Treatment with lapatinib sequesters EGFR to the cytosol in triple negative breast cancer cells.

<p>(A–C) Cells were subjected to 1 µM lapatinib treatment for 16 hours and EGFR location was assessed by subcellular fractionation. Lapatinib induced cytosolic translocation of EGFR in (A) MDA-MB-231, (B) MDA-MB-453 and (C) MDA-MB-468 cells. Histone H1 and α-tubulin were used to test the purity of the nuclear and cytosolic fractions respectively. Quantification of EGFR levels was performed via densitometry. Shown is the representative data of three independent experiments (mean +/− SEM, **p<0.01).</p

Lapatinib attenuates homologous recombination mediated DNA double strand break repair in triple negative breast cancer cells.

<p>(A–C) Homologous recombination (HR) repair capacity was measured in (A) MDA-MB-231, (B) MDA-MB-453 and (C) MDA-MB-468 triple negative breast cancer cell lines by assessing radiation-induced rad51 foci, a well characterized marker for HR repair. Briefly, cells were exposed to mock or 3 Gy irradiation (IR) and subsequently subjected to immunofluorescence staining for rad51 foci. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with rad51 foci (**p<0.01 compared to vehicle). (D) Chromosomal HR repair capacity was directly measured in MDA-MB-231DRGFP cells. MDA-MB-231DRGFP were treated with 1 µM lapatinib or vehicle control. 16 hours following the treatment period, cells were transfected with ISce-1 or control vector. 48 hours following transfection cells were subjected to flow cytometry for GFP expression. Shown is the representative fold induction in GFP (mean +/− SEM) from at least 3 independent experiments (**p<0.01 compared to vehicle). Inset is a representative figure depicting the DRGFP repair model.</p

Lapatinib interrupts the interaction between BRCA1 and EGFR in triple negative breast cancer cell lines.

<p>(A–B) Reciprocal immunoprecipitation with (A) EGFR and (B) BRCA1 was performed in (from left to right) MDA-MB-231, MDA-MB-453 and MDA-MB-468 cells following 16 hours of vehicle or 1 µM lapatinib treatment. The levels of BRCA1 and EGFR in immunocomplexes were normalized to the amount of the reciprocal protein that was pulled down. (C–H) Quantification of EGFR and BRCA1 levels was performed via densitometry. Shown is the representative data of three independent experiments (mean +/− SEM, **p<0.01).</p

Treatment with lapatinib sequesters BRCA1 away from its nuclear repair substrates to the cytosol in triple negative breast cancer cells.

<p>(A–C) Cells were subjected to 1 µM lapatinib treatment for 16 hours and BRCA1 location was assessed by subcellular fractionation. Lapatinib induced cytosolic translocation of BRCA1 in (A) MDA-MB-231, (B) MDA-MB-453 and (C) MDA-MB-468 cells. Histone H1 and α-tubulin were used to test the purity of the nuclear and cytosolic fractions respectively. Quantification of BRCA1 levels was performed via densitometry. Shown is the representative data of three independent experiments (mean +/− SEM, **p<0.01).</p

Combined EGFR and PARP inhibition delays the growth of orthotopic breast tumor xenografts in mice.

<p>MDA-MB-231 cells were injected into the mammary fat pads of mice. Once tumors were palpable, mice were treated with vehicle, 30 mg/kg/day lapatinib (b.i.d.), 100 mg/kg/day ABT-888 (b.i.d.), or combination of lapatinib and ABT-888. Treatment period was for 26 days and tumors were measured via caliper three times per week (n = 7, mean +/− SEM, ***p<0.001).</p

Cetuximab (C225) enhances cytotoxicity with the PARP inhibitor ABT-888 in head and neck cancer cells.

<p>(A) Combination C225 and ABT-888 reduces the viability of UM-SCC1, UM-SCC6, and FaDu head and neck cancer cells. Cells were treated with either vehicle or 2.5 µg/mL C225 for 16 hours and subsequently exposed to vehicle or 10 µM ABT-888. Twenty-four hours following ABT-888, cell viability was assayed with the ATPlite system (Perkin Elmer). Shown is the representative data of at least 3 independent experiments of the cell viability following various treatments as measured by relative ATP levels (mean +/− SEM, *p<0.01, **p<0.001 compared to vehicle control). (B–D) Combination C225 and ABT-888 reduces the colony forming ability of (B) UM-SCC1, (C) UM-SCC6, and (D) FaDu head and neck cancer cells. Cells were treated with either vehicle or 2.5 µg/mL C225 for 16 hours. Following the treatment period, cells were seeded for colony formation assays and subjected to various doses of ABT-888. Shown is the mean survival fraction (+/− SEM) from at least 3 independent colony formation assay experiments following treatment (**p<0.001).</p

Combination cetuximab (C225) and ABT-888 induces persistent DNA double strand break damage in head and neck cancer cells.

<p>(A–C) DNA damage 2, 24, and 48 hours following vehicle, C225, PARPi, or both C225+PARPi was assessed by γ-H2AX foci in (A) UM-SCC1, (B) UM-SCC6, and (C) FaDu cells. Cells were treated with vehicle or various doses of C225 for 16 hours and subsequently exposed to vehicle or various doses of ABT-888. At the indicated times following PARP inhibition, cells were processed for immunofluorescence staining for γ-H2AX foci. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with >10 foci (*p<0.05, **p<0.01 compared to vehicle control at each respective time point).</p

Cetuximab (C225) attenuates non-homologous end-joining (NHEJ) repair.

<p>C225 attenuates irradiation (IR)-induced DNA-Pk Thr2609 foci, well established markers of non-homologous end joining (NHEJ)-mediated DNA DSB repair in (A) UM-SCC1, (B) UM-SCC6, and (C) FaDu head and neck cancer cells. Cells were treated with vehicle, 2.5 µg/mL C225, or 5.0 µg/mL C225 for 16 hours and subsequently subjected to mock or 4 Gy IR. At the indicated time following IR, cells were processed for immunofluorescence staining for DNA-Pk Thr2609 foci. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with >10 foci (*p<0.05, **p<0.01 compared to cells not exposed to C225). (D) C225 reduces phospho-Thr2609 DNA-Pk levels in UM-SCC6 head and neck cancer cells. Cells were treated with vehicle or 2.5 µg/mL C225 for 16 hours and subsequently subjected to mock or 4 Gy IR. One hour following IR, cells were processed for Western blot analysis for phospho-Thr2609 DNA Pk levels. Total DNA Pk was also analyzed and tubulin was used as loading control.</p

Cetuximab (C225) attenuates homologous recombination (HR) repair.

<p>C225 attenuates IR-induced Rad51 foci, well characterized markers of homologous recombination (HR)-mediated DNA DSB repair in (A) UM-SCC1, (B) UM-SCC6, and (C) FaDu cells. Cells were treated with vehicle, 2.5 µg/mL C225, or 5.0 µg/mL C225 for 16 hours and subsequently subjected to mock or 4 Gy irradiation (IR). At the indicated times following IR, cells were processed for immunofluorescence staining for Rad51 foci. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with >10 foci (*p<0.05, **p<0.01 compared to vehicle at each respective time point). The inset in (A) is a representative image of UM-SCC1 cells exhibiting Rad51 foci following IR.</p

Cetuximab (C225) increases DNA damage by inhibiting DNA double strand break (DSB) repair in head and neck cancer cells.

<p>(A) C225 increases the number of cells with DSBs as evidenced by γ-H2AX foci, a commonly used marker for DSBs. Shown is the representative data of 3 independent experiments the % of cells (mean +/− SEM) with >10 foci (*p<0.05, **p<0.01 compared to vehicle control). (B) C225 increases γ-H2AX protein levels in treated cells. UM-SCC1, UM-SCC6, and FaDu cells were treated with vehicle, 2.5 µg/mL C225, or 5.0 µg/mL C225 for 16 hours. Following the treatment period, cells were processed for (A) immunofluorescence staining for γ-H2AX foci or (B) western blot analysis for γ-H2AX levels. Shown is the representative Western blot of 3 independent experiments.</p