The noncoding RNA MALAT1 is a critical regulator of the metastasis phenotype of lung cancer cells

Abstract

The long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1), also known as MALAT-1 or NEAT2 (nuclear-enriched abundant transcript 2), is a highly conserved nuclear noncoding RNA (ncRNA) and a predictive marker for metastasis development in lung cancer. To uncover its functional importance, we developed a MALAT1 knockout model in human lung tumor cells by genomically integrating RNA destabilizing elements using zinc finger nucleases. The achieved 1,000-fold MALAT1 silencing provides a unique loss-of-function model. Proposed mechanisms of action include regulation of splicing or gene expression. In lung cancer, MALAT1 does not alter alternative splicing but actively regulates gene expression including a set of metastasis-associated genes. Consequently, MALAT1-deficient cells are impaired in migration and form fewer tumor nodules in a mouse xenograft. Antisense oligonucleotides (ASO) blocking MALAT1 prevent metastasis formation after tumor implantation. Thus, targeting MALAT1 with ASOs provides a potential therapeutic approach to prevent lung cancer metastasis with this ncRNA serving as both predictive marker and therapeutic target. Finally, regulating gene expression, but not alternative splicing, is the critical function of MALAT1 in lung cancer metastasis. In summary, 10 years after the discovery of the lncRNA MALAT1 as a biomarker for lung cancer metastasis, our loss-of-function model unravels the active function of MALAT1 as a regulator of gene expression governing hallmarks of lung cancer metastasis.

Figure 1. MALAT1 specifically regulates gene expression but not splicing

The impact of MALAT1 on genome-wide gene expression and alternative splicing was analyzed by Exon Microarrays. Three WT (A549, WT, GFP) and three KO (KO1-3) lines were analyzed in biological duplicates. A) Spider web chart for the specificity of gene regulation (red) or alternative splicing (blue) alterations by loss of MALAT1. The exon array analysis revealed 459 significantly regulated transcripts and 3025 differentially spliced exons. To assess the specificity of the uncovered regulations, we permutated the dataset and repeated the analysis: first, the six cell lines were grouped into two groups according to their genotype (WT versus KO). Then, the six samples were randomly divided into two groups in all nine possible permutations. The genotype-based yielded both, significant hits at the exon and the transcript level (each set as 100%). At the exon level, all nine random sample permutations gave rise to significant hits. In stark contrast, none of the nine sample permutations gave rise to any significantly regulated transcripts indicating the specificity of the gene regulation. B) Analysis of alternative splicing. MALAT1-dependent, alternatively spliced exons were analyzed by qRT-PCR (n=3; mean ±SEM). Alternative splicing alterations were detected neither for previously identified cassette exons (22) (upper panel) nor for cassette exons from our own exon array analysis (lower panel). C) Validation of differentially expressed genes after loss of MALAT1. MALAT1 KO cells showed a significant deregulation of 459 genes (Supplementary Table S1). The differential expression of 23 metastasis-associated target genes was confirmed using qRT-PCR. All validation experiments were conducted in three independent RNA panels. Given is the relative expression of genes normalized to the mean of the WT cells. PPIA (Cyclophilin A) served as reference gene.

Figure 2. MALAT1 is important for cell migration in vitro

Scratch / wound healing assays were performed with MALAT1 WT and KO A549 cells. Shown are representative figures from three independent experiments. A) Wound fields were observed directly after removal of inserts (0h) and cell migration was followed for 24 h and 48 h. An obvious impact of MALAT1 loss on cell migration was detected at both time points. B) Statistical analysis of wound closure. Gap size at 0 h was set to 100 percent and percentage of closed wound was calculated after 24 h and 48 h after image analysis. WT clones efficiently migrated into the gap while KO clones uniformly displayed a significantly impaired wound closure (p≤0.001).

Figure 3. MALAT1 is a critical factor for lung cancer homing and nodule formation in vivo

A549 lung cancer cells or MALAT1 KO cells (KO2 and KO3) were analyzed for the metastatic potential in a mouse xenograft assay recapitulating major steps of metastasis development. 10–11 animals were analyzed per cell line. A) Lung cancer tumor nodule formation. After tail vein injection of MALAT1 WT or KO cells, formation of lung tumor nodules was analyzed. The lungs were resected, fixed and the total number of tumors and the tumor area were determined. The tumor burden was drastically reduced in MALAT1 KO cells (KO2 and KO3) compared to wild-type control cells. Representative pictures from the left lobe are shown. B) Statistical analysis. On average, 136 distinct nodules were counted in mice injected with A549 WT cells, whereas only 24 and 12 metastases were found in KO cells (t-test: p=0.007 and p=0.004, respectively). C) Histology of lung tumor nodules. HE staining (upper panel) shows an extensive growth of MALAT1 WT tumor cells, which destroyed the normal alveolar structure of the lung (left). MALAT1 KO cells either developed micrometastases (magnified inlet, mid) or tumor cells resided inside blood vessels (right). Staining of a consecutive section against human pan-cytokeratin (clone KL-1) proves the human origin of tumor cells that invaded into the mouse lung tissue (magnified inlets, lower panel). Scale bar = 200 µm for 4 × magnification and 50 µm for 20 × magnification.

Figure 4. Downregulation of MALAT1 in EBC-1 lung cancer cells and tumor stroma

Mice bearing human NSCLC EBC-1 cells were treated with ASOs by s.c. injection at 50 mg/kg, five times a week for five weeks. A) Accumulation of MALAT1 ASO in both tumor and tumor-associated stromal cells was demonstrated by IHC using an antibody specific for ASOs. B) Human and mouse MALAT1 RNA levels in EBC-1 tumor were measured by qRT-PCR using species-specific probe/primer sets and verified the downregulation of MALAT1 in human tumor and murine normal tissue after ASO application in vivo in the animal model. C) Reduction in MALAT1 RNA levels in both tumor and its surrounding stromal cells was visualized by the ‘ViewRNA’ ISH method using species-specific probes.

Figure 5. MALAT1 ASO inhibits the metastatic spread of EBC-1 tumors to the lung

EBC-1-derived primary tumors were induced by flank injections into nude mice. The animals were then treated with MALAT1 ASO. After five weeks, tumors were surgically excised from their primary sites and the animals were kept for the following seven weeks without ASO treatment. At week 12, lung tissues were collected and analyzed for tumor burden and histology. A) Animals treated with MALAT1 ASO had significantly fewer tumor nodules in the lung compared to control ASO-treated animals (p=0.038). B-C) A significant decrease in tumor volume in the MALAT1 ASO-treated group compared to the control ASO group was demonstrated by microCT scanning (p=0.038). ‘Purple’ indicates airway volumes and ‘green’ represents lung tissue volumes including tumor mass.