Heat shock factor 1 (HSF1), the transcriptional activator of the heat shock genes, is increasingly implicated in cancer. We have shown that HSF1 binds to the corepressor metastasis-associated protein 1 (MTA1) in vitro and in human breast carcinoma samples. HSF1-MTA1 complex formation was strongly induced by the transforming ligand heregulin and complexes incorporated a number of additional proteins including histone deacetylases (HDAC1 and 2) and Mi2a, all components of the NuRD corepressor complex. These complexes were induced to assemble on the chromatin of MCF7 breast carcinoma cells and associated with the promoters of estrogen-responsive genes. Such HSF1 complexes participate in repression of estrogen-dependent transcription in breast carcinoma cells treated with heregulin and this effect was inhibited by MTA1 knockdown. Repression of estrogen-dependent transcription may contribute to the role of HSF1 in cancer.

Proteasomal degradation of topoisomerase I (topoI) is one of the most remarkable cellular phenomena observed in response to camptothecin (CPT). Importantly, the rate of topoI degradation is linked to CPT resistance. Formation of the topoI-DNA-CPT cleavable complex inhibits DNA re-ligation resulting in DNA-double strand break (DSB). The degradation of topoI marks the first step in the ubiquitin proteasome pathway (UPP) dependent DNA damage response (DDR). Here, we show that the Ku70/Ku80 heterodimer binds with topoI, and that the DNA-dependent protein kinase (DNA-PKcs) phosphorylates topoI on serine 10 (topoI-pS10), which is subsequently ubiquitinated by BRCA1. A higher basal level of topoI-pS10 ensures rapid topoI degradation leading to CPT resistance. Importantly, PTEN regulates DNA-PKcs kinase activity in this pathway and PTEN deletion ensures DNA-PKcs dependent higher topoI-pS10, rapid topoI degradation and CPT resistance.

The age-adjusted mortality rate for cancer is about the same in the 21st century as it was 50 years ago. In the past decade, our understanding of oncogenesis at molecular level has increased greatly. This has resulted in the development of several targeted drugs and many more are in the pipeline. However, the lack of predictive biomarkers to stratify the patient population has restricted the benefits of these new developments. Camptothecins (CPTs) target topoisomeraseI (topoI) and represent a highly potent class of anticancer drugs. Two topoI inhibitors, topotecan and irrinotecan (CPT analogues), are in clinical use and several other topoI inhibitors are in different phases of development. SCLC, colon, ovarian, breast and cervical carcinomas are treated with topoI inhibitors. However, only 13% to 32% of patients respond to these drugs. TopoI mutations are rare in the patient population, and CPTs are not substrates for MDR transporters, therefore the rate of proteolytic degradation of topoI is a potential mechanism explaining CPT resistance. A differential rate of topoI degradation by the ubiquitin-proteosomal pathway (UPP) has been linked to CPT response, but the mechanism is not well understood. Using a functional proteomics approach, we have isolated a topoI interacting complex and have determined that: i) Ku70/Ku80/DNA-PKcs associates with topoI, and DNA-PKcs specifically phosphorylates topoI at S10, ii) S10-phosphorylated topoI is ubiquitinated by the BRCA1/BARD1 heterodimer, K-48 ubiquitin linkage indicates polyubiquitination and proteosomal degradation of topoI in response to CPT, iii) topoI S10 phosphorylation level determines the rate of topoI ubiquitination and proteosomal degradation in triple negative breast cancer (TNBC) and colon cancer cells. Higher levels of topoI pS10 ensures rapid degradation of topoI by the UPP and confers resistance to CPT. These cell based findings have further been tested in retrospective clinical studies. We have raised a monoclonal phosphospecific antibody against topoI-pS10 and shown its specificity in IF and IHC assays. In a retrospective study, we have performed IHC of FFPE tissues from metastatic CRC patients treated with FOLFIRI (combination therapy, 5FU, Leucovorin and Irinotecan). IHC analysis was performed in ten responders and ten non-responders tissues by anti topoI-pS10. Our preliminary data indicates that non-responders have very high level of topoI-pS10; in contrast the responders were negative for IHC staining. A larger retrospective study is underway to develop this predictive biomarker. Citation Format: Ankur K. Shah, Vibhu Sachdev, Julian Taylor Parker, Yevnegyia Malinkovich, Yara Hamade Tohme, Yiheng Hu, Jeffrey D. Parvin, Ajit K. Bharti. DNA-PKcs dependent phosphorylation of topoI-S10 determines its ubiquitination by BRCA1/BARD1, rate of topoI degradation and CPT response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4653. doi:10.1158/1538-7445.AM2013-4653 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.