β1 integrin mediates an alternative survival pathway in breast cancer cells resistant to lapatinib

Abstract

Introduction: The overexpression of human epidermal growth factor receptor (HER)-2 in 20% of human breast cancers and its association with aggressive growth has led to widespread use of HER2-targeted therapies, such as trastuzumab (T) and lapatinib (L). Despite the success of these drugs, their efficacy is limited in patients whose tumors demonstrate de novo or acquired resistance to treatment. The β1 integrin resides on the membrane of the breast cancer cell, activating several elements of breast tumor progression including proliferation and survival.

Methods: We developed a panel of HER2-overexpressing cell lines resistant to L, T, and the potent LT combination through long-term exposure and validated these models in 3D culture. Parental and L/T/LT-resistant cells were subject to HER2 and β1 integrin inhibitors in 3D and monitored for 12 days, followed by quantification of colony number. Parallel experiments were conducted where cells were either stained for Ki-67 and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or harvested for protein and analyzed by immunoblot. Results were subjected to statistical testing using analysis of variance and linear contrasts, followed by adjustment with the Sidak method.

Results: Using multiple cell lines including BT474 and HCC1954, we reveal that in L and LT resistance, where phosphorylation of EGFR/HER1, HER2, and HER3 are strongly inhibited, kinases downstream of β1 integrin--including focal adhesion kinase (FAK) and Src--are up-regulated. Blockade of β1 by the antibody AIIB2 abrogates this up-regulation and functionally achieves significant growth inhibition of L and LT resistant cells in 3D, without dramatically affecting the parental cells. SiRNA against β1 as well as pharmacologic inhibition of FAK achieve the same growth inhibitory effect. In contrast, trastuzumab-resistant cells, which retain high levels of phosphorylated EGFR/HER1, HER2, and HER3, are only modestly growth-inhibited by AIIB2.

Conclusions: Our data suggest that HER2 activity, which is suppressed in resistance involving L but not T alone, dictates whether β1 mediates an alternative pathway driving resistance. Our findings justify clinical studies investigating the inhibition of β1 or its downstream signaling moieties as strategies to overcome acquired L and LT resistance.

Figure 1

Phosphorylated levels of β1 downstream kinases are increased upon acquisition of resistance to lapatinib (L). (A) Parental (P) BT474 and (B) HCC1954 cells resistant to lapatinib (LRes), trastuzumab (TRes), and combination (LTRes) treatment strategies were developed by long-term exposure in 2D. Protein extracts were probed for β1, pHER2, pFAK, and pSrc, as well as totals.

Figure 2

β1 blockade overcomes lapatinib resistance in BT474 cells in 3D culture. (A) Parental and LRes cells were plated on lrECM and treated ± lapatinib. (B) The β1 inhibitory antibody AIIB2--or IgG control--was applied to parental and LRes cells on Day 0 of plating on lrECM and allowed to grow for 12 days. (C) Parental and LRes cells were plated in lrECM with appropriate inhibitors, allowed to grow for five days, stained with Ki67 antigen or TUNEL labeling, and imaged. (D) 3D cultures of BT474 parental and LRes cells treated with AIIB2 or IgG were allowed to propagate for five days, then harvested for protein and immunoblotting. Densitometry measurements were normalized to total levels--except β1, which was normalized to β-actin--and are representative of three independent experiments. (E) The FAK inhibitor PF 573228 was applied to parental and LRes cells on Day 0 of plating on lrECM and cultures were allowed to grow for 12 days.

Figure 3

HER2 and the β1 pathway play alternate roles in resistance to lapatinib-containing regimens, in comparison to trastuzumab. (A) Parental, LRes, and TRes cells were plated on lrECM in the presence of lapatinib and/or trastuzumab and assayed for response. (B) 3D cultures of parental, LRes, and TRes BT474 cells were harvested for protein and probed for phosphorylated and total HER receptors.

Figure 4

β1 inhibition impedes colony grow of BT474 LRes and LTRes cells, but not parental or TRes. (A) Parental, LRes, LTRes, and TRes cells were plated in lrECM, subjected to HER2 and/or β1 inhibitors on Day 0, and propagated for 10 to 12 days. (B) The Hs_ITGB1_5 siRNA was validated both in 2D and 3D (top), transfected at 40 nMsi into parental, LRes, LTRes, and TRes cells, which were then grown on lrECM for 10 days (bottom).

Figure 5

β1 inhibition overcomes resistance to potent LT therapy in HCC1954 cells. (A) HCC1954 parental and LTRes were plated on lrECM in the presence of LT and assayed for growth response. (B) Cells were plated on lrECM, subjected to either AIIB2 or IgG control, and imaged after 12 days. (C) Cultures were grown in the presence of appropriate inhibitors for five days and immunostained with Ki67 or TUNEL reagent as before.

Figure 6

AIIB2 neutralizes the upregulated pFAK and pSrc expression found in LTRes cells. (A) Parental and LTRes cells were grown on lrECM with AIIB2 or IgG, propagated for five days, and probed for markers. (B) The FAK inhibitor was applied to parental and LTRes cells on Day 0 of plating on lrECM and allowed to grow for 12 days. (C) 3D cultures were harvested for protein and probed for phospho- and total EGFR, HER2, and HER3.

Figure 7

β1 is critical for the HCC1954 LTRes phenotype, but not parental or TRes. (A) Parental, LTRes, and TRes cells were plated on lrECM, subjected to AIIB2, and allowed to grow for 12 days. (B) Cells were subjected to two consecutive rounds of siRNA transfection as before, validated both before and during the experiment (top), and plated in lrECM (bottom).