Rac inhibition as a novel therapeutic strategy for EGFR/HER2 targeted therapy resistant breast cancer

Abstract

Background: Even though targeted therapies are available for cancers expressing oncogenic epidermal growth receptor (EGFR) and (or) human EGFR2 (HER2), acquired or intrinsic resistance often confounds therapy success. Common mechanisms of therapy resistance involve activating receptor point mutations and (or) upregulation of signaling downstream of EGFR/HER2 to Akt and (or) mitogen activated protein kinase (MAPK) pathways. However, additional pathways of resistance may exist thus, confounding successful therapy.

Methods: To determine novel mechanisms of EGFR/HER2 therapy resistance in breast cancer, gefitinib or lapatinib resistant variants were created from SKBR3 breast cancer cells. Syngenic therapy sensitive and resistant SKBR3 variants were characterized for mechanisms of resistance by mammosphere assays, viability assays, and western blotting for total and phospho proteins.

Results: Gefitinib and lapatinib treatments reduced mammosphere formation in the sensitive cells, but not in the therapy resistant variants, indicating enhanced mesenchymal and cancer stem cell-like characteristics in therapy resistant cells. The therapy resistant variants did not show significant changes in known therapy resistant pathways of AKT and MAPK activities downstream of EGFR/HER2. However, these cells exhibited elevated expression and activation of the small GTPase Rac, which is a pivotal intermediate of GFR signaling in EMT and metastasis. Therefore, the potential of the Rac inhibitors EHop-016 and MBQ-167 to overcome therapy resistance was tested, and found to inhibit viability and induce apoptosis of therapy resistant cells.

Conclusions: Rac inhibition may represent a viable strategy for treatment of EGFR/HER2 targeted therapy resistant breast cancer.

Keywords: Breast cancer; EHop-016; MBQ-167; Rac inhibitors; Therapy resistance; Tyrosine kinase inhibitors (TKIs).

Fig. 1

Viability of therapy sensitive and resistant variants in the presence of TKIs. a SKBR3 therapy sensitive cells, and variant resistant to 0.1 μM lapatinib, b SKBR3 therapy sensitive and variants resistant to 0.1 gefitinib or 0.5 μM gefitinib, were subjected to a MTT cell viability assay to determine the IC₅₀ by exposing the cells to different concentrations of TKIs gefitinib and lapatinib. % Cell viability in response to gefitinib or lapatinib is shown for the therapy sensitive and resistant variants. N = 4 ± SEM

Fig. 2

EGFR and HER2 expression and phosphorylation in therapy sensitive and resistant variants. SKBR3 therapy sensitive or resistant (Gef.R 0.1 μM, Gef. R 0.5 μM and Lap. R 0.1 μM) cells treated with gefitinib or lapatinib for 24 h were lysed and western blotted for total and active (phospho) EGFR and HER2. a Representative western blots for pEGFR/EGFR (left) and pHER2/HER2 (right), with actin as a loading control, for cells treated with gefitinib or lapatinib for 24 h. b Fold change in EGFR and HER2 expression and phosphorylation for the therapy sensitive SKBR3 cells from positive bands quantified using image J software. c Representative western blots for pEGFR/EGFR and pHER2/HER2 in therapy sensitive (SKBR3) or resistant (Gef R, LapR variants, maintained in the indicated concentrations of gefitinib or lapatinib. d Fold change in EGFR expression, e Fold change in HER2 expression, f Fold change in EGFR phosphorylation, g Fold change in HER2 phosphorylation, N = 3 ± SEM. **** = p ≤ 0.001, *** = p ≤ 0.005, ** = p ≤ 0.01, * = p ≤ 0.05

Fig. 3

Apoptosis in therapy sensitive and resistant variants Apoptosis in therapy sensitive and resistant SKBR3 cell variants was detected by a Caspase-Glo 3/7 Assay. a Fold change in Caspase 3/7 activity in the therapy sensitive SKBR3 cell line following Gef or Lap treatment for 48 h compared to the vehicle controls. b Fold change in caspase 3/7 activity in the therapy resistant cell lines following treatment compared to non-treated cells. N = 3 ± SEM, * = p ≤ 0.05, *** = p ≤ 0.005

Fig. 4

Stem cell-like characteristics in therapy resistant variants. Mammosphere formation efficiency (MFE) of SKBR3 therapy sensitive and resistant variants was calculated by dividing the number of mammospheres formed by the number of cells seeded per well and multiplied by 100 for percentage. a Representative micrographs of mammosphere forming units. Fold changes of percentage are shown in: b MFE in gefitinib and lapatinib treated therapy sensitive cells relative to vehicle treated cells. c,d MFE in therapy resistant cells treated with (c) gefitinib or (d) lapatinib, relative to vehicle controls. e MFE of therapy resistant variants relative to therapy sensitive cells with no treatment. f Representative western blots of cancer stem cell markers integrin β3, CD133, and Nanog in SKBR3 therapy sensitive and resistant variants. N = 3 ± SEM,* = p ≤ 0.05 and,**** = p ≤ 0.001

Fig. 5

Akt and MAPK activities in therapy resistant variants. SKBR3 gefitinib and lapatinib sensitive and resistant cells were lysed and subjected to western blotting for expression and activity of a Akt/p-Akt^{S473, T308}, b p44/42 MAPK/p-MAPK^{T202, Y204} using total or phospho-specific antibodies to the active sites. c, d Average integrated density of p-Akt/Akt (c) or p-P44/42 MAPK/P44/42 MAPK (d), as quantified from Image J analysis of positive bands from western blots. N = 3

Fig. 6

Inhibition of upregulated Rac in therapy resistant variants. a Rac activation was determined by a pulldown assay using the p21-binding domain of p21-activated kinase (PAK) from lysates of therapy sensitive or resistant SKBR3 cells. Representative western blots for active Rac.GTP, total Rac, and actin are shown. b SKBR3 gefitinb and lapatinib sensitive and resistant cells were subjected to a MTT assay for cell viability following 24 h in the Rac inhibitor EHop-016 at 0, 5, or 10 μM. c SKBR3 lapatinib resistant cells were subjected to a MTT assay for cell viability following 24 h in vehicle (0), 0.1 μM lapatinib, 250 nM MBQ-167, or a combination of 0.1 μM lapatinib and 250 nM MBQ-167. d SKBR3 lapatinib resistant cells were subjected to a caspase3/7 assay for apoptosis following 24 h in vehicle (0), 0.1 μM lapatinib, 250 nM MBQ-167, or a combination of 0.1 μM lapatinib and 250 nM MBQ-167. e MDA-MB-435 laptinib resistant HER2+ cells were treated with 0.1 μM lapatinib, 250 nM MBQ-167, or a combination of 0.1 μM lapatinib and 250 nM MBQ-167 for 48 h and cell viability quantified by a MTT assay; fold change in cell viability relative to vehicle is shown. f MDA-MB-435 trastuzumab resistant HER2+ cells were treated with 5 or 10 μg/ml trastuzumab, 250 nM MBQ-167, or a combination of 5 μg/ml trastuzumab and 250 nM MBQ-167 for 48 h and cell viability quantified by a MTT assay; fold change in cell viability relative to vehicle is shown. N = 3 ± SEM * = p ≤ 0.05, ** = p ≤ 0.01**** = p ≤ 0.001