Dietary Protein Restriction Reprograms Tumor-Associated Macrophages and Enhances Immunotherapy.

PURPOSE: Diet and healthy weight are established means of reducing cancer incidence and mortality. However, the impact of diet modifications on the tumor microenvironment and antitumor immunity is not well defined. Immunosuppressive tumor-associated macrophages (TAMs) are associated with poor clinical outcomes and are potentially modifiable through dietary interventions. We tested the hypothesis that dietary protein restriction modifies macrophage function toward antitumor phenotypes. EXPERIMENTAL DESIGN: Macrophage functional status under different tissue culture conditions and in vivo was assessed by Western blot, immunofluorescence, qRT-PCR, and cytokine array analyses. Tumor growth in the context of protein or amino acid (AA) restriction and immunotherapy, namely, a survivin peptide-based vaccine or a PD-1 inhibitor, was examined in animal models of prostate (RP-B6Myc) and renal (RENCA) cell carcinoma. All tests were two-sided. RESULTS: Protein or AA-restricted macrophages exhibited enhanced tumoricidal, proinflammatory phenotypes, and in two syngeneic tumor models, protein or AA-restricted diets elicited reduced TAM infiltration, tumor growth, and increased response to immunotherapies. Further, we identified a distinct molecular mechanism by which AA-restriction reprograms macrophage function via a ROS/mTOR-centric cascade. CONCLUSIONS: Dietary protein restriction alters TAM activity and enhances the tumoricidal capacity of this critical innate immune cell type, providing the rationale for clinical testing of this supportive tool in patients receiving cancer immunotherapies.

Therapeutic Targeting of TFE3/IRS-1/PI3K/mTOR Axis in Translocation Renal Cell Carcinoma.

PURPOSE: Translocation renal cell carcinoma (tRCC) represents a rare subtype of kidney cancer associated with various TFE3, TFEB, or MITF gene fusions that are not responsive to standard treatments for RCC. Therefore, the identification of new therapeutic targets represents an unmet need for this disease. EXPERIMENTAL DESIGN: We have established and characterized a tRCC patient-derived xenograft, RP-R07, as a novel preclinical model for drug development by using next-generation sequencing and bioinformatics analysis. We then assessed the therapeutic potential of inhibiting the identified pathway using in vitro and in vivo models. RESULTS: The presence of a SFPQ-TFE3 fusion [t(X;1) (p11.2; p34)] with chromosomal break-points was identified by RNA-seq and validated by RT-PCR. TFE3 chromatin immunoprecipitation followed by deep sequencing analysis indicated a strong enrichment for the PI3K/AKT/mTOR pathway. Consistently, miRNA microarray analysis also identified PI3K/AKT/mTOR as a highly enriched pathway in RP-R07. Upregulation of PI3/AKT/mTOR pathway in additional TFE3-tRCC models was confirmed by significantly higher expression of phospho-S6 (P < 0.0001) and phospho-4EBP1 (P < 0.0001) in established tRCC cell lines compared with clear cell RCC cells. Simultaneous vertical targeting of both PI3K/AKT and mTOR axis provided a greater antiproliferative effect both in vitro (P < 0.0001) and in vivo (P < 0.01) compared with single-node inhibition. Knockdown of TFE3 in RP-R07 resulted in decreased expression of IRS-1 and inhibited cell proliferation. CONCLUSIONS: These results identify TFE3/IRS-1/PI3K/AKT/mTOR as a potential dysregulated pathway in TFE3-tRCC, and suggest a therapeutic potential of vertical inhibition of this axis by using a dual PI3K/mTOR inhibitor for patients with TFE3-tRCC.

Therapeutic Targeting of Sunitinib-Induced AR Phosphorylation in Renal Cell Carcinoma.

Androgen receptor (AR) plays a crucial role in the development and progression of prostate cancer. AR expression has also been reported in other solid tumors, including renal cell carcinoma (RCC), but its biological role here remains unclear. Through integrative analysis of a reverse phase protein array, we discovered increased expression of AR in an RCC patient-derived xenograft model of acquired resistance to the receptor tyrosine kinase inhibitor (RTKi) sunitinib. AR expression was increased in RCC cell lines with either acquired or intrinsic sunitinib resistance in vitro An AR signaling gene array profiler indicated elevated levels of AR target genes in sunitinib-resistant cells. Sunitinib-induced AR transcriptional activity was associated with increased phosphorylation of serine 81 (pS81) on AR. Additionally, AR overexpression resulted in acquired sunitinib resistance and the AR antagonist enzalutamide-induced AR degradation and attenuated AR downstream activity in sunitinib-resistant cells, also indicated by decreased secretion of human kallikrein 2. Enzalutamide-induced AR degradation was rescued by either proteasome inhibition or by knockdown of the AR ubiquitin ligase speckle-type POZ protein (SPOP). In vivo treatment with enzalutamide and sunitinib demonstrated that this combination efficiently induced tumor regression in a RCC model following acquired sunitinib resistance. Overall, our results suggest the potential role of AR as a target for therapeutic interventions, in combination with RTKi, to overcome drug resistance in RCC.Significance: These findings highlight the therapeutic potential of targeting the androgen receptor to overcome RCC resistance to receptor tyrosine kinase inhibitors. Cancer Res; 78(11); 2886-96. ©2018 AACR.

Radiosensitizing Pancreatic Cancer Xenografts by an Implantable Micro-Oxygen Generator.

Over the past decades, little progress has been made to improve the extremely low survival rates in pancreatic cancer patients. Extreme hypoxia observed in pancreatic tumors contributes to the aggressive and metastatic characteristics of this tumor and can reduce the effectiveness of conventional radiation therapy and chemotherapy. In an attempt to reduce hypoxia-induced obstacles to effective radiation treatment, we used a novel device, the implantable micro-oxygen generator (IMOG), for in situ tumor oxygenation. After subcutaneous implantation of human pancreatic xenograft tumors in athymic rats, the IMOG was wirelessly powered by ultrasonic waves, producing 30 µA of direct current (at 2.5 V), which was then utilized to electrolyze water and produce oxygen within the tumor. Significant oxygen production by the IMOG was observed and corroborated using the NeoFox oxygen sensor dynamically. To test the radiosensitization effect of the newly generated oxygen, the human pancreatic xenograft tumors were subcutaneously implanted in nude mice with either a functional or inactivated IMOG device. The tumors in the mice were then exposed to ultrasonic power for 10 min, followed by a single fraction of 5 Gy radiation, and tumor growth was monitored thereafter. The 5 Gy irradiated tumors containing the functional IMOG exhibited tumor growth inhibition equivalent to that of 7 Gy irradiated tumors that did not contain an IMOG. Our study confirmed that an activated IMOG is able to produce sufficient oxygen to radiosensitize pancreatic tumors, enhancing response to single-dose radiation therapy.

Lovastatin enhances adenovirus-mediated TRAIL induced apoptosis by depleting cholesterol of lipid rafts and affecting CAR and death receptor expression of prostate cancer cells.

Oncolytic adenovirus and apoptosis inducer TRAIL are promising cancer therapies. Their antitumor efficacy, when used as single agents, is limited. Oncolytic adenoviruses have low infection activity, and cancer cells develop resistance to TRAIL-induced apoptosis. Here, we explored combining prostate-restricted replication competent adenovirus-mediated TRAIL (PRRA-TRAIL) with lovastatin, a commonly used cholesterol-lowering drug, as a potential therapy for advanced prostate cancer (PCa). Lovastatin significantly enhanced the efficacy of PRRA-TRAIL by promoting the in vivo tumor suppression, and the in vitro cell killing and apoptosis induction, via integration of multiple molecular mechanisms. Lovastatin enhanced PRRA replication and virus-delivered transgene expression by increasing the expression levels of CAR and integrins, which are critical for adenovirus 5 binding and internalization. Lovastatin enhanced TRAIL-induced apoptosis by increasing death receptor DR4 expression. These multiple effects of lovastatin on CAR, integrins and DR4 expression were closely associated with cholesterol-depletion in lipid rafts. These studies, for the first time, show correlations between cholesterol/lipid rafts, oncolytic adenovirus infection efficiency and the antitumor efficacy of TRAIL at the cellular level. This work enhances our understanding of the molecular mechanisms that support use of lovastatin, in combination with PRRA-TRAIL, as a candidate strategy to treat human refractory prostate cancer in the future.

Androgen-independent molecular imaging vectors to detect castration-resistant and metastatic prostate cancer.

Prostate-specific promoters are frequently employed in gene-mediated molecular imaging and therapeutic vectors to diagnose and treat castration-resistant prostate cancer (CRPC) that emerges from hormone ablation therapy. Many of the conventional prostate-specific promoters rely on the androgen axis to drive gene expression. However, considering the cancer heterogeneity and varying androgen receptor status, we herein evaluated the utility of prostate-specific enhancing sequence (PSES), an androgen-independent promoter in CRPC. The PSES is a fused enhancer derived from the prostate-specific antigen (PSA) and prostate-specific membrane antigen gene regulatory region. We augmented the activity of PSES by the two-step transcriptional amplification (TSTA) system to drive the expression of imaging reporter genes for either bioluminescent or positron emission tomography (PET) imaging. The engineered PSES-TSTA system exhibits greatly elevated transcriptional activity, androgen independency, and strong prostate specificity, verified in cell culture and preclinical animal experimentations. These advantageous features of PSES-TSTA elicit superior gene expression capability for CRPC in comparison with the androgen-dependent PSA promoter-driven system. In preclinical settings, we showed robust PET imaging capacity of PSES-TSTA in a castrated prostate xenograft model. Moreover, intravenous administrated PSES-TSTA bioluminescent vector correctly identified tibial bone marrow metastases in 9 of 9 animals, whereas NaF- and FDG-PET was unable to detect the lesions. Taken together, this study showed the promising utility of a potent, androgen-independent, and prostate cancer-specific expression system in directing gene-based molecular imaging in CRPC, even in the context of androgen deprivation therapy.

An ultrasonically powered implantable micro-oxygen generator (IMOG).

In this paper, we present an ultrasonically powered implantable micro-oxygen generator (IMOG) that is capable of in situ tumor oxygenation through water electrolysis. Such active mode of oxygen generation is not affected by increased interstitial pressure or abnormal blood vessels that typically limit the systemic delivery of oxygen to hypoxic regions of solid tumors. Wireless ultrasonic powering (2.15 MHz) was employed to increase the penetration depth and eliminate the directional sensitivity associated with magnetic methods. In addition, ultrasonic powering allowed for further reduction in the total size of the implant by eliminating the need for a large area inductor. IMOG has an overall dimension of 1.2 mm × 1.3 mm × 8 mm, small enough to be implanted using a hypodermic needle or a trocar. In vitro and ex vivo experiments showed that IMOG is capable of generating more than 150 µA which, in turn, can create 0.525 µL/min of oxygen through electrolytic disassociation. In vivo experiments in a well-known hypoxic pancreatic tumor models (1 cm (3) in size) also verified adequate in situ tumor oxygenation in less than 10 min.

The role of interleukin-12 on modulating myeloid-derived suppressor cells, increasing overall survival and reducing metastasis.

Myeloid-derived suppressor cells (MDSC) are important to the tumour microenvironment as they actively suppress the immune system and promote tumour progression and metastasis. These cells block T-cell activation in the tumour microenvironment, preventing anti-tumour immune activity. The ability of a treatment to alter the suppressive function of these cells and promote an immune response is essential to enhancing overall therapeutic efficacy. Interleukin-12 (IL-12) has the potential not only to promote anti-tumour immune responses but also to block the activity of cells capable of immune suppression. This paper identifies a novel role for IL-12 as a modulator of MDSC activity, with implications for IL-12 as a therapeutic agent. Treatment with IL-12 was found to alter the suppressive function of MDSC by fundamentally altering the cells. Interleukin-12-treated MDSC exhibited up-regulation of surface markers indicative of mature cells as well as decreases in nitric oxide synthase and interferon-γ mRNA both in vitro and in vivo. Treatment with IL-12 was also found to have significant therapeutic benefit by decreasing the percentage of MDSC in the tumour microenvironment and increasing the percentage of active CD8(+) T cells. Treatment with IL-12 resulted in an increase in overall survival accompanied by a reduction in metastasis. The findings in this paper identify IL-12 as a modulator of immune suppression with significant potential as a therapeutic agent for metastatic breast cancer.

Osteoblasts can stimulate prostate cancer growth and transcriptionally down-regulate PSA expression in cell line models.

INTRODUCTION: To investigate the effect of bone environment on cellular proliferation, mature prostate-specific antigen (PSA) production and secretion, and PSA transcriptional regulation of prostate cancer cells. MATERIALS AND METHODS: Androgen-independent C4-2 prostate cancer cells were co-cultured with various osteoblastic cells in a transwell system. Proliferation was measured via cell counting and MTT assay. Lactate and PSA were determined in the conditioned media (CM). Transcriptional activity of the full-length PSA promoter (6.1 kilobases) and of 3 deletion constructs was determined via luciferase reporter assay upon exposure to CM from various osteoblastic cell lines. RESULTS: Osteoblastic bone cells and CM, but not control cells (fibroblast) or CM, reproducibly stimulated the proliferation of C4-2 cells. The co-culture system, PSA production by C4-2 cells transiently decreased when in co-culture with osteoblastic, but not with control cells. After abundant prostate cell proliferation, the secreted PSA levels rose exponentially. Addition of CM from osteoblastic cells, but not control cells, consistently decreased (about 3-fold) the transcriptional activity of the PSA promoter in C4-2 cells. Deletion construct analysis of the PSA promoter revealed that the transcriptional down-regulation is dually controlled by elements close to the TATA and upstream androgen responsive (ARE(III)) components. CONCLUSIONS: The osteoblastic environment stimulates prostate cancer cell proliferation but reduces PSA production initially. The mechanism of PSA down-regulation is transcriptional, most likely in response to soluble factors present in the osteoblastic bone stromal cell CM. Transcriptional down-regulation appears to be mediated by elements near both the TATA box and the ARE(III) component.

Docetaxel increases antitumor efficacy of oncolytic prostate-restricted replicative adenovirus by enhancing cell killing and virus distribution.

BACKGROUND: We explored multiple molecular mechanisms of the combination of docetaxel and an oncolytic prostate-restricted replication competent adenovirus (Ad) (PRRA) in advanced prostate cancer (PCa) models. The combinational therapy has potential to overcome the therapeutic limitations of poor virus distribution inside solid tumors. METHODS: We evaluated the effect of docetaxel on the antitumor efficacy and efficiency of virus transduction, transgene expression and virus distribution of PRRA in a prostate-specific antigen/prostate-specific membrane antigen-positive tumor xenograft model. We also evaluated the effect of docetaxel on apoptosis induction, cell killing and the efficiency of transgene expression and virus replication in vitro. RESULTS: Tumor growth inhibition was significantly enhanced when docetaxel was administrated before intratumor injection of PRRA. In vivo dual-photon microscopy and ex vivo fluorescence microscopy and immunohistochemistry showed that docetaxel increased transgene expression and expanded virus distribution. The combination of docetaxel and PRRA also increased cell apoptosis. In vitro, docetaxel significantly increased cell killing in PRRA-treated PCa cells. Docetaxel significantly increased Ad-mediated trangene expression independent of Ad binding receptors and replication capability. Docetaxel increased the activity of cytomegalovirus (CMV) promoter but not of a chimeric prostate-specific enhancer, resulting in higher transgene expression. The enhanced CMV promoter activity resulted from activation of p38 mitogen-activated protein kinase (MAPK) because inhibition of p38 MAPK blocked the docetaxel-induced increase in CMV promoter activity. CONCLUSIONS: Combining docetaxel with an oncolytic PRRA improved therapeutic potential by expanding virus distribution and enhancing cell apoptosis and killing. These studies suggested a novel mechanism for enhancing the effect of therapeutic genes delivered by a PRRA.

TRAIL recombinant adenovirus triggers robust apoptosis in multidrug-resistant HL-60/Vinc cells preferentially through death receptor DR5.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic because of its highly selective apoptosis-inducing action on neoplastic versus normal cells. However, some cancer cells express resistance to recombinant soluble TRAIL. To overcome this problem, we used a TRAIL adenovirus (Ad5/35-TRAIL) to induce apoptosis in a drug-sensitive and multidrug-resistant variant of HL-60 leukemia cells and determined the molecular mechanisms of Ad5/35-TRAIL-induced apoptosis. Ad5/35-TRAIL did not induce apoptosis in normal human lymphocytes, but caused massive apoptosis in acute myelocytic leukemia cells. It triggered more efficient apoptosis in drug-resistant HL-60/Vinc cells than in HL-60 cells. Treating the cells with anti-DR4 and anti-DR5 neutralizing antibodies (particularly anti-DR5) reduced, whereas anti-DcR1 antibody enhanced, the apoptosis triggered by Ad5/35-TRAIL. Whereas Ad5/35-TRAIL induced apoptosis in both cell lines through activation of caspase-3 and caspase-10, known to link the cell death receptor pathway to the mitochondrial pathway, it triggered increased mitochondrial membrane potential change (m) only in HL-60/Vinc cells. Ad5/35-TRAIL also increased the production of reactive oxygen species, which play an important role in apoptosis. Therefore, using Ad5/35-TRAIL may be an effective therapeutic strategy for eliminating TRAIL-resistant malignant cells and these studies may provide clues to treat and eradicate acute myelocytic leukemias.

Suppression of renal cell carcinoma growth and metastasis with sustained antiangiogenic gene therapy.

Renal cell carcinoma (RCC) is the third most common urologic neoplasm. This aggressive malignancy has proven refractory to conventional treatment options. Antiangiogenic agents have shown early success in treating metastatic disease. The highly vascular nature of RCC appears particularly susceptible to this approach. This study investigates the potential of sustained expression of an endostatin-angiostatin fusion protein in an early-stage model of RCC to inhibit tumor growth and metastasis. Subcutaneous RCC-29 tumors were induced in athymic nude mice. Once tumors reached volumes of 10 and 25 mm(3), subjects received intratumoral injections of a nonreplicating adenoviral vector every 20 days until the conclusion of the trial. The mice were randomly assigned to three treatment groups: saline control, viral Ad-GFP control, and Ad-EndoAngio. Tumor volumes were measured twice weekly for 80 days. During days 40-50 of the trial, subjects underwent dual-photon optical imaging of the tumor vasculature to ascertain angiogenic changes. All animals underwent postmortem histopathological analysis to assess for metastatic disease in the kidney, lung, liver, brain, and spleen. Results indicate that tumors treated with Ad-EndoAngio displayed 97% growth reduction compared with controls (p < 0.001). Further, in vivo tumor vascular imaging illustrated a reduction in blood vessel number and lumen diameter size. Kaplan-Meier analysis suggested dramatic survival advantage with Ad-EndoAngio treatment. Importantly, histopathological examination demonstrated marked lung and liver metastasis suppression in the treatment arms. These results suggest that sustained EndoAngio gene therapy has effective antiangiogenic action against human RCC tumors and possesses potential as a novel treatment for metastatic renal cell carcinoma.

Prostate-restricted replicative adenovirus expressing human endostatin-angiostatin fusion gene exhibiting dramatic antitumor efficacy.

PURPOSE: Our previous studies coadministering a replication-deficient adenovirus expressing endostatin and angiostatin fusion gene (EndoAngio) and a prostate-restricted, replication-competent adenovirus (PRRA) showed dramatic antitumor efficacy. This study integrated EndoAngio with an improved PRRA vector to make a single antiangiogenic PRRA, thereby exerting a similarly dramatic antitumor effect with feasibility for future clinical trials. EXPERIMENTAL DESIGN: We developed an antiangiogenic PRRA with structural improvements. The antitumor efficacy of EndoAngio-PRRA was evaluated in prostate-specific antigen/prostate-specific membrane antigen (PSA/PSMA)-positive, androgen-independent CWR22rv tumor models. The tumor vasculature and cell morphology were observed by dual-photon microscopy. The antiangiogenic effect of EndoAngio delivered by PRRA and the killing activity of EndoAngio-PRRA were evaluated in vitro. Virus-inactivated conditioned media from virus-infected PSA/PSMA-positive cells were tested for apoptosis induction in prostate cancer cells. RESULTS: Our novel EndoAngio-PRRA is a strong antiangiogenic and antitumor agent. Nine of 10 CWR22rv tumors treated by EndoAngio-PRRA completely regressed, with 1 tumor remaining in a dormant status for 26 weeks after treatment. Dual-photon microscopy revealed that EndoAngio-PRRA not only inhibited the development of tumor vasculature but also induced apoptosis in tumor cells. Subsequent in vitro study indicated that EndoAngio-PRRA exhibited stronger tumor-specific killing activity than enhanced green fluorescent protein-PRRA, which expresses enhanced green fluorescent protein instead of EndoAngio. Virus-inactivated conditioned medium from EndoAngio-PRRA-infected PSA/PSMA-positive cells induced apoptosis in C4-2 and CWR22rv cells. CONCLUSIONS: EndoAngio-PRRA uniquely combines three distinct antitumor effects to eliminate androgen-independent prostate cancer: antiangiogenesis, viral oncolysis, and apoptosis. This novel antiangiogenic PRRA represents a powerful agent feasible for future clinical trials for prostate cancer therapy.

Anti-angiogenic gene therapy for metastatic renal cell carcinoma produces tumor growth suppression in an athymic nude mouse model.

PURPOSE: We investigated the anti-angiogenic and antitumor properties of 2 adenoviral vectors expressing the endostatin-angiostatin fusion protein Ad-EndoAngio and the soluble, endothelium specific tyrosine kinase receptor Ad-Tie2 in a mouse renal cell carcinoma xenograft model. MATERIALS AND METHODS: A total of 29 bilateral subcutaneous renal cell carcinomas were induced in athymic nude mice. On days 2 and 10 following tumor establishment the mice were intratumorally injected with an adenoviral vector in the right flank only. Seven treatment groups were randomly assigned, including the control group of 7 mice, the Ad-GFP control group of 7, the Ad-Tie2 group of 9, the Ad-EndoAngio group of 8, the Ad-GFP plus Ad-Tie2 group of 7, the Ad-GFP plus Ad-EndoAngio group of 9 and the Ad-EndoAngio plus Ad-Tie2 group of 8. Tumor volume was measured biweekly for 60 days. Additionally, each treatment group was administered fluorescent rhodamine conjugated bovine serum albumin dye for vascular imaging. After establishing skin windows overlying the tumors dual photon optical imaging was used to qualitatively assess the tumor vasculature. RESULTS: Tumors treated with Ad-EndoAngio, Ad-GFP plus Ad-EndoAngio and Ad-EndoAngio plus Ad-Tie2 demonstrated 82%, 83% and 87% growth reduction, respectively, compared to controls (p <0.001). Furthermore, in vivo imaging revealed a decrease in the number of blood vessels, lumen diameter and flow velocity in these treatment groups. CONCLUSIONS: Adenoviral vectors expressing endostatin-angiostatin fusion protein have effective anti-angiogenic action against human renal cell carcinoma cells as well as potential as a novel treatment for metastatic renal cell carcinoma.

Long-term outcome of phase I/II clinical trial of Ad-OC-TK/VAL gene therapy for hormone-refractory metastatic prostate cancer.

We evaluated the long-term safety and efficacy of Ad-OC-TK (recombinant adenoviral vector carrying an osteocalcin promoter-driven herpes simplex virus thymidine kinase gene) plus VAL (valacyclovir) gene therapy for hormone-refractory prostate cancer. Ad-OC-TK/VAL therapy is the first in vivo adenovirus-mediated gene therapy to be used to treat metastatic prostate cancer, including bone metastasis. Six patients were enrolled in this trial, and two doses of Ad-OC-TK (2.5 x 10(9) or 2.5 x 10(10) plaque-forming units) were injected into locally recurrent tumor or bone metastasis on day 1 and day 8. Patients were also given VAL (3 g/day) for 21 days. Safety and efficacy were evaluated for at least 8 months in each patient. All patients tolerated this therapy with no serious adverse events. One prostate-specific antigen (PSA) response (from 318.3 to 4.9 ng/ml) was observed with a time to PSA progression (TTP) of 12 months. Docetaxel (30 mg/m2 per week) and estramustine (560 mg/day) combination chemotherapy (DE) was given to three docetaxel-naive patients on PSA failure after gene therapy. All three patients had a PSA response to DE therapy with 21, 7, and 4 months of TTP. These results suggest that additional trials are warranted.

Fas ligand delivery by a prostate-restricted replicative adenovirus enhances safety and antitumor efficacy.

PURPOSE: Recent studies showed that Fas ligand (FasL) induced apoptosis in tumor cells and suppressed the immune response in several types of tumors. However, the toxicity of FasL limited further administration. This study delivered FasL in prostate cancer cells using an improved prostate-restricted replicative adenovirus (PRRA), thereby improving the antitumor effect while decreasing systemic toxicity. EXPERIMENTAL DESIGN: We designed a FasL-armed PRRA, called AdIU3, by placing adenoviral E1a and E4 genes, FasL cDNA, and E1b gene under the control of two individual PSES enhancers. Tissue-specific viral replication and FasL expression were analyzed, and the tumor killing effect of AdIU3 was investigated both in vitro and in vivo using androgen-independent CWR22rv s.c. models via local administration and bone models via systemic administration. The safety of systemic administration of AdIU3 was evaluated. AdCMVFasL, in which FasL was controlled by a universal cytomegalovirus (CMV) promoter, was used as a control. RESULTS: AdIU3 enhanced FasL expression in prostate-specific antigen (PSA)/prostate-specific membrane antigen (PSMA)-positive cells but not in PSA/PMSA-negative cells. It induced apoptosis and killed PSA/PMSA-positive prostate cancer cells but spared normal human fibroblasts, hepatocytes, and negative cells. The increase in killing activity was confirmed to result in part from a bystander killing effect. Furthermore, AdIU3 was more effective than a plain PRRA in inhibiting the growth of androgen-independent prostate cancer xenografts and bone tumor formation. Importantly, systemic administration of AdIU3 resulted in undetectable toxicity, whereas the same doses of AdCMVFasL killed all mice due to multiviscera failure in 16 h. CONCLUSIONS: AdIU3 decreased the toxicity of FasL by controlling its expression with PSES, with greatly enhanced prostate cancer antitumor efficacy. The results suggested that toxic antitumor factors can be delivered safely by a PRRA.

Advances in preclinical investigation of prostate cancer gene therapy.

Treating recurrent prostate cancer poses a great challenge to clinicians. Research efforts in the last decade have shown that adenoviral vector-based gene therapy is a promising approach that could expand the arsenal against prostate cancer. This maturing field is at the stage of being able to translate many preclinical discoveries into clinical practices. At this juncture, it is important to highlight the promising strategies including prostate-targeted gene expression, the use of oncolytic vectors, therapy coupled to reporter gene imaging, and combined treatment modalities. In fact, the early stages of clinical investigation employing combined, multimodal gene therapy focused on loco-regional tumor eradication and showed promising results. Clinicians and scientists should seize the momentum of progress to push forward to improve the therapeutic outcome for the patients.

Current status of anti-angiogenesis therapy for prostate cancer.

Prostate cancer is the second leading cause of cancer mortality in American men and the single most diagnosed cancer in men. Despite advances in early detection and conventional treatment strategies, prostate cancer progresses and becomes resistant to treatment. Because tumor growth and establishment of metastases are dependent on angiogenesis, interest in the development of anti-angiogenesis therapies has grown. Preclinical studies and early clinical evaluation show promise in the adjunctive use of anti-angiogenesis to overcome the limitations of current therapeutic approaches. In this review, we outline the basic science principles of angiogenesis and their application in the development of anticancer therapies.

Future innovations in treating advanced prostate cancer.

Many novel techniques for the treatment of prostate cancer are being aggressively investigated because prostate cancer is prevalent in the population and the current treatments for advanced prostate cancer are woefully inadequate. Although the current treatment options prolong life, most patients will eventually experience local recurrence or develop advanced disease. A greater understanding of the molecular events underlying cancer has enabled investigators to explore gene therapy approaches that are targeted against these molecular events. This article discusses antiangiogenic therapy, immune based therapy, and gene therapy. Any of these experimental modalities could be developed to replace hormone ablation therapy which causes unpleasant side effects, decreases the quality of life of the patient, and only temporarily controls the disease.

Anti-tumor efficacy of a transcriptional replication-competent adenovirus, Ad-OC-E1a, for osteosarcoma pulmonary metastasis.

BACKGROUND: Osteosarcoma (OSA) is the most frequent type of primary malignant bone tumor and is apt to occur in children and young adults. Pulmonary metastasis (OSPM) is the major reason for its fatal outcome. Osteocalcin (OC) is a major noncollagenous bone protein whose expression is limited almost exclusively to bone marrow and osteotropic tumors. OC is also known to express in cell lines with bone metastasis feathers. Gene therapy strategies with the OC promoter directing the replication of adenovirus in a tumor-specific manner are a potential modality for OSPM therapy. METHODS: We detected OC mRNA expression by RNA in situ hybridization in OSA and OSPM samples from patients, and tested OC promoter transcriptional activity in OSA and non-OSA cell lines. Then we used a transcriptional replication-competent adenovirus, Ad-OC-E1a, to treat OSPM, and evaluated its tumor-specific replication and killing activities in vitro as well as anti-OSPM efficacy in vivo via systemic delivery. RESULTS: OC mRNA was detected in all types of OSA tissues, including OSPM tissues. The transcriptional activity of the OC promoter was much higher in a OSPM cell line SAOS-2LM7 and primary OSA cell line MG63 than in non-OSA cell lines, including cell lines from breast cancer, colon cancer, and liver cancer. Ad-OC-E1a expressed E1a protein only in MG63 and SAOS-2LM7, which indicated that adenovirus E1a was under strict control by the OC promoter. Ad-OC-E1a demonstrated killing and viral replication activity close to wild-type adenovirus levels in MG63 and SAOS-2LM7, but the killing and viral replication activities were attenuated significantly in cells expressing low OC transcriptional activity. To test whether Ad-OC-E1a could be used to target human OSPM in vivo, SAOS-2LM7 pulmonary metastasis models in nude mice were induced and treated by tail-vein injection with Ad-OC-E1a. Compared to tumor nodules in the lung in groups treated with PBS or control virus, the quantity of metastasized tumor nodules decreased significantly. Adenovirus-infected cells were stained immunohistochemically only inside and around the OSPM nodules but spared normal lung tissue and other organs. CONCLUSIONS: These data demonstrated that OC promoter could direct adenovirus replication by controlling the E1a gene to target human OSPM in a tumor-specific manner, providing an efficient tool to develop a feasible therapeutic modality for OSPM.