Neuroendocrine Tumors Tissues Research Articles

Network pharmacology analysis and molecular mechanism of paeoniflorin and its metabolite in prolactinoma cells.

Prolactinoma was the most common functionalpituitary neuroendocrine tumor tissue type, which was caused by excessive proliferation of pituitary prolactin (PRL) cells. Drug therapy of dopamine receptoragonists was generally considered as the prior treatment for prolactinoma patients. However, there were still prolactinoma patients who were resistant to dopamine agonists. Studies have been reported that paeoniflorin can inhibit the secretion of PRL in prolactinoma cells lacking dopamine D2 receptor (D2R) expression, and paeoniflorin can be metabolized into albiflorin by intestinal flora in rats. The effect of albiflorin on prolactinoma has not been reported yet. In this study, network pharmacology was used to analyze the mechanism of paeoniflorin and its metabolite albiflorin as multi-target therapy for prolactinoma, and the experimental verification was carried out. In order to clarify the complex relationship among paeoniflorin, albiflorin and prolactinoma, we constructed a component-target-disease network, and further constructed interaction network, MMP9, EGFR, FGF2, FGFR1 and LGALS3 were screened as the core targets. Kyoto encyclopedia of genes and genomes (KEGG) analysis showed that paeoniflorin and albiflorin may be involved in various pathways in the treatment of prolactinoma, included relaxin signaling pathway and PI3K-Akt signaling pathway. Molecular docking analysis showed that paeoniflorin and albiflorin had good binding activity with MMP9. Western blotting results showed that paeoniflorin and albiflorin could significantly reduce the expression of MMP9, and ELISA results showed that paeoniflorin and albiflorin could significantly reduce the concentration of PRL in GH3 cells, and the reduce degree of albiflorin was stronger than paeoniflorin at 50μM, which indicated that albiflorin might be a potential drug to treat prolactinoma, which can regulate prolactinoma through MMP9 and reduce the concentration of PRL. Our study provided a new therapeutic strategy for prolactinoma.

Cancer-Associated Fibroblasts Induce Proliferation and Therapeutic Resistance to Everolimus in Neuroendocrine Tumors through STAT3 Activation

Introduction: Cancer-associated fibroblasts (CAF) have been identified as relevant contributors to cancer progression and drug resistance in many tumors. Although neuroendocrine tumors (NET) are often associated with a strong stromal reaction, no study has addressed whether CAF are involved in progression and therapeutic resistance in NET. The aim of this study was to characterize the role of CAF in NET. Methods: We established primary CAF cultures derived from NET liver metastases to study the effect on NET cell lines NT-3 and BON. Immunohistochemistry was performed on tissue sections of primary and metastatic NET tissue. Results: Immunohistochemistry identified CAF dispersed in between tumor cells and within fibrotic bands separating tumor cell clusters in NET. Stimulating NET cells with CAF decreased expression of SSTR2 and chromogranin A and induced expression of CXCR4. CAF induced a 2.3-fold increase in proliferation and completely reversed the response to everolimus in NT-3 cells. We identified STAT3 as the main signaling pathway induced by CAF. STAT3 targeting by small interfering RNA knockdown and inhibitors prevented CAF-induced proliferation and restored everolimus responsiveness. STAT3 activation in NET tissue was associated with decreased chromogranin A expression, increased Ki-67 index, and decreased 5-year overall and progression-free survival. CAF directly influence proliferation and therapeutic response in NET cells. Conclusion: Identifying STAT3 as the contributing pathway of this so far neglected tumor-stroma interaction has the potential to become a new therapeutic target to halt tumor growth and to restore therapeutic responsiveness in NET.

Abstract 197: A systemic model derivation platform for generating 3D neuroendocrine tumor cell spheroids to accelerate cancer research

Abstract The Broad Cancer Cell Line Factory (CCLF) aims to increase the number and representation of in vitro/ex vivo cell models for common and rare cancer types. Neuroendocrine tumor (NET) cell model derivation is one of the CCLFs focus because it lacks well-characterized, publicly available models.Two major barriers existed in deriving NET cell models, including how to collect sufficient patient tumor tissue samples for the model derivation pilot and how to systematically iterate model derivation strategies since there is no prior knowledge for NET cell model generation success. Thus, we partnered with the MD Anderson Cancer Center, the Dana-Farber Cancer Institute, and the Rare Cancer Research Foundation to collect patient tissue samples. All patient’s NET tissues were sequenced with a targeted Pan-Cancer panel to ensure high tumor content. To reduce fibroblast outgrowth, we combined an empirical rich media matrix (HYBRID technology) with a 3D spheroid culture system to initiate one sample in 16-64 conditions. The growing cultures at passage 3-5 were genomically credentialed to ensure the driver events matched with the original patient tissue. So far, we have received more than 70 NET samples. While several derived models are still under culture, we successfully generated 5 genomically verified NET tumor models, including small intestinal, pancreas, and liver subtypes. To phenotypically characterize these NET models, neuroendocrine biomarkers such as chromogranin A, synaptophysin, SSTR2, and VMAT 1/2 were also evaluated using qRT-PCR and ELISA. We observed that these NET spheroid models display long doubling times (2-4 weeks) at later passages which limits their utility for large scale perturbation experiments and model sharing capability with the research community. While we are currently working on several strategies to improve the propagation ability in these models, 1 (out of 5) model has reached passage 15 with a 3 day doubling time. Genomic studies, such as RNAseq, will be performed to address the model transcriptome changes after overcoming growth plateaus. Here we showed that it is feasible to derive NET models from patient biospecimens using our HYBRID strategy. As we expand our NET cohort, we will further refine disease-specific model generation protocols for different NET types. Our goal is to share our model generation experience and make these tumor cell models publicly available to the research community in order to accelerate cancer research. Citation Format: Adel Attari, Madison Liistro, Barbara Van Hare, Jennifer Chan, Emma Coleman, Tim Heffernan, Bianca Amador, Matthew Meyerson, Jesse Boehm, William Sellers, Yuen-Yi (Moony) Tseng. A systemic model derivation platform for generating 3D neuroendocrine tumor cell spheroids to accelerate cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 197.

The role of HMGA1 protein in gastroenteropancreatic neuroendocrine tumors

ABSTRACT Neuroendocrine tumors (NETs) are neoplasms derived from neuroendocrine cells. One of their main features is to often remain asymptomatic and clinically undetectable. High Mobility Group A (HMGA) proteins belong to a family of non-histone chromatinic proteins able to modulate gene expression through the interaction with DNA and transcription factors. They are overexpressed in most of the human malignancies, playing a critical role in carcinogenesis. However, their expression levels and their role in neuroendocrine carcinogenesis has not been exhaustively evaluated until now. Therefore, in this study, we have addressed the validity of using the expression of HMGA1 as a diagnostic marker and have investigated its role in NET carcinogenesis. The expression of HMGA1 has been evaluated by qRT-PCR and immunohistochemistry, using NET tissue microarrays, in a cohort of gastroenteropancreatic (GEP)-NET samples. The expression levels of HMGA1 have been then correlated with the main clinical features of NET samples. Finally, the contribution of HMGA1 overexpression to NET development has been addressed as far as the modulation of proliferation and migration abilities of NET cells is concerned. Here, we report that HMGA1 is overexpressed in GEP-NET samples, at both mRNA and protein levels, and that the silencing of HMGA1 protein expression interferes with the ability of NET cells to proliferate and migrate through the downregulation of Cyclin E, Cyclin B1 and EZH2. These results propose the HMGA proteins as new diagnostic and prognostic markers.

Ex Vivo Modeling of Human Neuroendocrine Tumors in Tissue Surrogates.

Few models exist for studying neuroendocrine tumors (NETs), and there are mounting concerns that the currently available array of cell lines is not representative of NET biology. The lack of stable patient-derived NET xenograft models further limits the scientific community’s ability to make conclusions about NETs and their response to therapy in patients. To address these limitations, we propose the use of an ex vivo 3D flow-perfusion bioreactor system for culturing and studying patient-derived NET surrogates. Herein, we demonstrate the utility of the bioreactor system for culturing NET surrogates and provide methods for evaluating the efficacy of therapeutic agents on human NET cell line xenograft constructs and patient-derived NET surrogates. We also demonstrate that patient-derived NET tissues can be propagated using the bioreactor system and investigate the near-infrared (NIR) dye IR-783 for its use in monitoring their status within the bioreactor. The results indicate that the bioreactor system and similar 3D culture models may be valuable tools for culturing patient-derived NETs and monitoring their response to therapy ex vivo.

Transcriptome alteration landscape of pituitary neuroendocrine tumour tissue

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Medication for Acromegaly Reduces Expression of MUC16, MACC1 and GRHL2 in Pituitary Neuroendocrine Tumour Tissue.

Acromegaly is a disease mainly caused by pituitary neuroendocrine tumor (PitNET) overproducing growth hormone. First-line medication for this condition is the use of somatostatin analogs (SSAs), that decrease tumor mass and induce antiproliferative effects on PitNET cells. Dopamine agonists (DAs) can also be used if SSA treatment is not effective. This study aimed to determine differences in transcriptome signatures induced by SSA/DA therapy in PitNET tissue. We selected tumor tissue from twelve patients with somatotropinomas, with half of the patients receiving SSA/DA treatment before surgery and the other half treatment naive. Transcriptome sequencing was then carried out to identify differentially expressed genes (DEGs) and their protein–protein interactions, using pathway analyses. We found 34 upregulated and six downregulated DEGs in patients with SSA/DA treatment. Three tumor development promoting factors MUC16, MACC1, and GRHL2, were significantly downregulated in therapy administered PitNET tissue; this finding was supported by functional studies in GH3 cells. Protein–protein interactions and pathway analyses revealed extracellular matrix involvement in the antiproliferative effects of this type of the drug treatment, with pronounced alterations in collagen regulation. Here, we have demonstrated that somatotropinomas can be distinguished based on their transcriptional profiles following SSA/DA therapy, and SSA/DA treatment does indeed cause changes in gene expression. Treatment with SSA/DA significantly downregulated several factors involved in tumorigenesis, including MUC16, MACC1, and GRHL2. Genes that were upregulated, however, did not have a direct influence on antiproliferative function in the PitNET cells. These findings suggested that SSA/DA treatment acted in a tumor suppressive manner and furthermore, collagen related interactions and pathways were enriched, implicating extracellular matrix involvement in this anti-tumor effect of drug treatment.

Anti-SSTR2 antibody-drug conjugate for neuroendocrine tumor therapy.

Neuroendocrine (NE) tumors include a diverse spectrum of hormone-secreting neoplasms that arise from the endocrine and nervous systems. Current chemo- and radio- therapies have marginal curative benefits. The goal of this study was to develop an innovative antibody-drug conjugate (ADC) to effectively treat NE tumors (NETs). First, we confirmed that somatostatin receptor 2 (SSTR2) is an ideal cancer cell surface target by analyzing 38 patient-derived NET tissues, 33 normal organs, and 3 NET cell lines. Then, we developed a new monoclonal antibody (mAb, IgG1 and kappa) to target two extracellular domains of SSTR2, which showed strong and specific surface binding to NETs. The ADC was constructed by conjugating the anti-SSTR2 mAb and antimitotic monomethyl auristatin E. In vitro evaluations indicated that the ADC can effectively bind, internalize, release payload, and kill NET cells. Finally, the ADC was evaluated in vivo using a NET xenograft mouse model to assess cancer-specific targeting, tolerated dosage, pharmacokinetics, and anti-tumor efficacy. The anti-SSTR2 ADC exclusively targeted and killed NET cells with minimal toxicity and high stability in vivo. This study demonstrates that the anti-SSTR2 ADC has a high therapeutic potential for NET therapy.

Development of a novel microRNA-based signature for prediction of rectal neuroendocrine tumor invasion and metastasis

Abstract Background Rectal neuroendocrine tumors (NETs) are the most common of GI NETs, growing in incidence. Though NETs are capable of invasion and metastasis regardless of size, currently there is no molecular marker to predict them. Accumulating evidence indicates that miRNAs have been implicated as key regulators of carcinogenesis. Aim: We sought to develop a miRNA-based signature to predict rectal NETs invasion and metastasis using comprehensive miRNA-based analysis and examined its performance. We also analyzed associated mechanisms. Methods: We performed miRNA microarray using small (≤1cm) rectal NETs tissues resected endoscopically to compare lymphovascular invasion (LVI)(+) (n = 7) vs. LVI(-) (n = 7). Candidate miRNAs were assessed by qRT-PCR in an independent cohort (n = 10). We then evaluated the diagnostic performance of the marker by qRT-PCR for the other independent cohort (n = 22). Finally, we examined its metastasis-promoting mechanisms by pathway analysis program and in-vitro experiments using NET cell line H727. Results: Initially, we identified 5 most over-expressed miRNAs in LVI, of which miR-144/451 were successfully validated in an alternative cohort. The miRNA signature (miR-144/451) was able to accurately discriminate LVI patients (AUC=0.75; Sens:80%, Spec:82%). Mechanistically, we revealed miR-144/451 directly targeted PTEN/p19 and validated by immunohistochemistry. Transfection of miR-144/451 mimics into NET cells significantly increased invasion and migration. Similarly, knockdown of PTEN/p19 with siRNAs in NET cells significantly increased them. Conclusion: Using systematic and comprehensive biomarker discovery approach, we have developed and successfully validated the first novel and robust miRNA signature to detect rectal NETs invasion and metastasis and demonstrated the underlying mechanisms for invasion and metastasis. These data highlights great potential for not only the biomarker-guided treatment strategy, but therapeutic targets for rectal NET.

Abstract 31: Organoid derived from human operative sample of NET shows NET characteristics

Abstract Background: Neuroendocrine tumor is a rare neoplasm, then, it was difficult to examine using in vitro or animal models. Recently, patient-derived tumor organoid (PDTO) is considered as a promising method to analyze natural characteristics of human tumor, because PDTO is cultured in near-natural circumstances compared with cell lines. We established PDTO of neuroendocrine tumor (NET) utilizing operative samples from a pancreatic NET patient. Aim: Aim of this study is to establish organoid derived from an operative sample of pancreatic NET patient and to compare its characteristics with original tumor using immuno-histochemistry. Material and Method: A pancreatic NET tissue derived from operative sample was cut into small pieces and was treated by collagenase. Prepared tissues were 3-dimensionally cultured on the Matrigel in a serum-free medium (Advanced MEM/F12) with 4 growth factors (Respondin, EGF, Rock inhibitor, Noggin), then PDTO was established. To evaluate growth rate of PDTO, Major axis of the organoid was measured on the day 1, 2, 3, and 6. One week after primary culture of the organoid, subculture was performed. Then major axis of the 2nd, 6th and 7th filial organoid was also measured and compared with each filial. To evaluate characteristics of PDTO as neuroendocrine tumor, immunohistochemistry was performed for the 1st, 2nd and 3rd filial PDTO using anti-chromogranin-A antibody (Dako, mouse IgG), anti-synaptophysin antibody (Abcam, rabbit IgG), anti-NCAM antibody (Abcam mouse IgG) and Neuro-specific enolase (Dako, rabbit IgG). Results: Growth rate of the 2nd and 3rd filial on the day 6 did not show significant difference compared with that of the 1st filial. On the other hand, Growth rate of the 2nd and 3rd filial was significantly lower than that of the 1st filial. Expression of the 4 factors in the 1st, 2nd and 3rd filial PDTO was kept as much as the primary tumor. Conclusion: PDTO derived from neuroendocrine tumor showed the same characteristics as the primary tumor. The 6th and 7th filial PDTO might show cellular aging. Citation Format: Yasushi Ichikawa, Yukihiko Hiroshima, Noritoshi Kobayashi, Yusaku Masuda, Ayumu Goto, Motohiko Tokuhisa, Yuma Takeda, Takashi Ishikawa, Itaru Endo. Organoid derived from human operative sample of NET shows NET characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 31.

Abstract 4583: Versican accumulation and proteolysis in neuroendocrine tumors

Abstract Introduction: Neuroendocrine tumors (NETs) are a rare, understudied form of human cancer lacking robust preclinical models. Our laboratory has recently demonstrated the importance of the tumor microenvironment (TME) in T-cell exclusion from tumors. Here we examine the accumulation of the immunoregulatory proteoglycan, versican (VCAN), and its immunostimulatory proteolysis product, versikine (Vkine), produced by VCAN cleavage via ADAMTS proteins. Immunotherapeutics are increasingly being studied in NETs and investigation of the immune permissive nature of their TME is warranted. Methods: Human NET tissue microarrays (TMAs) were obtained from the University of Wisconsin Carbone Cancer Center TSB Biobank, containing human NET tissues across a broad spectrum of disease sites including the lung and gastrointestinal (GI) tract among others. Immunohistochemistry was performed using antibodies to VCAN, Vkine, and CD8+ T-cells. VCAN and Vkine levels were measured using a four-tiered relative intensity system (0/1/2/3), while CD8+ T-cell infiltration was measured by counting the CD8+ T-cells per high power field (hpf). Experimental groups were binned as low if VCAN or Vkine scored 0 or 1 and high if VCAN or Vkine scored 2 or 3. Results: Across all samples, high VCAN levels were observed in 20% of samples while high Vkine levels were observed in 55% of samples. Within the lung NET subset, which includes small cell lung cancer, 15% of samples were designated VCAN high and 65% were Vkine high. For the GI NET subset, 34% of samples scored as VCAN high while 50% scored as Vkine high. This study identifies a greater amount of Vkine high tumors present in NETs than we previously observed for colorectal, anal, pancreatic, and breast cancers. The proteolytic predominant phenotype (VCAN low and Vkine high) was present in 55% of lung NETs and 31% of GI NETs. CD8+ T cell infiltration correlated with the VCAN proteolysis predominant phenotype (VCAN low/Vkine High - CD8+ T-cell 16/hpf vs. all other VCAN/Vkine phenotypes 6.5/hpf, p<0.001). This trend persisted across all NET specimen types, including lung and GI primary NETs. Discussion: VCAN accumulation occurs in a subset of NETs, likely leading to immune exclusion and potential resistance to immunotherapies. Excitingly, we demonstrate that a large subset of these cancers has active proteolysis of VCAN in the TME leading to release of the immunostimulatory fragment Vkine. Those NETs with low levels of VCAN and high levels of Vkine are predicted to have a permissive TME for immunotherapies. Further studies are needed to discern the underlying mechanisms resulting in this proteolysis and the signaling consequences with Vkine production. Additionally, this study indicates that VCAN proteolysis should be investigated as a novel biomarker for immunotherapy response for NETs. Citation Format: Christopher P. Babiarz, Philip B. Emmerich, Carley M. Sprackling, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican accumulation and proteolysis in neuroendocrine tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4583.

Risk factors associated with the progression and metastases of hindgut neuroendocrine tumors: a retrospective study

BackgroundThe worldwide incidence of neuroendocrine tumors (NETs) has increased remarkably, with the hindgut being the second most common site for such tumors. However, the mechanisms underlying progression and metastasis of hindgut NETs are unclear. A retrospective study was conducted to elucidate these mechanisms.MethodsClinicopathological data of cases of hindgut NET between April 1996 and September 2015 were analyzed, retrospectively. Patients with neuroendocrine carcinoma were excluded. Formalin-fixed paraffin-embedded tissues of hindgut NET cases were subjected to detailed morphometric and immunohistochemical analyses. Statistical analyses were performed using the non-parametric Mann-Whitney U test, Spearman’s correlation coefficient, and chi-squared test. Multivariate logistic regression analysis was conducted as appropriate for the data set.ResultsFifty-six hindgut NET cases were considered. Microvessel density and lymphatic microvessel density were identified as significant risk factors for venous and lymphatic invasion. There was a positive correlation between microvessel density and the maximum tumor diameter. Multivariate logistic regression analysis revealed that the maximum tumor diameter alone was an independent predictor of lymph node metastasis, whereas lymphovascular invasion and MVD was not the predictor of lymph node metastasis. There were no significant correlations between the Ki-67 labeling index and any of the parameters evaluated including age, sex, the maximum tumor diameter, venous invasion, lymphatic invasion, microvessel density, lymphatic microvessel density, and lymph node metastasis.ConclusionsAngiogenic mechanisms may play important roles in the progression of hindgut NET. Otherwise, the maximum tumor diameter alone was an independent predictor of lymph node metastasis in hindgut NETs. Moreover, our study raises the question of whether the presence of lymphovascular invasion, in endoscopically obtained hindgut NET tissues, is an absolute indication for additional surgery or not.

Frequent silencing of RASSF1A by DNA methylation in thymic neuroendocrine tumours

ObjectivesAberrant methylation of promoter CpG islands (CGIs) of tumour suppressor genes is a common epigenetic mechanism underlying cancer pathogenesis. The methylation patterns of thymic tumours have not been studied in detail since such tumours are rare. Herein, we sought to identify genes that could serve as epigenetic targets for thymic neuroendocrine tumour (NET) therapy. Materials and methodsGenome-wide screening for aberrantly methylated CGIs was performed in three NET samples, seven thymic carcinoma (TC) samples, and eight type-B3 thymoma samples. The methylation status of thymic epithelial tumours (TETs) samples was validated by pyrosequencing in a larger cohort. The expression status was analysed by quantitative polymerase chain reaction (PCR) and immunohistochemistry. ResultsWe identified a CGI on a novel gene, RASSF1A, which was strongly hypermethylated in NET, but not in thymic carcinoma or B3 thymoma. RASSF1A was identified as a candidate gene statistically and bibliographically, as it showed frequent CGI hypermethylation in NET by genome-wide screening. Pyrosequencing confirmed significant hypermethylation of a RASSF1A CGI in NET. Low-grade NET tissue was more strongly methylated than high-grade NET. Quantitative PCR and immunohistochemical staining revealed that RASSF1A mRNA and protein expression levels were negatively regulated by DNA methylation. ConclusionsRASSF1A is a tumour suppressor gene epigenetically dysregulated in NET. Aberrant methylation of RASSF1A has been reported in various tumours, but this is the first report of RASSF1A hypermethylation in TETs. RASSF1A may represent an epigenetic therapeutic target in thymic NET.

Retinoblastoma-binding protein 2 (RBP2) is frequently expressed in neuroendocrine tumors and promotes the neoplastic phenotype.

Neuroendocrine tumors (NETs), which can have survival rates as low as 4%, currently have limited therapeutic interventions available highlighting the dire need for the identification of novel biological targets for use as new potential drug targets. One such potential target is retinoblastoma-binding protein 2 (RBP2), an H3K4 demethylase whose overexpression has been linked to cancer formation and metastasis in non-endocrine tumor types. We measured RBP2 mRNA and protein levels in enteropancreatic NETs by measuring RBP2 in matched human normal and NET tissue samples. Further, proliferation, migration, invasion and colony formation assays were performed in the physiologically relevant NET cell lines βlox5, H727 and QGP-1 to understand the role of RBP2 and its demethylase activity on end points of tumorigenesis. Our data indicate a strong correlation between RBP2 mRNA and protein expression in NET specimens. RBP2 was overexpressed relative to tissue-matched normal controls in 80% of the human tumors measured. In vitro studies showed RBP2 overexpression significantly increased proliferation, migration, invasion and colony formation, whereas knockdown significantly decreases the same parameters in a demethylase-independent manner. The cell cycle inhibitors p21 and p57 decreased with RBP2 overexpression and increased upon its depletion, suggesting a regulatory role for RBP2 in cellular proliferation. Taken together, our results support the hypothesis that the aberrant overexpression of RBP2 is a frequent contributing factor to tumor formation and metastasis in enteropancreatic NETs.

Abstract 1728: DCLK1 promotes tumor growth and invasion through Slug-mediated epithelial-mesenchymal transition in pancreatic neuroendocrine tumors

Abstract Introduction: Accumulating evidence suggests that Doublecortin-like kinase 1 (DCLK1) is a putative marker for intestinal and pancreatic cancer stem cells. (Nakanishi et al., Nat Genet 2013, Bailey et al., Gastroenterology 2013). Recently, we have reported that DCLK1 was highly and diffusely expressed in human rectal neuroendocrine tumors (NETs) (Ikezono et al., Oncol Lett, 2015). However, the function of DCLK1 has not yet been investigated in detail. Aims: The aims of the present study were to assess expression levels of DCLK1 in pancreatic NET (PNET) tissues and to identify critical functions of this molecule in PNET cells. Methods: Fifteen patients (8 male, 7 female; mean age, 56) with PNETs were enrolled in this study. The tumors were surgically resected between 1997 and 2012 in Kurume University Hospital. Immunohistochemistry (IHC) was employed to assess expression levels of DCLK1. QGP1, a human PNET cell line, was used in this study, and the cells were transfected with dclk1 cDNA to establish the DCLK1-overexpressing (QGP1-DOE) cells. The QGP1-DOE cells were subjected to dclk1 silencing to confirm acquired cellular characteristics by DCLK1 overexpression. Protein and mRNA expression levels were analyzed by Western blot and real-time PCR (ABI PRISM 7700), respectively. Results: In IHC, all of the 15 PNETs clearly and diffusely expressed DCLK1 in the tumor areas. QGP1-DOE cells robustly expressed full length of DCLK1, showing epithelial-mesenchymal transition (EMT)-like appearance. Of note, extremely high expression of the EMT regulator Slug was found in QGP1-DOE cells compared with control cells at both protein and mRNA levels. Expressions of N-cadherin and Vimentin were also upregulated, in concert with those of phospholylated (p-) FAK, Paxillin, and cyclin D1, in the QGP1-DOE cells. These cells increased cellular motility and proliferation. DCLK1 knockdown restored the above-mentioned expression profile of the EMT-associated molecules. Conclusion: We demonstrated robust expression of DCLK1 in human PNET tissues and PNET cells for the first time. Enforced expression of DCLK1 induced EMT via upregulating Slug expression and promoted proliferation and invasion probably through increasing expression levels of cyclin D1, pFAK, and Paxillin. Therefore, it is speculated that inhibition of DCLK1 expression is a novel therapeutic strategy for PNETs. Citation Format: Yu Ikezono, Hironori Koga, Jun Akiba, Mitsuhiko Abe, Fumitaka Wada, Toru Nakamura, Hideki Iwamoto, Atsutaka Masuda, Takahiko Sakaue, Hirohisa Yano, Osamu Tsuruta, takuji Torimura. DCLK1 promotes tumor growth and invasion through Slug-mediated epithelial-mesenchymal transition in pancreatic neuroendocrine tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1728.

Functional characterization of EZH2 in pancreatic carcinogenesis

ID: 1413. Dual specificity kinase TTK in pancreatic neuroendocrine cancer Marina Hutzler , Harald Schmidt , Brajesh Kaistha , Rita Lawlor , Aldo Scarpa , Thomas Gress , Malte Buchholz 1 Gastroenterology and Endocrinology, Philipps University Marburg, Marburg, Germany Dipartimento ad Attivit a Integrata (DAI) di Patologia e Diagnostica, University of Verona, Verona, Italy Introduction: Pancreatic neuroendocrine tumours are a rare but still very challenging disease, as despite ongoing research, surgical resection still is the only reliable cure. Therefore, about 80 % of patients have a terminal illness and their tumours are still difficult to manage with the currently available chemoand biotherapies obtaining tumour growth stabilization rather than inducing regression. Gene expression analyses of primary tissues from PNET patients identified a number of potential target genes for the development of new therapeutic strategies. Amongst others, we found strong upregulation of the dual specificity kinase TTK, which has been shown before to play an important role during mitosis by inhibiting transition to anaphase until all chromosomes have been attached to the spindle apparatus correctly. Aims: The aim of this study is to elucidate the role of TTK and its value for PNET therapy. Materials & methods: Quantitative realtime PCR, RNAi, cell proliferation and viability assays, Westernblots Results: TTK is strongly overexpressed in pancreatic neuroendocrine tumour tissue. Functional in vitro analyses by siRNA-mediated knockdown of TTK in human as well as in rat PNET cell lines resulted in decreased proliferation and viability of cells, leading to increased apoptosis rates which could be further confirmed by Westernblot analyses of key molecules of the classical apoptosis pathway (Caspase-3 and PARP cleavage). Conclusion: Overexpression of TTK dual specificity kinase in pancreatic neuroendocrine tumours is important for proliferative phenotype of tumour cells and inhibition of TTK leads to apoptotic cell death. Therefore TTK could be a promising target for the treatment of neuroendocrine tumours. Abstract ID: 1416, Oral-3.ID: 1416, Oral-3. Context-dependent epigenetic regulation of NFATc1 transcription in pancreatic plasticity Elisabeth Hessmann , Nai-ming Chen , Clara Lubeseder-Martellato , Jochen Gaedcke , Jens Siveke , Albrecht Neesse , Volker Ellenrieder 1 Department of Gastroenterology and Gastrointestinal Oncology, University Medical Center Goettingen, Germany 2 II. Med. Klinikum rechts der Isar, TU Munich, Germany Department of General, Visceral and Pediatric Surgery, University Medical Center Goettingen, Germany West German Cancer Center Essen, Germany Introduction: Cellular plasticity of the pancreas highly contributes to its enormous regenerative potential upon acinar insults, but also causes therapeutic resistance in pancreatic cancer (PDAC) treatment. On the molecular level, regulation of cellular plasticity involves epigenetic mechanisms that transcriptionally control key-signatures of tissue adaptation. Transcriptional activation of the oncogenic Nuclear Factor of Activated T-cell (NFAT) c1 transcription factor significantly contributes to PDAC cell plasticity. Nevertheless, how epigenetic alterations converge on transcriptional regulation of NFATc1 remains elusive. Aims: To investigate how epigenetic regulation impacts on NFATc1 transcription in pancreatic plasticity. Materials&methods: NFATc1 function and regulationwere studied in the regenerative pancreas upon caerulein-induced acinar cell damage and in various in vitroand in vivo PDAC-models. EZH2-dependency of NFATc1 expression was determined upon genetic or pharmacological EZH2Abstracts / Pancreato S10 inhibition. NFATc1 promoter activity was investigated by ChIP-Assays in pancreatic tissue and cells. Co-expression of EZH2 and NFATc1 was validated in micro-dissected PDAC cells from human samples. Results: Pancreatic tissue regeneration requires silencing of the NFATc1 promoter by the Polycomb-Repressor-Complex member EZH2 to circumvent organ atrophy and exocrine insufficiency. In contrast, EZH2 activation in PDAC shows inverse effects on NFATc1 promoter regulation and promotes PDAC cell plasticity by enhancing NFATc1 transcription. Oncogenic mutation of Kraswas identified as the crucial genetic hit responsible for the functional switch in EZH2-dependent regulation of NFATc1 transcription. Conclusion: These data complement previous descriptions of Polycomb-independent EZH2 functions and emphasize how epigenetic mechanisms integrate into genomic alterations in PDAC. Consequently, pharmacological inhibition of EZH2 represents a beneficial strategy to overcome oncogenic NFATc1 activity in PDAC.

Splenic scintigraphy for further differentiation of unclear (68) Ga-DOTATOC-PET/CT findings: Strengths and limitations.

Splenic scintigraphy has been described to be a powerful tool in unclear (68) Ga-DOTATOC-PET/CT findings, allowing differentiation between somatostatin receptor (Sst)-positive tissue deriving from neuroendocrine tumour (NET) and functioning splenic tissue. However, our own experiences sometimes show a lack of identification on splenic scintigraphy, especially in small lesions, leading to uncertainties regarding the safe identification of NET or splenic tissue. Here, we report on 10 cases with (68) Ga-DOTATOC-PET/CT and (99m) Tc-heat-denaturated red blood cell (HDRBC) scintigraphy and we illustrate the strengths and limitations of (99m) Tc-HDRBC scintigraphy in this context.

Blood and tissue neuroendocrine tumor gene cluster analysis correlate, define hallmarks and predict disease status.

A multianalyte algorithmic assay (MAAA) identifies circulating neuroendocrine tumor (NET) transcripts (n=51) with a sensitivity/specificity of 98%/97%. We evaluated whether blood measurements correlated with tumor tissue transcript analysis. The latter were segregated into gene clusters (GC) that defined clinical 'hallmarks' of neoplasia. A MAAA/cluster integrated algorithm (CIA) was developed as a predictive activity index to define tumor behavior and outcome. We evaluated three groups. Group 1: publically available NET transcriptome databases (n=15; GeneProfiler). Group 2: prospectively collected tumors and matched blood samples (n=22; qRT-PCR). Group 3: prospective clinical blood samples, n=159: stable disease (SD): n=111 and progressive disease (PD): n=48. Regulatory network analysis, linear modeling, principal component analysis (PCA), and receiver operating characteristic analyses were used to delineate neoplasia 'hallmarks' and assess GC predictive utility. Our results demonstrated: group 1: NET transcriptomes identified (92%) genes elevated. Group 2: 98% genes elevated by qPCR (fold change >2, P<0.05). Correlation analysis of matched blood/tumor was highly significant (R(2)=0.7, P<0.0001), and 58% of genes defined nine omic clusters (SSTRome, proliferome, signalome, metabolome, secretome, epigenome, plurome, and apoptome). Group 3: six clusters (SSTRome, proliferome, metabolome, secretome, epigenome, and plurome) differentiated SD from PD (area under the curve (AUC)=0.81). Integration with blood-algorithm amplified the AUC to 0.92±0.02 for differentiating PD and SD. The CIA defined a significantly lower SD score (34.1±2.6%) than in PD (84±2.8%, P<0.0001). In conclusion, circulating transcripts measurements reflect NET tissue values. Integration of biologically relevant GC differentiate SD from PD. Combination of GC data with the blood-algorithm predicted disease status in >92%. Blood transcript measurement predicts NET activity.

Abstract 1098: TIMP-1: A potential biomarker of neuroendocrine differentiation in metastatic castration-resistant prostate cancer (mCRPC)

Abstract BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a 28.5 kDa secreted glycoprotein that inhibits matrix metalloproteinase (MMP) activity. Recent studies have demonstrated that TIMP-1 promotes tumor growth in an MMP-independent manner. We have previously shown that elevated plasma TIMP-1 predicts worse survival in mCRPC patients; however, the mechanism underpinning this association is unknown. Normal and malignant neuroendocrine (NE) cells, from various organs, express TIMP-1 as measured by immunohistochemistry (IHC). We hypothesized that elevated blood levels of TIMP-1 in mCRPC patients results from androgen deprivation therapy-induced NE differentiation. METHODS: In this study, we used qPCR, ELISA and IHC to correlate the expression of TIMP-1 and NE markers such as chromogranin A (CGA) in 7 prostate cancer cell lines, 37 patient serum samples and prostate cancer (adenocarcinoma and NE cancer) tissue microarrays. RESULTS: We first examined TIMP-1 and NE marker (NSE and PTHLH) mRNA expression in 7 prostate cancer cell lines. Our results show that PC-3, PC-3M, DU145 cells express high levels of TIMP-1 as well as NE markers. On the other hand, LNCAP, VCAP and LAPC-4 cells had barely detectable levels of TIMP-1 and much lower expression of NE markers. We then measured serum TIMP-1 and CGA levels from CRPC patient samples by ELISA. Patients with castration resistant disease had significantly higher serum TIMP-1 levels (mean value of 373.5 ng/ml) than patients with hormone sensitive disease (mean value of 304.8 ng/ml) (p=0.01), suggesting that castration resistance in prostate cancer patients is associated with increased TIMP-1 production. Interestingly, when we divided patients using a cutoff of 100 ng/ml (based on cutoffs reported in published literature), serum TIMP-1 was significantly higher in the high CGA group (p=0.018), indicating an association between TIMP-1 production and existing NE markers. We also compared PSA values between low and high CGA groups and found that the high CGA group was associated with lower PSA levels (p=0.029). Next, we explored TIMP-1 expression in NE tumor tissues. We performed TIMP-1 and CGA IHC staining in archival liver neuroendocrine and thyroid cancers and found that TIMP-1 expression was associated with CGA expression. In prostate cancer specimens, normal prostate epithelial cells expressed high levels of TIMP-1, but the expression was lost in adenocarcinomas. In contrast, prostatic neuroendocrine cancers expressed high levels of TIMP-1 as well as the NE marker CGA. CONCLUSIONS: These observations support the further evaluation of TIMP-1 as a tissue and serum biomarker for NE differentiation in CRPC. Note: This abstract was not presented at the meeting. Citation Format: Yixuan Gong, Uma Chippada-Venkata, Xudong Dai, Matthew D. Galsky, Jiaoti Huang. TIMP-1: A potential biomarker of neuroendocrine differentiation in metastatic castration-resistant prostate cancer (mCRPC). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1098. doi:10.1158/1538-7445.AM2014-1098

Neuropeptide S receptor 1 (NPSR1) activates cancer-related pathways and is widely expressed in neuroendocrine tumors.

Neuroendocrine tumors (NETs) arise from disseminated neuroendocrine cells and express general and specific neuroendocrine markers. Neuropeptide S receptor 1 (NPSR1) is expressed in neuroendocrine cells and its ligand neuropeptide S (NPS) affects cell proliferation. Our aim was to study whether NPS/NPSR1 could be used as a biomarker for neuroendocrine neoplasms and to identify the gene pathways affected by NPS/NPSR1. We collected a cohort of NETs comprised of 91 samples from endocrine glands, digestive tract, skin, and lung. Tumor type was validated by immunostaining of chromogranin-A and synaptophysin expression and tumor grade was analyzed by Ki-67 proliferation index. NPS and NPSR1 expression was quantified by immunohistochemistry using polyclonal antibodies against NPS and monoclonal antibodies against the amino-terminus and carboxy-terminus of NPSR1 isoform A (NPSR1-A). The effects of NPS on downstream signaling were studied in a human SH-SY5Y neuroblastoma cell line which overexpresses NPSR1-A and is of neuroendocrine origin. NPSR1 and NPS were expressed in most NET tissues, with the exception of adrenal pheochromocytomas in which NPS/NPSR1 immunoreactivity was very low. Transcriptome analysis of NPSR1-A overexpressing cells revealed that mitogen-activated protein kinase (MAPK) pathways, circadian activity, focal adhesion, transforming growth factor beta, and cytokine–cytokine interactions were the most altered gene pathways after NPS stimulation. Our results show that NETs are a source of NPS and NPSR1, and that NPS affects cancer-related pathways.Electronic supplementary materialThe online version of this article (doi:10.1007/s00428-014-1602-x) contains supplementary material, which is available to authorized users.