Bone Metastatic Breast Cancer Cells Research Articles

Abstract PR07: Dissection of cancer cells extravasation through human vascularized 3D microfluidic model: The major role of talin-1

Abstract Cancer cells spread from a primary tumor to secondary loci is responsible for more than 90% of cancer related mortality. Hematogenous metastasis is a complex process [1]. It includes a chain of events that can be summarized as follows: migration from primary tumor site and intravasation of the primary tumor cancer cells into the blood flow, dissemination through the circulation, extravasation in different organs, survival in the new microenvironment and colonization with generation of a new tumor. Recently our group presented a microfluidic 3D model reproducing the effects of the CXCL5-CXCR2 interaction between bone cells and metastatic breast cancer cells observed in vivo [2]. We further developed a human 3D microfluidic model that enables the study of human metastatic breast cancer cell extravasation within a perfusable human microvascularized bone-mimicking microenvironment [3]. Understanding the cellular and molecular events implicated in extravasation could facilitate the design of new therapeutic strategies targeting cancer cells only in order to couple these new developed therapies with already existing treatments and finally fight cancer. Talin-1 is a cytoplasmic adaptor essential for integrin-mediated adhesion to the ECM. Talin-1 links the actin cytoskeleton to integrin at the plasma membrane, regulates the focal adhesion pathway in normal cells and it is up-regulated in triple negative breast cancer cells such as MDA-MB231 and it is mutated in sarcomas. Based on the above described models, we developed a human vascularized 3D microfluidic device where human perfusable capillary like structures were embedded in fibrin matrix, characterized by mature endothelium markers (VE-cadherin, ZO-1) and physiological permeability (1.5±0.76)*10-6cm/s. Since these models can provide quantitative data on cancer cell extravasation, we focused on the role of Talin-1 in extravasation through blood vessels and in early invasion in two cell lines whose Talin-1 expression was silenced by SiRNA: triple negative metastatic breast cancer cells (MDA-MB231) and the metastatic fibro-sarcoma cell line (HT1080). Talin-1 silencing was confirmed by western blotting with specific antibodies. Adhesion to the endothelial wall, extravasation and ensuing migration out from the wall within the extracellular matrix was monitored by means of confocal high resolution real time imaging analyses. We demonstrated that silencing of Talin-1 expression dramatically and in a statistically significant way reduced both the adhesion efficiency and extravasation in both MDA-MB 231 and HT1080. Cell migration was also strongly and statistically reduced by the SiRNA treatment in both analyzed cell lines. In summary, we are the first to dissect the role of Talin-1 in each step of extravasation, demonstrating that targeting this protein could be an effective strategy to block metastasis. These data identify Talin-1 as a promising target for the development of new anti-metastatic therapies based on Talin-1 inhibition.

Enhanced MAF Oncogene Expression and Breast Cancer Bone Metastasis.

Background:There are currently no biomarkers for early breast cancer patient populations at risk of bone metastasis. Identification of mediators of bone metastasis could be of clinical interest.Methods:A de novo unbiased screening approach based on selection of highly bone metastatic breast cancer cells in vivo was used to determine copy number aberrations (CNAs) associated with bone metastasis. The CNAs associated with bone metastasis were examined in independent primary breast cancer datasets with annotated clinical follow-up. The MAF gene encoded within the CNA associated with bone metastasis was subjected to gain and loss of function validation in breast cancer cells (MCF7, T47D, ZR-75, and 4T1), its downstream mechanism validated, and tested in clinical samples. A multivariable Cox cause-specific hazard model with competing events (death) was used to test the association between 16q23 or MAF and bone metastasis. All statistical tests were two-sided.Results:16q23 gain CNA encoding the transcription factor MAF mediates breast cancer bone metastasis through the control of PTHrP. 16q23 gain (hazard ratio (HR) for bone metastasis = 14.5, 95% confidence interval (CI) = 6.4 to 32.9, P < .001) as well as MAF overexpression (HR for bone metastasis = 2.5, 95% CI = 1.7 to 3.8, P < .001) in primary breast tumors were specifically associated with risk of metastasis to bone but not to other organs.Conclusions:These results suggest that MAF is a mediator of breast cancer bone metastasis. 16q23 gain or MAF protein overexpression in tumors may help to select patients at risk of bone relapse.

Abstract 3191: The role of rigidity in myeloid-tumor cell interactions in bone

Abstract Bone is one of the most common metastatic sites for breast cancer, where nearly 70% of patients that die from disease develop skeletal metastases. Skeletal metastases can cause hypercalcemia, fractures, and pain. With the use of 2D polyurethane films (PUR) that mimic tissue rigidity ranging from the basement membrane (10 MPa) to bone (2600 MPa), we have previously published that bone rigidity increases expression of Parathyroid hormone-related protein (PTHrP). This factor is important in tumor-induced bone disease in several cancer cell lines that metastasize to bone. Current in vivo studies implanting 3D scaffolds seeded with the bone metastatic breast cancer cells MDA-MB-231 GFP into athymic nude mice indicate that cells from the microenvironment infiltrate into the scaffolds, suggesting that these cells may play a role in tumor response to rigidity. Myeloid cells, which have been implicated in promoting tumor progression by altering the tumor microenvironment, are one of major infiltrating populations as determined by flow cytometry. Specifically CD11b+ myeloid cells made up 15% of overall cells within the scaffold, with the remaining population being mostly GFP+ tumor cells. Since few studies have examined the effect of rigidity on myeloid cells, we hypothesize that the physical rigidity of the bone promotes myeloid secretion of pro-tumorigenic factors that lead to an increase in tumor-expression of factors important in bone metastasis. In order to determine how rigidity influences myeloid cell differentiation and their effect on tumor progression, we seeded the macrophage/osteoclast cell line, RAW 264.7 cells on 2D PUR films. Differentiation of cells was monitored and conditioned media collected and used to treat MDA-MB-231 cells seeded on 2D PUR films. Surprisingly, we found that the conditioned media from RAW cells that were seeded onto the rigid films (bone-micking) decreased expression of PTHrP in tumor cells seeded on both rigid and soft films using RT-PCR. This effect was not seen from conditioned media from RAW cells seeded on soft films. Additionally, RAW cells seeded on soft films upregulate their expression of TGF-β, a factor important in regulating the expression of PTHrP, by 6-fold as compared to cells seeded on a rigid film. In contrast, PTHrP expression increases on rigid scaffolds in the absence of RAW conditioned media. Together these results suggest that myeloid cells sense changes in rigidity and alter their signaling profile accordingly to aid bone metastasis. Importantly, these studies highlight the need to perform studies that include more diverse cell populations, since the presence of myeloid cells or other cell types can alter gene expression within tumor cells. The use of 3D scaffolds will allow us to include multiple cell types and examine the interactions between tumor cells and cells in the bone microenvironment. Better understanding this interaction will help identify new therapeutic targets for the inhibition of tumor-induced bone disease. Citation Format: Denise Buenrostro, Ushashi Dadwal, Scott Guelcher, Julie Sterling. The role of rigidity in myeloid-tumor cell interactions in bone. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3191. doi:10.1158/1538-7445.AM2015-3191

New mechanistic insights of integrin β1 in breast cancer bone colonization.

Bone metastasis is a frequent and life-threatening complication of breast cancer. The molecular mechanisms supporting the establishment of breast cancer cells in the skeleton are still not fully understood, which may be attributed to the lack of suitable models that interrogate interactions between human breast cancer cells and the bone microenvironment. Although it is well-known that integrins mediate adhesion of malignant cells to bone extracellular matrix, their role during bone colonization remains unclear. Here, the role of β1 integrins in bone colonization was investigated using tissue-engineered humanized in vitro and in vivo bone models. In vitro, bone-metastatic breast cancer cells with suppressed integrin β1 expression showed reduced attachment, spreading, and migration within human bone matrix compared to control cells. Cell proliferation in vitro was not affected by β1 integrin knockdown, yet tumor growth in vivo within humanized bone microenvironments was significantly inhibited upon β1 integrin suppression, as revealed by quantitative in/ex vivo fluorescence imaging and histological analysis. Tumor cells invaded bone marrow spaces in the humanized bone and formed osteolytic lesions; osteoclastic bone resorption was, however, not reduced by β1 integrin knockdown. Taken together, we demonstrate that β1 integrins have a pivotal role in bone colonization using unique tissue-engineered humanized bone models.

Runx2, a target gene for activating transcription factor-3 in human breast cancer cells.

Activating transcription factor (ATF-3) is a stress response gene and is induced by transforming growth factor beta 1 (TGF-β1) in breast cancer cells. In this study, we dissected the functional role of ATF-3 gene in vitro by knocking down its expression stably in human bone metastatic breast cancer cells (MDA-MB231). Knockdown of ATF-3 expression in these cells decreased cell number, altered cell cycle phase transition, and decreased mRNA expression of cell cycle genes. Knockdown of ATF-3 expression in MDA-MB231 cells also decreased cell migration, and the expression levels of invasive and metastatic genes such as MMP-13 and Runx2 were found to be decreased in these cells. Most importantly, ATF-3 was associated with Runx2 promoter in MDA-MB231 cells and knockdown of ATF-3 expression decreased its association with Runx2 promoter. Hence, our results suggested that ATF-3 plays a role in proliferation and invasion of bone metastatic breast cancer cells in vitro and we identified for the first time that Runx2 is a target gene of ATF-3 in MDA-MB231 cell line.

Direct but not indirect co-culture with osteogenically differentiated human bone marrow stromal cells increases RANKL/OPG ratio in human breast cancer cells generating bone metastases.

BackgroundBone metastases arise in nearly 70% of patients with advanced breast cancer, but the complex metastatic process has not been completely clarified yet. RANKL/RANK/OPG pathway modifications and the crosstalk between metastatic cells and bone have been indicated as potential drivers of the process. Interactions between tumor and bone cells have been studied in vivo and in vitro, but specific effects of the direct contact between human metastatic cells and human bone cells on RANKL/RANK/OPG pathway have not been investigated.FindingsWe directly co-cultured bone metastatic human breast cancer cells (BOKL) with osteo-differentiated human mesenchymal cells (BMSCs) from 3 different donors. BMSCs and BOKL were then enzymatically separated and FACS sorted. We found a significant increase in the RANKL/OPG ratio as compared to control, which was not observed in BOKL cultured in medium conditioned by BMSCs, neither in BOKL directly cultured with fibroblasts or medium conditioned by fibroblasts. Direct co-culture with osteo-differentiated BMSCs caused BOKL aggregation while proliferation was not affected by co-culture. To more specifically associate RANKL expression to osteogenic differentiation degree of BMSCs, we determined their osteogenic markers expression and matrix calcification relative to osteoblasts and fibroblasts.ConclusionsIn conclusion, our co-culture model allowed to demonstrate for the first time that direct contact but not paracrine interactions between human metastatic breast cancer cells and bone cells has a significant effect on RANKL/OPG expression in bone metastatic cells. Furthermore, only direct contact with the bone microenvironment induced BOKL clustering without however significantly influencing their proliferation and migration.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-238) contains supplementary material, which is available to authorized users.

Abstract 130: Deadly KISS, Kisspeptin10 interaction with osteoblasts and breast cancer metastastic cells

Abstract Current cancer research focuses on blockading metastasis with the hope of increasing patient survival and prognoses. This research has spurred an interest in metastasis suppressor genes, such as KISS-1, whose role in breast cancer remains controversial. This study focuses on the interactions among one of the proteolytic processing products of the Kiss1 protein (kisspeptin10, KP10), its receptor, KISS1R (GPR54, AXOR12), osteoblasts (bone forming cells), and bone-metastatic breast cancer cells, MDA-MB-231. Previous research suggests that treatment with KP10 reduces the ability of the cancer cells to invade a reconstituted basement membrane (Matrigel) and to migrate in response to osteosarcoma cells. The aim of this present study was to determine if KP10 affected the interaction of MC3T3-E1 osteoblasts and bone-metastatic breast cancer cells. Our laboratory had previously determined that osteoblasts exhibit an inflammatory response when treated with conditioned medium from breast cancer cells. This inflammatory response may contribute to cancer cell proliferation in the bone. This response has not yet been characterized in the presence of KP10. I hypothesized that KP10 would down regulate the inflammatory response of osteoblasts to breast cancer cells. MDA-MB-231 breast cancer cells express theKISS1R; therefore, these cells were treated with KP10 (10-100 nM, 2hr), the medium was collected, and the osteoblasts were treated with it. KP10 significantly decreased the inflammatory response of osteoblasts to breast cancer conditioned media indicated by decreased levels of IL-6 as determined by ELISA. This research has important implications because it provides insight into how KP10 may be acting on either the breast cancer cells or the bone cells. In my future research, I will continue to characterize how KP10 can be used to prevent proliferation or activation of breast cancer metastases in the bone. This work was supported by an undergraduate research grant from the Eberly College of Science of Penn State University. Citation Format: Kaitlyn E. Leahey, Danny R. Welch, Yu-Chi Chen, Karen M. Bussard, Andrea M. Mastro. Deadly KISS, Kisspeptin10 interaction with osteoblasts and breast cancer metastastic cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 130. doi:10.1158/1538-7445.AM2014-130

Elevated Pentraxin 3 in bone metastatic breast cancer is correlated with osteolytic function

Pentraxin 3 (PTX3), a modulator of tumor-associated inflammation, is known to be positively correlated with tumor grade and severity of malignancies, but its exact role remains unclear. This study found that PTX3 expression was up-regulated in distant bone metastases of breast cancer compared to lung, liver, and brain metastases in 64 human breast cancer patients. Elevated expression of PTX3 was correlated with poor survival in patients with breast cancer. PTX3 expression was also up-regulated in a bone metastatic breast cancer cell line and further enhanced by pro-inflammatory cytokine TNFα. Administration of PTX3 promoted the migratory potential of breast cancer cells and the mobilization of macrophages, a precursor of osteoclasts (OCs), toward breast cancer cells. In addition, elevated expression of PTX3 by TNFα led to enhanced OC formation, implying the distinct role of PTX3 in osteolytic bone metastasis of breast cancer cells. Furthermore, PTX3 silencing using PTX3-specific siRNA prevented breast cancer cell migration, macrophage chemotaxis, and subsequent OC formation. These findings provide an important insight into the key role of PTX3 in inflammation-associated osteolytic complications of breast cancer.

A Novel Model of Dormancy for Bone Metastatic Breast Cancer Cells

Mortality of patients with breast cancer is due overwhelmingly to metastatic spread of the disease. Although dissemination is an early event in breast cancer, extended periods of cancer cell dormancy can result in long latency of metastasis development. Deciphering the mechanisms underlying cancer cell dormancy and subsequent growth at the metastatic site would facilitate development of strategies to interfere with these processes. A challenge in this undertaking has been the lack of models for cancer cell dormancy. We have established novel experimental systems that model the bone microenvironment of the breast cancer metastatic niche. These systems are based on 3D cocultures of breast cancer cells with cell types predominant in bone marrow. We identified conditions in which cancer cells are dormant and conditions in which they proliferate. Dormant cancer cells were able to proliferate upon transfer into supportive microenvironment or upon manipulation of signaling pathways that control dormancy. These experimental systems will be instrumental for metastasis studies, particularly the study of cellular dormancy.

Identification of MicroRNAs Inhibiting TGF-β-Induced IL-11 Production in Bone Metastatic Breast Cancer Cells

Development of bone metastases is dependent on the cancer cell-bone cell interactions in the bone microenvironment. Transforming growth factor β (TGF-β) is released from bone during osteoclastic bone resorption and induces production of osteolytic factors, such as interleukin 11 (IL-11), in breast cancer cells. IL-11 in turn increases osteolysis by stimulating osteoclast function, launching a vicious cycle of cancer growth and bone destruction. We aimed to identify and functionally characterize microRNAs (miRNAs) that mediate the bone metastatic process, focusing on miRNAs that regulate the TGF-β induction of IL-11. First, we profiled the expression of 455 miRNAs in a highly bone metastatic MDA-MB-231(SA) variant as compared to the parental MDA-MB-231 breast cancer cell line and found 16 miRNAs (3.5%) having a >3-fold expression difference between the two cell types. We then applied a cell-based overexpression screen with Pre-miRNA constructs to functionally identify miRNAs regulating TGF-β-induced IL-11 production. This analysis pinpointed miR-204, miR-211, and miR-379 as such key regulators. These miRNAs were shown to directly target IL11 by binding to its 3′ UTR. MiR-379 also inhibited Smad2/3/4-mediated transcriptional activity. Gene expression analysis of miR-204 and miR-379-transfected cells indicated that these miRNAs downregulated the expression of several genes involved in TGF-β signaling, including prostaglandin-endoperoxide synthase 2 (PTGS2). In addition, there was a significant correlation between the genes downregulated by miR-379 and a set of genes upregulated in basal subtype of breast cancer. Taken together, the functional evidence and clinical correlations imply novel mechanistic links between miRNAs and the key steps in the bone metastatic process in breast cancer, with potential clinical relevance.

Peroxiredoxin 2 specifically regulates the oxidative and metabolic stress response of human metastatic breast cancer cells in lungs

Little is known about metastatic pathways that are specific to the lung rather than other organs. We previously showed that antioxidant proteins such as peroxiredoxins were specifically upregulated in lung metastatic breast cancer cells. We hypothesize that cancer cells that live under aerobic conditions, as might be the case in lungs, protect themselves against the damage caused by reactive oxygen species (ROS). To examine this hypothesis, we studied the role of peroxiredoxin-2 (PRDX2) in lung vs bone metastasis formation. A metastatic variant of MDA-MB-435 breast cancer cells that specifically metastasize to lungs (435-L3) was transduced with short hairpin RNAs to specifically silence PRDX2. Conversely, a bone metastatic variant of MDA-MB-231 cells (BO2) was stably transfected to overexpress PRDX2. The 435-L3 cells silenced for PRDX2 were significantly more sensitive to H(2)O(2)-induced oxidative stress than the parental and scrambled transfected cells. BO2/PRDX2 cells produced less ROS than BO2/green fluorescent protein control cells under oxidative stress. Moreover, PRDX2 knockdown inhibited the growth of 435-L3 cells in the lungs, whereas lymph node metastasis remained unaffected. In contrast, PRDX2 overexpression in bone metastatic BO2 breast cancer cells led to drastic inhibition of the skeletal tumor burden and reduction of bone destruction. Furthermore, PRDX2 expression in breast cancer cells was associated with a glucose-dependent phenotype, different from bone metastatic cells. Overall, our results strongly suggest that PRDX2 is a targetable 'metabolic adaptor' driver protein implicated in the selective growth of metastatic cells in the lungs by protecting them against oxidative stress.

Erratum to: Enhanced serine production by bone metastatic breast cancer cells stimulates osteoclastogenesis

Erratum to: Enhanced serine production by bone metastatic breast cancer cells stimulates osteoclastogenesis

CCN3 Impairs Osteoblast and Stimulates Osteoclast Differentiation to Favor Breast Cancer Metastasis to Bone

Bone is a preferred site for breast cancer metastasis, causing pain, fractures, spinal cord compressions, and hypercalcemia, all of which can significantly diminish the patient's quality of life. We identified CCN3 as a novel factor that is highly expressed in bone metastatic breast cancer cells from a xenograft mouse model and in bone metastatic lesions from patients with breast cancer. We demonstrate that CCN3 overexpression enhances the ability of weakly bone metastatic breast cancer cells to colonize and grow in the bone without altering their growth in the mammary fat pad. We further demonstrated that human recombinant CCN3 inhibits osteoblast differentiation from primary bone marrow cultures, leading to a higher receptor activator of NF-κB ligand (RANKL)/osteoprotegerin (OPG) ratio. In conjunction with its ability to impair osteoblast differentiation, we uncovered a novel role for CCN3 in promoting osteoclast differentiation from RANKL-primed monocyte precursors. CCN3 exerts its pro-osteoclastogenic effects by promoting calcium oscillations and nuclear factor of activated T cells c1 (NFATc1) nuclear translocation. Together, these results demonstrate that CCN3 regulates the differentiation of bone resident cells to create a resorptive environment that promotes the formation of osteolytic breast cancer metastases.

Osteoblasts are a major source of inflammatory cytokines in the tumor microenvironment of bone metastatic breast cancer

Metastatic breast cancer cells co-opt the cells of the bone to increase their production of inflammatory cytokines. Here, we sought to identify key cytokines expressed by osteoblasts in vitro and in vivo in the presence of MDA-MB-231 metastatic breast cancer cells, including a bone-seeking variant. We hypothesized that osteoblast-derived cytokines increase in the presence of metastatic breast cancer cell conditioned medium (CM), act as chemoattractants for cancer cells, and enhance osteoclast formation. We detected increases in the concentrations of osteoblast-derived IL-6, MCP-1, VEGF, MIP-2, and KC in vitro in culture supernatants from MC3T3-E1 cells in the presence of metastatic breast cancer cell CM and from cancer-bearing femurs ex vivo. A comparison of cancer cell- and osteoblast-derived cytokines revealed that while breast cancer cells expressed the same or equivalent cytokines as the osteoblasts, the breast cancer cells only produced picogram quantities of MCP-1; osteoblasts expressed nanogram amounts. Bone-derived MCP-1 increased in the proximal metaphysis, an area where breast cancer cells preferentially trafficked following intracardiac inoculation in athymic mice. An MDA-MB-231 bone-seeking variant was not different from parental lines. Osteoblast CM was a potent chemoattractant for metastatic breast cancer cells. Furthermore, culture supernatants of osteoblasts treated with breast cancer cell CM enhanced osteoclast formation. These findings suggest that bone metastatic breast cancer cells utilize osteoblast-derived cytokines to facilitate breast cancer cell colonization and survival upon arrival in the bone microenvironment.

Enhanced serine production by bone metastatic breast cancer cells stimulates osteoclastogenesis

Since bone metastatic breast cancer is an incurable disease, causing significant morbidity and mortality, an understanding of the underlying molecular mechanisms would be highly valuable. Here, we describe in vitro and in vivo evidences for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Genome-wide gene expression profiling of this isogenic cell line pair revealed that all the three genes involved in the L: -serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L: -serine in mammalian tissues. Consistently, we observed that the proliferation of MDA-MB-231(SA) cells in serine-free conditions was dependent on PSAT1 expression. In addition, we observed that L: -serine is essential for the formation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L: -serine on osteoclastogenesis and cancer cell proliferation indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions.

Osteoblasts modulate Ca2+ signaling in bone-metastatic prostate and breast cancer cells

Metastatic prostate and breast cancers display a predilection for the skeleton. The high incidence of skeletal metastasis may be a reflection of favorable reciprocal interactions between the bone microenvironment and disseminated cancer cells. Here we show that bone-metastatic PC3-ML prostate cancer cells and MDA-231 breast cancer cells-when co-cultured with human osteoblasts-down-regulate the increase in cytosolic free calcium (Ca(2+)) induced by agonist stimulation. This osteoblast promoted alteration of Ca(2+) signaling develops and reverts in a time-dependent manner. Most importantly, the Ca(2+) responses of cancer cells lacking bone metastatic potential are not affected by osteoblasts. The limited increase in cytosolic Ca(2+) observed in bone-metastatic cells does not result from depleted intracellular Ca(2+) stores but rather a decreased entry of Ca(2+) from the extracellular space. Interestingly, the inhibition of histone deacetylase in cancer cells replicates the changes in Ca(2+) signaling induced by osteoblasts, suggesting the participation of epigenetic mechanisms. Finally, cancer cells harvested from skeletal metastases induced in mice showed Ca(2+) responses identical to cells co-cultured with osteoblasts. However, Ca(2+) signaling in cancer cells recovered from metastases to soft-tissues was not affected, emphasizing the role of the bone microenvironment in regulating the functional behavior of bone-metastatic cells. We propose that osteoblasts protect selected malignant phenotypes from cell death caused by an excessive increase in cytosolic Ca(2+), thereby facilitating their progression into macroscopic skeletal metastases.

Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca2+. Here, we show that extracellular Ca2+ (Ca2+(o)) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca2+(o) compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca2+(o) in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cbeta (PLCbeta) led to an abolition of the Ca2+(o)-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca2+(o)-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca2+(o) concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca2+-rich environment.

Up-regulation of bone marrow stromal protein 2 (BST2) in breast cancer with bone metastasis

BackgroundBone metastases are frequent complications of breast cancer. Recent literature implicates multiple chemokines in the formation of bone metastases in breast cancer. However, the molecular mechanism of metastatic bone disease in breast cancer remains unknown. We have recently made the novel observation of the BST2 protein expression in human breast cancer cell lines. The purpose of our present study is to investigate the expression and the role of BST2 in bone metastatic breast cancer.MethodscDNA microarray analysis was used to compare the BST2 gene expression between a metastatic to bone human breast cancer cell line (MDA-231BO) and a primary human breast cancer cell line (MDA-231). The BST2 expression in one bone metastatic breast cancer and seven non-bone metastatic breast cancer cell lines were also determined using real-time RT-PCR and Western blot assays. We then employed tissue array to further study the BST2 expression in human breast cancer using array slides containing 20 independent breast cancer tumors that formed metastatic bone lesions, 30 non-metastasis-forming breast cancer tumors, and 8 normal breast tissues. In order to test the feasibility of utilizing BST2 as a serum marker for the presence of bone metastasis in breast cancer, we had measured the BST2 expression levels in human serums by using ELISA on 43 breast cancer patients with bone metastasis, 43 breast cancer patients without bone metastasis, and 14 normal healthy controls. The relationship between cell migration and proliferation and BST2 expression was also studied in a human breast recombinant model system using migration and FACS analysis.ResultsThe microarray demonstrated over expression of the BST2 gene in the bone metastatic breast cancer cell line (MDA-231BO) compared to the primary human breast cancer cell line (MDA-231). The expression of the BST2 gene was significantly increased in the bone metastatic breast cancer cell lines and tumor tissues compared to non-bone metastatic breast cancer cell lines and tumor tissues by real time RT-PCR, Western blot and TMA. Furthermore, serum levels of BST2 measured by ELISA were also significantly higher among patients with breast cancer metastatic to bone compared to breast cancer patients without metastatic to bone (P < .0001). Most importantly, the breast cancer cell line that transfected with BST2 demonstrated increased BST2 expressions, which was associated with increased cancer cell migration and cell proliferation.ConclusionThese results provide novel data indicating the BST2 protein expression is associated with the formation of bone metastases in human breast cancer. We believe that BST2 may be a potential biomarker in breast cancer with bone metastasis.

Extracellular calcium promotes the migration of breast cancer cells through the activation of the calcium sensing receptor

Breast cancer is the most frequent form of cancer in women, with the highest incidence of metastasis to the bone. The reason for the preferential destination to the bone is believed to be due to chemoattractant factors released during bone resorption, which act on the cancer cells facilitating their metastasis. One of the factors released during osteolysis that may mediate breast cancer bone localization is Ca 2+. Here, we show that extracellular Ca 2+ (Ca 2+ o) acting via the calcium-sensing receptor (CaSR), greatly promotes the migration of bone-preferring breast cancer cells. In Boyden Chamber and Scratch Wound migration assays, an increase in breast cancer cell migration was observed at 2.5 mM and 5 mM Ca 2+ o compared to basal levels for three of the four breast cancer cell lines tested. However, a significantly greater migratory response was observed for the highly bone metastatic MDA-MB-231 cells, compared to the MCF7 and T47D, which have a lower metastatic potential in vivo. The BT474 cells, which do not metastasize to the bone, did not respond to elevated concentrations of Ca 2+ o in the migration assays. Inhibition of either ERK1/2 MAPK or phospholipase Cβ (PLCβ) led to an abolition of the Ca 2+ o-induced migration, implicating these pathways in the migratory response. Knockdown of the CaSR by siRNA resulted in an inhibition of the Ca 2+ o-induced migration, demonstrating the involvement of this receptor in the effect. These results suggest that the activation of the CaSR by elevated Ca 2+ o concentrations, such as those found near resorbing bone, produces an especially strong chemoattractant effect on bone metastatic breast cancer cells toward the Ca 2+-rich environment.

P26. MicroRNA expression profiling and functional screening in bone metastatic breast cancer cells

P26. MicroRNA expression profiling and functional screening in bone metastatic breast cancer cells