Evidence of Distinct Tumour-Propagating Cell Populations with Different Properties in Primary Human Hepatocellular Carcinoma

Increasing evidence that a number of malignancies are characterised by tumour cell heterogeneity has recently been published, but there is still a lack of data concerning liver cancers. The aim of this study was to investigate and characterise tumour-propagating cell (TPC) compartments within human hepatocellular carcinoma (HCC).After long-term culture, we identified three morphologically different tumour cell populations in a single HCC specimen, and extensively characterised them by means of flow cytometry, fluorescence microscopy, karyotyping and microarray analyses, single cell cloning, and xenotransplantation in NOD/SCID/IL2Rγ/⁻ mice.The primary cell populations (hcc-1, -2 and -3) and two clones generated by means of limiting dilutions from hcc-1 (clone-1/7 and -1/8) differently expressed a number of tumour-associated stem cell markers, including EpCAM, CD49f, CD44, CD133, CD56, Thy-1, ALDH and CK19, and also showed different doubling times, drug resistance and tumorigenic potential. Moreover, we found that ALDH expression, in combination with CD44 or Thy-1 negativity or CD56 positivity identified subpopulations with a higher clonogenic potential within hcc-1, hcc-2 and hcc-3 primary cell populations, respectively. Karyotyping revealed the clonal evolution of the cell populations and clones within the primary tumour. Importantly, the primary tumour cell population with the greatest tumorigenic potential and drug resistance showed more chromosomal alterations than the others and contained clones with epithelial and mesenchymal features.Individual HCCs can harbor different self-renewing tumorigenic cell types expressing a variety of morphological and phenotypical markers, karyotypic evolution and different gene expression profiles. This suggests that the models of hepatic carcinogenesis should take into account TPC heterogeneity due to intratumour clonal evolution

The Core Stem Genes SOX2, POU5F1/OCT4, and NANOG Are Expressed in Human Parathyroid Tumors and Modulated by MEN1, YAP1, and β-catenin Pathways Activation

Tumors of the parathyroid glands are the second most common endocrine neoplasia. Epigenetic studies revealed an embryonic signature involved in parathyroid tumorigenesis. Here, we investigated the expression of the stem core genes SOX2, POU5F1/OCT4, and NANOG. Rare cells within normal parathyroid glands expressed POU5F1/OCT4 and NANOG, while SOX2 was undetectable. Nuclear SOX2 expression was detectable in 18% of parathyroid adenomas (PAds, n = 34) involving 5–30% of cells, while OCT4 and NANOG were expressed at the nuclear level in a more consistent subset of PAds involving 15–40% of cells. Most parathyroid carcinomas expressed the core stem genes. SOX2-expressing cells co-expressed parathormone (PTH). In PAds-derived primary cultures, silencing of the tumor suppressor gene MEN1 induced the expression of SOX2, likely through a MEN1/HAR1B/SOX2 axis, while calcium-sensing receptor activation increased SOX2 mRNA levels through YAP1 activation. In addition, inducing nuclear β-catenin accumulation in PAds-derived primary cultures by short-term incubation with lithium chloride (LiCl), SOX2 and POU5F1/OCT4 expression levels increased, while NANOG transcripts were reduced, and LiCl long-term incubation induced an opposite pattern of gene expression. In conclusion, detection of the core stem genes in parathyroid tumors supports their embryogenic signature, which is modulated by crucial genes involved in parathyroid tumorigenesis

Yes-Associated Protein 1 Is a Novel Calcium Sensing Receptor Target in Human Parathyroid Tumors

The Hippo pathway is involved in human tumorigenesis and tissue repair. Here, we investigated the Hippo coactivator Yes-associated protein 1 (YAP1) and the kinase large tumor suppressor 1/2 (LATS1/2) in tumors of the parathyroid glands, which are almost invariably associated with primary hyperparathyroidism. Compared with normal parathyroid glands, parathyroid adenomas (PAds) and carcinomas show variably but reduced nuclear YAP1 expression. The kinase LATS1/2, which phosphorylates YAP1 thus promoting its degradation, was also variably reduced in PAds. Further, YAP1 silencing reduces the expression of the key parathyroid oncosuppressor multiple endocrine neoplasia type 1(MEN1), while MEN1 silencing increases YAP1 expression. Treatment of patient-derived PAds-primary cell cultures and Human embryonic kidney 293A (HEK293A) cells expressing the calcium-sensing receptor (CASR) with the CASR agonist R568 induces YAP1 nuclear accumulation. This effect was prevented by the incubation of the cells with RhoA/Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors Y27632 and H1152. Lastly, CASR activation increased the expression of the YAP1 gene targets CYR61, CTGF, and WNT5A, and this effect was blunted by YAP1 silencing. Concluding, here we provide preliminary evidence of the involvement of the Hippo pathway in human tumor parathyroid cells and of the existence of a CASR-ROCK-YAP1 axis. We propose a tumor suppressor role for YAP1 and LATS1/2 in parathyroid tumors

Sequence variants identification at the KCNQ1OT1:TSS differentially Methylated region in isolated omphalocele cases

Abstract Background Omphalocele is a congenital midline ventral body wall defect that can exist as isolated malformation or as part of a syndrome. It can be considered one of the major and most frequent clinical manifestation of Beckwith-Wiedemann Syndrome (BWS) in case of loss of methylation at KCNQ1OT1: Transcription Star Site-Differentially Methylated Region (TSS-DMR) or in presence of CDKN1C mutations. The isolated form of the omphalocele accounts approximately for about the 14% of the total cases and its molecular etiology has never been fully elucidated. Methods Given the tight relationship with BWS, we hypothesized that the isolated form of the omphalocele could belong to the heterogeneous spectrum of the BWS associated features, representing an endophenotype with a clear genetic connection. We therefore investigated genetic and epigenetic changes affecting BWS imprinted locus at 11p15.5 imprinted region, focusing in particular on the KCNQ1OT1:TSS DMR. Results We studied 21 cases of isolated omphalocele detected during pregnancy or at birth and identified the following rare maternally inherited variants: i) the non-coding variant G > A at nucleotide 687 (NR_002728.3) at KCNQ1OT1:TSS-DMR, which alters the methylation pattern of the imprinted allele, in one patient; ii) the deletion c.624-629delGGCCCC at exon 1 of CDKN1C, with unknown clinical significance, in two unrelated cases. Conclusions Taken together, these findings suggest that KCNQ1OT1:TSS-DMR could be a susceptibility locus for the isolated omphalocele

Beckwith–Wiedemann syndrome prenatal diagnosis by methylation analysis in chorionic villi

<div><p>Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder that can be prenatally suspected or diagnosed based on established clinical guidelines. Molecular confirmation is commonly performed on amniocytes. The possibility to use fresh (CVF) and cultured (CVC) chorionic villi has never been investigated. To verify whether CVF and CVC are reliable sources of DNA to study fetal methylation, we used pyrosequencing to test the methylation level of a number of differentially methylated regions (DMRs) at several imprinted loci (ICR1, ICR2, <i>H19,</i> PWS/AS-ICR, <i>GNASXL, GNAS1A, ZAC/PLAGL1,</i> and <i>MEST)</i> and at non-imprinted <i>MGMT</i> and <i>RASSF1A</i> promoters. We analyzed these regions in 19 healthy pregnancies and highlighted stable methylation levels between CVF and CVC at ICR1, ICR2, <i>GNASXL</i>, PWS/AS-ICR, and <i>MEST</i>. Conversely, the methylation levels at <i>H19</i> promoter, <i>GNAS1A and ZAC/PLAGL1</i> were different in CVC compared to fresh CV. We also investigated ICR1 and ICR2 methylation level of CVF/CVC of 2 BWS-suspected fetuses (P1 and P2). P1 showed ICR2 hypomethylation, P2 showed normal methylation at both ICR1 and ICR2. Our findings, although limited to one case of BWS fetus with an imprinting defect, can suggest that ICR1 and ICR2, but not <i>H19</i>, could be reliable targets for prenatal BWS diagnosis by methylation test in CVF and CVC. In addition, PWS/AS-ICR, <i>GNASXL,</i> and <i>MEST</i>, but not <i>GNAS1A and ZAC/PLAGL1,</i> are steadily hemimethylated in CV from healthy pregnancies, independently from culture. Thus, prenatal investigation of genomic imprinting in CV needs to be validated in a locus-specific manner.</p></div