Osteocytes and Paget's Disease of Bone.

PURPOSE OF REVIEW: To describe the contributions of osteocytes to the lesions in Paget's disease, which are characterized by locally overactive bone resorption and formation. RECENT FINDINGS: Osteocytes, the most abundant cells in bone, are altered in Paget's disease lesions, displaying increased size, decreased canalicular length, incomplete differentiation, and less sclerostin expression compared to controls in both patients and mouse models. Pagetic lesions show increased senescent osteocytes that express RANK ligand, which drives osteoclastic bone resorption. Abnormal osteoclasts in Paget's disease secrete abundant IGF1, which enhances osteocyte senescence, contributing to lesion formation. Recent data suggest that osteocytes contribute to lesion formation in Paget's disease by responding to high local IGF1 released from abnormal osteoclasts. Here we describe the characteristics of osteocytes in Paget's disease and their role in bone lesion formation based on recent results with mouse models and supported by patient data.

Pharmacologic targeting of the p62 ZZ domain enhances both anti-tumor and bone-anabolic effects of bortezomib in multiple myeloma.

Multiple myeloma (MM) is a malignancy of plasma cells whose antibody secretion creates proteotoxic stress relieved by the N-end rule pathway, a proteolytic system that degrades Narginylated proteins in the proteasome. When the proteasome is inhibited, protein cargo is alternatively targeted for autophagic degradation by binding to the ZZ-domain of p62/sequestosome-1. Here, we demonstrate that XRK3F2, a selective ligand for the ZZ-domain, dramatically improved two major responses to the proteasome inhibitor bortezomib by increasing: 1) killing of human MM cells by stimulating both bortezomib mediated apoptosis and necroptosis, a process regulated by p62; and 2) preservation of bone mass by stimulating osteoblasts differentiation and inhibiting osteoclastic bone destruction. Co-administration of bortezomib and XRK3F2 inhibited both branches of the bimodal N-end rule pathway exhibited synergistic anti-MM effects on MM cell lines and CD138+ cells from MM patients, and prevented stromal-mediated MM cell survival. In mice with established human MM, coadministration of bortezomib and XRK3F2 decreased tumor burden and prevented the progression of MM-induced osteolytic disease by inducing new bone formation more effectively than either single agent alone. The results suggest that p62-ZZ ligands enhance the anti-MM efficacy of proteasome inhibitors and can reduce MM morbidity and mortality by improving bone health.

Osteoclast-derived IGF1 induces RANKL production in osteocytes and contributes to pagetic lesion formation.

We previously reported that measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) of patients with Paget disease (PD) or targeted to the OCL lineage in MVNP-transgenic mice (MVNP mice) increases IGF1 production in osteoclasts (OCL-IGF1) and leads to development of PD OCLs and pagetic bone lesions (PDLs). Conditional deletion of Igf1 in OCLs of MVNP mice fully blocked development of PDLs. In this study, we examined whether osteocytes (OCys), key regulators of normal bone remodeling, contribute to PD. OCys in PDLs of patients and of MVNP mice expressed less sclerostin, and had increased RANKL expression compared with OCys in bones from WT mice or normal patients. To test whether increased OCL-IGF1 is sufficient to induce PDLs and PD phenotypes, we generated TRAP-Igf1 (T-Igf1) transgenic mice to determine whether increased IGF1 expression in the absence of MVNP in OCLs is sufficient to induce PDLs and pagetic OCLs. We found that T-Igf1 mice at 16 months of age developed PD OCLs, PDLs, and OCys, with decreased sclerostin and increased RANKL, similar to MVNP mice. Thus, pagetic phenotypes could be induced by OCLs expressing increased IGF1. OCL-IGF1 in turn increased RANKL production in OCys to induce PD OCLs and PDLs.

Osteoclast-derived IGF1 is required for pagetic lesion formation in vivo.

We report that transgenic mice expressing measles virus nucleocapsid protein (MVNP) in osteoclasts (OCLs) (MVNP mice) are Paget's disease (PD) models and that OCLs from patients with PD and MVNP mice express high levels of OCL-derived IGF1 (OCL-IGF1). To determine OCL-IGF1's role in PD and normal bone remodeling, we generated WT and MVNP mice with targeted deletion of Igf1 in OCLs (Igf1-cKO) and MVNP/Igf1-cKO mice, and we assessed OCL-IGF1's effects on bone mass, bone formation rate, EphB2/EphB4 expression on OCLs and osteoblasts (OBs), and pagetic bone lesions (PDLs). A total of 40% of MVNP mice, but no MVNP/Igf1-cKO mice, had PDLs. Bone volume/tissue volume (BV/TV) was decreased by 60% in lumbar vertebrae and femurs of MVNP/Igf1-cKO versus MVNP mice with PDLs and by 45% versus all MVNP mice tested. Bone formation rates were decreased 50% in Igf1-cKO and MVNP/Igf1-cKO mice versus WT and MVNP mice. MVNP mice had increased EphB2 and EphB4 levels in OCLs/OBs versus WT and MVNP/Igf1-cKO, with none detectable in OCLs/OBs of Igf1-cKO mice. Mechanistically, IL-6 induced the increased OCL-IGF1 in MVNP mice. These results suggest that high OCL-IGF1 levels increase bone formation and PDLs in PD by enhancing EphB2/EphB4 expression in vivo and suggest OCL-IGF1 may contribute to normal bone remodeling.

Small molecules inhibit ex vivo tumor growth in bone.

Bone is a common site of metastasis for breast, prostate, lung, kidney and other cancers. Bone metastases are incurable, and substantially reduce patient quality of life. To date, there exists no small-molecule therapeutic agent that can reduce tumor burden in bone. This is partly attributed to the lack of suitable in vitro assays that are good models of tumor growth in bone. Here, we take advantage of a novel ex vivo model of bone colonization to report a series of pyrrolopyrazolone small molecules that inhibit cancer cell invasion and ex vivo tumor growth in bone at single-digit micromolar concentration. We find that the compounds modulated the expression levels of genes associated with bone-forming osteoblasts, bone-destroying osteoclasts, cancer cell viability and metastasis. Our compounds provide chemical tools to uncover novel targets and pathways associated with bone metastasis, as well as for the development of compounds to prevent and reverse bone tumor growth in vivo.

The Role of Semaphorin 4D in Bone Remodeling and Cancer Metastasis.

Semaphorin 4D (Sema4D; CD100) is a transmembrane homodimer 150-kDa glycoprotein member of the Semaphorin family. Semaphorins were first identified as chemorepellants that guide neural axon growth. Sema4D also possesses immune regulatory activity. Recent data suggest other Sema4D functions: inactivation of platelets, stimulation of angiogenesis, and regulation of bone formation. Sema4D is a coupling factor expressed on osteoclasts that inhibits osteoblast differentiation. Blocking Sema4D may, therefore, be anabolic for bone. Sema4D and its receptor Plexin-B1 are commonly dysregulated in cancers, suggesting roles in cancer progression, invasion, tumor angiogenesis, and skeletal metastasis. This review focuses on Sema4D in bone and cancer biology and the molecular pathways involved, particularly Sema4D-Plexin-B1 signaling crosstalk between cancer cells and the bone marrow microenvironment-pertinent areas since a humanized Sema4D-neutralizing antibody is now in early phase clinical trials in cancers and neurological disorders.

Halofuginone inhibits TGF-ß/BMP signaling and in combination with zoledronic acid enhances inhibition of breast cancer bone metastasis.

More efficient therapies that target multiple molecular mechanisms are needed for the treatment of incurable bone metastases. Halofuginone is a plant alkaloid-derivative with antiangiogenic and antiproliferative effects. Here we demonstrate that halofuginone is an effective therapy for the treatment of bone metastases, through multiple actions that include inhibition of TGFß and BMP-signaling. Halofuginone blocked TGF-ß-signaling in MDA-MB-231 and PC3 cells showed by inhibition of TGF-ß-induced Smad-reporter, phosphorylation of Smad-proteins, and expression of TGF-ß-regulated metastatic genes. Halofuginone increased inhibitory Smad7-mRNA and reduced TGF-ß-receptor II protein. Proline supplementation but not Smad7-knockdown reversed halofuginone-inhibition of TGF-ß-signaling. Halofuginone also decreased BMP-signaling. Treatment of MDA-MB-231 and PC3 cells with halofuginone reduced the BMP-Smad-reporter (BRE)4, Smad1/5/8-phosphorylation and mRNA of the BMP-regulated gene Id-1. Halofuginone decreased immunostaining of phospho-Smad2/3 and phospho-Smad1/5/8 in cancer cells in vivo. Furthermore, halofuginone decreased tumor-take and growth of orthotopic-tumors. Mice with breast or prostate bone metastases treated with halofuginone had significantly less osteolysis than control mice. Combined treatment with halofuginone and zoledronic-acid significantly reduced osteolytic area more than either treatment alone. Thus, halofuginone reduces breast and prostate cancer bone metastases in mice and combined with treatment currently approved by the FDA is an effective treatment for this devastating complication of breast and prostate-cancer.

Bidirectional Notch Signaling and Osteocyte-Derived Factors in the Bone Marrow Microenvironment Promote Tumor Cell Proliferation and Bone Destruction in Multiple Myeloma.

In multiple myeloma, an overabundance of monoclonal plasma cells in the bone marrow induces localized osteolytic lesions that rarely heal due to increased bone resorption and suppressed bone formation. Matrix-embedded osteocytes comprise more than 95% of bone cells and are major regulators of osteoclast and osteoblast activity, but their contribution to multiple myeloma growth and bone disease is unknown. Here, we report that osteocytes in a mouse model of human MM physically interact with multiple myeloma cells in vivo, undergo caspase-3-dependent apoptosis, and express higher RANKL (TNFSF11) and sclerostin levels than osteocytes in control mice. Mechanistic studies revealed that osteocyte apoptosis was initiated by multiple myeloma cell-mediated activation of Notch signaling and was further amplified by multiple myeloma cell-secreted TNF. The induction of apoptosis increased osteocytic Rankl expression, the osteocytic Rankl/Opg (TNFRSF11B) ratio, and the ability of osteocytes to attract osteoclast precursors to induce local bone resorption. Furthermore, osteocytes in contact with multiple myeloma cells expressed high levels of Sost/sclerostin, leading to a reduction in Wnt signaling and subsequent inhibition of osteoblast differentiation. Importantly, direct contact between osteocytes and multiple myeloma cells reciprocally activated Notch signaling and increased Notch receptor expression, particularly Notch3 and 4, stimulating multiple myeloma cell growth. These studies reveal a previously unknown role for bidirectional Notch signaling that enhances MM growth and bone disease, suggesting that targeting osteocyte-multiple myeloma cell interactions through specific Notch receptor blockade may represent a promising treatment strategy in multiple myeloma.

FGF23 is elevated in multiple myeloma and increases heparanase expression by tumor cells.

Multiply myeloma (MM) grows in and destroys bone, where osteocytes secrete FGF23, a hormone which affects phosphate homeostasis and aging. We report that multiple myeloma (MM) cells express receptors for and respond to FGF23. FGF23 increased mRNA for EGR1 and its target heparanase, a pro-osteolytic factor in MM. FGF23 signals through a complex of klotho and a classical FGF receptor (FGFR); both were expressed by MM cell lines and patient samples. Bone marrow plasma cells from 42 MM patients stained positively for klotho, while plasma cells from 8 patients with monoclonal gammopathy of undetermined significance (MGUS) and 6 controls were negative. Intact, active FGF23 was increased 2.9X in sera of MM patients compared to controls. FGF23 was not expressed by human MM cells, but co-culture with mouse bone increased its mRNA. The FGFR inhibitor NVP-BGJ398 blocked the heparanase response to FGF23. NVP-BGJ398 did not inhibit 8226 growth in vitro but significantly suppressed growth in bone and induction of the osteoclast regulator RANK ligand, while decreasing heparanase mRNA. The bone microenvironment provides resistance to some anti-tumor drugs but increased the activity of NVP-BGJ398 against 8226 cells. The FGF23/klotho/heparanase signaling axis may offer targets for treatment of MM in bone.

The TGF-ß Signaling Regulator PMEPA1 Suppresses Prostate Cancer Metastases to Bone.

Transforming growth factor-ß (TGF-ß) regulates the expression of genes supporting breast cancer cells in bone, but little is known about prostate cancer bone metastases and TGF-ß. Our study reveals that the TGFBR1 inhibitor SD208 effectively reduces prostate cancer bone metastases. TGF-ß upregulates in prostate cancer cells a set of genes associated with cancer aggressiveness and bone metastases, and the most upregulated gene was PMEPA1. In patients, PMEPA1 expression decreased in metastatic prostate cancer and low Pmepa1 correlated with decreased metastasis-free survival. Only membrane-anchored isoforms of PMEPA1 interacted with R-SMADs and ubiquitin ligases, blocking TGF-ß signaling independently of the proteasome. Interrupting this negative feedback loop by PMEPA1 knockdown increased prometastatic gene expression and bone metastases in a mouse prostate cancer model.

Tumor-expressed adrenomedullin accelerates breast cancer bone metastasis.

INTRODUCTION: Adrenomedullin (AM) is secreted by breast cancer cells and increased by hypoxia. It is a multifunctional peptide that stimulates angiogenesis and proliferation. The peptide is also a potent paracrine stimulator of osteoblasts and bone formation, suggesting a role in skeletal metastases-a major site of treatment-refractory tumor growth in patients with advanced disease. METHODS: The role of adrenomedullin in bone metastases was tested by stable overexpression in MDA-MB-231 breast cancer cells, which cause osteolytic bone metastases in a standard animal model. Cells with fivefold increased expression of AM were characterized in vitro, inoculated into immunodeficient mice and compared for their ability to form bone metastases versus control subclones. Bone destruction was monitored by X-ray, and tumor burden and osteoclast numbers were determined by quantitative histomorphometry. The effects of AM overexpression on tumor growth and angiogenesis in the mammary fat pad were determined. The effects of AM peptide on osteoclast-like multinucleated cell formation were tested in vitro. A small-molecule AM antagonist was tested for its effects on AM-stimulated ex vivo bone cell cultures and co-cultures with tumor cells, where responses of tumor and bone were distinguished by species-specific real-time PCR. RESULTS: Overexpression of AM mRNA did not alter cell proliferation in vitro, expression of tumor-secreted factors or cell cycle progression. AM-overexpressing cells caused osteolytic bone metastases to develop more rapidly, which was accompanied by decreased survival. In the mammary fat pad, tumors grew more rapidly with unchanged blood vessel formation. Tumor growth in the bone was also more rapid, and osteoclasts were increased. AM peptide potently stimulated bone cultures ex vivo; responses that were blocked by small-molecule adrenomedullin antagonists in the absence of cellular toxicity. Antagonist treatment dramatically suppressed tumor growth in bone and decreased markers of osteoclast activity. CONCLUSIONS: The results identify AM as a target for therapeutic intervention against bone metastases. Adrenomedullin potentiates osteolytic responses in bone to metastatic breast cancer cells. Small-molecule antagonists can effectively block bone-mediated responses to tumor-secreted adrenomedullin, and such agents warrant development for testing in vivo.

Role of bone-anabolic agents in the treatment of breast cancer bone metastases.

Skeletal metastases are an incurable complication afflicting the majority of patients who die from advanced breast cancer. They are most often osteolytic, characterized by net bone destruction and suppressed new bone formation. Life expectancy from first diagnosis of breast cancer bone metastases is several years, during which time skeletal-related events - including pain, fracture, hypercalcemia, and spinal cord compression - significantly degrade quality of life. The bone marrow niche can also confer hormonal and chemo-resistance. Most treatments for skeletal metastases target bone-destroying osteoclasts and are palliative. Recent results from the Breast cancer trials of Oral Everolimus-2 trial suggest that agents such as the mammalian target of rapamycin inhibitor everolimus may have efficacy against breast cancer bone metastases in part via stimulating osteoblasts as well as by inhibiting tumor growth. Selective estrogen receptor modulators similarly inhibit growth of estrogen receptor-positive breast cancers while having positive effects on the skeleton. This review discusses the future role of bone-anabolic agents for the specific treatment of osteolytic breast cancer metastases. Agents with both anti-tumor and bone-anabolic actions have been tested in the setting of multiple myeloma, a hematological malignancy that causes severe osteolytic bone loss and suppression of osteoblastic new bone formation. Stimulation of osteoblast activity inhibits multiple myeloma growth - a strategy that might decrease breast cancer burden in osteolytic bone metastases. Proteasome inhibitors (bortezomib and carfilzomib) inhibit the growth of myeloma directly and are anabolic for bone. Drugs with limited anti-tumor activity but which are anabolic for bone include intermittent parathyroid hormone and antibodies that neutralize the WNT inhibitors DKK1 and sclerostin, as well as the activin A blocker sotatercept and the osteoporosis drug strontium ranelate. Transforming growth factor-beta inhibitors have little tumor antiproliferative activity but block breast cancer production of osteolytic factors and are also anabolic for bone. Some of these treatments are already in clinical trials. This review provides an overview of agents with bone-anabolic properties, which may have utility in the treatment of breast cancer metastatic to the skeleton.

Halofuginone inhibits the establishment and progression of melanoma bone metastases.

TGF-ß derived from bone fuels melanoma bone metastases by inducing tumor secretion of prometastatic factors that act on bone cells to change the skeletal microenvironment. Halofuginone is a plant alkaloid derivative that blocks TGF-ß signaling with antiangiogenic and antiproliferative properties. Here, we show for the first time that halofuginone therapy decreases development and progression of bone metastasis caused by melanoma cells through the inhibition of TGF-ß signaling. Halofuginone treatment of human melanoma cells inhibited cell proliferation, phosphorylation of SMAD proteins in response to TGF-ß, and TGF-ß-induced SMAD-driven transcription. In addition, halofuginone reduced expression of TGF-ß target genes that enhance bone metastases, including PTHrP, CTGF, CXCR4, and IL11. Also, cell apoptosis was increased in response to halofuginone. In nude mice inoculated with 1205 Lu melanoma cells, a preventive protocol with halofuginone inhibited bone metastasis. The beneficial effects of halofuginone treatment were comparable with those observed with other anti-TGF-ß strategies, including systemic administration of SD208, a small-molecule inhibitor of TGF-ß receptor I kinase, or forced overexpression of Smad7, a negative regulator of TGF-ß signaling. Furthermore, mice with established bone metastases treated with halofuginone had significantly less osteolysis than mice receiving placebo assessed by radiography. Thus, halofuginone is also effective in reducing the progression of melanoma bone metastases. Moreover, halofuginone treatment reduced melanoma metastasis to the brain, showing the potential of this novel treatment against cancer metastasis.

The stem cell niche as a pharmaceutical target for prevention of skeletal metastases.

Advanced cancers of the prostate and breast commonly progress by metastasizing to the skeleton, where they are incurable but cause serious morbidity and contribute to mortality. Growth of tumor in bone takes several years, opening a large window for pharmaceutical prevention of metastatic progression. Bone provides a unique microenvironment for tumor growth, including niches occupied by hematopoietic and mesenchymal stem cells. Recent data suggest that circulating tumor cells usurp these niches and compete with the normal stem cell occupants. Agents that encourage normal hematopoiesis or bone formation could inhibit colonization of bone by tumor stem cells and prevent or delay metastatic progression. It may be possible to develop high-throughput assays to test compounds for their ability to suppress tumor stem cell occupation of skeletal niches, thus decreasing metastatic progression in at-risk patients.

Regulation of postnatal trabecular bone formation by the osteoblast endothelin A receptor.

Endothelin-1 (ET-1) is a potent vasoconstrictor that also stimulates cells in the osteoblast lineage by binding to the endothelin A receptor (ETAR). ET-1 ligand is widely secreted, particularly by the vasculature. However, the contributions of ETAR signaling to adult bone homeostasis have not been defined. ETAR was inactivated in osteoblasts by crossing ETAR-floxed and osteocalcin-Cre mice. Histomorphometric analyses were performed on 4-, 8-, and 12-week-old osteoblast-targeted ETAR knockout (KO) and wild-type (WT) male and female mice. Tibial trabecular bone volume was significantly lower from 12 weeks in KO versus WT mice in both males and females. Bone-formation rate, osteoblast density, and in vitro osteoblast differentiation were reduced by targeted inactivation of ETAR. A separate longitudinal analysis was performed between 8 and 64 weeks to examine the effect of aging and castration on bone metabolism in ETAR KO mice. Hypogonadism did not change the rate of bone accrual in WT or KO females. However, eugonadal KO males had a significantly larger increase in tibial and femoral bone acquisition than WT mice. Male mice castrated at 8 weeks of age showed the reverse: KO mice had reduced rates of tibial and femoral BMD acquisition compared with WT mice. In vitro, ET-1 increased osteoblast proliferation, survival, and differentiation. Dihydrotestosterone also increased osteoblast differentiation using a mechanism distinct from the actions of ET-1. These results demonstrate that endothelin signaling in osteoblasts is an important regulator of postnatal trabecular bone remodeling and a modulator of androgen effects on bone.

Agents targeting prostate cancer bone metastasis.

Bone is the most common site for metastasis of advanced prostate cancers. Once housed in the skeleton, tumors are incurable and cause protracted morbidity, and bone metastases may contribute to mortality through unknown mechanisms. Bone provides a unique microenvironment whose local interactions with tumor cells offer novel targets for therapeutic interventions. Many standard cancer treatments cause bone loss, which may aggravate skeletal metastases, although this is preventable with approved agents. Improved bone-targeted treatments can decrease the serious skeletal morbidities associated with metastatic prostate cancer and may in the future improve overall survival. The development of such treatments requires preclinical evaluation in animal models of prostate cancer growth in bone.

Hypoxia and TGF-beta drive breast cancer bone metastases through parallel signaling pathways in tumor cells and the bone microenvironment.

BACKGROUND: Most patients with advanced breast cancer develop bone metastases, which cause pain, hypercalcemia, fractures, nerve compression and paralysis. Chemotherapy causes further bone loss, and bone-specific treatments are only palliative. Multiple tumor-secreted factors act on the bone microenvironment to drive a feed-forward cycle of tumor growth. Effective treatment requires inhibiting upstream regulators of groups of prometastatic factors. Two central regulators are hypoxia and transforming growth factor (TGF)- beta. We asked whether hypoxia (via HIF-1alpha) and TGF-beta signaling promote bone metastases independently or synergistically, and we tested molecular versus pharmacological inhibition strategies in an animal model. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed interactions between HIF-1alpha and TGF-beta pathways in MDA-MB-231 breast cancer cells. Only vascular endothelial growth factor (VEGF) and the CXC chemokine receptor 4 (CXCR4), of 16 genes tested, were additively increased by both TGF-beta and hypoxia, with effects on the proximal promoters. We inhibited HIF-1alpha and TGF-beta pathways in tumor cells by shRNA and dominant negative receptor approaches. Inhibition of either pathway decreased bone metastasis, with no further effect of double blockade. We tested pharmacologic inhibitors of the pathways, which target both the tumor and the bone microenvironment. Unlike molecular blockade, combined drug treatment decreased bone metastases more than either alone, with effects on bone to decrease osteoclastic bone resorption and increase osteoblast activity, in addition to actions on tumor cells. CONCLUSIONS/SIGNIFICANCE: Hypoxia and TGF-beta signaling in parallel drive tumor bone metastases and regulate a common set of tumor genes. In contrast, small molecule inhibitors, by acting on both tumor cells and the bone microenvironment, additively decrease tumor burden, while improving skeletal quality. Our studies suggest that inhibitors of HIF-1alpha and TGF-beta may improve treatment of bone metastases and increase survival.

Assessing new bone formation in neonatal calvarial organ cultures.

Bone formation is a complex process, and testing anabolic effects on the skeleton of agents is slow and expensive in animals. Neonatal mouse calvariae cultured ex vivo show strong anabolic or catabolic bone responses to 1-week treatments and can be analyzed by quantitative histomorphometry. Changes in new bone area and osteoblast number caused by added proteins, drugs, or transfected genes can be quantified and analyzed for statistical significance. The organ cultures preserve much of the skeletal architecture and cellular diversity present in vivo and offer greater physiological relevance than cell lines studied in vitro.

Intracellular proadrenomedullin-derived peptides decorate the microtubules and contribute to cytoskeleton function.

Adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP) are secretory hormones, but it is not unusual to find them in intracellular compartments. Using yeast-2 hybrid technology, we found interactions between AM and several microtubule-associated proteins (MAPs), and between PAMP and tubulin. Expression of fluorescent-tagged AM and PAMP as well as immunofluorescence for the native peptides showed a complete decoration of the microtubules and colocalization with other MAPs. PAMP, but not AM, bound to tubulin in vitro and destabilized tubulin polymerization. Down-regulation of the gene coding for both AM and PAMP through small interfering RNA technology resulted in morphological changes, microtubule stabilization, increase in posttranslational modifications of tubulin such as acetylation and detyrosination, reduction in cell motility, and partial arrest at the G2 phase of the cell cycle, when compared with cells transfected with the same vector carrying a scrambled sequence. These results show that PAMP is a novel MAP, whereas AM may be exerting more subtle effects in regulating cytoskeleton function.

Molecular biology of bone metastasis.

Metastasis is a final stage of tumor progression. Breast and prostate cancer cells preferentially metastasize to bone, wherein they cause incurable osteolytic and osteoblastic lesions. The bone matrix is rich in factors, such as transforming growth factor-beta and insulin-like growth factors, which are released into the tumor microenvironment by osteolysis. These factors stimulate the growth of tumor cells and alter their phenotype, thus promoting a vicious cycle of metastasis and bone pathology. Physical factors within the bone microenvironment, including low oxygen levels, acidic pH, and high extracellular calcium concentrations, may also enhance tumor growth. These elements of the microenvironment are potential targets for chemotherapeutic intervention to halt tumor growth and suppress bone metastasis.