Some Morphology Frontiers of Dysplasia in the Tubular Gastrointestinal Tract: The Rodger C. Haggitt Memorial Lecture.

This review, based on the content of the 2020 US Gastrointestinal Pathology Society's Rodger Haggitt Lecture, concerns an array of tubular gastrointestinal tract dysplastic or possible "predysplastic lesions" with an almost purely morphologic focus based on our collaborative efforts over the past few years. These processes include esophageal epidermoid metaplasia, Barrett esophagus-associated dysplasia, polypoid gastric dysplastic lesions, small intestinal dysplasia, and the ability of metastases to mimic it, the controversial "serrated epithelial change" encountered in the setting of long-standing ulcerative and Crohn colitis, and recently described anal columnar human papilloma virus-associated neoplasms.

Biomechanical Properties of Cancer Cells.

Since the crucial role of the microenvironment has been highlighted, many studies have been focused on the role of biomechanics in cancer cell growth and the invasion of the surrounding environment. Despite the search in recent years for molecular biomarkers to try to classify and stratify cancers, much effort needs to be made to take account of morphological and nanomechanical parameters that could provide supplementary information concerning tissue complexity adaptation during cancer development. The biomechanical properties of cancer cells and their surrounding extracellular matrix have actually been proposed as promising biomarkers for cancer diagnosis and prognosis. The present review first describes the main methods used to study the mechanical properties of cancer cells. Then, we address the nanomechanical description of cultured cancer cells and the crucial role of the cytoskeleton for biomechanics linked with cell morphology. Finally, we depict how studying interaction of tumor cells with their surrounding microenvironment is crucial to integrating biomechanical properties in our understanding of tumor growth and local invasion.

Expression of invadopodia markers can identify oral lesions with a high risk of malignant transformation.

Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of the oral cavity and is usually preceded by a range of premalignant tissue abnormalities termed oral potentially malignant disorders. Identifying malignant transformation is critical for early treatment and consequently improved survival and decreased morbidity. Invadopodia (INV) are specialized subcellular structures required for cancer cell invasion. We developed a new method to visualize INV in keratinocytes using fluorescent immunohistochemistry (FIHC) and semi-automated images analysis. The presence of INV was used to determine the risk of malignant transformation. We analyzed 145 formalin-fixed, paraffin-embedded (FFPE) oral biopsy samples from 95 patients diagnosed as nondysplastic, dysplastic, and OSCC including 49 patients whose lesions transformed to OSCC (progressing) and 46 cases that did not transform to OSCC (control). All samples were stained for Cortactin, tyrosine kinase substrate with five SH3 domains (Tks5) and matrix metallopeptidase 14 (MMP14) using FIHC, imaged using confocal microscopy and analyzed using a multichannel colocalization analysis. The areas of colocalization were used to generate an INV score. Using the INV score, we were able to identify progressing lesions with a sensitivity of 75-100% and specificity of 72-76%. A positive INV score was associated with increased risk of progression to OSCC. Our results suggest that INV markers can be used in conjunction with the current diagnostic standard for early detection of OSCC.

Malignant transformation of liver fatty acid binding protein-deficient hepatocellular adenomas: histopathologic spectrum of a rare phenomenon.

The molecular classification of hepatocellular adenomas highlights a distinctive genotype-phenotype correlation. Malignant transformation is an exceptionally rare complication of hepatocyte nuclear factor 1α (HNF1A)-inactivated hepatocellular adenomas. This subtype is characterized by loss of liver fatty acid binding protein immunoexpression. In this study, we characterized the histopathologic spectrum of 13 liver fatty acid binding protein-deficient hepatocellular adenoma cases showing malignant transformation from multiple centers. Clinicopathologic characteristics of these patients were evaluated. Stains for reticulin, liver fatty acid binding protein, beta-catenin and glutamine synthetase were applied to these lesions. Moreover, the findings were compared to patients with ß-catenin mutated hepatocellular adenoma. Liver fatty acid binding protein-deficient hepatocellular adenomas with borderline features/carcinoma were seen predominantly in females (77%) with an average age of 46 ± 18 years and multiple lesions (77%; five patients with adenomatosis). Meanwhile, ß-catenin mutated hepatocellular adenoma patients with malignant transformation were predominantly male (67%, p = 0.018) with single lesion (86%, p = 0.0009). The largest liver fatty acid binding protein-deficient hepatocellular adenoma nodule in each patient ranged from 4 to 15.5 cm. Loss of liver fatty acid binding protein by immunohistochemistry was noted in all adenoma and borderline/carcinoma components. Features of malignant transformation were pseudoglandular architecture (85%), cytologic atypia (85%), architectural atypia (100%) and lack of steatosis (100%). Other findings included myxoid change (39%), peliosis (46%) and sinusoidal dilatation (46%). Molecular studies confirmed somatic inactivation of HNF1A in 3 cases and absence of TERT promotor and exon 3 CTNNB1 mutations in five cases. To summarize, liver fatty acid binding protein-deficient hepatocellular adenoma with malignant transformation is most frequently seen in female patients with multiple lesions. Most of these lesions demonstrate pseudoglandular architecture, cytologic and architectural atypia, with lack of steatosis. The natural history of these lesions is relatively benign with the exception of disease recurrence in 1 patient.

Immunohistochemical assessment of Survivin and Bcl3 expression as potential biomarkers for NF-κB activation in the Barrett metaplasia-dysplasia-adenocarcinoma sequence.

Non-dysplastic Barrett's oesophagus (NDBE) occurs as a consequence of an inflammatory response triggered through prolonged gastro-oesophageal reflux and it may precede the development of oesophageal adenocarcinoma. NF-κB activation as a result of the inflammatory response has been shown in NDBE, but the possible mechanism involved in the process is unknown. The aim of this study was to assess, using immunohistochemistry, Survivin and Bcl3 expression as potential biomarkers for NF-κB activation along the oesophageal metaplasia-dysplasia-adenocarcinoma sequence. Survivin is an NF-κB-inducible anti-apoptotic protein, and Bcl3 is a negative regulator of NF-κB. There was progressive upregulation of Survivin expression along the oesophageal metaplasia-dysplasia-adenocarcinoma sequence. Bcl3 expression was upregulated in non-dysplastic Barrett's oesophagus, low-grade, high-grade dysplasia and oesophageal adenocarcinoma when compared to squamous group. The study shows the differential expression of Bcl3 between the squamous and Barrett's stage, suggesting that Bcl3 could be a surrogate marker for early event involving constitutive NF-κB activation. In addition, the study suggests that NF-κB activation may infer resistance to apoptosis through the expression of anti-apoptotic genes such as Survivin, which showed progressive increase in expression throughout the oesophageal metaplasia-dysplasia-adenocarcinoma sequence. This ability to avoid apoptosis may underlie the persistence and malignant predisposition of Barrett's metaplasia.

Bioorthogonal chemical imaging of metabolic changes during epithelial-mesenchymal transition of cancer cells by stimulated Raman scattering microscopy.

Study of metabolic changes during epithelial-mesenchymal transition (EMT) of cancer cells is important for basic understanding and therapeutic management of cancer progression. We here used metabolic labeling and stimulated Raman scattering (SRS) microscopy, a strategy of bioorthogonal chemical imaging, to directly visualize changes in anabolic metabolism during cancer EMT at a single-cell level. MCF-7 breast cancer cell is employed as a model system. Four types of metabolites (amino acids, glucose, fatty acids, and choline) are labeled with either deuterium or alkyne (C≡C) tag. Their intracellular incorporations into MCF-7 cells before or after EMT are visualized by SRS imaging targeted at the signature vibration frequency of C-D or C≡C bonds. Overall, after EMT, anabolism of amino acids, glucose, and choline is less active, reflecting slower protein and membrane synthesis in mesenchymal cells. Interestingly, we also observed less incorporation of glucose and palmitate acids into membrane lipids, but more of them into lipid droplets in mesenchymal cells. This result indicates that, although mesenchymal cells synthesize fewer membrane lipids, they are actively storing energy into lipid droplets, either through de novo lipogenesis from glucose or direct scavenging of exogenous free fatty acids. Hence, metabolic labeling coupled with SRS can be a straightforward method in imaging cancer metabolism.

From the Cover: Genomic Effects of Androstenedione and Sex-Specific Liver Cancer Susceptibility in Mice.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified. Short-term hepatic effects of A4 were compared with reference agonists of the estrogen receptor (ethinyl estradiol, EE) and glucocorticoid receptor (prednisone, PRED). Male B6C3F1 mice were exposed for 7 or 28 days to A4, EE, or PRED. EE increased and PRED suppressed hepatocyte proliferation, while A4 had no detectable effects. In a microarray analysis, EE and PRED altered >3000 and >670 genes, respectively, in a dose-dependent manner, whereas A4 did not significantly alter any genes. Gene expression was subsequently examined in archival liver samples from male and female B6C3F1 mice exposed to A4 for 90 days. A4 altered more genes in females than males and did not alter expression of genes linked to activation of the mitogenic xenobiotic receptors AhR, CAR, and PPARα in either sex. A gene expression biomarker was used to show that in female mice, the high dose of A4 activated the growth hormone-regulated transcription factor STAT5b, which controls sexually dimorphic gene expression in the liver. These findings suggest that A4 induces subtle age-related effects on STAT5b signaling that may contribute to the higher risk of liver cancer in males compared with females.

Histopathological evidence of neoplastic progression of von Meyenburg complex to intrahepatic cholangiocarcinoma.

Von Meyenburg complex (VMC) is generally thought to be benign, although its preneoplastic potential for intrahepatic cholangiocarcinoma (iCC) has been a subject of contention. We retrospectively reviewed 86 hepatectomy specimens with a diagnosis of iCC. Morphologically, an association between iCC and VMC was appreciated in 35% of cases that illustrated a gradual neoplastic progression from benign VMC to dysplasia and then to iCC. Among them, 24 cases had VMC lined by epithelial cells with low-grade biliary dysplasia and 13 with high-grade biliary dysplasia. VMC-associated iCCs were smaller in size and well to moderately differentiated, with features of anastomosing glandular architecture, ductal carcinoma in situ-like growth pattern, peritumoral lymphocytic infiltrate, central fibrous scar, and complete pushing border. They often presented as T1 tumors. In contrast, non-VMC-associated iCCs were moderately to poorly differentiated with solid, cribriform or papillary growth patterns. They likely exhibited necrosis, perineural invasion, positive surgical margin, lymphovascular invasion, and high T stage. Additionally, Ki67 and p53 immunostains support the continuing neoplastic evolution from benign VMC to dysplasia and then to iCC. VMC could become neoplastic, serving as an in situ carcinoma lesion to transform to iCC. The underlying molecular alteration and clinical implication of this neoplastic transformation deserves further investigation.

Alteration of PDGFRß-Akt-mTOR pathway signaling in fibrosarcomatous transformation of dermatofibrosarcoma protuberans.

Dermatofibrosarcoma protuberans (DFSP) is a cutaneous mesenchymal tumor of intermediate malignancy and fibroblastic/myofibroblastic differentiation. Fibrosarcomatous (FS) component is a high-grade component of DFSP. The detailed oncogenic difference between DFSP and FS components is not clear. We thus investigated the Akt-mTOR pathway in both components. We used 65 tumor samples obtained from 65 patients. The phosphorylation of Akt-mTOR pathway proteins (Akt, mTOR, 4EBP1, and S6RP) and PDGFRα/ß was assessed by immunohistochemical staining, the results of which were confirmed by Western blotting. The immunohistochemical results were as follows: in ordinary DFSP components, p-PDGFRα-positive tumors were 41.9% (18/43 cases), p-PDGFRß 55.8% (24/43 cases), p-Akt 51.2% (22/43 cases), p-mTOR 39.5% (17/43 cases), p-4EBP1 46.5% (20/43 cases), and p-S6RP 41.8% (18/43 cases); in DFSP components of FS-DFSP, 52.6% (10/19 cases), 47.4% (9/19 cases), 52.6% (10/19 cases), 36.8% (7/19 cases), 52.6% (10/19 cases), and 52.6% (10/19 cases); and in FS components, 45.5% (10/22 cases), 36.4% (8/22 cases), 72.7% (16/22 cases), 54.5% (12/22 cases), 72.7% (16/22 cases), and 68.2% (15/22 cases), respectively. There were significant positive correlations of the phosphorylation of most of the Akt-mTOR pathway proteins (p-Akt, p-mTOR, p-4EBP1, and p-S6RP) with each other (P < .05). Phospho-PDGFRß was well correlated with the phosphorylation of Akt-mTOR pathway proteins in DFSP components of ordinary and FS-DFSPs, but these correlations were weaker in FS components. This study suggested the association of activation of Akt-mTOR pathway proteins and PDGFR with the progression of DFSP to FS. The Akt-mTOR pathway is thus a potential therapeutic target in imatinib-resistant DFSP/FS.

Caveolin-1 expression in oral lichen planus, dysplastic lesions and squamous cell carcinoma.

Caveolin-1(Cav-1), the main part of caveolae structure, is supposed to play a role in pathogenesis of many human tumors. Since oral lichen planus (OLP) is considered as a potential premalignant disease, this study evaluated Cav-1 expression in OLP in comparison with benign hyperkeratosis, dysplastic epithelium and oral squamous cell carcinoma (OSCC), to investigate its possible role in pathogenesis and malignant transformation of OLP. In this cross-sectional retrospective study, immunohistochemical expression of Cav-1 in the epithelial component and stroma was evaluated in 81 samples, including 12 cases of hyperkeratosis, 24 OLP, 22 epithelial dysplasia, and 23 OSCC samples. Correlations between Cav-1 expression and clinicopathological variables were evaluated statistically. Positive Cav-1 staining was found in 58% of OLP, 91% of hyperkeratosis, 100% of epithelial dysplasia, and 95% of OSCC samples. OSCC showed the highest Cav-1 expression and OLP had the lowest (P=0.001). The intensity of staining was significantly increased in stepwise manner from OLP to OSCC (P=0.001). Expression of Cav-1 was related to the grade of samples in OSCC and dysplastic samples (P=0.04). Based on the findings, it was concluded that Cav-1 may play a role in the pathogenesis of OLP and carcinogenesis of SCC, but its role in malignant transformation of OLP is not confirmed. Further studies are needed to evaluate its potential therapeutic function in OLP and SCC.

[Immunohistochemical criteria for differential diagnostics of squamous epithelium changes in pyogenic granuloma and squamous cell carcinoma of the oral mucosa]. / Immunogistokhimicheskie kriterii differentsial'noi diagnostiki ploskogo épiteliia pri piogennoi granuleme i ploskokletochnom rake slizistoi obolochki rta.

The paper presents an example of differential diagnostic studies for pyogenic granuloma and oral squamous cell carcinoma (SCC). In the described case immunohistochemistry with antibodies to Ki-67 and Gli1 was used as conventional histological procedure proved to be inconvenient for adequate diagnostics. The immunohistochemical study established increased proliferative activity of epithelial cells specific both pyogenic granuloma and oral SCC, but intensive Gli1 protein expression in membranes and cytoplasm of epithelial cells with malignant transformation allowed differentiation of these neoplasms.

Progression of atypical extraventricular neurocytoma to anaplastic ganglioglioma.

We report a childhood case of thalamic atypical extraventricular neurocytoma that progressed to highly anaplastic ganglioglioma after 8 years of dormancy after subtotal resection and chemotherapy. The neurocytoma displayed immunoreactivity only for synaptophysin, ß-catenin, S100, and CD56. The ganglioglioma acquired strong immunoreactivity for chromogranin, glial fibrillary acidic protein, neuron-specific enolase, and p53 and showed a very high proliferation rate approaching 50% in some areas. Tumor transformation was associated with overexpression of components of the sonic hedgehog and Wnt developmental signaling pathways, which are known to regulate tumor-initiating cells in malignant brain neoplasms.

Malignant transformation of polyostotic fibrous dysplasia with aberrant keratin expression.

Malignant transformation of fibrous dysplasia (FD) is exceedingly rare, occurring in less than 1% of all FD cases, and has been described in both monostotic and polyostotic forms of this entity. We report a case of a large proximal femur mass arising in a 45-year-old man. The biopsy revealed a high-grade pleomorphic malignancy that focally expressed multiple keratins. Based on the presence of keratin immunoreactivity, the morphologic differential diagnosis included metastatic sarcomatoid carcinoma. However, review of the clinical information revealed a history of polyostotic FD, and imaging findings were compatible with malignant transformation of FD. The resected neoplasm was biphasic and composed of areas of conventional FD admixed with a high-grade pleomorphic malignancy. Activating GNAS mutations were identified in both components. To the best of our knowledge, this is the first description of keratin expression in malignant transformation of FD.

Mucinous adenocarcinoma arising from a residual supratentorial neurenteric cyst and expressing mutated KRAS: a case report.

Malignant transformation of intracranial neurenteric cysts (NCs) has been reported in only 7 cases, but the molecular characteristics leading to malignant transformation remain unclear. A 61-year-old woman presented with headache and dizziness. Imaging revealed a 10-cm, extra-axial cystic mass in both middle fossae. A partial resection was performed, and the residual mass size gradually decreased. She had repeated ventriculoperitoneal shunts to relieve symptoms of hydrocephalus. Eight years later, follow-up images revealed marked enlargement of the mass and a newly developed lesion. After a second partial resection, a mucinous adenocarcinoma infiltrating the brain was identified. Transitions from benign-looking cuboidal cells to dysplastic cells were observed. A KRAS mutation, which might be associated with malignant NC transformation and was not present in the initial specimen, was identified in the adenocarcinoma. In conclusion, KRAS-mutant mucinous adenocarcinoma may arise in a longstanding residual NC after partial resection.

Loss of expression of Plag1 in malignant transformation from pleomorphic adenoma to carcinoma ex pleomorphic adenoma.

PLAG1 (pleomorphic adenoma gene 1) is frequently activated in pleomorphic adenoma (PA). Carcinoma ex pleomorphic adenoma (CXPA) arises in PA, and PLAG1 expression is believed to be maintained from PA to CXPA, as it can contribute to the carcinogenesis process. To evaluate if PLAG1 is a good marker of malignant transformation from PA to CXPA as well as to evaluate if PLAG1 expression is associated with progression and histopathologic subtype of CXPA. Forty PAs, 21 residual PAs (without malignant transformation), and 40 CXPAs were analyzed by immunohistochemistry with PLAG1 antibody. The proportion of positive neoplastic cells was assessed according to a 2-tiered scale: >10% to 50%, and >50% positive cells. The CXPA group was classified according to histopathologic subtype and invasiveness degree. Thirty-seven PAs (92.5%), 15 residual PAs (71%), and 14 CXPAs (35%) were positive for PLAG1. In relation to the CXPA group, among the intracapsular cases, myoepithelial carcinoma and epithelial-myoepithelial carcinoma showed the highest level of PLAG1 expression. PLAG1 expression is lost when PA undergoes malignant transformation, possibly due to other pathway activation and different clone cells. In addition, PLAG1 expression seems to be present mainly in low-grade carcinomas and in cases with early phase of invasion, due to its regulation of oncogene-induced cell senescence. In CXPA, PLAG1 expression was most associated with myoepithelial differentiation. This way, loss of PLAG1 expression can be considered a hallmark of CXPA carcinogenesis, mainly when there is only epithelial differentiation.

Evaluation of hypoxia, angiogenesis, and lymphangiogenesis in actinic cheilitis.

BACKGROUND: Actinic cheilitis is a potentially malignant condition caused mainly by chronic sun exposure. Here we aim to evaluate the role of hypoxia, angiogenesis, and lymphatic density in the clinical and morphological progression of a series of cases of actinic cheilitis. MATERIALS AND METHODS: Immunohistochemistry was used to evaluate positivity to hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF)-C, and D2-40 in 40 cases of actinic cheilitis of the lower lip. RESULTS: The cases studied exhibited variable degrees of positivity to the markers. The median number of lymphatic vessels was 3.2, 2.4, and 3.0 in lesions showing no epithelial dysplasia (NED) and with mild (MED) and moderate (MOED) epithelial dysplasia, respectively. The median VEGF-C positivity index was 82.44% (NED), 92.74% (MED), and 82.83% (MOED), and the median HIF-1α positivity index was 11.57% (NED), 5.26% (MED), and 13.55% (MOED). No significant differences in lymphatic density or median VEGF-C and HIF-1α positivity indices were observed between histological grades or clinical presentations of actinic cheilitis (P > 0.05). CONCLUSIONS: Although representing early events in lip carcinogenesis, the present results suggest that hypoxia, angiogenesis, and lymphangiogenesis do not influence the morphological or clinical progression of actinic cheilitis.

High Relative Expression of Pannexin 3 (PANX3) in an Axillary Sweat Gland Carcinoma With Osteosarcomatous Transformation.

Primary cutaneous sweat gland carcinomas (SGCs) are rare tumors that commonly involve axillae, have a high local recurrence rate, and rarely show sarcomatoid transformation. A 68-year-old man presented with rapid enlargement of a previously stable, asymptomatic pea-sized nodule in the left axilla. Initial excision (with positive surgical margins) at another institution showed characteristic histologic features of a high-grade osteosarcoma and molecular analysis using a 92-gene real-time quantitative reverse transcription-polymerase chain reaction assay confirmed a diagnosis of osteosarcoma with 96% certainty. Notably, the molecular assay demonstrated consistently high relative expression of pannexin 3 (PANX3), a gene involved in normal osteoblast differentiation which, when highly expressed, strongly predicts osteosarcoma per the assay's algorithm. However, on further histologic review, the tumor also contained focal cystic areas, nests, and ducts composed of malignant epithelial cells reminiscent of SGC; these areas directly transitioned into the osteosarcomatous component and were strongly positive for pancytokeratin, CK7, and p63. Within 2 weeks, the lesion recurred and grew rapidly, prompting complete resection, histologic sections of which showed high-grade osteosarcoma without residual epithelial elements. This is the fifth report, to our knowledge, of osteosarcomatous transformation in a SGC, and the only report to date including molecular data. This case demonstrates that osteosarcoma arising from a SGC has a similar molecular profile to de novo primary osteosarcoma of bone. It also emphasizes the importance of histopathologic findings as the established diagnostic gold standard and the need to interpret molecular results within the clinical context.

Dissecting characteristics and dynamics of differentially expressed proteins during multistage carcinogenesis of human colorectal cancer.

AIM: To discover novel biomarkers for early diagnosis, prognosis or treatment of human colorectal cancer. METHODS: iTRAQ 2D LC-MS/MS analysis was used to identify differentially expressed proteins (DEPs) in the human colonic epithelial carcinogenic process using laser capture microdissection-purified colonic epithelial cells from normal colon, adenoma, carcinoma in situ and invasive carcinoma tissues. RESULTS: A total of 326 DEPs were identified, and four DEPs (DMBT1, S100A9, Galectin-10, and S100A8) with progressive alteration in the carcinogenic process were further validated by immunohistochemistry. The DEPs were involved in multiple biological processes including cell cycle, cell adhesion, translation, mRNA processing, and protein synthesis. Some of the DEPs involved in cellular process such as "translation" and "mRNA splicing" were progressively up-regulated, while some DEPs involved in other processes such as "metabolism" and "cell response to stress" was progressively down-regulated. Other proteins with up- or down-regulation at certain stages of carcinogenesis may play various roles at different stages of the colorectal carcinogenic process. CONCLUSION: These findings give insights into our understanding of the mechanisms of colorectal carcinogenesis and provide clues for further investigation of carcinogenesis and identification of biomarkers.

Phospho-proteomic analyses of B-Raf protein complexes reveal new regulatory principles.

B-Raf represents a critical physiological regulator of the Ras/RAF/MEK/ERK-pathway and a pharmacological target of growing clinical relevance, in particular in oncology. To understand how B-Raf itself is regulated, we combined mass spectrometry with genetic approaches to map its interactome in MCF-10A cells as well as in B-Raf deficient murine embryonic fibroblasts (MEFs) and B-Raf/Raf-1 double deficient DT40 lymphoma cells complemented with wildtype or mutant B-Raf expression vectors. Using a multi-protease digestion approach, we identified a novel ubiquitination site and provide a detailed B-Raf phospho-map. Importantly, we identify two evolutionary conserved phosphorylation clusters around T401 and S419 in the B-Raf hinge region. SILAC labelling and genetic/biochemical follow-up revealed that these clusters are phosphorylated in the contexts of oncogenic Ras, sorafenib induced Raf dimerization and in the background of the V600E mutation. We further show that the vemurafenib sensitive phosphorylation of the T401 cluster occurs in trans within a Raf dimer. Substitution of the Ser/Thr-residues of this cluster by alanine residues enhances the transforming potential of B-Raf, indicating that these phosphorylation sites suppress its signaling output. Moreover, several B-Raf phosphorylation sites, including T401 and S419, are somatically mutated in tumors, further illustrating the importance of phosphorylation for the regulation of this kinase.

Interferon-microRNA signalling drives liver precancerous lesion formation and hepatocarcinogenesis.

OBJECTIVE: Precancerous lesion, a well-established histopathologically premalignant tissue with the highest risk for tumourigenesis, develops preferentially from activation of DNA damage checkpoint and persistent inflammation. However, little is known about the mechanisms by which precancerous lesions are initiated and their physiological significance. DESIGN: Laser capture microdissection was used to acquire matched normal liver, precancerous lesion and tumour tissues. miR-484(-/-), Ifnar1(-/-) and Tgfbr2(△hep) mice were employed to determine the critical role of the interferon (IFN)-microRNA pathway in precancerous lesion formation and tumourigenesis. RNA immunoprecipitation (RIP), pull-down and chromatin immunoprecipitation (ChIP) assays were applied to explore the underlying mechanisms. RESULTS: miR-484 is highly expressed in over 88% liver samples clinically. DEN-induced precancerous lesions and hepatocellular carcinoma were dramatically impaired in miR-484(-/-) mice. Mechanistically, ectopic expression of miR-484 initiates tumourigenesis and cell malignant transformation through synergistic activation of the transforming growth factor-ß/Gli and nuclear factor-κB/type I IFN pathways. Specific acetylation of H3K27 is indispensable for basal IFN-induced continuous transcription of miR-484 and cell transformation. Convincingly, formation of precancerous lesions were significantly attenuated in both Tgfbr2(△hep) and Ifnar1(-/-) mice. CONCLUSIONS: These findings demonstrate a new protumourigenic axis involving type I IFN-microRNA signalling, providing a potential therapeutic strategy to manipulate or reverse liver precancerous lesions and tumourigenesis.