The combination of gefitinib and RAD001 inhibits growth of HER2 overexpressing breast cancer cells and tumors irrespective of trastuzumab sensitivity

<p>Abstract</p> <p>Background</p> <p>HER2-positive breast cancers exhibit high rates of innate and acquired resistance to trastuzumab (TZ), a HER2-directed antibody used as a first line treatment for this disease. TZ resistance may in part be mediated by frequent co-expression of EGFR and by sustained activation of the mammalian target of rapamycin (mTOR) pathway. Here, we assessed feasibility of combining the EGFR inhibitor gefitinib and the mTOR inhibitor everolimus (RAD001) for treating HER2 overexpressing breast cancers with different sensitivity to TZ.</p> <p>Methods</p> <p>The gefitinib and RAD001 combination was broadly evaluated in TZ sensitive (SKBR3 and MCF7-HER2) and TZ resistant (JIMT-1) breast cancer models. The effects on cell growth were measured in cell based assays using the fixed molar ratio design and the median effect principle. <it>In vivo </it>studies were performed in Rag2M mice bearing established tumors. Analysis of cell cycle, changes in targeted signaling pathways and tumor characteristics were conducted to assess gefitinib and RAD001 interactions.</p> <p>Results</p> <p>The gefitinib and RAD001 combination inhibited cell growth <it>in vitro </it>in a synergistic fashion as defined by the Chou and Talalay median effect principle and increased tumor xenograft growth delay. The improvement in therapeutic efficacy by the combination was associated <it>in vitro </it>with cell line dependent increases in cytotoxicity and cytostasis while treatment <it>in vivo </it>promoted cytostasis. The most striking and consistent therapeutic effect of the combination was increased inhibition of the mTOR pathway (<it>in vitro </it>and <it>in vivo</it>) and EGFR signaling <it>in vivo </it>relative to the single drugs.</p> <p>Conclusions</p> <p>The gefitinib and RAD001 combination provides effective control over growth of HER2 overexpressing cells and tumors irrespective of the TZ sensitivity status.</p

Setting Goals to Reduce Cardiovascular Risk : A Retrospective Chart Review of a Pharmacist-Led Initiative in the Workplace

Background: Cardiovascular diseases (CVD) are the second leading cause of death in Canada with many modifiable risk factors. Pharmacists at a Canadian university delivered a novel CVD risk management program, which included goal-setting and medication management. Aim: This study aimed to describe what CVD prevention goals are composed of in a workplace CVD risk reduction program, and how might these goals change over time. Methods: A longitudinal, descriptive qualitative study using a retrospective chart review of clinical care plans for 15 patients enrolled in a CVD prevention program. Data across 6 visits were extracted from charts (n = 5413 words) recorded from May 2019–November 2020 and analyzed using quantitative content analysis and descriptive statistics. Results: Behavioural goals were most popular among patients and were more likely to change over the 12-month follow-up period, compared to health measure goals. Behavioural goals included goals around diet, physical activity (PA), smoking, medication, sleep and alcohol; health measure goals centered on weight measures, blood pressure (BP) and blood lipid levels. The most common behavioural goals set by patients were for diet (n = 11) and PA (n = 9). Over time, goals around PA, medication, alcohol and weight were adapted while others were added (e.g. diet) and some only continued. Patients experienced a number of barriers to their goal(s) which informed how they adapted their goal(s). These included environmental limitations (including COVID-19) and work-related time constraints. Conclusions: This study found CVD goal-setting in the pharmacist-led workplace wellness program was complex and evolved over time, with goals added and/or adapted. More detailed qualitative research could provide further insights into the patient-provider goal-setting experience in workplace CVD prevention.Medicine, Faculty ofPharmaceutical Sciences, Faculty ofOther UBCEmergency Medicine, Department ofReviewedFacultyResearcherGraduat

Cytogenetic characterization and gene expression profiling of the trastuzumab-resistant breast cancer cell line JIMT-1.

Resistance to the HER-2 targeting drug trastuzumab can be observed clinically, but the lack of suitable experimental models hampers studies of resistance mechanisms. We characterized a HER-2–positive carcinoma cell line (JIMT-1) derived from a 62-year-old breast cancer patient which was clinically resistant to trastuzumab. Multicolor fluorescence in situ hybridization revealed a complex hyperdiploid karyotype with numerous marker chromosomes and unbalanced translocations. Comparative genomic hybridization (CGH) revealed numerous regions of copy number aberration (CNA). Further analysis by array CGH identified 27 regions of CNA (16 amplified, 11 deleted). Thirty-eight percent of the genes in the amplified regions were overexpressed, compared to only 9% in regions of normal copy number ratios (CNR). Accordingly, 26% of the genes in the deleted regions were underexpressed, compared to 10% in regions of normal CNR. Most amplified and overexpressed genes were located on chromosome 1 as well as on 8q, 12q14.1, 17q11not, vert, similarq21, and 20q13. In 17q11not, vert, similarq21, we identified two separate amplicons, the HER-2 amplicon and a previously unreported amplicon at 17q21.31. Several aberrant genes are implicated in cancer development (e.g., JUN, CDK4, and SLUG protooncogenes, as well as the drug/hormone–metabolizing genes GSTM1 and CYP24). We conclude that cytogenetic and expression profiling of JIMT-1 revealed several new features that need further characterization and may shed light on trastuzumab resistance

Induction of Autophagy Is an Early Response to Gefitinib and a Potential Therapeutic Target in Breast Cancer

Gefitinib (Iressa®, ZD1839) is a small molecule inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase. We report on an early cellular response to gefitinib that involves induction of functional autophagic flux in phenotypically diverse breast cancer cells that were sensitive (BT474 and SKBR3) or insensitive (MCF7-GFPLC3 and JIMT-1) to gefitinib. Our data show that elevation of autophagy in gefitinib-treated breast cancer cells correlated with downregulation of AKT and ERK1/2 signaling early in the course of treatment. Inhibition of autophagosome formation by BECLIN-1 or ATG7 siRNA in combination with gefitinib reduced the abundance of autophagic organelles and sensitized SKBR3 but not MCF7-GFPLC3 cells to cell death. However, inhibition of the late stage of gefitinib-induced autophagy with hydroxychloroquine (HCQ) or bafilomycin A1 significantly increased (p&lt;0.05) cell death in gefitinib-sensitive SKBR3 and BT474 cells, as well as in gefitinib-insensitive JIMT-1 and MCF7-GFPLC3 cells, relative to the effects observed with the respective single agents. Treatment with the combination of gefitinib and HCQ was more effective (p&lt;0.05) in delaying tumor growth than either monotherapy (p&gt;0.05), when compared to vehicle-treated controls. Our results also show that elevated autophagosome content following short-term treatment with gefitinib is a reversible response that ceases upon removal of the drug. In aggregate, these data demonstrate that elevated autophagic flux is an early response to gefitinib and that targeting EGFR and autophagy should be considered when developing new therapeutic strategies for EGFR expressing breast cancers

Effects of siRNA mediated EGFR silencing on downstream signaling and autophagy.

<p>(<b>A</b>) Following a transfection with EGFR siRNA, SKBR3 cells were treated for 72 h with vehicle or 5 µM gefitinib. (<b>B</b>) Following a double knockdown with the EGFR siRNA MCF7-GFPLC3 cells were treated with vehicle or 4 µM gefitinib for 24 h. Representative experiments are shown.</p

Gefitinib induces autophagic flux.

<p>(<b>A</b> - <b>D</b>) Autophagic flux assays performed in SKBR3 cells. (<b>A</b>) Western blot analysis of p62 expression in lysates derived from cells treated for 3 h with vehicle (0 µM gefitinib) or increasing concentrations of gefitinib (Gef) (<b>B</b>) Western blot analysis of LC3 levels in lysates derived from cells treated for 3 h with increasing concentrations of gefitinib in the absence or presence of 5 nM bafilomycin A1 (BAF). (<b>C</b>) HCA of TOA in cells treated for 72 h with increasing concentrations of gefitinib in the absence or presence of 10 mM 3-MA added for the last 3 h of treatment. The results are normalized to the vehicle control expressed as 1. Each bar represents the mean±SD from 3 replicate wells. Asterisks indicate statistically significant differences (p<0.05) between cells treated with gefitinib or 3-MA alone and cells treated with 3-MA in the presence of gefitinib. The results shown are representative of two experiments. (<b>D</b>) Western blot analysis of LC3-I and LC3-II levels in lysates derived from cells treated for 24 h with vehicle, 5 µM gefitinib, 5 mM 3-MA and the combination of gefitinib and 3-MA at the corresponding concentrations. Representative blots in (<b>A</b>), (<b>B</b>) and (<b>D</b>) are shown. (<b>E</b> - <b>G</b>) Autophagic flux assays performed in MCF7-GFPLC3 cells. (<b>E</b>) Western blot analysis of autophagic markers in lysates derived from cells treated for 18 h with increasing concentrations of gefitinib. Cleaved GFP is marked as GFP. (<b>F</b>) Western blot analysis of cleaved GFP levels in lysates derived from cells treated with vehicle or 10 µM gefitinib in the absence or presence of 10 mM 3-MA for 3 h (top panel), in the absence or presence of 50 nM bafilomycin A1 (BAF) for 24 h (middle panel), and in the absence or presence of 10 mg/ml lysosomal inhibitors (LYI; pepstatin A and E-64d) added for the last hour of treatment (bottom panel). Tubulin was used as loading control. Representative blots in (<b>E</b>) and (<b>F</b>) are shown. (<b>G</b>) HCA data showing the average GFPLC3 TOA/cell in MCF7-GFPLC3 cells treated for 3 h with increasing concentrations of gefitinib in the absence or presence of indicated autophagy inhibitors added for the last hour of treatment. The results are normalized to vehicle control expressed as 1. Each data point represents the mean±SD from 3 replicate wells and the results shown are representative of two experiments. 3-MA was used at 5 mM, bafilomycin A1 (BAF) was used at 5 nM and LYI were used at 10 µg/ml. Asterisks indicate statistically significant differences (p<0.05) between cells treated with gefitinib or autophagy inhibitors alone and cells treated with autophagy inhibitors in the presence of gefitinib. </p

<i>In</i><i>vivo</i> efficacy of HCQ alone and in combination with gefitinib.

<p>Mice bearing JIMT-1 tumors were treated (oral gavage) with the indicated agents (n = 6 mice per treatment group). Increase in tumor volume was plotted relative to the volume on the first day of treatment (indicated by arrows) expressed as 100%. (<b>A</b>) Efficacy of HCQ used at different doses. (<b>B</b>) Efficacy of the gefitinib and HCQ combination. An asterisk represents a significant (p<0.05) difference between vehicle and combination treated animals on the last day of treatment. </p

Gefitinib-induced autophagy is a reversible process.

<p>(<b>A</b>) Representative images of MCF7-GFPLC3 cells treated for 3 h with vehicle or 10 µM gefitinib (top images) obtained with IN Cell 1000 Analyzer. After the indicated treatment, cell cultures were washed twice with medium and then incubated in the absence of drug for the following 5 h (bottom images). GFPLC3-labeled autophagosomes appear as green fluorescent puncta in cellular cytoplasm. (<b>B</b>) MCF7-GFPLC3 cells were treated for 3 h with vehicle (0 µM) or increasing concentrations of gefitinib and washed as described in (<b>A</b>). Quantitation of GFPLC3 TOA/cell by HCA in MCF7-GFPLC3 cells; the data shown are normalized to vehicle-treated cells expressed as 1 (mean±SD, n = 3 replicate wells). (<b>C</b>) Clonogenic assay performed in MCF7-GFPLC3 cells treated for 3 h with vehicle (Veh) or 10 µM gefitinib (Gef) followed by drug removal and incubation in media for 17 days. The results are expressed as % plating efficiency representing the proportion of cells that are able to form colonies. Bar graphs represent colony count from 3 wells (mean±SD). A representative experiment is shown.</p