DNA damage tolerance facilitates the progression of replication forks that have encountered obstacles on the template strands. It involves either translesion DNA synthesis initiated by proliferating cell nuclear antigen monoubiquitination or less well-characterized fork reversal and template switch mechanisms. Herein, we characterize a novel tolerance pathway requiring the tumor suppressor p53, the translesion polymerase ι (POLι), the ubiquitin ligase Rad5-related helicase-like transcription factor (HLTF), and the SWI/SNF catalytic subunit (SNF2) translocase zinc finger ran-binding domain containing 3 (ZRANB3). This novel p53 activity is lost in the exonucleasedeficient but transcriptionally active p53(H115N) mutant. Wild-type p53, but not p53(H115N), associates with POLι in vivo. Strikingly, the concerted action of p53 and POLι decelerates nascent DNA elongation and promotes HLTF/ZRANB3-dependent recombination during unperturbed DNA replication. Particularly after cross-linkerinduced replication stress, p53 and POLι also act together to promote meiotic recombination enzyme 11 (MRE11)-dependent accumulation of (phospho-)replication protein A (RPA)-coated ssDNA. These results implicate a direct role of p53 in the processing of replication forks encountering obstacles on the template strand. Our findings define an unprecedented function of p53 and POLι in the DNA damage response to endogenous or exogenous replication stress.T he tumor suppressor protein p53 has been called the guardianof-the-genome due to its ability to transactivate downstream targets transcriptionally, which prevents S-phase entrance before facilitating DNA repair or eliminating cells with severe DNA damage via apoptosis (1). Interestingly, p53 also encodes an intrinsic 3′-5′ exonuclease activity located within its central DNA-binding domain (2-4). The contribution of the exonuclease proficiency to p53's function has largely remained obscure. Exonucleases are involved in DNA replication, DNA repair, and recombination, increasing the fidelity or efficiency of these processes. The 3′-5′ exonuclease activity of DNA polymerases (POLs) catalyzes the correction of replication errors, thereby preventing genomic instability and cancer (5-7). The potential involvement of p53's exonuclease in DNA repair has been ascribed to transcription-independent functions in nucleotide excision repair and base excision repair, in homologous recombination (HR), and in mitochondrial processes (8-10).Regarding HR, in particular, reports indicate a dual role for p53. On the one hand, it has been reported that p53 down-regulates unscheduled and excessive HR in response to severe genotoxic stress, like formation of DNA double-strand breaks (DSBs) (8-10). This antirecombinogenic effect of p53 has been linked to the blockage of continued strand exchange by interactions with recombinase RAD51, RAD54, and nascent HR intermediates carrying specific mismatches (11, 12). On the other hand, p53 stimulates spontaneous HR during S-phase to overcome replication fork stalling and to pr...

NKX3.1 is a haploinsufficient prostate cancer suppressor which has been shown to be reduced in most of the primary human prostate cancers. Reduced NKX3.1 expression in luminal epithelial cells was already observed in pre-neoplastic lesions like prostate intraepithelial neoplasia (PIN) and in early tumor stages. Since the NKX3.1 knock out mouse model displayed a higher incidence of PIN formation it is assumed that the loss of NKX3.1 is involved in the initiation of in prostate cancer. Another potential risk factor for the initiation of prostate cancer is a chronic inflammation of the prostate (prostatitis). However, a clear link between prostatitis and prostate cancer initiation has not yet been established. To estimate whether a loss of NKX3.1 in inflamed prostate tissue might be the basis for PIN formation and subsequent tumor initiation, we compared the NKX3.1 expression levels in postatitis, prostate cancer and normal tissue. We observed a pronounced reduction of NKX3.1 in areas which are positive for B cell and MPS markers CD20 and CD68, respectively. Stimulation of PCa cancer cell lines with cytokines and growth factors known to be expressed by those infiltrating cells revealed a distinct reduction of NKX3.1 expression in PCa cells stimulated with either TNFa or IL1a. Furthermore, the loss of NKX3.1 protein and mRNA was far more pronounced in PCa cells treated with the epidermal growth factor (EGF). Since EGF has a mitogenic function we also tested the impact of a combinatory stimulation of LNCaP cells with the strong mitogen phorbol-12-myristate-13-acetate (PMA) and the calcium ionophor Ionomycin on NKX3.1 expression. Similar to EGF stimulation, PMA+Ionomycin caused a dramatic reduction of NKX3.1 protein as well as mRNA level. NKX3.1 target genes were found to be derepressed upon EGF or P+I stimulation while androgen receptor mRNA expression was found to be decreased. EGF or P+I induced NKX3.1 loss in PCa cell lines appears to depend on two different mechanisms. While the decrease of NKX3.1 mRNA levels largely remained unaffected upon pretreatment with MG132 or Bortezomib, the stabilization of NKX3.1 protein after co-treatment with EGF/P+I and MG132 or Bortezomib suggests the participation of the proteasomal pathway in this process. In addition, the decline in NKX3.1 mRNA levels appears to be independent from a reduced NKX3.1 promoter activity as determined by luciferase reporter assays. Taken together our results imply that a mitogenic stimulus might be the cause for reduced NKX3.1 levels in luminal prostate epithelial cells. Since the production of mitogenic growth factors are increased in inflammatory sites the growth factor induced loss of NKX3.1 might be the connection between prostatitis and prostate cancer initiation. Citation Format: Josua Decker, Garima Jain, Philip Harazim, Tina Kießling, Peter Möller, Ralf Marienfeld. Prostatitis related mitogenic stimuli cause loss of NKX3.1: Increased risk for prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1667. doi:10.1158/1538-7445.AM2014-1667