Silencing of S-phase kinase-associated protein 2 enhances radiosensitivity of esophageal cancer cells through inhibition of PI3K/AKT signaling pathway.

We investigated the effect of S-phase kinase-associated protein 2 (SKP2) on radiosensitivity of esophageal cancer (EC) cells. Expression of SKP2, PI3K, AKT, Bcl-2 and Bax were assayed in EC. EC cells were transfected with SKP2-siRNA/IGF-1 to detect expression of SKP2, PI3K, AKT, Bcl-2 and Bax. At last, the radiosensitivity of cells in different doses of X (0, 2, 4, 6, 8 Gy) irradiation and cell apoptosis were also detected. EC cells displayed a higher positive expression rate of SKP2, elevated mRNA and protein expression of SKP2, PI3K, AKT, Bcl-2 and Bax, as well as higher extent of PI3K and AKT phosphorylation. SKP2 silencing downregulated mRNA and protein expression of PI3K, AKT and Bcl-2 but increased p27 protein expression, and inhibited the cell survival rate while promoting cell apoptosis. Taken together, silencing SKP2 can inhibit the PI3K/AKT signaling pathway, thereby increasing the radiosensitivity of EC cells.

Therapeutic targeting of the E3 ubiquitin ligase SKP2 in T-ALL.

Timed degradation of the cyclin-dependent kinase inhibitor p27Kip1 by the E3 ubiquitin ligase F-box protein SKP2 is critical for T-cell progression into cell cycle, coordinating proliferation and differentiation processes. SKP2 expression is regulated by mitogenic stimuli and by Notch signaling, a key pathway in T-cell development and in T-cell acute lymphoblastic leukemia (T-ALL); however, it is not known whether SKP2 plays a role in the development of T-ALL. Here, we determined that SKP2 function is relevant for T-ALL leukemogenesis, whereas is dispensable for T-cell development. Targeted inhibition of SKP2 by genetic deletion or pharmacological blockade markedly inhibited proliferation of human T-ALL cells in vitro and antagonized disease in vivo in murine and xenograft leukemia models, with little effect on normal tissues. We also demonstrate a novel feed forward feedback loop by which Notch and IL-7 signaling cooperatively converge on SKP2 induction and cell cycle activation. These studies show that the Notch/SKP2/p27Kip1 pathway plays a unique role in T-ALL development and provide a proof-of-concept for the use of SKP2 as a new therapeutic target in T-cell acute lymphoblastic leukemia (T-ALL).

Toxoplasma F-box protein 1 is required for daughter cell scaffold function during parasite replication.

By binding to the adaptor protein SKP1 and serving as substrate receptors for the SKP1 Cullin, F-box E3 ubiquitin ligase complex, F-box proteins regulate critical cellular processes including cell cycle progression and membrane trafficking. While F-box proteins are conserved throughout eukaryotes and are well studied in yeast, plants, and animals, studies in parasitic protozoa are lagging. We have identified eighteen putative F-box proteins in the Toxoplasma genome of which four have predicted homologs in Plasmodium. Two of the conserved F-box proteins were demonstrated to be important for Toxoplasma fitness and here we focus on an F-box protein, named TgFBXO1, because it is the most highly expressed by replicative tachyzoites and was also identified in an interactome screen as a Toxoplasma SKP1 binding protein. TgFBXO1 interacts with Toxoplasma SKP1 confirming it as a bona fide F-box protein. In interphase parasites, TgFBXO1 is a component of the Inner Membrane Complex (IMC), which is an organelle that underlies the plasma membrane. Early during replication, TgFBXO1 localizes to the developing daughter cell scaffold, which is the site where the daughter cell IMC and microtubules form and extend from. TgFBXO1 localization to the daughter cell scaffold required centrosome duplication but before kinetochore separation was completed. Daughter cell scaffold localization required TgFBXO1 N-myristoylation and was dependent on the small molecular weight GTPase, TgRab11b. Finally, we demonstrate that TgFBXO1 is required for parasite growth due to its function as a daughter cell scaffold effector. TgFBXO1 is the first F-box protein to be studied in apicomplexan parasites and represents the first protein demonstrated to be important for daughter cell scaffold function.

[Regulatory effect of Skp2 on the expression and transactivation of the androgen receptor in the progression of castration-resistant prostate cancer].

OBJECTIVE: To determine the expression of Skp2 in different prostate cancer (PCa) cell lines and tissues, and explore its influence on the androgen receptor (AR) signaling pathway and development of castration-resistant prostate cancer (CRPC). METHODS: The expression levels of Skp2 and AR in different PCa cell lines were detected by Western blot. After knockdown of Skp2 in the C4-2 and 22RV1 cells transfected with shRNA, the expressions of AR and P27 were determined and the activity of ARR3-Luc measured by dual-luciferase reporter gene assay following treatment with dihydrotestosterone (DHT). The expressions of AR and Skp2 in human naïve PCa or CRPC specimens were detected by immunohistochemical staining followed by analysis of their differences and correlation. RESULTS: The Skp2 protein expression level was significantly higher in the C4-2 or 22RV1 cells than in the LNCaP cells. DHT treatment increased the expression of Skp2 in the C4-2 cells, but knock-down of Skp2 significantly up-regulated the expression of the well-known downstream protein P27 and down-regulated that of AR. Consistently, DHT treatment increased the activity of ARR3-Luc, while knockdown of Skp2 remarkably decreased it in the C4-2 and 22RV1 cells (P < 0.05). In addition, significantly higher expressions of Skp2 and AR were observed in the CRPC than in the naïve specimens (P < 0.05), with a positive correlation between the two proteins (r = 0.658 1, P < 0.05). CONCLUSION: Skp2 can enhance the expression and transcription activity of the AR protein in CRPC cells or tissues and is promising to be a critical molecular therapeutic target.

Skp2 inhibits osteogenesis by promoting ubiquitin-proteasome degradation of Runx2.

Osteogenic transcription factor Runx2 is essential for osteoblast differentiation. The activity of Runx2 is tightly regulated at transcriptional as well as post-translational level. However, regulation of Runx2 stability by ubiquitin mediated proteasomal degradation by E3 ubiquitin ligases is little-known. Here, for the first time we demonstrate that Skp2, an SCF family E3 ubiquitin ligase negatively targets Runx2 by promoting its polyubiquitination and proteasome dependent degradation. Co-immunoprecipitation studies revealed that Skp2 physically interacts with Runx2 both in a heterologous as well as physiologically relevant system. Functional consequences of Runx2-Skp2 physical interaction were then assessed by promoter reporter assay. We show that Skp2-mediated downregulation of Runx2 led to reduced Runx2 transactivation and osteoblast differentiation. On the contrary, inhibition of Skp2 restored Runx2 levels and promoted osteoblast differentiation. We further show that Skp2 and Runx2 proteins are co-expressed and show inverse relation in vivo such as in lactating, ovariectomized and estrogen-treated ovariectomized animals. Together, these data demonstrate that Skp2 targets Runx2 for ubiquitin mediated degradation and hence negatively regulate osteogenesis. Therefore, the present study provides a plausible therapeutic target for osteoporosis or cleidocranial dysplasia caused by the heterozygous mutation of Runx2 gene.

microRNA-186 inhibits cell proliferation and induces apoptosis in human esophageal squamous cell carcinoma by targeting SKP2.

miR-186 has been demonstrated to have a significant role as a tumor suppressor in many types of cancers. Nevertheless, its biological function in esophageal squamous cell carcinoma (ESCC) remains unknown. In the present study, we found that the expression level of miR-186 was downregulated in ESCC in comparison with the adjacent normal tissues and was significantly associated with differentiation level, TNM stage, and lymph node metastasis of ESCC. Functional experiments revealed that enforced overexpression of miR-186 in ESCC cells suppressed the proliferation, invasion, and induced the apoptosis of cells. Luciferase reporter assay and western blotting analysis were performed to verify the target gene regulated by miR-186, SKP2. Our findings established that the miR-186 has a suppressive role in ESCC progression via SKP2-mediated pathway, and this implies that miR-186 could be a potential therapeutic target for ESCC.

[Over-expression of human S-phase kinase-associated protein 2 (Skp2) promotes proliferation of MCF-7 breast cancer cells and increases the number of S phase cells].

OBJECTIVE: To construct a eukaryotic expression vector for the human gene S-phase kinase-associated protein 2 (Skp2) with a FLAG tag (pcDNA3-FLAG-Skp2) and detect the effect of Skp2 over-expression on the cell growth and cell cycle in MCF-7 breast cancer cells. METHODS: Skp2 was amplified from MDA-MB-231 breast cancer cells by reverse-transcription polymerase chain reaction (RT-PCR) and then used to construct the eukaryotic expression vector pcDNA3-FLAG-Skp2. Integration of Skp2 into the vector was confirmed via restriction digest and sequencing; The pcDNA3-FLAG-Skp2 was then transfected into MCF-7 breast cancer cells. Expression of Skp2 protein was verified by Western blotting. Cell growth was assessed by Alamar blue proliferation assay, cell cycle analysis was carried out by flow cytometry. RESULTS: The PCR amplified fragment was matched up with the anticipated result and its sequence was the same as the data published on GenBank, indicating that the recombinant plasmid pcDNA3-FLAG-Skp2 was constructed successfully. Western blotting revealed that the expression of Skp2 protein was markedly up-regulated in the pcDNA3-FLAG-Skp2 transfected MCF-7 cells at 48 hours. Furthermore, cell growth was significantly promoted in Skp2 over-expressed MCF-7 cells, and the cell count in S phase were also raised. CONCLUSION: The recombinant eukaryotic expression vector pcDNA3-FLAG-Skp2 has been constructed and expressed in MCF-7 breast cancer cells successfully. Over-expression of Skp2 resulted in the increased cell growth and number of S phase cells in Skp2 transfected MCF-7 cells.

Wnt7a is a novel inducer of ß-catenin-independent tumor-suppressive cellular senescence in lung cancer.

Cellular senescence is an initial barrier for carcinogenesis. However, the signaling mechanisms that trigger cellular senescence are incompletely understood, particularly in vivo. Here we identify Wnt7a as a novel upstream inducer of cellular senescence. In two different mouse strains (C57Bl/6J and FVB/NJ), we show that the loss of Wnt7a is a major contributing factor for increased lung tumorigenesis owing to reduced cellular senescence, and not reduced apoptosis, or autophagy. Wnt7a-null mice under de novo conditions and in both the strains display E-cadherin-to-N-cadherin switch, reduced expression of cellular senescence markers and reduced expression of senescence-associated secretory phenotype, indicating a genetic predisposition of these mice to increased carcinogen-induced lung tumorigenesis. Interestingly, Wnt7a induced an alternate senescence pathway, which was independent of ß-catenin, and distinct from that of classical oncogene-induced senescence mediated by the well-known p16(INK4a) and p19(ARF) pathways. Mechanistically, Wnt7a induced cellular senescence via inactivation of S-phase kinase-associated protein 2, an important alternate regulator of cellular senescence. Additionally, we identified Iloprost, a prostacyclin analog, which initiates downstream signaling cascades similar to that of Wnt7a, as a novel inducer of cellular senescence, presenting potential future clinical translational strategies. Thus pro-senescence therapies using either Wnt7a or its mimic, Iloprost, might represent a new class of therapeutic treatments for lung cancer.

CCN5/WISP-2 promotes growth arrest of triple-negative breast cancer cells through accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation.

The matricellular protein CCN5/WISP-2 represents a promising target in triple-negative breast cancer (TNBC) because treatment or induced activation of CCN5 in TNBC cells promotes cell growth arrest at the G0/G1 phase, reduces cell proliferation and delays tumor growth in the xenograft model. Our studies found that the p27(Kip1) tumor suppressor protein is upregulated and relocalized to the nucleus from cytoplasm by CCN5 in these cells and that these two events (upregulation and relocalization of p27(Kip1)) are critical for CCN5-induced growth inhibition of TNBC cells. In the absence of CCN5, p27(Kip1) resides mostly in the cytoplasm, which is associated with the aggressive nature of cancer cells. Mechanistically, CCN5 inhibits Skp2 expression, which seems to stabilize the p27(Kip1) protein in these cells. On the other hand, CCN5 also recruits FOXO3a to mediate the transcriptional regulation of p27(Kip1). The recruitment of FOXO3a is achieved by the induction of its expression and activity through shifting from cytoplasm to the nucleus. Our data indicate that CCN5 blocks PI3K/AKT signaling to dephosphorylate at S318, S253 and Thr32 in FOXO3a for nuclear relocalization and activation of FOXO3a. Moreover, inhibition of α6ß1 receptors diminishes CCN5 action on p27(Kip1) in TNBC cells. Collectively, these data suggest that CCN5 effectively inhibits TNBC growth through the accumulation and trafficking of p27(Kip1) via Skp2 and FOXO3a regulation, and thus, activation of CCN5 may have the therapeutic potential to kill TNBC.

GsSKP21, a Glycine soja S-phase kinase-associated protein, mediates the regulation of plant alkaline tolerance and ABA sensitivity.

Plant SKP1-like family proteins, components of the SCF complex E3 ligases, are involved in the regulation of plant development and stress responses. Little is known about the precise function of SKP genes in plant responses to environmental stresses. GsSKP21 was initially identified as a potential stress-responsive gene based on the transcriptome sequencing of Glycine soja. In this study, we found that GsSKP21 protein contains highly conserved SKP domains in its N terminus and an extra unidentified domain in its C terminus. The transcript abundance of GsSKP21, detected by quantitative real-time PCR, was induced under the treatment of alkali and salt stresses. Overexpression of GsSKP21 in Arabidopsis dramatically increased plant tolerance to alkali stress. Furthermore, we found that overexpression of GsSKP21 resulted in decreased ABA sensitivity during both the seed germination and early seedling growth stages. GsSKP21 mediated ABA signaling by altering the expression levels of the ABA signaling-related and ABA-induced genes. We also investigated the tissue expression specificity and subcellular localization of GsSKP21. These results suggest that GsSKP21 is important for plant tolerance to alkali stress and plays a critical regulatory role in the ABA-mediated stress response.

Activation of AMPK inhibits pulmonary arterial smooth muscle cells proliferation.

The aims of the present study were to examine the effect of AMPK activation on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to address its potential mechanisms. ET-1 dose and time-dependently induced PASMCs proliferation, and this effect was suppressed by a selective AMPK activator metformin. The results of the study further indicated that the proliferation of PASMCs stimulated by ET-1 was associated with the increase of Skp2 and decrease of p27, and metformin reversed ET-1-induced Skp2 elevation and raised p27 protein level. Our study suggests that activation of AMPK suppresses PASMCs proliferation and has potential value in negatively modulating pulmonary vascular remodeling and therefore could prevent or treat the development of pulmonary arterial hypertension (PAH).

Brain tumor senescence might be mediated by downregulation of S-phase kinase-associated protein 2 via butylidenephthalide leading to decreased cell viability.

Developing an effective drug for treating human glioblastoma multiform (GBM) has been investigated persistently. A pure compound butylidenephthalide (BP), isolated from Angelica sinensis, has been shown the activities to arrest the growth and initiate apoptosis of GBM in our previous reports. In this study, we further demonstrated that BP treatment accelerates the cell senescence in a dose-dependent manner in vitro and in vivo. S-phase kinase-associated protein 2 (Skp2), a proto-oncogene, is generally upregulated in cancer. We found that it was downregulated in BP-treated GBM cells. The downregulation of Skp2 is parallel with increasing p16 and p21 expression which causes G0/G1 arrest and tumor cell senescence. We also found that restoring the Skp2 protein level by exogenous overexpression prevents the BP-induced cell senescence. Therefore, the linkage between cell senescence and Skp2 expression is strengthened. Promoter binding analysis further detailed that the BP-mediated SP1 reduction might involve in the Skp2 downregulation. In summary, these results emphasize that BP-triggered senescence in GBM cells is highly associated with its control on Skp2 regulation.

Mechanisms in cardiac fibroblast growth: an obligate role for Skp2 and FOXO3a in ERK1/2 MAPK-dependent regulation of p27kip1.

Cardiac fibroblast hyperplasia associated with enhanced matrix deposition is a major determinant of tissue remodeling in several disease states of the heart. However, mechanisms controlling cell cycle progression in cardiac fibroblasts remain unexplored. Identification of cell cycle regulatory elements in these cells is important to develop strategies to check adverse cardiac remodeling under pathological conditions. This study sought to probe the mechanisms underlying ERK1/2-mediated p27(Kip1) regulation in mitogenically stimulated cardiac fibroblasts. Addition of 10% fetal calf serum to quiescent cultures of adult rat cardiac fibroblasts promoted ERK1/2 activation, as evidenced by its phosphorylation status. Reduction in [(3)H]thymidine incorporation into DNA increased population doubling time, flow cytometry, and Western blot analysis showing reduced levels of cyclins D and A, p27(Kip1) induction, and retinoblastoma protein (Rb) hypophosphorylation in ERK1/2-inhibited cells indicated ERK1/2 dependence of G1-S transition in cardiac fibroblasts. Lack of p27(Kip1) protein in serum-stimulated, ERK1/2-active cells was associated with increased levels of Skp2, an E3 ubiquitin ligase for p27(Kip1), whose knockdown by RNA interference induced p27(Kip1) expression. Further, forced expression of Skp2 in ERK1/2-inhibited cells downregulated p27(Kip1). Transcriptional upregulation of p27(Kip1) mRNA in ERK1/2-inhibited cells, demonstrated by real-time PCR, correlated with forkhead box O 3a (FOXO3a) transcription factor activation, shown by gel shift assay. FOXO3a knockdown attenuated p27(Kip1) mRNA and protein expression in ERK1/2-inhibited cells. We provide evidence for the first time that, in cardiac fibroblasts, activated ERK1/2 regulates p27(Kip1) expression transcriptionally and posttranslationally via FOXO3a- and Skp2-dependent mechanisms. Additionally, this study uncovers interesting interactions between critical cell cycle regulatory elements that are only beginning to be understood.

HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

BACKGROUND & AIMS: Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2. METHODS: We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs. RESULTS: CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB. CONCLUSIONS: HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC.

The oncoprotein HBXIP up-regulates Skp2 via activating transcription factor E2F1 to promote proliferation of breast cancer cells.

Hepatitis B X-interacting protein (HBXIP) is a novel oncoprotein. In this study, we found that the expression levels of HBXIP were positively associated with those of S-phase kinase-associated protein 2 (Skp2) in clinical breast cancer tissues and cell lines. Moreover, we found that HBXIP was able to stimulate the promoter of Skp2 through binding to the -640/-443 region in Skp2 promoter involving activating E2F transcription factor 1 (E2F1). Skp2 plays crucial roles in HBXIP-enhanced proliferation of breast cancer cells in vitro and in vivo. We conclude that HBXIP up-regulates Skp2 via activating E2F1 to promote proliferation of breast cancer cells.

Foxo3a transcription factor is a negative regulator of Skp2 and Skp2 SCF complex.

Skp2 (S-phase kinase-associated protein-2) SCF complex displays E3 ligase activity and oncogenic activity by regulating protein ubiquitination and degradation, in turn regulating cell cycle entry, senescence and tumorigenesis. The maintenance of the integrity of Skp2 SCF complex is critical for its E3 ligase activity. The Skp2 F-box protein is a rate-limiting step and key factor in this complex, which binds to its protein substrates and triggers ubiquitination and degradation of its substrates. Skp2 is found to be overexpressed in numerous human cancers, which has an important role in tumorigenesis. The molecular mechanism by which the function of Skp2 and Skp2 SCF complex is regulated remains largely unknown. Here we show that Foxo3a transcription factor is a novel and negative regulator of Skp2 SCF complex. Foxo3a is found to be a transcriptional repressor of Skp2 gene expression by directly binding to the Skp2 promoter, thereby inhibiting Skp2 protein expression. Surprisingly, we found for the first time that Foxo3a also displays a transcription-independent activity by directly interacting with Skp2 and disrupting Skp2 SCF complex formation, in turn inhibiting Skp2 SCF E3 ligase activity and promoting p27 stability. Finally, we show that the oncogenic activity of Skp2 is repressed by Foxo3a overexpression. Our results not only reveal novel insights into how Skp2 SCF complex is regulated, but also establish a new role for Foxo3a in tumor suppression through a transcription-dependent and independent manner.

Roles of the Skp2/p27 axis in the progression of chronic nephropathy.

S-phase kinase-associated protein 2 (Skp2) is an F-box protein component of the Skp/Cullin/F-box-type E3 ubiquitin ligase that targets several cell cycle regulatory proteins for degradation through the ubiquitin-dependent pathway. Skp2-mediated degradation of p27, a cyclin-dependent kinase inhibitor, is involved in cell cycle regulation. Tubular epithelial cell proliferation is a characteristic feature of renal damage that is apparent in the early stages of nephropathy. The p27 level is associated with the progression of renal injury, and increased Skp2 expression in progressive nephropathy is implicated in decreases of p27 expression. In Skp2(-/-) mice, renal damage caused by unilateral ureteral obstruction (UUO) was ameliorated by p27 accumulation, mainly in tubular epithelial cells. However, the amelioration of UUO-induced renal injury in Skp2(-/-) mice was prevented by p27 deficiency in Skp2(-/-)/p27(-/-) mice. These results suggest that the Skp2-mediated reduction in p27 is a pathogenic activity that occurs during the progression of nephropathy. Here, we discuss the roles of the Skp2/p27 axis and/or related signaling pathways/components in the progression of chronic nephropathy.

Targeting the oncogenic E3 ligase Skp2 in prostate and breast cancer cells with a novel energy restriction-mimetic agent.

Substantial evidence supports the oncogenic role of the E3 ubiquitin ligase S-phase kinase-associated protein 2 (Skp2) in many types of cancers through its ability to target a broad range of signaling effectors for ubiquitination. Thus, this oncogenic E3 ligase represents an important target for cancer drug discovery. In this study, we report a novel mechanism by which CG-12, a novel energy restriction-mimetic agent (ERMA), down-regulates the expression of Skp2 in prostate cancer cells. Pursuant to our previous finding that upregulation of ß-transducin repeat-containing protein (ß-TrCP) expression represents a cellular response in cancer cells to ERMAs, including CG-12 and 2-deoxyglucose, we demonstrated that this ß-TrCP accumulation resulted from decreased Skp2 expression. Evidence indicates that Skp2 targets ß-TrCP for degradation via the cyclin-dependent kinase 2-facilitated recognition of the proline-directed phosphorylation motif (412)SP. This Skp2 downregulation was attributable to Sirt1-dependent suppression of COP9 signalosome (Csn)5 expression in response to CG-12, leading to increased cullin 1 neddylation in the Skp1-cullin1-F-box protein complex and consequent Skp2 destabilization. Moreover, we determined that Skp2 and ß-TrCP are mutually regulated, providing a feedback mechanism that amplifies the suppressive effect of ERMAs on Skp2. Specifically, cellular accumulation of ß-TrCP reduced the expression of Sp1, a ß-TrCP substrate, which, in turn, reduced Skp2 gene expression. This Skp2-ß-TrCP-Sp1 feedback loop represents a novel crosstalk mechanism between these two important F-box proteins in cancer cells with aberrant Skp2 expression under energy restriction, which provides a proof-of-concept that the oncogenic Csn5/Skp2 signaling axis represents a "druggable" target for this novel ERMA.

Oncogenic role of Skp2 and p27Kip1 in intraductal proliferative lesions of the breast.

OBJECTIVE: To investigate whether the connection of p27(Kip1) to S-phase kinase-associated protein 2 (Skp2) plays an oncogenic role in intraductal proliferative lesions of the breast. METHODS: Here we investigated the mechanism involved in association of Skp2's degradation of p27(Kip1) with the breast carcinogenesis by immunohistochemical method through detection of Skp2 and p27(Kip1) protein levels in 120 paraffin-embedded tissues of intraductal proliferative lesions including usual ductal hyperplasia (UDH, n=30), atypical ductal hyperplasia (n=30), flat epithelial atypia (FEA, n=30), and ductal carcinoma in situ (DCIS, n=30). Moreover, the expression status of Skp2 and p27(Kip1) in 30 cases of the normal breast paraffin-embedded tissues were explored. RESULTS: The DCIS group was with the highest Skp2 level and the lowest p27(Kip1) level, and the UDH group was with the lowest Skp2 level and the highest p27(Kip1) level.Both Skp2 and p27(Kip1) levels in the DCIS group were significantly different from those in the UDH group (all P<0.01).The levels of Skp2 and p27(Kip1) in the FEA group were significantly different from both the DCIS and UDH groups (all P<0.05).p27(Kip1) was negatively correlated with Skp2 in both the UDH group (r=-0.629, P=0.026) and DCIS group (r=-0.893, P=0.000). CONCLUSION: Overexpression of Skp2 might be the mechanism underlying p27(Kip1) over degradation.

Skp2 E3 ligase integrates ATM activation and homologous recombination repair by ubiquitinating NBS1.

The Mre11/Rad50/NBS1 (MRN) complex is thought to be a critical sensor that detects damaged DNA and recruits ATM to DNA foci for activation. However, it remains to be established how the MRN complex regulates ATM recruitment to the DNA foci during DNA double-strand breaks (DSBs). Here we show that Skp2 E3 ligase is a key component for the MRN complex-mediated ATM activation in response to DSBs. Skp2 interacts with NBS1 and triggers K63-linked ubiquitination of NBS1 upon DSBs, which is critical for the interaction of NBS1 with ATM, thereby facilitating ATM recruitment to the DNA foci for activation. Finally, we show that Skp2 deficiency exhibits a defect in homologous recombination (HR) repair, thereby increasing IR sensitivity. Our results provide molecular insights into how Skp2 and the MRN complex coordinate to activate ATM, and identify Skp2-mediatetd NBS1 ubiquitination as a vital event for ATM activation in response to DNA damage.