Article Figures & Data
Figures
- Figure 1.
AR-positive breast cancer cells are sensitive to NVP-BEZ235 treatment compared with AR-negative cells. A, four AR-positive breast cancer cell lines were treated with control or NVP-BEZ235 for 5 days. Cell growth rates were measured by MTT assay. Each data point represents a mean value of three independent experiments and the error bars indicate the SD, unless otherwise indicated. B, the remaining six breast cancer cell lines that are AR negative were treated with control or NVP-BEZ235 for 5 days. Cell growth rates were measured by MTT assays. C, GI50 values of NVP-BEZ235 in breast cancer cell lines treated with 5 days of treatment.
- Figure 2.
Activation of AR sensitized AR+/ER+ breast cancer cells to NVP-BEZ235 treatment. A, after control siRNA or AR knockdown, MCF-7 cells were treated with DHT and NVP-BEZ235. Growth rates of the cells were assessed by MTT assay, as previously described (46). B, clone colony formation in MCF-7 cells, treated with DHT and NVP-BEZ235, was measured by a clonogenic assay (Mann–Whitney test; *, P = 0.012; **, P = 0.0011). C, GI50 values of NVP-BEZ235 for AR-positive breast cancer cell lines with control or DHT (Mann–Whitney test; *, P < 0.01). D, MCF-7 cells were cultured in medium containing 10% or 25% FBS. After DHT or NVP-BEZ235 treatment for 48 hours, cell morphology changes were recorded by microscopy.
- Figure 3.
Reexpression of AR restored NVP-BEZ235 response in AR-negative breast cancer cells. A, six AR-negative cell lines were transfected with control or AR, and treated with 2 nmol/L NVP-BEZ235. Cell growth rates were assessed by MTT assay. B, GI50 values of NVP-BEZ23 for AR-negative breast cancer cell lines transfected with control or AR. C, morphologic changes of AU565 cells after DHT or NVP-BEZ235 treatment.
- Figure 4.
Combination of DHT and NVP-BEZ235 achieved better therapeutic effect in breast cancer xenografts by PTEN/KLLN upregulation. Nude mice bearing established MCF-7 (A) or MDA-MB-231 (B) xenografts were treated with vehicle (n = 26), 1 mg/kg/d NVP-BEZ235 (n = 7), 5 mg/kg/d NVP-BEZ235 (n = 8), DHT (n = 23), DHT/1 mg/kg/d NVP-BEZ235 (n = 8), or DHT/5 mg/kg/d NVP-BEZ235 (n = 8) for 5 weeks. Tumor volumes were measured on the indicated days. Combination treatment decreased tumor volumes below their initial sizes in AR-positive MCF-7 tumors but not in MDA-MB-231 xenografts. C, MCF-7 cells were transfected with ARE luciferase and treated with control or NVP-BEZ235 for 48 hours. Cell lysates were subjected to dual luciferase assay to evaluate AR activity. *, P < 0.01. D, MCF-7 cells were treated with control or NVP-BEZ235. After 48 hours, cell lysates were harvested for Western blot analysis. Tumors from mouse MCF-7 (E) and MBA-MB-231 (F) xenografts were dissected and subjected to hematoxylin and eosin (HE) staining and immunohistochemistry for the shown proteins.
Tables
- Table 1.
Subtypes and molecular status of breast cancer cell lines
Expression Mutation Cell lines Subtype AR ER PR HER2 PTEN PTEN PI3KCA p53 MCF-7a Luminal A + + + − + WT E545K WT MDA-MB-453a Unclassified + − − + + E307K H1047R Deletion SKBR3a HER2 + − − + + WT H1047R R175H BT-474a Luminal B + + + + + WT K111N E285K MDA-MB-468 TN − − − − − A72fs X5 WT R273H BT20 Basal − − − − + WT P539R K132Q H1047R MDA-MB-436 TN − − − − − WT WT 205 fs MDA-MB-231 TN − − − − + WT WT R280K AU565 HER2 − − − + + WT WT R175H HCC1395 Basal − + − − − N212fs WT R175H Abbreviation: TN, triple negative.
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↵aNVP-BEZ235 sensitive with cell death.
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- Table 2.
DHT/NVP-BEZ235 combination induces breast cancer response
NVP-BEZ235 (mg/kg/d) 0 1 5 10 25 50 Complete responsea Control 0/26 0/7 0/8 0/14 0/12 0/9 DHT 0/23 1/8 1/8 2/14 2/13 4/11 Partial responseb Control 0/26 0/7 0/8 0/14 0/12 0/9 DHT 0/23 5/8 6/8 4/14 10/13 7/11 Tumor growth (fold)c Control 3.36 2.84 1.7 2.58 1.66 1.38 DHT 1.69 0.46 0.36 0.58 0.41 0.3 -
↵aDenominators represent (number of complete response)/(total number of tumors) in MCF-7 xenograft models. Mantel–Cox test, P = 0.0016.
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↵bPartial response: at least 50% decrease in tumor volume after 5 weeks of treatment compared with initial measurement.
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↵cThe tumor volume of remaining MCF-7 xenografts after 5 weeks of treatment compared with initial measurement.
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Supplementary Data
Files in this Data Supplement:
- Supplementary Figures 1 - 7 - PDF file - 1622K, Figure S1.Combinaion of DHT and NVP-BEZ235 induced synergistic effect of growth inhibition in MCF cells growth. Figure S2. Combination of DHT and NVP-BEZ235 exposure affects expression of markers of epithelial to mesenchymal transitions (EMT) in MCF-7 cells. Figure S3. DHT did not inhibit AR-negative breast cancer cell growth. HCC1395 (AR-/ER+) and MDA-MB-231 (AR-/ER-) cells were treated with control, DHT, NVP-BEZ235 or DHT/BEZ235 combination for 5 days. Figure S4. Combination of DHT and NVP-BEZ235 achieved better therapeutic effects in breast cancer xenografts compared to either single agent. Figure S5. Tumors from mouse MCF-7 xenografts were dissected and subjected to immunohistochemistry for the shown proteins. Figure S6. DHT suppressed low level NVP-BEZ235-induced MYC expression and AKT phosphorylation in breast cancer. Figure S7. DHT decreased the known side effects of NVP-BEZ235 treatment.
Volume 13, Issue 2
