Kinase inhibitors of HER2/AKT pathway induce ERK phosphorylation via a FOXO-dependent feedback loop

Abstract

Inhibitors of the HER2/PI3K/AKT pathway are being developed, and shown promise in clinical trials for various types of cancers. However, development of drug resistance is a challenging problem for therapy. Elucidating various adaptive pathways leading to resistance or reduced sensitivity to drugs targeting the HER2/PI3K/AKT pathway may provide new insights into countering the resistance. Epidermal growth factor receptor (EGFR, aka HER1), which can dimerize with HER2, can activate a cascade consisting of Ras/RAF/MEK/ERK, promoting tumorigenesis. Lapatinib inhibits the kinase activity of both HER1 and HER2. In the current study, we found that repeated treatment of HER2+ breast cancer cells with HER1/2 inhibitor Lapatinib led to increased phosphorylation of RAF, MEK, and ERK, while suppressing HER1 phosphorylation and reduced the active form of Ras, indicating existence of factor(s) activating RAF/MEK/ERK by bypassing RAS activation. Notably, the Lapatinib treatment-induced phosphorylation of ERK was dependent on FOXO transcription factors, which are also activated by Lapatinib-mediated suppression of AKT. Moreover, the Lapatinib-induced phosphorylation of RAF and ERK is inhibited by a pan-PKC inhibitor. Furthermore, the Lapatinib induced increased ERK phosphorylation is correlated with increased stability of c-Myc, which is known to be stabilized by ERK-mediated phosphorylation. Together, these results suggest that chronic inhibition of the HER1/2 by Lapatinib triggers a feedback loop to activate RAF/MEK/ERK pathway, in a FOXO dependent but Ras-independent manner.

Keywords: ERK; HER2; Lapatinib; breast cancer.

Figure 1

Lapatinib treatment decreases EGFR phosphorylation but increases RAF, MEK and ERK phosphorylation. A: BT474 cells were treated daily with DMSO or 200 nM Lapatinib for four days, collected 18 hrs after last treatment for Western blot. B: BT474 cells were treated with either DMSO or 200 nM Lapatinib for the indicated times on first day, or treated daily with Lapatinib for four days and collected after the indicated hr after the last treatment in the last day, for Western blot, with indicated antibodies.

Figure 2

Lapatinib-induced ERK phosphorylation is independent of Ras activation. (A) BT474 cells were treated with either DMSO or Lapatinib daily for four days, collected 18 hrs after last treatment, or treated with 200 nM EGF for 30 min, and active Ras was assayed as described in materials and methods. (B) BT474 cells were treated with DMSO, 200 nM Lapatinib, 200 nM AZD6244 or a combination of 200 nM Lapatinib and 200 nM AZD6244 for 4 days, cells were collected 18 hours after last treatment and Western blot was performed (C) BT474 cells were treated with either DMSO or 200 nM MK2206 for four days, and collected 18 hrs after last treatment for Western blot. (D) The cells were treated with either DMSO or 200 nM MK2206 daily for four days, collected 18 hr after last treatment and active Ras assay was performed as described in (A).

Figure 3

FOXO knockdown reduces Lapatinib-induced ERK phosphorylation. BT474 cells were transduced with either scramble or FOXO1 and 3 shRNAs, followed by treatment with either DMSO or Lapatinib for four days, collected 18 hr after the last treatment for Western blot.

Figure 4

IRS2 knockdown does not affect Lapatinib-induced ERK phosphorylation. A: BT474 cells were transduced with either scramble or shRNAs targeting FOXO1 and 3, followed by treatment with Lapatinib daily for four days. The cells were collected 18 hr after the last treatment for Western blotting. B: BT474 cells were treated with DMSO or Lapatinib for three days, collcted 18 hrs after the last treatment for ChIP assay using primers for the IRS2 promoter region. C: BT474 cells were transduced with either scramble or shRNA targeting IRS2, and treated with either DMSO or lapatinib for four days, and collected 18 hrs after the last treatment for Western blotting with the indicated antibodies.

Figure 5

A pan-PKC inhibitor decreases Lapatinib-induced phosphorylation of RAF. BT474 cells were treated with either DMSO, Lapatinib, G06983 or combination of Lapatinib and G06983 daily for four days, and collected 18 hrs after the last treatment for Western blotting with the indicated antibody.

Figure 6

Lapatinib treatment increases stability of c-Myc protein. A: BT474 cells were treated daily with either DMSO or Lapatinib for 4 days and collected 18 hrs after the last treatment for Western blotting. B: BT474 cells were treated daily with either DMSO or Lapatinib for four days. Cycloheximide (20 μg/ml) was added and cells were collected at 0, 30, 60 and 90 min after treatment for Western blotting with the indicated antibodies. C: c-Myc protein was quantitated using Image J and plotted as % protein relative to 0 min. D: BT474 cells were treated with FOXOi without or with daily Lapatinib treatment for four days, followed by treatment with cycloheximide for 0, 30, 60 and 90 min. Cells were collected for Western blotting.

Figure 7

A working model for Lapatinib/AKTi-induced ERK phosphorylation and incresed c-Myc stability via a FOXO-mediated and a kinase dependent feedback loop.