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Keywords:

  • PAK4;
  • p57Kip2;
  • cell cycle;
  • ubiquitination;
  • degradation

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

The p21-activated kinases have been implicated in the control of cell cycle progression. However, the biological mechanism underlying the role of p21-activated kinase 4 (PAK4) in cell cycle control remains unknown. Here, by using quantitative RT–PCR and immunoblot analyses, we discovered that over-expression of PAK4 could suppress cyclin-dependent kinase inhibitor 1C (p57Kip2) expression in the MCF-7 human breast cancer cell line, whereas lentiviral vector-mediated small interfering RNA (siRNA) knockdown of PAK4 markedly promoted p57Kip2 expression in MCF-7 cells. Furthermore, PAK4-mediated down-regulation of p57Kip2 was reversed by MG132, a specific proteasome inhibitor. The ubiquitination assay confirmed that the activity of PAK4 attenuated p57Kip2 protein stability through the ubiquitin-proteasome pathway in MCF-7 cells. Moreover, a significant inverse correlation between PAK4 and p57Kip2 protein levels was observed in breast cancer tissues by immunohistochemical analysis. Taken together, our data demonstrate a novel function for PAK4 in regulating the stability of p57Kip2, possibly through the ubiquitin-proteasome pathway, leading to increased proliferation of breast cancer cells. Thus, PAK4 may be used as a potential diagnostic and therapeutic target for human breast cancer. Anat Rec, 296:1561–1567, 2013. © 2013 Wiley Periodicals, Inc.

The p21-activated kinases (PAKs) are members of a family of serine/threonine kinases. There are six mammalian PAKs that can be classified into two groups: group I PAKs (PAK1–3) and group II PAKs (PAK4–6). PAKs have been shown to play pivotal roles in various tumorigenic signaling pathways (Kumar et al., 2006). They have also been implicated as the downstream effectors of the small Rho GTPases Cdc42 and Rac1 (Li et al., 2010), which play important roles in cytoskeletal reorganization, cell survival, gene transcription, and tumorigenesis (Kumar et al., 2006; Li et al., 2010). PAK4 has been the most extensively studied member of the group II PAKs. It is known to be over-expressed in a variety of cancers and tumor cell lines. The PAK4 gene is localized to a region of chromosome 19, which is commonly amplified in a number of human colon, ovarian, and pancreatic cancers (Qu et al., 2001; Chen et al., 2008; Begum et al., 2009). More importantly, the in vivo over-expression of wild-type PAK4 led to tumor formation in athymic mice, while deletion of PAK4 abrogated tumor formation (Liu et al., 2008). Recently, several studies have demonstrated that PAK4 could regulate cell proliferation through the PAK4/c-Src/EGFR pathway that controls cyclin D1 expression (Siu et al., 2010) and may down-regulate p21 in the early G1 phase of the cell cycle. These data suggest that PAK4 plays a role in regulating the cell cycle that, when improperly expressed, can lead to oncogenesis (Nekrasova and Minden, 2011). However, the biological mechanism by which PAK4 can regulate the cell-cycle is not defined clearly.

The p57Kip2 protein, a cyclin-dependent kinase (CDK) inhibitor, may suppress tumor formation (Kavanagh et al., 2012). In addition, p57Kip2 has been shown to regulate the expression and kinase activity of cyclins or CDKs at the G1/S transition to inhibit the proliferation of tumor cells (Borriello et al., 2011). The down-regulation of p57kip2 expression may be an important molecular event in tumor growth and invasion (Guo et al., 2011). The p57kip2 gene has been suggested to be a tumor suppressor gene. It may be inactivated in various cancer types, linked to tumor progression and poor patient outcome. The p57kip2 protein is generally not mutated in cancer, but its expression is down-regulated through epigenetic changes, such as DNA methylation and repressive histone marks at the promoter (Kavanagh and Joseph, 2011). These data implicate p57kip2 as a tumor suppressor, but the relationship between p57kip2 and PAK4 in breast cancer has not been explored.

Here we demonstrated that PAK4 is a potent repressor of p57kip2. PAK4 regulated the stability of p57kip2 through the ubiquitin-proteasome pathway. Moreover, PAK4 expression was higher, but p57kip2 was expressed at relatively lower level in breast cancer tissues, as compared to normal breast tissues. This novel finding suggests that PAK4 promotes cell proliferation via the cell cycle inhibitor p57kip2, leading to cancer development.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

Cell Culture

MCF-7 cell lines were cultured at 37°C, 5% CO2 in Dulbecco's Modified Eagle Media (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin (Sigma).

Lentiviral Vector Construction for ShRNA-PAK4 and Infection

A lentiviral vector containing shRNA-PAK4 was purchased from Shanghai GeneChem (GeneChem, Shanghai, China). The empty shRNA lentiviral vector was used as a negative control. MCF-7 cells were transfected with recombinant lentivirus. After overnight incubation, the cells were maintained in selective medium containing puromycin (1 μg/ml) for 2–3 days before being harvested for immunoblotting analysis or RNA extraction.

Lentivirus Particles for PAK4 and Infection of MCF-7

PAK4 lentivirus particles encoding the FLAG epitope were constructed by Genechem, Ltd. (Shanghai, China). The complete cDNA sequence of PAK4 was generated by PCR, and inserted into pGC-FU-3×FLAG Vector, which was linearized with Age I and Nde I. The resultant 1776 bp fragment was confirmed by sequencing. Lentiviral vector were produced by cotransfected into HEK293T cells with helper construct. Titers of 5 × 108 TU/ml were routinely achieved. The cells were transiently transfected with control parent lentivirus or lentivirus expressing PAK4. After infection for 24 hr, the medium was changed, and the MCF-7 cells were further cultured in DMEM with 10% FBS. The efficiency of lentivirus infection of MCF-7 cells was evaluated by real-time PCR and Western Blotting.

Microarray GeneChip Expression

Total RNA was extracted from cells by the Trizol reagent method (Invitrogen, Shanghai, China). The control sample is RNA from MCF-7 cells transduced with the shRNA-control and the experimental sample is RNA from MCF-7 cells transfected with shRNA-PAK4. The microarray experiment was performed according to the GeneChip® 3′ IVT Express Kit protocol (Affymetrix, Santa Clara, CA).

Quantitative Reverse Transcriptase-Polymerase Chain Reaction

MCF-7 breast cancer cells were transiently transfected with increasing concentrations of Flag-PAK4. Cells were harvested following treatments for preparation of total RNA using Trizol reagent (Invitrogen, Shanghai, China). One microgram of RNA was used as a template for complementary DNA synthesis using Quantitect Reverse Transcription Kit (TaKaRa, Japan). Polymerase chain reaction (PCR) was performed in triplicate using an Mx3000P™ Real-Time PCR System by Agilent (Stratagene) and SYBR Green I detection (TaKaRa, Japan) according to the manufacturer's protocol. The following oligonucleotides (Invitrogen, Shanghai, China) were used for PCR amplification: PAK4 forward, 5′-CCA GGA TGA ACG AGG AGC AG-3′; PAK4 reverse, 5′-TAG GGA AGG CGG GAG ATG AG −3′. p57kip2 forward, 5′-GGG CTC TAA ATT GGC TCA CC-3′ and p57kip2 reverse, 5′-GCC TCT GAT CTC CGA TTT CTT-3′. The relative expression level of mRNA was normalized to β-actin levels with the following specific primers: β-actin forward, 5′-TCG TGC GTG ACA TTA AGG AG-3′; β-actin reverse, 5′-ATG CCA GGG TAC ATG GTG GT-3′. Relative gene expression was calculated with Mx3000P Software version 2.0 (Stratagene) by using the 2-ΔΔCt method. Statistical analysis significance was determined by parametric t tests using SPSS17.0 software (SPSS, Chicago, IL).

Ubiquitination Assay

Flag control or Flag-PAK4 lentivirus infected MCF-7 cells were treated with 10 μM MG132 for 24 h (Adams et al., 2009). Cell lysates prepared in NP-40 lysis buffer were precleared with isotypical preimmune IgG plus Protein A/G beads, followed by a brief centrifugation at 500g. Aliquots of the supernatants were incubated with anti-p57kip2 (Cell Signaling Technology, Beverley, MA) at 4°C for overnight and then mixed with protein A/G beads at 4°C for 3 h. Pellets harvested by brief centrifugation at 500g were washed with NP-40 buffer with descending Triton X-100 strength (0.1%) and subjected to SDA–PAGE followed by immunoblotting analysis with anti-ubiquitin (BD Biosciences) and anti-PAK4 (Cell Signaling Technology, Beverley, MA) antibodies.

Transfection and Immunoblotting Analysis

MCF-7 breast cancer cells were transfected with Lipofectamine™ 2000 (Invitrogen, Shanghai, China) according to the manufacture's protocol. Cells were treated with 10 μM MG132 (Sigma-Aldrich, Germany) or 10 mM CHX (Sigma-Aldrich, Germany) for indicated time. Cells were lysed with RIPA buffer (Sigma-Aldrich, Germany) and 30 μg of total protein was separated through electrophoresis on a SDS–PAGE gel and transferred to PVDF membranes (GE Healthcare). The membrane was blocked at RT for 1 hr in Tris-buffered saline (TBS) containing 0.1% Tween-20 (TBST) and 5% fat-free powdered milk, and incubated overnight with primary antibodies (PAK4 antibody, p57kip2 antibody, and GAPDH antibody) at 4°C. After incubation with the primary antibody, the membrane was then incubated with the secondary antibody for 1 hr, and washed three times for 10 min in TBST prior to chemiluminescence detection (GE Healthcare). The intensity of the bands was quantified by computerized densitometry using Quantity-One software version 4.62 (Bio-Rad). The relative optical density (OD) ratio was calculated by comparing to GAPDH.

Patients and Immunohistochemistry

We identified patients diagnosed with breast cancer who underwent surgical resection of tumor at the Pathology Department of China Medical University between 2002 and 2005. Slides of tissue sections were subjected to deparaffinization and antigen unmasking. The slides were then incubated with the antibody against PAK4 and p57kip2 at 4°C overnight. The slides were incubated with horseradish peroxidase-conjugated goat antirabbit IgG, and the color was developed with the DAB Horseradish Peroxidase Color Development Kit (Fuzhou Maixin, China). Multicenter ethical approval for data collection and tissue use was granted by the Human Research Ethics Committee of the above hospital.

Evaluation of Immunostaining

All the immunoreactions were separately evaluated by two independent pathologists. The percentage of positive PAK4 and p57KIP2 tumor cells (0% negative, <5% weak positive, 5–25% intermediate, 25–50% moderate, 50–100% strong) were assessed in at least 5 high power fields (400× magnification). The distribution of tumor cells in all experimental groups was determined: (0–5%) = lower expression and (5–100%) = higher expression.

Statistical Analysis

The data are expressed as the mean ± SD. The statistical analysis correlation of data was checked for significance by ANOVA and Student's χ2 test. Differences with P < 0.05 were considered significant. These analyses were performed using SPSS 17.0 software (SPSS, Chicago, IL).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

PAK4 Down-Regulates the mRNA and Protein Levels of p57Kip2 in Breast Cancer Cells

The PAK4 protein is predicted to have the function of regulating the cell cycle, but few PAK4 target genes have been identified. To identify novel PAK4 target genes, MCF-7 cells were stably infected with lentivirus harboring shRNA-control or shRNA-PAK4. Next, we performed cDNA microarray hybridization experiments using RNA from the transfected cell lines. We discovered that p57Kip2 mRNA was up-regulated by 7-fold, with concomitant down-regulation of PAK4. To confirm whether PAK4 regulates p57Kip2 mRNA expression, we carried out quantitative real-time PCR analysis. The mRNA expression level of p57Kip2 decreased with increasing levels of ectopic PAK4 expression. This indicated that PAK4 was able to down-regulate p57Kip2 mRNA expression in a dose-dependent manner (Fig. 1A). Knockdown of PAK4 by shRNA-PAK4 inversely promoted p57Kip2 mRNA expression (Fig. 1B). These findings support the notion that p57Kip2 is a target gene of PAK4.

image

Figure 1. PAK4 down-regulates the mRNA and protein levels of p57Kip2 in breast cancer cells. (A) Overexpression of PAK4 inhibits p57Kip2 mRNA expression. Real-time PCR analysis of PAK4 and p57Kip2 mRNA levels in MCF-7 cells. MCF-7 cells were transiently transfected with increasing concentrations of PAK4 (0.25–2 μg) for 24 hr. (B) Specific knockdown of PAK4 promotes p57Kip2 mRNA expression. MCF-7 cells were transfected with shRNA-control lentivirus or shRNA-PAK4 lentivirus. After 72 hr of transfection, the mRNA levels of PAK4 and p57Kip2 were measured by real-time PCR analysis. β-actin primers were used to normalize data. The results were the means ± SD of three individual experiments. *P < 0.05, ** P < 0.01. (C) MCF-7 cells were stably transfected with Flag or Flag-PAK4 constructs. Representative immunoblotting analysis for PAK4 and p57Kip2 are shown. (D) MCF-7 cells were transfected with shRNA-control or shRNA-PAK4 lentivirus. Representative immunoblotting analysis for PAK4 and p57Kip2 were shown. GAPDH was used as a loading control.

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To further test whether PAK4 regulates p57Kip2 protein expression, we performed immunoblotting analysis to detect p57Kip2 expression using Flag-PAK4 and Flag-control MCF-7 cells. Our results showed that over-expression of PAK4 down-regulated the p57Kip2 protein levels (Fig. 1C). In addition, knockdown of PAK4 by shRNA-PAK4 upregulated p57Kip2 protein expression in MCF-7 cells (Fig. 1D). Taken together, these results indicate that PAK4 regulates the levels of p57Kip2 mRNA and protein expression in breast cancer cells.

PAK4 Promotes p57Kip2 Ubiquitin-Mediated Proteasomal Degradation in MCF-7 Cells

To determine whether PAK4 regulates the stability of p57Kip2, we utilized cycloheximide (CHX), a specific protein synthesis inhibitor. After treatment with CHX, p57Kip2 had a shorter half-life of less than 6 hr in MCF-7 cells transfected with Flag-control vector. In MCF-7 cells transfected with Flag-PAK4, we observed a significant reduction in the half-life of p57Kip2 compared with the control vector (Fig. 2A). These results suggested that PAK4 promoted degradation of p57Kip2. To investigate how PAK4 over-expression promoted the degradation of p57Kip2, MCF-7 cells were treated with or without MG132, and the levels of p57Kip2 protein expression were examined. The p57Kip2 protein levels in Flag-PAK4 MCF-7 cells were decreased in the absence of MG132, but increased in the presence of MG132 (Fig. 2B). These results indicated that the proteasome proteolytic pathway was involved in the degradation of p57Kip2 induced by PAK4 in MCF-7 cells. Next, we attempted to determine whether PAK4 induced p57Kip2 degradation via p57Kip2 ubiquitination. To observe p57Kip2 ubiquitination, Flag-control or Flag-PAK4 transfected cells were treated with MG132 (10 μM) to prevent p57Kip2 degradation and were immunoprecipitated with anti-p57Kip2 antibodies (Fig. 2C). As expected, treatment with MG132 resulted in increased levels of ubiquitinated p57Kip2 in Flag-control transfected cells, indicating that the p57Kip2 degradation was ubiquitin dependent. Flag-PAK4 transfected cells dramatically increased p57Kip2 ubiquitination compared with Flag-control transfected cells in the presence of MG132. The data above indicate that PAK4 promotes the ubiquitination and degradation of p57Kip2 through the proteasome-dependent pathway in MCF-7 cells.

image

Figure 2. PAK4-mediated down-regulation of p57Kip2 depends on ubiquitin-proteasome pathway. (A) MCF-7 cells were transfected with Flag control or Flag-PAK4 vector. After 24 hr transfection, the cells were treated with CHX (10 mM) for indicated time and whole cell lysates were then subjected to SDS–PAGE and analyzed by immunoblotting analysis with anti-PAK4 and anti-p57Kip2 antibodies. (B) MCF-7 cells were transfected with Flag control or Flag-PAK4 vector in the presence or absence of MG132 (10 μM). Whole cell lysates were processed for SDS–PAGE and immunoblotting analysis using antibodies against PAK4 and p57Kip2 or GAPDH. GAPDH was used as a loading control. (C) Flag control or Flag-PAK4 transfected MCF-7 cells were treated with or without MG132 (10 μM) for 24 hr. Total cell lysates were subjected to ubiquitination assay with p57Kip2 antibody. The immuno complex was analyzed by immunoblotting analysis for ubiquitin antibody. The protein input was monitored for PAK4 antibody.

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PAK4 Expression is Negatively Correlated with p57Kip2 Expression in Human Breast Cancer

Because PAK4 is a regulator of p57Kip2, we further investigated the breast cancer tissue samples for the expression of PAK4 and p57Kip2. Three examples of p57Kip2 and PAK4 staining are shown in Fig. 3. The PAK4 expression level was found to be higher, whereas a relatively lower level expression of p57Kip2 was observed in breast cancer. We found that out of total 50 breast cancer tissue samples, 34 had higher expression levels of PAK4 protein (68%), while the remaining 16 had lower expression levels of PAK4 protein (32%). In addition, PAK4 expression was significantly higher in cancer tissues with decreased expression of p57Kip2 (P = 0.036) (Table 1). Taken together, these results suggest that PAK4 expression is inversely correlated with p57Kip2 expression in breast cancer.

image

Figure 3. Breast tissues from serial sections of three patients (specimen 1–3) were subjected to immunohistochemical staining of PAK4 (A–C, ×400) and p57Kip2 (D–F, ×400). (A and B) strong expression of PAK4, (C) intermediate expression of PAK4, (D and E) negative expression of p57Kip2, and (F) intermediate expression of p57Kip2. Each inset shows images taken at ×600 magnification. Scale bars = 50 μm.

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Table 1. Clinicopathological characteristics of 50 breast tumors
Clinicopathological characteristicsCases (n = 50)PAK4 lower expressionPAK4 higher expressionχ2-valueP-value
  1. Higher expression of PAK4 in breast cancer tissues was significantly correlated with negative expression of p57Kip2 (P < 0.05).

p57KIP2 positive2311 (47.8%)12 (52.2%)4.9030.036*
p57KIP2 negative275 (18.5%)22 (81.5%)  

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

Our data demonstrate that PAK4 could significantly down-regulate the mRNA and protein expression of p57Kip2. PAK4 promotes the degradation of p57Kip2 through the ubiquitin-mediated proteasomal pathway. More importantly, positive expression of PAK4 was significantly correlated with negative expression of p57Kip2 in breast cancer tissues. These findings may imply that the increase expression of PAK4 plays an essential role in breast cancer progression by regulating the p57Kip2 protein stability.

PAK4 has been shown to play an important role in cell-cycle regulation. The levels of PAK4 protein were highly up-regulated in the G1 phase of the cell cycle. In addition, PAK4 protein down-regulated the cell cycle inhibitor p21Cip1 but not p27Kip1, suggesting that PAK4 is involved in controlling initiation of the cell cycle (Nekrasova and Minden, 2011). We found that PAK4 controls the level of p57Kip2 in breast cancer cells. Next, we attempted to determine whether PAK4 affects p57Kip2 levels through single or multiple mechanisms. We found that the levels of p57Kip2 mRNA and protein were elevated in the absence of PAK4, suggesting that PAK4 may regulate the transcription and destabilization of p57Kip2.

Our data also revealed that PAK4 is important for the degradation of p57Kip2 protein. However, the regulation and function of p57Kip2 in cell-cycle control is largely unknown. Recently, it has been reported that p57Kip2 integrates stress signals into cell cycle progression to promote cell survival upon stress (Joaquin et al., 2012). The stability of the p57Kip2 protein is tightly regulated by ubiquitination and proteasome-mediated degradation during various stages of the cell cycle, either in steady state or in response to extracellular stimuli (Lu and Hunter, 2010). The p57Kip2 protein regulates the expression level and kinase activity of cyclins and CDK at the G1/S transition to inhibit the proliferation of tumor cells (Borriello et al., 2011).

The next question we addressed was whether the expression of PAK4 was associated with the expression of p57Kip2 in breast cancer cells. Down-regulation of p57Kip2, both transcriptionally and translationally, has been frequent demonstrated in many human cancers (Pateras et al., 2009), indicating that the level of p57Kip2 might be important to control cell-cycle progression (Besson et al., 2008). Signaling through the PI3K-Akt-mTOR pathway was necessary and sufficient for the increase in p57Kip2, whereas MEK-ERK activity suppressed this increase (Worster et al., 2012). P57kip2 is a candidate tumor suppressor gene in human breast cancer (Larson et al., 2008). The low expression level of p57Kip2 is associated with a poor prognosis in breast cancers (Yang et al., 2009). In our study, high PAK4 expression was associated with low p57Kip2 expression in breast cancer tissues, indicating that PAK4 protein expression is involved in the formation and proliferation of breast cancer.

PAK4 was first identified as a protein kinase that controls cell cycle progression by down-regulating the activity of p21. Furthermore, PAK4 activates the Raf-ERK pathway (Cammarano et al., 2005), which activates various transcriptional modifiers, including AP-1, c-Myc, and EZH-2. The histone methyltransferase EZH-2, which has been implicated in repression of p57Kip2 expression, is activated transcriptionally by ERK (Fujii et al., 2011) making it a potential mediator of p57Kip2 regulation. Another possible mechanism for the down-regulation of p57Kip2 by PAK4 may be due to the activity of the c-Myc-induced miR-221/222, which targets p57Kip2 (Fornari et al., 2008; Kim et al., 2010). Our previous study showed that nuclear PAK4 promotes β-catenin-mediated gene expression including c-Myc (Li et al., 2012). In the present study, our results suggested that PAK4 enhanced p57Kip2 protein degradation via ubiquitin-proteasome system in breast cancer cells. Therefore, it is likely that PAK4 regulates p57 Kip2 expression through multiple mechanisms.

In summary, our results revealed the oncogenic activity of PAK4 in cancer cell growth. The activity of the PAK4 protein inhibited p57Kip2 expression by the ubiquitin-proteasome pathway in breast cancer cells. This indicates that PAK4 may be an important factor contributing to breast tumorigenesis. In addition, the suppression of PAK4 expression may provide a potential target for therapeutic intervention in breast cancer.

ACKNOWLEDGEMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED

The authors would like to thank Dr. Liu Cao for helpful suggestions.

LITERATURE CITED

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGEMENTS
  7. LITERATURE CITED
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