Cancer Investigation
ISSN: 0735-7907 (Print) 1532-4192 (Online) Journal homepage: http://www.tandfonline.com/loi/icnv20
Downregulation of HOXC6 in Serous Ovarian Cancer David L. Tait, Zahra Bahrani-Mostafavi, C. Greer Vestal, Christine Richardson & M. Taghi Mostafavi To cite this article: David L. Tait, Zahra Bahrani-Mostafavi, C. Greer Vestal, Christine Richardson & M. Taghi Mostafavi (2015) Downregulation of HOXC6 in Serous Ovarian Cancer, Cancer Investigation, 33:7, 303-311, DOI: 10.3109/07357907.2015.1041641 To link to this article: http://dx.doi.org/10.3109/07357907.2015.1041641
Published online: 05 Jun 2015.
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Cancer Investigation, 33:303–311, 2015 ISSN: 0735-7907 print / 1532-4192 online C 2015 Informa Healthcare USA, Inc. Copyright DOI: 10.3109/07357907.2015.1041641
Downregulation of HOXC6 in Serous Ovarian Cancer David L. Tait,1 Zahra Bahrani-Mostafavi,2 C. Greer Vestal,3 Christine Richardson,3 and M. Taghi Mostafavi2
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Carolinas Medical Center, Levine Cancer Institute, Division of Gynecologic Oncology, Charlotte, North Carolina, USA,1 UNC-Charlotte, College of Computing and Informatics, Charlotte, North Carolina, USA,2 UNC-Charlotte, Department of Biological Sciences, Charlotte, North Carolina, USA3 gets or as biomarkers. These proteins are members of multiple super families characterized by their DNA binding domain motifs. One group of transcription factors that has become a subject of interest in cancer biology is the human homeobox class I (HOX) family. There are 39 HOX genes clustered on four chromosomal loci in humans, and expression of each HOX gene is tightly regulated (4). During development, HOX genes within each of the four clusters are expressed in sequence along the chromosome as the spatial arrangement of the developing tissue progresses from anterior to posterior. These genes direct cell differentiation and maintenance of cell identity and morphology from early embryonic development throughout adulthood. HOX proteins can act as either activators or repressors of transcription, and the role of individual HOX genes as either oncogenes or tumor suppressors in different cancers is well established (5). Research has shown altered expression levels of multiple HOX genes in cancers, including endometrial, cervical, pancreatic, thyroid, and lung (6–11). HOXC6 is expressed as multiple mRNAs that differ in their 5 regions but share the same DNA-binding homeodomains (12, 13). Transcripts co-exist in multiple cell types and tissues to include fibroblasts, skin, breast epithelium, stem cells of thymus, as well as myeloid progenitors of bone marrow, spinal cord, and oocytes (12–17). It has been reported that HOXC6 is regulated by MLL2, MLL3, hormones (i.e. estrogen and Lsh), and TGF (17–20). HOXC6 regulates genes with both oncogenic and tumor suppressor activities as well as epigenetic effects, regulating several genes such as CD44, CNTN1, DKK3, W1F1, MDR1, Bcl2, and the PI3K/AKT pathway (18–22). The activity of HOXC6 in ovarian development or maintenance has not been studied, although homozygous knockout HOXC6 −/− female mice are fertile (17). Elevated HOXC6 expression has been reported in meduloblastomas, osteosarcomas, breast, lung, gastrointestinal, head and neck squamous and prostate carcinomas, leukemia as well as normal trophoblast (4, 23–27). However,
Homeobox (HOX) genes encode transcription factors critical to morphogenesis and cell differentiation. Although dysregulation of several HOX genes in ovarian cancer has been reported, little is known about HOXC6 expression in epithelial ovarian cancer. In this report, analysis of laser capture microdissected samples determined HOXC6 expression patterns in normal versus malignant serous ovarian carcinoma tissues. HOXC6 protein was quantified by ELISA in parallel serum samples and further validated in a larger cohort of serum samples collected from women with and without serous ovarian carcinoma. These data demonstrate significant downregulation of HOXC6 in serous ovarian cancer. Keywords: Biomarker discovery, Homeobox genes, HOXC6, Serous ovarian cancer
INTRODUCTION Ovarian cancer is the most lethal gynecologic cancer and the fifth leading cause of cancer death for women in the United States. In 2014, the American Cancer Society estimated an annual diagnosis of more than 21,980 cases, with over 14,000 deaths (1). When diagnosed in early stages, 5year survival rates reach 90%; however, due to the lack of symptoms during the early phase of tumorigenesis, 75% of patients are diagnosed at later stages. Survival for these advanced stage patients is only 30%–40% (2). Understanding of the gene expression patterns and mechanisms associated with the pathogenesis of ovarian cancer are expected to lead to the development of new diagnostic and therapeutic modalities. Signaling pathways of tumor development have been extensively characterized leading to the elucidation of major paradigms in cancer biology and malignant transformation. Transcription factors are the final downstream effectors of signaling pathways localized in the nucleus (3) and thus they are an important focus of cancer research as therapeutic tar-
Correspondence to: M. Taghi Mostafavi, PhD, College of Computing and Informatics, University of North Carolina at Charlotte, Charlotte, NC 28223, USA. E-mail: [email protected] Received 27 November 2013; revised 28 February 2015; accepted 08 April 2015.
Downloaded by [Boston University] at 01:15 20 October 2015
D. L. Tait et al.
Figure 1. Transcriptional microarray analysis of HOX genes in serous ovarian carcinoma. Shown are relative expression levels of 37 HOX genes from three non-malignant ovarian and seven malignant ovarian samples. Unsupervised clustering analysis (hierarchical tree shown on left side) separated normal samples (NE, red bar) from malignant tumor samples (MT, orange bar).
little is known about HOXC6 expression or its activity in ovarian cancer (28–30). In an initial transcriptional microarray screen (n = 65) of ovarian cancer tissue samples, we noted that HOX genes were dysregulated, and HOXC6 was significantly down- regulated (28). To further characterize the expression of the HOXC6 gene in serous ovarian cancer, we now report evaluation of micro-dissected serous ovarian cancer and normal ovarian tissue samples as well as serum from individuals with and without ovarian cancer. We utilized DNA exon microarray, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) to analyze tissue and serum samples from women with normal ovaries and serous ovarian carcinomas. These studies demonstrate sustained high levels of HOXC6 in normal surface epithelial cells. Conversely, we describe this first report of significant down-regulation of HOXC6 gene and protein expression in patients with serous ovarian cancer. MATERIALS AND METHODS Tissue and serum collection Ovarian tissue specimens were obtained during surgery from patients with ovarian cancer or other gynecologic conditions according to an IRB-approved protocol at Carolinas Medical Center. All available patient data is presented in Table 1. All patients with serous carcinoma were stage III and IV, grade 3 and received platinum and taxane based chemotherapy after surgery. Tissue samples were placed in a R cryomold (Sakura Finetek USA, standard sized Cryomold Inc., Torrance, CA), covered with Optimal Cutting Temperature (OCT) compound (Sakura Finetek USA, Inc., Torrance, CA), frozen and stored at −80◦ C. Originally seven malignant matched and 11 non-malignant blood serum samples were R SSTTM serum separatcollected by using a BD Vacutainer
ing tube (BD Biosciences, San Jose, California) according to the manufacturer’s protocols, and stored at −80◦ C. To further validate the ELISA findings, 32 additional other serum samples were collected from pre- and postmenopausal women without ovarian cancer, and serum from an additional 14 patients with stage III and IV, grade 3 serous carcinoma of the ovary or fallopian tube—total of 21 malignant and 43 nonmalignant serum samples. Laser capture micro-dissection (LCM) OCT-embedded samples were serially sectioned into 8 μm R ∗/Plus Microscope sections using Fisherbrand Superfrost slides (Fisher Scientific, Pittsburgh, PA) by a Leica CM 1850 UV Cryostat (Leica Microsystems Inc., Bannockburn, IL). R LCM Sections were prepared for LCM using Histogene Frozen Section Staining kit (Applied Biosystems, Life Technologies, Co., Carlsbad, CA) according to the manufacturer’s protocols. After staining, samples were immediately microR R PixCell IIe LCM (Molecular Dedissected by an Arcturus vices, LLC, Sunnyvale, CA). Normal epithelium and tumor cells were separately collected from appropriate sections. The residual slide material was used to determine RNA quality (31). RNA preparation RNA extraction of LCM captured cells, and the residual slide R RNA Isolamaterials were performed using a PicoPure tion kit (Applied Biosystems, LifeTechnologies, Co., Carlsbad, CA) according to manufacturer’s protocols. RNA integrity was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) as described by the manufacturer (31). cDNA synthesis and amplification Complimentary DNA (cDNA) generation and amplification was performed using the Whole Transcriptome Cancer Investigation
HOXC6 in Serous Ovarian Cancer
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Table 1. Characteristics of Malignant Samples Used Sample #
Stage
Age
Collected Sample
Tumor Site
Assay
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21
III-B III-C III-C III-C III-C III-C III-C III-C III-C III-C III-C IV III-C III-C III-C III-C IV III-C IV III-B III-C
75 54 74 64 54 57 71 43 64 63 81 75 69 62 62 81 68 80 75 44 76
Tissue & Serum Tissue & Serum Tissue & Serum Tissue & Serum Tissue & Serum Tissue & Serum Tissue & Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Fallopian Tube Ovary Fallopian Tube Fallopian Tube Fallopian Tube Ovary Ovary Ovary Fallopian Tube Ovary Ovary
Microarray (MA), ELISA MA, IHC, ELISA MA, qPCR, IHC, ELISA MA, qPCR, IHC, ELISA MA, qPCR, IHC, ELISA MA, IHC, ELISA MA, IHC, ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA
All samples are high-grade serous carcinoma.
WT-OvationTM Pico RNA Amplification System Kit (NuGEN Technologies Inc., San Carlos, CA), according to manufacturer’s protocols. cDNA was quantified using a NanoDrop 1000 spectrophotometer (NanoDrop Products, Wilmington, DE) and used for microarray sample preparation and qPCR confirmation assays. Exon microarray sample preparation and hybridization Approximately 3 μg of single primer isothermal amplified (SPIA) cDNA was used to continue to sense–strand cDNA (ST-cDNA) conversion using the WT-OvationTM Exon Module (NuGEN Technologies Inc., San Carlos, CA). 5 μg ST-cDNA was fragmented and labeled with FLOvationTM cDNA Biotin Module V2 kit (NuGEN TechnoloR gies Inc., San Carlos, CA) then hybridized using GeneChip Affymetrix Human Exon 1.0 ST arrays (Affymetrix, Inc., Santa Clara, CA). Microarray hybridization was performed R R Hybridization Oven 640, GeneChip using a GeneChip R Fluidics Station 450, and GeneChip Scanner 3000 7G with Autoloader (Affymetrix, Inc., Santa Clara, CA). For the In Silico Quality Control, each array passed preliminary quality control including assessment of spike-in controls and the total distribution of intensities compared to manufacturer’s criteria (31). Quantitative RT-PCR RNA from primary ovarian tissue samples was prepared as described above. Normal ovarian surface epithelial cell line HOSE 6–3 (32) was used as reference sample. Ovarian cancer cell lines SKOV-3 and Caov-3, and other selected cell lines such as SW626, MDA-MB-231 [Breast], and HeLa [Cervix] (American Type Culture Collection, Manassas, VA) were used as cell culture model for this study. R Total RNA was isolated from 106 –107 cells using TRIzol Reagent (Invitrogen Co, Carlsbad, CA). Extracted RNA was C 2015 Informa Healthcare USA, Inc. Copyright
purified using RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). 50 ng RNA was reverse transcribed to cDNA in 20 μL total volume using QuantiTect Reverse Transcription Kit (Qiagen Inc., Valencia, CA). 50 ng cDNA was amplified R qPCR primers for HOXC6 using 500 nm of PrimeTime [Hs.PT.51.3113294)] and GAPDH [Hs.PT.51.2918858.g] (Integrated DNA Technologies Inc., San Jose, CA). GAPDH was used to normalize expression data. Primers were selected to amplify by real-time PCR the same transcripts as detected by microarray. Analysis of data was carried out according to the Comparative Ct method for relative quantitation of gene expression (33). Real-time quantitative PCR was carried out R 7500 Fast (ABI 7500 Fast) using the Applied Biosystems Real-Time PCR System (Applied Biosystem, Carlsbad, CA), R SYBR Green PCR kit (Qiagen, Inc., Vaand the QuantiTect lencia, CA) according to the manufacturer’s protocols. The ABI 7500 Fast instrument was operated under the following thermal cycling conditions: Initial heat activation of 95◦ C for 15 min, followed by 40 cycles of denaturation of 94◦ C for 15 s, annealing of 60◦ C for 30 s, and extension of 72◦ C for 30 s. The PCR products were checked using ethidium bromide-stained 2% agarose gels. Both standard and primary tissue samples were assayed in triplicate. Immunohistochemistry Tissue sections were incubated in 3% H2 O2 for 5 min at room temperature and washed with PBS, then incubated for 45 min with 50% fetal bovine serum blocking solution. After removal of blocking solution, sections were incubated overnight at 4◦ C in a 1:250 dilution of goat-anti-human HOXC6 antibody (Santa-Cruz Biotechnology Inc., Santa Cruz, CA). Sections were washed and incubated with a 1:250 dilution of biotinylated rabbit anti-goat secondary antibody for 45 min at room temperature (R&D systems Inc., Minneapolis, MN). A streptavidin-enzyme conjugate (BD Biosciences,
D. L. Tait et al.
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San Jose, California) was added to the slides according to the manufacturer’s protocols. Specific signals were visualized by incubation with streptavidin HRP followed with adding diaminobenzidine (DAB) as a chromogen (BD Biosciences, San Jose, California). Counterstaining was performed with Mayer’s hematoxylin (Sigma-Aldrige, St. Louis, MO). Images were captured and stored digitally for analysis. Indirect sandwich enzyme-linked immunosorbent assay (ELISA) The developed and optimized protocol is summarized as follows: Wells of a micro-titer plate (Nunc, Roskilde, Denmark) were coated with 100 μL of anti-HOXC6 capture antibody (B-7; Santa Cruz Biotechnology, Inc., CA) at 1 μg/mL in coating buffer (pH 9.6) of 0.1 M carbonate/bicarbonate buffer (Sigma- Aldrich, St. Louis, MO). The plates were incubated overnight at 4◦ C followed by washing 2× with PBS (SigmaAldridge, St. Louis) and were blocked with 200 μL of blocking buffer of PBS −1% (w/v) bovine serum albumin (BSA). The plates were incubated 2 h at room temperature followed by washing 2× with PBS. A 1:100 dilution of patient’s serum was added and incubated for 2 h at room temperature. To create the standard curve for HOXC6 protein, the HOXC6 partial recombinant protein (Novus Biological, LLC, Littleton, CO) at 1 μg/mL was used. Plates were washed, and a 1:100 dilution of goat anti-human HOXC6 primary antibody (N-13; Santa-Cruz Biotechnology, Inc., Santa Cruz, CA) at 200 μg/mL was added, and incubated at room temperature for 2 h. Plates were washed 4× and a 1:1000 dilution of bovine anti-goat IgG-HRP secondary antibody (Santa Cruz Biological, Inc. Santa Cruz, CA) at 400 μg/mL was added and incubated for 2 h at room temperature. The plates were washed 4× and 100 μL of 3,3 ,5,5 -tetramethylbenzidine (TMB) enzyme substrate (Thermo Scientific Inc., Barrington, IL) added to each well, followed by incubation at room temperature. Color development was stopped by addition of 2 M sulfuric acid (Sigma-Aldridge, St. Louis). Optical density was read at 450 nm using MultiskanTM GO Microplate Spectrophotometer (Thermo Scientific, Inc., Barrington, IL) according to manufacturer’s protocols (34). Based on the standard curve, the serum samples were analyzed in triplicate. Statistical methods After microarray hybridization, raw signal was normalized and gene expressions were calculated using GC Robust Multiarray Average (GCRMA). Significant difference in expression was calculated between nontumorous tissue samples and malignant tumor samples using Analysis Of Variance (ANOVA) and p-values from ANOVA were modified to false discovery rate (FDR). Genes were considered having statistically significant differential expression between nontumorous tissue and malignant tumor states if their FDR pvalues were less than or equal to 0.05. The algorithms and R Genomics SuiteTM 6.6. For methods were run using Partek qRT-PCR analysis of HOXC6 gene expression in tissue samples, Microsoft Excel 2007 was used to perform comparative Ct method for quantification of all mRNA transcripts.
IHC results were observed by two independent researchers to identify level of HOXC6 expression in each sample. For ELISA unity-based normalization was performed using the lowest levels of HOXC6 found (as the lowest range) to highest level of HOXC6 found (as the highest range) while considering the data from standard curve of each plate. SAS version 9.3 was used to analyze the normalized data and to compare data from tumor vs. non-tumorous samples. Two-sided t-test was performed to analyze the data. All tests had a statistical significance set at a value of p < 0.05. RESULTS In an initial transcriptional microarray screen of 65 ovarian cancer tissue samples, significant patterns of HOX gene dysregulation were observed, and HOXC6 was significantly down regulated (28). To further characterize the expression of the HOXC6 gene in serous ovarian cancer, transcriptional microarray analysis was performed on seven serous ovarian carcinoma tissue samples and three normal ovarian surface epithelium samples (Fig. 1). Use of laser-capture microdissection (LCM) in this study ensured specific collection of target tumor cells from malignant ovarian tissues (MT) or normal epithelium cells from non-malignant ovarian tissues (NE) and thus increased sample purity. Samples were categorized into normal and malignant serous ovarian origins according to pathological diagnosis. All study subjects were postmenopausal women at the time of surgery with mean age of 66 (Table 1). The age groups for non-malignant samples at the time of collection were 1/3 < 50 and 2/3 >50 (data not shown). Expression analysis was performed using ANOVA model (35). Fisher’s Least Significant Difference contrast(s) method (36) was performed to compare samples. Based on our previous ovarian cancer gene expression study showing significant patterns of HOX gene dysregulation (28), we used this sample set to determine HOX gene expression patterns of the existing 37 HOX gene probe sets in Affymetrix Human Exon 1.0 ST array. We determined the fold-change expression between serous malignant and normal ovarian surface epithelium samples of selected genes from the HOX family (Fig. 1). The most significantly up-regulated genes include HOXB2 and HOXB3, and the most significantly down regulated gene is HOXC6 (Table 2). We noted that several previously identified dysregulated HOX genes were not significantly altered (HOXA7, HOXA10, HOXD1; p-values> 0.05), likely due to the difference between bulk tissue extraction used previously and selective LCM dissection of tumor cells used here. To validate transcriptional microarray data, qRT-PCR was used. HOXC6 exhibited a significant decrease in expression in malignant serous ovarian cancer compared to normal ovarian surface epithelium (Figure 2). The number of samples tested for qRT-PCR was limited by the availability of cDNA from micro-dissected samples used for microarray. To confirm in vitro downregulation of HOXC6 in ovarian cancer tissue samples, 2 ovarian cancer cell lines SKOV3 and Caov-3 were analyzed by qRT-PCR. In addition, 3 cell lines, MDA-MB-231 (breast), HeLa (cervix), and SW262 Cancer Investigation
HOXC6 in Serous Ovarian Cancer Table 2. Up- and Downregulated HOX Genes Dysregulated in Serous Ovarian Carcinoma Generated from Microarray Analysis
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Gene Name Down regulated: HOXA4 HOXC6 HOXC9 HOXD8 Up regulated HOXB2 HOXB3 HOXB5 HOXB7 HOXB8 Not significant HOXA10 HOXA13 HOXA2 HOXA3 HOXA5 HOXA6 HOXA7 HOXA9 HOXB1 HOXB13 HOXB4 HOXB6 HOXB9 HOXC10 HOXC11 HOXC12 HOXC13 HOXC8 HOXD1 HOXD10 HOXD11 HOXD12 HOXD13 HOXD3 HOXD4 HOXD9 HOXA1 HOXA11
Gene ID
p-Value
Fold Change
NM NM NM NM
002141 004503 006897 019558
0.03483 0.00364 0.03245 0.03529
−1.3075 −2.1850 −1.8186 −1.7686
NM NM NM NM NM
002145 002146 002147 004502 024016
0.00240 0.01187 0.03546 0.03999 0.00917
1.7423 2.4289 1.9590 1.3667 2.7503
NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM
018951 000522 006735 153631 019102 024014 006896 152739 002144 006361 024015 018952 024017 017409 014212 173860 017410 022658 024501 002148 021192 021193 000523 006898 014621 014213 005522 005523
0.60377 0.80213 0.37518 0.41751 0.84787 0.89826 0.09294 0.65671 0.69206 0.22251 0.13328 0.23631 0.25668 0.33679 0.60687 0.72553 0.40956 0.05016 0.48361 0.93087 0.27080 0.15516 0.84913 0.70621 0.26843 0.07467 0.09329 0.07397
−1.1157 −1.0429 −1.1932 −1.1235 1.0441 −1.0210 −1.3266 −1.0564 −1.0793 −1.2476 1.2174 1.7481 1.3327 −1.1718 −1.1533 −1.0746 −1.1538 −1.4639 1.2490 1.0181 −1.2161 −1.3303 −1.0334 −1.2071 −1.3414 −1.5340 −1.3183 −1.3466
(colon metastasized to ovary (37)) were used to determine the specificity of HOXC6 in regard to ovarian cancer. HOXC6 was down-regulated in both ovarian cell lines as well as SW262 (Table 3). However, HOXC6 was highly expressed in both breast and cervical cell lines (Table 3). The variations in HOXC6 expression between the ovarian cell lines could be due to the invasive nature of SKOV-3 over other ovarian cell lines. The aggressiveness of SKOV-3 could be due to
Figure 2. Quantitative RT-PCR analysis of HOXC6 expression in serous ovarian carcinoma. Data are presented as means and standard deviation of three replicates for each. ∗∗ indicates statistically significant difference calculated between sample and HOSE 6-3 standard (M1 p-value = 0.0002, M2 p-value = 0.004, M3 p-value = 0.0030), using the comparative Ct method for quantification of all mRNA transcripts [33].
low and high production of Inhibin A and Activin, respectively (38), and activation of oncogenes such as nm23 and c-erbB-2 (39). To determine if decreased HOXC6 RNA levels correlate with decreased protein levels, immunohistochemistry (IHC) was performed on a subset of the serous ovarian cancer samples (n = 6) and non-malignant ovarian tissue samples (n = 8; 5 normal ovary and 3 benign ovarian tumor). IHC demonstrated that HOXC6 protein is present at high levels in normal ovarian surface epithelial cells, normally translocated to the nucleus. The normal ovarian surface epithelial layer stains positively for HOXC6 with absent staining in the underlying stroma. By contrast, HOXC6 staining is absent in the malignant ovarian tissue likely due to the disordered tissue
Table 3. Quantitative RT-PCR Analysis of HOXC6 Expression in Human Epithelial Cell Lines #
Cell-line
1 2 3
HOSE 6-3 SKOV-3 (HTB77) SW626 (HTB78)
4 5 6
CaOv-3 (HTB75) MDA-MB-231 (HTB26) HeLa (CCL2)
∗
From∗
Origin
Type
HOXC6 Fold Change
p-Value
S.W. Tsao [32] ATCC ATCC
Ovarian Surface epithelial Ovarian epithelial Colon-to-Ovarian epithelial
Normal Immortalized Adenocarcinoma Metastatic Adenocarcinoma (Grade III) Adenocarcinoma Adenocarcinoma Adenocarcinoma
N/A −12.5 −2.80
N/A 0.016939552 0.058767875
−1.37 530 3.21
0.283791563 0.00998836 0.01114571
ATCC ATCC ATCC
Ovarian epithelial Breast epithelial cell Cervix
ATCC: American Type Culture Collection.
C 2015 Informa Healthcare USA, Inc. Copyright
D. L. Tait et al.
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Figure 3. IHC analysis showing level of HOXC6 protein in normal and malignant ovarian tissues. Five normal and 6 tumorous ovarian samples are displayed. In addition a placenta sample is used as control. Observed by two independent viewers, >%50 HOXC6 present in 3 normal and placenta samples, 25% in one and
ISSN: 0735-7907 (Print) 1532-4192 (Online) Journal homepage: http://www.tandfonline.com/loi/icnv20
Downregulation of HOXC6 in Serous Ovarian Cancer David L. Tait, Zahra Bahrani-Mostafavi, C. Greer Vestal, Christine Richardson & M. Taghi Mostafavi To cite this article: David L. Tait, Zahra Bahrani-Mostafavi, C. Greer Vestal, Christine Richardson & M. Taghi Mostafavi (2015) Downregulation of HOXC6 in Serous Ovarian Cancer, Cancer Investigation, 33:7, 303-311, DOI: 10.3109/07357907.2015.1041641 To link to this article: http://dx.doi.org/10.3109/07357907.2015.1041641
Published online: 05 Jun 2015.
Submit your article to this journal
Article views: 41
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=icnv20 Download by: [Boston University]
Date: 20 October 2015, At: 01:15
Cancer Investigation, 33:303–311, 2015 ISSN: 0735-7907 print / 1532-4192 online C 2015 Informa Healthcare USA, Inc. Copyright DOI: 10.3109/07357907.2015.1041641
Downregulation of HOXC6 in Serous Ovarian Cancer David L. Tait,1 Zahra Bahrani-Mostafavi,2 C. Greer Vestal,3 Christine Richardson,3 and M. Taghi Mostafavi2
Downloaded by [Boston University] at 01:15 20 October 2015
Carolinas Medical Center, Levine Cancer Institute, Division of Gynecologic Oncology, Charlotte, North Carolina, USA,1 UNC-Charlotte, College of Computing and Informatics, Charlotte, North Carolina, USA,2 UNC-Charlotte, Department of Biological Sciences, Charlotte, North Carolina, USA3 gets or as biomarkers. These proteins are members of multiple super families characterized by their DNA binding domain motifs. One group of transcription factors that has become a subject of interest in cancer biology is the human homeobox class I (HOX) family. There are 39 HOX genes clustered on four chromosomal loci in humans, and expression of each HOX gene is tightly regulated (4). During development, HOX genes within each of the four clusters are expressed in sequence along the chromosome as the spatial arrangement of the developing tissue progresses from anterior to posterior. These genes direct cell differentiation and maintenance of cell identity and morphology from early embryonic development throughout adulthood. HOX proteins can act as either activators or repressors of transcription, and the role of individual HOX genes as either oncogenes or tumor suppressors in different cancers is well established (5). Research has shown altered expression levels of multiple HOX genes in cancers, including endometrial, cervical, pancreatic, thyroid, and lung (6–11). HOXC6 is expressed as multiple mRNAs that differ in their 5 regions but share the same DNA-binding homeodomains (12, 13). Transcripts co-exist in multiple cell types and tissues to include fibroblasts, skin, breast epithelium, stem cells of thymus, as well as myeloid progenitors of bone marrow, spinal cord, and oocytes (12–17). It has been reported that HOXC6 is regulated by MLL2, MLL3, hormones (i.e. estrogen and Lsh), and TGF (17–20). HOXC6 regulates genes with both oncogenic and tumor suppressor activities as well as epigenetic effects, regulating several genes such as CD44, CNTN1, DKK3, W1F1, MDR1, Bcl2, and the PI3K/AKT pathway (18–22). The activity of HOXC6 in ovarian development or maintenance has not been studied, although homozygous knockout HOXC6 −/− female mice are fertile (17). Elevated HOXC6 expression has been reported in meduloblastomas, osteosarcomas, breast, lung, gastrointestinal, head and neck squamous and prostate carcinomas, leukemia as well as normal trophoblast (4, 23–27). However,
Homeobox (HOX) genes encode transcription factors critical to morphogenesis and cell differentiation. Although dysregulation of several HOX genes in ovarian cancer has been reported, little is known about HOXC6 expression in epithelial ovarian cancer. In this report, analysis of laser capture microdissected samples determined HOXC6 expression patterns in normal versus malignant serous ovarian carcinoma tissues. HOXC6 protein was quantified by ELISA in parallel serum samples and further validated in a larger cohort of serum samples collected from women with and without serous ovarian carcinoma. These data demonstrate significant downregulation of HOXC6 in serous ovarian cancer. Keywords: Biomarker discovery, Homeobox genes, HOXC6, Serous ovarian cancer
INTRODUCTION Ovarian cancer is the most lethal gynecologic cancer and the fifth leading cause of cancer death for women in the United States. In 2014, the American Cancer Society estimated an annual diagnosis of more than 21,980 cases, with over 14,000 deaths (1). When diagnosed in early stages, 5year survival rates reach 90%; however, due to the lack of symptoms during the early phase of tumorigenesis, 75% of patients are diagnosed at later stages. Survival for these advanced stage patients is only 30%–40% (2). Understanding of the gene expression patterns and mechanisms associated with the pathogenesis of ovarian cancer are expected to lead to the development of new diagnostic and therapeutic modalities. Signaling pathways of tumor development have been extensively characterized leading to the elucidation of major paradigms in cancer biology and malignant transformation. Transcription factors are the final downstream effectors of signaling pathways localized in the nucleus (3) and thus they are an important focus of cancer research as therapeutic tar-
Correspondence to: M. Taghi Mostafavi, PhD, College of Computing and Informatics, University of North Carolina at Charlotte, Charlotte, NC 28223, USA. E-mail: [email protected] Received 27 November 2013; revised 28 February 2015; accepted 08 April 2015.
Downloaded by [Boston University] at 01:15 20 October 2015
D. L. Tait et al.
Figure 1. Transcriptional microarray analysis of HOX genes in serous ovarian carcinoma. Shown are relative expression levels of 37 HOX genes from three non-malignant ovarian and seven malignant ovarian samples. Unsupervised clustering analysis (hierarchical tree shown on left side) separated normal samples (NE, red bar) from malignant tumor samples (MT, orange bar).
little is known about HOXC6 expression or its activity in ovarian cancer (28–30). In an initial transcriptional microarray screen (n = 65) of ovarian cancer tissue samples, we noted that HOX genes were dysregulated, and HOXC6 was significantly down- regulated (28). To further characterize the expression of the HOXC6 gene in serous ovarian cancer, we now report evaluation of micro-dissected serous ovarian cancer and normal ovarian tissue samples as well as serum from individuals with and without ovarian cancer. We utilized DNA exon microarray, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) to analyze tissue and serum samples from women with normal ovaries and serous ovarian carcinomas. These studies demonstrate sustained high levels of HOXC6 in normal surface epithelial cells. Conversely, we describe this first report of significant down-regulation of HOXC6 gene and protein expression in patients with serous ovarian cancer. MATERIALS AND METHODS Tissue and serum collection Ovarian tissue specimens were obtained during surgery from patients with ovarian cancer or other gynecologic conditions according to an IRB-approved protocol at Carolinas Medical Center. All available patient data is presented in Table 1. All patients with serous carcinoma were stage III and IV, grade 3 and received platinum and taxane based chemotherapy after surgery. Tissue samples were placed in a R cryomold (Sakura Finetek USA, standard sized Cryomold Inc., Torrance, CA), covered with Optimal Cutting Temperature (OCT) compound (Sakura Finetek USA, Inc., Torrance, CA), frozen and stored at −80◦ C. Originally seven malignant matched and 11 non-malignant blood serum samples were R SSTTM serum separatcollected by using a BD Vacutainer
ing tube (BD Biosciences, San Jose, California) according to the manufacturer’s protocols, and stored at −80◦ C. To further validate the ELISA findings, 32 additional other serum samples were collected from pre- and postmenopausal women without ovarian cancer, and serum from an additional 14 patients with stage III and IV, grade 3 serous carcinoma of the ovary or fallopian tube—total of 21 malignant and 43 nonmalignant serum samples. Laser capture micro-dissection (LCM) OCT-embedded samples were serially sectioned into 8 μm R ∗/Plus Microscope sections using Fisherbrand Superfrost slides (Fisher Scientific, Pittsburgh, PA) by a Leica CM 1850 UV Cryostat (Leica Microsystems Inc., Bannockburn, IL). R LCM Sections were prepared for LCM using Histogene Frozen Section Staining kit (Applied Biosystems, Life Technologies, Co., Carlsbad, CA) according to the manufacturer’s protocols. After staining, samples were immediately microR R PixCell IIe LCM (Molecular Dedissected by an Arcturus vices, LLC, Sunnyvale, CA). Normal epithelium and tumor cells were separately collected from appropriate sections. The residual slide material was used to determine RNA quality (31). RNA preparation RNA extraction of LCM captured cells, and the residual slide R RNA Isolamaterials were performed using a PicoPure tion kit (Applied Biosystems, LifeTechnologies, Co., Carlsbad, CA) according to manufacturer’s protocols. RNA integrity was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) as described by the manufacturer (31). cDNA synthesis and amplification Complimentary DNA (cDNA) generation and amplification was performed using the Whole Transcriptome Cancer Investigation
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Table 1. Characteristics of Malignant Samples Used Sample #
Stage
Age
Collected Sample
Tumor Site
Assay
M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21
III-B III-C III-C III-C III-C III-C III-C III-C III-C III-C III-C IV III-C III-C III-C III-C IV III-C IV III-B III-C
75 54 74 64 54 57 71 43 64 63 81 75 69 62 62 81 68 80 75 44 76
Tissue & Serum Tissue & Serum Tissue & Serum Tissue & Serum Tissue & Serum Tissue & Serum Tissue & Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum Serum
Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Ovary Fallopian Tube Ovary Fallopian Tube Fallopian Tube Fallopian Tube Ovary Ovary Ovary Fallopian Tube Ovary Ovary
Microarray (MA), ELISA MA, IHC, ELISA MA, qPCR, IHC, ELISA MA, qPCR, IHC, ELISA MA, qPCR, IHC, ELISA MA, IHC, ELISA MA, IHC, ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA ELISA
All samples are high-grade serous carcinoma.
WT-OvationTM Pico RNA Amplification System Kit (NuGEN Technologies Inc., San Carlos, CA), according to manufacturer’s protocols. cDNA was quantified using a NanoDrop 1000 spectrophotometer (NanoDrop Products, Wilmington, DE) and used for microarray sample preparation and qPCR confirmation assays. Exon microarray sample preparation and hybridization Approximately 3 μg of single primer isothermal amplified (SPIA) cDNA was used to continue to sense–strand cDNA (ST-cDNA) conversion using the WT-OvationTM Exon Module (NuGEN Technologies Inc., San Carlos, CA). 5 μg ST-cDNA was fragmented and labeled with FLOvationTM cDNA Biotin Module V2 kit (NuGEN TechnoloR gies Inc., San Carlos, CA) then hybridized using GeneChip Affymetrix Human Exon 1.0 ST arrays (Affymetrix, Inc., Santa Clara, CA). Microarray hybridization was performed R R Hybridization Oven 640, GeneChip using a GeneChip R Fluidics Station 450, and GeneChip Scanner 3000 7G with Autoloader (Affymetrix, Inc., Santa Clara, CA). For the In Silico Quality Control, each array passed preliminary quality control including assessment of spike-in controls and the total distribution of intensities compared to manufacturer’s criteria (31). Quantitative RT-PCR RNA from primary ovarian tissue samples was prepared as described above. Normal ovarian surface epithelial cell line HOSE 6–3 (32) was used as reference sample. Ovarian cancer cell lines SKOV-3 and Caov-3, and other selected cell lines such as SW626, MDA-MB-231 [Breast], and HeLa [Cervix] (American Type Culture Collection, Manassas, VA) were used as cell culture model for this study. R Total RNA was isolated from 106 –107 cells using TRIzol Reagent (Invitrogen Co, Carlsbad, CA). Extracted RNA was C 2015 Informa Healthcare USA, Inc. Copyright
purified using RNeasy Mini Kit (Qiagen, Inc., Valencia, CA). 50 ng RNA was reverse transcribed to cDNA in 20 μL total volume using QuantiTect Reverse Transcription Kit (Qiagen Inc., Valencia, CA). 50 ng cDNA was amplified R qPCR primers for HOXC6 using 500 nm of PrimeTime [Hs.PT.51.3113294)] and GAPDH [Hs.PT.51.2918858.g] (Integrated DNA Technologies Inc., San Jose, CA). GAPDH was used to normalize expression data. Primers were selected to amplify by real-time PCR the same transcripts as detected by microarray. Analysis of data was carried out according to the Comparative Ct method for relative quantitation of gene expression (33). Real-time quantitative PCR was carried out R 7500 Fast (ABI 7500 Fast) using the Applied Biosystems Real-Time PCR System (Applied Biosystem, Carlsbad, CA), R SYBR Green PCR kit (Qiagen, Inc., Vaand the QuantiTect lencia, CA) according to the manufacturer’s protocols. The ABI 7500 Fast instrument was operated under the following thermal cycling conditions: Initial heat activation of 95◦ C for 15 min, followed by 40 cycles of denaturation of 94◦ C for 15 s, annealing of 60◦ C for 30 s, and extension of 72◦ C for 30 s. The PCR products were checked using ethidium bromide-stained 2% agarose gels. Both standard and primary tissue samples were assayed in triplicate. Immunohistochemistry Tissue sections were incubated in 3% H2 O2 for 5 min at room temperature and washed with PBS, then incubated for 45 min with 50% fetal bovine serum blocking solution. After removal of blocking solution, sections were incubated overnight at 4◦ C in a 1:250 dilution of goat-anti-human HOXC6 antibody (Santa-Cruz Biotechnology Inc., Santa Cruz, CA). Sections were washed and incubated with a 1:250 dilution of biotinylated rabbit anti-goat secondary antibody for 45 min at room temperature (R&D systems Inc., Minneapolis, MN). A streptavidin-enzyme conjugate (BD Biosciences,
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San Jose, California) was added to the slides according to the manufacturer’s protocols. Specific signals were visualized by incubation with streptavidin HRP followed with adding diaminobenzidine (DAB) as a chromogen (BD Biosciences, San Jose, California). Counterstaining was performed with Mayer’s hematoxylin (Sigma-Aldrige, St. Louis, MO). Images were captured and stored digitally for analysis. Indirect sandwich enzyme-linked immunosorbent assay (ELISA) The developed and optimized protocol is summarized as follows: Wells of a micro-titer plate (Nunc, Roskilde, Denmark) were coated with 100 μL of anti-HOXC6 capture antibody (B-7; Santa Cruz Biotechnology, Inc., CA) at 1 μg/mL in coating buffer (pH 9.6) of 0.1 M carbonate/bicarbonate buffer (Sigma- Aldrich, St. Louis, MO). The plates were incubated overnight at 4◦ C followed by washing 2× with PBS (SigmaAldridge, St. Louis) and were blocked with 200 μL of blocking buffer of PBS −1% (w/v) bovine serum albumin (BSA). The plates were incubated 2 h at room temperature followed by washing 2× with PBS. A 1:100 dilution of patient’s serum was added and incubated for 2 h at room temperature. To create the standard curve for HOXC6 protein, the HOXC6 partial recombinant protein (Novus Biological, LLC, Littleton, CO) at 1 μg/mL was used. Plates were washed, and a 1:100 dilution of goat anti-human HOXC6 primary antibody (N-13; Santa-Cruz Biotechnology, Inc., Santa Cruz, CA) at 200 μg/mL was added, and incubated at room temperature for 2 h. Plates were washed 4× and a 1:1000 dilution of bovine anti-goat IgG-HRP secondary antibody (Santa Cruz Biological, Inc. Santa Cruz, CA) at 400 μg/mL was added and incubated for 2 h at room temperature. The plates were washed 4× and 100 μL of 3,3 ,5,5 -tetramethylbenzidine (TMB) enzyme substrate (Thermo Scientific Inc., Barrington, IL) added to each well, followed by incubation at room temperature. Color development was stopped by addition of 2 M sulfuric acid (Sigma-Aldridge, St. Louis). Optical density was read at 450 nm using MultiskanTM GO Microplate Spectrophotometer (Thermo Scientific, Inc., Barrington, IL) according to manufacturer’s protocols (34). Based on the standard curve, the serum samples were analyzed in triplicate. Statistical methods After microarray hybridization, raw signal was normalized and gene expressions were calculated using GC Robust Multiarray Average (GCRMA). Significant difference in expression was calculated between nontumorous tissue samples and malignant tumor samples using Analysis Of Variance (ANOVA) and p-values from ANOVA were modified to false discovery rate (FDR). Genes were considered having statistically significant differential expression between nontumorous tissue and malignant tumor states if their FDR pvalues were less than or equal to 0.05. The algorithms and R Genomics SuiteTM 6.6. For methods were run using Partek qRT-PCR analysis of HOXC6 gene expression in tissue samples, Microsoft Excel 2007 was used to perform comparative Ct method for quantification of all mRNA transcripts.
IHC results were observed by two independent researchers to identify level of HOXC6 expression in each sample. For ELISA unity-based normalization was performed using the lowest levels of HOXC6 found (as the lowest range) to highest level of HOXC6 found (as the highest range) while considering the data from standard curve of each plate. SAS version 9.3 was used to analyze the normalized data and to compare data from tumor vs. non-tumorous samples. Two-sided t-test was performed to analyze the data. All tests had a statistical significance set at a value of p < 0.05. RESULTS In an initial transcriptional microarray screen of 65 ovarian cancer tissue samples, significant patterns of HOX gene dysregulation were observed, and HOXC6 was significantly down regulated (28). To further characterize the expression of the HOXC6 gene in serous ovarian cancer, transcriptional microarray analysis was performed on seven serous ovarian carcinoma tissue samples and three normal ovarian surface epithelium samples (Fig. 1). Use of laser-capture microdissection (LCM) in this study ensured specific collection of target tumor cells from malignant ovarian tissues (MT) or normal epithelium cells from non-malignant ovarian tissues (NE) and thus increased sample purity. Samples were categorized into normal and malignant serous ovarian origins according to pathological diagnosis. All study subjects were postmenopausal women at the time of surgery with mean age of 66 (Table 1). The age groups for non-malignant samples at the time of collection were 1/3 < 50 and 2/3 >50 (data not shown). Expression analysis was performed using ANOVA model (35). Fisher’s Least Significant Difference contrast(s) method (36) was performed to compare samples. Based on our previous ovarian cancer gene expression study showing significant patterns of HOX gene dysregulation (28), we used this sample set to determine HOX gene expression patterns of the existing 37 HOX gene probe sets in Affymetrix Human Exon 1.0 ST array. We determined the fold-change expression between serous malignant and normal ovarian surface epithelium samples of selected genes from the HOX family (Fig. 1). The most significantly up-regulated genes include HOXB2 and HOXB3, and the most significantly down regulated gene is HOXC6 (Table 2). We noted that several previously identified dysregulated HOX genes were not significantly altered (HOXA7, HOXA10, HOXD1; p-values> 0.05), likely due to the difference between bulk tissue extraction used previously and selective LCM dissection of tumor cells used here. To validate transcriptional microarray data, qRT-PCR was used. HOXC6 exhibited a significant decrease in expression in malignant serous ovarian cancer compared to normal ovarian surface epithelium (Figure 2). The number of samples tested for qRT-PCR was limited by the availability of cDNA from micro-dissected samples used for microarray. To confirm in vitro downregulation of HOXC6 in ovarian cancer tissue samples, 2 ovarian cancer cell lines SKOV3 and Caov-3 were analyzed by qRT-PCR. In addition, 3 cell lines, MDA-MB-231 (breast), HeLa (cervix), and SW262 Cancer Investigation
HOXC6 in Serous Ovarian Cancer Table 2. Up- and Downregulated HOX Genes Dysregulated in Serous Ovarian Carcinoma Generated from Microarray Analysis
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Gene Name Down regulated: HOXA4 HOXC6 HOXC9 HOXD8 Up regulated HOXB2 HOXB3 HOXB5 HOXB7 HOXB8 Not significant HOXA10 HOXA13 HOXA2 HOXA3 HOXA5 HOXA6 HOXA7 HOXA9 HOXB1 HOXB13 HOXB4 HOXB6 HOXB9 HOXC10 HOXC11 HOXC12 HOXC13 HOXC8 HOXD1 HOXD10 HOXD11 HOXD12 HOXD13 HOXD3 HOXD4 HOXD9 HOXA1 HOXA11
Gene ID
p-Value
Fold Change
NM NM NM NM
002141 004503 006897 019558
0.03483 0.00364 0.03245 0.03529
−1.3075 −2.1850 −1.8186 −1.7686
NM NM NM NM NM
002145 002146 002147 004502 024016
0.00240 0.01187 0.03546 0.03999 0.00917
1.7423 2.4289 1.9590 1.3667 2.7503
NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM NM
018951 000522 006735 153631 019102 024014 006896 152739 002144 006361 024015 018952 024017 017409 014212 173860 017410 022658 024501 002148 021192 021193 000523 006898 014621 014213 005522 005523
0.60377 0.80213 0.37518 0.41751 0.84787 0.89826 0.09294 0.65671 0.69206 0.22251 0.13328 0.23631 0.25668 0.33679 0.60687 0.72553 0.40956 0.05016 0.48361 0.93087 0.27080 0.15516 0.84913 0.70621 0.26843 0.07467 0.09329 0.07397
−1.1157 −1.0429 −1.1932 −1.1235 1.0441 −1.0210 −1.3266 −1.0564 −1.0793 −1.2476 1.2174 1.7481 1.3327 −1.1718 −1.1533 −1.0746 −1.1538 −1.4639 1.2490 1.0181 −1.2161 −1.3303 −1.0334 −1.2071 −1.3414 −1.5340 −1.3183 −1.3466
(colon metastasized to ovary (37)) were used to determine the specificity of HOXC6 in regard to ovarian cancer. HOXC6 was down-regulated in both ovarian cell lines as well as SW262 (Table 3). However, HOXC6 was highly expressed in both breast and cervical cell lines (Table 3). The variations in HOXC6 expression between the ovarian cell lines could be due to the invasive nature of SKOV-3 over other ovarian cell lines. The aggressiveness of SKOV-3 could be due to
Figure 2. Quantitative RT-PCR analysis of HOXC6 expression in serous ovarian carcinoma. Data are presented as means and standard deviation of three replicates for each. ∗∗ indicates statistically significant difference calculated between sample and HOSE 6-3 standard (M1 p-value = 0.0002, M2 p-value = 0.004, M3 p-value = 0.0030), using the comparative Ct method for quantification of all mRNA transcripts [33].
low and high production of Inhibin A and Activin, respectively (38), and activation of oncogenes such as nm23 and c-erbB-2 (39). To determine if decreased HOXC6 RNA levels correlate with decreased protein levels, immunohistochemistry (IHC) was performed on a subset of the serous ovarian cancer samples (n = 6) and non-malignant ovarian tissue samples (n = 8; 5 normal ovary and 3 benign ovarian tumor). IHC demonstrated that HOXC6 protein is present at high levels in normal ovarian surface epithelial cells, normally translocated to the nucleus. The normal ovarian surface epithelial layer stains positively for HOXC6 with absent staining in the underlying stroma. By contrast, HOXC6 staining is absent in the malignant ovarian tissue likely due to the disordered tissue
Table 3. Quantitative RT-PCR Analysis of HOXC6 Expression in Human Epithelial Cell Lines #
Cell-line
1 2 3
HOSE 6-3 SKOV-3 (HTB77) SW626 (HTB78)
4 5 6
CaOv-3 (HTB75) MDA-MB-231 (HTB26) HeLa (CCL2)
∗
From∗
Origin
Type
HOXC6 Fold Change
p-Value
S.W. Tsao [32] ATCC ATCC
Ovarian Surface epithelial Ovarian epithelial Colon-to-Ovarian epithelial
Normal Immortalized Adenocarcinoma Metastatic Adenocarcinoma (Grade III) Adenocarcinoma Adenocarcinoma Adenocarcinoma
N/A −12.5 −2.80
N/A 0.016939552 0.058767875
−1.37 530 3.21
0.283791563 0.00998836 0.01114571
ATCC ATCC ATCC
Ovarian epithelial Breast epithelial cell Cervix
ATCC: American Type Culture Collection.
C 2015 Informa Healthcare USA, Inc. Copyright
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Figure 3. IHC analysis showing level of HOXC6 protein in normal and malignant ovarian tissues. Five normal and 6 tumorous ovarian samples are displayed. In addition a placenta sample is used as control. Observed by two independent viewers, >%50 HOXC6 present in 3 normal and placenta samples, 25% in one and
