Abstract 1971: Inhibition of CXCR4 pathway augments trastuzumab sensitivity in HER2 positive breast cancer cells with intrinsic and acquired trastuzumab resistance

Abstract

The clinical benefit from trastuzumab (T) in HER2-positive breast cancers is limited by de novo or acquired resistance. By serial exposure of HER2+ breast cancer cell lines BT474 and SKBr3 to T at 200 µg/ml for 1 year and clonogenic selection, we have developed T-resistant cell lines and identified differentially expressing genes including consistent overexpression of CXCR4 at the mRNA and protein levels. CXCR4 is involved in breast cancer metastasis and proliferation and its overexpression in human breast cancer specimens is linked to poor prognosis. We sought to functionally validate CXCR4 as a resistance mechanism to T. CXCR4 mRNA was quantified using qRT-PCR, and CXCR4 protein expression was quantified by Western blotting. Stable clones overexpressing CXCR4 were selected by transfecting CXCR4 full length sequence-containing lentivirus into parental trastuzumab sensitive BT474 and SKBR3 cells. Stable CXCR4 knockdown clones were selected by transfecting three CXCR4 sequence specific shRNA containing lentivirus along with scrambled sequence as a control, into acquired T-resistant cell lines termed BT474R1 and SKBR3R1 and intrinsically T-resistant HCC-1419 cell line. Stable clones of BT474R1 with tetracycline controlled (tet-on) CXCR4 expression, were created using the pSLIK-hygro lentiviral system. CXCR4 expression was assessed by Western blot assay and by flow cytometry. MTT cell viability analysis was performed on these transfected cell lines to characterize T dose responsiveness with parental sensitive and T-resistant BT474R1 cells as control. BT474R cells demonstrated a 100-fold increase in IC50 to T treatment when compared to parental BT474 cells and also exhibited a 4-fold increase in CXCR4 mRNA and a corresponding increase in CXCR4 protein. Transfection of CXCR4 into sensitive BT474 and SkBR3 cell lines showed a greater than 100 fold increase in the IC50 value by MTT assay after treatment with T as compared to the parental cell lines. Semi-quantitative analysis by Western-blotting confirmed a 7 to 8 fold increase in CXCR4 expression in CXCR4-transfected cells as compared to parental cells. shRNA-mediated knockdown of CXCR4 in BT474R1 as well as primarily resistant HCC-1419 cell line showed a significantly improved responsiveness to trastuzumab with a 100 fold decrease in the IC50 value whereas a moderate response was seen in the SKBR3R1 cells. CXCR4 overexpression and activation of the CXCR4/CXCL-12 pathway contributes to trastuzumab resistance in human breast cancer cell lines. shRNA-mediated inhibition of the CXCR4 pathway in resistant cells significantly augments trastuzumab sensitivity. CXCR4 targeting may represent a therapeutic strategy to reverse de novo and acquired resistance and to improve the efficacy of trastuzumab in HER2-positive breast cancer. Citation Format: Arjun Mehta, Gloria Yang-Kolodji, Debu Tripathy. Inhibition of CXCR4 pathway augments trastuzumab sensitivity in HER2 positive breast cancer cells with intrinsic and acquired trastuzumab resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1971. doi:10.1158/1538-7445.AM2014-1971

Targeting CXCR4 abrogates resistance to trastuzumab by blocking cell cycle progression and synergizes with docetaxel in breast cancer treatment

“…In our previous studies, by establishing and using trastuzumab-resistant breast cancer cell models, we initially found upregulation of C-X-C motif chemokine receptor 4 (CXCR4), a G protein-coupled receptor of stromal cell-derived factor-1 (SDF-1; CXCL12) in trastuzumab-resistant breast cancer. Knockdown of CXCR4 with shRNA sensitized the cells to trastuzumab [18][19][20]. Consistent with our ndings, a very recent clinical study showed increased CXCR4 expression in trastuzumab-resistant breast cancer tissues and was associated with a higher risk of recurrence [21].…”

“…We previously showed upregulation of CXCR4 is involved as a driver of trastuzumab resistance in HER2+ breast cancer cells [18][19][20]. In the current study, we demonstrated that the increased CXCR4 expression is associated with cell cycle progression and reaches a peak in the G2/M phases.…”