Human Cancer Biology

Nonredundant Functions for Tumor D52-Like Support Specific Targeting of TPD52 Mona Shehata,1, 2 Ivan Bie' che,4,5 Rose Boutros,1, 2 Judith Weidenhofer,1Susan Fanayan,1, 2 Lisa Spalding,6 Nikolajs Zeps,6 Karen Byth,3 Robert K. Bright,7 Rosette Lidereau,4 and Jennifer A. Byrne1, 2

Abstract Purpose: Tu m o r p r o t e i n D 5 2 (TPD52 or D52) is frequently overexpressed in breast and other cancers and present at increased copy number. It is, however, unclear whether D52 ampli- fication and overexpression target specific functionalproperties of the encoded protein. ExperimentalDesign: The expression of D52-like and MAL2 was compared in breast tissues using quantitative reverse transcription-PCR.The functions of human D52 and D53 genes were then compared by stable expression in BALB/c 3T3 fibroblasts and transient gene knock- down in breast carcinoma cell lines.In situ D52 and MAL2 protein expression was analyzed in breast tissue samples using tissue microarray sections. Results: The D52 (8q21.13), D54 (20q13.33), and MAL2 (8q24.12) genes were significantly overexpressed in breast cancer tissue (n = 95) relative to normal breast (n =7;P V 0.005) unlike the D53 gene (6q22.31; P = 0.884).Subsequently, D52-expressing but not D53-expressing 3T3 cell lines showed increased proliferation and anchorage-independent growth capacity, and reduced D52 but not D53 expression in SK-BR-3 cells significantly increased apoptosis. High D52 but not MAL2 expression was significantly associated with reduced overall survival in breast carcinoma patients (log-rank test, P < 0.001; n = 357) and was an independent predictor of survival(hazard ratio, 2.274; 95% confidence interval,1.228-4.210; P =0.009;n = 328). Conclusion: D52 overexpression in cancer reflects specific targeting and may contribute to a more proliferative, aggressive tumor phenotype in breast cancer.

Chromosome 8q gain is one of the most frequent cytogenetic of the D52-like family of adaptor proteins (10, 11). Human aberrations in human cancer (1). Increasingly refined amplifi- D52 transcripts or protein are overexpressed in breast, prostate, cation mapping studies have indicated numerous target genes and ovarian cancer, with this being associated with increased along this chromosomal arm, including genes at gene copy number in a proportion of cases (6–9). Expression 8q21 (2–5). A critical chromosome 8q21.13 gene is indicated microarray studies also indicate that the D52 gene is amplified to be tumor protein D52 (TPD52 or D52; refs. 6–9), a member and/or overexpressed in multiple myeloma (12, 13), pancreatic cancer (14), and seminoma (15). The frequency with which D52 is overexpressed in cancer argues strongly for this playing a causal role. However, as 1 Authors’ Affiliations: Molecular Oncology Laboratory, Oncology Research Unit, extensive chromosome 8q regions are frequently gained (1), 2The University of Sydney Discipline of Paediatrics and Child Health,The Children’s Hospitalat Westmead; 3Westmead Millennium Institute, Westmead, New South and large numbers of 8q genes may be consequentially Wales, Australia; 4Laboratoire d’Oncoge¤ ne¤ tique, INSERM U735, Centre Rene¤ overexpressed (16), the significance of many overexpressed Huguenin, St-Cloud, France; 5Laboratoire de Ge¤ ne¤ tique Mole¤ culaire, UPRES 8q genes remains unclear. In the case of D52, this is INSERM U745, Faculte¤ des Sciences Pharmaceutiques et Biologiques, Universite¤ compounded by the fact that D52-like proteins have shared Rene¤ Descartes-Paris V, Paris, France; 6Department of Radiation Oncology, Sir Charles Gairdner Hospital, Nedlands,Western Australia, Australia; and7Department functions, including mutual interactions (17, 18), and binding of Microbiology and Immunology and the Southwest Cancer Treatment and a common partner MAL2 (19–21), which is itself overex- Research Center,TexasTech University Health Sciences Center, Lubbock,Texas pressed in breast and other cancers (22–24). Most studies Received 11/27/07; revised 3/5/08; accepted 3/19/08. reporting phenotypes associated with increased or decreased Grant support: Australian Postgraduate Award (M. Shehata), National Health and MedicalResearch Councilof AustraliaPeter Doherty Fellowship (S. Fanayan), D52 expression have also not compared D52 functions with Cancer Institute New South Wales Fellowship (J.A. Byrne), donations to the those of related proteins (25–29). It is therefore not known Oncology Department of the Children’s Hospital at Westmead, and Oncology whether D52-like proteins beyond D52 itself have similar roles Children’s Foundation. in enhancing cancer-associated phenotypes such as prolifera- The costs of publication of this article were defrayed in part by the payment of page tion and anchorage-independent growth (26, 28). There is charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. therefore still uncertainty whether D52 overexpression in Requests for reprints: Jennifer A. Byrne, Molecular Oncology Laboratory, cancer passively reflects its gene location or actively reflects Oncology Research Unit, The Children’s Hospital at Westmead, Locked Bag nonredundant functional properties that are specifically 4001,Westmead 2145, New South Wales, Australia. Phone: 61-2-9845-3027; targeted (10). Fax: 61-2-9845-3078; E-mail: JennifeB @chw.edu.au. F 2008 American Association for Cancer Research. To analyze this question, the present study has taken several doi:10.1158/1078-0432.CCR-07-4994 approaches. Firstly, we have directly compared the expression

Clin Cancer Res 2008;14(16) August 15, 2008 5050 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. SpecificTPD52 Targeting in Breast Cancer of three widely expressed D52-like genes and their common Total RNA extraction and cDNA synthesis. Total RNA was extracted partner MAL2 in normal breast and breast carcinoma. This using the acid-phenol guanidinium method, and RNA quality was identified that D52, D54, and MAL2 were significantly up- assessed using agarose gel electrophoresis and ethidium bromide  regulated in breast carcinoma and that these genes localize to staining. Reverse transcription reactions contained 1 reverse transcrip- A regions of the genome gained in breast and other cancers. As tion buffer [500 mol/L each deoxynucleotide triphosphate, 3 mmol/L MgCl , 75 mmol/L KCl, 50 mmol/L Tris-HCl (pH 8.3)], 20 units D52 and D53 represent examples of D52-like genes that are or 2 RNasin RNase inhibitor (Promega), 10 mmol/L DDT, 100 units are not overexpressed in breast cancer, we directly compared Superscript II RNase H- reverse transcriptase (Invitrogen), 3 Amol/L the effects of expressing these genes in BALB/c 3T3 fibroblasts, random hexamers (Pharmacia), and 1 Ag total RNA in 20 AL. Samples which are highly responsive to D52 expression (28). We also were incubated at 20jC for 10 min and 42jC for 30 min, and reverse examined the effects of transiently knocking down human D52 transcriptase was inactivated by heating at 99jC for 5 min and cooling or D53 expression in MCF-7 and SK-BR-3 breast carcinoma cell at 5jC for 5 min. lines, which endogenously express both proteins (18). These Real-time reverse transcription-PCR. Real-time reverse transcription- approaches identified both shared and nonredundant func- PCR analyses were done as described previously (30). Briefly, reactions tions for these proteins, with nonredundant functions indicat- were done using an ABI Prism 7700 Sequence Detection System using ing cancer-specific roles for D52. As D52 and MAL2 genes the SYBR Green PCR Core Reagents kit (Perkin-Elmer Applied were most significantly overexpressed in breast carcinoma Biosystems). The thermal cycling conditions comprised an initial denaturation step at 95jC for 10 min and 50 cycles at 95jC for 15 s relative to normal breast, we then analyzed D52 and MAL2 and 65jC for 1 min. Quantitative values were obtained from Ct values expression in breast tissue samples using tissue microarray using the Perkin-Elmer Biosystems analysis software according to the sections. This indicated that high D52 but not MAL2 manufacturer’s instructions. Results were expressed as fold differences expression was significantly associated with reduced overall in target gene expression relative to the endogenous RNA control TBP. survival in breast cancer patients. The present study has Gene expression values were then expressed relative to the median therefore obtained evidence that D52-like proteins have value obtained in the 7 normal breast samples, which was set at 1.0 for nonredundant cellular functions and that the proliferative each gene. functions of D52 may contribute to a more aggressive breast Cell culture and stable transfection of BALB/c 3T3 fibroblasts. Mouse j cancer phenotype. BALB/c 3T3 fibroblasts were grown at 37 C, 5% CO2 in DMEM supplemented with 10% fetal bovine serum (Invitrogen), 2% L-glutamine (Invitrogen), and 7.5% sodium bicarbonate (Sigma). For the stable expression of human D52, a SalI-BamHI cDNA fragment Materials and Methods including the full-length coding sequence was subcloned into the same sites of the PG307 vector (31). The PG307hD52 plasmid, the Breast tissue samples. Breast tissue samples analyzed by quantitative PG307hD53 plasmid (18), and the PG307 vector were stably trans- reverse transcription-PCR represented 7 normal breast samples, 14 fected into 3T3 fibroblasts by seeding cells at f60% confluence in benign breast lesions, and 95 invasive carcinomas. Normal breast six-well plates and transfecting 24 h later with 5 Ag DNA in 250 AL samples represented adjacent normal tissue from 4 breast cancer Opti-MEM (Invitrogen) and 6 AL LipofectAMINE 2000 (Invitrogen). patients and normal tissue from 3 women undergoing cosmetic breast After 24 h, cells were passaged into 100 mm dishes and 48 h post- surgery. The tumor group consisted of 11 grade 1 tumors, 12 grade 3 transfection, G418 (Invitrogen) was added (1 mg/mL), and drug tumors, 12 estrogen receptor (ER)–negative tumors, 12 ER-positive selection continued for 2 weeks. Selected G418-resistant clones were tumors, and 48 additional ER-positive tumors from tamoxifen-treated screened using Western blot analyses, and 6 cell lines expressing human patients, of which 24 relapsed and 24 remained disease-free (median D52, 5 cell lines expressing human D53, and 3 vector control cell lines follow-up, 86 months; range, 18-120 months). Tissue samples were were maintained in 1 mg/mL G418 medium for further analyses. obtained and stored before RNA extraction as described previously Transient small interfering RNA transfections. Human D52 and D53 (30), and total RNA was extracted from whole frozen specimens. Breast small interfering RNA (siRNA) duplexes were synthesized by Dharma- tissue microarray sections were purchased from the Western Australian con. The targeted sequences (sense strand; 5¶-3¶)weresi2-1 Research Tissue Network from samples obtained from the Royal Perth (GCGGAAACTTGGAATCAAT), si2-2 (GGAGAAGTCTTGAATTCGG) Hospital (RPH) Pathology Department under ethics approvals granted and si2-3 (AAGAAAAGGTCGAAAACTT) for D52 and si3-2 (TCA- by the RPH and Sir Charles Gairdner Hospital Human Research Ethics CAAGCCTCAAGACGAA), si3-3 (GCTAGAAGACGAAATTACA), and Committees. Samples were surgically removed at RPH between 1995 si3-4 (GCAAGAAGTTCGGAGACAT) for D53. Positive control and 2001, fixed in formaldehyde, and embedded in paraffin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) siRNA sections included samples of normal breast, in situ and invasive (TGGTTTACATGTTCCAATA) and nontargeting siControl siRNA carcinomas. Cores containing breast and prostate cancer cell lines were (TAGCGACTAAACACATCAA) were also purchased from Dharmacon. included in all array sections as controls. A total of 357 invasive The human breast cancer cell lines MCF-7 and SK-BR-3 were cultured in carcinomas were analyzed for in situ D52 expression, and a subset of RPMI supplemented with 10% fetal bovine serum (Invitrogen), 3% 320 cases was analyzed for in situ MAL2 expression. Patients were ages L-glutamine (Invitrogen), and 10 Ag/mL insulin (Sigma). Cells were 27 to 91 years at diagnosis (median, 59 years) and had no prior cultured to 70% confluence, trypsinized, and plated onto glass treatment before surgery. Breast carcinomas were analyzed for the coverslips, or 2  104 MCF-7 and 4  104 SK-BR-3 cells were seeded expression of established markers including ER by the RPH Pathology into wells of 24-well plates. After 24 h, cells were transfected with Department, with tumors with 10% ER-positive cells being considered 100 nmol/L siRNA duplexes using TransIT-TKO transfection reagent positive [ER-positive cases (n = 265), ER-negative cases (n = 85), and (Mirus) in complete medium following the manufacturer’s instructions unknown status (n = 7)]. Other clinical and histologic data available and analyzed 48 h later. included lymph node status at diagnosis [node-negative (n = 244) and Western blot analyses. Cells were washed twice in PBS and lysed in node-positive (n = 113)], tumor grade [grade 1 (n = 88), grade 2 SDS lysis buffer for total protein extracts as described (18). Protein (n = 159), grade 3 (n = 104), and unknown (n = 6)], and patient extracts (10 Ag/well) were resolved using SDS-PAGE on 12.5% survival (median follow-up, 55 months; range, 0.3-123 months). polyacrylamide gels and electrotransferred to nitrocellulose membranes Additional clinical information was provided by the Multidisciplinary (Millipore). Membranes were blocked overnight at 4jC in 5% skim Breast Service at RPH. milk powder in TBS. Membranes were washed twice with TBS and

www.aacrjournals.org 5051 Clin Cancer Res 2008;14(16) August 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Human Cancer Biology

Table 1. Expression of human D52-like and MAL2 genes in breast tissues using quantitative reverse transcription-PCR

Gene Normal breast Benign lesions Invasive carcinoma P* P* P* (cytogenetic location) (n = 7), (n = 14), (n = 95), (normal vs (benign vs (normal vs median (range) median (range) median (range) benign) carcinoma) carcinoma) D52 (8q21.13) 1.00 (0.50-1.26) 1.10 (0.46-2.53) 1.76 (0.2-14.4) 0.167 0.016 0.003 D53 (6q22.31) 1.00 (0.60-1.76) 1.56 (0.57-3.01) 1.05 (0.01-8.21) 0.117 0.155 0.884 D54 (20q13.33) 1.00 (0.77-1.19) 1.45 (0.93-1.94) 1.64 (0.45-11.93) 0.005 0.191 0.005 MAL2 (8q24.12) 1.00 (0.80-1.30) 1.67 (0.30-3.14) 3.21 (0.43-23.78) 0.057 <0.001 <0.001

*Mann-Whitney U tests, P values <0.01 are shown in bold.

incubated with affinity-purified rabbit polyclonal D52 antisera Crystal violet and apoptosis assays. SK-BR-3 or MCF-7 cells were (1:100),8 affinity-purified rabbit polyclonal human D53 antisera seeded in duplicate in 24-well plates and transfected with siRNA as (1:100; ref. 18), mouse monoclonal actin (1:2,000; a gift from J. described above. Cell density per well was determined after 48 h by Lessard), or mouse monoclonal GAPDH (1:5,000; Ambion) antibodies crystal violet assay. Briefly, following 15 min of cell fixation with 3% in 0.1% Tween 20 in TBS for 1 to 2 h. Membranes were washed three acetic acid/10% methanol, cells were stained with 0.4% crystal violet times in 0.1% Tween 20 in TBS and then incubated with a horseradish for 50 min and rinsed twice in water. Cells were air-dried and triplicates peroxidase–conjugated donkey anti-rabbit or anti-mouse secondary were taken using a DC 500 dissecting microscope and camera (Leica antibody (1:5,000; GE Biosciences) for 1 h. Membranes were finally Technologies). Cell areas were calculated using Metamorph (version washed four times and visualized by Western lightening chemilumi- 6.1; Molecular Devices) with manual thresholding. The means of cell nescent reagent (Perkin-Elmer). areas measured in triplicate from three independent experiments were Indirect immunofluorescence analyses. BALB/c 3T3 cell lines were used to generate data points and SE. DNA fragmentation produced cultured to near confluence, trypsinized, and plated onto glass 48 h post-transfection was quantified using the Cell Death ELISAPLUS coverslips overnight. Cells were washed twice with PBS, fixed in 4% kit according to the manufacturer’s instructions (Roche). Briefly, cells paraformaldehyde/PBS for 20 min, permeabilized with 0.1% saponin were lysed and centrifuged, and supernatants were transferred to for 10 min, washed twice with PBS, and incubated overnight with 96-well precoated plates to assay cytoplasmic histone-associated DNA affinity-purified human D52 (1:100; ref. 6) or human D53 (1:50) fragments. After incubations and washes, stop substrate was added and antisera in 0.1% bovine serum album in PBS. Cells were washed twice color development was measured at 405 nm. Samples were analyzed and incubated with secondary Cy3-conjugated donkey anti-rabbit in duplicate in three independent experiments. (1:500; Jackson Immunoresearch) antibody in 0.1% bovine serum Immunohistochemical analysis of paraffin-embedded breast tissue album in PBS for 1 h in the dark. For phalloidin staining, cells were microarrays. The D52 antisera employed for immunohistochemical washed twice and incubated with FITC-conjugated phalloidin (Sigma) analyses have been described previously (6), whereas the derivation of for 15 min in the dark. Cells were washed again and DNA was the rabbit polyclonal MAL2 antisera will be described elsewhere.9 Tissue counterstained with 10 nmol/L 4¶,6-diamino-2-phenylindole (Sigma). microarray slides were washed and rehydrated in xylene and ethanol After washing in PBS, cells were mounted in DABCO (Sigma) prepared before rehydrating in water and equilibrating in PBS. Slides were according to the manufacturer’s instructions. Images were taken using blocked in 10% normal goat serum for 20 min, rinsed in PBS, and an Olympus BX50 microscope equipped with a SPOT camera incubated for 2 h with affinity-purified D52 (1:100) or MAL2 (1:100) (Diagnostic Instruments). Between 50 and 100 cells from each antibodies in 2% normal goat serum in PBS. Primary antibody was transfected cell line were visually scored according to in situ D52 and omitted in control incubations. Biotinylated goat anti-rabbit secondary D53 expression levels or actin stress fibers, and Image ProPlus (Media (Jackson Immunoresearch) was added (1:500) to the slides and Cybernetics) was used to calculate cell area (Am2). incubated for 1 h. Slides were incubated with hydrogen peroxide for Cell proliferation assays. Parental cells or transfected 3T3 cell lines 30 min and stained with tertiary ABC reagent (Pierce) for 1 h. DAB (1 Â 103), 2 Â 103 MCF-7 cells, or 4 Â 103 SK-BR-3 cells were plated in (Sigma) was added for 2 to 5 min and slides counterstained with triplicate in 96-well plates. In all cases, 50 AL 3-(4,5-dimethylthiazol-2- Nuclear Fast Red (Fronine). Slides were scanned using the Virtual yl)-2,5-diphenyltetrazolium bromide reagent (Sigma) was added to Microscope ScanScope Unit and ScanScope Console program (Aperio each well either immediately (0 time point) or at the designated Technologies) at Â200 magnification. Tissue arrays were visualized number of days post-seeding and incubated at 37jC in the presence of using Image Scope and staining intensity was quantified within tissue

5% CO2 for 4 h. To stop each reaction, medium was replaced with cores of fixed and uniform diameter using the Positive Pixel Count 100 AL DMSO (Sigma) per well and mixed thoroughly. Absorbances at algorithm (Aperio Technologies). The number of strong pixels (defined 540 nm were taken using a Multiskan Ascent plate reader. Means of as pixels of 175-220 intensity) was measured per tissue core. Partial triplicate wells from four independent experiments were used to tissue cores, those with staining artifacts, or those without epithelial generate data points and SE. elements (normal or cancerous) were excluded from analyses. Soft-agar colony formation assays. Stably transfected or parental 3T3 Statistical analyses. The SPSS for Windows package (version 13; cell lines (5 Â 104) were seeded in duplicate into six-well dishes in 3 mL SPSS) was used in most analyses. Distributions of continuous variables complete medium containing 0.33% agar solution overlaying 0.5% were often skewed and summarized using medians and interquartile agar (Becton Dickson). Colonies containing >10 cells in 20 random ranges. Mann-Whitney U tests were used to test for differences in cell fields were counted at Â100 magnification 14 days after plating. Means size according to human D52 or D53 expression categories. Categorical of duplicate wells from three independent experiments were used to variables were summarized using percentages within each group. generate data points and SE. Spearman rank correlation and Fisher’s exact test were used to compare

8 Weidenhoferetal.,inpreparation. 9 S. Fanayan et al., submitted for publication.

Clin Cancer Res 2008;14(16) August 15, 2008 5052 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. SpecificTPD52 Targeting in Breast Cancer

protein expression and other variables. In patient samples, strong pixel using two-tailed, unequal variance Student’s t tests calculated using counts were compared in normal breast, in situ carcinoma, or breast Excel (Microsoft). carcinoma samples from the same patients using the Wilcoxon signed rank test. Survival distributions were estimated by the Kaplan-Meier method, and the significance of differences between overall survival Results rates was ascertained using the log-rank test. Best-fitting multiple Cox MAL2 proportional hazards models with backward stepwise selection were D52-like and gene transcript levels in breast tissues. used to identify independent predictors of survival from potential risk Quantitative reverse transcription-PCR was employed to study factors. Results of all cell proliferation, soft agar, crystal violet, and the relative expression of D52-like genes and MAL2 in a cohort apoptosis assays are expressed as mean F SE of three to four of 7 normal breast samples, 14 benign breast lesions, and 95 independent experiments. Comparisons between groups were made invasive carcinomas. Gene expression values were expressed

Fig. 1. Generation of human D52- and D53-expressing BALB/c 3T3 fibroblast cell lines.A and B, totalprotein extracts from parental cells (3T3), vector only cell lines (V1,V2, and V3), and cell lines stably transfected with human D52 (A) or D53 (B) expression constructs were separated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to Western blot analyses using non-species-specific D52 (A) or human D53 polyclonal antisera (B) and an anti-actin monoclonal to compare lane loading (A and B). Human D52 or D53 (right)were detected in relevant transfected cell lines and MCF-7 breast cancer cells but not in vector-transfected or parental cells. Left, positions of molecular weight standards (kDa). C, human D52 or D53 expression is associated with reduced cell area and actin stress fibers in 3T3 cells.Vector (V2 cell line), D52 (2-4 cell line), and D53 (3-2 cell line) cells were labeled with human D52 (top and middle left) or D53 polyclonal antisera (bottom left ; red) and FITC-phalloidin (right ; green). Nuclei were counterstained with 4¶,6-diamino-2-phenylindole (blue). Arrows, single examples of small cells with high D52 or D53 expression and poor actin stress fibers; arrowheads, single examples of larger cells with undetectable/low D52 or D53 expression and prominent actin stress fibers. Magnification, Â400. Bar, 20 Am.

www.aacrjournals.org 5053 Clin Cancer Res 2008;14(16) August 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Human Cancer Biology

Fig. 2. Significant negative associations between D52 or D53 expression status and both cell area and actin stress fibers. Between 50 and 100 cells were measured per cell line, and data from the six D52-transfected or five D53-transfected cell lines were pooled for analyses.A and B, box plots summarizing cell areas (Am2)according to in situ D52 (A) or D53 (B) expression status (negative/low versus moderate/high). Horizontallines, median cell areas;boxes, interquartile ranges; verticallines, 95% confidence intervals; open circles, outliers; asterisks, extreme values. C and D, graphicalrepresentations comparing the proportions of cellsexpressing negative, low, moderate, or high D52 (C) or D53 (D) levels in situ with prominent, moderate, or no/poor actin stress fibers.

relative to median values obtained in normal breast samples, relative to carcinoma, we compared their functions by stably which were set at 1.0 for each gene. The results of comparing expressing these in BALB/c 3T3 fibroblastic cells, which were median relative gene expression values in different tissue types shown previously to be highly responsive to increased mouse using Mann-Whitney tests are summarized in Table 1. Of the D52 expression (28). Six human D52-expressing cell lines (2-1, genes examined, D52, D54, and MAL2 transcripts were all 2-2, 2-3, 2-4, 2-5, and 2-6), 5 human D53-expressing cell lines detected at significantly increased levels in breast carcinoma (3-1, 3-2, 3-3, 3-4, and 3-5), and 3 vector control cell lines (V1, samples relative to normal breast (Table 1) and also in those 24 V2, and V3) were employed in further studies (Fig. 1A and B). ER-positive tumors from patients who subsequently relapsed Cells were immunofluorescently labeled for human D52 or after tamoxifen therapy compared with 24 tumors from D53 and phalloidin to examine the intracellular distribution of patients who remained disease-free (Mann-Whitney U test, these proteins and overall cell morphology. Whereas vector P < 0.001 for D52, P = 0.001 for D54, and P = 0.001 for MAL2; control cells not expressing D52 or D53 showed the expected n = 48). In addition, D54 transcripts were detected at fibroblastic morphology with prominent actin stress fibers significantly increased levels in benign lesions relative to (Fig. 1C, top row; data not shown), cells expressing moderate to normal breast, and MAL2 transcripts were detected at signifi- high levels of D52 or D53 were smaller, with dense phalloidin cantly increased levels in breast carcinoma samples relative to staining and few actin stress fibers (Fig. 1C, middle and lower benign lesions (Table 1). In contrast, D53 was not differentially rows, respectively). We consistently observed in situ hetero- expressed between the sample categories compared (Table 1). geneity of D52 and D53 levels within transfected cell lines, D52 and D53 expression is associated with reduced cell area with individual cells showing low/undetectable or moderate/ and actin stress fibers in 3T3 cells. As human D52 and D53 high levels of D52 or D53 (Fig. 1C, middle and bottom left, genes exhibited different expression patterns in normal breast respectively). Transfected cells expressing low/undetectable

Clin Cancer Res 2008;14(16) August 15, 2008 5054 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. SpecificTPD52 Targeting in Breast Cancer

D52 or D53 levels showed fewer alterations to actin stress fibers U test, P = 0.126, n = 1,167). Similarly, actin stress fibers were and were comparable with vector controls (Fig. 1C). significantly inversely correlated with D52 or D53 expression To quantitate these phenomena, 50 to 100 cells from each category (Spearman’s rank correlation test rs = -0.246, P < D52- or D53-expressing cell line were visually scored for both 0.001, n = 665 for D52 and rs = -0.308, P < 0.001, n = 502 for D52 or D53 expression (categorized as undetectable, low, D53; Fig. 2C and D). Future studies will be required to moderate, or high) and actin stress fibers (categorized as none/ determine whether D52 and D53 expression reduce fibroblast poor, moderate, or prominent). Cell area (Am2) was calculated cell area by altering actin stress fibers or whether this occurs using Image ProPlus software, and measurements from through another mechanism. individual D52 or D53 cells were grouped. Comparing median D52 expression increases proliferation and anchorage-indepen- cell areas according to D52 or D53 expression status dent growth in 3T3 cells. As previous analyses have identified (undetectable/low versus moderate/high) indicated that mod- D52 as a positive regulator of cell proliferation (26, 28), D52, erate/high D52 or D53 expressing cells were significantly D53, and vector control cell lines were compared using 3-(4,5- smaller than cells with low/undetectable D52 or D53 levels dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays (Mann-Whitney U test, P < 0.001, n = 665 for D52 and P < over 4 days. Assays were carried out in triplicate for each cell 0.001, n = 502 for D53; Fig. 2A and B). Cell area was also line on four independent occasions, with the results for significantly inversely correlated with D52 or D53 expression individual D52, D53, and vector cell lines being grouped for category (Spearman’s rank correlation test rs = -0.455, P < analyses. These analyses indicated significantly increased 0.001, n = 665 for D52 and rs = -0.326, P < 0.001, n = 502 for proliferation in D52-expressing but not D53-expressing cell D53), but the overall median cell areas of D52- versus D53- lines at 3 and 4 days, relative to vector controls (Fig. 3A and B). transfected cells were not significantly different (Mann-Whitney Colony formation assays were also carried out for all D52, D53,

Fig. 3. D52-expressing 3T3 cell lines show increased proliferation and growth in soft agar relative to vector controls. Graphs showing the results of 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (A and B) or colony formation assays (C and D)ofD52-expressing(A and C) or D53-expressing (B and D)cellsrelativeto vector controls. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were done for 6 D52-expressing cell lines, 5 D53 expressing cell lines, and 3 vector controls in triplicate over 4 d on four separate occasions. Soft-agar assays (soft-agar colony counts in 20 random fields at Â100 magnification) were done for the same cell lines in duplicate on three separate occasions. MeanF SE of individual cell lines from all experiments, grouped according to transfection status. *,P < 0.05; **, P < 0.001, Student’s t test.

www.aacrjournals.org 5055 Clin Cancer Res 2008;14(16) August 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Human Cancer Biology

Fig. 4. A, reduced D52 or D53 protein levels through transient transfection of siRNAs targeting D52 or D53 transcripts in MCF-7 cells.To p, siRNAs employed for each transfection as follows: si2-1, si2-2, si2-3 target D52; si3-2, si3-3, si3-4 target D53; siGAPDH targets GAPDH; siControlrepresents a nontargeting siRNA. TKO represents transfection reagent-treated cells and MCF-7 indicates nontransfected cells. Right, proteins detected in Western blot analyses, with GAPDH being used to confirm GAPDH knockdown where relevant or as a loading control. Left, positions of molecular weight standards (kDa). Representative of those obtained in four independent experiments. B, reduction of D52 expression is associated with reduced adherent MCF-7 and SK-BR-3 cell numbers. Bright-field images are shown of MCF-7 cells treatedwith individual D52, D53, or nontargeting siRNA orTKO transfection reagent only and fixed and stained with crystal violet after 48 h. Representative of those obtained in three independent experiments done in duplicate. Magnification, Â50. C, siRNA and TKO transfection reagent-treated cells were analyzed using Metamorph to quantify crystal violet stained cells at 48 h post-transfection in MCF-7 and SK-BR-3 cells, with three random images from each well being analyzed. MeanF SE of three independent experiments done in duplicate. *, P < 0.05; **, P < 0.001, Student’s t test. D, reduced D52 expression in SK-BR-3 cells is associated with increased apoptosis. SK-BR-3 and MCF-7 cells were treated with three differentD52 or D53 siRNAs, nontargeting siRNA (siControl), orTKO transfection reagent only and apoptosis assays were done 48 h post transfection. Comparing the combined results obtained for all D52 siRNA-treated SK-BR-3 cells showed increased apoptosis compared with that occurring in siControl-treated cells (**,P < 0.01, Student’s t test). Mean F SE of three independent experiments done in duplicate.

and vector control cell lines in duplicate on three separate control cell lines were grouped for analyses. This indicated occasions. After 14 days of growth, all visible colonies greater that D52- but not D53-expressing cell lines produced signifi- than 10 cells were counted in 20 random fields, and the cumu- cantly more colonies than vector controls (P < 0.001; Fig. 3C lative results from all experiments using D52, D53, and vector and D). Scratch wound-healing assays were subsequently

Clin Cancer Res 2008;14(16) August 15, 2008 5056 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. SpecificTPD52 Targeting in Breast Cancer carried out in all cell lines, but these did not reveal significant by GAPDH or nontargeting siRNA or TKO treatment only. differences in migration at 12 h post-wounding in four Reduced D52 levels were not associated with reduced D53 independent experiments (data not shown). levels or vice versa (Fig. 4A; data not shown). Reduced D52 levels were associated with reduced adherent When MCF-7 and SK-BR-3 cells transfected with D52 and MCF-7 and SK-BR-3 cell numbers and increased apoptosis in D53 siRNAs, crystal violet assays revealed reduced adherent cell SK-BR-3 cells. Endogenous D52 or D53 levels were transiently numbers 48 h post-transfection (Fig. 4B). Areas of crystal violet reduced in MCF-7 and SK-BR-3 breast cancer cells, which staining were quantified using Metamorph, and data from the detectably express both proteins (18). The SK-BR-3 cell line is three D52 and D53 siRNAs were pooled and compared with also amplified at the D52 locus (4, 6) and expresses high D52 that obtained from nontargeting siControl transfections. There levels (4, 6, 18). Three different D52 and D53 siRNAs were were significantly fewer adherent cells in D52 siRNA-treated employed as well as a siRNA targeting GAPDH as a positive MCF-7 and SK-BR-3 cells compared with nontargeting siCon- control and a non-targeting siRNA, and transfection reagent- trol-transfected cells (Student’s t test, P < 0.001; Fig. 4C). In treated cells were employed as negative controls (Fig. 4). At 48 contrast, reduced D53 levels were associated with reduced to 96 h after transfection, D52 or D53 protein levels were adherent cell numbers in MCF-7 cells only (Student’s t test, P < reduced by treatment with relevant siRNAs but were unaffected 0.05; Fig. 4C). As reduced cell numbers may reflect increased

Fig. 5. A,(1- 3 ), immunohistochemicaldetection of D52 ( black) within paraffin-embedded breast tissue core sections; (1, 2 ), high-level D52 staining within cancer cells in a ductalcarcinoma from a 66-year-oldpatient ( 1) and in a ductalcarcinoma in situ from a 62-year-old patient (2); (3), low-level D52 staining within cancer cells in a ductalcarcinoma from a 60-year-oldpatient; ( 4), detection of D52 in paraffin-embedded LnCaP prostate cancer cells included on tissue arrays as controls. D52 staining was highest in LnCaP cells relative to other breast and prostate cancer cell lines examined, including SK-BR-3 cells (data not shown). Sections were counterstained with Nuclear Fast Red. B to D, Kaplan-Meier plots showing that (B) high D52 expression was significantly associated with reduced overall patient survival in the overall breast carcinoma cohort (log-rank test, P < 0.001; n = 357). Different associations between D52 expression status and survivalwere measured in patients with ER-positive (C) versus ER-negative (D) tumors (log-rank test, P = 0.284, n =265ER-positivepatients;P < 0.001, n = 85 ER-negative patients). High D52 expression (black)was defined as >70,000 strong pixels of immunohistochemical staining within a tissue core. Gray, all other tumors (D52 low).Xaxis, overall patient survival (in months).

www.aacrjournals.org 5057 Clin Cancer Res 2008;14(16) August 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Human Cancer Biology cell death, we determined whether decreased D52 or D53 Discussion expression in MCF-7 and/or SK-BR-3 cells was associated with increased apoptosis using the Cell Death Detection ELISAPLUS This study was carried out to investigate whether D52 kit. Reduced D52 expression in SK-BR-3 cells was associated overexpression in breast cancer passively reflects its gene with significantly increased apoptosis at 48 h post-transfection location at chromosome 8q21.13 or specifically reflects relative to siControl-transfected cells (Student’s t test, P < 0.01; nonredundant functional properties of the D52 protein. Direct Fig. 4D). Although there was a similar trend in MCF-7 cells, this comparison of D52-like gene expression in breast tissue samples was not significant (Fig. 4D). Significant increases in apoptosis confirmed that genes overexpressed in breast cancer map to were not measured in D53 siRNA-transfected MCF-7 or SK- amplified genomic regions. We therefore directly compared the BR-3 cells (Fig. 4D). cellular functions of D52 and D53, representing genes that are D52 and MAL2 expression using immunohistochemical analysis and are not overexpressed in breast cancer, respectively. of breast tissue microarrays. As D52 and MAL2 overexpression Expressing D52 and D53 proteins in 3T3 cells produced both in cancer has been reported previously (6, 22–24), and these shared and distinct phenotypes. Common phenotypes included genes were most significantly overexpressed in breast carcinoma smaller, rounder cells with reduced actin stress fibers, and we in the present study (Table 1), we carried out immunohisto- also noted significant positive correlations between in situ D52 chemical analyses of D52 and MAL2 expression in a large or D53 and cyclin B1 expression (data not shown), as reported cohort of normal breast, in situ, and invasive carcinomas in for endogenous D53 expression in MCF-7 cells, and exogenous tissue microarray format. Both D52 and MAL2 immunoreac- D53 expression in MDA-MB-231-derived D53-H1 cells (32). tivity were detected predominantly in the cytoplasm as reported Distinct phenotypes represented increased proliferation and previously for in situ D52 expression in breast cancer samples anchorage-independent growth in D52-expressing cell lines (6). As predicted, compared with strong pixel counts measured only, in agreement with the findings of Lewis et al. (28), where in normal breast tissue cores, pixel counts were significantly mouse D52 was expressed in 3T3 cells, and of previous studies elevated in invasive breast tumors (Wilcoxon signed rank test, where the D52 isoform PC-1/PrLZ was expressed in different P < 0.001, n = 176 for D52 and n = 148 for MAL2) and in situ cell types (26, 29). A common limitation of the present and carcinomas (Wilcoxon signed rank test, P < 0.001, n = 95 for previous studies is the use of fibroblast cell lines, and analyses D52 and n = 82 for MAL2) from the same patients. of D52 overexpression in a breast cell line such as MCF10A may Cut-point determination using expression data distributions be more informative in terms of the role of D52 role in breast and decile analyses indicated that it was most relevant to cancer. However, the present study extends previous findings by compare the top 20% of tumors according to D52 or MAL2 showing that D53 expression does not similarly increase strong pixel counts to all other tumors (data not shown). We proliferation and anchorage-independent growth. Increased approximated this cut point to 70,000 pixels (top 20.4% of proliferation in response to D52 expression is therefore likely to tumors) for D52 and to 100,000 pixels (top 20.6% of tumors) be mechanistically independent of the morphologic phenotype for MAL2. Use of these cut points indicated that high in situ induced by both proteins. D52expression(Fig.5A),butnothighin situ MAL2 Transient knockdown of D52 and D53 protein expression in expression, was strongly associated with reduced overall MCF-7 and SK-BR-3 cells reinforced the existence of nonredun- patient survival (log-rank test, P < 0.001, n = 357; Fig. 5B). dant functions for these proteins in breast cancer cells. Notably, Significant associations between D52 expression and reduced reduced D52 levels were associated with significantly increased overall survival were also measured when cut points of apoptosis in SK-BR-3 cells only, which highly overexpress D52 100,000 and 50,000 pixels were employed (data not shown), in response to gene amplification (4, 6). This finding supports indicating a possible dose-response relationship between those of previous studies where reduced expression of other in situ D52 expression and patient survival. High D52 oncogenes increased apoptosis in amplified or overexpressing expression was significantly associated with reduced overall cell lines only (33–35). survival in patients with ER-negative tumors (log-rank test, P < Of the genes analyzed in the present study, D52 and MAL2 0.001, n = 85 ER-negative patients; P = 0.284, n = 265 ER- were maximally overexpressed in breast cancer and also showed positive patients; Fig. 5C and D) in node-positive patients (log- significantly positively correlated gene or protein expression rank test, P = 0.005, n = 113 node-positive patients; P = 0.393, values (data not shown). We therefore examined and compared n = 244 node-negative patients) and in patients with high- the clinical significance of D52 and MAL2 expression in a large grade tumors (histologic grade 3; log-rank test, P = 0.008, n = cohort of human breast cancers, applying digital image analysis 104 grade 3 tumors; P = 0.191 n = 247 grade 1/2 tumors). The to identify tumor subpopulations with high D52 and MAL2 proportions of tumors with high D52 expression also expression. These analyses showed that high D52 but not MAL2 significantly differed according to tumor grade, with 8 of 88 expression was associated with reduced overall survival in the (9%) grade 1 tumors, 29 of 159 (18%) grade 2 tumors, and breast cancer cohort examined, and particularly in ER-negative, 36 of 104 (35%) grade 3 tumors expressing high D52 levels node-positive, and high-grade tumor groups, and was an (Pearson’s m2 test, P < 0.001, n = 351). A significant positive independent predictor of patient survival. This indicates that correlation was also measured between strong D52 pixel high D52 expression may significantly promote further tumor counts and tumor grade (Spearman rank correlation coefficient progression in patients with ER-negative tumors and/or more rs = 0.244, P < 0.001, n = 351). Multivariate analyses identified aggressive disease at diagnosis. Overall survival analyses in high D52 expression as an independent predictor of survival patients grouped according to adjuvant treatment status did not (hazard ratio, 2.274; 95% confidence interval, 1.228-4.210; identify high D52 expression as predicting response to P = 0.009; n = 328) after adjustment for age at diagnosis, node radiotherapy, chemotherapy, or hormone therapy (data not and ER status, and tumor grade. shown). We therefore hypothesize that high D52 expression

Clin Cancer Res 2008;14(16) August 15, 2008 5058 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. SpecificTPD52 Targeting in Breast Cancer contributes to a more aggressive tumor phenotype, which is narrow gene amplification peaks at 81 Mb, corresponding to also supported by the fact that high D52 expression was the position of the D52 locus (2–5). Previous associations significantly more frequent in grade 3 tumors. High D52 between chromosome 8q21 gain in breast cancer and poor expression in breast tumors may therefore represent a clinically patient outcome (37–39) may therefore reflect high D52 useful marker following development of a reliable semiquan- expression in tumors. These combined data suggest that titative immunohistochemical assay (36). reducing D52 expression or inhibiting D52 function may Several studies have now linked poor outcome or adverse improve outcomes in patients with D52-amplified or D52- breast cancer histology with the gain of chromosome 8q21 overexpressing breast cancer and possibly in other cancer types (37–39), and D52 has been included in signatures associated where D52 is overexpressed. with adverse prognosis in breast (40, 41) and prostate cancer (42) and reduced survival in mantle cell lymphoma (43). Disclosure of Potential Conflicts of Interest Proliferation genes are emerging as a common driving force in prognostic cancer gene signatures (44). As D52 positively No potentialconflicts of interest were disclosed. regulates cell proliferation and anchorage-independent growth (26, 28, 29), the reduced survival associated with high D52 Acknowledgments expression in breast cancer likely reflects this contributing to a more proliferative and aggressive cancer phenotype. The We thank Prof. Peter Gunning (The Children’s Hospital atWestmead) for support and for providing the PG307 expression vector, Drs. Daniel Catchpoole, Geraldine identification of nonredundant cancer-promoting properties O’Neill, and Rosemary Balleine (Westmead Millennium Institute) for scientific for D52 also support the specific targeting of D52 expression in discussions, Angela Bailey and Jayne Hardy for excellent technical assistance, and cancer, as do the results of recent mapping studies indicating Jill Tinning (RPH) for providing clinical data associated with breast tissue arrays.

References 1. MyllykangasS,HimbergJ,BohlingT,NagyB, protein in pancreatic acinar cells. J Biol Chem 22. Pollack JR, SÖrlie T, Perou CM, et al. Microarray Hollmen J, Knuutila S. DNA copy number amplifica- 2002;277:35496 ^ 502. analysis reveals a major direct role of DNA copy num- tion profiling of human neoplasms. Oncogene 2006; 12. Tiacci E, Orvietani PL, Bigerna B, et al.Tumor protein ber alteration in the transcriptional program of human 25:7324 ^ 32. D52 (TPD52): a novel B-cell/plasma-cell molecule breast tumors. Proc NatlAcad Sci USA 2002;99: 2. vanDuinM,vanMarionR,VissersK,etal.High- with unique expression pattern and Ca(2+)-depen- 12 9 6 3 ^ 8 . resolution array comparative genomic hybridization of dent association with Annexin VI. Blood 2005;105: 23. Paik S, Kim C-Y,SongY-K, KimW-S.Technology in- chromosome arm 8q: evaluation of genetic progres- 2812^ 20. sight: application of molecular techniques to formalin- sion markers for prostate cancer. Genes Chromo- 13. Largo C, Alvarez S, Saez B, et al. Identification of fixed paraffin-embedded tissues from breast cancer. somes Cancer 2005;44:438 ^ 49. overexpressed genes in frequently gained/amplified NatClinPractOncol2005;2:246^54. 3. Hicks J, Krasnitz A, Lakshmi B, et al. Novel patterns chromosome regions in multiple myeloma. Haemato- 24. Heinzelmann-Schwarz VA, Gardiner-Garden M, of genome rearrangement and their association with logica 2006;91:184 ^ 91. Henshall SM, et al. Overexpression of the cell adhe- survivalin breast cancer. Genome Res 2006;16: 14. Loukopoulos P, Shibata T, Katoh H, et al. sion molecules DDR1, Claudin 3, and Ep-CAM in 14 6 5 ^ 79. (2007). Genome-wide array-based comparative metaplastic ovarian epithelium and ovarian cancer. 4. Rodriguez V, Chen Y, Elkahloun A, Dutra A, Pak E, genomic hybridization analysis of pancreatic ade- Clin Cancer Res 2004;10:4427 ^ 36. Chandrasekharappa S. BAC array nocarcinoma: identification of genetic indicators 25. ChenSL,ZhangXK,HalversonDO,etal.Character- comparative genomic hybridization and expression that predict patient outcome. Cancer Sci 2007; ization of human N8 protein. Oncogene 1997;15: analysis identify amplification and overexpression of 98:392^400. 2577 ^88. TRMT12 in breast cancer. Genes Can- 15. Skotheim RI, Autio R, Lind GE, et al. Novel genomic 26. Chang XT, Liang RX, Zhou JG, et al. Malignant cer 2007;46:694 ^ 707. aberrations in testicular germ cell tumors by array- transformation of NIH3T3 cells induced by ectopic ex- 5. Kim JH, Dhanasekaran SM, Mehra R, et al. Integra- CGH, associated gene expression changes. Cell Oncol pression of PC-1 gene [Chinese]. Zhonghua Bing Li tive analysis of genomic aberrations associated with 2006;28:315^ 26. Xue Za Zhi 2005;34:42^ 6. prostate cancer progression. Cancer Res 2007;67: 16. Jo« nsson G, Staaf J, Olsson E, et al. High-resolution 27. Zhou LQ, Zhang H, Gao XS, et al. Impact of PC-1 8229^ 39. genomic profiles of breast cancer cell lines assessed gene knockdown on the biological action of prostate 6. Balleine R, Schoenberg Fejzo M, Sathasivam P, by tiling BAC array comparative genomic hybridiza- cancer cell line C4-2 [Chinese]. Zhonghua Nan Ke Basset P, Clarke C, Byrne JA. The hD52 (TPD52) tion. Genes Chromosomes Cancer 2007;46: Xue 2005;11:256^60. gene is a candidate target gene for events resulting in 543^58. 28. Lewis JD, Payton LA, Whitford JG, et al. Induction increased 8q21copy number in human breast carcino- 17. Byrne JA, Nourse CR, Basset P, Gunning P.Identifi- of tumorigenesis and metastasis by the murine ortho- ma. Genes Chromosomes Cancer 2000;29:48 ^ 57. cation of homo- and heteromeric interactions be- logue of tumor protein D52. Mol Cancer Res 2007;5: 7. RubinMA,VaramballyS,BeroukhimR,etal.Over- tween members of the breast carcinoma-associated 13 3 ^ 4 4. expression, amplification, and androgen regulation of D52 protein family using the yeast two-hybrid system. 29. Zhang H,Wang J, Pang B, et al. PC-1/PrLZ contrib- TPD52 in prostate cancer. Cancer Res 2004;64: Oncogene 1998;16:873 ^ 81. utes to malignant progression in prostate cancer. Can- 3814^ 22. 18. Boutros R, BaileyAM,Wilson SH, ByrneJA. Alterna- cer Res 2007;67:8906 ^ 13. 8. Wang R, Xu J, Sarama« ki O, et al. PrLZ , a novel tive splicing as a mechanism for regulating 14-3-3 30. Bie' che I, Tozlu S, Girault I, Lidereau R. Identifica- prostate-specific and androgen-responsive gene of binding: interactions between hD53 (TPD52L1) and tion of a three-gene expression signature of poor- the TPD52 family, amplified in chromosome 8q21.1 14-3-3 proteins. J MolBiol2003;332:675 ^ 87. prognosis breast carcinoma. MolCancer 2004; and overexpressed in human prostate cancer. Cancer 19. RualJF,Venkatesan K, Hao T, et al.Towards a pro- 3:37. Res 2004;64:1589^94. teome-scale map of the human protein-protein inter- 31. Qin H, Gunning P.The 30-end of the human h-actin 9. Byrne JA, Balleine RL, Schoenberg Fejzo M, et al. action network. Nature 2005;437:1173 ^ 8. gene enhances activity of the h-actin expression vec- Tumor protein D52 (TPD52) is overexpressed and a 20. Wilson SH, Bailey AM, Nourse CR, Mattei MG, tor system: construction of improved vectors. J Bio- gene amplification target in ovarian cancer. Int J Can- Byrne JA. Identification of MAL2, a novelmember of chem Biophys Methods 1997;36:63 ^ 72. cer 2005;117:1049 ^ 54. the malproteolipid family, through interactions with 32. Boutros R, Byrne JA. D53 (TPD52L1) is a cell 10. Boutros R, Fanayan S, Shehata M, Byrne JA. The TPD52-like proteins in the yeast two-hybrid system. cycle-regulated protein maximally expressed at the tumor protein D52 family: many pieces, many puz- Genomics 2001;76:81 ^ 8. G2-M transition in breast cancer cells. Exp Cell Res zles. Biochem Biophys Res Commun 2004;325: 21. Cao Q, Chen J, Zhu L, et al. A testis-specific and 2005;310:152^ 65. 1115 ^ 2 1. testis developmentally regulated tumor protein D52 33. Kao J, Pollack JR. RNA interference-based func- 11. Thomas DD, Kaspar KM, Taft WB, Weng N, (TPD52)-like protein TPD52L3/hD55 interacts with tionaldissection of the 17q12 amplicon in breast can- Rodenkirch LA, Groblewski GE. Identification of TPD52 family proteins. Biochem Biophys Res Com- cer reveals contribution of coamplified genes. Genes Annexin VI as a Ca2+-sensitive CRHSP-28-binding mun 2006;344:798 ^ 806. Chromosomes Cancer 2006;45:761 ^ 9.

www.aacrjournals.org 5059 Clin Cancer Res 2008;14(16) August 15, 2008 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Human Cancer Biology

34. ParkJT,LiM,NakayamaK,etal.Notch3geneam- tures and prognosis. A study of 305 tumors by transcriptionalsignatures in cancer. Nat Genet 2006; plification in ovarian cancer. Cancer Res 2006;66: comparative genomic hybridization. Cancer Res 38:421^30. 6312^8. 2003;63:8861^8. 41. Liu R,Wang X, Chen GY,et al.The prognostic role of 35. LutterbachB,ZengQ,DavisLJ,etal.Lungcancer 38. Melchor L, Alvarez S, Honrado E, et al. The ac- a gene signature from tumorigenic breast-cancer cells. cell lines harboring METgene amplification are depen- cumulation of specific amplifications characterizes N EnglJ Med 2007;356:217^ 26. dent on Met for growth and survival. Cancer Res two different genomic pathways of evolution of 42. Bismar TA, Demichelis F, Riva A, et al. Defining 2007;67:2081 ^ 8. familial breast tumors. Clin Cancer Res 2005;11: aggressive prostate cancer using a 12-gene model. 36. PhillipsT, Murray G,Wakamiya K, et al. Development 8577^84. Neoplasia 2006;8:59 ^ 68. of standard estrogen and progesterone receptor im- 39. Han W, Han MR, Kang JJ, et al. Genomic alterations 43. Ma S. Principalcomponent analysis in linear regres- munohistochemicalassays for selection of patients identified by array comparative genomic hybridization sion survivalmodelwith microarray data. J Data Sci for antihormonaltherapy. ApplImmunohistochem as prognostic markers in tamoxifen-treated estrogen 2007;5:183 ^98. MolMorphol2007;15:325^ 31. receptor-positive breast cancer. BMC Cancer 2006; 44. Sotiriou C, Piccart MJ. Taking gene-expression 37. Rennstam K, Ahlstedt-Soini M, Baldetorp B, et al. 6:92. profiling to the clinic: when will molecular signatures Patterns of chromosomalimbalances defines sub- 40. Adler AS, Lin M, Horlings H, Nuyten DS, van de become relevant to patient care? Nat Rev Cancer groups of breast cancer with distinct clinical fea- Vijver MJ, Chang HY.Genetic regulators of large-scale 2007;7:545 ^ 53.

Clin Cancer Res 2008;14(16) August 15, 2008 5060 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. Nonredundant Functions for Tumor Protein D52-Like Proteins Support Specific Targeting of TPD52

Mona Shehata, Ivan Bièche, Rose Boutros, et al.

Clin Cancer Res 2008;14:5050-5060.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/14/16/5050

Cited articles This article cites 44 articles, 15 of which you can access for free at: http://clincancerres.aacrjournals.org/content/14/16/5050.full#ref-list-1

Citing articles This article has been cited by 10 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/14/16/5050.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/14/16/5050. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research.