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1 A B AP VP DLP H&E AP AP VP DLP p-akt wild-type prostate PTEN-null prostate Supplementary Fig. 1. Targeted deletion of PTEN in prostate epithelium resulted in HG-PIN in all three lobes. (A) The anatomy of mouse prostate: anterior prostate (AP), ventral prostate (VP), and dorsal-lateral prostate (DLP). Wild-type mouse prostate is shown at 12 weeks of age. (B) PTEN loss in prostate epithelium resulted in HG-PIN accompanied by activated downstream AKT signaling. Prostate lobes of PTEN lox/lox ; PbCre4 (i.e. PTEN-null) mouse were harvested at 12 weeks of age and stained with H&E and p-akt (S473).

2 a HG-PIN surgical castration Tumorigenesis of PTEN -/- prostates 8 wks weeks post castration (age) (wks) lymphocyte infiltration lymphocyte infiltration apoptosis invasion invasion b relative levels Testosterone In circulation (n 8) relative levels Testosterone Intra-prostate Human Mice Human Mice PIN CRPC PIN CRPC Intact Castrated Supplementary Fig. 2. a. Androgen deprivation potentiated disease progression from HG-PIN to invasive castration-resistant prostatic adenocarcinoma. Mice harboring HG-PIN were castrated at 8 weeks of age, and PTEN-null prostate tumors developed invasive castrationresistant prostate cancer (CRPC) over the time. b. Testosterone levels in CRPC dramatically decreased compared to intact PIN mice. Testosterone levels were measured in circulation (left panel) and intra-prostate (middle panel) in CRPC post castration 16 wks. Testosterone amounts in circulation in human vs mice with intact or castration conditions. (n 8) ne amount ulation Testostero in circu (ng/dl)

3 H&E SMA CK8 p63 Supplementary Fig. 3. Invasive CRPC lesions contained both luminal cells and basal-like cells. FFPE prostate tissues containing invasive lesions from Supplementary Fig.2 were stained with SMA (smooth muscle marker), CK8 (luminal cell marker) and p63 (basal cell marker).

4 Supplementary Fig. 4. AR expression increased in castration-resistant prostate cancer. Western blot of AR using prostates isolated from age-matched wide-type littermates (WT), PTEN-null HG-PIN (PIN), PTEN-null castration-resistant prostate cancer (CRPC). For CRPC samples, HG-PIN harboring mice were castrated at 8 weeks of age and CRPC samples were harvested 6 weeks or 16 weeks post castration. Note that there is a 1.7-fold increase of AR protein in VP and 2.3-fold increase in AP when CRPC compared to HG-PIN. Normalized quantification of AR expression at 16 weeks post castration is shown below.

5 post castration 6 weeks AP VP DLP WT HG-PIN CRPC WT HG-PIN CRPC WT HG-PIN CRPC p53 AR, c-19 Quantification p53 16 weeks 16 weeks p53 Vinculin SMAD4 a-tubulin ification Quanti SMAD4 Supplementary Fig. 5. Down-regulated expression of p53 and SMAD4 in castration-resistant prostate cancer. Western blot of p53 and SMAD4 using prostates isolated from age-matched wide-type littermate (WT), PTEN-null HG-PIN (PIN), PTEN-null castration-resistant prostate cancer (CRPC). In case of CRPC, HG-PIN harboring mice were castrated at 8 weeks of age and CRPC samples were harvested 6 weeks or 16 weeks post castration. Note the loss of p53 and down-regulation of SMAD4 at 16 weeks post castration (see red box). Normalized quantification of p53 and SMAD4 at 16 weeks post castration is shown (right).

6 a CRPC: forward b CRPC: reverse d HG-PIN: forward c CRPC allele-specific PCR amplicon: reverse 5 - T G T C C T A G A - 3 anti-sense strand sense strand 5 - T C T G G G A C A A C A G G A T C T - 5 sense strand 5 - Ser Gly Thr Thr Arg Ser - 5 Supplementary Fig. 6. p53 somatic point mutations in PTEN-null CRPC. HG-PIN harboring mice were castrated at 8 weeks of age, and CRPC samples were isolated 16 weeks post castration. Prostate genomic DNA was used to amplify p53 exons followed by Sanger sequencing. Conventional PCR amplicons using CRPC genomic DNA were sequenced using both p53 forward primer (a) and reverse primer (b). p53 mutant amplicons were enriched in a mutation allele-specific PCR using the same DNA in (a) and (b) prior to Sanger sequencing using reverse primers (c). Conventional PCR amplicons using prostate DNA of HG-PIN or wild-type were sequenced using p53 primers, where no point mutations ever found. A representative forward sequencing result is shown (d). Note the position of p53 somatic point mutation (red arrow) and the resulting amino acid change in mouse (yellow box), i.e. p53 G114R (Gly Arg), corresponding to human p53 G117R (Gly Arg).

7 p63 DAPI p63/dapi HG-PIN CRPC (intra-luminal) CRPC (invasive) SMA p63/sma p63/sma/dapi S l t Fi 7 A d d i ti l dt i d 63 iti ith li l ll i th lti t I fl Supplementary Fig. 7. Androgen deprivation led to increased p63-positive epithelial cells in the resulting tumors. Immunofluorescence staining: p63 (red), SMA (green) and DAPI (blue). Top panel (HG-PIN): note the disconnected thin layer of spindle-shaped p63-positive basal epithelial cells (arrow head). Middle panel: the enrichment of intra-luminal p63-positive cells in CRPC lesion (arrow). Lower panel: the enrichment of enlarged rounded nuclei of p63-positive epithelial cells in the invasive lesions lining above SMA-stained stromal layer (arrow), as compared to the spindle shaped smaller nuclei of basal epithelial cells (arrow head).

8 ** ** ** Sham operation Surgical castration Supplementary Fig. 8. Androgen deprivation led to increased p63-positive epithelial cells in the resulting tumors. p63- positive epithelial tumor cells were calculated in up to 100 ventral prostate glands / per group, n=12 mice per group. Two groups: sham operation vs. surgical castration.

9 a Pten f/f; Pb-Cre Placebo MDV AP VP DLP Prostate tumor weight (mg) p< p>0.05 Vehicle (n=9) MDV (n=13) p< AP VP DLP b CRPC H&E invasion AR invasion p63 invasion H& E AR p63 HG-PIN Supplementary Fig. 9. MDV3100 treatment also resulted ultimately in the progression to invasiveness of otherwise stable HG-PIN. Mice pp y g y p g harboring PTEN-null HG-PIN at 8 weeks of age were treated with MDV3100 or vehicle via oral gavage daily. a. Prostate tissues were harvested and weighted 16 weeks post chemical castration. b. Prostate tissues were stained with H&E, AR, and p63. Note the invasive lesions (black arrow) enrichment of p63-positive cells in CRPC. However, the HG-PIN (control tumor) is well localized within the boundary surrounded by basement membrane (H&E), and p63 IHC staining labels a thin, disconnected layer of p63-positive spindle shaped basal epithelial cells (blue arrow).

10 p110β dose PTEN -/- ; p110β +/+ PTEN -/- ; p110β +/- PTEN -/- ; p110β -/- Supplementary Fig. 10. Genetic ablation of p110β impairs prostate tumorigenesis in the AP in a dose-dependent manner. Heterozygous mice obtained during the breeding process showed that loss of a single copy of p110β impaired PTEN-controlled tumorigenesis in a dose-dependent manner. Left: H&E, Right: prostate weight (mg). Anterior prostates are shown at 12 weeks of age.

11 AP VP DLP PTEN-/-; p110β-/-; p110α-/- PTEN-/-; p110β-/-; p110α+/- Supplementary Fig. 11. In VP and DLP, genetic ablation of one p110α allele in the p110β null background restored the normal ductal structure in PTEN-/-; p110β-/- ; p110α+/- prostates. Prostates are shown at 12 weeks of age.

12 Placebo BEZ235 Placebo BEZ235 CRPC, PTEN-null Surgical castration Primary HG-PIN, PTEN-null Sham operation Supplementary Fig. 12. Inhibition of PI3K pathway only partially affected the proliferation of PTEN-null CRPC prostate tumors. Mice harboring PTEN-null HG-PIN at 8 weeks of age underwent either surgical castration (left panel) or sham operation (right panel) for 16 weeks. Mice were then treated with BEZ235 or placebo. Brdu was administered at 1-hour post treatment and samples harvested 5 hours later. Shown are Brdu immunohistochemical staining.

13 wild-type HG-PIN CRPC AP VP DLP AP VP DLP AP VP DLP pakt 6 weeks post castration perk1/2 Tubulin 1 2 Quantificatio on pakt on Quantificati perk1/2 Supplementary Fig 13 The gradual signaling shift between PI3K and MAPK pathways in castration-resistant prostate tumors Tissue Supplementary Fig. 13. The gradual signaling shift between PI3K and MAPK pathways in castration-resistant prostate tumors. Tissue western for prostates of wild-type (WT), Pten-null HG-PIN (PIN), PTEN-null castration-resistant prostate tumors (CRPC). Mice were castrated at 8 weeks of age, and CRPC samples harvested 6 weeks post castration. Normalized quantification of pakt and p-erk1/ in VP is shown.

14 per duct 100 * ** * BrdU- -positive cells Placebo BEZ235 Placebo BEZ235 Placebo BEZ235 AZD6244 BEZ235+ AZD6244 * Supplementary Fig. 14. Concurrent inhibition of PI3K and MAPK signaling pathways substantially suppressed cell division of CRPC. Mice harboring PTEN-null HG-PIN at 8 weeks of age underwent surgical castration for 16 weeks. Mice were then treated with placebo, BEZ235, AZD6244, or combo. BrdU was administered at 1-hour post treatment and prostate tissues were harvested 5 hours later. BrdU IHC staining was shown in Fig 4b, and the percentage of Brdu+ cells per duct was calculated here.

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