Systemic PPARγ Antagonism Reduces Metastatic Tumor Progression in Adipocyte-Rich Bone in Excess Weight Male Rodents

Primary tumors are widely associated with an excess in body fat. The role of adipose tissue on tumor cell homing to bone is yet poorly defined. In this study, we aimed to assess whether bone colonization by tumor cells is favored by an adipocyte-rich bone marrow. We delineated the accompanying alterations of the bone microenvironment and established a treatment approach that interferes with high fat diet (HFD)-induced bone metastasis formation. We were able to show that adipocytes affect skeletal tumor growth in a metastatic model of breast cancer in male rats and melanoma in male mice as well as in human breast cancer bone biopsies. Indeed, HFD-induced bone marrow adiposity was accompanied by accelerated tumor progression and increased osteolytic lesions. In human bone metastases, bone marrow adiposity correlated with tumor cell proliferation. By antagonization of the adipocyte differentiation and storage pathway linked to the peroxisome proliferator-activated receptor gamma (PPARγ) with bisphenol-A-diglycidylether (BADGE), we were able to decelerate tumor progression and subsequent osteolytic damage in the bones of two distinct metastatic animal models exposed to HFD. Overall these data show that adipose tissue is a critical factor in bone metastases and cancer-induced bone loss. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).     

Early detection of bone metastases in a murine model using fluorescent human breast cancer cells: application to the use of the bisphosphonate zoledronic acid in the treatment of osteolytic lesions.

A very common metastatic site for human breast cancer is bone. The traditional bone metastasis model requires human MDA-MB-231 breast carcinoma cell inoculation into the left heart ventricle of nude mice. MDA-MB-231 cells usually develop osteolytic lesions 3-4 weeks after intracardiac inoculation in these animals. Here, we report a new approach to study the formation of bone metastasis in animals using breast carcinoma cells expressing the bioluminescent jellyfish protein (green fluorescent protein [GFP]). We first established a subclone of MDA-MB-231 cells by repeated in vivo passages in bone using the heart injection model. On stable transfection of this subclone with an expression vector for GFP and subsequent inoculation of GFP-expressing tumor cells (B02/GFP.2) in the mouse tail vein, B02/GFP.2 cells displayed a unique predilection for dissemination to bone. Externally fluorescence imaging of live animals allowed the detection of fluorescent bone metastases approximately 1 week before the occurrence of radiologically distinctive osteolytic lesions. The number, size, and intensity of fluorescent bone metastases increased progressively with time and was indicative of breast cancer cell progression within bone. Histological examination of fluorescent long bones from B02/GFP.2-bearing mice revealed the occurrence of profound bone destruction. Treatment of B02/GFP.2-bearing mice with the bisphosphonate zoledronic acid markedly inhibited the progression of established osteolytic lesions and the expansion of breast cancer cells within bone. Overall, this new bone metastasis model of breast cancer combining both fluorescence imaging and radiography should provide an invaluable tool to study the effectiveness of pharmaceutical agents that could suppress cancer colonization in bone.     

A CTGF-RUNX2-RANKL Axis in Breast and Prostate Cancer Cells Promotes Tumor Progression in Bone

Metastasis to bone is a frequent occurrence in patients with breast and prostate cancers and inevitably threatens the patient's quality of life and survival. Identification of cancer-derived mediators of bone metastasis and osteolysis may lead to novel therapeutic strategies. In this study, we established highly bone-metastatic PC3 prostate and MDA-MB-231 (MDA) breast cancer cell sublines by in vivo selection in mice. In bone-metastatic cancer cells, the expression and secretion of connective tissue growth factor (CTGF) were highly upregulated. CTGF knockdown in bone-metastatic cells decreased invasion activity and MMP expression. RUNX2 overexpression in the CTGF knockdown cells restored the invasion activity and MMP expression. In addition, CTGF increased RUNX2 protein stability by inducing its acetylation via p300 acetyl transferase. The integrin αvβ3 receptor mediated these effects of CTGF. Furthermore, CTGF promoted RUNX2 recruitment to the RANKL promoter, resulting in increased RANKL production from the tumor cells and subsequent stimulation of osteoclastogenesis from precursor cells. In addition, animal model with injection of CTGF knocked-down prostate cancer cells into 6-week old BALB/c male mice showed reduced osteolytic lesions. More importantly, the expression levels of CTGF and RANKL showed a strong positive correlation in human primary breast tumor tissues and were higher in bone metastases than in other site metastases. These findings indicate that CTGF plays crucial roles for osteolytic bone metastasis both by enhancing invasiveness of tumor cells and by producing RANKL for osteoclastogenesis. Targeting CTGF may lead to the development of effective preventive and therapeutic strategies for osteolytic metastasis. © 2019 American Society for Bone and Mineral Research.     

The ubiquitin E3 ligase WWP1 decreases CXCL12-mediated MDA231 breast cancer cell migration and bone metastasis

Advanced breast cancers preferentially metastasize to bone where cells in the bone microenvironment produce factors that enhance breast cancer cell homing and growth. Expression of the ubiquitin E3 ligase WWP1 is increased in some breast cancers, but its role in bone metastasis has not been investigated. Here, we studied the effects of WWP1 and itch, its closest family member, on breast cancer bone metastasis. First, we immunostained a multi-tumor tissue microarray and a breast cancer tissue microarray and demonstrated that WWP1 and ITCH are expressed in some of breast cancer cases. We then knocked down WWP1 or itch in MDA-MB-231 breast cancer cells using shRNA and inoculated these cells and control cells into the left ventricle of athymic nude mice. Radiographs showed that mice given shWWP1 cells had more osteolytic lesions than mice given control MDA-MB-231 cells. Histologic analysis confirmed osteolysis and showed significantly increased tumor area in bone marrow of the mice. WWP1 knockdown did not affect cell growth, survival or osteoclastogenic potential, but markedly increased cell migration toward a CXCL12 gradient in vitro. Furthermore, WWP1 knockdown significantly reduced CXCL12-induced CXCR4 lysosomal trafficking and degradation. In contrast, itch knockdown had no effect on MDA-MB-231 cell bone metastasis. Taken together, these findings demonstrate that WWP1 negatively regulates cell migration to CXCL12 by limiting CXCR4 degradation to promote breast cancer metastasis to bone and highlight the potential utility of WWP1 as a prognostic indicator for breast cancer bone metastasis.     

Breast Cancer Metastasis to Bone: Mechanisms of Osteolysis and Implications for Therapy

The most common skeletal complication of breast cancer is osteolytic bone metastasis. Bone metastases are present in 80% of patients with advanced disease and cause significant morbidity. They are most often osteolytic, but can be osteoblastic or mixed. Tumor cells, osteoblasts, osteoclasts and bone matrix are the four components of a vicious cycle necessary for the initiation and development of bone metastases. Tumor cell gene expression is modified by interaction with bone-derived factors. For example, parathyroid hormone related protein (PTHrP), a tumor cell factor, is upregulated by bone-derived transforming growth factor β (TGFβ). Tumor cell factors, in turn, act upon bone cells to cause dysregulated bone destruction and formation. PTHrP increases osteoblast expression of RANK (receptor activator of NFκB) ligand which, in turn, activates osteoclasts. PTHrP-independent osteolytic factors, such as interleukin [IL]-11 and IL-8, also contribute to the vicious cycle. Other tumor-bone interactions, such as stimulation of tumor-homing through the CXCR4 chemokine receptor by its bone-derived ligand stromal-derived factor-1 (SDF-1), may be responsible for the site-specific predilection of breast cancer for bone. These factors and their roles in fueling the vicious cycle may identify novel targets for therapies to prevent metastasis.     

Breast cancer osteomimicry and its role in bone specific metastasis; an integrative,systematic review of preclinical evidence

Metastasis accounts for most of the deaths from breast cancer and the preference of invasive breast cancer metastasising to bone has been widely reported. However, the biological basis of breast cancer osteotropism is not fully understood. This paper provides, for the first time, an integrative, systematic review of evidence of molecular factors that have functional roles in the homing of metastatic breast cancer to the bone.Pubmed, Web of Science and EBSCOhost were searched using keywords and synonyms for molecular, metastasis, breast cancer and bone to identify articles published between January 2004 and August 2016. 4491 potentially relevant citations were retrieved. 63 articles met the inclusion criteria, which were primary studies reporting evidence of molecular factors that have functional roles in predisposing breast cancer bone metastasis in vivo. 12 of those 63 articles that additionally met quality criteria were included in the review. Extracted data were tabulated and key findings that indicated biological mechanisms involved in breast cancer metastasis to bone were synthesised.15 proteins expressed by breast cancer cells were identified as factors that mediate breast cancer bone metastasis: ICAM-1, cadherin-11, osteoactivin, bone sialoprotein, CCN3, IL-11, CCL2, CITED2, CXCR4, CTGF, OPN, CX₃CR1, TWIST1, adrenomedullin and Enpp1. Upregulation or overexpression of one or more of them by breast cancer cells resulted in increased breast cancer metastasis to bone in vivo, except for CCL2 where bone-metastatic cells showed a reduced expression of this factor. All factors identified, here expressed by breast cancer cells, are proteins that are normally expressed in the bone microenvironment and linked to physiologic bone functions. All have a functional role in one of more of the following: cell proliferation and differentiation, bone mineralization and remodelling, cell adhesion and/or chemokine signalling. Six of them (cadherin-11, ICAM-1, OPN, CX₃CR1, CCN3 and osteoactivin) have a reported function in cell adhesion and another eight (CCN3, osteoactivin, Enpp1, IL-11, CTGF, TWIST1, adrenomedullin and CITED2) are reported to be involved in cell proliferation and differentiation.This review collates and synthesises published evidence to increase our understanding of the biology of breast cancer osteomimicry in the development of bone metastasis. Findings of this review suggest that changes in expression of proteins in breast cancer cells that confer osteomimicry facilitate homing to bone to enable the development of bone metastasis.     

Low cell motility induced by hsp27 overexpression decreases osteolytic bone metastases of human breast cancer cells in vivo.

The mechanisms controlling the formation of osteolytic bone metastases in patients with breast cancer are still poorly understood. To explore the role of motility in the establishment of osteolytic bone metastases, we have used a model of bone metastasis in which MDA-MB-231 breast cancer cells exhibiting low (hsp27-transfectants) and high (control-transfectant) endogenous cell motility were compared. We found that MDA-MB-231 cells exhibiting low cell motility were less capable of establishing osteolytic lesions. The number and the area of the osteolytic lesions in mice inoculated with low motility cells were both significantly smaller. Histomorphometry of bone lesions also demonstrated less tumor area in mice bearing hsp27 transfectants although there was no difference in the osteoclast number per square millimeter of tumor-bone interface. These data suggest that cell motility may be an important mechanism in the metastatic cascade of breast cancer cells to the bone and that controlling cell motility may be a useful target to prevent the establishment of osteolytic bone metastases.     

Inhibiting activin‐A signaling stimulates bone formation and prevents cancer‐induced bone destruction in vivo

Cancers that grow in bone, such as myeloma and breast cancer metastases, cause devastating osteolytic bone destruction. These cancers hijack bone remodeling by stimulating osteoclastic bone resorption and suppressing bone formation. Currently, treatment is targeted primarily at blocking bone resorption, but this approach has achieved only limited success. Stimulating osteoblastic bone formation to promote repair is a novel alternative approach. We show that a soluble activin receptor type IIA fusion protein (ActRIIA.muFc) stimulates osteoblastogenesis (p < .01), promotes bone formation (p < .01) and increases bone mass in vivo (p < .001). We show that the development of osteolytic bone lesions in mice bearing murine myeloma cells is caused by both increased resorption (p < .05) and suppression of bone formation (p < .01). ActRIIA.muFc treatment stimulates osteoblastogenesis (p < .01), prevents myeloma‐induced suppression of bone formation (p < .05), blocks the development of osteolytic bone lesions (p < .05), and increases survival (p < .05). We also show, in a murine model of breast cancer bone metastasis, that ActRIIA.muFc again prevents bone destruction (p < .001) and inhibits bone metastases (p < .05). These findings show that stimulating osteoblastic bone formation with ActRIIA.muFc blocks the formation of osteolytic bone lesions and bone metastases in models of myeloma and breast cancer and paves the way for new approaches to treating this debilitating aspect of cancer. © 2010 American Society for Bone and Mineral Research.     

Selective tyrosine kinase inhibition of insulin‐like growth factor‐1 receptor inhibits human and mouse breast cancer–induced bone cell activity,bone remodeling,and osteolysis

Insulin‐like growth factor 1 (IGF‐1) plays an important role in both bone metabolism and breast cancer. In this study, we investigated the effects of the novel IGF‐1 receptor tyrosine kinase inhibitor cis‐3‐[3‐(4‐methyl‐piperazin‐l‐yl)‐cyclobutyl]‐1‐(2‐phenyl‐quinolin‐7‐yl)‐imidazo[1,5‐a]pyrazin‐8‐ylamine (PQIP) on osteolytic bone disease associated with breast cancer. Human MDA‐MB‐231 and mouse 4T1 breast cancer cells enhanced osteoclast formation in receptor activator of NF‐κB ligand (RANKL) and macrophage colony‐stimulating factor (M‐CSF) stimulated bone marrow cultures, and these effects were significantly inhibited by PQIP. Functional studies in osteoclasts showed that PQIP inhibited both IGF‐1 and conditioned medium–induced osteoclast formation by preventing phosphatidylinositol 3‐kinase (PI3K)/protein kinase B (Akt) activation without interfering with RANKL or M‐CSF signaling. Treatment of osteoblasts with PQIP significantly inhibited the increase in RANKL/osteoprotegerin (OPG) ratio by IGF‐1 and conditioned medium and totally prevented conditioned medium–induced osteoclast formation in osteoblast–bone marrow (BM) cell cocultures, thereby suggesting an inhibitory effect on osteoblast–osteoclast coupling. PQIP also inhibited IGF‐1–induced osteoblast differentiation, spreading, migration, and bone nodule formation. Treatment with PQIP significantly reduced MDA‐MB‐231 conditioned medium–induced osteolytic bone loss in a mouse calvarial organ culture system ex vivo and in adult mice in vivo. Moreover, once daily oral administration of PQIP significantly decreased trabecular bone loss and reduced the size of osteolytic bone lesions following 4T1 intratibial injection in mice. Quantitative histomorphometry showed a significant reduction in bone resorption and formation indices, indicative of a reduced rate of cancer‐associated bone turnover. We conclude that inhibition of IGF‐1 receptor tyrosine kinase activity by PQIP suppresses breast cancer–induced bone turnover and osteolysis. Therefore, PQIP, and its novel derivatives that are currently in advanced clinical development for the treatment of a number of solid tumors, may be of value in the treatment of osteolytic bone disease associated with breast cancer. © 2013 American Society for Bone and Mineral Research.     

Tumor‐derived syndecan‐1 mediates distal cross‐talk with bone that enhances osteoclastogenesis

Tumor‐stimulated bone resorption fuels tumor growth and marks a dramatic decline in the health and prognosis of breast cancer patients. Identifying mechanisms that mediate cross‐talk between tumor and bone remains a key challenge. We previously demonstrated that breast cancer cells expressing high levels of heparanase exhibit enhanced shedding of the syndecan‐1 proteoglycan. Moreover, when these heparanase‐high cells are implanted in the mammary fat pad, they elevate bone resorption. In this study, conditioned medium from breast cancer cells expressing high levels of heparanase was shown to significantly stimulate human osteoclastogenesis in vitro (p < .05). The osteoclastogenic activity in the medium of heparanase‐high cells was traced to the presence of syndecan‐1, intact heparan sulfate chains, and heat‐labile factor(s), including the chemokine interleukin 8 (IL‐8). The enhanced osteoclastogenesis promoted by the heparanase‐high cells results in a dramatic increase in bone resorption in vitro. In addition, the long bones of animals bearing heparanase‐high tumors in the mammary fat pad had significantly higher numbers of osteoclasts compared with animals bearing tumors expressing low levels of heparanase (p < .05). Together these data suggest that syndecan‐1 shed by tumor cells exerts biologic effects distal to the primary tumor and that it participates in driving osteoclastogenesis and the resulting bone destruction. © 2010 American Society for Bone and Mineral Research     

Skeletal Colonization by Breast Cancer Cells Is Stimulated by an Osteoblast and β2AR‐Dependent Neo‐Angiogenic Switch

The skeleton is a common site for breast cancer metastasis. Although significant progress has been made to manage osteolytic bone lesions, the mechanisms driving the early steps of the bone metastatic process are still not sufficiently understood to design efficacious strategies needed to inhibit this process and offer preventative therapeutic options. Progression and recurrence of breast cancer, as well as reduced survival of patients with breast cancer, are associated with chronic stress, a condition known to stimulate sympathetic nerve outflow. In this study, we show that stimulation of the beta 2‐adrenergic receptor (β2AR) by isoproterenol, used as a pharmacological surrogate of sympathetic nerve activation, led to increased blood vessel density and Vegf‐a expression in bone. It also raised levels of secreted Vegf‐a in osteoblast cultures, and accordingly, the conditioned media from isoproterenol‐treated osteoblast cultures promoted new vessel formation in two ex vivo models of angiogenesis. Blocking the interaction between Vegf‐a and its receptor, Vegfr2, blunted the increase in vessel density induced by isoproterenol. Genetic loss of the β2AR globally, or specifically in type 1 collagen‐expressing osteoblasts, diminished the increase in Vegf‐positive osteoblast number and bone vessel density induced by isoproterenol, and reduced the higher incidence of bone metastatic lesions induced by isoproterenol after intracardiac injection of an osteotropic variant of MDA‐MB‐231 breast cancer cells. Inhibition of the interaction between Vegf‐a and Vegfr2 with the blocking antibody mcr84 also prevented the increase in bone vascular density and bone metastasis triggered by isoproterenol. Together, these results indicate that stimulation of the β2AR in osteoblasts triggers a Vegf‐dependent neo‐angiogenic switch that promotes bone vascular density and the colonization of the bone microenvironment by metastatic breast cancer cells. © 2017 American Society for Bone and Mineral Research.     

PTHrP(12‐48) Modulates the Bone Marrow Microenvironment and Suppresses Human Osteoclast Differentiation and Lifespan

Bone is a common site for metastasis in breast cancer patients and is associated with a series of complications that significantly compromise patient survival, partially due to the advanced stage of disease at the time of detection. Currently, no clinically‐approved biomarkers can identify or predict the development of bone metastasis. We recently identified a unique peptide fragment of parathyroid hormone‐related protein (PTHrP), PTHrP(12‐48), as a validated serum biomarker in breast cancer patients that correlates with and predicts the presence of bone metastases. In this study, the biological activity and mode of action of PTHrP(12‐48) was investigated. Sequence‐based and structure‐based bioinformatics techniques predicted that the PTHrP(12‐48) fragment formed an alpha helical core followed by an unstructured region after residue 40 or 42. Thereafter, detailed structure alignment and molecular docking simulations predicted a lack of interaction between PTHrP(12‐48) and the cognate PTH1 receptor (PTHR1). The in silico prediction was confirmed by the lack of PTHrP(12‐48)‐stimulated cAMP accumulation in PTHR1‐expressing human SaOS2 cells. Using a specific human PTHrP(12‐48) antibody that we developed, PTHrP(12‐48) was immunolocalized in primary and bone metastatic human breast cancer cells, as well as within human osteoclasts (OCLs) in bone metastasis biopsies, with little or no localization in other resident bone or bone marrow cells. In vitro, PTHrP(12‐48) was internalized into cultured primary human OCLs and their precursors within 60 min. Interestingly, PTHrP(12‐48) treatment dose‐dependently suppressed osteoclastogenesis, via the induction of apoptosis in both OCL precursors as well as in mature OCLs, as measured by the activation of cleaved caspase 3. Collectively, these data suggest that PTHrP(12‐48) is a bioactive breast cancer–derived peptide that locally regulates the differentiation of hematopoietic cells and the activity of osteoclasts within the tumor–bone marrow microenvironment, perhaps to facilitate tumor control of bone. © 2017 American Society for Bone and Mineral Research.     

COX-2 induces IL-11 production in human breast cancer cells

BACKGROUND: Cyclooxygenase-2 (COX-2) is overexpressed in 40% of human invasive breast cancers. Interleukin-11 (IL-11), a potent mediator of osteoclastogenesis, is involved in breast cancer metastasis to bone. Since breast cancers that overexpress COX-2 are associated with a higher rate of metastasis to bone, we hypothesized that COX-2 expression in tumor cells would induce IL-11. MATERIALS AND METHODS: We transfected MCF-7 (poorly metastatic) and MDA-231 (highly metastatic) human breast cancer cell lines with COX-2 expression vectors. COX-2 overexpression was confirmed by Western blot and PGE(2) immunoassay, and IL-11 production was measured by immunoassay. We also used a nude mouse model to study COX-2 and IL-11 production from breast cancer cells that metastasized to bone. The bone-seeking clones (BSC) were isolated and cultured from the long bone metastases. RESULTS: COX-2 transfection caused an approximately 5- to 6-fold increase in IL-11 production in both MCF-7 and MDA-231 cells. MDA-435S-COX2-BSC (cells isolated from bone metastasis) produced elevated levels of IL-11 and PGE2 (an important mediator of COX-2) as compared to the parental MDA-435S-COX2 cells. Furthermore, a treatment with low 1- to 2-microm concentration NS-398 or Celecoxib significantly reduced the production of IL-11 in COX-2-transfected MDA-231 cells, thus confirming the involvement of COX-2 in IL-11 induction. CONCLUSION: COX-2-mediated production of IL-11 in breast cancer cells may be vital to the development of osteolytic bone metastases in patients with breast cancer, and a COX-2 inhibitor may be useful in inhibiting this process.     

内源性miR103A通过抑制RGS2的表达从而影响乳腺癌的转移

目的 探索内源性miR-103A通过作用于抑癌基因RGS2启动子进而抑制其表达从而影响乳腺癌的转移。方法 将从乳腺癌及正常乳腺组织中提取的RNA进行小分子RNA的深度测序检测乳腺癌中小分子RNA的表达,通过生物信息学分析miR103A的目标基因以及其作用位点,用miRNA103A的mimics转染乳腺癌细胞后用QPCR及Western-Blot检测受到影响的肿瘤相关基因,QPCR检测miR103A以及RGS2在乳腺癌及正常癌组织中的差异表达。结果 小分子RNA深度测序发现miR103A在乳腺癌及乳腺组织有明显的差异表达且在乳腺癌中高表达,miR103A被发现在伴有淋巴结转移以及HER2+的乳腺癌组织中高表达;生物信息学分析发现miR103A序列能与RGS2的启动子区匹配,且miR103A下调了RGS2 mRNA及蛋白水平;miR103A还上调肿瘤转移相关的MMP9、VGEF、snail、Vimentin的表达,下调了E-cadherin的表达;27例成对临床样本检测发现RGS2在乳腺癌低表达。结论 miR103A通过作用于抑癌基因RGS2的启动子从而下调其表达影响乳腺癌的转移。     

Monitoring metastatic behavior of human tumor cells in mice with species-specific polymerase chain reaction: elevated expression of angiogenesis and bone resorption stimulators by breast cancer in bone metastases.

Tumor-stroma interactions are of primary importance in determining the pathogenesis of metastasis. Here, we describe the application of sensitive competitive polymerase chain reaction (PCR) techniques for detection and quantitation of human breast cancer cells (MDA-MB-231) in an in vivo mouse model of experimental metastasis. Human-specific oligonucleotide primers in competitive PCR reactions were used to quantify the amount of MDA-MB-231 cells per tissue per organ. Using this species-specific (semi)quantitative PCR approach, gene expression patterns of (human) tumor cells or (mouse) stromal cells in metastatic lesions in the skeleton or soft tissues were investigated and compared. In all metastatic lesions, MDA-MB-231 cells express angiogenic factors (vascular endothelial growth factors [VEGFs]; VEGF-A, -B, and -C) and bone-acting cytokines (parathyroid hormone-related protein [PTHrP] and macrophage colony-stimulating factor [M-CSF]). In these metastases, PECAM-1-positive blood vessels and stromal cells of mouse origin are detected. The latter express angiogenic factors and markers of sprouting vessels (VEGF receptors flt-1/flk - 1/flk-4 and CD31/PECAM-1). Strikingly, steady-state messenger RNA (mRNA) levels of VEGF-A and -B and the major bone resorption stimulators PTHrP and M-CSF by tumor cells were elevated significantly in bone versus soft tissues (p < or = 0.05, p < or = 0.0001, p < or = 0.001, and p < or = 0.05, respectively), indicating tissue-specific expression of these tumor progression factors. In conclusion, MDA-MB-231 breast cancer cells express a variety of factors in vivo that have been implicated in metastatic bone disease and that correlate with poor survival of patients with breast cancer. We hypothesize that the observed up-regulated expression of angiogenic and bone-resorbing factors by the breast cancer cells in the skeleton underlie the clinically observed osteotropism of breast cancer cells and pathogenesis of osteolytic bone metastases. The application of the species-specific competitive PCR-based assay in vivo can provide new information concerning the involvement of gene families in tumor progression and metastatic disease and greatly facilitates the study of tumor-stroma interactions in cancer invasion and metastasis.     

Therapeutic potential of hyaluronan oligosaccharides for bone metastasis of breast cancer

Hyaluronan (HA) oligosaccharides were reported to have suppressive effects on various malignant tumors via disruption of receptor HA interactions. However, no studies have focused on the effects of HA oligosaccharides on bone metastasis of breast cancer. In this study, we clarified the effective size of HA oligosaccharides required to inhibit cell growth in the highly invasive breast cancer cell line, MDA‐MB‐231 cells. Based on the results of cell growth assay, we subsequently analyzed the effects of HA tetrasaccharides, HA decasaccharides, and high molecular weight HA on the other breast cancer cell behaviors in vitro and breast cancer bone metastasis in vivo. HA decasaccharides significantly inhibited cell growth, motility, and invasion, whereas tetrasaccharides did not. HAS2 mRNA expression was altered after the treatment with both tetrasaccharides and decasaccharides. Phosphorylation of Akt was suppressed after 1 h treatment with HA decasaccharides, and the effect was partially reversed by anti‐CD44 monoclonal antibody. In vivo, local application of HA decasaccharides inhibited the expansion of osteolytic lesions in tibia on soft X‐rays using mouse bone metastasis model of breast cancer. Histological analysis revealed HA accumulation in bone metastatic lesions was perturbed by decasaccharides. These results suggest that HA oligosaccharides suppressed progression of bone metastasis in breast cancer via interruption of endogenous HA–CD44 interaction, and as such, can be a novel therapeutic candidate to limit bone metastasis of breast cancer. © 2011 Orthopaedic Research Society. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 30:662–672, 2012     

Breast tumors induced by N‐methyl‐N‐nitrosourea are damaging to bone strength,structure, and mineralization in the absence of metastasis in rats

Current theory on the influence of breast cancer on bone describes metastasis of tumor cells to bone tissue, followed by induction of osteoclasts and bone degradation. Tumor influences on bone health in pre‐ or nonmetastatic models are unknown. Female rats (n = 48, 52 days old) were injected with N‐methyl‐N‐nitrosourea (MNU) to induce breast cancer. Animals were euthanized 10 weeks later, and tumors were weighed and classified histologically. Right femurs were extracted for testing of bone mineral density (BMD) by dual X‐ray absorptiometry (DXA), bone mechanical strength by three‐point bending and femoral neck bending tests, and structure by micro–computed tomography (µCT). Of 48 rats, 22 developed one or more tumors in response to MNU injection by 10 weeks. Presence of any tumor predicted significantly poorer bone health in 17 of 28 measures. In tumored versus nontumored animals, BMD was adversely affected by 3%, force at failure of the femoral midshaft by 4%, force at failure of the femoral neck by 12%, and various trabecular structural parameters by 6% to 27% (all p < .05). Similarly, greater tumor burden, represented by total tumor weight, adversely correlated with bone outcomes: r = ?0.51 for BMD, ?0.42 and ?0.35 for femur midshaft force and work at failure, and between 0.36 and 0.59 (absolute values) for trabecular architecture (all p < .05). Presence of MNU‐induced tumors and total tumor burden showed a negative association with bone health of the femur in rats in the absence of metastasis. Further study is required to elucidate mechanisms for this association. © 2011 American Society for Bone and Mineral Research.