From the AGA Journals

MicroRNA-375 may be key to fibrolamellar carcinoma

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Cancer needs workaround genetic approach

For several decades, fibrolamellar carcinoma was the enigmatic liver cancer. Neither etiology nor molecular causes were known. The breakthrough came when tumor sequencing identified a hitherto undescribed fusion gene in 15 out of 15 patients analyzed: A small portion of the heat shock protein DNAJB1 was fused to the catalytic subunit of protein kinase A (PKA, or PRKACA), which retained full kinase activity.

Dr. Morten Frödin

Underscoring the significance of this finding, the DNAJB1-PRKACA fusion gene was shown to be sufficient to elicit tumors similar to human fibrolamellar carcinoma when engineered in mice. The absence of conspicuous codriver genes makes DNAJB1-PRKACA a primary candidate for therapeutic target. However, PKA inhibitors would be problematic in the clinic because of the vital physiological functions of PKA. Consequently, the hunt is on to decipher the oncogenic signaling pathways emanating from DNAJB1-PRKACA with the hope to identify alternative targets among its downstream mediators.

In this work, the Sethupathy lab performed a thorough study on abnormally regulated microRNAs in fibrolamellar carcinoma tumors. Intriguingly, they identified several microRNAs controlled by DNAJB1-PRKACA that have oncogenic or tumor suppressor function in other cancers. In particular, the tumor suppressor microRNA-375 was massively down-regulated by DNAJB1-PRKACA. Furthermore, introducing a microRNA-375 mimic in fibrolamellar cancer cells suppressed proliferation and motility. Important studies like this open up new avenues aiming to manipulate cancer microRNAs as alternative or complementary approaches for targeting DNAJB1-PRKACA signaling in the highly fatal fibrolamellar carcinoma.

Morten Frödin, MSc, PhD, is an associate professor and group leader of the Biotech Research and Innovation Centre, University of Copenhagen.



Up-regulation of microRNA-375 may be a future therapeutic strategy for patients with fibrolamellar carcinoma (FLC), according to investigators.

Analysis of primary FLC tumors showed that microRNA-375 was the most abnormal microRNA, down-regulated 27-fold, reported lead author Timothy A. Dinh, MD, of the University of North Carolina at Chapel Hill and his colleagues. Overexpression of microRNA-375 in an FLC cell line suppressed cell migration and proliferation, hinting at therapeutic potential.

“Overall, our results show that miR-375 [microRNA-375] functions as a tumor suppressor in FLC and points toward future therapies based on miR-375 mimics that may provide a viable option for patients,” the investigators wrote in a Cellular and Molecular Gastroenterology and Hepatology.

FLC is an uncommon liver cancer in adolescents and young adults. Currently, surgery is the only effective treatment; unfortunately, many patients have metastatic disease at the time of diagnosis, disallowing surgical cure.

“The lack of knowledge of underlying disease mechanisms has hindered our understanding of this cancer and the development of novel therapeutics for FLC patients,” the investigators wrote.

Previous research has shown that almost all patients with FLC (80%-100%) have a heterozygous deletion mutation on chromosome 19. However, it is not a loss of genetic information that incites neoplasia; instead, the deletion causes a fusion of genes DNAJB1 and PRKACA. This fusion is capable of triggering liver tumors, a phenomenon confirmed through mouse models. The present study built on these findings, along with recent awareness that several microRNAs are dysregulated in FLC, compared with normal liver tissue.

First, the investigators performed small RNA-sequencing in six primary FLC tumors from The Cancer Genome Atlas (TCGA). They found that 30 microRNAs were up-regulated and 46 microRNAs were down-regulated. Among these, microRNA-375 was the most significantly down-regulated, at 27-fold (P = .009). To confirm these findings, the same process was repeated in 18 independent samples, with the same result.

The investigators explained that, in addition to magnitude of down-regulation, microRNA-375 deserved attention for at least three other reasons: It is down-regulated in numerous cancer types, it directly targets known oncogenes, and it is suppressed by the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signaling axis, which is overactive in FLC.

Further testing confirmed that microRNA-375 was consistently more down-regulated in samples of FLC, by up to 20-fold, than it was in nonmalignant liver tissue. To confirm that loss of microRNA-375 expression occurred in FLC tumor cells instead of other cell types, such as stromal cells, a patient-derived xenograft of FLC was compared with liver lineage cells, including adult hepatocytes, hepatoblasts, hepatic stem cells, and biliary tree stem cells. Again, microRNA-375 was down-regulated most in the FLC cells. Additional comparisons within the TCGA showed that microRNA-375 was more down-regulated in FLC than 21 out of 22 other tumor types (second only to melanoma).

“Taken together with our findings from primary tumor tissue, our results strongly suggest that miR-375 may function as a tumor suppressor in FLC,” the investigators wrote.

Having confirmed the ubiquity of microRNA-375 down-regulation in FLC, the investigators turned to the relationship between the DNAJB1-PRKACA fusion and microRNA-375. Using two methods – gene deletion with CRISPR/Cas9 and transposon injection – the investigators found that creating the DNAJB1-PRKACA fusion in cells of mice was sufficient to suppress microRNA-375 expression, which supports a downstream relationship.

Finally, the investigators showed that treating an FLC cell line with an microRNA-375 mimic suppressed the Hippo signaling pathway, including connective tissue growth factor (CTGF) and yes-associated protein 1 (YAP1). These events translated to reduced cellular activity, which suggests that up-regulating microRNA-375 could, indeed, control FLC.

“Importantly, introduction of a miR-375 mimic significantly reduced colony formation, EdU incorporation, and migration, indicative of reduced survival, proliferation, and metastatic potential, respectively,” the investigators wrote.

“With RNA-based therapies showing increasing promise, miR-375–based therapies merit future consideration for FLC therapeutics,” they concluded.

The study was funded by the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute on Alcohol Abuse and Alcoholism, and the Fibrolamellar Cancer Foundation. The investigators declared no conflicts of interest.

SOURCE: Dinh TA et al. Cell Mol Gastroenterol Hepatol. 2019 Feb 11. doi: 10.1016/j.jcmgh.2019.01.008.

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