Signaling and Transcriptional Changes Critical for Transformation of Human Cells by Simian Virus 40 Small Tumor Antigen Or Protein Phosphatase 2A B56␥ Knockdown

[CANCER RESEARCH 64, 6978–6988, October 1, 2004] Signaling and Transcriptional Changes Critical for Transformation of Human Cells by Simian Virus 40 Small Tumor Antigen or Protein Phosphatase 2A B56␥ Knockdown

Carlos S. Moreno,1,2 Sumathi Ramachandran,1,2 Danita G. Ashby,2,3 Noelani Laycock,1,2 Courtney A. Plattner,2,3 Wen Chen,4 William C. Hahn,4,5 and David C. Pallas2,3 1Department of Pathology and Laboratory Medicine, Emory University School of Medicine, 2Winship Cancer Institute, and 3Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia; 4Department of Medical Oncology, Dana-Farber Cancer Institute, Department of Medicine, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts; and 5Broad Institute, Cambridge, Massachusetts

ABSTRACT LXCXE motif in the central region of the protein that enables it to bind and inactivate members of the retinoblastoma family of tumor One set of genes sufficient for transformation of primary human cells suppressor proteins (4), helping to release cells from G arrest. The uses the combination of Ha-Ras-V12, the telomerase catalytic subunit 1 NH -terminal DnaJ domain is also necessary to functionally inactivate hTERT, SV40 large tumor antigen (LT), and SV40 small tumor antigen 2 (ST). Whereas SV40 LT inactivates the retinoblastoma protein and p53, retinoblastoma family tumor suppressors (5). In addition, the COOH- the contribution of ST is poorly understood. The essential helper function terminal domain of LT binds and inactivates the p53 tumor suppres- of ST requires a functional interaction with protein phosphatase 2A sor (6). (PP2A). Here we have identified changes in gene expression induced by ST The unique COOH-terminal half of ST encodes a domain that and show that ST mediates these changes through both PP2A-dependent enables binding to protein phosphatase 2A (PP2A), a heterotrimeric and PP2A-independent mechanisms. Knockdown of PP2A B56␥ subunit serine-threonine phosphatase that plays important roles in numerous can substitute for ST expression to fully transform cells expressing LT, cellular processes including apoptosis, cell cycle regulation, and sig- hTERT, and Ras-V12. We also identify those genes affected similarly in nal transduction (7, 8). ST provides critical helper functions to LT for two cell lines that have been fully transformed from a common parental transformation of both mouse (9) and human cells (10). In addition, line by two alternative mechanisms, namely ST expression or PP2A B56␥ ST binding to PP2A is essential for ST to provide its helper function subunit knockdown. ST altered expression of genes involved in proliferation, apoptosis, integrin signaling, development, immune responses, and for transformation of human cells (11–13). transcriptional regulation. ST reduced surface expression of MHC class I Efforts to delineate a defined set of genetic changes essential for molecules, consistent with a need for SV40 to evade immune detection. ST transformation of primary human cells has demonstrated that one expression enabled cell cycle progression in reduced serum and src phos- combination of genes sufficient to produce anchorage-independent phorylation in anchorage-independent media, whereas B56␥ knockdown growth in soft agar and tumors in nude mice includes SV40 LT and required normal serum levels for these phenotypes. Inhibitors of integrin ST, the catalytic subunit of human telomerase (hTERT), and the and src signaling prevented anchorage-independent growth of trans- constitutively activated V12 mutant of H-Ras (H-Ras-V12; refs. 11, formed cells, suggesting that integrin and src activation are key ST- 12, 14). A COOH-terminal deletion mutant, ST110, that encodes only mediated events in transformation. Our data support a model in which ST the first 110 residues of ST, including the DnaJ domain, cannot bind promotes survival through constitutive integrin signaling, src phosphoryl- to PP2A and cannot provide the essential helper function needed to ation, and nuclear factor ␬B activation, while inhibiting cell-cell adhesion pathways. transform human cells (12). To investigate the ST-induced changes in gene expression that are essential for human tumor formation, we used whole genome expres- INTRODUCTION sion profiling to compare the expression patterns of four human cell The study of DNA tumor viruses and the oncogenes that they lines. Each of these cell lines stably expresses hTERT, H-Ras-V12, encode has provided many critical insights into the essential steps for and LT (12). In addition to these three stably expressed genes, the oncogenesis. SV40 is a potently oncogenic DNA virus that can cause tumorigenic HEK-TERST cell line expresses wild-type SV40 ST, tumors in rodents (1) and possibly humans (2). The two oncogenic whereas the nontumorigenic HEK-TERST110 and HEK-TERV cell proteins responsible for the transforming activity of SV40 are known lines express the ST110 mutant or vector alone, respectively (12). as the large tumor antigen (LT) and small tumor antigen (ST). The Introduction of an antisense construct to the B56␥3 subunit of PP2A early region of the SV40 genome encodes the 82 kDa LT and the 20 into the HEK-TER cell line (HEK-TERASB56␥) nearly completely kDa ST as two alternatively spliced protein products that share a suppresses B56␥ expression at the protein level, permits anchorage- common NH2-terminal sequence of 82 amino acids with similarity to independent growth, and enables tumor formation in nude mice in a the DnaJ family of molecular chaperones (3). LT also contains an manner similar to cells expressing ST (15). Here, we show that introduction of ST or suppression of PP2A B56␥ subunits impacts Received 3/31/04; revised 6/9/04; accepted 7/23/04. expression of a small subset of genes involved in apoptosis, integrin Grant support: NIH Grant K22 CA96560 (C. S. Moreno), NIH Grant CA57327 signaling, transcriptional regulation, and cytoskeletal control. These (D. C. Pallas), NIH Grants K01 CA94223 and P01 CA50661, a Doris Duke Clinical gene expression and signaling changes may promote growth and Scientist Development Award, a Dunkin Donuts Rising Stars Award, and a Kimmel Scholar Award (W. C. Hahn). oppose apoptotic signals that prevent growth of normal cells in soft The costs of publication of this article were defrayed in part by the payment of page agar, enabling anchorage-independent growth and tumor formation. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Note: C. S. Moreno and S. Ramachandran contributed equally to this work. Supple- mentary data for this article can be found at http://morenolab.whitehead.emory.edu/pubs/ MATERIALS AND METHODS ST/. Raw microarray data can be accessed at Array Express, accession number E-MEXP- 156. Cell Lines, Culture, and Lysates. Stable human embryonic kidney (HEK) Requests for reprints: Carlos Moreno, Pathology and Laboratory Medicine, White- cell lines HEK-TERST, HEK-TERST110, and HEK-TERV have been de- head Room 105J, 615 Michael Street, Emory University, Atlanta, GA 30322. E-mail: ␥ [email protected], [email protected]. scribed previously (12). The HEK-TERASB56 cell line is described else- ©2004 American Association for Cancer Research. where (15). Cells were grown in ␣-MEM, 10% fetal bovine serum (FBS), 2 6978

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. TRANSFORMATION TARGETS OF SV40 ST AND B56␥ mmol/L L-glutamine, and 100 units/ml penicillin/streptomycin and serum- ␮mol/L PP1 (21) or PP3 inhibitor (22), or 1 ␮mol/L wortmannin. The c-src- starved in ␣-MEM, 0.1% FBS, 2 mmol/L L-glutamine, and 100 units/ml specific kinase inhibitor PP1 [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyra- penicillin/streptomycin for 24 hours. zolo(3,4-D)pyrimidine] was obtained from Biomol and the inactive structural Western Blots and Antibodies. Cell lysates were prepared as described analogue PP3 [4-amino-7-phenylpyrazol(3,4-D)pyrimidine] was purchased previously (16) or with the additional step of sonication before centrifugation from CalBiochem (San Diego, CA). Cell viability was determined using to generate a whole cell lysate. Antibodies to SV40 LT (monoclonal antibody 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye staining as 419), ST, H-Ras-V12, SERPINB2/PAI-2, plakoglobin (all from Santa Cruz described (23). Biotechnology Biotech, Santa Cruz, CA), IQGAP-2 (Upstate Biotechnology, Methylcellulose Assays. Methylcellulose assays were performed as de- Lake Placid, NY), thymidine kinase (QED Bioscience, San Diego, CA), cyclin scribed (24). Briefly, methylcellulose culture medium (final concentration of A (BD Transduction Laboratories, Lexington, KY), BNIP3, cyclin B (Onco- 1.3% w/v) was made by diluting autoclaved 2.6% methylcellulose (Sigma- gene Research, San Diego, CA), Src-phospho-Y418 (Biosource Intl, Ca- Aldrich, St. Louis, MO) in water with an equal volume of 2ϫ concentrated marillo, CA), survivin, and inhibitor of NF␬B(I␬B␣, Cell Signaling Technol- MEM followed by stirring overnight at 4°C. Essentially, 3 ϫ 106 cells were ogy, Beverly, MA) were used in immunoblots as described previously (16). incubated in 10 mL of methylcellulose culture medium in a flask at 37°C. After The src 327 monoclonal antibody was a generous gift from Joan Brugge (Ariad incubation for 24 hours, the cells were recovered by solubilizing the methyl- Pharmaceuticals, Inc., Cambridge, MA). cellulose medium with 5 volumes of chilled PBS followed by gently mixing Fluorescent-Activated Cell Sorting Analysis. For analysis of MHC class and centrifugation at 1,500 rpm for 5 minutes. I expression, 106 cells were harvested in PBS and 0.02% EDTA, incubated in 500 ␮L of PBS and 10 ␮g/ml W6/32 mouse monoclonal antibody (a generous gift of Dr. Charles A. Parkos, Emory University), washed, and then stained RESULTS with antimouse FITC-conjugated secondary antibody. For DNA content analysis, cells were incubated in ␣-MEM and 0.1% FBS for 24 hours and harvested SV40 Small Tumor Antigen Alters the Expression of 452 Genes. immediately or collected after 24 hours in 10% FBS. For cell synchronization, We used a whole genomic profiling approach of ST-expressing 0.5 ϫ 106 cells were treated with 10 ␮mol/L aphidicolin in 10% FBS for 24 (HEK-TERST), ST110 mutant-expressing (HEK-TERST110), PP2A hours and then harvested immediately or released into ␣-MEM and 0.1% FBS B56␥ knockdown (HEK-TERASB56␥), and vector control (HEK- without aphidicolin for 24 hours. Cells were fixed in 70% EtOH at Ϫ20°C TERV) cell lines to investigate the transcriptional changes in trans- overnight, stained with 10 ␮g/ml propidium iodide, and sorted on a FACS- formation of human cells. Each of these cell lines is actually a pool of calibur sorter (Becton-Dickinson, Franklin Lakes, NJ). hundreds of individual clones derived from retroviral infection of a Quantitative Real-Time PCR. Quantitative real-time PCR (QRT-PCR) HEK cell line that stably expresses hTERT, H-Ras-V12, and LT was performed in an I-cycler (Bio-Rad, Hercules, CA) using SYBR Green (HEK-TER; ref. 12). We reasoned that high levels of serum might (Molecular Probes, Eugene, OR). The critical cycle threshold was determined for each gene and the difference relative to the critical cycle threshold for cause some of the same changes in gene expression as ST expression ␥ glyceraldehyde-3-phosphate dehydrogenase or ⌬ cycle threshold was com- or B56 down-regulation. Therefore, all of the cell lines were grown puted for each RNA sample. Two independent RNA samples from each cell in low serum for 24 hours to prevent serum effects from masking the line were analyzed in quadruplicate, and the mean and SD were computed. impact of ST expression or B56␥ down-regulation on global gene Primers were designed using Primer Express software to amplify across splice expression patterns. Total RNA was prepared from two independent junctions. The sequences of the primer sets used are given in Supplementary biological replicates and analyzed using the Affymetrix U133 Human Table S7. Genome GeneChip Array Set. To compensate for multiple testing Microarrays and Data Analysis. Total RNA was prepared using the issues, we have used the Significance Analysis of Microarrays soft- RNeasy kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s ware (25), which computes false discovery rates. Significance Anal- instructions. RNA was reverse transcribed, and labeled probes were frag- ysis of Microarrays resulted in a total of 555 probe sets corresponding mented and hybridized to the Human Genome U133 Chip Set (Affymetrix Inc., Santa Clara, CA) according to the manufacturer’s protocols. Analysis of four to 452 unique genes that exhibited at least 1.5 fold-change between cell lines in duplicate on both the U133A and U133B GeneChips resulted in 16 HEK-TERST and HEK-TERV cell lines with a predicted false dis- microarray hybridizations that generated eight combined U133AB whole ge- covery rate of 3% (q Ͻ 0.03; see Materials and Methods and Sup- nome datasets (ST-1, ST-2, ST110–1, ST110–2, B56–1, B56–2, TERV-1, and plementary Table S1). Although a 1.5-fold change in mRNA levels TERV-2). Scanned images were analyzed, and all 12 of the possible compar- may not be biologically significant for some genes, microarrays often ison files against the TERV-1 and TERV-2 datasets were generated using underestimate the actual fold change when compared with QRT-PCR Microarray Suite 5.0 software. Each chip was normalized with a target value and Northern blots (26), and Significance Analysis of Microarrays of 150. Genes called absent in all of the hybridizations and genes that were produces fewer false positives than fold change criteria (25). More- called no change in more than one ST-TERV Affymetrix comparison file were over, a cutoff of 1.5-fold has been used in several studies (27, 28), and filtered out leaving 2,545 probes for Significance Analysis of Microarrays the data seen at the 1.5-fold level included genes such as cyclin B, analysis. Data from Affymetrix CEL files was then normalized using the robust multiarray average method (17). After data normalization, Significance Anal- which we confirmed at the protein level were induced much more than ysis of Microarrays analysis was performed on the remaining 2,545 probe sets 1.5-fold. using the following relevant parameters: ⌬ϭ0.26, fold-change ϭ 1.5, number We analyzed the GO consortium annotations of the genes that were permutations ϭ 1000, random number generation seed ϭ 1234567, median significantly affected by ST using the GOstat software (18), which false discovery rate ϭ 3%, significant genes ϭ 555, and predicted false finds GO terms that are statistically over-represented in a gene list positives ϭ 17. Hierarchical clustering with average linkage based on Euclid- compared with the rest of the genome. GOstat analysis of the genes ian distance was performed with Spotfire Decision Site 7.0 software. Anno- affected by ST expression found five major categories of genes that 6 tations were obtained from the NetAffx website and the April 2003 assembly were significantly over-represented relative to all of the annotated 7 of the Human Genome at University of California Santa Cruz. P values for gene human genes using the Hochberg and Benjamini correction for false ontology (GO) categories were computed using GOstat (18) and corrected to discovery rate (Supplementary Table S2; Fig. 1, A and B). Of the 452 false discovery rate using the method of Hochberg and Benjamini (19). Soft Agar Assays. Soft agar assays were performed essentially as de- unique genes, only 274 had GO annotations (Fig. 1A). Of the 274 scribed (14). Briefly, 10,000 cells were seeded in 0.3% Noble Agar and either annotated genes, 109 genes (or 40%) had GO annotations relating to 10 ␮g/ml RGD or RAD peptide (Biomol, Plymouth Meeting, PA; ref. 20), 10 cellular proliferation (Fig. 1B) compared with 4,622 of 19,085 total genes (or 24%) in these categories for the entire genome (Supplemen- ϭ ϫ Ϫ7 6 Internet address: http://www.affymetrix.com/analysis/index.affx. tary Table S2; P 2.55 10 ). The other major functional 7 Internet address: http://genome.ucsc.edu. categories significantly over-represented in the genes affected by ST 6979

Fig. 1. SV40 ST alters expression in genes involved in proliferation, development, transcriptional regulation, and inflammation. A, summary of number of GO annotations and annotated genes used for GOstat analysis. B, GO categories that were significantly over-represented in the 452 genes affected by ST expression as determined by GOstat analysis included genes involved in proliferation, inflammation, antigen presentation, development, and transcription. C, FACS analysis of HEK-TERV and HEK-TERST cells using the pan-HLA monoclonal antibody W6/32 shows decreased surface expression of MHC class I molecules in the HEK-TERST cells as a shift in the peaks to the left relative to the HEK-TERV cells. A representative result from three independent experiments is shown. D, FACS analysis of HEK-TERV and HEK-TERST110 cells as in C. E, FACS analysis of HEK-TERV and HEK-TERASB56␥ cells as in C. expression included development and morphogenesis CD24 were induced by ST, suggesting that the changed expression (P ϭ 4.8 ϫ 10Ϫ5), inflammation (P ϭ 3.5 ϫ 10Ϫ4), regulation of of these developmental transcription factors results in downstream transcription (P ϭ 6.2 ϫ 10Ϫ4), and antigen presentation differences between HEK-TERV and HEK-TERST cells. Whereas (P ϭ 2.6 ϫ 10Ϫ3). changes in mRNA levels alone do not necessarily translate into Although some of the 452 genes affected by ST such as os- phenotypic alterations, the coordinated alterations of so many teopontin (29) and thymidine kinase (30, 31) agree with earlier genes involved in similar processes, the alterations in cell cycle studies, the vast majority of these ST targets have not been re- progression reported here, and previous literature all support the ported previously (Supplementary Table S1). ST appears to affect hypothesis that ST-induced changes in mRNA expression likely proliferation, possibly through changes in developmental programs affect these cellular functions. that are controlled by sequence-specific transcription factors. Our ST may also repress immune responses and impair antigen presen- data show for the first time that ST induces expression of the tation by repressing expression of MHC class I molecules HLA-A, developmental HOX genes HOXA9, HOXB6, and HOXB3; the HLA-B, HLA-C, and ␤2-microglobulin; the CD74 invariant chain forkhead family factors FOXD1, FOXM1, and FOXG1b; and the necessary for folding of MHC class II heterodimers; and proinflam- cancer-associated developmental transcription factors c-MYC, matory cytokines such as interleukin (IL)-8, IL-1␤, consistent with the ETS1, ID2, and SMAD3. Our observation that ST induces c-myc need for SV40 to avoid immune detection. Fluorescence-activated cell mRNA complements recent findings that PP2A inhibition stabi- sorter (FACS) analysis of MHC class I expression in three independ- lizes c-myc at the protein level, contributing to transformation ent experiments demonstrated that surface expression of HLA mole- (32). Moreover, several early developmental markers from multi- cules was repressed in the HEK-TERST, HEK-TERST110, and HEK- ple tissue types such as endothelial cell-specific molecule 1, TERASB56␥ cell lines compared with the HEK-TERV line (Fig. 1, epithelial membrane protein 1, stathmin-like 3, osteopontin, and C–E). Whereas MHC class I expression was reduced in all three of the 6980

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. TRANSFORMATION TARGETS OF SV40 ST AND B56␥ lines, the repression was most effective in the cells that express wild-type ST. SV40 Small Tumor Antigen Alters the Expression of 171 Genes in a PP2A-Independent Manner and 281 Genes in a PP2A- Dependent Manner. Expression data from the HEK-TERST110 and HEK-TERASB56␥ cell lines was also compared with the HEK-TERV data, and signal-log ratios for all of the cell lines were then analyzed. Comparison of the HEK-TERST110 line with the HEK-TERV line identified 411 genes that were altered in expression levels (Supple- mentary Table S3). PP2A-independent genes were defined as the intersection of the 452 genes altered by ST expression and the set of 411 genes affected by ST110 expression that changed in the same direction relative to vector control cells. The intersection of these two gene sets resulted in a set of 171 genes that are represented by the magenta and cyan sections of the Venn diagram in Fig. 2A. The expression pattern of all 452 ST-regulated genes was analyzed by hierarchical clustering (Fig. 2B), and the expression pattern of the 171 PP2A-independent genes is shown in Fig. 2C. Strikingly, 37% of ST-regulated genes (Supplementary Table S4) can be regulated by the

NH2-terminal domain of SV40 ST independently of PP2A modulation. Because these changes are induced by ST in cells that already express LT, these data suggest that the NH2-terminal half of ST may have effects that LT, which also contains a DnaJ domain, does not. Given that the transforming helper function of ST is known to be PP2A dependent, genes that are affected by ST in a PP2A-dependent manner are of particular interest. The 281 genes affected by ST that were not similarly affected by the ST110 mutant represent this PP2A- dependent gene set (red and orange sections of Fig. 2A). Fig. 2B shows that many of the genes affected by ST are unaffected by down-regulation of the PP2A B56␥ subunit in the HEK-TERASB56␥ cell line or are even affected in the opposite direction. Thus, the subset of genes similarly affected by B56␥ antisense and ST expression (Fig. 2D) are PP2A-dependent via two different molecular mechanisms that transform human cells. The fact that knockdown of PP2A B56␥ subunits produced changes in 843 genes (green section of Fig. 2A) that are not affected by ST or ST110 is not surprising, given the many cellular functions of PP2A. It is difficult to know what mechanism would affect the 161 genes represented in blue in Fig. 2A that were affected by ST110 alone. One could speculate that the truncated NH2-terminal portion of ST could gain new functions that are not necessarily biologically relevant. More Fig. 2. Hierarchical clustering of gene subsets affected by SV40 ST expression. A, Venn diagram of gene sets analyzed. The entire set of 452 ST-regulated genes is likely, the effects of the COOH-terminal portion of ST on PP2A represented by the large red circle. Numbers indicate the number of unique genes in each activity could counteract or attenuate effects that are mediated by the subset. PP2A-independent genes, shown in magenta and cyan, were similar in ST110 and NH -terminal portion of ST. The 79 genes affected by both ST110 and ST. Strongly PP2A-dependent genes, shown in orange, were similar in ASB56␥ and ST 2 but not in ST110. Genes shown in cyan were affected similarly in ST, ST110, and B56␥ knockdown (but not by wild-type ST) are interesting (Supple- ASB56␥. B, hierarchical clustering of 555 probe sets affected at least 1.5-fold by mentary Table S5), because several of them are altered in human wild-type ST expression. Signal log ratios for all 12 possible comparisons of ST/TERV ␥ cancers (AMACR, TWIST, TGF␤R2, and MMP-10) or are involved in (HEK-TERST versus HEK-TERV cells), B56/TERV (HEK-TERASB56 versus HEK- TERV cells), and ST110/TERV (HEK-TERST110 versus HEK-TERV cells) were clus- the wnt signaling pathway (SFRP1, GSK3␤, and Frizzled). Two genes tered by gene and by sample. As an example, the dataset label of ST-1/TERV-1 corre- (SFRP1 and tropomysin 1) were repressed by ST110 and B56␥ sponds to a comparison of HEK-ST (repeat 1) versus HEK-TERV (repeat 1). Increased and decreased expression levels are indicated by red and green intensities, respectively. C, knockdown but induced by ST, whereas 1 gene (stem cell growth hierarchical clustering of 205 probe sets corresponding to 171 genes significantly affected factor) was induced by ST110 and B56␥ knockdown but repressed by by wild-type ST expression in a PP2A-independent manner. This gene set shows similar ST. expression effects for wild-type ST and the ST110-mutant. D, hierarchical clustering of 128 probe sets corresponding to 99 genes with similar expression patterns in fully Western Blotting and Real-Time PCR Data Confirm the Mi- transformed HEK-TERST and HEK-TERASB56␥ cells. croarray Data. To confirm our microarray observations, we prepared total RNA from independently treated samples and performed QRT-PCR on 29 PP2A-dependent genes and 12 PP2A-independent ratios, and in 22 of 25 (88%) of the genes in the ASB56␥ versus genes and compared expression changes relative to glyceraldehyde- TERV ratios. Thus, the QRT-PCR data confirmed our microarray 3-phosphate dehydrogenase. The magnitude of the changes observed observations in 96 of 107 comparisons, or 90% of the time. Among in the QRT-PCR assays varied somewhat from that observed in the the numerous novel ST targets that we confirmed by QRT-PCR are microarray experiments (Fig. 3, A–D and G–I). Nevertheless, the two thrombin protease-activated receptors, F2R and F2R2, which are directionality of the QRT-PCR changes confirmed the microarray expressed in prostate cancers and are implicated in motility, metasta- results for 40 of 41 (98%) of the genes assayed in the ST versus TERV sis, angiogenesis, and src kinase activation (33–35). ratios, in 34 of 41 (83%) of the genes in the ST110 versus TERV To confirm changes at the protein level, we first immunoblotted for 6981

Fig. 3. QRT-PCR and immunoblotting data confirm microarray observations. Expression ratios from QRT-PCR and microarray data are shown for 41 sample genes compared with mRNA levels in HEK-TERV cells. The average fold change for four independent samples are shown; bars, ϮSE. A. Expression ratios are shown for four genes up-regulated by ST in a PP2A-independent manner. B. Expression ratios are shown for 8 genes down-regulated by ST in a PP2A-independent manner. C. Expression ratios are shown for 10 genes up-regulated by ST in a PP2A -dpendent manner. D. Expression ratios are shown for 6 genes down-regulated by ST in a PP2A-dependent manner. E, immunoblot verification of microarray data at the protein level for plakoglobin, IQGAP2, and thymidine kinase. PP2A C subunit is shown as a loading control. IQGAP2 was difficult to detect, indicating that it is expressed at a very low level even in the HEK-TERV cells. Blotting of IQGAP2 immunoprecipitates from these cell lines (data not shown) confirmed the reduction in IQGAP2 protein seen in this figure. F. I␬B␣ levels are decreased in the tumorigenic HEK-TERST and HEK-TERASB56␥ cell lines, but not in HEK-TERST110 cells. Quantitation of three independent experiments (data not shown) by chemilumimager demonstrated that I␬B␣ levels were reduced to 55% and 28% of HEK-TERV levels in the HEK-TERST and HEK-TERASB56␥ cells, respectively, whereas I␬B␣ in the HEK-TERST110 cells was unchanged. Immunoblotting also confirms of expression changes at the protein level for BNIP3 and the NF␬B targets SERPINB2/PAI-2 and survivin. G. Expression ratios are shown in HEK-TERST, HEK-TERST110, and HEK-TERASB56␥ cell lines for 10 genes regulated by ST in a PP2A-independent manner. H. Expression ratios are shown in HEK-TERST, HEK-TERST110, and HEK-TERASB56␥ cell lines for 8 genes up-regulated by ST in a PP2A-dependent manner. I. Expression ratios are shown in HEK-TERST, HEK-TERST110, and HEK-TERASB56␥ cell lines for 7 genes down-regulated by ST in a PP2A-dependent manner. 6982

3 genes representative of different expression patterns. In agreement set of genes was similarly affected by the ST110 mutant and by with mRNA data, junction plakoglobin (JUP) decreased in a PP2A- knockdown of B56␥ suggests that these genes can be regulated by dependent manner, IQGAP2 decreased in a PP2A-independent man- both PP2A-dependent and PP2A-independent mechanisms. Of these ner, and thymidine kinase 1 (TK-1) increased in HEK-TERST110 99 newly described targets that are similarly affected by ST expres- cells and even more so in the HEK-TERST cells (Fig. 3E). We also sion and B56␥ knockdown, 62 genes (orange section of Fig. 2A) were confirmed changes at the protein level for BNIP3 and SERPINB2 differentially affected in the HEK-TERST110 cell line (orange bar, (Fig. 3F). Nevertheless, one of the limitations of microarray analyses Fig. 2D), suggesting that they are strongly dependent on ST-PP2A is that changes at the mRNA level are not always reflected at the interactions (Table 1). Among these genes were the transcription protein level, and, thus, it is not possible to be certain that the mRNA factors FOXD1 and HOXB3; the apoptosis-related genes, gelsolin, changes reported here are all reflected at the protein level. Protein ALDH1A3, and SERPINB2; and cell-cell adhesion molecule protocad- levels of LT and H-Ras-V12 were also measured in the HEK-TERST, herin ␥A11. HEK-TERST110, HEK-TERASB56␥, and HEK-TERV by immuno- It has been shown previously that ST activates nuclear factor ␬B blotting, quantitated using a chemiluminescence imager as described (NF␬B) via protein kinase C ␨ and phosphatidylinositol 3Ј-kinase (36), and determined to be within 2-fold for each of the matched lines (PI3K) signaling (37). Our observation that the antiapoptotic NF␬B (data not shown). targets ALDH1 and SERPINB2 were up-regulated in HEK-TERST Expression Profiling of PP2A B56␥ Knockdown Cells Identifies and HEK-TERASB56␥ cells (Table 1) prompted us to test the steady- 99 Potentially Critical Genes Correlated with Tumorigenicity. state protein levels of the inhibitor of NF␬B(I␬B␣). Quantitation of Antisense knockdown of PP2A B56␥ subunits has been shown pre- three independent immunoblots (Fig. 3F) by chemilumimager dem- viously to nearly completely suppress B56␥ expression at the protein onstrated that I␬B␣ levels are reduced to 55% and 28% of HEK- level (15). However, our comparison of microarray expression data in TERV levels in the HEK-TERST and HEK-TERASB56␥ cells, re- the HEK-TERASB56␥ and HEK-TERV cells showed only a 1.5-fold spectively, whereas I␬B␣ in the HEK-TERST110 cells was decrease of B56␥ at the mRNA level, suggesting that much of the loss unchanged. In addition, the NF␬B targets SERPINB2 and survivin are of B56␥ protein may be due to translational inhibition. Because increased at the protein level in HEK-TERST and HEK-TERASB56␥ down-regulation of the PP2A B56␥ subunit can substitute for ST (15), cells compared with the HEK-TERV controls (Fig. 3F). Activation of we reasoned that the set of genes similarly affected by ST expression NF␬B might be expected to be due to downstream effects of enhanced and B56␥ knockdown would include some of the ST targets that are integrin and src signaling (see below), because NF␬B mediates en- ␣ the most relevant to tumorigenesis. The intersection of the genes dothelial cell survival signals from the integrin v␤3–src pathway (38) affected by B56␥ knockdown with those genes affected by ST (shown or via inhibition of PP2A dephosphorylation of the I␬B kinase (39) or in orange and cyan in Fig. 2A) was 128 probe sets corresponding to 99 a combination of mechanisms. Thus, expression of ST and knock- unique genes (Supplementary Table S6 and Fig. 2D). Among these 99 down of B56␥ PP2A subunits both appear to result in PP2A-depend- genes were 37 genes (cyan section of Fig. 2A) that were similarly ent decreased I␬B␣ protein and subsequent NF␬B activation. Never- affected in the HEK-TERASB56␥, HEK-TERST, and HEK- theless, the increase in SERPINB2 and survivin expression in the TERST110 cell lines. These 37 genes included matrix metalloprotein- HEK-TERST110 line suggests that some antiapoptotic NF␬B targets ase MMP-1, the apoptosis-related genes TRAIL, MFGE8, and BNIP3, can also be activated by ST by PP2A-independent mechanisms. and the microfilament-associated protein palladin. The fact that this ST repressed expression of the proinflammatory NF␬B targets IL-8

Table 1 PP2A-dependent genes with similar profiles in tumorigenic HEK-TERST and HEK-TERASB56␥ lines that are clustered in Fig. 2D. Accession Symbol Fold change Accession Symbol Fold change NM_002575 SERPINB2 3.6 BE856336 C8orf13 1.6 NM_002276 KRT19 2.5 NM_032883 C20orf100 1.6 NM_032873 KIAA1959 2.3 AV724183 FLJ31362 1.6 NM_017580 TRABID 2.2 NM_001673 ASNS 1.6 H28999 FLJ36748 2.2 NM_152329 PPIL5 1.6 NM_007173 SPUVE 2.2 NM_022731 NUCKS 1.6 AI653107 ESTs 2.1 NM_020390 EIF5A2 1.6 NM_139072 DNER 2.1 NM_018482 DDEF1 1.6 AI143879 FLJ25677 2.0 NM_153689 FLJ38973 1.6 BG165011 ESTs 2.0 NM_018312 C11orf23 1.6 NM_000693 ALDH1A3 1.9 NM_015934 NOP5/NOP58 1.6 NM_007203 AKAP2 1.9 NM_005238 ETS1 1.5 NM_152322 FLJ33957 1.9 NM_002588 PCDHGC3 Ϫ1.5 NM_007107 SSR3 1.9 U20489 ESTs Ϫ1.5 AW664964 ESTs 1.8 NM_001560 IL13RA1 Ϫ1.6 NM_032333 MGC4248 1.8 NM_001946 DUSP6 Ϫ1.6 NM_006717 SPIN 1.8 NM_000393 COL5A2 Ϫ1.6 NM_002128 HMGB1 1.8 NM_018912 PCDHGA1 Ϫ1.6 NM_006572 GNA13 1.8 NM_018914 PCDHGA11 Ϫ1.6 NM_015440 DKFZP586G1517 1.8 NM_004995 MMP14 Ϫ1.6 AU144882 FLJ13545 1.8 NM_018916 PCDHGA3 Ϫ1.7 NM_004472 FOXD1 1.7 NM_001814 CTSC Ϫ1.7 NM_001034 RRM2 1.7 NM_006033 LIPG Ϫ1.7 NM_006559 KHDRBS1 1.7 NM_005610 RBBP4 Ϫ1.7 NM_005996 TBX3 1.7 NM_001219 CALU Ϫ1.7 NM_017829 CECR5 1.7 NM_004342 CALD1 Ϫ1.7 AW575374 FLJ22425 1.7 NM_000935 PLOD2 Ϫ1.9 NM_007368 GAP1IP4BP 1.7 U40053 ESTs Ϫ2.0 NM_052865 C20orf72 1.7 NM_006227 PLTP Ϫ2.0 NM_152330 C14orf31 1.7 NM_001901 CTGF Ϫ2.4 AK023585 FLJ13523 1.6 NM_004105 EFEMP1 Ϫ2.9 NOTE. Fold change values are for HEK-TERST relative to HEK-TERV. 6983

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. TRANSFORMATION TARGETS OF SV40 ST AND B56␥ and IL-1␤, whereas it enhanced expression of the antiapoptotic targets of NF␬B, ALDH1, survivin, and SERPINB2. QRT-PCR and Western blotting confirmed microarray observations for 22 common targets of ST and B56␥ including SERPINB2, TRABID, DNER, ALDH1, SMAD3, gelsolin, MMP1, FOXD1, CTGF, PRSS11, and ICAM1 (Fig. 3, F–I). Normally, NF␬B activation results in activation of both proinflammatory and antiapoptotic target genes. Consistent with this typical response, the knockdown of PP2A B56␥ subunits in HEK-TER- ASB56␥ cells increased expression of both proinflammatory (IL-8 and IL-1␤) and antiapoptotic targets of NF␬B (ALDH1, survivin, and SERPINB2). However, ST effects on expression of NF␬B targets were unusual in that proinflammatory and antiapoptotic targets are affected in opposite directions. These differences were due to the general repression by STs of immune system genes such as MHC class I genes, invariant chain (CD74), and tumor necrosis factor- related apoptosis-inducing ligand among others (Supplementary Table S1). Small Tumor Antigen Affects Expression of Cell Cycle Genes and Cell Cycle Progression in Low Serum. As expected, ST induced a general pattern of increased expression of genes associated with cell cycle progression and decreased expression of genes associated with cell cycle arrest. In agreement with published studies (30, 31) in which coexpression of SV40 LT and ST drives cells into S phase, thymidine kinase (TK-1) and dihydrofolate reductase (DHFR) were up-regulated by ST. Notably, some S-phase and cell cycle regulated genes, such as TK-1 (Fig. 3, A and C), were also up- regulated by ST110. Immunoblotting for cyclin A showed high expression in the HEK-TERV cells, demonstrating that LT and H-Ras- V12 can activate the cyclin A promoter in the absence of ST (Fig. 4A). Cyclin A levels were decreased in HEK-TERST and HEK-TERST110 cells compared with HEK-TERV cells at both the protein and mRNA levels (Fig. 4A; Supplementary Table S1). This may be because protein and mRNA were prepared after 24 hours in low serum, causing an arrest of HEK-TERV cells at the G1-S-phase transition, whereas HEK-TERST and HEK-TERST110 cells progressed into

G2-M. Consistent with this hypothesis, the HEK-TERST and HEK- TERST110 cells have elevated cyclin B levels, demonstrating the ability of these lines to progress through the cell cycle under conditions of low serum. The HEK-TERASB56␥ cells showed intermediate changes, with a partial decrease in cyclin A levels and partial increase in cyclin B levels, suggestive of fewer cells progressing through the cell cycle. Moreover, TK-1 and DHFR mRNA were not increased in HEK-TERASB56␥ cells (Supplementary Table S1), indicating that unlike ST or ST110 expression, the reduction of B56␥ is not sufficient to enable cell cycle progression in low serum even in the presence of activated Ras. To ascertain the cell cycle distribution of these cell lines after 24 hours in low serum, DNA content was measured by FACS analysis (Fig. 4B). HEK-TERST and HEK-TERST110 cells exhibited highly similar cell cycle distributions, with Ͼ35% in S phase and little sign of cell cycle arrest. However, the 4N peak composed of cells in G2-M dropped dramatically in HEK-TERASB56␥ cells, indicative of a Fig. 4. ST and ST110-expressing cells progress through S phase, whereas B56␥ strong dependence on serum. To additionally investigate the serum knockdown and vector control cell lines are serum-dependent. A. Immunoblots show decreased cyclin A levels and increased cyclin B levels in HEK-TERST and HEK- dependence of the four cell lines, all of the lines were synchronized in TERST110 cells. B. Cell cycle distribution of each cell line is shown as determined by the G1 phase by aphidicolin treatment in 10% FBS for 24 hours and propidium iodide staining and FACS. Peaks corresponding to cells with 2N DNA content then either harvested or released into low serum conditions without in G1 phase or 4N DNA content in G2-M are indicated on the X axis in arbitrary units. Sub-2N peaks presumably correspond to subpopulations of aneuploid cells in lines that aphidicolin. The HEK-TERST and HEK-TERST110 cells progressed express LT but not ST. Shown are cell populations corresponding to asynchronous cells through the cell cycle in a serum-independent manner, whereas most in 10% FBS (black) or to cell populations after 24 hours in low serum conditions (gray). of the HEK-TERV and HEK-TERASB56␥ cells remained arrested in C, similar FACS analysis to that shown in B except that all lines were synchronized in the G1 phase by 24 hours of aphidicolin treatment in 10% FBS and then immediately G1 in a serum-dependent manner (Fig. 4C). Thus, ST and ST110 can harvested (black) or released into low serum conditions without aphidicolin for an drive HEK cells through the cell cycle in the presence of LT and additional 24 hours (gray). activated Ras, whereas knockdown of B56␥ subunits cannot. 6984

Small Tumor Antigen-Induced Gene Expression Patterns Sug- dependence of HEK-ASB56␥ cells on serum for src phosphorylation gest Increased Integrin Signaling and Reduced Cell-Cell Adhe- is consistent with the observation that B56␥ knockdown does not sion. A large number of genes involved in cellular adhesion, cy- transform human cells as efficiently as ST transformation in soft agar toskeletal structure, and motility were affected by the presence of ST. or tumor formation assays (15). Src-Y418 was not phosphorylated in In particular, several genes known to affect the integrin signaling the HEK-TERV or HEK-TERST110 cells under any conditions, dem- pathway such as osteopontin, paxillin, f-spondin, gelsolin, and matrix onstrating that ST-induced activation of src is PP2A-dependent. metalloproteinase-1 (MMP-1) were changed in directions consistent Whereas it is not possible to determine whether the changes in src with integrin activation by microarray analysis (Supplementary Table phosphorylation are direct or indirect effects of ST based on these S1) and by QRT-PCR (Fig. 3, A and B). Our data confirmed previous data, we hypothesize that ST may indirectly activate src through studies (29) that SV40 activates expression of osteopontin (SPP1) and PP2A-dependent activation of integrins (Fig. 5D). show that ST does this in a PP2A-dependent manner. In addition, ST up-regulated expression of integrin signaling targets such as MMP-1, ␣ collagens, c-myc, and paxillin by both PP2A-dependent and PP2A- DISCUSSION independent mechanisms. These data suggest, but do not prove, that ST may activate integrin signaling directly or indirectly. Here we have identified the changes in gene expression that are In contrast to the apparent up-regulation of integrin signaling, generated by SV40 ST and determined that many of these targets are expression of many genes important for cell-cell adhesion such as highly relevant to cancer growth, survival, motility, and metastasis. ICAM-1 and VCAM-1 were down-regulated. Components of junc- Our data provide new insights that support a model (Fig. 5D) in which tional adhesion complexes were also repressed, such as ␤-catenin, ST promotes growth and prevents apoptosis through constitutive ␬ plakoglobin, junctional adhesion molecule 1(JAM), claudin 11, and integrin signaling and NF B activation while inhibiting components protocadherin ␥ family members. In addition, secreted-frizzled related of cell-cell adhesion pathways that might provide cell cycle arrest and protein 1, which binds directly to wingless and can inhibit wnt prodifferentiation signals. Whereas many of the effects of ST were signaling through destabilization of ␤-catenin (40), was up-regulated shown to be independent of PP2A binding and inhibition, the key by ST. changes shared by the tumorigenic HEK-TERST and HEK-TER- ␥ HEK-TERST Activation of Src Shows Less Serum Dependence ASB56 cell lines were largely PP2A-dependent and included up- Than HEK-ASB56␥. The tyrosine kinase c-src and the PI3K are regulation of antiapoptotic effectors like SERPINB2/PAI-2, as well as downstream components of integrin signaling pathways (41, 42), and developmental homeobox and forkhead box transcription factors. integrin activation of c-src can block proper assembly of cell-cell Moreover, ST appeared to be able to differentially regulate proinflam- contacts (43). To determine whether activation of integrin signaling matory and antiapoptotic targets of NF␬B. through c-src and PI3K is essential for the anchorage-independent The 137 genes that were affected by ST in a PP2A-independent growth phenotype of the HEK-TERST cell line, soft agar assays were manner but were unaffected by B56␥ knockdown may enhance, but performed in the presence of inhibitors of integrin, c-src,orPI3K not be essential, for transformation. Many of these genes were cell signaling. To test the effect of inhibition of integrin signaling on cycle regulated genes and may reflect the inability of the HEK- growth in soft agar, HEK-TERST and HEK-ASB56␥ cells were TERASB56␥ line to progress through S phase under conditions of low plated in the presence of 10 ␮g/mL of a circularized arginine-glycine- serum. In agreement with published literature (10, 30, 31, 44–48), ␣ coexpression of SV40 LT and ST drove cells into S phase in low aspartic acid (RGD) peptide that acts as an integrin v␤3 antagonist, or an equal concentration of a control arginine-alanine-aspartic acid serum, and we observed corresponding increases in expression of (RAD) peptide. To determine whether c-src signaling is essential for S-phase genes such as TK-1, DHFR, and G0S2. In contrast to earlier HEK-TERST and HEK-ASB56␥ growth in soft agar, a c-src-specific studies in which S-phase entry mediated by polyoma or SV40 LT and kinase inhibitor PP1 and an inactive structural analogue, PP3, were ST was PP2A-dependent (13, 49), we observed that the ST110 mutant used. HEK-TERST and HEK-ASB56␥ cells were also plated in the could also support S-phase entry, although to a lesser degree than presence of wortmannin to test for the requirement of PI3K signaling wild-type ST. One potential reason for the ability of the ST110 mutant in anchorage-independent growth. The integrin RGD inhibitor, c-src to support cell cycle progression in our model system is that our cell PP1 inhibitor, and wortmannin all dramatically interfered with the lines also express the constitutively activated H-Ras-V12 mutant.

HEK-TERST and the HEK-ASB56␥ cell line’s ability to form colo- Thus, in the presence of LT and activated Ras signaling, the NH2- nies in soft agar (Fig. 5A), whereas the control inhibitors had minimal terminal domain of ST appears to be sufficient to drive cells into S effects. These data show that integrin signaling is essential for trans- phase. Consistent with this hypothesis, mutations in the DnaJ domain formation by ST expression and by B56␥ knockdown and suggest that of polyoma ST has been shown to strongly inhibit activation of the both of these transformation events activate integrin signaling directly cyclin A promoter (50). or indirectly. The viability of HEK-TERST cells was determined by The set of 99 genes that were regulated similarly by ST and B56␥ 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye as- knockdown may represent some of the most critical ST-induced say after 2 weeks of treatment with each of these inhibitors in soft changes for transformation of human cells. Many of these expression agar, demonstrating that none of the inhibitors had a direct killing changes likely result from previously identified effects of SV40 ST in effect, but rather prevented colony growth (Fig. 5B). signal transduction pathways such as p27/Kip1 down-regulation (30), To determine whether ST expression or B56␥ knockdown affects AKT and telomerase activation (51), mitogen-activated protein kinase src activation and phosphorylation on tyrosine 418, HEK-TERST and pathway activation (52), protein kinase C ␨ activation of NF␬B (37, HEK-ASB56␥ cells were also grown in anchorage-independent 1.3% 53), and induction of cyclins (46, 47). The remainder of the expression methylcellulose in both 5% and 10% FBS. After 24 hours in anchor- alterations may result from still unidentified effects of ST on other age-independent media, cells were spun down and harvested as de- signal transduction pathways or from direct effects of ST on transcribed (24), lysed, and whole cell lysates were probed for the pres- scription factors. It is important to note that HEK-TERASB56␥ cells ence of phospho-Y418-src (Fig. 5C). ST induced phosphorylation and are serum-dependent, grow more slowly, and are less potently transactivation of src under all conditions, whereas src-Y418 phosphoryl- formed than the HEK-TERST cells. Thus, whereas the set of 99 genes ation in HEK-ASB56␥ cells was detected only in 10% serum. The are probably the most critical ones for tumorigenesis, other ST- 6985

regulated genes outside of this group may account for the rapidly proliferating, serum-independent phenotype of the HEK-TERST cells. Whereas 99 genes were affected similarly by ST and B56␥ knockdown, several hundred genes were not. Several different reasons could account for these observations. First, B56␥ antisense down-regulates B56␥ to a greater extent than ST (15). Second, ST is known to target PP2A isoforms other than B56 (52), although knockdown of B55 subunits cannot fully transform human cells as B56␥ knockdown does

(15). Third, the NH2-terminal domain of ST may influence the pattern of gene expression caused by PP2A inhibition. Finally, because our microarray experiments were performed in low serum and HEK- ASB56␥ cells are serum-dependent, more similarities with ST expression may be identified if these cells were compared in normal serum conditions. Several of the genes regulated by ST have roles in prevention or induction of apoptosis. ST up-regulated ALDH1, which protects cells by metabolizing oxidized lipids; moreover, inhibitors of ALDH1 can drive Bcl-2 overexpressing cells into apoptosis (54). ST also increased expression of SERPINB2, which inhibits tumor necrosis factor-induced apoptosis and strongly repressed expression of tumor necrosis factor-related apoptosis-inducing ligand. ST also inhibits apoptosis through repression of gelsolin, a regulator and effector of apoptosis. Gelsolin plays a key role in actin remodeling and motility, and ␣ associates with integrin v, c-src, focal adhesion kinase, PI3K, and paxillin in response to integrin activation by osteopontin (55). Using an RGD peptide inhibitor, we showed that integrin signaling was essential for anchorage independent growth of both HEK-TERST and HEK-TERASB56␥ cell lines. Besides increases in integrin signaling targets, we also observed increased expression of three protease-activated receptors (PAR-1, PAR-2, and PAR-3), which could also contribute toward activation of PI3K and AKT. Consistent with recent work showing that constitutive PI3K signaling can substitute for ST to fully transform human cells (56), we have shown that integrin signaling is critical for ST-helper function in tumorigenesis. Our data demonstrated that ST expression induces activation of src in low serum, whereas knockdown of B56␥ subunits results in serum- dependent src phosphorylation. Thus, the effects of ST on the integrin-src-PI3K pathway are critical for transformation of human cells. ␣ It has been shown recently that 6␤4 integrin signaling can confer resistance to apoptosis in mammary epithelium via NF␬B activation (57). It is known that ST activates NF␬B via protein kinase C ␨ and PI3K signaling (37) and that PP2A regulates NF␬B activation by dephosphorylation of the I␬B kinase (39). Thus, ST may be impacting NF␬B activation in multiple ways, by mimicking and/or stimulating growth factor and integrin signaling and by modulation of PP2A activity. ST affected expression of several developmental transcription factors, including HOXA9, HOXB3, HOXB6, Ets-1, FOXD1, FOXG1,

B, viability of HEK-TERST cells after 2 weeks in soft agar in the presence of treatments used in A as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The mean of four replicates are shown; bars, ϮSD. C, HEK-TERV, HEK- TERST110, HEK-TERST, and HEK-TERASB56 were grown in 1.3% methycellulose for 24 h in either 5% or 10% FBS and whole cell lysates prepared. Immunoblots were probed for total src and with antibodies specific to src kinase phosphorylated at tyrosine 418. PP2A C subunit is shown as a loading control. ST induced phosphorylation and activation of Src under all conditions, whereas in B56␥ knockdown cells src phosphorylation was detected only in 10% serum. Src was not phosphorylated in the HEK-TERV or HEK- Fig. 5. Integrin-src-PI3K signaling is essential for anchorage-independent growth. A, TERST110 cells under any conditions, demonstrating that ST-induced activation of src is soft agar assays of HEK-TERV, HEK-TERST110, HEK-TERST, and HEK-TERASB56␥ PP2A-dependent. D, model of the role of ST and B56␥ in anchorage-independent growth. cells either untreated or with an inhibitor of integrin signaling (RGD peptide), a control HEK-TERST cells: integrin signaling and ST together activate src and NF␬B to increase peptide (RAD), a src inhibitor (PP1), a control inhibitor (PP3), or a PI3K inhibitor expression of antiapoptotic genes. ST represses proinflammatory genes by an unknown (wortmannin). Results for the HEK-TERASB56␥ line were similar to the HEK-TERST mechanism. HEK-TERASB56 cells: lack of B56␥ together with integrin signaling and line except that HEK-TERASB56␥ colonies were generally smaller than HEK-TERST growth factors from serum enable src activation, NF␬B activation, and increases in both colonies (data not shown). The mean of data from six replicates are shown; bars, ϮSD. proinflammatory and antiapoptotic genes. 6986

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. TRANSFORMATION TARGETS OF SV40 ST AND B56␥ and FOXM1. Additional developmental markers induced by ST in- 17. Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization cluded markers of pre-B and pro-B lymphocytes, cardiac, epithelial, methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 2003;19:185–93. endothelial, and neuronal tissues. The expression of early differenti- 18. Beissbarth T, Speed T. GOstat: find statistically overrepresented gene ontologies ation makers from such a wide variety of tissue types (see Supple- within a group of genes. Bioinformatics 2004;20;1464–5. mentary Table S1) suggests that part of the helper function of ST 19. Hochberg Y, Benjamini Y. More powerful procedures for multiple significance testing. Stat Med 1990;9:811–8. results in a “dedifferentiated” phenotype of transformed cells. The 20. Castel S, Pagan R, Garcia R, et al. Alpha v integrin antagonists induce the disassem- observations that we report here suggest that ST may achieve this bly of focal contacts in melanoma cells. Eur J Cell Biol 2000;79:502–12. function in part by repression of components of junctional adhesion 21. Hanke JH, Gardner JP, Dow RL, et al. Discovery of a novel, potent, and Src ␤ family-selective tyrosine kinase inhibitor. Study of Lck- and FynT-dependent T cell complexes such as -catenin, plakoglobin, JAM1, and protocadherin activation. J Biol Chem 1996;271:695–701. family members. Our conclusions are consistent with reports that 22. Altmann C, Steenpass V, Czyborra P, Hein P, Michel MC. Comparison of signalling overexpression of plakoglobin can suppress tumorigenicity of SV40- mechanisms involved in rat mesenteric microvessel contraction by noradrenaline and sphingosylphosphorylcholine. Br J Pharmacol 2003;138:261–71. transformed cells (58) and that ST can alter distribution of and reduce 23. Kuppumbatti YS, Rexer B, Nakajo S, Nakaya K, Mira-y-Lopez R. CRBP suppresses levels of tight junction proteins such as occludin and claudin in breast cancer cell survival and anchorage-independent growth. Oncogene 2001;20: polarized epithelial cells (59). 7413–9. 24. Kume K, Jinno S, Miwatani H, et al. Oncogenic signal-induced ability to enter S In conclusion, we have identified the changes in gene expression phase in the absence of anchorage is the mechanism for the growth of transformed induced by ST in transformation of human cells and determined that NRK cells in soft agar. New Biol 1992;4:504–11. many of the critical changes are in genes that influence cellular 25. Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. 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