Targeting interleukin 6 signaling by monoclonal antibody siltuximab on cholangiocarcinoma.

BACKGROUND AND AIM: Cholangiocarcinoma has an unimproved prognosis. Interleukin 6 (IL-6) has an oncogenic potential in some cancer diseases. However, the role of IL-6 in cholangiocarcinoma carcinogenesis is not well understood. The current study investigated the role of IL-6 signaling in cholangiocarcinoma carcinogenesis and efficacy of siltuximab treatment on cholangiocarcinoma in vitro and in vivo. METHODS: The expression of IL-6 was analyzed on human cholangiocarcinoma cell lines and murine and human cholangiocarcinoma tissues, using reverse transcription real-time polymerase chain reaction and immunohistochemistry. In addition, the effect of anti-IL-6 chimeric monoclonal antibody, siltuximab, was investigated in vitro by proliferation, migration, and two-dimensional and three-dimensional invasion assays and in vivo by xenograft mouse model. Western blot was applied to study the molecular alteration. RESULTS: Our result shows high expression of IL-6 in human cholangiocarcinoma cells, and IL-6 stimulants enhance cholangiocarcinoma cell proliferation. In addition, murine and human cholangiocarcinoma tissues express significantly higher levels of IL-6, compared with adjacent non-tumor tissues. On the cholangiocarcinoma engineered mouse model, IL-6 level is associated with tumor volume. Taken together, our data indicate an oncogenic potential of IL-6 in cholangiocarcinoma carcinogenesis. Siltuximab sufficiently abrogates IL-6 signaling and inhibits cholangiocarcinoma progression in vitro and in vivo. The results additionally indicate a relative alteration of IL-6 signaling and its molecular targets, such as STAT3, Wnt/ß-catenin, and mesenchymal markers. CONCLUSIONS: Interleukin 6 plays an essential role in cholangiocarcinoma carcinogenesis, and siltuximab has the potential to be considered as a new treatment option for cholangiocarcinoma patients.

Hypoxia induced Sonic Hedgehog signaling regulates cancer stemness, epithelial-to-mesenchymal transition and invasion in cholangiocarcinoma.

Aberrant activation of Sonic Hedgehog (SHH) pathway has been implicated in a variety of cancers including cholangiocarcinoma (CC); however, the influencing factors are still unknown. Additionally, intratumoral hypoxia is known to contribute towards therapeutic resistance through modulatory effects on various pathways. In this study, we investigated the relationship between hypoxia and SHH pathway activation and the effect of this interplay on cancer stemness and epithelial-to- mesenchymal transition (EMT) during cholangiocarcinogenesis. Hypoxia promoted SHH pathway activation, evidenced by upregulated SHH and SMO levels, and enhanced glioma-associated oncogene homolog 1 (GLI1) nuclear translocation; whereas silencing of HIF-1α impaired SHH upregulation. Hypoxia also enhanced the expression of cancer stem cell (CSC) transcription factors (NANOG, Oct4, SOX2), CD133 and EMT markers (N-cadherin, Vimentin), thereby supporting invasion. Cyclopamine treatment suppressed hypoxia induced SHH pathway activation, consequently reducing invasiveness by downregulating the expression of CSC transcription factors, CD133 and EMT. Cyclopamine induced apoptosis in CC cells under hypoxia, suggesting that hypoxia induced activation of SHH pathway has modulatory effects on CC progression. Therefore, SHH signaling is proposed as a target for CC treatment, which is refractory to standard chemotherapy.

Silencing of Kangai 1 C-terminal interacting tetraspanin suppresses progression of cholangiocarcinoma.

Cholangiocarcinoma (CC) is the second most common primary hepatic malignancy. CC treatment options are very limited especially for patients with distant metastasis. Kangai 1 C-terminal interacting tetraspanin (KITENIN) is highly expressed in numerous cancers, but the role of KITENIN in CC remains unknown. Here, we have investigated for the first time the function of KITENIN in human CC cell lines (TFK-1, SZ-1), tissues and a CC mouse model (Alb-Cre/LSL-KRASG12D/p53L/L). KITENIN was expressed in 92.2% of human CC tissues, in murine CC samples and also in human CC cell lines. Knockdown of KITENIN by small interfering RNA (siRNA) effectively reduced proliferation, migration, invasion and colony formation in both intra- and extra-hepatic CC cells. The expression of epithelial-mesenchymal transition (EMT) markers like N-cadherin, Vimentin, Snail and Slug were suppressed in KITENIN knockdown CC cells. Our results indicate that KITENIN is crucial for cholangiocarcinogenesis and it might become a potential therapeutic target for human CC treatment.

Targeting c-MET by LY2801653 for treatment of cholangiocarcinoma.

Palliative treatment options for human cholangiocarcinoma (CCC) are quite limited and new therapeutic strategies are of utmost need. c-MET has been shown to be deregulated in many cancers, but the role of c-MET in the carcinogenesis of CCC remains unclear. The main purpose of this study is to evaluate the expression and also to investigate the role of c-MET and its effective inhibition for the treatment of CCC. In this study we investigated the effects of LY2801653, a small-molecule inhibitor with potent activity against MET kinase, in human CCC cell lines and in vivo using a xenograft mouse model. We have investigated the role of c-MET and its inhibitory effects on migration, invasion, colony formation, MET downstream targets, and CCC tumor growth. We also analyzed the role of apoptosis and senescence as well as the influence of hypoxia in this context. c-MET and p-MET were expressed in 72% and 12.5% of human CCC tissues and in TFK-1, SZ-1 cell lines. MET inhibition was achieved by blocking phosphorylation of MET with LY2801653 and subsequent down regulation of c-MET downstream targets. Treatment showed in a xenograft model potent anti-tumor activity. LY2801653 is an effective inhibitor and suppress the proliferation of CCC cells as well as the growth of xenograft tumors. Therefore, inhibition of c-MET could be a possible alternative approach for the treatment of human CCC. © 2016 Wiley Periodicals, Inc.

Subgroup analysis of patients with G2 gastroenteropancreatic neuroendocrine tumors.

Neuroendocrine tumors (NET) are malignancies with an increasing incidence rate. NETs are graded or classified by the expression level of Ki67, a proliferation marker in Grade 1 and 2 tumors. Out of 120 patients who visited our hospital between 2003 and 2012, 40 were classified as G2 NET. This study was mainly designed to investigate a new threshold for optimising the Ki67 system. Patients were subdivided into two new groups according to Ki67 (group 1 = 3-9%, group 2 = 10-20%). Twenty-five patients were allocated to group 1 and 15 to group 2. The primary tumor originated in 46% from the foregut and 68% NET were functionally active. Patients were treated in 88 versus 60% by surgery, 48 versus 80% by somatostatin analogs, 0 versus 20% by chemotherapy, 2,5 versus 0% by Everolimus and 32 versus 47% underwent peptide receptor radionuclide therapy. Group 1 patients showed a significantly (p = 0.01) better survival compared with group 2 and also a significant difference of Chromogranin A (p = 0.03) and alkaline phosphatase (p = 0.01). In addition, all patients with elevated lactate dehydrogenase showed a significantly (p = 0.03) shorter survival. Prognostic relevance of G2 NETs may be improved by using a new boundary. Patients with Ki67 of 3-9% showed a better response to current treatment methods and significantly longer survival compared to group 2. Thus, our data clearly show that patients with higher G2 proliferation index should be treated differently. Finally, LDH has been found to be a new prognostic factor in patients with G2 NET.

DCLK1 marks a morphologically distinct subpopulation of cells with stem cell properties in preinvasive pancreatic cancer.

BACKGROUND & AIMS: As in other tumor types, progression of pancreatic cancer may require a functionally unique population of cancer stem cells. Although such cells have been identified in many invasive cancers, it is not clear whether they emerge during early or late stages of tumorigenesis. Using mouse models and human pancreatic cancer cell lines, we investigated whether preinvasive pancreatic neoplasia contains a subpopulation of cells with distinct morphologies and cancer stem cell-like properties. METHODS: Pancreatic tissue samples were collected from the KC(Pdx1), KPC(Pdx1), and KC(iMist1) mouse models of pancreatic intraepithelial neoplasia (PanIN) and analyzed by confocal and electron microscopy, lineage tracing, and fluorescence-activated cell sorting. Subpopulations of human pancreatic ductal adenocarcinoma (PDAC) cells were similarly analyzed and also used in complementary DNA microarray analyses. RESULTS: The microtubule regulator DCLK1 marked a morphologically distinct and functionally unique population of pancreatic cancer-initiating cells. These cells displayed morphological and molecular features of gastrointestinal tuft cells. Cells that expressed DCLK1 also expressed high levels of ATAT1, HES1, HEY1, IGF1R, and ABL1, and manipulation of these pathways in PDAC cell lines inhibited their clonogenic potential. Pharmacological inhibition of γ-secretase activity reduced the abundance of these cells in murine PanIN in a manner that correlated with inhibition of PanIN progression. CONCLUSIONS: Human PDAC cells and pancreatic neoplasms in mice contain morphologically and functionally distinct subpopulations that have cancer stem cell-like properties. These populations can be identified at the earliest stages of pancreatic tumorigenesis and provide new cellular and molecular targets for pancreatic cancer treatment and/or chemoprevention.

Combined inhibition of Notch and JAK/STAT is superior to monotherapies and impairs pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a high rate of metastasis. Recent studies have indicated that Notch and janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) signaling pathways are both important for the initiation and progression of PDAC. The purpose of this study was to determine the outcome of targeting these two tumor signaling pathways simultaneously both in vitro and in vivo. We assessed the combinational effects of the γ-secretase inhibitor IX (GSI IX) and JAK2 inhibitor (AG-490) on growth and epithelial plasticity of human pancreatic cancer cell lines, and in a genetically engineered mouse model (Pdx1-Cre, LSL-KrasG12D, p53(lox/+)) of PDAC. Dual treatment with GSI IX and AG-490 significantly impaired cell proliferation, migration, invasion, soft agar growth and apoptosis when compared with monotherapies. Most importantly, combinational treatment significantly attenuates tumor progression in vivo and suppresses conversion from acinar-ductal-metaplasia to PDAC. Our results suggest that targeting Notch and JAK2/STAT3 signaling pathways simultaneously is superior to single inhibitions, supporting combined treatment by GSI IX and AG-490 as a potential therapeutic approach for PDAC. However, the study design limits the direct transfer into the clinic and the impact of dual treatment in patients with PDAC remains still to be determined.

Inhibition of hedgehog signaling attenuates carcinogenesis in vitro and increases necrosis of cholangiocellular carcinoma.

UNLABELLED: The Hedgehog signaling pathway plays a pivotal role during embryonic development, stem cell maintenance, and wound healing. Hedgehog signaling also is deregulated in many cancers. However, the role of this signaling pathway in the carcinogenesis of cholangiocarcinoma (CCC) is still unknown. In this study, we investigated the effects of Hedgehog inhibition by cyclopamine and 5E1 in cultured human CCC cell lines and in vivo using a xenograft mouse model. We also investigated the involvement of Hedgehog in epithelial to mesenchymal transition (EMT), migration, and CCC tumor growth. Sonic hedgehog (Shh) ligand was highly expressed in 89% of human CCC tissues and in CCC cell lines. Cyclopamine and 5E1 treatments effectively inhibited cell proliferation, migration, and invasion by down-regulating the Hedgehog target genes glioblastoma 1 and glioblastoma 2. In vitro and in vivo, we detected an increase in epithelial marker, E-cadherin, after Hedgehog inhibition. In addition, we saw an increase in necrotic areas and a decrease in mitotic figures in cyclopamine and 5E1-treated CCC xenograft tumors. CONCLUSION: This study supports the presence of autocrine Hedgehog signaling in human CCC, where CCC cells produce and respond to Shh ligand. Blocking the Hedgehog pathway inhibited EMT and decreased the viability of CCC cells. In addition, cyclopamine and 5E1 inhibited the growth of CCC xenograft tumors.

Analysis of second-line chemotherapies for ductal pancreatic adenocarcinoma in a German single-center cohort.

Pancreatic ductal adenocarcinoma (PDAC) is the third most common tumor of the gastrointestinal tract. At the time of diagnosis, the majority of PDACs shows already metastasis and does not qualify for curative surgery. Therefore, palliative chemotherapy has a very high priority, but recommendations after failure of first-line chemotherapies are quite limited. The aim of our analysis was to evaluate the efficacy of different second-line treatments after pretreatment with gemcitabine (57.5%), gemcitabine + erlotinib (25%), and platinum-based chemotherapy (17.5%). We included all patients with advanced PDAC treated with second-line chemotherapy in our department between 2005 and 2012. A total of 22 patients were treated with XELOX, 8 patients with FOLFOX, 6 patients with gemcitabine (+/- erlotinib) and 4 patients with FOLFIRI. On average, the patients received 4.2 cycles (standard deviation [SD] SD: 3.5) over a period of 2.5 months (SD: 2.6). The median overall survival (OS) for all patients was 5.4 months, progression-free survival was 3.5 months, and a tumor control was achieved in 21% of all cases. Toxicity profile was acceptable between the second-line chemotherapies and there was no significant difference in the other investigated end points. Interestingly, there was also no effect of the first-line treatment and their duration for the OS of the second-line therapy. According to our findings, second-line chemotherapies in advanced PDAC are beneficial and should be offered to patients, but we did not detect any superiority of a specific drug combination. More prospective, randomized and larger studies are necessary to evaluate new strategies for second-line chemotherapies.

Urine proteomic analysis differentiates cholangiocarcinoma from primary sclerosing cholangitis and other benign biliary disorders.

BACKGROUND: Diagnosis and curative treatment of cholangiocarcinoma (CC) often comes too late due to the lack of reliable tumour markers especially in patients with primary sclerosing cholangitis (PSC). The authors recently introduced bile proteomic analysis for CC diagnosis. Nevertheless, bile collection depends on invasive endoscopic retrograde cholangiography. The authors therefore evaluated urine proteomic analysis for non-invasive CC diagnosis. METHODS: Using capillary electrophoresis mass spectrometry the authors established a CC-specific peptide marker model based on the distribution of 42 peptides in 14 CC, 13 PSC and 14 benign biliary disorder (BBD) patients. RESULTS: In cross-sectional validation of 123 patients, the urine peptide marker model correctly classified 35 of 42 CC patients and 64 of 81 PSC and BBD patients with an area under the curve value of 0.87 (95% CI 0.80 to 0.92, p=0.0001, 83% sensitivity, 79% specificity). Evaluation of 101 normal controls resulted in 86% specificity. All 10 patients with CC on top of PSC were correctly classified. The majority of sequence-identified peptides are fragments of interstitial collagens with some of them also detected in blood indicating their extra-renal origin. Immunostaining of liver sections for matrix metallopeptidase 1 indicated increased activity of the interstitial collagenase in liver epithelial cells of CC patients. CONCLUSION: The urine test differentiates CC from PSC and other BBD and may provide a new diagnostic non-invasive tool for PSC surveillance and CC detection.

Novel Adiponectin Receptor Agonist Inhibits Cholangiocarcinoma via Adenosine Monophosphate-activated Protein Kinase.

BACKGROUND: Cholangiocarcinoma (CCA) has a poor prognosis and only limited palliative treatment options. The deficiency of adiponectin and adenosine monophosphate-activated protein kinase (AMPK) signaling was reported in several malignancies, but the alteration of these proteins in CCA is still unclear. OBJECTIVES: This study aimed to assess the role of adiponectin and AMPK signaling in CCA. Furthermore, AdipoRon, a novel adiponectin receptor (AdipoR) agonist, was evaluated in vitro and in vivo as a new anti-tumor therapy for CCA. METHODS: The expression of AdipoR1 and p-AMPKα in human tissue microarrays (TMAs) was evaluated by immunohistochemistry staining (IHC). The effect of 2-(4-Benzoylphenoxy)-N-[1-(phenylmethyl)- 4-piperidinyl]-acetamide (AdipoRon) was investigated in vitro with proliferation, crystal violet, migration, invasion, colony formation, senescence, cell cycle and apoptosis assays and in vivo using a CCA engineered mouse model (AlbCre/LSL-KRASG12D/p53L/L). RT-qPCR and western blot methods were applied to study molecular alterations in murine tissues. RESULTS: AdipoR1 and p-AMPKα were impaired in human CCA tissues, compared to adjacent non-tumor tissue. There was a positive correlation between the AdipoR1 and p-AMPKα levels in CCA tissues. Treatment with AdipoRon inhibited proliferation, migration, invasion and colony formation and induced apoptosis in a time- and dose-dependent manner in vitro(p<0.05). In addition, AdipoRon reduced the number of CCA and tumor volume, prolonged survival, and decreased metastasis and ascites in the treated group compared to the control group (p<0.05). CONCLUSIONS: AdipoR1 and p-AMPKα are impaired in CCA tissues, and AdipoRon effectively inhibits CCA in vitro and in vivo. Thus, AdipoRon may be considered as a potential anti-tumor therapy in CCA.

Epidemiological trends in incidence and mortality of hepatobiliary cancers in Germany.

OBJECTIVE: While marked changes in the frequency of hepatobiliary malignancies, most notably hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), have been observed in different populations, no such data have been reported for Germany. We aimed to provide epidemiological data on recent trends in liver-related mortality, specifically mortality from hepatobiliary malignancies, in Germany. MATERIAL AND METHODS: We used incidence and mortality data to determine changes in the frequency of malignant and non-malignant liver disease in Germany over the past 30 years. RESULTS: While overall liver disease mortality has slightly declined in Germany, deaths from hepatobiliary malignancies have declined in women, but remained constant in men. Among hepatobiliary malignancies, ICC stands out, because mortality has more than tripled both in men and women between 1998 and 2008. This is mirrored by a marked increase in new cases reported to local cancer registries, that is, incidence. Over the same time period, HCC and extrahepatic cholangiocarcinoma (ECC) have remained largely constant while gall bladder cancers (GBC) have declined twofold. The rapid rise in ICC is in line with finding from different regions worldwide, but in contrast to recent data from Denmark and France, two of Germany's direct neighbors. CONCLUSIONS: The incidence of and mortality from ICC are rising markedly in Germany. The risk factors underlying this trend are as yet unclear.

Inhibition of gamma-secretase activity inhibits tumor progression in a mouse model of pancreatic ductal adenocarcinoma.

BACKGROUND & AIMS: The Notch signaling pathway is required for the expansion of undifferentiated pancreatic progenitor cells during embryonic development and has been implicated in the progression of pancreatic ductal adenocarcinoma (PDAC). The interaction of Notch ligands with their receptors promotes a gamma-secretase-dependent cleavage of the Notch receptor and release of the Notch intracellular domain, which translocates to the nucleus and activates transcription. We investigated the role of this pathway in PDAC progression. METHODS: We tested the effects of a gamma-secretase inhibitor (GSI) that blocks Notch signaling in PDAC cell lines and a genetically engineered mouse model of PDAC (Kras p53 L/+ mice). RESULTS: Notch signaling was activated in PDAC precursors and advanced tumors. The GSI inhibited the growth of premalignant pancreatic duct-derived cells in a Notch-dependent manner. Additionally, in a panel of over 400 human solid tumor-derived cell lines, PDAC cells, as a group, were more sensitive to the GSI than any other tumor type. Finally, the GSI completely inhibited tumor development in the genetically engineered model of invasive PDAC (P < .005, chi2 test; compared with mice exposed to vehicle). CONCLUSIONS: These results suggest that Notch signaling is required for PDAC progression. Pharmacologic targeting of this pathway offers therapeutic potential in this treatment-refractory malignancy.