Decreased autocrine EGFR signaling in metastatic breast cancer cells inhibits tumor growth in bone and mammary fat pad

Breast cancer metastasis to bone triggers a vicious cycle of tumor growth linked to osteolysis. Breast cancer cells and osteoblasts express the epidermal growth factor receptor (EGFR) and produce ErbB family ligands, suggesting participation of these growth factors in autocrine and paracrine signaling within the bone microenvironment. EGFR ligand expression was profiled in the bone metastatic MDA-MB-231 cells (MDA-231), and agonist-induced signaling was examined in both breast cancer and osteoblast-like cells. Both paracrine and autocrine EGFR signaling were inhibited with a neutralizing amphiregulin antibody, PAR34, whereas shRNA to the EGFR was used to specifically block autocrine signaling in MDA-231 cells. The impact of these was evaluated with proliferation, migration and gene expression assays. Breast cancer metastasis to bone was modeled in female athymic nude mice with intratibial inoculation of MDA-231 cells, and cancer cell-bone marrow co-cultures. EGFR knockdown, but not PAR34 treatment, decreased osteoclasts formed in vitro (p<0.01), reduced osteolytic lesion tumor volume (p<0.01), increased survivorship in vivo (p<0.001), and resulted in decreased MDA-231 growth in the fat pad (p<0.01). Fat pad shEGFR-MDA-231 tumors produced in nude mice had increased necrotic areas and decreased CD31-positive vasculature. shEGFR-MDA-231 cells also produced decreased levels of the proangiogenic molecules macrophage colony stimulating factor-1 (MCSF-1) and matrix metalloproteinase 9 (MMP9), both of which were decreased by EGFR inhibitors in a panel of EGFR-positive breast cancer cells. Thus, inhibiting autocrine EGFR signaling in breast cancer cells may provide a means for reducing paracrine factor production that facilitates microenvironment support in the bone and mammary gland.     

Osteoprotegrin and the bone homing and colonization potential of breast cancer cells

Breast cancer cells preferentially metastasize to bone, leading to the formation of primarily osteolytic lesions. Osteoprotegerin (OPG) plays multifactorial roles in the development of osteolytic bone metastases. An increase in the ratio of receptor activator of nuclear factor kappaB ligand (RANKL) to OPG increases osteoclastogenesis within the bone microenvironment. OPG also acts as a survival factor for cancer cells by protecting them from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apoptosis. This study compares OPG production in vitro in a number of breast cancer cell lines exhibiting both differences in metastatic capacity and in preferential metastasis to bone. Our studies demonstrated that OPG expression by MDA-231, MDA-MET, and MDA-231/K cancer cells was directly correlated with bone specific homing and colonization potential but not with metastasis of cancer cells to other organs; both in IL-1 beta stimulated and control cells. We also demonstrated expression of other bone-related markers including type I collagen, osteocalcin, osteopontin, and Runx2 in these cells. However, the generally lower expression of these markers in the bone selective cell line MDA-MET suggested that increased OPG expression in the bone specific variant was not merely a consequence of enhanced osteomimicry by these cells but that it has a significant role in the metastatic process. Co-culture of breast cancer cells with osteoblastic cells (hFOB 1.19) led to an overall downregulation in OPG production, which was not affected by the bone homing and colonization potential of the cell lines, suggesting that OPG alone is not indicative of osteolytic bone activity by breast cancer cells.     

Role of prostaglandin E produced by osteoblasts in osteolysis due to bone metastasis

Prostaglandin E2 (PGE2) is produced in bone mainly by osteoblasts and stimulates bone resorption. Osteolytic bone metastasis of cancers is accompanied by bone resorption. In this study, we examined the roles of PGE2 in osteolysis due to bone metastasis of breast cancer. Injection of human breast cancer cells, MDA-MB-231 (MDA-231), into nude mice causes severe osteolysis in the femur and tibia. The expression of cyclo-oxygenase-2 (COX-2) and the receptor activator of NF-kappaB ligand (RANKL), a key molecule in osteoclast differentiation, mRNAs was markedly elevated in bone with metastasis. When MDA-231 cells were cocultured with mouse calvaria, COX-2-induced PGE2 production and bone resorption progressed. The contact with MDA-231 cells could induce the expression of COX-2 and RANKL in osteoblasts by mechanisms involving MAP kinase and NF-kappaB. The blockage of PGE2 signal by indomethacin and EP4 antagonist abrogated the osteoclast formation induced by the breast cancer cells. Here, we show a PGE-dependent mechanism of osteolysis due to bone metastasis.     

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.     

骨唾液酸蛋白基因沉默抑制乳腺癌细胞与骨基质粘附

旨在探索骨唾液酸蛋白 (Bone sialoprotein,BSP) 基因沉默对亲骨转移乳腺癌细胞 (MDA-MB-231BO) 与骨基质粘附能力的影响,为以BSP为靶点的乳腺癌骨转移预防和靶向治疗提供实验依据。体外检测BSP基因沉默对乳腺癌细胞与小鼠骨基质粘附能力的影响,MTS法检测细胞增殖能力;扫描电镜观察骨片表面肿瘤细胞粘附情况和骨吸收状况;ELISA法检测骨基质细胞粘附培养上清中TGF-β1和RANKL表达分泌量差异;左心室注射法构建裸鼠骨转移模型,检测不同细胞株在裸鼠体内转移能力。结果提示BSP     

Curcumin analogue UBS109 prevents bone loss in breast cancer bone metastasis mouse model: involvement in osteoblastogenesis and osteoclastogenesis

Bone metastasis of breast cancer typically leads to osteolysis, which causes severe pathological bone fractures and hypercalcemia. Bone homeostasis is skillfully regulated through osteoblasts and osteoclasts. Bone loss with bone metastasis of breast cancer may be due to both activation of osteoclastic bone resorption and suppression of osteoblastic bone formation. This study was undertaken to determine whether the novel curcumin analogue UBS109 has preventive effects on bone loss induced by breast cancer cell bone metastasis. Nude mice were inoculated with breast cancer MDA-MB-231 bone metastatic cells (10⁶ cells/mouse) into the head of the right and left tibia. One week after inoculation, the mice were treated with control (vehicle), oral administration (p.o.) of UBS109 (50 or 150 mg/kg body weight), or intraperitoneal administration (i.p.) of UBS109 (10 or 20 mg/kg body weight) once daily for 5 days per week for 7 weeks. After UBS109 administration for 7 weeks, hind limbs were assessed using an X-ray diagnosis system and hematoxylin and eosion staining to determine osteolytic destruction. Bone marrow cells obtained from the femurs and tibias were cultured to estimate osteoblastic mineralization and osteoclastogenesis ex vivo and in vitro. Remarkable bone loss was demonstrated in the tibias of mice inoculated with breast cancer MDA-MB-231 bone metastatic cells. This bone loss was prevented by p.o. administration of UBS109 (50 and 150 mg/kg body weight) and i.p. treatment of UBS109 (10 and 20 mg/kg) in vivo. Culture of bone marrow cells obtained from the bone tissues of mice with breast cancer cell bone metastasis showed suppressed osteoblastic mineralization and stimulated osteoclastogenesis ex vivo. These changes were not seen after culture of the bone marrow cells obtained from mice treated with UBS109. Moreover, UBS109 was found to stimulate osteoblastic mineralization and suppress lipopolysaccharide (LPS)-induced osteoclastogenesis in bone marrow cells obtained from normal nude mice in vitro. These findings suggest that the novel curcumin analogue UBS109 prevents breast cancer cell bone metastasis-induced bone loss by stimulating osteoblastic mineralization and suppressing osteoclastogenesis.     

cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-β. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.     

Activation of the RalGEF/Ral pathway promotes prostate cancer metastasis to bone

A hallmark of metastasis is organ specificity; however, little is known about the underlying signaling pathways responsible for the colonization and growth of tumor cells in target organs. Since tyrosine kinase receptor activation is frequently associated with prostate cancer progression, we have investigated the role of a common signaling intermediary, activated Ras, in prostate cancer metastasis. Three effector pathways downstream of Ras, Raf/extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase, and Ral guanine nucleotide exchange factors (RalGEFs), were assayed for their ability to promote the metastasis of a tumorigenic, nonmetastatic human prostate cancer cell line, DU145. Oncogenic Ras promoted the metastasis of DU145 to multiple organs, including bone and brain. Activation of the Raf/ERK pathway stimulated metastatic colonization of the brain, while activation of the RalGEF pathway led to bone metastases, the most common organ site for prostate cancer metastasis. In addition, loss of RalA in the metastatic PC3 cell line inhibited bone metastasis but did not affect subcutaneous tumor growth. Loss of Ral appeared to suppress expansive growth of prostate cancer cells in bone, whereas homing and initial colonization were less affected. These data extend our understanding of the functional roles of the Ral pathway and begin to identify signaling pathways relevant for organ-specific metastasis.     

TAK1-TAB2 signaling contributes to bone destruction by breast carcinoma cells

Advanced-stage breast cancers frequently metastasize to the bones and cause bone destruction, but the underlying mechanism is not fully understood. This study presents evidence that TGF-β-activated protein kinase 1 (TAK1) signaling in tumor cells promotes bone destruction by metastatic breast carcinoma cells, controlling expression of prometastatic factors including matrix metalloproteinase (MMP) 9 and COX2. Suppression of TAK1 signaling by dominant-negative TAK1 (dn-TAK1) in breast carcinoma MDA-MB-231 cells impairs bone colonization by carcinoma cells and bone osteolysis in the intracardiac injection model. Mechanistic studies showed that inhibition of TAK1 by dn-TAK1 or siRNA blocked expression of factors implicated in bone metastasis, such as MMP-9, COX2/PTGS2, parathyroid hormone-related protein (PTHrP) and interleukin 8 (IL-8), but did not affect activation of p38MAPK by TGF-β. TAK1 signaling is mediated by TAK1-binding partners TAB1, TAB2, and TAB3. Carcinoma cells express elevated mRNA levels of TAB2 and TAB3, whereas the TAB1 expression is noticeably low. Accordingly, depletion of TAB2 by siRNA reduced expression of MMP-9 and COX2. Together, these studies show that the TAK1-TAB2-TAB3 signaling axis is critical for carcinoma-induced bone lesions, mediating expression of proinvasive and osteolytic factors. These findings identify the TAK1-TAB2 axis as a potential therapeutic target in bone metastasis.     

Identification of NOG as a specific breast cancer bone metastasis-supporting gene

Metastasis requires numerous biological functions that jointly provide tumor cells from a primary site to seed and colonize a distant organ. Some of these activities are selected for in the primary site, whereas others are acquired at the metastatic niche. We provide molecular evidence showing that the BMP inhibitor, NOG, provides metastatic breast cancer cells with the ability to colonize the bone. NOG expression is acquired during the late events of metastasis, once cells have departed from the primary site, because it is not enriched in primary tumors with high risk of bone relapse. On the contrary, breast cancer bone metastatic lesions do select for high levels of NOG expression when compared with metastasis to the lung, liver, and brain. Pivotal to the bone colonization functions is the contribution of NOG to metastatic autonomous and nonautonomous cell functions. Using genetic approaches, we show that when NOG is expressed in human breast cancer cells, it facilitates bone colonization by fostering osteoclast differentiation and bone degradation and also contributes to metastatic lesions reinitiation. These findings reveal how aggressive cancer cell autonomous and nonautonomous functions can be mechanistically coupled to greater bone metastatic potential.     

Angiostatin inhibits bone metastasis formation in nude mice through a direct anti-osteoclastic activity

Bone is a very common metastatic site for breast cancer. In bone metastasis, there is a vicious circle wherein bone-residing metastatic cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone promote tumor growth. The contribution of tumor angiogenesis in the growth of bone metastases is, however, unknown. By using an experimental model of bone metastasis caused by MDA-MB-231/B02 breast cancer cells that quite closely mimics the conditions likely to occur in naturally arising metastatic human breast cancers, we demonstrate here that when MDA-MB-231/B02 cells were engineered to produce at the bone metastatic site an angiogenesis inhibitor, angiostatin, there was a marked inhibition in the extent of skeletal lesions. Inhibition of skeletal lesions came with a pronounced reduction in tumor burden in bone. However, although angiostatin produced by MDA-MB-231/B02 cells was effective at inhibiting in vitro endothelial cell proliferation and in vivo angiogenesis in a Matrigel implant model, we have shown that it inhibited cancer-induced bone destruction through a direct inhibition of osteoclast activity and generation. Overall, these results indicate that, besides its well known anti-angiogenic activity, angiostatin must also be considered as a very effective inhibitor of bone resorption, broadening its potential clinical use in cancer therapy.     

NUAK1通过影响F-actin聚合促进乳腺癌的侵袭转移

本课题组先前研究证明NUAK1/ARK5参与乳腺癌、胶质瘤侵袭转移,但机制尚不清楚.本文证明,NUAK1通过影响F-actin聚合促进乳腺癌的侵袭转移.应用小RNA干扰技术敲除乳腺癌细胞系MDA-MB-231中的NUAK1,用G418进行稳定筛选,并用Western印迹进行蛋白质表达检测,结果显示,成功敲除MDA-MB-231细胞中的NUAK1;采用Transwell侵袭实验检测NUAK1在乳腺癌细胞侵袭转移中的作用,结果表明,NUAK1被干扰的SiNUAK1/MDA-231细胞的侵袭能力明显减弱;应用免疫荧光法对细胞的F-actin进行荧光染色,半定量F-actin聚合分析结果显示,IGF-1在转染空载的细胞组（Scr/MDA-231）能诱导肌动蛋白短暂的聚合反应,而在敲除NUAK1的细胞组（SiNUAK1/MDA-231）肌动蛋白的聚合显著减少;用细胞因子IGF-1刺激乳腺癌细胞观察cofilin磷酸化,结果显示,在敲除NUAK1的细胞组（SiNUAK1/MDA-231）,IGF-1诱导的cofilin的磷酸化明显受抑制.上述结果表明,NUAK1能通过促进F-actin的聚合从而影响乳腺癌细胞的侵袭转移.     

BMP9 inhibits the bone metastasis of breast cancer cells by downregulating CCN2 (connective tissue growth factor,CTGF) expression

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-β superfamily, regulate a wide range of cellular responses including cell proliferation, differentiation, adhesion, migration, and apoptosis. BMP9, the latest BMP to be discovered, is reportedly expressed in a variety of human carcinoma cell lines, but the role of BMP9 in breast cancer has not been fully clarified. In a previous study, BMP9 was found to inhibit the growth, migration, and invasiveness of MDA-MB-231 breast cancer cells. In the current study, the effect of BMP9 on the bone metastasis of breast cancer cells was investigated. After absent or low expression of BMP9 was detected in the MDA-MB-231 breast cancer cells and breast non-tumor adjacent tissues using Western blot and immunohistochemistry, In our previous study, BMP9 could inhibit the proliferation and invasiveness of breast cancer cells MDA-MB-231 in vitro and in vivo. This paper shows that BMP9 inhibit the bone metastasis of breast cancer cells by activating the BMP/Smad signaling pathway and downregulating connective tissue growth factor (CTGF); however, when CTGF expression was maintained, the inhibitory effect of BMP9 on the MDA-MB-231 cells was abolished. Together, these observations indicate that BMP9 is an important mediator of breast cancer bone metastasis and a potential therapeutic target for treating this deadly disease.     

Tumor-supportive and Osteoclastogenic Changes Induced by Breast Cancer-derived Factors Are Reversed by Inhibition of γ-Secretase

During breast cancer metastasis to bone, tumor cells home to bone marrow, likely targeting the stem cell niche, and stimulate osteoclasts, which mediate osteolysis required for tumor expansion. Although osteoblasts contribute to the regulation of the hematopoietic stem cell niche and control osteoclastogenesis through production of proresorptive cytokine RANKL (receptor activator of NF-κB ligand), their role in cancer metastases to bone is not fully understood. C57BL/6J mouse bone marrow cells were treated for 3–12 days with ascorbic acid (50 μg/ml) in the presence or absence of 10% medium conditioned by breast carcinoma cells MDA-MB-231, 4T1, or MCF7. Treatment with cancer-derived factors resulted in a sustained 40–60% decrease in osteoblast differentiation markers, compared with treatment with ascorbic acid alone, and induced an osteoclastogenic change in the RANKL/osteoprotegerin ratio. Importantly, exposure of bone cells to breast cancer-derived factors stimulated the subsequent attachment of cancer cells to immature osteoblasts. Inhibition of γ-secretase using pharmacological inhibitors DAPT and Compound E completely reversed cancer-induced osteoclastogenesis as well as cancer-induced enhancement of cancer cell attachment, identifying γ-secretase activity as a key mediator of these effects. Thus, we have uncovered osteoblasts as critical intermediary of premetastatic signaling by breast cancer cells and pinpointed γ-secretase as a robust target for developing therapeutics potentially capable of reducing both homing and progression of cancer metastases to bone.     

Heparin-like polysaccharides reduce osteolytic bone destruction and tumor growth in a mouse model of breast cancer bone metastasis

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β-induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β-induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts.     

HGF and M-CSF modulate adhesion of MDA-231 breast cancer cell by increasing osteopontin secretion

Recent studies reported an increased expression of osteopontin (OPN) in metastatic breast cancer cells, but the mechanisms modulating OPN production and the interaction of the cells with the secreted protein are far from clear. In this work, we utilized as an experimental system the cell line MDA-231 and we showed that HGF and M-CSF significantly enhance their adhesion onto OPN. Furthermore, in the presence of HGF and M-CSF, MDA-231 cells can adhere when plated onto BSA via increased OPN secretion. Moreover HGF and M-CSF induce de novo synthesis of OPN. In conclusion, these data suggest that HGF and M-CSF stimulate OPN production by MDA-231 cells, and that OPN is subsequently used as a substrate for cell adhesion.     

T Cells?Induce Pre-Metastatic Osteolytic?Disease and Help Bone Metastases Establishment in a Mouse Model of Metastatic Breast Cancer

Bone metastases, present in 70% of patients with metastatic breast cancer, lead to skeletal disease, fractures and intense pain, which are all believed to be mediated by tumor cells. Engraftment of tumor cells is supposed to be preceded by changes in the target tissue to create a permissive microenvironment, the pre-metastatic niche, for the establishment of the metastatic foci. In bone metastatic niche, metastatic cells stimulate bone consumption resulting in the release of growth factors that feed the tumor, establishing a vicious cycle between the bone remodeling system and the tumor itself. Yet, how the pre-metastatic niches arise in the bone tissue remains unclear. Here we show that tumor-specific T cells induce osteolytic bone disease before bone colonization. T cells pro-metastatic activity correlate with a pro-osteoclastogenic cytokine profile, including RANKL, a master regulator of osteoclastogenesis. In vivo inhibition of RANKL from tumor-specific T cells completely blocks bone loss and metastasis. Our results unveil an unexpected role for RANKL-derived from T cells in setting the pre-metastatic niche and promoting tumor spread. We believe this information can bring new possibilities for the development of prognostic and therapeutic tools based on modulation of T cell activity for prevention and treatment of bone metastasis.     

Acquired TRAIL resistance in human breast cancer cells are caused by the sustained cFLIP(L) and XIAP protein levels and ERK activation

We established TRAIL-resistant MDA-231/TR cells from MDA-231 parent cells to understand the mechanism of TRAIL resistance in breast cancer cells. The selected TRAIL-resistant cells were cross-resistant to TNF-alpha/cycloheximide but remained sensitive to DNA-damage drugs such as oxaliplatin and etoposide. The expression levels of death receptors (DR4 and DR5), FADD, cIAP1, cIAP2, and Bcl-2 family were not changed in TRAIL-treated both cells. Significant down-regulation of XIAP and cFLIP was occurred after TRAIL treatment in MDA-231 cells whereas their levels were sustained in MDA-231/TR cells. TRAIL-mediated activation of ERK and JNK were also observed in parent MDA-231 cells but not in MDA-231/TR cells. However, TRAIL-resistant cells showed constitutive activation state after treatment with TRAIL. Pretreatment with PD98059 or transfection of MKK1-DN (dominant negative) expression vector attenuated TRAIL resistance in MDA-231/TR cells. Our findings provide the evidence that the sustained expression level of cFLIP(L) and XIAP protein and constitutive ERK activation may lead to acquired TRAIL resistance in breast cancer cells.     

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. J. Cell. Biochem. 111: 1138–1148, 2010. © 2010 Wiley‐Liss, Inc.     

Anti-tumor effect in human breast cancer by TAE226, a dual inhibitor for FAK and IGF-IR in vitro and in vivo

Focal adhesion kinase (FAK) is a 125-kDa non-receptor type tyrosine kinase that localizes to focal adhesions. FAK overexpression is frequently found in invasive and metastatic cancers of the breast, colon, thyroid, and prostate, but its role in osteolytic metastasis is not well understood. In this study, we have analyzed anti-tumor effects of the novel FAK Tyr³⁹⁷ inhibitor TAE226 against bone metastasis in breast cancer by using TAE226. Oral administration of TAE226 in mice significantly decreased bone metastasis and osteoclasts involved which were induced by MDA-MB-231 breast cancer cells and increased the survival rate of the mouse models of bone metastasis. TAE226 also suppressed the growth of subcutaneous tumors in vivo and the proliferation and migration of MDA-MB-231 cells in vitro. Significantly, TAE226 inhibited the osteoclast formation in murine pre-osteoclastic RAW264.7 cells, and actin ring and pit formation in mature osteoclasts. Moreover, TAE226 inhibited the receptor activator for nuclear factor κ B Ligand (RANKL) gene expression induced by parathyroid hormone-related protein (PTHrP) in bone stromal ST2 cells and blood free calcium concentration induced by PTHrP administration in vivo. These findings suggest that FAK was critically involved in osteolytic metastasis and activated in tumors, pre-osteoclasts, mature osteoclasts, and bone stromal cells and TAE226 can be effectively used for the treatment of cancer induced bone metastasis and other bone diseases.