Prostate cancer is the most common solid tumor in men, and it shares with all cancers the hallmark of elevated, nonhomeostatic cell proliferation. Here we have tested the hypothesis that the SONIC HEDGEHOG (SHH)-GLI signaling pathway is implicated in prostate cancer. We report expression of SHH-GLI pathway components in adult human prostate cancer, often with enhanced levels in tumors versus normal prostatic epithelia. Blocking the pathway with cyclopamine or anti-SHH antibodies inhibits the proliferation of GLI1 ؉ ͞PSA ؉ primary prostate tumor cultures. Inversely, SHH can potentiate tumor cell proliferation, suggesting that autocrine signaling may often sustain tumor growth. In addition, pathway blockade in three metastatic prostate cancer cell lines with cyclopamine or through GLI1 RNA interference leads to inhibition of cell proliferation, suggesting cell-autonomous pathway activation at different levels and showing an essential role for GLI1 in human cells. Our data demonstrate the dependence of prostate cancer on SHH-GLI function and suggest a novel therapeutic approach.S ONIC HEDGEHOG (SHH) signaling has been implicated in different aspects of animal development, acting through several components, including the transmembrane proteins PATCHED1 (PTCH1) and SMOOTHENED (SMOH), to activate the GLI zinc-finger transcription factors (1, 2). In addition, we and others have shown that SHH signaling is implicated in a number of tumors (reviewed in refs. 2 and 3), such as basal cell carcinomas (4-6), medulloblastomas (7,8), gliomas (7), sarcomas (9, 10), tumors of the digestive tract (11), small cell lung cancers (12,) and pancreatic carcinomas (13). To date there is no direct evidence linking SHH signaling to prostate cancer, the most common solid cancer in men (14), although we have found that sporadic prostate tumors express GLI1 (7), a reliable marker of SHH signaling (15,16). This observation allowed us to propose the hypothesis that the SHH-GLI pathway participates in prostate cancer (7). Consistently, Shh signaling has been found to be essential for prostate patterning and development (17)(18)(19)(20)(21)(22), and genetic mapping data has revealed that at least two key components of the SHH-GLI pathway [SMOH and SUPPRESSOR OF FUSED (SUFUH)] are located in chromosomal regions implicated in familial human prostate cancer (23,24). Here we have tested the involvement of SHH-GLI signaling in prostate cancer. MethodsCell Lines and Primary Cultures. The PC3, LNCaP, and DU145 cell lines (25-27) were purchased from American Type Culture Collection and grown as specified. All primary prostate tumors were obtained following approved protocols. Tumors in PBS were chopped with a razor blade and incubated with Papain for 1 h at 37°C, they were then dissociated by passing them through a fire-polished pipette and washed several times in serum containing media. All dissociated primary tumors were plated in polyornithinand laminin-treated p16 plates in DMEM-F12 with 10% FBS at Ϸ30,000 cells per p16 well. Primary cultures were ...

Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in Drosophila, we performed chromatin immunoprecipitation-sequencing analysis with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a) within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling.

The traditional definition of natural antibodies (NAbs) states that these antibodies are present prior to the body encountering cognate antigen, providing a first line of defense against infection thereby, allowing time for a specific antibody response to be mounted. The literature has a seemingly common definition of NAbs; however, as our knowledge of antibodies and B cells is refined, re-evaluation of the common definition of Nabs may be required. Defining Nabs becomes important as the function of NAb production is used to define B cell subsets (1) and as these important molecules are shown to play numerous roles in the immune system (Figure 1). Herein, we aim to briefly summarize our current knowledge of NAbs in the context of initiating a discussion within the field of how such an important and multifaceted group of molecules should be defined.

The Food and Drug Administration (FDA) held an open public workshop in June 2010 to discuss the current state of science related to antibody-mediated rejection (AMR) in kidney transplantation. Desensitization, acute AMR and chronic AMR (CAMR) were considered in the context of clinical trial design. Participants discussed experiences with HLA antibody detection and quantitation and the utility of monitoring donorspecific antibodies (DSAs) to inform the management of patients with AMR. The role for animal models was discussed. Diagnostic and prognostic features of histology were presented, followed by discussion of sensitivity and specificity of various criteria. The published literature on treatment of acute AMR was summarized, which consisted of case series and limited data from controlled clinical trials. Considerations for future clinical trials were presented, including endpoints and statistical evaluations of outcome. Although many issues need further consideration, the meeting enabled an important exchange of ideas between experts in the field.

We generated the 1E10 γ-type anti-idiotype mAb (Ab2) specific to an Ab1 mAb able to react specifically with N-glycolyl-containing gangliosides and with Ags expressed on human melanoma and breast carcinoma cells. This Ab2 mAb induced an Ab response in animal models sharing immunochemically defined idiotopes with the Ab1. The treatment of tumor-bearing mice with 1E10 mAb induced a strong antitumor activity. A clinical trial was conducted in 20 patients with advanced malignant melanoma. Patients were treated with six intradermal injections of aluminum hydroxide-precipitated 1E10 anti-Id mAb given at 2-wk intervals. Sixteen of the 17 patients who received at least four doses of the anti-Id vaccine develop Ab3 Abs capable of inhibiting Ab2 binding to Ab1 (Ab3Id+). In contrast to the incapacity of 1E10 mAb to generate Ab3 Abs with the same antigenic specificity as the Ab1 mAb in mice, a very specific and strong Ab3 response against N-glycolyl-containing gangliosides was induced in 16 patients (Ab3Ag+). No evidence of serious or unexpected adverse effects has been observed in this clinical trial. 1E10 anti-Id vaccine was safe, well tolerated, and immunologically effective, with most patients being able to generate a specific immune response against 1E10 and Neu-glycolyl-GM3 ganglioside.

Human antibody secreting cell (ASC) populations in circulation are not well studied. In addition to B-1 (CD20+CD27+CD38lo/intCD43+) cell and the conventional plasmablast (CD20-CD27hiCD38hi) cell populations, here we identified a novel B cell population termed 20+38hi B cells (CD20+CD27hiCD38hi) that spontaneously secretes antibody. At steady state, 20+38hi B cells are distinct from plasmablasts on the basis of CD20 expression, amount of antibody production, frequency of mutation, and diversity of B cell receptor repertoire. However, cytokine treatment of 20+38hi B cells induces loss of CD20 and acquisition of CD138, suggesting that 20+38hi B cells are precursors to plasmablasts, or pre-plasmablasts. We then evaluated similarities and differences between CD20+CD27+CD38lo/intCD43+ B-1 cells, CD20+CD27hiCD38hi 20+38hi B cells, CD20-CD27hiCD38hi plasmablasts, and CD20+CD27+CD38lo/intCD43- memory B cells. We found that B-1 cells differ from 20+38hi B cells and plasmablasts in numbers of ways, including antigen expression, morphological appearance, transcriptional profiling, antibody skewing, antibody repertoire, and secretory response to stimulation. In terms of gene expression, B-1 cells align more closely with memory B cells than with 20+38hi B cells or plasmablasts, but differ in that memory B cells do not express antibody secretion related genes. We found that, B-1 cell antibodies utilize Vh4-34, which is often associated with autoreactivity, 3 to 6-fold more often than other B cell populations. Along with selective production of IgM anti-PC, this data suggests that human B-1 cells might be preferentially selected for autoreactivity/natural-specificity. In sum, our results indicate that human healthy adult peripheral blood at steady state consists of 3 distinct ASC populations.

1E10 mAb is an anti-Id murine mAb (Ab2 mAb) specific for an Ab1 mAb that reacts with NeuGc-containing gangliosides, sulfatides, and Ags expressed in some human tumors. In preclinical studies, this Ab2 Ab was able to mimic NeuGc-containing gangliosides only in animals lacking expression of these Ags in normal tissues. In this study, we report on the immune responses elicited in 20 non-small cell lung cancer patients treated with 1 mg of aluminum hydroxide-precipitated 1E10 mAb. In the hyperimmune sera from 16 of 20 patients, a strong specific Ab response of both IgM and IgG isotypes against NeuGcGM3 ganglioside was observed. Patient immune sera were able to induce complement-independent cell death of NeuGcGM3-expressing X63 murine myeloma target cells. Significant immunoreactivity to NeuGcGM3 was still detected after the complete abrogation of the reactivity against 1E10 mAb by the adsorption of patient sera with this Ab. We hypothesize that Id−Ag+ Abs could reflect the activation of an autologous idiotypic cascade into the patients. Both Id+Ag+ and Id−Ag+ fractions were separated by affinity chromatography and characterized. Although IgG isotype Abs were found in both fractions, IgM isotype Abs were found only in the Id−Ag+ fraction. Both Id+Ag+ and Id−Ag+ Abs were able to specifically recognize and induce cell death in NeuGcGM3-expressing X63 myeloma target cells. Patients that developed IgG and/or IgM Abs against NeuGcGM3 showed longer median survival times.

Purpose: Racotumomab-alum is an anti-idiotype vaccine targeting the NeuGcGM3 tumor-associated ganglioside. This clinical trial was conducted to provide a preliminary estimate of efficacy and safety of racotumomab as switch maintenance for patients with advanced non-small cell lung cancer (NSCLC).Experimental design: Patients with stage IIIb/IV NSCLC who have at least stable disease after first-line chemotherapy were randomized 1:1 to racotumomab-alum (5 immunizations every 2 weeks and reimmunizations every 4 weeks) or placebo. Treatment was administered beyond progressive disease, until severe performance status worsening or toxicity. At progression, only five patients per group received further anticancer therapy. The primary endpoint was overall survival (OS).Results: One-hundred and seventy-six patients were randomized to racotumomab-alum (n ¼ 87) and placebo (n ¼ 89). Median OS was 8.23 and 6.80 months, respectively [HR, 0.63; 95% confidence interval (CI), 0.46-0.87; P ¼ 0.004]. Median progression-free survival (PFS) in vaccinated patients was 5.33 versus 3.90 months for placebo (HR, 0.73; 95% CI 0.53-0.99; P ¼ 0.039). The most common adverse events in the racotumomab-alum arm were burning and pain at the injection site, bone pain, and asthenia. A high antibody response of IgM and IgG isotype against the NeuGcGM3 ganglioside was obtained. Hyperimmune sera were able to specifically recognize and kill the NeuGcGM3-expressing L1210 cell line. Patients who developed anti-NeuGcGM3 antibodies capable to bind and kill 30% L1210 cells showed longer median survival times.Conclusions: Switch maintenance with racotumomab-alum is an effective and a well-tolerated treatment option for patients with advanced NSCLC. Clin Cancer Res; 20(14); 3660-71. Ó2014 AACR.