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Dissemination of tumour cells to the bone marrow is an early event in breast cancer, however cells may lie dormant for many years before bone metastases develop. Treatment for bone metastases is not curative, therefore new adjuvant therapies which prevent the colonisation of disseminated cells into metastatic lesions are required. There is evidence that cancer stem cells (CSCs) within breast tumours are capable of metastasis, but the mechanism by which these colonise bone is unknown. Here, we establish that bone marrow-derived IL1β stimulates breast cancer cell colonisation in the bone by inducing intracellular NFkB and CREB signalling in breast cancer cells, leading to autocrine Wnt signalling and CSC colony formation. Importantly, we show that inhibition of this pathway prevents both CSC colony formation in the bone environment, and bone metastasis. These findings establish that targeting IL1β-NFKB/CREB-Wnt signalling should be considered for adjuvant therapy to prevent breast cancer bone metastasis. In breast cancer, dormant cancer cells may develop into bone metastases. Here, the authors demonstrate that microenvironmental IL1β stimulates metastatic breast cancer cell colonisation in the bone via IL1β-NFKB/CREB-Wnt pathway activation and cancer stem cell colony formation

Background Late-stage breast cancer preferentially metastasises to bone; despite advances in targeted therapies, this condition remains incurable. The lack of clinically relevant models for studying breast cancer metastasis to a human bone microenvironment has stunted the development of effective treatments for this condition. To address this problem, we have developed humanised mouse models in which breast cancer patient-derived xenografts (PDXs) metastasise to human bone implants with low variability and high frequency. Methods To model the human bone environment, bone discs from femoral heads of patients undergoing hip replacement surgery were implanted subcutaneously into NOD/SCID mice. For metastasis studies, 7 patient-derived xenograft tumours (PDX: BB3RC32, ER+ PR+ HER2−; BB2RC08, ER+ PR+ ER2−; BB6RC37, ER− PR− HER2− and BB6RC39, ER+ PR+ HER2+), MDA-MB-231-luc2, T47D-luc2 or MCF7-Luc2 cells were injected into the 4th mammary ducts and metastases monitored by luciferase imaging and confirmed on histological sections. Bone integrity, viability and vascularisation were assessed by uCT, calcein uptake and histomorphometry. Expression profiling of genes/proteins during different stages of metastasis were assessed by whole genome Affymetrix array, real-time PCR and immunohistochemistry. Importance of IL-1 was confirmed following anakinra treatment. Results Implantation of femoral bone provided a metabolically active, human-specific site for tumour cells to metastasise to. After 4 weeks, bone implants were re-vascularised and demonstrated active bone remodelling (as evidenced by the presence of osteoclasts, osteoblasts and calcein uptake). Restricting bone implants to the use of subchondral bone and introduction of cancer cells via intraductal injection maximised metastasis to human bone implants. MDA-MB-231 cells specifically metastasised to human bone (70% metastases) whereas T47D, MCF7, BB3RC32, BB2RC08, and BB6RC37 cells metastasised to both human bone and mouse bones. Importantly, human bone was the preferred metastatic site especially from ER+ PDX (100% metastasis human bone compared with 20–75% to mouse bone), whereas ER-ve PDX developed metastases in 20% of human and 20% of mouse bone. Breast cancer cells underwent a series of molecular changes as they progressed from primary tumours to bone metastasis including altered expression of IL-1B, IL-1R1, S100A4, CTSK , SPP1 and RANK. Inhibiting IL-1B signalling significantly reduced bone metastasis. Conclusions Our reliable and clinically relevant humanised mouse models provide significant advancements in modelling of breast cancer bone metastasis.

Breast cancer bone metastasis is currently incurable, ~75% of patients with late-stage breast cancer develop disease recurrence in bone and available treatments are only palliative. We have previously shown that production of the pro-inflammatory cytokine interleukin-1B (IL-1B) by breast cancer cells drives bone metastasis in patients and in preclinical in vivo models. In the current study, we have investigated how IL-1B from tumour cells and the microenvironment interact to affect primary tumour growth and bone metastasis through regulation of the immune system, and whether targeting IL-1 driven changes to the immune response improves standard of care therapy for breast cancer bone metastasis. Using syngeneic IL-1B/IL1R1 knock out mouse models in combination with genetic manipulation of tumour cells to overexpress IL-1B/IL1R1, we found that IL-1B signalling elicited an opposite response in primary tumours compared with bone metastases. In primary tumours, IL-1B inhibited growth, by impairing the infiltration of innate immune cell subsets with potential anti-cancer functions but promoted enhanced tumour cell migration. In bone, IL-1B stimulated the development of osteolytic metastases. In syngeneic models of breast cancer, combining standard of care treatments (Doxorubicin and Zoledronic acid) with the IL-1 receptor antagonist Anakinra inhibited both primary tumour growth and metastasis. Anakinra had opposite effects on the immune response compared to standard of care treatment, and its anti-inflammatory signature was maintained in the combination therapy. These data suggest that targeting IL-1B signalling may provide a useful therapeutic approach to inhibit bone metastasis and improve efficacy of current treatments for breast cancer patients.

Triple-negative breast cancer (TNBC) is a highly aggressive and recurrent type of breast carcinoma that is associated with poor patient prognosis. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. The CXCR4-CXCL12 chemokine signaling axis guides cell migration in physiological and pathological processes, including breast cancer metastasis. Although targeted therapies to inhibit the CXCR4-CXCL12 axis are under clinical experimentation, still no effective therapeutic approaches have been established to block CXCR4 in TNBC. To unravel the role of the CXCR4-CXCL12 axis in the formation of TNBC early metastases, we used the zebrafish xenograft model. Importantly, we demonstrate that cross-communication between the zebrafish and human ligands and receptors takes place and human tumor cells expressing CXCR4 initiate early metastatic events by sensing zebrafish cognate ligands at the metastatic site. Taking advantage of the conserved intercommunication between human tumor cells and the zebrafish host, we blocked TNBC early metastatic events by chemical and genetic inhibition of CXCR4 signaling. We used IT1t, a potent CXCR4 antagonist, and show for the first time its promising anti-tumor effects. In conclusion, we confirm the validity of the zebrafish as a xenotransplantation model and propose a pharmacological approach to target CXCR4 in TNBC.

Neutrophilic inflammation with prolonged neutrophil survival is common to many inflammatory conditions, including chronic obstructive pulmonary disease (COPD). There are few specific therapies that reverse neutrophilic inflammation, but uncovering mechanisms regulating neutrophil survival is likely to identify novel therapeutic targets. Screening of 367 kinase inhibitors in human neutrophils and a zebrafish tail fin injury model identified ErbBs as common targets of compounds that accelerated inflammation resolution. The ErbB inhibitors gefitinib, CP-724714, erbstatin and tyrphostin AG825 significantly accelerated apoptosis of human neutrophils, including neutrophils from people with COPD. Neutrophil apoptosis was also increased in Tyrphostin AG825 treated-zebrafish in vivo. Tyrphostin AG825 decreased peritoneal inflammation in zymosan-treated mice, and increased lung neutrophil apoptosis and macrophage efferocytosis in a murine acute lung injury model. Tyrphostin AG825 and knockdown of egfra and erbb2 by CRISPR/Cas9 reduced inflammation in zebrafish. Our work shows that inhibitors of ErbB kinases have therapeutic potential in neutrophilic inflammatory disease. Chronic obstructive pulmonary disease (or COPD) is a serious condition that causes the lungs to become inflamed for long periods of time, leading to permanent damage of the airways. Immune cells known as neutrophils promote inflammation after an injury, or during an infection, to aid the healing process. However, if they are active for too long, they may also cause tissue damage and drive inflammatory diseases including COPD. To limit damage to the body, neutrophils usually have a very short lifespan and die by a regulated process known as apoptosis. Finding ways to stimulate apoptosis in neutrophils may be key to developing better treatments for inflammatory diseases. Cells contain many enzymes known as kinases that control apoptosis and other cell processes. Drugs that inhibit specific kinases are effective treatments for some types of cancer and other conditions, and new kinase-inhibiting drugs are currently being developed. However, it remains unclear which kinases regulate apoptosis in neutrophils or which kinase-inhibiting drugs may have the potential to treat COPD and other inflammatory diseases. To address these questions, Rahman et al. tested over 350 kinase-inhibiting drugs to identify ones that promote apoptosis in neutrophils. The experiments showed that human neutrophils treated with drugs that inhibit the ErbB family of kinases died by apoptosis more quickly than untreated neutrophils. Next, Rahman et al. used zebrafish with injured tail fins as models to study inflammation. Zebrafish treated with one of these drugs – known as Tyrphostin AG825 – had lower levels of inflammation and their neutrophils underwent apoptosis more frequently than untreated zebrafish. Since drugs can have off-target effects, Rahman et al. went on to show using gene-editing technology that reducing the activity of two genes that encode ErbB kinases in zebrafish also decreased the levels of inflammation in the fish. Further experiments used mice that develop inflammation in the lungs similar to COPD in humans. As expected, neutrophils in the lungs of mice treated with Tyrphostin AG825 underwent apoptosis more frequently than those in untreated mice. These dead neutrophils were effectively cleared by other immune cells called macrophages, which also helps limit damage caused by neutrophils. Together, these findings show that Tyrphostin AG825 and other drugs that inhibit ErbB kinases help to reduce inflammation by promoting the death of neutrophils. Since several of these drugs are already used to treat human cancers, it may be possible in the future to repurpose them for use in people with COPD and other long-term inflammatory diseases. Determining whether this is possible is an aim for future studies.

Bone is the most common site for advanced breast cancer to metastasise. The proinflammatory cytokine, interleukin-1β (IL-1β) plays a complex and contradictory role in this process. Recent studies have demonstrated that breast cancer patients whose primary tumours express IL-1β are more likely to experience relapse in bone or other organs. Importantly, IL-1β affects different stages of the metastatic process including growth of the primary tumour, epithelial to mesenchymal transition (EMT), dissemination of tumour cells into the blood stream, tumour cell homing to the bone microenvironment and, once in bone, this cytokine participates in the interaction between cancer cells and bone cells, promoting metastatic outgrowth at this site. Interestingly, although inhibition of IL-1β signalling has been shown to have potent anti-metastatic effects, inhibition of the activity of this cytokine has contradictory effects on primary tumours, sometimes reducing but often promoting their growth. In this review, we focus on the complex roles of IL-1β on breast cancer bone metastasis: specifically, we discuss the distinct effects of IL-1β derived from tumour cells and/or microenvironment on inhibition/induction of primary breast tumour growth, induction of the metastatic process through the EMT, promotion of tumour cell dissemination into the bone metastatic niche and formation of overt metastases.

CXC chemokine receptor 4 plays a critical role in chemotaxis and leukocyte differentiation. Furthermore, there is increasing evidence that links this receptor to angiogenesis. Using the well-established zebrafish- Mycobacterium marinum model for tuberculosis, angiogenesis was recently found to be important for the development of cellular aggregates called granulomas that contain the mycobacteria and are the hallmark of tuberculosis disease. Here, we found that initiation of the granuloma-associated proangiogenic programme requires CXCR4 signalling. The nascent granulomas in cxcr4b -deficient zebrafish embryos were poorly vascularised, which in turn also delayed bacterial growth. Suppressed infection expansion in cxcr4b mutants could not be attributed to an overall deficient recruitment of leukocytes or to different intramacrophage bacterial growth rate, as cxcr4b mutants displayed similar microbicidal capabilities against initial mycobacterial infection and the cellular composition of granulomatous lesions was similar to wildtype siblings. Expression of vegfaa was upregulated to a similar extent in cxcr4b mutants and wildtypes, suggesting that the granuloma vascularisation phenotype of cxcr4b mutants is independent of vascular endothelial growth factor.

Breast cancer bone metastasis is currently incurable. Evidence suggests that inhibiting IL-1 signalling with the IL1R antagonist, Anakinra, or the IL1β antibody, Canakinumab, prevents metastasis and almost eliminates breast cancer growth in the bone. However, these drugs increase primary tumour growth. We, therefore, investigated whether targeting other members of the IL-1 pathway (Caspase-1, IL1β or IRAK1) could reduce bone metastases without increasing tumour growth outside of the bone. Inhibition of IL-1 via MLX01 (IL1β secretion inhibitor), VRT043198/VX765 (Caspase-1 inhibitor), Pacritinib (IRAK1 inhibitor) or Anakinra (IL1R antagonist) on tumour cell viability, migration and invasion were assessed in mouse mammary E0771 and Py8119 cells in vitro and on primary tumour growth, spontaneous metastasis and metastatic outgrowth in vivo. In vitro, Inhibition of IL-1 signalling by MLX01, VRT043198 and Anakinra reduced migration of E0771 and Py8119 cells and reversed tumour-derived IL1β induced-increased invasion and migration towards bone cells. In vivo, VX765 and Anakinra significantly reduced spontaneous metastasis and metastatic outgrowth in the bone, whereas MLX01 reduced primary tumour growth and bone metastasis. Pacritinib had no effect on metastasis in vitro or in vivo. Targeting IL-1 signalling with small molecule inhibitors may provide a new therapeutic strategy for breast cancer bone metastasis.

The receptor tyrosine kinase (RTK) RON is linked to an aggressive metastatic phenotype of carcinomas. While gaining interest as a therapeutic target, RON remains unstudied in sarcomas. In Ewing sarcoma, we identified RON among RTKs conferring resistance to insulin-like growth factor-1 receptor (IGF1R) targeting. Therefore, we explored RON in pediatric sarcoma cell lines and an embryonic Tg(kdrl:mCherry) zebrafish model, using an shRNA-based approach. To examine RON–IGF1R crosstalk, we employed the clinical-grade monoclonal antibody IMC-RON8, alone and together with the IGF1R-antibody IMC-A12. RON silencing demonstrated functions in vitro and in vivo, particularly within micrometastatic cellular capacities. Signaling studies revealed a unidirectional IGF1-mediated cross-activation of RON. Yet, IMC-A12 failed to sensitize cells to IMC-RON8, suggesting additional mechanisms of RON activation. Here, RT-PCR revealed that childhood sarcomas express short-form RON, an isoform resistant to antibody-mediated targeting. Interestingly, in contrast to carcinomas, treatment with DNA methyltransferase inhibitor did not diminish but increased short-form RON expression. Thus, this first report supports a role for RON in the metastatic progression of Ewing sarcoma. While principal molecular functions appear transferrable between carcinomas, Ewing sarcoma and possibly more common sarcoma subtypes, RON highlights that specific regulations of cellular networks and isoforms require better understanding to successfully transfer targeting strategies.

Triple negative breast cancer (TNBC) is a highly aggressive and recurrent type of breast carcinoma that is associated with poor patient prognosis. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. The CXCR4-CXCL12 chemokine signaling axis guides cell migration in physiological and pathological processes including breast cancer metastases. Although targeted therapies to inhibit the CXCR4-CXCL12 axis are under clinical experimentation, still no effective therapeutic approaches have been established to block CXCR4 in TNBC. To unravel the role of the CXCR4-CXCL12 axis in TNBC early metastasis formation, we used the zebrafish xenograft model. Importantly, we demonstrate that cross communication between the zebrafish and human ligands and receptors takes place and human tumor cells expressing CXCR4 initiate early metastatic events by sensing zebrafish cognate ligands at the metastatic site. Taking advantage of the conserved intercommunication between human tumor cells and the zebrafish host, we blocked TNBC early metastatic events by chemical and genetic inhibition of CXCR4 signaling. We used IT1t, a potent CXCR4 antagonist, and show for the first time its promising anti-tumor effects. In conclusion, we confirm the validity of the zebrafish as a xenotransplantation model and propose a pharmacological approach to target CXCR4 in TNBC.