Background: Trastuzumab significantly improves survival in patients with HER2 positive breast cancer; however, de novo or acquired resistance ensues in the advanced setting. We have identified differentially expression genes in acquired trastuzumab resistance cell line models following long-term trastuzumab exposure and have demonstrated consistent overexpression of CXCR-4 at the mRNA and protein levels. The interaction between the cognate ligand CXCL-12 and its receptor CXCR-4 is known to promote malignant cell proliferation and angiogenesis via activation of Akt and MAPK pathways. CXCR-4 shows no to low expression in normal breast epithelium while its expression sequentially increases from precancerous lesions to carcinomas. CXCR-4 overexpression in human breast cancer is also associated with a worse outcome in small studies. We sought to explore the role of CXCR-4 expression on trastuzumab drug responsiveness.

Methods: Trastuzumab-resistant cell lines were generated by serial passaging of HER+ cells lines BT-474 and SKBR-3 for 1 year with continual exposure to 200 μg/ml of trastuzumab. For these resistant cell lines, termed BtRT and SkRT, CXCR-4 mRNA was quantified using qRT-PCR, and CXCR-4 protein expression was quantified by Western blot. Stable clones overexpressing CXCR-4 receptor were selected by transfecting CXCR-4 sequence-containing lentivirus into sensitive parental sensitive BT-474 and SKBR3 cells. Expression of CXCR-4 receptor was assessed by Western blot assay and by flow cytometry. MTT cell viability analysis was performed on these transfected cell lines to characterize trastuzumab dose responsiveness with parental cells as control.

Results: Trastuzumab-resistant cell line BtRT demonstrated a 100-fold increase in IC50 to trastuzumab treatment when compared to parental BT-474 cells and also exhibited a 3-fold further increase in CXCR-4 mRNA and a corresponding increase in CXCR-4 protein. CXCR-4-transfected sensitive BT474 cell lines showed a greater than 100 fold increase in the IC50 value on MTT assay after treatment with trastuzumab as compared to the parental cell line. Semi-quantitative analysis by Western-blot confirmed a 5-fold increase in CXCR-4 expression in CXCR-4-transfected cells as compared to parental cells. CXCR-4 expression in HER2+ human breast tumors and responsiveness in patients treated with neoadjuvant trastuzumab-based therapy will be presented.

Conclusion: CXCR-4 overexpression and activation of the CXCR-4/CXCL-12 pathway contributes to trastuzumab resistance in human breast cancer cell lines. CXCR-4 targeting may represent a therapeutic strategy to improve the efficacy of trastuzumab in HER2-positive breast cancer.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-04.