Ganwei Lu

Annexin II/Annexin II Receptor Axis Regulates Adhesion, Migration, Homing, and Growth of Prostate Cancer

One of the most life‐threatening complications of prostate cancer is skeletal metastasis. In order to develop treatment for metastasis, it is important to understand its molecular mechanisms. Our work in this field has drawn parallels between hematopoietic stem cell and prostate cancer homing to the marrow. Our recent work demonstrated that annexin II expressed by osteoblasts and endothelial cells plays a critical role in niche selection. In this study, we demonstrate that annexin II and its receptor play a crucial role in establishing metastasis of prostate cancer. Prostate cancer cell lines migrate toward annexin II and the adhesion of prostate cancer to osteoblasts and endothelial cells was inhibited by annexin II. By blocking annexin II or its receptor in animal models, short‐term and long‐term localization of prostate cancers are limited. Annexin II may also facilitate the growth of prostate cancer in vitro and in vivo by the MAPK pathway. These data strongly suggest that annexin II and its receptor axis plays a central role in prostate cancer metastasis, and that prostate cancer utilize the hematopoietic stem cell homing mechanisms to gain access to the niche. J. Cell. Biochem. 105: 370–380, 2008.

Combination Mammalian Target of Rapamycin Inhibitor Rapamycin and HSP90 Inhibitor 17-Allylamino-17-Demethoxygeldanamycin Has Synergistic Activity in Multiple Myeloma

Purpose: The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (mTOR) pathway and the heat shock protein family are up-regulated in multiple myeloma and are both regulators of the cyclin D/retinoblastoma pathway, a critical pathway in multiple myeloma. Inhibitors of mTOR and HSP90 protein have showed in vitro and in vivo single-agent activity in multiple myeloma. Our objective was to determine the effects of the mTOR inhibitor rapamycin and the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) on multiple myeloma cells. Experimental Design: Multiple myeloma cell lines were incubated with rapamycin (0.1-100 nmol/L) and 17-AAG (100-600 nmol/L) alone and in combination. Results: In this study, we showed that the combination of rapamycin and 17-AAG synergistically inhibited proliferation, induced apoptosis and cell cycle arrest, induced cleavage of poly(ADP-ribose) polymerase and caspase-8/caspase-9, and dysregulated signaling in the phosphatidylinositol 3-kinase/AKT/mTOR and cyclin D1/retinoblastoma pathways. In addition, we showed that both 17-AAG and rapamycin inhibited angiogenesis and osteoclast formation, indicating that these agents target not only multiple myeloma cells but also the bone marrow microenvironment. Conclusions: These studies provide the basis for potential clinical evaluation of this combination for multiple myeloma patients.

Alpha9beta1: a novel osteoclast integrin that regulates osteoclast formation and function.

We identified a previously unknown integrin, α9β1, on OCLs and their precursors. Antibody to α9 inhibited OCL formation in human marrow cultures, and OCLs from α9 knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity.

α9β1: A Novel Osteoclast Integrin That Regulates Osteoclast Formation and Function

We identified a previously unknown integrin, α9β1, on OCLs and their precursors. Antibody to α9 inhibited OCL formation in human marrow cultures, and OCLs from α9 knockout mice had a defect in actin ring reorganization and an impaired bone resorption capacity.

Proteomic Analysis of Waldenstrom Macroglobulinemia

To better understand the molecular changes that occur in Waldenstrom macroglobulinemia (WM), we employed antibody-based protein microarrays to compare patterns of protein expression between untreated WM and normal bone marrow controls. Protein expression was defined as a >2-fold or 1.3-fold change in at least 67% of the tumor samples. Proteins up-regulated by >2-fold included Ras family proteins, such as Rab-4 and p62DOK, and Rho family proteins, such as CDC42GAP and ROKalpha. Other proteins up-regulated by >1.3-fold included cyclin-dependent kinases, apoptosis regulators, and histone deacetylases (HDAC). We then compared the samples of patients with symptomatic and asymptomatic WM and showed similar protein expression signatures, indicating that the dysregulation of signaling pathways occurs early in the disease course. Three proteins were different by >2-fold in symptomatic versus asymptomatic, including the heat shock protein HSP90. Elevated protein expression was confirmed by immunohistochemistry and immunoblotting. Functional significance was validated by the induction of apoptosis and inhibition of proliferation using specific HDAC and HSP90 inhibitors. This study, therefore, identifies, for the first time, multiple novel proteins that are dysregulated in WM, which both enhance our understanding of disease pathogenesis and represent targets of novel therapeutics.

Annexin II stimulates RANKL expression through MAPK

We report that AX‐II, in addition to inducing GM‐CSF expression, also increases membrane‐bound RANKL synthesis by marrow stromal cells and does so through a previously unreported MAPK‐dependent pathway. Thus, both GM‐CSF and RANKL are required for AX‐II stimulation of OCL formation.

Cloning and Characterization of the Annexin II Receptor on Human Marrow Stromal Cells

Annexin II is a heterotetramer, consisting of two 11-kDa (p11) and two 36-kDa (p36) subunits, that is produced by osteoclasts and stimulates osteoclast formation. However, its receptor is unknown. We showed that annexin II binds to normal primary human marrow stromal cells and the Pagets marrow-derived PSV10 stromal cell line to induce osteoclast formation. 125I-Labeled annexin II binding assays with PSV10 cells demonstrated that there was a single class of annexin II receptors with a Kd of 5.79 nm and Bmax of 2.13 × 105 receptors/cell. Annexin III or annexin V did not bind this receptor. Using 125I-labeled annexin II binding to screen NIH3T3 transfected with a human marrow cDNA expression library, we identified a putative annexin II receptor clone, which encoded a novel 26-kDa type I membrane receptor protein when expressed in HEK 293 cells. HEK 293 cells transformed with the cloned annexin II receptor cDNA showed a similar binding affinity to annexin II as that observed in PSV10 cells. Chemical cross-linking experiments with biotinylated annexin II and intact PSV10 cells identified a 55-kDa band on Western blot analysis that reacted with both an anti-p11 antibody and streptavidin but not anti-p36 antibody. A rabbit polyclonal antibody raised against the putative recombinant annexin II receptor also recognized the same 26-kDa protein band detected in PSV10 cells. Importantly, the annexin II receptor antibody dose-dependently blocked the stimulatory effects of annexin II on human osteoclast formation, demonstrating that the receptor mediates the effects of annexin II on osteoclast formation.

ADAM8 enhances osteoclast precursor fusion and osteoclast formation in vitro and in vivo

ADAM8 expression is increased in the interface tissue around a loosened hip prosthesis and in the pannus and synovium of patients with rheumatoid arthritis, but its potential role in these processes is unclear. ADAM8 stimulates osteoclast (OCL) formation, but the effects of overexpression or loss of expression of ADAM8 in vivo and the mechanisms responsible for the effects of ADAM8 on osteoclastogenesis are unknown. Therefore, to determine the effects of modulating ADAM expression, we generated tartrate‐resistant acid phosphatase (TRAP)–ADAM8 transgenic mice that overexpress ADAM8 in the OCL lineage and ADAM8 knockout (ADAM8 KO) mice. TRAP‐ADAM8 mice developed osteopenia and had increased numbers of OCL precursors that formed hypermultinucleated OCLs with an increased bone‐resorbing capacity per OCL. They also had an enhanced differentiation capacity, increased TRAF6 expression, and increased NF‐κB, Erk, and Akt signaling compared with wild‐type (WT) littermates. This increased bone‐resorbing capacity per OCL was associated with increased levels of p‐Pyk2 and p‐Src activation. In contrast, ADAM8 KO mice did not display a bone phenotype in vivo, but unlike WT littermates, they did not increase RANKL production, OCL formation, or calvarial fibrosis in response to tumor necrosis factor α (TNF‐α) in vivo. Since loss of ADAM8 does not inhibit basal bone remodeling but only blocks the enhanced OCL formation in response to TNF‐α, these results suggest that ADAM8 may be an attractive therapeutic target for preventing bone destruction associated with inflammatory disease.