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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.
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- MATERIALS AND METHODS
- 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.