, Cussenot O, Hopwood L, Leduc A, Maitland N. Functional expression of sv40 in normal human prostatic epithelial and fibroblastic cells - differentiation pattern of nontumorigenic cell- lines. Int J Oncol. 1995 Feb;6(2):333-43. 3. Hayward SW, Dahiya R, Cunha GR, Bartek J, Deshpande N, Narayan P. Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1. In Vitro Cell Dev Biol Anim. 1995 Jan;31(1):14-24. 4. Maitland NJ, Macintosh CA, Hall J, Sharrard M, Quinn G, Lang S. In vitro models to study cellular differentiation and function in human prostate cancers. Radiat Res. 2001 Jan;155(1 Pt 2):133- 42. 5. Miki J, Furusato B, Li H, Gu Y, Takahashi H, Egawa S, et al. Identification of putative stem cell markers, CD133 and CXCR4, in hTERT-immortalized primary nonmalignant and malignant tumor- derived human prostate epithelial cell lines and in prostate cancer specimens. Cancer Res. 2007 Apr 1;67(7):3153-61. 6. Attard G, Rizzo S, Ledaki I, Clark J, Reid AH, Thompson A, et al. A novel, spontaneously immortalized, human prostate cancer cell line, Bob, offers a unique model for pre-clinical prostate cancer studies. Prostate. 2009 Oct 1;69(14):1507-20.

17