KIBRA (WWC1) Is a Metastasis Suppressor Gene Affected by

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A65. A646 A1471 A1129. Kibra expression/. Hprt chr11 retained chr11 loss ... B) The Cancer Cell Line Encyclopedia (CCLE) was used to investigate mRNA.
Cell Reports, Volume 22

Supplemental Information

KIBRA (WWC1) Is a Metastasis Suppressor Gene Affected by Chromosome 5q Loss in Triple-Negative Breast Cancer Jennifer F. Knight, Vanessa Y.C. Sung, Elena Kuzmin, Amber L. Couzens, Danielle A. de Verteuil, Colin D.H. Ratcliffe, Paula P. Coelho, Radia M. Johnson, Payman SamavarchiTehrani, Tina Gruosso, Harvey W. Smith, Wontae Lee, Sadiq M. Saleh, Dongmei Zuo, Hong Zhao, Marie-Christine Guiot, Ryan R. Davis, Jeffrey P. Gregg, Christopher Moraes, AnneClaude Gingras, and Morag Park

Wildtype mammary gland

4695 (MMTV-Met)

6030 (MMTV-Met) 1.0

1.0

0.5

0.5

0

0

Log (relative CN)

1.0 0.5 0 -0.5 -1.0 0

Log (relative CN)

2

4

6

8

12

10

-1.0

-1.0 0

4

6

8

0.5

1.0

0

0

0

2

4

8

12

10

8

10

12

10

12

0 -0.5

MCR

0

2

4

6

8

12

10

-1.0

MCR

0

A1471 (MMTV-Met;Trp53fl/+ ) 1.5

1.0

6

0.5

-2.0 6

4

1.0

-1.0 MCR

2

A3571 (Trp53fl/+)

2.0

-0.5

0

12

10

A4719 2/3L (Trp53fl/+)

A1034 (MMTV-Met;Trp53fl/+ )

Log (relative CN)

2

5482 (MMTV-Met_Trp53R245H)

1.0

-1.0

-0.5

-0.5

2

4

6

8

A1221 (MMTV-Met;Trp53fl/+) 2.0

1.0

0.5

1.0

0.5

0

0

0

-0.5

-0.5

-1.0

-1.0

MCR

0

2

4

6

8

10

Genomic position (x107 bases)

12

-1.0

MCR

0

2

4

6

8

10

12

Genomic position (x107 bases)

MCR

0

2

4

6

8

10

12

Genomic position (x107 bases)

Supplemental Figure S1. Loss of chromosome 11 is a frequent event in MMTV-Met;Trp53fl/+;Cre and Trp53fl/+;Cre mouse mammary tumors. Refers to figure 1 of the main manuscript. Examples of array-CGH (aCGH) profiles for mouse chromosome 11. Black dots indicate individual aCGH probes, red lines indicate segmented means for probe regions that deviate from a log copy number change of 0. A profile for chr11 in a normal wildtype mammary gland is shown, alongside profiles for 2 MMTV-Met model tumors (6030 and 4695), for which chr11 loss was an infrequent event (8/9 tumors showed no genomic loss). By contrast, loss of chr11 segments occurred frequently in tumors of the MMTV-Met;Trp53fl/+;Cre and Trp53fl/+;Cre models (18/19 tumors profiled), in addition to 1 MMTV-Met tumor with spontaneous Trp53 mutation (5482). The region of chr11 loss common to all tumors with loss was defined (referred to as the ‘minimal common region’ or MCR) and is highlighted in blue. This region spans from position chr11:18862572 to 49845204bp.

B

2 2.0

TCGA basal and claudin-low 5q33.2-35.3

0.6

mRNA z-score

0.4 0.2 0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2 108

W

W

C

Position on chromosome 5

Luminal Basal A Basal B

C

0

1.5 1.0 0.5 0 -0.5 -1.0 -1.5 -2.0 -2

C

Deletion frequency

5q

N G 1 1/ KI BR A C LI N T1

A

Cii

chr 11 retained

chr 11 loss

56 73 25 A1 00 A1 5 03 A6 4 5 A6 46 A1 47 A1 1 12 9

30

51

51

chr11 loss

60

5 4 3 2 1 0

54

chr11 retained

KIBRA

150kDa

ACTIN

9

1

A1 12

47

46

A1

5

A6

5

4

A6

03

00

A1

25

A1

73

56

50kDa

51

Kibra expression/Hprt

Ci

37kDa

Mouse tumor cell line Basal A (2) Basal B (3)

Basal B

150kDa

47

S 4 DA KB -M R3 B45 HC 3 HC C70 C1 95 4 BT 5 Hs 49 M 5 DA 7 -M 8T B23 1

50kDa

23 BM

D

A-

8T

H

s

57

9

19

54

BT

C

H

C

70

M H

C

C

3

A-

BR

D

M KIBRA

ACTIN

M

37kDa

M

BT

Basal A

1.2 1 0.8 0.6 0.4 0.2 0

SK

47

4

B-

Luminal

54

45

3

1

Luminal (3)

Dii

BT

KIBRA expression/GAPDH

Di

Human breast cancer cell line

Supplemental Figure S2. Loss of chromosome 5q in basal and claudin-low breast cancers is associated with low expression of KIBRA. Relates to figure 1 of the main manuscript. A) The Cancer Genome Atlas (TCGA) Invasive Breast Carcinoma single nucleotide polymorphism (SNP) array dataset, as analyzed by GISTIC, was used to investigate the frequency of gene loss on chromosome 5 among basal and claudin-low subtypes. Regions spanning the enitre long arm of chr5 (5q) occur in up to 60% of basal and claudin-low tumors. Region 5q33.2-35.3 is highlighted and represents the syntenic region of mouse chr11 that undergoes genomic loss in the transgenic breast cancer models used in this study. Loss of this region occurs in 40-55% of basal and claudin-low tumors. B) The Cancer Cell Line Encyclopedia (CCLE) was used to investigate mRNA expression of 3 genes (CCNG1, KIBRA, CLINT1) that undergo hemizygous deletion due to 5q loss (see main Figure 1). Human cell lines representative of the luminal, basal (’basal A’) and claudin-low (’basal B’) molecular subtypes were analysed. Low expression of KIBRA was specifically associated with basal B/claudin-low cell lines Ci) Quantitative real time PCR validated low Kibra mRNA expression in mouse mammary tumor cells with genomic loss of the syntenic region on mouse chr11. PCRs were performed in duplicate, error bars are SEM. Cii) Western blotting confirmed absence or low levels of KIBRA protein expression in mouse tumor cells affected by chr11 loss. Di) Quantitative real time PCR validated CCLE data showing reduced expression of KIBRA mRNA in basal B breast cancer cell lines compared to other subtypes. PCRs were performed in duplicate, error bars are SEM. Dii) Western blotting showed that KIBRA protein levels were low to absent in human cell lines belonging to the basal B/claudin-low subtype.

Ai Tumor volume (mm3)

400 350 300

pLKO group 11 SH3 group 11 SH4 group 11 pLKO group 22 SH3 group 22 SH4 group 22

250 200 150 100 50 0

0

5

10

15

20

n=10 n=8 n=7* n=3* n=3 n=3

p=0.994

p=0.872

* 1 mouse died post resection (no associated metastasis data)

25

Days post injection Aii

Aiii Final tumor volume at resection

Tumor bearing time before resection

SH3

SH4

350 300 250 200 150 100 50 0

p=0.580

p=0.071

40 Days

Tumor volume (mm3)

Days

p=0.111

pLKO

Time from resection to sacrifice

p=0.571

p=0.06 25 20 15 10 5 0

Aiv

p=0.582

30 20 10

pLKO

SH3

SH4

0

pLKO

SH3

SH4

Supplemental Figure S3. Kibra knockdown has no significant impact on the in vivo growth of MMTV-Met driven mammary tumor cells. Related to Figure 2 of the main manuscript. Ai) Primary tumour growth curves for mice presented in figure 2. Mammary fat pad injections were performed in nude mice using the MMTV-Met mouse mammary tumor cell line 5156-luc. Cells with Kibra knockdown (SH3, SH4) are compared to an empty vector control (pLKO). Two experimental groups containing the indicated number of mice are presented, mean values for all mice are shown, +/-SEM. Growth rates of pLKO and SH3/SH4 tumors were not statistically significant as determined by a Kruskal-Wallis One Way Analysis of Variance test. Aii-iv) Primary tumor resection data that accompanies results presented in Figure 2 of the main text. Tumor bearing time prior to resection, final tumor volume at resection and time from resection to sacrifice were equivalent between pLKO control and SH3, SH4 tumors. Mean values for all mice are shown, +/- SEM.

TWIST1

ZEB2

ZEB1

A

-2

WWC1

WWC1

VIM

SNAI1

SNAI2

WWC1

-1

WWC1

CDH1

CLDN1

Bii 2

n.s

**

1.5 ***

n.s

n.s

1

EV +Kibra

Ze b2

Ze b1

is Tw

t1 Tw is

Sn ai

1

0

t2

0.5

Expression/Hprt_Rpl13a

Bi

Expression/Hprt_Rpl13a

WWC1

WWC1

Expression/Hprt_Rpl13a

WWC1

WWC1

80

Cldn1 p=0.054

60 40 20 0

6.0

EV

+Kibra Cdh1

p=0.084

4.0 2.0 0.0

EV

+Kibra

Supplemental Figure S4. KIBRA mRNA levels correlate with expression of epithelial markers in human breast cancers and mouse model tumor cells. Relates to figure 3 of the main manuscript. A) Analysis of gene expression data for pooled basal and claudin-low tumors (TCGA, Nature 2012). Pearson correlation coefficients were calculated to determine the degree of correlation between mRNA levels of WWC1 (KIBRA) and a panel of genes associated with either mesenchymal (ZEB1/2, TWIST1, SNAI1/2, VIM) or epithelial (CLDN1, CDH1) phenotypes. X and Y axis values are mRNA Z-scores. The only significant correlation was with CDH1 (E-CADHERIN). B) RT-PCR data for mouse mammary tumor cells engineered to re-express Kibra. EV= empty vector control Bi) The only gene associated with a mesenchymal phenotype to significantly decrease following Kibra expression was Twist2 (p=0.008). Twist1 showed a compensatory increase (p=0.013). Bii) Kibra expression led to increases in the epithelial markers Cldn1 (Claudin 1) and Cdh1 (E-Cadherin). RT-PCR data were normalised to two housekeeping genes (Hprt and Rpl13a). The mean values for 3 independent experiments using two cell lines (A1005 and A1034) are shown. Error bars are SEM.

B

2.5 2 1.5 1 0.5 0

C 2li G ke lu -ri aP ch KC PD Z

Hs 578T *

*

*

T1 T2 Passage # BT549

T1

EV +KIBRA

*

**

**

T2 Passage #

C

KIBRA-WT Δ WW1/2 Δ PDZ Δ WW1/2/PDZ/aPKC Δ Glu-rich

T3

/P D

D

Δ PDZ, aPKC

T3

pL V KI X-G BR F ΔW A P ΔP W1 WT /2 D Δa Z Δ PK W C ΔG W1 /PD Z Δ lu-r /2 / C ic a 2 h P K

W W W W

C

C oi le d

-c oi l

2.5 2 1.5 1 0.5 0

Z

Hs 578T BT549

Relative SFE

+ KIBRA

Empty vector

Relative SFE

A

150kDa 50kDa 37kDa

KIBRA ACTIN

Δ C2

E

MDA-MB-231 + Empty Vector FLAG + DAPI

FLAG

FLAG + DAPI

YAP 5SA

TAZ S89A

pCMV control

FLAG

MDA-MB-231 + KIBRA

Supplemental Figure S5. KIBRA expression impairs tumorsphere formation in human basal B cell lines, a phenotype which requires the KIBRA WW-domains and can be rescued by expression of activated TAZ. Accompanies Figures 4 and 5 of the main manuscript. A) Representative images of tumorspheres formed by basal B cell lines Hs 578T and BT549 +/- KIBRA expression. Scale bars are 400 µm. B) Quantification of sphere forming efficiency (SFE) for Hs 578T and BT549 cells +/- KIBRA. Results mirror those obtained with MDAMB-231 cells as used in Figure 4 of the main manuscript (3 independent experiments, mean +/- SEM). C) Schematic showing GFP-tagged wildtype KIBRA (KIBRA-WT) and a series of KIBRA mutants lacking protein interaction and structural regions, including the WWdomains shown to be critical for the inhibition of tumorsphere formation (Figure 4) D) Western blotting showing expression of KIBRAWT and KIBRA mutants in MDA-MB-231 cells (Figure 4). E) Immunofluorescent labelling of MDA-MB-231 cells +/- KIBRA and transfected with FLAG-tagged TAZ S89A or YAP 5SA mutants or a pCMV empty vector control (see Figure 5). Nuclear localisation of FLAG confirms TAZ and YAP activity in control and KIBRA-expressing MDA-MB-231. Scale bars are 20 µm.

M

+K

EV

+K

EV

A

pt y C la n F1 e 0A

MDAMB-231

em

A1005

150kDa

KIBRA

150kDa

pLATS1/2 (Ser909/Ser872)

150kDa

LATS1

150kDa

LATS2

75kDa

MERLIN

50kDa

ACTIN

37kDa B Gene (frequency of loss*) LATS2 (35%) NF2 (17%) LATS1 (19%)

WWC1 (52%) WWC1 (52%) WWC1 (52%)

p-value

Log odds ratio

Association

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