Background
Breast cancer is the most common malignancy among women and represents an important public health issue [
1]. It is the major cause of cancer-related death in women, and treatment is particularly difficult in patients with tumor metastasis [
2]. Despite recent improvements in survival rates, many patients relapse, and the majority of these patients die from disseminated metastatic disease [
3].
The chromosome 1p36 locus is frequently mutated in many types of human tumors, especially in breast tumors. This lesion is associated with progression and lymph node metastasis [
4], poor prognosis [
5], higher rate of recurrence [
6], large tumor size and DNA aneuploidy [
7]. Deletion of this chromosome region occurs late in oncogenesis and is correlated with aggressive tumor growth, suggesting that one or more tumor-related genes may reside in this region [
8]. However, no specific tumor suppressor gene at 1p36 has yet been identified. Previous studies have identified
CHD5[
9] and
KIF1B[
10] as candidate tumor suppressor genes in this region, and more recently it was reported that disruption of
PER3 function may indicate the likelihood of tumor recurrence in patients with ERα-positive tumors [
11]. However, to date, no specific roles for these genes in breast cancer development have been demonstrated.
CDK11
p58 belongs to the large family of p34cdc2-related kinases [
12] and is located on human chromosome 1p36.33, a region frequently mutated in numerous human tumors, including breast cancer. CDK11
p58 is specifically expressed in the G2/M phase, and expression is closely associated with cell cycle arrest, oncogenesis and apoptosis [
13,
14]. Centrosome abnormalities are a common feature of breast cancer [
15,
16]. The overexpression of centrosome-related genes has been detected in premalignant lesions,
in situ breast tumors and over 70% of invasive breast tumors, and is associated with aneuploidy and tumor development [
17]. CDK11
p58 is a centrosome-associated mitotic kinase involved in centrosome maturation and bipolar spindle formation [
18] and is required for centriole duplication and Plk4 recruitment to mitotic centrosomes [
19]. Furthermore, we found that Thr-370 in CDK11
p58 is required for its autophosphorylation, dimerization and kinase activity [
13].
Previous studies have shown that CDK11
p58 is involved in the negative regulation of steroid receptors in a kinase activity-dependent manner, including androgen [
20], vitamin D [
21] and estrogen receptors (ERs) [
22]. We previously demonstrated that CDK11
p58 promotes the ubiquitin/proteasome-mediated degradation of ERα to repress its transcriptional activity [
22]. The sex steroid estrogen plays a major role in the development and progression of breast cancer and promotes breast cancer proliferation through a number of established pathways [
23]. Estrogen promotes breast cancer cell motility and invasion in ERα-positive breast cancer cells, largely through ERα via FAK and N-WASP [
24]. Additionally, the estrogen-ERα complex stimulates the transcriptional activity of MMP-26 and contributes to the survival of ERα-positive breast cancer patients [
25]. Because CDK11
p58 is involved in the negative regulation of the ERα signaling pathway, we speculated whether CDK11
p58 may function as a tumor suppressor in breast cancer via the inhibition of ERα.
In this study, we demonstrate that CDK11p58 inhibited the invasion of ERα-positive breast cancer cells by downregulating integrin β3 expression via ERα signaling. Moreover, the functions of CDK11p58 were highly dependent on its kinase activity. These data indicate that CDK11p58 plays an important role in the negative regulation of breast cancer invasion.
Methods
Patient samples
RNALater-preserved solid tissues, formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues and adjacent normal tissue (ANCT) were obtained from Fudan University Shanghai Cancer Center between 2002 and 2004. The tumors were assessed according to the WHO classification by two academic pathologists. This study was approved by the Ethics Committee of Fudan University Shanghai Cancer Center for Clinical Research. Written informed consent was obtained from all patients.
Immunohistochemical (IHC) analysis
A total of 250 FFPE blocks of breast cancer tissues and ANCT were collected for tissue microarrays. Two breast cancer tissue cores and two ANCT cores from the same patient’s FFPE blocks were arranged on a recipient paraffin block (with a 1-mm core per specimen). Paraffin sections (3-μm thick) were deparaffinized in xylene and rehydrated in a graded alcohol series, boiled with 10 mmol/L of citrate buffer (pH 6) for 15 min and pre-incubated in blocking solution (10% normal goat sera) for 1 h at room temperature. The steps were performed using the Envision two-step method. The Envision and DAB Color Kit was purchased from Gene Tech Company Limited (Shanghai, China). A rabbit anti-human polyclonal antibody against CDK11 was used at a 1:100 dilution. PBS (phosphate buffered saline) was used as a negative control. The tissue microarray slides were concurrently evaluated by two of the authors. Granular nuclear staining was assessed as positive. The staining index (SI, range 0–9) was considered as the product of the intensity score (0, no staining; 1+, faint/equivocal; 2+, moderate; 3+, strong) and the distribution score (0, no staining; 1+, staining of <10% of cells; 2+, between 10% and 50% of cells; and 3+, >50% of cells). For CDK11 protein in this study, a moderate/strong cytoplasm staining (SI = 3–9) was defined as positive staining, and a weak or negative staining (SI = 0–2) was defined as negative staining.
Cell culture and transfection
MCF-7, MDA-MB-231, ZR-75-30 and 293 T cells were grown using RPMI1640 supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin (Cat. 10378-016, Invitrogen, USA) at 37°C and 5% CO
2. The constructions of CDK11
p58 mutant plasmids T370A and T370D were mentioned in our previous report [
13]. Cell transfection was performed with Lipofectamine 2000 transfection reagent (Cat. 11668-019, Invitrogen) according to the manufacturer’s instructions.
RNA interference
Small interfering RNAs (siRNA) designed for CDK11 (three siRNAs, siRNA1–3) and ERα (siERα) were ordered from Shanghai GenePharma Co., Ltd. The sequences of the three siRNAs for siCDK11 were as follows: siRNA1, 5′-GAAGCAUGCUAGAGUGAAATT-3′, siRNA2, 5′-GGGAAUGGGAAAGACAGAATT-3′, and siRNA3, 5′-GCAGCAACAUGGACAAGAUTT-3′. The siERα sequence was 5′-GGAGAAUGUUGAAGCACAATT-3′.
Transwell invasion
Cell invasion was assayed using BD BioCoat Growth Factor Reduced (GFR) Matrigel Invasion Chambers (BD, CA). Transfected ZR-75-30 cells (0.5 ml; 2.5 × 104 cells/ml) were added to the inside of the inserts and incubated for 3 h. After incubation, non-invading cells were removed from the upper surface of the membrane using cotton-tipped swabs. The cells on the lower surface of the membrane were stained with Crystal violet and counted in the central field of triplicate membranes.
TaqMan® Array Metastasis 96-Well Plates were obtained from Life Technologies Corporation (California, USA) and contained lyophilized TaqMan® Gene Expression Assays. After converting total RNA to cDNA via reverse transcription, TaqMan® Array Plates and the associated reagents were used to quantitate mRNA expression levels. The results were analyzed using the RQ manager software. Genes altered in the Taqman Array gene expression assays were subsequently verified using a qRT-PCR assay system. Total RNA was extracted using TRIzol reagent (Invitrogen). After converting total RNA to cDNA in a reverse transcription (RT) reaction by Cdna Synthesis Kit (Cat. 6110A, TaKaRa, Dalian, China), qPCR were used to quantitate the mRNA expression levels by SYBR® Premix Ex Taq™(Cat. RR420Q, TaKaRa). The cycling conditions were as follows: 95°C for 5 minutes; 40 cycles of 95°C for 15 seconds and 60°C for 32 seconds. GAPDH was used as an endogenous control. Each qRT-PCR cycle was repeated three times to confirm the results.
Dual luciferase reporter gene assays
Dual luciferase assays were performed as previously described [
21]. Luciferase activity was measured using the dual luciferase reporter gene assay (Promega, USA), as previously reported [
20], and a SynergyHT Multi-Mode Microplate Reader (BioTek, USA).
Statistical analysis
The experimental data are expressed as the mean ± standard deviation, and the statistical significance between the different groups was determined using t-tests. The relationship between CDK11 expression and the clinicopathological features of breast cancer patients was analyzed using χ2 and Fisher’s exact tests. All statistical tests were two sided, and P values less than 0.05 were considered significant.
Discussion
CDK11
p58 is involved in a variety of important regulatory pathways in eukaryotic cells, including cell cycle control, apoptosis, neuronal physiology, differentiation and transcription [
26‐
28]. CDK11
p58 is a Ser/Thr kinase, and the majority of its functions are dependent on its kinase activity. In our previous study, we found that CDK11
p58 inhibited ERα transcriptional activity by promoting its degradation via the ubiquitin-proteasome pathway. In the present study, we demonstrate, for the first time, that CDK11
p58 expression is involved in the negative regulation of breast cancer invasion in a kinase-dependent manner.
Recent studies have reported the existence of various genomic alterations of 1p36 in ERα-positive breast cancers. CDK11
p58 is located on chromosome 1p36 and plays a role in the negative regulation of ERα signaling. Approximately 70–75% of breast cancers express ER and/or progesterone receptor. In recent years, we have witnessed tremendous advances in the understanding of ERα biology, revealing a complex process of ERα signaling that includes interactions with other signaling pathways [
29]. The ERα signaling pathway plays an important role in the development and progression of breast cancer, and we previously showed that CDK11
p58 interacts with ERα in breast cancer cells. Estrogens and ERα act as promoters of cell movement in different tissues, including breast tissue [
30]. Previous studies showed that ERα induced breast cancer cell migration and invasion via the phosphorylation of FAK and N-WASP [
24]. Another report revealed that within the broader range of actions of ERα, rapid extra-nuclear signaling to the actin cytoskeleton through the Gα13/RhoA/ROCK/moesin cascade is relevant for the determination of estrogen-dependent breast cancer cell movement and invasion, which are related to cancer metastasis [
31]. Previous studies also demonstrated that the MAPK-integrated signaling crosstalk between Ras and ERα leads to increased breast cancer invasion [
32]. Because CDK11
p58 plays a negative role in the regulation of ERα, we speculated that CDK11
p58 may participate in the progression of breast cancer via the regulation of the ERα pathway.
First we examined the expression of CDK11 in 250 breast cancer patients and found that high expression of CDK11 indicated a better prognosis, while low expression of CDK11 was related with a worse breast cancer node status, relapse and metastasis. These data suggest that CDK11
p58 is related to migration and invasion. As expected, CDK11
p58 inhibited the migration and invasion of ZR-75-30 ERα-positive breast cancer cells in a kinase-dependent manner. To examine the mechanism by which CDK11
p58 inhibited breast cancer cell invasion, we specifically examined CDK11
p58-dependent gene expression changes in invasion signaling pathways using TaqMan array human metastasis plates. We observed that the expression of integrin β3 was significantly attenuated in CDK11
p58-transfected cells. Integrins are cell surface glycoproteins that control cell attachment to the extracellular matrix [
33]. Integrins play a role in mammary gland biology, with expression in all cell types within the gland [
34], and activate intracellular signaling pathways that control proliferation, differentiation, apoptosis, cell motility, migration and survival [
35,
36]. Integrin β3 is expressed on the surface of endothelial cells, smooth muscle cells, monocytes and platelets [
37]. Expression is predominantly associated with tumor metastasis and has been reported to increase the metastatic potential of melanoma, breast cancer and lymphoma cells. Interestingly, the opposite effect is observed in ovarian cancer [
38].
In this study, we found that integrin β3 promoter activity was significantly repressed by CDK11
p58 and that this repression was dependent on CDK11
p58 kinase activity. ERα belongs to the superfamily of nuclear receptors, comprising structurally conserved transcription factors that enhance the transcription of specific genes upon hormone binding [
39]. Upon E2 binding, ERα specifically interacts with ERE sequences in target promoters and stimulates the transcription of a variety of E2-responsive genes [
40]. As previously published, CDK11
p58 is capable of repressing the transcriptional activity of ERα and promotes its degradation. In the present study, we further demonstrate that the promoter activity of integrin β3 is stimulated by ERα and that the inhibition of integrin β3 by CDK11
p58 is dependent on ERα.
Decreased expression of ERα correlated with a decrease in integrin β3 protein. Furthermore, the induction of ERα expression following transfection with ERα plasmids or the activation of ERα signaling with E2 was correlated with the enhanced expression of integrin β3 protein. Overall, CDK11p58 and T370D were capable of repressing the expression and transactivation of ERα and ultimately inhibiting the expression of integrin β3, thereby inhibiting the invasion of breast cancer cells. The kinase-dead mutants T370A failed to inhibit the expression of ERα and integrin β3, and were incapable of inhibiting cancer invasion. Taken together, we conclude that CDK11p58 may inhibit breast cancer cell invasion via the inhibition of the ERα signaling pathway, leading to the inhibition of integrin β3. Furthermore, metastasis-promoting MMP2, MMP3 and MMP9 proteins were subsequently inhibited. Thus, CDK11p58 inhibits the invasion and metastasis of breast cancer via the inhibition of integrin β3.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
JW and XZ conceived and designed the study. YC and SH performed the experiments. LW and RZ analyzed the data. LS, XX, DL and YC contributed reagents, materials and analysis tools. YC wrote the paper. All authors read and approved the final manuscript.