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Smokers develop metastatic prostate cancer more frequently than nonsmokers, suggesting that a tobacco-derived factor is driving metastatic progression. To identify smoking-induced alterations in human prostate cancer, we analyzed gene and protein expression patterns in tumors collected from current, past, and never smokers. By this route, we elucidated a distinct pattern of molecular alterations characterized by an immune and inflammation signature in tumors from current smokers that were either attenuated or absent in past and never smokers. Specifically, this signature included elevated immunoglobulin expression by tumor-infiltrating B cells, NF-κB activation, and increased chemokine expression. In an alternate approach to characterize smoking-induced oncogenic alterations, we also explored the effects of nicotine in human prostate cancer cells and prostate cancer–prone TRAMP mice. These investigations showed that nicotine increased glutamine consumption and invasiveness of cancer cells in vitro and accelerated metastatic progression in tumor-bearing TRAMP mice. Overall, our findings suggest that nicotine is sufficient to induce a phenotype resembling the epidemiology of smoking-associated prostate cancer progression, illuminating a novel candidate driver underlying metastatic prostate cancer in current smokers. Cancer Res; 76(5); 1055–65. ©2015 AACR.

Supplementary Table S1: Clinical characteristics of the study population by smoking status. Supplementary Table S2: Immunoglobulin expression in prostate tumors by smoking status of the patients. Supplementary Table S3: Genes differently expressed between current and never smokers in prostate tumors. Supplementary Table S4: Genes differently expressed between current and never/past smokers in 67 prostate tumors.

Supplementary Table S11: Differentially expressed genes comparing prostate tumors from nicotine-treated TRAMP mice with tumors from untreated mice (reference).

Supplementary Table 9: Genes differently expressed between nicotine-treated and untreated 22Rv1 cells.

Supplementary Table S6: Increased expression of genes regulating synaptic signal transduction in the cancerous prostate of nicotine�treated TRAMP mice.

Supplemetary Table 5: Genes differently expressed between current and never and between current and past/never smokers in the combined cohort of 67 prostate tumors

Supplementary Table 10: Genes differently expressed between nicotine-treated and untreated LNCaP cells.

Supplementary Table 7: Genes differently expressed between current and never smokers in microdissected prostate tumors.

Supplementary Figure S1: Weight curves for untreated TRAMP mice and TRAMP mice treated with nicotine for 80 days. Supplementary Figure S2: Ig lambda� and Ig kappa�positive B lymphocytes in cancerous prostate tissue by in�situ hybridization. Supplementary Figure S3: qRT�PCR validation of six genes up�regulated among current smokers. Supplementary Figure S4: Analysis of prostate tumors using Bioconductor limma R. Supplementary Figure S5: Gene Set Enrichment Analysis (GSEA) highlighting common features between smoking�related gene signatures in prostate tumors, nicotine�induced gene signatures in human prostate cancer cells, and gene signatures archived in the GSEA database. Supplementary Figure S6: Expression of various nicotinic acetylcholine receptor (AChR) subunits in human prostate cancer cell lines, prostate tumors, and adjacent non�tumor prostate tissues. Supplementary Figure S7: Nicotine�mediated Akt activation in human immortalized prostate epithelial cells and prostate cancer cell lines. Supplementary Figure S8: Effect of nicotine on the mobility of human prostate cancer cell lines. Supplementary Figure S9: Nicotine enhances cell surface integrin expression and extracellular matrix binding (ECM) of 22Rv1 human prostate cancer cells. Supplementary Figure S10: Nicotine enhances proliferation of RWPE�1 in complete K�SFM medium but not in K�SFM medium without EGF and bovine pituitary extract. Supplementary Figure S11: Nicotine did not enhance proliferation of 22Rv1 and PC�3 human prostate cancer cells. Supplementary Figure S12: Lymphotoxin�β plasma levels in prostate cancer patients (Case) and population�based controls (Control) by smoking status.

Supplemetary Table 8: Genes differently expressed between current and never in microdissected tumors using the Bioconductor limma R package.

Purpose: To determine whether mesothelin, a cell surface protein highly expressed in mesothelioma and ovarian cancer, is shed into serum and if so to accurately measure it. Experimental Design: We developed a sandwich ELISA using antibodies reacting with two different epitopes on human mesothelin. To quantitate serum mesothelin levels, a standard curve was generated using a mesothelin-Fc fusion protein. Sera from 24 healthy volunteers, 95 random hospital patients, 56 patients with mesothelioma, and 21 patients with ovarian cancer were analyzed. Serum mesothelin levels were also measured before and after surgical cytoreduction in six patients with peritoneal mesothelioma. Results: Elevated serum mesothelin levels were noted in 40 of 56 (71%) patients with mesothelioma and in 14 of 21 (67%) patients with ovarian cancer. Serum mesothelin levels were increased in 80% and 75% of the cases of mesothelioma and ovarian cancer, respectively, in which the tumors expressed mesothelin by immunohistochemistry. Out of the six patients with peritoneal mesothelioma who underwent surgery, four had elevated serum mesothelin levels before surgery. Out of these four patients, three had cytoreductive surgery and the serum mesothelin level decreased by 71% on postoperative day 1 and was undetectable by postoperative day 7. Conclusions: We developed a serum mesothelin assay that shows that mesothelin is elevated in patients with mesothelioma and ovarian cancer. The rapid decrease in mesothelin levels after surgery in patients with peritoneal mesothelioma suggests that serum mesothelin may be a useful test to monitor treatment response in mesothelin-expressing cancers.

The development of immunotherapy for prostate cancer based on the induction of autoimmunity to prostate-specific differentiation antigens is an attractive concept. In this study, we constructed novel virus-like particles vector (VLPV) platform derived from live attenuated human TC-83 IND vaccine against Venezuelan equine encephalitis virus. We tested the ability of VLPV-PSA particles to target mouse CD11c+ dendritic cells (DCs) using in vitro infection assay. Incubation of mouse splenic DCs with VLPV-PSA resulted in robust accumulation of PSA in the cytoplasm of CD11c+ cells as detected by immunofluorescent staining and confocal microscopy. We also tested the immunogenicity and anti-tumor effect of the VLPV-PSA vaccine in a stringent mouse model of prostate cancer in which tolerance to human PSA expressed as a ‘self’ antigen in the prostate was established in a context of the HLA-DR2b chimeric transgene (DR2bxPSA F1 mice). Preventive vaccination with VLPV-PSA induced strong PSA-specific CD8+ T-cell response as detected by IFNγ ELISPOT assay and intracellular cytokine staining. Potent expansion of PSA-specific CD8+ effector T-cells was found in peripheral blood and spleens of vaccinated mice using PSA peptide-loaded MHC class I dextramers. VLPV-PSA vaccination also induced PSA-specific antibody production in immunized mice with predominance of Th1-associated IgG2a sub-isotype. Injection of TRAMP tumor cells expressing PSA into vaccinated mice resulted in elevated infiltration of tumor site with CD8+ T-cells and rapid elimination of PSA-expressing tumor cells during first several weeks after tumor challenge. Tumor survival analysis revealed significant delay of tumor growth in VLPV-PSA vaccinated mice, especially at the early time-points (p=0.044, Gehan-Breslow test). Interestingly, the combination treatment with VLPV-PSA and the blockade of the CTLA-4-mediated checkpoint did not enhance further CD8 T cell response to PSA, and did not show synergistic anti-tumor effect compared to the VLPV-PSA treatment alone. Our data demonstrate that VLPV-PSA are able to infect mouse DCs and induce production of heterologous antigen (PSA) resulting in efficient break of immunological tolerance to PSA and induction of anti-tumor response in a stringent mouse model of prostate cancer. Citation Format: Vladimir Ryabov, Peter Pushko, Irina Tretyakova, Rikka Saito, Richard B. Alexander, Elena N. Klyushnenkova. Novel alphavirus-based vaccine targets dendritic cells and efficiently breaks immunological tolerance to “self” tumor-associated antigen (PSA) in an HLA-DR mouse model of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2880. doi:10.1158/1538-7445.AM2014-2880

Macrophage infiltration is observed in many types of human and mouse malignant tumors. Though some studies reported anti-tumor activity of tumor-associated macrophages (TAM) the majority of reports revealed contribution of TAM to tumor growth, invasion and metastasis. The phenotype and functions of TAM in prostate cancer are not well known. However, their abundance in the tumor suggests an important role in the course of malignant progression. In our novel humanized mouse model of prostate cancer, the expression of human HLA-DR*1501 (DR2b) transgene drastically impairs immune response to prostate tumor and induces excessive accumulation of TAM, which predominate in tumor tissue. In parallel, substantial accumulation of CD4+CD25+FOXP3+ T cells is observed in the tumor mass. The initial gene expression profiling of TAM in DR2b+ mice revealed their mixed M1/M2 phenotype since these cells expressed typical M2-associated genes TGFβ, IL10, arginase I, stabilin-1 and M1-associated genes IL1β, TNFα and IL6. Moreover, TAM strongly expressed chemokines CCL2, CCL3, CCL4, CCL5 and CXCL10, which are capable to recruit myeloid, NK and T cells as well as immune checkpoint receptor PDL-1. Interestingly, compared to DR2b- mice, TAM from DR2b+ animals had reduced expression of important NK and T cell survival factor IL15. Immunohistochemical investigation of the tumor tissue revealed that CD68+ TAM co-localized with T cells on the periphery of the tumor and expressed M2-associated marker stabilin-1. Moreover, T cells were found exclusively on the tumor periphery, whereas TAM were observed inside tumor mass. Altogether, present data suggest that TAM in DR2b model of prostate cancer have potential to recruit various immune cells to the tumor mass. However, elevated expression of immunosuppressive factors TGFβ, IL10, arginase I, PDL-1 and close association between TAM and T cells on tumor periphery may impair T cell activation and induce generation of Tregs. Citation Format: Vladimir Ryabov, David Kim, Rikka Saito, Richard B. Alexander, Elena Klyushnenkova. Characterization of tumor-infiltrating macrophages in humanized mouse model of prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1537. doi:10.1158/1538-7445.AM2013-1537

It is clearly established that the successful cancer immunotherapy will critically depend on the overcoming tolerance to “self” and negative immunoregulatory mechanisms. We have recently described a novel model of prostate cancer in double transgenic mice expressing human leukocyte antigen (HLA)-DR (HLA-DRB1*1501(DR2b)) and human prostate specific antigen (PSA) as a “self” antigen. In DR2bxPSA F1 mice, immunological tolerance to the transgenic mouse adenocarcinoma of prostate (TRAMP) tumor cells expressing PSA was established in a context of a “permissive” HLA-DR2b allele, and could be reversed by depleting CD25+ regulatory T cells (Tregs) prior to tumor inoculation. Several studies suggested that simultaneous targeting of both Tregs and negative regulatory pathway mediated by the T-cell inhibitory receptor CTL-associated antigen-4 (CTLA-4) may potentiate anti-tumor immune responses. We studied the effect of the combinatory treatment with anti-CD25 mAb and anti-CTLA-4 mAb on the growth of TRAMP-PSA tumor cells in DR2bxPSA F1 male mice. Single administration of anti-CD25 mAb (clone PC61) three days prior to tumor inoculation significantly increased PSA-specific CD8 T cell responses to the TRAMP-PSA tumor cells and delayed tumor growth compared to mice treated with isotype control antibodies. Similarly, treatment with anti-CTLA-4 mAb (clone 9D9) administered on days 3, 6 and 9 after tumor inoculation also significantly increased the levels CD8 T cells producing IFN-γ in response to the immunodominant H-2Db-restricted epitope PSA65–73 and delayed tumor growth. A combinatory treatment with both anti-CD25 and anti-CTLA-4 mAbs further enhanced anti-tumor immunity and caused a more profound delay in the tumor growth compared to each treatment alone. There were no tumor-free survivors among mice treated with anti-CD25 mAb alone (0 out of 21 mice), while 2 out of 21 mice treated with anti-CTLA-4 mAb alone (9.5%) and 3 out of 10 mice treated with a combination of anti-CD25 and anti-CTLA-4 mAbs (30%) survived tumor-free. Mice that survived a primary tumor challenge for 20 weeks demonstrated strong polyfunctional CD8 T cell memory responses to the secondary challenge with TRAMP-PSA tumors that was characterized by the significantly increased levels of PSA-specific IFN-γ+TNF-α+ CD8 T cells. Our data suggest that a combinatory treatment targeting Tregs and CTLA-4-mediated inhibitory pathways may be a promising approach for the prostate cancer immunotherapy. Citation Format: Elena N. Klyushnenkova, Richard B. Alexander. A combinatory treatment targeting CD25+ regulatory T cells and CTL-associated antigen-4 blockage enhanced antitumor immunity in the HLA-DR transgenic mouse model of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr A13.

We have recently described a novel mouse model of prostate cancer in transgenic mice engineered to express Human Leukocyte Antigen (HLA)-DRB1*1501 (DR2b in old nomenclature) (J. Immunol. 2009, 182:1242-1246). Using these mice, we investigated the impact of HLA-DR2b on the growth of prostate-specific antigen (PSA)-expressing Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) tumor cells. We demonstrated that a “permissive” HLA class II allele could change the pattern of anti-tumor immune response resulting in failure of tumor rejection. The simple presence of HLA-DR2b was all that was required in our model for the development of strong humoral immune responses to the tumor antigen (PSA), suppression of CD8 T cell responses and enhanced growth of the tumor. In contrast, mice bearing class II allele that did not support CD4 T cell response to PSA (I-Ab), developed strong CD8 T cell response in the absence of antibody response, and rejected PSA-expressing tumors. One of the major mechanisms by which the presence of CD4 T cell epitope in PSA can negatively affect anti-tumor immunity may include CD4+CD25+ regulatory T cells (Treg) that can be selectively activated in an antigen-specific manner in DR2b+ mice. We found that the depletion of CD4+CD25+ Treg cells prior to tumor inoculation resulted in the significant enhancement of PSA-specific CD8 T cell response in DR2b+ mice. Moreover, we also found that depletion of CD25+ T cells prior to tumor inoculation significantly delayed TRAMP-PSA tumor growth (p=0.01, log rank test). These results indicated that low level of CD8 T cell-mediated IFN-γ response against TRAMP-PSA tumors in DR2b+ mice is an active process of immune suppression mediated by CD4+CD25+ Treg cells, and that the activation of Treg cells can be involved in tumor progression in our model. We also analyzed a composition of the tumor-infiltrating leukocytes (TIL) in DR2b+ and DR2b- mice bearing TRAMP-PSA tumors by flow cytometry. The analysis of infiltrating CD3+ T cells revealed significant strain-specific differences. In DR2b+ mice, CD4+ T cell were accumulated in larger number compared to DR2b- mice, and a significant proportion of CD4+ cells expressed CD25. These cells were also foxp3+. These observations are consistent with other data in our model, and confirm that 1) HLA-DR2b-dependent Treg cell activation may be involved in tumor progression in DR2b+ mice, and 2) lack of such activation in DR2b- mice can result in the efficient CD8 T cell response and tumor rejection. Supported by a grant from the US Department of Veterans Affairs and a pilot grant from the Baltimore Research and Education Foundation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5318.