1) The document summarizes research into combining AR and NOTCH inhibitors to treat ERG-positive prostate cancer. It finds that dual inhibition more strongly reduces cell survival, motility, invasion, and epithelial-mesenchymal transition compared to either inhibitor alone.
2) Experiments on VCaP prostate cancer cells show that combining GSI-1, a NOTCH inhibitor, with bicalutamide or enzalutamide, AR inhibitors, further decreases colony formation and HES-1 expression than single inhibitors.
3) Combination treatment also induces more expression of epithelial markers and reduces EMT compared to single inhibitors, suggesting it may better inhibit tumor progression. The study concludes dual inhibition results in greater anti
1. Mulenga Chileshe
Senior, Biology Major
University of the District of Columbia
Mentor: Dr. Ahmed Mohamed
Manuscript focus:
Evaluation of the synergistic effects of AR and NOTCH
inhibitors in ERG positive prostate cancer cells
Confidential and Proprietary, CPDR, HJF, USUHS
2. Background Information
• The ETS Related Gene (ERG) is a member of the ETS
family of transcription factors, usually found fused to
TMPRSS2 in the majority of prostate cancer patients
• TMRPSS2-ERG is an ideal therapeutic target for
Prostate Cancer, because of the following;
1) It is prostate cancer specific
2) It is essential for the initiation and progression of
prostate cancer
3. AR inhibitors to block expression of TMPRSS2-ERG:
• Bicalutamide : Non steroidal Androgen receptor
antagonist.
• Enzalutamide: A synthetic, non-steroidal which is
considered a pure antiandrogen that binds to AR
with greater affinity than Bicalutamide.
7. Notch Transcription Factors/Signaling Pathway:
• Notch signaling plays a key role in the normal
development of many tissues and cell types by
regulating cell differentiation, proliferation, survival,
and apoptosis.
1) Evidence suggests that the notch signaling pathway
is frequently deregulated in human malignancies.
2) Use of Notch inhibitors may be a potential approach
to the treatment of ERG positive cancers.
9. Hypothesis & Aim
• Hypothesis: A Combination of ERG & Notch contribute to:
increased cell motility & invasion, and inhibition of cell
differentiation which leads to the growth of prostate
cancer.
• Aim: To target both AR and Notch signaling by inhibitors of
AR and Notch, therefore preventing the cell growth and
survival of the prostate cancer.
A
10. 10K cells in 6-well dishes
After 48h cells were treated with
the following concentrations
After 48h of treated medium, cells were replaced with
regular growth medium and allowed to grow for 10 days
After 10 days cells were fixed with 100% MethOH/10minutes
Then stained with Crystal violet/10minutes, washed, air dried, count colonies and took pictures
Experimental design
11. 0µM 1µM_GSI-1 1µM 5µM 10µM
0µM 1µM_GSI-1
Bicalutamide
GSI-1+ Bicalutamide
1+1µM 1+5µM 1+10µM
Combination of NOTCH and AR inhibitors
reduce survival colony formation of VCaP cells
12. 0µM 1µM_GSI-1 1µM 5µM 10µM
0µM 1µM_GSI-1
Enzalutamide
GSI-1+ Enzalutamide
1+1µM 1+5µM 1+10µM
Combination of GSI-1 and Enzalutamide
reduce survival colony formation of VCaP cells
16. A
TM ERG ERG
NOTCH1,2
Cell invasion/motility
EMT
Differentiation
Drug sensitivity
AR
Rx
ARi
Rx
GSI
X
X
X
X
X
CaP
Conclusion
Inhibition of AR combined with inhibition of NOTCH signaling
results in a greater decrease in cell motility, invasion, EMT and
increased differentiation of cells and drug sensitivity, thus
inhibiting tumor development and/or progression
17. Acknowledgements
• Dr. Shiv Srivastava
• Dr. David G. McLeod
• Dr. Deepak Kumar
• Dr. Taduru Sreenath
• Dr. Ahmed Mohammed
• Dr. Charles Xavier
• The CPDR staff
• Department of Defense ( CDMRP- DOD) for the HBCU grant
Editor's Notes
Activation of Notch signaling. Notch is expressed on the cell surface as a ligand-accessible form of the receptor. In the absence of ligand binding the Notch receptors are inactive. When Notch ligand binds to Notch receptor on an adjacent cell, a series of proteolytic cleavages occurs (referred to as S2 and S3 cleavages), resulting in release of the Notch intracellular domain (NotchIC) that subsequently translocates into the nucleus where it binds to it’s targets and thus cause activation of transcription of various Notch target genes including those belonging to the HES and HRT (HEY) families.