PI3K inhibition impairs BRCA1/2 expression and sensitizes BRCA-proficient triple-negative breast cancer to PARP inhibition

Abstract

PARP inhibitors are active in tumors with defects in DNA homologous recombination (HR) due to BRCA1/2 mutations. The phosphoinositide 3-kinase (PI3K) signaling pathway preserves HR steady state. We hypothesized that in BRCA-proficient triple-negative breast cancer (TNBC), PI3K inhibition would result in HR impairment and subsequent sensitization to PARP inhibitors. We show in TNBC cells that PI3K inhibition leads to DNA damage, downregulation of BRCA1/2, gain in poly-ADP-ribosylation, and subsequent sensitization to PARP inhibition. In TNBC patient-derived primary tumor xenografts, dual PI3K and PARP inhibition with BKM120 and olaparib reduced the growth of tumors displaying BRCA1/2 downregulation following PI3K inhibition. PI3K-mediated BRCA downregulation was accompanied by extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of an active form of MEK1 resulted in ERK activation and downregulation of BRCA1, whereas the MEK inhibitor AZD6244 increased BRCA1/2 expression and reversed the effects of MEK1. We subsequently identified that the ETS1 transcription factor was involved in the ERK-dependent BRCA1/2 downregulation and that knockdown of ETS1 led to increased BRCA1/2 expression, limiting the sensitivity to combined BKM120 and olaparib in 3-dimensional culture.

Significance: Treatment options are limited for patients with TNBCs. PARP inhibitors have clinical activity restricted to a small subgroup of patients with BRCA mutations. Here, we show that PI3K blockade results in HR impairment and sensitization to PARP inhibition in TNBCs without BRCA mutations, providing a rationale to combine PI3K and PARP inhibitors in this indication. Our findings could greatly expand the number of patients with breast cancer that would benefit from therapy with PARP inhibitors. On the basis of our findings, a clinical trial with BKM120 and olaparib is being initiated in patients with TNBCs.

Figure 1

BRCA downregulation and γH2AX staining following PI3K inhibition in vitro. A, fluorescence-activated cell-sorting (FACS) analysis showing staining of γH2AX during the 3 phases of the cell cycle (G₁, S, G₂) in MDA-MB-468 cells transfected with control, BRCA1, or PIK3CA siRNAs and treated with either dimethyl sulfoxide (DMSO) or 1 μmol/L olaparib. B, immunofluorescence of γH2AX in MDA-MB-468 cells transfected with control, BRCA1, or PIK3CA siRNAs and treated with either DMSO or 1 μmol/L of olaparib. Percentages of positive nuclei (10 foci or more per nucleus being considered positive) from 10 fields per sample are indicated. C, Western blot analysis of cell lysates (7 days posttransfection) from MDA-MB-468 cells transfected with control or PIK3CA siRNAs using the indicated antibodies. Tubulin was used as loading control. D, Western blot analysis of lysates from MDA-MB-468 treated with BKM120 (750 nmol/L) for 7 days using the indicated antibodies. Total ERK (tERK) was used as a loading control. E, quantitative real-time PCR (qRT-PCR) measuring both BRCA1 and BRCA2 mRNA levels in MDA-MB-468 treated with BKM120. Measurements were normalized to 18S mRNA levels and expressed as fold change compared with controls (log₂ scale). Data are shown as mean ± SE of 3 independent replicates for each condition.

Figure 2

Combined PI3K and PARP suppression in vitro. A, viability (assayed by Cell Titer-Glo) of MDA-MB-468 cells transfected with control, BRCA1, or PIK3CA siRNAs and treated with either dimethyl sulfoxide (DMSO) or olaparib for 7 days. IC₅₀ values were calculated using the GraphPad Prism program. B, MDA-MB-468 cells plated in anchorage-independent conditions treated with BKM-120 (750 nmol/L), olaparib (4 μmol/L), or the combination for 45 days, with weekly media changes. Cell colonies were stained with crystal violet. C, MDA-MB-231, HCC1143, and HCC70 cells plated in anchorage-dependent conditions treated with BKM-120 (750 nmol/L), olaparib (4 μmol/L), GDC-0941 (500 nmol/L), or the combination for 7 days. Magnification, ×10.

Figure 3

γH2AX staining and BRCA downregulation following PI3K inhibition in vivo. A, representative immunofluorescence staining for γH2AX (red) comparing placebo versus BKM120-treated (27.5 mg/kg) TNBC xenografts. Nuclei are stained with DAPI (blue). Quantifications of γH2AX staining are from 6 different tumors for each condition. *, P < 0.001. Magnification, ×20. B, qRT-PCR measuring both BRCA1 and BRCA2 mRNA levels of patient-derived TNBC1 and TNBC2 treated with BKM120. Measurements were normalized to 18S mRNA levels and expressed as fold change compared with controls (log₂ scale). Data are shown as mean ± SE of 3 independent replicates for each condition. *, P < 0.001. C, Western blot analysis of patient-derived xenografts of 2 independent tumors from different mice treated for 21 days with vehicle or BKM120 (27.5 mg/kg) using the indicated antibodies. Total ERK (tERK) is used as loading control.

Figure 4

Combined PI3K and PARP suppression in vivo. Tumor growth of TNBC1, TNBC2, and TNBC3 xenografts treated with vehicle control, BKM120 (27.5 mg/kg), olaparib (Olap; 50 mg/kg), or the combination of both agents. Relative tumor volumes are displayed as mean ± SE of a minimum of 6 tumors per arm. *, P < 0.001 combination versus BKM120 arm.

Figure 5

ERK-dependent BRCA downregulation. A, Western blot analysis of protein lysates from MDA-MB-468 cells constitutively overexpressing control or MEK1 plasmids and treated with the MEK inhibitor AZD6244 (500 nmol/L) for 4 days using the indicated antibodies. Total ERK (tERK) is used as loading control. B, qRT-PCR measuring both BRCA1 and BRCA2 mRNA levels in MDA-MB-468-MEK1 cells treated with AZD6244. Measurements were normalized to 18S mRNA levels and expressed as fold change compared to controls (log₂ scale). Data are shown as mean ± SE of 3 independent replicates for each condition. C, Western blot analysis of protein lysates from MDA-MB-468 cells treated with BKM120 (750 nmol/L), AZD6244 (500 nmol/L), or the combination of both for 4 days using the indicated antibodies. Total ERK (tERK) is used as loading control. D, qRT-PCR measuring both BRCA1 and BRCA2 mRNA levels in MDA-MB-468 cells treated with BKM120 and AZD6244. Measurements were normalized to 18S mRNA levels and expressed as fold change compared with controls (log₂ scale). Data are shown as mean ± SE of 3 independent replicates for each condition. E, Western blot analysis of the indicated proteins in 2 independent TNBC1 tumors treated for 4 days with BKM120 (50 mg/kg), AZD6244 (10 mg/kg), or the combination of both. Total ERK (tERK) is used as loading control. F, qRT-PCR measuring both BRCA1 and BRCA2 mRNA levels in tumor grafts. Measurements were normalized to 18S mRNA levels and expressed as fold change compared with controls (log₂ scale). Data are shown as mean ± SE of 3 tumors for each condition.

Figure 6

ETS1 knockdown and resistance to the combination of BKM120 and olaparib. A, Western blot analysis of protein lysates from MDA-MB-231 shRNA-ETS1–infected cells treated with doxycycline (20 ng/mL) for 4 days using the indicated antibodies. Total ERK (tERK) is used as loading control. B, qRT-PCR measuring both BRCA1 and BRCA2 mRNA levels in MDA-MB-231-shETS1 cells treated with doxycycline. Measurements were normalized to 18S mRNA levels and expressed as fold change compared with controls (log₂ scale). Data are shown as mean ± SE of 3 independent replicates for each condition. C, Western blot analysis of protein lysates from BT20 shRNA-ETS1–infected cells treated with doxycycline (20 ng/mL) for 4 days using the indicated antibodies. Total ERK (tERK) is used as loading control. D, qRT-PCR measuring both BRCA1 and BRCA2 mRNA levels in BT20-shETS1 cells treated with doxycycline. E, MDA-MB-231-shETS1 cells plated in anchorage-dependent conditions were treated with/without doxycycline (20 ng/mL) and BKM-120 (750 nmol/L), olaparib (Olap; 4 μmol/L), GDC-0941 (500 nmol/L), or the combination for 14 days. Magnification, ×10.