Supplementary Data from Functional Analysis of Secreted Caveolin-1 in Mouse Models of Prostate Cancer Progression

Supplementary Figure Legends 1-5 from <i>Glioma Pathogenesis-Related Protein 1</i> Exerts Tumor Suppressor Activities through Proapoptotic Reactive Oxygen Species–c-Jun–NH₂ Kinase Signaling

Supplementary Materials and Methods from <i>Glioma Pathogenesis-Related Protein 1</i> Exerts Tumor Suppressor Activities through Proapoptotic Reactive Oxygen Species–c-Jun–NH₂ Kinase Signaling

Supplementary Figure Legends 1-5 from Glioma Pathogenesis-Related Protein 1 Exerts Tumor Suppressor Activities through Proapoptotic Reactive Oxygen Species–c-Jun–NH2 Kinase Signaling

OBJECTIVES:The objectives of this study are to explore the potential benefits of combining AdGlipr1 (or AdGLIPR1) gene therapy with radiotherapy using subcutaneous prostate and bladder cancer models. MATERIALS AND METHODS:Combination adenoviral vector-mediated gene therapy and radiotherapy were applied to 178-2 BMA and TSU-Pr1 cells in vitro and colony formation and apoptosis were analyzed. In addition, combination therapies were administered to mice bearing subcutaneous 178-2 BMA and TSU-Pr1 tumors, and tumor growth suppression and survival extension were compared with the monotherapies (AdGlipr1/AdGLIPR1 and radiotherapy) or control vector Adv/CMV/βgal, as well as single-cycle treatment with 2-cycle treatment. RESULTS:Combination treatment significantly suppressed colony formation and increased apoptosis in vitro. In vivo, combination therapy produced significant 178-2 BMA and TSU-Pr1 tumor growth suppression and survival extension compared with the monotherapies or the control. Further tumor growth suppression and survival extension were observed after 2 cycles of the combination treatment. CONCLUSIONS:Combining AdGlipr1 (AdGLIPR1) with radiotherapy may achieve additive or synergistic tumor control in selected prostate and bladder tumors, and additional therapeutic effects may result with repeated treatment cycles.

Abstract Previously, we reported that caveolin-1 (cav-1) is overexpressed in metastatic prostate cancer and that virulent prostate cancer cells secrete biologically active cav-1. We also showed that cav-1 expression leads to prosurvival activities through maintenance of activated Akt and that cav-1 is taken up by other cav-1–negative tumor cells and/or endothelial cells, leading to stimulation of angiogenic activities through PI-3-K-Akt-eNOS signaling. To analyze the functional consequences of cav-1 overexpression on the development and progression of prostate cancer in vivo, we generated PBcav-1 transgenic mice. Adult male PBcav-1 mice showed significantly increased prostatic wet weight and higher incidence of epithelial hyperplasia compared with nontransgenic littermates. Increased immunostaining for cav-1, proliferative cell nuclear antigen, P-Akt, and reduced nuclear p27Kip1 staining occurred in PBcav-1 hyperplastic prostatic lesions. PBcav-1 mice showed increased resistance to castration-induced prostatic regression and elevated serum cav-1 levels compared with nontransgenic littermates. Intraprostatic injection of androgen-sensitive, cav-1–secreting RM-9 mouse prostate cancer cells resulted in tumors that were larger in PBcav-1 mice than in nontransgenic littermates (P = 0.04). Tail vein inoculation of RM-9 cells produced significantly more experimental lung metastases in PBcav-1 males than in nontransgenic male littermates (P = 0.001), and in cav-1+/+ mice than in cav-1−/− mice (P = 0.041). Combination treatment with surgical castration and systemic cav-1 antibody dramatically reduced the number of experimental metastases. These experimental data suggest a causal association of secreted cav-1 and prostate cancer growth and progression. (Mol Cancer Res 2009;7(9):1446–55)

Abstract Glioma pathogenesis-related protein 1 (GLIPR1), a novel p53 target gene, is down-regulated by methylation in prostate cancer and has p53-dependent and -independent proapoptotic activities in tumor cells. These properties suggest an important tumor suppressor role for GLIPR1, yet direct genetic evidence of a tumor suppressor function for GLIPR1 is lacking and the molecular mechanism(s), through which GLIPR1 exerts its tumor suppressor functions, has not been shown. Here, we report that the expression of GLIPR1 is significantly reduced in human prostate tumor tissues compared with adjacent normal prostate tissues and in multiple human cancer cell lines. Overexpression of GLIPR1 in cancer cells leads to suppression of colony growth and induction of apoptosis. Mice with an inactivated Glipr1 gene had significantly shorter tumor-free survival times than either Glipr1+/+ or Glipr1+/− mice in both p53+/+ and p53+/− genetic backgrounds, owing to their development of a unique array of malignant tumors. Mechanistic analysis indicated that GLIPR1 up-regulation increases the production of reactive oxygen species (ROS) leading to apoptosis through activation of the c-Jun–NH2 kinase (JNK) signaling cascade. Thus, our results identify GLIPR1 as a proapoptotic tumor suppressor acting through the ROS-JNK pathway and support the therapeutic potential for this protein. [Cancer Res 2008;68(2):434–43]

Caveolin-1 (cav-1) is a major structural protein of caveolae, small invaginations of the plasma membrane that integrate and regulate signaling pathways involved in cell growth and differentiation. We previously generated a genetically engineered mice that are homozygous for a null mutation in exon 2 of cav-1 and documented increased incidence of urolithiasis in young male cav-1(-/-) mice. We attributed this, in part, to improper localization of plasma membrane calcium/calmodulin-dependent calcium ATPase in the distal convoluted tubules of the kidney. To document pathologies related to cav-1 function, we maintained cav-1(-/-) and control cav-1(+/+) mice for an extended time period. We report here that cav-1(-/-) mice demonstrate organ-specific growth-related disorders in stromal cells that normally have high levels of cav-1 expression. In many of these organs, epithelial cell growth/differentiation abnormalities were also observed, yet in most of these sites the epithelial cells normally express low to non-detectable levels of cav-1. We propose that loss of cav-1 function in stromal cells of various organs directly leads to a disorganized stromal compartment that, in turn, indirectly promotes abnormal growth and differentiation of adjacent epithelium.

To investigate the immunomodulatory effects of interleukin-12 (IL-12) for treatment of metastatic prostate cancer, we administered adult bone marrow cells (BMC) that were genetically modified by retroviral vector-mediated IL-12 gene transduction in an experimental mouse model of prostate cancer metastasis. This therapy produced significant anti-metastatic effects in bone and lung and prolonged animal survival. Flow cytometric analysis indicated donor BMC could effectively home to bone and lung after treatment. Intensive infiltration of CD4 and CD8T cells in lung metastases and increased systemic natural killer and cytotoxic T lymphocyte activities indicated induction of a significant anti-metastatic immune response after treatment with IL-12 transduced BMC. Our results demonstrate the therapeutic potential of gene-modified BMC gene therapy.

This chapter will discuss the potential for delivery of cytokine molecules using neo-adjuvant/adjuvant gene therapy strategies to achieve antitumor efficacy. It focuses on two approaches for delivery of cytokine genes to achieve effective therapy; in situ delivery using adenoviral vectors also termed "active vaccination," and cell based approaches using specific immune cells modified with cytokine genes. These approaches have potential advantages for prostate cancer therapy and possibly other genitourinary malignancies.

Introduction: A program combining in situ gene therapy and intensity-modulated radiation therapy (IMRT) was implemented for the treatment of prostate cancer because the complementary mechanisms of cytotoxicity may have an enhanced efficacy. The in situ gene therapy comprised of adenoviral vector mediated Herpes Simplex Virus-thymidine kinase (AdHSVtk) + valacyclovir (VCV). We are currently conducting clinical trials using this approach. This study explores long-term immune responses following combined radio-gene therapy compared to gene therapy as mono-therapy. Methods: The study design included two separate clinical trials in the Baylor Prostate Cancer SPORE Program: Trial A gene therapy in combination with IMRT for prostate cancer (31 patients) and Trial B gene therapy as mono-therapy for local recurrent prostate cancer after failing initial radiotherapy (36 patients). Heparinized blood was collected at the time of vector injection and at selected intervals afterward until 12 months. Peripheral blood lymphocytes were analyzed by fluorescent antibody cell sorting (FACS) after the incubation with dual color labeled antibody pairs: CD45/CD14, CD3/CD19, CD3/CD8, CD3/CD4, CD8/HLA-DR, CD4/HLA-DR, CD3/HLA-DR, and CD3/CD56+CD16. Results: The pre-treatment mean percentage of activated CD8+ T cells (DR+CD8+ T cells) was 14.7% and 12.2% (Trials A and B, respectively). Two weeks after the vector injection, this increased to 31.7% and 21.9% (Trials A and B, respectively), and these increases were statistically significant (P < 0.0001 and P = 0.0188, respectively). Only in Trial A were significant increases seen at 4 weeks, 12 weeks, 4 months, 6 months, 8 months and 10 months (P < 0.0001, P = 0.0002, 0.0464, 0.0016, 0.0125 and 0.0354, respectively). In addition, activated CD4+ T cells were noted to increase significantly after the vector injection from 2 weeks till 12 months in Trial A only. (P = 0.0013 and 0.0069, respectively). Conclusions: This is the first report of long-term systemic immune responses following radio-gene therapy compared to gene therapy as mono-therapy. We present evidence showing long-term (up to 12 months) systemic T cell responses to two consecutive AdHSVtk injections during combination in situ gene therapy and IMRT for prostate cancer. These results suggest the potential for sustained activation of cell-mediated immune responses and may have enhanced activities against cancer. This combined approach may maximize tumor control, both local-regional and systemic through radio-gene therapy induced cytotoxicity and anti-tumor immunity.

We previously identified a novel p53 target gene, RTVP-1, that possesses unique cytotoxic and immunostimulatory activities which make it potentially useful for cancer gene therapy. To test the therapeutic potential of RTVP-1 in a gene-modified tumor cell-based vaccine model, we used an adenoviral vector capable of efficient transduction and expression of RTVP-1 (AdRTVP-1), together with a highly metastatic mouse prostate cancer cell line (178-2 BMA). A vaccine was prepared with 178-2 BMA cells transduced with AdRTVP-1 or a control adenoviral vector expressing β-galactosidase (Adβgal). After irradiation of the cells, syngeneic 129/Sv mice were vaccinated three times at weekly intervals. After 3 weeks, they were challenged with orthotopic 178-2 BMA cells. After 21 days, fewer than 60% of the RTVP-1-cell-vaccinated mice developed tumors compared to 100% of the control mice. The RTVP-1-cell vaccine significantly reduced primary tumor wet weight compared with control Adβgal-cell vaccine (P<0.0001 at 7 and 14 days). Experimental metastasis to lung was also significantly reduced (P=0.0377), and survival significantly increased (P=0.0002). In addition, significantly increased NK and CTL activities were demonstrated in the AdRTVP-1-cell-vaccinated mice. These findings indicate that RTVP-1 gene-modified cell-based vaccines may be useful in the prevention of recurrent prostate cancer.

Purpose: To explore long-term immune responses after combined radio-gene-hormonal therapy.Methods and Materials: Thirty-three patients with prostate specific antigen 10 or higher or Gleason score of 7 or higher or-clinical stage T2b to T3 were treated with gene therapy that consisted of 3 separate intraprostatic injections of AdHSV-tk on Days 0, 56, and 70. Each injection was followed by 2 weeks of valacyclovir. Intensity-modulated radiation therapy was delivered 2 days after the second AdHSV-tk injection for 7 weeks. Hormonal therapy was initiated on Day 0 and continued for 4 months or 2.3 years. Blood samples were taken before, during, and after treatment. Lymphocytes were analyzed by fluorescent antibody cell sorting (FACS).Results: Median follow-up was 26 months (range, 4-48 months). The mean percentages of DR(+)CD8(+) T cells were increased at all timepoints up to 8 months. The mean percentages of DR(+)CD4(+) T cells were increased later and sustained longer until 12 months. Long-term (2.3 years) use of hormonal therapy did not affect the percentage of any lymphocyte population.Conclusions: Sustained long-term (up to 8 to 12 months) systemic T-cell responses were noted after combined radio-gene-hormonal therapy for prostate cancer. Prolonged use of hormonal therapy does not suppress this response. These results suggest the potential for sustained activation of cell-mediated immune responses against cancer. (c) 2006 Elsevier Inc.

Caveolin-1 (cav-1) is a major scaffolding component of cell membrane invaginations (caveolae). It is involved in sequestering numerous effectors and signaling molecules and has antiapototic activities in prostate cancer. Perineural invasion (PNI) is associated with decreased apoptosis of cancer cells both in human tissues and the in vitro PNI model. We show here that stromal (perineurium) production of cav-1 is involved in a paracrine antiapoptotic loop in PNI. Transforming growth factor-beta1 is up-regulated in the cancer cells as they approach the nerve and is thought to up-regulate cav-1 in the perineurium of nerves with prostate cancer. Cav-1 is then secreted into the microenvironment and used by prostate cancer cells to inhibit apoptosis. In the in vitro PNI model, this phenomenon is partially reversed by neutralizing cav-1 antibodies or using ganglia from cav-1 knockout mice. Our results show a novel paracrine mechanism used by the prostate cancer in PNI to increase their proliferative activity and decrease apoptosis.

Introduction: Clinical trial of combination adenoviral vector mediated Herpes Simplex Virus-thymidine kinase (HSV-tk) + valacyclovir (VCV) in situ gene therapy with radiotherapy (RT) and hormonal therapy was conducted for Stage D1 prostate cancer. We now report the safety, efficacy, and immune responses using this approach. Methods: Five patients with Stage D1 prostate cancer were treated as follows: Gene therapy: 3 separate intraprostatic injections of AdHSV-tk were performed on days 0, 56, and 70. Each injection was followed by 2 weeks of VCV. RT to pelvic lymphatics and prostate was delivered 2 days after the second AdHSV-tk injection for 7 weeks. Hormonal therapy: lupron and flutamide were initiated on day 0 for 4 months or 2 years. Toxicity was assessed using CTEP and RTOG toxicity scores. Peripheral blood lymphocytes were analyzed by fluorescent antibody cell sorting (FACS) after the incubation with dual color labeled antibody pairs: CD45/CD14, CD3/CD19, CD3/CD8, CD3/CD4, CD8/HLA-DR, CD4/HLA-DR, CD3/HLA-DR, and CD3/CD56+CD16. PSA, prostate biopsy, and FACS samples were taken at selected intervals. ASTRO consensus definition was used for PSA failure. Results: Median age was 50 years and median follow-up for the entire group was 29 months. Mean and median pre-treatment PSA were 97.3 and 16.9 ng/ml (range; 2.5–374 ng/ml). Gleason score was 8 in 2 patients and 9 in the other 3 patients. Three of the 5 patients had biochemical failure at 3, 3, and 7.7 months: 2 patients had distant failure in the bones and 1 patient had failure in the para- aortic lymph nodes outside radiation treatment portal. Prostate biopsies however revealed no evidence of residual carcinoma in all cores. The pre-treatment mean percentages of activated CD8+ (DR+CD8+) T cells and activated CD4+ (DR+CD4+) T cells were 12.0% and 5.3% respectively. Four months after the initial treatment, the mean percentages of DR+CD8+ T cells and DR+CD4+ T cells increased to 32.7% and 18.8% (P=0.0431) respectively. No grade 3 or higher toxicity was seen. The GU and GI toxicity profiles are no different from those of patients treated with RT alone. Conclusions: We present evidence of safety and efficacy using this combined radio-gene-hormonal therapy approach for Stage D1 prostate cancer. These results also suggest the potential for activation of cell-mediated immune response using this combined therapy. Current efforts are aimed to improve systemic control by maximizing activation of anti-tumor immune response in these patients via radio-gene therapy.