Abstract A17: Tumor Snail knockdown reduces tumor burden and metastases by inducing antitumor immune responses in lung cancer
Authors: Minu K. Srivastava, Sherven Sharma, Li Zhu, Min Huang, Åsa Andersson, Marni Harris-White, Upendra K. Kar, Longsheng Hong, Michael Fishbein, Steven Dubinett
Journal: Cancer Research (2011)

Abstract Lung cancer remains the leading cause of cancer death in Unites States. Metastatic spread of tumors is the most common cause of therapy failure and mortality for lung cancer. We are defining genetic programs in lung cancer that modulate immune responses that regulate tumor growth and metastases. Activating immune effectors for tumor destruction has the potential for long-term protection and survival. Cancer cells acquire the ability to progress, invade, and metastasize by undergoing the process of epithelial-mesenchymal transition (EMT), by activating transcription factors (for example, Snail, Twist, Zeb, Slug) that repress E-cadherin, a transmembrane glycoprotein essential for epithelial cell-cell adhesion. These transcriptional repressors are normally active during embryogenesis where they program EMT to enable various morphogenetic steps. EMT is involved in tumor progression. Snail expression in primary NSCLCs has been associated with a shorter overall survival. Recent studies have demonstrated the importance of Snail in tumor EMT-induced metastases in melanoma. In this study we are evaluating the mechanistic role of tumor Snail expression on tumor growth and metastases. Our data demonstrates that tumor Snail expression mediates tumor growth and metastasis by modulating immune responses. Genetic knockdown of endogenous tumor Snail expression in the murine lung cancer cell lines [Lewis Lung Cancer (3LL) that spontaneously arose in the C57BL/6 mice] with lentiviral Snail shRNA was validated by qRTPCR and Western blot analyses. With the Snail shRNA we have achieved stable 80–90% Snail knockdown. No differences were noted in growth kinetics of Snail knockdown or controls in vitro. Flow cytometry was utilized to quantify changes in tumor and spleen leukocytic populations. Lung cancer Snail knockdown cells compared to controls: 1) decreased invasiveness (3-fold) and tumor-derived TGFβ (3.3-fold); 2) reduced subcutaneous tumor growth (4-fold), lung metastases (2-fold), tumor nodule TGFβ by ELISA(5-fold), MMP9, and VEGF (2-fold) by qRTPCR; 3) increased tumor infiltrates of CD4 (2-fold) and CD8 (1.5-fold) T lymphocytes that elaborated enhanced IFNγ≈2.5-fold) but reduced levels of IL-10 (2-fold); 4) increased expression of the CD107 (≈7-fold) cytolytic marker in tumor-infiltrating CD8 T cells; 5) augmented the frequencies of innate NK effectors and dendritic cells (DC) (7-fold); 6) reduced the immune suppressors MDSC (2-fold); and 7) reduced MDSC as well as the non-MDSC populations intracellular expression of arginase in the tumors (≈3–4 fold). Although MDSC from the tumor Snail knockdown group was comparable to the controls in suppressing anti-CD3/CD28 T cell proliferation, its suppressive effects on DC antigen processing and presentation activity (APC) to an antigen-specific CD8 T cell line was reduced (2-fold) in comparison to controls. The suppressive effects of MDSC on T cell proliferation and APC activity was reversed (≈2-fold) by catalase or N-acetyl cysteine, respectively. Further experiments will mechanistically delineate the genetic program(s) induced by tumor Snail knockdown that alter the balance of immune effectors and suppressors in the tumor. An adequate understanding of the genetic signatures in the tumor and tumor-host interactions that induce immune suppression and promote tumor growth, invasion and metastases will be crucial for the development of immune-based therapies for lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A17.