pik3ca mutation drives prostate cancer · pik3ca mutation drives prostate cancer 3 supplementary...

Pik3ca mutation drives prostate cancer

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Supplementary Material

Supplementary Methods

QRT-PCR: Prostate RNA was isolated using the RNeasy mini purification kit (#74134,

Qiagen), TURBO DNase treated (#AM1907, Ambion by Life Technologies), and reverse

transcribed with the Transcriptor First Strand cDNA Synthesis kit (#04897030001, Roche)

according to manufacturer’s instructions. Amplification of cDNA was performed on a

LightCycler 480 Instrument (Roche) and samples were normalized to Gapdh (1). Fold change

was calculated using the 2–ΔΔCt method. Pbsn and Nkx3.1 mRNA was detected using primers

published previously (2).

Western blotting: Protein lysates (20 g) were loaded onto a 4-12% gradient pre-cast gel

(#NW04122BOX, Thermo Fisher Scientific) in 4x BOLTTM LDS sample buffer (#B0007,

Thermo Fisher Scientific). Using the iBlotTM 2 gel transfer device (#IB21001, Thermo Fisher

Scientific), proteins were transferred to PVDF membrane (#IB24002, Thermo Fisher

Scientific) and blocked in blocking buffer 1 (BB1, Zeptosens) before incubating overnight with

primary antibodies (Supplementary Table 6) diluted 1:1,000 in assay buffer CAB1 (Zeptosens).

Membranes were washed in TBS/T (0.1% Tween 20) and incubated with 1:20,000 IRDye®

800CW goat-anti-rabbit IgG (H+L) secondary antibody (#925-32211, LI-COR Biosciences)

and bands detected using the LI-COR Odyssey CLx infrared imaging system.

Supplementary Table Legends

Supplementary Table 1: PIK3CA mutation and CNA frequency in prostate cancer

patients. Table displaying the frequency of PIK3CA genetic mutation and copy number

alteration in prostate cancer patient genomic datasets (3-10). Data was accessed using the


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online cBioPortal cancer genomics platform (11,12). All samples were filtered for those that

have undergone sequencing and CNA analysis. A GISTIC putative copy number algorithm

was applied by the cBioPortal platform to establish high- and low-level copy number thresholds

to define samples with PIK3CA amplification or gain respectively (11,12).

Supplementary Table 2: Clinicopathological features of TCGA prostate adenocarcinoma

provisional dataset. Table summarizing key clinicopathological features of the TCGA

provisional prostate cancer patient dataset (obtained from the TCGA data portal https://tcga-

data.nci.nih.gov/).

Supplementary Table 3: PIK3CA mutation/amplification/gain frequency within the

TCGA prostate adenocarcinoma provisional dataset. Table outlining the correlation

between PIK3CA mutation and amplification/gain CNA with key clinicopathological features

in prostate cancer patient samples. Data was obtained from the TCGA data portal (https://tcga-

data.nci.nih.gov/). PIK3CA amplification/gain criteria; Log R ratio ≥ 0.135, probe number ≥

10. Silent mutations were excluded.

Supplementary Table 4: PTEN mutation/loss frequency within the TCGA prostate

adenocarcinoma provisional dataset. Table outlining the correlation between PTEN mutation

and CNA loss with key clinicopathological features in prostate cancer patient samples. Data

was obtained from the TCGA data portal (https://tcga-data.nci.nih.gov/). PTEN copy number

loss criteria; Log R ratio ≤ -0.48 for deletion, probe number ≥ 10. Silent mutations were

excluded.

https://tcga-data.nci.nih.gov/






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Supplementary Table 5: Phenotype summary of mouse histopathology. Table outlines the

histopathological phenotypes observed in Wt, Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl

cohorts at 56, 100, 200, 300 and 400 d of age.

Supplemental Table 6: RPPA antibody list. Table showing RPPA primary antibodies

categorised into signaling pathways.

Supplementary Table 7: RPPA analysis of prostate carcinoma response to castration

in Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl transgenic mice. Table indicates

proteins/phospho-proteins with statistically significant differences in Log2 normalised

mean RPPA signal intensities between the indicated genotype/treatment cohorts (unpaired

two-tail Welch’s t-test, P < 0.05, n = 3).

Supplementary Figure Legends

Supplementary Figure 1: PIK3CA mutations are predominantly missense mutations and

PTEN mutation/loss predicts for poor prostate cancer patient survival. (A) Pie chart

depicting the frequency of missense/nonsense mutations, in-frame deletions and fusion events

in PIK3CA identified in the 9 prostate cancer genomic datasets assessed in Fig. 1A (3-10). (B)

Kaplan-Meier plot comparing TCGA provisional prostate adenocarcinoma dataset with PTEN

homozygous deletion, loss or mutation compared to the general population. PTEN age-adjusted

COXPH HR: 0.47, P = 0.0026* (n = 492, samples with sequencing and CNA data only). Data

was obtained from the TCGA data portal (https://tcga-data.nci.nih.gov/). PTEN copy number

loss criteria; Log R ratio ≤ -0.48, probe number ≥ 10. Silent mutations were excluded.



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Supplementary Figure 2: Heterozygous Pik3caH1047R oncogenic mutation causes invasive

prostate cancer in mice that is phenotypically distinct to Pten-null prostate cancer. (A)

Sequencing cDNA isolated from PBiCre+/-;Pik3ca+/HR prostate tissue confirmed

heterozygosity at known silent base changes within mutant exon 20 adjacent to exon 19,

indicating recombination has occurred. (B) allele-specific PCR of cDNA isolated from

PBiCre+/- prostate tissue expressing either Pik3ca+/+ (Wt) or Pik3ca+/HR alleles (as previously

described (13)) revealed the presence of the mutant exon 20 in PBiCre+/-;Pik3ca+/HR prostate

cDNA, but not in PBiCre+/-;Wt prostate cDNA. (C) Representative H&E images of PBiCre+/-;

Wt, Pik3ca+/HR and Ptenfl/fl ventral and anterior prostate epithelium at 400 d (scale bar: 100 m).

Phenotype incidence plots for PBiCre+/-; Wt, Pik3ca+/HR and Ptenfl/fl ventral (D) and anterior

(E) prostate lobes. VP = Ventral prostate, AP = anterior prostate, PIN = prostate intraepithelial

neoplasia. (F) IHC to detect SMA in Wt, Pik3ca+/HR and Ptenfl/fl mice at 400 d (scale bar: 50

m). (G) Quantitation of PCNA-positive nuclei in PBiCre+/-; Pik3ca+/HR and Ptenfl/fl prostate

hyperplastic lesions. *P <0.001, one-way ANOVA with Tukey’s multiple comparison test, n =

3. Error bars: SEM.

Supplementary Figure 3: Characterization of Pik3ca-mutated and Pten-deleted prostate

hyperplasia and carcinoma. (A) IHC to detect the apoptotic marker Cleaved-Caspase-3

(CC3) in Wt, Pik3ca+/HR and Ptenfl/fl mice at 400 d (scale bar: 50 m). (B) Quantitation of CC3-

positive nuclei in Wt, Pik3ca+/HR and Ptenfl/fl prostate epithelium (n = 3, *P <0.05 compared to

Wt, or as indicated, one-way ANOVA with Tukey’s multiple comparison test, ns = not

significant. Error bars: SEM). (C) IHC to detect CK5 and CK8 in Pik3ca+/HR and Ptenfl/fl

carcinomas (representative images from 3 prostates per genotype, scale bar: 50 m). (D)

Representative IHC images to detect PTEN, mTORC1 signaling components (p-AKT Thr308,

p-RPS6 Ser235/236 and p-4E-BP1 Thr37/46) and mTORC2 substrates (p-AKT Ser473 and p-


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NDRG1 Thr346) in Pik3ca+/HR and Ptenfl/fl hyperplastic lesions (n = 3, scale bar: 50 m). IHC

quantitation for (E) p-AKT Thr308, (F) p-RPS6 Ser235/236, (G) p-4E-BP1 Thr37/46, (H) p-

AKT Ser473 and (I) p-NDRG1 Thr346 in Pik3ca+/HR and Ptenfl/fl prostate hyperplastic lesions

(n = 3, Error bars: SEM, *P < 0.05, unpaired, two-tailed t-test).

Supplementary Figure 4: Pik3caH1047R heterozygous oncogenic mutation causes p110-

dependent prostate cancer. Representative H&E images (scale bar: 100 m) for Pik3ca+/HR

and Ptenfl/fl dorsolateral prostate and histograms displaying phenotype incidence for anterior

(B) and ventral (C) prostate lobes from Pik3ca+/HR and Ptenfl/fl mice treated with vehicle, p110

inhibitor (A66), p110 inhibitor (TGX-221), pan-PI3K inhibitor (BKM120) or A66 + TGX-

221 for 4 weeks. ND = not done. A66 and TGX-221 were generated in house by P.R.S.

(University of Auckland, New Zealand) (14) and BKM120 was obtained from SYNkinase

(Australia).

Supplementary Figure 5: Pik3caH1047R mutation and Pten-deletion synergize to promote

prostate cancer by increasing mTORC1/2 signaling. Histograms displaying phenotype

incidence for anterior (A) and ventral (B) prostate lobes at 56 and 100 days of age. (C)

Representative IHC images of Pik3ca+/HR;Ptenfl/fl prostate tumors at 100 d stained to detect

CK8, CK5 and SMA (n = 3, scale bar: 50 m). (D) Bar chart displaying total prostate weight

normalised to body weight for Wt (n = 7), Pik3ca+/HR (n = 8), Ptenfl/fl (n = 8) and

Pik3ca+/HR;Ptenfl/fl (n = 7) 100 d old mice. Error bars: SEM, *P <0.05 compared to Wt or as

indicated, one-way ANOVA with Tukey’s multiple comparison test. (E) Quantitation of the

apoptotic marker Cleaved-Caspase-3 (CC3) positive nuclei and (F) representative IHC images

of CC3 staining in Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl stage-matched invasive prostate

carcinoma (scale bar: 50 m, n = 3, one-way ANOVA with Tukey’s multiple comparison test.


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Error bars: SEM). (G) Representative IHC images of Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl

stage-matched invasive prostate carcinoma stained to detect p-AKT Thr308, p-RPS6

Ser235/236, p-4E-BP1 Thr37/46, p-AKT Ser473 and p-NDRG1 Thr346 (scale bar: 50 m).

(H) Representative images of RNA in situ hybridisation (ISH) to detect positive (housekeeping

gene PPIB, peptidylprolyl isomerase B) and negative (bacterial gene dapB) control probes to

confirm RNA quality and the absence of background signal respectively (scale bar: 50 m,

insert scale bar: 5 m).

Supplementary Figure 6: Pik3ca+/HR and Ptenfl/fl prostate cancers acquire CRPC, while

Pik3ca+/HR;Ptenfl/fl mutants are resistant to castration. (A) Representative H&E images of

Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl anterior (AP) and ventral (VP) prostate lobes post-

castration (scale bar: 50 m, n = 3). (B) Representative IHC images of Pik3ca+/HR, Ptenfl/fl and

Pik3ca+/HR;Ptenfl/fl prostate tissue stained to detect Androgen receptor (AR) 2 weeks post-

castration compared to uncastrated, age-matched controls (scale bar: 50 m, n = 3). Mice were

castrated when prostate carcinoma was prevalent; Pik3ca+/HR = 400 d old, Ptenfl/fl = 200 d old

and Pik3ca+/HR;Ptenfl/fl = 100 d old. Insert displays positive AR nuclei (arrows) in

Pik3ca+/HR;Ptenfl/fl compound mutants (scale bar: 5 m). (C) Bar chart displaying total prostate

weight normalised to body weight for Pik3ca+/HR mice 0, 2 and 42 weeks post-castration (n =

8, 7 and 7, respectively). Error bars: SEM, *P <0.05 compared to 0 weeks post-castration, or

as indicated, one-way ANOVA with Tukey’s multiple comparison test. (D) Representative

H&E images of Pik3ca+/HR mice 0, 2 and 42 weeks post-castration (scale bar: 100 m). Mice

were castrated at 100 d of age.

Supplementary Figure 7: Characterization of Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl

prostate tumors. Representative images of IHC to detect (A) PCNA and (B) Cleaved-caspase


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3 (CC3) in Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl prostate tissue 2 weeks post-castration

compared to uncastrated, age-matched controls (scale bar: 50 m, n = 3). Mice were castrated

when prostate carcinoma was prevalent; Pik3ca+/HR = 400 d old, Ptenfl/fl = 200 d old and

Pik3ca+/HR;Ptenfl/fl =100 d old. Quantitative RT-PCR to detect (C) Nkx3.1 and (D) Pbsn mRNA

in Wt prostate and Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl stage-matched prostate

carcinomas (n = 5). Error bars: SEM, *P <0.05 compared to Wt, or as indicated, one-way

ANOVA with Tukey’s multiple comparison test. (E) Western Blotting of protein lysates

isolated from Wt prostate and stage-matched Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl

prostate carcinomas to detect total AKT, p-AKT Thr308 and p-AKT Ser473 (n = 3).

Supplementary References

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Supplementary Table 1. PIK3CA mutation/CNA frequency in prostate cancer patients

Alteration No. of patients (%)

Neuroendocrine prostate cancer, Beltran et al, Nat Med 2016 (n = 77)

Mutation 1 (1.3%)

Amplification 22 (28.6%)

Gain 26 (33.8%)

Total 49 (63.6%)

Prostate adenocarcinoma, Kumar et al, Nat Med 2016 (n = 54)

Mutation 2 (3.7%)


Gain 19 (35.2%)

Total 25 (46.3%)

Prostate adenocarcinoma vs. Metastatic, Grasso et al, Nature 2012 (n = 59)

Mutation 0 (0.0%)


Gain 14 (23.7%)

Total 20 (33.9%)

Prostate adenocarcinoma, Baca et al, Cell 2013 (n = 56)

Mutation 2 (3.6%)


Gain 8 (14.3%)

Total 12 (21.4%)

TCGA prostate adenocarcinoma provisional dataset (n = 492)

Mutation 14 (2.9%)


Gain 60 (12.2%)

Total 86 (17.7%)* *2 patients displayed 2 PIK3CA mutations (E542A+N345I and R108C+L569I), 1 case had E542K mutation and gain.

Prostate adenocarcinoma TCGA, Cell 2015 (n = 333)

Mutation 6 (1.8%)**


Gain 47 (14.1%)

Total 58 (17.4%)

**1 patient displayed 2 PIK3CA mutations (case 1: E542A+N345I)

Prostate adenocarcinoma, Barbieri et al, Nat Genetics 2012 (n = 109)

Mutation 4 (3.7%)


Gain 14 (12.8%)

Total 19 (17.4%)

Prostate adenocarcinoma, Taylor et al, Cancer Cell 2010 (n = 103)

Mutation 3 (2.9%)


Gain 9 (8.7%)

Total 12 (11.7%)

Metastatic prostate cancer, Robinson et al, Cell 2015 (n = 150)

Mutation 6 (4.0%)


Gain 0 (0.0%)

Fusion + amplification 1 (0.7%)

Total 8 (5.3%)

Supplementary Table 2 Clinicopathological features of TCGA prostate adenocarcinoma provisional dataset

Characteristic

Sample size (n)

Follow-up (months)

492

Mean 20.9

Median 14.6

Range 0.03-151

Age (years)

<50 27

50-60 175

60-70 240

>70 50

Post-treatment PSA (ng/ml)

<4 409

4-10 10

10-20 11

>20 5

N/A 57

pT category

pT2 187

pT3a 156

pT3b 132

pT4 10

N/A 7

Gleason grade

≤3+3 44

3+4 154

4+3 106

≥4+4 188

Regional lymph node metastasis

pN0 343

pN+ 77

N/A 72

Supplementary Table 3 PIK3CA mutation/amplification/gain analysis of TCGA prostate cancer provisional dataset.

†P-value = Fisher’s exact test between PIK3CA mutation/amplification/gain and the general population. ††Two mutations occurred in the same patient in 2 cases (R108C+L569I and E542A+N345I). †††One PIK3CA mutant carrier also displayed a gain in PIK3CA copy number.

Characteristic n

PIK3CA

mutation

n (%)

PIK3CA

amplification/gain

n (%)

PIK3CA mutation/

amplification/gain

n (%)

General

population

n (%)

P value†

All Samples 492 14 (3%)†† 73 (15%)††† 86 (17%) 406 (83%)

Regional lymph node

metastasis

pN0 10 (71%) 43 (59%) 52 (60%) 291 (72%) 0.0004*

pN+ 1 (7%) 25 (34%) 26 (30%) 51 (13%)

N/A 3 (21%) 5 (7%) 8 (9%) 64 (16%)

pT category

pT2 3 (21%) 12 (16%) 15 (17%) 172 (42%) <0.0001*

pT3a 6 (43%) 25 (34%) 31 (36%) 125 (31%)

pT3b 5 (36%) 30 (41%) 34 (40%) 98 (24%)

pT4 0 (0%) 4 (5%) 4 (5%) 6 (1%)

N/A 0 (0%) 2 (3%) 2 (2%) 5 (1%)

Gleason grade

≤3+3 0 (0%) 1 (1%) 1 (1%) 43 (11%) <0.0001*

3+4 3 (21%) 8 (11%) 11 (13%) 143 (35%)

4+3 1 (7%) 11 (15%) 12 (14%) 94 (23%)

≥4+4 10 (71%) 53 (73%) 62 (72%) 126 (31%)

Post-treatment PSA

(ng/ml)

<4 10 (71%) 56 (77%) 65 (76%) 344 (85%) 0.05

4-10 0 (0%) 3 (4%) 3 (3%) 7 (2%)

10-20 1 (7%) 1 (1%) 2 (2%) 9 (2%)

>20 0 (0%) 3 (4%) 3 (3%) 2 (0%)

N/A 3 (21%) 10 (14%) 13 (15%) 44 (11%)

Supplementary Table 4 PTEN mutation/loss analysis of TCGA prostate cancer provisional dataset.

†P-value = Fisher’s exact test between PTEN mutation/loss and the general population. ††Seven patients carried a PTEN mutation and PTEN copy number loss.

Characteristic n

PTEN

mutation

n (%)

PTEN

loss

n (%)

PTEN

mutation/loss

n (%)

General

population

n (%)

P value†

All Samples† 492 17 (3%) 95 (19%) 105 (21%)†† 387 (79%)

Regional lymph node

metastasis

pN0 14 (82%) 64 (67%) 73 (70%) 270 (70%) 0.0028*

pN+ 2 (12%) 25 (26%) 25 (24%) 52 (13%)

N/A 1 (6%) 6 (6%) 7 (7%) 65 (17%)

pT category

pT2 7 (41%) 16 (17%) 21 (20%) 166 (43%) <0.0001*

pT3a 6 (35%) 36 (38%) 38 (36%) 118 (30%)

pT3b 4 (24%) 40 (42%) 43 (41%) 89 (23%)

pT4 0 (0%) 2 (2%) 2 (2%) 8 (2%)

N/A 0 (0%) 1 (1%) 1 (1%) 6 (2%)

Gleason grade

≤3+3 0 (0%) 5 (5%) 5 (5%) 39 (10%) 0.0006*

3+4 6 (35%) 17 (18%) 22 (21%) 132 (34%)

4+3 3 (18%) 19 (20%) 20 (19%) 86 (22%)

≥4+4 8 (47%) 54 (57%) 58 (55%) 130 (34%)

Post-treatment PSA

(ng/ml)

<4 14 (82%) 79 (83%) 87 (83%) 322 (83%) 0.24

4-10 0 (0%) 3 (3%) 3 (3%) 7 (2%)

10-20 1 (6%) 2 (2%) 2 (2%) 9 (2%)

>20 0 (0%) 3 (3%) 3 (3%) 2 (1%)

N/A 2 (12%) 8 (8%) 10 (10%) 47 (12%)

Age Genotype N

Prostate

hyperplasia

N (%)

Prostate

neoplasia

N (%)

Prostate

carcinoma

N (%)

Seminal vesicle

neoplasia

N (%)

Urethra

neoplasia

N (%)

Adrenal

pheochromocytoma

N (%)

56 d Wt 8 0 0 0 0 0 0

Pik3ca+/HR 9 0 0 0 0 0 0

Ptenfl/fl 7 7/7 (100%) 0 0 0 0 0

Pik3ca+/HR;Ptenfl/fl 5 0 1/5 (20%) 4/5 (80%) 1/5 (20%) 0 5/5 (100%)

100 d Wt 7 0 0 0 0 0 0

Pik3ca+/HR 8 6/8 (75%) 0 0 0 0 0

Ptenfl/fl 8 0 8/8 (100%) 0 0 0 3/8 (38%)

Pik3ca+/HR;Ptenfl/fl 7 0 0 7/7 (100%) 7/7 (100%) 5/7 (71%) 7/7 (100%)

200 d Wt 8 0 0 0 0 0 0

Pik3ca+/HR 8 7/8 (88%) 0 0 0 0 0

Ptenfl/fl 7 0 2/7 (29%) 5/7 (71%) 1/7 (14%) 0 4/7 (57%)

300 d Wt 7 0 0 0 0 0 0

Pik3ca+/HR 8 0 2/8 (25%) 6/8 (75%) 0 0 0

Ptenfl/fl 7 0 1/7 (14%) 6/7 (86%) 4/6 (67%) 1/6 (17%) 5/6 (83%)

400 d Wt 17 0 0 0 0 0 0

Pik3ca+/HR 17 2/17 (12%) 0 15/17 (88%) 2/17 (12%) 0 0

Ptenfl/fl 8 0 0 8/8 (100%) 5/8 (63%) 2/8 (25%) 7/8 (88%)

Supplementary Table 5: Phenotype summary of mouse histopathology

Antibody Source Cat.# Host

PI3K/mTOR Pathway

AKT Cell Signaling Technology 9272 Rabbit

p-AKT (S473) Cell Signaling Technology 4060 Rabbit

p-AKT (T308) Cell Signaling Technology 2965 Rabbit

AMPK Cell Signaling Technology 8208 Rabbit

p-AMPK Cell Signaling Technology 8208 Rabbit

4E-BP1 Cell Signaling Technology 9452 Rabbit

p-4E-BP1 (T37/T46) Cell Signaling Technology 9459 Rabbit

FOXO3A Cell Signaling Technology 2497 Rabbit

p-FOXO3A (S253) Cell Signaling Technology 9466 Rabbit

p-FOXO3A (S318/S321) Cell Signaling Technology 9465 Rabbit

GSK-3 Cell Signaling Technology 9315 Rabbit

p-GSK-3S9 Cell Signaling Technology 9336 Rabbit

PI3K p110 Cell Signaling Technology 4255 Rabbit

PI3K p110(clone C73F8) Cell Signaling Technology 4249 Rabbit

PI3K p110 Cell Signaling Technology 3011 Rabbit

p-PDK1 (S241) Cell Signaling Technology 3061 Rabbit

p-PKC (pan) (II S660) Cell Signaling Technology 9371 Rabbit

p-PKC Cell Signaling Technology 9378 Rabbit

PTEN Cell Signaling Technology 9552 Rabbit

p-PTEN (S380/T382/T383) Cell Signaling Technology 9554 Rabbit

MTOR Cell Signaling Technology 2972 Rabbit

p-MTOR (S2448) Cell Signaling Technology 2971 Rabbit

NDRG1 Cell Signaling Technology 5196 Rabbit

p-NDRG1 (T346) Cell Signaling Technology 3217 Rabbit

RPS6 Cell Signaling Technology 2217 Rabbit

p-RPS6 (S235/S236) Cell Signaling Technology 2211 Rabbit

p-RPS6 (S240/S244) Cell Signaling Technology 2215 Rabbit

SGK1 Cell Signaling Technology 12103 Rabbit

TSC2 Cell Signaling Technology 3612 Rabbit

p-TSC2 (T1462) Cell Signaling Technology 3617 Rabbit

Tyrosine kinase-mediated pathways

EGFR Cell Signaling Technology 2232 Rabbit

p-EGFR (Y1173) Cell Signaling Technology 4407 Rabbit

p-ErbB2/HER2 (Y1248)/EGFR (Y1173) Cell Signaling Technology 2244 Rabbit

p-ErbB2/HER2 (Y877) Cell Signaling Technology 2241 Rabbit

Supplementary Table 6. RPPA antibody list

IRS-1 Cell Signaling Technology 2382 Rabbit

p-IRS-1 (S636/S639) Cell Signaling Technology 2388 Rabbit

IGF-1R Cell Signaling Technology 3018 Rabbit

p-SHP-2 (Y542) Cell Signaling Technology 3751 Rabbit

p-MET (Y1234, Y1235) Cell Signaling Technology 3129 Rabbit

SRC Cell Signaling Technology 2109 Rabbit

p-SRC family (Y416) Cell Signaling Technology 2101 Rabbit

MAPK Pathway

ERK1/2 Cell Signaling Technology 9102 Rabbit

p-ERK1/2 (T202/Y204) Cell Signaling Technology 9101 Rabbit

p-c-RAF (S338) Cell Signaling Technology 9427 Rabbit

p-SHC (Y317) Cell Signaling Technology 2431 Rabbit

p-SHC (Y239/Y240) Cell Signaling Technology 2434 Rabbit

p-c-JUN (S73) Cell Signaling Technology 9164 Rabbit

p38 MAPK Cell Signaling Technology 9212 Rabbit

p-p38 MAPK (T180/Y182) Cell Signaling Technology 4631 Rabbit

JAK/STAT signaling

p-STAT1 (Y701) Cell Signaling Technology 7649 Rabbit

STAT3 Cell Signaling Technology 4904 Rabbit



Hippo Signaling

TAZ Cell Signaling Technology 4883 Rabbit

YAP Cell Signaling Technology 4912 Rabbit

p-YAP (S127) Cell Signaling Technology 13008 Rabbit

NF-B signaling

IB Cell Signaling Technology 4812 Rabbit

p-IB(S32) Cell Signaling Technology 2859 Rabbit

NF-B p65 Cell Signaling Technology 4764 Rabbit

p-NF-B p65 (S536) Cell Signaling Technology 3031 Rabbit

p-IKK/(S176/S180) Cell Signaling Technology 2694 Rabbit

Wnt Signalling

-catenin Cell Signaling Technology 9562 Rabbit

p--catenin (S33/S37/T41) Cell Signaling Technology 9561 Rabbit

p--catenin (T41/S45) Cell Signaling Technology 9565 Rabbit

DNA repair

ATM Cell Signaling Technology 2873 Rabbit

p-ATM/ATR (S/T) Substrate Cell Signaling Technology 2851 Rabbit

Apoptosis Pathway

Caspase-3 Cell Signaling Technology 9662 Rabbit

Cleaved Caspase-3 (D175) Cell Signaling Technology 9664 Rabbit

Cleaved Caspase-7 (D198) Cell Signaling Technology 9491 Rabbit

p-BAD (S112) Cell Signaling Technology 9291 Rabbit

p-BAD (S136) Cell Signaling Technology 9295 Rabbit

PARP Cell Signaling Technology 9542 Rabbit

Cell Cycle regulator

p21 (WAF1/CIP1) Cell Signaling Technology 2947 Rabbit

p27 Cell Signaling Technology 3686 Rabbit

p53 Cell Signaling Technology 9282 Rabbit

p-p53 (S15) Cell Signaling Technology 9284 Rabbit

p-AURORA A/B/C (T288;T232;T198) Cell Signaling Technology 2914 Rabbit

CDK2 Cell Signaling Technology 2546 Rabbit

Cyclin D1 Cell Signaling Technology 2978 Rabbit

p-CDC2 (Y15) Cell Signaling Technology 9111 Rabbit

c-MYC Cell Signaling Technology 5605 Rabbit

Other

-Actin Cell Signaling Technology 4970 Rabbit

CK2 Cell Signaling Technology 2656 Rabbit

CREB Cell Signaling Technology 9197 Rabbit

E-cadherin Cell Signaling Technology 3195 Rabbit

FAK Cell Signaling Technology 3285 Rabbit

p-FAK (Y397) Cell Signaling Technology 3283 Rabbit

p-HDAC 4/5/7 (S246;S259;S155) Cell Signaling Technology 3443 Rabbit

ROCK1 Cell Signaling Technology 4035 Rabbit

RAP1A/RAP1B Cell Signaling Technology 4938 Rabbit

p-SMAD2/3 (S465/S467;S423/S425) Cell Signaling Technology 8828 Rabbit

p-VEGFR2 (Y1059) Cell Signaling Technology 3817 Rabbit

p-VEGFR2 (Y1175) Cell Signaling Technology 2478 Rabbit

ZAP-70 Cell Signaling Technology 2705 Rabbit

Supplementary Table 7. RPPA analysis of prostate carcinoma response to castration in

Pik3ca+/HR, Ptenfl/fl and Pik3ca+/HR;Ptenfl/fl transgenic mice.

Protein/phospho-protein

Difference

in means†

P-value††

Pathway

Pik3ca+/HR vs Ptenfl/fl (uncastrated)

p-AKT (S473) 2.4437 0.00107 PI3K/mTOR p-AKT (T308) 1.4475 0.00211 PI3K/mTOR

p-FOX03A (S318/S321) 0.8597 0.04955 PI3K/mTOR

P-GSK-3 (S9) 2.0837 0.01230 PI3K/mTOR

PI3K p110 0.5628 0.02644 PI3K/mTOR

PI3K p110 (C73F8) 0.9666 0.00704 PI3K/mTOR

p-PDK1 (S241) 0.7877 0.03018 PI3K/mTOR PTEN 1.1412 0.03575 PI3K/mTOR

p-NDRG1 (T346) 1.5167 0.00478 PI3K/mTOR p-RPS6 (S240/S244) 1.0442 0.04917 PI3K/mTOR

TSC2 0.5981 0.04178 PI3K/mTOR

p-TSC2_(T1462) 1.5832 0.00414 PI3K/mTOR p-EGFR (Y1173) 0.7856 0.03456 Tyrosine kinase

IGF-1R 0.4104 0.02872 Tyrosine kinase p-SHP-2 (Y542) 2.3159 0.02446 Tyrosine kinase

p-SRC family (Y416) 2.7902 0.00627 Tyrosine kinase ERK1/2 0.8449 0.03442 MAPK

p-ERK1/2 (T202/Y204) 0.8334 0.03396 MAPK

p38 MAPK 1.4115 0.00122 MAPK p-p38 MAPK (T180, Y182) 0.9041 0.01873 MAPK

STAT3 0.8745 0.00396 JAK/STAT

IkB 0.9134 0.00712 NF-kB

NF-B p65 1.0817 0.03051 NF-kB

p--catenin (T41, S45) 0.5404 0.01435 Wnt

Caspase 3 0.9378 0.02699 Apoptosis Cleaved Caspase 3 (D175) 2.1374 0.03710 Apoptosis

p-BAD (S136) 0.7902 0.03906 Apoptosis p53 0.5471 0.02037 Cell Cycle

p-p53 (S15) 1.0223 0.02844 Cell Cycle

p-CDC2 (Y15) 1.9320 0.02961 Cell Cycle c-MYC 0.8257 0.00658 Cell Cycle

-Actin 2.2433 0.03791 Other p-VEGFR2 (Y1175) 1.6929 0.04150 Other

Pik3ca+/HR; Ptenfl/fl vs Pik3ca+/HR (uncastrated)

p-AKT (S473) 2.7871 0.01497 PI3K/mTOR

p-GSK-3 (S9) 1.8298 0.02440 PI3K/mTOR

p-NDRG1 (T346) 2.5913 0.00520 PI3K/mTOR

p-RPS6 (S240, S244) 9.3709 0.00605 PI3K/mTOR TSC2 0.7634 0.03142 PI3K/mTOR

p-ErbB2/HER2 (Y1248)/EGFR (Y1173) 10.1726 0.00424 Tyrosine kinase

p-SHP-2 (Y542) 11.7934 0.00134 Tyrosine kinase

SRC 1.3873 0.01029 Tyrosine kinase p-SRC (Y416) 13.009 0.00092 Tyrosine kinase

p38 MAPK 1.5542 0.01955 MAPK STAT3 0.8734 0.02893 JAK/STAT

p-STAT3 (Y705) 4.4402 0.02730 JAK/STAT

YAP 0.7721 0.02979 Hippo

NF-B p65 1.1489 0.03293 NF-kB

p-NF-B p65 (S536) 1.0470 0.03711 NF-kB

Caspase 3 0.7702 0.04155 Apoptosis

Cleaved Caspase 7 (D198) 7.6335 0.01754 Apoptosis p-Bad (S136) 1.3859 0.04272 Apoptosis

CDK2 0.8615 0.00224 Cell Cycle p-CDC2 (Y15) 11.0603 0.03132 Cell Cycle

-Actin 3.5345 0.01278 Other E-Cadherin 1.0714 0.03975 Other

Pik3ca+/HR; Ptenfl/fl vs Ptenfl/fl (uncastrated) AKT 0.5279 0.02408 PI3K/mTOR PTEN 0.8471 0.01546 PI3K/mTOR p-NDRG1 (T346) 1.0746 0.02542 PI3K/mTOR

p-RPS6 (S240, S244) 8.3267 0.01044 PI3K/mTOR

p-SHP-2 (Y542) 9.4775 0.00265 Tyrosine kinase p-SRC (Y412) 10.2188 <0.0001 Tyrosine kinase p-SHC (Y317) 0.8568 0.03593 Tyrosine kinase p-c-JUN (S73) 8.9696 0.00265 MAPK TAZ 0.4354 0.03794 Hippo Cleaved Caspase 7 (D198) 10.1273 0.00046 Apoptosis p-Bad (S136) 2.1761 0.01242 Apoptosis p-CDC2 (Y15) 9.1283 0.04929 Cell Cycle

-Actin 1.2912 0.00203 Other

E-Cadherin 1.5434 0.02583 Other

p-VEGFR2 (Y1059) 2.0316 0.00677 Other

p-VEGFR2 (Y1175) 3.4998 0.01987 Other

Pik3ca+/HR uncastrated vs Pik3ca+/HR castrated

mTOR 1.0502 0.01399 PI3K/mTOR

p-RPS6 (S240/S244) 5.9486 0.00896 PI3K/mTOR

p-TSC2 (T1462) 0.9590 0.01251 PI3K/mTOR

IGF-1R 0.4778 0.03151 Tyrosine kinase

IB 0.9337 0.03718 NF-B

p-NF-kB p65 (S536) 0.8596 0.04852 NF-B

p53 0.4184 0.04522 Cell cycle

CDK2 0.5662 0.01951 Cell cycle

RAP1A/RAP1B 0.9213 0.03866 Other

Ptenfl/fl uncastrated vs Ptenfl/fl castrated

p-AMPK 0.9451 0.04425 PI3K/mTOR

p-FOXO3A (S318/S321) 0.7328 0.02036 PI3K/mTOR

GSK-3 0.6393 0.03958 PI3K/mTOR

p-GSK-3S9 0.5814 0.04957 PI3K/mTOR

PI3K p110 (C73F8) 0.5006 0.04358 PI3K/mTOR

p-PKC 0.8070 0.04234 PI3K/mTOR

p-TSC2 (T1462) 0.7835 0.04773 PI3K/mTOR

IRS-1 1.0606 0.03694 Tyrosine kinase

ERK1/2 0.6510 0.04475 MAPK

p-c-RAF (S338) 0.5344 0.03642 MAPK

p38 MAPK 0.6484 0.0479 MAPK

Caspase 3 0.6075 0.03098 Apoptosis

CDK2 0.6562 0.02779 Cell Cycle

Pik3ca+/HR;Ptenfl/fl uncastrated vs Pik3ca+/HR;Ptenfl/fl castrated

p-SHP-2 (Y542) 9.2513 0.00054 Tyrosine kinase

p-SRC (Y416) 9.3157 0.00063 Tyrosine kinase

YAP 0.3805 0.03192 Hippo

CK2 0.3346 0.00985 Other

Pik3ca+/HR vs Ptenfl/fl (castrated)

AKT 1.5906 0.02614 PI3K/mTOR

AMPK 1.1520 0.03527 PI3K/mTOR

FOXO3A 2.3281 0.00609 PI3K/mTOR


PI3K p110 0.3448 0.02069 PI3K/mTOR

PTEN 2.0117 0.02173 PI3K/mTOR

p-NDRG1 (T346) 1.3790 <0.0001 PI3K/mTOR

IRS-1 1.7688 0.01342 Tyrosine Kinase

SRC 1.6332 0.03586 Tyrosine Kinase

p-ERK1/2 (T202, Y204) 1.9048 0.02239 MAPK

p-c-RAF (S338) 0.8342 0.03993 MAPK

p-SHC (Y317) 1.3861 0.03662 MAPK

p-STAT3 (Y705) 2.3954 0.04674 JAK/STAT

p-NF-B p65(S536) 1.0298 0.04484 NF-kB

p-ATM/ATR (S/T) Substrate 2.5925 0.04121 DNA repair

CDK2 0.7670 0.00730 Cell Cycle

CK2 1.2165 0.00651 Other


p-VEGFR2 (Y1175) 3.0430 0.02767 Other

Pik3ca+/HR; Ptenfl/fl vs Pik3ca+/HR (castrated)

p-AKT (473) 1.9109 0.03118 PI3K/mTOR

FOXO3A 1.2664 0.02862 PI3K/mTOR

MTOR 1.1556 0.00943 PI3K/mTOR

p-NDRG1 (T346) 2.3525 0.00012 PI3K/mTOR

SRC 1.9918 0.02073 Tyrosine kinase

p-SRC (Y416) 3.3642 0.02960 Tyrosine kinase

p-STAT3 (Y705) 3.2727 0.01735 JAK/STAT

PARP 1.8056 0.02269 Apoptosis

p53 0.3799 0.04869 Cell Cycle

p-CDC2 (Y15) 4.8288 0.01469 Cell Cycle

-Actin 2.1633 0.03093 Other

E-Cadherin 2.1185 0.00483 Other


Pik3ca+/HR; Ptenfl/fl vs Ptenfl/fl (castrated)

p-AMPK 1.3287 0.02707 PI3K/mTOR

FOXO3A 1.0617 0.01107 PI3K/mTOR


p-PKC 0.8743 0.04833 PI3K/mTOR

PTEN 1.0459 0.02067 PI3K/mTOR

NDRG1 1.1792 0.02267 PI3K/mTOR

p-NDRG1 (T346) 0.9735 0.00145 PI3K/mTOR

RPS6 1.8945 0.00819 PI3K/mTOR

p-RPS6 (S240/244) 4.6657 0.03675 PI3K/mTOR

p-ErbB2/HER2 (Y1248)/EGFR (Y1173) 7.0385 0.00244 Tyrosine kinase

p-SHC (Y317) 1.2112 0.01602 MAPK

p-c-JUN (S73) 9.8366 0.00011 MAPK

TAZ 0.4362 0.03212 Hippo

ATM 8.5576 0.00651 DNA repair

p-BAD (S136) 1.0201 0.01941 Apoptosis

-Actin 2.2330 0.02836 Other

CK2 0.7012 0.04958 Other †Absolute means difference calculated from Log2 normalised and median centred data. ††P-value <0.05 filtered data; unpaired, two-tailed Welch’s t test (n = 3).

pik3ca mutation drives prostate cancer · pik3ca mutation drives prostate cancer 3 supplementary...

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