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Function of Non-coding RNAs in Lung Cancer

Final Report Summary - NCRNALUNGCANCER (Function of Non-coding RNAs in Lung Cancer)

Function of Non-coding RNAs in Lung Cancer

Cancer has been recognized as a disease of the genome that can be caused by mutations, amplifications, deletions or deregulation of genomic products. Only about 2% of the human genome are transcribed and translated into proteins while up to 70% of the human genome are transcribed into non-protein-coding RNA (ncRNA). Thus, as a starting hypothesis, long ncRNAs could play important roles in lung tumorigenesis and progression. The aim of this project was the characterization of the functional relevance of long, intergenic non-coding RNAs in lung cancer.
The ncRNA MALAT1 was in the focus of the grant proposal. During my PhD thesis, we had discovered MALAT1 as a marker for the development of distant metastasis and poor prognosis in early stages of non-small cell lung cancer (NSCLC) (Ji*, Diederichs* et al., Oncogene 2003), so that MALAT1 took center stage in our project.

MALAT1 Knock-out model in human lung cancer
MALAT1 is not only a highly conserved nuclear ncRNA, it is also highly abundant with transcript numbers exceeding most common housekeeping genes. Hence, the knockdown of MALAT1 was difficult and of limited effectiveness with 20% MALAT1 expression remaining and giving rise to divergent phenotypes. Thus, it appeared questionable whether this knock-down efficiency would be sufficient to create loss-of-function phenotypes of MALAT1. Hence, we decided to aim for a knock-out instead of a knock-down strategy for MALAT1 in human lung cancer cells. Therefore, we developed a novel strategy to create functional knock-outs for long ncRNAs in human cells based on genome editing. Using a specifically engineered, synthetic Zinc Finger Nuclease targeting the 5’-end of MALAT1, we stably and biallelically integrated different RNA Destabilizing Elements (RDE) into the genome of human lung cancer cells. This strategy resulted in a more than 1000-fold silencing of MALAT1 and allowed an in-depth functional characterization of the cellular loss-of-function phenotype of MALAT1 (Gutschner et al., Genome Res 2011).

MALAT1 function in lung cancer metastasis
This novel loss-of-function model was employed to study the cellular function of MALAT1: The MALAT1-deficient cells were viable and did not show any cell cycle aberrations in contrast to previous, RNAi-based studies. We also established a knock-out mouse model in cooperation with Martin Zörnig (Georg-Speyer-Haus, Frankfurt), which surprisingly did not show any gross developmental or histological abnormalities and thus corroborated the viability data in our human cell line model (Eißmann et al., RNA Biol 2012). Since we discovered MALAT1 as a marker for lung cancer metastasis, we then focused on its potential role in cell migration, invasion and metastasis. Indeed, cells lacking MALAT1 were severely impaired in cell migration. In xenograft assays, MALAT1-depleted cells gave rise to significantly fewer and smaller tumor nodules after tail-vein injection recapitulating the second half of the metastatic cascade. Thus, ten years after its discovery, we could prove that MALAT1 was not only a marker but an active player in lung cancer metastasis (Gutschner et al., Cancer Res 2013).

Molecular mechanisms of MALAT1 action in lung cancer
Since multiple different molecular mechanisms had been proposed for MALAT1 including the epigenetic regulation of gene expression and the regulation of gene expression, we focused on the dissection of these two processes. Using exon microarrays to analyze the expression and splicing patterns in the MALAT1-deficient lung cancer cells (Gutschner et al., Genome Res 2011), we found a strong regulation of gene expression, but not of alternative splicing caused by the loss of MALAT1. MALAT1 induced a specific gene signature including many genes previously linked to cell migration, invasion and metastasis. Hence, MALAT1 did not act through the regulation of a single target gene, but through the concerted induction of a gene signature leading to a metastatic phenotype (Gutschner et al., Cancer Res 2013).

MALAT1 as a therapeutic target in metastasis prevention
Since we identified MALAT1 as an important player in lung cancer metastasis, we wanted to determine whether it could also serve as a therapeutic target. In close cooperation with the company ISIS Pharmaceuticals and the lab of David Spector (Cold Spring Harbor), we tested antisense oligonucleotides (ASO) as potential MALAT1 inhibitors. Indeed, these ASOs effectively silenced MALAT1 expression in vitro and in vivo. To recapitulate the entire metastatic cascade, we tested these ASOs in a xenograft mouse model with an established subcutaneous tumor from a human lung tumor cell line and then analyzed the impact of the ASOs on the metastatic spread from this primary tumor. Indeed, MALAT1 inhibition caused a dramatic decrease in metastasis. Thus, MALAT1 could not only be a marker and active player in lung cancer metastasis, but also a promising target for a metastasis prevention therapy (Gutschner et al., Cancer Res 2013). Since metastasis is by far the most frequent cause of (lung) cancer related death, these findings are of broad medical and socio-economic interest.

Non-coding RNA Profiling in lung cancer
Based on a collaboration with the Pathology and the Thoraxklinik in Heidelberg greatly facilitating our local integration, we have now profiled the expression of 17000 long ncRNAs significantly extending our profiling efforts beyond MALAT1 and generating a comprehensive map of long ncRNA expression and regulation in lung cancer. In a set of 27 lung adenocarcinomas and matched non-malignant tissues from the same patients, we identified 479 long ncRNAs significantly and at least twofold differentially expressed between lung cancer and normal lung tissue. These ncRNAs are prime candidates for future functional studies in our laboratory. Additionally, we identified ncRNAs differentially expressed between metastasizing and non-metastasizing primary lung tumors which will be further analyzed for their potency as biomarkers (manuscript in preparation).

In summary, the MC-IRG has allowed me to delve into the fascinating world of long ncRNAs in lung cancer by providing the necessary consumables for the study of MALAT1 and for the discovery of novel lncRNAs in lung cancer. For the model ncRNA MALAT1, we established a loss-of-function knock-out model in lung cancer (Gutschner et al., Genome Res 2011) as well as a knock-out mouse model (Eißmann et al., RNA Biol 2012) and established its important and active role in lung cancer metastasis, where it regulates a metastasis gene signature epigenetically and might serve as therapeutic target for metastasis prevention (Gutschner et al., Cancer Res 2013). The data generated in this MC-IRG - especially the discovery of yet unpublished, novel lncRNAs associated with lung cancer - will provide the competitive basis for future funding applications.