Supporting InformationAhmad et al. 10.1073/pnas.1101263108SI Materials and MethodsMice. The ARR2Probasin-Cre transgenic line, PB-Cre4, has the Crerecombinase under the control of a modified rat prostate-specificprobasin promoter (1). In the Her2KI transgenic strain, the firstcoding exon of the endogenous Her2 (neu) gene is replaced by anoncogenicHer2 gene containing an activating point mutation in thetransmembrane domain that is Cre-inducible, resulting in low levelsof Her2 up-regulation (2). The Ptenfl/fl mice carry a conditionalinactivatable Pten allele (where exons 4 and 5 are flanked by lox psites) (3). In the Z/EGFP reporter mouse, the Z/EGFP transgeneresults in the expression of β-galactosidase in most tissues via aβ-geo insert that is flanked by lox p sites. The presence of Crerecombinase results in the excision of the β-geo insert, activatingthe constitutive expression of GFP in the prostate epithelium (4).

Immunohistochemistry.For each genotype, we stained at least threesamples from different mice and took representative images forthis manuscript. We used antibodies against Ki-67 (VP-RM04;Vector Labs; 1:100, citrate buffer and water bath antigen re-trieval, 50 min at 99 °C), BrdU (347580; BD Biosciences; 1:500,citrate buffer and microwave antigen retrieval), PTEN (9559; CellSignaling; 1:100, citrate buffer and water bath antigen retrieval,50 min at 99 °C), pAKT (Ser473) (3787; Cell Signaling; 1:50, cit-rate buffer andmicrowave antigen retrieval), pERK1/2 (9101; CellSignaling; 1:100, citrate buffer and microwave antigen retrieval),p21 (M19; Santa Cruz Biotechnology; 1:500, citrate buffer andwater bath antigen retrieval, 50 min at 99 °C), p16 (M-156; SantaCruz Biotechnology; 1:25, citrate buffer and microwave antigenretrieval), p63 (clone 4A4; Thermo Scientific; 1:200, citrate bufferand microwave antigen retrieval), HER3 (MS-303-PABX cloneH3.105.5; Neomarkers; 1:20, no antigen retrieval),HER2 (K5204;HercepTest; Dako; was performed with the protocol outlinedin the kit, except we used a microwave-based antigen-retrievaltechnique and incubated with the primary antibody at 4 °Covernight), p-mTOR (Ser2448) (2976; Cell Signaling; 1:200, cit-rate buffer and microwave antigen retrieval), p-S6K (Thr421/Ser424) (9204; Cell Signaling; 1:100, citrate buffer and microwaveantigen retrieval), p53 (CM5; Vector Labs; 1:200, citrate bufferand microwave antigen retrieval), and cleaved caspase 3 andmyeloperoxidase (MPO).Senescence-associated β-galactosidase staining (SAβGal)

(9860; Cell Signaling) was used to assess levels of senescence.

Human Tissue Microarray. Cores of prostate cancer tissue (3 × 0.5-mm2), as identified by pathologists, were removed from repre-sentative areas of the formalin-fixed, paraffin-embedded blocks.All samples had been taken from prostate cancer patients at thetime of transurethral resection of the prostate. These samplesconsisted of Gleason 3 (n = 123), 4 (n = 49), and 5 (n = 37)cancers, as determined independently by two consultant path-ologists (M.S. and R.J.B.).

Histoscore. Protein expression levels were scored by two in-dependent observers blinded to clinical parameters, using aweighted histoscore method, also known as the H-score, atmagnification 40× (I.A. and L.B.S.) (5). Each cellular location(membranes, cytoplasm, and nuclei) was scored separately. Theweighted histoscore method assesses the staining intensity andthe percentage of cells stained with that intensity for the fullslide. It is calculated by (1 × % cells staining weakly positive) +(2 ×% cells staining moderately positive) + (3 ×% cells stainingstrongly positive). This provides a semiquantitative classificationof staining intensity, with the maximum score being 300 (if 100%of cells stain strongly positive) and the minimum score being 0 (if100% of cells are negative). The weighted histoscore method is awell-established method for scoring tissue that has heteroge-neous staining.

Plasmids, siRNA, and Cell Culture. Human prostate cancer cell linesDU145 and PC3were authenticated by Laboratory ofGovernmentScientists standards and grown in RPMI (Gibco) containing 10%serum supplement. For primary tumor cell culture, tumor tissuewas cut into small pieces and digested for 2 h in digestion medium(DMEM with 20% FBS, penicillin-G/streptomycin solution 1 mg/mL, collagenase 1 mg/mL, and hyaluronidase 1 mg/mL). The tu-mor cells were pelleted after passing the digested mix througha 40-μm cell strainer and cultured in DMEM containing 20% FBSand penicillin-G/streptomycin solution (1 mg/mL). The PTENsiRNA (6251) was obtained from Cell Signaling. The PTEN ex-pression construct (EGFP-PTEN) was generated by cloningPTEN cDNA (OriGene) into EcoRI/SalI-cut EGFPC2 (Clontech)vector. The HER2 expression construct was obtained fromAddgene (plasmid 16257) (6). The siRNA and plasmids weretransfected using nucleofector kits from Lonza (1001 and 1003).

Immunoblotting. Tumors were homogenized in lysis buffer (50 mMTris, pH 7.6, 150 mMNaCl, 1% Triton X-100, 0.5% deoxycholate,0.1% SDS, 1 mM sodium orthovanadate, 5 mM sodium fluoride,and protease inhibitor mixture; Calbiochem) using a Precellystissue homogenizer (Bertins Technology). Lysates were resolvedby SDS/PAGE on 4–12% gradient polyacrylamide gels (In-vitrogen) at 200 V for 1 h and transferred electrophoretically ontoPVDF membranes (Millipore) at 200 mA for 1 h. Blots wereblocked for 30 min with 5% skimmilk, rinsed, and probed with therespective antibodies (in 5% BSA, 0.1% Tween-20 containingTBS) overnight at 4 °C. After incubation with HRP-conjugatedsecondary antibody, bands were detected using ECL detectionreagent (GE Healthcare). Immunoblotting was performed withthe following antibodies: p-AKTser473 (Cell Signaling; 9271),AKT (Cell Signaling; 9272), p-mTOR (Cell Signaling; 2971),mTOR (Cell Signaling; 2983), p-ERK (Cell Signaling; 4377), ERK(Cell Signaling; 4695), actin (Cell Signaling; 4968), p-PTENser/threo (Cell Signaling; 9554), GAPDH (Sigma; G9295), HER2(Cell Signaling; 2165), and HER3 (Cell Signaling; 4754).

1. Wu X, et al. (2001) Generation of a prostate epithelial cell-specific Cre transgenicmouse model for tissue-specific gene ablation. Mech Dev 101:61e69.

2. Andrechek ER, et al. (2000) Amplification of the neu/erbB-2 oncogene in a mousemodel of mammary tumorigenesis. Proc Natl Acad Sci USA 97:3444e3449.

3. Lesche R, et al. (2002) Cre/loxP-mediated inactivation of the murine Pten tumorsuppressor gene. Genesis 32:148e149.

4. Novak A, Guo C, Yang W, Nagy A, Lobe CG (2000) Z/EG, a double reporter mouse linethat expresses enhanced green fluorescent protein upon Cre-mediated excision.Genesis 28:147e155.

5. Kirkegaard T, et al. (2006) Observer variation in immunohistochemical analysis ofprotein expression, time for a change? Histopathology 48:787e794.

6. Li YM, et al. (2004) Upregulation of CXCR4 is essential for HER2-mediated tumormetastasis. Cancer Cell 6:459e469.

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Fig. S1. HER2/3 up-regulation correlates with reduced PTEN and up-regulation of pAKTSer473. Immunohistochemistry (IHC) of prostate cancers from ourhuman tissue microarray revealed up-regulation of HER2 (A) and HER3 (C), with loss of PTEN signal (E ) and up-regulation of pAKT (G). In comparison, thebenign prostatic hypertrophic (BPH) samples demonstrated minimal HER2 (B), HER3 (D), and pAKT (H), with a demonstrable PTEN signal (F ). (Scale bars,100 μm.)

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Fig. S2. Comparison of HER2, HER3, PTEN, and pAKTSer473 IHC between BPH and CaP. Boxplots representing the comparison of histoscores between BPH andGleason grade 3–5 prostate cancers. This revealed a significant increase in cytoplasmic and membranous HER2 (A and B) and HER3 (C and D) as well as cy-toplasmic pAKT (F) in CaP samples compared with BPH. We also noticed a reduction in cytoplasmic PTEN (E) in CaP compared with BPH.

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Fig. S3. Histology of 12-mo-old wild-type, PB-Cre4: Her2KI, and PB-Cre4: Ptenfl/fl prostates. H&E (A–C) of the PB-Cre4: Ptenfl/fl prostate revealed an obvioustumor compared with wild-type and PB-Cre4: Her2KI. A minimal Pten D-FD-F epithelial staining was observed in PB-Cre4: Ptenfl/fl prostates (with an intactstromal signature) compared with wild-type and PB-Cre4: Her2KI. Her2 (G–I) staining was up-regulated in the PB-Cre4: Her2KI glands compared with PB-Cre4:Ptenfl/fl and wild-type. In Her3 (J–L) IHC, a minimal up-regulation was observed in PB-Cre4: Her2KI and PB-Cre4: Ptenfl/fl glands compared with wild-type. Wealso showed an intact PTEN signal in the cytoplasm of stromal cells of PB-Cre4: Ptenfl/fl mice (M). In MMTV-NIC Ptenfl/fl mammary tumors, which coupledexpression of activated Her2 and Pten loss in the same mammary epithelial cell (MMTV-NIC), we demonstrated a significant up-regulation of Her2 in thetumors in comparison with the minimal up-regulation in PB-Cre4: Her2KI prostate glands (N and O). (Scale bars, 200 μm.)

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Fig. S4. PB-Cre4: Ptenfl/fl Her2KI prostate tumors demonstrate a reduction in p63 as well as microinvasion. We observed a loss in continuity of both p63 andsmooth muscle actin (SMA) in PB-Cre4: Ptenfl/fl Her2KI compared with PB-Cre4: Ptenfl/fl prostate tumors by IHC, as well as a quantifiable reduction in percentageof p63, suggesting that the tumors have become locally invasive (A–E, respectively). Similarly, we observed a reduction in levels of androgen receptor (AR)(F–H). (Scale bars, 200 μm.)

Fig. S5. In vivo imaging of prostate tumors. (A–D) Imaging of 200-d-old wild-type (bright-field) (A) and OV100 imaging of wild-type (B), PB-Cre4: Ptenfl/fl (C),and PB-Cre4: Ptenfl/fl Her2KI (D) mice (centered on pelvic region) revealed that at this stage, signal is only demonstrable in PB-Cre4:Ptenfl/fl Her2KI mice. (E) IHCfor GFP in tumor indicated recombination. (F) Curve of age at which prostate tumors were detected with OV100 imaging demonstrated that tumors could beidentified earlier in PB-Cre4: Ptenfl/fl Her2KI compared with PB-Cre4: Ptenfl/fl tumors (P = 0.002). (G) Ultrasound (US) of a murine prostate tumor revealeddiscrete lesions. (Scale bars, 100 μm.)

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Fig. S6. Comparison of Ki67, BrdU, p16, and p21 IHC between PB-Cre4: Ptenfl/fl and PB-Cre4: Ptenfl/fl Her2KI cohorts and HER2/HER3 activation by heregulindecreased senescence in cultured tumor cells from PB-Cre4: Ptenfl/fl mice. Boxplots represent histoscores demonstrating up-regulation of proliferative markersKi67 (A) and BrdU (B) and down-regulation of senescence markers p16 (C) and 21 (D) in the cohort PB-Cre4: Ptenfl/fl Her2KI compared with PB-Cre4: Ptenfl/fl.Quantification of Ki67, BrdU, p16, and p21 in the cohort PB-Cre4: Ptenfl/fl Her2KI compared with PB-Cre4: Ptenfl/fl (E). Representative images and quantificationof SAβGal (senescence marker)- and cytokeratin (epithelial marker)-positive PB-Cre4: Ptenfl/fl tumor cells cultured with or without heregulin for 7 d. Wild-typemouse embryonic fibroblasts (MEFs) were used as negative control for cytokeratin staining (F).

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Fig. S7. Comparison of pERK1/2, pAKT, p-mTOR, pS6K, and HER3 in vehicle- or MEK inhibitor-treated tumors. Levels of pERK1/2, pAKT, p-mTOR, and pS6Kwere assayed by IHC. PB-Cre4: Ptenfl/fl tumors demonstrated minimal pERK1/2 (B), up-regulation of pAKT (F), p-mTOR (J), and pS6K (N). Vehicle-treated PB-Cre4: Ptenfl/fl Her2KI demonstrated up-regulation of pERK1/2 (C) and down-regulation of pAKT (G), p-mTOR (K), and pS6K (O). When PB-Cre4: Ptenfl/fl Her2KI

mice were treated with 7 d of MEK inhibitor (PD184352), the tumors demonstrated down-regulation of pERK1/2 (D) and a corresponding up-regulation ofpAKT (H), p-mTOR (L), and p-S6K (P). Wild-type controls of pERK1/2, pAKT, p-mTOR, and pS6K are also represented (A, E, I, andM, respectively). (Scale bars, 100μm.) To test the effect of Her2KI on Her3 levels, lysates from wild-type prostates, Ptenfl/fl, and Ptenfl/fl Her2KI tumors were analyzed by Western blotting. Her3levels were only increased in Ptenfl/fl Her2KI tumors. MEK inhibitor-treated Ptenfl/fl Her2KI tumors showed decreased Her3 levels compared with vehicle-treatedtumors (Q).

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Fig. S8. Comparison of Ki67, BrdU, p16, p21, MPO, and cleaved caspase 3 IHC between vehicle- and MEK inhibitor (MEKi)-treated tumors. Boxplots representhistoscores demonstrating up-regulation of pAKT (A) with no change in total AKT (B) in MEK inhibitor-treated PB-Cre4: Ptenfl/fl Her2KI tumors compared withvehicle. Similarly, up-regulationof p-mTOR (C), p-S6K (D), andp53 (E) was observed inMEK inhibitor-treated PB-Cre4: Ptenfl/flHer2KI tumors comparedwith vehicle.We also observed up-regulation of pERK1/2 (F) with no change in total ERK (G) inMEK inhibitor-treated PB-Cre4: Ptenfl/flHer2KI tumors comparedwith vehicle.Weshowed down-regulation of Ki67 (I) and BrdU (J), with up-regulation of p16 (K) and p21 (L) inMEK inhibitor-treated PB-Cre4: Ptenfl/flHer2KI cohorts comparedwithvehicle.We observed a difference inweight between vehicle- andMEK-treatedmouse tumors (H). A significant increase in neutrophil marker,MPO, and apoptosismarker cleaved caspase 3 IHCwas observed in tumors fromMEK-treated cohorts comparedwith vehicle-treated (M–O, and P–R respectively). [Scale bars, 200 μm(Mand N), 100 μm (P and Q).]

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Fig. S9. IHC of PB-Cre4: Ptenfl/fl Her2KI vehicle-treated tumors and correlation between HER2 and pERK1/2. IHC from PB-Cre4: Ptenfl/fl Her2KI tumors phe-nocopied age-matched PB-Cre4: Ptenfl/fl Her2KI tumors with respect to high levels of Ki67 (A) and BrdU (B), with minimal staining for p16 (C), p21 (D), andSAβGal (E). (Scale bars, 200 μm.) Correlation scatterplot of HER2 and pERK1/2 revealed strong correlation [Pearson’s correlation coefficient (cc) = 0.412,P < 0.0001] (F).

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Fig. S10. Mechanism of tumorigenesis in PB-Cre4: Ptenfl/fl Her2KI mice. PB-Cre4: Ptenfl/fl tumors demonstrated up-regulation of the PI3K pathway, which up-regulated p53 and led to a senescent phenotype (A). In PB-Cre4: Ptenfl/fl Her2KI tumors, up-regulation of MAPK “outweighed” PI3K up-regulation, leading toa proliferative phenotype (B). When these mice were treated with a MEK1/2 inhibitor, the proliferative phenotype via the MAPK pathway was suppressed andthe senescent phenotype (via the PI3K pathway) predominated (C).

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