Login to Access Video or Poster Abstract: MP99-05
Sources of Funding: 9R01CA186780 and 1P50CA180995

Introduction

Androgens are known to protect prostate cancer cells from DNA damage. Recent studies showed regulation of DNA repair genes by androgen receptor (AR) signaling in prostate cancers. We recently reported that androgen-regulated protein and potential tumor suppressor ELL2-associated factor 2 (EAF2) can enhance DNA repair through Ku70/Ku80 in the prostate. ELL2 (elongation factor, RNA polymerase II, 2), a component of the super elongation complex (SEC), is an important factor for RNA Pol II transiting from promoter-proximal paused state into elongation state. ELL2 is also regulated by androgens and frequently down-regulated in prostate cancer. These observations led to our hypothesis that ELL2 can regulate DNA repair through Ku70/Ku80 in prostate cancer cells.

Methods

Prostate cancer cells, in the presence or absence of siRNA of ELL2, were treated with ?-irradiation or doxorubicin and then collected for detecting the level of DNA damage marker ?H2ax or neutral comet assay. Nonhomologous end-joining (NHEJ) and homologous recombination (HR) assays were used to test the role of ELL2 in these two double-strand break (DSB) repair pathways. Co-immunoprecipitation was used to determine the interaction between ELL2 and NHEJ pathway proteins Ku70 and Ku80. We examined the effect of ELL2 knockdown on Ku70 and Ku80 recruitment in response to laser microirradiation.

Results

Knockdown of ELL2 sensitized prostate cancer cells to DNA damage and overexpression of ELL2 protected prostate cancer cells from DNA damage. Knockdown of ELL2 impaired NHEJ repair but not HR. Transfected ELL2 co-immunoprecipitated with both Ku70 and Ku80 proteins. ELL2 could binds to and co-accumulated with Ku70/Ku80 proteins at sites of DNA damage. Knockdown of ELL2 dramatically/significantl inhibited Ku70 and Ku80 accumulation and retention at DSB sites in prostate cancer cells, and also impaired recruitment of Ku70/Ku80 to DSB sites. The impaired recruitment of Ku70 and Ku80 proteins to DNA damage sites upon ELL2 knockdown was rescued by re-expression of an ELL2 transgene insensitive to siELL2.

Conclusions

This study suggests that ELL2 is an important factor mediating androgen protection of DNA damage via Ku70/Ku80 in prostate cancer cells.

Funding

9R01CA186780 and 1P50CA180995