Promotes Breast Cancer Bone Metastasis Research Articles

Lymphotoxin-β promotes breast cancer bone metastasis colonization and osteolytic outgrowth.

Bone metastasis is a lethal consequence of breast cancer. Here we used single-cell transcriptomics to investigate the molecular mechanisms underlying bone metastasis colonization-the rate-limiting step in the metastatic cascade. We identified that lymphotoxin-β (LTβ) is highly expressed in tumour cells within the bone microenvironment and this expression is associated with poor bone metastasis-free survival. LTβ promotes tumour cell colonization and outgrowth in multiple breast cancer models. Mechanistically, tumour-derived LTβ activates osteoblasts through nuclear factor-κB2 signalling to secrete CCL2/5, which facilitates tumour cell adhesion to osteoblasts and accelerates osteoclastogenesis, leading to bone metastasis progression. Blocking LTβ signalling with a decoy receptor significantly suppressed bone metastasis in vivo, whereas clinical sample analysis revealed significantly higher LTβ expression in bone metastases than in primary tumours. Our findings highlight LTβ as a bone niche-induced factor that promotes tumour cell colonization and osteolytic outgrowth and underscore its potential as a therapeutic target for patients with bone metastatic disease.

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.

Circular RNA circIKBKB promotes breast cancer bone metastasis through sustaining NF-\u03baB/bone remodeling factors signaling

BackgroundBreast cancer (BC) has a marked tendency to spread to the bone, resulting in significant skeletal complications and mortality. Recently, circular RNAs (circRNAs) have been reported to contribute to cancer initiation and progression. However, the function and mechanism of circRNAs in BC bone metastasis (BC-BM) remain largely unknown.MethodsBone-metastatic circRNAs were screened using circRNAs deep sequencing and validated using in situ hybridization in BC tissues with or without bone metastasis. The role of circIKBKB in inducing bone pre-metastatic niche formation and bone metastasis was determined using osteoclastogenesis, immunofluorescence and bone resorption pit assays. The mechanism underlying circIKBKB-mediated activation of NF-κB/bone remodeling factors signaling and EIF4A3-induced circIKBKB were investigated using RNA pull-down, luciferase reporter, chromatin isolation by RNA purification and enzyme-linked immunosorbent assays.ResultsWe identified that a novel circRNA, circIKBKB, was upregulated significantly in bone-metastatic BC tissues. Overexpressing circIKBKB enhanced the capability of BC cells to induce formation of bone pre-metastatic niche dramatically by promoting osteoclastogenesis in vivo and in vitro. Mechanically, circIKBKB activated NF-κB pathway via promoting IKKβ-mediated IκBα phosphorylation, inhibiting IκBα feedback loop and facilitating NF-κB to the promoters of multiple bone remodeling factors. Moreover, EIF4A3, acted acting as a pre-mRNA splicing factor, promoted cyclization of circIKBKB by directly binding to the circIKBKB flanking region. Importantly, treatment with inhibitor eIF4A3-IN-2 reduced circIKBKB expression and inhibited breast cancer bone metastasis effectively.ConclusionWe revealed a plausible mechanism for circIKBKB-mediated NF-κB hyperactivation in bone-metastatic BC, which might represent a potential strategy to treat breast cancer bone metastasis.

MAPK1/3 kinase-dependent ULK1 degradation attenuates mitophagy and promotes breast cancer bone metastasis

ABSTRACT The function of mitophagy in cancer is controversial. ULK1 is critical for induction of macroautophagy/autophagy and has a more specific role in mitophagy in response to hypoxia. Here, we show that ULK1 deficiency induces an invasive phenotype of breast cancer cells under hypoxia and increases osteolytic bone metastasis. Mechanistically, ULK1 depletion attenuates mitophagy ability during hypoxia. As a result, the accumulation of damaged, ROS-generating mitochondria leads to activation of the NLRP3 inflammasome, which induces abnormal soluble cytokines secretion, then promotes the differentiation and maturation of osteoclasts, and ultimately results in bone metastasis. Notably, phosphorylation of ULK1 by MAPK1/ERK2-MAPK3/ERK1 kinase triggers its interaction with BTRC and subsequent K48-linked ubiquitination and proteasome degradation. Also, a clearly negative correlation between the expression levels of ULK1 and p-MAPK1/3 was observed in human breast cancer tissues. The MAP2K/MEK inhibitor trametinib is sufficient to restore mitophagy function via upregulation of ULK1, leading to inhibition of NLRP3 inflammasome activation, thereby reduces bone metastasis. These results indicate that ULK1 knockout-mediated mitophagy defect promotes breast cancer bone metastasis and provide evidence to explore MAP2K/MEK- MAPK1/3 pathway inhibitors for therapy, especially in cancers displaying low levels of ULK1. Abbreviations: ATG: autophagy-related; Baf A1: bafilomycin A1; BTRC/β-TrCP: beta-transducin repeat containing E3 ubiquitin protein ligase; CHX: cycloheximide; CM: conditioned media; FBXW7/FBW7: F-box and WD repeat domain containing 7; MAPK1: mitogen-activated protein kinase 1; MTDR: MitoTracker Deep Red; mtROS: mitochondrial reactive oxygen species; microCT: micro-computed tomography; mtROS: mitochondrial reactive oxygen species; OCR: oxygen consumption rate; SQSTM1: sequestosome 1; ACP5/TRAP: acid phosphatase, tartrate resistant; ULK1: unc-51 like autophagy activating kinase 1

Abstract 3844: EZH2 engages TGF-β signaling to promote breast cancer bone metastasis

Abstract Metastasis, the dissemination of cancer cells to second organs, is an important hallmark of cancer. About 90% of cancer patients die of metastasis and approximately 55% of breast cancer patients develop bone metastases. However, prevention or treatment of bone metastasis remain challenging. To improve the clinical outcome and the quality of life of breast cancer patients with bone metastasis, the mechanisms of bone metastasis needs to be better understood. Enhancer of zeste homolog 2 (EZH2) is the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2). EZH2 is a histone methyl transferase that generally suppresses downstream target genes through tri-methylation of H3K27, but it also can work as a transcription co-activator. It is frequently over expressed in breast cancer and in bone metastasis of prostate cancer. To explore the function of EZH2 in breast cancer bone metastasis, we knocked down EZH2 by shRNAs in a MDA-MB-231 bone-seeking subline (231.1566. shEZH#3) and intra-cardiaclly injected into nude mice with 231.1566.shEZH#3 and its control cells (231.1566.shCtrl). Mice injected with 231.1566.shEZH#3 have longer bone metastasis free survival than the control mice. Furthermore, we generated a MDA.MB.231 subline that had CRISPR/Cas9-mediated EZH2-knockout (231.KO) and stably re-expressed in the 231.KO cells the wild-type EZH2 (231.KO.EZH2), or the pLenti control vector (231.KO.pLenti). These two derived sublines were intracardiaclly injected into nude mice, and the mice injected with 231.KO.EZH2 had poorer bone metastasis free survival than the control mice (231.KO.pLenti). Both loss-of-function and gain-of-function experiments demonstrated that EZH2 promotes breast cancer bone metastasis. To gain initial insight on the underlying mechanism of EZH2-induced bone metastasis, we co-cultured MDA-MB-231 cells or 231.KO with pre-osteoclasts (RAW264.7) and osteoblasts (MC3T3) under TGF-β treatment (triple co-culture) to mimic bone metastasis tumor microenvironment. We examined osteoclast maturation and breast cancer cell proliferation because osteolysis induced by mature osteoclasts is critical for breast cancer cell growth in the bone metastasis. In triple co-culture of MDA.MB.231 cells, more RAW264.7 pre-osteoclasts were differentiated to mature osteoclasts, as measured by TRAP+ staining, a marker of mature osteoclasts, and MDA.MB.231 cells grew faster than the 231.KO cells in triple co-culture. In addition, we found that the mRNA level of PTHrP, a master regulator of the “vicious cycle” of bone destruction, was expressed at significantly higher level in MDA.MB.231 cells than that in 231.KO cells under TGF-β stimulation. We also detected higher p-Smad2, the downstream of TGF-β in MDA.MB.231 cells than that in 231.KO cells. The data indicated that EZH2 promotes breast cancer bone metastasis through TGF-β/p-Smad2/PTHrP pathway. Currently, we are exploring how EZH2 regulates p-Smad2 in breast cancer cells to promote bone metastasis. Our studies may reveal novel biomarkers and/or therapeutic targets for bone metastasis. Citation Format: Yu-Wen Huang, Lin Zhang, Jingkun Qu, Zhifen Zhou, Dihua Yu. EZH2 engages TGF-β signaling to promote breast cancer bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3844.

Serum- and Glucocorticoid-inducible Kinase 1 is Essential for Osteoclastogenesis and Promotes Breast Cancer Bone Metastasis.

Bone metastasis is a severe complication associated with various carcinomas. It causes debilitating pain and pathologic fractures and dramatically impairs patients' quality of life. Drugs aimed at osteoclast formation significantly reduce the incidence of skeletal complications and are currently the standard treatment for patients with bone metastases. Here, we reported that serum- and glucocorticoid-inducible kinase 1 (SGK1) plays a pivotal role in the formation and function of osteoclasts by regulating the Ca2+ release-activated Ca2+ channel Orai1. We showed that SGK1 inhibition represses osteoclastogenesis in vitro and prevents bone loss in vivo Furthermore, we validated the effect of SGK1 on bone metastasis by using an intracardiac injection model in mice. Inhibition of SGK1 resulted in a significant reduction in bone metastasis. Subsequently, the Oncomine and the OncoLnc database were employed to verify the differential expression and the association with clinical outcome of SGK1 gene in patients with breast cancer. Our data mechanistically demonstrated the regulation of the SGK1 in the process of osteoclastogenesis and revealed SGK1 as a valuable target for curing bone metastasis diseases.

Overexpression of CENPF correlates with poor prognosis and tumor bone metastasis in breast cancer

BackgroundCentromere Protein F (CENPF) associates with the centromere–kinetochore complex and influences cell proliferation and metastasis in several cancers. The role of CENPF in breast cancer (BC) bone metastasis remains unclear.MethodsUsing the ONCOMINE database, we compared the expression of CENPF in breast cancer and normal tissues. Findings were confirmed in 60 BC patients through immunohistochemical (IHC) staining. Microarray data from GEO and Kaplan–Meier plots were used analyze the overall survival (OS) and relapse free survival (RFS). Using the GEO databases, we compared the expression of CENPF in primary lesions, lung metastasis lesions and bone metastasis lesions, and validated our findings in BALB/C mouse 4T1 BC models. Based on gene set enrichment analysis (GSEA) and western blot, we predicted the mechanisms by which CENPF regulates BC bone metastasis.ResultsThe ONCOMINE database and immunohistochemical (IHC) showed higher CENPF expression in BC tissue compared to normal tissue. Kaplan–Meier plots also revealed that high CENPF mRNA expression correlated to poor survival and shorter progression-free survival (RFS). From BALB/C mice 4T1 BC models and the GEO database, CENPF was overexpressed in primary lesions, other target organs, and in bone metastasis. Based on gene set enrichment analysis (GSEA) and western blot, we predicted that CENPF regulates the secretion of parathyroid hormone-related peptide (PTHrP) through its ability to activate PI3K–AKT–mTORC1.ConclusionCENPF promotes BC bone metastasis by activating PI3K–AKT–mTORC1 signaling and represents a novel therapeutic target for BC treatment.

Abstract 2739: EZH2 promotes breast cancer bone metastasis

Abstract Metastasis causes about 90% of cancer deaths and approximately 55% of breast cancer patients develop bone metastases. Although significant advances in the cure of breast cancer, effective therapies targeted bone metastases are still not available. To develop novel therapies for patients with bone metastasis, better understanding of underlying mechanisms of bone metastasis is critical. EZH2 is a classic histone methyl transferase that generally suppresses downstream target genes through tri-methylation of H3K27. EZH2 is considered as a breast cancer oncogene and is frequently over expressed in breast cancer, but the effect of EZH2 on bone metastasis is unknown. To explore the function of EZH2 in bone metastasis, we intracardially injected into nude mice control MDA.MB.231 breast cancer cells expressing readily detectable EZH2 or a MDA.MB.231 subline having EZH2 knocked out (231.EZH2.ko). We found that the mice injected with 231.EZH2.ko had prolonged bone metastasis-free survival compared to the control group of mice. To gain initial insight on the underlying mechanism of EZH2-mediated bone metastasis, we co-cultured MDA.MB.231 cells or 231.EZH2.ko with pre-osteoclasts (RAW264.7) and osteoblasts (MC3T3) under TGF-β treatment (triple co-culture) to mimic bone metastasis tumor microenvironment. We examined osteoclast maturation and breast cancer cell proliferation because osteolysis induced by mature osteoclasts is critical for breast cancer cell growth in the bone metastasis. In triple co-culture of MDA.MB.231 cells, more RAW264.7 pre-osteoclasts were differentiated to mature osteoclasts, as measured by TRAP+ staining, a marker of mature osteoclasts, and MDA.MB.231 cells grew faster than the triple co-culture of 231.EZH2.ko cells. Furthermore, we found that the mRNA level of PTHrP, a master regulator of bone metastasis and a TGF-β downstream cytokine, was expressed at significantly higher level in MDA.MB.231 cells than that in 231.EZH2.ko cells under TGF-β stimulation. PTHrP was known to promote the “vicious cycle” of bone destruction in bone metastasis. The data suggests EZH2 promotes breast cancer bone metastasis through PTHrP. Currently, we are further exploring how EZH2 regulates PTHrP in breast cancer cells to promote bone metastasis. Our studies may reveal novel biomarkers and/or therapeutic targets for bone metastasis. Citation Format: Jingkun Qu, Lin Zhang, Zhifen Zhou, Dihua Yu. EZH2 promotes breast cancer bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2739.

RUNX2 promotes breast cancer bone metastasis by increasing integrin α5-mediated colonization

Runt-related transcription factor 2 (RUNX2) is regarded as an important contributor to breast cancer bone metastasis. However, previous studies did not provide direct clinical evidence for a role of RUNX2 in bone-specific metastasis in breast cancer, and the mechanism of RUNX2 in cancer cell recruitment and adhesion to the bone remains unclear. In this study, we showed that RUNX2 expression is positively correlated with the risk of bone-specific metastasis in lymph node-negative breast cancer patients. Then, we identified ITGA5 as a transcriptional target of RUNX2 from multiple candidate genes encoding adhesion molecules or chemokine receptors. We further provided experimental and clinical evidence that RUNX2, in an integrin α5-dependent manner, promotes the attraction and adhesion of breast cancer cells to the bone and confers cancer cell survival and bone colonization advantages. Overall, our findings clarify an adhesion-dependent mechanism of RUNX2 for the osteotropism and bone colonization of breast cancer cells and implicate RUNX2 and integrin α5 as potential molecular markers for the prediction of bone metastasis and therapeutic targets for the treatment of breast cancer bone metastasis.

RANKL-Independent Osteoclastogenesis: A Long-Standing Controversy.

RANKL-Independent Osteoclastogenesis: A Long-Standing Controversy.

Gut-derived serotonin induced by depression promotes breast cancer bone metastasis through the RUNX2/PTHrP/RANKL pathway in mice.

Breast cancer metastasizes to the bone in a majority of patients with advanced disease resulting in bone destruction. The underlying mechanisms are complex, and both processes are controlled by an interaction between locally and systemically derived signals. Clinically, breast cancer patients with depression have a higher risk of bone metastasis, yet the etiology and mechanisms are yet to be elucidated. MDA‑MB‑231 breast cancer cells were used to establish a bone metastasis model by using intracardiac injection in nude mice. Chronic mild stress (CMS) was chosen as a model of depression in mice before and after inoculation of the cells. Knockdown of the RUNX‑2 gene was performed by transfection of the cells with shRNA silencing vectors against human RUNX‑2. A co‑culture system was used to test the effect of the MDA‑MB‑231 cells on osteoclasts and osteoblasts. RT‑PCR and western blotting were used to test gene and protein expression, respectively. We confirmed that depression induced bone metastasis by promoting osteoclast activity while inhibiting osteoblast differentiation. Free serotonin led to an increase in the expression of RUNX2 in breast cancer cells (MDA‑MB‑231), which directly inhibited osteoblast differentiation and stimulated osteoclast differentiation by the PTHrP/RANKL pathway, which caused bone destruction and formed osteolytic bone lesions. Additionally, the interaction between depression and breast cancer cells was interrupted by LP533401 or RUNX2 knockdown. In conclusion, depression promotes breast cancer bone metastasis partly through increasing levels of gut‑derived serotonin. Activation of RUNX2 in breast cancer cells by circulating serotonin appears to dissociate coupling between osteoblasts and osteoclasts, suggesting that the suppression of gut‑derived serotonin decreases the rate of breast cancer bone metastasis induced by depression.

ITGBL1 Is a Runx2 Transcriptional Target and Promotes Breast Cancer Bone Metastasis by Activating the TGFβ Signaling Pathway.

Bone metastasis affects more than 70% of advanced breast cancer patients, but the molecular mechanisms of this process remain unclear. Here, we present clinical and experimental evidence to clarify the role of the integrin β-like 1 (ITGBL1) as a key contributor to bone metastasis of breast cancer. In an in vivo model system and in vitro experiments, ITGBL1 expression promoted formation of osteomimetic breast cancers, facilitating recruitment, residence, and growth of cancer cells in bone microenvironment along with osteoclast maturation there to form osteolytic lesions. Mechanistic investigations identified the TGFβ signaling pathway as a downstream effector of ITGBL1 and the transcription factor Runx2 as an upstream activator of ITGBL1 expression. In support of these findings, we also found that ITGBL1 was an essential mediator of Runx2-induced bone metastasis of breast cancer. Overall, our results illuminate how bone metastasis occurs in breast cancer, and they provide functional evidence for new candidate biomarkers and therapeutic targets to identify risk, to prevent, and to treat this dismal feature of advanced breast cancer.

Does Our “Flight and Fight” Response Have a Link With Cancer?

Does Our “Flight and Fight” Response Have a Link With Cancer?

Stimulation of Host Bone Marrow Stromal Cells by Sympathetic Nerves Promotes Breast Cancer Bone Metastasis in Mice

Author SummaryImproved detection programs and better drugs to eradicate breast tumors have increased survival in women with breast cancer. However, pain and metastasis to distant organs, including bone, remain significant clinical problems. Understanding why and how metastatic cancer cells colonize specific organs is therefore critical if we are to further improve morbidity and mortality for these patients. Using a mouse model of breast cancer bone metastasis, we present evidence that activation of sympathetic nerves, which is typical in chronic stress or depression, promotes the colonization and establishment of metastatic cancer cells within the bone marrow, leading to an increase in bone osteolytic lesions. We show that this effect is mediated via a β-adrenergic receptor-dependent response of the host bone marrow stroma to catecholamines, that are released upon sympathetic activation, and via the pro-migratory activity of RANKL, a cytokine that is well known to promote bone resorption. Of importance clinically, blocking sympathetic activation with a β-blocker, or blocking RANKL signaling in cancer cells, inhibited the stimulatory effect of sympathetic activation on bone metastasis in this mouse model. Stress-induced sympathetic activation may thus explain, at least in part, the reduced survival rate of breast cancer patients experiencing severe depression. The data also support the use of β-blockers or RANKL blockade as possible adjuvant therapy for women with breast cancer.

Abstract 3323: Hyaluronan synthase 2 promotes breast cancer bone metastasis by stimulating interaction of cancer stem-like cells with tumor-associated macrophage

Abstract The molecular mechanism of metastatic progression, particularly the roles of microenvironment of the metastasized organs, is as yet poorly understood. In this study, we have isolated cancer stem cell-like cells (CSC) from human breast cancer cell lines, MDA-MB231, MDA231BoM and MDA231BrM. CSC were isolated from each line using surface markers (CD24-, CD44+, ESA+) and their tumor initiating abilities were tested in animal. We then performed an analysis of global expression profile for these CSCs using the Affymetrix expression array and found that among all up-regulated genes, two genes (HAS2 and MMP1) were significantly correlated with overall- and metastasis-free survival when they were cross-examined in GEO database. To further examine the role of HAS2 in tumor metastasis in vivo, we prepared CSC from MDA231BoM with or without carrying a shRNA expression vector for HAS2. The CSC were then implanted into nude mice by intra-cardiac injection followed by oral administration of 4MU, a specific inhibitor of HAS genes. We found that the injection of CSC of MDA231BoM which expresses shRNA to HAS2 significantly improved the survival rate of animals. In addition, administration of 4MU significantly suppressed tumor metastasis of CSC, suggesting that inhibition of HAS2 can indeed reduce the incidence of metastasis in vivo. We also examined the effect of tumor associated macrophage (TAM) on growth of CSC in animals by injecting CSC of MDA231-BoM with or without TAM into the tibial bone. We found that co-injection of TAM with CSC significantly augmented the growth of tumor in their legs. Moreover, the interaction between cancer cell and TAMs enhanced secretion of PDGF-BB from TAMs which then activated stromal cells and in turn enhanced self-renewal of CSCs by secretion of FGF9 and FGF7. We also found that treatment of bone marrow stem cell (BMSC) with PDGF followed by co-culture of CSC significantly promoted proliferation of CSC. Collectively, our results suggest that TAM and BMSC promote self-renewal of CSC in bone microenvironment via direct interaction between hyaluronan and CD44. Our results also provide strong rationale to target HAS2 by using 4MU in order to block the metastatic progression of CSC in breast cancer. 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 3323. doi:1538-7445.AM2012-3323