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Whole-Transcriptome Study Finds Chimeric Transcript in Rare Liver Cancer

NEW YORK (GenomeWeb News) – By comparing tumor and normal tissue, researchers in New York City have identified a chimeric transcript that is expressed in fibrolamellar hepatocellular carcinomas, as they reported in this week's edition of Science.

Researchers led by Rockefeller University's Sanford Simon performed whole-transcriptome and whole-genome sequencing on more than two dozen samples of primary tumors, recurrences, metastases, and matched normal tissue from patients with FL-HCC. In the RNA-seq data, they found evidence of a chimeric transcript present in the cancerous tissue that was absent in the normal tissue. The chimeric transcript, they reported, appears to be due to an in-frame fusion of one end of the DNAJB1 gene to part of the PRKACA gene. The protein resulting from the fusion, they noted, is expressed and retains some kinase activity.

"Evidence supporting the presence of the DNAJB1-PRKACA chimeric transcript in 100 [percent] of the FL-HCCs examined suggests that this genetic alteration contributes to tumor pathogenesis," Simon and his colleagues said.

Other institutions involved in the work include Memorial Sloan-Kettering Cancer Center, the New York Genome Center, and the Dalton School.

FL-HCC is a rare liver tumor that typically develops in adolescents and young adults. The tumors don't usually respond well to chemotherapy, and the five-year overall survival is between 30 percent and 45 percent.

To try to understand the molecular basis of FL-HCC, Simon and his colleagues conducted whole-genome and whole-transcriptome sequencing of paired tumor and normal samples. Based on the RNA-seq results from their set of 29 samples of primary tumors, recurrences, metastases, and matched normal tissue from 11 patients, the researchers uncovered a single recurrent candidate chimeric transcript that was present in each tumor sample.

The transcript, they noted, appears to result from an in-frame fusion of exon 1 of the DNAJB1 gene to exons 2 through 10 of the PRKACA gene. DNAJB1, they noted, encodes a member of the heat shock 40 protein family, while PRKACA encodes the adenosine 3', 5'monophosphate-dependent protein kinase A catalytic subunit alpha.

As compared to normal tissues, the researchers found that PRKACA transcript levels were increased in tumor samples from the nine patients they examined. Further, they noted that in all nine, the expression levels of exons 2 through 10 was elevated in tumor samples as compared to normal tissue samples, but the expression level of exon 1, which is not part of the chimera, remained the same in both tissue sets.

Two patients, the researchers added, had an additional splice variant affecting PRKACA and DNAJB1. Samples from those patients also contained reads starting in the middle of exon 2 of DNAJB1 that joined with the start of exon 2 of PRKACA.

Simon and his colleagues confirmed the chimeric transcripts they saw using RT-PCR coupled with Sanger sequencing.

They also noted that tumor tissue appeared to retain a few transcripts of the full-length PRKACA, indicating that some cells still harbored a wild-type copy of the gene.

The researchers also searched for structural variations within the FL-HCC genome by sifting through whole-genome sequencing data from the paired tumor and normal samples. In eight of the 10 samples they analyzed, the researchers uncovered paired-end reads longer than about 400 kilobases that mapped to both PRKACA and DNAJB1. While the precise breakpoint of the deletion varied, it was consistent with a deletion in the first intron or second exon of DNAJB1 and ending in the first intron of PRKACA for each sample.

Through western blot analysis of proteins isolated from tumor and normal tissue, the researchers found that the chimeric transcript was still expressed. By transfecting chimeric, wild-type, or control protein plasmids into a human kidney cell line, the researchers found that cells with the chimeric or wild-type protein plasmids had higher PKA activity than the cells containing empty control plasmids. This, the researchers said, demonstrated that the chimeric proteins keep their PKA activity.

"This chimera is predicted to incorporate the J domain of DNAJB1 and the catalytic domain of PRKACA," they noted. "The promoter is from the DNAJB1 gene, which could explain why the chimeric transcript is expressed at higher levels than the WT PRKACA transcript."

The chimeric protein is also predicted to lack the domain that binds to PKA regulators. Usually, PKA phosphorylates a number of substrates, including ones involved in estrogen signaling, apoptosis, and other pathways. PKA is also involved in signaling through the endothelial growth factor receptor and in regulating aromatase expression, both of which, the researchers said, can be overexpressed in FL-HCC.

While there isn't a molecular diagnostic that tests for FL-HCC, the researchers said this chimeric transcript could be a marker for the disease. They also suggested that though the role of the chimera in disease pathogenesis remains to be worked out, that it might contribute to tumor development.