Abstract A21: MALAT-1 is essential for lung cancer metastasis in a novel human knockout model

The highly conserved long non-coding RNA MALAT-1 (Metastasis-Associated in Lung Adenocarcinoma Transcript 1) had been discovered as a prognostic marker associated with poor survival and development of distant metastasis in lung adenocarcinoma. Since then, it has been found to be deregulated in numerous tumor entities and has been linked to splicing. However, its functional relevance in tumor cells remains to be elucidated. Knockdown models for MALAT1 have been described but suffer from insufficient silencing efficiency of the highly abundant, nuclear non-coding RNA (ncRNA). In this project, we have developed a novel strategy to create ncRNA knockouts in human cancer cell lines. We have successfully used a synthetic Zinc Finger Nuclease engineered to target the 5′-region of MALAT1 to stably and biallelically integrate RNA-destabilizing elements into the genome of human lung cancer cells (A549). This approach resulted in a specific and more than 1000-fold silencing of MALAT1 in individual clones compared to a less than 5-fold silencing using siRNAs. Thus, this approach can be used to create functional knockouts of coding as well as non-coding genes also in human tumor cell lines allowing loss-of-function studies also of non-conserved ncRNAs in the future. Phenotypically, the MALAT1-Knockout cells (KO) greatly differ from their parental cell line and wildtype clones (WT): Next to morphological changes, the migration of the KO cells is largely impaired as shown in scratch assays. In xenograft assays after i.v. injection, the KO cells form significantly fewer and smaller lung metastases than their WT counterparts. Since no large difference was observed after subcutaneous injection of the WT and the KO cells, this indicates a specific, active and essential function of MALAT1 in metastasis. Exon microarrays of the WT and KO cell lines have revealed multiple migration- or metastasis-associated transcripts deregulated by loss of MALAT1 and hence potential target genes. These analyses also uncovered, that splicing is likely not the only functional mechanism of MALAT1 in the nucleus. Taken together, we have developed a novel, highly effective approach for the knockout of genes that can be used for non-coding as well as coding RNAs in human tumor cells as well as cells from other species. Knockout of MALAT1 in human lung cancer cells revealed its essential function in metastasis as well as genes directly and indirectly targeted by MALAT1. Citation Format: Tony Gutschner, Moritz Eismann, Monika Hammerle, Marion Stentrup, Catherina Hildenbrandt, Matthias Gros, Martin Zornig, Sven Diederichs. MALAT-1 is essential for lung cancer metastasis in a novel human knockout model [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A21.