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01.08.2011 | Research article | Ausgabe 4/2011 Open Access

Breast Cancer Research 4/2011

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

Zeitschrift:
Breast Cancer Research > Ausgabe 4/2011
Autoren:
Catherine Huang, Catherine C Park, Susan G Hilsenbeck, Robin Ward, Mothaffar F Rimawi, Yen-chao Wang, Jiang Shou, Mina J Bissell, C Kent Osborne, Rachel Schiff
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​bcr2936) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CH conducted the molecular studies presented in this manuscript, including 3D culturing, application of pharmacological and genetic approaches to inhibit β1, immunostaining, protein extraction, and immunoblotting. CP, MR, and MJB contributed substantially to the conception and design of the data. SH performed all statistical analyses necessary for each molecular study. RW and YW developed the lapatinib, trastuzumab, and LT resistant cell lines through long term exposure of parental cells to each inhibitor or set of inhibitors. CKO and RS conceived of the study and its design and implementation. All authors read and approved the final manuscript.

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.
Zusatzmaterial
Additional file 1: A second siRNA sequence applied to BT474 and HCC1954 cells in 3D lrECM confirms that LRes and LTRes cells depend more critically on β1 than their parental counterparts. Double, consecutive rounds of siRNA transfection at 40 nM were executed and cells plated directly onto lrECM for 10 days, followed by quantification. (TIFF 303 KB)
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Additional file 2: Phosphorylated levels of the β1 downstream kinases FAK and Src are increased in additional HER2-overexpressing cell line models upon acquisition of resistance to lapatinib (L)-containing HER-targeted therapies. (A) Parental (P) AU565 and (B) HCC202 cells resistant to lapatinib (LRes) 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. (TIFF 98 KB)
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Additional file 3: β1 blockade overcomes resistance to lapatinib-containing regimens in AU565 and HCC202 cells and abrogates upregulated pFAK and pSrc expression. (A) and (C) Cells were propagated in 3D lrECM and treated with respective inhibitors and/or AIIB2. Statistical analysis was conducted as in Figure 2. ( B and D, left) 3D extracts of AU565 cells exhibit upregulated protein expression of β1, pFAK, and pSrc upon acquisition of resistance to lapatinib. These effects are neutralized upon application of the β1 inhibitory antibody AIIB2. Expression of phosphorylated levels of MAPK and AKT are decreased in LRes cells in comparison to their parental counterparts. ( B and D, right) The HER receptor layer is effectively inhibited in L- and LT-Res cells but remains active in both parental and TRes cells. (TIFF 265 KB)
Additional file 5: Doubling the dose of lapatinib in cells resistant to lapatinib-containing regimens does not dramatically affect growth. BT474 LRes and HCC1954 LTRes cells were first primed in 2D with 2 μM lapatinib (twice the usual dose) for five days. Cells were then plated onto lrECM, propagated for 12 days, and quantified. (TIFF 219 KB)
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Authors’ original file for figure 1
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