Localization Of p27Kip1 Research Articles

Knockdown of UBE2I inhibits tumorigenesis and enhances chemosensitivity of cholangiocarcinoma via modulating p27kip1 nuclear export.

The asymptomatic nature of cholangiocarcinoma (CCA), particularly during its early stages, in combination with its high aggressiveness and chemoresistance, significantly compromises the efficacy of current therapeutic options, contributing to a dismal prognosis. As a tumor suppressor that inhibits the cell cycle, abnormal cytoplasmic p27kip1 localization is related to chemotherapy resistance and often occurs in various cancers, including CCA. Nevertheless, the underlying mechanism is unclear. SUMOylation, which is involved in regulating subcellular localization and the cell cycle, is a posttranslational modification that regulates p27kip1 activity. Here, we confirmed that UBE2I, as the only key enzyme for SUMOylation, was highly expressed and p27kip1 was downregulated in CCA tissues, which were associated with poor outcomes in CCA. Moreover, UBE2I silencing inhibited CCA cell proliferation, delayed xenograft tumor growth in vivo,and sensitized CCA cells to the chemotherapeutics, which may be due to cell cycle arrest induced by p27kip1 nuclear accumulation. According to the immunoprecipitation result, we found that UBE2I could bind p27kip1, and the binding amount of p27kip1 and SUMO-1 decreased after UBE2I silencing. Moreover, nuclear retention of p27kip1 was induced by UBE2I knockdown and SUMOylation or CRM1 inhibition, further suggesting that UBE2I could cooperate with CRM1 in the nuclear export of p27kip1. These data indicate that UBE2I-mediated SUMOylation is a novel regulatory mechanism that underlies p27kip1 export and controls CCA tumorigenesis, providing a therapeutic option for CCA treatment.

The AMPK/p27Kip1 Pathway as a Novel Target to Promote Autophagy and Resilience in Aged Cells.

Once believed to solely function as a cyclin-dependent kinase inhibitor, p27Kip1 is now emerging as a critical mediator of autophagy, cytoskeletal dynamics, cell migration and apoptosis. During periods of metabolic stress, the subcellular location of p27Kip1 largely dictates its function. Cytoplasmic p27Kip1 has been found to be promote cellular resilience through autophagy and anti-apoptotic mechanisms. Nuclear p27Kip1, however, inhibits cell cycle progression and makes the cell susceptible to quiescence, apoptosis, and/or senescence. Cellular location of p27Kip1 is regulated, in part, by phosphorylation by various kinases, including Akt and AMPK. Aging promotes nuclear localization of p27Kip1 and a predisposition to senescence or apoptosis. Here, we will review the role of p27Kip1 in healthy and aging cells with a particular emphasis on the interplay between autophagy and apoptosis.

Akt/p27kip1 Pathway Is Not Involved in Human Insulinoma Tumorigenesis.

Insulinomas are pancreatic neuroendocrine tumors (pNET), usually benign. Akt/p27kip1 is an intracellular pathway overexpressed in many pNET. There are no data regarding its expression in human insulinomas. We aimed to investigate the expression of Akt and p27kip1 in 24 human insulinomas and to compare them to their expression in normal surrounding islets. Staining was performed on embedded paraffin tissue using polyclonal antibodies against total Akt, p-Akt, p27kip1, and pp27kip1. p-Akt was the predominant form in insulinomas; they presented lower Akt and p-Akt expression than normal islets in 83.3% and 87.5% of tumors, respectively. p27kip1 and pp27kip1 were mainly cytoplasmic in both insulinomas and normal tissue. Cytoplasmic pp27kip1 staining was higher in insulinomas and surprisingly nearly half of the insulinomas also presented nuclear p27kip1 (p = 0.029). No differences were observed in the subcellular localization of p27kip1 and activation of Akt between benign and malignant insulinomas. The low expression of Akt seen in insulinomas might explain the usual benign behavior of this type of pNET. Cytoplasmic p27kip1 in both insulinomas and normal islet cells could reflect the low rate of replication of beta cells, while nuclear p27kip1 would seem to indicate stabilization and nuclear anchoring of the cyclin D-Cdk4 complex. Our data seem to suggest that the Akt pathway is not involved in human insulinoma tumorigenesis.

Cip2a promotes cell cycle progression in triple-negative breast cancer cells by regulating the expression and nuclear export of p27Kip1.

Triple-negative breast cancer (TNBC) is very aggressive and currently has no specific therapeutic targets; as a consequence, TNBC exhibits poor clinical outcome. In this study, we showed that cancerous inhibitor of protein phosphatase 2A (Cip2a) represents a promising target in TNBC because Cip2a was highly expressed in TNBC cells and tumor tissues, and its expression showed an inverse correlation with overall survival in patients with TNBC. We found that inhibition of Cip2a in TNBC cells induced cell cycle arrest at the G2/M phase, inhibited cell proliferation and delayed tumor growth in the xenograft model. Moreover, Cip2a markedly decreased the expression and nuclear localization of p27Kip1 and this is critical for the ability of Cip2a to promote TNBC progression. Mechanistically, our studies showed that Cip2a promoted p27Kip1 phosphoration at Ser10 via inhibiting Akt-associated PP2A activity, which seems to relocalize p27Kip1 to the cytoplasm in TNBC cells. On the other hand, Cip2a also recruited c-myc to mediate the transcriptional inhibition of p27Kip1. Notably, we observed negative correlation between Cip2a and p27Kip1 expression in TNBC specimens. In addition, our data showed that Cip2a depletion could sensitize TNBC to PARP inhibition. Collectively, these data suggested that Cip2a effectively promotes TNBC cell cycle progression and tumor growth via regulation of PP2A/c-myc/p27Kip1 signaling, which could serve as a potential therapeutic target for TNBC patients.

The B56γ3 regulatory subunit-containing protein phosphatase 2A outcompetes Akt to regulate p27KIP1 subcellular localization by selectively dephosphorylating phospho-Thr157 of p27KIP1

The B56γ-containing protein phosphatase 2A (PP2A-B56γ) has been postulated to have tumor suppressive functions. Here, we report regulation of p27KIP1 subcellular localization by PP2A-B56γ3. B56γ3 overexpression enhanced nuclear localization of p27KIP1, whereas knockdown of B56γ3 decreased p27KIP1 nuclear localization. B56γ3 overexpression decreased phosphorylation at Thr157 (phospho-Thr157), whose phosphorylation promotes cytoplasmic localization of p27KIP1, whereas B56γ3 knockdown significantly increased the level of phospho-Thr157. In vitro, PP2A-B56γ3 catalyzed dephosphorylation of phospho-Thr157 in a dose-dependent and okadaic acid-sensitive manner. B56γ3 did not increase p27KIP1 nuclear localization by down-regulating the upstream kinase Akt activity and outcompeted a myristoylated constitutively active Akt (Aktca) in regulating Thr157 phosphorylation and subcellular localization of p27KIP1. In addition, results of interaction domain mapping revealed that both the N-terminal and C-terminal domains of p27 and a domain at the C-terminus of B56γ3 are required for interaction between p27 and B56γ3. Furthermore, we demonstrated that p27KIP1 levels are positively correlated with B56γ levels in both non-tumor and tumor parts of a set of human colon tissue specimens. However, positive correlation between nuclear p27KIP1 levels and B56γ levels was found only in the non-tumor parts, but not in tumor parts of these tissues, implicating a dysregulation in PP2A-B56γ3-regulated p27KIP1 nuclear localization in these tumor tissues. Altogether, this study provides a new mechanism by which the PP2A-B56γ3 holoenzyme plays its tumor suppressor role.

P27Kip1 signaling: Transcriptional and post-translational regulation

p27Kip1 is an inhibitor of a broad spectrum of cyclin-dependent kinases (CDKs), and the loss of a single p27Kip1 allele is thereby sufficient to increase tumor incidence via CDK-mediated cell cycle entry. As such, down-regulation of p27Kip1 protein levels, in particular nuclear expressed p27Kip1, is implicated in both disease progression and poor prognosis in a variety of cancers. p27Kip1 expression is positively regulated by the transcription factor MENIN, and inhibited by oncogenic transcription factors MYC and PIM. However, regulation of p27Kip1 protein expression and function is predominantly through post-translational modifications that alter both the cellular localization and the extent of E3 ubiquitin ligase-mediated degradation. Phosphorylation of p27Kip1 at Thr187 and Ser10 is a prerequisite for its degradation via the E3 ubiquitin ligases SKP2 (nuclear) and KPC (cytoplasmic), respectively. Additionally, Ser10 phosphorylated p27Kip1 is predominantly localized in the cytoplasm due to the nuclear export protein CRM1. Another E3 ubiquitin ligase, PIRH2, degrades p27Kip1 in both the cytoplasm and nucleus independent of phosphorylation state. As such, inhibition of cell cycle entry and progression in a variety of cancers may be achieved with therapies designed to correct p27Kip1 localization and/or block its degradation.

Nrdp1-mediated ErbB3 degradation inhibits glioma cell migration and invasion by reducing cytoplasmic localization of p27(Kip1).

We previously reported that loss of Nrdp1 contributes to human glioma progression by reducing apoptosis. However, the role of Nrdp1 in glioma migration and invasion has not been investigated. Here, we report that ErbB3, a substrate of Nrdp1, is undetectable in normal brain tissues and grade II/III glioma tissues, but is abundant in a certain percentage of grade IV glioma tissues and is associated with the loss of Nrdp1. This suggests that Nrdp1 may be involved in glioma migration and invasion by regulating ErbB3. Thus, the role of Nrdp1/ErbB3 signaling in glioma cell migration and invasion was investigated using Nrdp1 loss- and gain-of-function. The results show that down-regulation of Nrdp1 by use of short hairpin RNA promoted glioma cell migration and invasion. In contrast, overexpression of Nrdp1 significantly inhibited glioma cell migration and invasion. Further investigation on molecular targets revealed that Nrdp1 decreased the level of ErbB3, which resulted in decreasing p-AKT thereby reducing cytoplasmic p27(Kip1). Taken together, these findings suggest that Nrdp1-mediated ErbB3 degradation suppresses glioma migration and invasion and that loss of Nrdp1 may amplify ErbB3 signaling to contribute to glioma migration and invasion. These findings suggest that Nrdp1 may be a target for glioma therapy.

Increased Cytoplasmic Localization of p27kip1 and Its Modulation of RhoA Activity during Progression of Chronic Myeloid Leukemia

The role of p27kip1 in Chronic Myeloid Leukemia (CML) has been well studied in relation to its function as a cell cycle inhibitor. However, its cytoplasmic function especially in CML remains to be seen. We studied the localization of p27kip1 and its function during the progression of CML from chronic to blast phase. Our investigations revealed an increased localization of p27kip1 in the cytoplasm of CD34+ cells in the blast phase compared to chronic phase. Cytoplasmic p27kip1 was found to modulate RhoA activity in CD34+ stem and progenitor cells. Further, RhoA activity was shown to be dependent on cytoplasmic p27kip1 which in turn was dependent on p210Bcr-Abl kinase activity. Interestingly, RhoA activity was observed to affect cell survival in the presence of imatinib through the SAPK/JNK pathway. Accordingly, inhibition of SAPK/JNK pathway using SP600125 increased apoptosis of K562 cells in presence of imatinib. Our results, for the first time, thus reveal a crucial link between cytoplasmic p27kip1, RhoA activity and SAPK/JNK signalling. To this effect we observed a correlation between increased cytoplasmic p27kip1, increased RhoA protein levels, decreased RhoA-GTP levels and increased SAPK/JNK phosphorylation in blast phase CD34+ cells compared to chronic phase CD34+ cells.

BRAF V600E associates with cytoplasmatic localization of p27kip1 and higher cytokeratin 19 expression in papillary thyroid carcinoma

The genetic alterations are responsible for the altered protein expression in tumors. The knowledge of the link between the altered protein expression and genetic alterations may provide potentially important biological and clinical information. In this study, the expression of some protein markers (Gal-3, p21Kip1, CK19) known to be associated to the papillary thyroid carcinoma (PTC) was assessed in a series of surgical samples by immunohistochemistry, and the association between expression of these markers and the BRAF (V600E) mutation was investigated. Gal-3 positive staining was evident in 26 % of benign nodules. The BRAF (V600E) mutation and Gal-3 expression, were found in 55.5 and 87 % of PTC respectively, and were unlinked. The expression of CK19 in benign nodules was weak and limited to scattered follicular cells. Diffuse cytoplasmatic expression of CK19 was present in malignant tumors in a variable percentage of cells. A higher percentage of CK19 expressing cells was associated with BRAF (V600E) (P ≤ 0.001). All benign nodules displayed nuclear p27kip1 in more than 15 % of the cells. Twenty-nine PTC showed a cytoplasmatic staining with negative nuclei. PTC with cytoplasmatic or 0-5 % of cells with nuclear staining, 6-15 % or >15 % of cells with nuclear staining were 72 (66.7 %), 24 (22.2 %), and 12 (11.1 %) respectively. In BRAF (V600E) positive tumors, the cytoplasmatic localization of p27kip1 was significantly more frequent (P = 0.024). In conclusion, we provide evidences that BRAF (V600E) is non-associated with Gal-3 expression, whereas it is associated with cytoplasmatic localization of p27kip1 and higher CK19 expression in PTC.

Phosphorylation and Subcellular Localization of p27Kip1 Regulated by Hydrogen Peroxide Modulation in Cancer Cells

The Cyclin-dependent kinase inhibitor 1B (p27Kip1) is a key protein in the decision between proliferation and cell cycle exit. Quiescent cells show nuclear p27Kip1, but this protein is exported to the cytoplasm in response to proliferating signals. We recently reported that catalase treatment increases the levels of p27Kip1 in vitro and in vivo in a murine model. In order to characterize and broaden these findings, we evaluated the regulation of p27Kip1 by hydrogen peroxide (H2O2) in human melanoma cells and melanocytes. We observed a high percentage of p27Kip1 positive nuclei in melanoma cells overexpressing or treated with exogenous catalase, while non-treated controls showed a cytoplasmic localization of p27Kip1. Then we studied the levels of p27Kip1 phosphorylated (p27p) at serine 10 (S10) and at threonine 198 (T198) because phosphorylation at these sites enables nuclear exportation of this protein, leading to accumulation and stabilization of p27pT198 in the cytoplasm. We demonstrated by western blot a decrease in p27pS10 and p27pT198 levels in response to H2O2 removal in melanoma cells, associated with nuclear p27Kip1. Melanocytes also exhibited nuclear p27Kip1 and lower levels of p27pS10 and p27pT198 than melanoma cells, which showed cytoplasmic p27Kip1. We also showed that the addition of H2O2 (0.1 µM) to melanoma cells arrested in G1 by serum starvation induces proliferation and increases the levels of p27pS10 and p27pT198 leading to cytoplasmic localization of p27Kip1. Nuclear localization and post-translational modifications of p27Kip1 were also demonstrated by catalase treatment of colorectal carcinoma and neuroblastoma cells, extending our findings to these other human cancer types. In conclusion, we showed in the present work that H2O2 scavenging prevents nuclear exportation of p27Kip1, allowing cell cycle arrest, suggesting that cancer cells take advantage of their intrinsic pro-oxidant state to favor cytoplasmic localization of p27Kip1.

H 2O 2 scavenging inhibits G1/S transition by increasing nuclear levels of p27 KIP1

The aim of the present study was to evaluate cell cycle regulation by scavenging H 2O 2 in tumor cells. A significant arrest in the G1 phase of the cell cycle was demonstrated in CH72-T4 carcinoma cells exposed to catalase, associated with a decrease in cyclin D1 and an increase in the CDK inhibitory protein p27 KIP1. Moreover, we found a differential intracellular distribution of p27 KIP1, which remained in the nucleus after catalase treatment. In vivo experiments showed an increase in nuclear levels of p27 KIP1 associated with the inhibition of tumor growth by H 2O 2 scavenging, confirming in vitro results. To conclude, H 2O 2 scavenging may induce cell cycle arrest through the modulation of cyclin D1 and p27 KIP1 levels and nuclear localization of p27 KIP1. To our knowledge, this is the first report that demonstrates that the modulation of ROS alters the intracellular localization of a key regulatory protein of G1/S transition.

Rheb activates AMPK and reduces p27Kip1 levels in Tsc2-null cells via mTORC1-independent mechanisms: implications for cell proliferation and tumorigenesis

Tuberous sclerosis complex (TSC) is an autosomally inherited disorder that causes tumors to form in many organs. It is frequently caused by inactivating mutations in the TSC2 tumor-suppressor gene. TSC2 negatively regulates the activity of the GTPase Rheb and thereby inhibits mammalian target of rapamycin complex 1 (mTORC1) signaling. Activation of mTORC1 as a result of lack of TSC2 function is observed in TSC and sporadic lymphangioleiomyomatosis (LAM). TSC2 deficiency has recently been associated with elevated AMP-activated protein kinase (AMPK) activity, which in turn correlated with cytoplasmic localization of p27Kip1 (p27), a negative regulator of cyclin-dependent kinase 2 (Cdk2). How AMPK in the absence of TSC2 is stimulated is not fully understood. In this study, we demonstrate that Rheb activates AMPK and reduces p27 levels in Tsc2-null cells. Importantly, both effects occur largely independent of mTORC1. Furthermore, increased p27 levels following Rheb depletion correlated with reduced Cdk2 activity and cell proliferation in vitro, and with inhibition of tumor formation by Tsc2-null cells in vivo. Taken together, our data suggest that Rheb controls proliferation of TSC2-deficient cells by a mechanism that involves regulation of AMPK and p27, and that Rheb is a potential target for TSC/LAM therapy.

W1896 Combination Therapy of Gemcitabine and Irinotecan in Advanced Biliary Tract Cancer: Phase II Trial

3, 7 (9.2%): Ki67 negative, 33 (43.4%); positive, 43 (56.6%). The staining intensity of Skp2 and Cks1 showed significant linear correlation (Pearson's R 0.427, p < 0.01). Each staining intensity of Skp2 and Cks1 also showed significant linear correlations with the staining intensity of p27kip1, respectively (Pearson's R 0.314, p < 0.01 for Skp2 and p27kip1, Pearson's R 0.403, p < 0.01 for Cks1 and p27kip1). The positive immunostaining group for Ki67 showed significantly stronger immunostaining intensity for Skp2 (p < 0.01), Cks1 (p < 0.01) and cytoplasmic p27kip1 (p = 0.033). By Kaplan-Meier analyses, the higher IS (p = 0.018) and stronger immunostaining intensity (p < 0.01) of Skp2 and Cks1 were significantly associated with shorter survival for patients with extrahepatic cholangiocarcinoma. Conclusions: Overexpression of Skp2/Cks1 and aberrant localization of p27kip1 into cytoplasm might be useful predictors of prognosis in patients with extrahepatic bile duct cancer.

W1894 Elucidating Gene Regulatory Network of COOH-Terminal Truncated Hbx Associated With Hepatocellular Carcinoma

3, 7 (9.2%): Ki67 negative, 33 (43.4%); positive, 43 (56.6%). The staining intensity of Skp2 and Cks1 showed significant linear correlation (Pearson's R 0.427, p < 0.01). Each staining intensity of Skp2 and Cks1 also showed significant linear correlations with the staining intensity of p27kip1, respectively (Pearson's R 0.314, p < 0.01 for Skp2 and p27kip1, Pearson's R 0.403, p < 0.01 for Cks1 and p27kip1). The positive immunostaining group for Ki67 showed significantly stronger immunostaining intensity for Skp2 (p < 0.01), Cks1 (p < 0.01) and cytoplasmic p27kip1 (p = 0.033). By Kaplan-Meier analyses, the higher IS (p = 0.018) and stronger immunostaining intensity (p < 0.01) of Skp2 and Cks1 were significantly associated with shorter survival for patients with extrahepatic cholangiocarcinoma. Conclusions: Overexpression of Skp2/Cks1 and aberrant localization of p27kip1 into cytoplasm might be useful predictors of prognosis in patients with extrahepatic bile duct cancer.

Abstract 5273: The cyclin dependent kinase inhibitor p27KIP1 inhibits neuroblastoma cell migration and invasion

Abstract Neuroblastoma (NB) is the most common extra-cranial pediatric tumor. Over 70% of NB patients are diagnosed with metastasis, and the survival probability of these patients is significantly lower than patients without metastasis. While the role of p27KIP1 in cell cycle regulation is well documented and conserved in all cell types, there are contradictory reports regarding its function in regulating cell migration and invasion. The purpose of the present study was to elucidate the role of p27KIP1 in regulating migration and invasion of NB cells with MYCN over-expression. Western blot analysis, migration and invasion assays were performed on NB cells with and without over-expression of MYCN. The expression of p27KIP1 was down-regulated using siRNA, and the migration rate was determined on NB cells with MYCN over-expression. Also, the cytoplasmic localization of p27KIP1 in NB cells with and without MYCN over-expression was determined. The results from the current study show that MYCN over-expression increases migration and invasion of NB cells, compared to cells without MYCN over-expression via a process that involves p27KIP1. In addition to its role as a cell cycle regulator, p27KIP1 also regulates NB migration and invasion. This dual role of p27KIP1 as a tumor repressor suggests that it may be an attractive therapeutic target for NB. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5273.

Localization of p27kip1 in the developing avian retina: Sustained expression in the mature tissue

p27kip1 is a cyclin/CDK inhibitor that is expressed in cells that exit cell cycle and turn post-mitotic. Here, we characterized the expression and localization of p27kip1 during the development of the chick embryo retina. Expression of p27kip1 in this tissue begins at embryonic day 5 (E5), increasing as development proceeds. In contrast to the expression in the developing rat retina that markedly decreases after postnatal day 6, expression of p27kip1 in the chick retina decreases only slightly (∼30%) after E12. Thereafter, it remains highly expressed in the tissue. p27kip1 expression increases in an orderly succession. By E5, immunoreactivity was observed over β-tubulin III (TUJ-1) positive cell bodies located in the prospective Ganglion Cell Layer. By E7, p27kip1 was also detected over elongated cell nuclei located in the inner and outer portions of the Neuroblastic Layer and over cell bodies in the middle of the Inner Plexiform Layer. By E12, besides labeled cell bodies, labeled processes from amacrine cells and from cells at the GCL in the IPL were identified. In retinas from post-hatched chicken, immunoreactivity was observed over cell bodies located at all nuclear layers. Several differentiated ChAT positive cholinergic cells were labeled for p27kip1. Our data suggest that, as in the retina of other species, p27kip1 is expressed in cells that are exiting cell cycle and differentiating in the early developing chick embryo retina. However, as opposed to rodents and amphibians, neuronal expression of p27kip1 is sustained in the adult chick retina, indicating that its expression is differently regulated during development in this specie.

Tuberous Sclerosis Complex Suppression in Cerebellar Development and Medulloblastoma: Separate Regulation of Mammalian Target of Rapamycin Activity and p27Kip1 Localization

Abstract During development, proliferation of cerebellar granule neuron precursors (CGNP), candidate cells-of-origin for the pediatric brain tumor medulloblastoma, requires signaling by Sonic hedgehog (Shh) and insulin-like growth factor (IGF), the pathways of which are also implicated in medulloblastoma. One of the consequences of IGF signaling is inactivation of the mammalian target of rapamycin (mTOR)–suppressing tuberous sclerosis complex (TSC), comprised of TSC1 and TSC2, leading to increased mRNA translation. We show that mice, in which TSC function is impaired, display increased mTOR pathway activation, enhanced CGNP proliferation, glycogen synthase kinase-3α/β (GSK-3α/β) inactivation, and cytoplasmic localization of the cyclin-dependent kinase inhibitor p27Kip1, which has been proposed to cause its inactivation or gain of oncogenic functions. We observed the same characteristics in wild-type primary cultures of CGNPs in which TSC1 and/or TSC2 were knocked down, and in mouse medulloblastomas induced by ectopic Shh pathway activation. Moreover, Shh-induced mouse medulloblastomas manifested Akt-mediated TSC2 inactivation, and the mutant TSC2 allele synergized with aberrant Shh signaling to increase medulloblastoma incidence in mice. Driving exogenous TSC2 expression in Shh-induced medulloblastoma cells corrected p27Kip1 localization and reduced proliferation. GSK-3α/β inactivation in the tumors in vivo and in primary CGNP cultures was mTOR-dependent, whereas p27Kip1 cytoplasmic localization was regulated upstream of mTOR by TSC2. These results indicate that a balance between Shh mitogenic signaling and TSC function regulating new protein synthesis and cyclin-dependent kinase inhibition is essential for the normal development and prevention of tumor formation or expansion. [Cancer Res 2009;69(18):7224–34]

Mel-18 Negatively Regulates INK4a/ARF-Independent Cell Cycle Progression via Akt Inactivation in Breast Cancer

Mel-18, a polycomb group (PcG) protein, has been suggested as a tumor suppressor in human breast cancer. Previously, we reported that Mel-18 has antiproliferative activity in breast cancer cells. However, its functional mechanism has not been fully elucidated. Here, we investigated the role of Mel-18 in human breast cancer. We saw an inverse correlation between Mel-18 and phospho-Akt, which were expressed at low and high levels, respectively, in primary breast tumor tissues from 40 breast cancer patients. The effect of Mel-18 on cell growth was examined in two breast cancer cell lines, SK-BR-3 and T-47D, which express relatively low and high levels of endogenous Mel-18, respectively. On Mel-18 overexpression in SK-BR-3 cells, cell growth was attenuated and G(1) arrest was observed. Likewise, suppression of Mel-18 by antisense expression in T-47D cells led to enhanced cell growth and accelerated G(1)-S phase transition. In these cells, cyclin-dependent kinase (Cdk)-4 and Cdk2 activities were affected by Mel-18, which were mediated by changes in cyclin D1 expression and p27(Kip1) phosphorylation at Thr(157), but not by INK4a/ARF genes. The changes were both dependent on the phosphatidylinositol 3-kinase/Akt signaling pathway. Akt phosphorylation at Ser(473) was reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 suppression in T-47D cells. Akt-mediated cytoplasmic localization of p27(Kip1) was inhibited by Mel-18 in SK-BR-3 cells. Moreover, Mel-18 overexpression showed reduced glycogen synthase kinase-3beta phosphorylation, beta-catenin nuclear localization, T-cell factor/lymphoid enhancer factor promoter activity, and cyclin D1 mRNA level. Taken together, we established a linear relationship between Mel-18-->Akt-->G(1) phase regulators.

Infertility caused by retardation of follicular development in mice with oocyte-specific expression of Foxo3a

In recent years, mammalian oocytes have been proposed to have important roles in the orchestration of ovarian follicular development and fertility. To determine whether intra-oocyte Foxo3a, a component of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, influences follicular development and female fertility, a transgenic mouse model was generated with constitutively active Foxo3a expressed in oocytes. We found that the female transgenic mice were infertile, which was caused by retarded oocyte growth and follicular development, and anovulation. Further mechanistic studies revealed that the constitutively active Foxo3a in oocytes caused a dramatic reduction in the expression of bone morphogenic protein 15 (Bmp15), connexin 37 and connexin 43, which are important molecules for the establishment of paracrine and gap junction communications in follicles. Foxo3a was also found to facilitate the nuclear localization of p27(kip1) in oocytes, a cyclin-dependent kinase (Cdk) inhibitor that may serve to inhibit oocyte growth. The results from the current study indicate that Foxo3a is an important intra-oocyte signaling molecule that negatively regulates oocyte growth and follicular development. Our study may therefore give some insight into oocyte-borne genetic aberrations that cause defects in follicular development and anovulation in human diseases, such as premature ovarian failure.

P27kip1 siRNA Induces Proliferation in Corneal Endothelial Cells from Young but Not Older Donors

To determine whether small interfering (si)RNA downregulation of the cyclin-dependent kinase inhibitor p27kip1 overcomes G(1)-phase arrest and promotes cell-cycle progression in human corneal endothelial cells (HCECs) from young (<30 years old) and older (>60 years old) donors. Transfection of siRNA was confirmed by incubating confluent cultures of HCECs with FITC-labeled nonsilencing siRNA. Confluent cultures were transfected for 48 hours with p27kip1 siRNA (2.5, 5, 25, or 100 nM) or nonsilencing siRNA, with a lipid transfection reagent. As a comparison, cultures were also transfected for 48 hours with p27kip1 antisense (AS) or missense (MS) oligonucleotides (oligo). At various times after transfection, cells were fixed for immunocytochemical localization of p27kip1 or extracted for Western blot analysis to assess relative p27kip1 protein levels. Cultures were also prepared for ZO-1 immunolocalization, to assess the effect of transfection on the morphology of the monolayer. The number of cells was counted at 0, 48, 96, 144, and 192 hours after incubation, and a cell-viability assay was performed. A dose-dependent decrease in p27kip1 protein level was observed in Western blot analysis, and nuclear staining for p27kip1 was greatly reduced in HCECs incubated with p27kip1 siRNA. No change in p27kip1 levels or in nuclear staining was observed in the nonsilencing control. p27kip1 siRNA (25 nM) appeared to be quantitatively more efficient than antisense oligonucleotide (500 nM) in reducing p27kip1 protein levels. Viability was less affected by siRNA treatment than by AS oligo transfection. ZO-1 staining showed no effect on morphology of the monolayer. The number of HCECs from young donors (<30 years old) transfected with p27kip1 siRNA increased up to 144 hours after incubation, whereas no change in the number of cells was observed in HCECs transfected with nonsilencing siRNA. In contrast to the results from young donors, no change in the number of cells was observed at any time point tested in HCECs from older donors (>60 years old) after p27kip1 siRNA transfection. Transfection of p27kip1 siRNA was sufficient to promote proliferation in confluent cultures of HCECs from younger, but not older donors. These results suggest that inhibition of proliferation in older donors is regulated by other mechanisms in addition to p27kip1.