RETRACTED ARTICLE: Overexpression of Cks1 is associated with poor survival by inhibiting apoptosis in breast cancer

Abstract

Purpose

The present study was undertaken to examine the amplification and expression status of Cks1 in breast cancer and its significance.

Methods

The amplification and expression status of Cks1 gene was examined by FISH, real-time PCR and immunohistochemistry, respectively. RNA interference was used to detect the effects of Cks1 on migration, invasion, cell cycle progress and apoptosis of breast cancer cells.

Results

Cks1 gene amplification was highly correlated with protein overexpression. Overexpression of Cks1 was strongly associated with lymph node metastasis and poor prognosis (P = 0.000, 95% CI (0.00–0.02); P = 0.008, 95% CI (0.001–0.05), respectively). Knockdown of Cks1 expression by RNA interference inhibited the cell cycle progress, migration and invasion ability of breast cancer cells. Moreover, overexpression of Cks1 inhibited apoptosis of breast cancer cells through MEK-Erk pathway.

Conclusion

Cks1 may be considered as potential novel prognostic markers and targets for the future development of specific therapeutic interventions in breast cancer.

Electronic supplementary material

The online version of this article (doi:10.1007/s00432-009-0582-8) contains supplementary material, which is available to authorized users.

Introduction

The identification of the molecular mechanisms responsible for aggressive tumor behavior is important for understanding disease progression, guiding disease management, and developing potentially novel treatment strategies. The prognosis and clinical management of patients with breast cancer are commonly determined by traditional clinicopathological factors such as tumor size and grade and lymph node status, and some biological markers such as hormone receptors, bcl-2, p53 mutations, c-erbB2, Ki-67 and nuclear DNA ploidy (Mori et al. 1997). However, these markers are not sufficient to guide physician in diagnosis and treatment. So some new markers should be found. The Cks1 gene is located at chromosome 1q21, a region that is commonly overrepresented in breast cancer (Orsetti et al. 2006), prostate cancer (Valeri et al. 1999), and bladder cancer (Huang et al. 2002). The Cks1 protein is a member of the highly conserved family of Cks/Suc1 proteins which interact with Cdks and was found to be an essential cofactor for efficient Skp2-dependent ubiquitination of p27. Some studies have investigated the expression of Cks1 in different human cancers and its relation to Skp2 and p27 expression (Ganoth et al. 2001; Shapira et al. 2005; Sitry et al. 2002). However, the exact mechanism of Cks1 action in breast cancer remained poorly understood until recently. In the present study, we investigated the amplification and expression status of Cks1 in breast cancer. A positive correlation was found between Cks1 amplification and overexpression, suggesting that protein overexpression was the result of gene amplification. In addition, overexpression of Cks1 was associated with lymph node metastasis and poor prognosis in breast cancer. Function experiments showed that decreased expression of Cks1 by siRNA inhibited MDA-MB-231 cells migration and invasion. Knockdown of Cks1 expression promoted the apoptosis of breast cancer cells and overexpression of Cks1 by positive transfection inhibited apoptosis. This effect was mediated by the MEK-ERK signal pathway. Our results suggest that Cks1 is an oncogene in the 1q21 amplicon that plays an important role in breast cancer development. Cks1 thus may be a potential gene therapy target for breast cancer.

Materials and methods

Tissue specimens

Fresh tissues containing breast cancer and adjacent histological normal tissue were procured from surgical resection specimens collected by the Department of Pathology in Cancer Hospital, Shandong province, China. Primary tumor regions and the corresponding histological normal tissue from the same patients were separated by experienced pathologists, and immediately stored at −70°C until use. None of the patients received treatment before surgery. Number of patients received adjuvant chemotherapy and hormonal therapy were 98 (65.3) and 121 (80.7%), respectively. All patients signed informed consent forms for sample collection. Using of patient samples of tumor and adjacent histologically normal tissues had been approved by our institutional ethics committee.

Fluorescence in situ hybridization (FISH)

Tumor tissues were scraped and then digested with 0.2% type II collagenase (Sigma). Cells were incubated in 0.075 M KCl hypotonic buffer at 37°C for 60 min and fixed in methanol/acetic acid (3:1, v/v) at 4°C. Single-cell suspensions were dropped on cool wet slides. After being air dried overnight, slides were sequentially treated with RNase and pepsin. Denaturation was in 70% formamide, 2 × SSC, pH 7 for 3 min at 75°C. A BAC clones of Cks1 (RP11-95I19, Invitrogen) and 1p36 locus (RP5-963K15) were labeled by random primer technique with cy3-dUTP and FITC-dUTP, respectively. The probe was denatured at 74°C for 8 min and then hybridized to denatured slides at 37°C for 48 h. Post-hybridization washes were carried out in 50% formamide for 15 min and twice in 2 × SSC. Slides were counterstained with 4,6-diamidino-2-phenylindole (DAPI). Grey images were captured with a cooled charged-coupled device camera (Princeton Inc.) equipped with an Opton fluorescence microscope. The images were analyzed using the MetaMorph Imaging System (Universal Imaging Corp). Probes were tested by FISH on interphase nuclei and metaphase spreads from normal lymphocytes and yielded two signals. 100 interphase cell nuclei of each tumor sample were analyzed and ratios (target BAC/control) >2 were assessed as amplification (Sen et al. 2002).

Immunohistochemical staining

A total of 150 formalin-fixed, paraffin-embedded primary breast carcinomas and the corresponding normal control were collected for immunohistochemical analysis. The slides were deparaffinized, rehydrated, then immersed in 3% hydrogen peroxide solution for 10 min, heated in citrate buffer (pH 6.0) at 95°C for 25 min, and then cooled at room temperature for 60 min. The slides were blocked by 10% normal goat serum at 37°C for 30 min, and then incubated with rabbit polyclonal antibody against Cks1 (1:200, Santa Cruz), estrogen receptor (ER) (1:500, Santa Cruz), progesterone receptor (PR) (1:500, Santa Cruz), Her2 (1:500, Santa Cruz) and Ki 67(1:500, Santa Cruz) for 3 h at 37°C, respectively. After being washed with PBS, the slides were incubated with biotinylated second antibody (diluted 1:100) for 30 min at 37°C, followed by a streptavidin–peroxidase (1:100 dilution) incubation at 37°C for 30 min. Immunolabeling was visualized with a mixture of DAB solution. Counterstaining was carried out with hematoxylin. The immunohistochemical slides were scored according to the percentage of tumor cells exhibiting nuclear staining. The extent of positively Cks1 stained nuclei was scored into three grades: 0 for <10%, 1 for 10–30%, 2 for >30%.

Western blot analysis

The proteins were separated by SDS-PAGE and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). Blots were blocked and then probed with antibodies against Cks1 (1:1,000 dilution, Santa Cruz), Akt (1:1,000 dilution, Santa Cruz), p-Akt (Ser473, 1:400 dilution, Santa Cruz), Erk (1:1,000 dilution, Santa Cruz), p-Erk (Tyr204, 1:400 dilution, Santa Cruz), caspase 3 (1:500 dilution, Santa Cruz) and PARP (1:500 dilution, CST) and beta-actin (1:5,000 dilution, Sigma). After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibodies and visualized by super ECL detection reagent (Applygen, Beijing, China).

Cell cultures

Human breast cancer cells (MDA-MB-231 and MCF-7) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Biological Industries) supplemented with 10% fetal bovine serum and cultured at 37°C in 5% CO₂. Expression level of Cks1 is higher in MDA-MB-231 cell and lower in MCF-7 cell (Slotky et al. 2005).

Plasmids and transfection

Vector pSUPPRESSOR generates biologically active siRNAs from the U6 promoter (Imagenex, San Diego, CA). A synthetic double-strand oligonucleotide (5′-GGGACATAGCCAAGCTGGTCgagtactgcGACCAGCTTGGCTATGTCC-3′) was introduced into pSUPPRESSOR. Oligonucleotide sequences correspond to a 19-nt sequence from Cks1 (nucleotide 78–96) which is separated by a 9-nt linker from the reverse complement of the same 19-nt sequence. We used a circular control plasmid, which contains a scrambled sequence that does not show significant homology to rat, mouse, or human gene sequences, as a control. Mutant pcDNA3.1-Cks1 preserving the same amino acid sequence, but containing three point mutations in the target nucleotide sequence and therefore resistant to Cks1-shRNA, was produced by GeneChem company(Shanghai, China).The wild-type Cks1 was cloned into pcDNA3.1 to create the overexpression vector. Cell transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacture’s instruction. In transient transfection, 100 nM Cks1 siRNA or scrambled siRNA were used, and cells were harvested 48 h after transfections. In the stable transfection, cells were selected with 200 μg/ml G418 and clones were isolated by serial dilution. The pool of Cks1 RNAi clones were chosen for subsequent experiments.

Flow cytometry

Cells were harvested 48 h post-transfection for apoptosis detection using the annexin V-FITC apoptosis detection kit (Sigma) and subsequently analyzed by flow cytometry. Mitogen-activated protein/ERK kinase (MEK) inhibitor PD98059 (2-amino-3-methoxyflavone, Calbiochem, 40 μmol/L) were used in the apoptosis assay.

Migration and invasion assays

For haptotactic cell migration assay, 15 × 10⁴ Cks1 RNAi, Scrambled-RNAi, and parental MDA-MB-231 cells were seeded on a fibronectin-coated polycarbonate membrane insert (6.5 mm in diameter with 8.0-μm pores) in a transwell apparatus (Costar) and cultured in RPMI 1640. FBS was added to the lower chamber. After incubation for 12 h at 37°C in a CO₂ incubator, the insert was washed with PBS and cells on the top surface of the insert were removed by wiping with a cotton swab. For the Matrigel chemoinvasion assay, the procedure was similar to the haptotactic cell migration assay, except that the transwell membrane was coated with 300 ng/μl Matrigel (BD Biosciences) and the cells were incubated for 24 h at 37°C. Cells that migrated to the bottom surface of the insert were fixed with methanol and stained with 0.4% crystal violet and then subjected to microscopic inspection. Cells were counted based on five field digital images taken randomly at 200×.

Statistical analysis

All statistical analyses were performed using SPSS13.0 software. We statistically evaluated experimental results using the Kruskal–Wallis test, Mann–Whitney test and ANOVA test. Survival curves were constructed using the Kaplan–Meier method. P < 0.05 was considered statistically significant.

Results

Overexpression of Cks1 in breast cancer and correlation between Cks1 gene amplification and protein expression

Amplification of Cks1 gene was observed in 37 out of 60 (61.7%) tumors. Signals of 1p36 probe (RP5-963K15) as control indicated that the increase in Cks1 copy number did not result from polyploidy of chromosome 1 (Fig. 1). We did not find any case showing chromosome 1 polyploidy in the present series. Amplification of Cks1 was associated with lymph node metastasis and stage of TNM classification (Table 1).

Fig. 1.

Amplification and overexpression of Cks1 and Ki67 expression in breast cancer. FISH analysis in one representative cell (Cks1 in red, RP5-963K15 as a control in green) from several tumors in which Cks1 gene amplification was detected (left) and IHC analysis in the same corresponding cases (right). With the increase of Cks1 signal number, the expression of Cks1 and Ki67 protein also increased

Table 1.

Gene amplification and tumor clinicopathologic features

Clinicopathologic features	Number of cases	No amplification	Amplification	P
Age
≥50 years	32	13 (21.7)	19 (31.7)	0.696†
<50 years	28	10 (16.7)	18 (30)
TNM classification
pT
pT1	12	5 (8.3)	7 (11.7)	0.214‡
pT2	12	6 (10)	6 (10)
pT3	14	4 (6.7)	10 (16.7)
pT4	22	8 (13.3)	14 (23.3)
Lymph node metastasis
N0	29	20 (33.3)	9 (15)	0.018†
N1	31	3 (5)	28 (46.7)
Stage
I	5	3 (5)	2 (3.3)	0.009‡
II	8	5 (8.3)	3 (5)
III	20	8 (13.3)	12 (20)
IV	27	7 (11.7)	20 (33.3)
Grade
G1	23	9 (15)	14 (23.3)	0.401‡
G2	18	8 (13.3)	10 (16.7)
G3	19	6 (10)	13 (21.7)
ER
Negative	23	10 (16.7)	13 (21.7)	0.518†
Positive	37	13 (21.7)	24 (40.0)
PR
Negative	21	8 (13.3)	13 (21.7)	0.978†
Positive	39	15 (25.0)	24 (40.0)
Her-2
Negative	37	11 (18.3)	26 (28.3)	0.082†
Positive	23	12 (20.0)	11 (18.3)

^†Kruskal–Wallis test

^‡Mann–Whitney test

To determine whether amplification of the Cks1 gene affected protein expression in breast cancer, immunohistochemistry was performed on the samples that had been analyzed by FISH. Overexpression of Cks1 was observed in almost all tumors with gene amplification (33 out of 36 cases with amplification), while lack of overexpression in tumors without amplification was observed in 79.2% (19 out of 24) of the cases. Significant positive correlation was found between Cks1 gene amplification and protein overexpression (data not shown). In addition, the expression level of Cks1 and Ki67 increased with an increase of Cks1 signal (Fig. 1).

We then detected the expression of Cks1 in 150 tumor samples including above samples. Statistical analysis showed that overexpression of Cks1 was associated with lymph node metastasis and stage of TNM classification. No correlation was found between Cks1 expression with ER, PR and Her-2 expression (Table 2). Significantly negative correlation was found between Cks1 expression and prognosis (P < 0.05) (Supplemental Fig. 1). Multivariate Cox regression analysis showed that Cks1 expression (P = 0.012) and regional lymph node metastasis (P = 0.035) were independent prognostic factors (Table 3).

Table 2.

Protein overexpression and tumor clinicopathologic features

Clinicopathologic features	Number of cases	Cks1 expression			P
		Negativea n (%)	Positivea n (%)	Strongly positivea n (%)
Age
≥50 years	90	49 (32.7)	16 (10.7)	25 (16.7)	0.104†
<50 years	60	24 (16)	19 (12.7)	17 (11.3)
TNM classification
pT
pT1	35	21 (14)	8 (5.3)	6 (4)	0.121‡
pT2	54	34 (22.7)	10 (6.7)	10 (6.7)
pT3	61	18 (12)	17 (11.3)	26 (17.3)
Lymph node metastasis
N0	70	55 (36.7)	10 (6.7)	5 (3.3)	0.000†
N1	80	18 (12)	25 (16.7)	37 (24.7)
Stage
I	24	19 (12.7)	2 (1.3)	3 (2)	0.014‡
II	24	8 (5.3)	8 (5.3)	8 (5.3)
III	40	25 (16.7)	7 (4.7)	8 (5.3)
IV	62	21 (14)	18 (12)	23 (15.3)
Grade
G1	55	19 (12.7)	10 (6.7)	26 (17.3)	0.329‡
G2	47	28 (18.7)	12 (8)	7 (4.7)
G3	48	26 (18.7)	13 (8.7)	9 (6)
ER
Negative	69	30 (20.0)	19 (12.7)	20 (13.3)	0.473†
Positive	81	43 (28.7)	20 (13.3)	18 (12.0)
PR
Negative	72	36 (24.0)	15 (10.0)	21 (14.0)	0.783†
Positive	78	37 (24.7)	20 (13.3)	21 (14.0)
Her-2
Negative	54	21 (14.0)	13 (8.7)	20 (13.3)	0.102†
Positive	96	52 (34.7)	22 (14.7)	22 (14.7)

^†Kruskal–Wallis test

^‡Mann–Whitney test

^aFor definition see text

Table 3.

Multivariate analysis of prognostic factors by the Cox proportional hazards model in 100 breast cancer patients

Variables	Relative risk	95% confidence interval	P value
Lymph nodes metastasis	3.221	1.131–4.462	0.035
Tumor stage	0.841	0.240–2.431	0.674
Cks1 expression	4.271	1.249–4.260	0.012

Decreased expression of Cks1 inhibit MDA-MB-231 cells invasion and migration

Knockdown of Cks1 expression inhibited the ability of cells to invade surrounding tissues, as determined by Matrigel assay. We also examined cell migration and MDA-MB-231 cells treated with Cks1 RNAi demonstrated a decreased ability to migrate through membranes of 8-μm pore size that were not coated with Matrigel (Supplemental Fig. 2).

Knockdown of Cks1 promoted apoptosis of breast cancer cells

As shown in Fig. 2a, the total percent (early and late apoptosis) of apoptotic Cks1 RNAi cells was higher than that of the control and parental cells in the assessment by annexin V-FITC/PI staining. We then examined the effects of Cks1 knockdown on caspase 3 activity (a central mediator of apoptosis). Decreased Cks1 expression markedly increased the activity of caspase 3 compared to parental or scrambled RNAi controls (Fig. 2c).

Fig. 2.

Silencing of Cks1 promoted apoptosis of MDA-MB-231 cells. a Representative images of flow cytometry analysis of apoptotic cells by annexin V-FITC/PI staining. b Flow cytometry results were plotted as mean ± SD from triplicate experiments. P < 0.01 versus scrambled RNAi or parental MDA-MB-231 cells. c Down-regulation of Cks1 led to increased activation of caspase 3, as evident from increased levels of Cleaved-PARP. d Expression levels of Cks1 in parental, Cks1 RNAi and rescue groups. e Percentage of apoptosis cells in rescue group was significantly lower than that of Cks1 RNAi group. f Flow cytometry results were plotted as mean ± SD from triplicate experiments

To verify that the effects of RNAi described above are specific, we prepared a Cks1 construct bearing triple-point mutation in the 19-bp sequence that served as target for shRNA-mediated knockdown in our experiments. The mutation conserved the amino acid sequence of the Cks1 protein, but rendered its expression insensitive to inhibition by the shRNA. Transfection of this mutant shRNA into the cells with stable expression of Cks1-shRNA, indeed, restored the ability of Cks1 to inhibit apoptosis (Fig. 2e, f).

Effects of Cks1 on apoptosis were mediated by MEK-ERK pathway

We subsequently examined the potential effect of Cks1 on the activation of several known pathways associated with apoptosis. Only phosphorylated ERK was down-regulated in Cks1 siRNA cells (Fig. 3a). We next treated the cells with the MEK inhibitor PD98059. As shown in Fig. 3b, c, the percentage of apoptosis cells in Cks1 RNAi group after adding PD98059 was not significantly different with those of control groups, suggesting that MEK-ERK pathway is the downstream target of Cks1.

Fig. 3.

Effects of Cks1 on apoptosis were mediated by MEK-ERK pathway. a Down-regulation of Cks1 led to decreased expression of p-ERK. No changes in p-AKT expression were observed. b Representative images of flow cytometry analysis of apoptotic cells by annexin V-FITC/PI staining. c Flow cytometry results were plotted as mean ± SD from triplicate experiments. d Increased expression of Cks1 led to increased levels of p-ERK. e Forced overexpression of Cks1 inhibited apoptosis of MCF-7 cells. Representative images of flow cytometry analysis of apoptotic cells by annexin V-FITC/PI staining. f Flow cytometry results were plotted as mean ± SD from triplicate experiments

To further confirm the above results, we examined the apoptosis of MCF-7 cells in which the expression of Cks1 was low after transfection with wild-type Cks1 expression vector. The p-ERK level was increased in the Cks1 + pcDNA3.1 group (Fig. 3d). Moreover, apoptosis of MCF-7 cells was significantly inhibited by overexpression of Cks1 (Fig. 3e, f). These data further confirmed that Cks1 can protect breast cancer cells from apoptosis via MEK-ERK pathway.

Discussion

Multiple genetic changes have been found in breast cancer, but little is known about the major oncogenes and tumor suppressor genes involved in the tumorigenesis of the disease. Proto-oncogenes are frequently activated by genomic amplification with consequent overexpression, which play an important role in the development of human cancers. Characterization of genes with copy number increase and overexpression in tumor tissues will facilitate the identification of tumor-specific oncogenes. Some studies previously identified chromosome 1q as a frequently amplified region in many carcinomas by comparative genomic hybridization (CGH) (Albertson 2003; Borg et al. 1992; Wei et al. 2002). In the present study, we found that the Cks1 gene is amplified in primary breast cancer. Moreover, the status of amplification was significantly associated with lymph node metastasis and stage of TNM classification.

Clues for discrimination between amplified genes which are critical to carcinogenesis from those that are bystanders have emerged from analysis of the correlations of gene amplification/expression, functional characterization, or association with clinical parameters. On that basis, we used the same samples to evaluate the prevalence of Cks1 genomic amplification by FISH and Cks1 protein expression by IHC. When the results of IHC and FISH were compared, correlation between protein overexpression and gene amplification was significant, supporting the hypothesis that gene amplification was indeed an important mechanism for Cks1 overexpression. To further examine the relationship between Cks1 expression and clinical parameters, immunohistochemistry was done in 150 tumor samples. Further statistical analysis revealed that Cks1 overexpression was significantly associated with lymph node metastasis and poor prognosis in breast cancer. Other studies also reported that increased Cks1 expression was associated with poor prognosis in colorectal carcinoma (Shapira et al. 2005) and breast cancer (Slotky et al. 2005). Therefore, we suggest that amplification and consequent overexpression of Cks1 might play an important role in breast cancer tumorigenesis and can be an independent prognostic marker. Targeting this molecule may be a promising therapeutic option for breast cancer.

Metastasis to lymph nodes is a major obstacle for successful treatment of cancer. Our results suggested that knockdown of Cks1 expression inhibited the migration and invasion activities in MDA-MB-231 cells, suggesting that Cks1 may be a useful diagnostic biomarker for breast cancer metastatic potential. We next examined the effects of Cks1 on breast cancer cells’ apoptosis. Our data showed that ERK phosphorylation decreased after Cks1 RNAi transfection and no changes were observed in p-AKT phosphorylation. Moreover, knockdown of Cks1 had no more effects on apoptosis after treated cells with MEK-ERK pathway inhibitor and forced overexpression of Cks1 inhibited apoptosis of MCF-7 cells. Thus, it is MEK-ERK pathway, not PI3K-AKT signaling, contributed to the effects of Cks1 on breast cancer cells’ apoptosis. Lan et al. recently reported that knockdown of Cks1 expression in malignant prostate tumor cells inhibited proliferation, anchorage-independent growth and migration activities, whereas knockdown of Cks2 expression induced programmed cell death (Lan et al. 2008).

Taken together, our data suggest that Cks1 is an oncogene in the 1q21 amplicon which exerts functions on tumor metastasis in breast cancer. Future studies will need to address whether targeted therapies directed against Cks1 will inhibit metastasis in breast cancer.