Jianxiang Wang

Suppression of caveolin expression induces androgen sensitivity in metastatic androgen-insensitive mouse prostate cancer cells

Although prostate cancer cells are often initially sensitive to androgen ablation, they eventually lose this response and continue to survive, grow and spread in the absence of androgenic steroids. The mechanism(s) that underlie resistance to androgen ablation therapy remain mostly unknown. We have demonstrated that elevated caveolin protein levels are associated with human prostate cancer progression in pathological specimens. Here we show that suppression of caveolin expression by a stably transfected antisense caveolin-1 cDNA vector converted androgen-insensitive metastatic mouse prostate cancer cells to an androgen-sensitive phenotype. Orthotopically grown tumors and low-density cell cultures derived from antisense caveolin clones had increased apoptosis in the absence of androgenic steroids, whereas similarly grown tumors and cells from vector (control) clones and parental cells were not sensitive to androgens. Studies using a representative antisense caveolin clone showed that selection for androgen resistance in vivo correlated with increased caveolin levels, and that adenovirus-mediated caveolin expression blocked androgen sensitivity. Our results identify a new candidate gene for hormone-resistant prostate cancer in man and indicate that androgen insensitivity can be an inherent property of metastatic prostate cancer.

Adenovirus-mediated interleukin-12 gene therapy for prostate cancer: Suppression of orthotopic tumor growth and pre-established lung metastases in an orthotopic model

Interleukin-12 (IL-12) can elicit potent antitumoral effects that involve the recruitment of specific immune effector cells. We investigated the efficacy of a single injection of a recombinant adenovirus expressing murine IL-12 (AdmIL-12) directly into orthotopic mouse prostate carcinomas generated from a poorly immunogenic cell line (RM-9) derived from the mouse prostate reconstitution system. Significant growth suppression (>50% reduction of tumor weight) and increased mean survival time (23.4 to 28.9 days) were observed compared with controls. Suppression of pre-established lung metastases was also observed following the injection of AdmIL-12 into the orthotopic tumor. Cytolytic natural killer cell activity was markedly enhanced 1–2 days after virus injection. Immunohistochemical analysis showed significantly elevated intratumoral infiltration of CD4+ and CD8+ T cells 7 days after virus injection. However, splenocyte-derived cytotoxic T lymphocytes were not detected during the 14 days following treatment. Increased numbers of nitric oxide synthase-positive macrophages were seen in the AdmIL-12 treated group 7 days following injection. Systemic inhibition of natural killer cells with anti-asialo-GM1 serum led to increased numbers of lung metastases in AdmIL-12-treated orthotopic tumors but did not affect local tumor growth. In this model system the antitumor effects of a single injection of adenovirus-mediated IL-12 appears to be based to a large extent on the activation of nitric oxide synthase in macrophages and possibly T cell activities, whereas the relatively early cytolytic activity of natural killer cells are largely but not exclusively responsible for the antimetastatic effects.

Caveolin-1 is regulated by c-myc and suppresses c-myc-induced apoptosis

Recent data indicating that overexpression of caveolin-1 as well as c-myc are relatively common features of advanced prostate cancer prompted us to test for potential cooperative interactions between caveolin-1 and c-myc that would be consistent with malignant progression. We used the well-characterized Rat1AmycERTM cells to show that the caveolin-1 gene is down-regulated at the level of transcription by c-myc. By maintaining relatively high levels of caveolin-1 with an adenoviral vector or in stably transfected clones we show that caveolin-1 can suppress c-myc-induced apoptosis. Further we established human prostate cancer cell lines with the mycERTM construct and show that clones with increased caveolin-1 are more resistant to myc-induced apoptosis and have increased capacity for growth in soft agar when c-myc is activated.

mRTVP-1, a novel p53 target gene with proapoptotic activities

ABSTRACT We identified a novel mouse gene, mRTVP-1, as a p53 target gene using differential display PCR and extensive promoter analysis. The mRTVP-1 protein has 255 amino acids and differs from the human RTVP-1 (hRTVP-1) protein by two short in-frame deletions of two and nine amino acids. RTVP-1 mRNA was induced in multiple cancer cell lines by adenovirus-mediated delivery of p53 and by gamma irradiation or doxorubicin both in the presence and in the absence of endogenous p53. Analysis of RTVP-1 expression in nontransformed and transformed cells further supported p53-independent gene regulation. Using luciferase reporter and electrophoretic mobility shift assays we identified a p53 binding site within intron 1 of the mRTVP-1 gene. Overexpression of mRTVP-1 or hRTVP-1 induced apoptosis in multiple cancer cell lines including prostate cancer cell lines 148-1PA, 178-2BMA, PC-3, TSU-Pr1, and LNCaP, a human lung cancer cell line, H1299, and two isogenic human colon cancer cell lines, HCT116 p53+/+ and HCT116 p53−/−, as demonstrated by annexin V positivity, phase-contrast microscopy, and in selected cases 4′,6′-diamidino-2-phenylindole staining and DNA fragmentation. Deletion of the signal peptide from the N terminus of RTVP-1 reduced its apoptotic activities, suggesting that a secreted and soluble form of RTVP-1 may mediate, in part, its proapoptotic activities.

Adenoviral Vector-Mediated mRTVP-1 Gene Therapy for Prostate Cancer

We previously identified the mouse RTVP-1 (mRTVP-1; related to testes-specific, vespid, and pathogenesis proteins) gene as a direct target of p53 with proapoptotic activities in various cancer cell lines, including prostate cancer. To test the therapeutic potential of mRTVP-1 we constructed an adenoviral vector capable of efficient transduction and expression of mRTVP-1 (AdmRTVP-1) and used this vector in an orthotopic, metastatic mouse model of prostate cancer. A single intratumoral administration of AdmRTVP-1 gene therapy significantly reduced primary tumor wet weight compared with control Adbetagal-injected tumors at two time points after injection with two different vector doses (p < or = 0.01 at 7 and 14 days). Spontaneous metastasis to lung was also significantly reduced (p < or = 0.02). Evaluation of treated tumors revealed increased apoptosis and lower microvessel density counts. In a rat aortic ring sprouting assay, AdmRTVP-1 inhibited endothelial cell sprouting compared with Adbetagal, confirming its antiangiogenic activity. These therapeutic activities were associated with a significant increase in survival from 22.9 to 26.8 days (p = 0.003) in this aggressive model of prostate cancer. Interestingly, there were significant increases in the infiltration of tumor-associated macrophages, dendritic cells, and CD8+ T cells, which persisted at 14 days posttreatment in the AdmRTVP-1-treated tumors compared with Adbetagal control-treated tumors. In addition, significantly increased natural killer and cytotoxic T lymphocyte activities were demonstrated in the mice with AdmRTVP-1-treated tumors. The unique therapeutic properties of AdmRTVP-1 gene therapy demonstrated in this study provide new opportunities for gene and immunotherapy of prostate cancer and potentially other malignancies.

Combination gene therapy with adenoviral vector-mediated HSV- tk +GCV and IL-12 in an orthotopic mouse model for prostate cancer

We previously demonstrated significant therapeutic activities associated with adenoviral vector-mediated Herpes Simplex Virus/thymidine kinase (AdHSV-tk) with ganciclovir (GCV) in situ gene therapy in the RM-1 orthotopic mouse prostate cancer model and interleukin-12 (AdmIL-12) in situ gene therapy in the RM-9 orthotopic mouse prostate model for prostate cancer. In both protocols, local cytotoxicity and activities against pre-established lung metastases were demonstrated. To test whether combined AdHSV-tk+GCV+IL-12 gene therapy would lead to enhanced therapeutic effects when compared to either treatment alone, we used RM-9 mouse prostate cancer cells in both orthotopic and pre-established lung metastases models of prostate cancer. Combined treatment with a single injection of optimal doses of AdHSV-tk+GCV or AdmIL-12 led to significantly increased suppression of orthotopic tumor growth. IL-12 gene therapy alone was more effective than AdHSV-tk+GCV in suppressing spontaneous lymph node metastases and pre-established lung metastases but combination gene therapy did not result in additional anti-metastatic activities. Combination gene therapy also did not achieve significantly better animal survival compared to AdHSV-tk+GCV or AdmIL-12 alone. Analysis of localized antitumor activities demonstrated that AdHSV-tk+GCV therapy induced higher levels of necrosis compared to AdmIL-12 or combination therapy. However, both treatments alone and combination therapy produced similar increases in apoptotic index. To address the possible mechanisms of locally co-operative cytotoxic activities, we analyzed the systemic natural killer (NK) response and the numbers of tumor-infiltrating immune cells using quantitative immunohistochemical analysis. AdHSV-tk+GCV therapy alone led to detectable increases in iNOS-positive cells, CD4+and CD8+T-cells and moderately increased numbers of F4/80 (macrophage selective)-positive cells within treated tumors. In contrast, AdmIL-12 elicited a highly robust pattern of tumor infiltration for all four of these immune cells that was in general mimicked by combination therapy. Further analysis of the accumulation of transforming growth factor-β1 (TGF-β1) immunohistochemical staining demonstrated that AdHSV-tk+GCV treatment, but not AdmIL-12 treatment, produced cancer cell-associated increases in this cytokine relative to control Ad-β-gal injections. Interestingly, local injection with AdHSV-tk+GCV induced significant splenocyte-derived NK cell cytolytic activities with maximal response 7 days following treatment, whereas AdmIL-12 injection produced significantly higher NK activity with maximal response 2 days following injection. The combined treatment produced a higher systemic NK response over the 14-day treatment period. Depletion of NK cells in vivo demonstrated that this immunocyte subpopulation was responsible for early locally cytotoxic activities induced by AdHSV-tk+GCV but not AdmIL-12 and that NK activities were largely responsible for activities against pre-established metastases generated by both gene therapy protocols.

Gene therapy for prostate cancer: toxicological profile of four HSV- tk transducing adenoviral vectors regulated by different promoters

Adenoviral vector delivery of the Herpes simplex virus thymidine kinase (HSV-tk) gene in combination with the prodrug ganciclovir (GCV) has been tested in phase I clinical trials for prostate cancer and found to exhibit a satisfactory toxicity profile. We have developed additional adenoviral vectors with differing promoters to optimize the expression profile and in the present study evaluate the potential systemic toxicity of these vectors. Four recombinant adenoviral vectors that express the HSV-tk gene were generated using three different promoters: CMV (leftward orientation); RSV (both rightward and leftward orientation); and the mouse caveolin-1 (cav-1) promoter (leftward orientation). Efficacy was determined in vitro by cytotoxicity assays in a mouse prostate cancer cell line, RM-9, and in vivo by treating orthotopic tumors. Potential toxicity was evaluated from liver histology and apoptotic cell counts and enzyme levels in the serum following intravenous adenoviral vector injection. Although there were differences in HSV-tk expression at the protein level among the four vectors there were no significant differences in in-vitro cytotoxicity studies with GCV or in vivo in tumor growth suppression of an orthotopic mouse prostate cancer model in GCV treated mice. Intravenous delivery of high doses of all adenoviral vectors lead to abnormalities in liver function as measured by specific serum markers and histological evaluation of liver tissue and increased levels of apoptosis in the liver. These abnormalities were most prevalent with the vector containing the CMV promoter and the rightward oriented RSV promoter. They were least prevalent in the vector regulated by the cav-1 promoter. Upregulation of specific chemokines, MIP-2 and MIP-1β was correlated with apoptotic counts. Our results demonstrate that comprehensive toxicological analysis of adenoviral vectors provides internally consistent information that can differentiate vectors with comparable efficacy based on toxicity. In these studies vectors with the cav-1 promoter-driven and leftward RSV-driven HSV-tk gene demonstrated minimal toxicities with cytotoxic effectiveness comparable to more toxic vectors. Our studies further suggest that promoter selection can influence the toxic effects of an adenoviral gene therapy vector.

In situ gene therapy for prostate cancer: Immunomodulatory approaches

The development of effective treatments for prostate cancer is thwarted by the natural history of the disease. The biological and clinical potential of most individual cancers is uncertain. In many cases the disease will not progress to clinical significance but experimental and clinical studies indicate that prostate cancer can and may metastasise early in the course of the disease from relatively small foci (i.e., not necessarily the largest or index cancer). Localised prostate cancer is potentially curable with localised therapies (radical prostatectomy or irradiation therapy). However, there are no curative therapies for metastatic prostate cancer. Gene therapy, especially those approaches with an immunomodulatory component, may provide additional therapeutic options with the potential to affect both localised and systemic disease. We have pioneered the development and application of in situ gene therapy protocols using adenoviral vectors to transduce specific genes that generate cytotoxic activity and/or a systemic antitumour immune response. In addition we have completed initial studies that demonstrate the therapeutic potential of adenoviral vector-mediated gene modified cell-based vaccines. Our review discusses preclinical studies focused on the development of immunostimulatory in situ gene therapy approaches that hopefully will provide novel and effective treatments for localised and metastatic prostate cancer.

Therapeutic effects of gelatin matrix-embedded IL-12 gene-modified macrophages in a mouse model of residual prostate cancer

We evaluated the potential use of intraoperative gelatin matrix hemostatic sealant (GMHS; FloSeal; Baxter Healthcare) embedded with macrophages (Mφ) transduced with murine interleukin (IL)-12 recombinant adenoviral vector (G/Mφ/AdmIL-12) for prevention of recurrence of prostate cancer following radical prostatectomy. Application of G/Mφ/AdmIL-12 resulted in significant suppression of tumor growth and spontaneous lung metastases, a statistically significant survival advantage of the G/Mφ/AdmIL-12-treated animals, more efficient trafficking of Mφ to lymph nodes draining from the prostate and generation of systemic natural killer cell activity and tumor-specific cytolytic T lymphocyte responses compared to the controls in a preclinical mouse model of residual prostate cancer. Our data recommend this treatment as a novel adjuvant for prevention of local recurrence of prostate cancer following radical prostatectomy.

GLIPR1-ΔTM protein as a systemic therapeutic approach for prostate cancer

GLIPR1 is a p53 target gene known to be downregulated in prostate cancer, and increased endogenous GLIPR1 expression has been associated with increased production of reactive oxygen species, increased apoptosis, decreased c‐Myc protein levels and increased cell cycle arrest. Recently, we found that upregulation of GLIPR1 in prostate cancer cells increases mitotic catastrophe through interaction with heat shock cognate protein 70 (Hsc70) and downregulation of Aurora kinase A and TPX2. In this study, we evaluated the mechanisms of recombinant GLIPR1 protein (glioma pathogenesis‐related protein 1‐transmembrane domain deleted [GLIPR1‐ΔTM]) uptake by prostate cancer cells and the efficacy of systemic GLIPR1‐ΔTM administration in a prostate cancer xenograft mouse model. GLIPR1‐ΔTM was selectively internalized by prostate cancer cells, leading to increased apoptosis through reactive oxygen species production and to decreased c‐Myc protein levels. Interestingly, GLIPR1‐ΔTM was internalized through clathrin‐mediated endocytosis in association with Hsc70. Systemic administration of GLIPR1‐ΔTM significantly inhibited VCaP xenograft growth. GLIPR1‐ΔTM showed no evidence of toxicity following elimination from mouse models 8 hr after injection. Our results demonstrate that GLIPR1‐ΔTM is selectively endocytosed by prostate cancer cells, leading to increased reactive oxygen species production and apoptosis, and that systemic GLIPR1‐ΔTM significantly inhibits growth of VCaP xenografts without substantial toxicity.