Targeting CXCR2 enhances chemotherapeutic response, inhibits mammary tumor growth, angiogenesis, and lung metastasis

Abstract

Breast cancer is one of the leading causes of cancer deaths among females. Many challenges exist in the current management of advanced stage breast cancer as there are fewer recognized therapeutic strategies, often because of therapy resistance. How breast cancer cells evade chemotherapy and the underlying mechanism remains unclear. We and others have observed that malignant cells that survive initial chemo- and radiation therapy express higher levels of CXCR2 ligands, which may provide a survival benefit leading to therapy resistance. In this report, we test the hypothesis that CXCR2-dependent signaling in malignant cells may be critical for chemotherapy resistance and targeting this signaling axis may enhance the antitumor and antimetastatic activity of chemotherapeutic drugs and limit their toxicity. We used Cl66-wt, 4T1-wt, Cl66sh-CXCR2, and 4T1sh-CXCR2 cells expressing differential levels of the CXCR2 receptor to evaluate the role of targeting CXCR2 on chemotherapeutic responses. Knockdown of CXCR2 enhances paclitaxel and doxorubicin-mediated toxicity at suboptimal doses. Moreover, we observed an increase in the expression of CXCL1, a CXCR2 ligand in paclitaxel and doxorubicin-treated mammary tumor cells, which were inhibited following CXCR2 knockdown. Knockdown of CXCR2 enhanced antitumor activity of paclitaxel in an in vivo mammary tumor model. We observed significant inhibition of spontaneous lung metastases in animals bearing CXCR2 knockdown tumors and treated with paclitaxel as compared with the control group. Our data suggest the novel role of CXCR2 and its ligands in maintaining chemotherapy resistance and provide evidence that targeting CXCR2 signaling in an adjuvant setting will help circumvent chemotherapy resistance.

Figure 1. Effect of chemotherapeutic agents on the expression of CXCR2 ligands

A) PCR results showing expression of CXCR1, CXCR2 and CXCL8 in different breast cancer cell lines. B &C) ELISA results showing the expression of CXCL1 and CXCL8 in the supernatants of paclitaxel treated MDA-MB-231, MDA-MET or SKBR3 cells. D&E) Chemical structures of paclitaxel and doxorubicin drugs (adapted from NCBI PubChem Substance).

Figure 2. CXCR2 knockdown enhances sensitivity of mammary tumor cells toward chemotherapeutic agents

A & B) Cell toxicity determined by MTT assay in CXCR2 knockdown Cl66 and 4T1 cells (Cl66sh-CXCR2, 4T1sh-CXCR2) and wild type cells transfected with vector alone (Cl66-wt, 4T1-wt) at lower doses of paclitaxel (p ≤0.05) and (C & D) doxorubicin (p ≤0.05).

Figure 3. Effect of chemotherapeutic agents on the expression of CXCR2 ligands

A) Apoptosis in CXCR2 knockdown cells after treatment with 10 nM dose of paclitaxel or doxorubicin and vector transfected Cl66 cells evaluated using CaspACE^™ FITC-VAD-FMK in Situ marker. B) Quantification of apoptosis after treatment with paclitaxel or doxorubicin of Cl66-wt and Cl66 sh-CXCR2 cells (p ≤0.05). C & D) Expression of CXCL1 in the supernatants of Cl66-wt, Cl66sh-CXCR2, 4T1-wt and 4T1sh-CXCR2 cells after treatment with paclitaxel demonstrated by ELISA (p ≤0.05).

Figure 4. Paclitaxel treatment along with CXCR2 knockdown reduces tumor growth and metastasis

A) Growth of tumors formed by Cl66-wt and Cl66sh-CXCR2 cells injected in the mammary fat pad of Balb/c mice. Mice were treated with two different doses of paclitaxel, 10mg/Kg or 25mg/Kg body weight. Control group tumors were treated with PBS. Tumor size was measured twice a week and treatment with drugs was started 2 weeks after injection of tumor cells. p≤0.05 was considered significant. B) Lungs from mice harboring Cl66-wt tumor or Cl66sh-CXCR2 tumor treated with 10mg/Kg paclitaxel showing metastatic nodules which were quantitated and represented as a bar graph (p<0.05). C) Quantification of the metastatic nodules from mice bearing Cl66sh-CXCR2 tumors treated with PBS or 10 mg/Kg of paclitaxel.

Figure 5. Expression of CXCL1 in tumors

A & B) qRT-PCR of CXCL1 in primary tumors formed by Cl66-wt or Cl66sh-CXCR2 cells treated with PBS or 10mg/Kg paclitaxel. C & D) Expression of CXCL1 in tumors evaluated by ELISA.

Figure 6. Angiogenesis in tumors formed by cells varying the expression of CXCR2

A) Immunohistochemical analysis of tumor sections for Isolectin-B4 to evaluate micro-vessel density in tumor formed by CXCR2 knockdown Cl66 cells (Cl66sh-CXCR2) treated with PBS or 10mg/Kg paclitaxel. B) Quantification of micro-vessels in CXCR2 knockdown tumors after paclitaxel treatment (p<0.05).