Bone metastasis: mechanisms, therapies, and biomarkers.

Skeletal metastases are frequent complications of many cancers, causing bone complications (fractures, bone pain, disability) that negatively affect the patient's quality of life. Here, we first discuss the burden of skeletal complications in cancer bone metastasis. We then describe the pathophysiology of bone metastasis. Bone metastasis is a multistage process: long before the development of clinically detectable metastases, circulating tumor cells settle and enter a dormant state in normal vascular and endosteal niches present in the bone marrow, which provide immediate attachment and shelter, and only become active years later as they proliferate and alter the functions of bone-resorbing (osteoclasts) and bone-forming (osteoblasts) cells, promoting skeletal destruction. The molecular mechanisms involved in mediating each of these steps are described, and we also explain how tumor cells interact with a myriad of interconnected cell populations in the bone marrow, including a rich vascular network, immune cells, adipocytes, and nerves. We discuss metabolic programs that tumor cells could engage with to specifically grow in bone. We also describe the progress and future directions of existing bone-targeted agents and report emerging therapies that have arisen from recent advances in our understanding of the pathophysiology of bone metastases. Finally, we discuss the value of bone turnover biomarkers in detection and monitoring of progression and therapeutic effects in patients with bone metastasis.

MiR-662 is associated with metastatic relapse in early-stage breast cancer and promotes metastasis by stimulating cancer cell stemness.

BACKGROUND: Breast cancer (BC) metastasis, which often occurs in bone, contributes substantially to mortality. MicroRNAs play a fundamental role in BC metastasis, although microRNA-regulated mechanisms driving metastasis progression remain poorly understood. METHODS: MiRome analysis in serum from BC patients was performed by TaqMan™ low-density array. MiR-662 was overexpressed following MIMIC-transfection or lentivirus transduction. Animal models were used to investigate the role of miR-662 in BC (bone) metastasis. The effect of miR-662-overexpressing BC cell conditioned medium on osteoclastogenesis was investigated. ALDEFLUOR assays were performed to study BC stemness. RNA-sequencing transcriptomic analysis of miR-662-overexpressing BC cells was performed to evaluate gene expression changes. RESULTS: High levels of hsa-miR-662 (miR-662) in serum from BC patients, at baseline (time of surgery), were associated with future recurrence in bone. At an early-stage of the metastatic disease, miR-662 could mask the presence of BC metastases in bone by inhibiting the differentiation of bone-resorbing osteoclasts. Nonetheless, metastatic miR-662-overexpressing BC cells then progressed as overt osteolytic metastases thanks to increased stem cell-like traits. CONCLUSIONS: MiR-662 is involved in BC metastasis progression, suggesting it may be used as a prognostic marker to identify BC patients at high risk of metastasis.

IL-1ß in breast cancer bone metastasis.

Bone is the most common site for advanced breast cancer to metastasise. The proinflammatory cytokine, interleukin-1ß (IL-1ß) plays a complex and contradictory role in this process. Recent studies have demonstrated that breast cancer patients whose primary tumours express IL-1ß are more likely to experience relapse in bone or other organs. Importantly, IL-1ß 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-1ß 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-1ß on breast cancer bone metastasis: specifically, we discuss the distinct effects of IL-1ß 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.

Development of clinically relevant in vivo metastasis models using human bone discs and breast cancer patient-derived xenografts.

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.

The IL-1/IL-1R axis induces greater fibroblast-derived chemokine release in human papillomavirus-negative compared to positive oropharyngeal cancer.

Human papillomavirus (HPV) is now recognised as a major aetiological agent in the pathogenesis of oropharyngeal carcinoma (OPC). HPV-positive tumours are associated with better outcomes compared to HPV-negative tumours, possibly due to differences in their aetiology and/or the tumour microenvironment. Increased numbers of tumour-associated leukocytes have been observed in many cancers including OPC, with variable influence on prognosis depending on the leukocyte subpopulation investigated. Whether HPV status influences leukocyte recruitment to OPC remains unknown. This in-vitro study examined differences in the chemoattractant capacity of HPV-positive and HPV-negative OPC cell lines. Gene and protein expression analysis demonstrated that whilst both monocultures of HPV-positive and HPV-negative cell lines, along with normal tonsillar fibroblasts (NTF), expressed low chemokine levels, NTF cultured with conditioned medium from HPV-negative OPC cells expressed significantly higher levels of all chemokines tested compared to NTF incubated with the medium from HPV-positive OPC cell lines. HPV-negative OPC lines expressed IL-1ß mRNA whereas HPV-positive cells did not, and NTF constitutively expressed IL-1R1. Pre-treatment with the IL-R antagonist, anakinra or siRNA to IL-1R1 significantly reduced chemokine secretion from NTF stimulated with conditioned medium from HPV-negative tumour cells or recombinant IL-1ß (p < 0.05). These data suggest that secretion of chemokines is driven by the interaction between HPV-negative OPC cells and stromal fibroblasts through an IL-1/IL-1R-mediated mechanism that is less prominent within the HPV-positive tumour microenvironment. These observations may explain differences in leukocyte sub-populations recruited to HPV-positive versus negative OPC and indicate that HPV status is a key determinant in controlling the inflammatory tumour microenvironment.

Parathyroid Hormone (PTH) Increases Skeletal Tumour Growth and Alters Tumour Distribution in an In Vivo Model of Breast Cancer.

Breast cancer cells colonize the skeleton by homing to specific niches, but the involvement of osteoblasts in tumour cell seeding, colonization, and progression is unknown. We used an in vivo model to determine how increasing the number of cells of the osteoblast lineage with parathyroid hormone (PTH) modified subsequent skeletal colonization by breast cancer cells. BALB/c nude mice were injected for five consecutive days with PBS (control) or PTH and then injected with DiD-labelled breast cancer cells via the intra-cardiac route. Effects of PTH on the bone microenvironment and tumour cell colonization and growth was analyzed using bioluminescence imaging, two-photon microscopy, and histological analysis. PTH treatment caused a significant, transient increase in osteoblast numbers compared to control, whereas bone volume/structure in the tibia was unaffected. There were no differences in the number of tumour cells seeding to the tibias, or in the number of tumours in the hind legs, between the control and PTH group. However, animals pre-treated with PTH had a significantly higher number of tumour colonies distributed throughout skeletal sites outside the hind limbs. This is the first demonstration that PTH-induced stimulation of osteoblastic cells may result in alternative skeletal sites becoming available for breast cancer cell colonization.

Mitotic quiescence, but not unique "stemness," marks the phenotype of bone metastasis-initiating cells in prostate cancer.

This study aimed to identify subpopulations of prostate cancer cells that are responsible for the initiation of bone metastases. Using rapidly dividing human prostate cancer cell lines, we identified mitotically quiescent subpopulations (<1%), which we compared with the rapidly dividing populations for patterns of gene expression and for their ability to migrate to the skeletons of athymic mice. The study used 2-photon microscopy to map the presence/distribution of fluorescently labeled, quiescent cells and luciferase expression to determine the presence of growing bone metastases. We showed that the mitotically quiescent cells were very significantly more tumorigenic in forming bone metastases than fast-growing cells (55 vs. 15%) and had a unique gene expression profile. The quiescent cells were not uniquely stem cell like, with no expression of CD133 but had the same level expression of other putative prostate stem cell markers (CD44 and integrins α2/ß1), when compared to the rapidly proliferating population. In addition, mitotic quiescence was associated with very high levels of C-X-C chemokine receptor type 4 (CXCR4) production. Inhibition of CXCR4 activity altered the homing of quiescent tumor cells to bone. Our studies suggest that mitotic dormancy is a unique phenotype that facilitates tumor cell colonization of the skeleton in prostate cancer.

Lysophosphatidic acid receptor type 1 (LPA1) plays a functional role in osteoclast differentiation and bone resorption activity.

Lysophosphatidic acid (LPA) is a natural bioactive lipid that acts through six different G protein-coupled receptors (LPA1-6) with pleiotropic activities on multiple cell types. We have previously demonstrated that LPA is necessary for successful in vitro osteoclastogenesis of bone marrow cells. Bone cells controlling bone remodeling (i.e. osteoblasts, osteoclasts, and osteocytes) express LPA1, but delineating the role of this receptor in bone remodeling is still pending. Despite Lpar1(-/-) mice displaying a low bone mass phenotype, we demonstrated that bone marrow cell-induced osteoclastogenesis was reduced in Lpar1(-/-) mice but not in Lpar2(-/-) and Lpar3(-/-) animals. Expression of LPA1 was up-regulated during osteoclastogenesis, and LPA1 antagonists (Ki16425, Debio0719, and VPC12249) inhibited osteoclast differentiation. Blocking LPA1 activity with Ki16425 inhibited expression of nuclear factor of activated T-cell cytoplasmic 1 (NFATc1) and dendritic cell-specific transmembrane protein and interfered with the fusion but not the proliferation of osteoclast precursors. Similar to wild type osteoclasts treated with Ki16425, mature Lpar1(-/-) osteoclasts had reduced podosome belt and sealing zone resulting in reduced mineralized matrix resorption. Additionally, LPA1 expression markedly increased in the bone of ovariectomized mice, which was blocked by bisphosphonate treatment. Conversely, systemic treatment with Debio0719 prevented ovariectomy-induced cancellous bone loss. Moreover, intravital multiphoton microscopy revealed that Debio0719 reduced the retention of CX3CR1-EGFP(+) osteoclast precursors in bone by increasing their mobility in the bone marrow cavity. Overall, our results demonstrate that LPA1 is essential for in vitro and in vivo osteoclast activities. Therefore, LPA1 emerges as a new target for the treatment of diseases associated with excess bone loss.

OPG-Fc inhibits ovariectomy-induced growth of disseminated breast cancer cells in bone.

Dormant disseminated tumour cells can be detected in the bone marrow of breast cancer patients several years after resection of the primary tumour. The majority of these patients will remain asymptomatic, however, ∼ 15% will go on to develop overt bone metastases and this condition is currently incurable. The reason why these dormant cells are stimulated to proliferate and form bone tumours in some patients and not others remains to be elucidated. We have recently shown that in an in vivo model, increasing bone turnover by ovariectomy stimulated proliferation of disseminated tumour cells, resulting in formation of bone metastasis. We now show for the first time that osteoclast mediated mechanisms induce growth of tumours from dormant MDA-MB-231 cells disseminated in the bone. We also show that disruption of RANK-RANKL interactions following administration of OPG-Fc inhibits growth of these dormant tumour cells in vivo. Our data support early intervention with anti-resorptive therapy in a low-oestrogen environment to prevent development of bone metastases.

The differential anti-tumour effects of zoledronic acid in breast cancer - evidence for a role of the activin signaling pathway.

BACKGROUND: Neo-adjuvant breast cancer clinical trials of zoledronic acid (ZOL) have shown that patients with oestrogen negative (ER-ve) tumours have improved disease outcomes. We investigated the molecular mechanism behind this differential anti-tumour effect according to ER status, hypothesising it may in part be mediated via the activin signaling pathway. METHODS: The effects of activin A, its inhibitor follistatin and zoledronic acid on proliferation of breast cancer cells was evaluated using either an MTS proliferation assay or trypan blue. Secretion of activin A and follistatin in conditioned medium (CM) from MDA-MB-231, MDA-MB-436, MCF7 and T47D cell lines were measured using specific ELISAs. The effects of ZOL on phosphorylation domains of Smad2 (pSmad2c + pSmad2L) were evaluated using immunofluorescence. Changes seen in vitro were confirmed in a ZOL treated subcutaneous ER-ve MDA-MB-436 xenograft model. RESULTS: Activin A inhibits proliferation of both ER-ve and oestrogen positive (ER + ve) breast cancer cells, an effect impaired by follistatin. ZOL significantly inhibits proliferation and the secretion of follistatin from ER-ve cells only, which increases the biological activity of the canonical activin A pathway by significantly increasing intracellular pSmad2c and decreasing nuclear accumulation of pSmad2L. In vivo, ZOL significantly decreases follistatin and pSmad2L expression in ER-ve subcutaneous xenografts compared to saline treated control animals. CONCLUSIONS: This is the first report showing a differential effect of ZOL, according to ER status, on the activin pathway and its inhibitors in vitro and in vivo. These data suggest a potential molecular mechanism contributing to the differential anti-tumour effects reported from clinical trials and requires further evaluation in clinical samples.

The role of IL-1B in breast cancer bone metastasis.

Interleukin-1B (IL-1B) is a potent pro-inflammatory cytokine that plays multiple, pivotal roles, in the complex interplay between breast cancer cells and the bone microenvironment. IL-1B is involved in the growth of the primary tumours, regulation of inflammation within the tumour microenvironment, promotion of epithelial to mesenchymal transition (EMT), migration and invasion. Moreover, when breast cancer cells arrive in the bone microenvironment there is an upregulation of IL-1B which promotes the creation of a conducive niche for metastatic breast cancer cells as well as stimulating initiation of the vicious cycle of bone metastasis. Pre-clinical studies have demonstrated that inhibition of IL-1 signalling reduces bone metastasis from oestrogen receptor positive/triple-negative breast cancers in various mouse models. However, effects on primary tumours and soft tissue metastasis remain controversial with some studies showing increased tumour growth in these sites, whilst others show no effects. Notably, combining anti-IL-1 therapy with standard-of-care treatments, such as chemotherapy and immunotherapy, has been demonstrated to reduce the growth of primary tumours, bone metastasis, as well as metastatic outgrowth in other organs. This review focuses on the mechanisms by which IL-1B promotes breast cancer bone metastasis.

The CDK4/6 Inhibitor Palbociclib Inhibits Estrogen-Positive and Triple Negative Breast Cancer Bone Metastasis In Vivo.

CDK 4/6 inhibitors have demonstrated significant improved survival for patients with estrogen receptor (ER) positive breast cancer (BC). However, the ability of these promising agents to inhibit bone metastasis from either ER+ve or triple negative BC (TNBC) remains to be established. We therefore investigated the effects of the CDK 4/6 inhibitor, palbociclib, using in vivo models of breast cancer bone metastasis. In an ER+ve T47D model of spontaneous breast cancer metastasis from the mammary fat pad to bone, primary tumour growth and the number of hind limb skeletal tumours were significantly lower in palbociclib treated animals compared to vehicle controls. In the TNBC MDA-MB-231 model of metastatic outgrowth in bone (intracardiac route), continuous palbociclib treatment significantly inhibited tumour growth in bone compared to vehicle. When a 7-day break was introduced after 28 days (mimicking the clinical schedule), tumour growth resumed and was not inhibited by a second cycle of palbociclib, either alone or when combined with the bone-targeted agent, zoledronic acid (Zol), or a CDK7 inhibitor. Downstream phosphoprotein analysis of the MAPK pathway identified a number of phosphoproteins, such as p38, that may contribute to drug-insensitive tumour growth. These data encourage further investigation of targeting alternative pathways in CDK 4/6-insensitive tumour growth.

Loss of plakoglobin promotes decreased cell-cell contact, increased invasion, and breast cancer cell dissemination in vivo.

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 inhibits development and progression of mammary tumours in immunocompetent mice.

We have determined the effect of combining the chemotherapy agent doxorubicin and the anti-resorptive drug zoledronic acid on the early stages of spontaneous mammary tumour development using the immunocompetent PyMT mouse model that closely mimics human breast cancer development. 6-week-old PyMT mice were treated weekly for 6 weeks with PBS, 2 mg/kg doxorubicin, 100 µg/kg zoledronic acid or doxorubicin followed 24 h later by zoledronic acid (n = 15/group). Untreated, 6-week-old animals were used for comparison of tumour development. Tumour volume, apoptosis and angiogenesis were analysed on H&E, caspase 3, CD31 and CD34 stained histological sections. Proliferation was measured by BrdU incorporation and Ki67 staining and tumour macrophage infiltration assessed by F4/80 immunohistochemistry. Animals treated with doxorubicin followed by zoledronic acid did not develop palpable mammary tumours, whereas in all other treatment groups tumours progressed to late stage adenocarcinomas. Tumours from the combination treatment group were of comparable size to those in 6-week-old untreated animals, remaining pre-malignant with well-differentiated acinar arrangements and with tumour volume only reaching on average 26% of that in the PBS control group. Tumour cell apoptosis and proliferation was significantly reduced compared to control, single agent or untreated 6-week-old mice. Significantly fewer circulating tumour cells were present in animals following sequential treatment compared to all other groups. Combination treatment with doxorubicin followed by zoledronic acid inhibits development and progression of spontaneously occurring mammary tumours.

Novel Methods of Targeting IL-1 Signalling for the Treatment of Breast Cancer Bone Metastasis.

Breast cancer bone metastasis is currently incurable. Evidence suggests that inhibiting IL-1 signalling with the IL1R antagonist, Anakinra, or the IL1ß 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, IL1ß or IRAK1) could reduce bone metastases without increasing tumour growth outside of the bone. Inhibition of IL-1 via MLX01 (IL1ß 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 IL1ß 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 kinetochore protein Cenp-F is a potential novel target for zoledronic acid in breast cancer cells.

The anti-resorptive agent zoledronic acid inhibits key enzymes in the mevalonate pathway, disrupting post-translational modification and thereby correct protein localization and function. Inhibition of prenylation may also be responsible for the reported anti-tumour effects of zoledronic acid, but the specific molecular targets have not been identified. Cenp-F/mitosin, a kinetochore-associated protein involved in the correct separation of chromosomes during mitosis, has been shown to undergo post-translational prenylation and may therefore be a novel target contributing to the anti-tumour effects of zoledronic acid. We investigated whether zoledronic acid causes loss of Cenp-F from the kinetochore in breast cancer cells, to determine if the reported anti-tumour effects may be mediated by impairing correct chromosome separation. MDA-MB-436, MDA-MB-231 and MCF-7 breast cancer cells and MCF-10A non-malignant breast epithelial cells were treated with zoledronic acid in vitro, and the effect on Cenp-F localization was analysed by immunoflourescence microscopy. Zoledronic acid caused loss of Cenp-F from the kinetochore, accompanied by an increase in the number of cells in pro-, /prometa- and metaphase in all of the cancer cell lines. There was also a significant increase in the number of lagging chromosomes in mitotic cells. The effects of zoledronic acid could be reversed by inclusion of an intermediary of the mevalonate pathway, showing that the loss of Cenp-F from the kinetochore was caused by the inhibition of farnesylation. In contrast, no effect was seen on Cenp-F in non-malignant MCF-10A cells. This is the first report showing a specific effect of zoledronic acid on a protein involved in the regulation of chromosome segregation, identifying Cenp-F as a potential new molecular target for NBPs in tumour cells.

Transcriptomic Profiling Reveals Novel Candidate Genes and Signalling Programs in Breast Cancer Quiescence and Dormancy.

Metastatic recurrence, the major cause of breast cancer mortality, is driven by reactivation of dormant disseminated tumour cells that are defined by mitotic quiescence and chemoresistance. The molecular mechanisms underpinning mitotic quiescence in cancer are poorly understood, severely limiting the development of novel therapies for removal of residual, metastasis-initiating tumour cells. Here, we present a molecular portrait of the quiescent breast cancer cell transcriptome across the four main breast cancer sub-types (luminal, HER2-enriched, basal-like and claudin-low) and identify a novel quiescence-associated 22-gene signature using an established lipophilic-dye (Vybrant® DiD) retention model and whole-transcriptomic profiling (mRNA-Seq). Using functional association network analysis, we elucidate the molecular interactors of these signature genes. We then go on to demonstrate that our novel 22-gene signature strongly correlates with low tumoural proliferative activity, and with dormant disease and late metastatic recurrence (≥5 years after primary tumour diagnosis) in metastatic breast cancer in multiple clinical cohorts. These genes may govern the formation and persistence of disseminated tumour cell populations responsible for breast cancer recurrence, and therefore represent prospective novel candidates to inform future development of therapeutic strategies to target disseminated tumour cells in breast cancer, eliminate minimal residual disease and prevent metastatic recurrence.

Oestradiol Contributes to Differential Antitumour Effects of Adjuvant Zoledronic Acid Observed Between Pre- and Post-Menopausal Women.

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.

Increased Abundance of Tumour-Associated Neutrophils in HPV-Negative Compared to HPV-Positive Oropharyngeal Squamous Cell Carcinoma Is Mediated by IL-1R Signalling.

The incidence of human papillomavirus (HPV)-associated cancer is increasing and HPV is now implicated in the aetiology of more than 60% of all oropharyngeal squamous cell carcinomas (OPSCC). In OPSCC, innate immune cells such as neutrophils and macrophages generally correlate with poor prognosis, whilst adaptive immune cells, such as lymphocytes, tend to correlate with improved prognosis. This may, in part, be due to differences in the immune response within the tumour microenvironment leading to the recruitment of specific tumour-associated leukocyte sub-populations. In this study, we aimed to examine if differences exist in the levels of infiltrated leukocyte sub-populations, with particular emphasis on tumour-associated neutrophils (TAN), and to determine the mechanism of chemokine-induced leukocyte recruitment in HPV-positive compared to HPV-negative OPSCC. Immunohistochemical analysis showed that HPV-negative OPSCC contained significantly more neutrophils than HPV-positive tumours, whilst levels of CD68+ macrophages and CD3+ lymphocytes were similar. Using a 3D tissue culture model to represent tumour-stromal interactions, we demonstrated that HPV-negative tumour-stromal co-cultures expressed significantly higher levels of CXCL8, leading to increased neutrophil recruitment compared to their HPV-positive counterparts. HPV-negative OPSCC cells have previously been shown to express higher levels of IL-1 than their HPV-positive counterparts, indicating that this cytokine may be responsible for driving increased chemokine production in the HPV-negative 3D model. Inhibition of IL-1R in the tumour-stromal models using the receptor-specific antagonist, anakinra, dramatically reduced chemokine secretion and significantly impaired neutrophil and monocyte recruitment, suggesting that this tumour-stromal response is mediated by the IL-1/IL-1R axis. Here, we identify a mechanism by which HPV-negative OPSCC may recruit more TAN than HPV-positive OPSCC. Since TAN are associated with poor prognosis in OPSCC, our study identifies potential therapeutic targets aimed at redressing the chemokine imbalance to reduce innate immune cell infiltration with the aim of improving patient outcome.