DLK1-DIO3 Genomic Imprinted MicroRNA Cluster at 14q32.2 Defines a Stemlike Subtype of Hepatocellular Carcinoma Associated with Poor Survival

Abstract

Hepatocellular carcinoma (HCC) is a heterogeneous and highly aggressive malignancy, for which there are no effective cures. Identification of a malignant stemlike subtype of HCC may offer patients with a dismal prognosis a potential targeted therapy using c-MET and Wnt pathway inhibitors. MicroRNAs (miRNAs) show promise as diagnostic and prognostic tools for cancer detection and stratification. Using a TRE-c-Met-driven transgenic HCC mouse model, we identified a cluster of 23 miRNAs that is encoded within the Dlk1-Gtl2 imprinted region on chromosome 12qF1 overexpressed in all of the isolated liver tumors. Interestingly, this region is conserved among mammalian species and maps to the human DLK1-DIO3 region on chromosome 14q32.2. We thus examined the expression of the DLK1-DIO3 miRNA cluster in a cohort of 97 hepatitis B virus-associated HCC patients and identified a subgroup (n ‫؍‬ 18) of patients showing strong coordinate overexpression of miRNAs in this cluster but not in other cancer types (breast, lung, kidney, stomach, and colon) that were tested. Expression levels of imprinted gene transcripts from neighboring loci in this 14q32.2 region and from a subset of other imprinted sites were concomitantly elevated in human HCC. Interestingly, overexpression of the DLK1-DIO3 miRNA cluster was positively correlated with HCC stem cell markers (CD133, CD90, EpCAM, Nestin) and associated with a high level of serum ␣-fetoprotein, a conventional biomarker for liver cancer, and poor survival rate in HCC patients. In conclusion, our findings suggest that coordinate up-regulation of the DLK1-DIO3 miRNA cluster at 14q32.2 may define a novel molecular (stem cell-like) subtype of HCC associated with poor prognosis.

Induction of hepatocellular carcinoma by in vivo gene targeting

“…Although it would be interesting to establish whether the paternal or maternal imprinted alleles were dysregulated by gene targeting, this could not be determined in the inbred mouse strain used in our experiments. The HCCs produced by Rian gene targeting were remarkably similar to the C3 subclass of human HCCs identified by micro-RNA profiling (17,18), demonstrating the accuracy of in vivo gene targeting for modeling human cancer. These similarities included an aggressive phenotype with vascular invasion, AFP and EPCAM expression, activated PI3K/AKT signaling, and an identical domain of increased gene expression within the imprinted BEGAIN-DIO3 locus.…”

“…1A). A recent report showed that c-Met overexpression in transgenic mice can also induce Afp + HCCs that express Dlk1 and a subset (23 of 42) of the Rian-Mirg microRNAs (18). However, in this model additional secondary mutations are required for HCC formation (39), and c-Met was not overexpressed in the mouse tumors we produced by gene targeting (Table S3).…”

“…Humans have an imprinted locus on chromosome 14 syntenic to mouse chromosome 12, and molecular profiling has identified a poor prognosis subclass of human HCCs (type C3) characterized by increased microRNA expression from this locus (17,18). These human C3 HCCs produced high serum AFP levels, expressed DLK1 and an Epcam-related gene signature, activated PI3K/AKT signaling, and exhibited frequent vascular invasion, demonstrating their similarity to mouse tumors produced by Rian gene targeting.…”

“…These small RNAs could regulate a large number of target genes, and the human homologs of some of these microRNAs have been proposed to both stimulate and inhibit tumorigenesis in other types of malignancies (13)(14)(15)(16). Two recent studies found that a subset of human HCCs have elevated expression of this microRNA cluster (17,18).…”

Pyogenic granuloma, an impaired wound healing process, linked to vascular growth driven by FLT4 and the nitric oxide pathway

“…The latter are part of the mir-379/mir-656 miRNA cluster, a chromosomal region that appears increasingly more relevant to normal placental development [17][18][19] and fetal growth, 20 stem cell differentiation 21 and various pathological conditions. [22][23][24] Further statistical analyses identified additional imprinted genes as specifically related to the placenta and hemangioma: PLAGL1, DIO3OS, TFPI2, GRB14, H19 and MEG3. If the role of imprinted genes in placental development was already well established, it is, to our knowledge, the first assessment of them in infantile hemangiomas.…”