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1.  A Drosophila Model Identifies a Critical Role for Zinc in Mineralization for Kidney Stone Disease 
PLoS ONE  2015;10(5):e0124150.
Ectopic calcification is a driving force for a variety of diseases, including kidney stones and atherosclerosis, but initiating factors remain largely unknown. Given its importance in seemingly divergent disease processes, identifying fundamental principal actors for ectopic calcification may have broad translational significance. Here we establish a Drosophila melanogaster model for ectopic calcification by inhibiting xanthine dehydrogenase whose deficiency leads to kidney stones in humans and dogs. Micro X-ray absorption near edge spectroscopy (μXANES) synchrotron analyses revealed high enrichment of zinc in the Drosophila equivalent of kidney stones, which was also observed in human kidney stones and Randall’s plaques (early calcifications seen in human kidneys thought to be the precursor for renal stones). To further test the role of zinc in driving mineralization, we inhibited zinc transporter genes in the ZnT family and observed suppression of Drosophila stone formation. Taken together, genetic, dietary, and pharmacologic interventions to lower zinc confirm a critical role for zinc in driving the process of heterogeneous nucleation that eventually leads to stone formation. Our findings open a novel perspective on the etiology of urinary stones and related diseases, which may lead to the identification of new preventive and therapeutic approaches.
doi:10.1371/journal.pone.0124150
PMCID: PMC4430225  PMID: 25970330
2.  Targeting cMET with INC280 impairs tumour growth and improves efficacy of gemcitabine in a pancreatic cancer model 
BMC Cancer  2015;15:71.
Background
Expression and activation of the cMET receptor have been implicated in tumor progression and resistance to chemotherapy in human pancreatic cancer. In this regard we assessed the effects of targeting cMET in pancreatic cancer models in vitro and in vivo.
Methods
Human (L3.6pl, BxP3, HPAF-II, MiaPaCa2) and murine (Panc02) pancreatic cancer cell lines, endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) were used for the experiments. Furthermore, the human pancreatic cancer cell line MiaPaCa2 with acquired resistance to gemcitabine was employed (MiaPaCa2(G250)). For targeting the cMET receptor, the oral available, ATP-competitive inhibitor INC280 was used. Effects of cMET inhibition on cancer and stromal cells were determined by growth assays, western blotting, motility assays and ELISA. Moreover, orthotopic xenogeneic and syngeneic mouse (BALB-C nu/nu; C57BL/6) models were used to assess in vivo efficacy of targeting cMET alone and in combination with gemcitabine.
Results
Treatment with INC280 impairs activation of signaling intermediates in pancreatic cancer cells and ECs, particularly when cells were stimulated with hepatocyte growth factor (HGF). Moreover, motility of cancer cells and ECs in response to HGF was reduced upon treatment with INC280. Only minor effects on VSMCs were detected. Interestingly, MiaPaCa2(G250) showed an increase in cMET expression and cMET inhibition abrogated HGF-induced effects on growth, motility and signaling as well as DFX-hypoxia HIF-1alpha and MDR-1 expression in vitro. In vivo, therapy with INC280 alone led to inhibition of orthotopic tumor growth in xenogeneic and syngeneic models. Similar to in vitro results, cMET expression was increased upon treatment with gemcitabine, and combination of the cMET inhibitor with gemcitabine improved anti-neoplastic capacity in an orthotopic syngeneic model. Immunohistochemical analysis revealed a significant inhibition of tumor cell proliferation (Ki67) and tumor vascularization (CD31). Finally, combination of gemcitabine with INC280 significantly prolonged survival in the orthotopic syngeneic tumor model even when treatment with the cMET inhibitor was initiated at an advanced stage of disease.
Conclusions
These data provide evidence that targeting cMET in combination with gemcitabine may be effective in human pancreatic cancer and warrants further clinical evaluation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12885-015-1064-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12885-015-1064-9
PMCID: PMC4340491  PMID: 25884642
Pancreatic cancer; cMET; Resistance; Survival
3.  Oxaliplatin-based versus irinotecan-based hyperthermic intraperitoneal chemotherapy (HIPEC) in patients with peritoneal metastasis from appendiceal and colorectal cancer: a retrospective analysis 
BMC Cancer  2014;14:807.
Background
Cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) provide an effective treatment option for selected patients with colorectal peritoneal metastasis with encouraging survival results. Many different drug combinations and HIPEC regimens including bidirectional, i.e. synchronous intravenous and intraperitoneal, drug application have been used. However, there is still no standardization of the HIPEC regimen.
Methods
Between 05/2007 and 04/2010 190 patients underwent CRS and HIPEC at the University Hospital Regensburg. Thirty-two patients with peritoneal metastasis arising from colorectal or appendiceal cancer underwent complete macroscopic cytoreduction (CC-0/1) and bidirectional HIPEC and completed at least 3-year follow-up. Twenty patients received oxaliplatin-based (OX) and twelve patients received irinotecan-based HIPEC (IRI). Group-specific perioperative morbidity and 3-year survival has been determined.
Results
The grade 3/4 morbidity rate according to CTCAE v4 was 35.0% in the OX group vs. 33.3% in the IRI group (p = 1.000). There was no perioperative mortality in both groups. Median survival was 26.8 months (95% CI 15.7-33.1 months) in the IRI group and has not yet been reached in the OX group during a median follow-up of 39.4 months. Three-year survival rates were 65.0% in the OX group vs. 41.7% in the IRI group (p = 0.295).
Conclusions
The morbidity and toxicity rates of bidirectional irinotecan-based and oxaliplatin-based HIPEC are comparable. Nevertheless, in the absence of contraindications oxaliplatin-based HIPEC might be preferred due to the positive trend regarding 3-year and median survival.
doi:10.1186/1471-2407-14-807
PMCID: PMC4228082  PMID: 25369730
Peritoneal carcinomatosis; HIPEC; Irinotecan; Oxaliplatin; Morbidity; Survival
4.  The inhibition of tyrosine kinase receptor signalling in leiomyosarcoma cells using the small molecule kinase inhibitor PTK787/ZK222584 (Vatalanib®) 
International Journal of Oncology  2014;45(6):2267-2277.
Leiomyosarcomas remain challenging tumors to manage and novel therapy strategies besides radiation and conventional chemotherapy are needed. Targeting angiogenesis by inhibition of vascular endothelial growth factor (VEGF) receptor tyrosine kinases (RTKs) of the tumor vasculature with small molecules is a promising new therapy. It has been shown recently that these receptors are not only expressed on tumor endothelium but also on tumor cells themselves. Thus, we investigated the expression of members of the VEGF receptor (VEGFR) family and corresponding growth factors in leiomyosarcoma tissue specimens and in the leiomyosarcoma cell lines SK-LMS-1 and SK-UT-1. We evaluated the influence of the VEGFR inhibitor PTK787/ZK222584 (PTK787) on cell growth, migration, apoptosis and phosphorylation of intracellular signalling molecules. In human leiomyosarcoma tissue specimens VEGFR-1/-2 and platelet-derived growth factor receptor (PDGFR-β) were strongly expressed. Both leiomyosarcoma cell lines expressed VEGFR-1/-3 and PDGFR-β but VEGFR-2 protein expression was positive only in SK-UT-1. SK-LMS-1 and SK-UT-1 cells secreted high and low amounts of VEGF-A, respectively, whereas PDGF-BB secretion was similar in both cell lines. Application of PTK787 led to partial inhibition of PDGF-BB-activated AKT/p90RSK and ERK1/2 signalling pathways. In contrast, protein phosphorylation was not affected by PTK787 in VEGF-A-treated cells. PTK787 turned out to inhibit cell migration even though no effects were observed upon stimulation with VEGF-A or PDGF-BB. In line, cell growth in leiomyosarcoma cell lines remained unchanged upon PTK787 treatment alone and with subsequent VEGF-A- or PDGF-BB-stimulation. However, VEGF-A, but not PDGF-BB-treated cells showed increased cell death upon PTK787 treatment. VEGFR family members are expressed in leiomyosarcomas in vivo and in vitro. Upon receptor stimulation, PTK787 is able to inhibit subsequent phosphorylation events and influences cell survival but not metabolic activity and migration. Thus, the inhibitor is possibly an additional option in the treatment of leiomyosarcomas.
doi:10.3892/ijo.2014.2683
PMCID: PMC4215578  PMID: 25340839
leiomyosarcoma; receptor tyrosine kinase; small molecule inhibitor; PTK787/ZK222584; tumor; angiogenesis; VEGF; VEGF receptor
5.  New Aspects of an Old Drug – Diclofenac Targets MYC and Glucose Metabolism in Tumor Cells 
PLoS ONE  2013;8(7):e66987.
Non-steroidal anti-inflammatory drugs such as diclofenac exhibit potent anticancer effects. Up to now these effects were mainly attributed to its classical role as COX-inhibitor. Here we show novel COX-independent effects of diclofenac. Diclofenac significantly diminished MYC expression and modulated glucose metabolism resulting in impaired melanoma, leukemia, and carcinoma cell line proliferation in vitro and reduced melanoma growth in vivo. In contrast, the non-selective COX inhibitor aspirin and the COX-2 specific inhibitor NS-398 had no effect on MYC expression and glucose metabolism. Diclofenac significantly decreased glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and monocarboxylate transporter 1 (MCT1) gene expression in line with a decrease in glucose uptake and lactate secretion. A significant intracellular accumulation of lactate by diclofenac preceded the observed effect on gene expression, suggesting a direct inhibitory effect of diclofenac on lactate efflux. While intracellular lactate accumulation impairs cellular proliferation and gene expression, it does not inhibit MYC expression as evidenced by the lack of MYC regulation by the MCT inhibitor α-cyano-4-hydroxycinnamic acid. Finally, in a cell line with a tetracycline-regulated c-MYC gene, diclofenac decreased proliferation both in the presence and absence of c-MYC. Thus, diclofenac targets tumor cell proliferation via two mechanisms, that is inhibition of MYC and lactate transport. Based on these results, diclofenac holds potential as a clinically applicable MYC and glycolysis inhibitor supporting established tumor therapies.
doi:10.1371/journal.pone.0066987
PMCID: PMC3706586  PMID: 23874405
6.  A prospective multicenter phase II study evaluating multimodality treatment of patients with peritoneal carcinomatosis arising from appendiceal and colorectal cancer: the COMBATAC trial 
BMC Cancer  2013;13:67.
Background
Peritoneal carcinomatosis is regarded as a common sign of advanced tumor stage, tumor progression or local recurrence of appendiceal and colorectal cancer and is generally associated with poor prognosis. Although survival of patients with advanced stage CRC has markedly improved over the last 20 years with systemic treatment, comprising combination chemotherapy +/− monoclonal antibodies, the oncological outcome—especially of the subgroup of patients with peritoneal metastases—is still unsatisfactory. In addition to systemic therapy, cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC) are specific treatment options for a selected group of these patients and may provide an additional therapeutic benefit in the framework of an interdisciplinary treatment concept.
Methods/design
The COMBATAC trial is a prospective, multicenter, open-label, single-arm, single-stage phase II trial investigating perioperative systemic polychemotherapy including cetuximab in combination with CRS and HIPEC patients with histologically proven wild-type KRAS colorectal or appendiceal adenocarcinoma and synchronous or metachronous peritoneal carcinomatosis. The planned total number of patients to be recruited is 60. The primary endpoint is progression-free survival (PFS). Secondary endpoints include overall survival (OS), perioperative morbidity and treatment-associated toxicity, feasibility of the combined treatment regimen, quality of life (QoL) and histopathological regression after preoperative chemotherapy.
Discussion
The COMBATAC trial is designed to evaluate the feasibility and efficacy of the combined multidisciplinary treatment regimen consisting of perioperative systemic combination chemotherapy plus cetuximab and CRS plus bidirectional HIPEC with intraperitoneal oxaliplatin.
Trial registration
ClinicalTrials.gov Identifier: NCT01540344, EudraCT number: 2009-014040-11
doi:10.1186/1471-2407-13-67
PMCID: PMC3575316  PMID: 23391248
Cytoreductive surgery; HIPEC; Perioperative chemotherapy; Cetuximab; Colorectal cancer; Peritoneal carcinomatosis
7.  STAT5b as Molecular Target in Pancreatic Cancer—Inhibition of Tumor Growth, Angiogenesis, and Metastases12 
Neoplasia (New York, N.Y.)  2012;14(10):915-925.
The prognosis of patients suffering from pancreatic cancer is still poor and novel therapeutic options are urgently needed. Recently, the transcription factor signal transducer and activator of transcription 5b (STAT5b) was associated with tumor progression in human solid cancer. Hence, we assessed whether STAT5b might serve as an anticancer target in ductal pancreatic adenocarcinoma (DPAC). We found that nuclear expression of STAT5b can be detected in approximately 50% of DPAC. Blockade of STAT5b by stable shRNA-mediated knockdown showed no effects on tumor cell growth in vitro. However, inhibition of tumor cell motility was found even in response to stimulation with epidermal growth factor or interleukin-6. These findings were paralleled by a reduction of prometastatic and proangiogenic factors in vitro. Subsequent in vivo experiments revealed a strong growth inhibition on STAT5b blockade in subcutaneous and orthotopic models. These findings were paralleled by impaired tumor angiogenesis in vivo. In contrast to the subcutaneous model, the orthotopic model revealed a strong reduction of tumor cell proliferation that emphasizes the meaning of assessing targets in an appropriate microenvironment. Taken together, our results suggest that STAT5b might be a potential novel target for human DPAC.
PMCID: PMC3479837  PMID: 23097626
8.  TRC40 can deliver short secretory proteins to the Sec61 translocon 
Journal of Cell Science  2012;125(15):3612-3620.
Summary
Whilst the co-translational translocation of nascent proteins across the mammalian endoplasmic reticulum (ER) is well defined, the capacity of this organelle for post-translational translocation is poorly delineated. Here we identify two human secretory protein precursors, apelin and statherin, as bona fide substrates for post-translational translocation across the ER membrane. Further studies, in combination with Hyalophora cecropia preprocecropin A (ppcecA), show that all three proteins bind to TRC40 and can utilise this component for their delivery to the ER membrane in a well-established in vitro system. However, ppcecA is not an obligate TRC40 substrate, and it can also be delivered to the ER by an alternative TRC40-independent pathway. Upon arrival at the ER membrane, these short secretory proteins appear to be ubiquitously transported across the ER membrane through the Sec61 translocon, apparently irrespective of their delivery route. We speculate that the post-translational translocation of secretory proteins in higher eukaryotes is more prevalent than previously acknowledged.
doi:10.1242/jcs.102608
PMCID: PMC3445324  PMID: 22505607
Asna 1; Endoplasmic reticulum; Post-translational translocation; Tail-anchored protein; WRB protein; Eeyarestatin I
9.  Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms 
In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.
doi:10.3791/2730
PMCID: PMC3196168  PMID: 21775954
10.  Tumor development in murine ulcerative colitis depends on MyD88 signaling of colonic F4/80+CD11bhighGr1low macrophages  
The Journal of Clinical Investigation  2011;121(5):1692-1708.
Patients with prolonged ulcerative colitis (UC) frequently develop colorectal adenocarcinoma for reasons that are not fully clear. To analyze inflammation-associated colonic tumorigenesis, we developed a chronic form of oxazolone-induced colitis in mice that, similar to UC, was distinguished by the presence of IL-13–producing NKT cells. In this model, the induction of tumors using azoxymethane was accompanied by the coappearance of F4/80+CD11bhighGr1low M2 macrophages, cells that undergo polarization by IL-13 and are absent in tumors that lack high level IL-13 production. Importantly, this subset of macrophages was a source of tumor-promoting factors, including IL-6. Similar to dextran sodium sulfate–induced colitis, F4/80+CD11bhighGr1intermediate macrophages were present in the mouse model of chronic oxazolone-induced colitis and may influence tumor development through production of TGF-β1, a cytokine that inhibits tumor immunosurveillance. Finally, while robust chronic oxazolone-induced colitis developed in myeloid differentiation primary response gene 88–deficient (Myd88–/–) mice, these mice did not support tumor development. The inhibition of tumor development in Myd88–/– mice correlated with cessation of IL-6 and TGF-β1 production by M2 and F4/80+CD11bhighGr1intermediate macrophages, respectively, and was reversed by exogenous IL-6. These data show that an UC-like inflammation may facilitate tumor development by providing a milieu favoring development of MyD88-dependent tumor-supporting macrophages.
doi:10.1172/JCI42540
PMCID: PMC3083803  PMID: 21519141
11.  Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (Hsp90) inhibition 
BMC Cancer  2010;10:668.
Background
Activating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer.
Methods
Regulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression.
Results
The results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer.
Conclusion
In conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor.
doi:10.1186/1471-2407-10-668
PMCID: PMC3003660  PMID: 21129190
12.  ENMD-1198, a novel tubulin-binding agent reduces HIF-1alpha and STAT3 activity in human hepatocellular carcinoma(HCC) cells, and inhibits growth and vascularization in vivo 
BMC Cancer  2008;8:206.
Background
Hepatocellular carcinoma (HCC) represents a highly vascularized tumor entity and the process of angiogenesis is essential for the growth of HCC. Importantly, the pro-angiogenic transcription factors HIF-1α and STAT3 have been implicated in HCC progression, thus representing interesting targets for molecular targeted therapy. We hypothesized that therapeutic inhibition of HIF-1α could be achieved by using a novel tubulin-binding agent (ENMD-1198). ENMD-1198 is an analog of 2-methoxyestradiol (2ME2) with antiproliferative and antiangiogenic activity.
Methods
The human HCC cell lines HUH-7 and HepG2 were used for experiments. Effects of ENMD-1198 on constitutive and inducible (hypoxia, growth factors) activation of signaling cascades, including HIF-1α and STAT3, were investigated by Western blotting. Changes in VEGF expression were determined by real-time PCR. Effects of ENMD-1198 on cancer cell migration and invasion were evaluated in in vitro-assays. The growth-inhibitory effects of ENMD-1198 (200 mg/kg/day) were determined in a subcutaneous tumor model (HUH-7).
Results
ENMD-1198 inhibited the phosphorylation of MAPK/Erk, PI-3K/Akt and FAK. Moreover, activation of HIF-1α and STAT3 was dramatically reduced by ENMD-1198, which resulted in lower VEGF mRNA expression (P < 0.05). In addition, tumor cell migratory and invasive properties were significantly inhibited (P < 0.05, for both). In vivo, treatment with ENMD-1198 led to a significant reduction in tumor growth, tumor vascularization, and numbers of proliferating tumor cells (P < 0.05 for all).
Conclusion
The novel microtubule destabilizing agent ENMD-1198 is suitable for inhibiting HIF-1α and STAT3 in human HCC cells and leads to reduced tumor growth and vascularization in vivo. Hence, inhibition of HIF-1α and STAT3 could prove valuable for therapy of hepatocellular carcinoma.
doi:10.1186/1471-2407-8-206
PMCID: PMC2496914  PMID: 18651980

Results 1-12 (12)