Corrigendum: SPOCK1 promotes the development of hepatocellular carcinoma.

[This corrects the article DOI: 10.3389/fonc.2022.819883.].

SPOCK1 Promotes the Development of Hepatocellular Carcinoma.

The extracellular matrix proteoglycan SPOCK1 is increasingly recognized as a contributor to the development and progression of cancers. Here, we study how SPOCK1, which is present in non-tumorous hepatocytes at low concentrations, promotes the development and progression of malignant hepatocellular tumors. Although SPOCK1 is an extracellular matrix proteoglycan, its concentration increases in the cytoplasm of hepatocytes starting with very low expression in the normal cells and then appearing in much higher quantities in cells of cirrhotic human liver and hepatocellular carcinoma. This observation is similar to that observed after diethylnitrosamine induction of mouse hepatocarcinogenesis. Furthermore, syndecan-1, the major proteoglycan of the liver, and SPOCK1 are in inverse correlation in the course of these events. In hepatoma cell lines, the cytoplasmic SPOCK1 colocalized with mitochondrial markers, such as MitoTracker and TOMM20, a characteristic protein of the outer membrane of the mitochondrion and could be detected in the cell nucleus. SPOCK1 downregulation of hepatoma cell lines by siRNA inhibited cell proliferation, upregulated p21 and p27, and interfered with pAkt and CDK4 expression. A tyrosine kinase array revealed that inhibition of SPOCK1 in the liver cancer cells altered MAPK signaling and downregulated several members of the Sarc family, all related to the aggressivity of the hepatoma cell lines. These studies support the idea that SPOCK1 enhancement in the liver is an active contributor to human and rodent hepatocarcinogenesis and cancer progression. However, its mitochondrial localization raises the possibility that it has a currently unidentified physiological function in normal hepatocytes.

Alterations in protein expression and site-specific N-glycosylation of prostate cancer tissues.

Identifying molecular alterations occurring during cancer progression is essential for a deeper understanding of the underlying biological processes. Here we have analyzed cancerous and healthy prostate biopsies using nanoLC-MS(MS) to detect proteins with altered expression and N-glycosylation. We have identified 75 proteins with significantly changing expression during disease progression. The biological processes involved were assigned based on protein-protein interaction networks. These include cellular component organization, metabolic and localization processes. Multiple glycoproteins were identified with aberrant glycosylation in prostate cancer, where differences in glycosite-specific sialylation, fucosylation, and galactosylation were the most substantial. Many of the glycoproteins with altered N-glycosylation were extracellular matrix constituents, and are heavily involved in the establishment of the tumor microenvironment.

Overexpression of Human Syndecan-1 Protects against the Diethylnitrosamine-Induced Hepatocarcinogenesis in Mice.

Although syndecan-1 (SDC1) is known to be dysregulated in various cancer types, its implication in tumorigenesis is poorly understood. Its effect may be detrimental or protective depending on the type of cancer. Our previous data suggest that SDC1 is protective against hepatocarcinogenesis. To further verify this notion, human SDC1 transgenic (hSDC1+/+) mice were generated that expressed hSDC1 specifically in the liver under the control of the albumin promoter. Hepatocarcinogenesis was induced by a single dose of diethylnitrosamine (DEN) at an age of 15 days after birth, which resulted in tumors without cirrhosis in wild-type and hSDC1+/+ mice. At the experimental endpoint, livers were examined macroscopically and histologically, as well as by immunohistochemistry, Western blot, receptor tyrosine kinase array, phosphoprotein array, and proteomic analysis. Liver-specific overexpression of hSDC1 resulted in an approximately six month delay in tumor formation via the promotion of SDC1 shedding, downregulation of lipid metabolism, inhibition of the mTOR and the ß-catenin pathways, and activation of the Foxo1 and p53 transcription factors that lead to the upregulation of the cell cycle inhibitors p21 and p27. Furthermore, both of them are implicated in the regulation of intermediary metabolism. Proteomic analysis showed enhanced lipid metabolism, activation of motor proteins, and loss of mitochondrial electron transport proteins as promoters of cancer in wild-type tumors, inhibited in the hSDC1+/+ livers. These complex mechanisms mimic the characteristics of nonalcoholic steatohepatitis (NASH) induced human liver cancer successfully delayed by syndecan-1.

Syndecan-1 Promotes Hepatocyte-Like Differentiation of Hepatoma Cells Targeting Ets-1 and AP-1.

Syndecan-1 is a transmembrane heparan sulfate proteoglycan which is indispensable in the structural and functional integrity of epithelia. Normal hepatocytes display strong cell surface expression of syndecan-1; however, upon malignant transformation, they may lose it from their cell surfaces. In this study, we demonstrate that re-expression of full-length or ectodomain-deleted syndecan-1 in hepatocellular carcinoma cells downregulates phosphorylation of ERK1/2 and p38, with the truncated form exerting an even stronger effect than the full-length protein. Furthermore, overexpression of syndecan-1 in hepatoma cells is associated with a shift of heparan sulfate structure toward a highly sulfated type specific for normal liver. As a result, cell proliferation and proteolytic shedding of syndecan-1 from the cell surface are restrained, which facilitates redifferentiation of hepatoma cells to a more hepatocyte-like phenotype. Our results highlight the importance of syndecan-1 in the formation and maintenance of differentiated epithelial characteristics in hepatocytes partly via the HGF/ERK/Ets-1 signal transduction pathway. Downregulation of Ets-1 expression alone, however, was not sufficient to replicate the phenotype of syndecan-1 overexpressing cells, indicating the need for additional molecular mechanisms. Accordingly, a reporter gene assay revealed the inhibition of Ets-1 as well as AP-1 transcription factor-induced promoter activation, presumably an effect of the heparan sulfate switch.

Decreased Expression of ZNF554 in Gliomas is Associated with the Activation of Tumor Pathways and Shorter Patient Survival.

Zinc finger protein 554 (ZNF554), a member of the Krüppel-associated box domain zinc finger protein subfamily, is predominantly expressed in the brain and placenta in humans. Recently, we unveiled that ZNF554 regulates trophoblast invasion during placentation and its decreased expression leads to the early pathogenesis of preeclampsia. Since ZNF proteins are immensely implicated in the development of several tumors including malignant tumors of the brain, here we explored the pathological role of ZNF554 in gliomas. We examined the expression of ZNF554 at mRNA and protein levels in normal brain and gliomas, and then we searched for genome-wide transcriptomic changes in U87 glioblastoma cells transiently overexpressing ZNF554. Immunohistochemistry of brain tissues in our cohort (n = 62) and analysis of large TCGA RNA-Seq data (n = 687) of control, oligodendroglioma, and astrocytoma tissues both revealed decreased expression of ZNF554 towards higher glioma grades. Furthermore, low ZNF554 expression was associated with shorter survival of grade III and IV astrocytoma patients. Overexpression of ZNF554 in U87 cells resulted in differential expression, mostly downregulation of 899 genes. The "PI3K-Akt signaling pathway", known to be activated during glioma development, was the most impacted among 116 dysregulated pathways. Most affected pathways were cancer-related and/or immune-related. Congruently, cell proliferation was decreased and cell cycle was arrested in ZNF554-transfected glioma cells. These data collectively suggest that ZNF554 is a potential tumor suppressor and its decreased expression may lead to the loss of oncogene suppression, activation of tumor pathways, and shorter survival of patients with malignant glioma.

Proteomic identification of Placental Protein 1 (PP1), PP8, and PP22 and characterization of their placental expression in healthy pregnancies and in preeclampsia.

INTRODUCTION: Placental Protein 1 (PP1), PP8, and PP22 were isolated from the placenta. Herein, we aimed to identify PP1, PP8, and PP22 proteins and their placental and trophoblastic expression patterns to reveal potential involvement in pregnancy complications. METHODS: We analyzed PP1, PP8, and PP22 proteins with LC-MS. We compared the placental behaviors of PP1, PP8, and PP22 to the predominantly placenta-expressed PP5/TFPI-2. Placenta-specificity scores were generated from microarray data. Trophoblasts were isolated from healthy placentas and differentiated; total RNA was isolated and subjected to microarray analysis. We assigned the placentas to the following groups: preterm controls, early-onset preeclampsia, early-onset preeclampsia with HELLP syndrome, term controls, and late-onset preeclampsia. After histopathologic examination, placentas were used for tissue microarray construction, immunostaining with anti-PP1, anti-PP5, anti-PP8, or anti-PP22 antibodies, and immunoscoring. RESULTS: PP1, PP8, and PP22 were identified as 'nicotinate-nucleotide pyrophosphorylase', 'serpin B6', and 'protein disulfide-isomerase', respectively. Genes encoding PP1, PP8, and PP22 are not predominantly placenta-expressed, in contrast with PP5. PP1, PP8, and PP22 mRNA expression levels did not increase during trophoblast differentiation, in contrast with PP5. PP1, PP8, and PP22 immunostaining were detected primarily in trophoblasts, while PP5 expression was restricted to the syncytiotrophoblast. The PP1 immunoscore was higher in late-onset preeclampsia, while the PP5 immunoscore was higher in early-onset preeclampsia. DISCUSSION: PP1, PP8, and PP22 are expressed primarily in trophoblasts but do not have trophoblast-specific regulation or functions. The distinct dysregulation of PP1 and PP5 expression in either late-onset or early-onset preeclampsia reflects different pathophysiological pathways in these preeclampsia subsets.

The Protective Role of Decorin in Hepatic Metastasis of Colorectal Carcinoma.

Decorin, the prototype member of the small leucine-rich proteoglycan gene family of extracellular matrix (ECM) proteins, acts as a powerful tumor suppressor by inducing the p21Waf1/Cip1 cyclin-dependent kinase inhibitor, as well as through its ability to directly bind and block the action of several tyrosine kinase receptors. Our previous studies suggested that the lack of decorin promotes hepatic carcinogenesis in mice. Based on this, we set out to investigate whether excess decorin may protect against the liver metastases of colon carcinoma. We also analyzed the effect of decorin in tissue microarrays of human colon carcinoma liver metastasis and examined whether the tumor cells can directly influence the decorin production of myofibroblasts. In humans, low levels of decorin in the liver facilitated the development of colon carcinoma metastases in proportion with more aggressive phenotypes, indicating a possible antitumor action of the proteoglycan. In vitro, colon carcinoma cells inhibited decorin expression in LX2 hepatic stellate cells. Moreover, liver-targeted decorin delivery in mice effectively attenuated metastasis formation of colon cancer. Overexpressed decorin reduced the activity of multiple receptor tyrosine kinases (RTKs) including the epidermal growth factor receptor (EGFR), an important player in colorectal cancer (CRC) pathogenesis. Downstream of that, we observed weakened signaling of ERK1/2, PLCγ, Akt/mTOR, STAT and c-Jun pathways, while p38 MAPK/MSK/CREB and AMPK were upregulated culminating in enhanced p53 function. In conclusion, decorin may effectively inhibit metastatic tumor formation in the liver.

Two ways of epigenetic silencing of TFPI2 in cervical cancer.

OBJECTIVE: Comparison of human mRNA microarray results from tumor-associated and normal cervical fibroblasts revealed significant TFPI2 downregulation in tumor-associated fibroblasts isolated from cervical cancer, indicating that TFPI2 downregulation may play an important role in the pathogenesis of the disease. In the present work, we investigated the mechanism of TFPI2 downregulation in tumor-associated fibroblasts and tumor cells. METHODS: In vitro models of monocultures and co-cultures were established with tumor cells and fibroblasts to explore the changes of TFPI-2 expression and epigenetic modifications of the TFPI2 gene. RESULTS: The TFPI2 gene was hypermethylated only in tumor cells. Reduction of TFPI-2 protein levels in tumor-associated fibroblasts, although the gene was not methylated, suggested alternative regulatory mechanisms of gene expression, such as inhibition by microRNAs. The expression pattern of miR-23a, a gene thought to inhibit TFPI2 translation, showed changes strongly correlated to detected TFPI-2 protein alterations. Transfections with miR-23a mimics resulted in a decrease of TFPI-2 protein expression whereas miR-23a inhibitors increased the TFPI-2 amount. Due to downregulation of miR-23a expression by HPV in cancer cells, TFPI2 was silenced by promoter methylation. In contrary, miR-23a was active in HPV-free fibroblasts and inactivated TFPI2. CONCLUSION: These results indicate dual epigenetic inhibition of TFPI2 on the transcription level by promoter methylation in cancer cells and on the translation level by miR-23a in tumor-associated fibroblasts. As a consequence, inactivation of the TFPI2 gene plays a strategic role in the progression of cervical cancer.

Aberrant Expression of Syndecan-1 in Cervical Cancers.

Syndecan-1, is a transmembrane heparan/chondroitin sulfate proteoglycan necessary for cell-cell and cell-matrix interactions. Its decreased level on the cell surface correlates with poor prognosis in several tumor types. Aberrant stromal localization of syndecan-1 is also considered an unfavorable prognostic factor in various human malignancies. In the presented work the question was addressed if changes in syndecan-1 expression are related to the prognosis of cervical cancer. Immunohistochemistry for syndecan-1 extracellular domain was performed on surgical specimens of primary cervical cancer. To follow the communication between tumor cells and stromal fibroblasts, their mono-and co-cultures were studied, detecting the expression of syndecan-1, smooth muscle actin, vimentin, and desmin. Immunohistochemistry of tumorous specimens revealed that while cell surface syndecan-1 expression was reduced on cancer cells, it appeared on the surface of tumor-associated fibroblasts. Until year 7, the cohort with high cell surface syndecan-1 expression had significantly longer survival. No difference in the same time-period could be detected when stromal syndecan-1 expression was analyzed. In vitro analysis revealed, that tumor cells can induce syndecan-1 expression on fibroblast, and fibroblasts showed that fibroblast-like cells are built by two cell types: (a) syndecan-1 positive, cytokeratin negative real fibroblasts, and (b) syndecan-1 and cytokeratin positive epithelial-mesenchymal transformed tumor cells. Syndecan-1 on the surface of cancer cells appears to be a positive prognostic marker. Although syndecan-1 positive fibroblasts promote tumor cell proliferation in vitro, we failed to detect their cancer promoting effect in vivo.

Demethylation Status of Somatic DNA Extracted From Pituitary Neuroendocrine Tumors Indicates Proliferative Behavior.

BACKGROUND: Cytosine intermediaries 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), epigenetic hallmarks, have never been investigated in pituitary neuroendocrine tumors (PitNET). OBJECTIVE: To examine methylation-demethylation status of global deoxyribonucleic acid (DNA) in PitNET tissues and to assess its correlation with clinical and biological parameters. MATERIALS AND METHODS: Altogether, 57 PitNET and 25 corresponding plasma samples were collected. 5mC and 5hmC were investigated using liquid chromatography-tandem mass spectrometry. Expression of DNA methyltransferase 1 (DNMT1); tet methylcytosine dioxygenase 1 through 3 (TET1-3); and ubiquitin-like, containing PHD and RING finger domains 1 and 2 (UHRF1-2) were measured by reverse transcription-polymerase chain reaction. Levels of 5hmC and UHRF1-2 were explored by immunohistochemistry. Effect of demethylating agent decitabine was tested on pituitary cell lines. RESULTS: 5hmC/5mC ratio was higher in less differentiated PitNET samples. A negative correlation between Ki-67 proliferation index and 5hmC, 5hmC to 5mC ratio were revealed. Higher 5mC was observed in SF-1 + gonadotroph adenomas with a higher Ki-67 index. Expressions of TET2 and TET3 were significantly higher in adenomas with higher proliferation rate. UHRF1 showed gradually increased expression in higher proliferative adenoma samples, and a significant positive correlation was detected between UHRF2 expression and 5hmC level. Decitabine treatment significantly decreased 5mC and increased 5hmC levels in both cell lines, accompanied with decreased cell viability and proliferation. CONCLUSION: The demethylation process negatively correlated with proliferation rate and the ratio of 5hmC to 5mC was higher in less differentiated adenomas. Therefore, epigenetic markers can be potential biomarkers for PitNET behavior. Altering the epigenome in adenoma cells by decitabine decreased proliferation, suggesting that this treatment might be a novel medical treatment for PitNET.

Syndecan-1 in Liver Diseases.

Liver diseases such as liver cirrhosis, primary and metastatic liver cancers are still a major medical challenge. Syndecan-1 is one of the most important proteoglycans in the liver. Syndecan-1 is normally expressed on the surfaces of hepatocytes and cholangiocytes. Due to liver diseases the amount of syndecan-1 increases in the liver. Despite the emerging data of the biological function of syndecan-1, the clinical usefulness of this proteoglycan is still unknown. In our study we correlated syndecan-1 expression to clinico-pathological data. We found that syndecan-1 proved to be a good marker for hepatitis C virus based hepatocellular carcinoma and increased with liver dysfunction.

Proteomic identification of membrane-associated placental protein 4 (MP4) as perlecan and characterization of its placental expression in normal and pathologic pregnancies.

BACKGROUND: More than 50 human placental proteins were isolated and physico-chemically characterized in the 70-80s by Hans Bohn and co-workers. Many of these proteins turned to have important role in placental functions and diagnostic significance in pregnancy complications. Among these proteins was membrane-associated placental protein 4 (MP4), for which identity or function has not been identified yet. Our aim was to analyze the sequence and placental expression of this protein in normal and complicated pregnancies including miscarriage, preeclampsia and HELLP syndrome. METHODS: Lyophilized MP4 protein and frozen healthy placental tissue were analyzed using HPLC-MS/MS. Placental tissue samples were obtained from women with elective termination of pregnancy (first trimester controls, n = 31), early pregnancy loss (EPL) (n = 13), early preeclampsia without HELLP syndrome (n = 7) and with HELLP syndrome (n = 8), late preeclampsia (n = 8), third trimester early controls (n = 5) and third trimester late controls (n = 9). Tissue microarrays were constructed from paraffin-embedded placentas (n = 81). Slides were immunostained with monoclonal perlecan antibody and evaluated using light microscopy and virtual microscopy. Perlecan was also analyzed for its expression in placentas from normal pregnancies using microarray data. RESULTS: Mass spectrometry-based proteomics of MP4 resulted in the identification of basement membrane-specific heparan sulfate proteoglycan core protein also known as perlecan. Immunohistochemistry showed cytoplasmic perlecan localization in syncytiotrophoblast and cytotrophoblasts of the villi. Perlecan immunoscore decreased with gestational age in the placenta. Perlecan immunoscores were higher in EPL compared to controls. Perlecan immunoscores were higher in early preeclampsia without and with HELLP syndrome and lower in late preeclampsia than in respective controls. Among patients with preeclampsia, placental perlecan expression positively correlated with maternal vascular malperfusion and negatively correlated with placental weight. CONCLUSION: Our findings suggest that an increased placental perlecan expression may be associated with hypoxic ischaemic injury of the placenta in miscarriages and in early preeclampsia with or without HELLP syndrome.

MRE11 as a Predictive Biomarker of Outcome After Radiation Therapy in Bladder Cancer.

PURPOSE: Organ-confined muscle-invasive bladder cancer is treated with cystectomy or bladder preservation techniques, including radiation therapy. There are currently no biomarkers to inform management decisions and aid patient choice. Previously we showed high levels of MRE11 protein, assessed by immunohistochemistry (IHC), predicted outcome after radiation therapy, but not cystectomy. Therefore, we sought to develop the MRE11 IHC assay for clinical use and define its relationship to clinical outcome in samples from 2 major clinical trials. METHODS AND MATERIALS: Samples from the BCON and BC2001 randomized controlled trials and a cystectomy cohort were stained using automated IHC methods and scored for MRE11 in 3 centers in the United Kingdom. RESULTS: Despite step-wise creation of scoring cards and standard operating procedures for staining and interpretation, there was poor intercenter scoring agreement (kappa, 0.32; 95% confidence interval, 0.17-0.47). No significant associations between MRE11 scores and cause-specific survival were identified in BCON (n = 132) and BC2001 (n = 221) samples. Reoptimized staining improved agreement between scores from BCON tissue microarrays (n = 116), but MRE11 expression was not prognostic for cause-specific survival. CONCLUSIONS: Manual IHC scoring of MRE11 was not validated as a reproducible biomarker of radiation-based bladder preservation success. There is a need for automated quantitative methods or a reassessment of how DNA-damage response relates to clinical outcomes.

Increased placental expression of Placental Protein 5 (PP5) / Tissue Factor Pathway Inhibitor-2 (TFPI-2) in women with preeclampsia and HELLP syndrome: Relevance to impaired trophoblast invasion?

INTRODUCTION: Placental Protein 5 (PP5)/Tissue Factor Pathway Inhibitor-2 (TFPI-2) is an extracellular matrix-associated protein mainly expressed by the syncytiotrophoblast that may regulate trophoblast invasion. Our aim was to study placental PP5/TFPI-2 expression and its relation to placental pathology in various forms of preeclampsia and HELLP syndrome. METHODS: Placental and maternal blood specimens were collected at the time of delivery from the same women in the following groups: 1) early controls; 2) early preeclampsia; 3) early preeclampsia with HELLP syndrome; 4) late controls; and 5) late preeclampsia. After histopathological examination, placental specimens were immunostained with polyclonal anti-PP5/TFPI-2 antibody on Western blot and tissue microarray immunohistochemistry. Placental PP5/TFPI-2 immunoscores were assessed manually and with a semi-automated method. Maternal sera were immunoassayed for PP5/TFPI-2. RESULTS: PP5/TFPI-2 was localized to the cytoplasm of syncytiotrophoblast. Manual and semi-automated PP5/TFPI-2 immunoscores were higher in early preeclampsia with or without HELLP syndrome but not in late preeclampsia than in respective controls. In patients with preeclampsia, the correlation of placental PP5/TFPI-2 expression with maternal vascular malperfusion score of the placenta was positive while it was negative with birthweight and placental weight. Maternal serum PP5/TFPI-2 concentration was higher in early preeclampsia and it tended to be higher in early preeclampsia with HELLP syndrome than in early controls. DISCUSSION: Our findings suggest that an increased placental PP5/TFPI-2 expression may be associated with abnormal placentation in early preeclampsia, with or without HELLP syndrome.

The Presence of ALK Alterations and Clinical Relevance of Crizotinib Treatment in Pediatric Solid Tumors.

Soft tissue sarcomas (STS) and neuroblastomas (NBL), are childhood malignancies still associated with poor prognoses despite the overall improvement in childhood tumor survival of the past decades. Anaplastic lymphoma kinase (ALK) inhibition is promising new strategy to improve the outcome of these pediatric tumors. Eighteen histologic samples of pediatric STS and 19 NBL patients were analyzed for ALK abnormalities using fluorescent in situ hybridization (FISH) with break-apart probes and immunohistochemistry (IHC). ALK alterations were presented in 20 of the 37 sections. The presence of ALK alteration in NBL samples were detected using IHC in 84,2% of all cases compared to 21,1% FISH positivity. In STS cases the results were less different (IHC 16,7% vs FISH 22,2%). The difference can be explained by the different type of molecular alterations. FISH method detected translocation and amplification, but not the point mutation of ALK gene. IHC confirmed the diagnosis by detecting the expression of ALK protein.After ALK positivity was proven, the effectiveness and safety of the crizotinib therapy was examined in 4 patients (1 alveolar rhabdomyosarcoma (RMA), 1 embryonal rhabdomyosarcoma (RME), 1 inflammatory myofibroblastic tumor (IMT), 1 NBL). We observed continuous remission of the IMT patient, all other cases the inhibitor treatment was not curative.Our findings underline the importance of screening the ALK status parallel with both IHC and FISH. Crizotinib treatment had a long-term effect in ALK positive IMT patients, however itwas only temporary efficient in relapsed, progressive STS and NBL.

Survivin as a potential therapeutic target of acetylsalicylic acid in pituitary adenomas.

Acetylsalicylic acid (ASA) is known as a cancer preventing agent, but there is no data available regarding the effect of ASA on pituitary cells. We investigated 66 nonfunctioning (NFPA) and growth hormone (GH)-producing adenomas and 15 normal pituitary samples. Functional assays (cell viability, proliferation, flow cytometry cell cycle analysis, caspase-3 activation and DNA degradation) were applied to explore the effect of ASA, YM155 (survivin inhibitor), survivin-targeting siRNA and TNF-related apoptosis-inducing ligand (TRAIL) in RC-4B/C and GH3 cells. Pituitary adenoma xenografts were generated in immunocompromised mice. We found that survivin was overexpressed and TRAIL was downregulated in NFPAs compared to normal pituitary tissue. ASA decreased proliferation but did not induce apoptosis in pituitary cells. Additionally, ASA treatment decreased cells in S phase and increased cells in G2/M phase of the cell cycle. Inhibition of survivin using an inhibitor or siRNA-mediated silencing reversed the ASA-induced growth inhibition partially. In addition, we also found survivin-independent effects of ASA on the cell cycle that were mediated through inhibition of cyclin A, cyclin dependent kinase 2 (CDK2) and phospho-CDK2. We also aimed to test the effect of acetylsalicylic acid in an animal model using RC-4 B/C cells, but in contrast to GH3 cells, RC-4 B/C cells failed to adhere and grow a xenograft. We concluded that ASA inhibited the growth of pituitary adenoma cells. Survivin inhibition is a key mechanism explaining its antineoplastic effects. Our results suggest that inhibition of survivin with small molecules or ASA could serve as potential therapeutic agents in NFPA.

Sequencing of prostate cancers identifies new cancer genes, routes of progression and drug targets.

Prostate cancer represents a substantial clinical challenge because it is difficult to predict outcome and advanced disease is often fatal. We sequenced the whole genomes of 112 primary and metastatic prostate cancer samples. From joint analysis of these cancers with those from previous studies (930 cancers in total), we found evidence for 22 previously unidentified putative driver genes harboring coding mutations, as well as evidence for NEAT1 and FOXA1 acting as drivers through noncoding mutations. Through the temporal dissection of aberrations, we identified driver mutations specifically associated with steps in the progression of prostate cancer, establishing, for example, loss of CHD1 and BRCA2 as early events in cancer development of ETS fusion-negative cancers. Computational chemogenomic (canSAR) analysis of prostate cancer mutations identified 11 targets of approved drugs, 7 targets of investigational drugs, and 62 targets of compounds that may be active and should be considered candidates for future clinical trials.

Appraising the relevance of DNA copy number loss and gain in prostate cancer using whole genome DNA sequence data.

A variety of models have been proposed to explain regions of recurrent somatic copy number alteration (SCNA) in human cancer. Our study employs Whole Genome DNA Sequence (WGS) data from tumor samples (n = 103) to comprehensively assess the role of the Knudson two hit genetic model in SCNA generation in prostate cancer. 64 recurrent regions of loss and gain were detected, of which 28 were novel, including regions of loss with more than 15% frequency at Chr4p15.2-p15.1 (15.53%), Chr6q27 (16.50%) and Chr18q12.3 (17.48%). Comprehensive mutation screens of genes, lincRNA encoding sequences, control regions and conserved domains within SCNAs demonstrated that a two-hit genetic model was supported in only a minor proportion of recurrent SCNA losses examined (15/40). We found that recurrent breakpoints and regions of inversion often occur within Knudson model SCNAs, leading to the identification of ZNF292 as a target gene for the deletion at 6q14.3-q15 and NKX3.1 as a two-hit target at 8p21.3-p21.2. The importance of alterations of lincRNA sequences was illustrated by the identification of a novel mutational hotspot at the KCCAT42, FENDRR, CAT1886 and STCAT2 loci at the 16q23.1-q24.3 loss. Our data confirm that the burden of SCNAs is predictive of biochemical recurrence, define nine individual regions that are associated with relapse, and highlight the possible importance of ion channel and G-protein coupled-receptor (GPCR) pathways in cancer development. We concluded that a two-hit genetic model accounts for about one third of SCNA indicating that mechanisms, such haploinsufficiency and epigenetic inactivation, account for the remaining SCNA losses.

Cell cycle dependent RRM2 may serve as proliferation marker and pharmaceutical target in adrenocortical cancer.

Adrenocortical cancer (ACC) is a rare, but agressive malignancy with poor prognosis. Histopathological diagnosis is challenging and pharmacological options for treatment are limited. By the comparative reanalysis of the transcriptional malignancy signature with the cell cycle dependent transcriptional program of ACC, we aimed to identify novel biomarkers which may be used in the histopathological diagnosis and for the prediction of therapeutical response of ACC. Comparative reanalysis of publicly available microarray datasets included three earlier studies comparing transcriptional differences between ACC and benign adrenocortical adenoma (ACA) and one study presenting the cell cycle dependent gene expressional program of human ACC cell line NCI-H295R. Immunohistochemical analysis was performed on ACC samples. In vitro effects of antineoplastic drugs including gemcitabine, mitotane and 9-cis-retinoic acid alone and in combination were tested in the NCI-H295R adrenocortical cell line. Upon the comparative reanalysis, ribonucleotide reductase subunit 2 (RRM2), responsible for the ribonucleotide dezoxyribonucleotide conversion during the S phase of the cell cycle has been validated as cell cycle dependently expressed. Moreover, its expression was associated with the malignancy signature, as well. Immunohistochemical analysis of RRM2 revealed a strong correlation with Ki67 index in ACC. Among the antiproliferative effects of the investigated compounds, gemcitabine showed a strong inhibition of proliferation and an increase of apoptotic events. Additionally, RRM2 has been upregulated upon gemcitabine treatment. Upon our results, RRM2 might be used as a proliferation marker in ACC. RRM2 upregulation upon gemcitabine treatment might contribute to an emerging chemoresistance against gemcitabine, which is in line with its limited therapeutical efficacy in ACC, and which should be overcome for successful clinical applications.