Rad51 over-expression contributes to chemoresistance in human STS; A role for p53/AP2 transcriptional regulation J. Hannay, J. Liu, S. Bolshakov, D. Yu, A. Lazar, R. Pollock, D. Lev Sarcoma Research Center University of Texas, MD Anderson Cancer Center

Overall 5YS rate – 46.5%

Have we made much progress? Overall 5YS – 50%

STS are chemoresistant: Modest Response Rates; Significant Toxicity DTIC 15%, IFF 20%, DOX 20-30% Combination therapies 20-50% - (short lived responses, only limited improvement in survival): DOX/IFF combinations Sequential hi dose IFF/hi dose DOX + DTIC + /- CY MAID + growth factor Gem + taxotere Tumor cell properties/ tumor microenvironment

The complexity of fighting chemoresistance: Tumors are heterogeneous Multiple chemoresistance mechanisms may be operative in any specific tumor cell Tumor cells are genetically unstable New molecular mechanisms develop during the progression of the tumor

p53 and STS chemoresistance p53 gene mutation or dysfunction (e.g., MDM2) are the most frequent known alteration in STS Found more frequently: metastases vs. primary tumor, high-grade vs. low-grade. Significant negative impact on both overall and sarcoma-specific survival Studies from other tumor systems suggest a connection between mutp53 and resistance to chemotherapy

Re-expression of wtp53 in mutp53 STS cell lines results in significant sensitization to Dox – in vitro

And in vivo…

Mut. p53 PKC-α P-gp Midkine Rad-51 TKR EGFR PDGFR Hs27 SKLMS1 SW872 HT1080 SKLMS1 HUVEC NHF A204 RD SW684 Rad-51 Hs27 SKLMS1 SW872 A204 TKR EGFR PDGFR SK HT RD A204 SW684 SW 872 HUVEC Anti-Rad51

STS chemoresistance: Rad51 Homologous recombination (HR) is crucial for the repair of complex forms of DNA damage such as double-strand breaks (DSBs) Over-expression of Rad51, the key factor of HR has been observed in a number of tumors such as breast and pancreatic cancer to correlate with chemoresistance

Rad-51 is overexpressed in STS Cell lines, Xenografts and Tumors Normal rat testis SKLMS-1 xenograft SKLMS-1 SW684 SW872 HT1080 RD A204 Rad51 β-actin MFH - p MFH - p ASPS - r FS - m RMS - p RMS - m LPS - p LPS - r

Prognostic marker? Rad-51 expression possibly correlates with survival: High and moderate expressers had a 31% and 47% 5YS rate, whereas 66% of low expressers survived five years (Fisher’s exact; p < 0.05). In most of the IHC positive tumors Rad-51 could be identified in the cytoplasm Hs27 SKLMS1 SW872 A204

Rad-51 impacts on STS chemosensitivity: SK-SHAR51-c1 β-actin Rad51 HARad51 Lipofect’ alone 14-3-3ζ +ve cont scram -ve cont 100nM 200nM 400nM siRad51 A B 20nM 10nM 1nM 50nM

Rad51 translocates to the nucleus within 1 hour of Dox exposure in SKLMS-1 No DOX 1µM DOX – 1h Rad51 DAPI

No Dox, t=0 24h Dox 48h Dox Dox 24h 48h Rad51 β-actin

p53/Rad51 p53 mutant cells exhibit elevated rates of spontaneous and induced HR and increased Rad51 expression Rad51 regulation by wt p53 is thought to be transcription-independent; binding of wt p53 to Rad51 inhibits its function and results in increased degradation of the protein complex

Induction of wt p53 leads to suppression of Rad51 protein levels - RD ß actin p21 Rad51 FLAGp53 500 100 50 10 1 AdEV AdFLAGp53 MOI Induction of wt p53 leads to suppression of Rad51 protein levels AdFLAGp53 - RD AdFLAGp53 - A204 MOI 1 10 50 100 500 Rad51 suppression by wt p53 does not appear to be strictly due to enhanced proteosomal degradation ß - actin Rad51 FLAGp53 mk Hours AdLacZ 2 4 6 DMSO MG132 AdFLAGp53

Induction of wt p53 leads to suppression of Rad51 mRNA transcript generation SK SK SK neo neo neo Ala10 Ala10 Ala10 Ala14 Ala14 Ala14 Ala21 Ala21 Ala21 AdLacZ AdLacZ AdFLAGp53 AdFLAGp53 wt wt ° ° ° C C C 32 32 32 38 38 38 32 32 32 38 38 38 32 32 32 38 38 38 32 32 32 38 38 38 32 32 32 38 38 38 VP:cell VP:cell mock mock 20 20 200 200 2000 2000 20 20 200 200 2000 2000 Rad51 Rad51 Rad51 Rad51 Rad51 p21 p21 p21 p21 p21 GAPDH GAPDH GAPDH GAPDH GAPDH Next we showed the wt p53 did not shorten Rad51 mRNA half life Does wt p53 transcriptionally represses Rad51 promoter?

wt p53 leads to suppression of rad51 promoter activity - - - 403 403 403 - - - 295 295 295 - - - 185 185 185 - - - 50 50 50 +1 +1 +1 +63 +63 +63 Genebank sequence of the Rad51 promoter was screened (DNASys software): no classical p53 binding sites were found Rad51/Luc Rad51/Luc Rad51/Luc Potential AP Potential AP Potential AP - - - 2 binding site 2 binding site 2 binding site Potential SP Potential SP Potential SP - - - 1 binding site 1 binding site 1 binding site Potential core enhancer Potential core enhancer Potential core enhancer Potential Ets Potential Ets Potential Ets - - - 1 binding site 1 binding site 1 binding site Potential E2A binding site Potential E2A binding site Potential E2A binding site Reported p53 responsive element Reported p53 wt p53 leads to suppression of rad51 promoter activity 40 SKLMS1/38C 35 SKLMS1/32C Ala14/38C 30 Ala14/32C 25 Relative luciferase activity 20 15 10 5 pRad-403Luc

Reintroduction of wtp53 increases AP-2 binding to the rad51 promoter Using truncation mutants of the rad51 promoter we were further able to show that the p53 responsive element is in the –295 to –185 region NE + AP2 consensus Water + probe NE + probe NE + probe + XS c.c. NE + probe + AP2 ab Z.NE + probe p53WT.NE + probe This region contains an AP-2 binding site which when mutated, the wtp53 effect is eliminated, suggesting that the wtp53 repressive effect is mediated via AP-2 αAP2/AP2/ probe AP2/ probe Reintroduction of wtp53 increases AP-2 binding to the rad51 promoter Free probe

Future investigations: How do p53 and AP-2 cooperate to repress the Rad-51 promoter? Does AP-2 play a role in STS chemoresistance?

Many thanks to: Theresa Nguyen Jeffery Liu Parimal Das Jonathan Hannay Svetlana Bolshakov Borys Korchin Dhana Kotillingam Wen-Hong Ren Zeming Jin Raph Pollock Alex Lazar