Breast cancer cells induce osteoblast apoptosis: a possible contributor to bone degradation

Breast cancer cells exhibit a predilection for metastasis to bone. There, the metastases usually bring about bone loss with accompanying pain and loss of function. One way that breast cancer cells disrupt the normal pattern of bone remodeling is by activating osteoclasts, the bone degrading cells. Nevertheless, targeting the osteoclasts does not cure the disease or result in bone repair. These observations indicate that osteoblast function also may be compromised. The objective of this study was to investigate the interaction of metastatic breast cancer cells with osteoblasts. Human metastatic breast cancer cells, MDA-MB-435 or MDA-MB-231, or their conditioned media were co-cultured with a human osteoblast line hFOB1.19. The breast cancer cells caused an increase in the prevalence of apoptotic osteoblasts. Apoptotic osteoblasts detected by the TUNEL assay or by caspase activity increased approximately two to fivefold. This increase was not seen with non-metastatic MDA-MB-468 cells. In an investigation of the mechanism, it was determined that the hFOB1.19 cells expressed fas and that fas was functional. Likewise the hFOB1.19 cells were susceptible to TNF-alpha, but this cytokine was not detected in the conditioned medium of the breast cancer cells. This study indicates that osteoblasts are the target of breast cancer cell-induced apoptosis, but fas/fas-ligand and TNF-alpha, two common initiators of cell death, are probably not involved in this aspect of the metastases/bone cell axis. There are several mechanisms that remain to be explored in order to determine how breast cancer cells bring about osteoblast apoptosis. Even though the specific initiator of apoptosis remains to be identified, the results of this study suggest that the mechanism is likely to be novel.     

Osteoprotegerin production by breast cancer cells is suppressed by dexamethasone and confers resistance against TRAIL‐induced apoptosis

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF‐κB ligand (RANKL) and TNF‐related apoptosis‐inducing ligand (TRAIL). While RANKL is essential for osteoclastogenesis and facilitates breast cancer migration into bone, TRAIL promotes breast cancer apoptosis. We analyzed the expression of OPG and TRAIL and its modulation in estrogen receptor‐positive MCF‐7 cells and receptor‐negative MDA‐MB‐231 cells. In both cells, OPG mRNA levels and protein secretion were dose‐ and time‐dependently enhanced by interleukin (IL)‐1β and suppressed by dexamethasone. In contrast to MCF‐7 cells, MDA‐MB‐231 abundantly expressed TRAIL mRNA, which was enhanced by IL‐1β and inhibited by dexamethasone. TRAIL activated pro‐apoptotic caspase‐3, ‐7, and poly‐ADP‐ribose polymerase and decreased cell numbers of MDA‐MB‐231, but had no effect on MCF‐7 cells. Gene silencing siRNA directed against OPG resulted in a 31% higher apoptotic rate compared to non‐target siRNA‐treated MDA‐MB‐231 cells. Furthermore, TRAIL induced significantly less apoptosis in cells cultured in conditioned media (containing OPG) compared to cells exposed to TRAIL in fresh medium lacking OPG (P < 0.01) and these protective effects were reversed by blocking OPG with its specific ligand RANKL (P < 0.05). The association between cancer cell survival and OPG production by MDA‐MB‐231 cells was further supported by the finding, that modulation of OPG secretion using IL‐1β or dexamethasone prior to TRAIL exposure resulted in decreased and increased rate of apoptosis, respectively (P < 0.05). Thus, OPG secretion by breast cancer cells is modulated by cytokines and dexamethasone, and may represent a critical resistance mechanism that protects against TRAIL‐induced apoptosis. J. Cell. Biochem. 108: 106–116, 2009. © 2009 Wiley‐Liss, Inc.     

Metastatic breast cancer induces an osteoblast inflammatory response

Breast cancer preferentially metastasizes to the skeleton, a hospitable environment that attracts and allows breast cancer cells to thrive. Growth factors released as bone is degraded support tumor cell growth, and establish a cycle favoring continued bone degradation. While the osteoclasts are the direct effectors of bone degradation, we found that osteoblasts also contribute to bone loss. Osteoblasts are more than intermediaries between tumor cells and osteoclasts. We have presented evidence that osteoblasts contribute through loss of function induced by metastatic breast cancer cells. Metastatic breast cancer cells suppress osteoblast differentiation, alter morphology, and increase apoptosis. In this study we show that osteoblasts undergo an inflammatory stress response in the presence of human metastatic breast cancer cells. When conditioned medium from cancer cells was added to human osteoblasts, the osteoblasts were induced to express increased levels of IL-6, IL-8, and MCP-1; cytokines known to attract, differentiate, and activate osteoclasts. Similar findings were seen with murine osteoblasts and primary murine calvarial osteoblasts. Osteoblasts are co-opted into creating a microenvironment that exacerbates bone loss and are prevented from producing matrix proteins for mineralization. This is the first study implicating osteoblast produced IL-6, IL-8 (human; MIP-2 and KC mouse), and MCP-1 as key mediators in the osteoblast response to metastatic breast cancer cells.     

Aberrant methylation of WD‐repeat protein 41 contributes to tumour progression in triple‐negative breast cancer

WD‐repeat proteins are implicated in a variety of biological functions, most recently in oncogenesis. However, the underlying function of WD‐repeat protein 41 (WDR41) in tumorigenesis remains elusive. The present study was aimed to explore the role of WDR41 in breast cancer. Combined with Western blotting and immunohistochemistry, the results showed that WDR41 was expressed at low levels in breast cancer, especially in triple‐negative breast cancer (TNBC). Using methylation‐specific PCR (MSP), we observed that WDR41 presented hypermethylation in MDA‐MB‐231 cells. Methylation inhibitor 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) management increased the expression of WDR41 in MDA‐MB‐231 cells, but not in MCF‐10A (normal mammary epithelial cells) or oestrogen receptor‐positive MCF‐7 breast cancer cells. WDR41‐down‐regulation promoted, while WDR41‐up‐regulation inhibited the tumour characteristics of TNBC cells including cell viability, cell cycle and migration. Further, WDR41‐up‐regulation dramatically suppressed tumour growth in vivo. Mechanistically, WDR41 protein ablation activated, while WDR41‐up‐regulation repressed the AKT/GSK‐3β pathway and the subsequent nuclear activation of β‐catenin in MDA‐MB‐231 cells, and 5‐aza‐dC treatment enhanced this effect. After treatment with the AKT inhibitor MK‐2206, WDR41‐down‐regulation‐mediated activation of the GSK‐3β/β‐catenin signalling was robustly abolished. Collectively, methylated WDR41 in MDA‐MB‐231 cells promotes tumorigenesis through positively regulating the AKT/GSK‐3β/β‐catenin pathway, thus providing an important foundation for treating TNBC.     

Modulation of protein expression levels and DNA methylation status of breast cancer metastasis genes by anthracycline-based chemotherapy and the demethylating agent decitabine

Epigenetic drugs are promising add‐ons to cancer treatment; still, adverse effects concerning tumour promotion have been reported occasionally. In this in vitro study, we investigated the effect of combination treatment of decitabine with anthracycline‐based chemotherapy [5‐fluorouracil plus epirubicine plus cyclophosphamide (FEC)] on viability and metastatic activity of breast cancer cell lines, MDA‐MB‐231 (estrogen receptor‐negative) and MCF‐7 (estrogen receptor‐positive). The effect of decitabine and its combined treatment with FEC on viability of both cancer cell lines was assessed using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide and adenosine triphosphate (ATP) cell survival assays. DNA methylation specific real‐time polymerase chain reaction (PCR) (Methylight®) was employed to document the methylation status of the metastasis‐relevant urokinase‐type plasminogen activator (uPA) and plasminogen activator inhibitor‐I (PAI‐1) genes. Additionally, protein expression levels of uPA and PAI‐1 were determined using enzyme‐linked immunosorbent assays. Invasion capacity of cells was assayed using Matrigel® invasion assay. Decitabine lowered the viability of MCF‐7 cells, although MDA‐MB‐231 cells were not affected. Decitabine did not augment FEC‐mediated cytotoxicity in both cell lines. In MCF‐7 cells, methylation of the uPA and PAI‐1 gene promoter was significantly reduced by decitabine or decitabine plus FEC. Protein levels of uPA and PAI‐1 were induced by all treatments. Decitabine significantly induced the invasion capacity of MCF‐7 cells, whereas all of the drugs resulted in decreased invasion capacity of MDA‐MB‐231. Our results suggest differential effects of single‐dose decitabine and its combination with FEC on the metastatic capacity and survival of breast cancer cell lines endowed with different metastatic behaviour. Copyright © 2011 John Wiley & Sons, Ltd.     

EGF-like ligands stimulate osteoclastogenesis by regulating expression of osteoclast regulatory factors by osteoblasts: implications for osteolytic bone metastases

Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. Recent in vivo mouse models suggest this signaling network plays an essential role in bone metabolism. Using a coculture system containing bone marrow macrophage and osteoblastic cells, here we report that EGF-like ligands stimulate osteoclastogenesis by acting on osteoblastic cells. This stimulation is not a direct effect because osteoclasts do not express functional EGF receptors (EGFRs). Further studies reveal that EGF-like ligands strongly regulate the expression of two secreted osteoclast regulatory factors in osteoblasts by decreasing osteoprotegerin (OPG) expression and increasing monocyte chemoattractant protein 1 (MCP1) expression in an EGFR-dependent manner and consequently stimulate TRAP-positive osteoclast formation. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands, while addition of a neutralizing antibody against MCP-1 exhibited partial inhibition. Coculture with bone metastatic breast cancer MDA-MB-231 cells had similar effects on the expression of OPG and MCP1 in the osteoblastic cells, and those effects could be partially abolished by the EGFR inhibitor PD153035. Because a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone.     

Androgens increase osteoblast-stimulating activity of human breast cancer cells in vitro

Bone metastases of breast cancers produce not only osteolytic but also osteosclerotic lesions. The latter are often observed after androgenic treatment of the tumor. Potential production of osteoblast stimulating activity (ObSA) in breast cancer cell lines, and possible androgen control of this activity have been investigated. Conditioned media (CM) collected from 4 breast cancer cell lines (MCF-7, ZR75, MDA-MB 231, BT20) was tested in vitro on ROS 17/2,8 osteoblast-like cells and on osteoblasts derived from human bone biopsies. The parameters monitored in osteoblasts were [3H]thymidine incorporation, alkaline phosphatase activity, and osteocalcin secretion. Serum-free media conditioned during 24 h by MCF-7 cells presented the highest ObSA. CM decreased thymidine incorporation in DNA and increased alkaline phosphatase activity in a dose-dependent manner. Bone GLA protein (osteocalcin) secretion by human osteoblasts was not increased however in the presence of CM. MCF-7 cells were cultured in the presence of dihydrotestosterone (DHT) [1-100 nM] for 5 days. Serum-free, DHT-free CM collected after an additional 24 h, contained alkaline-phosphatase stimulating activity which was DHT dose-dependent. Estradiol and 1,25(OH)2D3 failed to elicit a comparable increase of the ObSA in the CM. In conclusion, MCF-7 cells product factor(s) that interfere with bone remodeling. The DHT modulation of ObSA parallels the estradiol control of MCF-7 cells osteolytic lesions in relation with Prostaglandin E secretion. Sex hormones at physiological and pharmacological levels might thus control both osteosclerotic and osteolytic lesions observed in bone deposits of hormone dependent cancers.     

Dynamic interaction between breast cancer cells and osteoblastic tissue: comparison of two- and three-dimensional cultures

Breast cancer cell colonization of osteoblast monolayers grown in standard tissue culture (2D) is compared to colonization of a multi-cell-layer osteoblastic tissue (3D) grown in a specialized bioreactor. Colonization of 3D tissue recapitulates events observed in clinical samples including cancer penetration of tissue, growth of microcolonies, and formation of "Single cell file" commonly observed in end-stage pathological bone tissue. By contrast, adherent cancer cell colonies did not penetrate 2D tissue and did not form cell files. Thus, it appears that 3D tissue is a more biologically (clinically) relevant model than 2D monolayers in which to study cancer cell interactions with osteoblastic tissue. This direct comparison of 2D and 3D formats is implemented using MC3T3-E1 murine osteoblasts and MDA-MB-231 human metastatic breast cancer cells, or the metastasis-suppressed line, MDA-MB-231BRMS1, for comparison. When osteoblasts were co-cultured with metastatic cells, production of osteocalcin (a mineralization marker) decreased and secretion of the pro-inflammatory cytokine IL-6 increased in both 2D and 3D formats. Cancer cell penetration of the 3D tissue coincided with a changed osteoblast morphology from cuboidal to spindle-shaped, and with osteoblasts alignment parallel to the cancer cells. Metastasis-suppressed cells did not penetrate 3D tissue, did not cause a change in osteoblast morphology or align in rows. Moreover, they proliferated much less in the 3D culture than in the 2D culture in a manner similar to their growth in bone. In both systems, the cancer cells proliferated to a greater extent with immature osteoblasts compared to more mature osteoblasts.     

Profilin‐1 overexpression inhibits proliferation of MDA‐MB‐231 breast cancer cells partly through p27kip1 upregulation

Profilin‐1 (Pfn1), a ubiquitously expressed actin‐binding protein, has gained interest in epithelial‐derived cancer because of its downregulation in expression in various adenocarcinoma. Pfn1 overexpression impairs tumorigenic ability of human breast cancer xenografts thus suggesting that Pfn1 could be a tumor‐suppressor protein. The objective of the present study was to determine how Pfn1 overexpression affects cell‐cycle progression of breast cancer cells. We show that Pfn1 overexpression in MDA‐MB‐231 breast cancer cells causes cell‐cycle arrest in G1 phase and dramatically reduced proliferation in culture. Pfn1 overexpression results in increased protein stability of p27^kip1 (p27—a major cyclin‐dependent kinase inhibitor) and marked elevation in the overall cellular level of p27. Proliferation defect of Pfn1 overexpressers can be partly rescued by silencing p27 expression thus suggesting a critical role of p27 in Pfn1‐induced growth inhibition of MDA‐MB‐231 cells. Finally, Pfn1 overexpression was found to sensitize MDA‐MB‐231 cells to apoptosis in response to cytotoxic stimulus thus suggesting for the first time that survival of breast cancer cells can also be negatively influenced by Pfn1 upregulation. These findings may provide novel insights underlying Pfn1's tumor‐suppressive action. J. Cell. Physiol. 223:623–629, 2010. © 2010 Wiley‐Liss, Inc.     

Human ether à-gogo K(+) channel 1 (hEag1) regulates MDA-MB-231 breast cancer cell migration through Orai1-dependent calcium entry

Breast cancer (BC) has a poor prognosis due to its strong metastatic ability. Accumulating data present ether à go‐go (hEag1) K⁺ channels as relevant player in controlling cell cycle and proliferation of non‐invasive BC cells. However, the role of hEag1 in invasive BC cells migration is still unknown. In this study, we studied both the functional expression and the involvement in cell migration of hEag1 in the highly metastatic MDA‐MB‐231 human BC cells. We showed that hEag1 mRNA and proteins were expressed in human invasive ductal carcinoma tissues and BC cell lines. Functional activity of hEag1 channels in MDA‐MB‐231 cells was confirmed using astemizole, a hEag1 blocker, or siRNA. Blocking or silencing hEag1 depolarized the membrane potential and reduced both Ca²⁺ entry and MDA‐MB‐231 cell migration without affecting cell proliferation. Recent studies have reported that Ca²⁺ entry through Orai1 channels is required for MDA‐MB‐231 cell migration. Down‐regulation of hEag1 or Orai1 reduced Ca²⁺ influx and cell migration with similar efficiency. Interestingly, no additive effects on Ca²⁺ influx or cell migration were observed in cells co‐transfected with sihEag1 and siOrai1. Finally, both Orai1 and hEag1 are expressed in invasive breast adenocarcinoma tissues and invaded metastatic lymph node samples (LNM⁺). In conclusion, this study is the first to demonstrate that hEag1 channels are involved in the serum‐induced migration of BC cells by controlling the Ca²⁺ entry through Orai1 channels. hEag1 may therefore represent a potential target for the suppression of BC cell migration, and thus prevention of metastasis development. J. Cell. Physiol. 227: 3837–3846, 2012. © 2012 Wiley Periodicals, Inc.     

Apoptosis‐inducing effect of garcinol is mediated by NF‐κB signaling in breast cancer cells

Garcinol, obtained from Garcinia indica in tropical regions, is used for its numerous biological effects. Its anti‐cancer activity has been suggested but the mechanism of action has not been studied in‐detail, especially there is no report on its action against breast cancer cells. Here we tested our hypothesis that garcinol may act as an anti‐proliferative and apoptosis‐inducing agent against breast cancer cell lines. Using multiple techniques such as MTT, Histone‐DNA ELISA, Annexin V‐PI staining, Western blot for activated caspases and cleaved PARP, homogenous caspase‐3/7 fluorometric assay and EMSA, we investigated the mechanism of apoptosis‐inducing effect of garcinol in ER‐positive MCF‐7 and ER‐negative MDA‐MB‐231 cells. We found that garcinol exhibits dose‐dependent cancer cell‐specific growth inhibition in both the cell lines with a concomitant induction of apoptosis, and has no effect on non‐tumorigenic MCF‐10A cells. Our results suggested induction of caspase‐mediated apoptosis in highly metastatic MDA‐MB‐231 cells by garcinol. Down‐regulation of NF‐κB signaling pathway was observed to be the mechanism of apoptosis‐induction. Garcinol inhibited constitutive NF‐κB activity, which was consistent with down‐regulation of NF‐κB‐regulated genes. This is the first report on anti‐proliferative and apoptosis‐inducing action of garcinol against human breast cancer cells and the results suggest that this natural compound merits investigation as a potential chemo‐preventive/‐therapeutic agent, especially against breast cancer. J. Cell. Biochem. 109: 1134–1141, 2010. © 2010 Wiley‐Liss, Inc.     

Breast cancer cell line MDA-MB 231 exerts a potent and direct anti-apoptotic effect on mature osteoclasts

Cancer cells metastasized to bone stimulate osteoclastogenesis resulting in bone destruction. However, the influence of tumor cells on fully differentiated osteoclasts is much less known. We postulated that breast cancer cells directly stimulate the survival of mature osteoclasts. We thus tested the effect of conditioned media (CM) prepared from MDA-MB-231 cells on the activity and apoptosis of osteoclasts isolated from 10-day-old rabbit long bones. First, we demonstrated that CM increased the bone resorbing activity in our cell model of rabbit mature osteoclasts. Using a highly purified osteoclast cell population, we found that MDA-MB-231 CM dramatically inhibited osteoclast apoptosis. In the presence of 20% CM, apoptosis was decreased by approximately 60%. LY294002, a PI3 kinase inhibitor, strongly prevented the CM anti-apoptotic effect. Neutralizing experiments with human antibody revealed that macrophage-colony stimulating factor originating from MDA-MB 231 cells was possibly involved in the CM anti-apoptotic effect. These results suggest that breast cancer cells, in addition to stimulating osteoclastogenesis, potently inhibit mature osteoclast apoptosis, a mechanism which may greatly contribute to their osteolytic potential.     

Human umbilical cord Wharton's jelly stem cell (hWJSC) extracts inhibit cancer cell growth in vitro

Umbilical cord mesenchymal stem cells (MSCs) have been shown to inhibit breast cancer cell growth but it is not known whether this effect is specific to only breast cancer cells. We compared the effects of human Wharton's jelly stem cell (hWJSC) extracts [conditioned medium (hWJSC‐CM) and cell lysate (hWJSC‐CL)] on breast adenocarcinoma (MDA‐MB‐231), ovarian carcinoma (TOV‐112D), and osteosarcoma (MG‐63) cells. The cells were treated with either hWJSC‐CM (50%) or hWJSC‐CL (15 µg/ml) for 48–72 h and changes in cell morphology, proliferation, cycle, gene expression, migration, and cell death studied. All three cancer cell lines showed cell shrinkage, blebbing, and vacuolations with hWJSC‐CL and hWJSC‐CM compared to controls. MTT and BrdU assays showed inhibition of cell growth by 2–6% and 30–60%, while Transwell migration assay showed inhibition by 20–26% and 31–46% for hWJSC‐CM and hWJSC‐CL, respectively, for all three cancer cell lines. Cell cycle assays showed increases in sub‐G1 and G2/M phases for all three cancer cell lines suggestive of apoptosis and metaphase arrest. AnnexinV‐FITC and TUNEL positive cells seen in TOV‐112D and MDA‐MB‐231 suggested that inhibition was via apoptosis while the presence of anti‐BECLIN1 and anti‐LC3B antibodies seen with MG‐63 indicated autophagy. Upregulation of pro‐apoptotic BAX and downregulation of anti‐apoptotic BCL2 and SURVIVIN genes were observed in all three cancer cell lines and additionally the autophagy genes (ATG5, ATG7, and BECLIN1) were upregulated in MG‐63 cells. hWJSCs possess tumor inhibitory properties that are not specific to breast cancer cells alone and these effects are mediated via agents in its extracts. J. Cell. Biochem. 113: 2027–2039, 2012. © 2012 Wiley Periodicals, Inc.     

Breast cancer cell line MDA-231 stimulates osteoclastogenesis and bone resorption in human osteoclasts

Breast cancers commonly cause osteolytic metastases in bone, a process that is dependent upon osteoclast-mediated bone resorption, but the mechanism responsible for tumor-mediated osteoclast activation has not yet been clarified. In the present study we utilized a well-known human breast cancer cell line (MDA-231) in order to assess its capability to influence osteoclastogenesis in human bone marrow cultures and bone resorption in fully differentiated osteoclasts. We demonstrated that conditioned medium (CM) harvested from MDA-231 increased the formation of multinucleated TRAP-positive cells in bone marrow cultures. Bone resorption activity of fully differentiated human osteoclasts and of osteoclast-like cell lines, from giant cell tumors of bone (GCT), was highly increased by the presence of MDA-231 CM. Moreover, while MDA-231 by themselves did not produce IL-6 tumor cell, CM increased the secretion of IL-6 by primary human osteoclasts and GCT cell lines compared to untreated controls. These data suggest that MDA-231 produce osteoclastic activating factor(s) that increase both osteoclast formation in bone marrow culture and bone resorption activity by mature cells. Moreover, breast cancer cells stimulate IL-6 secretion by osteoclasts that is one of the factors known to supports osteoclastogenesis.