Activation of Akt characterizes estrogen receptor positive human breast cancers which respond to anthracyclines

Abstract

Anthracyclines are key components of human breast cancer chemotherapy. Here, we explored the role of Akt signaling in anthracycline resistance.The antitumor activity of doxorubicin and Akt inhibitor A-443654 alone or combined was examined in estrogen receptor (ER) positive and negative human breast cancer cell lines. Further, we examined mRNA changes induced by anthracyclines in locally advanced breast cancers biopsied before and after treatment in two clinical trials.Doxorubicin increased Akt phosphorylation in ER positive MCF7 and T47D cell lines, with no effect in ER negative MDA-MB231 breast cancer cells. A-443654 was significantly more cytotoxic in doxorubicin-resistant compared to doxorubicin-naïve MCF7. This difference was not observed in MDA-MB231. Among 24 patients, AKT1 gene expression increased 24 hrs after the initial epirubicin exposure in ER positive tumors responding to therapy (n=6), as compared to ER positive non-responders (n=7) or ER negative tumors (n=11). In contrast, AKT1 mRNA changes after 16 weeks of doxorubicin were unrelated to clinical response and ER status (n=30).In conclusion, rapid Akt activation was observed in ER positive breast cancers which responded to anthracyclines. Increased cytotoxicity of A-443654 in doxorubicin-resistant MCF7 cells indicates a possible role for Akt inhibitors in ER positive breast cancers where chemoresistance evolves.

Keywords: Akt; breast cancer; doxorubicin; drug resistance; estrogen receptor.

Figure 1. Doxorubicin treatment of doxorubicin-naïve and doxorubicin-resistant human breast cancer cell lines

(A-B) Western blots of PTEN and Akt-mTOR-S6K signaling in MB231 and MCF7 breast cancer cells in vitro, either doxorubicin-naïve (A-B) or doxorubicin-resistant (C-D, dox-res). Drug exposure lasted 24 hrs, at either 1.5 μM for MB231 and 2 μM for MCF7 or an equivalent volume of DMSO (stock solvent for doxorubicin) for control wells, three independent experiments per group. Whole cell lysate, 30 μg protein loaded per lane. (E-F) Densitometries for western blots (A-D) depict the relative protein expression, normalized to actin. Phosphorylated Akt (p-Akt) and mTOR (p-mTOR) were normalized to actin and thereafter to total Akt and mTOR, respectively. Bars represent the mean protein expression for experiments performed in triplicate ± SEM. **p<0.01

Figure 2. Akt inhibitor treatment of doxorubicin-naïve and doxorubicin-resistant human breast cancer cell lines

(A-B) Western blots of Akt phosphorylation induced by increasing doses of the Akt inhibitor A-443654, 0-10 μM, 2 hrs exposure in doxorubicin-naïve or doxorubicin-resistant MB231 (A) and MCF7 (B) human breast cancer cells in vitro. Whole cell lysate, 30 μg protein loaded per lane. Densitometries for western blots (A-B) depict the relative protein expression, normalized to actin and total Akt. Phosphorylated Akt increased significantly in doxorubicin-naïve (dox-naïve), compared to doxorubicin-resistant MCF7 cells (dox-res), at AKTi concentrations above 0.5 μM. *p<0.05. (C-D) Western blot analysis of Akt and downstream signaling in doxorubicin-naïve or doxorubicin-resistant MB231 (C) and MCF7 (D) human breast cancer cells, after 24 hrs exposure to A-443654 (MB231: 1 μM, MCF7 0.5 μM) in vitro. Densitometries for western blots (C-D) depict the relative protein expression, normalized to actin. Bars represent the mean protein expression in experiments performed in triplicate ± SEM.

Figure 3. Cytotoxicity of A-443654 and doxorubicin in doxorubicin-naïve and doxorubicin-resistant human breast cancer cell lines

In vitro cytotoxicity of doxorubicin (1 μM), Akt inhibitor A-443654 at IC30 concentration, or the combination, in doxorubicin-naïve or resistant MB231 (A) and MCF7 (B) cells, after 24 hrs drug exposure. WST-1 cell proliferation assay, absorbance read at optical density (OD) 450 nm, normalized to readings in control wells exposed to equivalent volumes of DMSO (doxorubicin stock solvent) and HPMC (dissolvent for A-443654). Bars depict the mean ± SEM.**p<0.01, ***p<0.001

Figure 4. The influence of A-443654 and doxorubicin on tumor growth in vivo

(A-B) Tumor growth of MB231 and MCF7 breast cancer in NOD/SCID mice, given doxorubicin (DOX) 1.25 mg/kg i.p. qW twice (red arrows), Akt inhibitor A-443654 (AKTi) 3.75 mg mg/kg BID 14 days (green lines) or the combination. AKTi treatment commenced either at the first (A) or at the second (B) doxorubicin injection. Tumor volume is displayed as the mean ± SEM for each group, relative to tumor volume on the day treatment started. *p<0.05, **p<0.01. (C-D) Western blots for PTEN and Akt-mTOR-S6K signaling in MB231 (C) and MCF7 (D) tumors, harvested the last day of A-443654 treatment. Whole cell lysate, 30 μg protein loaded per lane. The sample order on the blot pictures has been rearranged to enhance readability. Densitometries for western blots (C-D) depict the relative protein expression, normalized to actin. Phosphorylated Akt (p-Akt) and mTOR (p-mTOR) were normalized to actin and thereafter to total Akt and mTOR, respectively. Bars represent the mean protein expression for experiments performed in duplicate (D) or triplicate (C) ± SEM. *p<0.05

Figure 5. AKT1, PTEN and S6K gene expression in human breast cancers before and 24 hrs after epirubicin exposure

(A) Box plots of gene expression of PTEN, AKT1 and S6K normalized to RPLP2 in human breast cancer samples, before and 24 hrs after the first epirubicin dose, from patients in the dose dense trial. (B) Box plots from the same patient cohort as in (A), but depicted separately for estrogen receptor (ER) positive and ER negative breast cancers. *p<0.05

Figure 6. AKT1 and PTEN gene expression in human breast cancers before and after 16 weeks of doxorubicin treatment

(A) Box plots of gene expression of PTEN and AKT1 normalized to RPLP2 in human breast cancer samples, before and after 16 weeks of doxorubicin treatment, from patients in the doxorubicin trial. (B) Box plots from the same patient cohort as in (A), but depicted separately for estrogen receptor (ER) positive and negative breast cancers. *p<0.05