Abstract
The significance of Cdk5 in cell-cycle control and cancer biology has gained increased attention. Here we report the inverse correlation between the protein levels of Cdk5 and p21CIP1 from cell-based and clinical analysis. Mechanistically, we identify that Cdk5 overexpression triggers the proteasome-dependent degradation of p21CIP1 through a S130 phosphorylation in a Cdk2-independent manner. Besides, the evidence from cell-based and clinical analysis shows that Cdk5 primarily regulates nuclear p21CIP1 protein degradation. S130A-p21CIP1 mutant enables to block either its protein degradation or the increase of cancer cell growth caused by Cdk5. Notably, Cdk5-triggered p21CIP1 targeting primarily appears in S-phase, while Cdk5 overexpression increases the activation of Cdk2 and its interaction with DNA polymerase δ. The in vivo results show that Cdk2 might play an important role in the downstream signaling to Cdk5. In summary, these findings suggest that Cdk5 in a high expression status promotes cancer growth by directly and rapidly releasing p21CIP1-dependent cell-cycle inhibition and subsequent Cdk2 activation, which illustrates an oncogenic role of Cdk5 potentially applied for future diagnosis and therapy. Cancer Res; 76(23); 6888–900. ©2016 AACR.
Introduction
Cdk5 activation has been reported in numerous types of tumors and high Cdk5 expression is detrimental to patients' survival (1) and poor prognosis (2). The single-nucleotide polymorphisms in the promoter and amplification of the CDK5 gene increase the risk of human cancers (3, 4), while Cdk5 activation correlates to prostate cancer metastasis and pancreatic cancer progression (5, 6). Our previous studies show that Cdk5 activates STAT3 (7) or androgen receptor (AR; ref. 8) in different cancer cells and supervises the cooperation of AR and STAT3 for prostate cancer growth (9). We also show the cell-cycle arrest or apoptosis of various cancer cells by drug-triggered Cdk5 activation (10–12). The involvements of Cdk5 in microtubule depolymerization, tumor cell mitosis (13), and Retinoblastoma-related thyroid cancer proliferation (14) were reported. These observations strongly suggest the relevant contribution of Cdk5 to cell-cycle control and cancer growth.
p21CIP1 is a member of Cdk inhibitor family containing conserved N-terminal regions for Cdk-binding, which suppresses cell-cycle progression (15). Loss of p21CIP1 causes carcinogenesis and spontaneous tumor development (16). The study indicates that the dual roles p21CIP1 plays in cell-cycle inhibition and anti-apoptosis depend on its subcellular localization (17). Nuclear p21CIP1 inhibits Cdks to restrain cell cycle; cytoplasmic p21CIP1 resists apoptosis by binding with proapoptotic proteins, such as ASK1 or caspase 3/8/10 (18). As the phosphorylation regulation of p21CIP1 determines its protein stability and potentially regulates its subcellular localization (19), it is of interest to investigate the upstream kinases to p21CIP1 in cell-cycle control.
Here, we provide evidence demonstrating the inverse correlation of Cdk5 and p21CIP1 protein levels and observed the novel and specific phosphorylation-dependent degradation of p21CIP1 by Cdk5. The Cdk5 impact on p21CIP1 was primarily restricted to the nucleus, followed by subsequent Cdk2 activation and promotion of cancer growth. Suppression of Cdk5 by RNAi in a xenograft model increased p21CIP1 protein levels and inhibited tumor growth. This study demonstrates the relevance of Cdk5 in p21CIP1-related regulation and sheds light on the potential targeting of Cdk5 in future cancer therapy.
Materials and Methods
Cell culture, transfection, and protein analysis
Breast cancer (MCF-7 and MDA-MB-231), prostate cancer (LNCaP, 22Rv1, DU145, and PC-3), lung cancer (A549), and fibroblast-like (COS-1) cell lines were all purchased from the cell bank (Bioresource Collection and Research Center, Taiwan), that performed cell line characterizations (by STR analysis) and passaged in the laboratory for fewer than 6 months after receipt or resuscitation. MCF-7 and DU145 cells were grown in 10% FBS (Gibco)-containing MEM supplemented with 0.1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, and penicillin/streptomycin (P/S). MDA-MB-231 and COS-1 cells were maintained in DMEM supplemented with 10% FBS and P/S. LNCaP and 22Rv1 cells were cultured in RPMI1640 supplemented with 10% FBS, 10 mmol/L HEPES, 1 mmol/L sodium pyruvate, and P/S. PC-3 and A549 cells were cultured in Ham F-12 medium containing P/S with 7% and 10% FBS, respectively. All cells were incubated under 5% CO2 at 37°C. To establish stable human cancer cell lines, the coding sequences of empty-vector (EV), wild-type (WT) Cdk5, kinase-dead Cdk5 with D144N mutation (KD; ref. 14) were cloned into mammalian expression plasmids and then transfected into cells. The transfected cells were subsequently selected in the presence of G418 (TOKU-E) for at least 4 weeks. The expression plasmid or siRNA were premixed within Lipofectamine 2000 or Lipofectamine RNAiMAX transfection reagent (Invitrogen) according to the manufacturer's instructions, and then the liposome/nucleic acids complex was transfected into cells with culture medium for 2 days. Commercial products of siRNA-Cdk5 (siCdk5) and nonspecific control siRNA (siCon) were purchased from Dharmacon (SMARTpool). Small hairpin RNAs, shCdk5 (TRCN0000021467) and shCdk2 (TRCN0000195003), for specific protein knockdown were obtained from National RNAi Core Facility of Academia Sinica (Taiwan). The 3×FLAG tagged p21CIP1 expression plasmid was kindly provided by Professor Hideo Nishitani, Graduate School of Life Science, University of Hyogo (Hyogo, Japan; ref. 20). The kinase-dead mutant pcDNA3-D144N-Cdk5 (KD) and 3×FLAG-S130A-p21CIP1 were cloned using the QuickChange Lightning Site-Directed Mutagenesis Kit (Stratagene) according to its standard instruction manual. Clinical specimens from breast cancer patients who underwent surgical resection and the procedure was approved by the Institutional Review Boards of Chang Bing Show Chwan Memorial Hospital (No. IRB-D016-B). The details of Western blot analysis and immunoprecipitation were referred to previous publication (8).
Immunostaining
Cells on coverslips were sequentially fixed, permeabilized, and blocked as described previously (8). After primary antibody binding, samples were incubated with Alexa Fluor 488- or 546-conjugated secondary antibodies (Molecular Probes). DAPI fluorescent dye was used to stain cell nuclei. The coverslips were mounted and examined by fluorescent microscopy (Olympus). The human paraffin-embedded tissue arrays (prostate cancer:PR952 and PR803a; lung cancer: LC1201; breast cancer: BR1921a) were purchased from Biomax Co. and stained by procedures of Vectastain ABC Kit and DAB Peroxidase Substrate Kit (Vector Laboratories). The slides were counterstained with hematoxylin solution (Merck) to indicate cell nuclei. The specimens were mounted and imaged by light microscopy (Olympus). Two experts blinded to the image sources evaluated the specimens in accordance with a scoring system that was based on the intensity of staining signals (Supplementary Fig. S3 for the details).
Quantitative real-time PCR
Total RNA extraction (Genemark) following by reverse transcription PCR (Applied Biosystems) was performed using SYBR Green (Roche Applied Science)-dependent real-time quantification as described previously (21). Data were analyzed as Ct values and were adjusted relative to the levels of actin housekeeping gene. Primers used in this study were listed in Supplementary Methods.
In vitro kinase assay
Recombinant full-length human p21CIP1 proteins (wild-type or S130A mutant) were expressed in the pQE30 expression system and the proteins were purified following the Qiagen manufacturer's protocol. Active Cdk5/p35 protein complex were purchased from Millipore. The kinase assays of immunoprecipitated or recombinant Cdk5 or Cdk2 were performed with histone H1 (Millipore) or recombinant p21CIP1 as the substrates like previous description (8, 22). [γ-32P]-ATP (Perkin Elmer) or cold ATP (Sigma) with phosSTOP phosphatase inhibitor cocktail (Roche) were used and the phospho-signals were detected by isotope visualization or Western blotting with specific antibody.
Identification of phosphorylation sites by mass spectrometry
The product of in vitro Cdk5-dependent phosphorylated p21CIP1 was resolved by SDS-PAGE and analyzed by LC/MS-MS (Q-Exactive mass spectrometer, Thermo Fisher Scientific), which was performed by the Mass Solutions Technology Co., Ltd.
Cell-cycle synchronization
MCF-7 cells were synchronized at different stages of the cell cycle [G1 phase: serum starvation (24 hours); S-phase: hydroxyurea treatment (3 μmol/L, 24 hours); G2–M phase: nocodazole treatment (50 ng/mL, 24 hours)] in accordance with a modified protocol (23, 24).
Tumor xenograft
Approximately 1 × 107 MDA-MB-231 or 2 × 107 22Rv1 cells were subcutaneously injected into the flanks of 5-week-old male BALB/c nude mice. The mice were randomly grouped after tumor volumes reached over 100 mm3 and intratumorally injected with a mixture of 10 μg pcDNA3-empty-vector, shCdk5, shCdk2, or pcDNA3-Cdk5 plasmids by in vivo jet-PEI transfection reagent (Polyplus) every 3 days. Tumor volumes were measured and calculated using the formula L×W×W×3.14/6, where L and W are the longest and shortest tumor axis, respectively. All animal experiments were performed according to the protocol approved by the Institutional Animal Care and Use Committee of National Chung Hsing University.
Statistical analysis
Data are presented as the mean ± SEM. The paired Student t test was used for statistical evaluation of data from the cell proliferation experiments. One-way ANOVA was utilized for animal experiments. The correlation between Cdk5 levels and p21CIP1 levels in human cancer specimens was analyzed using Fisher exact test. Statistical significance was noted with ∗, P < 0.05; ∗∗, P < 0.01. n.s. stands for no significance.
Results
Cdk5 reduces p21CIP1 protein levels in cancer cells by increasing proteasome-mediated degradation
Figure 1A showed that p21CIP1 protein was decreased after Cdk5 overexpression in MCF-7 cells (Fig. 1A), while Cdk5 knockdown by specific siRNA resulted in increased p21CIP1 protein levels (Fig. 1B). The kinase activity of Cdk5 was in accordance with its protein levels (Supplementary Fig. S1A). The expression levels of other Cdks (including Cdk1, 2, 4, 6) were also observed with Cdk5 manipulations. Interestingly, the expression of Cdk2 after Cdk5 knockdown was increased implying the putative functional compensation between Cdk5 and Cdk2. On the basis of p21CIP1 increase with Cdk2 upregulation after Cdk5 knockdown, the authors suggested that Cdk5 might play a specific and prominent role in p21CIP1 protein regulation. Although p21CIP1 protein levels were affected by Cdk5, p21CIP1 mRNA expression was unaffected (Fig. 1C). The distribution of Cdk5 protein levels in 39 breast cancer patients' tumor specimens was shown in Fig. 1D for the quantified results and Supplementary Fig. S2A for the blotting images. The mutual growth and decline between Cdk5 and p21CIP1 protein levels in the 29 patients' tumor specimens with Cdk5 overexpression are shown in Fig. 1E (see images in Supplementary Fig. S2B), in which the Cdk5 protein levels from 29 patients were categorized into three grades. Besides, the cancer tissue arrays from 397 patients (including cancers of prostate, lung, and breast) were examined by IHC and the representative images with high Cdk5 level (low p21CIP1) and low Cdk5 level (high p21CIP1) were presented in Fig. 1F. Table 1 indicated the negative correlations between the levels of Cdk5 and p21CIP1 in the tumor sections of the prostate (P < 0.001), lung (P = 0.021), and breast (P = 0.031; analyzed by the Fisher exact test). The scoring system was characterized in Supplementary Fig. S3. Cdk5 overexpression facilitated p21CIP1 protein degradation in stable cancer cell lines identified by chase experiments with cycloheximide (CHX) treatment (Fig. 1G). The Cdk5-dependent p21CIP1 degradation was apparently rescued by treatments with proteasome inhibitors, MG132 or lactacystin (Fig. 1H and Supplementary Fig. S4A). These data suggest that the Cdk5-related decline of p21CIP1 protein is likely regulated through promoting the proteasome-mediated degradation.
. | Cdk5 Level (%) . | . | . | . | |||
---|---|---|---|---|---|---|---|
p21CIP1 Level in human cancers (%) . | Negative . | Low . | Moderate . | High . | Total, n . | P . | Pearson's R . |
Prostate cancer | P < 0.001a | R = −0.22 | |||||
Negative | 1 (0.69%) | 5 (3.45%) | 0 (0.00%) | 3 (2.07%) | 9 (6.21%) | ||
Low | 7 (4.83%) | 21 (14.48%) | 22 (15.17%) | 13 (8.97%) | 63 (43.45%) | ||
Moderate | 9 (6.21%) | 26 (17.93%) | 3 (2.07%) | 3 (2.07%) | 41 (28.27%) | ||
High | 4 (2.76%) | 22 (15.17%) | 1 (0.69%) | 5 (3.45%) | 32 (22.07%) | ||
Total, n | 21 (14.48%) | 74 (51.04%) | 26 (17.93%) | 24 (16.55%) | 145 (100%) | ||
Lung cancer | P = 0.021a | R = −0.12 | |||||
Negative | 7 (7.29%) | 12 (12.50%) | 8 (8.33%) | 4 (4.17%) | 31 (32.29%) | ||
Low | 3 (3.13%) | 4 (4.17%) | 9 (9.38%) | 11 (11.46%) | 27 (28.13%) | ||
Moderate | 6 (6.25%) | 9 (9.38%) | 1 (1.04%) | 2 (2.08%) | 18 (18.75%) | ||
High | 7 (7.29%) | 5 (5.21%) | 5 (5.21%) | 3 (3.13%) | 20 (20.83%) | ||
Total, n | 23 (23.96%) | 30 (31.25%) | 23 (23.96%) | 20 (20.83%) | 96 (100%) | ||
Breast cancer | P = 0.031a | R = −0.07 | |||||
Negative | 5 (3.21%) | 19 (12.18%) | 10 (6.41%) | 8 (5.13%) | 42 (26.92%) | ||
Low | 3 (1.92%) | 13 (8.33%) | 24 (15.38%) | 15 (9.62%) | 55 (35.26%) | ||
Moderate | 10 (6.41%) | 15 (9.62%) | 8 (5.13%) | 7 (4.49%) | 40 (25.64%) | ||
High | 3 (1.92%) | 8 (5.13%) | 3 (1.92%) | 5 (3.21%) | 19 (12.18%) | ||
Total, n | 21 (13.46%) | 55 (35.26%) | 45 (28.85%) | 35 (22.43%) | 156 (100%) |
. | Cdk5 Level (%) . | . | . | . | |||
---|---|---|---|---|---|---|---|
p21CIP1 Level in human cancers (%) . | Negative . | Low . | Moderate . | High . | Total, n . | P . | Pearson's R . |
Prostate cancer | P < 0.001a | R = −0.22 | |||||
Negative | 1 (0.69%) | 5 (3.45%) | 0 (0.00%) | 3 (2.07%) | 9 (6.21%) | ||
Low | 7 (4.83%) | 21 (14.48%) | 22 (15.17%) | 13 (8.97%) | 63 (43.45%) | ||
Moderate | 9 (6.21%) | 26 (17.93%) | 3 (2.07%) | 3 (2.07%) | 41 (28.27%) | ||
High | 4 (2.76%) | 22 (15.17%) | 1 (0.69%) | 5 (3.45%) | 32 (22.07%) | ||
Total, n | 21 (14.48%) | 74 (51.04%) | 26 (17.93%) | 24 (16.55%) | 145 (100%) | ||
Lung cancer | P = 0.021a | R = −0.12 | |||||
Negative | 7 (7.29%) | 12 (12.50%) | 8 (8.33%) | 4 (4.17%) | 31 (32.29%) | ||
Low | 3 (3.13%) | 4 (4.17%) | 9 (9.38%) | 11 (11.46%) | 27 (28.13%) | ||
Moderate | 6 (6.25%) | 9 (9.38%) | 1 (1.04%) | 2 (2.08%) | 18 (18.75%) | ||
High | 7 (7.29%) | 5 (5.21%) | 5 (5.21%) | 3 (3.13%) | 20 (20.83%) | ||
Total, n | 23 (23.96%) | 30 (31.25%) | 23 (23.96%) | 20 (20.83%) | 96 (100%) | ||
Breast cancer | P = 0.031a | R = −0.07 | |||||
Negative | 5 (3.21%) | 19 (12.18%) | 10 (6.41%) | 8 (5.13%) | 42 (26.92%) | ||
Low | 3 (1.92%) | 13 (8.33%) | 24 (15.38%) | 15 (9.62%) | 55 (35.26%) | ||
Moderate | 10 (6.41%) | 15 (9.62%) | 8 (5.13%) | 7 (4.49%) | 40 (25.64%) | ||
High | 3 (1.92%) | 8 (5.13%) | 3 (1.92%) | 5 (3.21%) | 19 (12.18%) | ||
Total, n | 21 (13.46%) | 55 (35.26%) | 45 (28.85%) | 35 (22.43%) | 156 (100%) |
aP < 0.05 indicates statistical significance.
p21CIP1 is a novel substrate of Cdk5
The interaction between endogenous Cdk5 and p21CIP1 was barely detectable due to degradation in normal condition; however, the interaction became prominent after MG132 treatment in three cancer cell lines (Fig. 2A). The effects of MG132 or lactacystin on p21CIP1 mRNA was shown in Supplementary Fig. S5. These results imply that Cdk5-bound p21CIP1 was unstable and rapidly degraded by proteasome. The in vitro phosphorylation of recombinant p21CIP1 by Cdk5 was detected in a dose-dependent manner (Fig. 2B). LC/MS-MS analysis revealed that Ser130 of p21CIP1 was the single site of Cdk5-dependent phosphorylation (Fig. 2C). Further confirmation of p21CIP1 phosphorylation was performed using Western blotting and the dose-dependent phosphorylation by different amounts of Cdk5/p35 complex was presented in Fig. 2D. In addition, the Ser130 phosphorylation of p21CIP1 was dose dependently reduced by in vitro treatment with a Cdk5 inhibitor, roscovitine (Fig. 2E). By using histone H1 as a substrate for in vitro Cdk5 kinase assay, WT p21CIP1 but not S130A-mutant p21CIP1 could dose dependently decrease histone H1 phosphorylation (Fig. 2F). Altogether, we determine that p21CIP1 is a novel substrate of Cdk5.
Cdk5 induces p21CIP1 protein degradation through Ser130 phosphorylation in a Cdk2-independent manner
With MG132 treatment, the levels of phospho-Ser130-p21CIP1 were correspondent to Cdk5 activity, while the total p21CIP1 protein was escaped from proteasomal degradation (Fig. 2G). FLAG-SA-p21CIP1 was resistant to Cdk5-triggered degradation in different cancer cell lines (Fig. 2H). The chase experiments illustrated that SA-p21CIP1 exhibited higher protein stability than WT-p21CIP1 in different lines of Cdk5-overexpressing cells (Fig. 2I). As Cdk2 is also responsible to p21CIP1 Ser130 phosphorylation (25), Cdk5/p21CIP1 regulation was monitored in the condition of Cdk2 knockdown. The results showed that Cdk5 still significantly reduced p21CIP1 protein levels while Cdk2 was knocked down (Fig. 3A). The effects on cell growth were correspondent to the levels of p21CIP1 protein (Fig. 3B). Supplementary Fig. S6A and S6B showed the efficiency of different shRNA clones for Cdk5 and Cdk2. Altogether, by using either Cdk5 knockdown or SA-p21CIP1 to inhibit Cdk5-dependent phosphorylation, we reveal that Ser130 phosphorylation by Cdk5 is essential for p21CIP1 protein degradation in a Cdk2-independent manner.
Cdk5 primarily targets nuclear p21CIP1
The results of cellular protein fractionation revealed that Cdk5 primarily repressed p21CIP1 protein in the nucleus of cancer cells (Fig. 4A). Oppositely, Cdk5 knockdown increased nuclear p21CIP1 protein (Supplementary Fig. S7). The nuclear distribution of endogenous p21CIP1 was significantly diminished in WT Cdk5-, but not in kinase-dead (KD) Cdk5-, overexpressing cells (Fig. 4B). Treatments of MG132 or lactacystin restored the WT-Cdk5-induced degradation of nuclear p21CIP1 protein (Fig. 4B and Supplementary Fig. S4B). The results of immunostaining showed that inhibition of Cdk5-dependent p21CIP1 phosphorylation using KD-Cdk5 or the SA-p21CIP1 mutant stabilized nuclear p21CIP1 levels (top, Fig. 4C; bottom for the quantified results). In clinical study, the tissue array data and the representative images of three cancer types showed the inverse correlation between nuclear distributions of Cdk5 and p21CIP1 (in total 397 patients, P < 0.001 for prostate cancer; P = 0.006 for lung cancer; P < 0.001 for breast cancer, Fig. 4D and E). Altogether, these results suggest that Cdk5 primarily targets the nuclear p21CIP1 protein through Ser130 phosphorylation-related degradation.
Cdk5-related p21CIP1 reduction and Cdk2 activation contribute to cancer cell proliferation
Cdk5 overexpression significantly promoted the growth of 22Rv1 cells with the reduction of p21CIP1 protein levels (Fig. 5A). In Fig. 5B, p21CIP1 significantly inhibited cell growth (bar 3 vs. bar 1), and Cdk5 overexpression rescued p21CIP1-dependent growth inhibition (bar 4 vs. bar 3; Supplementary Fig. S8 for protein levels). However, Cdk5 overexpression was unable to rescue the growth inhibition induced by SA-p21CIP1 (bar 5 vs. bar 3). These results imply that Cdk5 promotes cancer cell growth by eliminating p21CIP1 protein. Moreover, Cdk5 overexpression in a MDA-MB-231 xenograft model also showed a significant increase of tumor growth (Fig. 5C) with the reduction of p21CIP1 proteins, whereas p27KIP1 was unaffected (top, Fig. 5D; bottom for the quantified data). In synchronized MCF-7 stable cells, WT-Cdk5 but not KD-Cdk5 decreased p21CIP1 protein levels primarily in S-phase (Fig. 5E). As Cdk2 activity is important to the progression of S-phase and regulated by p21CIP1 (26), we observed that Cdk2 was activated in WT-Cdk5-overexpressing cells (but not in KD-Cdk5-overexpressing cells) when p21CIP1 was targeted (Fig. 5F). As the activated Cdk2 interacts with DNA polymerase δ to promote the process of DNA replication, the protein interaction between Cdk2 and DNA polymerase δ was significantly enhanced by Cdk5 overexpression (Fig. 5G). Our findings suggest that Cdk5 might expedite S-phase progression and promote cell growth by degrading nuclear p21CIP1 protein and subsequently activating Cdk2.
Cdk2 is important to Cdk5-promoted tumor growth
Cdk5 knockdown significantly retarded tumor growth in a 22Rv1 xenograft model (Fig. 6A) with p21CIP1 protein elevation (Fig. 6B). To clarify the involvement of Cdk2 in Cdk5-regulated tumor growth, Cdk2 knockdown was performed in the xenograft model while Cdk5 was overexpressed. The results showed that Cdk2 knockdown significantly blocked the Cdk5-promoted tumor growth (Fig. 6C and D for the correspondent protein levels). The significant efficiency of Cdk2 knockdown was shown in Supplementary Fig. S6C. It suggests the important role of Cdk2 in the downstream signaling to Cdk5. Besides, Cdk5 still increased tumor growth with Cdk2 knockdown (Fig. 6C), implying that Cdk5 might promote tumor growth through a Cdk2-independent pathway, which echoed to the authors' previous reports, for example, STAT3 (7, 9). Finally, we hypothesize that Cdk5 targets nuclear p21CIP1 protein and leads to downstream Cdk2 activation. Therefore the transition of G1–S phase in cell cycle is promoted and tumor growth is accelerated (Fig. 6E).
Discussion
By using in vitro, in vivo, and clinical evidence, this study explored the oncogenic role of Cdk5 in a high-expressing status might cause the degradation of the cell-cycle inhibitor p21CIP1 in nucleus by Ser130 phosphorylation, subsequently activate Cdk2, and finally promote cancer growth. Therefore Cdk5 might play an oncogenic role in cancer biology and potentially exhibited as a future diagnostic marker. Cdk5 targeting could also become a future strategy for cancer treatment.
p21CIP1 expression and protein stability are tightly regulated by multiple mechanisms and contributed to cell-cycle control. The phosphorylation regulation on p21CIP1 protein controls its protein subcellular localization, partner binding affinity, and protein stability (27). This study demonstrates that p21CIP1 was a novel target of Cdk5 phosphorylation and followed by the decrease of p21CIP1 protein stability primarily in the nucleus of cancer cells (Figs. 1–4). However, the Ser130 residue of p21CIP1 can be phosphorylated by several kinases under different conditions for discrepant effects. ERK2 phosphorylates p21CIP1 at Thr57/Ser130 and induces cytoplasmic localization for protein degradation (28). Cdk2 phosphorylates p21CIP1 at Ser130 in vitro and recruits the E3 ligase complex, which contributes to p21CIP1 degradation and the release from cell-cycle inhibition (25). Pozo and colleagues found that KD-Cdk5 decreased p21CIP1 expression in medullary thyroid cancer cells, which belong to a category of neuroendocrine cancer that has distinct features compared with other types of cancer (29). Cdk5 plays unique roles in both tumor cells and neuronal cells, in which Cdk5 regulates the opposite functions of growth and differentiation, respectively. In addition to expression regulation, the issue of p21CIP1 subcellular localization under Cdk2 regulation was not commented. On the other hand, Cdk6 phosphorylation of p21CIP1 at Ser130 has minor effects on p21CIP1 protein stability or localization and fails to interact with (or inhibits) Cdk2 (30). TGFβ1 activates the stress-related JNK1 and p38α pathway and stabilizes p21CIP1 protein through Ser130 phosphorylation (31). Thus, the regulations of p21CIP1 under distinct circumstances would promote its unclear roles instead of a cell-cycle inhibitor, and the protein stability and subcellular localization of p21CIP1 regulated by kinases remain controversial. Under the inhibition of proteasomal degradation or the SA-p21CIP1 mutant (Fig. 4B and C; Supplementary Fig. S4 and S9), we showed that p21CIP1 protein was unaffected by Cdk5 and primarily localized in the nucleus of cancer cells. These results suggest that Cdk5 primarily targets nuclear p21CIP1 and contributes to cell-cycle progression, whereas cytoplasmic transport of p21CIP1 is not correlated with Cdk5 regulation.
It has been reported that p21CIP1 directly binds to the C8 subunit of the 20S proteasome and is turned over without ubiquitination-mediated targeting (32). This ubiquitination-independent degradation of p21CIP1 is accelerated by oncogenic proteins or phosphorylation on specific sites (33–35). Depletion of the 20S proteasome activator REGγ leads to the increase of nuclear p21CIP1 protein and alters the cell-cycle progression of thyroid carcinoma cells (36). Notably, the structure of the 20S proteasome contains a nuclear localization signal and can be trapped by the nuclear envelope (37). The 20S proteasome returns to the cytoplasm after nuclear envelop breakdown during mitosis and this nuclear translocation is frequently observed during the cell-cycle progression of cancerous cells and may be required for the degradation of some nuclear proteins (37). In our study, although Cdk5 degrades p21CIP1 protein via proteasomal degradation in cancer cells (Fig. 1H and Supplementary Fig. S4), no change of ubiquitinated-p21CIP1 was observed (Supplementary Fig. S10). As Bornstein and colleagues claimed that ubiquitination is not necessary after p21CIP1 phosphorylation (25), the 20S proteasome-mediated proteolysis is possibly involved in Cdk5-triggered nuclear p21CIP1 degradation in cancer cells.
Lindqvist and colleagues (38) and Zhang and colleagues (39) back-to-back raised the evidence showing the indirect regulation between Cdk5 and p21CIP1 with the connections of Akt activation and p53 downregulation under Cdk5 knockdown status, which is considerable as a cellular stress compared to high Cdk5 expression in cancer patients' tissues. Notably, our data indicates that Cdk5 still significantly reduced p21CIP1 protein under Akt inhibition (Supplementary Fig. S11). With respect to subcellular localization of p21CIP1, Hung and colleagues found that Her2 induces cytosolic localization of p21CIP1 to play anti-apoptotic role through activations of PI3K/Akt (19) and MDM2/p53 (40). Li and colleagues identified that Akt phosphorylates p21CIP1 and enhances its protein stability to promote anti-apoptosis and cell survival (41). While Cdk5 has chances to individually regulate Her2 (42), PI3K/Akt (38), and p53 (43), the network among these proteins with the issue of p21CIP1 subcellular localization becomes complicated to illustrate in one simple hypothesis. Therefore, we simply propose one direct and rapid regulation between Cdk5 (in a high expression status) and p21CIP1 in fast-growing tumors, which is independent to the findings under different pathological events (e.g., cellular stress). We believe that these findings provide a new and important perspective to the whole picture of Cdk5 regulation on p21CIP1 in cell biology.
p21CIP1 nuclear localization has also been reported to inhibit Cdk activation and PCNA function in response to DNA damage-induced cell-cycle arrest at G1–S transition (15). Harper and colleagues indicate that p21CIP1 effectively inhibits the activities of Cdk2, Cdk3, Cdk4, and Cdk6 (Ki 0.5–15 nmol/L) but poorly to Cdk5 (Ki >2 μmol/L; ref. 44). In our in vitro analysis, recombinant p21CIP1 protein significantly reduced the phosphorylation of histone H1, which is an index of Cdk5 activity (Fig. 2F). However, p21CIP1 Ser130 phosphorylation was increased opposite to the decrease of histone H1 phosphorylation, suggesting that the role of Cdk5 substrate in the reaction was from the original histone H1 shifted to the increasing p21CIP1. This finding is consistent with Harper and colleagues (44) and suggests that Cdk5 phosphorylates the substrate p21CIP1 instead of histone H1 in vitro. However, Hengst and colleagues demonstrate that a single p21CIP1 molecule is sufficient for Cdk2 inhibition and p21CIP1-saturated complex contains only one stably bound inhibitor molecule (26), suggesting that tiny change of cellular p21CIP1 protein level is critical to the regulation of cell-cycle progression. In our study, the Cdk5-reduced p21CIP1 was observed in early S-phase after hydroxyurea treatment (Fig. 5E), suggesting that Cdk5, unlike other Cdks, helps cells escape from p21CIP1 inhibition on Cdk2 by phosphorylation-dependent degradation and therefore contributes to promote S-phase progression. Interestingly, we found that the protein interaction between KD-Cdk5 and p21CIP1 were increased (Supplementary Fig. S12). The interaction between SA-p21CIP1 mutant and Cdk5 in the nucleus were also increased (Supplementary Fig. S13). These results suggest that Cdk5 might be capable of trapping p21CIP1 and relieving the inhibition of Cdk2 even when Cdk5 was inactive or p21CIP1 phosphorylation was absent. This finding leads us to conclude that Cdk5 counters the inhibitory function on p21CIP1 in cell-cycle control through activation-dependent or -independent regulations. p35 and p39 have been believed as two major physiologic regulators for Cdk5 kinase activity in recent literature. The authors previously found that Cdk5 and p35 can be two independent factors regulating the downstream Ser727 STAT3 phosphorylation and the growth of prostate cancer cells by in vitro cell culture experiments as well as clinical analysis (9).This prospect potentially highlights Cdk5 as an independent marker in future clinical analysis irrespective of the status of its regulators.
In conclusion, we had presented in vitro, in vivo, and clinical evidence demonstrating that Cdk5, as a novel and direct p21CIP1 regulator, decreases nuclear p21CIP1 protein through phosphorylation/proteasome-dependent degradation. Consistent with other recent evidence (1, 2, 5, 8, 9, 13, 14, 45, 46), we provide a new model that the aberrant elevation of Cdk5 expression in malignancy might contribute to cancer growth by promoting escape of p21CIP1 inhibition and Cdk2 activation while more work is needed to confirm the role of Cdk2 in this process (Fig. 6E). These findings shed light on the roles of Cdk5 in future cancer research for either diagnosis or therapy.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: P.-H. Huang, H. Lin
Development of methodology: P.-H. Huang
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P.-H. Huang, C.-H. Chang, Y.-C. Wang, C.-H. Lai, J.-H. Wang, E. Lin
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P.-H. Huang, M.-C. Chen, W.-H. Kao, C.-H. Chang, C.-H. Yue, H. Lin
Writing, review, and/or revision of the manuscript: P.-H. Huang, M.-C. Chen, W.-H. Kao, J.-T. Hsieh, H. Lin
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): P.-H. Huang, Y.-C. Wang, J.-T. Hsieh, Y.-T. Lee, H.-Y. Wang, H. Lin
Study supervision: H. Lin
Other (performed the animal experiments): Y.-T. Peng
Other (conducted some preliminary results): S.-C. You
Acknowledgments
The authors thank Dr. H. C. Chen, Dr. C. M. Chen, Dr. C. M. Hsueh, and Dr. L. C. Lin (National Chung Hsing University, Taiwan) for help with animal experiments; Dr. H. C. Hung, Dr. Y. L. Liu, Dr. H. C. Cheng, Dr. J. W. Chen, Dr. H. L. Su, and Dr. C. C. Lai (National Chung Hsing University, Taiwan) for technical supports; Dr. Z. F. Chang (National Yang Ming university, Taiwan), Dr. H. Nishitani (University of Hyogo, Japan), and Dr. D. J. Mann (Imperial College London, UK) for providing p21CIP1 constructs; Dr. M. T. Lai (Chang Bing Show Chawn Memorial Hospital, Taiwan) for instructing the clinical analysis.
Grant Support
This work was supported by The Ministry of Science and Technology, Taiwan (101-2320-B005-004-MY3 and 103-2911-I-005-507 to H. Lin); joint grant of Taichung Veterans General Hospital and National Chung Hsing University (TCVGH-NCHU1047604 to H. Lin); and Chang Bing Show Chwan Memorial Hospital, Taiwan (CBSH9905001 to E. Lin; CBBC001 to Y.-T. Lee).
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