Receptor activator of NF-κB (RANK)-mediated induction of metastatic spread and association with poor prognosis in renal cell carcinoma.

BACKGROUND: Inhibition of the receptor activator of NF-κB ligand (RANKL) has become a standard of care supportive treatment to prevent skeletal related events in cancer patients. Moreover, RANKL inhibition has been implicated with better survival outcome in lung cancer, while RANKL expression induces tumor progression and metastatic spread in vivo in breast cancer. Whether RANK/RANKL may have an impact on the pathogenesis of clear cell renal cell carcinoma (ccRCC) is currently unknown. PATIENTS AND METHODS: A retrospective tissue micro array (TMA)-study was carried out determining the expression of RANK/RANKL in primary tumors of 306 ccRCC patients. Additionally, 24 ccRCC cell lines were employed for in vitro analyses of the RANK/RANKL axis including cell proliferation, migration and anchorage independent growth. RESULTS: RANK (+) vs. RANK (-) tumors had both worse cancer specific survival (CSS) (6.3 vs. 1.3 years; p < 0.001) and recurrence free survival (RFS) (9.9 vs. 5.8 years; p < 0.001). RANK (+) (HR 2.21; p < 0.001) was an independent prognostic factor for CSS and RFS (HR 4.98; p < 0.001). RANKL treatment resulted in increased proliferation, soft agar growth, and colony formation of RANK (+) RCC cell lines, which could be reversed by treatment with an NF-κB inhibitor and with a combination of osteoprotegrin and RANKL in vitro. CONCLUSIONS: RANK is expressed in ccRCC tissue, correlates with clinicopathological features, survival outcome, and when stimulated with RANKL can induce ccRCC progression in vitro. Consequently, RANKL inhibition combined with standard of care treatment may be a promising approach to improve ccRCC patient's survival.

RANK rewires energy homeostasis in lung cancer cells and drives primary lung cancer.

Lung cancer is the leading cause of cancer deaths. Besides smoking, epidemiological studies have linked female sex hormones to lung cancer in women; however, the underlying mechanisms remain unclear. Here we report that the receptor activator of nuclear factor-kB (RANK), the key regulator of osteoclastogenesis, is frequently expressed in primary lung tumors, an active RANK pathway correlates with decreased survival, and pharmacologic RANK inhibition reduces tumor growth in patient-derived lung cancer xenografts. Clonal genetic inactivation of KRasG12D in mouse lung epithelial cells markedly impairs the progression of KRasG12D -driven lung cancer, resulting in a significant survival advantage. Mechanistically, RANK rewires energy homeostasis in human and murine lung cancer cells and promotes expansion of lung cancer stem-like cells, which is blocked by inhibiting mitochondrial respiration. Our data also indicate survival differences in KRasG12D -driven lung cancer between male and female mice, and we show that female sex hormones can promote lung cancer progression via the RANK pathway. These data uncover a direct role for RANK in lung cancer and may explain why female sex hormones accelerate lung cancer development. Inhibition of RANK using the approved drug denosumab may be a therapeutic drug candidate for primary lung cancer.

Poor prognosis of patients with triple-negative breast cancer can be stratified by RANK and RANKL dual expression.

PURPOSE: As clinical studies have correlated RANK expression levels with survival in breast cancer, and that RANK signaling is dependent on its cognate ligand RANKL, we hypothesized that dual protein expression further stratifies the poor outcome in TNBC. METHODS: RANK mRNA and protein expression was evaluated in TNBC using genomic databases, cell lines and in a tissue microarray of curated primary tumor samples derived from 87 patients with TNBC. RANK expression was evaluated either by Mann-Whitney U test on log-normalized gene expression data or by Student's t test on FACS data. Analysis of RANK and RANKL immunostaining was calculated by H-score, and correlations to clinical factors performed using χ 2 or Fisher's exact test. Associations with RFS and OS were assessed using univariate and multivariate Cox proportional hazard models. Survival estimates were generated using the Kaplan-Meier method. RESULTS: In three distinct datasets spanning 684 samples, RANK mRNA expression was higher in primary tumors derived from TNBC patients than from those with other molecular subtypes (P < 0.01). Cell surface-localized RANK protein was consistently higher in TNBC cell lines (P = 0.037). In clinical samples, TNBC patients that expressed both RANK and RANKL proteins had significantly worse RFS (P = 0.0032) and OS (P = 0.004) than patients with RANK-positive, RANKL-negative tumors. RANKL was an independent, poor prognostic factor for RFS (P = 0.04) and OS (P = 0.01) in multivariate analysis in samples that expressed both RANK and RANKL. CONCLUSIONS: RANK and RANKL co-expression is associated with poor RFS and OS in patients with TNBC.

Distribution of RANK and RANK Ligand in Normal Human Tissues as Determined by an Optimized Immunohistochemical Method.

The expression and tissue distribution of RANK (Receptor Activator of Nuclear Factor κ B) and RANK Ligand (RANKL) are of critical interest in relation to efficacy and safety of antibodies against RANK or RANKL that are approved or under consideration as potential therapeutic agents. Data from the literature using protein or mRNA analyses of rodent and human tissues or immunohistochemical (IHC) studies with a variety of antibodies and methods have provided some background of the distribution of RANK and RANKL but have yielded inconsistent findings. The present study reports the generation of carefully validated antibodies to RANK and RANKL and the development of an optimized IHC method, with confirmatory data from 2 well-validated alternative protocols that were developed and performed in separate laboratories at USC and at Amgen. Tissue expression of RANK and RANKL is reported for the optimized IHC assay.

RANK ligand as a potential target for breast cancer prevention in BRCA1-mutation carriers.

Individuals who have mutations in the breast-cancer-susceptibility gene BRCA1 (hereafter referred to as BRCA1-mutation carriers) frequently undergo prophylactic mastectomy to minimize their risk of breast cancer. The identification of an effective prevention therapy therefore remains a 'holy grail' for the field. Precancerous BRCA1(mut/+) tissue harbors an aberrant population of luminal progenitor cells, and deregulated progesterone signaling has been implicated in BRCA1-associated oncogenesis. Coupled with the findings that tumor necrosis factor superfamily member 11 (TNFSF11; also known as RANKL) is a key paracrine effector of progesterone signaling and that RANKL and its receptor TNFRSF11A (also known as RANK) contribute to mammary tumorigenesis, we investigated a role for this pathway in the pre-neoplastic phase of BRCA1-mutation carriers. We identified two subsets of luminal progenitors (RANK(+) and RANK(-)) in histologically normal tissue of BRCA1-mutation carriers and showed that RANK(+) cells are highly proliferative, have grossly aberrant DNA repair and bear a molecular signature similar to that of basal-like breast cancer. These data suggest that RANK(+) and not RANK(-) progenitors are a key target population in these women. Inhibition of RANKL signaling by treatment with denosumab in three-dimensional breast organoids derived from pre-neoplastic BRCA1(mut/+) tissue attenuated progesterone-induced proliferation. Notably, proliferation was markedly reduced in breast biopsies from BRCA1-mutation carriers who were treated with denosumab. Furthermore, inhibition of RANKL in a Brca1-deficient mouse model substantially curtailed mammary tumorigenesis. Taken together, these findings identify a targetable pathway in a putative cell-of-origin population in BRCA1-mutation carriers and implicate RANKL blockade as a promising strategy in the prevention of breast cancer.

RANK-ligand (RANKL) expression in young breast cancer patients and during pregnancy.

INTRODUCTION: RANKL is important in mammary gland development during pregnancy and mediates the initiation and progression of progesterone-induced breast cancer. No clinical data are available on the effect of pregnancy on RANK/RANKL expression in young breast cancer patients. METHODS: We used our previously published dataset of 65 pregnant and 130 matched young breast cancer patients with full clinical, pathological, and survival information. 85% of patients had available transcriptomic data as well. RANK/RANKL expression by immunohistochemistry using H-score on the primary tumor and adjacent normal tissue was performed. We examined the difference in expression of RANK/RANKL between pregnant and non-pregnant patients and their association with clinicopathological features and prognosis. We also evaluated genes and pathways associated with RANK/RANKL expression on primary tumors. RESULTS: RANKL but not RANK expression was more prevalent in the pregnant group, both on the tumor and adjacent normal tissue, independent of other clinicopathological factors (both P <0.001). 18.7% of pregnant and 5.3% of non-pregnant patients had tumors showing ≥10% of cells with 3+ RANKL expression. RANKL expression was significantly higher in progesterone receptor-positive, and luminal A-like tumors, with negative correlation with Ki-67 (all P <0.001). On the contrary, RANK expression was higher in triple negative tumors (P <0.001). Using false discovery rate <0.05, 151 and 1,207 genes were significantly correlated with tumor-expressed RANKL and RANK expression by immunohistochemistry, respectively. High RANKL expression within primary tumor was associated with pathways related to mammary gland development, bone resorption, T-cell proliferation and regulation of chemotaxis, while RANK expression was associated with immune response and proliferation pathways. At a median follow-up of 65 months, neither RANK nor RANKL expression within tumor was associated with disease free survival in pregnant or non-pregnant group. CONCLUSIONS: Pregnancy increases RANKL expression both in normal breast and primary tumors. These results could guide further development of RANKL-targeted therapy.

RANK and RANK ligand expression in primary human osteosarcoma.

Receptor activator of nuclear factor kappa-B ligand (RANKL) is an essential mediator of osteoclast formation, function and survival. In patients with solid tumor metastasis to the bone, targeting the bone microenvironment by inhibition of RANKL using denosumab, a fully human monoclonal antibody (mAb) specific to RANKL, has been demonstrated to prevent tumor-induced osteolysis and subsequent skeletal complications. Recently, a prominent functional role for the RANKL pathway has emerged in the primary bone tumor giant cell tumor of bone (GCTB). Expression of both RANKL and RANK is extremely high in GCTB tumors and denosumab treatment was associated with tumor regression and reduced tumor-associated bone lysis in GCTB patients. In order to address the potential role of the RANKL pathway in another primary bone tumor, this study assessed human RANKL and RANK expression in human primary osteosarcoma (OS) using specific mAbs, validated and optimized for immunohistochemistry (IHC) or flow cytometry. Our results demonstrate RANKL expression was observed in the tumor element in 68% of human OS using IHC. However, the staining intensity was relatively low and only 37% (29/79) of samples exhibited≥10% RANKL positive tumor cells. RANK expression was not observed in OS tumor cells. In contrast, RANK expression was clearly observed in other cells within OS samples, including the myeloid osteoclast precursor compartment, osteoclasts and in giant osteoclast cells. The intensity and frequency of RANKL and RANK staining in OS samples were substantially less than that observed in GCTB samples. The observation that RANKL is expressed in OS cells themselves suggests that these tumors may mediate an osteoclastic response, and anti-RANKL therapy may potentially be protective against bone pathologies in OS. However, the absence of RANK expression in primary human OS cells suggests that any autocrine RANKL/RANK signaling in human OS tumor cells is not operative, and anti-RANKL therapy would not directly affect the tumor.

Intravital imaging reveals distinct responses of depleting dynamic tumor-associated macrophage and dendritic cell subpopulations.

Tumor-infiltrating inflammatory cells comprise a major part of the stromal microenvironment and support cancer progression by multiple mechanisms. High numbers of tumor myeloid cells correlate with poor prognosis in breast cancer and are coupled with the angiogenic switch and malignant progression. However, the specific roles and regulation of heterogeneous tumor myeloid populations are incompletely understood. CSF-1 is a major myeloid cell mitogen, and signaling through its receptor CSF-1R is also linked to poor outcomes. To characterize myeloid cell function in tumors, we combined confocal intravital microscopy with depletion of CSF-1R-dependent cells using a neutralizing CSF-1R antibody in the mouse mammary tumor virus long-terminal region-driven polyoma middle T antigen breast cancer model. The depleted cells shared markers of tumor-associated macrophages and dendritic cells (M-DCs), matching the phenotype of tumor dendritic cells that take up antigens and interact with T cells. We defined functional subgroups within the M-DC population by imaging endocytic and matrix metalloproteinase activity. Anti-CSF-1R treatment altered stromal dynamics and impaired both survival of M-DCs and accumulation of new M-DCs, but did not deplete Gr-1(+) neutrophils or block doxorubicin-induced myeloid cell recruitment, and had a minimal effect on lung myeloid cells. Nevertheless, prolonged treatment led to delayed tumor growth, reduced vascularity, and decreased lung metastasis. Because the myeloid infiltrate in metastatic lungs differed significantly from that in mammary tumors, the reduction in metastasis may result from the impact on primary tumors. The combination of functional analysis by intravital imaging with cellular characterization has refined our understanding of the effects of experimental targeted therapies on the tumor microenvironment.

Apo2L/TRAIL and the death receptor 5 agonist antibody AMG 655 cooperate to promote receptor clustering and antitumor activity.

Death receptor agonist therapies have exhibited limited clinical benefit to date. Investigations into why Apo2L/TRAIL and AMG 655 preclinical data were not predictive of clinical response revealed that coadministration of Apo2L/TRAIL with AMG 655 leads to increased antitumor activity in vitro and in vivo. The combination of Apo2L/TRAIL and AMG 655 results in enhanced signaling and can sensitize Apo2L/TRAIL-resistant cells. Structure determination of the Apo2L/TRAIL-DR5-AMG 655 ternary complex illustrates how higher order clustering of DR5 is achieved when both agents are combined. Enhanced agonism generated by combining Apo2L/TRAIL and AMG 655 provides insight into the limited efficacy observed in previous clinical trials and suggests testable hypotheses to reconsider death receptor agonism as a therapeutic strategy.

RANKL expression in normal and malignant breast tissue responds to progesterone and is up-regulated during the luteal phase.

The receptor activator of nuclear factor-κB ligand (RANKL) acts as a paracrine factor in progesterone-induced mammary epithelial proliferation and tumorigenesis. This evidence comes mainly from mouse models. Our aim was to examine whether RANKL expression in human normal and malignant breast is under the control of progesterone throughout the menstrual cycle. Breast epithelial samples were obtained by random fine needle aspiration (rFNA) of the contralateral unaffected breasts (CUB) of 18 breast cancer patients, with simultaneous serum hormone measurements. Genes correlated with serum progesterone levels were identified through Illumina microarray analysis. Validation was performed using qRT-PCR in rFNA samples from CUB of an additional 53 women and using immunohistochemistry in tissue microarrays of 61 breast cancer samples. Expression of RANKL, DIO2, and MYBPC1 was correlated with serum progesterone in CUB, and was significantly higher in luteal phase. RANKL and MYBPC1 mRNA expression were highly correlated between CUB and matched tumor samples. RANKL protein expression was also significantly increased in the luteal phase and highly correlated with serum progesterone levels in cancer samples, especially in hormone receptor positive tumors. The regulatory effects of progesterone on the expression of RANKL, DIO2, and MYBPC1 were confirmed in three-dimensional cultures of normal breast organoids. In normal breast and in breast cancer, RANKL mRNA and protein expression fluctuate with serum progesterone with highest levels in the luteal phase, suggesting that RANKL is a modulator of progesterone signaling in normal and malignant breast tissue and a potential biomarker of progesterone action and blockade.

RANK expression as a prognostic and predictive marker in breast cancer.

RANK ligand (RANKL) is crucial for the development of mouse mammary glands during pregnancy. RANKL functions as a major paracrine effector of the mitogenic action of progesterone in mammary epithelium via its receptor RANK and has a role in expansion and regenerative potential of mammary stem cells. Pharmacologic inhibition of RANKL attenuates the development of mammary carcinoma and inhibits metastatic progression in multiple mouse models. Primary breast carcinoma samples from the neoadjuvant GeparTrio study were analyzed to correlate the expression of human RANK and RANKL with pathological complete response (pCR), disease-free (DFS), and overall (OS) survival. Pre-treatment FFPE core biopsies (n = 601) were analyzed for percentage and intensity of immunohistochemical RANK and RANKL expression. Antibodies against human RANK (N-1H8; Amgen) and human RANKL (M366; Amgen) were used. RANK protein was expressed in 160 (27 %) patients. Increased RANK expression was observed in 14.5 % of patients and correlated with high tumor grade (p < 0.023) and negative hormone receptor (HR) status (p < 0.001). Patients with high RANK expression showed a higher pCR rate (23.0 % vs. 12.6 %, p = 0.010), shorter DFS (p = 0.038), and OS (p = 0.011). However, prognostic and predictive information was not an independent parameter. Only 6 % of samples expressed RANKL, which was not correlated with any clinical features. Higher RANK expression in the primary tumor is associated with a higher sensitivity to chemotherapy, but also a higher risk of relapse and death. Our study provides a basis for further exploration of the antitumor activity of clinical antibodies against RANKL.

Progestin effects on cell proliferation pathways in the postmenopausal mammary gland.

INTRODUCTION: Menopausal hormone therapies vary widely in their effects on breast cancer risk, and the mechanisms underlying these differences are unclear. The primary goals of this study were to characterize the mammary gland transcriptional profile of estrogen + progestin therapy in comparison with estrogen-alone or tibolone and investigate pathways of cell proliferation in a postmenopausal primate model. METHODS: Ovariectomized female cynomolgus macaque monkeys were randomized into the following groups: placebo (Con), oral conjugated equine estrogens (CEE), CEE with medroxyprogesterone acetate (MPA) (CEE + MPA), and tibolone given at a low or high dose (Lo or Hi Tib). All study treatment doses represented human clinical dose equivalents and were administered in the diet over a period of 2 years. RESULTS: Treatment with CEE + MPA had the greatest effect on global mRNA profiles and markers of mammary gland proliferation compared to CEE or tibolone treatment. Changes in the transcriptional patterns resulting from the addition of MPA to CEE were related to increased growth factors and decreased estrogen receptor (ER) signaling. Specific genes induced by CEE + MPA treatment included key members of prolactin receptor (PRLR)/signal transducer and activator of transcription 5 (STAT5), epidermal growth factor receptor (EGFR), and receptor activator of nuclear factor kappa B (RANK)/receptor activator of nuclear factor kappa B ligand (RANKL) pathways that were highly associated with breast tissue proliferation. In contrast, tibolone did not affect breast tissue proliferation but did elicit a mixed pattern of ER agonist activity. CONCLUSION: Our findings indicate that estrogen + progestin therapy results in a distinct molecular profile compared to estrogen-alone or tibolone therapy, including upregulation of key growth factor targets associated with mammary carcinogenesis in mouse models. These changes may contribute to the promotional effects of estrogen + progestin therapy on breast cancer risk.

RANKL inhibition combined with tamoxifen treatment increases anti-tumor efficacy and prevents tumor-induced bone destruction in an estrogen receptor-positive breast cancer bone metastasis model.

Tumor cells in bone can induce the activation of osteoclasts, which mediate bone resorption and release of growth factors and calcium from the bone matrix, resulting in a cycle of tumor growth and bone breakdown. Targeting the bone microenvironment by the inhibition of RANKL, an essential mediator of osteoclast function, not only prevents tumor-induced osteolysis but also decreases skeletal tumor burden in preclinical models. The inhibition of skeletal tumor progression after the inhibition of osteoclasts is via interruption of the "vicious cycle" of tumor/bone interactions. The majority of breast cancer patients at risk for bone metastases harbor estrogen receptor-positive (ER+) tumors. We developed a mouse model for ER+ breast cancer bone metastasis and evaluated the effect of RANKL inhibition on tumor-induced osteolysis and skeletal tumor growth both alone and in combination with tamoxifen. Luciferase-labeled MCF-7 cells (MCF-7Luc) formed metastatic foci in the hind limbs following intracardiac injection and caused mixed osteolytic/osteoblastic lesions. RANKL inhibition by OPG-Fc treatment blocked osteoclast activity and prevented tumor-induced osteolysis, as well as caused a marked decrease in skeletal tumor burden. Tamoxifen as a single agent reduced MCF-7Luc tumor growth in the hind limbs. In a combination experiment, OPG-Fc plus tamoxifen resulted in significantly greater tumor growth inhibition than either single agent alone. Histologic analysis revealed a decrease in the proliferation of tumor cells by both single agents, which was enhanced in the combination treatment. Upon treatment with OPG-Fc alone or in combination with tamoxifen, there was a complete absence of osteolytic lesions, demonstrating the ability of RANKL inhibition to prevent skeletal related morbidity in an ER+ model. The combination approach of targeting osteoclasts and the bone microenvironment by RANKL inhibition and the tumor directly via hormonal therapy may provide additional benefit to reducing skeletal tumor progression in ER+ breast cancer patients.

Denosumab induces tumor reduction and bone formation in patients with giant-cell tumor of bone.

PURPOSE: Giant-cell tumor of bone (GCTB) is a locally aggressive, benign osteolytic tumor in which bone destruction is mediated by RANK ligand (RANKL). The RANKL inhibitor denosumab is being investigated for treatment of GCTB. We describe histologic analyses of GCTB tumor samples from a phase II study of denosumab in GCTB. EXPERIMENTAL DESIGN: Adult patients with recurrent or unresectable GCTB received subcutaneous denosumab 120 mg every 4 weeks (with additional doses on days 8 and 15). The primary histologic efficacy endpoint was the proportion of patients who had a 90% or more elimination of giant cells from their tumor. Baseline and on-study specimens were also evaluated for overall tumor morphology and expression of RANK and RANKL. RESULTS: Baseline tumor samples were typically composed of densely cellular proliferative RANKL-positive tumor stromal cells, RANK-positive rounded mononuclear cells, abundant RANK-positive tumor giant cells, and areas of scant de novo osteoid matrix and woven bone. In on-study samples from 20 of 20 patients (100%), a decrease of 90% or more in tumor giant cells and a reduction in tumor stromal cells were observed. In these analyses, thirteen patients (65%) had an increased proportion of dense fibro-osseous tissue and/or new woven bone, replacing areas of proliferative RANKL-positive stromal cells. CONCLUSIONS: Denosumab treatment of patients with GCTB significantly reduced or eliminated RANK-positive tumor giant cells. Denosumab also reduced the relative content of proliferative, densely cellular tumor stromal cells, replacing them with nonproliferative, differentiated, densely woven new bone. Denosumab continues to be studied as a potential treatment for GCTB.

Conatumumab, a fully human agonist antibody to death receptor 5, induces apoptosis via caspase activation in multiple tumor types.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4 and 5 (DR4, DR5) to transduce apoptotic signals. Conatumumab (AMG 655) is an investigational, fully human monoclonal agonist antibody (IgG(1)) to human DR5, which induces apoptosis via caspase activation. In this study, we demonstrate that conatumumab binds to DR5, activating intracellular caspases in vitro in the presence of a cross-linker. We also show that conatumumab has activity in vivo and inhibits tumor growth in colon (Colo205 and HCT-15), lung (H2122) and pancreatic (MiaPaCa2/T2) xenograft models. Conatumumab also enhances the antitumor activity of chemotherapeutics in vivo. Caspase activation in Colo205 tumors is dose-dependent and correlated with serum concentrations of conatumumab. We demonstrate for the first time that increases in serum caspase-3/7 activity and levels of M30 (neoepitope of caspase-cleaved cytokeratin-18) are linked to activation of the extrinsic apoptotic pathway using conatumumab in a preclinical model. These data suggest that conatumumab has potential as a therapeutic agent for treating patients with multiple tumor types, and that serum caspase-3/7 and M30 levels may serve as biomarkers of conatumumab activity.

Safety, pharmacokinetics, and pharmacodynamics of AMG 102, a fully human hepatocyte growth factor-neutralizing monoclonal antibody, in a first-in-human study of patients with advanced solid tumors.

PURPOSE: The aims were to assess the safety, pharmacokinetics, maximum tolerated dose, and antitumor activity of AMG 102, a fully human hepatocyte growth factor/scatter factor (HGF/SF)-neutralizing monoclonal antibody, in patients with solid tumors. EXPERIMENTAL DESIGN: Patients (N = 40) with refractory advanced solid tumors were enrolled into six sequential dose-escalation cohorts (0.5, 1, 3, 5, 10, or 20 mg/kg AMG 102 i.v. every 2 weeks) and a dose-expansion cohort (20 mg/kg AMG 102 every 2 weeks). Safety, anti-AMG 102 antibody formation, pharmacokinetics, tumor response, and exploratory biomarkers were assessed. RESULTS: AMG 102 was well tolerated up to the planned maximum dose of 20 mg/kg, and the maximum tolerated dose was not reached. Treatment-related adverse events were generally mild and included fatigue (13%), constipation (8%), nausea (8%), vomiting (5%), anorexia (5%), myalgia (5%), and hypertension (5%). Two patients experienced dose-limiting toxicities: one patient (0.5 mg/kg cohort) experienced grade 3 hypoxia and grade 3 dyspnea and one patient (1 mg/kg cohort) experienced grade 3 upper gastrointestinal hemorrhage. No anti-AMG 102 antibodies were detected, and AMG 102 had linear pharmacokinetics within the dose range investigated. Sixteen of 23 (70%) evaluable patients had a best response of stable disease with progression-free survival ranging from 7.9 to 40 weeks. Circulating levels of the biomarker HGF/SF (bound and unbound) increased in a dose-dependent manner, whereas soluble c-Met concentrations were generally similar across doses. CONCLUSIONS: AMG 102 is safe and well tolerated, has a favorable pharmacokinetic profile, and will be further investigated as a monotherapy and in combination with other agents.

Receptor activator of NF-kappa B ligand inhibition suppresses bone resorption and hypercalcemia but does not affect host immune responses to influenza infection.

Receptor activator of NF-kappaB (RANK) and its ligand (RANKL) are essential for osteoclast formation, function, and survival. Osteoprotegerin (OPG) inhibits RANK signaling by sequestering RANKL. This study evaluated the antiosteoclast and immunoregulatory effects of mouse rRANK-Fc, which, similar to OPG, can bind RANKL. The effect of RANKL inhibition by RANK-Fc on osteoclast function was determined by inhibition of vitamin D(3) (1,25(OH)(2)D(3))-induced hypercalcemia. Mice were injected with a single dose of 0, 10, 100, 500, or 1000 microg of RANK-Fc; 100 microg of OPG-Fc; or 5 microg of zoledronate 2 h before 1,25(OH)(2)D(3) challenge on day 0, and sacrificed on days 1, 2, 4, 6, 8, 12, 16, and 20. RANK-Fc doses of 100 or 500 microg were tested in a mouse respiratory influenza virus host-resistance model. A single dose of RANK-Fc > or =100 microg suppressed elevation of serum calcium levels and suppressed the bone turnover marker serum pyridinoline at day 4 and later time points, similar to those observed with OPG-Fc and zoledronate (p < or = 0.01 vs controls). By day 6, both immature and mature osteoclasts were depleted by high doses of RANK-Fc (500 and 1000 microg) or 100 microg of OPG-Fc. RANK-Fc doses of 100 or 500 microg had no detectable effect on immune responses to influenza infection, as measured by activation of cytotoxic T cell activity, influenza-specific IgG response, and virus clearance. RANK-Fc inhibition of RANKL has antiosteoclast activity at doses that have no detectable immunoregulatory activity, suggesting that RANKL inhibitors be further studied for their potential to treat excess bone loss.

Expression of anti-apoptotic factors modulates Apo2L/TRAIL resistance in colon carcinoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) selectively induces apoptosis in transformed cells. Normal cells and certain tumor cells can evade Apo2L/TRAIL induced cell death, but the determinants of Apo2L/TRAIL sensitivity are poorly understood. To better understand the factors that contribute to Apo2L/TRAIL resistance, we characterized two colon carcinoma lines with pronounced differences in Apo2L/TRAIL sensitivity. Colo205 cells are highly sensitive to Apo2L/TRAIL whereas Colo320 cells are unresponsive. Components of the DISC (death inducing signaling complex) could be immunoprecipitated from both cell lines in response to Apo2L/TRAIL. Sensitizing agents including a proteasome inhibitor conferred Apo2L/TRAIL sensitivity in Colo320 cells, indicating that the apoptotic machinery was intact and functional. We specifically suppressed the expression of Bcl-2, FLIP or XIAP in Colo320 cells. Downregulation of either FLIP or XIAP but not Bcl-2 restored sensitivity of Colo320 cells to Apo2L/TRAIL. Moreover, stable knockdown of XIAP expression in Colo320 subcutaneous tumors resulted in suppression of tumor growth and sensitivity to Apo2L/TRAIL in vivo. Our results indicate that only a specific subset of anti-apoptotic proteins can confer resistance to Apo2L/TRAIL in Colo320 cells. Elucidation of the factors that contribute to Apo2L/TRAIL resistance in tumor cells may provide insight into combination therapies with Apo2L/TRAIL in a clinical setting.

RANK overexpression in transgenic mice with mouse mammary tumor virus promoter-controlled RANK increases proliferation and impairs alveolar differentiation in the mammary epithelia and disrupts lumen formation in cultured epithelial acini.

RANK and RANKL, the key regulators of osteoclast differentiation and activation, also play an important role in the control of proliferation and differentiation of mammary epithelial cells during pregnancy. Here, we show that RANK protein expression is strictly regulated in a spatial and temporal manner during mammary gland development. RANK overexpression under the control of the mouse mammary tumor virus (MMTV) promoter in a transgenic mouse model results in increased mammary epithelial cell proliferation during pregnancy, impaired differentiation of lobulo-alveolar structures, decreased expression of the milk proteins beta-casein and whey acidic protein, and deficient lactation. We also show that treatment of three-dimensional in vitro cultures of primary mammary cells from MMTV-RANK mice with RANKL results in increased proliferation and decreased apoptosis in the luminal area, resulting in bigger acini with filled lumens. Taken together, these results suggest that signaling through RANK not only promotes proliferation but also inhibits the terminal differentiation of mammary epithelial cells. Moreover, the increased proliferation and survival observed in a three-dimensional culture system suggests a role for aberrant RANK signaling during breast tumorigenesis.

Nectin-like protein 2 defines a subset of T-cell zone dendritic cells and is a ligand for class-I-restricted T-cell-associated molecule.

Dendritic cells (DCs) are a phenotypically and functionally heterogenous population of leukocytes with distinct subsets serving a different set of specialized immune functions. Here we applied an in vitro whole cell panning approach using antibody phage display technology to identify cell-surface epitopes specifically expressed on human blood BDCA3(+) DCs. A single-chain antibody fragment (anti-1F12 scFv) was isolated that recognizes a conserved surface antigen expressed on both human BDCA3(+) DCs and mouse CD8alpha(+) DCs. We demonstrate that anti-1F12 scFv binds Nectin-like protein 2 (Necl2, Tslc1, SynCaM, SgIGSF, or Igsf4), an adhesion molecule involved in tumor suppression, synapse formation, and spermatogenesis. Thus, Necl2 defines a specialized subset of DCs in both mouse and human. We further show that Necl2 binds Class-I-restricted T-cell-associated molecule (CRTAM), a receptor primarily expressed on activated cytotoxic lymphocytes. When present on antigen presenting cells, Necl2 regulates IL-22 expression by activated CD8(+) T-cells. We propose that Necl2/CRTAM molecular pair could regulate a large panel of cell/cell interactions both within and outside of the immune system.