The bisphosphonate ibandronate promotes apoptosis in MDA-MB-231 human breast cancer cells in bone metastases

Bisphosphonate (BP), a specific inhibitor of osteoclasts, has been widely used as a beneficial agent for the treatment of bone metastases in patients with breast cancer. It is well recognized that BP reduces osteolysis by promoting apoptosis in osteoclasts. However, recent animal and human data suggest that BPs not only reduce osteolysis associated with metastatic breast cancer, but also decrease tumor burden in bone. The mechanisms by which tumor burden is decreased following BP administration are unknown. Here we examined the effects of the BP ibandronate on MDA-231 human breast cancer cells in bone metastases in a well-characterized animal model of bone metastasis. Ibandronate, which was administered (s.c. daily; 4 microg/mouse/day) after bone metastases were established, inhibited the progression of established osteolytic bone metastases as assessed by radiographic analysis. Histological and histomorphometrical examination revealed that ibandronate reduced osteoclastic bone resorption, with increased apoptosis in osteoclasts. Furthermore, ibandronate also significantly decreased the MDA-231 tumor burden, with increased apoptosis in MDA-231 breast cancer cells in bone metastases. In contrast, ibandronate failed to inhibit MDA-231 tumor formation with no effects on apoptosis in MDA-231 breast cancer cells in the orthotopic mammary fat pads. These data suggest that the effects of ibandronate on apoptosis in MDA-231 breast cancer cells are restricted in bone in which ibandronate selectively deposits. Consistent with these in vivo results, a relatively high concentration of ibandronate (100 microM) increased caspase-3 activity and induced DNA fragmentation in MDA-231 breast cancer cells in culture. Moreover, a caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone, blocked ibandronate-induced DNA fragmentation in MDA-231 cells, suggesting an involvement of caspase-3 in ibandronate-induced apoptosis. Our results suggest that BP suppresses bone metastases through promotion of apoptosis in metastatic cancer cells as well as in osteoclasts. However, it still remains open whether BP has direct anticancer actions in vivo.     

Role of RANKL-induced osteoclast formation and MMP-dependent matrix degradation in bone destruction by breast cancer metastasis

Bone metastasis of breast cancer induces severe osteolysis with increased bone resorption. Osteoclast differentiation regulated by the receptor activator of NF-kappaB ligand (RANKL) in osteoblasts and matrix degradation induced by matrix metalloproteinases (MMPs) are thought to be involved in the process of bone resorption. When nude mice were inoculated with human breast cancer cells, MDA-MB-231(MDA-231), numerous osteoclasts resorbed bone and the degradation of the bone matrix markedly progressed in the femur and tibia with metastasis of the MDA-231 tumour. The expression of RANKL, MMP-13 and membrane-type 1-MMP mRNA was markedly elevated in bone with metastasis. When MDA-231 cells were cocultured with mouse calvaria, MDA-231 markedly induced bone resorption measured by calcium release from the calvaria, and the expression of RANKL, MMP-2 and MMP-13 was elevated in the calvaria after the coculture. The separation of MDA-231 from the calvaria using filter insert showed decreased bone resorption, suggesting that cell-to-cell interaction is essential for cancer-induced bone resorption. Adding MDA-231 cells to bone marrow cultures markedly induced osteoclast formation, and the expression of RANKL in osteoblasts was enhanced by contact with the cell surface of MDA-231 cells. These results indicate that RANKL-induced osteoclast formation and MMP-dependent matrix degradation are associated with osteolysis because of bone metastasis of breast cancer.     

Bone morphogenetic protein 7 in the development and treatment of bone metastases from breast cancer

Bone morphogenetic protein 7 (BMP7) counteracts the physiological epithelial-to-mesenchymal transition (EMT), a process that is indicative of epithelial plasticity. Because EMT is involved in cancer, we investigated whether BMP7 plays a role in breast cancer growth and metastasis. In this study, we show that decreased BMP7 expression in primary breast cancer is significantly associated with the formation of clinically overt bone metastases in patients with > or = 10 years of follow-up. In line with these clinical observations, BMP7 expression is inversely related to tumorigenicity and invasive behavior of human breast cancer cell lines. Moreover, BMP7 decreased the expression of vimentin, a mesenchymal marker associated with invasiveness and poor prognosis, in human MDA-MB-231 (MDA-231)-B/Luc(+) breast cancer cells under basal and transforming growth factor-beta (TGF-beta)-stimulated conditions. In addition, exogenous addition of BMP7 to TGF-beta-stimulated MDA-231 cells inhibited Smad-mediated TGF-beta signaling. Furthermore, in a well-established bone metastasis model using whole-body bioluminescent reporter imaging, stable overexpression of BMP7 in MDA-231 cells inhibited de novo formation and progression of osteolytic bone metastases and, hence, their metastatic capability. In line with these observations, daily i.v. administration of BMP7 (100 mug/kg/d) significantly inhibited orthotopic and intrabone growth of MDA-231-B/Luc(+) cells in nude mice. Our data suggest that decreased BMP7 expression during carcinogenesis in the human breast contributes to the acquisition of a bone metastatic phenotype. Because exogenous BMP7 can still counteract the breast cancer growth at the primary site and in bone, BMP7 may represent a novel therapeutic molecule for repression of local and bone metastatic growth of breast cancer.     

Protein C inhibitor inhibits breast cancer cell growth, metastasis and angiogenesis independently of its protease inhibitory activity

Protein C inhibitor (PCI) regulates the anticoagulant protein C pathway and also inhibits urinary plasminogen activator (uPA), a mediator of tumor cell invasion. In the present study, we evaluated the effect of human PCI and its inactive derivatives on tumor growth and metastasis of human breast cancer (MDA-231) cells, and on angiogenesis in vivo. The invasiveness of MDA-231 cells was inhibited by recombinant intact PCI, but not by reactive site-modified PCI (R354APCI) or by the N-terminal fragment of protease-cleaved PCI (NTPCI). The in vitro invasiveness of MDA-231 cells expressing intact PCI (MDA-PCI) was significantly decreased as compared to MDA-231 cells expressing R354APCI (MDA-R354APCI) or NTPCI (MDA-NTPCI). Further, in vivo growth and metastatic potential of MDA-PCI, MDA-R354APCI and MDA-NTPCI cells in severe combined immunodeficient (SCID) mice were significantly decreased as compared to MDA-Mock cells. Angiogenesis was also significantly decreased in Matrigel implant containing MDA-PCI, MDA-R354APCI or MDA-NTPCI cells as compared to that containing MDA-Mock cells. In vivo angiogenesis in rat cornea and in vitro tube formation were also inhibited by recombinant intact PCI, R354APCI and NTPCI. Furthermore, the anti-angiogenic activity of PCI was strong as cleaved antithrombin (AT), and slightly stronger than that of plasminogen activator inhibitor (PAI)-1 and pigment epithelium-derived factor (PEDF). Overall, this study showed that, in addition to a reactive site-dependent mechanism, PCI may also regulate tumor growth and metastasis independently of its protease inhibitory activity by inhibiting angiogenesis.     

COX-2 overexpression increases motility and invasion of breast cancer cells

Cyclooxygenase-2 (COX-2), an inducible enzyme involved in prostaglandin (including PGE(2)) biosynthesis, is overexpressed in several epithelial malignancies including breast cancer. We tested the hypothesis that COX-2 overexpression in breast cancer cells results in increased cell motility and invasion. COX-2 overproducing cells were generated by stable transfection of several human breast cancer cells with pSG5-COX2 vector. We confirmed the overexpression of COX-2 protein by western blotting, and by measuring PGE(2) in the medium with an immunoassay. We measured cell motility by counting the number of cells crossing an 8-micron pore size PET membrane, and cell invasion by counting the number of cells invading through a Matrigel-coated membrane that simulates basement membrane. COX-2 transfected MDA-231 cells produced 30-43-fold more PGE2 as compared to parental cells. COX-2 overexpression increased cell migration approximately 2.2-fold and cell invasion through Matrigel approximately 5.1-fold. Addition of 50 microM NS-398, a COX-2 inhibitor, inhibited Matrigel invasion of MDA-231 cells by 54% as compared to solvent confirming the role of COX-2 in cell invasion. It is known that an increase in cell migration and invasion can be brought about by cytoskeletal alterations and basement membrane degradation due to increased expression of pro-urokinase plasminogen activator (pro-uPA). To investigate the mechanism of our observed increase in cell invasion by COX-2, we found by western blotting that the level of pro-uPA was significantly higher (approximately 5-fold) in COX-2 transfected MDA-231 cells than untransfected MDA-231 cells. Similar to our observations in cell culture, we found evidence that increased COX-2 activity correlates with uPA in a mouse model of breast cancer metastasis to bone. In this study, we conclude that COX-2 overexpression in human breast cancer cells enhances cell motility and invasiveness thus suggesting a mechanism of COX-2 mediated metastasis.     

Ipriflavone inhibits osteolytic bone metastasis of human breast cancer cells in a nude mouse model

Osteolytic bone metastasis is a frequent problem in the treatment of cancer. Ipriflavone, a synthetic isoflavone that inhibits osteoclastic bone resorption, has been used for the treatment of osteoporosis in some countries. Some other isoflavones also exhibit an antitumor effect in vitro and in vivo. Here, we studied the effects of ipriflavone on osteolytic bone metastasis of MDA-231 human breast cancer cells injected intracardially into athymic nude mice (ICR-nu/nu). Daily oral administration of ipriflavone at 12 mg/mouse significantly inhibited the development of new osteolytic bone metastases (p < 0.05) and the progression of established osteolytic lesions (p = 0.01), prolonging the life of tumor-bearing mice (p = 0.01 vs. control). In addition, ipriflavone reduced the number of osteoclasts at the bone-cancer interface with no severe adverse effects on the host. In vitro, ipriflavone inhibited the proliferation and DNA synthesis of MDA-231 cells and blocked the ligand-induced phosphorylation of Tyr(845) of the EGFR. Ipriflavone did not promote apoptosis of MDA-231 cells. Our results show that ipriflavone not only directly inhibits the growth of cancer cells but also reduces osteoclasts to prevent the soft tissue tumor burden and osteolytic bone metastases. These findings raise the possibility that ipriflavone may be of use as a therapeutic agent against osteolytic bone metastasis.     

C-SRC tyrosine kinase activity is associated with tumor colonization in bone and lung in an animal model of human breast cancer metastasis

The proto-oncogene, c-src, has been implicated in the tumorigenesis in breast cancer. However, the relationship of c-src with distant metastasis is unclear. Moreover, the role of c-src in organ-preferential metastasis of breast cancer is unknown. Because breast cancer has a strong predilection for metastasizing to bone, we examined the role of c-src in bone metastases using an animal model in which inoculation of the MDA-231 human breast cancer cells into the left cardiac ventricle preferentially developed osteolytic bone metastases in female nude mice. A clone of the MDA-231 with the increased capacity of bone metastasis exhibited elevated c-src tyrosine kinase (TK) activity compared with parental cells. MDAsrc527 cells caused significantly increased size of the osteolytic bone metastases with increased number of osteoclasts and mitotic cancer cells compared with MDA-231EV or MDAsrcWT. In contrast, MDAsrc295 cells caused impaired metastases to bone. Of note, mice inoculated with MDAsrc295 cells via tail vein developed reduced lung metastases and prolonged survival compared with mice with MDA-231EV cells, suggesting that c-src TK is unlikely to play a specific role in bone metastases. The growth in vitro and in vivo and production of parathyroid hormone-related protein, a key cytokine in the pathogenesis of osteolytic bone metastases in breast cancer, were promoted in MDAsrc527 and diminished in MDAsrc295. These results suggest that c-src TK is associated with the capacity of breast cancer to metastasize to bone through regulating cell growth and parathyroid hormone-related protein production. Our results together with the fact that c-src is an essential molecule for bone resorption by osteoclasts, which are central players in osteolytic bone metastases, support the notion that c-src TK is a potential target molecule for designing novel therapeutic interventions, especially for bone metastases in breast cancer.     

Knockdown of the transforming growth factor-beta type III receptor impairs motility and invasion of metastatic cancer cells

The transforming growth factor-beta (TGF-beta) signaling pathway plays dual roles in epithelial cell tumorigenesis. TGF-beta is initially growth inhibitory, but as tumorigenesis progresses, TGF-beta becomes prometastatic. Although the role of the types I and II TGF-beta receptors is fairly well established, the role of the ubiquitously expressed TGF-beta type III receptor (TbetaRIII) in tumorigenesis is less defined. To examine the role of TbetaRIII in breast cancer cells, we stably expressed short hairpin RNAs specific to TbetaRIII in MDA-231 human breast cancer cells and mouse mammary carcinoma cells expressing the polyomavirus middle T oncogene (PMTLuc). MDA-231 and PMTLuc cells with down-regulated TbetaRIII expression (231-kd; PMTLuc-kd) exhibited decreased growth rate, motility, and invasion into Matrigel, as well as an increase in apoptosis, compared with control cells. MDA-231 xenografts established in nude mice metastasized, whereas tumors made by 231-kd cells did not. Nuclear factor-kappaB (NF-kappaB) activity, which is known to regulate cell growth and motility, was lower in the MDA-231 and PMTLuc knockdown cells compared with control cells. Transfection of an expression vector encoding constitutively active IKK2 into the 231-kd cells restored the ability of TbetaRIII-deficient cells to invade Matrigel and decreased their basal level of apoptosis. These data indicate that TbetaRIII differentially regulates cell growth, motility, and invasion in tumorigenic MDA-231 and PMTLuc cells and that these growth changes occur through the modulation of NF-kappaB activity.     

Short hairpin RNA-mediated inhibition of matrix metalloproteinase-1 in MDA-231 cells: effects on matrix destruction and tumor growth

Increased matrix metalloproteinase-1 (MMP-1) expression is associated with advanced stages of breast cancer and may be a predictive marker for the development of invasive disease. In this report, we used short hairpin RNA (shRNA) molecules to investigate whether MMP-1 production in MDA-231 breast cancer cells contributed to the degradation of a collagen matrix or tumor formation in nude mice. We created two groups of MDA-231 cell lines. MDA-231 cells containing a vector producing shRNA specific for MMP-1 had a >90% decrease in MMP-1 mRNA and protein compared with cells containing an empty vector, and blocking MMP-1 expression inhibited the in vitro collagenolytic activity of MDA-231 cells. When the cells were injected into the mammary fat pad, there was no difference in the frequency of tumor formation in mice. However, the average tumor size was larger in mice injected with cells containing the empty vector (1,216 +/- 334 mm3) than in mice injected with cells expressing the MMP-1 shRNA (272 +/- 117 mm3; P = 0.027). We conclude that MMP-1 expression is essential for the ability of MDA-231 cells to invade and destroy a collagen matrix and in vivo experiments suggest an important role for MMP-1 in breast tumor growth.     

Inhibition of gp130 signaling in breast cancer blocks constitutive activation of Stat3 and inhibits in vivo malignancy

The cytokine receptor gp130 is the common signaling subunit of receptors used by the interleukin (IL)-6 cytokine family. gp130 is widely expressed in breast cancer cell lines and primary tumors. The role of gp130 in breast cancer in vivo is unknown. To study the effect of gp130 inhibition in breast cancer, endogenous gp130 signaling in breast cancer cell lines was blocked with a dominant-negative gp130 protein (DN gp130). DN gp130 inhibited constitutive Stat3 activation in breast cancer cells. Both gp130 and epidermal growth factor receptor (EGFR) have been implicated in constitutive Stat3 activation in breast cancer. There are known physical and functional interactions between gp130 and EGFR. Consistent with this, we show that DN gp130 inhibits signaling downstream of the EGFR in breast cancer cells. The effect of DN gp130 on breast cancer in vivo was assessed with an orthotopic nude mouse model. DN gp130 MDA-231 cells had markedly decreased engraftment, size, and metastasis compared with control cells. These results are particularly striking considering that DN gp130-expressing breast cancer cells grow faster in vitro. We hypothesized that DN gp130 expression results in inhibition of invasion and metastasis in vivo. Marked angiogenesis was present in tumors from control animals and was absent in tumors from DN gp130 animals. We additionally show that tissue inhibitor of metalloproteinase-3, an inhibitor of tumor invasion and angiogenesis, is up-regulated in both MDA-231 DN gp130 cells and tumors. These results, in light of the availability of several potential pharmacological inhibitors of gp130, suggest novel approaches to breast cancer therapy.     

Caveolin-1 inhibits breast cancer growth and metastasis

Caveolin-1 was identified in a screen for genes involved in breast cancer progression. Caveolin-1 is the major protein component of caveolae, flask-shaped invaginations found in a number of different cell types. Using an orthotopic model of spontaneous breast cancer metastasis, caveolin-1 was found to be expressed in low and non-metastatic primary tumors, but at much lower levels in highly metastatic 4T1.2 and 4T1.13 tumors. Exogenous expression of caveolin-1 at moderate levels in 4T1.2 cells was sufficient to suppress primary tumor growth after inoculation of cells into the mammary gland. Expression of high levels of caveolin-1 also inhibited subsequent metastasis to distant organs. Cells expressing high levels of caveolin-1 showed reduced capacity to invade Matrigel, diminished response to laminin-1 stimulation and decreased metastasis to lung and bone. This study provides the first functional evidence that caveolin-1 regulates primary breast tumor growth and spontaneous metastasis of breast cancer.     

The effect of the bisphosphonate ibandronate on breast cancer metastasis to visceral organs

Bisphosphonate (BPs), specific inhibitors of osteoclastic bone resorption, are widely used therapeutic agents for bone metastases in breast cancer patients. Nevertheless, the effects of BPs on visceral metastases are controversial. Here we specifically studied the effects of the BP ibandronate on visceral metastases of breast cancer using two animal models. In the first set of experiments, we examined the effects of ibandronate on lung metastasis using 4T1 mouse mammary tumor that developed pulmonary and bone metastases following orthotopic inoculation in syngeneic female Balb/c mice. In the second set of experiments, we examined the effects of ibandronate on adrenal metastasis using a clone of the MDA-MB-231 (MDA-231) human breast cancer (MDA-231AD cells) that developed adrenal and bone metastases following intracardiac inoculation in female nude mice. These breast cancer cells were stably transfected with a firefly luciferase cDNA to facilitate quantification of the metastatic tumor burden in visceral organs. Ibandronate (4 µg/day, sc, daily) was given either after metastases were established (therapeutic administration) or at the time of tumor cell inoculation (preventative administration). In both models with each protocol, ibandronate reproducibly reduced bone metastases, establishing that BPs are effective pharmacological agents for the treatment of bone metastases in breast cancer. In the 4T1 model, neither the preventative nor therapeutic administration of ibandronate caused any effects on lung metastases. In the MDA-231 model, the preventative administration of ibandronate significantly increased adrenal metastases. However, no increase in the adrenal metastases was observed when an anti-cancer agent doxorubicin was co-administered. Therapeutic administration of ibandronate showed no effects on the adrenal metastases. Our results suggest that BPs cause no adverse effects on visceral metastases when administered in the manners that breast cancer patients usually receive.     

Interleukin-2 differentially affects the proliferation of a hormone-dependent and a hormone-independent human breast cancer cell line in vitro and in vivo

Direct in vitro and in vivo effects of the lymphokine, interleukin-2 (IL-2), on hormone-dependent (MCF-7) and- independent (MDA-231) human breast cancer cell proliferation were investigated. In vitro, picomolar concentrations of IL-2 directly inhibited MCF-7 cell proliferation after 12 days of culture, while nanomolar doses of IL-2 significantly stimulated MCF-7 cell growth over the same time period. In addition, micromolar concentrations of IL-2 had virtually no effect on the in vitro proliferation of MCF-7 cells. In parallel in vitro growth experiments, the hormone-independent cells, MDA-231, were not affected by IL-2 regardless of concentration. IL-2 treatment of overiectomized, estrogen-treated nude mice, burdened with MCF-7 or MDA-231 tumors, inhibited MCF-7 tumor growth, but had no effect on MDA-231 tumors. Examination of T, B and natural killer (NK) cell function in these animals indicated that the interleukin-2-mediated effect on MCF-7 cell growth in vivo is independent of the proliferative abilities of these lymphoid cells, suggesting that IL-2 may directly affect the growth of these hormone-dependent human breast cancer cells.     

Effect of a newly developed bisphosphonate,YH529, on osteolytic bone metastases in nude mice

YH529, [1-hydroxy-2-(imidazo [1,2-a] pyridin-3-yl) ethylidene]-bisphosphonic acid monohydrate, is a newly developed third-generation bisphosphonate with a potent inhibitory activity toward osteoclastic bone resorption. The primary cellular mechanism of osteolysis associated with metastatic cancer is osteoclast-mediated. It is likely that bisphosphonates would be efficacious in this situation. In the present study, we examined the effect of YH529 in a nude mice bone metastasis model, in which the intracardiac injection of a human breast cancer cell line, MDA-MB-231(MDA-231), leads to osteolytic bone metastases. To examine whether YH529 would prevent such bone metastasis, we administered YH529 s.c. to nude mice simultaneously with cancer cell inoculation through the entire experimental period (protocol 1) or performed short-term prophylactic administration before inoculation of the MDA-231 cells (protocol 2). In addition, to examine the possible therapeutic effects of the drug on established bone metastases, we injected YH529 after radiographically small but distinct osteolytic bone metastases had been detected (protocol 3). In all protocols, YH529 (2 μg/mouse/day) markedly inhibited bone metastases as well as the progression of established metastatic foci that were quantified on the radiographs. Histological examination and histomorphometrical analysis revealed that YH529 markedly reduced the number of osteoclasts and the size of the tumor at the metastatic bone sites. Our results suggest that YH529 may suppress metastasis formation and tumor growth in bone through inhibition of osteoclastic bone resorption. Int. J. Cancer 77:279–285, 1998.© 1998 Wiley-Liss, Inc.     

Over-expression of bone sialoprotein enhances bone metastasis of human breast cancer cells in a mouse model

Bone sialoprotein (BSP) is a major non-collagenous protein found almost exclusively in bone and other mineralized tissues including enamel, dentin and cementum. Although a role for BSP in mineralization has been indicated, BSP also appears to function in patho-physiological processes, including the metastasis of breast and prostate cancer cells to bone. The purpose of this study was to determine the role of BSP in the homing of cancer cells and to provide insights into the role of BSP in physiological as well as pathological processes. We established cultures of MDA-231 breast cancer cells stably transfected with DNA constructs of pIRES2-EGFP (green fluorescent protein) expressing human BSP (hBSP) cDNA (231BSP) under a CMV promoter, or with an antisense sequence of hBSP cDNA (231BSPAS), or with an empty vector as a control (231EV). These 3 cell groups were selected for neomycin resistance using G418 and analyzed by flow cytometry for GFP expression. The resultant cultured cells expressed different levels of hBSP as detected by RT-PCR and Western blot. Among the three, 231BSP expressed the highest levels of hBSP while 231BSPAS expressed the lowest. The capacity of the tumor cells to metastasize to bone was determined in nude mice (5 in each group) by intra-cardiac injection of the cells from the 3 different groups. Four weeks after inoculation, radiological examination revealed that all the 5 mice in the 231BSP cell group had developed osteolytic bone metastases. In the 231BSPAS group only 1 mouse demonstrated metastatic bone lesions while 3 out of 5 mice in the control group (231EV) developed metastatic lesions in the bone. These results strongly suggest that BSP over-expression in human tumor cells can enhance bone metastasis of MDA-231 cells whereas repressed expression of BSP, using antisense BSP cDNA, inhibits this effect in a mouse model.     

Tumor alphavbeta3 integrin is a therapeutic target for breast cancer bone metastases

In breast cancer bone metastasis, tumor cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone stimulate tumor growth. The alphavbeta3 integrin is an adhesion receptor expressed by breast cancer cells and osteoclasts. It is implicated in tumor cell invasion and osteoclast-mediated bone resorption. Here, we hypothesized that the therapeutic targeting of tumor alphavbeta3 integrin would prevent bone metastasis formation. We first showed that, compared with mock-transfected cells, the i.v. inoculation of alphavbeta3-overexpressing MDA-MB-231 breast cancer cells in animals increased bone metastasis incidence and promoted both skeletal tumor burden and bone destruction. The direct inoculation of alphavbeta3-overexpressing transfectants into the tibial bone marrow cavity did not however enhance skeletal tumor burden and bone destruction, suggesting that alphavbeta3 controls earlier events during bone metastasis formation. We next examined whether a nonpeptide antagonist of alphavbeta3 (PSK1404) exhibits meaningful antitumor effects in experimental breast and ovarian cancer bone metastasis. A continuous PSK1404 treatment, which inhibited osteoclast-mediated bone resorption in an animal model of bone loss, substantially reduced bone destruction and decreased skeletal tumor burden. Importantly, a short-term PSK1404 treatment that did not inhibit osteoclast activity also decreased skeletal tumor burden and bone destruction. This dosing regimen caused a profound and specific inhibition of bone marrow colonization by green fluorescent protein, alphavbeta3-expressing tumor cells in vivo and blocked tumor cell invasion in vitro. Overall, our data show that tumor alphavbeta3 integrin stands as a therapeutic target for the prevention of skeletal metastases.     

Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell aggressive phenotype with PBR expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol

Aberrant cell proliferation and increased invasive and metastatic behavior are hallmarks of the advancement of breast cancer. Numerous studies implicate a role for cholesterol in the mechanisms underlying cell proliferation and cancer progression. The peripheral-type benzodiazepine receptor (PBR) is an Mr 18,000 protein primarily localized to the mitochondria. PBR mediates cholesterol transport across the mitochondrial membranes in steroidogenic cells. A role for PBR in the regulation of tumor cell proliferation has also been shown. In this study, we examined the expression, characteristics, localization, and function of PBR in a battery of human breast cancer cell lines differing in their invasive and chemotactic potential as well as in several human tissue biopsies. Expression of PBR ligand binding and mRNA was dramatically increased in the highly aggressive cell lines, such as MDA-231, relative to nonaggressive cell lines, such as MCF-7. PBR was also found to be expressed at high levels in aggressive metastatic human breast tumor biopsies compared with normal breast tissues. Subcellular localization with both antibodies and a fluorescent PBR drug ligand revealed that PBR from the MDA-231 cell line as well as from aggressive metastatic human breast tumor biopsies localized primarily in and around the nucleus. This localization is in direct contrast to the largely cytoplasmic localization seen in MCF-7 cells, normal breast tissue, and to the typical mitochondrial localization seen in mouse tumor Leydig cells. Pharmacological characterization of the receptor and partial nucleotide sequencing of PBR cDNA revealed that the MDA-231 PBR is similar, although not identical, to previously described PBR. Addition of high affinity PBR drug ligands to MDA-231 cells increased the incorporation of bromodeoxyuridine into the cells in a dose-dependent manner, suggesting a role for PBR in the regulation of MDA-231 cell proliferation. Cholesterol uptake into isolated MDA-231 nuclei was found to be 30% greater than into MCF-7 nuclei. High-affinity PBR drug ligands regulated the levels of cholesterol present in MDA-231 nuclei but not in MCF-7. In addition, the PBR-dependent MDA-231 cell proliferation was found to highly correlate (r = -0.99) with the PBR-mediated changes in nuclear membrane cholesterol levels. In conclusion, these data suggest that PBR expression, nuclear localization, and PBR-mediated cholesterol transport into the nucleus are involved in human breast cancer cell proliferation and aggressive phenotype expression, thus participating in the advancement of the disease.