Abstract
Triple negative breast cancer (TNBC) represents approximately 10–15% of all breast cancers and has a poor outcome as it lacks a receptor target for therapy, and TNBC is frequently associated with a germline mutation of BRCA1. Poly (ADP-ribose) polymerase inhibitor (PARPi) drugs have demonstrated some effectiveness in treating BRCA1 or BRCA2 mutated breast and ovarian cancers but resistance to PARPi is common. Published results found that resistance to Olaparib, a PARPi, can be due to downregulation of EMI1 and the consequent upregulation of the RAD51 recombinase. Using a tissue culture-based cell viability assay, we extended those observations to another PARPi and to other chemotherapy drugs that affect DNA repair or the cell cycle. As we expected, EMI1 downregulation resulted in resistance to another PARPi drug, Talazoparib. EMI1 downregulation also led to resistance to other cytotoxic drugs, Cisplatin and CHK1 inhibitor. Notably, increasing the RAD51 protein expression only recapitulated some, but not all, of the effects of EMI1 depletion in conferring to the cell resistance to different PARPi and the other cytotoxic drugs. These results suggest that the downstream effects of EMI1 downregulation that contribute to PARPi resistance are increasing the concentration of RAD51 protein in the cell and blocking mitotic entry. We found that combining CHK1 inhibitor with olaparib results in restoration of sensitivity even when EMI1 expression is downregulated. This combination therapy may be a means to overcome the PARPi resistance in BRCA1-deficient TNBC cells.
Highlights
The prognosis of breast cancer depends on several characteristic features, namely, estrogen receptor (ER), progesterone receptor (PR), and HER2 receptor expression and mutation status
In experiments using the siRNA-1 targeting EMI1, the IC50 was reached at the lowest concentration of olaparib tested (1.25 μM) in the cells receiving the control siRNA; cells receiving the EMI1 specific siRNA were comparatively resistant at these concentrations
Extending the results from Marzio et al to a second Poly (ADP-ribose) polymerase inhibitors (PARPi), talazoparib, which has a higher propensity than olaparib to have a bound moiety trapped on the DNA [24], we found that depletion of EMI1 by transfection of either of the two siRNAs from MDA-MB-436 cells rendered the cells resistant to talazoparib and the IC50 changed from less than 0.05 μM with normal EMI1 levels to about 10 μM with EMI1 levels reduced by transfection of specific siRNA (Fig 1C), consistent with the expectation of PARPi resistance when the EMI1 levels were depleted
Summary
The prognosis of breast cancer depends on several characteristic features, namely, estrogen receptor (ER), progesterone receptor (PR), and HER2 receptor expression and mutation status. BRCA1 or BRCA2 mutant breast tumor cells are deficient in the repair of DNA double strand breaks (DSB) via the homology directed repair (HDR) mechanism. (ADP-ribose) polymerase inhibitors (PARPi) block the base excision repair mechanism for single strand breaks, but PARPi in combination with a BRCA1 or BRCA2 mutation are synthetic lethal, and such cells are sensitive to PARPi [3,4]. PARP inhibitors have shown promise in cancer therapy via a mechanism dependent on synthetic lethality; the inhibition of PARP results in the accumulation of a significant amount of double-strand breaks (DSB) by interfering with replication fork progression at the site of DNA damage [6]. There are multiple mechanisms of resistance to PARPi chemotherapy, some of them are targetable for therapy including the modulation of EMI1 and RAD51 expression levels [8,9]
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