LKB1 signaling is activated in CTNNB1-mutated HCC and positively regulates ß-catenin-dependent CTNNB1-mutated HCC.

Hepatocellular carcinomas (HCCs) are known to be highly heterogenous. Within the extensive histopathological and molecular heterogeneity of HCC, tumors with mutations in CTNNB1, encoding ß-catenin (CTNNB1-mutated HCC), constitute a very homogeneous group. We previously characterized a distinctive metabolic and histological phenotype for CTNNB1-mutated HCC. They were found to be well-differentiated, almost never steatotic, and often cholestatic, with a microtrabecular or acinar growth pattern. Here, we investigated whether LKB1, which controls energy metabolism, cell polarity, and cell growth, mediates the specific phenotype of CTNNB1-mutated HCC. The LKB1 protein was overexpressed in CTNNB1-mutated HCC and oncogenic activation of ß-catenin in human HCC cells induced the post-transcriptional accumulation of the LKB1 protein encoded by the LKB1 (STK11) gene. Hierarchical clustering, based on the expression of a murine hepatic liver Lkb1 (Stk11) signature in a human public dataset, identified a HCC cluster, composed of almost all the CTNNB1-mutated HCC, that expresses a hepatic liver LKB1 program. This was confirmed by RT-qPCR of an independent cohort of CTNNB1-mutated HCC and the suppression of the LKB1-related profile upon ß-catenin silencing of CTNNB1-mutated human hepatoma cell lines. Previous studies described an epistatic relationship between LKB1 and CTNNB1 in which LKB1 acts upstream of CTNNB1. Thus, we also analyzed the consequences of Lkb1 deletion on the zonation of hepatic metabolism, known to be the hallmark of ß-catenin signaling in the liver. Lkb1 was required for the establishment of metabolic zonation in the mouse liver by positively modulating ß-catenin signaling. We identified positive reciprocal cross talk between the canonical Wnt pathway and LKB1, both in normal liver physiology and during tumorigenesis that likely participates in the amplification of the ß-catenin signaling by LKB1 and the distinctive phenotype of the CTNNB1-mutated HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Generation of Mice with Hepatocyte-Specific Conditional Deletion of Notum.

BACKGROUND: Fine tuning of the Wnt/ß-catenin signaling pathway is essential for the proper development and function of the liver. Aberrant activation of this pathway is observed in 20%-40% of hepatocellular carcinomas (HCC). Notum encodes a secreted Wnt deacylase that inhibits Wnt activity and thereby restricts the zone of activation of Wnt/ß-catenin signaling. An important role of NOTUM has been described in development in drosophila, planaria and zebrafish, but its role in the mammalian liver is unknown. Notum is required for spatial control of the Wnt/ß-catenin signaling in several animal models and the Wnt/ß-catenin pathway contributes to liver patterning involved in metabolic zonation. Therefore, Notum may be involved in the liver patterning induced by the Wnt/ß-catenin signaling during the adult stage. METHODOLOGY/PRINCIPAL FINDINGS: We generated a conditional Notum knockout mouse mutant to study the effect of the deletion of Notum in the liver. We show that Notum is a direct target of the Wnt/ß-catenin signaling in the liver. Liver-specific deletion of Notum did not modify liver zonation, but Notum deletion had a long-term effect on mouse physiology. In particular, male mutant mice developed metabolic disorders. CONCLUSION: We show that Notum is not a key actor of Wnt/ß-catenin-dependent liver patterning of adult mice, but has role in liver glucose homeostasis. Male mice deficient in Notum specifically in the liver develop metabolic dysfunctions implicating Notum in the development of Type 2 diabetes.

Antitumour activity of an inhibitor of miR-34a in liver cancer with ß-catenin-mutations.

OBJECTIVE: Hepatocellular carcinoma (HCC) is the most prevalent primary tumour of the liver. About a third of these tumours presents activating mutations of the ß-catenin gene. The molecular pathogenesis of HCC has been elucidated, but mortality remains high, and new therapeutic approaches, including treatments based on microRNAs, are required. We aimed to identify candidate microRNAs, regulated by ß-catenin, potentially involved in liver tumorigenesis. DESIGN: We used a mouse model, in which ß-catenin signalling was overactivated exclusively in the liver by the tamoxifen-inducible and Cre-Lox-mediated inactivation of the Apc gene. This model develops tumours with properties similar to human HCC. RESULTS: We found that miR-34a was regulated by ß-catenin, and significantly induced by the overactivation of ß-catenin signalling in mouse tumours and in patients with HCC. An inhibitor of miR-34a (locked nucleic acid, LNA-34a) exerted antiproliferative activity in primary cultures of hepatocyte. This inhibition of proliferation was associated with a decrease in cyclin D1 levels, orchestrated principally by HNF-4α, a target of miR-34a considered to act as a tumour suppressor in the liver. In vivo, LNA-34a approximately halved progression rates for tumours displaying ß-catenin activation together with an activation of caspases 2 and 3. CONCLUSIONS: This work demonstrates the key oncogenic role of miR-34a in liver tumours with ß-catenin gene mutations. We suggest that patients diagnosed with HCC with ß-catenin mutations could be treated with an inhibitor of miR-34a. The potential value of this strategy lies in the modulation of the tumour suppressor HNF-4α, which targets cyclin D1, and the induction of a proapoptotic programme.

Diagnosis and molecular aspects of solid-pseudopapillary neoplasms of the pancreas.

Solid-pseudopapillary neoplasm of the pancreas (SPN) is an uncommon low-grade malignant neoplasm occurring mostly in young women. In addition to its distinctive pathological appearance of pseudopapillae with poorly cohesive neoplastic cells, rare variants exist raising the differential diagnosis especially with neuroendocrine neoplasms. The overall prognosis for patients with SPNs is excellent after surgical resection. Nevertheless, 10% of cases may have malignant behavior characterized by tumor recurrence and/or metastasis. Despite numerous studies, the histogenesis of this neoplasm remains unclear. Distinctive molecular alterations such as the presence of CTNNB1 mutations are observed in nearly all cases, while mutations classically observed in ductal adenocarcinoma, such as KRAS, TP53, and SMAD4, are not observed in SPNs, reinforcing its distinct nature compared to all other pancreatic neoplasms. Recent transcriptional studies have shown that activation of the Wnt/beta-catenin pathway in these tumors is associated with the upregulation of genes belonging to Notch, Hedgehog, and androgen receptor signaling pathways.

Combined hepatocellular-cholangiocarcinomas exhibit progenitor features and activation of Wnt and TGFß signaling pathways.

Intrahepatic malignant tumours include hepatocellular carcinomas (HCC), cholangiocarcinomas (CC) and combined hepatocholangiocarcinomas (cHCC-CC), a group of rare and poorly characterized tumours that exhibit both biliary and hepatocytic differentiation. The aim of the study was to characterize the molecular pathways specifically associated with cHCC-CC pathogenesis. We performed a genome-wide transcriptional analysis of 20 histologically defined cHCC-CC and compared them with a series of typical HCC and of CC. Data were analysed by gene set enrichment and integrative genomics and results were further validated in situ by tissue microarray using an independent series of 152 tumours. We report that cHCC-CC exhibit stem/progenitor features, a down-regulation of the hepatocyte differentiation program and a commitment to the biliary lineage. TGFß and Wnt/ß-catenin were identified as the two major signalling pathways activated in cHCC-CC. A ß-catenin signature distinct from that observed in well-differentiated HCC with mutant ß-catenin was found in cHCC-CC. This signature was associated with microenvironment remodelling and TGFß activation. Furthermore, integrative genomics revealed that cHCC-CC share characteristics of poorly differentiated HCC with stem cell traits and poor prognosis. The common traits displayed by CC, cHCC-CC and some HCC suggest that these tumours could originate from stem/progenitor cell(s) and raised the hypothesis of a potential continuum between intrahepatic CC, cHCC-CC and poorly differentiated HCC.

EpCAM, a new marker for cancer stem cells in hepatocellular carcinoma.

BACKGROUND & AIMS: Cancer progression/metastases and embryonic development share many properties including cellular plasticity, dynamic cell motility, and integral interaction with the microenvironment. We hypothesized that the heterogeneous nature of hepatocellular carcinoma (HCC), in part, may be owing to the presence of hepatic cancer cells with stem/progenitor features. METHODS: Gene expression profiling and immunohistochemistry analyses were used to analyze 235 tumor specimens derived from 2 recently identified HCC subtypes (EpCAM(+) alpha-fetoprotein [AFP(+)] HCC and EpCAM(-) AFP(-) HCC). These subtypes differed in their expression of AFP, a molecule produced in the developing embryo, and EpCAM, a cell surface hepatic stem cell marker. Fluorescence-activated cell sorting was used to isolate EpCAM(+) HCC cells, which were tested for hepatic stem/progenitor cell properties. RESULTS: Gene expression and pathway analyses revealed that the EpCAM(+) AFP(+) HCC subtype had features of hepatic stem/progenitor cells. Indeed, the fluorescence-activated cell sorting-isolated EpCAM(+) HCC cells displayed hepatic cancer stem cell-like traits including the abilities to self-renew and differentiate. Moreover, these cells were capable of initiating highly invasive HCC in nonobese diabetic, severe combined immunodeficient mice. Activation of Wnt/beta-catenin signaling enriched the EpCAM(+) cell population, whereas RNA interference-based blockage of EpCAM, a Wnt/beta-catenin signaling target, attenuated the activities of these cells. CONCLUSIONS: Taken together, our results suggest that HCC growth and invasiveness is dictated by a subset of EpCAM(+) cells, opening a new avenue for HCC cancer cell eradication by targeting Wnt/beta-catenin signaling components such as EpCAM.

Gene expression profiling provides insights into the pathways involved in solid pseudopapillary neoplasm of the pancreas.

Solid-pseudopapillary neoplasms (SPNs) are rare human pancreatic neoplasms usually associated with a good prognosis. In contrast to other pancreatic tumours, aberrant activation of the Wnt-beta-catenin pathway appears to be a constant feature in SPN. Aside from activation of the Wnt-beta-catenin pathway, little is known about biological pathways deregulated in SPN. We carried out transcriptome profiling of SPN to gain insights into the pathogenesis of these tumours. As expected, the over-expression of AXIN2, TBX3, SP5 and NOTUM demonstrated activation of the beta-catenin pathway. Members of the Notch pathway (HEY1, HEY2, NOTCH2) were also up-regulated, relative to their expression in ductal adenocarcinomas (DAC) or pancreatic endocrine tumours (PET). Other genes, such as EDN3, HAND2, netrin-G2 and the receptor netrin-G1 ligand, involved in neural crest differentiation, were also identified as altered. Increased levels of SOX10 and TuJ-1 proteins were also indicative of neural-like differentiation. In conclusion, SPN display a complex expression profile, distinct from that observed in PET and DAC and involving both the beta-catenin and Notch pathways, together with expression of neural differentiation markers.

Wnt/beta-catenin pathway in hepatocellular carcinoma pathogenesis and liver physiology.

The Wnt/beta-catenin pathway is a key developmental pathway for which alterations have been described in various human cancers. The aberrant activation of this pathway is a major event in human hepatocellular carcinoma. Several laboratories have shown that the Wnt/beta-catenin pathway plays an essential role in all phases of liver development and maturation, and is required for the metabolic function of this organ. In this review, we summarize current knowledge regarding the role of the Wnt/beta-catenin pathway in hepatocellular carcinoma pathogenesis and liver biology, and the possibilities for developing new therapeutic interventions based on this knowledge.

Genome-wide differences in hepatitis C- vs alcoholism-associated hepatocellular carcinoma.

AIM: To look at a comprehensive picture of etiology-dependent gene abnormalities in hepatocellular carcinoma in Western Europe. METHODS: With a liver-oriented microarray, transcript levels were compared in nodules and cirrhosis from a training set of patients with hepatocellular carcinoma (alcoholism, 12; hepatitis C, 10) and 5 controls. Loose or tight selection of informative transcripts with an abnormal abundance was statistically valid and the tightly selected transcripts were next quantified by qRTPCR in the nodules from our training set (12 + 10) and a test set (6 + 7). RESULTS: A selection of 475 transcripts pointed to significant gene over-representation on chromosome 8 (alcoholism) or -2 (hepatitis C) and ontology indicated a predominant inflammatory response (alcoholism) or changes in cell cycle regulation, transcription factors and interferon responsiveness (hepatitis C). A stringent selection of 23 transcripts whose differences between etiologies were significant in nodules but not in cirrhotic tissue indicated that the above dysregulations take place in tumor but not in the surrounding cirrhosis. These 23 transcripts separated our test set according to etiologies. The inflammation-associated transcripts pointed to limited alterations of free iron metabolism in alcoholic vs hepatitis C tumors. CONCLUSION: Etiology-specific abnormalities (chromo-some preference; differences in transcriptomes and related functions) have been identified in hepatocellular carcinoma driven by alcoholism or hepatitis C. This may open novel avenues for differential therapies in this disease.

Impaired E-cadherin expression and glutamine synthetase overexpression in solid pseudopapillary neoplasm of the pancreas.

OBJECTIVES: To analyze in solid pseudopapillary neoplasm of the pancreas (SPNP) the consequences of the deregulated Wnt pathway by studying the expression of Wnt target glutamine synthetase (GLUL), cyclin D1, and E-cadherin, which is one of the beta-catenin binding partner in cell adhesion. METHODS: The expression of cyclin D1 and GLUL was studied at the protein and/or messenger RNA levels, and the immunolocalization for E-cadherin was analyzed in 28 SPNPs screened for beta-catenin mutations. Expression of cyclin D1, GLUL, and beta-catenin was also assessed in pancreatic endocrine tumors as controls. RESULTS: Cytosolic and/or nuclear accumulation of beta-catenin was observed in all tumors; an activating beta-catenin mutation was identified in 21 (91%) of 23 tumors analyzed. E-cadherin expression is lost from the membrane and is observed in intracytosolic "dotlike" structures. Whereas cyclin D1 expression is observed widely in SPNP and endocrine tumors, GLUL expression is restricted to SPNP (100%) and rare endocrine tumors (10%) displaying Wnt activation. CONCLUSIONS: The activation of the Wnt/beta-catenin pathway in SPNP has 2 main consequences. First, E-cadherin expression moved from membranous to intracytoplasmic localization. Second, GLUL expression is highly correlated with Wnt/beta-catenin activation, demonstrating its faithfulness as a Wnt target gene.

Rapid identification of promoter hypermethylation in hepatocellular carcinoma by pyrosequencing of etiologically homogeneous sample pools.

Aberrant DNA methylation patterns have been identified in a variety of human diseases, particularly cancer. Pyrosequencing has evolved in recent years as a sensitive and accurate method for the analysis and quantification of the degree of DNA methylation in specific target regions. However, the number of candidate genes that can be analyzed in clinical specimens is often restricted by the limited amount of sample available. Here, we present a novel screening approach that enables the rapid identification of differentially methylated regions such as promoters by pyrosequencing of etiologically homogeneous sample pools after bisulfite treatment. We exemplify its use by the analysis of five genes (CDKN2A, GSTP1, MLH1, IGF2, and CTNNB1) involved in the pathogenesis of human hepatocellular carcinoma using pools stratified for different parameters of clinical importance. Results were confirmed by the individual analysis of the samples. The screening identified all genes displaying differential methylation successfully, and no false positives occurred. Quantitative comparison of the pools and the samples in the pool analyzed individually showed a deviation of approximately 1.5%, making the method ideally suited for the identification of diagnostic markers based on DNA methylation while saving precious DNA material.

Mutations of beta-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors.

Adrenocortical cancer is a rare cancer with a very poor prognosis. The genetic alterations identified to date in adrenocortical tumors are limited. Activating mutations of the Wnt signaling pathway have been observed in more frequent cancers, particularly digestive tract tumors. We investigated whether Wnt pathway activation is involved in adrenocortical tumorigenesis. In a series of 39 adrenocortical tumors, immunohistochemistry revealed abnormal cytoplasmic and/or nuclear accumulation of beta-catenin in 10 of 26 adrenocortical adenomas and in 11 of 13 adrenocortical carcinomas. An activating somatic mutation of the beta-catenin gene was shown in 7 of 26 adrenocortical adenomas and in 4 of 13 adrenocortical carcinomas; these mutations were observed only in adrenocortical tumors with abnormal beta-catenin accumulation and most were point mutations altering the Ser45 of exon 3 (in the consensus GSK3-beta/CK1 phosphorylation site). Functional studies showed that the activating Ser45 beta-catenin mutation found in the adrenocortical cancer H295R cell line leads to constitutive activation of T-cell factor-dependent transcription. This is the first molecular defect to be reported with the same prevalence in both benign (27%) and malignant (31%) adrenocortical tumors. beta-Catenin mutations are also the most frequent genetic defect currently known in adrenocortical adenomas. In adrenocortical adenomas, beta-catenin alterations are more frequent in nonfunctioning tumors, suggesting that beta-catenin pathway activation might be mostly involved in the development of nonsecreting adrenocortical adenomas and adrenocortical carcinomas. The very frequent and substantial accumulation of beta-catenin in adrenocortical carcinomas suggests that other alterations might also be involved. This finding may contribute to new therapeutic approaches targeting the Wnt pathway in malignant adrenocortical tumors, for which limited medical therapy is available.

Conditional hepatocarcinogenesis in mice expressing SV 40 early sequences.

We closely mimicked the in vivo setting in which sporadic hepatocarcinoma occurs by establishing a transgenic mouse model carrying regulatable SV40 early sequences under the control of the regulatory sequences of the human antithrombin III gene that confer hepatic expression. In this system, floxed dormant oncogenic sequences became functional after excision due to adenoviral expression of Cre recombinase or the stable transgenic expression in liver of a tamoxifen-inducible Cre. Hepatic oncogene expression was switched on by both methods, leading to the development of hepatocellular carcinoma. This model could be useful for investigating the key steps of the preneoplastic process, to identify suitable targets for the testing of new therapies.

The expression of TA and DeltaNp63 are regulated by different mechanisms in liver cells.

The TP63 gene, a member of the TP53 gene family, encodes several isoforms with (TAp63) or without (DeltaNp63) transactivating properties. Whereas the role of p63 in the normal development of squamous epithelia is well established, its function in other cell types remains to be elucidated. Here, we have analysed the expression of TA and DeltaNp63 isoforms in liver cells, by using both primary hepatocytes from wild type and p53-null mice and three human hepatocellular carcinoma (HCC) cell lines, according to the transformation state and the TP53 status of the cells. We observed the expression of DeltaNp63 isoforms only in a p53-null context. On the other hand, the expression of TAp63 isoforms was restricted to the HCC cell lines, whatever the TP53 status. We then studied the expression of TP63 upon genotoxic treatment. When treated with UVB or H(2)O(2), hepatocytes did not exhibit any change in p63 mRNA level. At the opposite, upon treatment with topoisomerase II inhibitors (doxorubicin or etoposide), the expression of TAp63 isoforms was clearly induced, independently of the TP53 status of cells. The same treatment did not induce any variation in the expression of DeltaNp63 isoforms, both at mRNA and protein levels. In HCC cell lines, doxorubicin or etoposide treatment also resulted in an increase of TAp63 transcripts only. This increase was accompanied by an increase in the intracellular level of TAp63 alpha protein. In parallel, we observed an upregulation of some p53-target genes related to cell cycle regulation, such as WAF1/CIP1, PIG3, 14-3-3sigma or GADD45, independently of the TP53 status of cells. In conclusion, we report for the first time that TA and DeltaNp63 alpha proteins are present in liver cells. Furthermore, our results suggest that p63 may partially substitute for wild-type p53, in counteracting uncontrolled liver cell proliferation in response to certain forms of DNA-damage.

Identification of the leukocyte cell-derived chemotaxin 2 as a direct target gene of beta-catenin in the liver.

To clarify molecular mechanisms underlying liver carcinogenesis induced by aberrant activation of Wnt pathway, we isolated the target genes of beta-catenin from mice exhibiting constitutive activated beta-catenin in the liver. Adenovirus-mediated expression of oncogenic beta-catenin was used to isolate early targets of beta-catenin in the liver. Suppression subtractive hybridization was used to identify the leukocyte cell-derived chemotaxin 2 (LECT2) gene as a direct target of beta-catenin. Northern blot and immunohistochemical analyses demonstrated that LECT2 expression is specifically induced in different mouse models that express activated beta-catenin in the liver. LECT2 expression was not activated in livers in which hepatocyte proliferation was induced by a beta-catenin-independent signal. We characterized by mutagenesis the LEF/TCF site, which is crucial for LECT2 activation by beta-catenin. We further characterized the chemotactic property of LECT2 for human neutrophils. Finally, we have shown an up-regulation of LECT2 in human liver tumors that expressed aberrant activation of beta-catenin signaling; these tumors constituted a subset of hepatocellular carcinomas (HCC) and most of the hepatoblastomas that were studied. In conclusion, our results show that LECT2, which encodes a protein with chemotactic properties for human neutrophils, is a direct target gene of Wnt/beta-catenin signaling in the liver. Since HCC develops mainly in patients with chronic hepatitis or cirrhosis induced by viral or inflammatory factors, understanding the role of LECT2 in liver carcinogenesis is of interest and may lead to new therapeutic perspectives.

Effect of Bcl-2 expression on hepatic preneoplasia in mice.

In a transgenic model of hepatocellular carcinoma induced by the expression of SV40 early sequences (TAg mice), deregulation of hepatocyte proliferation induces an apoptotic process whose decrease coincides with the appearance of neoplastic foci. Mating these mice with transgenic mice overexpressing Bcl-2 led to a dramatic reduction in the number of apoptotic hepatocytes during preneoplasia, resulting in an enlargement of the liver. This decrease in apoptosis was followed, 2 weeks later, by a reduction in hepatocellular proliferation. Sequential reduction in apoptosis and proliferation rate suggests that the anti-apoptotic and the anti-mitotic activities of Bcl-2 might be operative in distinct stages of preneoplasia.