Mutations Causing Loss of Tumor Suppressor Gene Increase Aggressiveness of Rare Bile Duct Cancer

A team of Japanese cancer researchers identified a biomarker, that when absent or reduced, predicts the likely outcome of the rare cancer of the bile duct, intrahepatic cholangiocarcinoma (ICC).

Cholangiocarcinoma is rare in the Western world, with estimates of it occurring in fewer than two people per 100,000 per year. Rates are higher in South-East Asia where liver flukes are common. In parts of Thailand, for example, there are around 60 cases per 100,000 persons per year. ICC is typically incurable at diagnosis, which is made by examination of the tumor under a microscope. Overall mean duration of survival is less than six months in people with metastatic disease.

Genomic analyses have recently discovered the malignant subtype of human ICC characterized by frequent mutations of the chromatin remodeling gene ARID1A (AT-rich interactive domain-containing protein 1A); however, the biological and molecular functions of these mutations remain obscure. To help clarify these functions, investigators at the Tokyo Medical and Dental University (Japan) examined the clinical and biological effects of ARID1A deficiency in human ICC.

In one set of experiments, the investigators established ARID1A-knockout (KO) cells by using the CRISPR/Cas9 gene editing tool to modify two human cholangiocarcinoma cell lines.

CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. Since 2013, the CRISPR/Cas9 system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 enzyme and appropriate guide RNAs (sgRNAs) into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR/Cas9 system from Streptococcus pyogenes is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides that shepherd the Cas9 protein to the target gene on a DNA strand.

Results of the CRISPR/Cas9 study revealed that the modified ARID1A-KO cells were significantly more malignant than untreated ICC cells and exhibited enhanced migration, invasion, and sphere formation activity. Microarray analysis showed that ALDH1A1, a stemness gene, was the most significantly elevated gene in ARID1A-KO cells.

"We found that ARID1A suppresses the stemness gene ALDH1A1, which has been implicated in other cancers. Removing ARID1A causes the gene to become highly overactive in ICC cells," said first author Jun Yoshino, a graduate researcher at the Tokyo Medical and Dental University. "We found that when this cancer stemness gene is overactive, it causes the cells to act much more aggressively. For example, the cells can migrate and invade neighboring groups of cells, a behavior that is very common in highly-active cancers."

The investigators then turned to patients with ICC, measuring levels of ARID1A in tissue samples. In addition, they followed the patients’ medical histories to observe how the disease progressed. Results revealed that patients who were ARID1A-negative had much poorer outcomes: five years after their diagnosis, less than 20% of ARID1A-negative patients had survived, compared with over 50% of ARID1A-positive patients. These results suggested that loss of ARID1A, which could be detected by immunohistochemical examination of biopsy specimens, was an independent prognostic factor in ICC patients.

"ARID1A is known to take part in complexes that control gene expression," said Jun Yoshino. "This hinted that loss of ARID1A could play a role in ICC by causing certain genes to turn on or off. However, it was not clear which genes might be affected or how their expression might impact the ICC disease course."

The ICC study was published in the October 30, 2019, online edition of the journal Carcinogenesis.

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Tokyo Medical and Dental University