Abstract P5-03-04: Targeting the tumor microenvironment by CXCR4 inhibition to abrogate trastuzumab resistance in HER2-positive breast cancer

Xie

et al. 2023

Preprint

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.

Section: Introductionsupporting

confidence: 88%

“…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.…”

Section: Discussionsupporting

confidence: 62%

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

Xie

et al. 2023

Preprint

“…Amplification or mutation of HER2 and upregulation of downstream PI3K/AKT and MAPK pathway are usually proposed as the mechanism of drug resistance [ 9 – 17 ]. We previously reported that CXCR4 is involved as a driver of trastuzumab resistance in HER2 + breast cancer cells [ 18 – 20 ] with an unknown mechanism. In the current study, we demonstrated that the increased CXCR4 expression in trastuzumab-resistant tumor cells is associated with cell cycle progression and reaches a peak in the G2/M phases, contributing to cell proliferation and survival.…”

Section: Discussionmentioning

confidence: 99%

“…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 (GPCR) of stromal cell-derived factor-1 (SDF-1α; CXCL12) in trastuzumab-resistant breast cancer. Knockdown of CXCR4 with shRNA sensitized the cells to trastuzumab [ 18 – 20 ]. Consistent with our findings, 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 ].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2023

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.

“…CXCR4 activation is additionally associated with endocrine therapy resistance through the downregulation of estrogen receptor expression (30). CXCR4 signaling is implicated in other areas of drug resistance: In breast cancer, CXCR4 silencing sensitizes TNBC cells to cisplatin therapy (31), silencing of CXCR4 and SDF-1 sensitizes breast cancer cells to paclitaxel (32), and CXCR4 inhibition abrogates trastuzumab resistance in HER2-positive breast cancer (33). We previously demonstrated that CXCR4 expression mediates estrogen-independent tumorigenesis, metastasis, and resistance to endocrine therapies through increased MAPK signaling (34,35).…”

Section: Introductionmentioning

confidence: 99%

Application of a small molecule inhibitor screen approach to identify CXCR4 downstream signaling pathways that promote a mesenchymal and fulvestrant‑resistant phenotype in breast cancer cells

et al. 2021

Chemokine receptor 4 (CXCR4) and its ligand stromal-derived factor 1 (SDF-1) have well-characterized functions in cancer metastasis; however, the specific mechanisms through which CXCR4 promotes a metastatic and drug-resistant phenotype remain widely unknown. The aim of the present study was to demonstrate the application of a phenotypic screening approach using a small molecule inhibitor library to identify potential CXCR4-mediated signaling pathways. The present study demonstrated a new application of the Published Kinase Inhibitor Set (PKIS), a library of small molecule inhibitors from diverse chemotype series with varying levels of selectivity, in a phenotypic medium-throughput screen to identify potential mechanisms to pursue. Crystal violet staining and brightfield microscopy were employed to evaluate relative cell survival and changes to cell morphology in the screens. 'Hits' or lead active compounds in the first screen were PKIS inhibitors that reversed mesenchymal morphologies in CXCR4-activated breast cancer cells without the COOH-terminal domain (MCF-7-CXCR4-ΔCTD) and in the phenotypically mesenchymal triple-negative breast cancer cells (MDA-MB-231, BT-549 and MDA-MB-157), used as positive controls. In a following screen, the phenotypic and cell viability screen was used with a positive control that was both morphologically mesenchymal and had acquired fulvestrant resistance. Compounds within the same chemotype series were identified that exhibited biological activity in the screens, the 'active' inhibitors, were compared with inactive compounds. Relative kinase activity was obtained using published datasets to discover candidate kinase targets responsible for CXCR4 activity. MAP4K4 and MINK reversed both the mesenchymal and drug-resistant phenotypes, NEK9 and DYRK2 only reversed the mesenchymal morphology, and kinases, including ROS, LCK, HCK and LTK, altered the fulvestrant-resistant phenotype. Oligoarray experiments revealed pathways affected in CXCR4-activated cells, and these pathways were compared with the present screening approach to validate our screening tool. The oligoarray approach identified the integrin-mediated, ephrin B-related, RhoA, RAC1 and ErbB signaling pathways to be upregulated in MCF-7-CXCR4-ΔCTD cells, with ephrin B signaling also identified in the PKIS phenotypic screen. The present screening tool may be used to discover potential mechanisms of targeted signaling pathways in solid cancers.