DYRK1B blockade promotes tumoricidal macrophage activity in pancreatic cancer.

OBJECTIVE: Highly malignant pancreatic ductal adenocarcinoma (PDAC) is characterised by an abundant immunosuppressive and fibrotic tumour microenvironment (TME). Future therapeutic attempts will therefore demand the targeting of tumours and stromal compartments in order to be effective. Here we investigate whether dual specificity and tyrosine phosphorylation-regulated kinase 1B (DYRK1B) fulfil these criteria and represent a promising anticancer target in PDAC. DESIGN: We used transplantation and autochthonous mouse models of PDAC with either genetic Dyrk1b loss or pharmacological DYRK1B inhibition, respectively. Mechanistic interactions between tumour cells and macrophages were studied in direct or indirect co-culture experiments. Histological analyses used tissue microarrays from patients with PDAC. Additional methodological approaches included bulk mRNA sequencing (transcriptomics) and proteomics (secretomics). RESULTS: We found that DYRK1B is mainly expressed by pancreatic epithelial cancer cells and modulates the influx and activity of TME-associated macrophages through effects on the cancer cells themselves as well as through the tumour secretome. Mechanistically, genetic ablation or pharmacological inhibition of DYRK1B strongly attracts tumoricidal macrophages and, in addition, downregulates the phagocytosis checkpoint and 'don't eat me' signal CD24 on cancer cells, resulting in enhanced tumour cell phagocytosis. Consequently, tumour cells lacking DYRK1B hardly expand in transplantation experiments, despite their rapid growth in culture. Furthermore, combining a small-molecule DYRK1B-directed therapy with mammalian target of rapamycin inhibition and conventional chemotherapy stalls the growth of established tumours and results in a significant extension of life span in a highly aggressive autochthonous model of PDAC. CONCLUSION: In light of DYRK inhibitors currently entering clinical phase testing, our data thus provide a novel and clinically translatable approach targeting both the cancer cell compartment and its microenvironment.

Histone H3K27 demethylase KDM6A is an epigenetic gatekeeper of mTORC1 signalling in cancer.

OBJECTIVE: Large-scale genome sequencing efforts of human tumours identified epigenetic modifiers as one of the most frequently mutated gene class in human cancer. However, how these mutations drive tumour development and tumour progression are largely unknown. Here, we investigated the function of the histone demethylase KDM6A in gastrointestinal cancers, such as liver cancer and pancreatic cancer. DESIGN: Genetic alterations as well as expression analyses of KDM6A were performed in patients with liver cancer. Genetic mouse models of liver and pancreatic cancer coupled with Kdm6a-deficiency were investigated, transcriptomic and epigenetic profiling was performed, and in vivo and in vitro drug treatments were conducted. RESULTS: KDM6A expression was lost in 30% of patients with liver cancer. Kdm6a deletion significantly accelerated tumour development in murine liver and pancreatic cancer models. Kdm6a-deficient tumours showed hyperactivation of mTORC1 signalling, whereas endogenous Kdm6a re-expression by inducible RNA-interference in established Kdm6a-deficient tumours diminished mTORC1 activity resulting in attenuated tumour progression. Genome-wide transcriptional and epigenetic profiling revealed direct binding of Kdm6a to crucial negative regulators of mTORC1, such as Deptor, and subsequent transcriptional activation by epigenetic remodelling. Moreover, in vitro and in vivo genetic epistasis experiments illustrated a crucial function of Deptor and mTORC1 in Kdm6a-dependent tumour suppression. Importantly, KDM6A expression in human tumours correlates with mTORC1 activity and KDM6A-deficient tumours exhibit increased sensitivity to mTORC1 inhibition. CONCLUSION: KDM6A is an important tumour suppressor in gastrointestinal cancers and acts as an epigenetic toggle for mTORC1 signalling. Patients with KDM6A-deficient tumours could benefit of targeted therapy focusing on mTORC1 inhibition.

Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network (EVNet) to fuel tumour progression.

OBJECTIVE: Intratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour. DESIGN: We have studied the spontaneous flow of extracellular vesicles (EVs) between subpopulations of cancer cells: cancer stem cells (CSC) and non-stem cancer cells (NSCC). To determine the biological significance of the most frequent communication route, we used pancreatic ductal adenocarcinoma (PDAC) orthotopic models, patient-derived xenografts (PDXs) and genetically engineered mouse models (GEMMs). RESULTS: We demonstrate that PDAC tumours establish an organised communication network between subpopulations of cancer cells using EVs called the EVNet). The EVNet is plastic and reshapes in response to its environment. Communication within the EVNet occurs preferentially from CSC to NSCC. Inhibition of this communication route by impairing Rab27a function in orthotopic xenographs, GEMMs and PDXs is sufficient to hamper tumour growth and phenocopies the inhibition of communication in the whole tumour. Mechanistically, we provide evidence that CSC EVs use agrin protein to promote Yes1 associated transcriptional regulator (YAP) activation via LDL receptor related protein 4 (LRP-4). Ex vivo treatment of PDXs with antiagrin significantly impairs proliferation and decreases the levels of activated YAP.Patients with high levels of agrin and low inactive YAP show worse disease-free survival. In addition, patients with a higher number of circulating agrin+ EVs show a significant increased risk of disease progression. CONCLUSION: PDAC tumours establish a cooperation network mediated by EVs that is led by CSC and agrin, which allows tumours to adapt and thrive. Targeting agrin could make targeted therapy possible for patients with PDAC and has a significant impact on CSC that feeds the tumour and is at the centre of therapy resistance.

Targeting cancer-associated fibroblast-secreted WNT2 restores dendritic cell-mediated antitumour immunity.

OBJECTIVE: Solid tumours respond poorly to immune checkpoint inhibitor (ICI) therapies. One major therapeutic obstacle is the immunosuppressive tumour microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a key component of the TME and negatively regulate antitumour T-cell response. Here, we aimed to uncover the mechanism underlying CAFs-mediated tumour immune evasion and to develop novel therapeutic strategies targeting CAFs for enhancing ICI efficacy in oesophageal squamous cell carcinoma (OSCC) and colorectal cancer (CRC). DESIGN: Anti-WNT2 monoclonal antibody (mAb) was used to treat immunocompetent C57BL/6 mice bearing subcutaneously grafted mEC25 or CMT93 alone or combined with anti-programmed cell death protein 1 (PD-1), and the antitumour efficiency and immune response were assessed. CAFs-induced suppression of dendritic cell (DC)-differentiation and DC-mediated antitumour immunity were analysed by interfering with CAFs-derived WNT2, either by anti-WNT2 mAb or with short hairpin RNA-mediated knockdown. The molecular mechanism underlying CAFs-induced DC suppression was further explored by RNA-sequencing and western blot analyses. RESULTS: A negative correlation between WNT2+ CAFs and active CD8+ T cells was detected in primary OSCC tumours. Anti-WNT2 mAb significantly restored antitumour T-cell responses within tumours and enhanced the efficacy of anti-PD-1 by increasing active DC in both mouse OSCC and CRC syngeneic tumour models. Directly interfering with CAFs-derived WNT2 restored DC differentiation and DC-mediated antitumour T-cell responses. Mechanistic analyses further demonstrated that CAFs-secreted WNT2 suppresses the DC-mediated antitumour T-cell response via the SOCS3/p-JAK2/p-STAT3 signalling cascades. CONCLUSIONS: CAFs could suppress antitumour immunity through WNT2 secretion. Targeting WNT2 might enhance the ICI efficacy and represent a new anticancer immunotherapy.

RIPK3 acts as a lipid metabolism regulator contributing to inflammation and carcinogenesis in non-alcoholic fatty liver disease.

OBJECTIVE: Receptor-interacting protein kinase 3 (RIPK3) is a key player in necroptosis execution and an emerging metabolic regulator, whose contribution to non-alcoholic fatty liver disease (NAFLD) is controversial. We aimed to clarify the impact of RIPK3 signalling in the pathogenesis of human and experimental NAFLD. DESIGN: RIPK3 levels were evaluated in two large independent cohorts of patients with biopsy proven NAFLD diagnosis and correlated with clinical and biochemical parameters. Wild-type (WT) or Ripk3-deficient (Ripk3-/-) mice were fed a choline-deficient L-amino acid-defined diet (CDAA) or an isocaloric control diet for 32 and 66 weeks. RESULTS: RIPK3 increased in patients with non-alcoholic steatohepatitis (NASH) in both cohorts, correlating with hepatic inflammation and fibrosis. Accordingly, Ripk3 deficiency ameliorated CDAA-induced inflammation and fibrosis in mice at both 32 and 66 weeks. WT mice on the CDAA diet for 66 weeks developed preneoplastic nodules and displayed increased hepatocellular proliferation, which were reduced in Ripk3-/- mice. Furthermore, Ripk3 deficiency hampered tumourigenesis. Intriguingly, Ripk3-/- mice displayed increased body weight gain, while lipidomics showed that deletion of Ripk3 shifted hepatic lipid profiles. Peroxisome proliferator-activated receptor γ (PPARγ) was increased in Ripk3-/- mice and negatively correlated with hepatic RIPK3 in patients with NAFLD. Mechanistic studies established a functional link between RIPK3 and PPARγ in controlling fat deposition and fibrosis. CONCLUSION: Hepatic RIPK3 correlates with NAFLD severity in humans and mice, playing a key role in managing liver metabolism, damage, inflammation, fibrosis and carcinogenesis. Targeting RIPK3 and its intricate signalling arises as a novel promising approach to treat NASH and arrest disease progression.

TREM-2 defends the liver against hepatocellular carcinoma through multifactorial protective mechanisms.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a prevalent and aggressive cancer usually arising on a background of chronic liver injury involving inflammatory and hepatic regenerative processes. The triggering receptor expressed on myeloid cells 2 (TREM-2) is predominantly expressed in hepatic non-parenchymal cells and inhibits Toll-like receptor signalling, protecting the liver from various hepatotoxic injuries, yet its role in liver cancer is poorly defined. Here, we investigated the impact of TREM-2 on liver regeneration and hepatocarcinogenesis. DESIGN: TREM-2 expression was analysed in liver tissues of two independent cohorts of patients with HCC and compared with control liver samples. Experimental HCC and liver regeneration models in wild type and Trem-2-/- mice, and in vitro studies with hepatic stellate cells (HSCs) and HCC spheroids were conducted. RESULTS: TREM-2 expression was upregulated in human HCC tissue, in mouse models of liver regeneration and HCC. Trem-2-/- mice developed more liver tumours irrespective of size after diethylnitrosamine (DEN) administration, displayed exacerbated liver damage, inflammation, oxidative stress and hepatocyte proliferation. Administering an antioxidant diet blocked DEN-induced hepatocarcinogenesis in both genotypes. Similarly, Trem-2-/- animals developed more and larger tumours in fibrosis-associated HCC models. Trem-2-/- livers showed increased hepatocyte proliferation and inflammation after partial hepatectomy. Conditioned media from human HSCs overexpressing TREM-2 inhibited human HCC spheroid growth in vitro through attenuated Wnt ligand secretion. CONCLUSION: TREM-2 plays a protective role in hepatocarcinogenesis via different pleiotropic effects, suggesting that TREM-2 agonism should be investigated as it might beneficially impact HCC pathogenesis in a multifactorial manner.

miR-541 potentiates the response of human hepatocellular carcinoma to sorafenib treatment by inhibiting autophagy.

OBJECTIVE: Autophagy participates in the progression of hepatocellular carcinoma (HCC) and the resistance of HCC cells to sorafenib. We investigated the feasibility of sensitising HCC cells to sorafenib by modulating miR-541-initiated microRNA-autophagy axis. DESIGN: Gain- and loss-of-function assays were performed to evaluate the effects of miR-541 on the malignant properties and autophagy of human HCC cells. Autophagy was quantified by western blotting of LC3, transmission electron microscopy analyses and confocal microscopy scanning of mRFP-GFP-LC3 reporter construct. Luciferase reporter assays were conducted to confirm the targets of miR-541. HCC xenograft tumours were established to analyse the role of miR-541 in sorafenib-induced lethality. RESULTS: The expression of miR-541 was downregulated in human HCC tissues and was associated with malignant clinicopathologic phenotypes, recurrence and survival of patients with HCC. miR-541 inhibited the growth, metastasis and autophagy of HCC cells both in vitro and in vivo. Prediction software and luciferase reporter assays identified autophagy-related gene 2A (ATG2A) and Ras-related protein Rab-1B (RAB1B) as the direct targets of miR-541. Consistent with the effects of the miR-541 mimic, inhibition of ATG2A or RAB1B suppressed the malignant phenotypes and autophagy of HCC cells. Furthermore, siATG2A and siRAB1B partially reversed the enhancement of the malignant properties and autophagy in HCC cells mediated by the miR-541 inhibitor. More interestingly, higher miR-541 expression predicted a better response to sorafenib treatment, and the combination of miR-541 and sorafenib further suppressed the growth of HCC cells in vivo compared with the single treatment. CONCLUSIONS: Dysregulation of miR-541-ATG2A/RAB1B axis plays a critical role in patients' responses to sorafenib treatment. Manipulation of this axis might benefit survival of patients with HCC, especially in the context of the highly pursued strategies to eliminate drug resistance.

[Cholangiocarcinoma-diagnosis, classification, and molecular alterations]. / Cholangiokarzinome ­ Übersicht zur aktuellen anatomischen, histomorphologischen und morphomolekularen Klassifikation.

BACKGROUND: The entity cholangiocarcinoma comprises various malignant epithelial neoplasms of different morphology, etiology, and pathogenesis. AIM: In this review, the etiology, pathogenesis, diagnostic criteria, classification, and molecular alterations of intra- and extrahepatic cholangiocarcinomas are summarized. MATERIAL AND METHODS: This review is based on the currently available literature and personal knowledge. RESULTS AND DISCUSSION: Cholangiocarcinomas are morphologically and molecularly diverse neoplasms that can develop ubiquitously in the biliary tract. Since there is a significant histological and immunohistochemical overlap with frequent liver metastases, a definite diagnosis can often only be rendered in the clinical context. Cholangiocarcinomas are subclassified into intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA) tumors according to macro-anatomical criteria. Recent studies show that there are distinctive molecular differences in particular between iCCA and extrahepatic CCA (eCCA, including pCCA and dCCA). In addition, morphologically well-characterized precursor lesions have been identified, which in various frequencies can be assigned not only to the anatomical classification, but also to certain etiologies. An exact classification is therefore essential, especially with regard to the development of innovative, targeted therapeutic approaches.

RSPO2 gene rearrangement: a powerful driver of ß-catenin activation in liver tumours.

OBJECTIVE: We aimed at the identification of genetic alterations that may functionally substitute for CTNNB1 mutation in ß-catenin-activated hepatocellular adenomas (HCAs) and hepatocellular carcinoma (HCC). DESIGN: Large cohorts of HCA (n=185) and HCC (n=468) were classified using immunohistochemistry. The mutational status of the CTNNB1 gene was determined in ß-catenin-activated HCA (b-HCA) and HCC with at least moderate nuclear CTNNB1 accumulation. Ultra-deep sequencing was used to characterise CTNNB1wild-type and ß-catenin-activated HCA and HCC. Expression profiling of HCA subtypes was performed. RESULTS: A roof plate-specific spondin 2 (RSPO2) gene rearrangement resulting from a 46.4 kb microdeletion on chromosome 8q23.1 was detected as a new morphomolecular driver of ß-catenin-activated HCA. RSPO2 fusion positive HCA displayed upregulation of RSPO2 protein, nuclear accumulation of ß-catenin and transcriptional activation of ß-catenin-target genes indicating activation of Wingless-Type MMTV Integration Site Family (WNT) signalling. Architectural and cytological atypia as well as interstitial invasion indicated malignant transformation in one of the RSPO2 rearranged b-HCAs. The RSPO2 gene rearrangement was also observed in three ß-catenin-activated HCCs developing in context of chronic liver disease. Mutations of the human telomerase reverse transcriptase promoter-known to drive malignant transformation of CTNNB1-mutated HCA-seem to be dispensable for RSPO2 rearranged HCA and HCC. CONCLUSION: The RSPO2 gene rearrangement leads to oncogenic activation of the WNT signalling pathway in HCA and HCC, represents an alternative mechanism for the development of b-HCA and may drive malignant transformation without additional TERT promoter mutation.

Stromal biology and therapy in pancreatic cancer: ready for clinical translation?

Pancreatic ductal adenocarcinoma (PDA) is notoriously aggressive and hard to treat. The tumour microenvironment (TME) in PDA is highly dynamic and has been found to promote tumour progression, metastasis niche formation and therapeutic resistance. Intensive research of recent years has revealed an incredible heterogeneity and complexity of the different components of the TME, including cancer-associated fibroblasts, immune cells, extracellular matrix components, tumour vessels and nerves. It has been hypothesised that paracrine interactions between neoplastic epithelial cells and TME compartments may result in either tumour-promoting or tumour-restraining consequences. A better preclinical understanding of such complex and dynamic network systems is required to develop more powerful treatment strategies for patients. Scientific activity and the number of compelling findings has virtually exploded during recent years. Here, we provide an update of the most recent findings in this area and discuss their translational and clinical implications for basic scientists and clinicians alike.

Genetic Alterations of Periampullary and Pancreatic Ductal Adenocarcinoma: An Overview.

Pancreatic Ductal AdenoCarcinoma (PDAC) is one of the most lethal malignancies of all solid cancers. Precancerous lesions for PDAC include PanIN, IPMNs and MCNs. PDAC has a poor prognosis with a 5-year survival of approximately 6%. Whereas Periampulary AdenoCarcinoma (PAC) having four anatomic subtypes, pancreatic, Common Bile Duct (CBD), ampullary and duodenum shows relative better prognosis. The highest incidence of PDAC has been reported with black with respect to white population. Similarly, incidence rate of PAC also differs with different ethnic populations. Several lifestyle, environmental and occupational exposures including long-term diabetes, obesity, and smoking, have been linked to PDAC, however, for PAC the causal risk factors were poorly described. It is now clear that PDAC and PAC are a multi-stage process resulting from the accumulation of genomic alterations in the somatic DNA of normal cells as well as inherited mutations. Approximately 10% of PDAC have a familial inheritance. Germline mutations in CDKN2A, BRCA2, STK11, PALB2, PRSS1, etc., as well as certain syndromes have been well associated with predisposition to PDAC. KRAS, CDKN2A, TP53 and SMAD4 are the 4 "mountains" (high-frequency driver genes) which have been known to earliest somatic alterations for PDAC while relatively less frequent in PAC. Our understanding of the molecular carcinogenesis has improved in the last few years due to extensive research on PDAC which was not well explored in case of PAC. The genetic alterations that have been identified in PDAC and different subgroups of PAC are important implications for the development of genetic screening test, early diagnosis, and prognostic genetic markers. The present review will provide a brief overview of the incidence and prevalence of PDAC and PAC, mainly, increased risk in India, the several kinds of risk factors associated with the diseases as well as required genetic alterations for disease initiation and progression.

hTERT promotes the invasion of gastric cancer cells by enhancing FOXO3a ubiquitination and subsequent ITGB1 upregulation.

BACKGROUND AND AIMS: Human telomerase reverse transcriptase (hTERT) plays an important role in cancer invasion, but the relevant mechanism is not well known. This study aims to investigate the role and mechanism of hTERT in gastric cancer metastasis. DESIGN: Proteomics analysis, qPCR and western blotting were used to screen for hTERT-regulated candidate molecules in gastric cancer invasion. Chromatin immunoprecipitation (ChIP) qPCR was performed to identify the binding sites of hTERT at the regulatory region of the integrin ß1 (ITGB1) gene. ChIP assays were further applied to elucidate the transcription factors that bound to the regulatory region. The interactions between hTERT and the transcription factors were tested by co-immunoprecipitation (Co-IP) and glutathione S-transferase (GST) pull-down experiments. Moreover, the revealed pathway was verified in tumour-bearing nude mice and human gastric cancer tissues. RESULTS: ITGB1 was identified as a downstream gene of hTERT, and there were two hTERT-binding regions within this gene. hTERT alleviated the binding of forkhead box O3 (FOXO3a) to FOXO3a binding element (+9972∼+9978), but it enhanced the binding of forkhead box M1 (FOXM1) to FOXM1 binding element (-1104∼-1109) in ITGB1 gene. Importantly, FOXO3a played a major role in hTERT-induced ITGB1 expression, and the hTERT/murine double minute 2 (MDM2) complex promoted the ubiquitin-mediated degradation of FOXO3a. Moreover, hTERT increased ITGB1 expression in xenograft gastric cancer, and the level of hTERT was positively correlated with that of ITGB1 in human gastric cancer tissues. CONCLUSIONS: The hTERT/MDM2-FOXO3a-ITGB1 pathway markedly contributes to hTERT-promoted gastric cancer invasion, suggesting that this pathway might be a novel target for the prevention and treatment of gastric cancer metastasis.

Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification.

BACKGROUND & AIMS: Our increasing understanding of hepatocellular carcinoma (HCC) biology holds promise for personalized care, however its translation into clinical practice requires a precise knowledge of its relationship to tumour phenotype. METHODS: We aimed at investigating molecular-phenotypic correlations in a large series of HCC. To this purpose, 343 surgically resected HCC samples were investigated by pathological review, immunohistochemistry, gene expression profiling and sequencing. RESULTS: CTNNB1 (40%) and TP53 (21%) mutations were mutually exclusive and defined two major groups of HCC characterized by distinct phenotypes. CTNNB1 mutated tumours were large (p=0.002), well-differentiated (p<0.001), cholestatic (p<0.001), with microtrabecular (p<0.001) and pseudoglandular (p<0.001) patterns and without inflammatory infiltrates (p<0.001). TP53 mutated tumours were poorly differentiated (p<0.001) with a compact pattern (p=0.02), multinucleated (p=0.01) and pleomorphic (p=0.02) cells and frequent vascular invasion (p=0.02). World Health Organization (WHO) classification of histological subtypes were also strongly related to molecular features. The scirrhous subtype was associated with TSC1/TSC2 mutations (p=0.005), epithelial-to-mesenchymal transition and a progenitor expression profile. The steatohepatitic subtype showed frequent IL-6/JAK/STAT activation without CTNNB1, TERT and TP53 pathway alterations (p=0.01). Pathological review identified a novel subtype, designated as "macrotrabecular-massive" associated with poor survival (p<0.001), high alpha-fetoprotein serum level (p=0.02), vascular invasion (p<0.001), TP53 mutations (p<0.001) and FGF19 amplifications (p=0.02), features also validated in The Cancer Genome Atlas (TCGA) data. Finally, integration of HCC pathological characteristics with its transcriptomic classification showed phenotypically distinct tumour subclasses closely related to G1-G6 subgroups. CONCLUSION: HCC phenotypes are tightly associated with gene mutations and transcriptomic classification. These findings may help in translating our knowledge of HCC biology into clinical practice. Lay summary: HCC is a very heterogenous tumour, both at the pathological and molecular levels. We show here that HCC phenotype is tightly associated to its molecular alterations and underlying oncogenic pathways.

Molecular Patterns and Biology of HPV-Associated HNSCC.

Head and neck cancer is the sixth most common cancer worldwide. The large majority are squamous cell carcinomas (HNSCC) that develop in the mucosal linings of the upper aerodigestive tract. These tumors develop either by exogenous carcinogen exposure (smoking, alcohol drinking) or by human papillomavirus (HPV) infection, particularly those in the oropharynx (OPSCC). HPV-positive (HPV+ve) and HPV-negative (HPV-ve) OPSCC are considered different disease entities. HPV+ve tumors are different at the molecular level and likely as a consequence have a much more favorable prognosis than HPV-ve tumors, despite their generally advanced stage at presentation. In general, HNSCCs develop in precancerous mucosal changes, and the apparent lack of precancerous HPV+ve mucosal changes is therefore remarkable. In this Chapter, head and neck carcinogenesis is discussed and the molecular differences between HPV+ve and HPV-ve tumors are outlined.

Genotoxic and cytotoxic effects of the environmental pollutant 3-nitrobenzanthrone on bladder cancer cells.

3-Nitrobenzanthrone (3-NBA), a potential human carcinogen, is present in diesel exhaust. The main metabolite of 3-NBA, 3-aminobenzanthrone, was detected in urine of miners occupationally exposed to diesel emissions. Environmental and occupational factors play an important role in development of bladder cancer (BC), one of the most frequent malignancies. It is expected that exposure of urothelium to 3-NBA and its metabolites may induce BC initiation and/or progression. To test this hypothesis, we studied geno- and cytotoxicity of 3-NBA using an in vitro BC model. 3-NBA induced higher levels of DNA adducts, reactive oxygen species and DNA breaks in aggressive T24 cells than in more differentiated RT4 cells. To understand the nature of this difference we examined the role of several enzymes that were identified as 3-NBA bio activators. However, the difference in DNA adduct formation cannot be directly linked to the different activity of any of the examined enzymes. Conversely, the difference of tested cell lines in p53 status can partly explain the distinct levels of 3-NBA-DNA adducts and DNA damage induced by 3-NBA. Therefore, we assume that more aggressive T24 cells are more predisposed for DNA adduct formation, DNA damage and, possibly, mutations and as a result further tumorigenesis.

Advances in Understanding of Penile Carcinogenesis: The Search for Actionable Targets.

Penile cancer (PeCa) is a rare malignancy with potentially devastating effects. Squamous cell carcinoma is the most common variant with distinct precancerous lesions before development into invasive disease. Involvement of the inguinal lymph nodes is the most important prognostic factor in PeCa, and once disease is present outside the groin, prognosis is poor. Metastatic PeCa is challenging to treat and often requires multidisciplinary approaches in management. Due to its rarity, molecular understanding of the disease continues to be limited with most studies based on small, single center series. Thus far, it appears PeCa has diverse mechanisms of carcinogenesis affecting similar molecular pathways. In this review, we evaluate the current landscape of the molecular carcinogenesis of PeCa and explore ongoing research on potential actionable targets of therapy. The emergence of anti-epidermal growth factor receptor (EGFR) and other immunotherapeutic strategies may improve outcomes for PeCa patients.

[The genetic markers and targets of target therapy of melanoma.]

Melanoma is the most dangerous malignant disease of skin with high risk of relapsing and metastasis dissemination. The molecular biological studies implemented during last decade drastically altered our concepts about mechanisms of carcinogenesis of melanocytes. The review considers both hereditary factors of predisposition to melanoma (rare alleles of genes CDKN2A и CDK4, mutations MITF and BAP1) and somatic genetic disorders involved into carcinogenesis of melanoma. These mutations in genes causing hyper-activation of RAS-MAPK (BRAF, NRAS, MEK, NF1) и PI3K- (PTEN, AKT) of signaling pathways and also genes of tyrosine-kinase receptors KIT, ERBB4 activating transfer of signal in cell. Also, the role of сAMP and NF-κB in melanomagenesis is considered. The detection of activating mutations of key signaling pathways in oncogenes permitted to apply medications of target action many of which demonstrated a good therapeutic effect. The combined treatment of melanoma in aggregate with immune therapy is especially perspective.

Neoangiogenesis-related genes are hallmarks of fast-growing hepatocellular carcinomas and worst survival. Results from a prospective study.

OBJECTIVE: The biological heterogeneity of hepatocellular carcinoma (HCC) makes prognosis difficult. We translate the results of a genome-wide high-throughput analysis into a tool that accurately predicts at presentation tumour growth and survival of patients with HCC. DESIGN: Ultrasound surveillance identified HCC in 78 (training set) and 54 (validation set) consecutive patients with cirrhosis. Patients underwent two CT scans 6 weeks apart (no treatment in-between) to determine tumour volumes (V0 and V1) and calculate HCC doubling time. Baseline-paired HCC and surrounding tissue biopsies for microarray study (Agilent Whole Human Genome Oligo Microarrays) were also obtained. Predictors of survival were assessed by multivariate Cox model. RESULTS: Calculated tumour doubling times ranged from 30 to 621 days (mean, 107±91 days; median, 83 days) and were divided into quartiles: ≤53 days (n=19), 54-82 days (n=20), 83-110 days (n=20) and ≥111 days (n=19). Median survival according to doubling time was significantly lower for the first quartile versus the others (11 vs 41 months, 42, and 47 months, respectively) (p<0.0001). A five-gene transcriptomic hepatic signature including angiopoietin-2 (ANGPT2), delta-like ligand 4 (DLL4), neuropilin (NRP)/tolloid (TLL)-like 2 (NETO2), endothelial cell-specific molecule-1 (ESM1), and nuclear receptor subfamily 4, group A, member 1 (NR4A1) was found to accurately identify rapidly growing HCCs of the first quartile (ROC AUC: 0.961; 95% CI 0.919 to 1.000; p<0.0001) and to be an independent factor for mortality (HR: 3.987; 95% CI 1.941 to 8.193, p<0.0001). CONCLUSIONS: The hepatic five-gene signature was able to predict HCC growth in individual patient and the consequent risk of death. This implies a role of this molecular tool in the future therapeutic management of patients with HCC. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT01657695.

Carbonic anhydrase IV inhibits colon cancer development by inhibiting the Wnt signalling pathway through targeting the WTAP-WT1-TBL1 axis.

OBJECTIVE: We found that carbonic anhydrase IV (CA4), a member of the carbonic anhydrases, is silenced in colorectal cancer (CRC). We analysed its epigenetic inactivation, biological effects and prognostic significance in CRC. DESIGN: The biological functions of CA4 were determined by in vitro and in vivo tumorigenicity assays. The CA4 co-operator was identified by immunoprecipitation and mass spectrometry. CA4 downstream effectors and signalling pathways were elucidated by promoter luciferase assay, electrophoretic mobility shift assay and chromatin immunoprecipitation. The clinical impact of CA4 was assessed in 115 patients with CRC. RESULTS: CA4 was silenced in all nine CRC cell lines and 92.6% of CRC tumours. The promoter hypermethylation contributed to the inactivation of CA4, and it was detected in 75.7% of the patients with CRC. After a median follow-up of 49.3 months, multivariate analysis showed that the patients with CA4 hypermethylation had a recurrence of Stage II/III CRC. The re-expression of CA4 inhibited cell proliferation, induced apoptosis and cell cycle arrest in the G1 phase. CA4 inhibited the activity of the Wnt signalling pathway and mediated the degradation of ß-catenin. CA4 interacted with Wilms' tumour 1-associating protein (WTAP) and induced WTAP protein degradation through polyubiquitination. Moreover, CA4 promoted the transcriptional activity of Wilms' tumour 1 (WT1), an antagonist of the Wnt pathway, which resulted in the induction of transducin ß-like protein 1 (TBL1) and the degradation of ß-catenin. CONCLUSIONS: CA4 is a novel tumour suppressor in CRC through the inhibition of the Wnt signalling pathway by targeting the WTAP-WT1-TBL1 axis. CA4 methylation may serve as an independent biomarker for the recurrence of CRC.