The androgen-regulated Calcium-Activated Nucleotidase 1 (CANT1) is commonly overexpressed in prostate cancer and is tumor-biologically relevant in vitro.

Previously, we identified the calcium-activated nucleotidase 1 (CANT1) transcript as up-regulated in prostate cancer. Now, we studied CANT1 protein expression in a large cohort of nearly 1000 prostatic tissue samples including normal tissue, prostatic intraepithelial neoplasia (PIN), primary carcinomas, metastases, and castrate-resistant carcinomas, and further investigated its functional relevance. CANT1 displayed predominantly a Golgi-type immunoreactivity with additional and variable cytoplasmic staining. In comparison to normal tissues, the staining intensity was significantly increased in PIN lesions and cancer. In cancer, high CANT1 levels were associated with a better prognosis, and castrate-resistant carcinomas commonly showed lower CANT1 levels than primary carcinomas. The functional role of CANT1 was investigated using RNA interference in two prostate cancer cell lines with abundant endogenous CANT1 protein. On CANT1 knockdown, a significantly diminished cell number and DNA synthesis rate, a cell cycle arrest in G(1) phase, and a strong decrease of cell transmigration rate and wound healing capacity of CANT1 knockdown cells was found. However, on forced CANT1 overexpression, cell proliferation and migration remained unchanged. In summary, CANT1 is commonly overexpressed in the vast majority of primary prostate carcinomas and in the precursor lesion PIN and may represent a novel prognostic biomarker. Moreover, this is the first study to demonstrate a functional involvement of CANT1 in tumor biology.

A pooled analysis of sequential therapies with sorafenib and sunitinib in metastatic renal cell carcinoma.

OBJECTIVE: To evaluate the optimal sequence for the receptor tyrosine kinase inhibitors (rTKIs) sorafenib and sunitinib in metastatic renal cell cancer. METHODS: We performed a retrospective analysis of patients who had received sequential therapy with both rTKIs and integrated these results into a pooled analysis of available data from other publications. Differences in median progression-free survival (PFS) for first- (PFS1) and second-line treatment (PFS2), and for the combined PFS (PFS1 plus PFS2) were examined using weighted linear regression. RESULTS: In the pooled analysis encompassing 853 patients, the median combined PFS for first-line sunitinib and 2nd-line sorafenib (SuSo) was 12.1 months compared with 15.4 months for the reverse sequence (SoSu; 95% CI for difference 1.45-5.12, p = 0.0013). Regarding first-line treatment, no significant difference in PFS1 was noted regardless of which drug was initially used (0.62 months average increase on sorafenib, 95% CI for difference -1.01 to 2.26, p = 0.43). In second-line treatment, sunitinib showed a significantly longer PFS2 than sorafenib (average increase 2.66 months, 95% CI 1.02-4.3, p = 0.003). CONCLUSION: The SoSu sequence translates into a longer combined PFS compared to the SuSo sequence. Predominantly the superiority of sunitinib regarding PFS2 contributed to the longer combined PFS in sequential use.

Golgi phosphoprotein 2 (GOLPH2) expression in liver tumors and its value as a serum marker in hepatocellular carcinomas.

UNLABELLED: Hepatocellular carcinomas (HCCs) and bile duct carcinomas (BDCs) have a poor prognosis. Therefore, surveillance strategies including sensitive and specific serum markers for early detection are needed. Recently, Golgi Phosphoprotein 2 (GOLPH2) has been proposed as a serum marker for HCC, but GOLPH2 expression data in liver tissues was not available. Using tissue microarrays and immunohistochemistry, we semiquantitatively analyzed GOLPH2 protein expression in patients with HCC (n = 170), benign liver tumors (n = 22), BDC (n = 114) and normal liver tissue (n = 105). A newly designed sandwich enzyme-linked immunoassay (ELISA) was used to analyze GOLPH2 levels in the sera of patients with HCC (n = 62), hepatitis C virus (HCV) (n = 29), BDC (n = 10), and healthy control persons (n = 12). By immunohistochemistry 121/170 (71%) of HCC showed strong GOLPH2 expression, which was significantly associated with a higher tumor grade (P = 0.01). A total of 97/114 (85%) BDCs showed a strong GOLPH2 expression which proved to be an independent prognostic factor for overall survival (P < 0.05). Serum levels of GOLPH2 measured by ELISA were significantly elevated in patients with HCC with underlying HCV infection (median 18 mg/L, P < 0.05) and patients with BDC (median = 14.5 mg/L, P < 0.01) in comparison to healthy controls (median 4 mg/L). CONCLUSION: GOLPH2 protein is highly expressed in tissues of HCC and BDC. GOLPH2 protein levels are detectable and quantifiable in sera by ELISA. In patients with hepatitis C, serial ELISA measurements in the course of the disease appear to be a promising complementary serum marker in the surveillance of HCC. GOLPH2 should be further evaluated as a serum tumor marker in BDC on a larger scale.

Therapeutic Targeting of Golgi Phosphoprotein 2 (GOLPH2) with Armed Antibodies: A Preclinical Study of Anti-GOLPH2 Antibody Drug Conjugates in Lung and Colorectal Cancer Models of Patient Derived Xenografts (PDX).

BACKGROUND: Golgi phosphoprotein 2 (GOLPH2) has been shown to be involved in chronic inflammatory processes and carcinogenesis. GOLPH2 is prominently overexpressed in hepatocellular carcinoma, melanoma, glioblastoma, prostate, lung, and colorectal cancer. With a low and tightly regulated expression in non-malignant tissues, GOLPH2 has been proposed as an attractive target for cancer therapy. However, GOLPH2 is predominantly located intracellularly and when situated outside of the cell it is proteolytically cleaved and shed from the cell surface. Until now, GOLPH2 has been regarded as an "undruggable" target. OBJECTIVE: We sought to create antibodies that specifically bind to GOLPH2 overexpressing tumor cells. PATIENTS AND METHODS: Antibodies binding to membranous GOLPH2 despite shedding of the protein were generated from a scFV library screening. These antibodies target the part of GOLPH2 that remains at the cell surface after proteolytic cleavage. These antibodies were then tested in vitro and in vivo. RESULTS: Two candidates (G2-1 and G2-2) showed target specific binding in vitro. Utilizing a tumor array (n = 128 tumors) with G2-2 and a reference antibody, a GOLPH2 expression scoring system was established. Rapid internalization of the antibodies was noted so this was exploited to deliver a toxic payload of pyrrolobenzodiazepine (PBD). In two patient-derived xenograft (PDX)-models, colorectal and lung cancer, the G2-2 antibody drug conjugate (ADC) displayed high efficacy with significant tumor responses (P = 0.001; P = 0.013) and improved survival (P = 0.0001; P = 0.0011) compared with controls. CONCLUSIONS: Treatment with GOLPH2-directed antibodies induces durable responses in colorectal and lung cancer models. With a robust companion assay for GOLPH2 positivity at hand our findings prepare for the translation into a clinical trial.

Targeted therapeutic approach for an anaplastic thyroid cancer in vitro and in vivo.

Anaplastic thyroid carcinoma (ATC) is among the most aggressive human malignancies, being responsible for the majority of thyroid cancer-related deaths. Despite multimodal therapy including surgery, chemotherapy, and radiotherapy, the outcome of ATC is poor. The human ATC cell line MB1, derived from tumor tissue of a 57-year-old man with thyroid cancer and pronounced neutrophilia, was established from surgically excised tumor tissue. The karyotype of the cell line shows many chromosomal abnormalities. Preclinical investigations have shown antitumor activity and effectiveness of the BRAF kinase inhibitor Sorafenib and the proteasome inhibitor Bortezomib. After establishment of the MB1 cell line these agents were applied in vitro and, showing activity in a cell culture model, were also used for in vivo treatment. Sorafenib had some clinical effect, namely normalization of leucocytosis, but had no sustained impact on subsequent tumor growth and development of distant metastasis. Molecular diagnostics of the tumor demonstrated no BRAF mutations in exons 11 and 15 concordant with a rather modest effect of Sorafenib on MB1 cell growth. Clinical benefit was seen with subsequent bortezomib therapy inducing a temporary halt to lymph node growth and a progression-free interval of 7 weeks. Our observations together with previous data from preclinical models could serve as a rationale for selecting those patients suffering from ATC most likely to benefit from targeted therapy. A prospective controlled randomized trial integrating kinase and proteasome inhibitors into a therapeutic regime for ATC is warranted.

Severe neutropenia during cabazitaxel treatment is associated with survival benefit in men with metastatic castration-resistant prostate cancer (mCRPC): A post-hoc analysis of the TROPIC phase III trial.

BACKGROUND: Cabazitaxel significantly improves overall survival (OS) in men with metastatic castration-resistant prostate cancer (mCRPC) progressing during or after docetaxel, but is associated with a higher rate of grade ≥3 neutropenia compared with docetaxel. We thus examined the relationship between cabazitaxel-induced grade ≥3 neutropenia, baseline neutrophil-lymphocyte ratio (NLR) and treatment outcomes. METHODS: Data from the experimental arm of the TROPIC phase 3 trial which randomly assigned men with mCRPC to cabazitaxel or mitoxantrone every 3 weeks, both combined with daily prednisone, were analysed. The influence on OS (primary end-point) and progression-free survival (PFS) of at least one episode of grade ≥3 neutropenia during cabazitaxel therapy was investigated using Cox regression models, adjusted for pain at baseline. The relationships with prostate-specific antigen (PSA) responses during cabazitaxel therapy and baseline NLR were also analysed. FINDINGS: The occurrence of grade ≥3 neutropenia during cabazitaxel therapy was associated with a prolonged OS (median 16.3 versus 14.0 months, hazard ratio (HR) [95% confidence interval] = 0.65 [0.43-0.97], p = 0.035), a twice longer PFS (median 5.3 versus 2.6 months, HR = 0.56 [0.40-0.79], p = 0.001) and a higher confirmed PSA response ≥50% (49.8% versus 24.4%, p = 0.005), as compared with patients who did not develop grade ≥3 neutropenia. Grade ≥3 neutropenia was more common in case of NLR <3 as compared with NLR ≥3 at baseline (88.8% versus 75.3%, p = 0.002). Combining low NLR at baseline and grade ≥3 neutropenia during therapy was associated with the longest OS (median 19.2 months) while high NLR at baseline and no grade ≥3 neutropenia was associated with a poor OS (median 12.9 months, HR 0.46 [0.28-0.76], p = 0.002). In the subgroup of neutropenic patients the median OS was 19.7 months in those treated with granulocyte colony-stimulating factor (G-CSF) and 16 months on those without G-CSF support. INTERPRETATION: This post-hoc analysis of TROPIC suggests that the occurrence of grade ≥3 neutropenia with cabazitaxel is associated with improved OS and PFS. Patients with a low NLR at baseline were more likely to develop grade ≥3 neutropenia during cabazitaxel therapy and showed the longest OS. High NLR at baseline and no grade ≥3 neutropenia during therapy was associated with poor outcomes which may suggest insufficient drug exposure or a limited impact on the tumour-associated immune response. Primary or secondary prophylactic use of G-CSF had no adverse impact for outcome. If prospectively confirmed, these results would justify maintaining the intended cabazitaxel dose of 25 mg/m(2) whenever possible.

Daily Pomegranate Intake Has No Impact on PSA Levels in Patients with Advanced Prostate Cancer - Results of a Phase IIb Randomized Controlled Trial.

Pomegranate has been shown to prolong PSA doubling time in early prostate cancer, but no data from a placebo controlled trial has been published yet. The objective of this study was to prospectively evaluate the impact of pomegranate juice in patients with prostate cancer. We conducted a phase IIb, double blinded, randomized placebo controlled trial in patients with histologically confirmed prostate cancer. Only patients with a PSA value ≥ 5ng/ml were included. The subjects consumed 500 ml of pomegranate juice or 500 ml of placebo beverage every day for a 4 week period. Thereafter, all patients received 250 ml of the pomegranate juice daily for another 4 weeks. PSA values were taken at baseline, day 14, 28 and on day 56. The primary endpoint was the detection of a significant difference in PSA serum levels between the groups after one month of treatment. Pain scores and adherence to intervention were recorded using patient diaries. 102 patients were enrolled. The majority of patients had castration resistant prostate cancer (68%). 98 received either pomegranate juice or placebo between October 2008 and May 2011. Adherence to protocol was good, with 94 patients (96%) completing the first period and 87 patients (89%) completing both periods. No grade 3 or higher toxicities occurred within the study. No differences were detected between the two groups with regard to PSA kinetics and pain scores. Consumption of pomegranate juice as an adjunct intervention in men with advanced prostate cancer does not result in significant PSA declines compared to placebo.

RP1 is a phosphorylation target of CK2 and is involved in cell adhesion.

RP1 (synonym: MAPRE2, EB2) is a member of the microtubule binding EB1 protein family, which interacts with APC, a key regulatory molecule in the Wnt signalling pathway. While the other EB1 proteins are well characterized the cellular function and regulation of RP1 remain speculative to date. However, recently RP1 has been implicated in pancreatic cancerogenesis. CK2 is a pleiotropic kinase involved in adhesion, proliferation and anti-apoptosis. Overexpression of protein kinase CK2 is a hallmark of many cancers and supports the malignant phenotype of tumor cells. In this study we investigate the interaction of protein kinase CK2 with RP1 and demonstrate that CK2 phosphorylates RP1 at Ser(236) in vitro. Stable RP1 expression in cell lines leads to a significant cleavage and down-regulation of N-cadherin and impaired adhesion. Cells expressing a Phospho-mimicking point mutant RP1-ASP(236) show a marked decrease of adhesion to endothelial cells under shear stress. Inversely, we found that the cells under shear stress downregulate endogenous RP1, most likely to improve cellular adhesion. Accordingly, when RP1 expression is suppressed by shRNA, cells lacking RP1 display significantly increased cell adherence to surfaces. In summary, RP1 phosphorylation at Ser(236) by CK2 seems to play a significant role in cell adhesion and might initiate new insights in the CK2 and EB1 family protein association.

Characterization of the human GARP (Golgi associated retrograde protein) complex.

The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the "orphan" SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells.

CADD, a Chlamydia protein that interacts with death receptors.

We report here the identification of a bacterial protein capable of interacting with mammalian death receptors in vitro and in vivo. The protein is encoded in the genome of Chlamydia trachomatis and has homologues in other Chlamydia species. This protein, which we refer to as "Chlamydia protein associating with death domains" (CADD), induces apoptosis in a variety of mammalian cell lines when expressed by transient gene transfection. Apoptosis induction can be blocked by Caspase inhibitors, indicating that CADD triggers cell death by engaging the host apoptotic machinery. CADD interacts with death domains of tumor necrosis factor (TNF) family receptors TNFR1, Fas, DR4, and DR5 but not with the respective downstream adaptors. In infected epithelial cells, CADD is expressed late in the infectious cycle of C. trachomatis and co-localizes with Fas in the proximity of the inclusion body. The results suggest a role for CADD modulating the apoptosis pathways of cells infected, revealing a new mechanism of host-pathogen interaction.

Structure of the Chlamydia protein CADD reveals a redox enzyme that modulates host cell apoptosis.

The Chlamydia protein CADD (Chlamydia protein associating with death domains) has been implicated in the modulation of host cell apoptosis via binding to the death domains of tumor necrosis factor family receptors. Transfection of CADD into mammalian cells induces apoptosis. Here we present the CADD crystal structure, which reveals a dimer of seven-helix bundles. Each bundle contains a di-iron center adjacent to an internal cavity, forming an active site similar to that of methane mono-oxygenase hydrolase. We further show that CADD mutants lacking critical metal-coordinating residues are substantially less effective in inducing apoptosis but retain their ability to bind to death domains. We conclude that CADD is a novel redox protein toxin unique to Chlamydia species and propose that both its redox activity and death domain binding ability are required for its biological activity.