Homing Of Breast Cancer Cells Research Articles

From breast cancer cell homing to the onset of early bone metastasis: The role of bone (re)modeling in early lesion formation.

Breast cancer often metastasizes to bone, causing osteolytic lesions. Structural and biophysical changes are rarely studied yet are hypothesized to influence metastasis. We developed a mouse model of early bone metastasis and multimodal imaging to quantify cancer cell homing, bone (re)modeling, and onset of metastasis. Using tissue clearing and three-dimensional (3D) light sheet fluorescence microscopy, we located enhanced green fluorescent protein-positive cancer cells and small clusters in intact bones and quantified their size and spatial distribution. We detected early bone lesions using in vivo microcomputed tomography (microCT)-based time-lapse morphometry and revealed altered bone (re)modeling in the absence of detectable lesions. With a new microCT image analysis tool, we tracked the growth of early lesions over time. We showed that cancer cells home in all bone compartments, while osteolytic lesions are only detected in the metaphysis, a region of high (re)modeling. Our study suggests that higher rates of (re)modeling act as a driver of lesion formation during early metastasis.

Pre-osteoblasts and endothelial progenitors differently affect homing of breast cancer cells to the bone marrow

Pre-osteoblasts and endothelial progenitors differently affect homing of breast cancer cells to the bone marrow

An Emerging and Previously Unrecognized Paradigm of Cxcl12/Cxcr4 Pathway Implication in Breast Cancer Metastasis

Abstract The Cxcl12/Cxcr4 signaling axis promotes metastasis in mouse models of breast carcinoma and is linked with increased metastatic risk in humans. Prior studies have linked Cxcl12/Cxcr4 to breast cancer cell homing and survival at metastatic sites. However, the precise mechanism via which Cxcr4+ breast cancer cells escape primary tumors, which also express Cxcl12, is poorly understood. By using a novel imaging method for visualizing chemokine gradients, we show that Cxcl12 is concentrically expressed around cancer cell intravasation doorways, known as Tumor Microenvironment of Metastasis (TMEM). Using distance analysis algorithms, we also show that TMEM-mediated Cxcl12 gradients contextually associate with Cxcr4+ breast cancer cells migrating towards TMEM doorways. As such, pharmacological inhibition of the Cxcl12/Cxcr4 axis significantly abrogates translocation of Cxcr4+ cancer cells to TMEM doorways, thus suppressing metastatic dissemination. However, the targeted elimination of Cxcr4 from breast cancer cells paradoxically results in a suboptimal response compared to pharmacologic inhibition, implying the existence of a compensatory mechanism. Using clodronate-mediated macrophage depletion, we demonstrate that, in the absence of Cxcr4 expression in tumor cells, accompanying Cxcr4+ macrophages may still “read” TMEM-generated Cxcl12 chemotactic gradients. Indeed, Pdgfrb+ stromal cells may express low levels of Cxcl12, but TMEM-generated Cxcl12 gradients are primarily linked to a subset of Iba1+ perivascular tumor-associated macrophages. Overall, our data reveal a new paradigm for the implication of Cxcl12/Cxcr4 in early stages of metastasis and propose an avenue for rationalized antimetastatic treatments. Supported by Recruitment Funds RECR 3A3218

Physical Cues in the Microenvironment Regulate Stemness-Dependent Homing of Breast Cancer Cells.

Tissue-specific microenvironmental factors contribute to the targeting preferences of metastatic cancers. However, the physical attributes of the premetastatic microenvironment are not yet fully characterized. In this research, we develop a transwell-based alginate hydrogel (TAH) model to study how permeability, stiffness, and roughness of a hanging alginate hydrogel regulate breast cancer cell homing. In this model, a layer of physically characterized alginate hydrogel is formed at the bottom of a transwell insert, which is placed into a matching culture well with an adherent monolayer of breast cancer cells. We found that breast cancer cells dissociate from the monolayer and home to the TAH for continual growth. The process is facilitated by the presence of rich serum in the upper chamber, the increased stiffness of the gel, as well as its surface roughness. This model is able to support the homing ability of MCF-7 and MDA-MB-231 cells drifting across the vertical distance in the culture medium. Cells homing to the TAH display stemness phenotype morphologically and biochemically. Taken together, these findings suggest that permeability, stiffness, and roughness are important physical factors to regulate breast cancer homing to a premetastatic microenvironment.

The bone metastasis niche in breast cancer: potential overlap with the haematopoietic stem cell niche in vivo

Background: Bone metastasis is one of the most common complications of advanced breast cancer. During dissemination to bone, breast cancer cells locate in a putative ‘metastatic niche’, a microenvironment that regulates the colonisation, maintenance of tumour cell dormancy and subsequent tumour growth. The precise location and composition of the bone metastatic niche is not clearly defined. We have used in vivo models of early breast cancer dissemination to provide novel evidence that demonstrates overlap between endosteal, perivascular, HSC and the metastatic niche in bone.Methods: Estrogen Receptor (ER) +ve and -ve breast cancer cells were labelled with membrane dyes Vybrant-DiD and Vybrant-CM-DiI and injected via different routes in BALBc/nude mice of different ages. Two-photon microscopy was used to detect and quantitate tumour cells and map their location within the bone microenvironment as well as their distance to the nearest bone surface compared to the nearest other tumour cell. To investigate whether the metastatic niche overlapped with the HSC niche, animals were pre-treated with the CXCR4 antagonist AMD3100 to mobilise hematopoietic (HSCs) prior to injection of breast cancer cells.Results: Breast cancer cells displayed a characteristic pattern of homing in the long bones, with the majority of tumour cells seeded in the trabecular regions, regardless of the route of injection, cell-line characteristics (ER status) or animal age. Breast cancer cells located in close proximity to the nearest bone surface and the average distance between individual tumour cells was higher than their distance to bone. Mobilisation of HSCs from the niche to the circulation prior to injection of cell lines resulted in increased numbers of tumour cells disseminated in trabecular regions.Conclusion: Our data provide evidence that homing of breast cancer cells is independent of their ER status and that the breast cancer bone metastasis niche is located within the trabecular region of bone, an area rich in osteoblasts and microvessels. The increased number of breast cancer cells homing to bone after mobilisation of HSCs suggests that the HSC and the bone metastasis niche overlap.

Abstract P1-05-01: Breast cancer cell-derived IL-1B drives metastasis and colonisation of the bone microenvironment

Abstract 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.

Biomimetic strategies to recapitulate organ specific microenvironments for studying breast cancer metastasis.

The progression of breast cancer from the primary tumor setting to the metastatic setting is the critical event defining Stage IV disease, no longer considered curable. The microenvironment at specific organ sites is known to play a key role in influencing the ultimate fate of metastatic cells; yet microenvironmental mediated-molecular mechanisms underlying organ specific metastasis in breast cancer are not well understood. This review discusses biomimetic strategies employed to recapitulate metastatic organ microenvironments, particularly, bone, liver, lung and brain to elucidate the mechanisms dictating metastatic breast cancer cell homing and colonization. These biomimetic strategies include in vitro techniques such as biomaterial-based co-culturing techniques, microfluidics, organ-mimetic chips, bioreactor technologies, and decellularized matrices as well as cutting edge in vivo techniques to better understand the interactions between metastatic breast cancer cells and the stroma at the metastatic site. The advantages and disadvantages of these systems are discussed. In addition, how creation of biomimetic models will impact breast cancer metastasis research and their broad utility is explored.

Mechanisms involved in breast cancer liver metastasis.

Liver metastasis is a frequent occurrence in patients with breast cancer; however, the available treatments are limited and ineffective. While liver-specific homing of breast cancer cells is an important feature of metastasis, the formation of liver metastases is not random. Indeed, breast cancer cell factors contribute to the liver microenvironment. Major breakthroughs have been achieved recently in understanding breast cancer liver metastasis (BCLM). The process of liver metastasis consists of multiple steps and involves various factors from breast cancer cells and the liver microenvironment. A further understanding of the roles of breast cancer cells and the liver microenvironment is crucial to guide future work in clinical treatments. In this review we discuss the contribution of breast cancer cells and the liver microenvironment to liver metastasis, with the aim to improve therapeutic efficacy for patients with BCLM.

Breast cancer cell homing to bone in vivo is modulated by zoledronic acid (676.6)

Cancer metastasis to the skeleton causes mortality in breast cancer, however the mechanisms involved in bone metastases development remain unclear. A metastatic initiating cell (MIC) phenotype CD44+/CD24‐/ALDHhigh has been identified in breast cancer. Zoledronic acid (zol), a bone anti‐resorptive bisphosphonate is currently the only agent with proven benefit in breast cancer. This study investigates whether zol modulates MIC homing in the early stages of breast cancer bone metastasis using the Dorsal Skinfold Chamber (DSC) metatarsal model in vivo.Following DSC metatarsal implantation, mice were treated weekly either before or after intra‐cardiac MDA‐MB‐231 tumour cell injection (1x105) for 4 weeks (Zol: 25µg/kg/injection=100μg/kg or PBS control). MDA‐MB‐231 cells were sorted into ALDHhigh and ALDHlow subpopulations using the ALDEFLUORkit and then injected into groups of mice (n=6), treated with zol before tumour cell injection. Chamber tissue and metatarsal recordings were made every 48hr for up to 4 weeks to identify tumour cell homing. At the end of the study, tissue was harvested and processed for µCT, multi‐photon analysis, histology and immunohistochemistry.Zol treatment reduces MDA‐MB‐231 cells homing to bone by 76% (before) and 62% (after) compared to control animals. ALDHhigh cells home to bone in significantly (p<0.05) higher numbers than ALDHlow cells (Day 25: 10.7±1.3 cells vs. 1.1±0.6 cells). Week zol administration (before tumour cell injection) does not reduce MDA‐MB‐231 cells bone‐homing.Weekly treatment with zol reduces breast tumour cell homing to bone, whereas ALDHhigh cell homing appears resistant to zol treatment in this model.

Different molecular profiles are associated with breast cancer cell homing compared with colonisation of bone: evidence using a novel bone-seeking cell line

Advanced breast cancer is associated with the development of incurable bone metastasis. The two key processes involved, tumour cell homing to and subsequent colonisation of bone, remain to be clearly defined. Genetic studies have indicated that different genes facilitate homing and colonisation of secondary sites. To identify specific changes in gene and protein expression associated with bone-homing or colonisation, we have developed a novel bone-seeking clone of MDA-MB-231 breast cancer cells that exclusively forms tumours in long bones following i.v. injection in nude mice. Bone-homing cells were indistinguishable from parental cells in terms of growth rate in vitro and when grown subcutaneously in vivo. Only bone-homing ability differed between the lines; once established in bone, tumours from both lines displayed similar rates of progression and caused the same extent of lytic bone disease. By comparing the molecular profile of a panel of metastasis-associated genes, we have identified differential expression profiles associated with bone-homing or colonisation. Bone-homing cells had decreased expression of the cell adhesion molecule fibronectin and the migration and calcium signal binding protein S100A4, in addition to increased expression of interleukin 1B. Bone colonisation was associated with increased fibronectin and upregulation of molecules influencing signal transduction pathways and breakdown of extracellular matrix, including hRAS and matrix metalloproteinase 9. Our data support the hypothesis that during early stages of breast cancer bone metastasis, a specific set of genes are altered to facilitate bone-homing, and that disruption of these may be required for effective therapeutic targeting of this process.

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 biological subtypes and protein expression predict for the preferential distant metastasis sites: a nationwide cohort study

IntroductionSome molecular subtypes of breast cancer have preferential sites of distant relapse. The protein expression pattern of the primary tumor may influence the first distant metastasis site.MethodsWe identified from the files of the Finnish Cancer Registry patients diagnosed with breast cancer in five geographical regions Finland in 1991-1992, reviewed the hospital case records, and collected primary tumor tissue. Out of the 2,032 cases identified, 234 developed distant metastases after a median follow-up time of 2.7 years and had the first metastatic site documented (a total of 321 sites). Primary tumor microarray (TMA) cores were analyzed for 17 proteins using immunohistochemistry and for erbB2 using chromogenic in situ hybridization, and their associations with the first metastasis site were examined. The cancers were classified into luminal A, luminal B, HER2+ enriched, basal-like or non-expressor subtypes.ResultsA total of 3,886 TMA cores were analyzed. Luminal A cancers had a propensity to give rise first to bone metastases, HER2-enriched cancers to liver and lung metastases, and basal type cancers to liver and brain metastases. Primary tumors that gave first rise to bone metastases expressed frequently estrogen receptor (ER) and SNAI1 (SNAIL) and rarely COX2 and HER2, tumors with first metastases in the liver expressed infrequently SNAI1, those with lung metastases expressed frequently the epidermal growth factor receptor (EGFR), cytokeratin-5 (CK5) and HER2, and infrequently progesterone receptor (PgR), tumors with early skin metastases expressed infrequently E-cadherin, and breast tumors with first metastases in the brain expressed nestin, prominin-1 and CK5 and infrequently ER and PgR.ConclusionsBreast tumor biological subtypes have a tendency to give rise to first distant metastases at certain body sites. Several primary tumor proteins were associated with homing of breast cancer cells.

Abstract 3338: Capacity of breast cancer cell homing to distant organs is altered by acquisition of stem cell properties

Abstract It is hypothesized that migration of tumor cells to distant organs is mediated by E-selectin expressed on vascular endothelium of a secondary tissue and its counter receptors expressed on circulating breast tumor cells. Previously, our group has characterized glycosphingolipids and CD44 as E-selectin binding molecules expressed on BT-20 and MDA-MB-468 breast cancer cell lines. We now report that the ZR-75-1 breast cancer cell line possesses E-selectin ligand activity, yet does not express any of the known E-selectin ligands found on colon cancer cells, leukocyte, or BT-20 and MDA-MB-468 breast cancer cells. A systematic investigation to identify novel E-selectin ligands expressed by ZR-75-1 cells was performed. Molecules isolated from whole cell lysate by immunoprecipitation with mouse E-selectin/ human Fc chimera (E-Ig chimera) were analyzed by mass spectrometry, which revealed that a glycoprotein known as Mac-2 binding protein (Mac-2bp) is an E-selectin ligand. Immunoprecipitation of Mac-2bp from whole cell lysate by using anti-Mac-2bp antibody followed by western blotting with E-Ig chimera and HECA-452 antibody demonstrated that Mac-2bp is a sialofucosylated E-selectin ligand. Dual staining of ZR-75-1 cells with anti-Mac-2bp antibody and E-Ig chimera imaged by fluorescence microscopy showed that Mac-2bp is a cell surface glycoprotein that is co-localized with E-selectin binding epitopes. Collectively, these results demonstrated that Mac-2bp is a novel E-selectin ligand expressed by ZR-75-1 breast cancer cells. In addition to the three breast cancer cell lines above, we tested four other breast cancer cell lines for their E-selectin ligand activity. Among the panel of cell lines examined, MDA-MB-231 and Hs-578t cells showed very weak activity for E-selectin binding as compared to the other five cell lines. Surprisingly, these were the only cells among all tested that were characterized by the CD44+/CD24-/low phenotype (>80% of cells). Staining with E-Ig chimera with subsequent detection by flow cytometry revealed differential E-selectin ligand activities of cancer stem versus non-stem cell phenotypes. Further, BT-20 cells (predominantly CD44+/CD24+) were treated with the metastatic niche factor TGF-β to force EMT and transformation into cancer stem cells, and change(s) in E-selectin ligand expression and function were assessed. Based on these results, we propose a new paradigm for the metastasis cascade, whereby breast cancer cells home to distant organs via an E-selectin-mediated pathway followed by transformation into breast cancer stem cells using metastatic niche factors. This model is consistent with the notion that cancer cell plasticity might have a significant role in metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3338. doi:10.1158/1538-7445.AM2011-3338

Identification of prospective factors promoting osteotropism in breast cancer: a potential role for CITED2

Breast cancer metastases develop in the bone more frequently than any other site and are a common cause of morbidity in the form of bone pain, pathological fractures, nerve compression and life-threatening hypercalcemia. Despite ongoing research efforts, the molecular and cellular mechanisms that regulate breast cancer cell homing to and colonization of the bone as well as resultant pathological bone alteration remain poorly understood. To identify key mediators promoting breast cancer metastasis to bone, we utilized an immunocompetent, syngeneic murine model of breast cancer metastasis employing the mammary tumor cell line NT2.5. Following intracardiac injection of NT2.5 cells in neu-N mice, metastases developed in the bone, liver and lung, closely mimicking the anatomical distribution of metastases in patients with breast cancer. Using an in vivo selection process, we established NT2.5 sublines demonstrating an enhanced ability to colonize the bone and liver. Genome-wide cDNA microarray analysis comparing gene expression between parental NT2.5 cells and established sublines revealed both known and novel mediators of bone metastasis and osteolysis, including the transcriptional co-activator CITED2. In further studies, we found that expression of CITED2 was elevated in human primary breast tumors and bone metastasis compared to normal mammary epithelium and was highest in breast cancer cell lines that cause osteolytic bone metastasis in animal models. In addition, reducing CITED2 expression in NT2.5 cells inhibited the establishment of bone metastasis and osteolysis in vivo, suggesting a potential role for CITED2 in promoting breast cancer bone metastasis.

Chemokine receptors in advanced breast cancer: differential expression in metastatic disease sites with diagnostic and therapeutic implications

Background: We investigated the expression of CXCR4, CCR7, estrogen receptor (ER), progesterone receptor (PR) and HER2-neu in human metastatic breast cancers to determine whether these biological biomarkers were preferentially expressed in any organ-specific metastases.Materials and methods: CXCR4, CCR7, ER, PR and HER2-neu expression levels were evaluated by immunohistochemical staining using paraffin-embedded tissue sections of metastatic breast cancers (n=41) obtained by either diagnostic biopsy or surgical resection.Results: The metastatic sites included the following: bone (n=15), brain (n=14), lung (n=6), liver (n=2), and omental metastases (n=2). CXCR4 was expressed in 41% of cases, CCR7 expression was demonstrated in 10%, and HER2-neu overexpression was present in 27%. CXCR4 was more likely to be expressed in bone metastases than visceral metastases (67% versus 26%, P=0.020). Visceral sites demonstrated a lower rate of CXCR4 positivity (33% and 23%, respectively, for lung and brain metastases). Similarly, CCR7 was more likely to be found in bone metastases than visceral sites (27% versus 0%, P=0.037).Conclusions: These results indicate that CXCR4 can contribute to the homing of breast cancer cells to the bone. This finding might have important clinical implications since patients with metastatic bone disease may achieve the highest benefit from a CXCR4-targeted therapy.

Plasma Stromal Cell–Derived Factor-1: Host Derived Marker Predictive of Distant Metastasis in Breast Cancer

Homing of breast cancer cells to metastatic sites may be regulated by the production of stromal cell-derived factor (SDF)-1 by specific target organs, which attracts CXCR4-expressing breast cancer cells. We investigated the value of SDF-1 as a predictive blood marker of distant metastasis in breast cancer, together with a common polymorphism of SDF-1, SDF-1-3'A. Plasma samples were collected prospectively for 270 consecutive primary breast cancer patients with a median follow-up of 3.3 years. Plasma SDF-1 levels were measured using an ELISA, and the polymorphism was identified via PCR-RFLP analysis. Plasma SDF-1 levels were divided into two groups, low and high, based on the median SDF-1 value of 2,661 pg/mL. Patients with low SDF-1 showed an increased risk of developing distant metastasis (relative risk, 1.94; P = 0.02) and poorer breast cancer-specific survival [adjusted hazard ratio (AHR), 3.92; P = 0.007]. Patients with both low plasma SDF-1 levels and the SDF-1-3'A polymorphism showed a poorer breast cancer-specific survival (AHR, 3.98; P = 0.001) and distant disease-free survival (AHR, 2.88; P = 0.003). In a separate cohort of 22 breast cancer patients, we found no significant difference in SDF-1 levels before and posttumor resection. We found that low plasma SDF-1 is an independent host-derived predictive marker of distant metastasis in breast cancer. The prognostic value of the combination of a low plasma SDF-1 level and the SDF-1-3'A polymorphism identifies a cohort of patients with an intrinsic susceptibility for poorer survival.

Bone sialoprotein and its transcriptional regulatory mechanism

Bone sialoprotein is a mineralized tissue-specific noncollagenous protein that is glycosylated, phosphorylated and sulfated. The temporo-spatial deposition of bone sialoprotein into the extracellular matrix of bone, and the ability of bone sialoprotein to nucleate hydroxyapatite crystal formation, indicates a potential role for bone sialoprotein in the initial mineralization of bone, dentin and cementum. Bone sialoprotein is also expressed in breast, lung, thyroid and prostate cancers. We used osteoblast-like cells (rat osteosarcoma cell lines ROS17/2.8 and UMR106, rat stromal bone marrow RBMC-D8 cells and human osteosarcoma Saos2 cells), and breast and prostate cancer cells to investigate the transcriptional regulation of bone sialoprotein. To determine the molecular basis of the transcriptional regulation of the bone sialoprotein gene, we conducted northern hybridization, transient transfection analyses with chimeric constructs of the bone sialoprotein gene promoter linked to a luciferase reporter gene and gel mobility shift assays. Bone sialoprotein transcription is regulated by hormones, growth factors and cytokines through tyrosine kinase, mitogen-activated protein kinase and cAMP-dependent pathways. Microcalcifications are often associated with human mammary lesions, particularly with breast carcinomas. Expression of bone sialoprotein by cancer cells could play a major role in the mineral deposition and in preferred bone homing of breast cancer cells. Bone sialoprotein protects cells from complement-mediated cellular lysis, activates matrix metalloproteinase 2 and has an angiogenic capacity. Therefore, regulation of the bone sialoprotein gene is potentially important in the differentiation of osteoblasts, bone matrix mineralization and tumor metastasis. This review highlights the function and transcriptional regulation of bone sialoprotein.

Genes involved in breast cancer metastasis to bone.

Metastasis to bone occurs frequently in advanced breast cancer and is accompanied by debilitating skeletal complications. Current treatments are palliative and new therapies that specifically prevent the spread of breast cancer to bone are urgently required. While our understanding of interactions between breast cancer cells and bone cells has greatly improved, we still know little about the molecular determinants that regulate specific homing of breast cancer cells to the bone. In this review, we focus on genes that have been implicated in migration and adhesion of breast cancer cells to bone, as well as genes that promote tumor cell proliferation in the bone microenvironment. In addition, the review discusses new technologies, including better animal models, that will further assist with the identification of the molecular determinants of bone metastasis and will guide the development of new therapies.

Calcitonin receptors, bone sialoprotein and osteopontin are expressed in primary breast cancers.

Several human breast cancer cell lines express the calcitonin receptor (CTR), but this has not been demonstrated previously in clinical breast cancers. We examined 18 primary breast cancers by reverse transcription-PCR, for expression of CTR and of the bone proteins osteopontin (OPN) and bone sialoprotein (BSP). OPN and CTR were expressed by each of the tumours, and 7 (39%) additionally expressed an alternate form of CTR, whilst BSP was expressed by 13 tumours (72%). In situ hybridisation confirmed that expression of OPN and CTR was confined to the tumour cells. Expression of CTR, BSP and OPN may prove to be a useful marker for breast cancers, and their role in the homing of breast cancer cells to bone remains to be investigated.

H-31 human breast cancer cells stimulate type I collagenase production in osteoblast-like cells and induce bone resorption.

Bone is one of the most common sites of metastasis in breast cancer. For metastasis to occur in bone, tumor cells must induce osteolysis by osteoclasts. Degradation of the osteoid layer by type I collagenase is a necessary process before osteolysis can occur because the osteoid layer hinders osteoclasts from adhering to bone. In this study, we investigated the function of H-31 human breast cancer cells in inducing type I collagenase production and in enhancing bone resorption. H-31 cells did not themselves produce type I collagenase whereas MG-63 human osteoblast-like cells and MC3T3-E1 mouse osteoblast cells constantly produced type I collagenase. When these osteoblast-like cells were cocultured with H-31 cells, type I collagenase production was enhanced. The same enhancement occurred when the conditioned medium of H-31 cells was added to the osteoblast-like cells. The activity of this type I collagenase was inhibited by EDTA and minocyclin, an inhibitor of matrix metalloproteinases, hence it was identified as matrix metalloproteinase-1 (MMP-1). H-31 cells exhibited chemotactic migration towards collagen; therefore, collagen degraded by MMP-1 may play an important role in the localisation of breast cancer cells like H-31 to bone. In an organ culture system using newborn mouse calvaria, the conditioned medium of H-31 cells increased the concentration of calcium in the medium, and this effect was inhibited by minocyclin, indicating that bone resorption occurred in this system. Based on these observations, we speculate that type I collagenase produced by osteoblast cells in response to breast cancer cells (exemplified by H-31) may facilitate degradation of the osteoid layer and the homing of breast cancer cells to bone. This can lead to osteolysis by osteoclasts, a crucial event for bone metastasis.