Homology-Directed Repair Is Diminished in PTEN-Positive Breast Tumor Cells.
Authors: Murphy C. , Singh J. , Brunet E. , Jackson T. , She Q. , Jasin M. , Moynahan M. 
Published: December 2010

BackgroundThe tumor suppressor gene PTEN is mutated or lost in a substantial proportion of human cancers, including basal-like breast cancer (BLBC). Recently, aside from its lipid phosphatase activity, PTEN has been implicated in DNA repair via a regulatory role of RAD51, the central repair protein in homology-directed repair (HDR). As BLBCs are transcriptionally similar to HDR-deficient BRCA1-associated breast cancers, it is hypothesized that loss of PTEN may extend a repair-deficient phenotype to BRCA1 wild-type BLBC. Tumors which exhibit impaired DNA double-strand break (DSB) repair are potential targets for chemotherapy with DNA-damaging agents, and novel strategies such as poly(ADP)ribose polymerase (PARP) inhibition. We therefore set out to quantify the repair capability of an induced DSB by HDR in PTEN-deficient breast tumor cells, and to assess their sensitivity to agents targeting DNA repair.MethodsTo assess HDR of DNA DSBs we used several assays developed to rapidly quantify cellular repair capability – including the I-SceI-induced DSB DR-GFP assay (Pierce et al., 1999) and a newly developed assay measuring GFP-gene targeting to a zinc finger nuclease (ZFN) induced DSB (Brunet et al., PNAS 2009). PTEN-deficient MDA-MB-468 cells stably transfected with tetracycline-inducible PTEN (She et al., 2003) underwent transient co-transfection with an I-SceI expression vector and DR-GFP. Assessment of gene targeting to an induced break in the p84 gene on chromosome 19 was also assessed as an alternative HDR assay by co-transfection of GFP p84 and ZFNp84 constructs. PTEN and RAD51 protein expression in doxycycline supplemented and unsupplemented cells were assessed by Western blot. We performed clonogenic survival assays following PARP inhibitor continuous exposure at concentrations of 10-5 to 10-8 M for 7-14 days.ResultsPTEN induction following doxycycline exposure was confirmed by Western blot. RAD51 expression was similar in PTEN-positive and PTEN-negative cells. Cell cycle analysis of PTEN-induced cells indicated that similar proportions of PTEN-positive and PTEN-negative cells were in S-phase of the cell cycle. PTEN-positive cells demonstrated decreased HDR of an induced DSB relative to PTEN-negative cells using the DR-GFP reporter system with a mean ratio of GFP-positive cells (PTEN positive: PTEN negative) of 0.52 (SD 0.30). The ratio was statistically significant by log-transformed paired t-test (p=0.02, 95% CI 0.08, 0.60). Preliminary experiments demonstrate a two-fold reduction in gene targeting to a ZFN-induced chromosome break in PTEN positive cells as compared to PTEN negative cells. Initial clonogenic assays demonstrate significantly increased sensitivity to PARP inhibition in the PTEN-positive cells.ConclusionIn contrast with the expectation that nuclear PTEN promotes RAD51 expression, direct assessment of HDR DSB repair demonstrates decreased repair proficiency as well as increased sensitivity to PARP inhibition in PTEN-positive as compared to cells with PTEN loss. Further work will focus on HDR assessment in additional PTEN-deficient cell lines to determine whether this is an MDA-MB-468-specific phenomenon.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 1120.