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.

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.

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.

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.

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.

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.

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.

Gene therapy for prostate cancer by controlling adenovirus E1a and E4 gene expression with PSES enhancer.

PSES is a chimeric enhancer containing enhancer elements from prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) genes that are prevalently expressed in androgen-independent prostate cancers. PSES shows strong activity equivalent to cytomegalovirus (CMV) promoter, specifically in PSA/PSMA-positive prostate cancer cells, the major cell types in prostate cancer in the absence of androgen. We developed a recombinant adenovirus (AdE4PSESE1a) by placing adenoviral E1a and E4 genes under the control of the bidirectional enhancer PSES and enhanced green fluorescent protein gene for the purpose of intratumoral virus tracking under the control of CMV promoter. Because of PSES being very weak in nonprostatic cells, including HEK293 and HER911 that are frequently used to produce recombinant adenovirus, AdE4PSESE1a can only be produced in the HER911E4 cell line which expresses both E1 and E4 genes. AdE4PSESE1a showed similar viral replication and tumor cell killing activities to wild-type adenovirus in PSA/PSMA-positive prostate cancer cells. The viral replication and tumor cell killing activities were dramatically attenuated in PSA/PSMA-negative cells. To test whether AdE4PSESE1a could be used to target prostate tumors in vivo, CWR22rv s.c. tumors were induced in nude mice and treated with AdE4PSESE1a via intratumoral and tail vein injection. Compared to tumors treated with control virus, the growth of CWR22rv tumors was dramatically inhibited by AdE4PSESE1a via tail vein injection or intratumoral injection. These data show that adenoviral replication can be tightly controlled in a novel fashion by controlling adenoviral E1a and E4 genes simultaneously with a single enhancer.

Combination therapy of androgen-independent prostate cancer using a prostate restricted replicative adenovirus and a replication-defective adenovirus encoding human endostatin-angiostatin fusion gene.

Although prostate-restricted replicative adenovirus has exhibited significant antitumor efficacy in preclinical studies, it is necessary to develop more potent adenoviruses for prostate cancer gene therapy. We evaluated the synergistic killing effect of prostate-restricted replicative adenovirus and AdEndoAngio, a replication-defective adenovirus expressing the endostatin-angiostatin fusion protein (EndoAngio). When coadministered with AdEndoAngio, prostate-restricted replicative adenovirus significantly elevated EndoAngio expression, suggesting that AdEndoAngio coreplicates with prostate-restricted replicative adenovirus. Conditioned medium from prostate cancer cells infected by prostate-restricted replicative adenovirus plus AdEndoAngio inhibited the growth, tubular network formation, and migration of human umbilical vein endothelial cells better than conditioned medium from prostate cancer cells infected by AdEndoAngio alone. Furthermore, in vivo animal studies showed that the coadministration of prostate-restricted replicative adenovirus plus AdEndoAngio resulted in the complete regression of seven out of eight treated androgen-independent CWR22rv tumors, with a tumor nodule maintaining a small size for 14 weeks. The residual single tumor exhibited extreme pathologic features together with more endostatin-reactive antibody-labeled tumor cells and fewer CD31-reactive antibody-labeled capillaries than the AdEndoAngio-treated tumors. These results show that combination therapy using prostate-restricted replicative adenovirus together with antiangiogenic therapy has more potent antitumor effects and advantages than single prostate-restricted replicative adenovirus and deserves more extensive investigation.

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.

A novel targeting modality to enhance adenoviral replication by vitamin D(3) in androgen-independent human prostate cancer cells and tumors.

We report the development of a novel replication-competent adenoviral vector, Ad-hOC-E1, containing a single bidirectional human osteocalcin (hOC) promoter to drive both the early viral E1A and E1B gene. This vector selectively replicated in OC-expressing but not non-OC-expressing cells, with viral replication enhanced at least 10-fold on vitamin D(3) exposure. Both the artificial TATA-box and hOC promoter element in this bidirectional promoter construct were controlled by a common OC regulatory element which selectively activated OC expression in cells. The expression ofE1A and E1B gene by Ad-hOC-E1 can be markedly induced by vitamin D(3). Unlike Ad-sPSA-E1, an adenoviral vector with viral replication controlled by a strong super prostate-specific antigen (sPSA) promoter which only replicates in PSA-expressing cells with androgen receptor (AR), Ad-hOC-E1 retarded the growth of both androgen-dependent and androgen-independent prostate cancer cells irrespective of their basal level of AR and PSA expression. A single i.v. administration of 2 x 10(9) plaque-forming units of Ad-hOC-E1 inhibited the growth of previously established s.c. DU145 tumors (an AR- and PSA-negative cell line). Viral replication is highly enhanced by i.p. administration of vitamin D(3). Ultimately, enhancing Ad-hOC-E1 viral replication by vitamin D(3) may be used clinically to treat localized and osseous metastatic prostate cancer in men.

Transcriptional targeting modalities in breast cancer gene therapy using adenovirus vectors controlled by alpha-lactalbumin promoter.

The breast-specific antigen alpha-lactalbumin is expressed in >60% of breast cancer tissues. To evaluate the effect of gene therapy for breast cancer by controlling adenovirus replication with human alpha-lactalbumin promoter, we investigated the activity of a 762-bp human alpha-lactalbumin promoter. Alpha-lactalbumin promoter showed significantly higher activity in MDA-MB-435S and T47D breast cancer cells than in normal breast cell lines or other tumor cell lines. We then developed two novel breast cancer-restricted replicative adenoviruses, AdALAE1a and AdE1aALAE1b. In AdALAE1a, expression of adenoviral E1a gene is under the control of alpha-lactalbumin promoter, and in AdE1aALAE1b, expression of both E1a and E1b genes is under the control of a single alpha-lactalbumin promoter. Both breast cancer-restricted replicative adenoviruses showed viral replication efficiency and tumor cell-killing capability similar to wild-type adenovirus in MDA-MB-435S and T47D cells. The replication efficiency and tumor cell-killing capability of both viruses were attenuated significantly in cells that did not support alpha-lactalbumin promoter. AdE1aALAE1b showed better breast cancer-restricted replication than AdALAE1a, suggesting that a transcriptional targeting modality with alpha-lactalbumin promoter controlling both E1a and E1b gene expression is superior to alpha-lactalbumin promoter controlling only E1a gene expression. Importantly, we found that AdE1aALAE1b could be used to target hormone-independent breast tumors in vivo by inhibiting the growth of MDA-MB-435S s.c. tumors. These data showed that alpha-lactalbumin promoter could regulate the replication of adenovirus to target hormone-independent breast cancers, suggesting that alpha-lactalbumin promoter can be used to develop a novel therapeutic modality for hormone-independent breast cancer.

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.

NFATc1 with AP-3 site binding specificity mediates gene expression of prostate-specific-membrane-antigen.

Prostate-specific-membrane-antigen (PSMA) is a marker protein expressed primarily in prostate epithelium. Its prostate-specific expression is conferred by PSMA enhancer (PSME), localized within the third intron of PSMA-encoding gene FOLH1. We recently reported that the 5'-end 90 bp of PSME harbored crucial enhancer elements for high PSMA expression. Deletion of this 90 bp sequence, called PSME(del3), significantly diminished PSME activity. We have further analyzed the regulatory elements in this 90 bp by transient transfection of linker scanning mutants. Two mutants, LN17 and 18, which harbored an AP-1 site and an AP-3 site, respectively, exhibited significantly lower enhancer activity. Subsequent site-directed mutagenesis changing the AP-3 site abolished the enhancer activity of PSME but not AP-1, indicating that AP-3 was the key cis-element enabling high PSMA expression. In addition, a 12 bp AP-3 site was able to enhance PSME(del3) activity by almost 40% higher compared to full-length PSME. However, AP-3 alone retained just the basal level of activity, indicating that the action by AP-3 was mediated by cooperation with other transcription factors binding to the PSME(del3) region. Transcription factor NFATc1 isoforms in nuclear extract were co-precipitated with the biotinylated AP-3 site by immobilized agarose beads and the genomic DNA containing PSME was precipitated by antibodies reactive to NFATc1, demonstrating that NFATc1 isoforms bound to the AP-3 site in PSME in vivo. Furthermore, ionomycin (calcium ionophore) and TPA augmented the enhancer activity of PSME, implying that calcium is an important regulator for PSMA expression in prostate cancer cell.

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.

Phase I dose escalation clinical trial of adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase in localized and metastatic hormone-refractory prostate cancer.

Osteocalcin (OC), a major noncollagenous bone matrix protein, is expressed prevalently in prostate cancer epithelial cells, adjacent fibromuscular stromal cells, and osteoblasts in locally recurrent prostate cancer and prostate cancer bone metastasis [Matsubara, S., Wada, Y., Gardner, T.A., Egawa, M., Park, M.S., Hsieh, C.L., Zhau, H.E., Kao, C., Kamidono, S., Gillenwater, J.Y., and Chung, L.W. (2001). Cancer Res. 61, 6012-6019]. We constructed an adenovirus vector carrying osteocalcin promoter-driven herpes simplex virus thymidine kinase (Ad-OC-hsv-TK) to cotarget prostate cancer cells and their surrounding stromal cells. A phase I dose escalation clinical trial of the intralesional administration of Ad-OC-hsv-TK followed by oral valacyclovir was conducted at the University of Virginia (Charlottesville, VA) in 11 men with localized recurrent and metastatic hormone-refractory prostate cancer (2 local recurrent, 5 osseous metastasis, and 4 lymph node metastasis) in order to determine the usefulness of this vector for the palliation of androgen-independent prostate cancer metastasis. This is the first clinical trial in which therapeutic adenoviruses are injected directly into prostate cancer lymph node and bone metastasis. Results show that (1). all patients tolerated this therapy with no serious adverse events; (2). local cell death was observed in treated lesions in seven patients (63.6%) as assessed by TUNEL assay, and histomorphological change (mediation of fibrosis) was detected in all posttreated specimens; (3). one patient showed stabilization of the treated lesion for 317 days with no alternative therapy. Of the two patients who complained of tumor-associated symptoms before the treatment, one patient with bone pain had resolution of pain, although significant remission of treated lesions was not observed by image examination; (4). CD8-positive T cells were predominant compared with CD4-positive T cells, B cells (L26 positive), and natural killer cells (CD56 positive) in posttreated tissue specimens; (5). levels of HSV TK gene transduction correlated well with coxsackie-adenovirus receptor expression but less well with the titers of adenovirus injected; and (6). intrinsic OC expression and the efficiency of HSV TK gene transduction affected the levels of HSV TK protein expression in clinical specimens. Our data suggest that this form of gene therapy requires further development for the treatment of androgen-independent prostate cancer metastasis although histopathological and immunohistochemical evidence of apoptosis was observed in the specimens treated. Further studies including the development of viral delivery will enhance the efficacy of Ad-OC-hsv-TK.

Tumor-specific gene therapy for uterine cervical cancer using MN/CA9-directed replication-competent adenovirus.

Although gene therapies using tissue-specific promoters have been reported to be a promising tool for treating cancers, few studies have explored this possibility for uterine cervical cancer. MN/CA9 is a transmembrane glycoprotein that was first identified in the human cervical carcinoma cell line, HeLa. Since MN/CA9 protein is highly expressed in uterine cervical cancer tissues, but not in normal cervix, we constructed a tumor-specific replication-competent adenoviral vector utilizing MN/CA9 promoter (Ad-MN/CA9-E1a), which can replicate only in MN/CA9-expressing cells. Infection of Ad-MN/CA9-E1a to MN/CA9-positive uterine cervical cancer cells (HeLa, C-33 A and SiHa) resulted in much stronger Ad5 E1a protein expressions compared with MN/CA9-negative cells (SK-RC-29), suggesting a tissue-specific replication of this recombinant adenovirus. In vitro cytotoxicity assay revealed that the growth of MN/CA9-positive cells was significantly inhibited with 0.01-1 MOI of Ad-MN/CA9-E1a, but the growth of MN/CA9-negative cells (SK-RC-29) could only be inhibited by as many as 100 MOI. Intratumoral injection of Ad-MN/CA9-E1a effectively induced growth delay of HeLa tumors in nude mice. These results suggest that a novel replication-competent adenoviral vector mediated by MN/CA9 promoter, Ad-MN/CA9-E1a, can selectively replicate in MN/CA9-expressing tumors with cytotoxic effects and may be utilized for the treatment of uterine cervical cancer.