HNF1A recruits KDM6A to activate differentiated acinar cell programs that suppress pancreatic cancer.

Defects in transcriptional regulators of pancreatic exocrine differentiation have been implicated in pancreatic tumorigenesis, but the molecular mechanisms are poorly understood. The locus encoding the transcription factor HNF1A harbors susceptibility variants for pancreatic ductal adenocarcinoma (PDAC), while KDM6A, encoding Lysine-specific demethylase 6A, carries somatic mutations in PDAC. Here, we show that pancreas-specific Hnf1a null mutant transcriptomes phenocopy those of Kdm6a mutations, and both defects synergize with KrasG12D to cause PDAC with sarcomatoid features. We combine genetic, epigenomic, and biochemical studies to show that HNF1A recruits KDM6A to genomic binding sites in pancreatic acinar cells. This remodels the acinar enhancer landscape, activates differentiated acinar cell programs, and indirectly suppresses oncogenic and epithelial-mesenchymal transition genes. We also identify a subset of non-classical PDAC samples that exhibit the HNF1A/KDM6A-deficient molecular phenotype. These findings provide direct genetic evidence that HNF1A deficiency promotes PDAC. They also connect the tumor-suppressive role of KDM6A deficiency with a cell-specific molecular mechanism that underlies PDAC subtype definition.

BRAFV600E and BRAF-WT Specific Antitumor Immunity in Papillary Thyroid Cancer.

One feature of papillary thyroid cancer (PTC) is the frequently present somatic BRAFV600E mutation. PTCs are also characterized by a lymphocytic infiltration, which may correlate with an improved clinical outcome. The objective of the study was the characterization of BRAFV600E specific anti-immunity in PTC patients and correlation analyses with the clinical outcome. Fourteen HLA A2 positive PTC patients were included into the study of whom tumor tissue samples were also available. Of those, 8 PTC patients revealed a somatic BRAFV600E mutation. All PTC patients were also MHC class II typed. Tetramer analyses for detection of MHC class I and MHC class II-restricted, BRAFV600E epitope-specific T cells using unstimulated and peptide-stimulated T cells were performed; correlation analyses between MHC phenotypes, T cell immunity, and the clinical course were performed. In regard to unstimulated T cells, a significantly higher amount of BRAFV600E epitope specific T cells was detected compared to a control tetramer. Importantly, after overnight peptide stimulation a significantly higher number of BRAFV600E positive and BRAF WT epitope-specific T cells could be seen. In regard to the clinical course, however, no significant differences were seen, neither in the context of the initial tumor size, nor in the context of lymph node metastases or peripheral metastastic spread. In conclusion, we clearly demonstrated a BRAF-specific tumor immunity in PTC-patients which is, however, independent of a BRAFV600E status of the PTC patients.

Prognostic value of indoleamine 2,3 dioxygenase in patients with higher-risk myelodysplastic syndromes treated with azacytidine.

Hypomethylating agents (HMAs) are widely used in patients with higher-risk myelodysplastic syndromes (MDS) not eligible for stem cell transplantation; however, the response rate is <50%. Reliable predictors of response are still missing, and it is a major challenge to develop new treatment strategies. One current approach is the combination of azacytidine (AZA) with checkpoint inhibitors; however, the potential benefit of targeting the immunomodulator indoleamine-2,3-dioxygenase (IDO-1) has not yet been evaluated. We observed moderate to strong IDO-1 expression in 37% of patients with high-risk MDS. IDO-1 positivity was predictive of treatment failure and shorter overall survival. Moreover, IDO-1 positivity correlated inversely with the number of infiltrating CD8+ T cells, and IDO-1+ patients failed to show an increase in CD8+ T cells under AZA treatment. In vitro experiments confirmed tryptophan catabolism and depletion of CD8+ T cells in IDO-1+ MDS, suggesting that IDO-1 expression induces an immunosuppressive microenvironment in MDS, thereby leading to treatment failure under AZA treatment. In conclusion, IDO-1 is expressed in more than one-third of patients with higher-risk MDS, and is predictive of treatment failure and shorter overall survival. Therefore, IDO-1 is emerging as a promising predictor and therapeutic target, especially for combination therapies with HMAs or checkpoint inhibitors.

The atypical cadherin Dachsous1 localizes to the base of the ciliary apparatus in airway epithelia.

Mucociliary clearance requires the distinct orientation and coordinated movement of airway cilia, which is established through planar cell polarity signaling (PCP). The atypical cadherin Dachsous1 (Dchs1) is a transmembrane protein that regulates PCP in D. melanogaster. However, little is known about Dchs1 expression and its potential role in PCP in mammalian adult tissues. Here, we show that Dchs1 is ubiquitously expressed in mouse embryos, but exhibits a highly restricted expression to lung tissues in the adult stage. Strikingly, human Dchs1 localized exclusively to the base of the ciliary apparatus in cultured human respiratory epithelial cells with differentiated motile 9 + 2 cilia. This localization could be functionally important as we observed aberrant DCHS1 mRNA expression in human non-small cell lung cancer tissue. In sum, we establish Dchs1 as a component of the membrane domain surrounding the ciliary base. This suggests a specific role of Dchs1 in PCP-dependent organization of ciliary function and a possible role in lung disease.

Tenascin-C affects invasiveness of EGFR-mutated lung adenocarcinoma through a putative paracrine loop.

Tenascin C (TNC) is an extracellular matrix (ECM) protein and a potential biomarker affecting progression of different tumor types, such as pancreatic and lung cancer. Alternative splicing variants of TNC are known to have an impact on interaction partners like other ECM proteins or cell surface receptors, including epidermal growth factor receptor (EGFR), leading to numerous and sometimes opposite roles of TNC in tumor cell dissemination and proliferation. Only little is known about the impact of TNC on biologic characteristics of lung cancer, such as invasion and metastatic potential. In the present study, we could link an increased expression of TNC in lung adenocarcinoma (LUAD) tissues with an unfavorable clinical outcome of patients. Furthermore, we investigated the functional role of TNC in LUAD. Immunohistochemical staining of TNC revealed a significant increase of TNC levels in primary tumours and metastases compared to normal lung tissue. Additionally, a significant correlation between TNC mRNA expression and EGFR copy number and protein expression levels has been determined. Moreover, inhibition of TNC in lung fibroblasts led to reduced invasiveness of LUAD cells harboring EGFR-activating mutations and to a shorter lamellipodia perimeter and a reduced lamellipodia area on the surface of LUAD cells. This study provides the evidence that TNC expression might be a biological relevant factor in LUAD progression in an EGFR-dependent manner and that it regulates tumor cell invasion by rearrangement of the actin cytoskeleton, especially affecting lamellipodia formation.

Communicator-Driven Data Preprocessing Improves Deep Transfer Learning of Histopathological Prediction of Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer is a fatal malignancy with poor prognosis and limited treatment options. Early detection in primary and secondary locations is critical, but fraught with challenges. While digital pathology can assist with the classification of histopathological images, the training of such networks always relies on a ground truth, which is frequently compromised as tissue sections contain several types of tissue entities. Here we show that pancreatic cancer can be detected on hematoxylin and eosin (H&E) sections by convolutional neural networks using deep transfer learning. To improve the ground truth, we describe a preprocessing data clean-up process using two communicators that were generated through existing and new datasets. Specifically, the communicators moved image tiles containing adipose tissue and background to a new data class. Hence, the original dataset exhibited improved labeling and, consequently, a higher ground truth accuracy. Deep transfer learning of a ResNet18 network resulted in a five-class accuracy of about 94% on test data images. The network was validated with independent tissue sections composed of healthy pancreatic tissue, pancreatic ductal adenocarcinoma, and pancreatic cancer lymph node metastases. The screening of different models and hyperparameter fine tuning were performed to optimize the performance with the independent tissue sections. Taken together, we introduce a step of data preprocessing via communicators as a means of improving the ground truth during deep transfer learning and hyperparameter tuning to identify pancreatic ductal adenocarcinoma primary tumors and metastases in histological tissue sections.

Detection of cytogenetic changes and chromosomal aneuploidy with fluorescent in situ hybridization in cytological specimens of oral cancers in Fanconi anemia-Proof of concept.

OBJECTIVES: Fanconi anemia (FA) is a rare inherited DNA instability disorder with a remarkably elevated risk of neoplasia compared with the general population, mainly leukemia and squamous cell carcinoma (SCC). Two thirds of the SCCs arise in the oral cavity and are typically preceded by visible lesions. These lesions can be classified with brush biopsy-based cytological methods regarding their risk of a malignant transformation. As a proof of concept, this study aims to investigate genetic changes and chromosomal aneuploidy using fluorescent in situ hybridization (FISH) on oral squamous cells derived from FA affected individuals. MATERIAL AND METHODS: Five FA oral SCC (OSCC) tumor cell lines, one FA OSCC cervical lymph node metastasis as well as tumor-negative and atypical smears from oral brush biopsies were analyzed with FISH probes covering 5p15.2, MYC, EGFR, TERC, 9q34.1, CCND1, 9p21 and centromeres of chromosomes 3, 6, 7, 9, 11, and 17. RESULTS: OSCC specimens showed gains of all analyzed chromosomal regions. Chromosomal aneuploidy was observed in five of the six OSCC specimens in two multicolor FISH assays with panels of four probes each. Five out of six OSCC specimens displayed a relative deletion of 9p21. Applied on atypical brush biopsy-based smears, chromosomal aneuploidy was detected in malignant lesions but not in the smear derived from a severe parodontitis. CONCLUSIONS: As proof of concept, FISH was able to detect genetic changes and chromosomal aneuploidy discriminating oral cancer from noncancerous lesions in individuals with FA. This supports its application on oral brush biopsy-based cytology.

EZH2 Loss Drives Resistance to Carboplatin and Paclitaxel in Serous Ovarian Cancers Expressing ATM.

Mechanisms of intrinsic resistance of serous ovarian cancers to standard treatment with carboplatin and paclitaxel are poorly understood. Seventeen primary serous ovarian cancers classified as responders or nonresponders to standard treatment were screened with DigiWest protein array analysis for 279 analytes. Histone methyl transferase EZH2, an interaction partner of ataxia telangiectasia mutated (ATM), was found as one of the most significantly represented proteins in responsive tumors. Survival analysis of 616 patients confirmed a better outcome in patients with high EZH2 expression, but a worse outcome in patients with low EZH2 and high-ATM-expressing tumors compared with patients with low EZH2 and low-ATM-expressing tumors. A proximity ligation assay further confirmed an association between ATM and EZH2 in tumors of patients with an increased disease-free survival. Knockdown of EZH2 resulted in treatment-resistant cells, but suppression of both EZH2 and ATM, or ATM alone, had no effect. DigiWest protein analysis of EZH2-knockdown cells revealed a decrease in proteins involved in mitotic processes and checkpoint regulation, suggesting that deregulated ATM may induce treatment resistance. IMPLICATIONS: Ovarian cancer is a malignancy with high mortality rates, with to date, no successful molecular characterization strategies. Our study uncovers in a comprehensive approach the involvement of checkpoint regulation via ATM and EZH2, potentially providing a new therapeutic perspective for further investigations.

Epigenetic loss of putative tumor suppressor SFRP3 correlates with poor prognosis of lung adenocarcinoma patients.

Secreted frizzled related protein 3 (SFRP3) contains a cysteine-rich domain (CRD) that shares homology with Frizzled CRD and regulates WNT signaling. Independent studies showed epigenetic silencing of SFRP3 in melanoma and hepatocellular carcinoma. Moreover, a tumor suppressive function of SFRP3 was shown in androgen-independent prostate and gastric cancer cells. The current study is the first to investigate SFRP3 expression and its potential clinical impact on non-small cell lung carcinoma (NSCLC). WNT signaling components present on NSCLC subtypes were preliminary elucidated by expression data of The Cancer Genome Atlas (TCGA). We identified a distinct expression signature of relevant WNT signaling components that differ between adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC). Of interest, canonical WNT signaling is predominant in LUAD samples and non-canonical WNT signaling is predominant in LUSC. In line, high SFRP3 expression resulted in beneficial clinical outcome for LUAD but not for LUSC patients. Furthermore, SFRP3 mRNA expression was significantly decreased in NSCLC tissue compared to normal lung samples. TCGA data verified the reduction of SFRP3 in LUAD and LUSC patients. Moreover, DNA hypermethylation of SFRP3 was evaluated in the TCGA methylation dataset resulting in epigenetic inactivation of SFRP3 expression in LUAD, but not in LUSC, and was validated by pyrosequencing of our NSCLC tissue cohort and in vitro demethylation experiments. Immunohistochemistry confirmed SFRP3 protein downregulation in primary NSCLC and indicated abundant expression in normal lung tissue. Two adenocarcinoma gain-of-function models were used to analyze the functional impact of SFRP3 on cell proliferation and regulation of CyclinD1 expression in vitro. Our results indicate that SFRP3 acts as a novel putative tumor suppressor gene in adenocarcinoma of the lung possibly regulating canonical WNT signaling.

Abundant NDRG2 Expression Is Associated with Aggressiveness and Unfavorable Patients' Outcome in Basal-Like Breast Cancer.

NDRG2, a member of the N-myc downstream-regulated gene family, is thought to be a putative tumor suppressor gene with promising clinical impact in breast cancer. Since breast cancer comprises heterogeneous intrinsic subtypes with distinct clinical outcomes we investigated the pivotal role of NDRG2 in basal-type breast cancers. Based on subtype classified tumor (n = 45) and adjacent normal tissues (n = 17) we examined NDRG2 mRNA expression and CpG-hypermethylation, whose significance was further validated by independent data sets from The Cancer Genome Atlas (TCGA). In addition, NDRG2 protein expression was evaluated immunohistochemically using a tissue micro array (TMA, n = 211). In vitro, we investigated phenotypic effects caused by NDRG2 silencing in the basal A-like HCC1806 as well as NDRG2 over-expression in basal A-like BT20 compared to luminal-type MCF7 breast cancer cells. Our tissue collections demonstrated an overall low NDRG2 mRNA expression in breast cancer subtypes compared to normal breast tissue in line with an increased CpG-hypermethylation in breast cancer tissue. Independent TCGA data sets verified a significant (P<0.001) expression loss of NDRG2 in breast tumors. Of interest, basal-like tumors more frequently retained abundant NDRG2 expression concordant with a lower CpG-hypermethylation. Unexpectedly, basal-like breast cancer revealed an association of NDRG2 expression with unfavorable patients' outcome. In line with this observation, in vitro experiments demonstrated reduced proliferation and migration rates (~20%) in HCC1806 cells following NDRG2 silencing. In contrast, NDRG2 over-expressing luminal-type MCF7 cells demonstrated a 26% decreased proliferation rate. Until now, this is the first study investigating the putative role of NDRG2 in depth in basal-type breast cancer. Our data indicate that the described putative tumor suppressive function of NDRG2 may be confined to luminal- and basal B-type breast cancers.

Low expression of ITIH5 in adenocarcinoma of the lung is associated with unfavorable patients' outcome.

Inter-α-trypsin inhibitor heavy chain 5 (ITIH5) is supposed to be involved in extracellular matrix stability and thus may play a key role in the inhibition of tumor progression. The current study is the first to analyze in depth ITIH5 expression and DNA methylation, as well as its potential clinical impact in non-small-cell lung carcinoma (NSCLC). We examined ITIH5 mRNA expression in tumor and adjacent normal lung tissue specimens of NSCLC patients. In addition, methylation frequency of the ITIH5 promoter was investigated using methylation-specific PCR and pyrosequencing. Significance of our data was validated by independent data sets from The Cancer Genome Atlas and the Kaplan-Meier Plotter platform. Furthermore, ITIH5 protein expression was evaluated by immunohistochemistry utilizing a tissue microarray with 385 distinct lung tissue samples. Based on our tissue collections, ITIH5 mRNA expression was significantly decreased in NSCLC compared to normal lung tissue in line with an increased methylation frequency in lung cancer tissue. Independent TCGA data confirmed significant expression loss of ITIH5 in lung cancer concordant with ITIH5 promoter hypermethylation in NSCLC. Of interest, low ITIH5 mRNA expression was particularly found in the magnoid and squamoid ADC expression subtype, concordant with an unfavorable patients' outcome in squamoid as well as tobacco smoking ADC patients. In conclusion, ITIH5 may be a novel putative tumor suppressor gene in NSCLC with a potential molecular significance in the squamoid ADC subtype and further clinical impact for risk stratification of adenocarcinoma patients. In addition, ITIH5 may serve as a novel biomarker for prognosis of tobacco smoking ADC patients.

BDNF is associated with SFRP1 expression in luminal and basal-like breast cancer cell lines and primary breast cancer tissues: a novel role in tumor suppression?

Secreted frizzled related protein 1 (SFRP1) functions as an important inhibitor of the Wnt pathway and is a known tumor suppressor gene, which is epigenetically silenced in a variety of tumors e.g. in breast cancer. However, it is still unclear how SFRP1 exactly affects the Wnt pathway. Our aim was to decipher SFRP1 involvement in biochemical signaling in dependency of different breast cancer subtypes and to identify novel SFRP1-regulated genes. We generated SFRP1 over-expressing in vitro breast cancer models, reflecting the two major subtypes by using basal-like BT20 and luminal-like HER2-positive SKBR3 cells. DNA microarray expression profiling of these models revealed that SFRP1 expression potentially modulates Bone morphogenetic protein- and Smoothened signaling (p<0.01), in addition to the known impact on Wnt signaling. Importantly, further statistical analysis revealed that in dependency of the cancer subtype model SFRP1 may affect the canonical and non-canonical Wnt pathway (p<0.01), respectively. While SFRP1 re-expression generally mediated distinct patterns of transcriptionally induced or repressed genes in BT20 and SKBR3 cells, brain derived neurotrophic factor (BDNF) was identified as a SFRP1 induced gene in both cell lines. Although BDNF has been postulated as a putative oncogene, the co-regulation with SFRP1 indicates a potential suppressive function in breast cancer. Indeed, a positive correlation between SFRP1 and BDNF protein expression could be shown (p<0.001) in primary breast cancer samples. Moreover, TCGA dataset based analysis clearly underscores that BDNF mRNA is down-regulated in primary breast cancer samples predicting a poor prognosis of these patients. In line, we functionally provide evidence that stable BDNF re-expression in basal-like BT20 breast cancer cells blocks tumor cell proliferation. Hence, our results suggest that BDNF might rather mediate suppressive than promoting function in human breast cancer whose mode of action should be addressed in future studies.