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Novel Long Non-Coding RNA Affects Metastasis in Colon Cancer

This article has been updated to reflect the contribution of the Rigoutsos lab.

NEW YORK (GenomeWeb) – A primate-specific long non-coding RNA contributes to metastasis in colon cancer, according to a new study.

Levels of N-BLR, a novel lncRNA, are associated with tumor stage, invasion potential, and overall survival among colorectal cancer patients, researchers led by MD Anderson's George Calin and Thomas Jefferson University's Isidore Rigoutsos found. They further reported in Genome Biology that a short pyknon motif appeared to mediate these effects by affecting crosstalk between N-BLR and E-cadherin and ZEB1, both of which are involved in the epithelial-to-mesenchymal transition (EMT).

"The primate-specific N-BLR is a novel molecular contributor to the complex mechanisms that underlie metastasis in colorectal cancer and a potential novel biomarker for this disease," Calin, Rigoutsos, and their colleagues wrote in their paper.

The researchers were initially investigating pyknons — a class of short DNA motifs — and whether they were active or passive. In a qRT-PCR assay of some 11 pyknons in regions associated with loss of heterozygosity thought to include functionally relevant sequences, they found that transcription of all 11 genomic pyknon regions varied among the seven colorectal cancer cell lines they used.

Following up on that, they noted that levels of a particular pyknon — pyk-reg-90 — correlated with tumor stage and poor overall survival in a set of 81 tumor samples, a finding they confirmed in an independent cohort of patients and a set of patient-derived xenografts.

Calin, Rigoutsos, and their colleagues traced this pyknon to a specific region of chromosome 3 and cloned N-BLR, the novel lncRNA. With custom probes and a tissue microarray containing normal, adenocarcinoma, metastatic, benign, and colitis samples from the colon, they found that N-BLR was more highly expressed in the cancerous samples than in the normal, colitis, or benign samples. This suggested to the researchers that N-BLR overexpression was specific to epithelial malignant cells.

In a series of cell-based assays, the researchers found that knocking down N-BLR led to increased levels of cleaved PARP-1, a substrate of activated cell death proteases, and eliminated expression of the X-lined inhibitor of apoptosis, XIAP. Down-regulation of N-BLR likewise led to an increase in apoptosis.

At the same time, they reported that cells with decreased N-BLR also had decreased invasion and migration abilities. They found that reduced levels of N-BLR upregulated E-cadherin, while it downregulated vimentin, both of which are involved in the EMT cell motility in human colon carcinoma.

Levels of N-BLR also varied with levels of members of the miR-200 family, which regulates EMT through the ZEB1 and ZEB2 transcription factors, the researchers found. Knocking down N-BLR led to an increase of miR-141-3p and miR-200c-3p in a colorectal cancer cell line. Further, they reported that low miR-141-3p and miR-200c-3p levels were associated with low overall survival among colorectal cancer patients, as was high levels of N-BLR.

In turn, they noted that miR-200c-3p expression downregulated XIAP expression, which makes colorectal cancer cell lines more susceptible to 5-FU-induced apoptosis.

While cells overexpressing N-BLR had an increased ability to migrate and invade, cells overexpressing N-BLR with its pyknon motif removed had no increase in migration or invasion, the researchers reported. This and other analyses suggested to the researchers that the pyknon motif has a critical role in N-BLR's function.

Calin, Rigoutsos, and their colleagues added that N-BLR, miR-141-3p, and miR-200c-3p, along with E-cadherin, vimentin, and ZEB1 appeared to make up a new part of signaling interactions in the EMT pathway, and that overexpression of N-BLR in colon cancer boosted cells' ability to metastasize and invade.

"Our analyses indicate that N-BLR is a novel molecular player in the mechanisms underlying the metastatic potential in CRC," the researchers wrote. "This, together with our pyknon microarray findings, suggests that N-BLR and likely other transcripts … could prove important to our understanding of key molecular processes and might potentially find uses as novel biomarkers or novel therapeutics in human cancers and other diseases."