Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis

  • Authors:
    • Masato Komatsu
    • Tetsuro Yoshimaru
    • Taisuke Matsuo
    • Kazuma Kiyotani
    • Yasuo Miyoshi
    • Toshihito Tanahashi
    • Kazuhito Rokutan
    • Rui Yamaguchi
    • Ayumu Saito
    • Seiya Imoto
    • Satoru Miyano
    • Yusuke Nakamura
    • Mitsunori Sasa
    • Mitsuo Shimada
    • Toyomasa Katagiri
  • View Affiliations

  • Published online on: December 18, 2012     https://doi.org/10.3892/ijo.2012.1744
  • Pages: 478-506
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Abstract

Triple negative breast cancer (TNBC) has a poor outcome due to the lack of beneficial therapeutic targets. To clarify the molecular mechanisms involved in the carcinogenesis of TNBC and to identify target molecules for novel anticancer drugs, we analyzed the gene expression profiles of 30 TNBCs as well as 13 normal epithelial ductal cells that were purified by laser-microbeam microdissection. We identified 301 and 321 transcripts that were significantly upregulated and downregulated in TNBC, respectively. In particular, gene expression profile analyses of normal human vital organs allowed us to identify 104 cancer-specific genes, including those involved in breast carcinogenesis such as NEK2, PBK and MELK. Moreover, gene annotation enrichment analysis revealed prominent gene subsets involved in the cell cycle, especially mitosis. Therefore, we focused on cell cycle regulators, asp (abnormal spindle) homolog, microcephaly-associated (Drosophila) (ASPM) and centromere protein K (CENPK) as novel therapeutic targets for TNBC. Small-interfering RNA-mediated knockdown of their expression significantly attenuated TNBC cell viability due to G1 and G2/M cell cycle arrest. Our data will provide a better understanding of the carcinogenesis of TNBC and could contribute to the development of molecular targets as a treatment for TNBC patients.

Introduction

Breast cancer is one of the most common solid malignant tumors among women worldwide. Breast cancer is a heterogeneous disease that is currently classified based on the expression of estrogen receptor (ER), progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2) (1,2). For patients with ER- or PgR-positive breast cancer, approximately five years of adjuvant endocrine therapy reduces the annual breast cancer death rate by approximately 30% (3). The addition of HER2-antagonist trastuzumab to adjuvant chemotherapy has improved the prognosis of HER2-positive breast cancer patients (46). In contrast, triple negative breast cancer (TNBC), defined as tumors that are negative for ER, PgR and HER2 overexpression, accounts for at least 15–20% of all breast cancers, and the prognosis for TNBC patients is poor because of its propensity for recurrence and metastasis and a lack of clinically-established targeted therapies (7,8). Therefore, only neoadjuvant chemotherapy with conventional cytotoxic agents yield an excellent outcome for TNBC patients who have a complete pathological response, but the outcome for the vast majority with residual disease after chemotherapy is relatively poor compared to non-TNBC patients (6,7). Thus, because the heterogeneity of breast cancer makes it difficult to treat many subtypes, including TNBC, the molecular mechanisms of the carcinogenesis of TNBC must be elucidated to develop novel molecular-targeted therapies that improve the clinical outcome of TNBC patients.

Current ‘omics’ technology including DNA microarray analysis can provide very helpful information that can be used to categorize the characteristics of various malignant tumors and identify genes that may be applicable for the development of novel molecular targets for therapeutic modalities (9). To this end, we analyzed the gene expression profile of 30 TNBC cells and normal breast ductal cells that were purified by laser-microbeam microdissection and identified a number of cancer-specific genes that might contribute to the carcino genesis of TNBC. TNBC gene expression profiling analysis can provide comprehensive information on the molecular mechanism underlying the carcinogenesis of TNBC and possibly lead to the development of novel effective therapies.

Materials and methods

Clinical samples and cell lines

A total of 48 TNBC (18 cases did not entry DNA microarray analysis) and 13 normal mammary tissues were obtained with informed consent from patients who were treated at Tokushima Breast Care Clinic, Tokushima, Japan. This study, as well as the use of all clinical materials described above, was approved by the Ethics Committee of The University of Tokushima. Clinical information was obtained from medical records and tumors were diagnosed as triple-negative by pathologists when immunohistochemical staining was ER-negative, PR-negative, and HER2 (0 or 1+). The clinicopathological features of each patient are summarized in Table I. Samples were immediately embedded in TissueTek OCT compound (Sakura, Tokyo, Japan), frozen, and stored at −80°C. Human TNBC cell lines MDA-MB-231, BT-20, BT-549, HCC1143, and HCC1937 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). The human normal breast epithelial cell line, MCF10A, was purchased from Cambrex Bioscience, Inc. All cells were cultured under the conditions recommended by their respective depositors.

Table I.

Clinicopathological features of 48 TNBC patients.

Table I.

Clinicopathological features of 48 TNBC patients.

IDAgeHistologyTNMStageER/PgR/HER2MicroarrayRT-PCR
144 Papillo-tubularT0N3M1IV−/−/0DoneDone
879DCIST1N0M0I−/−/0Not doneDone
1057 Papillo-tubularT1N0M0I−/−/1+Not doneDone
1963Solid-tubularT1N0M0I−/−/0Not doneDone
2760Solid-tubularT2N1M0II−/−/0DoneDone
4259Solid-tubularT2N0M0II−/−/0Not doneDone
4479 Papillo-tubularRecurrence-−/−/1+Not doneDone
5355 Papillo-tubularT1N0M0I−/−/0Not doneDone
5477Solid-tubularT1N1M0II−/−/0Not doneDone
5628ScirrhousT2N1M0II−/−/0DoneDone
5758Solid-tubularT1N1M0II−/−/0Not doneDone
6054Solid-tubularT2N1M0II−/−/0DoneDone
6460 Papillo-tubularT2N0M0II−/−/0Not doneDone
6659Special typeT2N1M0II−/−/0Not doneDone
7845Solid-tubularT2N1M0II−/−/0DoneDone
8944 Papillo-tubularRecurrence-−/−/0Not doneDone
9560Solid-tubularT1N0M0I−/−/0Not doneDone
10160ScirrhousT2N1M0II−/−/0Not doneDone
11077ScirrhousT2N1M0II−/−/1+Not doneDone
11670Solid-tubularT2N1M0II−/−/0DoneDone
15536Solid-tubularT1N1M0II−/−/0DoneDone
22549 Papillo-tubularT2N1M0II−/−/1+Not doneDone
25249Solid-tubularT2N1M0II−/−/1+DoneDone
25349ScirrhousT2N1M0II−/−/0DoneDone
26580ScirrhousT1N1M0II−/−/0-1+DoneDone
31353ScirrhousT3N2M0III−/−/0DoneDone
33742Solid-tubularT2N1M0II−/−/1+DoneDone
35955 Papillo-tubularT2N0M0II−/−/0DoneDone
36237 Papillo-tubularT2N1M0II−/−/0DoneDone
36369 Papillo-tubularT2N0M0II−/−/0DoneDone
36661Special typeT2N1M0II−/−/0-1+DoneDone
38432 Papillo-tubularT3N0M0II−/−/0DoneDone
39246 Papillo-tubularT1N1M0II−/−/0DoneDone
41460 Papillo-tubularT2N1M0II−/−/1+Not doneDone
41554Solid-tubularT2N0M0II−/−/1+DoneDone
42041Solid-tubularT3N0M0II−/−/0DoneDone
42370Solid-tubularT2N0M0II−/−/0DoneDone
43863Solid-tubularT3N0M0II−/−/0DoneDone
44539Solid-tubularT2N1M0II−/−/0DoneDone
45350Solid-tubularT2N1M0II−/−/0DoneDone
48159Solid-tubularT3N1M0III−/−/0DoneDone
52855Solid-tubularT2N1M0II−/−/0DoneDone
53558Solid-tubularT2N1M0II−/−/0Not doneDone
55371Solid-tubularT0N1M0II−/−/1+Not doneDone
55856Solid-tubularT2N1M0II−/−/0DoneDone
56264ScirrhousT2N0M0II−/−/0DoneDone
56652Solid-tubularT3N1M0III−/−/0DoneDone
65145ScirrhousT2N1M0II−/−/0DoneDone

[i] DCIS, ductal carcinoma in situ; papillo-tubular, papillo-tubular adenocarcinoma; solid-tubular, solid-tubular adenocarcinoma; scirrhous, scirrhous carcinoma; special type ID 66, adenocarcinoma with squamous cell carcinoma; ID 366, osseous metaplasia; case 44, axillary lymph node metastasis was diagnosed 8 months after the first surgery followed by the dissection of metastatic lymph nodes; case 89, local recurrence in residual breast occurred after 2 years of the first surgery followed by a lumpectomy. All information was judged according to the General Rules for Clinical and Pathological Recording of Breast Cancer (The Japanese Breast Cancer Society). T, tumor stage; N, lymph node metastasis status; M, distant metastasis.

Laser-microbeam microdissection (LMM), RNA extraction, RNA amplification, and hybridization

Frozen specimens were serially sectioned in 8-μm slices with a cryostat (Leica, Herborn, Germany) and stained with hematoxylin and eosin to define the analyzed regions. We purified 48 TNBC and 13 normal ductal cells using the LMM system (Carl Zeiss, Jena, Germany) according to the manufacturer’s instructions. Dissected cancer and normal ductal cells were dissolved in RLT lysis buffer (Qiagen, Valencia, CA, USA) containing 1% β-mercaptoethanol. The extracted total RNA was purified with an RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. For RNA amplification and labeling, we used an Agilent Low-Input QuickAmp labeling kit according to the manufacturer’s instructions. Briefly, 100 ng of total RNA from each sample was amplified using T7 RNA polymerase with simultaneous Cy3-labeled CTP incorporation. Then, 2 μg of Cy3-labeled cRNA was fragmented, hybridized onto the Agilent Whole Human Genome Microarray 4×44K slide (Agilent Technologies, Palo Alto, CA, USA) and then incubated with rotation at 65°C for 18 h. Then slides were washed and scanned by the Agilent Microarray scanner system in an ozone protection fume hood.

Microarray analysis

The features of scanned image files containing the Cy3-fluorescence signals of the hybridized Agilent Microarrays were extracted using the Agilent Feature Extraction (version 9.5) (Agilent Technologies). The data were analyzed using GeneSpring (version 11.5). We normalized the microarray data across all chips and genes by quantile normalization, and baseline transformed the signal values to the median in all samples. Finally, we performed quality control and filtering steps based on flags and expression levels. To identify genes that were significantly alternated between TNBC and normal ductal cells the mean signal intensity values in each analysis were compared. In this experiment, we applied Mann-Whitney (unpaired) t-test and random permutation test 10,000 times for each comparison and adjusted for multiple comparisons using the Benjamini Hochberg false discovery rate (FDR). Gene expression levels were considered significantly different when the FDR (corrected P-value) <5×10−4 (when comparing normal ductal cells and TNBC) and the fold change was ≥5.0. Data from this microarray analysis has been submitted to the NCBI Gene Expression Omnibus (GEO) archive as series GSE38959.

Functional gene annotation clustering

The Database for Annotation, Visualization and Integrated Discovery (DAVID 6.7) was approved to detect functional gene annotation clusters based on gene expression profiling by gene annotation enrichment analysis (http://david.abcc.ncifcrf.gov/) (10,11). The clusters from the gene annotation enrichment analysis were selected in this study based on a previous report (12).

Quantitative reverse transcription-PCR (qRT-PCR) analysis

Total RNA was extracted from each TNBC cell line and clinical sample using an RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. Purified RNA from each clinical sample and cell line, as well as poly-A RNA from normal human heart, lung, liver, and kidney (Takara, Otsu, Japan) was reverse transcribed for single-stranded cDNA using oligo(dT)12–18 primers with Superscript II reverse transcriptase (Invitrogen, Life Technologies, Carlsbad, CA, USA). qRT-PCR analysis was performed using an ABI PRISM 7500 Real-Time PCR system (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) and SYBR Premix Ex Taq (Takara) according to the manufacturer’s instructions. The PCR primer sequences were as follows: 5′-GCAGGTCTCC TTTCCTTTGCT-3′ and 5′-CTCGGCCTTCTTTGAGT GGT-3′ for ASPM; 5′-CACTCACCGATTCAAATG CTC-3′ and 5′-ACCACCGTTGTTCCCTTTCT-3′ for CENPK; 5′-AAC TTAGAGGTGGGGAGCAG-3′ and 5′-CACAACCATGCC TTACTTTATC-3′ for β2 microglobulin (β2-MG) as a quantitative control.

Gene-silencing effect by RNA interference

Targeted sequences for ASPM and CENPK were determined using an siRNA Targeted Finder (Applied Biosystems, Life Technologies; http://www.ambion.com/techlib/misc/siRNA_finder.html). The siRNA targeting sequences were 5′-CATACAGAAGT GCGAGAAA-3′ for ASPM, 5′-CTCAGTCAATGGC AGAAAA-3′ for CENPK and 5′-GCAGCACGACTTCT TCAAG-3′ for EGFP as a control siRNA. Human TNBC cell lines, HCC1937, MDA-MB-231 and BT-20, were plated at a density of 1×104 cells per well in 12-wells for the MTT assay and 3×104 cells per well in 6-well plates for flow cytometry and RT-PCR analyses. Cells were transfected with 16.6 nM of each siRNA using Lipofectamine RNAiMAX Reagent (Invitrogen). To evaluate the gene-silencing effects of the siRNAs by qRT-PCR, total RNA was extracted from the siRNA-transfected cells as described above after the indicated times. The following specific qRT-PCR primer sets were used: 5′-CGGAAAAGAAAGAGCGATGG-3′ and 5′-ACCACCAAGTGAAGCCCTGT-3′ for ASPM and 5′-GG GTGCCATCATTTTCTGGT-3′ and 5′-CCACCGTTGTT CCCTTTCTAAG-3′ for CENPK. To evaluate cell viability, the MTT assay was performed using the cell counting kit-8 reagent (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Absorbance at 450 nm was measured with a micro-plate reader infinite 200 (Tecan, Männedorf, Switzerland). These experiments were performed in triplicate.

Colony formation assay

Vector-based shRNAs and the psiU6BX3 expression system were constructed as previously described (13). The shRNA target sequences were the same as those of the siRNA oligonucleotides. The DNA sequences of all constructs were confirmed by DNA sequencing. BT-20 and MDA-MB-231 cells were plated in 10-cm dishes (1×106 cells/dish) and transfected with 6 μg of psiU6BX3.0-ASPM or psiU6BX3.0-CENPK and psiU6BX3.0-EGFP as a control using Fugene-6 (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were re-seeded for a colony formation assay (5.0×105 cells/10-cm dish) and RT-PCR (5.0×105 cells/10-cm dish). We selected psiU6BX3.0-transfected cells using selection medium containing 0.6 mg/ml of neomycin for BT-20 cells and 1.4 mg/ml for MDA-MB-231 cells. Total RNA was extracted from the cells after a 7-day incubation with neomycin, and then the knockdown effects of the siRNAs were examined by qRT-PCR. The specific primer sets for quantitative RT-PCR were the same as those for the siRNA oligonucleotides. Nineteen days after transfection, the cells were fixed with 4% paraformaldehyde for 10 min and stained with Giemsa solution (Merck, Darmstadt, Germany).

Cell cycle analysis

For flow cytometric analysis, adherent and detached cells were harvested and fixed with 70% ethanol at room temperature for 30 min. After washing with PBS (−), the cells were incubated at 37°C for 30 min with 1 mg/ml RNase I in PBS (−) and stained with 20 μg propidium iodide at room temperature for 30 min in the dark. A total of 10,000 cells were analyzed for DNA content using flow cytometry and CellQuest software (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). Assays were performed in duplicate.

Immunocytochemical staining analysis

HCC1937 and MDA-MB-231 cells were plated onto a 2-well glass slide (Thermo Fisher Scientific, Rochester, NY, USA) at a density of 1.0×104/well and incubated for 24 h before siRNA trans fection. Forty-eight hours post-transfection, the cells were fixed with 4% paraformaldehyde for 30 min at 4°C and then permeablized with 0.1% Triton X-100 for 2 min at room temperature. Subsequently, the cells were covered with 3% bovine serum albumin for 60 min at room temperature and then incubated with an anti-α/β tubulin antibody (Cell Signaling, Beverly, MA, USA) diluted 1:50 for 1 h. After washing with PBS (−), the cells were stained with an Alexa 488-conjugated anti-rabbit secondary antibody (Molecular Probes, Eugene, OR, USA) diluted 1:1,000 for 1 h. The nuclei were counterstained with 4′,6′-diamidine-2′-phenylindole dihydrochloride (DAPI). Fluorescent images were obtained using an IX71 microscope (Olympus, Tokyo, Japan).

Statistical analysis

Statistical significance was calculated by Mann-Whitney t-test using Stat View 5.0 J software (SAS Institute, Inc., Cary, NC, USA) to compare the gene expression levels between TNBC cells and normal ductal cells, and by Student’s two-sided t-test using Microsoft® Excel 2008 to assess cell proliferation, gene expression, and alteration of cell cycle. A difference of P<0.05 was considered statistically significant.

Results

Identification of genes upregulated or downregulated in TNBCs

To obtain precise expression profiles of TNBC cells, we used LMM to avoid contamination of non-cancer cells, such as adipocytes, fibroblasts, and inflammatory cells from the tissue sections (Fig. 1A, upper panels). Because breast cancer originates from normal breast ductal cells, we used similarly purified populations of normal duct cells as controls (Fig. 1A, lower panels). The precise gene-expression profiles of TNBC by DNA microarray identified 301 genes that were upregulated >5-fold in TNBC compared to 13 normal ductal cells, and 321 genes that were downregulated to <1/5 of the normal ductal cells (Fig. 1B). Table II lists the 301 upregulated genes in TNBC, including ubiquitin-conjugating enzyme E2C (UBE2C) (14), S100 calcium binding protein P (S100P) (15), ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase) (UCHL1) (16), pituitary tumor-transforming 1 (PTTG1) (17), ubiquitinconjugating enzyme E2T (UBE2T) (13), ubiquitin-like with PHD and ring finger domains 1 (UHRF1) (18), SIX homeobox 1 (SIX1) (19), and protein regulator of cytokinesis 1 (PRC1) (20), which were previously reported to be overexpressed in breast cancer and involved in mammary carcinogenesis. In particular, topoisomerase (DNA) IIα (TOP2A) (21,22), HORMA domain containing 1 (HORMAD1) (23), ATPase family, Fatty acid binding protein 5 (psoriasis-associated) (FABP5) (24), and AAA domain containing 2 (ATAD2) (25) were previously reported to be potentially involved in the carcinogenesis of TNBC, and to serve as prognostic markers or therapeutic targets for TNBC.

Table II.

Genes significantly upregulated in TNBC compared with normal ductal cells.

Table II.

Genes significantly upregulated in TNBC compared with normal ductal cells.

Probe IDAccession no.SymbolGene nameFold change (log)P-value
A_24_P334130NM_054034FN1Fibronectin 15.331.26E-04
A_24_P940678N/AN/A5.071.26E-04
A_23_P367618NM_003412ZIC1Zic family member 1 (odd-paired homolog, Drosophila)5.011.26E-04
A_23_P118834NM_001067TOP2ATopoisomerase (DNA) IIα 170 kDa4.761.26E-04
A_32_P119154BE138567N/A4.751.26E-04
A_23_P35219NM_002497NEK2NIMA (never in mitosis gene a)-related kinase 24.671.26E-04
A_23_P166360NM_206956PRAMEPreferentially expressed antigen in melanoma4.641.26E-04
A_24_P332314NM_198947FAM111BFamily with sequence similarity 111, member B4.631.26E-04
A_24_P413884NM_001809CENPACentromere protein A4.591.26E-04
A_23_P68610NM_012112TPX2TPX2, microtubule-associated, homolog (Xenopus laevis)4.581.26E-04
A_23_P58266NM_005980S100PS100 calcium binding protein P4.571.26E-04
A_24_P297539NM_181803UBE2C Ubiquitin-conjugating enzyme E2C4.491.26E-04
A_23_P401NM_016343CENPFCentromere protein F, 350/400 ka (mitosin)4.441.26E-04
A_23_P57379NM_003504CDC45LCDC45 cell division cycle 45-like (S. cerevisiae)4.441.26E-04
A_23_P118815NM_001012271BIRC5Baculoviral IAP repeat-containing 54.431.26E-04
A_23_P210853NM_021067GINS1GINS complex subunit 1 (Psf1 homolog)4.411.26E-04
A_23_P258493NM_005573LMNB1Lamin B14.311.26E-04
A_24_P119745NM_212482FN1Fibronectin 14.311.26E-04
A_24_P680947BC044933KIF18BKinesin family member 18B4.31.26E-04
A_32_P92642N/AN/A4.31.26E-04
A_23_P356684NM_018685ANLNAnillin, actin binding protein4.291.26E-04
A_24_P314571BU616832N/A4.241.26E-04
A_23_P98580NM_004265FADS2Fatty acid desaturase 24.21.26E-04
A_23_P52017NM_018136ASPMasp (abnormal spindle) homolog, microcephaly associated (Drosophila)4.171.26E-04
A_24_P20607NM_005409CXCL11Chemokine (C-X-C motif) ligand 114.162.33E-04
A_32_P199884NM_032132HORMAD1HORMA domain containing 14.132.33E-04
A_23_P70007NM_012484HMMRHyaluronan-mediated motility receptor (RHAMM)4.111.26E-04
A_23_P22378NM_003108SOX11SRY (sex determining region Y)-box 114.11.26E-04
A_23_P259586NM_003318TTKTTK protein kinase4.091.26E-04
A_23_P200310NM_017779DEPDC1DEP domain containing 14.081.26E-04
A_24_P378331NM_170589CASC5Cancer susceptibility candidate 54.061.26E-04
A_23_P111888NM_138455CTHRC1Collagen triple helix repeat containing 14.051.26E-04
A_23_P48835NM_138555KIF23Kinesin family member 234.051.26E-04
A_23_P115872NM_018131CEP55Centrosomal protein 55 kDa4.031.26E-04
A_23_P132956NM_004181UCHL1Ubiquitin carboxyl-terminal esterase L1 (ubiquitin thiolesterase)4.031.26E-04
A_24_P911179NM_018136ASPMasp (abnormal spindle) homolog, microcephaly associated (Drosophila)4.021.26E-04
A_23_P408955NM_004091E2F2E2F transcription factor 24.021.26E-04
A_23_P7636NM_004219PTTG1Pituitary tumor-transforming 141.26E-04
A_23_P204941NM_004004GJB2Gap junction protein, β2, 26 kDa41.26E-04
A_23_P18452NM_002416CXCL9Chemokine (C-X-C motif) ligand 93.942.33E-04
A_24_P96780NM_016343CENPFCentromere protein F, 350/400 ka (mitosin)3.921.26E-04
A_23_P69537NM_006681NMUNeuromedin U3.91.26E-04
A_24_P14156NM_006101NDC80NDC80 homolog, kinetochore complex component (S. cerevisiae)3.861.26E-04
A_23_P254733NM_024629MLF1IPMLF1 interacting protein3.851.26E-04
A_23_P74115NM_003579RAD54LRAD54-like (S. cerevisiae)3.841.26E-04
A_23_P50108NM_006101NDC80NDC80 homolog, kinetochore complex component (S. cerevisiae)3.841.26E-04
A_24_P150160NM_004265FADS2Fatty acid desaturase 23.831.26E-04
A_23_P155815NM_022346NCAPGNon-SMC condensin I complex, subunit G3.821.26E-04
A_23_P125278NM_005409CXCL11Chemokine (C-X-C motif) ligand 113.811.26E-04
A_23_P51085NM_020675SPC25SPC25, NDC80 kinetochore complex component, homolog (S. cerevisiae)3.811.26E-04
A_23_P133123NM_032117MND1Meiotic nuclear divisions 1 homolog (S. cerevisiae)3.81.26E-04
A_32_P62997NM_018492PBKPDZ binding kinase3.81.26E-04
A_23_P256956NM_005733KIF20AKinesin family member 20A3.791.26E-04
A_24_P933613N/AN/A3.781.26E-04
A_23_P212844NM_006342TACC3Transforming, acidic coiled-coil containing protein 33.781.26E-04
A_24_P254705NM_020394ZNF695Zinc finger protein 6953.761.26E-04
A_23_P115482NM_014176UBE2T Ubiquitin-conjugating enzyme E2T (putative)3.751.26E-04
A_32_P201723N/AN/A3.731.26E-04
A_23_P256425NM_014479 ADAMDEC1ADAM-like, decysin 13.731.26E-04
A_23_P432352NM_001017978CXorf61Chromosome X open reading frame 613.731.26E-04
A_23_P208880NM_013282UHRF1Ubiquitin-like with PHD and ring finger domains 13.721.26E-04
A_23_P323751NM_030919FAM83DFamily with sequence similarity 83, member D3.711.26E-04
A_23_P48669NM_005192CDKN3Cyclin-dependent kinase inhibitor 33.711.26E-04
A_24_P234196NM_001034RRM2Ribonucleotide reductase M23.691.26E-04
A_23_P253791NM_004345CAMPCathelicidin antimicrobial peptide3.691.26E-04
A_23_P76914NM_005982SIX1SIX homeobox 13.674.43E-04
A_23_P94571NM_004432ELAVL2ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2 (Hu antigen B)3.671.26E-04
A_23_P200222NM_033300LRP8Low density lipoprotein receptor-related protein 8, apolipoprotein E receptor3.671.26E-04
A_24_P416079NM_016359NUSAP1Nucleolar and spindle associated protein 13.661.26E-04
A_23_P104651NM_080668CDCA5Cell division cycle associated 53.651.26E-04
A_23_P150667NM_031217KIF18AKinesin family member 18A3.641.26E-04
A_24_P859859N/AN/A3.634.43E-04
A_23_P312150NM_001956EDN2Endothelin 23.611.26E-04
A_23_P375NM_018101CDCA8Cell division cycle associated 83.591.26E-04
A_32_P68525BC035392N/A3.581.26E-04
A_23_P43490NM_058197CDKN2ACyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)3.561.26E-04
A_23_P1691NM_002421MMP1Matrix metallopeptidase 1 (interstitial collagenase)3.551.26E-04
A_23_P117852NM_014736 KIAA0101KIAA01013.541.26E-04
A_24_P319613NM_002497NEK2NIMA (never in mitosis gene a)-related kinase 23.531.26E-04
A_23_P10385NM_016448DTLDenticleless homolog (Drosophila)3.531.26E-04
A_32_P1173NM_138441 C6orf150Chromosome 6 open reading frame 1503.511.26E-04
A_23_P94422NM_014791MELKMaternal embryonic leucine zipper kinase3.51.26E-04
A_23_P340909BC013418SKA3Spindle and kinetochore associated complex subunit 33.481.26E-04
A_23_P385861NM_152562CDCA2Cell division cycle associated 23.471.26E-04
A_23_P124417NM_004336BUB1Budding uninhibited by benzimidazoles 1 homolog (yeast)3.471.26E-04
A_24_P257099NM_018410HJURPHolliday junction recognition protein3.431.26E-04
A_24_P270460NM_005532IFI27Interferon, α-inducible protein 273.412.33E-04
A_23_P206059NM_003981PRC1Protein regulator of cytokinesis 13.391.26E-04
A_23_P74349NM_145697NUF2NUF2, NDC80 kinetochore complex component, homolog (S. cerevisiae)3.361.26E-04
A_24_P302584NM_003108SOX11SRY (sex determining region Y)-box 113.364.43E-04
A_24_P68088NR_002947TCAM1Testicular cell adhesion molecule 1 homolog (mouse)3.352.33E-04
A_24_P605612NM_003247THBS2Thrombospondin 23.341.26E-04
A_24_P366033NM_018098ECT2Epithelial cell transforming sequence 2 oncogene3.341.26E-04
A_23_P93258NM_003537 HIST1H3BHistone cluster 1, H3b3.331.26E-04
A_23_P211762N/ACOL8A1Collagen, type VIII, α13.294.43E-04
A_23_P77493NM_006086TUBB3Tubulin, β33.291.26E-04
A_23_P204947NM_004004GJB2Gap junction protein, β2, 26 kDa3.291.26E-04
A_23_P149668NM_014875KIF14Kinesin family member 143.291.26E-04
A_23_P34325NM_033300LRP8Low density lipoprotein receptor-related protein 8, apolipoprotein E receptor3.281.26E-04
A_32_P56154N/AN/A3.281.26E-04
A_32_P10403BU618641 SERPINE1Serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 13.271.26E-04
A_23_P138507NM_001786CDC2Cell division cycle 2, G1→S and G2→M3.241.26E-04
A_23_P48513NM_005532IFI27Interferon, α-inducible protein 273.231.26E-04
A_23_P49972NM_001254CDC6Cell division cycle 6 homolog (S. cerevisiae)3.221.26E-04
A_24_P306896XR_040656 LOC283711Hypothetical protein LOC2837113.221.26E-04
A_23_P44684NM_018098ECT2Epithelial cell transforming sequence 2 oncogene3.211.26E-04
A_24_P161773N/AN/A3.21.26E-04
A_23_P100344NM_014321ORC6LOrigin recognition complex, subunit 6 like (yeast)3.21.26E-04
A_32_P162183NM_000063C2Complement component 23.181.26E-04
A_23_P163481NM_001211BUB1BBudding uninhibited by benzimidazoles 1 homolog β (yeast)3.171.26E-04
A_32_P113784N/AN/A3.161.26E-04
A_32_P87849N/AN/A3.161.26E-04
A_24_P397107NM_001789CDC25ACell division cycle 25 homolog A (S. pombe)3.151.26E-04
A_23_P209200NM_001238CCNE1Cyclin E13.151.26E-04
A_32_P16625N/AN/A3.151.26E-04
A_23_P58321NM_001237CCNA2Cyclin A23.151.26E-04
A_24_P37903N/ALOXLysyl oxidase3.121.26E-04
A_32_P64919NM_001042517DIAPH3Diaphanous homolog 3 (Drosophila)3.121.26E-04
A_23_P379614NM_007280OIP5Opa interacting protein 53.121.26E-04
A_23_P206441NM_000135FANCAFanconi anemia, complementation group A3.091.26E-04
A_23_P16915NM_012413QPCTGlutaminyl-peptide cyclotransferase3.091.26E-04
A_23_P137173NM_021992TMSB15AThymosin β 15a3.071.26E-04
A_24_P313504NM_005030PLK1Polo-like kinase 1 (Drosophila)3.071.26E-04
A_23_P251421NM_031942CDCA7Cell division cycle associated 73.061.26E-04
A_23_P252292NM_006733CENPICentromere protein I3.041.26E-04
A_23_P158725NM_001042422SLC16A3Solute carrier family 16, member 3 (monocarboxylic acid transporter 4)3.041.26E-04
A_23_P57417NM_005940MMP11Matrix metallopeptidase11 (stromelysin 3)3.031.26E-04
A_24_P291044N/AN/A3.021.26E-04
A_23_P343927NM_175065 HIST2H2ABHistone cluster 2, H2ab3.011.26E-04
A_23_P63789NM_032997ZWINTZW10 interactor3.011.26E-04
A_23_P123596NM_000170GLDCGlycine dehydrogenase (decarboxylating)31.26E-04
A_23_P88731NM_002875RAD51RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)31.26E-04
A_23_P161474NM_182751MCM10Minichromosome maintenance complex component 102.991.26E-04
A_24_P303354NM_021064 HIST1H2AGHistone cluster 1, H2ag2.981.26E-04
A_23_P10518NM_016521TFDP3Transcription factor Dp family, member 32.981.26E-04
A_24_P247660NM_001002033HN1Hematological and neurological expressed 12.971.26E-04
A_23_P134910NM_003878GGHγ-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase)2.971.26E-04
A_32_P7193N/AN/A2.971.26E-04
A_23_P49878NM_019013FAM64AFamily with sequence similarity 64, member A2.961.26E-04
A_24_P359231BC014312 HIST1H2BJHistone cluster 1, H2bj2.951.26E-04
A_32_P140262N/AN/A2.951.26E-04
A_23_P55270NM_002988CCL18Chemokine (C-C motif) ligand 18 (pulmonary and activation-regulated)2.951.26E-04
A_24_P462899NM_001012507 C6orf173Chromosome 6 open reading frame 1732.941.26E-04
A_23_P502520NM_172374IL4I1Interleukin 4 induced 12.941.26E-04
A_23_P253762N/AN/A2.941.26E-04
A_23_P214908AY374131N/A2.941.26E-04
A_24_P225534NM_017821RHBDL2Rhomboid, veinlet-like 2 (Drosophila)2.941.26E-04
A_23_P203419NM_013402FADS1Fatty acid desaturase 12.941.26E-04
A_23_P150935NM_005480TROAPTrophinin associated protein (tastin)2.941.26E-04
A_24_P412088NM_182751MCM10Minichromosome maintenance complex component 102.941.26E-04
A_23_P71727NM_001827CKS2CDC28 protein kinase regulatory subunit 22.931.26E-04
A_23_P217236NM_005342HMGB3High-mobility group box 32.921.26E-04
A_32_P109296NM_152259 C15orf42Chromosome 15 open reading frame 422.911.26E-04
A_23_P89509NM_006461SPAG5Sperm associated antigen 52.911.26E-04
A_24_P563068N/AN/A2.911.26E-04
A_23_P416468NM_025049PIF1PIF1 5′-to-3′ DNA helicase homolog (S. cerevisiae)2.911.26E-04
A_24_P38895NM_002105H2AFXH2A histone family, member X2.91.26E-04
A_23_P52278NM_004523KIF11Kinesin family member 112.891.26E-04
A_24_P144543N/AN/A2.891.26E-04
A_24_P71468NM_012413QPCTGlutaminyl-peptide cyclotransferase2.882.33E-04
A_23_P116123NM_001274CHEK1CHK1 checkpoint homolog (S. pombe)2.881.26E-04
A_32_P106235N/AN/A2.871.26E-04
A_24_P139152AL359062COL8A1Collagen, type VIII, α12.874.43E-04
A_23_P36831NM_003979GPRC5AG protein-coupled receptor, family C, group 5, member A2.871.26E-04
A_23_P387471NM_005931MICBMHC class I polypeptide-related sequence B2.851.26E-04
A_23_P9574NM_018098ECT2Epithelial cell transforming sequence 2 oncogene2.841.26E-04
A_24_P535256AK001903INHBAInhibin, βA2.841.26E-04
A_24_P76521AK056691GSG2germ cell associated 2 (haspin)2.831.26E-04
A_23_P103795NM_138959VANGL1vang-like 1 (van gogh, Drosophila)2.831.26E-04
A_32_P74409NM_001145033 LOC387763Hypothetical protein LOC3877632.831.26E-04
A_23_P100632NM_001002033HN1Hematological and neurological expressed 12.831.26E-04
A_23_P126212NM_022111CLSPNClaspin homolog (Xenopus laevis)2.831.26E-04
A_24_P659113NM_152523CCNYL1Cyclin Y-like 12.831.26E-04
A_24_P367227NM_001144755MYBL1v-myb myeloblastosis viral oncogene homolog (avian)-like 12.821.26E-04
A_23_P162719NM_030932DIAPH3Diaphanous homolog 3 (Drosophila)2.811.26E-04
A_32_P221799NM_003514 HIST1H2AMHistone cluster 1, H2am2.811.26E-04
A_23_P60120NM_031415GSDMCGasdermin C2.812.33E-04
A_24_P902509NM_018193FANCIFanconi anemia, complementation group I2.81.26E-04
A_23_P50096NM_001071TYMSThymidylate synthetase2.791.26E-04
A_32_P143245NM_001012507 C6orf173Chromosome 6 open reading frame 1732.791.26E-04
A_23_P155969NM_014264PLK4Polo-like kinase 4 (Drosophila)2.791.26E-04
A_23_P62021N/AN/A2.781.26E-04
A_32_P183218NM_153695ZNF367Zinc finger protein 3672.771.26E-04
A_23_P46118NM_001821CHMLChoroideremia-like (Rab escort protein 2)2.762.33E-04
A_23_P327643N/AN/A2.751.26E-04
A_23_P375104NM_018193FANCIFanconi anemia, complementation group I2.751.26E-04
A_23_P1823NM_000280PAX6Paired box 62.751.26E-04
A_23_P168014NM_021066 HIST1H2AJHistone cluster 1, H2aj2.741.26E-04
A_24_P413126NM_020182PMEPA1Prostate transmembrane protein, androgen induced 12.741.26E-04
A_23_P80032NM_005225E2F1E2F transcription factor 12.741.26E-04
A_23_P215976NM_057749CCNE2Cyclin E22.722.33E-04
A_32_P231415AF132203SCDStearoyl-CoA desaturase (δ-9-desaturase)2.721.26E-04
A_23_P370989NM_005914MCM4Minichromosome maintenance complex component 42.721.26E-04
A_23_P216429NM_017680ASPNAsporin2.711.26E-04
A_24_P195621NR_027288 LOC341056SUMO-1 activating enzyme subunit 1 pseudogene2.711.26E-04
A_32_P151800NM_207418FAM72DFamily with sequence similarity 72, member D2.71.26E-04
A_23_P122197NM_031966CCNB1Cyclin B12.71.26E-04
A_23_P34788NM_006845KIF2CKinesin family member 2C2.71.26E-04
A_32_P206698NM_001826CKS1BCDC28 protein kinase regulatory subunit 1B2.71.26E-04
A_23_P99292NM_006479 RAD51AP1RAD51 associated protein 12.71.26E-04
A_23_P133956NM_002263KIFC1Kinesin family member C12.691.26E-04
A_32_P143496N/AN/A2.691.26E-04
A_32_P163858NM_005063SCDStearoyl-CoA desaturase (δ-9-desaturase)2.691.26E-04
A_32_P175557R01145N/A2.691.26E-04
A_23_P63618NM_005063SCDStearoyl-CoA desaturase (δ-9-desaturase)2.691.26E-04
A_23_P88630NM_000057BLMBloom syndrome, RecQ helicase-like2.681.26E-04
A_24_P276102NM_183404RBL1Retinoblastoma-like 1 (p107)2.681.26E-04
A_23_P135385N/AN/A2.681.26E-04
A_23_P57658NM_020386HRASLSHRAS-like suppressor2.671.26E-04
A_23_P23303NM_003686EXO1Exonuclease 12.671.26E-04
A_23_P88691NM_000745CHRNA5Cholinergic receptor, nicotinic, α52.671.26E-04
A_24_P923381NR_002219EPR1Effector cell peptidase receptor 1 (non-protein coding)2.661.26E-04
A_23_P24444NM_001360DHCR7 7-dehydrocholesterol reductase2.651.26E-04
A_23_P43157NM_001080416MYBL1v-myb myeloblastosis viral oncogene homolog (avian)-like 12.652.33E-04
A_23_P88740NM_018455CENPNCentromere protein N2.641.26E-04
A_23_P131866NM_198433AURKAAurora kinase A2.641.26E-04
A_23_P259641NM_004456EZH2Enhancer of zeste homolog 2 (Drosophila)2.641.26E-04
A_32_P72341NM_173084TRIM59Tripartite motif-containing 592.621.26E-04
A_24_P227091NM_004523KIF11Kinesin family member 112.611.26E-04
A_23_P145238NM_080593 HIST1H2BKHistone cluster 1, H2bk2.611.26E-04
A_23_P136805NM_014783 ARHGAP11ARho GTPase activating protein 11A2.61.26E-04
A_23_P167997NM_003518 HIST1H2BGHistone cluster 1, H2bg2.61.26E-04
A_23_P63402NM_013296GPSM2G-protein signaling modulator 2 (AGS3-like, C. elegans)2.61.26E-04
A_24_P192994NM_013402FADS1Fatty acid desaturase 12.591.26E-04
A_23_P25559NM_005845ABCC4ATP-binding cassette, sub-family C (CFTR/MRP), member 42.593.41E-04
A_23_P309381NM_001040874 HIST2H2AA4Histone cluster 2, H2aa42.591.26E-04
A_23_P35871NM_024680E2F8E2F transcription factor 82.581.26E-04
A_23_P207307N/AN/A2.581.26E-04
A_24_P399888NM_001002876CENPMCentromere protein M2.581.26E-04
A_23_P360754NM_005099ADAMTS4ADAM metallopeptidase with thrombospondin type 1 motif, 42.573.41E-04
A_23_P21706NM_001905CTPSCTP synthase2.571.26E-04
A_24_P174924NM_003537 HIST1H3BHistone cluster 1, H3b2.571.26E-04
A_23_P155989NM_022145CENPKCentromere protein K2.571.26E-04
A_23_P103981NM_001040874 HIST2H2AA4Histone cluster 2, H2aa42.561.26E-04
A_23_P571NM_006516SLC2A1Solute carrier family 2 (facilitated glucose transporter), member 12.561.26E-04
A_23_P420551NM_007174CITCitron (rho-interacting, serine/threonine kinase 21)2.561.26E-04
A_23_P411335NM_152524SGOL2Shugoshin-like 2 (S. pombe)2.541.26E-04
A_32_P147090NM_199357 ARHGAP11ARho GTPase activating protein 11A2.541.26E-04
A_23_P70448NM_005325 HIST1H1AHstone cluster 1, H1a2.531.26E-04
A_23_P43484NM_058197CDKN2ACyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)2.521.26E-04
A_24_P85539NM_212482FN1Fibronectin 12.521.26E-04
A_32_P28704N/AN/A2.521.26E-04
A_23_P107421NM_003258TK1Thymidine kinase 1, soluble2.511.26E-04
A_23_P502425NM_020409MRPL47Mitochondrial ribosomal protein L472.51.26E-04
A_24_P351466NM_020890 KIAA1524KIAA15242.51.26E-04
A_23_P211910NM_182943PLOD2Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 22.51.26E-04
A_24_P9321NM_003533 HIST1H3IHistone cluster 1, H3i2.491.26E-04
A_24_P334248NM_014996PLCH1Phospholipase C, eta 12.481.26E-04
A_24_P819890NM_001005210LRRC55Leucine rich repeat containing 552.484.43E-04
A_23_P146456NM_001333CTSL2Cathepsin L22.482.33E-04
A_24_P242440NM_003780B4GALT2UDP-Gal:βGlcNAc β 1,4-galactosyltransferase, polypeptide 22.471.26E-04
A_23_P88331NM_014750DLGAP5Discs, large (Drosophila) homolog-associated protein 52.471.26E-04
A_23_P216068NM_014109ATAD2ATPase family, AAA domain containing 22.461.26E-04
A_32_P31021N/AN/A2.461.26E-04
A_23_P373119NR_002165HMGB3L1High-mobility group box 3-like 12.461.26E-04
A_23_P361419NM_018369DEPDC1BDEP domain containing 1B2.451.26E-04
A_23_P10870NM_014908DOLKDolichol kinase2.441.26E-04
A_23_P420692NM_015053PPFIA4Protein tyrosine phosphatase, receptor type, f polypeptide (PTPRF), interacting protein (liprin), α42.431.26E-04
A_23_P146284NM_003129SQLESqualene epoxidase2.431.26E-04
A_32_P159254AK123584N/A2.432.33E-04
A_23_P25626NM_024808 C13orf34Chromosome 13 open reading frame 342.431.26E-04
A_23_P59005NM_000593TAP1Transporter 1, ATP-binding cassette, sub-family B (MDR/TAP)2.432.33E-04
A_24_P49747XM_929965 LOC646993Similar to high mobility group box 32.431.26E-04
A_23_P252740NM_024094DSCC1Defective in sister chromatid cohesion 1 homolog (S. cerevisiae)2.421.26E-04
A_23_P397341NM_152341PAQR4Progestin and adipoQ receptor family member IV2.421.26E-04
A_23_P59045NM_021052 HIST1H2AEHistone cluster 1, H2ae2.421.26E-04
A_23_P140316NM_001099652GPR137CG protein-coupled receptor 137C2.421.26E-04
A_23_P207520Z74615COL1A1Collagen, type I, α12.411.26E-04
A_24_P920968NM_182625GEN1Gen homolog 1, endonuclease (Drosophila)2.411.26E-04
A_23_P366216NM_003524 HIST1H2BHHistone cluster 1, H2bh2.411.26E-04
A_23_P217049NM_014286FREQFrequenin homolog (Drosophila)2.412.33E-04
A_32_P194264NM_001008708CHAC2ChaC, cation transport regulator homolog 2 (E. coli)2.42.33E-04
A_32_P35839N/AN/A2.41.26E-04
A_23_P154894NM_000100CSTBCystatin B (stefin B)2.41.26E-04
A_24_P340066NM_001421ELF4E74-like factor 4 (ets domain transcription factor)2.41.26E-04
A_24_P857404NM_001093725MEX3Amex-3 homolog A (C. elegans)2.41.26E-04
A_24_P133488NM_017955CDCA4Cell division cycle associated 42.41.26E-04
A_23_P339240NM_014996PLCH1Phospholipase C, eta 12.392.33E-04
A_23_P52410NM_145307RTKN2Rhotekin 22.391.26E-04
A_23_P59877NM_001444FABP5Fatty acid binding protein 5 (psoriasis-associated)2.391.26E-04
A_23_P29594NM_052969RPL39LRibosomal protein L39-like2.381.26E-04
A_23_P11984NM_201649SLC6A9Solute carrier family 6 (neurotransmitter transporter, glycine), member 92.382.33E-04
A_23_P200866NM_203401STMN1Stathmin 12.371.26E-04
A_32_P182135N/AN/A2.361.26E-04
A_24_P323598NM_001017420ESCO2Establishment of cohesion 1 homolog 2 (S. cerevisiae)2.361.26E-04
A_23_P39574NM_001080539CCDC150Coiled-coil domain containing 1502.361.26E-04
A_24_P275386AK025766BRI3BPBRI3 binding protein2.361.26E-04
A_23_P85460NM_078626CDKN2CCyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4)2.351.26E-04
A_23_P57306NM_005441CHAF1BChromatin assembly factor 1, subunit B (p60)2.351.26E-04
A_23_P335329NM_004485GNG4Guanine nucleotide binding protein (G protein), γ42.352.33E-04
A_23_P92441NM_002358MAD2L1MAD2 mitotic arrest deficient-like 1 (yeast)2.351.26E-04
A_24_P13390NM_032814RNFT2Ring finger protein, transmembrane 22.351.26E-04
A_23_P362046NM_138779 C13orf27Chromosome 13 open reading frame 272.341.26E-04
A_23_P24716NM_017870 TMEM132ATransmembrane protein 132A2.341.26E-04
A_23_P91900NM_005496SMC4structural maintenance of chromosomes 42.331.26E-04
A_24_P105102NM_182687PKMYT1Protein kinase, membrane associated tyrosine/threonine 12.331.26E-04
A_24_P244420NM_018367ACER3alkaline ceramidase 32.332.33E-04
A_23_P112673NM_017975ZWILCHZwilch, kinetochore associated, homolog (Drosophila)2.331.26E-04
A_23_P87769NM_017915 C12orf48Chromosome 12 open reading frame 482.331.26E-04
A_24_P296254NM_014783 ARHGAP11ARho GTPase activating protein 11A2.321.26E-04
A_23_P166306NM_000071CBS Cystathionine-β-synthase2.321.26E-04

[i] N/A, not annotated; P-value, Benjamini-Hochberg false discovery rate of random permutation test; log fold change, between groups.Gene symbol, accession number and gene name were exported from GeneSpring (from the NCBI databases).

On the other hand, Table III lists the 321 genes that were downregulated to <1/5 of normal ductal cells. Among these significantly downregulated genes, prolactin-induced protein (PIP) and dynein, axonemal, light intermediate chain 1 (DNALI1) were previously shown to be downregulated in TNBC (26). In particular, suppression of WNT inhibitory factor 1 (WIF1) (27) and signal peptide, CUB domain, EGF-like (SCUBE2) (28), both of which function as tumor suppressors, were among the genes that were downregulated as malignancy progressed. These data suggest that silencing or depletion of these genes might lead to the carcinogenesis of TNBC.

Table III.

Significantly downregulated genes in TNBC compared with normal ductal cells.

Table III.

Significantly downregulated genes in TNBC compared with normal ductal cells.

Probe IDAccession no.SymbolGene nameFold change (log)P-value
A_23_P127781NM_006552SCGB1D1Secretoglobin, family 1D, member 1−6.771.26E-04
A_32_P234405CK570316N/A−6.621.26E-04
A_23_P150555NM_006551SCGB1D2Secretoglobin, family 1D, member 2−6.511.26E-04
A_23_P12533NM_052997 ANKRD30AAnkyrin repeat domain 30A−6.441.26E-04
A_23_P8702NM_002652PIPProlactin-induced protein−6.341.26E-04
A_23_P501010NM_000494COL17A1Collagen, type XVII, α1−5.691.26E-04
A_24_P844984NM_002644PIGRPolymeric immunoglobulin receptor−5.551.26E-04
A_32_P216520NM_007191WIF1WNT inhibitory factor 1−5.531.26E-04
A_23_P71364NM_015886PI15Peptidase inhibitor 15−5.331.26E-04
A_24_P273756NM_003722TP63Tumor protein p63−5.111.26E-04
A_23_P132619NM_000916OXTROxytocin receptor−4.891.26E-04
A_32_P111873BQ432543N/A−4.881.26E-04
A_32_P23272N/AN/A−4.851.26E-04
A_24_P643776N/AN/A−4.741.26E-04
A_23_P136777NM_001647APODApolipoprotein D−4.711.26E-04
A_23_P9711NM_006040HS3ST4Heparan sulfate (glucosamine) 3-O-sulfotransferase 4−4.581.26E-04
A_23_P305292NR_027180 LOC728264Hypothetical LOC728264−4.571.26E-04
A_23_P159974NM_033495KLHL13Kelch-like 13 (Drosophila)−4.551.26E-04
A_23_P105144NM_020974SCUBE2Signal peptide, CUB domain, EGF-like 2−4.511.26E-04
A_32_P14253N/AN/A−4.471.26E-04
A_23_P327380NM_003722TP63Tumor protein p63−4.451.26E-04
A_23_P337270AK057247N/A−4.431.26E-04
A_23_P420442NM_153618SEMA6DSema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6D−4.341.26E-04
A_23_P8812N/AN/A−4.31.26E-04
A_23_P160377NM_003462DNALI1Dynein, axonemal, light intermediate chain 1−4.261.26E-04
A_24_P92680AK093340 LOC100132116Hypothetical LOC100132116−4.231.26E-04
A_23_P216779NM_001007097NTRK2Neurotrophic tyrosine kinase, receptor, type 2−4.231.26E-04
A_23_P148249NM_024817THSD4Thrombospondin, type I, domain containing 4−4.181.26E-04
A_23_P206920NM_001040114MYH11Myosin, heavy chain 11, smooth muscle−4.131.26E-04
A_32_P154473NM_004522KIF5CKinesin family member 5C−4.131.26E-04
A_23_P128362NM_206819MYBPC1Myosin binding protein C, slow type−4.113.41E-04
A_23_P83381NM_001143962CAPN8Calpain 8−4.081.26E-04
A_23_P397208NM_000848GSTM2Glutathione S-transferase mu 2 (muscle)−4.071.26E-04
A_23_P503072NM_148672CCL28Chemokine (C-C motif) ligand 28−4.031.26E-04
A_23_P143068NM_024726IQCA1IQ motif containing with AAA domain 1−4.011.26E-04
A_24_P829209AK096334LOC285944Hypothetical protein LOC285944−3.992.33E-04
A_23_P394246GPR81G protein-coupled receptor 81−3.961.26E-04
A_24_P34186NM_004010DMDDystrophin−3.961.26E-04
A_23_P303087NM_002825PTNPleiotrophin−3.951.26E-04
A_24_P243749NM_002612PDK4Pyruvate dehydrogenase kinase, isozyme 4−3.941.26E-04
A_32_P39944AK095791N/A−3.821.26E-04
A_23_P217379NM_033641COL4A6Collagen, type IV, α6−3.81.26E-04
A_23_P407565NM_001337CX3CR1Chemokine (C-X3-C motif) receptor 1−3.761.26E-04
A_23_P373464NM_002285AFF3AF4/FMR2 family, member 3−3.751.26E-04
A_32_P183765NM_005235ERBB4v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian)−3.751.26E-04
A_23_P145514NM_014432IL20RAInterleukin 20 receptor, α−3.751.26E-04
A_24_P870620NM_002825PTNPleiotrophin−3.742.33E-04
A_32_P154361N/AN/A−3.731.26E-04
A_24_P330633NM_000353TATTyrosine aminotransferase−3.721.26E-04
A_23_P360777NM_013960NRG1Neuregulin 1−3.721.26E-04
A_23_P253982NM_002141HOXA4Homeobox A4−3.691.26E-04
A_32_P114475N/AN/A−3.681.26E-04
A_32_P221774BX099483N/A−3.661.26E-04
A_23_P212608NM_022131CLSTN2Calsyntenin 2−3.662.33E-04
A_23_P254165NM_021785RAI2Retinoic acid induced 2−3.651.26E-04
A_24_P794447NR_024430 LOC399959Hypothetical LOC399959−3.641.26E-04
A_23_P149517NM_002644PIGRPolymeric immunoglobulin receptor−3.641.26E-04
A_24_P904484NR_024344 LOC283174Hypothetical LOC283174−3.621.26E-04
A_32_P194423N/AN/A−3.621.26E-04
A_23_P371495NM_175861TMTC1Transmembrane and tetratricopeptide repeat containing 1−3.62.33E-04
A_23_P134162NM_016356DCDC2Doublecortin domain containing 2−3.581.26E-04
A_32_P232455NM_178840C1orf64Chromosome 1 open reading frame 64−3.581.26E-04
A_24_P318160NM_014903NAV3Neuron navigator 3−3.571.26E-04
A_23_P59388NM_001723DSTDystonin−3.561.26E-04
A_23_P399217NM_153445OR5P3Olfactory receptor, family 5, subfamily P, member 3−3.561.26E-04
A_23_P309739NM_000125ESR1Estrogen receptor 1−3.531.26E-04
A_24_P608007AK022390N/A−3.531.26E-04
A_23_P501538NM_153631HOXA3Homeobox A3−3.521.26E-04
A_24_P602871NM_001030060SAMD5Sterile α motif domain containing 5−3.521.26E-04
A_23_P136433N/AN/A−3.511.26E-04
A_23_P30294NM_001801CDO1Cysteine dioxygenase, type I−3.481.26E-04
A_23_P218928NM_016613FAM198BFamily with sequence similarity 198, member B−3.471.26E-04
A_23_P154627XM_002345419TSHZ2Teashirt zinc finger homeobox 2−3.471.26E-04
A_23_P303833NM_174934SCN4BSodium channel, voltage-gated, type IV, β−3.451.26E-04
A_24_P930088XM_002342181 LOC100286909Hypothetical protein LOC100286909−3.451.26E-04
A_32_P81623AA514833N/A−3.421.26E-04
A_24_P923028BC020707TATTyrosine aminotransferase−3.411.26E-04
A_23_P58869NR_002932 LOC442245Glutathione S-transferase mu 2 pseudogene−3.41.26E-04
A_23_P2271NM_198965PTHLHParathyroid hormone-like hormone−3.41.26E-04
A_32_P43664−3.391.26E-04
A_32_P16007NM_207355POTEBPOTE ankyrin domain family, member B−3.391.26E-04
A_23_P94840NM_130897DYNLRB2Dynein, light chain, roadblock-type 2−3.381.26E-04
A_24_P5153NM_024817THSD4Thrombospondin, type I, domain containing 4−3.381.26E-04
A_32_P223675N/AN/A−3.371.26E-04
A_24_P904845AK095791N/A−3.371.26E-04
A_23_P403209N/AN/A−3.361.26E-04
A_23_P215382N/AN/A−3.353.41E-04
A_24_P209710NM_004816 FAM189A2Family with sequence similarity 189, member A2−3.351.26E-04
A_23_P167168NM_144646IGJImmunoglobulin J polypeptide, linker protein for immunoglobulin α and mu polypeptides−3.341.26E-04
A_24_P70183NM_001040113MYH11Myosin, heavy chain 11, smooth muscle collagen, type XIV, α1−3.321.26E-04
A_23_P216361NM_021110COL14A1−3.321.26E-04
A_23_P113351NM_004684SPARCL1SPARC-like 1 (hevin)−3.311.26E-04
A_32_P17145N/AN/A−3.311.26E-04
A_23_P35414NM_005398PPP1R3CProtein phosphatase 1, regulatory (inhibitor) subunit 3C−3.291.26E-04
A_23_P31945NM_033439IL33Interleukin 33−3.271.26E-04
A_23_P204630NM_021229NTN4Netrin 4−3.261.26E-04
A_23_P501831NM_032385C5orf4Chromosome 5 open reading frame 4−3.261.26E-04
A_23_P200015NM_174858AK5Adenylate kinase 5−3.261.26E-04
A_24_P802145NM_005544IRS1Insulin receptor substrate 1−3.261.26E-04
A_24_P251969NM_000800FGF1Fibroblast growth factor 1 (acidic)−3.241.26E-04
A_32_P228618NM_001003793RBMS3RNA binding motif, single stranded interacting protein−3.231.26E-04
A_23_P125233NM_001299CNN1Calponin 1, basic, smooth muscle−3.222.33E-04
A_23_P500998NM_152739HOXA9Homeobox A9−3.192.33E-04
A_23_P83838NM_004056CA8Carbonic anhydrase VIII−3.191.26E-04
A_24_P911950N/AN/A−3.171.26E-04
A_23_P159952NM_018476BEX1Brain expressed, X-linked 1−3.171.26E-04
A_23_P45185NM_004469FIGFc-fos induced growth factor (vascular endothelial growth factor D)−3.162.33E-04
A_23_P14083NM_181847AMIGO2Adhesion molecule with Ig-like domain 2−3.161.26E-04
A_24_P920366N/AN/A−3.141.26E-04
A_24_P167668NM_000428LTBP2Latent transforming growth factor β binding protein 2−3.121.26E-04
A_32_P161033BC043411N/A−3.111.26E-04
A_23_P348159NM_020388DSTDystonin−3.111.26E-04
A_32_P89415N/AN/A−3.11.26E-04
A_23_P165778NM_024101MLPHMelanophilin−3.081.26E-04
A_32_P168701N/AN/A−3.073.41E-04
A_32_P78491NM_004956ETV1ets variant 1−3.061.26E-04
A_24_P87036NM_018043ANO1Anoctamin 1, calcium activated chloride channel−3.061.26E-04
A_24_P912799NM_003966SEMA5ASema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A−3.061.26E-04
A_23_P315364NM_002089CXCL2Chemokine (C-X-C motif) ligand 2−3.051.26E-04
A_24_P71341NM_001461FMO5Flavin containing monooxygenase 5−3.052.33E-04
A_32_P199796NM_004023DMDDystrophin−3.052.33E-04
A_32_P179998NM_033053DMRTC1DMRT−like family C1−3.041.26E-04
A_32_P17984N/AN/A−3.041.26E-04
A_23_P138938NM_000926PGRProgesterone receptor−3.041.26E-04
A_23_P18559NM_003866INPP4BInositol polyphosphate-4-phosphatase, type II, 105 kDa−3.031.26E-04
A_23_P124946NM_153610CMYA5Cardiomyopathy associated 5−3.031.26E-04
A_23_P212241NM_006614CHL1Cell adhesion molecule with homology to L1CAM (close homolog of L1)−3.031.26E-04
A_23_P156402NM_003551NME5Non-metastatic cells 5, protein expressed in (nucleoside-diphosphate kinase)−3.021.26E-04
A_23_P150053NM_001613ACTA2Actin, α2, smooth muscle, aorta−3.021.26E-04
A_32_P58912N/AN/A−3.021.26E-04
A_32_P216841NM_145263SPATA18Spermatogenesis associated 18 homolog (rat)−3.012.33E-04
A_23_P257087NM_002612PDK4Pyruvate dehydrogenase kinase, isozyme 4−3.011.26E-04
A_23_P110686NM_003714STC2Stanniocalcin 2−31.26E-04
A_23_P369994NM_004734DCLK1Doublecortin-like kinase 1−2.992.33E-04
A_23_P422831NM_004816 FAM189A2Family with sequence similarity 189, member A2−2.981.26E-04
A_24_P325992NM_002310LIFRLeukemia inhibitory factor receptor α−2.981.26E-04
A_23_P387000NM_173683XKR6XK, Kell blood group complex subunit-related family, member 6−2.983.41E-04
A_32_P83811NM_001136570FAM47EFamily with sequence similarity 47, member E−2.981.26E-04
A_32_P44210BX538299N/A−2.971.26E-04
A_24_P918317NM_015881DKK3Dickkopf homolog 3 (Xenopus laevis)−2.974.43E-04
A_23_P203957NM_175861TMTC1Transmembrane and tetratricopeptide repeat containing 1−2.963.41E-04
A_23_P30217NM_052863SCGB3A1Secretoglobin, family 3A, member 1−2.961.26E-04
A_23_P77066NM_022807SNRPNSmall nuclear ribonucleoprotein polypeptide N−2.941.26E-04
A_32_P109242AK055302CSRNP3 Cysteine-serine-rich nuclear protein 3−2.911.26E-04
A_24_P937265N/AN/A−2.911.26E-04
A_32_P97968N/AN/A−2.91.26E-04
A_32_P85684AA069768N/A−2.891.26E-04
A_23_P385067NM_053277CLIC6Chloride intracellular channel 6−2.894.43E-04
A_23_P82868NM_000930PLATPlasminogen activator, tissue−2.881.26E-04
A_32_P108396N/AN/A−2.881.26E-04
A_23_P148345NM_194463RNF128Ring finger protein 128−2.871.26E-04
A_24_P314477NM_178012TUBB2BTubulin, β 2B−2.871.26E-04
A_24_P895836N/AN/A−2.871.26E-04
A_23_P171074NM_004867ITM2AIntegral membrane protein 2A−2.851.26E-04
A_23_P9135NM_033655CNTNAP3Contactin associated protein-like 3−2.854.43E-04
A_23_P372234NM_001218CA12Carbonic anhydrase XII−2.831.26E-04
A_23_P393099NM_003226TFF3Trefoil factor 3 (intestinal)−2.822.33E-04
A_23_P113701NM_002607PDGFAPlatelet-derived growth factor α polypeptide−2.821.26E-04
A_23_P10995NM_014483RBMS3RNA binding motif, single stranded interacting protein−2.821.26E-04
A_24_P269006NM_001182ALDH7A1Aldehyde dehydrogenase 7 family, member A1−2.811.26E-04
A_23_P415533AK054879N/A−2.811.26E-04
A_23_P216225NM_004430EGR3Early growth response 3−2.81.26E-04
A_24_P101282N/AN/A−2.81.26E-04
A_32_P72541N/AN/A−2.82.33E-04
A_24_P299474NM_001122679ODZ2odz, odd Oz/ten-m homolog 2 (Drosophila)−2.81.26E-04
A_23_P416395NM_003714STC2Stanniocalcin 2−2.81.26E-04
A_23_P40415NM_007038ADAMTS5ADAM metallopeptidase with thrombospondin type 1 motif, 5−2.81.26E-04
A_32_P3545XM_002345868 LOC100131504Hypothetical LOC100131504−2.794.43E-04
A_23_P106405NM_002487NDNNecdin homolog (mouse)−2.791.26E-04
A_23_P405129NM_000428LTBP2Latent transforming growth factor β binding protein 2−2.791.26E-04
A_24_P237804NM_174981POTEDPOTE ankyrin domain family, member D−2.781.26E-04
A_23_P89780NM_198129LAMA3Laminin, α3−2.781.26E-04
A_23_P213415NM_003966SEMA5ASema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A−2.773.41E-04
A_24_P397386NM_002310LIFRLeukemia inhibitory factor receptor α−2.771.26E-04
A_23_P73297NM_004742MAGI1Membrane associated guanylate kinase, WW and PDZ domain containing 1−2.771.26E-04
A_23_P165783NM_024101MLPHMelanophilin−2.761.26E-04
A_23_P212061NM_007289MMEMembrane metallo-endopeptidase−2.761.26E-04
A_23_P75056NM_001002295GATA3GATA binding protein 3−2.761.26E-04
A_24_P748377CR749529−2.752.33E-04
A_24_P810476NTRK3Neurotrophic tyrosine kinase, receptor, type 3−2.743.41E-04
A_32_P60606AL713753 DKFZp667F0711Hypothetical protein DKFZp667F0711−2.741.26E-04
A_32_P200697NM_181709FAM101AFamily with sequence similarity 101, member A−2.734.43E-04
A_24_P84220NR_027995 LOC284232Ankyrin repeat domain 20 family, member A2 pseudogene−2.731.26E-04
A_23_P157914NM_153267MAMDC2MAM domain containing 2−2.711.26E-04
A_24_P393596N/AN/A−2.711.26E-04
A_32_P25419N/AN/A−2.71.26E-04
A_24_P169873N/AN/A−2.71.26E-04
A_24_P358534N/AN/A−2.693.41E-04
A_32_P34750AV702101N/A−2.691.26E-04
A_32_P9941NM_007191WIF1WNT inhibitory factor 1−2.682.33E-04
A_23_P335143U81001SNRPNSmall nuclear ribonucleoprotein polypeptide N−2.671.26E-04
A_23_P56855NM_001137671POTECPOTE ankyrin domain family, member C−2.671.26E-04
A_32_P59837AK091914N/A−2.651.26E-04
A_24_P737553AK023774N/A−2.652.33E-04
A_23_P204286NM_000900MGPMatrix Gla protein−2.651.26E-04
A_24_P725895BE218249N/A−2.631.26E-04
A_32_P4337N/AN/A−2.631.26E-04
A_23_P154400NM_001042467MLPHMelanophilin−2.621.26E-04
A_23_P29800NM_005602CLDN11Claudin 11−2.611.26E-04
A_23_P156025NM_033267IRX2Iroquois homeobox 2−2.611.26E-04
A_32_P193091N/AN/A−2.611.26E-04
A_23_P83857NM_000240MAOAMonoamine oxidase A−2.61.26E-04
A_32_P355396NM_014844TECPR2Tectonin β-propeller repeat containing 2−2.61.26E-04
A_32_P214565BU928689N/A−2.61.26E-04
A_24_P468950AK021439N/A−2.61.26E-04
A_24_P683583N/AN/A−2.61.26E-04
A_23_P203558NM_000518HBBHemoglobin, β−2.62.33E-04
A_32_P140153N/AN/A−2.61.26E-04
A_32_P124461AK129743N/A−2.591.26E-04
A_23_P136026AK128476N/A−2.591.26E-04
A_23_P28295NM_004525LRP2Low density lipoprotein-related protein 2−2.594.43E-04
A_24_P586712NM_198485TPRG1Tumor protein p63 regulated 1−2.581.26E-04
A_23_P139500NM_030762BHLHE41Basic helix-loop-helix family, member e41−2.581.26E-04
A_23_P121480NM_001004196CD200CD200 molecule−2.581.26E-04
A_23_P32577NM_080759DACH1Dachshund homolog 1 (Drosophila)−2.581.26E-04
A_23_P315815NM_004495NRG1Neuregulin 1−2.581.26E-04
A_23_P93772NM_019102HOXA5Homeobox A5−2.581.26E-04
A_32_P150748CR749529N/A−2.581.26E-04
A_32_P204959N/AN/A−2.581.26E-04
A_23_P363149N/AN/A−2.574.43E-04
A_23_P41487NM_015130TBC1D9TBC1 domain family, member 9 (with GRAM domain)−2.571.26E-04
A_23_P257296NM_003226TFF3Trefoil factor 3 (intestinal)−2.563.41E-04
A_23_P250735NM_175709CBX7Chromobox homolog 7−2.561.26E-04
A_24_P189516NM_001609ACADSBacyl-coenzyme A dehydrogenase, short/branched chain−2.561.26E-04
A_23_P253012NM_017577GRAMD1CGRAM domain containing 1C−2.561.26E-04
A_24_P179244XM_001723863 LOC100128979Hypothetical protein LOC100128979−2.551.26E-04
A_32_P117846N/AN/A−2.551.26E-04
A_32_P42224BX097190N/A−2.552.33E-04
A_24_P119665NM_001128933 SYNPO2Synaptopodin 2−2.541.26E-04
A_32_P105825NM_001584 MPPED2 Metallophosphoesterase domain containing 2−2.543.41E-04
A_24_P225679NM_005544IRS1Insulin receptor substrate 1−2.541.26E-04
A_32_P226907N/AN/A−2.541.26E-04
A_23_P356581NM_022370ROBO3Roundabout, axon guidance receptor, homolog 3 (Drosophila)−2.531.26E-04
A_32_P221096AW015426N/A−2.531.26E-04
A_23_P106016NM_002742PRKD1Protein kinase D1−2.521.26E-04
A_32_P210193N/AN/A−2.521.26E-04
A_32_P38436N/AN/A−2.521.26E-04
A_24_P512775N/AN/A−2.521.26E-04
A_23_P151529NR_023938 C14orf132Chromosome 14 open reading frame 132−2.521.26E-04
A_32_P235568AK125221N/A−2.521.26E-04
A_23_P71270NM_001185AZGP1α-2-glycoprotein 1, zinc-binding−2.524.43E-04
A_24_P650425N/AN/AMatrilin 2−2.511.26E-04
A_23_P71328NM_030583MATN2ras homolog gene family, member J−2.512.33E-04
A_24_P153803NM_020663RHOJ−2.511.26E-04
A_24_P912730N/AN/A−2.511.26E-04
A_24_P347624NM_022804SNURFSNRPN upstream reading frame−2.51.26E-04
A_32_P52785NM_015345DAAM2Dishevelled associated activator of morphogenesis 2−2.53.41E-04
A_23_P61042N/AN/A−2.51.26E-04
A_23_P67661NM_001864 COX7A1Cytochrome c oxidase subunit VIIa polypeptide 1 (muscle)−2.491.26E-04
A_23_P213486N/APARP8Poly(ADP-ribose) polymerase family, member 8−2.491.26E-04
A_23_P18713NM_004827ABCG2ATP-binding cassette, sub-family G (WHITE), member 2−2.484.43E-04
A_23_P76658NM_052818 N4BP2L1NEDD4 binding protein 2-like 1−2.481.26E-04
A_23_P96590NM_014710 GPRASP1G protein-coupled receptor associated sorting protein 1−2.481.26E-04
A_24_P460763AK022443N/A−2.481.26E-04
A_23_P85672NM_006610MASP2Mannan-binding lectin serine peptidase 2−2.481.26E-04
A_24_P416489N/AN/A−2.471.26E-04
A_24_P321525NM_032918RERGRAS-like, estrogen-regulated, growth inhibitor−2.471.26E-04
A_24_P256526BC005914SP2Sp2 transcription factor−2.471.26E-04
A_24_P261417NM_015881DKK3Dickkopf homolog 3 (Xenopus laevis)−2.471.26E-04
A_23_P98369NM_000829GRIA4Glutamate receptor, ionotrophic, AMPA 4−2.471.26E-04
A_23_P6818NM_020163 SEMA3GSema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3G−2.463.41E-04
A_32_P100379N/AN/A−2.461.26E-04
A_23_P30163NR_026804 FLJ13197Hypothetical FLJ13197−2.461.26E-04
A_24_P206328NM_005020PDE1CPhosphodiesterase 1C, calmodulin-dependent 70 kDa−2.461.26E-04
A_24_P93948AB210045N/A−2.461.26E-04
A_32_P52414N/AN/A−2.451.26E-04
A_23_P123228NM_000111 SLC26A3Solute carrier family 26, member 3−2.451.26E-04
A_24_P666553N/AN/A−2.451.26E-04
A_24_P916816N/AN/A−2.441.26E-04
A_23_P134734NM_017786 GOLSYNGolgi-localized protein−2.441.26E-04
A_24_P296772NM_033256 PPP1R14AProtein phosphatase 1, regulatory (inhibitor) subunit 14A−2.431.26E-04
A_24_P267523NM_144613 COX6B2Cytochrome c oxidase subunit VIb polypeptide 2 (testis)−2.431.26E-04
A_23_P133517NM_002310LIFRLeukemia inhibitory factor receptor α−2.431.26E-04
A_24_P787680N/AN/A−2.431.26E-04
A_32_P52829N/AN/A−2.433.41E-04
A_23_P162047NM_015881DKK3Dickkopf homolog 3 (Xenopus laevis)−2.431.26E-04
A_32_P185140BX648171TPM1Tropomyosin 1 (α)−2.431.26E-04
A_24_P319892NM_198274SMYD1SET and MYND domain containing 1−2.431.26E-04
A_24_P226322NM_031469 SH3BGRL2SH3 domain binding glutamic acid-rich protein like 2−2.421.26E-04
A_23_P86012NM_001017402LAMB3Laminin, β3−2.421.26E-04
A_23_P62255NM_005314GRPRGastrin-releasing peptide receptor−2.411.26E-04
A_24_P141520N/AN/A−2.412.33E-04
A_23_P114883NM_002023FMODFibromodulin−2.411.26E-04
A_23_P300033NM_006206 PDGFRAPlatelet-derived growth factor receptor, α polypeptide−2.412.33E-04
A_24_P108311NM_015277 NEDD4LNeural precursor cell expressed, developmentally downregulated 4-like−2.411.26E-04
A_23_P345746NM_199261TPTETransmembrane phosphatase with tensin homology−2.411.26E-04
A_23_P418083NM_181714LCA5Leber congenital amaurosis 5−2.411.26E-04
A_32_P208341N/AN/A−2.411.26E-04
A_24_P930337N/AN/A−2.411.26E-04
A_24_P915095NM_017577 GRAMD1CGRAM domain containing 1C−2.41.26E-04
A_32_P4792AK057820N/A−2.41.26E-04
A_24_P82032NM_020663RHOJras homolog gene family, member J−2.392.33E-04
A_23_P204296NM_032918RERGRAS-like, estrogen-regulated, growth inhibitor−2.381.26E-04
A_24_P920712N/AN/A−2.382.33E-04
A_24_P401185NM_001042784 CCDC158Coiled-coil domain containing 158−2.381.26E-04
A_32_P109604XM_001715342 LOC100132733Similar to FLJ00310 protein−2.371.26E-04
A_24_P131173NM_024709 C1orf115Chromosome 1 open reading frame 115−2.372.33E-04
A_24_P64241NM_001012421 ANKRD20A2Ankyrin repeat domain 20 family, member A2−2.371.26E-04
A_32_P58437N/AN/A−2.371.26E-04
A_24_P602348N/AN/A−2.371.26E-04
A_24_P135856NM_016124RHDRh blood group, D antigen−2.371.26E-04
A_23_P333038NM_025145 C10orf79Chromosome 10 open reading frame 79−2.372.33E-04
A_23_P352266NM_000633BCL2B-cell CLL/lymphoma 2−2.361.26E-04
A_23_P207699NM_016835MAPT Microtubule-associated protein tau−2.361.26E-04
A_23_P392529NR_027270 C21orf81Ankyrin repeat domain 20 family, member A3 pseudogene−2.361.26E-04
A_23_P904NM_024603BEND5BEN domain containing 5−2.361.26E-04
A_23_P115785NM_145235FANK1Fibronectin type III and ankyrin repeat domains 1−2.351.26E-04
A_32_P146844N/AN/A−2.351.26E-04
A_23_P26865NM_002470MYH3Myosin, heavy chain 3, skeletal muscle, embryonic−2.351.26E-04
A_32_P100641XM_001714998 LOC100128139Hypothetical LOC100128139−2.352.33E-04
A_24_P930727AK091677N/A−2.351.26E-04
A_23_P406341NM_001001936 AFAP1L2Actin filament associated protein 1-like 2−2.351.26E-04
A_24_P54863NM_152400 C4orf32Chromosome 4 open reading frame 32−2.341.26E-04
A_23_P133120NM_018342 TMEM144Transmembrane protein 144−2.341.26E-04
A_32_P86705BC040577N/A−2.341.26E-04
A_24_P833256N/AN/A−2.331.26E-04
A_23_P401106NM_002599PDE2APhosphodiesterase 2A, cGMP-stimulated−2.331.26E-04
A_24_P102119AF264623N/A−2.331.26E-04
A_23_P358714NM_020775 KIAA1324KIAA1324−2.321.26E-04
A_32_P162494N/AN/A−2.323.41E-04
A_23_P326931NM_145170TTC18Tetratricopeptide repeat domain 18−2.321.26E-04

[i] N/A, not annotated; P-value, Benjamini-Hochberg false discovery rate of random permutation test; log fold change, between groups. Gene symbol, accession number and gene name were exported from GeneSpring (from the NCBI databases).

Identification of cancer-specific genes

Next, to develop novel therapeutic targets for TNBC with a minimum risk of adverse events, we performed a DNA microarray analysis of normal human vital organs consisting of the heart, lung, liver and kidney as well as TNBC cases and attempted to identify genes whose expression was exclusively upregulated in TNBC, but not expressed in normal vital organs. We identified 104 genes, which were specifically upregulated in TNBC, including cancer-specific molecules such as NIMA-related kinase 2 (NEK2) (29,30), PDZ binding kinase (PBK) (31), denticleless homolog (Drosohila) (DTL) (32), maternal leucine zipper kinase (MELK) (33), and kinesin family member C (KIF2C) (34), which have previously been shown to be involved in breast carcinogenesis (Fig. 1C and Table IV).

Table IV.

Genes specifically expressed in TNBC, but not expressed in normal human vital organs.

Table IV.

Genes specifically expressed in TNBC, but not expressed in normal human vital organs.

Probe IDAccession no.SymbolGene nameFold change (log)P-value
A_23_P118834NM_001067TOP2ATopoisomerase (DNA) IIα 170 kDa4.761.26E-04
A_32_P119154BE138567N/A4.751.26E-04
A_23_P35219NM_002497NEK2NIMA (never in mitosis gene a)-related kinase 24.671.26E-04
A_23_P166360NM_206956PRAMEPreferentially expressed antigen in melanoma4.641.26E-04
A_24_P332314NM_198947 FAM111BFamily with sequence similarity 111, member B4.631.26E-04
A_24_P413884NM_001809CENPACentromere protein A4.591.26E-04
A_23_P68610NM_012112TPX2TPX2, microtubule-associated, homolog (Xenopus laevis)4.581.26E-04
A_23_P401NM_016343CENPFCentromere protein F, 350/400 ka (mitosin)4.441.26E-04
A_23_P57379NM_003504 CDC45LCDC45 cell division cycle 45-like (S. cerevisiae)4.441.26E-04
A_23_P356684NM_018685ANLNAnillin, actin binding protein4.291.26E-04
A_23_P52017NM_018136ASPMasp (abnormal spindle) homolog, microcephaly associated (Drosophila)4.171.26E-04
A_32_P199884NM_032132 HORMAD1HORMA domain containing 14.132.33E-04
A_23_P259586NM_003318TTKTTK protein kinase4.091.26E-04
A_23_P200310NM_017779 DEPDC1DEP domain containing 14.081.26E-04
A_23_P115872NM_018131CEP55Centrosomal protein 55 kDa4.031.26E-04
A_24_P911179NM_018136ASPMasp (abnormal spindle) homolog, microcephaly associated (Drosophila)4.021.26E-04
A_24_P96780NM_016343CENPFCentromere protein F, 350/400 ka (mitosin)3.921.26E-04
A_24_P14156NM_006101NDC80NDC80 homolog, kinetochore complex component (S. cerevisiae)3.861.26E-04
A_23_P254733NM_024629 MLF1IPMLF1 interacting protein3.851.26E-04
A_23_P74115NM_003579 RAD54LRAD54-like (S. cerevisiae)3.841.26E-04
A_23_P50108NM_006101NDC80NDC80 homolog, kinetochore complex component (S. cerevisiae)3.841.26E-04
A_23_P155815NM_022346NCAPGNon-SMC condensin I complex, subunit G3.821.26E-04
A_23_P51085NM_020675SPC25SPC25, NDC80 kinetochore complex component, homolog (S. cerevisiae)3.811.26E-04
A_32_P62997NM_018492PBKPDZ binding kinase3.81.26E-04
A_23_P256956NM_005733 KIF20AKinesin family member 20A3.791.26E-04
A_23_P212844NM_006342TACC3Transforming, acidic coiled-coil containing protein 33.781.26E-04
A_24_P254705NM_020394 ZNF695Zinc finger protein 6953.761.26E-04
A_23_P432352NM_001017978 CXorf61Chromosome X open reading frame 613.731.26E-04
A_23_P48669NM_005192CDKN3Cyclin-dependent kinase inhibitor 33.711.26E-04
A_23_P94571NM_004432 ELAVL2ELAV (embryonic lethal, abnormal vision, Drosophila)-like 2 (Hu antigen B)3.671.26E-04
A_23_P150667NM_031217 KIF18AKinesin family member 18A3.641.26E-04
A_32_P68525BC035392N/A3.581.26E-04
A_24_P319613NM_002497NEK2NIMA (never in mitosis gene a)-related kinase 23.531.26E-04
A_23_P10385NM_016448DTLDenticleless homolog (Drosophila)3.531.26E-04
A_23_P94422NM_014791MELKMaternal embryonic leucine zipper kinase3.51.26E-04
A_23_P340909BC013418SKA3Spindle and kinetochore associated complex subunit 33.481.26E-04
A_23_P124417NM_004336BUB1Budding uninhibited by benzimidazoles 1 homolog (yeast)3.471.26E-04
A_24_P257099NM_018410HJURPHolliday junction recognition protein3.431.26E-04
A_23_P74349NM_145697NUF2NUF2, NDC80 kinetochore complex component, homolog (S. cerevisiae)3.361.26E-04
A_24_P302584NM_003108SOX11SRY (sex determining region Y)-box 113.364.43E-04
A_24_P68088NR_002947TCAM1Testicular cell adhesion molecule 1 homolog (mouse)3.352.33E-04
A_24_P366033NM_018098ECT2Epithelial cell transforming sequence 2 oncogene3.341.26E-04
A_23_P93258NM_003537 HIST1H3BHistone cluster 1, H3b3.331.26E-04
A_23_P149668NM_014875KIF14Kinesin family member 143.291.26E-04
A_23_P34325NM_033300LRP8Low density lipoprotein receptor-related protein 8, apolipoprotein E receptor3.281.26E-04
A_32_P56154N/AN/A3.281.26E-04
A_23_P138507NM_001786CDC2Cell division cycle 2, G1→S and G2→M3.241.26E-04
A_23_P49972NM_001254CDC6Cell division cycle 6 homolog (S. cerevisiae)3.221.26E-04
A_24_P306896XR_040656 LOC283711Hypothetical protein LOC2837113.221.26E-04
A_23_P44684NM_018098ECT2Epithelial cell transforming sequence 2 oncogene3.211.26E-04
A_23_P100344NM_014321ORC6LOrigin recognition complex, subunit 6 like (yeast)3.21.26E-04
A_23_P163481NM_001211BUB1BBudding uninhibited by benzimidazoles 1 homolog β (yeast)3.171.26E-04
A_32_P87849N/AN/A3.161.26E-04
A_24_P397107NM_001789 CDC25ACell division cycle 25 homolog A (S. pombe)3.151.26E-04
A_23_P209200NM_001238CCNE1Cyclin E13.151.26E-04
A_32_P16625N/AN/A3.151.26E-04
A_24_P37903N/ALOXLysyl oxidase3.121.26E-04
A_24_P313504NM_005030PLK1Polo-like kinase 1 (Drosophila)3.071.26E-04
A_23_P252292NM_006733CENPICentromere protein I3.041.26E-04
A_23_P161474NM_182751MCM10Minichromosome maintenance complex component 102.991.26E-04
A_23_P253762N/AN/A2.941.26E-04
A_24_P225534NM_017821 RHBDL2Rhomboid, veinlet-like 2 (Drosophila)2.941.26E-04
A_24_P412088NM_182751MCM10Minichromosome maintenance complex component 102.941.26E-04
A_32_P109296NM_152259 C15orf42Chromosome 15 open reading frame 422.911.26E-04
A_24_P76521AK056691GSG2Germ cell associated 2 (haspin)2.831.26E-04
A_23_P126212NM_022111CLSPNClaspin homolog (Xenopus laevis)2.831.26E-04
A_23_P60120NM_031415GSDMCGasdermin C2.812.33E-04
A_24_P902509NM_018193FANCIFanconi anemia, complementation group I2.81.26E-04
A_23_P155969NM_014264PLK4Polo-like kinase 4 (Drosophila)2.791.26E-04
A_32_P183218NM_153695 ZNF367Zinc finger protein 3672.771.26E-04
A_23_P46118NM_001821CHML Choroideremia-like (Rab escort protein 2)2.762.33E-04
A_23_P327643N/AN/A2.751.26E-04
A_23_P215976NM_057749CCNE2Cyclin E22.722.33E-04
A_32_P151800NM_207418 FAM72DFamily with sequence similarity 72, member D2.71.26E-04
A_23_P34788NM_006845KIF2CKinesin family member 2C2.71.26E-04
A_23_P133956NM_002263KIFC1Kinesin family member C12.691.26E-04
A_23_P88630NM_000057BLMBloom syndrome, RecQ helicase-like2.681.26E-04
A_24_P276102NM_183404RBL1 Retinoblastoma-like 1 (p107)2.681.26E-04
A_23_P23303NM_003686EXO1Exonuclease 12.671.26E-04
A_23_P88691NM_000745 CHRNA5Cholinergic receptor, nicotinic, α52.671.26E-04
A_32_P72341NM_173084 TRIM59Tripartite motif-containing 592.621.26E-04
A_24_P227091NM_004523KIF11Kinesin family member 112.611.26E-04
A_23_P136805NM_014783 ARHGAP11ARho GTPase activating protein 11A2.61.26E-04
A_23_P63402NM_013296GPSM2G-protein signaling modulator 2 (AGS3-like, C. elegans)2.61.26E-04
A_23_P35871NM_024680E2F8E2F transcription factor 82.581.26E-04
A_23_P207307N/AN/A2.581.26E-04
A_24_P399888NM_001002876CENPMCentromere protein M2.581.26E-04
A_23_P155989NM_022145CENPKCentromere protein K2.571.26E-04
A_23_P411335NM_152524SGOL2Shugoshin-like 2 (S. pombe)2.541.26E-04
A_23_P43484NM_058197 CDKN2ACyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)2.521.26E-04
A_32_P28704N/AN/A2.521.26E-04
A_24_P351466NM_020890 KIAA1524KIAA15242.51.26E-04
A_24_P334248NM_014996PLCH1Phospholipase C, eta 12.481.26E-04
A_23_P88331NM_014750 DLGAP5Discs, large (Drosophila) homolog-associated protein 52.471.26E-04
A_32_P31021N/AN/A2.461.26E-04
A_23_P361419NM_018369 DEPDC1BDEP domain containing 1B2.451.26E-04
A_23_P397341NM_152341PAQR4Progestin and adipoQ receptor family member IV2.421.26E-04
A_23_P140316NM_001099652 GPR137CG protein-coupled receptor 137C2.421.26E-04
A_23_P217049NM_014286FREQFrequenin homolog (Drosophila)2.412.33E-04
A_32_P35839N/AN/A2.41.26E-04
A_24_P857404NM_001093725MEX3Amex-3 homolog A (C. elegans)2.41.26E-04
A_24_P323598NM_001017420ESCO2Establishment of cohesion 1 homolog 2 (S. cerevisiae)2.361.26E-04
A_23_P112673NM_017975 ZWILCHZwilch, kinetochore associated, homolog (Drosophila)2.331.26E-04
A_24_P296254NM_014783 ARHGAP11ARho GTPase activating protein 11A2.321.26E-04

[i] N/A, not annotated; P-value, Benjamini-Hochberg false discovery rate of random permutation test; log fold change, between groups. Gene symbol, accession number and gene name were exported from GeneSpring (from the NCBI databases).

Functional gene annotation clustering analysis

To elucidate the biological processes and pathways characterized in TNBC, we performed a functional analysis of these upregulated or downregulated genes in 30 TNBC cases using the gene annotation clustering of the DAVID algorithm. We identified the most prominent cluster (cluster 1; gene enrichment score, 29.90) composed of various functional annotation terms consisting of 87 upregulated genes in TNBC (Table V). Cluster 1 consisted almost entirely of cell cycle-associated genes as represented by nuclear division (fold enrichment, 15.04), mitosis (fold enrichment, 15.04), M phase of the mitotic cell cycle (fold enrichment, 14.78), organelle fission (fold enrichment, 14.45), and M phase (fold enrichment, 12.90) (Fig. 2). These findings suggest that most of the upregulated genes in TNBC might be functionally responsible for cell cycle progression.

Table V.

Genes listed in cluster 1 and cluster 2.

Table V.

Genes listed in cluster 1 and cluster 2.

No. of genesGenes
Cluster 1 (enrichment score, 29.90)
87BLM, CKS1B, CKS2, CHEK1, E2F1, E2F2, E2F8, FANCA, FANCI, H2AFX, HORMAD1, HJURP, MAD2L1, NDC80, NEK2, NUF2, OIP5, PBK, RAD51, RAD54L, SPC25, TPX2, TTK, ZWINT ZWILCH, ANLN, ASPM, AURKA, BIRC5, BUB1, BUB1B, CASC5, CDC25A, CDC6, CDCA2, CDCA5, CDCA8, CENPA, CENPF, CEP55, CHAF1B, SKA3, C13orf34, CIT, CLSPN, CCNA2, CCNB1, CCNE1, CCNE2, CDKN2A, CDKN2C, CDKN3, DSCC1, DLGAP5, ESCO2, EXO1, FAM83D, GSG2, INHBA, KIF11, KIF14, KIF18A, KIF18B, KIF20A, KIF23, KIF2C, KIFC1, LMNB1, MND1, NCAPG, NUSAP1, PTTG1, PLK1, PLK4, PKMYT1, PRC1, RBL1, SGOL2 SPAG5, STMN1, SMC4, TMSB15A, TOP2A, TACC3, TUBB3, UBE2C, UHRF1
Cluster 2 (enrichment score, 6.43)
45ADAMTS5, MAMDC2, SPARCL1, WIF1, AZGP1, APOD, FIGF, CHL1, CCL28, CXCL2, COL4A6, COL14A1, COL17A1, CNTNAP3, DKK3, DST, FGF1, FMOD, HS3ST4, IGJ, IL33, LAMA3, LAMAB, LTBP2, LIFR, LRP2, MASP2, MATN2, MGP, NTN4, NRG1, PTHLH, PI15, PLAT, PDGFA, PTN, PIGR, PIP, SCGB1D1, SCGB1D2, SCGB3A1, SEMA3G, STC2, THSD4, TFF3

[i] Genes enriched in cluster 1 and cluster 2 according to DAVID.

On the other hand, we also identified the most prominent cluster functionally deactivated in TNBC based on down-regulated genes in TNBC (cluster 2; enrichment score, 6.43). As shown in Table V and Fig. 2, cluster 2 consisted of functions induced by extracellular matrix-cell adhesion-associated genes such as latent transforming growth factor β binding protein 2 (LTBP2), laminin α3 (LAMA3) and cell adhesion molecule with homology to L1CAM (close homolog of L1) (CHL1), which have been reported to be downregulated in various tumors (3537). These results suggest that loss of cell-cell or matrix-cell interactions might be a key mechanism in TNBC progression.

Identification of ASPM and CENPK as novel molecular targets for TNBC therapy

Because the upregulated genes were mainly included in the cell cycle-associated gene cluster as described above, we directed our focus to two cancer-specific genes that function as cell cycle regulators, asp (abnormal spindle) homolog, microcephaly associated (Drosophila) (ASPM), which is fundamental for cytokinesis (38) and centromere protein K (CENPK), which is essential for proper kinetochore assembly during mitosis (39), as novel therapeutic targets for TNBC. qRT-PCR experiments confirmed that ASPM and CENPK genes were significantly upregulated in 48 clinical TNBC cases (Fig. 3A) and five cell lines derived from TNBC (Fig. 3B), but undetectably expressed in a mixture of 13 microdissected normal mammary ductal cells and the normal mammary epithelial cell line MCF10A as well as normal human vital organs.

To ascertain the possible roles of ASPM and CENPK in TNBC cell growth, we knocked down the expression of endogenous ASPM and CENPK in three TNBC cell lines, HCC1937, BT-20 and MDA-MB-231 cells, which highly express both of these genes (Fig. 3), using RNAi. qRT-PCR experiments showed that ASPM and CENPK were significantly knocked down in cells transfected with siASPM and siCENPK, but not with siEGFP as a control (Fig. 4A). In concordance with their knockdown, the MTT assay clearly revealed growth suppression of breast cancer cells in a time-dependent manner by siASPM and siCENPK, compared with a control siEGFP, which showed no knockdown (Fig. 4B). In addition, a colony formation assay also confirmed that introducing both shRNA-ASPM and -CENPK constructs remarkably suppressed the growth of BT-20 and MDA-MB-231 cells, respectively, compared with shEGFP-transfected cells (Fig. 4C), suggesting that both genes are likely indispensable for breast cancer cell growth. Furthermore, we investigated the phenotypic alterations of TNBC cells transfected with ASPM and CENPK siRNAs showing significant knockdown effects. FACS analysis revealed that depleting ASPM caused a cell cycle arrest at the G2/M phase in HCC1937 cells (siEGFP:siASPM, 24.4:34.0%) at 2 days after transfection, and a subsequent increase in the sub-G1 population (siEGFP:siASPM, 9.86:43.68%) at 6 days (Fig. 5A). On the other hand, reduced CENPK expression resulted in an increase in the proportion of G0/G1 phase cells (siEGFP:siCENPK, 56.49:72.2%) in MDA-MB-231 after 2 days of transfection, and a subsequent increase in the sub-G1 population (siEGFP:siCENPK, 12.73:30.96%) at 6 days (Fig. 5B). Interestingly, we observed an enlarged size of HCC1937 cells, which was likely due to abnormal tubulin formation due to decreased ASPM expression (Fig. 5C, arrowheads). In addition, we observed a disruption in the structural integrity of tubulin in CENPK-depleted MDA-MB-231 cells (Fig. 5D, arrowheads), compared with those in siEGFP-transfected cells.

These results suggest that the absence of ASPM and CENPK caused an arrest in the G2/M and G0/G1 phases, respectively, and then induced cell death. Taken together, these findings strongly suggest that ASPM and CENPK have indispensable roles in cell proliferation and mitosis, especially in the G2/M and G0/G1 phases, in TNBC cells.

Discussion

TNBC patients do not benefit from endocrine therapy and trastuzumab. Conventional chemotherapy is currently the mainstay of systemic medical treatment, although TNBC patients have a worse outcome after chemotherapy than patients with other breast subtypes. In particular, because cytotoxic drugs often cause severe adverse effects, it is obvious that thoughtful selection of novel target molecules based on the detailed molecular mechanisms of TNBC carcinogenesis should be very helpful to develop effective anticancer drugs with a minimum risk of side effects. To this end, we performed DNA microarray using the microdissected TNBC and normal ductal cells, and normal human vital organs including the heart, lung, liver and kidney and identified 104 genes that were significantly upregulated in TNBC compared to normal duct cells, but not expressed in normal human vital organs. They included cancer specific kinases, such as NEK2, PBK, and MELK, which might serve as druggable targets for new therapeutic agents against TNBC.

NEK2, a member of the NIMA-related serine/threonine kinase family, is involved in cell division and the mitotic regulation by centrosome splitting, and is upregulated in a wide variety of human cancers including breast cancer (40). siRNA-mediated depletion of NEK2 expression results in growth suppression of breast and colorectal cancers (29,30). PBK, a mitotic serine/threonine kinase, is significantly upregulated in the majority of breast cancers. siRNA-mediated knockdown of PBK expression also results in significant suppression of cell growth due to cytokinetic failure (31). MELK, a member of the snf1/AMPK serine-threonine kinase family, is involved in mammalian embryonic development and is also frequently upregulated in breast cancers and brain tumors (33,41). Suppression of MELK expression by siRNA significantly inhibits the growth of human breast cancer cells (33). These findings strongly suggest that these cancer-specific kinases, NEK2, PBK and MELK, are promising therapeutic targets for TNBC.

Furthermore, we performed a gene-annotation enrichment analysis using DAVID based on gene expression profiling to elucidate the biological processes and pathways associated with each gene cluster. We found that the vast majority of genes upregulated in TNBC are functionally responsible for cell cycle progression involved in nuclear division, microtubule organization, kinetochore, and chromosome segregation, and that most inactivated functions closely related to TNBC progression are involved in cell-cell or cell-matrix interactions, which is consistent with epithelial mesenchymal transition (EMT) features as a phenotype of TNBC (42).

To further the development of novel anticancer drugs with minimum adverse effects, we focused on the cancer-specific cell-cycle associated genes ASPM and CEPNK as novel molecular targets for TNBC therapy. ASPM has been reported to play an essential role in nucleating microtubules at centrosomes, to localize to the spindle poles during mitosis (39) and to contribute to glioblastoma cell growth (43), but has not been associated with breast carcinogenesis, especially TNBC. Here, we confirmed that ASPM is upregulated in clinical samples and TNBC cell lines (Fig. 3) and that siRNA-mediated knockdown of endogenous ASPM results in the loss of nucleating microtubules through mitosis by impeding centrosome function, resulting in G2/M cell cycle arrest and subsequent apoptosis. These results suggest that aberrant ASPM expression might be involved in the carcinogenesis of TNBC and that ASPM targeting might be an attractive therapeutic option with less adverse effects. CENPK is known to be a subunit of the CENPH-I complex, and essential for proper kinetochore assembly (39), but little is known about the roles of CENPK in human cancer growth, progression, and carcinogenesis. We also confirmed that CENPK is upregulated in clinical samples and TNBC cell lines, and that siRNA-mediated knockdown also causes cell growth inhibition through G0/G1 cell cycle arrest due to a loss of correct tubulin structures (Figs. 35). Interestingly, we determined that other centromere or kinetochore-associated proteins, CENPA, CENPF, CENPI, CENPM, NDC80 and HJURP, were also significantly overexpressed in TNBC cases, but not expressed in normal vital organs (Fig. 1C and Table IV). Human CENPA was first identified based on autoantibodies found in patients suffering from scleroderma (44) and is over-expressed in colorectal cancers (45). CENPF is also reportedly upregulated in head and neck squamous cell carcinomas and pancreatic ductal carcinomas (46,47). NDC80 and HJURP are reportedly overexpressed in breast cancers and associated with tumor grade and poor prognosis (48,49). These findings suggest that aberrant regulation of kinetochore assembly and centromere function through mitosis might contribute to the carcinogenesis of TNBC and that destroying one component of the kinetochore, such as targeting CENPK, might be a novel molecular target for TNBC treatment.

TNBC is a heterogeneous subgroup of breast cancers; therefore oncologists, pathologists, and geneticists had tried to clarify TNBC by means of gene expression profiling and immunohistochemical analyses. We also applied unsupervised 2-dimensional hierarchical clustering analysis to groups of genes based on similarities in the expression pattern, but there is no clustering for TNBC based on gene expression patterns, probably due to the small sample size (data not shown). However, the information provided in this study will facilitate the development of novel and attractive molecular drug targets without adverse events.

Acknowledgements

We thank Dr Tomoya Fukawa and Dr Le Tan Dat for helpful and constructive discussions and Ms. Hitomi Kawakami for technical assistance in microdissection. This work was supported in part by a grant from Health Labour Research Grant ‘Third Term Comprehensive Control Research for Cancer (H24-3rd-Gan-Ippan-006), and Kobayashi Foundation for cancer Research (2009) (TK).

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February 2013
Volume 42 Issue 2

Print ISSN: 1019-6439
Online ISSN:1791-2423

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APA
Komatsu, M., Yoshimaru, T., Matsuo, T., Kiyotani, K., Miyoshi, Y., Tanahashi, T. ... Katagiri, T. (2013). Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis. International Journal of Oncology, 42, 478-506. https://doi.org/10.3892/ijo.2012.1744
MLA
Komatsu, M., Yoshimaru, T., Matsuo, T., Kiyotani, K., Miyoshi, Y., Tanahashi, T., Rokutan, K., Yamaguchi, R., Saito, A., Imoto, S., Miyano, S., Nakamura, Y., Sasa, M., Shimada, M., Katagiri, T."Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis". International Journal of Oncology 42.2 (2013): 478-506.
Chicago
Komatsu, M., Yoshimaru, T., Matsuo, T., Kiyotani, K., Miyoshi, Y., Tanahashi, T., Rokutan, K., Yamaguchi, R., Saito, A., Imoto, S., Miyano, S., Nakamura, Y., Sasa, M., Shimada, M., Katagiri, T."Molecular features of triple negative breast cancer cells by genome-wide gene expression profiling analysis". International Journal of Oncology 42, no. 2 (2013): 478-506. https://doi.org/10.3892/ijo.2012.1744