Downregulation Of Skp2 Research Articles

Apigenin induces apoptosis via downregulation of S‐phase kinase‐associated protein 2‐mediated induction of p27Kip1 in primary effusion lymphoma cells

The mechanisms that regulate mitogenic and antiapoptotic signals in primary effusion lymphoma (PEL) are not well known. In efforts to identify novel approaches to block the proliferation of PEL cells, we assessed the effect of apigenin (4',5,7-trihydroxyflavone), a flavonoid on a panel of PEL cell lines. We studied the effect of apigenin on four PEL cell lines. Apoptosis was measured by annexin V/PI dual staining and DNA laddering. Protein expression was measured by immunoblotting. Apigenin induced apoptosis in PEL cell lines in a dose dependent manner. Such effects of apigenin appeared to result from suppression of constitutively active kinase AKT resulting in down-regulation of SKP2, hypo-phosphorylation of Rb and accumulation of p27Kip1. Apigenin treatment of PEL cells caused dephosphorylation of p-Bad protein leading to down regulation of the anti-apoptotic protein, Bcl-2 and an increase in Bax/Bcl2 ratio. Apigenin treatment also triggered Bax conformational change and subsequently translocation from cytosole to mitochondria causing loss of mitochondrial membrane potential with subsequent release of cytochrome c. Released cytochrome c onto the cytosole activated caspase-9 and caspase-3, followed by polyadenosin-5'-diphosphate-ribose polymerase (PARP) cleavage. Finally, treatment of PEL cells with apigenin down-regulated the expression of inhibitor of apoptosis protein (IAPs). Altogether, these data suggest a novel function for apigenin, acting as a suppressor of AKT/PKB pathway in PEL cells, and raise the possibility that this agent may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of the AKT/PKB pathway.

Aberrant expression of p27Kip1-interacting cell-cycle regulatory proteins in ovarian clear cell carcinomas and their precursors with special consideration of two distinct multistage clear cell carcinogenetic pathways

We have previously reported that alterations of p27(Kip1)-interacting cell-cycle proteins frequently occur during the development of endometriosis-associated ovarian clear cell adenocarcinoma (CCA; Yamamoto et al., Histopathology in press, 20). However, CCA also occurs in association with clear cell adenofibroma (CCAF). In this study, the expressions of p27(Kip1)-interacting proteins, i.e., p27(Kip1), Skp2, Cks1, cyclin A, cyclin E, and the Ki-67 labeling index (LI), were analyzed in 25 CCAFs (11 benign and 14 borderline) and 15 CCAF-associated CCAs, and compared with the expression status of each protein in the 23 previously studied endometriosis-associated CCAs. Although aberrant expression of all p27(Kip1)-interacting proteins was more frequent in the CCAF-associated CCAs than in the benign CCAFs, statistical significance was found only for Cks1 overexpression. The frequencies of p27(Kip1) downregulation and overexpression of Skp2 and cyclin A were significantly lower in CCAF-associated than in endometriosis-associated CCAs (P < 0.05, respectively). The frequencies of p27(Kip1) downregulation and Skp2 overexpression in borderline CCAFs were significantly lower than those in atypical endometriosis components in endometriosis-associated CCAs (P < 0.05, respectively). Mean Ki-67 LI increased significantly through benign (4.9%) to borderline (11.1%) CCAF and to CCAF-associated CCA (30.6%), but the latter two values were significantly lower than those in atypical endometriosis (21.4%) and endometriosis-associated CCA (46.9%; P < 0.05, respectively). These data suggest that accumulated alterations of p27(Kip1)-interacting proteins may accelerate the development of CCAs regardless of their carcinogenetic pathways, but that tumor cells in the CCAF-associated pathway appear to show slower cell-cycle progression than those in the endometriosis-associated pathway, possibly accounting for the distinct clinicopathological features of the two CCA subtypes.

P27-Associated G1 arrest induced by hinokitiol in human malignant melanoma cells is mediated via down-regulation of pRb, Skp2 ubiquitin ligase, and impairment of Cdk2 function

Increasing evidence has confirmed that hinokitiol (β-thujaplicin), a tropolone-related compound, exhibits anticancer activity in a variety of cancers through inhibition of cell proliferation. The present study indicates that hinokitiol selectively inhibits cell growth and DNA synthesis in FEM human melanoma cells. Hinokitiol-induced growth inhibition was associated with strong G1 cell cycle arrest. Consistent with blocking the G1–S-phase transition, hinokitiol markedly increased p27 protein levels, but caused only a moderate increase in p21, in addition to a decrease in Cdk2, cyclin E, and phosphorylated Rb. In addition, hinokitiol increased the stability of the p27 protein by inhibiting p27 phosphorylation at Thr 187 and by down-regulating Skp2 expression. siRNA knockdown of p27 abrogated hinokitiol-mediated growth inhibition, while knockdown of Skp2 exacerbated the G1 arrest. In addition to increasing Cdk inhibitor levels and decreasing cyclin A expression, hinokitiol also impaired Cdk2 function by inhibiting Cdk2 kinase activity, impeding cyclin E or A/Cdk2 binding, and inducing translocation of the Cdk2 protein complex. Taken together, our data demonstrate that the novel anticancer mechanism of hinokitiol involves accumulation of p27, down-regulation of Skp2, and impairment of Cdk2 function in FEM human melanoma cells. The therapeutic potential of hinokitiol may lead to novel cell-cycle-based anticancer strategies for malignant melanoma.

WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells

Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G(1) arrest. The G(1) arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G(1) arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and beta-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G(1) arrest include (a) SKP2 down-regulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer.

Inhibitor of growth 4 is involved in melanomagenesis and induces growth suppression and apoptosis in melanoma cell line M14

The objective of this study is as follows. Inhibitor of growth (ING) 4 is a novel member of the ING family. It has been thought to play an inhibitory role in several malignancies through its involvment in gene transcription, apoptosis, cell cycle control, and tumor angiogenesis. The involvement of ING4 in melanomagenesis remains unknown. The purpose of this study was to investigate the inhibitory effects of ING4 on melanoma and its mechanisms. The method used was to construct recombinant plasmid pcDNA3.1-ING4 and transfect it into the human melanoma cell line M14. The effects and mechanisms of ING4 on proliferation and apoptosis of M14 cells were analyzed in vitro according to MTT assay, colony formation assay, and TUNEL assay. The detection of the expression of cell cycle or apoptosis regulators in transfected M14 cells was carried out by western blot analysis. Moreover, the level of ING4 in melanoma tissues was examined by immunohistochemistry. The expression of ING4 was markedly reduced in cutaneous melanoma tissues. Overexpression of ING4 could induce growth suppression and apoptosis enhancement in M14 cells, and also induce the upregulation of p27, Bax and Cyt-c, and the downregulation of cyclinD1, SKP2, Bcl-2, and caspase-3. In conclusion, ING4, as a novel tumor suppressor, has a potential role in growth suppression and apoptosis enhancement of melanoma M14 through the activation of the mitochondrial-induced apoptotic pathway and the hindrance of cell cycle progression. The deregulation of ING4 might be involved in melanomagenesis.

Inverse correlation between Skp2 and p27Kip1 in normal endometrium and endometrial carcinoma

Cyclin-dependent-kinase (cdk) inhibitor, p27(Kip1) (p27), has been shown to participate in progestin-induced growth suppression of normal endometrial glands. To analyse the molecular mechanisms regulating p27 protein, we examined immunohistochemical expression of the SCF(Skp2) (Skp1-Cullin-F-box protein) complex factors, i.e. Skp1, Cul1 and Skp2, and compared them with that of p27, steroid receptors and Ki-67. In normal endometrial glands, the expression of Skp2 was observed in the proliferative phase, whereas that of p27 was observed in the secretory phase. Cultured normal endometrial glandular cells showed that progesterone induced the down-regulation of Skp2 along with up-regulation of p27. In endometrial carcinomas, the inverse topological correlation between Skp2 and p27 was evident in 39/66 (59%) cases, and the expression of Skp2 showed a strong correlation with Ki-67. These findings suggest that the expression of SCF(Skp2) complex changes during the menstrual cycle in normal endometrium and the SCF(Skp2) ubiquitin-proteasome pathway may also work in endometrial carcinomas.

Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53

Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G(1) arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2, which then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G(2)-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation, which then led to a G(2)-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G(2)-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G(2)-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer.

Absence of SKP2 expression attenuates BCR-ABL–induced myeloproliferative disease

AbstractBCR-ABL is proposed to impair cell-cycle control by disabling p27, a tumor suppressor that inhibits cyclin-dependent kinases. We show that in cell lines p27 expression is inversely correlated with expression of SKP2, the F-box protein of SCFSKP2 (SKP1/Cul1/F-box), the E3 ubiquitin ligase that promotes proteasomal degradation of p27. Inhibition of BCR-ABL kinase causes G1 arrest, down-regulation of SKP2, and accumulation of p27. Ectopic expression of wild-type SKP2, but not a mutant unable to recognize p27, partially rescues cell-cycle progression. A similar regulation pattern is seen in cell lines transformed by FLT3-ITD, JAK2V617F, and TEL-PDGFRβ, suggesting that the SKP2/p27 conduit may be a universal target for leukemogenic tyrosine kinases. Mice that received transplants of BCR-ABL–infected SKP2−/− marrow developed a myeloproliferative syndrome but survival was significantly prolonged compared with recipients of BCR-ABL-expressing SKP2+/+ marrow. SKP2−/− leukemic cells demonstrated higher levels of nuclear p27 than SKP2+/+ counterparts, suggesting that the attenuation of leukemogenesis depends on increased p27 expression. Our data identify SKP2 as a crucial mediator of BCR-ABL–induced leukemogenesis and provide the first in vivo evidence that SKP2 promotes oncogenesis. Hence, stabilization of p27 by inhibiting its recognition by SCFSKP2 may be therapeutically useful.

EGCG Stabilizes p27kip1 in E2-Stimulated MCF-7 Cells through Down-Regulation of the Skp2 Protein

Loss of p27Kip1 is associated with a poor prognosis in breast cancer. According to previous findings, a decrease in p27Kip1 levels is mainly the result of enhanced proteasome-dependent degradation mediated by its specific ubiquitin ligase subunit S-phase kinase protein 2 (Skp2). Epigallocatechin-3-gallate (EGCG), the main constituent of green tea, was found to stabilize p27Kip1 levels in breast cancer, but whether this effect is mediated through changes in Skp2 expression remains unclear. Here we investigated the mechanisms involved in EGCG's growth inhibition of estrogen-responsive human breast cancer MCF-7 cells. In our results, EGCG increased p27Kip1 and decreased Skp2 in a time- and dose-dependent manner, suggesting that p27Kip1 and Skp2 may be involved in the growth inhibition by EGCG in estrogen-stimulated MCF-7 cells. Interestingly, mRNA levels of p27Kip1 and Skp2 did not significantly change in estrogen-stimulated MCF-7 cells after EGCG treatments. Moreover, overexpression of Skp2 in MCF-7 cells prevented accumulation of p27Kip1 and promoted resistance to the antiproliferative effects of EGCG. This suggests that the down-regulation of the F-box protein Skp2 is the mechanism underlying p27Kip1 accumulation. Furthermore, both tamoxifen and paclitaxel significantly and synergistically enhanced the growth inhibition of MCF-7 cells by EGCG through the down-regulation of Skp2 protein. However, the down-regulation of Skp2 was not always correlate with the up-regulation of p27, suggesting that EGCG-dependent Skp2 down-regulation can influence cell growth in several ways. The therapeutic strategies designed to reduce Skp2 may therefore play an important clinical role in treatment of breast cancer cells.

Growth inhibition of a tongue squamous cell carcinoma cell line (Tca8113) in vitro and in vivo via siRNA-mediated down-regulation of skp2

Human S-phase kinase-associated protein (skp) 2, the specific ubiquitin ligase subunit that targets the negative regulator p27 Kip1 of the cell cycle and brings about its degradation, is overexpressed in various cancers, including human oral squamous cell carcinomas; its expression levels are inversely related to those of p27 Kip1 in these cells. Recently, a technique known as RNA interference (RNAi) has successfully been applied to mammalian cells. In order to evaluate the applications of the RNAi strategy for cancer gene therapy, the authors treated Tca8113 cells expressing high levels of Skp2 with a small interfering RNA (siRNA) expression plasmid vector targeting skp2 in vitro and in vivo. They demonstrated that the Skp2 protein levels decreased, while the p27 Kip1 protein levels increased in Tca8113 cells transfected with skp2siRNA. Further, skp2siRNA inhibited growth in the Tca8113 cells in vitro and suppressed tumor proliferation in vivo. These results suggest that siRNA-mediated silencing of the skp2 gene may represent a useful strategy for gene therapy of tumors expressing high levels of Skp2.

Bortezomib (Velcade) Induces p27Kip1 Expression through S-Phase Kinase Protein 2 Degradation in Colorectal Cancer

S-phase kinase protein 2 (SKP2), an F-box protein, targets cell cycle regulators including cycle-dependent kinase inhibitor p27Kip1 via ubiquitin-mediated degradation. SKP2 is frequently overexpressed in a variety of cancers. We investigated the role of SKP2 and its ubiquitin-proteasome pathway in colorectal carcinoma using a panel of cell lines, clinical samples, and the NUDE mouse model. Using immunohistochemical analysis on a large tissue microarray of 448 samples, an inverse association of SKP2 expression with p27Kip1 protein levels was seen. A colorectal cancer (CRC) subset with high level of SKP2 and low level of p27Kip1 showed a decreased overall survival (P = 0.0057). Treatment of CRC cell lines with bortezomib or expression of small interfering RNA of SKP2 causes down-regulation of SKP2 and accumulation of p27Kip1. Furthermore, treatment of CRC cells with bortezomib causes apoptosis by involving the mitochondrial pathway and activation of caspases. In addition, treatment of CRC cells with bortezomib down-regulated the expression of XIAP, cIAP1, and survivin. Finally, treatment of CRC cell line xenografts with bortezomib resulted in growth inhibition of tumors in NUDE mice via down-regulation of SKP2 and accumulation of p27Kip1. Altogether, our results suggest that SKP2 and the ubiquitin-proteasome pathway may be potential targets for therapeutic intervention for treatment of CRC.

Ing4 induces Cell Growth Inhibition in Human Lung Adenocarcinoma A549 Cells by Means of Wnt‐1/β‐Catenin Signaling Pathway

ING4, as a novel candidate tumor suppressor gene, has been implicated in several human malignances by tumor growth inhibition and apoptosis enhancement. The mechanism of ING4 remains largely unknown. The purpose of this study was to investigate the inhibitory tumor growth effects of ING4 on lung adenocarcinoma, and its mechanism, by ING4 cDNA transduction into A549 cells. Furthermore, the expression level of ING4 in lung adenocarcinoma tissues was examined. The expression of ING4 was markedly reduced in human lung adenocarcinoma tissues. Overexpression of ING4 can induce growth inhibition in A549 cells both in vitro and in vivo, and also induce up-regulation of p27, down-regulation of cyclinD1, SKP2, and Cox2, and inactivation of the Wnt-1/beta-catenin pathway. Moreover, overexpression of ING4 can enhance the sensitivity of A549 cells to radiotherapy and chemotherapy. Thus, ING4 may play an inhibitory role on A549 cell proliferation and tumor growth in lung adenocarcinoma by up-regulation or down-regulation of cell proliferation-regulating proteins such as p27, cyclinD1, SKP2, and Cox2 by means of inactivation of Wnt-1/beta-catenin signaling.

Knockdown of S-phase Kinase-associated Protein-2 Expression in MCF-7 Inhibits Cell Growth and Enhances the Cytotoxic Effects of Epirubicin

S-phase kinase-associated protein-2 (Skp2) plays a key role in ubiquitin-mediated proteolysis, resulting in the progression of cells from a quiescent to a proliferative state. Skp2 is overexpressed in a variety of tumors and inversely correlated with p27 expression, including breast cancer. In this study, we used small interfering RNAs (siRNAs) to inhibit Skp2 expression in human breast cancer MCF-7 cells and thereby investigate if knockdown of Skp2 by RNA interference (RNAi) would inhibit breast cancer cell growth and influence the effects of epirubicin on MCF-7 cells. Three Skp2 siRNA constructs were recombined and transiently transfected human breast cancer MCF-7 cells. Down-regulation of Skp2 was confirmed by reverse transcription-polymerase chain reaction and Western blot analysis. We showed that Skp2 siRNA transfection decreased Skp2 protein and induced the accumulation of p27 protein in MCF-7 cells. Skp2 siRNA inhibited the cell proliferation both in vitro and in vivo. Knockdown of Skp2 by RNAi increased cellular apoptosis in vitro. Treatment with Skp2 siRNA followed by treatment with epirubicin further inhibited the proliferation of cancer cell lines, compared with control siRNA followed by epirubicin. Therefore, knockdown of Skp2 expression by RNAi inhibits breast cancer cell growth and enhances the effect of epirubicin. siRNA-mediated gene silencing of Skp2 could be a novel cancer gene therapy for the suppression of p27 down regulation.

Inhibition of Ubiquitin-Proteasome Pathway Causes Growth Suppression and Induces Apoptosis in Diffuse Large B Cell Lymphoma.

SKP2, an F-box protein targets cell cycle regulators including cycle-dependent kinase inhibitor p27KIP1 via ubiquitin-mediated degradation. SKP2 is frequently overexpressed in variety of cancer cells and has been implicated in oncogenesis, however its role in diffuse large B-cell lymphoma (DLBCL) has not been elucidated. Therefore, we investigated the role of SKP2 and its ubiquitin-proteasome pathway in DLBL using a panel of cell lines and clinical samples. MG132, a reversible and specific inhibitor of ubiquitin-proteasome pathway that acts through blocking ubiquitin-mediated proteolysis via binding to 20S and 26S proteasomes. Our study showed that treatment of DLBCL cell lines (SUDHL4, SUDHL5, SUDHL8 and OCILY19) with MG132 suppressed growth and induced apoptosis. Treatment of DLBCL cells with MG132 caused downregulation of SKP2, accumulation and stabilization of p27KIP1 leading to the apoptosis. Further downstream, MG132 induced activation of caspase-8 and cleavage of Bid subsequently leading to loss of mitochondrial membrane potential and release of cytochrome c from mitochondria into cytosol, resulting in activation of caspase-3 and cleavage of PARP. zVAD-fmk, a universal inhibitor of caspases prevented caspase-3 activation and abrogated apoptosis induced by MG132 treatment. Finally, using immunohistochemistry, SKP2 was detected in 94 (32.9%) of 286 DLBCL tumors. Furthermore, patients with high SKP2 expression were associated with high expression of Ki67, a proliferative marker. Altogether, these results suggest that SKP2 and ubiquitin-proteasome pathway may be a potential target for therapeutic intervention in diffuse large B-cell lymphoma.

Cell adhesion induces p27Kip1-associated cell-cycle arrest through down-regulation of the SCFSkp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

AbstractMounting evidence suggests that dynamic interactions between a tumor and its microenvironment play a critical role in tumor development, cell-cycle progression, and response to therapy. In this study, we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stromal cells resulted in a reversible G1 arrest associated with elevated p27Kip1 and p21 (WAF1) proteins. The adhesion-mediated p27Kip1 and p21 increases were posttranslationally regulated via the down-regulation of Skp2, a subunit of SCFSkp2 ubiquitin ligase. Overexpression of Skp2 in MCL decreased p27Kip1, whereas inhibition of Skp2 by siRNA increased p27Kip1 and p21 levels. Furthermore, we found cell adhesion up-regulated Cdh1 (an activating subunit of anaphase-promoting complex [APC] ubiquitin ligase), and reduction of Cdh1 by siRNA induced Skp2 accumulation and hence p27Kip1 degradation, thus implicating Cdh1 as an upstream effector of the Skp2/p27Kip1 signaling pathway. Overall, this report, for the first time, demonstrates that cell-cell contact controls the tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHLs. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches for modifying tumor cell growth and response to therapy.

Thiazolidinediones Modulate the Expression of β-Catenin and Other Cell-Cycle Regulatory Proteins by Targeting the F-Box Proteins of Skp1-Cul1-F-box Protein E3 Ubiquitin Ligase Independently of Peroxisome Proliferator-Activated Receptor γ

Considering the role of aberrant beta-catenin signaling in tumorigenesis, we investigated the mechanism by which the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist troglitazone facilitated beta-catenin down-regulation. We demonstrate that troglitazone and its more potent PPARgamma-inactive analogs Delta2TG and STG28 mediated the proteasomal degradation of beta-catenin in prostate cancer cells by up-regulating the expression of beta-transducin repeat-containing protein (beta-TrCP), an F-box component of the Skp1-Cul1-F-box protein E3 ubiquitin ligase. Evidence indicates that although small interfering RNA-mediated beta-TrCP knockdown protected cells against STG28-facilitated beta-catenin ablation, ectopic beta-TrCP expression enhanced the degradation. The involvement of beta-TrCP in beta-catenin degradation was also corroborated by the pull-down analysis and the concurrent down-regulation of known beta-TrCP substrates examined, including Wee1, Ikappabetaalpha, cdc25A, and nuclear factor-kappaB/p105. Furthermore, glycogen synthase kinase-3beta represented a key regulator in the effect of these thiazolidinedione derivatives on beta-catenin proteolysis even though these agents increased its phosphorylation level. It is noteworthy that this drug-induced beta-TrCP up-regulation was accompanied by the concomitant down-regulation of Skp2 and Fbw7, thereby affecting many of the target proteins of these two F-box proteins (such as p27 and cyclin E). As a consequence, the ability of troglitazone to target these F-box proteins provides a molecular basis to account for its reported effect on modulating the expression of aforementioned cell-cycle regulatory proteins. Despite this complicated mode of pharmacological actions, normal prostate epithelial cells, relative to LNCaP cells, were less susceptible to the effects of STG28 on modulating the expression of beta-catenin and beta-TrCP, suggesting the translation potential of using STG28 as a scaffold to develop more potent chemopreventive agents.

Cell Cycle Regulation Targets of MYCN Identified by Gene Expression Microarrays

Background: We have previously shown that MYCN knockdown causes a G1 arrest in MYCN amplified (MNA), p53 wild type (wt) and p53 mutant MNA neuroblastoma cell lines, with increases in p21WAF1 and hypo RB in p53 wt cell lines. 1 Hypothesis: MYCN acts by inhibiting p21WAF1, and also by p21 independent mechanisms to override the G1 checkpoint in exponentially growing cells. Methods: Genes potentially regulated by MYCN were identified using gene expression microarrays in p53 wt MNA IMR-32 and p53 mutant MNA SKNBE(2c) neuroblastoma cell lines treated with MYCN or scrambled siRNA. Results were validated using qRT-PCR and confirmed using the regulatable MYCN expression system (SHEP Tet21N). Results: MYCN knockdown altered the expression of several cell cycle related genes. SKP2 was down regulated in both cell lines, and up regulated in MYCN+ Tet21N cells. Expression of the WNT antagonist DKK3 increased in both cell lines and decreased in MYCN+ Tet21N cells. Expression of CDKN1C (p57cip2) and TP53INP1 also increased after MYCN knockdown. Conclusions: MYCN may override the G1 checkpoint through down-regulation of SKP2 and TP53INP1 resulting in reduced p21WAF1 expression in p53 wt cell lines, and in addition may act through the WNT signalling pathway in a p53 independent manner.

Proteosome Inhibitor Treatment Down-Regulates S-Phase Kinase-Associated Protein 2 Causing Inhibition of Proliferation and Induces Apoptosis in Primary Effusion Lymphoma.

Proteosome inhibition is a novel approach for treating malignancy and has been approved for clinical use. The proteosome is the primary proteolytic mechanism in eukaryotic cells and inhibition of its catalytic activity initiates a cascade of events affecting cell cycle and apoptotic activities. These activities ultimately lead to cell cycle arrest and apoptosis in malignant cells however, the normal counterpart of these cells are spared. In this study, we used a panel of primary effusion lymphoma cell lines (BC1, BC3, BCBL1 and HBL6) to study the effects of proteosome inhibitor, MG132 on cell proliferation and apoptosis. Our data showed that proteosome inhibitor MG132 decreased cell viability as well as induced apoptosis in a dose dependent manner ranging from 0.5–10μM. Furthermore, treatment with 2.5μM MG132 for 24hours induced 41% apoptosis in BC1, 51% in BC3, 41% in BCBL1 and 48% in HBL6 cell lines as detected by annexinV/PI dual staining. S-phase kinase-associated protein 2 (skp-2) is a proto-oncogene and over expressed in various types of tumors. We sought to determine the role of Skp-2 following proteosome inhibition in PELs. MG132 treatment of PEL cell lines resulted in down-regulation of SKP-2 protein in a dose dependent manner whereas the expression of p-27 was up-regulated demonstrating an inverse relationship between these two proteins. Furthermore, MG132 treatment of PELs led to conformational changes in Bax protein and translocation to the mitochondria leading to the loss of mitochondrial membrane potential with subsequent release of cytochrome c from mitochondria into cytosol. Cytochrome c release caused activation of caspase-3 followed by polyadenosin-5′-diphosphate-ribose polymerase (PARP) cleavage. In addition, proteosome inhibitor treatment also caused down-regulation of inhibitor of apoptosis protein, XIAP. Taken together, our findings show that proteosome inhibition causes down-regulation of skp-2, up-regulation of p-27, inhibition of proliferation as well as caspase-dependent apoptosis in primary effusion lymphoma cells suggesting a role of proteosome inhibitors in the treatment of these aggressive cancers.

IKKα controls p52/RelB at the skp2 gene promoter to regulate G1- to S-phase progression

The IkappaB-inducing kinase (IKK) is composed of two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma. IKK-regulated signaling pathways are believed to promote the proliferation of normal cells as well as the aberrant proliferation of cancer cells. The molecular mechanisms linking the IKK signaling pathway components to the cell cycle machinery are not entirely understood. To study the function(s) of the catalytic subunits of the IKK complex, we used pancreatic cancer cells, with constitutive IKK activity. We show that the G1 phase of the cell cycle is specifically regulated by the IKKalpha subunit, which regulates the stability of the cyclin-dependent kinase inhibitor p27(Kip1). Increased p27(Kip1) protein levels following the transfection of IKKalpha-specific siRNAs are a result of the downregulation of the F-box protein S-phase kinase-associated protein 2 (skp2). Additionally, we demonstrate that IKKalpha signaling regulates the transcription of the skp2 gene by controlling the composition of a RelB-containing NF-kappaB complex. Together, this work defines a novel IKKalpha-regulated growth pathway involving the p52/RelB-dependent transcriptional regulation of the skp2 gene.

Up-regulation of Skp2 after Prostate Cancer Cell Adhesion to Basement Membranes Results in BRCA2 Degradation and Cell Proliferation

Aberrant interaction of carcinoma cells with basement membranes (BM) is a fundamental pathophysiological process that initiates a series of events resulting in cancer cell invasion and metastasis. In this report, we describe the results of our investigations pertaining to the events triggered by the adhesion of normal (PNT1A) and highly metastatic (PC-3) prostate cells onto BM proteins. Unlike PNT1A, PC-3 cells adhered avidly to Matrigel BM matrix as well as to isolated collagen type IV, laminin, and heparan sulfate proteoglycan perlecan, main BM components. This aberrantly increased cancer cell adhesion resulted in sustained BRCA2 protein depletion and vigorous cell proliferation, a cascade triggered by beta1 integrin-mediated phosphatidylinositol 3-kinase activation leading to BRCA2 degradation in the proteasome. This latter effect was orchestrated by phosphatidylinositol 3-kinase-dependent up-regulation of Skp2, a subunit of the Skp1-Cul1-F-box protein ubiquitin complex that directly associates with BRCA2 as demonstrated by coimmunoprecipitation assays, determines its ubiquitination, and ultimately targets it for proteasomal degradation. Inhibition of Skp2 expression by small interference RNA prevented BRCA2 depletion and inhibited the trophic effect upon cell proliferation. These results provide additional evidence on the role of BRCA2 as a modulator of cancer cell growth and elucidate the molecular mechanisms involved in its down-regulation in cancer cells when interacting with BM, a crucial step in the biology of metastasis. Furthering the understanding of this molecular pathway may prove valuable in designing new therapeutic strategies aimed at modifying the natural history of prostate carcinoma.