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STAT3 silencing enhances the efficacy of the HSV.tk suicide gene in gastrointestinal cancer therapy

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Abstract

Aberrant activation of Signal Transducer and Activator of Transcription 3 (STAT3) signaling has been shown to be associated with uncontrolled cell proliferation and suppression of host-immune surveillance. Conversely, silencing STAT3 can have the dual effects of inhibiting cancer cell proliferation and inducing anti-tumor immune responses. Here, we report on the effects of STAT3 silencing on suicide gene therapy with thymidine kinase (tk). STAT3 silencing by siRNA inhibited the proliferation of AGS human gastric cancer cells through G1 cell cycle arrest, decreased levels of immune-suppressive cytokines, and increased levels of immune-activating cytokines. CT26 mouse colon adenocarcinoma cells, in which STAT3 expression was knocked-down by a STAT3 shRNA-containing lentivirus, grew more slowly in syngenic model Balb/c mice than control CT26 cells. Moreover, we found that STAT3 silencing augmented the efficacy of suicide gene therapy in CT26 cell xenografted mice. When we administrated adenoviruses harboring the herpes simplex virus thymidine kinase gene (Ad5.CMV.HSV.tk) into STAT3-silenced CT26 cell tumors, extensive apoptosis was observed and there was a significant reduction in the size of CT26 cell tumors. STAT3 silencing also enhanced the recruitment and cytotoxic activity of CD3+CD8+ T-cells, and changed the cytokine expression pattern of CT26 cell tumors, reflecting augmentation of anti-cancer immune responses. We conclude that combining suicide gene therapy with STAT3 silencing can result in enhanced anti-cancer effects.

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Abbreviations

GCV:

Ganciclovir

HSV.tk:

Herpes simplex virus thymidine kinase

STAT3:

Signal Transducer and Activator of Transcription-3

7-AAD:

7-Aminoactinomycin D

PE:

Phycoerythrin

MOI:

Multiplicity of infection

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Acknowledgments

We thank Ki-Youl Han for technical support. This study was supported by a grant from the National Cancer Center of Republic of Korea (#NCC-0910150) and a grant from the Basic Science Research Program (2010-1031790) through the National Research Foundation (NRF) funded by the Ministry of Education, Science, and Technology, Republic of Korea.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine , 250 Sungsan-ro, Seodaemun-gu, Seoul, 120-752, Republic of Korea

    Kyung-Hee Chun

  2. Gastric Cancer Branch, Division of Translational & Clinical Research I, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, 410-769, Republic of Korea

    Ye-Hyeon Ahn, Hwajung Yi, Ji-Young Shin & Kang-Duck Lee

  3. Genitourinary Cancer Branch, Division of Translational & Clinical Research II, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang-si, Gyeonggi-do, Republic of Korea

    Seung-Pil Shin & Sang-Jin Lee

  4. Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea

    Ye-Hyeon Ahn & Jaewhan Song

Authors
  1. Ye-Hyeon Ahn
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  2. Hwajung Yi
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  3. Ji-Young Shin
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Correspondence to Kyung-Hee Chun.

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Supplementary Fig. 1

Sequences and effects of STAT3-specific siRNAs. STAT3 siRNA sequences are shown in table (a) and decreased expression of STAT3 and its downstream target cyclin D1 after STAT3 specific-siRNA treatment are shown in (b) (TIFF 73 kb)

Supplementary Fig. 2

Lentiviral STAT3 shRNA construct. Two primers for STAT3 shRNA were annealed and inserted into the pLL3.7 vector to produce a lentivirus capable of inhibiting both human and mice STAT3 expression by expression of STAT3-specific shRNA (TIFF 84 kb)

Supplementary Fig. 3

Genes regulated by inhibition of STAT3 expression in CT26 mouse colon cancer cells. CT26 cells were harvested after treatment with the indicated siRNAs and RT-PCR analysis revealed a decreased STAT3 expression, b up-regulated expression of pro-inflammatory immune response-related genes and c down-regulated expression of anti-inflammatory immune response-related genes. Beta-actin was used as a loading control (TIFF 87 kb)

Supplementary Fig. 4

Ad5.CMV.HSV.tk adenoviral construct (TIFF 59 kb)

Supplementary Table 1

Sequences of primers used for quantitative PCR (Fig. 6c) (TIFF 65 kb)

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Ahn, YH., Yi, H., Shin, JY. et al. STAT3 silencing enhances the efficacy of the HSV.tk suicide gene in gastrointestinal cancer therapy. Clin Exp Metastasis 29, 359–369 (2012). https://doi.org/10.1007/s10585-012-9458-4

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  • DOI: https://doi.org/10.1007/s10585-012-9458-4

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