Feedback upregulation of HER3 (ErbB3) expression and activity attenuates antitumor effect of PI3K inhibitors

Abstract

We examined the effects of an inhibitor of PI3K, XL147, against human breast cancer cell lines with constitutive PI3K activation. Treatment with XL147 resulted in dose-dependent inhibition of cell growth and levels of pAKT and pS6, signal transducers in the PI3K/AKT/TOR pathway. In HER2-overexpressing cells, inhibition of PI3K was followed by up-regulation of expression and phosphorylation of multiple receptor tyrosine kinases, including HER3. Knockdown of FoxO1 and FoxO3a transcription factors suppressed the induction of HER3, InsR, IGF1R, and FGFR2 mRNAs upon inhibition of PI3K. In HER2(+) cells, knockdown of HER3 with siRNA or cotreatment with the HER2 inhibitors trastuzumab or lapatinib enhanced XL147-induced cell death and inhibition of pAKT and pS6. Trastuzumab and lapatinib each synergized with XL147 for inhibition of pAKT and growth of established BT474 xenografts. These data suggest that PI3K antagonists will inhibit AKT and relieve suppression of receptor tyrosine kinase expression and their activity. Relief of this feedback limits the sustained inhibition of the PI3K/AKT pathway and attenuates the response to these agents. As a result, PI3K pathway inhibitors may have limited clinical activity overall if used as single agents. In patients with HER2-overexpressing breast cancer, PI3K inhibitors should be used in combination with HER2/HER3 antagonists.

Fig. 1.

XL147 inhibits cell growth in a dose-dependent manner. (A) Breast cancer cell lines with different lesions in the PI3K pathway (as noted within parentheses) were treated with 0 to 20 μM XL147 and counted on the days indicated. Each bar represents the mean ± SE of six replicates (^a,b,cP < 0.05 vs. 0 μM XL147, paired t test). (B) Cells were cultured in Matrigel with or without 0 to 20 μM XL147 and photographed (magnification 10×) on indicated days.

Fig. 2.

XL147-mediated inhibition of PI3K associates with induction of HER3 and pHER3. Cells lines were harvested after overnight treatment with 0 to 20 μM XL147 in serum-free medium followed by immunoblot analysis with indicated antibodies.

Fig. 3.

XL147-induced up-regulation of HER3 transcription is dependent on FoxO. (A) BT474 cells were treated with 6 μM XL147 for the indicated times before RNA isolation and real-time qPCR with HER3-specific primers. (B) BT474 cells were treated with 2 μM 5J8, 20 μM LY294002, and 50 nM rapamycin for 10 h before RNA isolation and qPCR for HER3. (C) MDA453 and SKBR3 cells were treated with 6 μM XL147 for as long as 48 h before RNA isolation and qPCR for HER3. For A–C, each bar represents the mean ± SE of three wells. (D) BT474 and MDA453 cells were treated with DMSO, 6 μM XL147, or 2 μM 5J8 for 4 h. Nuclear and cytoplasmic extracts were subjected to immunoblot analysis with FoxO1 and FoxO3a antibodies. Loading controls for nuclear extracts: HDAC3; for cytoplasmic extracts: RhoA (BT474) and MEK1/2 (MDA453). Arrow indicates the FoxO3a-specific band. (E and F) BT474, MDA453, and SKBR3 cells were transfected with control or FoxO1- and FoxO3a-specific siRNA duplexes followed by treatment with XL147 for 6 h before harvesting, RNA isolation and qPCR for HER3 (E) or FoxO1 and FoxO3a (F). Each bar represents the mean ± SE of triplicate wells. (G) BT474 cells were transfected with control or FoxO1 and FoxO3a siRNA and treated with XL147 for 6 h. Cell lysates were used for hybridization with pRTK arrays.

Fig. 4.

Knockdown of compensatory feedback to HER3 sensitizes to PI3K inhibitor. (A) BT474 cells were treated with 6 μM XL147 for as long as 72 h and subjected to immunoblot analysis. (B) Immunoblot of lysates from BT474 cells treated with 6 or 20 μM XL147 for 0 to 48 h. (C and D) BT474 cells were treated with 0 to 20 μM XL147 for 24 h and lysed. Cell lysates (0.5 mg) were subjected to immunoprecipitation with a p85 antibody followed by immunoblot for p85 and pTyr (C) or p85 and HER3 (D). Arrows in C indicate p85-associated pTyr bands. (E and F) BT474 cells were transfected with control or HER3-specific siRNA duplexes and treated with 6 μM XL147 for 24 h. Cell lysates (0.5 mg) were subjected to immunoprecipitation with a p85 antibody followed by immunoblot with HER3 (E), pHER3^Y1197 (F), and p85 (E and F) antibodies. (G and H) BT474 cells were transfected with HER3-specific siRNA and treated with DMSO or 2 μM XL147. Cells were harvested for counting (G) or crystal violet staining (H) on day 6. In G, each bar represents the mean ± SE of six replicates (*P < 0.05, paired t test).

Fig. 5.

Recovery of HER3 phosphorylation depends on HER2 and is limited by HER2 inhibitors. (A and B) BT474 cells were treated with 2 μM XL147 alone or in combination with 0.1 μM lapatinib (Lap) (A) or 10 μg/mL trastuzumab (Tras) (B) and counted after 6 d (A) or 8 d (B). Each bar represents mean ± SE of six replicates. (C) Immunoblot of biomarkers of apoptosis and G₁–S phase transition with lysates from BT474 cells treated with the indicated inhibitors for 72 h. (D) Real-time qPCR for HER3 mRNA in cells treated with XL147 (6 μM), Lap (1 μM), Tras (10 μg/mL), or the indicated combinations for 10 h. Each bar represents mean ± SE of triplicate wells. (E) Total HER3 and pHER3 immunoblot of lysates from BT474 cells treated over a time course (0–24 h) with the indicated inhibitors at similar concentrations as in D.

Fig. 6.

Combined inhibition of HER2 and PI3K is synergistic in vivo. (A) Tumor growth curve from nude mice transplanted with BT474 cells and treated with vehicle (Ctrl), XL147, lapatinib, trastuzumab, or the indicated drug combinations for 28 d. Each data point represents the mean tumor volume (in mm³) ± SE of eight mice per treatment (CR, complete response to treatment; *P < 0.05 for trastuzumab vs. trastuzumab plus XL147 and lapatinib vs. lapatinib plus XL147, two-way ANOVA). (B) Histoscore (H-score) analysis of immunohistochemical sections. Each bar represents mean ± SE (*P < 0.05 vs. control, unpaired t test).

Fig. 7.

Inhibition of PI3K induces RTKs other than HER3. (A) BT474 cells were transfected with HER3 siRNA duplexes and treated with XL147 for 24 h. Cell lysates (0.5 mg) were precipitated with a p85 antibody, followed by immunoblot analysis with antibodies indicated to the right. Cell lysates from BT474 cells treated with 1 μM lapatinib for 6 h were used as positive controls (lanes 1 and 2). (B and C) BT474 cells were treated with 6 μM XL147 over a time course to 24 h as indicated. Cell lysates were prepared and 0.2 mg (lower sensitivity; B) or 0.5 mg (higher sensitivity; C) of total protein were applied to pRTK arrays. Arrows indicate RTKs whose phosphorylation was up-regulated on treatment with the PI3K inhibitor. (D) Real-time qPCR analysis of the indicated RTKs in RNA collected from cells treated with DMSO or 10 μM XL147 for 6 h. (E) Analysis of IGF1R, InsR, and FGFR2 mRNA by qPCR in RNA extracted from BT474 cells transfected with FoxO1 and FoxO3a siRNA and then treated with 10 μM XL147 for 6 h. Each bar represents the mean ± SE of triplicate wells.