Inhibition of BRCT(BRCA1)-phosphoprotein interaction enhances the cytotoxic effect of olaparib in breast cancer cells: a proof of concept study for synthetic lethal therapeutic option
Authors: Amarnath Natarajan
Ziyan Yuan Pessetto
Ying Yan
Tadayoshi Bessho
Journal: Breast cancer research and treatment (2012)
Discipline: CLINICAL MEDICINE
Keywords: Synthetic lethal; therapeutic strategy; PARP inhibitor; olaparib; BRCA1
Affiliations: Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE 68198, USA. 

Abstract:  Synthetic lethal therapeutic strategy using poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitor olaparib in carriers of BRCA1 or BRCA2 mutation has shown promise in clinical settings. Since &lt;5 % of patients are BRCA1 or BRCA2 mutation carriers, small molecules that functionally mimic BRCA1 or BRCA2 mutations will extend the synthetic lethal therapeutic option for non-mutation carriers. Here we provide proof of principle for this strategy using a BRCA1 inhibitor peptide 2 that targets the BRCT(BRCA1)-phosphoprotein interaction and mimics the M177R/K BRCA1 mutation. Reciprocal immunoprecipitation and immunoblotting of BRCA1 and Abraxas was used to demonstrate inhibitor 2 targets BRCT(BRCA1)-Abraxas interface. Immunostaining of γH2AX, cell cycle analysis and homologous recombination (HR) assays were conducted to confirm that inhibitor 2 functionally mimics a chemosensitizing BRCA1 mutation. The concept of synthetic lethal therapeutic strategy with the BRCA1 inhibitor 2 and the PARP inhibitor Olaparib was explored in HeLa, MDA-MB-231, and HCC1937 cell lines. The results show that inhibition of BRCA1 by 2 sensitizes HeLa and MDA-MB-231 cells but not HCC1937 to Olaparib mediated growth inhibition and apoptosis. These results provide the basis for developing high affinity BRCT(BRCA1) inhibitors as adjuvants to treat sporadic breast and ovarian cancers. 

Retracted: No

PubPeer comments:
 There is an erratum for this paper, issued in February 2016, four months after this issue was reported to the journal. The text of the erratum reads:  Figure 1c in the original publication had nuclear lamin B controls inserted in both the cytoplasm and nucleus sets. The revised Fig. 1c show the correct GAPDH controls. The authors regret this error. For the record, here is the original Figure 1C with the duplications marked in red and green boxes. ![file] , 
 Interesting correction (below compared to original). It shows that the purported effects of the inhibitor were due to inconsistent gel loading (top), and what the bottom panel was meant to show is now completely lost owing to removal of loading controls. It is difficult to extract any scientific meaning here. ![file] , 
 #2 We claimed inhibition of the interaction only in the nuclear fraction (due to the loading control in the +IR cytoplasmic fraction). The text from the manuscript is pasted below. The loading control for the bottom panel is the same. In retrospect we should have arranged the panels one below the other instead of side by side to avoid confusion. The conclusion from this figure is  inhibitor 2 only affects BRCA1u2013Abraxas interaction, in the nuclear fractions and in the presence of IR Hope this helps  HeLa cells were treated with increasing concentrations (0, 12.5, 25, 50 and 100 lM) of inhibitor 2 in the presence or absence of IR (10 Gy). The cells were lysed, the cytoplasmic and nuclear fractions were separated and subjected to reciprocal IP and immunoblotting (IB) with BRCA1 and Abraxas antibodies (Fig. 1c). The bottom panel shows reciprocal IP and IB with BRCA1 and Abraxas antibodies of nuclear fractions subjected to IR with increasing concentrations of 2. Western blot analyses showed that inhibitor 2 had no effect on BRCA1u2014Abraxas interaction in the cytoplasm in the presence or absence of IR and in the nucleus in the absence of IR (Fig. 1c). However, in IR treated nuclear fractions, inhibition of BRCA1u2014Abraxas interaction by 2 was observed (Fig. 1c). ,