Cancer targeted gene therapy of BikDD inhibits orthotopic lung cancer growth and improves long-term survival

Abstract

Lung cancer is a leading cause of cancer death due to the high incidence of metastasis; therefore, novel and effective treatments are urgently needed. A current strategy is cancer-specific targeted gene therapy. Although many identified that cancer-specific promoters are highly specific, they tend to have low activity compared with the ubiquitous cytomegalovirus (CMV) promoter, limiting their application. We developed a targeted gene therapy expression system for lung cancer that is highly specific with strong activity. Our expression vector uses the survivin promoter, highly expressed in many cancers but not normal adult tissues. We enhanced the survivin promoter activity comparable to the CMV promoter in lung cancer cell lines using an established platform technology, whereas the survivin promoter remained weak in normal cells. In mouse models, the transgene was specifically expressed in the lung tumor tissue, compared with the CMV promoter that was expressed in both normal and tumor tissues. In addition, the therapeutic gene BikDD, a mutant form of pro-apoptotic Bcl2 interacting killer, induced cell killing in vitro, and inhibited cell growth and prolonged mouse survival in vivo. Importantly, there was virtually no toxicity when BikDD was expressed with our expression system. Thus, the current report provides a therapeutic efficacy and safe strategy worthy of development in clinical trials treating lung cancer.

1

The VISA system can selectively enhance transgene expression driven by the Survivin promoter in lung cancer cell lines, but not in normal cells. (A) Schematic diagram of engineered promoter driving luciferase constructs. (B) The transcriptional activity of the Survivin, and CMV promoters was measured in lung cancer cell lines and normal cells by cotransfection with indicated plasmid DNA and pRL-TK in dual luciferase assay. The relative luciferase activity shown here represents the dual luciferase activity ratio (firefly versus renilla luciferase) in Survivin-VISA vector relative to that of the CMV promoter by setting CMV activity as 1. (C) The transcriptional activity of the Survivin-VISA, and CMV promoters was measured in lung cancer cell lines and normal cells as (B). The p53 status is listed under the indicated lung cancer cell lines. W indicated wild type; M indicated mutant; N/A indicates not available.

2

Survivin-VISA vector selectively expressed luciferase in the lung tumor tissue of SCID mice carrying orthotopic lung cancer xenograft, while CMV promoter strongly expressed luciferase in both of lung and lung tumor tissue of tumor-bearing mice. Fifty µg plasmid plus HLDC liposome were administrated into mice by tail vein injection, and the luciferase activity was detected with the noninvasive imaging system (IVIS imaging system, xenogen) after 48 hours. (A) The promoter specificity of CMV and Survivin-VISA (SV) was detected by driving luciferase expression in tumor-free and tumor-bearing mice (upper panel). The organs from mice were then dissected for ex vivo imaging (lower panel). The quantified signal from lungs and livers is also shown under the lower panel. (B) The lung organs from tumor-bearing mice were fixed and processed for immunohistochemical analysis of firefly luciferase expression by using anti-Luc antibody. The luciferase protein was stained in red and nucleus in blue.

3

The therapeutic gene BikDD was expressed in lung cancer cells and inhibited lung cancer cells growth. (A) The schematic diagram of therapeutic constructs. (B) BikDD driven by CMV and SV was detected in F4 lung cancer cell lysate after 24h plasmids transient transfection by Western blot. (C) In vitro cell killing effect of BikDD. Indicated therapeutic plasmids and pRL-TK were co-transfected into cells by lipofetamine 2000. The renilla luciferase activity was detected after 48 hours. Relative cell viability was measured by setting Ctrl as 100% cell viability. The p values between lung cancer cell lines and normal cell lines with CMV-BikDD and SV-BikDD treatment were 0.19 and 0.006, respectively.

4

The therapeutic gene, BikDD driven by Survivin-VISA and CMV promoter inhibited tumor growth and prolonged survival time than Ctrl group in the F4-Luc lung cancer orthotopic xenograft model. (A) SCID mice with intrathoracic injection of F4-Luc lung cancer cells were intravenously injected with 25 µg of liposomal plasmid DNA. Arrows indicate the therapy time points. The photon signals were quantified with Xenogen’s living imaging system to reflect the tumor size. Error bars indicate SEM. (B) Kaplan-Meier survival analysis was performed. The mean survival time was 36±2, 59±5, and 64±5 days in Ctrl, CMV-BikDD, and SV-BikDD groups, respectively. (C) BikDD driven by CMV or Survivin-VISA promoter was detected in the lung tissue from the mouse bearing lung cancers by RT-PCR two days later after the last treatment. ** indicated p value < 0.01.

5

The therapeutic gene, BikDD driven by Survivin-VISA promoter inhibited tumor growth and prolonged survival time than that driven by CMV promoter in H1299-Luc lung cancer orthotopic xenograft models. (A) SCID mice with intrathoracic injection of H1299-Luc lung cancer cells were intravenously injected with 25 µg of liposomal plasmid DNA. Arrows indicate the therapy time points. The photon signals were quantified with Xenogen’s living imaging system to reflect the tumor size. Error bars indicate SEM. (B) Kaplan-Meier survival analysis was performed. The mean survival time is 42±2, 53±6, and 60±5 days in Ctrl, CMV-BikDD, and SV-BikDD groups, respectively. (C) SCID mice with intravenous injection of H1299-Luc lung cancer cells were treated with 25 µg of liposomal plasmid DNA as panel A. Each group has 10 mice. The survival curve was presented in Kaplan-Meier survival analysis. The mean survival time is 57±5, 73±5, and 78±3 days in Ctrl, CMV-BikDD, and SV-BikDD groups, respectively. * indicated p value < 0.05.

6

SV-BikDD treatment had no systemically acute toxicity compared with CMV-BikDD treatment in immunity complete mice. Male Balb/c mice were given single dose of 50 µg plasmid DNA in a liposomal complex via the tail vein injection (n = 5 mice/group). (A) Serum level of AST and ALT in mice was monitored after plasmid DNA injection. Error bars indicate SEM. (B) In vivo apoptosis of tissue specimens was detected by TUNEL assay and was quantified % of apoptotic cells from 4 fields. ** indicated p value < 0.01.