Background
Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. Unraveling the molecular and cellular mechanisms underlying breast cancer progression and metastasis is necessary for development of therapeutic agents to treat this disease.
PDZ-binding kinase (PBK) (also named T-lymphokine-activated killer cell–originated protein kinase (TOPK)) is a 322 amino acid MAPKK-like serine/threonine protein kinase that was identified as an interleukin-2 induced gene in T-lymphokine-activated killer cells and as an interaction partner with the human tumor suppressor hDlg [
1]. PBK is overexpressed in multiple types of cancer, including breast, prostate, colon, bladder, and lung cancer, but is undetectable in normal tissues except germ cells in the testis and several fetal tissues [
2-
5]. PBK mediates UVB-induced JNK activation and facilitates H-Ras-induced cell transformation [
6]. In addition, PBK serves as an oncogenic kinase that exerts positive feedback on ERK2 to promote colorectal cancer formation
in vitro and
in vivo [
7]. Furthermore, PBK physically interacts with the DBD domain of p53 and regulates the tumor suppressor function of p53 [
8]. PBK stimulates AKT-dependent cell migration/invasion by relieving the PTEN-dependent suppressive effect, indicating its crucial role in cancer metastasis [
9]. PBK phosphorylates histone H3 at Ser10
in vitro and
in vivo, and to function as molecular marker in breast cancer [
10]. Taken together, overexpression of PBK is correlated with oncogenesis.
Statins are a class of specific inhibitors of HMG-CoA reductase, a rate-limiting enzyme in the mevalonate pathway [
11]. The mevalonate pathway is biologically important because the metabolites of the mevalonate pathway play vital roles in protein post-translational modifications such as geranylgeranylation and farnesylation, cell membrane integrity, respiration electronic chain reaction, and cholesterol synthesis [
12]. As potent blockers of biosynthesis of cholesterol, statins have long been used in clinic to treat hypercholesterolemia and prevent cardiovascular diseases [
12]. Because cellular function of many cell growth signaling molecules, particularly small GTPases, is dependent on prenylation that is blocked by statins, treatment of cancer cells with statins inhibits cell proliferative, migration, and invasion, and induces apoptosis. Thus, statins become a promising cancer therapeutic agent against many types of cancers, including breast cancer [
12,
13]. It has been demonstrated that statins trigger tumor-specific apoptosis by blocking geranylgeranylation of the Rho family GTPase [
12,
14], resulting in disorganization of actin stress fibers [
15]. Simvastatin was shown to foster enhanced expression of mutant p53 to down-regulate CD44 expression, therefore preventing breast cancer cell metastasis to bone [
16]. Furthermore, simvastatin inactivates NF-κB, leading to de-repression of PTEN and repression of Bcl-xl to prevent breast cancer cell growth [
17]. Notably, it was recently demonstrated that the mevalonate pathway is necessary and sufficient to maintain the malignant state of breast cancer cells in 3D culture [
18]. However, specific antitumor targets and mechanisms of atorvastatin (AS) are poorly understood.
The Hippo pathway, with the transcriptional coactivator Yes-associated protein (YAP) as its downstream effector, is highly conserved throughout evolution [
19]. The mammalian Hippo pathway consists of a core kinase cascade in which MST1/2 (the Hippo analog in
Drosophila) phosphorylates the LATS1/2 kinases (Warts in Drosophila) [
20]. Activated LATS subsequently phosphorylates YAP (at Ser127) and its paralog TAZ (at Ser89) (Yorkie in Drosophila), leading to inactivation of the transcriptional activity [
21-
23]. YAP promotes tumor metastasis through interacting with the TEAD/TEF transcription factors (Scalloped in Drosophila). Increased YAP/TEAD activity was observed in cancer progression and metastasis [
24]. Upregulation of YAP and its nuclear localization strongly correlate with poor prognosis and tumor progression in multiple cancers, including breast [
25], lung, colorectal, ovarian, and liver carcinomas [
26]. Overexpression of YAP in a conditional YAP transgenic mouse model led to tissue overgrowth and tumorigenesis [
27]. Together, these studies highlight a pivotal role of the Hippo–YAP pathway in cancer development and progression.
In this report, we demonstrated that atorvastatin or the geranylgeranyltransferase (GGTase) I inhibitor GGTI-298 inhibited proliferation of the estrogen receptor (ER)-negative breast cancer MDA-MB-231 cells and down-regulated PBK, indicating that PBK is a target gene of geranylgeranylation signaling. Consistent with the effect of atorvastatin or GGTI-298, knockdown of PBK or inhibition of PBK significantly impaired MDA-MB-231 cell proliferation. Furthermore, we found that knockdown of YAP down-regulated expression of PBK, suggesting that PBK is a target gene of YAP. Our studies therefore identified PBK as a down-stream effector of geranylgeranylation signaling and a target gene of YAP, and defined a PBK signaling pathway activated by geranylgeranylation and the Hippo signaling for breast cancer growth.
Discussion
Breast cancer is the leading cause of cancer death in females worldwide. Unraveling the molecular and cellular mechanisms underlying breast cancer progression and metastasis is important for development of targeting drugs for breast cancer therapy. In this report, we have shown that atorvastatin inhibits breast cancer cell proliferation through impairing geranylgeranylation. Our data indicate that geranylgeranylation signaling controls expression of PBK, a gene whose product regulates cell mitosis, thus is important for cancer cell proliferation. Furthermore, we found that PBK is a target gene of YAP, a transcription co-activator in the Hippo pathway, suggesting that geranylgeranylation signaling activates YAP to regulate expression of PBK. Finally, we have shown that PBK is essential for breast cancer cell proliferation. However, expression of PBK is responsive to geranylgeranylation signaling only in ER- breast cancer MDA-MB-231 cells, not in ER+ breast cancer MCF7 cells. This observation raises a possibility that linkage of geranylgeranylation signaling to expression of PBK is established during breast cancer progression.
Our findings are consistent with recent studies that discovered the role of geranylgeranylation signaling in regulating the Hippo-YAP/TAZ pathway for breast cancer cell proliferation and migration [
28]. The geranylgeranylation signaling interplays with the Hippo pathway in regulating YAP/TAZ transcriptional activity. Currently the geranylgeranylated proteins that transduce signaling to the Hippo-YAP/TAZ pathway have not been identified. Prenylation, including farnesylation and geranylgeranylation, is essential for proper localization and activity of the RAS superfamily of small GTPases and heterotrimeric G-protein gamma subunit. The Rho GTPase subfamily is known to be geranylgeranylated and closely linked to cancer progression [
31]. Particularly, RhoA are thought to play a role in cell proliferation. Recent studies have shown that GPCR signaling activates RhoA GTPase that leads to inhibition of the Hippo kinases and activation of YAP/TAZ [
28,
32]. We speculate that Rho GTPase might be the geranylgeranylated protein signaling to the Hippo pathway to activate YAP and expression of
PBK.
Atorvastatin is one of the most popular medicines for reduction of cholesterol. The anti-breast cancer effect of atorvastatin has a huge impact on prevention of breast cancer. Our studies provide fundamental knowledge to help clinicians to use atorvastatin in breast cancer therapy and prevention. Our data suggest that atorvastatin can induce cytotoxicity only to certain type of breast cancer cells. The breast cancer cell must have a signaling context of geranylgeranylation signaling and the Hippo-YAP/TAZ pathway that confers the effect of atorvastatin on down-regulation of PBK. In addition, establishment of linkage of geranylgeranylation signaling to the Hippo-YAP/TAZ pathway in breast cancer cells seems developed in breast cancer progression, because our recent studies found that ER- breast cancer cells are more sensitive to atorvastatin than ER+ breast cancer cells [
28]. Thus, statins might be an effective anti-cancer drug for advanced stage breast cancer. We will further investigate the role of geranylgeranylation signaling in promoting cancer progression and validate geranylgeranylation signaling as a key targeting pathway in advanced breast cancer therapy.
PBK has been identified as a kinase regulating mitosis by phosphorylation of GPSM2 (G-protein signaling modulator 2) in breast cancer cells [
33]. PBK also interacts with p53 to regulate expression of cell cycle genes [
8]. Multiple studies have found that PBK overexpresses in breast, prostate, colon, bladder, and lung cancers and is a prognostic biomarker for poor outcomes [
2-
5]. Previous studies have shown that expression of PBK is regulated by Myc and E2F1 transcriptional factors [
34]. Immunofluorescent staining in Figure
3B has shown that PBK is localized in nuclei, confirming that PBK is a nuclear kinase and functions in phosphorylation of nuclear proteins. Furthermore, our results indicate that PBK is a target gene of YAP (Figure
5). Interestingly, it has been observed that E2F1 is also a target gene of YAP [
35]. These studies suggest that E2F1 may mediate YAP-activated expression of PBK in MDA-MB-231 cells. Furthermore, our studies have shown that PBK is connected to geranylgeranylation signaling most likely in advanced stage breast cancer, and essential for breast cancer cell proliferation, confirming that PBK is an important molecular target for breast cancer therapy. Thus, down-regulation and inhibition of PBK by either targeting geranylgeranylation signaling and the Hippo-YAP/TAZ pathway or directly impairing the kinase activity are promising approaches for breast cancer therapy, particularly advanced stage breast cancer therapy.
Materials and methods
Materials
Geranylgeraniol (G3278), GGTI-298 (G5169) and HI-TOPK-032 (SML0796) were purchased from Sigma-Aldrich. Atorvastatin calcium was from WuXi Sigma. Anti- PBK/TOPK (C-term) antibody (ab75987) was from Abcam, anti-Tubulin (G436) from Bioworld, and anti-β-actin (ac-15) from Sigma.
Cell culture
Human kidney cell line HEK293T and human breast cancer cell lines MCF-7 and MDA-MB-231 were grown in Dulbecco modified Eagle Medium (DMEM) medium(HyClone)supplemented with 10% fetal bovine serum (Excell bio), 100 U/mL penicillin, and 100 mg/mL streptomycin in 5% CO2 at 37°C. Transfection of plasmids was performed with Lipofectin transfection reagent according to the manufacturer’s protocol.
Reverse transcription PCR (RT-PCR) analysis
Total RNA was extracted using RNAiso Plus (TaKaRa) kit and reverse-transcribed into cDNA by RevertAid First Strand cDNA Synthesis Kit (TaKaRa) according to the manufacturer’s instructions. Quantitative PCR analysis was performed using iTaq Universal SYBR Green supermix (2×) (BIO-RAD). The primer pairs used for quantitative PCR are: PBK (human) forward primer: 5′-CCTTTGGCCTTACTTTGTG -3′; PBK (human) reverse primer: 5′-ACGATCTTTAGGGTCTTCAT-3′; GAPDH (human) forward primer: 5′-AACGGATTTGGTCGTATTG-3′; GAPDH (human) reverse primer: 5′- GGAAGATGGTGATGGGGAT -3′.
Preparation of cell lysates and immunoblotting
Culture medium was removed and cells were washed with cold PBS and lysed in precooled mammalian cell lysis buffer (40 mM HEPES, pH 7.4, 1% Triton X-100, 100 mM NaCl, 1 mM EDTA, 25 mM Beta-Glycerolphosphate, 1 mM Na-orthovanadate, 10ug/ml Leupeptin and 10ug/ml Aprotinin). The SDS-PAGE samples were prepared by addition of 5 × SDS sample buffer directly to the lysates, followed by rigorous vortex and denatured at 100°C for 10 min. Electrophoresis was run on 10% NuPAGE Bis-Tris SDS gels, and separated proteins were transferred onto Immobilon PVDF-FL (Millipore) membranes. The membranes were incubated with primary antibodies overnight at 4°C, followed by incubating with secondary antibodies for 1 h at room temperature. The protein bands were visualized by Enhanced Chemiluminescence Plus reagent (Millipore). The density of the bands was quantified using Quantity One software (MiNiCHEMI).
Immunofluorescent staining
The cells were cultured in glass coverslip-bottomed culture dishes (MatTek, Ashland, MA) to 50-80% confluence. After the culture medium was aspirated, the cells were rinsed with PBS twice, fixed with 3.7% paraformaldehyde at 25°C for 30 min, and permeabilized with 0.2% Triton X-100 in PBS at 25°C for 20 min. After washing with TBST, the cells were incubated with primary antibody at 37°C for 1 h. Then the cells were washed with TBST three times and incubated with secondary antibody that was conjugated with a fluorescent dye at 37°C for 1 h. Finally, the cells were washed with TBST three times, and the immunofluorescence staining was visualized under a Nikon inverted fluorescent microscope. The nuclei were stained with DAPI.
Lentiviral shRNA cloning, production, and infection
Knockdown of PBK or YAP was carried out by infection of cells with lentiviral vector-loaded shPBK or shYAP. The PBK shRNA target sequence is CTCTTCTCTGTATGCACTAAT; and the YAP shRNA targeting sequences is CCCAGTTAAATGTTCACCAAT. To produce the lentiviral particles, the pLKO. 1-tet-puro vector was co-transfected with the packaging plasmids psPAX2 and pMD2.G into HEK 293 T cells, and the cultured supernatant containing the viral particles was collected at 24, 48 and 72 hrs after transfection. This supernatant was used as the shRNA viral stock solution. For lentiviral infection, the shRNA viral stock solution was added into MDA-MB-231 cell culture medium for 24 hrs in the presence of 4 μg/mL polybrene. In general, the viral infection efficiency was about 80%. After the infection, cells were cultured in DMEM plus 10% FBS and 2 μg/ml puromycin. Selection of puromycin-resistant cell colonies was carried out 72 h after transfection. The cell colonies resistant to puromycin were selected and cultured in DMEM plus 10% FBS, 1 μg/ml tetracycline and 2 μg/ml puromycin. The knockdown efficiency was evaluated by detection of protein level with immunoblotting or mRNA level with qRT-PCR.
Statistical analysis
The experimental data are analyzed statistically using Student’s t-test for two-treatment comparisons. P < 0.05 is considered as significant.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XD performed most of the experiments and participated in preparation of the manuscript; JW carried out a part of RT-PCR experiments; AS carried out a part of PBK knockdown experiments; GS participated in supervision and design of experiments; CC carried out immunofluorescent staining experiments; WY participated in design of experiments, analysis of data and preparation of the manuscript; QL supervised and designed experiments, analyzed data and prepared the manuscript. All authors read and approved the final manuscript.
XD, JW, and AS are currently graduate students in the Tumor Molecular Biology program at School of Medical Sciences and Laboratory Medicine, Jiangsu University. GS is an associate professor at School of Medical Sciences and Laboratory Medicine, Jiangsu University. CC is a Research Technician III at Weis Center for Research. WY is a Staff Scientist at Weis Center for Research and a professor at School of Medical Sciences and Laboratory Medicine, Jiangsu University. His research group has found that geranylgeranylation signaling promotes breast cancer cell proliferation and migration through the hippo-YAP/TAZ pathway. QL currently is a professor of Tumor Molecular Biology at School of Medical Sciences and Laboratory Medicine, Jiangsu University. The major interest of hers research group is oncogenic function of biochemical modifications, particularly phosphorylation, ubiquitination, methylation and geranylgeranylation, in lung, breast and gastric cancer cells.