Gene Therapy (2015) 22, 305315; doi:10.1038/gt.2014.122; published online 15 January 2015

C-SLu1,7, J-LHsieh2,7, C-YLin1, H-WTsai1, B-HSu1, G-SShieh3, Y-CSu1, C-HLee4, M-YChang5, C-LWu1 and A-LShiau6

Hypoxia is a common feature of growing solid tumors. Adaptation to low oxygen condition in cells results in the transcriptional activation of more than 100 genes that regulate key aspects of tumorigenesis, including angiogenesis, metabolism, proliferation, invasion and metastasis.1 Promoters containing hypoxia response element (HRE) can be transactivated by hypoxia and drive-related gene expressions, leading to defective vasculogenesis and abnormal metabolic activity when tumors progress. Hypoxia-inducible factor (HIF)-1, which is an oxygen-sensitive transcriptional activator, primarily mediates this response. HIF-1 consists of two subunits, namely an oxygen-regulated subunit HIF-1 (or its paralogs HIF-2 and HIF-3) and a constitutively expressed subunit HIF-1.2 HIF-1 is degraded via the ubiquitin-proteasome pathway in normoxia. Whereas the HIF-1 subunit becomes stable and regulates the expression of target genes in hypoxia. Furthermore, unusual overexpression of HIF-1 has been found in various cancers. HIF-1 preferentially induces genes encoding glycolytic enzymes, whereas HIF-2 induces genes involved in tumor invasion, such as matrix metalloproteinases and the initiation of cancer stem cell (CSC) factors.3, 4, 5 HIF-2 activates signaling pathways such as Oct4 and Notch, which control the self-renewal and multipotency of CSCs.6 All of the altered characteristics of tumors may impair effective cancer treatment.

Replication-selective oncolytic adenoviruses are an attractive strategy for cancer therapy because they are able to infect, replicate in and lyse tumor cells. Viruses can be modified in various ways to improve their selectivity and therapeutic efficacy. First, viruses have mutated genes that are essential for viral replication in non-tumor cells but can be selectively compensated by specific cellular mutations in cancer cells.7 Second, the enhancer or promoter region of the E1A gene, which is required for adenovirus replication, can be modified with tumor- or tissue-specific promoters. 8, 9, 10, 11, 12 On these modifications, oncolytic adenoviruses are capable of replicating and lysing tumor cells while sparing non-tumor cells. These viruses have shown acceptable anti-tumor activity and overall safety in various cancers, including bladder cancer.13, 14, 15 In the past decade, numerous clinical trials have been conducted to assess the potential of oncolytic viruses for cancer therapy. 16 An oncolytic vaccinia virus, Pexa-Vec (formerly known as JX-594), was engineered to selectively replicate in cells with alterations of the RAS pathway and to express human granulocyte-macrophage colony-stimulating factor (hGM-CSF). Pexa-Vec was employed in clinical trials for the treatment of hepatocellular carcinoma (phase I) and colorectal cancer (phase II).17 An oncolytic herpes simplex virus type 1, Talimogene laherparepvec (T-VEC), which was manipulated to express hGM-CSF, has been tested for the treatment of unresected Stage IIIB, IIIC or IV melanoma. Results from the clinical trials indicated that these oncolytic viruses hold promise as anticancer agents.18, 19

Oct4 is a transcriptional factor that is a key regulator of pluripotency and self-renewal in embryonic stem cells and is also expressed in bladder cancer.20, 21 We have demonstrated that Oct4 expression reflects tumor progression and regulates motility of bladder cancer cells.11 We generated an E1B 55-kDa-deleted adenovirus, designated Ad9OC, which is driven by nine copies of the Oct4 response element (ORE) ligated to a human cytomegalovirus minimal (CMVmini) promoter.10 In addition to Ad9OC, we have also generated another oncolytic adenovirus, named AdWS4, under the control of the Oct4 promoter.11 These two Oct4-regulated oncolytic adenoviruses can specifically kill bladder cancer cells overexpressing Oct4 and exert potent antitumor activity in animal tumor models. 10, 11

Limited viral replication is one of the major obstacles to reaching a highly therapeutic effect. 22 A level of hypoxia similar to that found within solid tumors reduces the replication of adenoviral vectors by reducing E1A expression and hence oncolytic potentials.23, 24 Hypoxia has been exploited to drive the replication of oncolytic adenoviruses aiming at increasing therapeutic efficacy for solid tumors exhibiting significant areas of hypoxia.25, 26, 27 Currently designed oncolytic adenoviruses may require additional modifications to target tumor cells in hypoxic regions. To overcome such drawbacks, in the present study, we generated a new hypoxia-activated oncolytic adenovirus, designated AdLCY, which contains a dual hypoxia/Oct4-responsive promoter composed of the CMVmini promoter ligated with six copies of the HRE and nine copies of the ORE.

Bladder cancer is the most common cancer in the urinary system in the United States.28 Progression to or presentation with muscle-invasive disease usually worsens the survival rate of patients and requires more aggressive therapy. Oct4 and Sox2, which are stemness markers expressed in bladder cancer, have been implicated to be responsible for proliferation and differentiation of CSCs and are correlated to disease prognosis.29, 30 We found that using HRE/ORE segments to transcriptionally regulate adenoviral replication in Oct4-overexpressing cancer cells increased viral replication and oncolytic activities in hypoxic environments, thereby improving antitumor activity against bladder cancer. As Oct4 is expressed in a broad spectrum of cancer and tumor hypoxia increases malignant progression and metastasis,1, 31 Oct4 and hypoxia dual-regulated oncolytic adenoviruses may be broadly applicable.

We first used quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR) analysis to examine Oct4 mRNA levels in various human and murine bladder cancer cells under normoxic and hypoxic conditions. Levels of Oct4 mRNA expression were higher in hypoxic than in normoxic conditions in all the cells tested (Figure 1a). In human bladder cancer cells, hypoxia induced Oct4 mRNA expression by 4- to 23-folds. Accordingly, higher levels of Oct4, HIF-1 and HIF-2 proteins were also detected in these cell lines under hypoxia than under normoxia (Figure 1b). In the murine MBT-2 cell line and its two sublines, hypoxia also induced Oct4 mRNA expression, albeit at lower levels of induction compared with those in human bladder cancer cells (Figure 1a). Regarding protein expression levels, MBT-2 and MBT-2-LM7 cells under hypoxic conditions expressed higher levels of Oct4, HIF-1 and HIF-2 proteins than those under normoxic conditions (Figure 1b). However, only HIF-2, but not Oct4 and HIF-1 proteins, was elevated following hypoxic induction ( Figure 1b). We next used three different reporter constructs to examine the promoter activities of the CMVmini promoter ligated with either 6 HRE or 9 ORE, or both in hypoxic or normoxic TCC-SUP and MBT-2 cells. As shown in Figure 1c, activities of the three promoters increased when TCC-SUP (upper panel) and MBT-2 (lower panel) cells were under hypoxic conditions. Moreover, the CMVmini promoter ligated with both 6 HRE and 9 ORE exerted higher transcriptional activity than that conjugated with either 6 HRE or 9 ORE. Collectively, these results indicate that the CMVmini-6 HRE-9 ORE promoter was highly responsive to endogenous Oct4 and HIFs in hypoxic cells.

The CMVmini-6 HRE-9 ORE promoter was highly responsive to endogenous Oct4 and HIFs in hypoxic human and murine bladder cancer cells. (a) Expression of Oct4 mRNA in bladder cancer cells under normoxic and hypoxic conditions for 48h, as determined by quantitative real-time RT-PCR. (b) Detection of HIF-1, HIF-2 and Oct4 expression in bladder cancer cells after exposure to normoxia (N) or hypoxia (H) for 48h. The expression of -actin served as the loading control. (c) Determination of promoter activities. TCC-SUP and MBT-2 cells were transfected with single dual-luciferase reporter constructs, which contained the CMVmini promoter ligated with either 6 HRE or 9 ORE, or both to drive firefly luciferase, as well as the CMV promoter to drive Renilla luciferase, and then exposed to normoxia or hypoxia for 48h. Promoter activities were determined by a dual-luciferase reporter assay. The ratio of firefly luciferase activity to Renilla luciferase activity was expressed as relative light units (RLU) (n=4). Values are the means.e.m. of the mean. ***P<0.001; **P<0.01; *P<0.05.

As Oct4 has been identified as a HIF-2-specific target gene,5 we next tested whether silencing HIF-2 expression reduces Oct4 expression and thereby decreases the transcriptional activity of the CMVmini-9 ORE promoter in hypoxic tumor cells. TCC-SUP and MBT-2 cells that have been transfected with a reporter construct containing the CMVmini-9 ORE promoter were transfected with short hairpin RNA (shRNA) constructs specific to HIF-2 (shHIF-2) or green fluorescent protein (GFP) (shGFP) for 24h and then exposed to hypoxia or normoxia for additional 24h. Knockdown of HIF-2 expression resulted in decreased Oct4 expression in TCC-SUP ( Figure 2a, left panel) and MBT-2 ( Figure 2b, left panel) cells under hypoxic conditions. Furthermore, the transcriptional activity of the CMVmini-9 ORE was also downregulated in hypoxic TCC-SUP ( Figure 2a, right panel) and MBT-2 ( Figure 2b, right panel) cells after transduction with shRNA specific to HIF-2. These results confirmed that HIF-2 is involved not only in HRE-dependent transactivation, but also regulates Oct4 transactivation. Given that the transcriptional activity of the CMVmini-6 HRE-9 ORE promoter was higher than that of the CMVmini-9 ORE promoter (Figure 1c), we generated AdLCY, which is an Oct4 and hypoxia dual-regulated oncolytic adenovirus, by adding six copies of the HRE upstream of the CMVmini-9 ORE for driving adenovirus E1A gene expression in the context of the E1B 55-kDa-deleted adenovirus Ad9OC.10

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Gene Therapy - Potent antitumor activity of Oct4 and ...