Purpose: 1) To investigate expression of the E3 ligase, RNF126, in human invasive breast cancer (BC)and its links with BC outcomes. 2) To test the hypothesis that RNF126 determines the efficacy ofinhibitors targeting the cell cycle checkpoint kinase, CHK1.

Experimental Design: A retrospective analysis by immunohistochemistry (IHC) compared RNF126staining in 110 invasive BC and 78 paired adjacent normal tissues with clinicopathologic data. WhetherRNF126 controls CHK1 expression was determined by chromatin immunoprecipitation and a CHK1promoter driven luciferase reporter. Staining for these two proteins by IHC using tissue microarrays wasalso conducted. Cell killing/replication stress induced by CHK1 inhibition was evaluated in cells, with orwithout RNF126 knockdown, by MTT/colony formation, replication stress biomarker immunostainingand DNA fiber assays.

Results: RNF126 protein expression was elevated in BC tissue samples. RNF126 was associated witha poor clinical outcome after multivariate analysis and was an independent predictor. RNF126promotes CHK1 transcript expression. Critically, a strong correlation between RNF126 and CHK1proteins was identified in BC tissue and cell lines. The inhibition of CHK1 induced a greater cell killingand a higher level of replication stress in BC cells expressing RNF126 compared to RNF126 depletedcells.

Conclusions:  RNF126 protein is highly expressed in invasive BC tissue. The high expression ofRNF126 is an independent predictor of a poor prognosis in invasive BC and is considered a potentialbiomarker of a cancer’s responsiveness to CHK1 inhibitors. CHK1 inhibition targets BC cells expressinghigher levels of RNF126 by enhancing replication stress.