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

XRK3F2 inhibits p62 signaling and augments myeloma killing by proteasome inhibitors

Background: Despite advancements in therapy, multiple myeloma (MM) remains an incurable blood cancer. Our mission is to maximize the efficacy of a primary treatment for myeloma, proteasome inhibitors (PIs) which cause intracellular waste buildup, leading to ER stress and cell death. p62(sequestosome-1) provides an alternate pathway when the proteasome is inhibited, by breaking down cytotoxic material via autophagy. Upregulation of p62 is associated with PI resistance. We identified a small molecule, XRK3F2, that binds to the ZZ domain of p62 and inhibits its autophagic function. We hypothesized that XRK3F2 would improve MM killing when combined with PIs. Methods Used: We tested XRK3F2 and PI combinations in vitro, in ex vivo co-cultures, and in a human MM xenograft model. We tested XRK3F2 plus bortezomib in vitro and in ex vivo myeloma: bone cocultures and analyzed effects on tumor burden in a prior mouse xenograft experiment. Results: XRK3F2 induced cell death in various human MM cell lines, with a IC50s of 3-6 M. When combined with carfilzomib, the most potent approved PI, at physiologically relevant doses, there was strong synergy (Combinatorial index of 0.4 to 0.6, by Chou-Talalay analysis). The combination of the two agents significantly increased tumor killing in a tumor: bone co-culture model, where the microenvironment of the tumor provides MM survival signals and potential drug resistance. Enhanced tumor killing was further confirmed in a plasmacytoma model of the human MM cell line RPMI-8226 in NSG mice. We also identified soluble BCMA (B-cell maturation antigen, sBCMA) as a sensitive biomarker for tumor burden, which allowed for serial tumor measurements in all tested models. Conclusion and Potential Impact: Combining the p62-ZZ domain inhibitor XRK3F2 with PIs shows great promise in improving the killing of MM. Work is ongoing to validate the combination in xenograft models, where tumor cells colonize bones, and in immunocompetent models. Further mechanistic studies using primary MM cells from patients are also ongoing. sBCMA is a cheap, specific, and sensitive tool for serial tumor measurement and should be further validated for preclinical and clinical usage

IMMUNOHISTOCHEMISTRY EXPRESSION OF KLOTHO IN BONE MARROW BIOPSIES FROM NORMAL, MGUS, AND PLASMA CELL MYELOMA

poster abstractKlotho is an anti-aging gene, which has been shown to inhibit the insulin and insulin-like growth factor 1 (IGF-1) pathways in mice hepatocytes and myocytes. Immunochemistry analysis of Klotho expression in breast tissue arrays revealed high expression in normal breast, but very low expression in breast cancer. In this study we examined eight normal bone marrow, eight MGUS (monoclonal gammopathy of undetermined significance), and forty-two cases of plasma cell myeloma by immunohistochemistry with the Klotho antibody. The immunostaining of the Klotho antibody was localized in the cyto-plasm and as punctate granular staining of myeloma cells in the marrow. In the accompanying bone marrow clots, Klotho was seen as strong punctate granules on myeloma cells and not on other peripheral white blood cells. There was no staining of plasma cells in the eight normal bone marrow cas-es. Slight cytoplasmic staining was seen in myeloid series of cells in the normal bone marrow and in megakaryocytes. In the eight MGUS cases, there was very minimal cytoplasmic staining in a few of the myeloma cells. Minimal staining was seen in the myeloid series of cells in the marrow in these cases. Klotho was highly expressed in the myeloma cases and no staining in the normal and MGUS cases. In conclusion, Klotho was highly expressed in patients with myeloma in myelomas cells in the bone marrow. This project was sponsored by the Life Health Science Internship Progra

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

Multifocal Gastric Ulcers Caused by Diffuse Large B Cell Lymphoma in a Patient With Significant Weight Loss

Primary gastrointestinal (GI) lymphoma is a heterogeneous disease with varied clinical presentations. The stomach is the most common GI site and accounts for 70% to 75% of GI lymphomas. We present a patient with gastric diffuse large B cell lymphoma (DLBCL) who presented with significant weight loss, early satiety, and multifocal ulcerated gastric lesions. Esophagoduodenoscopy should be performed in patients presenting with warning symptoms as in our case. Diagnosis is usually made by endoscopic biopsies. Multiple treatment modalities including surgery, radiotherapy, and chemotherapy have been used. Advancements in endoscopic and pathologic technology decrease turnaround time for diagnosis and treatment initiation, thus reducing the need for surgery. Health care providers should maintain a high level of suspicion and consider gastric DLBCL as part of the differential diagnosis, especially in those with warning symptoms such as weight loss and early satiety with abnormal endoscopic findings

The novel histone deacetylase inhibitor, AR-42, inhibits gp130/Stat3 pathway and induces apoptosis and cell cycle arrest in multiple myeloma cells

Multiple myeloma (MM) remains incurable with current therapy, indicating the need for continued development of novel therapeutic agents. We evaluated the activity of a novel phenylbutyrate-derived histone deacetylase inhibitor, AR-42, in primary human myeloma cells and cell lines. AR-42 was cytotoxic to MM cells at a mean LC(50) of 0.18 ± 0.06 μmol/l at 48 hr and induced apoptosis with cleavage of caspases 8, 9 and 3, with cell death largely prevented by caspase inhibition. AR-42 downregulated the expression of gp130 and inhibited activation of STAT3, with minimal effects on the PI3K/Akt and MAPK pathways, indicating a predominant effect on the gp130/STAT-3 pathway. AR-42 also inhibited interleukin (IL)-6-induced STAT3 activation, which could not be overcome by exogenous IL-6. AR-42 also downregulated the expression of STAT3-regulated targets, including Bcl-xL and cyclin D1. Overexpression of Bcl-xL by a lentivirus construct partly protected against cell death induced by AR-42. The cyclin dependent kinase inhibitors, p16 and p21, were also significantly induced by AR-42, which together with a decrease in cyclin D1, resulted in G(1) and G(2) cell cycle arrest. In conclusion, AR-42 has potent cytotoxicity against MM cells mainly through gp130/STAT-3 pathway. The results provide rationale for clinical investigation of AR-42 in MM

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

Cell adhesion molecule CD166 drives malignant progression and osteolytic disease in multiple myeloma

Multiple myeloma (MM) is incurable once osteolytic lesions have seeded at skeletal sites, but factors mediating this deadly pathogenic advance remain poorly understood. Here we report evidence of a major role for the cell adhesion molecule CD166, which we discovered to be highly expressed in MM cell lines and primary bone marrow (BM) cells from patients. CD166+ MM cells homed more efficiently than CD166− cells to the BM of engrafted immunodeficient NSG mice. CD166 silencing in MM cells enabled longer survival, a smaller tumor burden and less osteolytic lesions, as compared to mice bearing control cells. CD166 deficiency in MM cell lines or CD138+ BM cells from MM patients compromised their ability to induce bone resorption in an ex vivo organ culture system. Further, CD166 deficiency in MM cells also reduced formation of osteolytic disease in vivo after intra-tibial engraftment. Mechanistic investigation revealed that CD166 expression in MM cells inhibited osteoblastogenesis of BM-derived osteoblast progenitors by suppressing RUNX2 gene expression. Conversely, CD166 expression in MM cells promoted osteoclastogenesis by activating TRAF6-dependent signaling pathways in osteoclast progenitors. Overall, our results define CD166 as a pivotal director in MM cell homing to the BM and MM progression, rationalizing its further study as a candidate therapeutic target for MM treatment