Supplementary Data from Mining Tissue Microarray Data to Uncover Combinations of Biomarker Expression Patterns that Improve Intermediate Staging and Grading of Clear Cell Renal Cell Cancer

The identification of cell surface accessible biomarkers enabling diagnosis, disease monitoring, and treatment of renal cell carcinoma (RCC) is as challenging as the biology and progression of RCC is unpredictable.A hallmark of most RCC is the loss-of-function of the von Hippel-Lindau (pVHL) protein by mutation of its gene (VHL).Using the cell surface capturing (CSC) technology, we screened and identified cell surface N-glycoproteins in pVHL-negative and positive 786-O cells.One hundred six cell surface N-glycoproteins were identified.Stable isotope labeling with amino acids in cell culture-based quantification of the CSC screen revealed 23 N-glycoproteins whose abundance seemed to change in a pVHL-dependent manner.Targeted validation experiments using transcriptional profiling of primary RCC samples revealed that nine glycoproteins, including CD10 and AXL, could be directly linked to pVHL-mediated transcriptional regulation.Subsequent human tumor tissue analysis of these cell surface candidate markers showed a correlation between epithelial AXL expression and aggressive tumor phenotype, indicating that pVHL-dependent regulation of glycoproteins may influence the biologic behavior of RCC.Functional characterization of the metalloprotease CD10 in cell invasion assays demonstrated a diminished penetrating behavior of pVHL-negative 786-O cells on treatment with the CD10-specific inhibitor thiorphan.Our proteomic surfaceome screening approach in combination with transcriptional profiling and functional validation suggests pVHL-dependent cell surface glycoproteins as potential diagnostic markers for therapeutic targeting and RCC patient monitoring.

BACKGROUND:Renal cell carcinoma (RCC) is characterized by a number of diverse molecular aberrations that differ among individuals. Recent approaches to molecularly classify RCC were based on clinical, pathological as well as on single molecular parameters. As a consequence, gene expression patterns reflecting the sum of genetic aberrations in individual tumors may not have been recognized. In an attempt to uncover such molecular features in RCC, we used a novel, unbiased and integrative approach.METHODS:We integrated gene expression data from 97 primary RCC of different pathologic parameters, 15 RCC metastases as well as 34 cancer cell lines for two-way nonsupervised hierarchical clustering using gene groups suggested by the PANTHER Classification System. We depicted the genomic landscape of the resulted tumor groups by means of Single Nuclear Polymorphism (SNP) technology. Finally, the achieved results were immunohistochemically analyzed using a tissue microarray (TMA) composed of 254 RCC.RESULTS:We found robust, genome wide expression signatures, which split RCC into three distinct molecular subgroups. These groups remained stable even if randomly selected gene sets were clustered. Notably, the pattern obtained from RCC cell lines was clearly distinguishable from that of primary tumors. SNP array analysis demonstrated differing frequencies of chromosomal copy number alterations among RCC subgroups. TMA analysis with group-specific markers showed a prognostic significance of the different groups.CONCLUSION:We propose the existence of characteristic and histologically independent genome-wide expression outputs in RCC with potential biological and clinical relevance.

The extracellular matrix N-glycoprotein periostin is thought to enhance tumor invasion. In this study, the expression patterns of periostin and its splice isoforms were investigated in renal cell carcinoma (RCC). Periostin mRNA expression patterns were characterized in 30 fresh-frozen RCCs in normal fetal and adult renal tissues by both isoform-specific and nonspecific RT-PCR and by gene expression array analysis. Its protein expression was analyzed by immunohistochemistry, using tissue microarrays. with tissue from 1007 RCC patients. Periostin mRNA in RCC was increased, as observed in both RT-PCR and gene microarray analyses, with significantly higher expression in the clear cell than in the papillary subtype. Four of eight periostin isoforms, identified in fetal kidney by direct sequencing, have not been described to date. Three isoforms could be detected in both RCC and matched non-neoplastic tissue, and one of them was expressed more frequently in RCC. Periostin protein was detected in both mesenchymal cells of the tumor stroma and epithelial tumor cells. Greater amounts of periostin in tumor epithelia correlated with the presence of sarcomatoid differentiation, higher tumor stage, lymph node metastases, and poor overall survival in the clear cell subtype. In conclusion, periostin expression in tumor epithelia may contribute to sarcomatoid differentiation and more aggressive behavior of RCC. The presence of a tumor-associated periostin isoform suggests splice-specific regulation in RCC tissue. (Am J Pathol 2011, 179:1513-1521; DOI: 10.1016/j.ajpath.2011.05.035)

Abstract Purpose: Tumor stage and nuclear grade are the most important prognostic parameters of clear cell renal cell carcinoma (ccRCC). The progression risk of ccRCC remains difficult to predict particularly for tumors with organ-confined stage and intermediate differentiation grade. Elucidating molecular pathways deregulated in ccRCC may point to novel prognostic parameters that facilitate planning of therapeutic approaches. Experimental Design: Using tissue microarrays, expression patterns of 15 different proteins were evaluated in over 800 ccRCC patients to analyze pathways reported to be physiologically controlled by the tumor suppressors von Hippel-Lindau protein and phosphatase and tensin homologue (PTEN). Tumor staging and grading were improved by performing variable selection using Cox regression and a recursive bootstrap elimination scheme. Results: Patients with pT2 and pT3 tumors that were p27 and CAIX positive had a better outcome than those with all remaining marker combinations. A prolonged survival among patients with intermediate grade (grade 2) correlated with both nuclear p27 and cytoplasmic PTEN expression, as well as with inactive, nonphosphorylated ribosomal protein S6. By applying graphical log-linear modeling for over 700 ccRCC for which the molecular parameters were available, only a weak conditional dependence existed between the expression of p27, PTEN, CAIX, and p-S6, suggesting that the dysregulation of several independent pathways are crucial for tumor progression. Conclusions: The use of recursive bootstrap elimination, as well as graphical log-linear modeling for comprehensive tissue microarray (TMA) data analysis allows the unraveling of complex molecular contexts and may improve predictive evaluations for patients with advanced renal cancer. Clin Cancer Res; 16(1); 88–98

Metal nanoparticles have distinctly different chemical and physical properties than currently investigated oxides. Since pure metallic nanoparticles are igniting at air, carbon stabilized copper nanoparticles were used as representative material for this class. Using copper as a representative example, we compare the cytotoxicity of copper metal nanoparticles stabilized by a carbon layer to copper oxide nanoparticles using two different cell lines. Keeping the copper exposure dose constant, the two forms of copper showed a distinctly different response. Whilst copper oxide had already been reported to be highly cytotoxic, carbon-coated copper nanoparticles were much less cytotoxic and more tolerated. Measuring the two material's intra- and extracellular solubility in model buffers explained this difference on the basis of altered copper release when supplying copper metal or the corresponding oxide particles to the cells. Control experiments using pure carbon nanoparticles were used to exclude significant surface effects. Reference experiments with ionic copper solutions confirmed a similar response of cultures if exposed to copper oxide nanoparticles or ionic copper. These observations are in line with a Trojan horse-type mechanism and illustrate the dominating influence of physico-chemical parameters on the cytotoxicity of a given metal.

Abstract Purpose: The paired box gene 2, PAX2, encodes for a transcription factor that is up-regulated during nephrogenesis and becomes silenced in mature epithelium of the glomeruli, the proximal, and distal tubules. Reactivation of PAX2 has been frequently observed in clear cell renal cell carcinoma (ccRCC), a tumor type characterized by loss of von Hippel-Lindau (VHL) tumor suppressor function. The regulation of PAX2 expression in ccRCC is unknown. Experimental Design: We applied reporter gene assays to investigate PAX2 promoter regulation. Furthermore, PAX2 expression was determined in ccRCC cell lines under normoxic and hypoxic condition in a VHL wild-type and mutated background. PAX2 expression was also assessed in 831 human ccRCC and correlated with hypoxia-inducible factor α (HIFα) and clinical parameters. Results: Here, we show that both loss of VHL protein (pVHL) function and hypoxia leads to strong PAX2 reexpression. Using luciferase reporter gene assays, no induction was obtained in spite of six hypoxia response element motifs identified in the promoter of PAX2. Comprehensive immunohistochemical analyses showed significant correlations between PAX2, HIF1α, and HIF2α—target CCND1 expression patterns in ccRCC patients. Notably, PAX2 expression was highly associated with early-stage, well-differentiated ccRCC and, consequently, better clinical outcome (P < 0.0001 each). Additional analyses indicated that PAX2 repressor WT1 and cancer-linked hypomethylation are not important for transcriptional regulation of PAX2 in ccRCC. Conclusion: We conclude that in ccRCC, PAX2 reactivation is driven by HIF-dependent mechanisms following pVHL loss.

Many genes that are required at specific points in the cell cycle exhibit cell cycle-dependent expression. In the early-diverging model eukaryote and important human pathogen Trypanosoma brucei, regulation of gene expression in the cell cycle and other processes is almost entirely post-transcriptional. Here, we show that the T. brucei RNA-binding protein PUF9 stabilizes certain transcripts during S-phase. Target transcripts of PUF9--LIGKA, PNT1 and PNT2--were identified by affinity purification with TAP-tagged PUF9. RNAi against PUF9 caused an accumulation of cells in G2/M phase and unexpectedly destabilized the PUF9 target mRNAs, despite the fact that most known Puf-domain proteins promote degradation of their target mRNAs. The levels of the PUF9-regulated transcripts were cell cycle dependent, peaking in mid- to late- S-phase, and this effect was abolished when PUF9 was targeted by RNAi. The sequence UUGUACC was over-represented in the 3' UTRs of PUF9 targets; a point mutation in this motif abolished PUF9-dependent stabilization of a reporter transcript carrying the PNT1 3' UTR. LIGKA is involved in replication of the kinetoplast, and here we show that PNT1 is also kinetoplast-associated and its over-expression causes kinetoplast-related defects, while PNT2 is localized to the nucleus in G1 phase and redistributes to the mitotic spindle during mitosis. PUF9 targets may constitute a post-transcriptional regulon, encoding proteins involved in temporally coordinated replicative processes in early G2 phase.

The onconeuronal cerebellar degeneration-related antigen Cdr2 is associated with paraneoplastic syndromes. Neoplastic expression of Cdr2 in ovary and breast tumors triggers an autoimmune response that suppresses tumor growth by developing tumor immunity, but culminates in cerebellar degeneration when Cdr2-specific immune cells recognize neuronal Cdr2. We identified Cdr2 as a novel interactor of the hypoxia-inducible factor (HIF) prolyl-4-hydroxylase PHD1 and provide evidence that Cdr2 might represent a novel important tumor antigen in renal cancer. Strong Cdr2 protein expression was observed in 54.2% of papillary renal cell carcinoma (pRCC) compared with 7.8% of clear-cell RCC and no staining was observed in chromophobe RCC or oncocytoma. High Cdr2 protein levels correlated with attenuated HIF target gene expression in these solid tumors, and Cdr2 overexpression in tumor cell lines reduced HIF-dependent transcriptional regulation. This effect was because of both attenuation of hypoxic protein accumulation and suppression of the transactivation activity of HIF-1α. pRCC is known for its tendency to avascularity, usually associated with a lower pathological stage and higher survival rates. We provide evidence that Cdr2 protein strongly accumulates in pRCC, attenuates the HIF response to tumor hypoxia and may become of diagnostic importance as novel renal tumor marker.

Background Despite recent progress in the identification of genetic and molecular alterations in prostate cancer, markers associated with tumor progression are scarce. Therefore precise diagnosis of patients and prognosis of the disease remain difficult. This study investigated novel molecular markers discriminating between low and highly aggressive types of prostate cancer. Results Using 52 microdissected cell populations of low- and high-risk prostate tumors, we identified via global cDNA microarrays analysis almost 1200 genes being differentially expressed among these groups. These genes were analyzed by statistical, pathway and gene enrichment methods. Twenty selected candidate genes were verified by quantitative real time PCR and immunohistochemistry. In concordance with the mRNA levels, two genes MAP3K5 and PDIA3 exposed differential protein expression. Functional characterization of PDIA3 revealed a pro-apoptotic role of this gene in PC3 prostate cancer cells. Conclusions Our analyses provide deeper insights into the molecular changes occurring during prostate cancer progression. The genes MAP3K5 and PDIA3 are associated with malignant stages of prostate cancer and therefore provide novel potential biomarkers.

Periostin (POSTN), also termed osteoblast-specific factor 2, is a secreted stromal protein of 93 kDa. Six different splice isoforms exist and have been reported, but only four of them have been sequenced. Periostin shares homology with the insect cell adhesion molecule fasciclin I and with human beta IgH3 and is induced by TGFbeta and BMP-2. Periostin promotes integrin-dependent cell adhesion and motility and supports osteoblastic cell lines attachment and spreading. It is expressed in few normal tissues (cardiac valves, placenta, embryonic periosteum and periodontal ligament) and is overexpressed in many cancerous tissues (lung, kidney, breast, ovarian, colon, prostate, head and neck cancer). It has been shown that high POSTN expression levels in serum correlate with tumor aggressivenes. Periostin also promotes epithelial mesenchymal transition of carcinoma cells which leads to invasion and metastasis. Therefore it is considered as a predictive marker and a potential target for tumor therapy. In our previous study periostin expression was analysed in a tissue microarray of 538 non-small cell lung cancers. We established that POSTN overexpression is associated with increasing TNM stage and grade. This study aims at the analysis of expression and localization of periostin isoforms in lung and kidney cancer and at their functional characterization. We performed: isoform-specific PCR; quantitative RT-PCR and immunoblot analysis on frozen tissues of 30 non-small cell lung cancer patients (adenocarcinoma and squamous cell carcinoma) and on frozen tissues of 30 kidney cancer patients (clear cell carcinoma and papillary carcinoma) and their matched non-neoplastic tissues. Furthermore we cloned and sequenced the region of periostin mRNA that undergoes alternative splicing (from exon 17 to exon 21), giving rise to different isoforms. Our current study identified four periostin isoforms in the lung and three isoforms in the kidney. Each of the isoforms was coexpressed in both tumor and matched non-neoplastic tissue. The cloning analysis of renal periostin isoforms revealed a new isoform which has to date not been reported in other human organs. The western blot analysis of lung and kidney cancer tissue and pleural effusions from lung cancer patients showed the presence of a cleaved form of periostin of 55 kDa. These results suggest potential novel tissue-specific functions of periostin isoforms in renal cell and lung carcinoma and open up the possibility of organ-specific targeted cancer therapy. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4968.

In eukaryotes, proteins containing RNA Recognition Motifs (RRMs) are involved in many different RNA processing reactions, RNA transport, and mRNA decay. Kinetoplastids rely extensively on post-transcriptional mechanisms to control gene expression, so RRM domain proteins are expected to play a prominent role. We here describe the results of an RNA interference screen targeting 37 of the 72 RRM-domain proteins of Trypanosoma brucei. RNAi targeting 8 of the genes caused clear growth inhibition in bloodstream trypanosomes, and milder effects were seen for 9 more genes. The small, single-RRM protein TbRBP3 specifically associated with 10 mRNAs in trypanosome lysates, but RBP3 depletion did not affect the transcriptome.

AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA 2775 The VHL tumor suppressor gene is somatically inactivated in the majority of clear cell Renal Cell Carcinoma (ccRCC). It encodes two protein isoforms pVHL30 and pVHL19. Both are known to play a crucial role in the proteasomal degradation of Hypoxia Inducible Factor α (HIFα). Deregulation of cell membrane proteins e.g. CAIX and CXCR4 as a result of the loss of pVHL followed by accumulation of HIFα is a hallmark in ccRCC progression. Due to their subcellular localization and release into body fluids cell surface proteins represent potential candidates in search of reliable molecular markers in ccRCC. Our study therefore aimed at the identification of novel VHL-regulated cellular membrane proteins in ccRCC. By applying LC-MS/MS qualitative and quantitative expression profiling of cellular membrane proteins was performed for VHL null 786-O and two VHL re-expressing stable transfectants 786-O-pVHL19 and 786-O-pVHL30. Using Taqman Low Density Arrays and Affymetrix gene chips, expression patterns of the identified proteins were further validated in different RCC and normal renal epithelial cell lines as well as human RCC specimen. These experiments identified CD10 as a novel pVHL-regulated gene on both the protein and RNA level. In silico sequence analysis of the CD10 promoter revealed two potential HIF-binding sites. Functional studies with transfectants expressing naturally occurring type 2B-mutated pVHL as well as hypoxia experiments with RCC cells expressing wildtype pVHL revealed a correlated expression of CD10, HIF2α and HIF target genes (GLUT-1, CAIX). Tissue Microarrays (TMA) were used to analyze the expression of CD10 in 831 human ccRCC specimen. These analyses revealed a positive correlation between CD10, GLUT-1 and CAIX. In summary, the application of proteomics and transcriptomics allowed the identification of the known ccRCC marker CD10 as a yet unknown VHL-/HIF-regulated cell surface protein in renal cancer.

ABSTRACT In the African trypanosome Trypanosoma brucei nearly all control of gene expression is posttranscriptional; sequences in the 3′-untranslated regions of mRNAs determine the steady-state mRNA levels by regulation of RNA turnover. Here we investigate the roles of two related proteins, Tb UBP1 and Tb UBP2, containing a single RNA recognition motif, in trypanosome gene expression. Tb UBP1 and Tb UBP2 are in the cytoplasm and nucleus, comprise ca. 0.1% of the total protein, and are not associated with polysomes or RNA degradation enzymes. Overexpression of Tb UBP2 upregulated the levels of several mRNAs potentially involved in cell division, including the CFB1 mRNA, which encodes a protein with a cyclin F-box domain. CFB1 regulation was mediated by the 3′-untranslated region and involved stabilization of the mRNA. Depletion of Tb UBP2 and Tb UBP1 inhibited growth and downregulated expression of the cyclin F box protein gene CFB2 ; trans splicing was unaffected. The results of pull-down assays indicated that all tested mRNAs were bound to Tb UBP2 or Tb UBP1, with some preference for CFB1 . We suggest that Tb UBP1 and Tb UBP2 may be relatively nonspecific RNA-binding proteins and that specific effects of overexpression or depletion could depend on competition between various different proteins for RNA binding.

Isoenzymes of phosphoglycerate kinase in Trypanosoma brucei are differentially expressed in its two main life stages. This study addresses how the organism manages to make sufficient amounts of the isoenzyme with the correct localization, which processes (transcription, splicing, and RNA degradation) control the levels of mRNAs, and how the organism regulates the switch in isoform expression. For this, we combined new quantitative measurements of phosphoglycerate kinase mRNA abundance, RNA precursor stability, trans splicing, and ribosome loading with published data and made a kinetic computer model. For the analysis of regulation we extended regulation analysis. Although phosphoglycerate kinase mRNAs are present at surprisingly low concentrations (e. g. 12 molecules per cell), its protein is highly abundant. Substantial control of mRNA and protein levels was exerted by both mRNA synthesis and degradation, whereas splicing and precursor degradation had little control on mRNA and protein concentrations. Yet regulation of mRNA levels does not occur by transcription, but by adjusting mRNA degradation. The contribution of splicing to regulation is negligible, as for all cases where splicing is faster than RNA precursor degradation.

The genomes of Trypanosoma brucei, Leishmania major and Trypanosoma cruzi each encode 10 proteins with PUF domains. PUF domain proteins from yeast and metazoa have been shown to bind RNA and to regulate mRNA stability and translation. Phylogenetic analysis suggested that the PUF proteins were duplicated and diverged early in evolution, and that most PUF proteins were lost during the evolution of mammals. Depletion of any of the first nine T. brucei PUF protein mRNAs by RNA interference had no effect on cell growth; combined depletion of PUF1 and PUF3, PUF3 and PUF4, and PUF1 and PUF4 mRNAs also had no effect. In conflict with a previous report, procyclic trypanosomes lacking PUF1 genes grew normally and we could find no evidence that PUF1 is required for growth of trypanosomes in culture. Depletion or elimination of PUF1 mRNA did not affect the abundances of any other mRNAs, as detected in microarray analysis, and also had minimal effects on the proteome. (In control experiments, treatment of bloodstream and procyclic cells with 100 ng/ml tetracycline also had no detectable effects on the transcriptome and proteome.) PUF1 preferentially bound to retroposon RNAs and was not associated with polysomes. We suggest that, as in yeast, there may be functional redundancy among the Kinetoplastid PUF proteins, or they may be involved in fine-tuning gene expression together with other proteins. Alternatively, PUF proteins may be needed in differentiating trypanosomes or in non-culturable life-cycle stages.

We describe developmentally regulated genes in two strains of Trypanosoma brucei: the monomorphic strain Lister 427 and the pleomorphic strain TREU927. Expression patterns were obtained using an array of 24,567 genomic fragments. Probes were prepared from bloodstream-form or procyclic-form trypanosomes. Fourteen procyclic-specific and 77 bloodstream-specific signals were obtained from sequences matching variant surface glycoprotein or associated genes, and a further 17 regulated sequences were repetitive or transposable-element-related. Two hundred and eighty-six regulated spots corresponded to mRNAs from other protein-coding genes; these spots represent 191 different proteins. Regulation of 113 different genes (79 from procyclic forms, 34 from bloodstream-forms) was supported by at least two independent experiments or criteria; of these, about 60 were novel. Only two genes -- encoding HSP83 and an importin-related protein -- appeared to be regulated in the TREU927 strain only. Our results confirmed previous estimates that 2% of trypanosome genes show developmental regulation at the mRNA level.