p27Kip1 Mediates Addiction of Ovarian Cancer Cells to MYCC (c-MYC) and Their Dependence on MYC Paralogs

The MYCC (c-MYC) gene is amplified in 30–60% of human ovarian cancers. We assessed the functional significance of MYCC amplification by siRNA inhibition of MYCC or MYC paralogs in a panel of ovarian cancer cell lines expressing varying levels of MYCC. Inactivation of MYCC inhibited cell proliferation and induced replicative senescence only in lines with amplified MYCC, indicating that these cells are addicted to continued MYCC overexpression. In contrast, siRNA knockdown of all three MYC isoforms inhibited proliferation of MYCC non-amplified ovarian cancer cells without inducing replicative senescence, and did not inhibit the proliferation of telomerase-immortalized ovarian surface epithelial cells. The arrest induced by MYCC knockdown was accompanied by an increase in the level of the Cdk inhibitor p27^Kip1 and a decrease in cyclin A expression and Cdk2 activity, and could be reversed by RNAi knockdown of p27^Kip1 or Rb, or by overexpression of cyclin A/Cdk2. The arrest induced by knockdown of all three MYC isoforms could similarly be reversed by p27^Kip1 knockdown. Our findings indicate that the addiction of MYCC-amplified ovarian cancer cells to MYCC differs from the dependence of MYCC non-amplified cancer cells on MYC paralogs, but both are mediated, at least in part, by p27^Kip1. They also suggest that growth of ovarian cancers may be blocked by inhibition of MYCC or MYC paralogs.     

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) delays cellular senescence by promoting p27(Kip1) degradation in human diploid fibroblasts

WW domain-containing E3 ubiquitin protein ligase 1 (WWP1) plays an important role in the proliferation of tumor cells and the lifespan of Caenorhabditis elegans. However, the role of WWP1 in cellular senescence is still unknown. Here, we show that the expression patterns of p27(Kip1) and WWP1 are inversely correlated during cellular senescence. Moreover, the overexpression of WWP1 delayed senescence, whereas the knockdown of WWP1 led to premature senescence in human fibroblasts. Furthermore, we demonstrate that WWP1 repressed endogenous p27(Kip1) expression through ubiquitin-proteasome-mediated degradation. Additionally, WWP1 had a strong preference for catalyzing the Lys-48-linked polyubiquitination of p27(Kip1) in vitro. Finally, we demonstrate that WWP1 markedly inhibited the replicative senescence induced by p27(Kip1) by promoting p27(Kip1) degradation. Therefore, our study provides a new molecular mechanism for the regulation of cellular senescence.     

Enhanced NOLC1 promotes cell senescence and represses hepatocellular carcinoma cell proliferation by disturbing the organization of nucleolus

The nucleolus is a key organelle that is responsible for the synthesis of rRNA and assembly of ribosomal subunits, which is also the center of metabolic control because of the critical role of ribosomes in protein synthesis. Perturbations of rRNA biogenesis are closely related to cell senescence and tumor progression; however, the underlying molecular mechanisms are not well understood. Here, we report that cellular senescence‐inhibited gene (CSIG) knockdown up‐regulated NOLC1 by stabilizing the 5′UTR of NOLC1 mRNA, and elevated NOLC1 induced the retention of NOG1 in the nucleolus, which is responsible for rRNA processing. Besides, the expression of NOLC1 was negatively correlated with CSIG in the aged mouse tissue and replicative senescent 2BS cells, and the down‐regulation of NOLC1 could rescue CSIG knockdown‐induced 2BS senescence. Additionally, NOLC1 expression was decreased in human hepatocellular carcinoma (HCC) tissue, and the ectopic expression of NOLC1 repressed the proliferation of HCC cells and tumor growth in a HCC xenograft model.     

Regulating the stability and localization of CDK inhibitor p27Kip1 via CSN6-COP1 axis

The COP9 signalosome subunit 6 (CSN6), which is involved in ubiquitin-mediated protein degradation, is overexpressed in many types of cancer. CSN6 is critical in causing p53 degradation and malignancy, but its target in cell cycle progression is not fully characterized. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase associating with COP9 signalosome to regulate important target proteins for cell growth. p27 is a critical G1 CDK inhibitor involved in cell cycle regulation, but its upstream regulators are not fully characterized. Here, we show that the CSN6-COP1 link is regulating p27^Kip1 stability, and that COP1 is a negative regulator of p27^Kip1. Ectopic expression of CSN6 can decrease the expression of p27^Kip1, while CSN6 knockdown leads to p27^Kip1 stabilization. Mechanistic studies show that CSN6 interacts with p27^Kip1 and facilitates ubiquitin-mediated degradation of p27^Kip1. CSN6-mediated p27 degradation depends on the nuclear export of p27^Kip1, which is regulated through COP1 nuclear exporting signal. COP1 overexpression leads to the cytoplasmic distribution of p27, thereby accelerating p27 degradation. Importantly, the negative impact of COP1 on p27 stability contributes to elevating expression of genes that are suppressed through p27 mediation. Kaplan-Meier analysis of tumor samples demonstrates that high COP1 expression was associated with poor overall survival. These data suggest that tumors with CSN6/COP1 deregulation may have growth advantage by regulating p27 degradation and subsequent impact on p27 targeted genes.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21^/Cip and p27^/Kip1. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.     

Attenuation of cell cycle regulator p27<Superscript>Kip1</Superscript> expression in vertebrate epithelial cells mediated by extracellular signals in vivo and in vitro

Cell cycle arrest in potentially dividing cells is often mediated by inhibitors of G1/S-phase cyclin-dependent kinases. The cyclin E/CDK2-inhibitor p27^Kip1 has been implicated in this context in epithelial cells. We cloned and sequenced p27^Kip1 of ducklings (Anas platyrhynchos) and used an in vitro assay system to study the mechanism of p27^Kip1 downregulation in the nasal gland which precedes an increase in proliferation rate upon initial exposure of the animals to osmotic stress. Western blot studies revealed that p27^Kip1 is downregulated during 24 h of osmotic stress in ducklings with the steepest decline in protein levels between 5 and 8 h. As indicated by the results of Northern blot and semi-quantitative PCR studies, protein downregulation is not accompanied by similar changes in mRNA levels indicating that Kip1 is regulated mainly at the translational (synthesis) or posttranslational level (degradation). Using recombinant duck Kip1 protein expressed in E. coli, we showed that Kip1 is subject to polyubiquitinylation by cytosolic enzymes from nasal gland cells indicating that loss of Kip1 may be regulated, at least in part, by acceleration of protein degradation. In cultured nasal gland tissue, attenuation of Kip1 expression could be induced by activation of the muscarinic acetylcholine receptor indicating that mAChR-receptor signalling may play a role in the re-entry of quiescent gland cells into the cell cycle.     

Adhesion to fibronectin induces p27Kip1 nuclear accumulation through down-regulation of Jab1 and contributes to cell adhesion-mediated drug resistance (CAM-DR) in RPMI 8,226 cells

Mounting evidence has been shown that integrin-mediated cellular adhesion confers resistance to chemotherapy of multiple myeloma. The molecular mechanism underlying cell adhesion-mediated drug resistance (CAM-DR) is, however, poorly understood. In this report, we demonstrated that RPMI 8,226 cells accumulated p27^Kip1 in the nucleus when they were adhered to fibronectin (FN). The adhesion-mediated p27^Kip1 nuclear recruitment was regulated via the down-regulation of Jab1, a negative regulator of cell cycle. Overexpression of Jab1 reversed the elevated p27^Kip1 in the nucleus, which needed phosphorylation of p27^Kip1 on Serine 10, whereas inhibition of Jab1 by siRNA further increased the elevated p27^Kip1. Furthermore, we found overexpression of Jab1 did not affect 8,226 cells adhesion to FN, but reversed doxorubicin or mitoxantrone-induced CAM-DR phenotype. In conclusion, our data suggest that Jab1 plays an important role in CAM-DR, which depends on pSer10-p27^Kip1-mediated subcellular localization of p27^Kip1. The understanding of this novel molecular mechanism may prove valuable in designing new therapeutic approaches for CAM-DR in Multiple myeloma.     

MicroRNA-200c is involved in proliferation of gastric cancer by directly repressing p27Kip1

P27^Kip1, also known as Cyclin-dependent kinase inhibitor 1B, is an important check-point protein in the cell cycle. It has been identified that although as a tumor suppressor, P27^Kip1 is expressed in different cancer cell types, which shows the therapeutic potential in tumor genesis. In this study, we examined the upstream regulatory mechanism of P27^Kip1 at the microRNA (miRNA) level in gastric carcinogenesis. We used bioinformatics to predict that microRNA-200c (miR-200c) might be a direct upstream regulator of P27^Kip1. It was also verified in gastric epithelial-derived cell lines that overexpression of miR-200c significantly inhibited the expression levels of P27^Kip1, whereas knockdown of miR-200c promoted P27^Kip1 expression in AGS and BGC-823 cells. Furthermore, we identified the direct binding of miR-200c on the P27^Kip1 3′ -UTR sequence by luciferase assay. MiR-200c could enhance the colony formation of cells by repressing P27^Kip1 expression. In addition, the negative correlation between P27^Kip1 and miR-200c in human gastric cancer tissues and matched normal tissues further supported the tumor-promoting action of miR-200c in vivo. Our finding suggested that miR-200c directly regulates the expression of P27^Kip1 and promotes cell growth in gastric cancer as an oncogene, which may provide new clues to treatment.     

Cytoplasmic p27Kip1 counteracts the pro-apoptotic function of the open conformation of PTEN by retention and destabilization of PTEN outside of the nucleus

The tumor suppressor activity of p27Kip1 takes place in the cell nucleus by inhibitory binding to cyclin/CDK complexes. p27Kip1 can also be localized in the cytoplasm, where it has been proposed to have oncogenic properties. Here, we describe a novel role for cytoplasmic p27Kip1 which could account for its activity as an oncoprotein by negative regulation of the PTEN tumor suppressor. p27Kip1 physically interacted with the open conformation of PTEN, which is competent to enter the nucleus. In mammalian cells, cytoplasmic p27Kip1 retained to nuclear-targeted PTEN in the cytoplasm. This retention was exerted by the C-terminal p27Kip1 region, and was independent of cyclin/CDK-binding. The nuclear accumulation of PTEN triggered by pro-apoptotic TNFα treatment was abolished by cytoplasmic p27Kip1. Furthermore, conformationally-open PTEN displayed diminished protein stability and pro-apoptotic activity in the presence of cytoplasmic p27Kip1. Our results support a conformationally-dependent model of cytoplasmic retention and negative regulation of the activity of nuclear PTEN by oncogenic cytoplasmic p27Kip1, and suggest the existence of reciprocal mechanisms to regulate the levels of both p27Kip1 and PTEN.     

Loss of retinoblastoma but not p16 function allows bypass of replicative senescence in human fibroblasts

Current models envision replicative senescence to be under dual control by the p53 and retinoblastoma (RB) tumour-suppressor pathways. The role of the p16^INK4a–RB pathway is controversial, and the function of RB in human cells has not been tested directly. We used targeted homologous recombination to knock out one copy of RB in presenescent human fibroblasts. During entry into senescence, RB^+/− cells underwent spontaneous loss of heterozygosity and the resultant RB^−/− clones bypassed senescence. The extended lifespan phase was eventually terminated by a crisis-like state. The same phenotype was documented for p21 ^CIP1/WAF1 and p53 heterozygous cells, indicating that loss of function of all three genes results in failure to establish senescence. By contrast, the abolition of p16 function by the expression of a p16-insensitive cyclin-dependent kinase 4 protein or siRNA-mediated knockdown provided only minimal lifespan extension that was terminated by senescence. We propose that p53, p21 and RB act in a linear genetic pathway to regulate cell entry into replicative senescence.     

MARCKS Signaling Differentially Regulates Vascular Smooth Muscle and Endothelial Cell Proliferation through a KIS-, p27kip1- Dependent Mechanism

Background

Overexpression of the myristolated alanine-rich C kinase substrate (MARCKS) occurs in vascular proliferative diseases such as restenosis after bypass surgery. MARCKS knockdown results in arrest of vascular smooth muscle cell (VSMC) proliferation with little effect on endothelial cell (EC) proliferation. We sought to identify the mechanism of differential regulation by MARCKS of VSMC and EC proliferation in vitro and in vivo.

Methods and Results

siRNA-mediated MARCKS knockdown in VSMCs inhibited proliferation and prevented progression from phase G₀/G₁ to S. Protein expression of the cyclin-dependent kinase inhibitor p27^kip1, but not p21^cip1 was increased by MARCKS knockdown. MARCKS knockdown did not affect proliferation in VSMCs derived from p27^kip1-/- mice indicating that the effect of MARCKS is p27^kip1-dependent. MARCKS knockdown resulted in decreased phosphorylation of p27^kip1 at threonine 187 and serine 10 as well as, kinase interacting with stathmin (KIS), cyclin D1, and Skp2 expression. Phosphorylation of p27^kip1 at serine 10 by KIS is required for nuclear export and degradation of p27^kip1. MARCKS knockdown caused nuclear trapping of p27^kip1. Both p27^kip1 nuclear trapping and cell cycle arrest were released by overexpression of KIS, but not catalytically inactive KIS. In ECs, MARCKS knockdown paradoxically increased KIS expression and cell proliferation. MARCKS knockdown in a murine aortic injury model resulted in decreased VSMC proliferation determined by bromodeoxyuridine (BrdU) integration assay, and inhibition of vascular wall thickening. MARCKS knockdown increased the rate of re-endothelialization.

Conclusions

MARCKS knockdown arrested VSMC cell cycle by decreasing KIS expression. Decreased KIS expression resulted in nuclear trapping of p27^kip1 in VSMCs. MARCKS knockdown paradoxically increased KIS expression in ECs resulting in increased EC proliferation. MARCKS knockdown significantly attenuated the VSMC proliferative response to vascular injury, but accelerated reestablishment of an intact endothelium. MARCKS is a novel translational target with beneficial cell type-specific effects on both ECs and VSMCs.     

Tyrosine phosphatase SHP-2 is a regulator of p27Kip1 tyrosine phosphorylation

Tyrosine phosphorylation of the cell cycle regulator p27^Kip1 plays a crucial role in its binding to cyclin dependent kinases and its subcellular localization. While Src and Bcr-Abl were shown to be responsible for tyrosine phosphorylation, no data are available on the dephosphorylation of p27^Kip1 and the phosphatase involved. Considering the associated dephosphorylation as a pivotal event in the regulation of cell cycle proteins, we focused on the tyrosine phosphatase SHP-2, which is regulated in promyelocytic leukemia cells on G-CSF stimulation. SHP-2 was thus found in association with p27^Kip1 and the G-CSF receptor, and we observed a nuclear translocation of SHP-2 on G-CSF stimulation. Using a catalytically inactive form of SHP-2 and siRNA directed against SHP-2, we could demonstrate the involvement of SHP-2 in tyrosine dephosphorylation of p27^Kip1. Moreover, SHP-2 was strongly activated on G-CSF stimulation and specifically dephosphorylated p27^Kip1 in vitro. Most importantly, we could illustrate that SHP-2 modulates p27^Kip1 stability and contributes to p27^Kip1-mediated cell cycle progression. Taken together, our results demonstrate that SHP-2 is a key regulator of p27^Kip1 tyrosine phosphorylation.