Accumulating evidence suggests that cellular subcomponents with stem cell properties may drive tumor growth and metastasis (1
). Furthermore, by virtue of their relative resistance to current therapeutic modalities, these cells may contribute to treatment resistance and relapse (2
). If this is the case, then the development of strategies to effectively target the CSC population will be required to improve therapeutic outcome. We describe such an approach based on blocking the CXCR1 cytokine receptor, which is expressed on breast CSCs. Experiments in a number of systems have demonstrated that cytokine networks play an important role in tumorigenesis. Furthermore, there is evidence that several of these cytokines may regulate stem cell behavior. IL-4 is capable of regulating self-renewal of pancreatic CSCs (30
), and IL-6 can regulate CSCs in colon and breast cancer (31
). The role of IL-8 in mediating tumor invasion and metastasis has previously been demonstrated (14
). In addition, IL-8 increases neural stem cell self-renewal during wound healing in the brain (32
). Recently, lung CSCs were described as expressing the chemokine receptor CXCR1 (33
). Consistent with these findings, we demonstrated that the CXCR1+
population was almost exclusively contained within the ALDEFLUOR+
population in breast cancer cell lines and primary xenografts as well as in normal human mammary cells. We found that this chemokine receptor was overexpressed in ALDEFLUOR+
breast cancer cell populations (13
). In breast cancers, IL-8 is produced in the tumor microenvironment by a number of cell types, including inflammatory cells, vascular endothelial cells, tumor-associated fibroblasts, and mesenchymal stem cells (21
). Because cytokine networks may mediate interaction among these cell types, we sought to target CSCs through blockade of the IL-8 receptor CXCR1.
Using in vitro assays, we demonstrated that blockade of CXCR1, but not of the alternative IL-8 receptor CXCR2, reduced the breast CSC population. This was followed by induction of apoptosis in the entire remaining cell population, which lacks CXCR1 expression. In addition to CXCR1-blocking antibodies, we demonstrated that repertaxin, a CXCR1/2 inhibitor, induced similar effects by targeting the CXCR1+ population. In contrast to its direct effects on the CXCR1-expressing CSC population, repertaxin had no direct effect on the bulk tumor cell population, which lacks CXCR1 expression. This suggested that CXCR1 blockade in CXCR1+ cells induced cell death in CXCR1– cells via a bystander effect. We confirmed this hypothesis and identified the FASL/FAS pathway as the mediator of this bystander killing effect. This phenomenon explains the efficacy of repertaxin treatment in inducing massive apoptosis in the entire cell population despite the fact that the CXCR1+ population represents less than 1% of the cell population. The role of FASL was demonstrated by the effective blocking of bystander killing by anti-FASL antibody.
The FASL/FAS death pathway may play a role in normal mammary physiology as well as in mediating chemotherapy-induced cell death. In the normal breast, the rapid decrease in hormone levels after lactation leads to massive apoptosis in the mammary gland that is largely mediated by FASL-induced FAS apoptosis (19
). Interestingly, involuting differentiated mammary cells undergoing apoptosis also secrete increased levels of IL-8 (19
). Based on our current studies, we speculate that this secreted IL-8 interacts with CXCR1 on normal mammary stem cells to increase their self-renewal as well as protecting them from FAS-mediated apoptosis. This process ensures sufficient regeneration of the mammary stem cell pool to support future pregnancy/lactation cycles.
Similar cytokine interactions may occur in tumors exposed to cytotoxic chemotherapy. Chemotherapy may directly induce cellular apoptosis in differentiated tumor cells as well as induce the production of FASL by these dying cells that in turn induces apoptosis in surrounding tumor cells via a FAS-mediated bystander effect. However, concomitant with the production of FASL, these injured cells also secrete increased levels of IL-8 in a process resembling mammary involution or wound healing. As is the case in the involuting mammary gland, this IL-8 may stimulate breast CSCs as well as protecting them from apoptosis. This may contribute to the relative increase in CSCs observed after chemotherapy in preclinical models (6
) and neoadjuvant clinical trials (7
). The effects of chemotherapy on apoptosis and self-renewal pathways in tumors are shown in Figure .
IL-8/CXCR1 signaling in CSCs treated with chemotherapy alone or in combination with repertaxin.
To determine whether CXCR1 blockade could target breast CSCs in vivo, we compared the effects of the cytotoxic agent docetaxel with those of repertaxin on the CSC compartment and on tumor growth in NOD/SCID mice. We chose docetaxel because it is one of the most effective chemotherapeutic agents currently used to treat women with breast cancer. We assessed the CSC populations by the ALDEFLUOR assay and by serial transplantation in NOD/SCID mice. Using these assays, we determined that chemotherapy treatment alone resulted in either no change or a relative increase in the CSC populations. In contrast, repertaxin treatment alone or in combination with chemotherapy significantly reduced the CSC population. Despite the significant reduction in the tumor-initiating populations, use of repertaxin alone did not result in a significant reduction in tumor size. This suggests that, unlike its in vitro effects, use of repertaxin in vivo failed to induce significant bystander killing in the bulk nontumor population. This may be caused by survival signals generated by the tumor microenvironment in vivo. Nevertheless, the combination of repertaxin plus chemotherapy resulted in significant reductions in tumor size and in the CSC population, which suggests that a strategy of combining these agents to target both CSCs and bulk tumor cell populations may maximize the efficacy of these treatments.
To elucidate the mechanism of action of repertaxin, we analyzed the pathways downstream from CXCR1. CXCR1 has previously been shown to act through the FAK/AKT signaling pathway (21
). We confirmed the interaction among CXCR1, FAK, and AKT and demonstrated that CXCR1 blockade acted specifically through FAK and AKT activation. We have previously shown that AKT activation regulates normal and malignant breast stem cell self-renewal through phosphorylation of GSK3β, resulting in the activation of the WNT pathway (26
). This may explain why cells with PTEN knockdown were resistant to repertaxin. An additional important function of AKT is the regulation of cell survival through phosphorylation of FOXO3A. AKT phosphorylation of FOXO3A results in its cytoplasmic sequestration. In contrast, we demonstrated that CXCR1 blockade led to decreased AKT activation, which results in the translocation of FOXO3A in the nucleus and subsequent induction of a number of genes, including FASL
). FASL induced via CXCR1 blockade is in turn responsible for the bystander killing effects that we observed in the present study. These pathways are illustrated in Figure .
In addition to its role in CXCR1 signaling, FAK mediates the interactions of cells with extracellular matrix components through integrin receptors (21
). We previously demonstrated a role for FAK signaling in regulating the self-renewal of normal and malignant mouse mammary stem cells in transgenic models (34
). FAK activation also promotes cell survival by blocking apoptosis mediated by FAS-associated protein with death domain (FADD) and receptor-interacting protein (RIP) (35
). This may explain the resistance of the CSC population to the FASL/FAS-induced apoptosis.
We and others have previously demonstrated that breast CSCs may play an important role in tumor invasion and metastasis (13
). Furthermore, there is evidence that IL-8 and CXCR1 also play important roles in these processes. Therefore, we analyzed the effects of CXCR1 blockade using repertaxin on the formation of experimental metastasis. We demonstrated that CXCR1 blockade reduced the development of metastasis when administered after intracardiac injection of breast cancer cells.
IL-8, in addition to other cytokines such has IL-6, has been implicated in tissue inflammation and repair as well as carcinogenesis (21
). In fact, repertaxin was originally developed to block IL-8 activity to reduce tissue damage after myocardial infarction or stroke (15
). Clinical phase I studies using this compound have demonstrated a lack of toxicity. This suggests that strategies aimed at interfering with cytokine regulatory loops such as IL-8 and CXCR1 may represent a novel strategy to target breast CSCs. Because these cells may drive tumor progression and metastasis, such strategies may lead to improving outcomes for women with advanced breast cancer.