INSM1 promoter-driven adenoviral herpes simplex virus thymidine kinase cancer gene therapy for the treatment of primitive neuroectodermal tumors

Abstract

The INSM1 gene encodes a developmentally regulated zinc finger transcription factor. INSM1 expression is normally absent in adult tissues, but is reactivated in neuroendocrine tumor cells. In the present study, we analyzed the therapeutic potential of an adenoviral INSM1 promoter-driven herpes simplex virus thymidine kinase (HSV-tk) construct in primitive neuroectodermal tumors (PNETs). We constructed an adenoviral INSM1 promoter-driven HSV-tk gene for therapy in PNETs. The PNET-specific adeno-INSM1 promoter HSV-tk construct was tested both in vitro and in vivo in a nude mouse tumor model. Northern blot analysis and transient transfection of an INSM1 promoter-driven luciferase reporter gene indicated that the INSM1 promoter was active in neuroblastoma (IMR-32), retinoblastoma (Y79), and medulloblastoma (D283 Med) cells, but not in glioblastoma (U-87 MG) cells. After Ad-INSM1p-HSV-tk infection, the levels of HSV-tk protein expression were consistent with INSM1 promoter activities. Furthermore, in vitro multiplicity of infection and ganciclovir (GCV) sensitivity studies indicated that the INSM1 promoter could mediate specific expression of the HSV-tk gene and selective killing of INSM1-positive PNETs. In vivo intratumoral adenoviral delivery demonstrated that the INSM1 promoter could direct HSV-tk gene expression in a nude mouse tumor model and effectively repressed tumor growth in response to GCV treatment. Taken together, our data show that the INSM1 promoter is specific and effective for targeted cancer gene therapy in PNETs.

FIG. 1.

Northern blot analysis of INSM1 gene expression and INSM1 promoter activity. (A) Total RNAs (20 μg) from human fetal and adult brain tissue were subjected to Northern blot analysis. Each blot was probed with a ³²P-labeled INSM1 cDNA probe, stripped, and reprobed with actin (loading control). (B) Northern blot analysis of human brain tumor cell lines IMR-32 (neuroblastoma), Y79 (retinoblastoma), D283 Med (medulloblastoma), and U-87 MG (glioblastoma). (C) Transient transfections of INSM1 promoter (bp −1661 to +40)-linked luciferase constructs into various tumor cell lines. Luciferase activities from various cell lines were compared with pGL3-Basic vector and presented as relative fold increase. All activities were normalized with β-galactosidase control and averaged from three different experiments. Data are presented as averages ± SEM.

FIG. 2.

In vitro efficacy of Ad-INSM1p-HSV-tk, Ad-RSV-HSV-tk, and Ad-SV40p-HSV-tk therapy. IMR-32, D283 Med, Y79, and U-87 MG cells were seeded at 2 × 10⁴ cells per well in a 96-well dish. Cells were transduced with 0, 1, 10, 50, or 100 MOI of Ad-INSM1p-HSV-tk (open diamonds), Ad-SV40p-HSV-tk (solid squares), or Ad-RSV-HSV-tk (solid circles), or with control Ad-CMV-LacZ (shaded triangles) or Ad-RSV-ntLacZ (mults). Twenty-four hours after viral infection, the cells were treated with 100 μM GCV for 5 days. MTT proliferation assays were performed in triplicate. The percentage cell viability was calculated relative to no virus addition from three different experiments. Top of each graph: Each cell line was transduced with adenovirus (MOI of 100) for 24 hr and the cell lysates were harvested for Western blot analysis using anti-HSV-tk antibody to reveal the expression levels of thymidine kinase protein.

FIG. 3.

Prodrug dose–response cell survival curves in various PNET cell lines. IMR-32, Y79, D283 Med, and U-87 MG cell lines were infected with Ad-INSM1p-HSV-tk (open diamonds), Ad-SV40p-HSV-tk (solid squares), Ad-RSV-HSV-tk (solid circles), Ad-RSV-ntLacZ (mults), or Ad-CMV-LacZ at an MOI of 100 and exposed to a range of concentrations (0–100 μM) of GCV. After 6 days, cell viability was measured in an MTT assay. Measurements were performed in triplicate and results are presented as the average of three experiments. The cell viability curve was calculated using zero GCV as 100%.

FIG. 4.

In vivo subcutaneous xenograft nude mouse model. D283 Med cells (5 × 10⁷) were injected subcutaneously into the flank of athymic nude mice. After tumor establishment, approximately 1 × 10⁹ IFU of Ad-CMV-LacZ (n = 4), Ad-INSM1p-HSV-tk (n = 5), Ad-RSV-HSV-tk (n = 4), or Ad-RSV-ntLacZ (n = 3) was directly injected intratumorally. Twenty-four hours later, GCV (50 mg/kg body weight) was injected intraperitoneally daily. Tumor growth was measured with calipers every other day for 18 days. Tumor volume was calculated according to the following formula: tumor volume (mm³) = 4/3 × πr³ (r = sum of the two diameters divided by 4). Data are expressed as size relative to initial tumor volume on the day before initiation of GCV administration.

FIG. 5.

In vitro and in vivo adenoviral transduction efficiency in D283 Med cells. (A) To determine the transduction efficiency of the adenovirus, 2 × 10⁵ D283 Med cells were infected with Ad-RSV-ntLacZ virus at MOIs of 0, 1, 10, 50, and 100. Forty-eight hours postinfection, the cells were fixed and stained to detect β-galactosidase activity. (B) To assess the distribution of virus within D283 tumor, 1 × 10⁹ IFU of Ad-RSV-ntLacZ virus was injected directly into subcutaneous D283 Med tumor. Forty-eight hours postinjection, the tumors were removed, fixed, and stained for β-galactosidase activity to localize the adenovirus within the tumor mass. Blue staining represents β-galactosidase activity. The tumor samples were counterstained with nuclear fast red to visualize the tumor. Color images available online at www.liebertonline.com/hum.

FIG. 6.

Ex vivo viral transduction of D283 Med cells in nude mouse tumor model. D283 Med cells were transduced with Ad-INSM1p-HSV-tk virus or Ad-CMV-LacZ virus (MOI of 200) ex vivo 24 hr before transplantation. The virally transduced cells were collected and injected subcutaneously into the flanks of mice (six mice in each group). Daily GCV injections were continued for 8 weeks until tumor growth was detected. At the end of the study, the tumors were removed and weighed for comparison. Five of six mice in the control group grew tumors and the average tumor weight was 0.4 g. In the Ad-INSM1p-HSVtk group only three of six mice grew tumors and the average tumor weight was 0.03 g (7% of the control). The mice that did not have visible tumors were monitored for an additional 4 weeks without any tumor growth.