p-rpS6 is a robust post-treatment indicator of HER2 pathway-targeted therapy resistance.

Background: Despite the fact that trastuzumab alongwithotherHER2-targetingdrugshavesignificantlyimprovedthe survival of patients with HER2 overexpression breast cancers (HER2+BC), resistance to trastuzumab is a clinical challenge in HER2+BC. Discerning actionable mechanisms of resistance to trastuzumab remains an important unmet need. WepreviouslyreporteddysregulationofCXCR4involvedintrastuzumab-resistance,butitscausal roleandthe associated mechanismsremainunknown. Methods We established trastuzumab-resistant (TR) human breast cancer HER2+ cell lines by continuously exposing cells to trastuzumab (20 μg/ml) for at least 6 months. CXCR4 expressionwasassessed in TR cells or parental cells with Westernblot.QuantitativedensitometricanalysisofthedensitywasperformedwithAlphaViewSAsoftware.Relevant cell phenotypes were measured, including mammosphere formation, in vitro antibody-dependent cellular cytotoxicity (ADCC) assay, and cell invasion induced by 10% fetal bovine serum with or without 100ng/ml stromal cell-derived factor-1α (SDF-1α, CXCL12), the ligand of CXCR4. ANOVAwasusedtotest differencesbetweenmorethantwogroups,whilethedifferencesbetweentwogroupswereassessedbasedon pairedt-test. Results: To better capture the heterogeneity of HER2+BC, we chose two trastuzumab-sensitive cell lines, BT474 (HER2+/HR+) and SKBR3 (HER2+/HR-) and an intrinsically trastuzumab-resistant cell line, HCC1419 (HER2+/HR-). We found much higher CXCR4 expression levels in cells with intrinsically trastuzumab-resistant cells compared to trastuzumab-sensitive cells. Upregulation of CXCR4 expression was found in each of the acquired TR cell lines compared to their parental cells. Dysregulation of CXCR4 significantly enhanced mammosphere formation and cell invasion (P < 0.001, respectively). SDF-1α induced cell invasion and clumping. Down-regulation of CXCR4 with shRNA significantly increased trastuzumab induced–antibody-dependent cellular cytotoxicity (2.17 folds of control cells, P < 0.01). Targeting CXCR4 with its approved inhibitor AMD3100 significantly decreased mammosphere formation and invasion of HER2+BC with TR (P < 0.01; P < 0.0001 respectively). Conclusion: Our results suggest that the SDF-1-CXCR4 axis plays a critical role in resistance to trastuzumab. Targeting CXCR4 signaling may lead to novel combinational therapies to overcome intrinsic or acquired resistance to trastuzumab in advanced HER2+BC, including postulated effects of trastuzumab on signal transduction, differentiation and immune activation. Citation Format: Liu S, Xie SM, Yang-Kolodji G, Tripathy D. Targeting the tumor microenvironment by CXCR4 inhibition to abrogate trastuzumab resistance in HER2-positive breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-03-04.

Background Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. Methods Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4 expression. Three-dimensional co-culture (tumor cells/breast cancer-associated fibroblasts/human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effects of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. Results Using a panel of cell lines and patient breast cancer samples, we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using a panel of trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. Conclusions Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.

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

Background: Although trastuzumab and other HER2-targeted therapies have significantly improved survival in patients with HER2 overexpressed or amplified (HER2+) breast cancer, a significant proportion of patients do not respond or eventually develop clinical resistance. Strategies to reverse trastuzumab resistance remain a high clinical priority. We were the first to report the role of CXCR4 in trastuzumab resistance. The present study aims to explore the therapeutic potential of targeting CXCR4 and better understand the associated mechanisms. Methods: Immunofluorescent staining, confocal microscopy analysis, and immunoblotting were used to analyze CXCR4 expression. BrdU incorporation assays and flow cytometry were used to analyze dynamic CXCR4expression. Three-dimensional co-culture (tumor cells/ breast cancer-associated fibroblasts / human peripheral blood mononuclear cells) or antibody-dependent cellular cytotoxicity assay was used to mimic human tumor microenvironment, which is necessary for testing therapeutic effect of CXCR4 inhibitor or trastuzumab. The FDA-approved CXCR4 antagonist AMD3100, trastuzumab, and docetaxel chemotherapy were used to evaluate therapeutic efficacy in vitro and in vivo. Reverse phase protein array and immunoblotting were used to discern the associated molecular mechanisms. Results: Using multiple cell lines and patient breast cancer samples we confirmed CXCR4 drives trastuzumab resistance in HER2+ breast cancer and further demonstrated that the increased CXCR4 expression in trastuzumab-resistant cells is associated with cell cycle progression with a peak in the G2/M phases. Blocking CXCR4 with AMD3100 inhibits cell proliferation by downregulating mediators of G2-M transition, leading to G2/M arrest and abnormal mitosis. Using multiple trastuzumab-resistant cell lines and an in vivo established trastuzumab-resistant xenograft mouse model, we demonstrated that targeting CXCR4 with AMD3100 suppresses tumor growth in vitro and in vivo, and synergizes with docetaxel. Conclusions: Our findings support CXCR4 as a novel therapeutic target and a predictive biomarker for trastuzumab resistance in HER2+ breast cancer.