PI3K inhibition results in enhanced HER signaling and acquired ERK dependency in HER2-overexpressing breast cancer

Abstract

There is a strong rationale to therapeutically target the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway in breast cancer since it is highly deregulated in this disease and it also mediates resistance to anti-HER2 therapies. However, initial studies with rapalogs, allosteric inhibitors of mTORC1, have resulted in limited clinical efficacy probably due to the release of a negative regulatory feedback loop that triggers AKT and ERK signaling. Since activation of AKT occurs via PI3K, we decided to explore whether PI3K inhibitors prevent the activation of these compensatory pathways. Using HER2-overexpressing breast cancer cells as a model, we observed that PI3K inhibitors abolished AKT activation. However, PI3K inhibition resulted in a compensatory activation of the ERK signaling pathway. This enhanced ERK signaling occurred as a result of activation of HER family receptors as evidenced by induction of HER receptors dimerization and phosphorylation, increased expression of HER3 and binding of adaptor molecules to HER2 and HER3. The activation of ERK was prevented with either MEK inhibitors or anti-HER2 monoclonal antibodies and tyrosine kinase inhibitors. Combined administration of PI3K inhibitors with either HER2 or MEK inhibitors resulted in decreased proliferation, enhanced cell death and superior anti-tumor activity compared with single agent PI3K inhibitors. Our findings indicate that PI3K inhibition in HER2-overexpressing breast cancer activates a new compensatory pathway that results in ERK dependency. Combined anti-MEK or anti-HER2 therapy with PI3K inhibitors may be required in order to achieve optimal efficacy in HER2-overexpressing breast cancer. This approach warrants clinical evaluation.

Figure 1

PI3K-pathway inhibition induces ERK phosphorylation in HER2-overexpressing cells. (a) Immunoblots of BT474 and MCF7-HER2 treated with BEZ235 (0–500 n) for 24 h. (b) Immunoblots of MCF7-HER2 cells treated for 24 h with different PI3K-pathway inhibitors at the indicated concentrations (all in n). (c) Immunoblots of p110-α knockdown MCF7-HER2 cells. SCR, scramble control; p110-α, siRNA for p110-α. Lapatinib treatment for 24 h. (d) Immunohistochemistry (IHC) of BT474-trastuzumab resistant (BT474-Tr) xenografts treated with BEZ235 (40 mg/kg, 4 × QD) with the indicated phospho-proteins. IHC pictures are representative of the 6-h time point.

Figure 2

PI3K-pathway inhibition induces HER receptors expression and phosphorylation in HER2-overexpressing cells. (a) Phospho-RTK Array of cell lysates from BT474 cells treated with either DMSO (Control) or 500 n BEZ235 (BEZ500) for 24 h. The array contains 42 duplicated spots for specific P-RTKs as well as positive and negative controls. (b) Immunoblots of protein lysates from BT474 and MCF7-HER2 cells treated with either BEZ235 alone (0–500 n) or the combination of BEZ235 (0–500 n) and the tyrosine kinase inhibitor lapatinib (LAP, 500 n) for 24 h. (c) EGFR, HER2 and HER3 mRNA levels (related to β actin, ACTB) measured by qRT–PCR. BT474 cells were treated with 500 n BEZ235 for 24 h. *P<0.05; **P<0.01.

Figure 3

BEZ235 increases HER2/HER3 heterodimerization and receptor signaling. (a) Immunoblots of lysates from BT474 and MCF7-HER2 cells treated with DMSO (c) or BEZ235 (b, 500 n) for 24 h and subjected to membrane cross-link (Xlink). Cross-linked protein lysates were immunoprecipitated with (IP) and immunoblotted (IB) against the indicated antibodies. Whole-cell lysate (WCL) serve as input controls. (b) Immunoblots of lysates from MCF7-HER2 cells treated with BEZ235 (b, 0–500 n), lapatinib (L, 500 n) or the combination (B+L, both at 500 n) for 24 h. Total lysates were immunoprecipitated with (IP) and immunoblotted (IB) against the indicated antibodies.

Figure 4

BEZ235-induced P-ERK pathway is prevented with agents inhibiting HER2 and MEK1/2 activity. (a) Immunoblots of lysates from BT474 and MCF7-HER2 cells treated with DMSO (c), BEZ235 (B, 500 n) and lapatinib (L, 500 n) or the combination of both inhibitors for 24 h. (b) Immunoblots of lysates from BT474 and MCF7-HER2 cells treated with DMSO (c), trastuzumab (T, 50 n, 72 h) and BEZ235 (B, 100 n, 24 h) or the combination of both. (c) Immunoblots of lysates from BT474 and MCF7-HER2 cells treated with AZD6244 and BEZ235 used at the indicated concentrations for 24 h. (d) Immunoblots of MCF7-HER2 cells treated with DMSO (c), BEZ235 (B, 500 n) and lapatinib (L, 500 n) as well as with erlotinib (E, 500 n) and the indicated combinations for 24 h. All protein lysates were immunoblotted with the indicated antibodies.

Figure 5

The combination of BEZ235 with either anti-HER2 agents or an MEK inhibitor reduces cell proliferation and induces apoptosis. (a) Cell proliferation assay of BT474, SKBR3 and BT474-Tr cells treated with BEZ235 (B, 10 n), lapatinib (L, 20 n), trastuzumab (T, 1 n), AZD6244 (A, 2 μ) or the indicated combinations for 15 days. Cell proliferation was quantified by crystal violet. The fold difference of viable cells is shown relative to the viable cells treated with BEZ235. In order to better visualize the differences between BEZ235-treated cells as single agent or in combination with the aforementioned agents, proliferation of control, trastuzumab and AZD6244-treated cells has been omitted. (b) Cell death expressed as sub-G0/G1 of BT474, SKBR3 and BT474-Tr cells treated with BEZ235 (B, 100 n), lapatinib (L, 100 n), trastuzumab (T, 50 n), AZD6244 (A, 2 μ) or the indicated combinations for 72 h. The subG0/G1 population was quantified by FACS analysis and related to the amount of apoptotic cells upon BEZ235 treatment. Error bars indicate SE of two (a) or three (b) independent experiments. *P<0.05.

Figure 6

Combined inhibition of PI3K/mTOR and HER2 or PI3K/mTOR and MEK results in greater inhibition of tumor growth and prevents ERK transactivation in vivo. (a) Left panel: tumor growth of BT474-Tr-derived xenografts treated with placebo (c), BEZ235 (B, 20 mg/kg, QD), trastuzumab (T, 20 mg/kg, BIW) or the combination of both. Right panel: tumor growth of BT474-Tr xenografts treated with placebo (c), BEZ235 (B, 25 mg/kg, QD) as single agent or combined with AZD6244 (A, 8 mg/kg, QD). *P<0.05. Error bars indicate SE of at least 10 mice. (b) Immunofluorescence of BT474-Tr xenografts treated with placebo (c), BEZ235 (40 mg/kg, 4 × QD), lapatinib (120 mg/kg, 3 × QD), trastuzumab (20 mg/kg, days 1 and 3), AZD6244 (15 mg/kg, 4 × QD) or in combination as indicated. Red indicates primary antibodies; blue indicates DAPI staining. White bars, 200 μm. (c) Images were quantified with AQUA™. Red indicates nuclear stain; blue indicates cytoplasmic stain. *P<0.05; **P<0.01. (d) Cartoon outlining the PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways. Inhibition of PI3K/AKT causes nuclear relocalization of the FoxO3a transcription factor and increase of HER3 mRNA and protein. The augmented HER3 is able to heterodimerize with HER2, therefore enhancing receptor phosphorylation and signaling to the PI3K and ERK pathways.