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Summary of the effects of IL-1B on breast cancer progression to metastasis and growth in the bone microenvironment.

Suppression of IL-1 signalling affects bone integrity and vasculature.

Supplementary figure legends

Effects of IL-1B on osteoclast and osteoblast activity in vivo.

Elevated plasma concentrations of soluble VEGFA isoforms are associated with poor prognosis in parallel with improved response to treatment with the anti-VEGFA antibody bevacizumab. To uncover the underlying mechanism to these observations, we administered anti-VEGFA therapy to mice bearing luminescent mouse fibrosarcomas expressing single VEGFA isoforms or their wild-type counterparts expressing all isoforms (fs120, fs164, fs188, or fsWT). Expression of the more soluble isoforms conferred an advantage for lung metastasis from subcutaneous tumors (fs120/164 vs. fs188/WT); fs120 cells also produced more lung colonies than fs188 cells when injected intravenously. Metastasis from subcutaneous fs120 tumors was more sensitive than fs188 to treatment with the anti-VEGFA antibody B20-4.1.1. Despite elevated plasma levels of VEGFA in fs120 tumor-bearing mice and a dependence on VEGF receptor 1 activity for metastasis to the lung, B20-4.1.1 did not affect survival in the lung on intravenous injection. B20-4.1.1 inhibited subcutaneous tumor growth and decreased vascular density in both fs120 and fs188 tumors. However, migration of fs120, but not fs188 cells, in vitro was inhibited by B20-4.1.1. The greater survival of fs120 cells in the lung was associated with VEGFR1-dependent accumulation of CD11b-positive myeloid cells and higher expression of the VEGFR1 ligand, PlGF2, by the fs120 cells in vitro and in the plasma and lungs of fs120 tumor-bearing mice. We conclude that soluble VEGFA isoform expression increases fibrosarcoma metastasis through multiple mechanisms that vary in their sensitivity to anti-VEGF/VEGFR inhibition, with VEGFA-targeted therapy suppressing metastasis through effects on the primary tumor rather than the metastatic site. Cancer Res; 77(10); 2633–46. ©2017 AACR.

Incubation with high concentrations of exogenous IL-1B modestly increases tumour cell migration and invasion.

In vivo model of spontaneous human breast cancer metastasis to human bone implants

Purpose:Breast cancer bone metastases are incurable, highlighting the need for new therapeutic targets. After colonizing bone, breast cancer cells remain dormant, until signals from the microenvironment stimulate outgrowth into overt metastases. Here we show that endogenous production of IL1B by tumor cells drives metastasis and growth in bone.Experimental Design:Tumor/stromal IL1B and IL1 receptor 1 (IL1R1) expression was assessed in patient samples and effects of the IL1R antagonist, Anakinra, or the IL1B antibody canakinumab on tumor growth and spontaneous metastasis were measured in a humanized mouse model of breast cancer bone metastasis. Effects of tumor cell–derived IL1B on bone colonization and parameters associated with metastasis were measured in MDA-MB-231, MCF7, and T47D cells transfected with IL1B/control.Results:In tissue samples from >1,300 patients with stage II/III breast cancer, IL1B in tumor cells correlated with relapse in bone (HR = 1.85; 95% CI, 1.05–3.26; P = 0.02) and other sites (HR = 2.09; 95% CI, 1.26–3.48; P = 0.0016). In a humanized model of spontaneous breast cancer metastasis to bone, Anakinra or canakinumab reduced metastasis and reduced the number of tumor cells shed into the circulation. Production of IL1B by tumor cells promoted epithelial-to-mesenchymal transition (altered E-Cadherin, N-Cadherin, and G-Catenin), invasion, migration, and bone colonization. Contact between tumor and osteoblasts or bone marrow cells increased IL1B secretion from all three cell types. IL1B alone did not stimulate tumor cell proliferation. Instead, IL1B caused expansion of the bone metastatic niche leading to tumor proliferation.Conclusions:Pharmacologic inhibition of IL1B has potential as a novel treatment for breast cancer metastasis.

The supplementary methods file contains additional detailed information regarding: 1. The production of cell lines stably expressing luciferase. 2. Mouse strains and pre-clinical models of tumor growth and metastasis. 3. Acquisition and analysis of bioluminescence data from preclinical and in vitro studies. 4. Acquisition and analysis of in vitro migration data. 5. Antibodies and immuno-techniques used within this study.

Tumour-derived IL-1B predicts distant recurrence and relapse in bone in patients with stage II and III breast cancer.

Stable transfection of breast cancer cells with IL-1B.

The supplementary data file contains the following data and figures: Fig S1- CD31 and CD34 co-localize to the vasculature in fs120-LS and fs188-LS subcutaneous tumors. Fig S2 - VEGFR1 activity has no effect on tumor growth or vascular density of subcutaneous fs120-LS or fs188-LS tumors. Fig S3 - fs120-LS and fs188-LS cells metastasize to the lung early during subcutaneous tumor growth. Fig S4 - fs120-LS cells express higher levels of PlGF2 than fs188-LS cells in vitro, with no difference in levels of PLGF2 detected in subcutaneous tumors. Fig S5 - Expression of laminin and collagen-I in lysates of fs120-LS and fs188-LS subcutaneous tumors. Fig S6 - Quantification of single cell migration using live video microscopy. Fig S7 - Localization of CD11b cells with respect to intravenously injected fibrosarcoma cells within the lung. Fig S8 - Summary of results showing key differences between fibrosarcomas expressing VEGF120 and VEGF188 and the effects of B20-4.1.1 and stromal VEGFR1 activity. Fig S9 - Effect of cediranib on survival of fs120-LS cells in the lung 48 h after iv injection.

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.

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.

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.

Clinical trials have demonstrated that adding zoledronic acid (Zol) to (neo)adjuvant standard of care has differential antitumour effects in pre- and post-menopausal women: Both benefit from reduced recurrence in bone; however, while postmenopausal women also incur survival benefit, none is seen in premenopausal women treated with adjuvant bisphosphonates. In the current study, we have used mouse models to investigate the role of oestradiol in modulating potential antitumour effects of Zol. Pre-, peri-, and post-menopausal concentrations of oestradiol were modelled in BALB/c wild-type, BALB/c nude, and C57BL/6 mice by ovariectomy followed by supplementation with oestradiol. Mice also received 40 mg/kg/day goserelin to prevent ovariectomy-induced increases in follicle-stimulating hormone (FSH). Metastasis was modelled following injection of MDA-MB-231, 4T1, or E0771 cells after ovariectomy and saline or 100 μg/kg Zol administered weekly. Supplementing ovariectomised mice with 12.5 mg/ml, 1.38 mg/ml, and 0 ng/ml oestradiol, in the presence of goserelin, resulted in serum concentrations of 153.16 ± 18.10 pg/ml, 48.64 ± 18.44 pg/ml, and 1.00 ± 0.27 pg/ml oestradiol, which are equivalent to concentrations found in pre-, peri-, and post-menopausal humans. Osteoclast activity was increased 1.5–1.8-fold with peri- and post-menopausal compared with premenopausal oestradiol, resulting in a 1.34–1.69-fold reduction in trabecular bone. Zol increased trabecular bone in all groups but did not restore bone to volumes observed under premenopausal conditions. In tumour-bearing mice, Zol reduced bone metastases in BALB/c (wild-type and nude), with greatest effects seen under pre- and post-menopausal concentrations of oestradiol. Zol did not affect soft tissue metastases in immunocompetent BALB/c mice but increased metastases 3.95-fold in C57BL/6 mice under premenopausal concentrations of oestradiol. In contrast, Zol significantly reduced soft tissue metastases 2.07 and 4.69-fold in immunocompetent BALB/c and C57BL/6 mice under postmenopausal oestradiol, mirroring the results of the clinical trials of (neo)adjuvant bisphosphonates. No effects on soft tissue metastases were observed in immunocompromised mice, and differences in antitumour response did not correlate with musculoaponeurotic fibrosarcoma (MAF), macrophage capping protein (CAPG), or PDZ domain containing protein GIPC1 (GIPC1) expression. In conclusion, oestradiol contributes to altered antitumour effects of Zol observed between pre- and post-menopausal women. However, other immunological/microenvironmental factors are also likely to contribute to this phenomenon.

IκB kinase subunit epsilon (IKKε), a key component of NFκB and interferon signalling, has been identified as a breast cancer oncogene. Here we report that the IKKε/TBK1 axis plays a role in the initiation and progression of breast cancer osteolytic metastasis. Cancer-specific knockdown of IKKε in the human MDA-MB-231-BT cells and treatment with the verified IKKε/TBK1 inhibitor Amlexanox reduced skeletal tumour growth and osteolysis in mice. Inaddition, combined administration of Amlexanox with Docetaxel reduced mammary tumour growth of syngeneic 4T1 cells, inhibited metastases and improved survival in mice after removal of the primary tumour. Functional and mechanistic studies in breast cancer cells, osteoclasts and osteoblasts revealed that IKKƐ inhibition reduces the ability of breast cancercells to grow, move and enhance osteoclastogenesis by engaging both IRF and NFκB signalling pathways. Thus, therapeutic targeting of the IKKƐ/TBK1 axis may be of value in the treatment of advanced triple negative breast cancer.

Background: Breast cancer bone metastases are incurable and new therapeutic targets needed. After homing and colonising bone, cancer cells remain dormant, until signals from the microenvironment stimulate their proliferation to form overt metastases. We have recently identified interleukin-1B (IL-1B) as a potential marker for predicting breast cancer patients at increased risk of skeletal relapse and established a role for IL-1 signalling in tumour dormancy in bone. Here we present novel data to support that tumour cell-derived IL-1B plays major roles in breast cancer metastasis and growth in bone. Methods: Tumour/stromal IL-B and IL-1R1 expression was assessed in samples from the AZURE study following immunohistochemical staining. A humanised mouse model of MDA-MB-231 bone metastasis was used to assess effects of the IL-1R antagonist, Anakinra, on tumour growth and spontaneous metastasis. Effects of tumour cell-derived IL-1B on parameters associated with epithelial to mesenchymal transition and metastasis were measured by ELISA, QPCR, Western blot, transwell and scratch assays in MDA-MB-231, MCF7 and T47D cells transfected with IL-1B/control. Homing of breast cancer cells to bone was monitored in BALB/c nude mice intra venously injected with IL-1B overexpressing/control cells. To assess anti-tumour effects of IL-1 inhibition combined with standard of care, BALB/c or C57BL/6 mice were injected with 4T1 or E0771 cells (intra cardiac or intra ductal) 7-days prior to administration of Anakinra, doxorubicin, zoledronic acid (ZA) or placebo, alone or in combination for 14 days. Results: In tissue samples from >1300 patients with stage II/III breast cancer, active IL-1B in tumour cells correlated with relapse in bone (hazard ratio 1.85; 95% CI 1.05-3.26; P=0.02) and other sites (hazard ratio 2.09; 95% CI 1.26-3.48; P=0.0016). In a model of spontaneous human breast cancer metastasis to human bone, Anakinra significantly reduced metastasis to bone (from 80% in control animals to, 20% in Anakinra treated mice) and reduced the number of tumour cells shed into the circulation. Genetic manipulation of breast cancer cells to overexpress IL-1B demonstrated that exogenous production of IL-1B promoted EMT (decreased E-Cadherin, N-Cadherin and G-Catenin), invasion, migration and organ-specific homing in ER-ve (MDA-MB-231) and ER+ve (T47D and MCF7) cells in vitro and in vivo. Contact between tumour cells and osteoblasts or bone marrow cells increased IL-1B secretion from all three cell types. Exposure of tumour cells to IL-1B in the absence of bone cells did not stimulate tumour cell proliferation. Instead, elevated concentrations of IL-1B caused expansion of the bone metastatic niche (increased osteoblasts and blood vessels) that in turn stimulated tumour proliferation. Adding Anakinra to chemotherapy and ZA to mice injected with E0771 cells completely prevented bone metastasis formation and reduced extra-skeletal metastasis by 33%. In mice injected with 4T1 cells, the triple combination therapy reduced bone metastasis by 50% and extra-skeletal metastases by 65%. Conclusion: Our novel data demonstrate that IL-1B/IL-1R1 signalling plays an important role in breast cancer metastasis to bone. Pharmacological inhibition of IL-1B has potential as a novel treatment. Citation Format: Ottewell PD, Lefley DV, Freeman K, Gregory WM, Hanby AM, Spicer-Hadlington AR, Liu X, Bradbury SM, Hambley L, Allocca G, Cookson V, Kruithof de-Julio M, Coleman RE, Brown JE, Holen I, Tulotta C. Breast cancer cell-derived IL-1B drives metastasis and colonisation of the bone microenvironment [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-01.

Purpose: Breast cancer bone metastases are incurable, highlighting the need for new therapeutic targets. After colonizing bone, breast cancer cells remain dormant, until signals from the microenvironment stimulate outgrowth into overt metastases. Here we show that endogenous production of IL1B by tumor cells drives metastasis and growth in bone. Experimental Design: Tumor/stromal IL1B and IL1 receptor 1 (IL1R1) expression was assessed in patient samples and effects of the IL1R antagonist, Anakinra, or the IL1B antibody canakinumab on tumor growth and spontaneous metastasis were measured in a humanized mouse model of breast cancer bone metastasis. Effects of tumor cell–derived IL1B on bone colonization and parameters associated with metastasis were measured in MDA-MB-231, MCF7, and T47D cells transfected with IL1B/control. Results: In tissue samples from >1,300 patients with stage II/III breast cancer, IL1B in tumor cells correlated with relapse in bone (HR = 1.85; 95% CI, 1.05–3.26; P = 0.02) and other sites (HR = 2.09; 95% CI, 1.26–3.48; P = 0.0016). In a humanized model of spontaneous breast cancer metastasis to bone, Anakinra or canakinumab reduced metastasis and reduced the number of tumor cells shed into the circulation. Production of IL1B by tumor cells promoted epithelial-to-mesenchymal transition (altered E-Cadherin, N-Cadherin, and G-Catenin), invasion, migration, and bone colonization. Contact between tumor and osteoblasts or bone marrow cells increased IL1B secretion from all three cell types. IL1B alone did not stimulate tumor cell proliferation. Instead, IL1B caused expansion of the bone metastatic niche leading to tumor proliferation. Conclusions: Pharmacologic inhibition of IL1B has potential as a novel treatment for breast cancer metastasis.

// Ingunn Holen 1 , Diane V. Lefley 1 , Sheila E. Francis 2 , Sarah Rennicks 1 , Steven Bradbury 1 , Robert E. Coleman 1 , Penelope Ottewell 1 1 Academic Unit of Clinical Oncology, Department of Oncology and Metabolism, Mellanby Centre for Bone Research, University of Sheffield, Sheffield S10 2RX, UK 2 Department of Infection Immunity and Cardiovascular Disease, University of Sheffield, Sheffield S10 2RX, UK Correspondence to: Penelope Ottewell, email: P.D.Ottewell@sheffield.ac.uk Keywords: breast cancer, bone metastasis, IL-1B, IL-1R, anakinra Received: June 01, 2016 Accepted: September 15, 2016 Published: September 27, 2016 ABSTRACT Background: We have recently identified interleukin 1B (IL-1B) as a potential biomarker for predicting breast cancer patients at increased risk for developing bone metastasis. In mouse models, IL-1B and its receptor (IL-1R1) are upregulated in breast cancer cells that metastasise to bone compared with cells that do not. We have now investigated the functional role of IL-1 by blocking IL-1R signalling with the clinically licensed antagonist, anakinra. Methodology: 6-week old female BALB/c mice received a subcutaneous or intra-venous injection of MDA-MB-231-IV or MCF7 cells. Anakinra (1mg/kg/day) or placebo was administered 3 days before (preventative) or 7 days later (treatment). Tumour volume, apoptosis (TUNEL, Caspase 3), proliferation (Ki67) and angiogenesis (CD34, VEGF and endothelin) were analysed. Effects on bone were measured by uCT, and TRAP, P1NP, IL-1B, TNF alpha and IL-6 ELISA. Results: Anakinra significantly reduced growth of MDA-MB-231-IV tumours in bone from 6.50+/3.00mm 2 (placebo) to 2.56+/-1.07mm 2 (treatment) and 0.63+/-0.18mm 2 (preventative). Anakinra also reduced the number of mice that developed bone metastasis from 90% (placebo) to 40% (treatment) and 10% (preventative). Anti-tumour effects were not confined to bone, subcutaneous tumour volumes reduced from 656.68mm 3 (placebo) to 160.47mm 3 (treatment) and 31.08mm 3 (preventative). Anakinra did not increase tumour cell apoptosis but reduced proliferation and angiogenesis in addition to exerting significant effects on the tumour environment reducing bone turnover markers, IL-1B and TNF alpha. Conclusions: Our novel data demonstrate a functional role of IL-1 signalling in breast tumour progression and metastasis, supporting that anakinra could be repurposed for the treatment of breast cancer bone metastasis.

Elevated plasma concentrations of soluble VEGFA isoforms are associated with poor prognosis in parallel with improved response to treatment with the anti-VEGFA antibody bevacizumab. To uncover the underlying mechanism to these observations, we administered anti-VEGFA therapy to mice bearing luminescent mouse fibrosarcomas expressing single VEGFA isoforms or their wild-type counterparts expressing all isoforms (fs120, fs164, fs188, or fsWT). Expression of the more soluble isoforms conferred an advantage for lung metastasis from subcutaneous tumors (fs120/164 vs. fs188/WT); fs120 cells also produced more lung colonies than fs188 cells when injected intravenously. Metastasis from subcutaneous fs120 tumors was more sensitive than fs188 to treatment with the anti-VEGFA antibody B20-4.1.1. Despite elevated plasma levels of VEGFA in fs120 tumor-bearing mice and a dependence on VEGF receptor 1 activity for metastasis to the lung, B20-4.1.1 did not affect survival in the lung on intravenous injection. B20-4.1.1 inhibited subcutaneous tumor growth and decreased vascular density in both fs120 and fs188 tumors. However, migration of fs120, but not fs188 cells

Background/Aims: The cytotoxic agent paclitaxel and the anti-resorptive drug zoledronic acid are used in the early and advanced breast cancer setting, respectively. Both agents have been demonstrated to have anti-tumour and anti-endothelial actions. Combining paclitaxel with zoledronic acid induces a synergistic increase in apoptotic breast cancer cell death in vitro, suggesting an increased anti-tumour effect in vivo, but any specific effects on the normal microvasculature and potential side-effects of this combination remain to be established. Methods: The effects of zoledronic acid and paclitaxel were investigated, alone and in combination, on human microvascular endothelial cells in vitro, using functional assays including proliferation, migration, tubule formation and apoptosis. The in vivo effect of the drugs on the normal microvasculature was determined using the dorsal microcirculation chamber model. Results/Conclusion: Zoledronic acid reduced human dermal microvascular endothelial cell (HuDMEC) proliferation, caused accumulation of cells in S phase, and inhibited migration, tube formation and Rap1a prenylation. Paclitaxel significantly inhibited tube formation and proliferation, and increased endothelial necrosis; the combination induced HuDMEC apoptosis and further enhanced the inhibition of tube formation and migration. The combination caused minimal effects on the normal microvasculature in vivo, suggesting that this potential therapeutic strategy is not associated with deleterious microvascular side-effects.

Patients with advanced breast cancer frequently develop bone metastases, and at this stage, the disease is considered incurable. Here, we show that a 6-week course of weekly administration of doxorubicin (2 mg/kg), followed 24 hours later by the bisphosphonate zoledronic acid (100μg/kg), causes substantial inhibition of MDA-MB-436 breast tumor burden in bone of immunocompromised mice, compared with administration of the single agents. Molecular analysis of tumors from animals treated sequentially with doxorubicin followed by zoledronic acid showed reduced numbers of proliferating tumor cells and decreased expression of cyclins E1, B, D1, and D3 as well as cdk2 and cdk4. Tumors from the sequential treatment group also displayed increased levels of apoptosis, increased expression of bcl2-associated X protein, decreased expression of B-cell chronic lymphocytic leukemia/lymphoma 2, and activation of caspase 3, 8, and 9. Zoledronic acid caused a small reduction in tumor volume, reduced tumor cell proliferation, and decreased expression of cyclins D1 and D3, compared with tumors from animals treated with saline or doxorubicin. Doxorubicin had no effect on tumor growth, cell cycle, or apoptosis in vivo, but did cause increased accumulation of a bisphosphonate in MDA-MB-436 cells in vitro, suggesting that doxorubicin may affect subsequent uptake of zoledronic acid. In support of this, accumulation of unprenylated Rap1A, a surrogate marker of zoledronic acid, was only detected in tumors following sequential treatment, and not following treatment with zoledronic acid alone. Our data are the first to show the specific molecular pathways by which sequential treatment with doxorubicin and zoledronic acid induce tumor cell apoptosis and inhibit proliferation in an in vivo model of breast tumor growth in bone. [Mol Cancer Ther 2009;8(10):2821–32]

Vascular endothelial growth factor-A (VEGF) is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120) on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188) or wild type controls (fswt) were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively) were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine kinase receptor activation. VEGF isoforms are emerging as potential biomarkers for anti-VEGF therapies. Our results reveal novel roles of individual isoforms associated with cancer growth and metastasis and highlight the importance of understanding their diverse actions.

Antiangiogenic therapy based on blocking the actions of vascular endothelial growth factor-A (VEGF) can lead to "normalization" of blood vessels in both animal and human tumors. Differential expression of VEGF isoforms affects tumor vascular maturity, which could influence the normalization process and response to subsequent treatment. Fibrosarcoma cells expressing only VEGF120 or VEGF188 isoforms were implanted either subcutaneously (s.c.) or in dorsal skin-fold "window" chambers in SCID mice. VEGF120 was associated with vascular fragility and hemorrhage. Tumor-bearing mice were treated with repeat doses of SU5416, an indolinone receptor tyrosine kinase inhibitor with activity against VEGFR-2 and proven preclinical ability to induce tumor vascular normalization. SU5416 reduced vascularization in s.c. implants of both VEGF120 and VEGF188 tumors. However, in the window chamber, SU5416 treatment increased red cell velocity in VEGF120 (representing vascular normalization) but not VEGF188 tumors. SU5416 treatment had no effect on growth or necrosis levels in either tumor type but tended to counteract the increase in interstitial fluid pressure seen with growth of VEGF120 tumors. SU5416 pretreatment resulted in the normally fragile blood vessels in VEGF120-expressing tumors becoming resistant to the vascular damaging effects of the tubulin-binding vascular disrupting agent (VDA), combretastatin A4 3-O-phosphate (CA4P). Thus, vascular normalization induced by antiangiogenic treatment can reduce the efficacy of subsequent VDA treatment. Expression of VEGF120 made tumors particularly susceptible to vascular normalization by SU5416, which in turn made them resistant to CA4P. Therefore, VEGF isoform expression may be useful for predicting response to both antiangiogenic and vascular-disrupting therapy.

Introduction The majority of deaths from breast cancer are a result of metastases; however, little is understood about the genetic alterations underlying their onset. Genetic profiling has identified the adhesion molecule plakoglobin as being three-fold reduced in expression in primary breast tumors that have metastasized compared with nonmetastatic tumors. In this study, we demonstrate a functional role for plakoglobin in the shedding of tumor cells from the primary site into the circulation. Methods We investigated the effects of plakoglobin knockdown on breast cancer cell proliferation, migration, adhesion, and invasion in vitro and on tumor growth and intravasation in vivo. MCF7 and T47D cells were stably transfected with miRNA sequences targeting the plakoglobin gene, or scramble vector. Gene and protein expression was monitored by quantitative polymerase chain reaction (qPCR) and Western blot. Cell proliferation, adhesion, migration, and invasion were measured by cell counting, flow cytometry, and scratch and Boyden Chamber assays. For in vivo experiments, plakoglobin knockdown and control cells were inoculated into mammary fat pads of mice, and tumor growth, shedding of tumor cells into the bloodstream, and evidence of metastatic bone lesions were monitored with caliper measurement, flow cytometry, and microcomputed tomography (μCT), respectively. Results Plakoglobin and γ-catenin expression were reduced by more than 80% in all knockdown cell lines used but were unaltered after transfection with the scrambled sequence. Reduced plakoglobin resulted in significantly increased in MCF7 and T47D cell proliferation in vitro and in vivo, compared with control, with significantly more tumor cells being shed into the bloodstream of mice bearing plakoglobin knockdown tumors. In addition, plakoglobin knockdown cells showed a >250% increase in invasion through basement membrane and exhibited reduced cell-to-cell adhesion compared with control cells. Conclusion Decreased plakoglobin expression increases the invasive behavior of breast cancer cells. This is the first demonstration of a functional role for plakoglobin/γ-catenin in the metastatic process, indicating that this molecule may represent a target for antimetastatic therapies.

Combination therapy, using agents that target the microenvironment as well as the cancer cells, is common in the treatment of advanced breast cancer. Here, we show that a 6-week course of weekly sequential administration of the cytotoxic drug doxorubicin (2 mg/kg), followed 24 hr later by the antiresorptive agent zoledronic acid (100 microg/kg), causes substantial inhibition of subcutaneous MDA-MB-436 breast tumor growth in immunocompromised mice, leading to significantly increased survival. Tumor growth did not resume following withdrawal of treatment after 6 weeks, with 60% of the animals in this group surviving for more than 160 days. In comparison, animals receiving single-agent therapy all died within 50 days. Molecular analysis of the tumors showed no effect on cell cycle or apoptosis following administration of 100 microg/kg zoledronic acid or 2 mg/kg doxorubicin alone. When doxorubicin was administered 24 hr before zoledronic acid, tumors displayed decreased expression of CYCLINS E1, B, D1 and D3 as well as CDK2, CDC2, CDK4 and CDK7, indicative of cell-cycle inhibition. Tumors from animals receiving sequential treatment also showed induction of both intrinsic- and extrinsic-apoptotic pathways, with increased expression of BAX, decreased expression of BCL-2 and activation of CASPASE 3, 8 and 9. Accumulation of the unprenylated form of RAP1a, a surrogate marker for uptake of zoledronic acid, was only detected in tumors from animals treated with doxorubicin 24 hr before zoledronic acid. Our data are the first to show a sustained antitumor effect in vivo following a limited course of sequential administration of doxorubicin followed by zoledronic acid.

Combination treatment using the chemotherapy drug doxorubicin and the anti-resorptive agent zoledronic acid has shown to be very effective in inducing apoptosis in breast cancer cells, and also to eradicate breast tumour growth in vivo. Here, we investigated whether apoptotic cell death is increased when zoledronic acid and doxorubicin are given in sequence or in combination in prostate cancer cells in vitro. PC3, DU145 and LNCaP prostate cancer cells were treated with zoledronic acid or doxorubicin alone, in sequence or in combination, and apoptosis was measured by evaluation of nuclear morphology following staining with Hoechst and PI. The involvement of the mevalonate pathway in the induction of apoptosis was assessed through the addition of the mevalonate pathway intermediate geranylgeraniol. Both agents induced PC3 cell death, with 5 μM zoledronic acid inducing 1.73% apoptosis and 50 nM doxorubicin 3.60% apoptosis following 24 h of exposure. In contrast, sequential exposure (doxorubicin followed by zoledronic acid) caused 8.87% apoptosis. Doxorubicin followed by zoledronic acid induced 4.77% apoptosis in LNCaP cells, compared to 1.53% caused by zol alone, 2.23% by dox alone and 2.5% following the reverse sequence (P

Bisphosphonates (BPs) are effective inhibitors of tumor-induced bone resorption. Recent studies have demonstrated that BPs inhibit growth, attachment and invasion of cancer cells in culture and promote apoptosis. The mechanisms responsible for the observed anti-tumor effects of BPs are beginning to be elucidated. Recently, we reported that nitrogen-containing bisphosphonates (N-BPs) induce formation of a novel ATP analog (ApppI) as a consequence of the inhibition of farnesyl diphosphate synthase in the mevalonate pathway. Similar to AppCp-type metabolites of non-N-BPs, ApppI is able to induce apoptosis. This study investigated BP-induced ATP analog formation and its effect on cancer cell growth. To evaluate zoledronic acid (a N-BP)-induced ApppI accumulation, inhibition of protein prenylation and clodronate (a non-N-BP) metabolism to AppCCl2p, MCF-7 and MDA-MB-436 breast cancer cells, MCF-10A nonmalignant breast cells, PC-3 prostate cancer cells, MG-63 osteosarcoma cells, RPMI-8226, and NCI-H929 myeloma cells were treated with 25 micromol/l zoledronic acid or 500 micromol/l clodronate for 24 h. The inhibition of cell growth by zoledronic acid and clodronate was studied in MCF-7, MDA-MB-436, and RPMI-8226 cells by exposing the cells with 1-100 micromol/l zoledronic acid or 10-2000 micromol/l clodronate for 72 h. Marked differences in zoledronic acid-induced ApppI formation and clodronate metabolism between the cancer cell lines were observed. The production of cytotoxic ATP analogs in tumor cells after BP treatment is likely to depend on the activity of enzymes, such as farnesyl diphosphate synthase or aminoacyl-tRNA synthetases, responsible for ATP analog formation. Additionally, the potency of clodronate to inhibit cancer cell growth corresponds to ATP analog formation.

Breast cancer patients commonly receive a combination of different therapies; however, our understanding of how such combined treatments work is incomplete. We have previously shown that sequential administration of the cytotoxic agent doxorubicin (dox) (Pharmachemie BV, Haarlem, The Netherlands) followed by the antiresorptive agent zoledronic acid (zol) (Novartis Pharma, Basel, Switzerland) synergistically increased tumour cell apoptosis in vitro, and also increased tumour cell apoptosis, decreased tumour cell proliferation and reduced subcutaneous in breast tumour growth in vivo. In contrast, pretreating the cells with zol before dox or adding both drugs simultaneously did not cause synergy. The aim of the present study was to determine the mechanism by which sequential administration of dox followed by zol exerts the increased antitumour effects.

We aimed to determine the influence of differential tumor cell expression of vascular endothelial growth factor A (VEGF) isoforms on lung metastasis and response to radiotherapy. We hypothesized that vascular and other adaptations of the tumor microenvironment, in response to tumor cell expression of individual VEGF isoforms, would impact on tumor progression and treatment response. Mouse fibrosarcoma cells that exclusively express either VEGF120, 164 or 188 (fs120, fs164 and fs188 cells respectively) and cells expressing all three isoforms (fsWT) were grown as sub-cutaneous implants in SCID mice. Tumor sections were stained for vascularity (CD31), oxygenation status (pimonidazole protein adducts), activated fibroblasts/pericytes (FAPalpha/alpha-sma), necrosis (H&E), apoptosis (TUNEL) and extra-cellular matrix proteins (collagen-1, laminin, fibronectin). Interstitial fluid pressure (IFP) was measured by the ‘wick-in-needle’ technique. Parallel single VEGF isoform-expressing cell lines were generated to stably express luciferase2 and mStrawberry (LS) for analysis of lung metastasis from sub-cutaneous implants. Luminescence was measured in lung tissue lysates, using an IVIS Lumina II optical imaging system. Sub-cutaneous tumors were irradiated with a single or fractionated 20 Gy dose of X-rays and tumor volume response measured. All tumors were well-vascularized but IFP was lower in untreated fs188 tumors than in fs120 tumors, consistent with a lower vascular permeability, as previously reported. Fs188 tumors were also significantly less hypoxic (P < 0.05) than fs120 tumors (approximately 20 versus 30% pimonidazole staining). Perivascular alpha-SMA was most prevalent in fs188 and fsWT tumors. FAPalpha was lowest in fs120 tumours. Lungs from fs120-LS and fs164-LS but not fs188-LS and fsWT-LS tumor-bearing mice were significantly more luminescent than control lungs (910 ± 283 and 1107 ± 401 versus 8 ± 3 luminescence units respectively). Laminin staining was significantly greater in the more metastatic tumor types (fs120 and fs164) but no other matrix protein showed any correlation. Fs188 tumors were significantly more sensitive to both single and fractionated radiotherapy (growth and necrosis end-points) than fs120 tumors, despite similar radio-sensitivities of the cells irradiated in vitro. However, TUNEL staining in CD31+ cells was greater in fs120 tumors than in fs188 tumors, suggesting that fs120-associated vasculature was more radio-sensitive than fs188-associated vasculature. Results showed that tumor cell expression of individual VEGF isoforms had a profound effect on the tumor microenvironment. The more metastatic fs120 tumors had relatively high IFP and hypoxia, factors known to influence tumor progression. Less pericyte investiture in fs120 and fs164 tumors could also facilitate tumor cell escape into the circulation for these more metastatic types, which may also relate to high levels of laminin in the extra-cellular matrix. The radio-sensitivity of fs188 tumors is most likely explained by relatively low hypoxia, as oxygen is a classic radio-sensitizer. The fact that blood vessels in the fs120 tumors were actually more sensitive that those in fs188 tumors suggests that the response of blood vessels has only a minor influence on the overall tumor response to radio-therapy. These results suggest the potential for differential VEGF isoform expression (or down-stream consequences of expression) as prognostic and predictive tumor bio-markers. This work was supported by a Programme Grant from Cancer Research UK. Citation Format: William R. English, Sarah Jane Lunt, Matthew Fisher, Jack E. Hurrell, Diane V. Lefley, Debayan Mukherjee, Rachel Daniel, Scott K. Lyons, Chryso Kanthou, Gillian M. Tozer. Differential tumor cell expression of VEGF isoforms impacts on the tumor microenvironment, metastasis and radiation response in a mouse fibrosarcoma model. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A57. doi:10.1158/1538-7445.CHTME14-A57

Background Murine fibrosarcoma cell lines that express single VEGF isoforms (VEGF120 VEGF165 and VEGF188) and a wild-type control (VEGFWT) have been developed. The most extreme differences in vascular properties are observed between VEGF120 and VEGF188 tumors, with VEGF120 tumors developing more immature vessels. These differences impact on therapeutic outcome. We hypothesised that differential signaling through VEGF receptors 1 and 2 underlie the isoform-specific differences. Methods VEGFWT, VEGF120 and VEGF164 tumors were implanted sub-cutaneously (s/c), and VEGF188 tumors s/c or in dorsal skin-flap window-chambers (WC) in SCID mice. Mice were treated with ImClone neutralizing antibodies against VEGFR-1 (MF-1) or VEGFR-2 (DC101). Excised s/c tumors were stained for blood vessels (CD31), pericytes (αSMA), necrosis (H&E) or apoptosis (Caspase III). Tumor vascular development in WCs was imaged using intra-vital microscopy. Results VEGFWT and VEGF188 tumors were most sensitive to growth inhibition following treatment with DC101, whereas MF-1 had no effect on growth of any tumor type. In vitro, DC101 had no effect on proliferation of any tumor cell types. Immunohistochemical studies showed no changes in necrosis or apoptosis in VEGF120 tumors, but a significant decrease in vascular area with DC101. VEGF188 tumors showed no change in apoptosis, but both treatments increased necrosis. Vascular area was significantly reduced post DC101 treatment but significantly increased post MF-1 treatment. Both antibodies significantly increased αSMA staining, primarily due to myofibroblast infiltration. WC studies using VEGF188 tumors supported the effects observed with DC101, showing a significantly reduced blood vessel number and length. Furthermore, MF-1 treatment (either immediately post tumor implantation or to established tumors) resulted in more hemorrhagic tumors with large peripheral feeder vessels. Early DC101 treatment impaired tumor development, and once tumors developed on cessation of treatment, the vessels were highly chaotic. Conclusions DC101-induced tumor growth retardation, and corresponding vascular effects in VEGF188 tumors, confirms a role for VEGFR2 signaling in tumor progression and vascular development. However, the lack of growth effect in VEGF120 and VEGF164 tumors following treatment with either antibody suggests a level of receptor redundancy. MF-1 increased angiogenesis in VEGF188 tumors, despite no change in growth rate, and these neo-vessels appeared hemorrhagic, implying increased permeability. These data suggest a role for VEGFR-1 in tumor angiogenesis, involving vascular maturation, at least where VEGF188 is highly expressed. This may occur via specific VEGFR-1 signaling and/or sequestration of VEGF to control signaling via VEGFR-2. Acknowledgements Supported by CR-UK We thank ImClone Systems for providing MF-1 and DC101. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5296. doi:1538-7445.AM2012-5296

Abstract #2151 Background: Recent reports suggest that zoledronic acid (Zometa®) may have anti-tumour effects outside the skeleton. We have previously shown that administration of doxorubicin (dox) 24h prior to zoledronic acid (zol) is critical for the inhibition of growth of established breast tumours implanted subcutaneously and intra-osseously. Inhibition of tumour growth was associated with reduced tumour cell proliferation and increased apoptosis in vivo. This is the first report of the potential molecular mechanisms by which doxorubicin and zoledronic acid exert their sequence-specific anti-tumour effects. Materials and methods: MDA-MB-436-GFP cells were inoculated into the flank (n=10/group) or tibia (n=8/group) of female MF1 nude mice. Mice were treated 1x per week for 6 weeks with saline, 2mg/kg dox, 100μg/kg zol, dox and zol simultaneously, dox followed 24h later by zol, or zol followed 24h later by dox. Animals were sacrificed 24h following the final treatment and tumour RNA prepared. Biotin labelled tumour RNA from each treatment group was hybridised to a GEArray cell cycle and an apoptosis pathway specific microarray (n=3 tumours/array). Changes in gene expression of 2 fold or greater were confirmed by qPCR and Western blotting. Results: Sequential treatment with dox 24h prior to zol reduced sub-cutaneous and intra-tibial tumour volume. Molecular analysis of these tumours showed equivalent effects on cell cycle and apoptosis related genes compared with tumours treated with dox or zol alone, sequential administration caused a cell cycle block at G2, G2/M, G1 and G1/S, with an increased expression of p53 and p21 accompanied by a reduction in cyclins E1, B, D1 and D3 as well as their related cyclin dependent kinases CDK2, CDC2, CDK4 and CDK7. Tumours treated sequentially with dox followed by zol also showed an induction in both extrinsic and intrinsic apoptotic pathways, with increased expression of Bax, decreased expression of Bcl-2 and an increase in caspase 8, caspase 9 and caspase 3 cleavage. In sub-cutaneous tumours administration of 100g/μkg zol showed no effect on tumour cell cycle or apoptosis related genes. 2mg/kg dox caused a cell cycle block at G1-S with a reduction in expression of cyclin E/CDK2, whereas apoptosis-related genes were unaffected. Treatment of intra-tibial tumours with 100μg/kg zol resulted in a reduction in tumour cell proliferation as well as reduced expression of cyclins D1 and D3. No alterations in cell cycle or apoptosis related gene expression were detected in intra-tibial tumours treated with 2mg/kg dox. Conclusions: This is the first report showing that sequential treatment of both subcutaneous and intra-tibial breast tumours in vivo with doxorubicin followed by zoledronic acid induces changes in expression of a number of specific genes and proteins associated with regulation of the cell cycle and apoptosis, which were unaffected by treatment with the single agents. Our data suggest that breast cancer patients may benefit from receiving zoledronic acid 24h after administration of anthracycline therapy. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2151.

Breast cancer patients commonly receive a combination of different therapies; for patients with late-stage breast cancer involving metastasis to the bone, a chemotherapeutic agent is usually given in combination with the antiresorptive drug zoledronic acid (zol) (Novartis Pharma, Basel, Switzerland). We have previously reported that administration of doxorubicin (dox) (Pharachemie BV, Haarlem, The Netherlands) 24 hours prior to zol inhibits subcutaneous breast tumour growth, inhibits tumour cell proliferation and increases apoptosis in vivo. The aims of the present study were to determine the mechanisms by which dox and zol exert their synergistic antitumour effects.

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