<p>This document contains supplemental details such as antibodies and primers, list of genes overrepresented in TWIST1 and TWIST1 mutants, IHC staining for TWIST1 in mouse prostate development, IHC staining for TWIST1 and HOXA9 in various mouse models of prostate cancer, correlation of TWIST1 and HOXA9 alteration with poor survival in human prostate cancer patients, IHC for TWIST1 and HOXA9 on primary tumors and bone metastasis from prostate cancer patients, IHC for HOXA9 on cell pellets expressing HOXA9 or control and on adult mouse prostate, data showing sufficiency of HOXA9 for some pro-metastatic behaviour in prostate cancer cells, data showing requirement of WDR5 and HOTTIP for TWIST1-dependent pro-metastatic behavior, ChIP data showing binding of TWIST1 and HOXA9 at the HOXA9 promoter, data showing effect of drugs that can inhibit HOXA9 on prostate cancer cell viability and pro metastatic behaviour.</p>
<p>This document contains supplemental details such as antibodies and primers, list of genes overrepresented in TWIST1 and TWIST1 mutants, IHC staining for TWIST1 in mouse prostate development, IHC staining for TWIST1 and HOXA9 in various mouse models of prostate cancer, correlation of TWIST1 and HOXA9 alteration with poor survival in human prostate cancer patients, IHC for TWIST1 and HOXA9 on primary tumors and bone metastasis from prostate cancer patients, IHC for HOXA9 on cell pellets expressing HOXA9 or control and on adult mouse prostate, data showing sufficiency of HOXA9 for some pro-metastatic behaviour in prostate cancer cells, data showing requirement of WDR5 and HOTTIP for TWIST1-dependent pro-metastatic behavior, ChIP data showing binding of TWIST1 and HOXA9 at the HOXA9 promoter, data showing effect of drugs that can inhibit HOXA9 on prostate cancer cell viability and pro metastatic behaviour.</p>
<p>PDF file - 1004K, S1. The Twist box domain is required for full Twist1 transcriptional activity. S2. Quantification of immunofluorescence from isogenic prostate cancer cell lines stably expressing Twist1 and Twist1-F191G. S3. The Twist box domain is required for full Twist1-induced EMT marker phenotypes of prostate cancer cells. S4. Twist1 overexpression induces temporal changes in the material properties of prostate cancer cells during their migration in a wound healing assay. S5. The overexpression of Twist1 or Twist1-F191G does not increase cellular proliferation of prostate cancer cells in vitro. S6. The Twist box domain is required for full Twist1-induced cellular migration in PC3 cells. S7. Twist1 overexpression increases cell traction forces of individual androgenindependent PC3 prostate cancer cells. S8. Twist1 overexpression confers radioresistance to prostate cancer cells which is attenuated by mutation of the Twist box domain. S9. The Twist box domain is required for Twist1-induced soft agar anchorageindependent growth of 22Rv1 prostate cancer cells. S10. Twist1 overexpression does not confer prostate cancer cells increased primary tumorigenicity and slows primary tumor cell growth in vivo. S11. The Twist box domain is required for full Twist1-induced expression of Hoxa9/HOXA9.</p>
Outcomes for poor-risk localized prostate cancers treated with radiation are still insufficient. Targeting the "non-oncogene" addiction or stress response machinery is an appealing strategy for cancer therapeutics. Heat-shock-protein-90 (Hsp90), an integral member of this machinery, is a molecular chaperone required for energy-driven stabilization and selective degradation of misfolded "client" proteins, that is commonly overexpressed in tumor cells. Hsp90 client proteins include critical components of pathways implicated in prostate cancer cell survival and radioresistance, such as androgen receptor signaling and the PI3K-Akt-mTOR pathway. We examined the effects of a novel non-geldanamycin Hsp90 inhibitor, AUY922, combined with radiation (RT) on two prostate cancer cell lines, Myc-CaP and PC3, using in vitro assays for clonogenic survival, apoptosis, cell cycle distribution, γ-H2AX foci kinetics and client protein expression in pathways important for prostate cancer survival and radioresistance. We then evaluated tumor growth delay and effects of the combined treatment (RT-AUY922) on the PI3K-Akt-mTOR and AR pathways in a hind-flank tumor graft model. We observed that AUY922 caused supra-additive radiosensitization in both cell lines at low nanomolar doses with enhancement ratios between 1.4–1.7 (p < 0.01). RT-AUY922 increased apoptotic cell death compared with either therapy alone, induced G2-M arrest and produced marked changes in client protein expression. These results were confirmed in vivo, where RT-AUY922 combination therapy produced supra-additive tumor growth delay compared with either therapy by itself in Myc-CaP and PC3 tumor grafts (both p < 0.0001). Our data suggest that combined RT-AUY922 therapy exhibits promising activity against prostate cancer cells, which should be investigated in clinical studies.
