Skip to main content
  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

AACR logo

  • Register
  • Log in
  • My Cart
Advertisement

Main menu

  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • CCR Focus Archive
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • Breast Cancer
      • Clinical Trials
      • Immunotherapy: Facts and Hopes
      • Editors' Picks
      • "Best of" Collection
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

  • AACR Publications
    • Blood Cancer Discovery
    • Cancer Discovery
    • Cancer Epidemiology, Biomarkers & Prevention
    • Cancer Immunology Research
    • Cancer Prevention Research
    • Cancer Research
    • Clinical Cancer Research
    • Molecular Cancer Research
    • Molecular Cancer Therapeutics

User menu

  • Register
  • Log in
  • My Cart

Search

  • Advanced search
Clinical Cancer Research
Clinical Cancer Research
  • Home
  • About
    • The Journal
    • AACR Journals
    • Subscriptions
    • Permissions and Reprints
  • Articles
    • OnlineFirst
    • Current Issue
    • Past Issues
    • CCR Focus Archive
    • Meeting Abstracts
    • Collections
      • COVID-19 & Cancer Resource Center
      • Breast Cancer
      • Clinical Trials
      • Immunotherapy: Facts and Hopes
      • Editors' Picks
      • "Best of" Collection
  • For Authors
    • Information for Authors
    • Author Services
    • Best of: Author Profiles
    • Submit
  • Alerts
    • Table of Contents
    • Editors' Picks
    • OnlineFirst
    • Citation
    • Author/Keyword
    • RSS Feeds
    • My Alert Summary & Preferences
  • News
    • Cancer Discovery News
  • COVID-19
  • Webinars
  • Search More

    Advanced Search

Translational Cancer Mechanisms and Therapy

Attenuation of SRC Kinase Activity Augments PARP Inhibitor–mediated Synthetic Lethality in BRCA2-altered Prostate Tumors

Goutam Chakraborty, Nabeela Khan Patail, Rahim Hirani, Subhiksha Nandakumar, Ying Z. Mazzu, Yuki Yoshikawa, Mohammad Atiq, Lina E. Jehane, Konrad H. Stopsack, Gwo-Shu Mary Lee, Wassim Abida, Michael J. Morris, Lorelei A. Mucci, Daniel Danila and Philip W. Kantoff
Goutam Chakraborty
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Goutam Chakraborty
  • For correspondence: kantoff@mskcc.org chakrabg@mskcc.org
Nabeela Khan Patail
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rahim Hirani
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Rahim Hirani
Subhiksha Nandakumar
2Center for Molecular Oncology, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ying Z. Mazzu
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yuki Yoshikawa
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Mohammad Atiq
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Mohammad Atiq
Lina E. Jehane
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Konrad H. Stopsack
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Konrad H. Stopsack
Gwo-Shu Mary Lee
3Dana-Farber Cancer Institute, Boston, Massachusetts.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Wassim Abida
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Michael J. Morris
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lorelei A. Mucci
4Department of Epidemiology, Harvard T.H. Chan School of Public Health, Boston, Massachusetts.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Daniel Danila
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Philip W. Kantoff
1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: kantoff@mskcc.org chakrabg@mskcc.org
DOI: 10.1158/1078-0432.CCR-20-2483 Published March 2021
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Additional Files
  • Figure 1.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 1.

    Activation of SRC signaling pathway in BRCA2-deleted prostate cancer. A, BRCA2 alteration status of samples in the Kumar and colleagues, 2016 mCRPC cohort. Data from patients with complete mutation and copy-number alteration information were analyzed (134 samples from 54 patients). B, Volcano plot shows genes that are altered in BRCA2 deleted (homozygous + heterozygous) compared with samples with wild-type BRCA2. C, The bar graph shows the oncogenic pathways that are altered in BRCA2-deleted (homozygous + heterozygous) mCRPC tumors in Kumar and colleagues' cohort. Pathway analyses were performed using GSEA (c6 oncogenic signature). The 20 most upregulated and downregulated (on the basis of NES) pathways are shown. D, Enrichment plots show the represented oncogenic pathways, including upregulation of SRC oncogenic signatures (NES = 1.78; P = 0.003; Q = 0.05). E and F, The bar graph and enrichment plots represent the hallmark signaling pathways that are significantly altered in BRCA2-deleted (homozygous + heterozygous) mCRPC tumors in the Kumar and colleagues' cohort. Pathway analyses were performed using GSEA (hallmark signature). G, The bar graph shows the drugs which are significantly associated with the genes that are upregulated in BRCA2-deleted mCRPC described in B. The gene–drug association analysis was performed using ToppGene Suite.

  • Figure 2.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 2.

    BRCA2 deletion induces SRC kinase activation and increases sensitivity of prostate cancer cells to dasatinib. A and B, LNCaP cells were transduced with three different gRNAs targeting BRCA2. Cells infected with scrambled (SCR) gRNA were used as control. CAS9, total SRC, GAPDH, and RhoGDI served as loading controls. C, Levels of phosphorylated SRC at tyrosine 416 (pSRC Y416), total SRC, androgen receptor (AR), and E-cadherin were assessed by Western blotting in prostate cancer cell lines. GAPDH was used as loading control. D, Cells were treated with 0.3 and 3 μmol/L dasatinib for 7 days. The equivalent volume of DMSO was used as placebo treatment. After 7 days, cells were treated with 0.5 mg/mL MTT and micrographed in 40 × magnification (top). The bar graph shows the changes in cell growth percentage compared with scrambled gRNA- and DMSO-treated samples (bottom). P values were calculated by Student t test. E, LNCaP scramble control and BRCA2-gRNA 2 cells were counted and plated in 96-well plates (2,500 cells/well in 100-μL media). Cells were treated with indicated concentration of bosutinib (top) or saracatinib (bottom) for 7 days. The equivalent volume of DMSO was used as a placebo treatment. Cells were treated with 0.5 mg/mL MTT and the graphs represent cell viability (MTT count, % control). P values were calculated by Student t test. F, Indicated cells (LNCaP scramble control and BRCA2-gRNA 2) were counted and plated in 96-well plates (2,500 cells/well in 100-μL media) and treated with 0.1 and 1 μmol/L concentration of ipatasertib for 7 days. The equivalent volume of DMSO was used as a placebo treatment. Cells were treated with 0.5 mg/mL MTT, measured in a plate reader at 570 nm, and represented in the form of a bar graph. P values were calculated by Student t test. G, Samples from patients in TCGA Firehose Legacy cohort were divided into four quartiles based on levels of phospho-SRC at Y416, reverse-phase protein arrays (RPPA). Kaplan–Meier curves were used to compare disease-free survival. Log-rank test was performed to examine significance. H, Volcano plot shows genes that were altered in phospho-SRC Y416 high (top quartile, RPPA value) cases compared with samples with phospho-SRC Y416 low (bottom quartile, RPPA value). The bar graph to the right of the volcano plot shows the oncogenic pathways that were altered in phospho-SRC Y416–high versus -low localized prostate cancer tumors of TCGA Firehose Legacy cohort. Pathway analyses were performed using GSEA (c6 oncogenic signature). The altered (based on NES) pathways are shown.

  • Figure 3.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 3.

    Synergistic effect of dasatinib and PARPis on BRCA2-null prostate cancer cell viability. A and B, PC3M and LNCaP-abl cells were treated with indicated drugs alone or in combination for 4 days. Drug synergy was calculated using Chou-Talalay Method/CompuSyn. Graphs show the combination index plot for dasatinib (dasa) with olaparib (ola)/talazoparib (tala) combination obtained from CompuSyn (left). Tables show dose-dependent combination index and average effect (Fa) values (right). C and D, LNCaP scramble controls and BRCA2-gRNA 2 cells were counted and plated in 96-well plates (2,500 cells/well in 100-μL media). Cells were treated with dasatinib (0.3 μmol/L) and olaparib (3 μmol/L) alone or in combination. The equivalent volume of DMSO was used as a placebo treatment. After indicated days, cells were treated with 0.5 mg/mL MTT. The graphs represent the inhibition of cell viability by combination treatment. P values were calculated by Student t test. E, MTT-treated LNCaP-BRCA2-gRNA 2 cells from D were photographed at 40× magnification. Representative images are shown. F, LNCaP-abl cells were counted and plated in 96-well plates (2,500 cells/well in 100 μL media). Cells were treated with bosutinib (0.3 μmol/L) and talazoparib (0.03 μmol/L) alone or in combination. The equivalent volume of DMSO was used as a placebo treatment. After 7 days, cells were treated with 0.5 mg/mL MTT, measured in a plate reader at 570 nm, and represented as a bar graph. G, LNCaP scramble (SCR) controls and BRCA2-gRNA 2 cells were counted and plated in 96-well plates (2,500 cells/well in 100-μL media). Cells were treated with dasatinib (0.3 μmol/L) and cisplatin (0.1 μmol/L; dissolved in DMSF) alone or in combination. The equivalent volume of DMSO was used as a placebo treatment. After the indicated number of days, cells were treated with 0.5 mg/mL MTT. The graph represents the inhibition of cell viability by single agent and combination treatment. P values were calculated by Student t test. H, MTT-treated cells from G (DMSO and combination treatment) were photographed at 40× magnification. Representative images are shown.

  • Figure 4.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 4.

    Dasatinib induces DNA damage in BRCA2-null prostate cancer cells. A, PC3M cells treated with dasatinib (0.3 μmol/L) for 24 hours. The equivalent volume of DMSO was used as a placebo treatment. Dot plots show the percentage of cells with phospho-ATM, phospho-H2A.X, and both phospho-ATM and phospho-H2A.X (left). Bar graphs compare the percentages of cells between DMSO- and dasatinib-treated conditions (± SD; right). DSB represents the percentage of cells with dual activation of ATM and H2A.X. Total DNA damage represents the percentage of cells with either activated ATM, activated H2A.X, or dual activation. P values were calculated by Student t test. B and C, LNCaP SCR-gRNA and BRCA2-gRNA2 cl7 cells were treated with dasatinib (0.3 μmol/L) for 24 hours. The equivalent volume of DMSO was used as a placebo treatment. Dot plots show the percentage of cells with phospho-ATM, phospho-H2A.X, and both phospho-ATM and phospho-H2A.X (left). Total DNA damage represents the percentage of cells with either activated ATM, activated H2A.X, or dual activation (right). P values were calculated by Student t test. D, Representative IHC images of paraffin-embedded sections of normal prostate from wild-type C57BL/6J mice and localized prostate tumors from PB-Tag mice. Sections were stained IHC with indicated antibodies (brown). Nuclei were costained with hematoxylin (blue). The micrographs were captured in 200 × magnification. Note that prostate tumors of PB-Tag mice exhibit higher DNA damage (increased phosphorylation of γH2AX) and Src activation (Src y416 phosphorylation) compared with normal mice prostate. E and F, TRAMP-C2 cells (2,500 cells/well in 100-μL media) were treated with dasatinib/bosutinib (0.3 μmol/L) and talazoparib (0.03 μmol/L) alone or in combination for 7 days. DMSO was used as a placebo treatment. Cell viability (± SD) was measured by MTT assay. G, TRAMP-C2 cells were treated with dasatinib (0.3 μmol/L) and cisplatin (0.1 μmol/L; dissolved in DMSF) alone or in combination for 5 and 8 days. The equivalent volume of DMSO was used as a placebo treatment. After indicated number of days, cells were treated with 0.5 mg/mL MTT. The graphs represent the inhibition of cell viability by single agent and combination treatment. P values were calculated by Student t test.

  • Figure 5.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 5.

    SRC activation leads to PARPi resistance. A, Viability (± SD) of 22RV1 cells treated with dasatinib (0.3 μmol/L) ± olaparib (3.0 μmol/L), as assessed by MTT assay. DMSO was used as a placebo control. B, Viability (± SD) of PC3M cells subjected to periodic treatment with talazoparib, followed by dasatinib, or vice versa, as assessed by MTT assay. DMSO was used as a placebo control. See Supplementary Fig. S5A for experimental schematic. P values were calculated by Student t test. C and D, PC3M cells were incubated in either olaparib (3 μmol/L) or talazoparib (0.03 μmol/L) supplemented media for 15 days to develop partial PARPi resistance. PARPi-resistant cells were counted and treated with dasatinib (see Supplementary Fig. S5B for experimental schematic). Cell viability was assessed by MTT assay. Viability (± SD) of PC3M cells after 7 days of dasatinib treatment is shown in C. Representative images of MTT-treated cells are shown in D. P values were calculated by Student t test. E, SRC or scrambled SMARTpool siRNA-transfected PC3M cells were cultured in olaparib (3 μmol/L) or talazoparib (0.03 μmol/L) supplemented medium for 4 days posttransfection. The equivalent volume of DMSO was used as a placebo treatment. Cell viability (± SD) was measured by MTT assay. P values were determined by Student t test. F, PC3M cells that transiently overexpressed Y527F SRC (constitutively active SRC) were treated with olaparib (3 μmol/L) or talazoparib (0.03 μmol/L) for 7 days. Control cells were transfected with GFP-expressing empty vector. Viability (± SD) of cells at 7 days, as determined by MTT assay is shown. P values determined by Student t test.

  • Figure 6.
    • Download figure
    • Open in new tab
    • Download powerpoint
    Figure 6.

    Synergistic effect of dasatinib (dasa) and PARPis on 3D prostate cancer organoid growth. A, PC3M cells (103 cells/well) were mixed with Matrigel, and 3D cell cultures (organoids) were grown in growth factor–enriched media. (see Supplementary Fig. S6A for schematic representation.) The number of 3D organoids (>200-μm diameter, ± SD) is shown. Each point represents the number of organoids (A) and average diameter of PC3M 3D organoids in B grown from 103 cells in each well. P value was determined by Student t test. B, Images of wells plate at day 21 (top). Organoids at 40× magnification (middle). Organoids at 100× magnification (bottom). Cells invading through Matrigel are indicated by arrows. C, Average number of 3D organoids (>100-μm diameter, ± SD) derived from LNCaP-abl cells (plated at 5 × 103 cells/well). P value was determined by Student t test.D, Activation of SRC-FAK pathway in mCRPC-derived organoids (MSKPCa1–3) was analyzed by Western blotting, using indicated antibodies. Lysate from parental LNCaP cells was used as cell line control. RhoGDI was used as loading control. E, Organoids [MSKPCa1 (bottom) and MSKPCa 3 (top)] were counted, plated on collagen-coated (collagen I 50 μg/mL) 96-well plates (5,000 cells/well in 100-μL media), and treated with dasatinib (0.3 μmol/L) and talazoparib (tala; 0.03 μmol/L) alone or in combination (combo). The equivalent volume of DMSO was used as a placebo treatment. After 10 days, cells were treated with 0.5 mg/mL MTT. The graphs represent the inhibition of cell viability by single agent and combination treatment. P values were calculated by Student t test. F, MSKPCa3 cells (5 × 103 cells/well) were mixed with Matrigel, and 3D cell cultures (organoids) were grown in growth factor–enriched media and treated with dasatinib (0.3 μmol/L) and talazoparib (0.03 μmol/L) alone or in combination. Images of MSKPCa3 cell–derived 3D organoid in 24-well plate at day 21 (left). Organoids at 200× magnification (right). Cells invading through Matrigel are indicated by arrows. G, The number of 3D organoids (>150 μm diameter, ± SD) derived from MSKPCa3 (top) and MSKPCa1 (bottom) are shown. Each point represents the number of organoids grown from 5 × 103 cells in each well of 24-well plate (after 4 weeks of plating the cells). P value was determined by Student t test.

Additional Files

  • Figures
  • Supplementary Data

    • Supplementary Appendix - Supplementary tables (except Table S3) and figure captions
    • Table S3 - Table S3
    • Figure S1 - Figure S1
    • Figure S2 - Figure S2
    • Figure S3 - Figure S3
    • Figure S4 - Figure S4
    • Figure S5 - Figure S5
    • Figure S6 - Figure S6
PreviousNext
Back to top
Clinical Cancer Research: 27 (6)
March 2021
Volume 27, Issue 6
  • Table of Contents
  • Table of Contents (PDF)
  • About the Cover
  • Editorial Board (PDF)

Sign up for alerts

View this article with LENS

Open full page PDF
Article Alerts
Sign In to Email Alerts with your Email Address
Email Article

Thank you for sharing this Clinical Cancer Research article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
Attenuation of SRC Kinase Activity Augments PARP Inhibitor–mediated Synthetic Lethality in BRCA2-altered Prostate Tumors
(Your Name) has forwarded a page to you from Clinical Cancer Research
(Your Name) thought you would be interested in this article in Clinical Cancer Research.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Citation Tools
Attenuation of SRC Kinase Activity Augments PARP Inhibitor–mediated Synthetic Lethality in BRCA2-altered Prostate Tumors
Goutam Chakraborty, Nabeela Khan Patail, Rahim Hirani, Subhiksha Nandakumar, Ying Z. Mazzu, Yuki Yoshikawa, Mohammad Atiq, Lina E. Jehane, Konrad H. Stopsack, Gwo-Shu Mary Lee, Wassim Abida, Michael J. Morris, Lorelei A. Mucci, Daniel Danila and Philip W. Kantoff
Clin Cancer Res March 15 2021 (27) (6) 1792-1806; DOI: 10.1158/1078-0432.CCR-20-2483

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Share
Attenuation of SRC Kinase Activity Augments PARP Inhibitor–mediated Synthetic Lethality in BRCA2-altered Prostate Tumors
Goutam Chakraborty, Nabeela Khan Patail, Rahim Hirani, Subhiksha Nandakumar, Ying Z. Mazzu, Yuki Yoshikawa, Mohammad Atiq, Lina E. Jehane, Konrad H. Stopsack, Gwo-Shu Mary Lee, Wassim Abida, Michael J. Morris, Lorelei A. Mucci, Daniel Danila and Philip W. Kantoff
Clin Cancer Res March 15 2021 (27) (6) 1792-1806; DOI: 10.1158/1078-0432.CCR-20-2483
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Jump to section

  • Article
    • Abstract
    • Introduction
    • Materials and Methods
    • Results
    • Discussion
    • Authors' Disclosures
    • Authors' Contributions
    • Acknowledgments
    • Footnotes
    • References
  • Figures & Data
  • Info & Metrics
  • PDF
Advertisement

Related Articles

Cited By...

More in this TOC Section

  • Molecular Profiling of Metastatic Breast Cancer
  • Preclinical Evaluation of AMG 160 in mCRPC Models
  • Therapeutic Targeting NRG1 Fusion Cancers with Seribantumab
Show more Translational Cancer Mechanisms and Therapy
  • Home
  • Alerts
  • Feedback
  • Privacy Policy
Facebook  Twitter  LinkedIn  YouTube  RSS

Articles

  • Online First
  • Current Issue
  • Past Issues
  • CCR Focus Archive
  • Meeting Abstracts

Info for

  • Authors
  • Subscribers
  • Advertisers
  • Librarians

About Clinical Cancer Research

  • About the Journal
  • Editorial Board
  • Permissions
  • Submit a Manuscript
AACR logo

Copyright © 2021 by the American Association for Cancer Research.

Clinical Cancer Research
eISSN: 1557-3265
ISSN: 1078-0432

Advertisement