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.     

Combining high selectivity of replication with fiber chimerism for effective adenoviral oncolysis of CAR-negative melanoma cells

Oncolytic adenoviruses constitute a new and promising tool for cancer treatment that has been rapidly translated into clinical trials. However, minimal or absent expression of the adenovirus serotype 5 (Ad5) receptor CAR (coxsackievirus and adenovirus receptor) on cancer cells represents a major limitation for Ad5-based oncolysis. Here, we report on the resistance of CAR-negative primary melanoma cells to cell killing by wild-type Ad5 (Ad5wt) even after high titer infection, thus underlining the need for tropism-modification of oncolytic adenoviruses. We engineered a new generation of oncolytic adenoviruses that exhibit both efficient target cell infection by swapping Ad5 fiber domains with those of Ad serotype 3, which binds to a receptor distinct from CAR, and targeted virus replication. Fiber chimerism resulted in efficient cytopathicity to primary melanoma cells, which was at least 10(4)-fold increased relative to Ad5wt. Since viral infectivity mediated by such modified viral capsids was not cell type-specific, it was pivotal to carefully restrict adenoviral replication to target cells. Towards this end, we replaced both E1A and E4 promoters of fiber chimeric viruses by tyrosinase enhancer/promoter constructs. The resulting viruses showed melanoma-specific expression of E1A and E4 and combined efficient virus replication and cell killing in melanoma cell lines and primary melanoma cells with a remarkable specificity profile that implements strong attenuation in nonmelanoma cells, including normal fibroblasts and keratinocytes.     

Use of synthetic vectors for neutralising antibody resistant delivery of replicating adenovirus DNA

Use of synthetic vectors to deliver genomes of conditionally replicating lytic viruses combines the strengths of viral and non-viral approaches by enabling neutralising antibody resistant deployment of cancer virotherapy. Adenovirus is particularly suitable for this application since all proteins essential for replication can be expressed from the input DNA, although the presence of terminal protein (TP) covalently linked to the 5' termini of the input virus genomes both improves expression of transgenes encoded in the input DNA and also enhances replication. These roles of TP were distinguished in experiments where E1-deleted Ad(GFP)DNA bearing TP (Ad(GFP)DNA-TP), delivered with DOTAP, gave a two-fold greater frequency of transduction than Ad(GFP)DNA(without TP) in non-complementing A549 cells, while in 293 cells (which support replication of E1-deleted viruses) the presence of TP mediated a much greater differential transgene expression, commensurate with its ability to promote replication. Subsequent studies using AdDNA for virotherapy, therefore, included covalently linked TP. AdDNA-TP delivered to A549 cells using a synthetic polyplex vector was shown to be resistant to levels of neutralising antisera that completely ablated infection by wild-type adenovirus, enabling polyplex/Ad(wild type)DNA-TP to mediate a powerful cytopathic effect. Similarly in vivo, direct injection of a polyplex/Ad(wild type)DNA-TP into A549 tumours was neutralising antibody-resistant and enabled virus replication, whereas intact virus was neutralised by the antibody and failed to infect. The delivery of adenovirus genomes-TP using synthetic vectors should provide a strategy to bypass neutralising antibodies and facilitate clinical application of replicating adenovirus for cancer virotherapy.     

E1A- and E1B-Double mutant replicating adenovirus elicits enhanced oncolytic and antitumor effects

Gene-modified replication-competent adenoviruses (Ads) are emerging as a promising new modality for the treatment of cancer. We have previously shown that E1B 19kDa and E1B 55kDa gene-deleted Ad (Ad-DeltaE1B19/55) exhibits improved tumor-specific replication and cell lysis, leading to an enhanced antitumor effect. In an effort to increase cancer cell selectivity of a replicating adenovirus, we first generated 11 E1A mutant Ads (Ad-E1mt1 to Ad-E1mt11) with deletion or substitution in retinoblastoma (pRb)-binding sites of E1A. Of these, Ad-E1mt7 demonstrated significant improvement in cytopathic effect (CPE) and viral replication in a cancer cell-specific manner. To further enhance the cancer cell specificity of Ad-E1mt7, Ad-DeltaE1Bmt7 was generated, in which both the E1B 19kDa and E1B 55kDa genes were deleted. As assessed in CPE assay and immunoblot analysis for Ad fiber expression, Ad-DeltaE1Bmt7 exerted marked enhancement in cancer cell-specific killing as well as viral replication in comparison with its comparative controls (Ad-E1mt7, Ad-DeltaE1B55). Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-DeltaE1Bmt7. In summary, we have developed an oncolytic adenovirus with a significantly improved therapeutic profile for cancer treatment.     

稳定沉默Rictor表达的前列腺癌CWR22RV1的构建

目的应用RNA沉默技术下调前列腺癌CWR22RV1细胞株的Rictor基因表达,并筛选具有稳定转染Rictor-shRNA的细胞株,为进一步研究Rictor在前列腺癌中的作用打下基础。方法针对Rictor基因设计合成siRNA序列;鉴定、测序并转染293T细胞观察基因表达情况;构建Rictor-shRNA慢病毒载体;进行Rictor-shRNA慢病毒包装及滴度测定;筛选稳定表达Rictor-shRNA的前列腺癌CWR22RV1细胞株;Western blot检测稳定转染前列腺癌CWR22RV1细胞株Rictor的表达水平。结果 Rictor-shRNA慢病毒成功包装后转染前列腺癌CWR22RV1细胞,进行嘌呤霉素筛选,获得沉默Rictor基因的前列腺癌CWR22RV1细胞株,Western blot检测显示该细胞株Rictor水平明显降低（P〈0.01）。结论成功构建了稳定沉默Rictor表达的前列腺癌CWR22RV1细胞株,为后续的实验了打下坚实的基础。     

Sensitizing hormone-refractory prostate cancer cells to drug treatment by targeting 14-3-3sigma

Advanced and hormone-refractory prostate cancer has long been considered as a chemoresistant disease. Recently, it was found that 14-3-3sigma expression increases as prostate tumor progresses, and that 14-3-3sigma contributes significantly to drug resistance in breast cancers. We, thus, hypothesized that advanced and hormone-refractory prostate cancers may have an increased level of 14-3-3sigma, which in turn may contribute to drug resistance in advanced and hormone-refractory prostate cancers. In this study, we tested this hypothesis and found that, indeed, the expression level of 14-3-3sigma in androgen-independent prostate cancer cell lines DU145, PC3, and CWR22RV are much higher than that in the androgen-dependent cell line LNCaP, and that the androgen-independent cells are more resistant to mitoxantrone and Adriamycin than the androgen-dependent cells. Depleting 14-3-3sigma expression in DU145 and CWR22RV by RNA interference significantly sensitized these cells to mitoxantrone and Adriamycin by abrogating G2-M checkpoint and increasing apoptosis, whereas restoring 14-3-3sigma expression in LNCaP cells enhanced drug resistance. We also showed that 14-3-3sigma deficiency caused nuclear localization of Cdc2 and dephosphorylation of the Tyr15 residue upon DNA damage. Based on these studies, we propose that therapeutic intervention targeting 14-3-3sigma may be useful for sensitizing hormone-refractory prostate cancers to chemotherapy by both G2-M checkpoint abrogation and apoptosis enhancement.     

In vitro primary cell culture as a physiologically relevant method for preclinical testing of human oncolytic adenovirus

Ad[I/PPT-E1A] is an oncolytic adenovirus that specifically kills prostate cells via restricted replication by a prostate-specific regulatory element. Off-target replication of oncolytic adenoviruses would have serious clinical consequences. As a proposed ex vivo test, we describe the assessment of the specificity of Ad[I/PPT-E1A] viral cytotoxicity and replication in human nonprostate primary cells. Four primary nonprostate cell types were selected to mimic the effects of potential in vivo exposure to Ad[I/PPT-E1A] virus: bronchial epithelial cells, urothelial cells, vascular endothelial cells, and hepatocytes. Primary cells were analyzed for Ad[I/PPT-E1A] viral cytotoxicity in MTS assays, and viral replication was determined by hexon titer immunostaining assays to quantify viral hexon protein. The results revealed that at an extreme multiplicity of infection of 500, unlikely to be achieved in vivo, Ad[I/PPT-E1A] virus showed no significant cytotoxic effects in the nonprostate primary cell types apart from the hepatocytes. Transmission electron microscopy studies revealed high levels of Ad[I/PPT-E1A] sequestered in the cytoplasm of these cells. Adenoviral green fluorescent protein reporter studies showed no evidence for nuclear localization, suggesting that the cytotoxic effects of Ad[I/PPT-E1A] in human primary hepatocytes are related to viral sequestration. Also, hepatocytes had increased amounts of coxsackie adenovirus receptor surface protein. Active viral replication was only observed in the permissive primary prostate cells and LNCaP prostate cell line, and was not evident in any of the other nonprostate cells types tested, confirming the specificity of Ad[I/PPT-E1A]. Thus, using a relevant panel of primary human cells provides a convenient and alternative preclinical assay for examining the specificity of conditionally replicating oncolytic adenoviruses in vivo.     

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.     

Targeting the replication of adenoviral gene therapy vectors to lung cancer cells: the importance of the adenoviral E1b-55kD gene

It has been proposed that an adenovirus with the E1b-55kD gene deleted has a selective advantage in replicating in cancer cells that have mutations in the p53 gene (Bischoff et al., 1996). We have explored this hypothesis in several lung cancer cell lines, and evaluated potential mechanisms that might regulate the replication of Ad338, an E1b-55kD-deleted virus, with the objective of developing a rational approach for targeting gene therapy to lung tumors. Our data show that Ad338 replicates poorly in three lung cancer cell lines with various p53 mutations (H441, H446, and Calu1), yet this virus replicates to a high level in a lung cancer cell line with wild-type p53 (A549) and in a normal lung fibroblast line (IMR90). Viral DNA replication, expression of viral proteins, and shutoff of host cell proteins were not important variables in limiting the replication of the E1b-55kD-deleted virus. However, the cell lines resistant to host cell protein shutoff were also the most resistant to the cytopathic effect induced by mutant and wild-type virus and the only cells to survive for 8 days following infection. The E1b-55kD protein clearly has an important role in viral replication beyond its interaction with p53. Thus, an E1b-55kD-deleted virus cannot be used to specifically target viral replication to p53-mutated lung cancer cells.     

Modulation of protein phosphatase 2A activity alters androgen-independent growth of prostate cancer cells: therapeutic implications

Earlier we identified PPP2CA, which encodes for the α-isoform of protein phosphatase 2A (PP2A) catalytic subunit, as one of the downregulated genes in androgen-independent prostate cancer. PP2A is a serine/threonine phosphatase and a potent tumor suppressor involved in broad cellular functions; however, its role in prostate cancer has not yet been determined. Here, we have investigated the effect of PP2A activity modulation on the androgen-independent growth of prostate cancer cells. Our data show that the PPP2CA expression and PP2A activity is downregulated in androgen-independent (C4-2) prostate cancer cells as compared with androgen-dependent (LNCaP) cells. Downregulation of PP2A activity by pharmacologic inhibition or short interfering RNA-mediated PPP2CA silencing sustains the growth of LNCaP cells under an androgen-deprived condition by relieving the androgen deprivation-induced cell-cycle arrest and preventing apoptosis. Immunoblot analyses reveal enhanced phosphorylation of Akt, extracellular signal-regulated kinase (ERK), BAD, increased expression of cyclins (A1/D1), and decreased expression of cyclin inhibitor (p27) on PP2A downregulation. Furthermore, our data show that androgen receptor (AR) signaling is partially maintained in PP2A-inhibited cells through increased AR expression and ligand-independent phosphorylation. Pharmacologic inhibition of Akt, ERK, and AR suggest a role of these signaling pathways in facilitating the androgen-independent growth of LNCaP cells. These observations are supported by the effect of ceramide, a PP2A activator, on androgen-independent C4-2 cells. Ceramide inhibited the growth of C4-2 cells on androgen deprivation, an effect that could be abrogated by PP2A downregulation. Altogether, our findings suggest that modulation of PP2A activity may represent an alternative therapeutic approach for the treatment of advanced androgen-independent prostate cancer.     

Gene therapy for prostate cancer delivered by ovine adenovirus and mediated by purine nucleoside phosphorylase and fludarabine in mouse models

A gene-directed enzyme pro-drug therapy (GDEPT) based on purine nucleoside phosphorylase (PNP), that converts the prodrug, fludarabine to 2-fluoroadenine, has been described, but studies are limited compared with other GDEPTs. We investigated the in vitro and in vivo efficacies of PNP-GDEPT for treating androgen-independent (AI) prostate cancer. The PNP gene controlled by Rous sarcoma virus (RSV) constitutive promoter was delivered using a recombinant ovine adenovirus vector (OAdV220) that uses a different receptor from human adenovirus type 5. In vitro, OAdV220 provided increased transgene expression over a comparable human Ad5 vector in infected AI, murine RM1 prostate cancer cells. Subsequent in vivo testing was therefore confined to OAdV220. Transduction of RM1 cells with OAdV220 before implantation in immunocompetent mice dramatically inhibited subcutaneous (s.c.) tumor growth when fludarabine phosphate was administered systemically and increased mouse survival in a dose-dependent manner. In tumor-bearing C57BL/6 mice, a single intratumoral injection of OAdV220 produced detectable PNP activity for at least 6 days and with prodrug, retarded the growth of aggressive RM1 s.c. tumors by 35% at day 14. There was a consistent trend to reduction of pre-established intraprostatic RM1 tumors. A similar regimen induced significant therapeutic efficacy in human PC3 xenografts. Thus, ovine adenovirus-mediated GDEPT using the PNP system was effective in vivo against AI prostate cancers, the aggressive murine RM1, and the human PC3 lines. Methods that improve viral dissemination and stimulate the immune system in vivo may further improve efficacy.     

Characterization of infectivity-enhanced conditionally replicating adenovectors for prostate cancer radiovirotherapy

Abstract Prostate cancer (PCa) is the second most commonly diagnosed and sixth leading cause of cancer death in American men and one for which no curative therapy exists after metastasis. To meet this need for novel therapies, our laboratory has previously generated conditionally replicating adenovirus (CRAd) vectors expressing the sodium iodide symporter (hNIS). This virus transduced PCa cells and induced functional NIS expression, allowing for noninvasive tumor imaging and combination therapy with radioiodide, referred to as radiovirotherapy. We have now generated two new modified vectors to further improve efficacy. Ad5/3PB-ADP-hNIS and Ad5/3PB-hNIS include a hybrid Ad5/3 fiber knob to improve transduction efficiency, and express NIS from the endogenous major late promoter to restrict NIS expression to target cells. Additionally, Ad5/3PB-ADP-hNIS includes the adenovirus death protein (ADP), which hastens the release of viral particles after assembly. These two vectors specifically induce radioisotope uptake, cytopathic effect, and viral replication in androgen receptor-expressing PCa cell lines with Ad5/3PB-ADP-hNIS showing earlier (131)I uptake and cytolysis at low multiplicity of infection. SPECT-CT imaging of xenograft tumors infected with Ad5/3PB-hNIS showed steady uptake, whereas infection with Ad5/3PB-ADP-hNIS led to increasing uptake, indicating viral spread. Radiovirotherapy of xenograft LNCaP tumors with Ad5/3PB-ADP-hNIS showed the most significant survival extension versus control tumors (p=0.001), but the benefit of radiovirotherapy was not statistically significant compared with virotherapy alone in this model. These results show the potential of Ad5/3PB-ADP-hNIS as a vector for treatment of prostate cancer.     

The effect of hypoxia on the uptake, replication and lytic potential of group B adenovirus type 3 (Ad3) and type 11p (Ad11p)

Replicating, tumor selective viruses are being tested as potential treatments for human cancers. Hypoxia is a pathophysiological cancer condition that alters the lytic potential of the replication-competent adenovirus serotype 5 (Ad5) virus by a mechanism independent of receptor levels or internalization rates. We extend these initial studies to examine the potential effects of hypoxia on the group B adenoviruses (Ads), adenovirus type 3 (Ad3) (group B1) and adenovirus type 11p (Ad11p) (group B2). Receptor expression (CD46) is not altered by hypoxia. However, the lytic potential is compromised in a cell-dependent fashion. Consequently, our study suggests that group B replicating Ad-based treatments, like the group C Ad-5-based viruses, will need to be modified in order to effectively treat hypoxic components of human tumors.     

A novel attenuated replication-competent adenovirus for melanoma therapy

To generate a replication-competent adenovirus (Ad) with specificity for melanoma, we constructed a tissue-specific promoter restricting E1A expression to melanoma cells. The combination of four copies of a mouse tyrosinase enhancer element (TE) fused to the human tyrosinase promoter (TP) yielded up to 2000-fold higher luciferase reporter activity in tyrosinase-expressing melanoma cells than in nonmelanoma cells. Insertion of the composite TETP construct upstream of the E1A gene was combined with deleting as far as possible the intertwined endogenous Ad enhancer/promoter (EP). The resulting AdDeltaEP-TETP vector, also deleted for the E3 region, was found to replicate in tyrosinase-positive melanoma cells, such as SK-Mel23 as efficiently as wild-type Ad5, but at a more than 50-fold reduced level in nonmelanoma tumour cells and primary human cells. Injection of AdDeltaEP-TETP into xenotransplanted melanomas, but not into HeLa-derived tumours led to long-lasting tumour regression in nude mice. This AdDeltaEP-TETP virus might be useful for the treatment of accessible lesions in advanced melanoma patients.     

Treatment of prostate cancer with Ad5/3Delta24hCG allows non-invasive detection of the magnitude and persistence of virus replication in vivo

Hormone refractory metastatic prostate cancer is a deadly disease that currently lacks curative treatments. Conditionally replicating adenoviruses (CRAds) are promising new agents against cancer due to their innate capability to cause oncolysis of tumor cells. Their antitumor effect is determined in part by their capacity for infecting cancer cells. However, the respective primary receptor, the coxsackie-adenovirus receptor (CAR), is variably expressed in many cancer types. We created Ad5/3Delta24hCG, a novel CRAd retargeted to the adenovirus serotype 3 receptor, which has been reported to be highly expressed in tumors. Furthermore, we added a transgene for the beta-chain of human chorionic gonadotropin (hCGbeta), whose expression was tightly coupled to virus replication. Ad5/3Delta24hCG was found effective in killing prostate cancer cells, and oncolysis was seen in concordance with hCGbeta production. In a s.c. in vivo model of hormone refractory prostate cancer, Ad5/3Delta24hCG treatment resulted in statistically significant tumor growth inhibition. Moreover, i.v. injection of Ad5/3Delta24hCG prolonged the survival of mice with hormone refractory prostate cancer metastatic to the lung. Detection of hCGbeta in serum samples confirmed viral replication in vivo. Infection of human clinical samples of cancerous and normal prostatic tissue resulted in effective hCGbeta production in cancer tissue, whereas it remained low in nonmalignant tissue, suggesting cancer-specific replication. These results suggest that Ad5/3Delta24hCG is a potent virus for the treatment of hormone refractory prostate cancer in vitro and in vivo. These preclinical data set the stage for translation into clinical studies.     

A tumor-targeted and conditionally replicating oncolytic adenovirus vector expressing TRAIL for treatment of liver metastases.

We have constructed a new capsid-modified adenovirus (Ad) vector that specifically replicates in tumor cells and expresses TNF-related apoptosis-inducing ligand (TRAIL). The Ad capsid contains short-shafted fibers derived from Ad serotype 35, which allow for efficient infection of malignant tumor cells, and largely avoids innate toxicity after intravenous application. Replication-dependent homologous recombination in Ad genomes was used to achieve tumor-specific expression of Ad E1a (to mediate viral replication) and TRAIL (to mediate apoptosis and enhance release of progeny virus from infected cells). We demonstrated that our oncolytic vector (Ad5/35.IR-E1A/TRAIL) induced apoptosis in human tumor cell lines derived from colorectal, lung, prostate, and liver cancer. Both in vitro and in vivo tumor models showed efficient intratumoral spread of this vector. In a model for metastatic colon cancer, tail vein infusion of Ad5/35.IR-E1A/TRAIL resulted in elimination of preestablished liver metastases. Intravenous injection of this vector caused a transient elevation of serum glutamic pyruvic transaminase in tumor-bearing mice, which we attributed to factors released from apoptotic tumor cells. Liver histology analyzed at day 14 after virus injection did not show signs of hepatocellular damage. This new oncolytic vector represents a potentially efficient means for gene therapy of metastatic cancer.