Introduction
The centromere has long been recognized as a locus important for proper cell division and accurate partitioning of chromosomes into daughter cells [
1‐
3]. Centromeres are the chromatin regions associated with kinetochores, which are massive multi-protein complexes that mediate chromosome segregation and the mitotic checkpoint [
4]. There is mounting evidence that kinetochores become functionally unstable during oncogenesis resulting in segregation defects, chromosome instability, and cancer development [
4‐
6].
Holliday Junction Recognition Protein (HJURP, also known as hFLEG1), which is a newly discovered gene, was reported to be overexpressed in lung cancer cells through genome-wide expression profile analysis [
7]. By quantitative RT-PCR, Valente et al found that the
HJURP expression levels significantly differ between glioblastoma resection tumor and non-neoplastic white matter [
8]. Additionally it was observed that the expression level of
HJURP in glioblastoma was changed about nine fold compared to typically benign pilocytic astrocytomas by microarray profile analysis [
9]. It has also been reported that
HJURP is involved in DNA double-strand break repair pathway through interaction with
MSH5 and
NBS1 [
7]. Recently two groups have shown that
HJURP functions at the level of the centromere, and is required for centromere protein A (CENPA) centromeric localization, for loading of new CENPA nucleosomes, and for accurate chromosomal segregation [
10‐
12]. A majority of cancer cells tend to gain and lose chromosomes at each mitotic division and are found to be aneuploid and chromosomally instable. Thus these findings support the hypothesis that alterations in
HJURP might play an important role in cancer development. We investigated whether altered expression levels of
HJURP are associated with adverse clinical outcomes using cohorts of patients with breast cancer.
Materials and methods
Cell lines and cell lysates
The names of cell lines used in our investigations are listed in Table
1. The derivation, sources, and maintenance of most of the breast cancer cell lines used in this study have been reported previously [
13] or were provided in Table
2. These cell lines have been previously analyzed for genomic aberrations by comparative genomic hybridization (CGH) and for gene-expression profiles using Affymetrix microarrays (Santa Clara, CA, USA) [
13]. The information on growth conditions of additional cell lines was listed in Table
2. Cells at 50% to 75% confluence were washed in ice-cold phosphate buffered saline (PBS). Then cells were extracted with a lysis buffer (containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 25 mM β-glycerophosphate, 25 mM NaF, 5 mM EGTA, 1 mM EDTA, 15 mM pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium molybdate, 1% Nonidet-P40, 10 mg/ml leupeptin, 10 mg/ml aprotinin, and 1 mM PMSF). Cell lysates were then clarified by centrifugation and frozen at -80°C. Protein concentrations were determined using the Bio-Rad BCA protein assay kit (Cat# 23227, Pierce Biotechnology, Rockford, IL, USA).
Table 1
The list of breast cancer cell lines and immortalized non-malignant mammary epithelial cells used in these investigations.
1 | SKBR3 | 1 | SKBR3 | 1 | SKBR3 | 1 | SKBR3 | 1 | SKBR3 | 1 | SKBR3 | 1 | SKBR3 |
2 | MCF12A | 2 | MCF12A | 2 | MCF12A | 2 | MCF12A | 2 | MCF12A | 2 | MCF12A | 2 | MCF12A |
3 | 600MPE | 12 | MDAMB134 | 21 | BT483 | 30 | 184A1N4 | 39 | DU4475 | 48 | HCC1395 | 57 | MX-1 |
4 | AU565 | 13 | MDAMB157 | 22 | HCC70 | 31 | 184B5 | 40 | SUM1315M02 | 49 | HCC1428 | 58 | SUM102 |
5 | BT20 | 14 | MDAMB175 | 23 | HCC1187 | 32 | HCC38 | 41 | HCC1954 | 50 | HCC1806 | 59 | SUM190 |
6 | BT474 | 15 | MDAMB231 | 24 | HCC1500 | 33 | HCC202 | 42 | SUM44PE | 51 | HCC1937 | 60 | HCC1419 |
7 | BT549 | 16 | MDAMB361 | 25 | MCF10A | 34 | HCC1143 | 43 | SUM52PE | 52 | HCC2185 | 61 | HCC3153 |
8 | CAMA1 | 17 | MDAMB415 | 26 | MDAMB453 | 35 | HCC1569 | 44 | SUM149PT | 53 | HCC2218 | 62 | S1 |
9 | HBL100 | 18 | MDAMB435 | 27 | ZR751 | 36 | HCC1599 | 45 | SUM159PT | 54 | HCC1599 | 63 | T4 |
10 | Hs578T | 19 | T47D | 28 | ZR7530 | 37 | LY2 | 46 | SUM185PE | 55 | UACC893 | 64 | MDAMB231-Gray |
11 | MCF7 | 20 | UACC812 | 29 | ZR75B | 38 | SUM225 | 47 | SUM225CWN | 56 | SUM229 | 65 | MDAMB231-ATCC |
Table 2
Additional cell line growth conditions and subtypes
30 | 184A1N4 | N | MEGMa | 37°C, 5% CO2 |
31 | 184B5 | N | MEGMa | 37°C, 5% CO2 |
36 | HCC1599 | Basal A | RPMI1640+10%FBSb | 37°C, 5% CO2 |
48 | HCC1395 | Basal B | RPMI1640+10%FBS | 37°C, 5% CO2 |
50 | HCC1806 | Basal A | RPMI1640+10%FBS | 37°C, 5% CO2 |
53 | HCC2218 | Luminal | RPMI1640+10%FBS | 37°C, 5% CO2 |
54 | HCC1599 | Basal A | RPMI1640+10%FBS | 37°C, 5% CO2 |
55 | UACC893 | Luminal | DMEM+10% FBS | 37°C, 5% CO2 |
56 | SUM229PE | N/A | Ham's F12+5% FBS+IHc | 37°C, 5% CO2 |
57 | MX-1 | N/A | RPMI1640+10%FBS | 37°C, 5% CO2 |
58 | SUM102PT | Basal A | Ham's F12+IHEd | 37°C, 5% CO2 |
60 | HCC1419 | Luminal | RPMI1640+10%FBS | 37°C, 5% CO2 |
62 | S1 | N | H14 medium +10 ng/ml EGF | 37°C, 5% CO2 |
63 | T4 | Basal B | H14 mediume | 37°C, 5% CO2 |
64 | MDAMB231-Gray | Basal B | DMEM+10% FBS | 37°C, 5% CO2 |
65 | MDAMB231-ATCC | Basal B | DMEM+10% FBS | 37°C, 5% CO2 |
Western blot
For Western blots, 10 μg of protein extracts per lane were electrophoresed with denaturing sodium doedecyl sulfate (SDS)-polyacrylamide gels (4% to 12%), transferred to PVDF membranes (Millipore, Temecula, CA, USA), and incubated with HJURP antibody 1:500 (Rabbit, HPA008436, Sigma-Aldrich, St. Louis, MO, USA) and actin (goat, sc-1616, Santa Cruz Biotechnology, Santa Cruz, CA, USA) diluted with blocking buffer (927-40000, LI-COR Biosciences, Lincoln, NE, USA) The membranes were washed four times with TBST and treated with 1:10,000 dilution of Alex Fluor 680 donkey anti-rabbit (A10043, Invitrogen, Carlsbad, CA, USA) and IRDye 800CW conjugated donkey anti-goat (611-731-127, Rockland, Gilbertsville, PA, USA) to detect HJURP and actin respectively. The signals were detected by infrared imaging (LI-COR Biosciences, Lincoln, NE, USA). Images were recorded as TIFF files for quantification.
Protein quantification
Protein levels were measured by quantifying infrared imaging recorded from labeled antibodies using Scion Image [
14]. For each protein, the blots were made for 7 sets of 11 cell lines, each set including the same pair (SKBR3 and MCF12A) to permit intensity normalization across sets. A basic multiplicative normalization was carried out by fitting a linear mixed effects model to log intensity values, and adjusting within each set to equalize the log intensities of the pair of reference cell lines across the sets.
Tumor samples
Detailed patient information has been described in our previous studies [
15]. This analysis is based on previously reported comparative genomic hybridization (CGH) and a gene expression profile of 130 tumors from UC San Francisco and the California Pacific Medical Center collected between 1989 and 1997.
Validation
The association of
HJURP expression levels and survival among patients with breast tumors was examined in existing microarray data sets of primary tumor samples that had been profiled with an Affymetrix microarray assay (either HG-U133A or HG U133 Plus 2.0) ((GEO:GSE1456), (GEO:GSE7390), (GEO:GSE2034), (GEO:GSE4922)) or Agilent oligo microarray (Santa Clara, CA, USA)(Table
3). Probe 218726_at and 20366 (GenBank: NM_018410) were used to measure
HJURP expression in Affymetrix and Agilent GeneChip, respectively. The process data from GEO website were downloaded for analysis.
Table 3
Information of gene expression datasets used in this study
1 | GSE1456 | Not available | |
2 | GSE7390 | Not available | |
3 | | 82.4% patients | |
4 | GSE2034 | 86.7% patients | |
5 | GSE4922 | Not available | |
HJURPshRNA construct
The shRNA sequences were (forward) 5'-GATCCCC GAGCGATTCATCTTCATCA TTCAAGAGA TGATGAAGATGAATCGCTC TTTTTGGAAA-3' and (reverse) 5'-AGCT TTTCCAAAAA GAGCGATTCATCTTCATCA TCTCTTGAA TGATGAAGATGAATCGCTC GGG-3' synthesized from IDT (Integrated DNA Technologies, Inc., San Diego, CA, USA). HJURP shRNA was cloned into BglII and HindIII cleavage sites of pSUPER.retro.puro vector based on manufactory's instruction (OligoEngine, Seattle, WA, USA). HJURP shRNA expression vector were confirmed by direct DNA sequencing.
Retroviral packaging and infection
HJURP shRNA (or empty) retroviral vectors along with packaging system pHit60 and pVSVG vectors were then co-transfected into the HEK 293 Phoenix ampho packaging cells (ATCC, Manassas, VA, USA) by using FuGENE6 transfection reagent (Roche, Lewes, UK) according to the instruction to produce retroviral supernatants. Forty-eight hours after transfection, the virus-containing supernatant was filtered through a 0.45 μm syringe filter. Retroviral infection was performed by adding filtered supernatant to a MDAMB231 cell line cultured on 10 cm dishes with 50% confluent in the presence 4 ug/ml of polybrene (Sigma, St. Louis, MO, USA). Six hours after infection, the medium was changed with fresh medium. After 48 hours, infected cells were selected by adding 5 μg/ml puromycin (Sigma) to the culture medium for 72 hours and then maintained in complete medium with 2 μg/ml puromycin. Down-regulation of HJURP expression was confirmed by Western blot analysis.
High content imaging to assess cell number and apoptotic cells
The effects on cell growth and apoptosis were assessed by a Cellomics high-content image screening system (Cellomics, Thermo Fisher Scientfic Inc., Pittsburgh, PA, USA) after breast cancer cells exposed to a single dose of 0 (sham), 1, 2, 4, 6, 8 or 10 Gy X-ray radiation emitted from an irradiator (model 43855F, Faxitron X-ray Corporation, Lincolnshire, IL, USA). Live cells in 96 well plates with six replicates from each treatment were stained with 1 μmol/L YO-PRO-1 positive cells.
Statistical analysis
Spearman's correlation coefficient and test were used to examine the relationship between HJURP mRNA level and its protein level in the cell line studies, and the relationship with age, tumor size in the tumor studies, and CENPA mRNA level. The association between HJURP mRNA level and clinical factors, such as estrogen receptor (ER), progesterone receptor (PR), ERBB2 and lymph node status, pathological stage, Scarff-Bloom-Richardson (SBR) grade, was analyzed by Mann-Whitney U (for two groups) or Kruskal-Wallis H (for more than two groups) test. Kaplan-Meier plots were constructed and a long-rank test was used to determine differences among disease free and overall survival curves according to HJURP expression level or radiotherapy. Multivariate analyses were carried out to examine whether HJURP expression is an independent prognostic factor for survival when adjusting for other covariates (age, ER, PR, lymph node, pathologic stage, SBR grade, tumor size) or the molecular subtypes (normal like, luminal, Erbb2 and Basal) using Cox proportional-hazard regression. In addition, the relation between HJURP expression and survival was explored in microarray data sets by dividing the cases from each cohort into a group with high (top one-third), moderate (middle one-third), and low (bottom one-third) level of expression. All analyses were performed by SPSS 11.5.0 for Windows. A two-tailed P-value of less than 0.05 was considered to indicate statistical significance.
Discussion
The current study is the first to report that HJURP is overexpressed in breast cancer cell lines and primary human breast cancer compared to non-malignant human mammary epithelial cells and normal breast tissues. High HJURP mRNA expression is significantly associated with both shorter disease-free and overall survival which were validated in five independent clinical datasets for breast cancer. Furthermore, HJURP is a predictive marker for sensitivity of radiotherapy, indicating levels of HJURP mRNA and protein in breast cancer patients are clinically relevant.
Although we found
HJURP mRNA levels were not associated with ERBB2 status, the mRNA levels of
HJURP was still found significantly higher in triple-negative (ER negative, PR negative, ERBB2/HER2/neu not overexpressed) breast cancer, possibly due to the fact that a higher
HJURP mRNA level is significantly associated with ER or PR negative status. Triple negative breast cancer has distinct clinical and pathological features, and also has relatively poor prognosis and aggressive behavior [
18‐
20], consistent with our finding that high
HJURP expression is associated with a bad prognosis. Furthermore, our studies showed that the prognostic effect of
HJURP mRNA level on survival is independent of the clinical factors, such as age, lymph node, pathological stage, SBR grade, ER, PR, tumor size, and the molecular subtypes. In addition, we found there is a significant correlation between
HJURP expression and Ki67 proliferation indices; however,
HJURP expression is a better biomarker than Ki67 proliferation indices for the predication of prognosis.
It is very interesting to find that the
HJURP mRNA level is a predictive marker for radiotherapy sensitivity. Our results showed that patients with low mRNA levels of
HJURP already had a good prognosis and could not get further benefit from radiotherapy, suggesting these patients may not necessarily benefit from receiving radiotherapy. However, patients with high
HJURP mRNA levels could increase their survival with radiotherapy, but they still had a worse prognosis than those with low levels as found in Dataset 3 (Figure
4c) and Dataset 4 (Figure
5a) where almost all patients received radiotherapy with or without additional benefit. Thus a high level of
HJURP is overall associated with poor prognosis. Although we note our findings will require replication in additional independent and larger cohorts, our
in vitro studies further confirmed that breast cancer cells with high levels of
HJURP are more sensitive to radiation treatment, and even more convincingly, knock down of
HJURP by shRNA reduces the sensitivity to radiation. The radiation induced more apoptosis in these cells, consistent with clinical findings. A previous report showed that
HJURP interacts with proteins hMSH5 and NBS1, suggesting
HJURP is involved in the DNA double-strand break repair process [
7]. The understanding of the roles that
HJURP plays in DNA repair and cell death in response to DNA damage may provide new insights into the molecular mechanisms of breast tumor development and may help to improve breast cancer therapies. In addition, we found that cells with
HJURP shRNA grew slowly (data not shown), which is consistent with the finding that the double time of cell lines was negatively correlated with
HJURP protein level, indicating
HJURP plays an important role in cell proliferation. Thus one of the reasons why the ability of
HJURP to act as a marker for prognosis and response to radiotherapy may be linked to its control of cell proliferation.
HJURP has recently been reported to interact with CENP-A for the purpose of localizing CENP-A and loading new CENP-A nucleosomes on the centromere [
11,
12]. CENP-A is the key determinant of centromere formation and kinetochore assembly, which regulate the complex job of attaching chromosomes to the mitotic spindle; ensuring that those attachments are correct; signalling a delay in mitotic progression if they are not, and regulating the movements of the chromosomes towards the spindle poles in anaphase. Thus overexpression of
HJURP in human breast cancer may be similar to overexpression of mitotic kinases, such as Aurora kinases, which induce genomic instability that is one of the hallmarks for tumor development. In this study we showed that
HJURP mRNA levels are highly significantly correlated with CENPA mRNA levels in human breast cancer cell lines and primary breast tumors. Such correlation is also found in other types of human cancer, such as cancers from lung, ovary, prostate (data not shown), suggesting that compatible mRNA levels of
HJURP and CENPA might be required for tumor progression. Further investigation of the interaction between
HJURP and CENPA for breast cancer development will be carried out in our future studies.
Acknowledgements
The research was supported by the National Institutes of Health, National Cancer Institute grant R01 CA116481 (JHM); by the Director, Office of Science, Office of Biological & Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231, by the National Institutes of Health, National Cancer Institute grants P50 CA 5820, the P30 CA 82103, and the U54 CA 112970 (JWG).
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
ZH and GH contributed equally. ZH, GH, SG, MP and NB performed in vitro studies. JHM and GH performed statistical analysis. AS and MEL provided microarray expression and survival data. JHM, EAB, ZH and JWG designed the study, and drafted and revised the paper. All authors read, commented, and approved the final manuscript.