reparixin and Breast-Neoplasms

Designing a multi-target nanomedicine without a carrier is pivotal for successful cancer nanotherapy. This study details a novel four-in-one RRX/BMS/CA4/PTX nanomedicine by simple nanoprecipitation. In this multi-target pure nanomedicine, paclitaxel (PTX) causes the immunogenic cell death of 4T1 tumour cells and the differentiation of marrow-derived suppressor cells (MDSCs) into dendritic cells (DCs) at low dose; repertaxin (RRX) selectively depletes cancer stem cells (CSCs) that are not killed by paclitaxel to inhibit lung metastasis from the breast; BMS-1 blocks the PD-1/PD-L1 pathway for proliferating effector T cells; and combretastatin A4 (CA4) targets tumour microvessels to cut off the blood supply in the tumour microenvironment. The synergy of multi-target therapies results in excellent antitumour effects. The tumour inhibition rate of 4T1 tumours is 92.5%, and the lung metastasis suppression rate exceeds 90%; no relapse is observed at 46 days after the treatment endpoint, and the survival of 50% of mice is prolonged by 95 days. Due to the low dose of PTX administration, the systemic toxicity of the RRX/BMS/CA4/PTX nanomedicine is not found. Our results suggest a strategy for designing multi-target pure nanomedicines with simple construction and efficacious therapeutic responses that present potential for clinical transformation.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Drug Screening Assays, Antitumor; Female; Mice; Mice, Inbred BALB C; Molecular Structure; Nanomedicine; Paclitaxel; Stilbenes; Sulfonamides

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment have been associated with tumor progression in breast cancer. Although crosstalk between breast cancer cells and CAFs has been studied, the effect of CAFs on non-neoplastic breast epithelial cells is not fully understood to date. Here, we investigated the effect of CAFs on aggressive phenotypes in non-neoplastic MCF10A breast epithelial cells. CAFs induced epithelial-to-mesenchymal transition (EMT) and invasive phenotype in MCF10A cells. S100A8, a potential prognostic marker in several cancers, was markedly increased in MCF10A cells by CAFs. S100A8 was crucial for CAFs-induced invasive phenotype of MCF10A cells. Among cytokines increased by CAFs, interleukin (IL)-8 induced S100A8 through transcription factors p65 NF-κB and C/EBPβ. In a xenograft mouse model with MCF10A cells and CAFs, tumor was not developed, suggesting that coinjection with CAFs may not be sufficient for in vivo tumorigenicity of MCF10A cells. Xenograft mouse tumor models with MDA-MB-231 breast carcinoma cells provided an in vivo evidence for the effect of CAFs on breast cancer progression as well as a crucial role of IL-8 in tumor growth and S100A8 expression in vivo. Using a tissue microarray of human breast cancer, we showed that S100A8 expression was correlated with poor outcomes. S100A8 expression was more frequently detected in cancer-adjacent normal human breast tissues than in normal breast tissues. Together, this study elucidated a novel mechanism for the acquisition of invasive phenotype of non-neoplastic breast cells induced by CAFs, suggesting that targeting IL-8 and S100A8 may be an effective strategy against breast cancer.

Topics: Animals; Breast Neoplasms; Calgranulin A; Cancer-Associated Fibroblasts; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Cell Movement; Coculture Techniques; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Interleukin-8; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Paracrine Communication; Phenotype; Signal Transduction; Sulfonamides; Transcription Factor RelA; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Cancer stem cells are responsible for tumor recurrence and metastasis. A new highly reproducible procedure for human breast cancer MCF-7 stem cells (BCSC) isolation and selection was developed by using a combination of hypoxia/hypoglycemia plus taxol and adriamycin for 24h. The BCSC enriched fraction (i) expressed (2-15 times) the typical stemness protein markers CD44+, ALDH1A3 and Oct 3/4; (ii) increased its clonogenicity index (20-times), invasiveness profile (>70%), migration capacity (100%) and ability to form mammospheres, compared to its non-metastatic MCF-7 counterpart. This isolation and selection protocol was successful to obtain stem cell enriched fractions from A549, SiHa and medulloblastoma cells. Since the secretion of HPI/AMF cytokine seems involved in metastasis, the effects of erytrose-4-phosphate (E4P) and 6-phosphogluconate (6PG), potent HPI inhibitors, on the acquisition of the breast stem cell-like phenotype were also evaluated. The presence of E4P during the BCSC selection deterred the development of the stemness phenotype, whereas both extracellular E4P (5-250nM) and 6PG (1μM) as well as siRNA HPI/AMF depressed the BCSC invasiveness ability (>90%), clonogenicity index (>90%) and contents (50-96%) of stemness (CD44, ALDH1A), pluripotency (p38 MAPK, Oct3/4, wnt/β-catenin) and EMT (SNAIL, MMP-1, vimentin) markers. The cytokine inhibitor repertaxin (10nM) or the anti-IL-8 or anti-TGF-β monoclonal antibodies (10μg/mL) did not significantly affect the BCSC metastatic phenotype. E4P also diminished (75%) the formation and growth of MCF-7 stem cell mammospheres. These results suggested that E4P by directly interacting with extracellular HPI/AMF may be an effective strategy to deter BCSC growth and progression.

Topics: Breast Neoplasms; Cell Hypoxia; Cell Movement; Cell Proliferation; Doxorubicin; Epithelial-Mesenchymal Transition; Female; Gluconates; Humans; Intracellular Signaling Peptides and Proteins; MCF-7 Cells; Neoplasm Metastasis; Neoplastic Stem Cells; Paclitaxel; RNA, Small Interfering; Sugar Phosphates; Sulfonamides

Stromal cells, infiltrating immune cells, paracrine factors and extracellular matrix have been extensively studied in cancers. However, autocrine factors produced by tumor cells and communications between autocrine factors and intracellular signaling pathways in the development of drug resistance, cancer stem-like cells (CSCs) and tumorigenesis have not been well investigated, and the precise mechanism and tangible approaches remain elusive. Here we reveal a new mechanism by which cytokines produced by breast cancer cells after chemotherapy withdrawal activate both Wnt/β-catenin and NF-κB pathways, which in turn further promote breast cancer cells to produce and secrete cytokines, forming an autocrine inflammatory forward-feedback loop to facilitate the enrichment of drug-resistant breast cancer cells and/or CSCs. Such an unexpected autocrine forward-feedback loop and CSC enrichment can be effectively blocked by inhibition of Wnt/β-catenin and NF-κB signaling. It can also be diminished by IL8-neutralizing antibody or blockade of IL8 receptors CXCR1/2 with reparixin. Administration of reparixin after chemotherapy withdrawal effectively attenuates tumor masses in a human xenograft model and abolishes paclitaxel-enriched CSCs in the secondary transplantation. These results are partially supported by the latest clinical data set. Breast cancer patients treated with chemotherapeutic drugs exhibited poor survival rate (66.7 vs 282.8 months, P=0.00071) and shorter disease-free survival time if their tumor samples expressed high level of IL8, CXCR1, CXCR2 genes and Wnt target genes. Taken together, this study provides new insights into the communication between autocrine niches and signaling pathways in the development of chemotherapy resistance and CSCs; it also offers a tangible approach in breast cancer treatment.

Topics: Animals; Antineoplastic Agents, Phytogenic; Autocrine Communication; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Disease-Free Survival; Drug Resistance, Neoplasm; Female; Humans; Interleukin-8; Mice; Mice, Nude; Neoplastic Stem Cells; NF-kappa B; Paclitaxel; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Signal Transduction; Sulfonamides; Transplantation, Heterologous

In breast cancer it has been proposed that the presence of cancer stem cells may drive tumor initiation, progression and recurrences. IL-8, up-regulated in breast cancer, and associated with poor prognosis, increases CSC self-renewal in cell line models. It signals via two cell surface receptors, CXCR1 and CXCR2. Recently, the IL-8/CXCR1 axis was proposed as an attractive pathway for the design of specific therapies against breast cancer stem cells. Reparixin, a powerful CXCR1 inhibitor, was effective in reducing in vivo the tumour-initiating population in several NOD/SCID mice breast cancer models, showing that the selective targeting of CXCR1 and the combination of reparixin and docetaxel resulted in a concomitant reduction of the bulk tumour mass and CSC population. The available data indicate that IL-8, expressed by tumour cells and induced by chemotherapeutic treatment, is a key regulator of the survival and self-renewal of the population of CXCR1-expressing CSC. Consequently, this investigation on the mechanism of action of the reparixin/paclitaxel combination, was based on the observation that reparixin treatment contained the formation of metastases in several experimental models. However, specific data on the formation of breast cancer brain metastases, which carry remarkable morbidity and mortality to a substantial proportion of advanced breast cancer patients, have not been generated. The obtained data indicate a beneficial use of the drug combination reparixin and paclitaxel to counteract brain tumour metastasis due to CSC, probably due to the combined effects of the two drugs, the pro-apoptotic action of paclitaxel and the cytostatic and anti-migratory effects of reparixin.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Blotting, Western; Brain Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Female; Flow Cytometry; Fluorescent Antibody Technique; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplastic Stem Cells; Paclitaxel; Receptors, Interleukin-8A; Signal Transduction; Sulfonamides; Xenograft Model Antitumor Assays

Recent evidence suggests that breast cancer and other solid tumors possess a rare population of cells capable of extensive self-renewal that contribute to metastasis and treatment resistance. We report here the development of a strategy to target these breast cancer stem cells (CSCs) through blockade of the IL-8 receptor CXCR1. CXCR1 blockade using either a CXCR1-specific blocking antibody or repertaxin, a small-molecule CXCR1 inhibitor, selectively depleted the CSC population in 2 human breast cancer cell lines in vitro. Furthermore, this was followed by the induction of massive apoptosis in the bulk tumor population via FASL/FAS signaling. The effects of CXCR1 blockade on CSC viability and on FASL production were mediated by the FAK/AKT/FOXO3A pathway. In addition, repertaxin was able to specifically target the CSC population in human breast cancer xenografts, retarding tumor growth and reducing metastasis. Our data therefore suggest that CXCR1 blockade may provide a novel means of targeting and eliminating breast CSCs.

Topics: Animals; Breast Neoplasms; Cell Division; Cell Line, Tumor; Cell Survival; Fas Ligand Protein; Female; Humans; Mice; Neoplastic Stem Cells; Receptors, Interleukin-8A; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stem Cells; Sulfonamides; Transplantation, Heterologous

Two recent studies show that epigenetics and inflammation play a relevant role in the regulation of transformation and cancer cell self-renewal in breast tumours, opening up the possibility that cancer progression can be controlled by interfering with inflammation cascades. Struhl's group showed that transient activation of the Src oncoprotein induces transformation and self-renewal of immortal cells via an epigenetic switch involving NF-kappaB, Lin28, Let-7 microRNA and IL-6. Concomitantly, Wicha's laboratory developed a strategy to selectively target cancer stem cells, retarding tumour growth and reducing metastasis by blocking the IL-8 receptor CXCR1 using either an inhibitor, repertaxin or a specific blocking antibody.

Topics: Anti-Inflammatory Agents; Antibodies, Blocking; Antineoplastic Agents; Breast Neoplasms; Cell Line; Cell Transformation, Neoplastic; Disease Progression; Epigenesis, Genetic; Genes, Switch; Humans; Inflammation; Interleukin-6; Neoplastic Stem Cells; Oncogene Protein pp60(v-src); Receptors, Interleukin-8A; Sulfonamides