Riko Nishimura

A Bone‐Seeking Clone Exhibits Different Biological Properties from the MDA‐MB‐231 Parental Human Breast Cancer Cells and a Brain‐Seeking Clone In Vivo and In Vitro

Breast cancer has a predilection for spreading to bone. The mechanism of preferential metastasis of breast cancer to bone is unknown. We hypothesize that breast cancer cells that develop bone metastases have the capacity to facilitate their colonization in bone. To examine this hypothesis, we established bone‐seeking (MDA‐231BO) and brain‐seeking (MDA‐231BR) clones of the human breast cancer cell line MDA‐MB‐231 by repeated sequential passages in nude mice and in vitro of metastatic cells obtained from bone and brain metastases, respectively. These clones were examined for distinguishing biological characteristics and compared with the MDA‐231 parental cells (MDA‐231P) in vivo and in vitro. Both the MDA‐231BR and the MDA‐231BO showed identical tumorigenicity to MDA‐231P at the orthotopic site. MDA‐231P that was inoculated into the heart developed metastases in bone, brain, ovary, and adrenal glands. On the other hand, MDA‐231BO exclusively metastasized to bone with larger osteolytic lesions than MDA‐231P. MDA‐231BR exclusively disseminated to brain and failed to develop bone metastases. In culture, MDA‐231BO produced greater amounts of parathyroid hormone‐related protein (PTH‐rP) than MDA‐231BR and MDA‐231P in the absence or presence of transforming growth factor β (TGF‐β). Furthermore, the anchorage‐independent growth of MDA‐ 231BO in soft agar was not inhibited by TGF‐β, whereas TGF‐β profoundly inhibited the growth of MDA‐231P and MDA‐231BR. Insulin‐like growth factor I (IGF‐I) markedly promoted the anchorage‐independent growth of MDA‐231BO, whereas marginal or no stimulation was observed in MDA‐231BR or MDA‐231P, respectively. Our data suggest that these phenotypic changes allow breast cancer cells to promote osteoclastic bone resorption, survive, and proliferate in bone, which consequently leads to the establishment of bone metastases.

BMP2 Regulates Osterix through Msx2 and Runx2 during Osteoblast Differentiation

Osterix/Sp7, a member of the Sp1 transcription factor family, plays an essential role in bone formation and osteoblastogenesis. Although Osterix has been shown to be induced by BMP2 in a mesenchymal cell line, the molecular basis of the regulation, expression and function of Osterix during osteoblast differentiation, is not fully understood. Thus we examined the role of BMP2 signaling in the regulation of Osterix using the mesenchymal cell lines C3H10T1/2 and C2C12. Osterix overexpression induced alkaline phosphatase activity and osteocalcin expression in C2C12 cells and stimulated calcification of murine primary osteoblasts. Considering that Runx2 overexpression induces Osterix, these results suggest that Osterix functions as downstream of Runx2. Surprisingly, BMP2 treatment induced Osterix expression and alkaline phosphatase activity in mesenchymal cells derived from Runx2-deficient mice. Furthermore, overexpression of Smad1 and Smad4 up-regulated Osterix expression, and an inhibitory Smad, Smad6, markedly suppressed BMP2-induced Osterix expression in the Runx2-deficient cells. Moreover, overexpression of a homeobox gene, Msx2, which is up-regulated by BMP2 and promotes osteoblastic differentiation, induced Osterix expression in the Runx2-deficient cells. Knockdown of Msx2 clearly inhibited induction of Osterix by BMP2 in the Runx2-deficient mesenchymal cells. Interestingly, microarray analyses using the Runx2-deficient cells revealed that the role of Osterix was distinct from that of Runx2. These findings suggest that Osterix is regulated via both Runx2-dependent and -independent mechanisms, and that Osterix controls osteoblast differentiation, at least in part, by regulating the expression of genes not controlled by Runx2.

Requirement of BMP-2-induced phosphatidylinositol 3-kinase and Akt serine/threonine kinase in osteoblast differentiation and Smad-dependent BMP-2 gene transcription.

The mechanism by which bone morphogenetic protein-2 (BMP-2) induces osteoblast differentiation is not precisely known. We investigated the involvement of the phosphatidylinositol (PI) 3-kinase/Akt signal transduction pathway in modulation of this process. BMP-2 stimulated PI 3-kinase activity in osteogenic cells. Inhibition of PI 3-kinase activity with the specific inhibitor Ly-294002 prevented BMP-2-induced alkaline phosphatase, an early marker of osteoblast differentiation. Expression of dominant-negative PI 3-kinase also abolished osteoblastic induction of alkaline phosphatase in response to BMP-2, confirming the involvement of this lipid kinase in this process. BMP-2 stimulated Akt serine/threonine kinase activity in a PI 3-kinase-dependent manner in osteoblast precursor cells. Inhibition of Akt activity by a dominant-negative mutant of Akt blocked BMP-2-induced osteoblastic alkaline phosphatase activity. BMP-2 stimulates its own expression during osteoblast differentiation. Expression of dominant-negative PI 3-kinase or dominant-negative Akt inhibited BMP-2-induced BMP-2 transcription. Because all the known biological activities of BMP-2 are mediated by transcription via BMP-specific Smad proteins, we investigated the involvement of PI 3-kinase in Smad-dependent BMP-2 transcription. Smad5 stimulated BMP-2 transcription independent of addition of the ligand. Dominant-negative PI 3-kinase or dominant-negative Akt inhibited Smad5-dependent transcription of BMP-2. Furthermore dominant-negative Akt inhibited translocation of BMP-specific Smads into nucleus. Together these data provide the first evidence that activation of BMP receptor serine/threonine kinase stimulates the PI 3 kinase/Akt pathway and define a role for this signal transduction pathway in BMP-specific Smad function during osteoblast differentiation.

Signalling mediated by the endoplasmic reticulum stress transducer OASIS is involved in bone formation

Eukaryotic cells have signalling pathways from the endoplasmic reticulum (ER) to cytosol and nuclei, to avoid excess accumulation of unfolded proteins in the ER. We previously identified a new type of ER stress transducer, OASIS, a bZIP (basic leucine zipper) transcription factor, which is a member of the CREB/ATF family and has a transmembrane domain. OASIS is processed by regulated intramembrane proteolysis (RIP) in response to ER stress, and is highly expressed in osteoblasts. OASIS−/− mice exhibited severe osteopenia, involving a decrease in type I collagen in the bone matrix and a decline in the activity of osteoblasts, which showed abnormally expanded rough ER, containing of a large amount of bone matrix proteins. Here we identify the gene for type 1 collagen, Col1a1, as a target of OASIS, and demonstrate that OASIS activates the transcription of Col1a1 through an unfolded protein response element (UPRE)-like sequence in the osteoblast-specific Col1a1 promoter region. Moreover, expression of OASIS in osteoblasts is induced by BMP2 (bone morphogenetic protein 2), the signalling of which is required for bone formation. Additionally, RIP of OASIS is accelerated by BMP2 signalling, which causes mild ER stress. Our studies show that OASIS is critical for bone formation through the transcription of Col1a1 and the secretion of bone matrix proteins, and they reveal a new mechanism by which ER stress-induced signalling mediates bone formation.

Bone morphogenetic protein receptor signaling is necessary for normal murine postnatal bone formation

Functions of bone morphogenetic proteins (BMPs) are initiated by signaling through specific type I and type II serine/threonine kinase receptors. In previous studies, we have demonstrated that the type IB BMP receptor (BMPR-IB) plays an essential and specific role in osteoblast commitment and differentiation. To determine the role of BMP receptor signaling in bone formation in vivo, we generated transgenic mice, which express a truncated dominant-negative BMPR-IB targeted to osteoblasts using the type I collagen promoter. The mice are viable and fertile. Tissue-specific expression of the truncated BMPR-IB was demonstrated. Characterization of the phenotype of these transgenic mice showed impairment of postnatal bone formation in 1-mo-old homozygous transgenic mice. Bone mineral density, bone volume, and bone formation rates were severely reduced, but osteoblast and osteoclast numbers were not significantly changed in the transgenic mice. To determine whether osteoblast differentiation is impaired, we used primary osteoblasts isolated from the transgenic mice and showed that BMP signaling is blocked and BMP2-induced mineralized bone matrix formation was inhibited. These studies show the effects of alterations in BMP receptor function targeted to the osteoblast lineage and demonstrate a necessary role of BMP receptor signaling in postnatal bone growth and bone formation in vivo.

Regulation of endoplasmic reticulum stress response by a BBF2H7-mediated Sec23a pathway is essential for chondrogenesis

Many tissues have a specific signal transduction system for endoplasmic reticulum (ER) dysfunction; however, the mechanisms underlying the ER stress response in cartilage remain unclear. BBF2H7 (BBF2 human homologue on chromosome 7), an ER-resident basic leucine zipper transcription factor, is activated in response to ER stress and is highly expressed in chondrocytes. In this study, we generated Bbf2h7−/− mice to assess the in vivo function of BBF2H7. The mice showed severe chondrodysplasia and died by suffocation shortly after birth because of an immature chest cavity. The cartilage showed a lack of typical columnar structure in the proliferating zone and a decrease in the size of the hypertrophic zone, resulting in a significant reduction of extracellular matrix proteins. Interestingly, proliferating chondrocytes showed abnormally expanded ER, containing aggregated type II collagen (Col2) and cartilage oligomeric matrix protein (COMP). We identified Sec23a, which encodes a coat protein complex II component responsible for protein transport from the ER to the Golgi, as a target of BBF2H7, which directly bound to a CRE-like sequence in the promoter region of Sec23a to activate its transcription. When Sec23a was introduced to Bbf2h7−/− chondrocytes, the impaired transport and secretion of cartilage matrix proteins was totally restored, indicating that by activating protein secretion the BBF2H7–Sec23a pathway has a crucial role in chondrogenesis. Our findings provide a new link by which ER stress is converted to signalling for the activation of ER-to-Golgi trafficking.

Functional gene screening system identified TRPV4 as a regulator of chondrogenic differentiation.

Sox9 is a transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expression. However, the precise mechanism of Sox9 activation during chondrogenesis is not fully understood. To investigate this mechanism, we performed functional gene screening to identify genes that activate SOX9-dependent transcription, using full-length cDNA libraries generated from a murine chondrogenic cell line, ATDC5. Screening revealed that TRPV4 (transient receptor potential vanilloid 4), a cation channel molecule, significantly elevates SOX9-dependent reporter activity. Microarray and quantitative real time PCR analyses demonstrated that during chondrogenesis in ATDC5 and C3H10T1/2 (a murine mesenchymal stem cell line), the expression pattern of TRPV4 was similar to the expression patterns of chondrogenic marker genes, such as type II collagen and aggrecan. Activation of TRPV4 by a pharmacological activator induced SOX9-dependent reporter activity, and this effect was abolished by the addition of the TRPV antagonist ruthenium red or by using a small interfering RNA for TRPV4. The SOX9-dependent reporter activity due to TRPV4 activation was abrogated by both EGTA and a calmodulin inhibitor, suggesting that the Ca2+/calmodulin signal is essential in this process. Furthermore, activation of TRPV4 in concert with insulin activity in ATDC5 cells or in concert with bone morphogenetic protein-2 in C3H10T1/2 cells promoted synthesis of sulfated glycosaminoglycan, but activation of TRPV4 had no effect alone. We showed that activation of TRPV4 increased the steady-state levels of SOX9 mRNA and protein and SOX6 mRNA. Taken together, our results suggest that TRPV4 regulates the SOX9 pathway and contributes to the process of chondrogenesis.

Evidence that tumor necrosis factor plays a pathogenetic role in the paraneoplastic syndromes of cachexia, hypercalcemia, and leukocytosis in a human tumor in nude mice.

Recently, we have established a human squamous cell carcinoma of the maxilla (called MH-85) associated with hypercalcemia, leukocytosis, and cachexia in culture. MH-85 tumor cells caused the same paraneoplastic syndromes in tumor-bearing nude mice. We found that there was a sixfold increase in splenic size in MH-85 tumor-bearing mice. This increase paralleled tumor growth and was reversed by surgical removal of the tumor. Splenectomy in nude mice 1 wk before or 6 wk after tumor inoculation resulted in a decrease in tumor growth, and impairment of hypercalcemia, leukocytosis, and cachexia. In MH-85 tumor-bearing animals that had been pretreated by splenectomy, intravenous injection of fresh normal spleen cells caused an immediate reversal of leukocytosis, hypercalcemia, and cachexia. Since the presence of cachexia in both the patient and the mice carrying the tumor suggested tumor necrosis factor (TNF) may be overproduced, we injected polyclonal neutralizing antibodies raised against murine TNF into tumor-bearing mice. There was a rapid and reproducible decrease in blood ionized calcium, accompanied by suppression of osteoclast activity. No changes in blood ionized calcium were seen in mice injected with normal immune sera. In addition, there was an increase in body weight and decrease in white cell count. Plasma immunoreactive TNF was increased almost fourfold in tumor-bearing nude mice compared with control nude mice. Although TNF activity was undetectable in MH-85 culture supernatants, cells of the macrophage lineage, including spleen cells, released increased amounts of TNF when cultured with MH-85 tumor-conditioned media. These results suggest that splenic cytokines such as TNF may influence the development of the paraneoplastic syndromes of hypercalcemia, leukocytosis, and cachexia in these animals, as well as tumor growth. They also show that paraneoplastic syndromes may be due to factors produced by normal host cells stimulated by the presence of the tumor.

Regulation of bone and cartilage development by network between BMP signalling and transcription factors

Bone morphogenetic protein(s) (BMP) are very powerful cytokines that induce bone and cartilage formation. BMP also stimulate osteoblast and chondrocyte differentiation. During bone and cartilage development, BMP regulates the expression and/or the function of several transcription factors through activation of Smad signalling. Genetic studies revealed that Runx2, Osterix and Sox9, all of which function downstream of BMP, play essential roles in bone and/or cartilage development. In addition, two other transcription factors, Msx2 and Dlx5, which interact with BMP signalling, are involved in bone and cartilage development. The importance of these transcription factors in bone and cartilage development has been supported by biochemical and cell biological studies. Interestingly, BMP is regulated by several negative feedback systems that appear necessary for fine-tuning of bone and cartilage development induced by BMP. Thus, BMP harmoniously regulates bone and cartilage development by forming network with several transcription factors.

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.