Sequence requirement for nuclear localization and growth inhibition of p27Kip1R,a degradation-resistant isoform of p27Kip1

p27(Kip1R) is an isoform of p27(Kip1), having a distinct C-terminus. The sequences of p27(Kip1R) required for nuclear localization and growth inhibition were determined in HeLa cells using a green fluorescence protein (GFP) as a reporter molecule. Region 153-168 and residues K168 and I169 were determined to play a critical role in the nuclear localization of p27(Kip1R). Aliphatic amino acid was found to be a substitute for the basic residue in the typical nuclear localization signal, while its functional substitution was incomplete, thereby causing a significant cytoplasmic retention of p27(Kip1R). p27(Kip1R) is thus the first example of an atypical bipartite nuclear localization signal with aliphatic amino acid as a functional residue. Despite cytoplasmic retention, p27(Kip1R) inhibited the cell growth as well as p27(Kip1), while GFP alone had no effect. The mutants lacking an N-terminus containing the binding regions for cyclins and cyclin-dependent kinases also showed a significant degree of nuclear localization, but failed to inhibit cell growth. The growth inhibition by p27(Kip1R) as well as p27(Kip1) was thus suggested to originate in the common N-terminal region.     

Inappropriate P-cadherin expression in the mouse mammary epithelium is compatible with normal mammary gland function

Cadherins comprise a family of cell-cell adhesion proteins critical to the architecture and function of tissues. Expression of family members E-, N-, and P-cadherin is regulated in a spatial and temporal fashion in the developing and adult organism. Using in vivo and in vitro experimental systems, perturbation of cadherin expression by genetic deletion, overexpression, mutant dominant-negative constructs, and, to a lesser degree, expression of an inappropriate cadherin have all been shown to alter embryogenesis, tissue architecture, and cell behavior. Here we studied how expression of an inappropriate cadherin affects the adult mouse mammary gland. Human P-cadherin was expressed in mammary epithelial cells under control of the mouse mammary tumor virus (MMTV) promoter, and the effect on mammary gland behavior was studied. Typically, E-cadherin is expressed by mammary epithelial cells, whereas P-cadherin is found in myoepithelial cells and cap cells of the ductal terminal end bud. However, breast cancers frequently express P-cadherin, even though they are thought to arise from epithelial cells, and it is a marker of poor prognosis. We developed two independent transgenic mouse lines that exhibited high levels of P-cadherin protein expression in the mammary epithelium. P-cadherin was detected in most, but not all, luminal epithelial cells, and was appropriately localized to cell-cell borders. It was detected in the mammary glands of virgin, pregnant, lactating, post-lactation, and aged parous female mice. Despite the robust and widespread expression of an inappropriate cadherin, no effect was observed on mammary gland morphogenesis, architecture, lactation, or involution in transgenic mice compared to wild-type mice. No mammary tumors formed spontaneously in either wild-type or transgenic mice. Moreover, mammary tumors induced by the neu oncogene, which was introduced by a breeding strategy, showed no differences between mice with or without hP-cadherin. Surprisingly, however, none of the tumors expressed hP-cadherin protein. Together, our studies show no apparent effect on adult mammary gland or tumor behavior by inappropriate expression of P-cadherin in normal mammary epithelial cells.     

p27Kip1 serine 10 phosphorylation determines its metabolism and interaction with cyclin-dependent kinases

p27Kip1 is a critical modulator of cell proliferation by controlling assembly, localization and activity of cyclin-dependent kinase (CDK). p27Kip1 also plays important roles in malignant transformation, modulating cell movement and interaction with the extracellular matrix. A critical p27Kip1 feature is the lack of a stable tertiary structure that enhances its “adaptability” to different interactors and explains the heterogeneity of its function. The absence of a well-defined folding underlines the importance of p27Kip1 post-translational modifications that might highly impact the protein functions. Here, we characterize the metabolism and CDK interaction of phosphoserine10-p27Kip1 (pS10- p27Kip1), the major phosphoisoform of p27Kip1. By an experimental strategy based on specific immunoprecipitation and bidimensional electrophoresis, we established that pS10-p27Kip1 is mainly bound to cyclin E/CDK2 rather than to cyclin A/CDK2. pS10- p27Kip1 is more stable than non-modified p27Kip1, since it is not (or scarcely) phosphorylated on T187, the post-translational modification required for p27Kip1 removal in the nucleus. pS10-p27Kip1 does not bind CDK1. The lack of this interaction might represent a mechanism for facilitating CDK1 activation and allowing mitosis completion. In conclusion, we suggest that nuclear p27Kip1 follows 2 almost independent pathways operating at different rates. One pathway involves threonine-187 and tyrosine phosphorylations and drives the protein toward its Skp2-dependent removal. The other involves serine-10 phosphorylation and results in the elongation of p27Kip1 half-life and specific CDK interactions. Thus, pS10-p27Kip1, due to its stability, might be thought as a major responsible for the p27Kip1-dependent arrest of cells in G1/G0 phase.     

p27Kip1 inhibits the cell cycle through non-canonical G1/S phase-specific gatekeeper mechanism

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 has been shown to regulate cellular proliferation via inhibition of CDK activities. It is now recognized that p27Kip1 can regulate cellular processes through non-canonical, CDK-independent mechanisms. We have developed an inducible p27Kip1 model in cultured cells to explore CDK-independent p27Kip1 regulation of biological processes. We present evidence that p27Kip1 can function in a CDK-independent manner to inhibit entry and/or progression of S phase. Even though this p27Kip1 mechanism is non-canonical it does requires the intact cyclin-binding motif in p27Kip1. We suggest a mechanism similar to that proposed in post-mitotic neural cells whereby p27Kip1 functions to coordinate growth arrest and apoptosis. Our hypothesis supports the concept that p27Kip1 is a gatekeeper for the entry and progression of S phase through interaction with specific protein(s) or via binding to specific DNA sequences in a CDK-independent manner.     

p27~(Kip1)和CyclinD1在非小细胞肺癌中表达及意义

为了探讨p27Kip1蛋白和CyclinD1蛋白在非小细胞肺癌(NSCLC)中的表达及意义,收集临床手术切除的非小细胞肺癌组织蜡块64例及正常肺组织10例,应用免疫组化(S-P法)检测组织中p27Kip1蛋白和CyclinD1蛋白的表达,结合临床病理资料和随访资料进行回顾性研究。实验发现NSCLC组织中p27Kip1蛋白表达和CyclinD1蛋白表达均明显不同于正常肺组织(P<0.01)。p27Kip1蛋白表达降低与NSCLC肿瘤大小、病理分级、分期增加、淋巴结转移之间有相关性(P<0.05),但与肿瘤组织学分型无相关性(P>0.05)。CyclinD1蛋白过表达与组织学分型、肿瘤大小、病理分级、临床分期、淋巴结转移无相关性(P>0.05)。p27Kip1蛋白表达与CyclinD1蛋白表达之间呈显著负相关(P<0.01)。cox单因素及多因素分析,p27Kip1蛋白低表达及CyclinD1过表达是影响NSCLC患者预后的主要因素。实验结果显示,NSCLC组织中,p27Kip1蛋白表达降低,而CyclinD1过表达,二者与NSCLC的发生发展机制有关,可作为预后指标,有利于NSCLC患者预后判断及个体化治疗。     

Cell-permeable carboxyl-terminal p27(Kip1) peptide exhibits anti-tumor activity by inhibiting Pim-1 kinase

The incidence and death rate of prostate cancer is increasing rapidly. In addition, the low sensitivity of prostate cancer to chemotherapy makes it difficult to treat this condition. The serine/threonine kinase Pim-1 plays an important role in cell cycle progression and apoptosis inhibition, resulting in prostate tumorigenesis. Therefore, Pim-1 inhibition has been expected to be an attractive target for developing new anti-cancer drugs. However, no small compounds targeting Pim-1 have progressed to clinical use because of their lack of specificity. Here, we have reported a new cell-permeable Pim-1 inhibitory p27(Kip1) peptide that could interfere with the binding of Pim-1 to its substrates and act as an anti-cancer drug. The peptide could bind to Pim-1 and inhibit phosphorylation of endogenous p27(Kip1) and Bad by Pim-1. Treatment of prostate cancer with the peptide induces G(1) arrest and subsequently apoptosis in vitro. However, the peptide showed almost no growth inhibitory or apoptosis-inducing effects in normal cells. The peptide could inhibit tumor growth in in vivo prostate cancer xenograft models. Moreover, the peptide treatment could overcome resistance to taxol, one of the first line chemotherapeutic agents for prostate cancer, and a combination of the peptide with taxol synergistically inhibited prostate cancer growth in vivo. These results indicate that a Pim-1 inhibitory p27(Kip1) peptide could be developed as an anti-cancer drug against prostate cancer.     

Modified p27 Kip1 is efficient in suppressing HER2-mediated tumorigenicity

Cyclin-dependent kinase (CDK) inhibitor p27 Kip1, a haplo-insufficient tumor suppressor, is downregulated by oncogenic signal of HER2, a receptor tyrosine kinase oncogene. HER2 promotes mitogenic growth and transformation of cancer cells. HER2 signaling can enhance p27 Kip1 ubiquitination, thereby promoting p27 degradation and subsequent activation of CDK activity. p27 ubiquitination and degradation is enhanced by JAB1 binding as well as by phosphorylation on Thr187. In this study, we generated modified p27 proteins, which are mutated at Thr 187 or deleted at JAB1 binding domain. We applied these modified p27 genes as novel anticancer agents for HER2-overexpressing cells under the control of a tetracycline (tet)-regulated gene expression system. Induction of p27 T187A and p27 T187A DeltaJAB inhibits HER2-activated cell growth, CDK2 activity, cell proliferation, and transformation. Significantly, a modified protein (p27 T187ADeltaJAB) reduced the tumor volume in a HER2-overexpressing tumor model efficiently. These findings demonstrate the applicability of employing modified p27 proteins as a therapeutic intervention in HER2-overexpressing cancers.     

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.     

Interaction of the stress protein p8 with Jab1 is required for Jab1-dependent p27 nuclear-to-cytoplasm translocation

p8 is an 80 amino-acid polypeptide identified because of its remarkable over-expression in the stressed pancreas. This protein, apparently devoid of enzymatic activity, is a powerful regulator of several intracellular pathways, suggesting that it has to interact with several molecular partners to modulate their activity. We used two-hybrid screening of a pre-transformed human testes cDNA library to identify some of these partners. One of them was the multifunctional protein Jab1, its interaction with p8 being confirmed by His6-pull down and co-immunoprecipitation assays. In addition, we could show that the two proteins co-localized in the cell. Our functional data demonstrate that Jab1 requires direct interaction with p8 to induce the translocation of p27 from nucleus to cytoplasm and its subsequent degradation. Experiments showing that the knock-down of p8 expression results in a strong inhibition of Jab1 activity confirmed that the mechanism by which Jab1 promotes cell growth by decreasing p27 level is p8-dependent.     

Long non-coding RNA UCA1 promotes breast tumor growth by suppression of p27 (Kip1)

Functional genomics studies have led to the discovery of a large amount of non-coding RNAs from the human genome; among them are long non-coding RNAs (lncRNAs). Emerging evidence indicates that lncRNAs could have a critical role in the regulation of cellular processes such as cell growth and apoptosis as well as cancer progression and metastasis. As master gene regulators, lncRNAs are capable of forming lncRNA–protein (ribonucleoprotein) complexes to regulate a large number of genes. For example, lincRNA-RoR suppresses p53 in response to DNA damage through interaction with heterogeneous nuclear ribonucleoprotein I (hnRNP I). The present study demonstrates that hnRNP I can also form a functional ribonucleoprotein complex with lncRNA urothelial carcinoma-associated 1 (UCA1) and increase the UCA1 stability. Of interest, the phosphorylated form of hnRNP I, predominantly in the cytoplasm, is responsible for the interaction with UCA1. Moreover, although hnRNP I enhances the translation of p27 (Kip1) through interaction with the 5′-untranslated region (5′-UTR) of p27 mRNAs, the interaction of UCA1 with hnRNP I suppresses the p27 protein level by competitive inhibition. In support of this finding, UCA1 has an oncogenic role in breast cancer both in vitro and in vivo. Finally, we show a negative correlation between p27 and UCA in the breast tumor cancer tissue microarray. Together, our results suggest an important role of UCA1 in breast cancer.     

Increased expression of MDM2, cyclin D1, and p27Kip1 in carcinogen-induced rat mammary tumors

It is thought that environmental pollutants, such as polycyclic aromatic hydrocarbons (PAH), contribute to human breast tumorigenesis, yet their roles remain incompletely elucidated. The prototypical PAH 7,12-dimethylbenz(alpha)anthracene (DMBA) specifically and effectively induces mammary tumor formation in rodent models. In an attempt to explore the molecular mechanisms by which PAH initiates and promotes mammary tumorigenesis, we examined the expression of several cell cycle regulators in rat mammary tumors induced by DMBA. Expression of cyclin D1, murine double minute-2 (MDM2), and Akt was up-regulated in tumors in comparison to normal mammary glands, as indicated by RT-PCR, Western blot analysis, and immunohistochemical staining. Expression of p27Kip1 protein was also elevated in the tumors with increased cytoplasmic localization. However, RB protein remained hyperphosphorylated. To directly test the effects of DMBA, the MCF-7 human breast cancer cells were treated. DMBA induced MDM2 expression in a dose- and time-dependent fashion in the MCF-7 cells, and this activation appeared to be p53 dependent. These data suggest that activation of cyclin D1, MDM2, and AKT as well as increased expression and cytoplasmic localization of p27Kip1 may play a role in this model of environmental pollutant-induced mammary tumorigenesis.     

Robust expression of p27Kip1 induced by viral infection is critical for antiviral innate immunity

Influenza A virus (IAV) infection regulates the expression of numerous host genes. However, the precise mechanism underlying implication of these genes in IAV pathogenesis remains largely unknown. Here, we employed isobaric tags for relative and absolute quantification (iTRAQ) to identify host proteins regulated by IAV infection. iTRAQ analysis of mouse lungs infected or uninfected with IAV showed a total of 167 differentially upregulated proteins in response to the viral infection. Interestingly, we observed that p27Kip1, a potent cyclin‐dependent kinase inhibitor, was markedly induced by IAV both at mRNA and protein levels through in vitro and in vivo studies. Furthermore, it was shown that innate immune signalling positively regulated p27Kip1 expression in response to IAV infection. Ectopic expression of p27Kip1 in A549 cells dramatically inhibited IAV replication, whereas, p27Kip1 knockdown significantly enhanced the virus replication. in vivo experiments demonstrated that p27Kip1 knockout (KO) mice were more susceptible to IAV than wild‐type (WT) mice: exhibiting higher viral load in lung tissue, faster body‐weight loss, reduced survival rate and more severe organ damage. Moreover, we found that p27Kip1 overexpression facilitated the degradation of viral NS1 protein, caused a dramatic STAT1 activation and promoted the expression of IFN‐β and several critical antiviral interferon‐stimulated genes (ISGs). Increased p27Kip1 expression also restricted infections of several other viruses. Conversely, IAV‐infected p27Kip1 KO mice exhibited a sharp increase in NS1 protein accumulation, reduced level of STAT1 activation and decreased expression of IFN‐β and the ISGs in the lung compared to WT animals. These findings reveal a key role of p27Kip1 in enhancing antiviral innate immunity.     

Colony stimulating factor-1 is required to recruit macrophages into the mammary gland to facilitate mammary ductal outgrowth

Mammary gland development initiates postnatally with the development of terminal end buds (TEBs) at the end of the rudimentary ducts. These grow out through the fat pad and bifurcate to lay down the rudimentary ductal tree. At the initiation of their development, TEBs recruit to their surrounding stroma a substantial population of macrophages. Using mice homozygous for a null mutation in the gene for the macrophage growth factor, colony stimulating factor-1 (CSF-1), that are severely depleted in macrophages, we demonstrated that CSF-1-regulated macrophages are required for normal branching morphogenesis in the mammary gland. However, these mice have a pleiotropic phenotype as a result of the generalized macrophage deficiency. To test that the effect of the mutation observed in the mammary gland was organ-autonomous, we developed a tetracycline-binary system whereby CSF-1 was specifically expressed in the mammary epithelium under the regulation of the MMTV-promoter. This restored mammary macrophage populations but not those in other tissues and corrected the branching morphogenesis defect. Inhibition of CSF-1 expression by tetracycline treatment for varying periods suggested that CSF-1-regulated macrophages are required throughout early mammary gland development. These data show that macrophages acting locally are required for branching morphogenesis of the mammary gland.     

Leukemia inhibitory factor induces apoptosis of the mammary epithelial cells and participates in mouse mammary gland involution

Leukemia inhibitory factor (LIF) is a multifunctional glycoprotein that displays multiple biological activities in different cell types, but to date there has been no report on its expression in the normal mammary gland. In this study we found that LIF is expressed at low but detectable levels in postpubertal, adult virgin, and pregnant mouse mammary glands. However, LIF expression drops after parturition to become almost undetectable in lactating glands. Interestingly, LIF expression shows a steep increase shortly after weaning that is maintained for the following 3 days. During this period, known as the first stage of mammary gland involution, the lack of suckling induces local factors that cause extensive epithelial cell death. It has been shown that Stat3 is the main factor in signaling the initiation of apoptosis, but the mechanism of its activation remains unclear. Herein, we show that LIF expression in the gland is induced by milk stasis and not by the decrease of circulating lactogenic hormones after weaning. Implantation of LIF containing pellets in lactating glands results in a significant increase in epithelium apoptosis. In addition, this treatment also induces Stat3 phosphorylation. We conclude that LIF regulated expression in the mouse mammary gland may play a relevant role during the first stage of mammary gland involution. Our results also show that LIF-induced mammary epithelium apoptosis could be mediated, at least partially, by Stat3 activation.