Absent in melanoma 2 enhances anti‐tumour effects of CAIX promotor controlled conditionally replicative adenovirus in renal cancer

Abstract Conditionally replicative adenoviruses (CRAds) were promising approach for solid tumour treatment, but its oncolytic efficiency and toxicity are still not satisfactory for further clinical application. Here, we developed the CAIX promotor (CAIXpromotor)‐controlled CRAd armed with a tumour suppressor absent in melanoma 2 (AIM2) to enhance its oncolytic potency. The CAIXpromotor‐AIM2 adenoviruses (Ad‐CAIXpromotor‐AIM2) could efficiently express E1A and AIM2 in renal cancer cells. Compared with Ad‐CAIXpromotor, Ad‐CAIXpromotor‐AIM2 significantly inhibited cell proliferation and enhanced cell apoptosis and cell killing, thus resulting in the oncolytic efficiency in 786‐O cells or OSRC‐2 cells. To explore the therapeutic effect, various Ads were intratumourally injected into OSRC‐2‐xenograft mice. The tumour growth was remarkably inhibited in Ad‐CAIXpromotor‐AIM2‐treated group as demonstrated by reduced tumour volume and weight with a low toxicity. The inflammasome inhibitor YVAD‐CMK resulted in the reduction of anti‐tumour activity by Ad‐CAIXpromotor‐AIM2 in vitro or in vivo, suggesting that inflammasome activation response was required for the enhanced therapeutic efficiency. Furthermore, lung metastasis of renal cancer mice was also suppressed by Ad‐CAIXpromotor‐AIM2 treatment accompanied by the decreased tumour fossil in lung tissues. These results indicated that the tumour‐specific Ad‐CAIXpromotor‐AIM2 could be applied for human renal cancer therapy. The therapeutic strategy of AIM2‐based CRAds could be a potential and promising approach for the therapy of primary solid or metastasis tumours.


| INTRODUC TI ON
Conditionally replicative adenoviruses (CRAds) were dependent on tumour-specific promotors by the control of virus gene (such as E1A) expression for selective replication. 1,2 The direct consequence of Ad replication results in tumour cell killing. 3 In contrast to other agents for current cancer therapies, CRAds possess the advantages of conditional selective replication viral self-spreading, highly transduction efficacy and subsequently augment anti-tumour effects in cancer cells. 4,5 Many attempts of CRAds have been made great development in cancer gene therapies, but treatment of solid or metastasis tumours remains a challenge. [6][7][8] Thus, novel therapeutic strategies for cancer should be developed to enhance the oncolytic efficiency with a low toxicity.
Tumour-specific promotors can control the expression of viral genes or therapeutic genes to select replication in tumour cells instead of normal cells. 9,10 The cell surface protein, carbonic anhydrase IX (CAIX), is a tumour-sassociated antigen of renal cancer. 11 CAIX expression is almost not detected on normal kidney cells or other normal cells, but an increasing expression is detected on 90% of renal cancer lesions. 12 CAIX antigen shows apparent tumour specificity in renal cancer, and is involved in tumour cell proliferation, oncogenesis and tumour progression. 13,14 Therefore, CAIX promotor (CAIX promotor ) could serve as an ideal target to construct tumour-specific Ad for renal cancer treatment. To increase the safety of Ad, some modifications have been introduced to restrict Ad replication to tumour cells. 15 We have previously constructed a novel oncolytic Ad with E1A gene controlled by CAIX promotor . 1 This modification is to replace promotors for essential viral genes with specific promotor that perform activation only in tumour cells. 16 Ads assembled with therapeutic genes can amplify the capable of lysing tumour cells selectively. 2,17 Absent in melanoma 2 (AIM2) is a member of the interferon-inducible HIN-200 protein family, and is associated with inflammation and cancer pathology. 18,19 AIM2 as a DNA sensor can form inflammasome complex by binding to cytosolic DNA and subsequently activate caspase-1 to produce IL-1β for regulating inflammatory responses. 20 Of note, AIM2 as a tumour suppressor is a prognostic marker and an independent predictor of disease progression in renal cell cancer, and is also used as the therapy target for renal cancer therapy. 21 Previous studies demonstrated that overexpression of AIM2 can exert anti-tumoural activities through enhancing the inflammasome activation to induce cell apoptosis or autophagy. 21,22 Thus, AIM2 might be applied as a therapeutic gene to enhance the potential efficacy of CARd for renal cancer treatment.
In the present study, we constructed CAIX promotor controlled oncolytic adenovirus expressing AIM2 (Ad-CAIX promotor -AIM2) and evaluated its efficacy and toxicity in renal cancer cells or tumour models. Ad-CAIX promotor -AIM2 decreased cell viability, and induced cell apoptosis and cell killing in human renal cancer cells.
Additionally, Ad-CAIX promotor -AIM2 displayed potent anti-tumour activity in OSRC-2-xenograft model or lung metastasis model. Our results indicated that the oncolytic Ad carrying a tumour therapeutic gene might be effective and safe for primary solid or metastasis tumour therapy.

| Animals
4-6 weeks old male nude mice (BALB/c-nu/nu) were purchased from Vital River Laboratory and maintained under pathogen-free conditions. All procedures for animal experiments were approved by the Committee on the Use and Care of Animals and performed in accordance with the guidelines of the Laboratory Animal Ethical Committee of Xuzhou Medical University.

| Cytotoxicity assay
Cells were seeded at a density of 3 × 10 5 per well in a six-well culture plate. After 24 h, cells were infected with Ad-Ctrl, Ad-AIM2, Ad-CAIX promotor and Ad-CAIX promotor -AIM2, respectively, at various MOIs: 0, 0.1, 1, 10 and 100. Five days after infection, the cells were exposed to 2% crystal violet in 20% methanol for 15 min, washed with distilled water (ddH 2 O) and photographed.

| Colony formation assay
Cells were trypsinized, counted and seeded in 6-well plate at a density of 1 × 10 3 cells per well. After 24 h, cells were infected with various Ads, respectively, at a 10 MOI. After 7 days of incubation, survival colonies were fixed with 4% paraformaldehyde for 10 minutes and stained with 0.5% of crystal violet and counted under the microscope.

| Cell apoptosis detection
The treated cells were collected and stained with an Annexin V-FITC/PI apoptosis detection kit (BD Biosciences) according to the manufacturer's instructions. Cell apoptosis was analysed by flow cytometry.

| Real-time PCR
Total RNA were extracted from collected cells by Trizol (Invitrogen) and reverse-transcribed (RT) using a cDNA synthesis kit (TaKaRa, China) according to the manufacturer's instructions. The expression of the genes encoding AIM2, ASC, caspase-1 and IL-1β was quantified by real-time PCR using 7900HT qPCR system thermal cycler (Applied Biosystems) and SYBR Green system (TaKaRa) normalized by GAPDH expression following the manufacturer's protocol.
Primers were used as following in Table 1.
Briefly, for cell samples, 2-10 × 10 6 cells were lysed in 50-100 μl lysis buffer on ice for 10 min. For tissue samples, 3-10 mg tissues were added to 100 μl lysis and were homogenated with a tissue homogenizer. The supernatant from extracts was collected by centrifuged at 12 000 g for 10 min. Protein concentrations were detected using the Bradford method, ensuring that the protein concentration was 1-3 μg/μl. A standard curve was prepared using the pNA standard.
10-35 μl volume of supernatant was incubated with 5 μl 2 mM Ac-YVAD-pNA substrate at 37°C. Then, the absorbance values of pNA were read on a spectrophotometer at the optical density 405nm (OD 405 ). The changes of caspase-1 activity were calculated by the radio of OD 405 of the experimental wells to that of the control wells.

| ELISA measurement of cytokines
To assess protein levels of IL-1β, IL-6 concentrations in culture supernatant or in homogenized tumour tissues of mice, enzyme-linked immunosorbent assays (ELISA) were performed according to the manufacturer's instructions (eBioscience). Animals were killed and tumour tissues were surgically excised from the mice. Tumour weight was evaluated.
In metastasis model, mice were intravenously injected with 4 × 10 6 OSRC-2 cells from the tail vein at day 0. Mice were divided into four groups and injected with various Ads at a dose of 4 × 10 8 PFU/ mouse every other day, with a total dosage of 2 × 10 9 pfu of Ads. 28 days after tumour inoculation, mice were killed and lungs were removed, and the metastatic nodules were quantified.

| Pathological analyses
For routine histological analyses, murine tissues were surgically resected and fixed in 4% paraformaldehyde (Sigma-Aldrich), embedded in paraffin and cut into sections 5 μm sections. H&E staining was performed according to the manufacturer's instructions, and pictures were acquired with microscope (Nikon). The numbers of metastatic nodules or the quantification of metastatic foci were assessed in the sections by a pathologist blinded to treatment group.

| Statistical analysis
Statistical analysis was performed with GraphPad Prism (Version 5.01) software and expressed as means ± SD. Statistical significance was evaluated using two-tailed Student's t test. Multiple comparisons were performed using one-way ANOVA. The statistical significance level was set as *P < .05; **P < .01; and ***P < .001.
CAIX promotor could serve as an ideal target to construct tumour-specific adenovirus for renal cancer treatment. Therefore, a novel CAIX promotor controlled oncolytic adenovirus was constructed by introduced the therapeutic gene of AIM2 for enhancing its oncolytic potency ( Figure 2C). For evaluation of Ad-CAIX promotor -AIM2 for cancer-specific expression, E1A or AIM2 expression was detected by flow cytometry in OSRC-2 cells infected with Ad-Ctrl, Ad-AIM2, Ad-CAIX promotor or Ad-CAIX promotor -AIM2, respectively. As shown in Figure 1D,E, the MFI analysis showed that a significant increasing expression of AIM2 was observed in Ad-AIM2 or Ad-CAIX promotor -AIM2-infected cells compared with the control cells, and indicated AIM2 gene was successfully expressed in Ad-AIM2 or Ad-CAIX promotor -AIM2-infected renal cancer cells. E1A is a prerequisite for Ad replication and an indicator of the potential of an adenovirus to lyse infected cells. 23 Accordingly, E1A protein confirmed by the MFI also could be efficiently expressed in Ad-CAIX promotor or Ad-CAIX promotor -AIM2-infected renal cancer cells ( Figure 1F,G), and indicated Ad-CAIX promotor or Ad-CAIX promotor -AIM2 efficiently droved the E1A expression in CAIX antigen-positive cells.
These results indicated that Ad-CAIX promotor -AIM2 had a higher selectivity in CAIX antigen-positive renal cancer cell lines.  (Fig. S1).

| Ad-CAIX promotor -AIM2 enhanced the oncolytic efficiency in renal cancer cells
Taken together, these results indicated that Ad-CAIX promotor -AIM2 could efficiently enhance the oncolytic effect by inhibiting renal cancer cell proliferation and increasing cell apoptosis.

| The inflammasome activation was required for enhanced oncolytic effect of Ad-CAIX promotor -AIM2
Recent evidence suggests that AIM2 inflammasome activation plays an important role in suppressing the progression of tumour cells. 24,25 To further validate our hypothesis that Ad-CAIX promotor -AIM2 suppressed the growth of renal cancer cells by enhancing the inflammasome activation, 786-O or OSRC-2 was infected with Ad-CAIX promoter -AIM2 or control Ads followed with or without the downstream inflammatory caspase inhibitor YVAD-CMK. Compared with control group, the expression of inflammasome components (AIM2, caspase-1 and IL-1β) ( Figure 4A,B), caspase-1 activation ( Figure 4C) and the protein levels of IL-1β ( Figure 4D) were robustly elevated in Ad-CAIX promotor -AIM2 group, and suggested that the inflammasome was activated in Ad-CAIX promotor -AIM2-infected cells.
The functional effect of AIM2-mediated inflammasome activation was inhibited by YVAD-CMK inflammasome inhibitor in Ad-AIM2 or Ad-CAIX promotor -AIM2-infected cells, and the levels of caspase-1 and IL-1β remarkably were reduced ( Figure 4A-D). Furthermore, the enhancement of cell apoptosis by Ad-CAIX promotor -AIM2 also suppressed in YVAD-CMK-treated Ad-CAIX promotor -AIM2 cells ( Figure 4H). The inhibition ability of cell migration by Ad-CAIX promotor -AIM2 in renal cancer cells was blocked by the YVAD-CMK inhibitor (Fig. S2). These data indicated that the oncolytic efficacy of Ad-CAIX promotor -AIM2 is depended on the inflammasome activation.

| The anti-tumour effect by Ad-CAIX promoter -AIM2 was increased in OSRC-2-xenograft mice
To All experiments were carried out in triplicate. Data are shown as means ± SD. The different significance was set at *P < .05, **P < .01 and ***P < .001 cytokines, like IL-6 in serum ( Figure 5G). There is a non-specific immune response demonstrated by normal pathological tissue of heart, liver and kidney in Ad-CAIX promotor -AIM2-treated mice ( Figure 5H), and indicated its low toxicity. Taken together, these data indicated that Ad-CAIX promotor -AIM2 treatment with low toxicity could effectively prevent the aggravation of tumour in renal cancer model.

| Ad-CAIX promotor -AIM2 promoted the inhibition of lung metastasis in OSRC-2-lung metastasis model
To assess whether Ad-CAIX promotor -AIM2 could also protect mice from tumour metastasis, we established a murine lung metastasis model of renal cancer. After OSRC-2 cell inoculation, mice were intravenously injected with Ad-Ctrl, Ad-AIM2, Ad-CAIX promotor or Ad-CAIX promotor -AIM2. Mice were killed and lungs were removed on day 28 after tumour inoculation, and the visible metastases were counted. Ad-CAIX promotor -AIM2 treatment dramatically reduced the number of lung metastatic nodules compared with the Ad-CAIX promotor treatment ( Figure 6A,B). The protective efficacy of Ad-CAIX promotor -AIM2 was further confirmed by and the quantification of metastatic foci from H&E staining of lung tissues of lung metastasis model ( Figure 6C,D). These results indicated that Ad- The function of Ads depends on the replication in tumour cells and killing them through the lytic replication cycle, 27  Data are means ± SD, *P < .05, **P < .01 and ***P < .001 regulatory functions. 31,32 Previous study showed that Akt is a pivotal serine/threonine kinase and promotes epithelial-mesenchymal transition process through NF-κB and GSK-3β in cell biological progression including cell proliferation, apoptosis and metabolism.
AIM2 inflammasome activation impaired cell invasion and metastasis via inhibiting Akt pathway in colorectal cancer. 33 Furthermore, overexpression of AIM2 in human renal cancer suppressed cell metastasis via enhancing autophagy induction. 21 Our results indicated that inflammasome inhibitor YVAD-CMK resulted in the reduction of oncolytic effect by Ad-CAIX promotor -AIM2, implied that this efficiency was dependent on AIM2 inflammasome activation responses.
To further explore the therapeutic effect of Ad-CAIX promotor -AIM2, OSRC-2 xenograft mice were injected subcutaneously into nude mice. The tumour growth inhibition curve accompanied by inflammasome activation in tumour and indicated that the oncolytic adenovirus could effectively inhibit the growth of primary solid tumour. This effect by AIM2 inflammasome signal was confirmed by blocking of inflammasome activation by VAD-CMK in vivo. However, the xenograft could not be eliminated totally only by Ad-CAIX promotor -AIM2, which might be due to immune escape from the tumour, such as the expression of PD-L1 inhibitory signals or immunosuppressive cells. In future study, we would further investigate that the potent way is combination of the Ad with blocking of PD-L1 or immunosuppressive cells to completely eliminate tumour cells in tumour microenvironment. Of note, the Ad expressed AIM2, which was under the control of CAIX promotor , thus ensuring no harm was done to the normal surrounding cells. Our results showed that Ad-CAIX promotor -AIM2 could not significantly elevate the level of IL-6 cytokine in serum and reduce the bodyweight, and indicated that there is a non-specific immune response and low toxicity.
In conclusion, a novel Ad was engineered with the E1A gene controlled by the -CAIX promotor and AIM2 gene. Ad-CAIX promotor -AIM2 showed the enhanced oncolysis in CAIX-positive renal cancer cells compared with the control. Ad-CAIX promotor -AIM2 indicated the potent growth inhibition effects on renal cancer models of primary or lung metastasis. The therapeutic strategy of AIM2-based Ads might be a potential and promising approach for the therapy of primary solid or metastasis tumours.