Fig. 1 MycTG and loss of PTEN cooperate for aggressive PCa development, resulting in decreased survival. (A) Total dehydrated prostate weights from 6- and 10-week-old mice were averaged for each genotype (n = 3 to 6 mice per arm, mean ± SEM). wild-type, WT. (B) Phenotypical penetrance percentages for low-grade prostatic intraepithelial neoplasia (LgPIN), HgPIN, and cancer in anterior prostate tissues from 6- and 10-week-old mice evaluated by hematoxylin and eosin (H&E) staining. (C) Left: Representative ultrasound images of prostate tumors at 7 months outlined in yellow from indicated genotypes; scale bars, 9 mm. Right: Quantification of prostate tumor size in mice with an average age of 8 months (n = 5 mice per arm, mean ± SEM). (D) Kaplan-Meier survival curves for mice with the indicated genotypes. Dotted line highlights the median life span of 75 weeks for Ptenfl/fl;MycTg mice. (E) Top: Representative bright-field images of three-dimensional organoid structures 6 days after seeding; scale bars, 50 μm. Bottom: Western blot analyzing the organoids, showing P-AKT, PTEN, GFP for MycTg, and β-actin. (F) Newly synthesized proteins measured by 35S methionine/cysteine incorporation in organoids (left panel), with quantification relative to WT littermates (right panel) (n = 5, mean ± SEM). *P < 0.05, **P < 0.01, ***P < 0.001, t test.
Fig. 2 The cooperation of MYC and loss of PTEN selectively activates the adaptive PERK–P-eIF2α arm of the UPR. (A) Left: Representative IF images of P-PERK/cytokeratin 5 (CK5) or P-eIF2α/CK5 co-staining with DAPI (4′,6-diamidino-2-phenylindole) used to visualize the nuclei within anterior prostate tissue from 10-week-old mice; scale bars, 100 μm. P-PERK or P-eIF2α expression quantified relative to DAPI (n = 3 mice per arm, with four images averaged per mouse, mean ± SEM). (B) Representative IF images of P-eIF2α/CK5 co-staining with DAPI in anterior prostate tissue from 6-week-old mice (scale bars, 100 μm) (left panel) and directly within areas of PIN (right panel). Lower panel depicts a model showing the timeline of PCa development within Ptenfl/fl;MycTg mice, highlighting when P-eIF2α is expressed. **P < 0.01, t test.
Fig. 3 Inhibition of P-eIF2a activity rebalances protein synthesis and prevents PCa progression. (A) Representative Western blot highlighting PERK signaling in organoid cultures. (B) Left: Total newly synthesized proteins measured by 35S methionine/cysteine incorporation and Western blot showing P-eIF2α and ATF4 in organoids treated with dimethyl sulfoxide (DMSO) or ISRIB (500 nM) for 6 hours. Right: Quantification of radioactive pulse relative to loading, depicted as percent over WT (n = 3, mean ± SEM). (C) Schematic of preclinical trial for escalating dosage and MRI schedule over 6 weeks. (D) Representative scans of two ISRIB-treated mice after 1 and 6 weeks of treatment for comparison. Tumor is outlined in red, and arrows highlight the seminal vesicles (SV) surrounding the tumor. (E) Quantification of tumor size as fold change relative to baseline volume at 3- and 6-week time points (mean ± SEM). **P < 0.01, ***P < 0.001, t test.
Fig. 4 High P-eIF2α expression in human prostate tumors with loss of PTEN function is associated with increased risk of metastasis or death after surgery. (A) Representative Western blot showing PTEN, MYC, P-PERK, P-eIF2α, and total eIF2α expression with β-actin loading control (Ctrl) in human prostatic RWPE-1 cell lines. (B) Quantification of annexin V–positive cells analyzed by flow cytometry relative to control cells after treatment with DMSO or 500 nM ISRIB for 9 hours (n = 3, mean ± SEM) *P < 0.05, t test. (C) Kaplan-Meier analysis of clinical progression–free survival [progression defined as visceral or bone metastasis or PCSM] for patients with normal PTEN expression versus PTEN loss and relative MYC expression identified by IF from the TMA. (D) Kaplan-Meier analysis of MET/PCSM for patients with normal PTEN expression versus PTEN loss grouped by eIF2α phosphorylation. (E) Cox proportional hazards regression results are shown in a Forest plot of hazard ratios and 95% CI for factors associated with risk of clinical progression after surgery. Independent factors are tumor with PTEN loss/low P-eIF2α or PTEN loss/high P-eIF2α versus a reference group with normal PTEN expression; age in years; PSA in nanograms per milliliter; Gleason score > 7 versus 6; and pathological stage T3-T4 versus T2 at the time of prostatectomy.
Fig. 5 Inhibition of P-eIF2α axis results in tumor regression and prolongs survival in a humanized model of metastatic PCa. (A) Schematic highlighting origin of PDX tumors from primary (pPCa) or lymph node metastasis (mPCa). Tumors from selected patients with high-risk features, based on Gleason score and clinical stage or with lymph node metastases determined by 68Ga–PSMA-11 positron emission tomography (PET) scans, were used to generate PDXs. Primary and metastatic tumors were confirmed from tissue collected at the time of surgery and immediately implanted into immunodeficient NOD scid gamma (NSG) mice. (B) Representative IF images of MYC/CK8 (epithelial cell marker), P-AKT/CK8, or P-eIF2α/CK8, co-staining with DAPI from benign (Ben) tissue adjacent to pPCa or mPCa tumors; scale bars, 50 μm. Right: Quantification of protein expression as relative mean IF intensity normalized to adjacent stromal tissue. (C) Kaplan-Meier tumor survival curves for mice bearing pPCa or mPCa tumors treated with ISRIB (10 mg/kg) or vehicle (n = 8, per cohort; **P = 0.01, log-rank test). The survival curves represent mice euthanized when tumors reached an end point of 2 cm or when the mice showed clear signs of morbidity. (D) Representative tumor sizes after 10 days of treatment. (E) Representative TUNEL staining and quantification of PDX tumors treated with vehicle or ISRIB (10 mg/kg); scale bars, 100 μm (n = 3, ***P < 0.001, t test). (F) Quantification of newly synthesized proteins in vivo, assessed by incorporation of OP-Puro within PDX treated with ISRIB (10 mg/kg) or vehicle (n = 3 to 4 per arm, mean ± SEM; *P < 0.05, t test). n.s., not significant. MFI, mean fluorescence intensity.
Fig. 6 ISRIB treatment decreases metastatic progression in an advanced castration-resistant PCa PDX model. (A) Representative H&E staining and IF of P-eIF2α, PTEN, and MYC expression at the primary site of implantation (mCRPC tumor), left kidney, and distant metastatic lesions in the liver; scale bars, 200 μm (top left); 100 μm (bottom left); and 50 μm for IF images. (B) Schematic of preclinical trial for mCRPC tumor growth and PET/CT schedule. Representative 68Ga–PSMA-11 PET/CT scans on day 0 (time of treatment) and on day 7 are shown for the control versus ISRIB-treated cohorts. Uptake of the PSMA-targeted radiotracer agent is observed in the liver, lymph node, and at the site of primary tumor implantation in the left kidney capsule. Physiologic uptake of the PSMA-targeted radiotracer is also seen in the contralateral kidney and bladder because it is excreted in the urinary tract. Histologic confirmation of liver metastasis is shown by H&E staining at the time of euthanasia with dashed outlines around metastatic lesions. (C) Kaplan-Meier survival curve for mice bearing mCRPC tumors treated once per day with ISRIB (10 mg/kg) or vehicle (n = 3, per cohort); *P = 0.02, log-rank test. The survival curves represent mice euthanized when PSMA 68Ga PET/CT showed progression from one distant metastatic lesion to two or more sites or when the mice showed signs of becoming moribund. (D) Quantification of visible metastatic lesions on the left medial lobe of the liver at the time of euthanasia in the cohorts (n = 3 per cohort); ***P = 0.001.
- Table 1 Characteristics of patients included in the TMA.
Baseline characteristics of the TMA cohort consisting of 424 tumor samples, where 58 years is the average age at diagnosis. More than 50% of the cohort had pathological Gleason grade 7 or higher, and 75% had organ-confined disease (pathological stage T2). Median follow-up was 10 years.
Patient characteristics
of TMAValue n (%) Race/ethnicity Native American 1 0 Asian/Pacific Islander 13 3 African-American 14 3 Caucasian 359 85 Mixed 25 6 Unknown 12 3 Biopsy Gleason grade 3 + 3 263 64 3 + 4 95 23 4 + 3 25 6 8 − 10 29 7 Missing 12 — Clinical T stage T2 296 98 T3 5 2 T4 2 1 Missing 121 — Pathologic Gleason grade 3 + 3 184 43 3 + 4 173 41 4 + 3 45 11 8 − 10 22 5 Pathologic T stage T2 313 75 T3 102 24 T4 5 1 Missing 4 — Pathologic N stage NX 200 48 N0 208 50 N1 7 2 Missing 9 — Surgical margins No 354 83 Yes 70 17 Adverse path (Gleason Grade
≥ 4 + 3 or pT3a/pN1)No 291 69 Yes 133 31
Supplementary Materials
www.sciencetranslationalmedicine.org/cgi/content/full/10/439/eaar2036/DC1
Materials and Methods
Fig. S1. MycTg and PTEN loss cooperate for aggressive PCa development in mice.
Fig. S2. The UPR is activated in murine PCa.
Fig. S3. PERK loss blocks PCa progression and decreases P-eIF2α expression.
Fig. S4. Loss of P-eIF2α activity by ISRIB shows no toxicity and does not substantially alter infiltrating immune cells.
Fig. S5. Inhibition of P-eIF2α activity by ISRIB does not affect human prostatic cell lines’ growth.
Fig. S6. PCa tissue from TMA shows specificity of protein expression in benign and tumor cells.
Fig. S7. Treatment with ISRIB or ATF4 siRNA results in increased apoptosis within metastatic tumor.
Fig. S8. A PDX was generated to recapitulate mCRPC.
Table S1. MRI tumor volumes during treatment in GEMMs.
References (49–54)
Additional Files
- Supplementary Material for:
Development of a stress response therapy targeting aggressive prostate cancer
Hao G. Nguyen, Crystal S. Conn,* Yae Kye, Lingru Xue, Craig M. Forester, Janet E. Cowan, Andrew C. Hsieh, John T. Cunningham, Charles Truillet, Feven Tameire, Michael J. Evans, Christopher P. Evans, Joy C. Yang, Byron Hann, Constantinos Koumenis, Peter Walter, Peter R. Carroll, Davide Ruggero*
*Corresponding author. Email: crystal.conn{at}ucsf.edu (C.S.C.); davide.ruggero{at}ucsf.edu (D.R.)
Published 2 May 2018, Sci. Transl. Med. 10, eaar2036 (2018)
DOI: 10.1126/scitranslmed.aar2036This PDF file includes:
- Materials and Methods
- Fig. S1. MycTg and PTEN loss cooperate for aggressive PCa development in mice.
- Fig. S2. The UPR is activated in murine PCa.
- Fig. S3. PERK loss blocks PCa progression and decreases P-eIF2α expression.
- Fig. S4. Loss of P-eIF2α activity by ISRIB shows no toxicity and does not substantially
alter infiltrating immune cells.
- Fig. S5. Inhibition of P-eIF2α activity by ISRIB does not affect human prostatic
cell lines’ growth.
- Fig. S6. PCa tissue from TMA shows specificity of protein expression in benign
and tumor cells.
- Fig. S7. Treatment with ISRIB or ATF4 siRNA results in increased apoptosis
within metastatic tumor.
- Fig. S8. A PDX was generated to recapitulate mCRPC.
- References (49–54)
Other Supplementary Material for this manuscript includes the following:
- Table S1 (Microsoft Excel format). MRI tumor volumes during treatment in GEMMs.
- Materials and Methods
