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Supplementary Data from Skp2 Directs Myc-Mediated Suppression of p27Kip1 yet Has Modest Effects on Myc-Driven Lymphomagenesis

Objective The role of gastrointestinal (GI) hormones in the pathophysiology of obesity is unclear, although they are involved in the regulation of satiation and glucose metabolism. To (i) examine glucagon-like peptide 1 (GLP-1), amylin, ghrelin, and glucagon responses to a meal in obese adolescents and to (ii) test which GI peptides are associated with insulin resistance are presented. Methods A total of 16 obese (body mass index (BMI) ≥ 97th percentile for age and gender) and 14 control (BMI between 25th and 75th percentiles) adolescents were included. Subjects were instructed to eat a test meal (490 kcal). Plasma samples were collected for hormone and glucose analysis. Results Obese adolescents were insulin resistant as expressed by the Homeostasis Model Assessment (HOMA) index and had significantly increased fasting glucagon and amylin levels compared to the control group (P = 0.003 and 0.044, respectively). In response to the meal, the increase in GLP-1 levels was reduced in obese adolescents (P

Cks1 is an activator of the SCF(Skp2) ubiquitin ligase complex that targets the cell cycle inhibitor p27(Kip1) for degradation. The loss of Cks1 results in p27(Kip1) accumulation and decreased proliferation and inhibits tumorigenesis. We identify here a function of Cks1 in mammalian cell cycle regulation that is independent of p27(Kip1). Specifically, Cks1(-/-); p27(Kip1-/-) mouse embryonic fibroblasts retain defects in the G(1)-S phase transition that are coupled with decreased Cdk2-associated kinase activity and defects in proliferation that are associated with Cks1 loss. Furthermore, concomitant loss of Cks1 does not rescue the tumor suppressor function of p27(Kip1) that is manifest in various organs of p27(Kip1-/-) mice. In contrast, defects in mitotic entry and premature senescence manifest in Cks1(-/-) cells are p27(Kip1) dependent. Collectively, these findings establish p27(Kip1)-independent functions of Cks1 in regulating the G(1)-S transition.

The role of gastrointestinal (GI) hormones in the pathophysiology of obesity is unclear, although they are involved in the regulation of satiation and glucose metabolism. To (i) examine glucagon-like peptide 1 (GLP-1), amylin, ghrelin, and glucagon responses to a meal in obese adolescents and to (ii) test which GI peptides are associated with insulin resistance are presented.A total of 16 obese (body mass index (BMI) ≥ 97th percentile for age and gender) and 14 control (BMI between 25th and 75th percentiles) adolescents were included. Subjects were instructed to eat a test meal (490 kcal). Plasma samples were collected for hormone and glucose analysis.Obese adolescents were insulin resistant as expressed by the Homeostasis Model Assessment (HOMA) index and had significantly increased fasting glucagon and amylin levels compared to the control group (P = 0.003 and 0.044, respectively). In response to the meal, the increase in GLP-1 levels was reduced in obese adolescents (P 0.001). In contrast, amylin secretion was significantly increased in the obese population compared to the control group (P 0.005).Obese adolescents have increased fasting glucagon and amylin levels and attenuated post-prandial GLP-1 concentrations compared with the control group. These factors could contribute to the metabolic syndrome.

Studies of mechanisms responsible for the persistence of hepatitis B virus (HBV) infection have been hindered by a lack of appropriate animal models. HBV genomes can be delivered to livers of mice using hydrodynamic injection or high doses of an adenoviral vector; these lead to clearance of HBV. We found that infection of immunocompetent mice with low doses of an adenoviral vector resulted in persistent HBV infection; the mice neither underwent seroconversion to production of antibodies against HBV nor developed a strong HBV-specific effector T-cell response. As in patients with chronic HBV infection, DNA vaccination failed to generate T cells that cleared infection. This model of persistent HBV infection could be used to study the pathogenesis of chronic HBV infection and develop new therapeutic strategies.

Pleiotrophin (Ptn) is strongly expressed by stromal cells which maintain HSCs. However, in vivo, Ptn deficiency does not alter steady-state hematopoiesis. However, knockdown of Ptn (PtnKD) in stromal cells increases production of hematopoietic progenitors as well as HSC activity in cocultures, suggesting that Ptn may have a role in HSC activation. Indeed, transplantations of wild-type (Ptn+/+) HSCs into Ptn−/− mice show increased donor cell production in serial transplantations and dominant myeloid regeneration caused by Ptn-dependent regulation of HSC repopulation behavior. This regulation of Lin−Kit+Sca1+ function is associated with increased proliferation and, on a molecular level, with up-regulated expression of cyclin D1 (Ccnd1) and C/EBPα (Cepba), but reduced of PPARγ. The known HSC regulator β-catenin is, however, not altered in the absence of Ptn. In conclusion, our results point to different Ptn-mediated regulatory mechanisms in normal hemostasis and in hematopoietic regeneration and in maintaining the balance of myeloid and lymphoid regeneration. Moreover, our results support the idea that microenvironmental Ptn regulates hematopoietic regeneration through β-catenin–independent regulation of Ccnd1 and Cebpa.

The universal cyclin-dependent kinase inhibitor p27Kip1 functions as a tumor suppressor, and reduced levels of p27Kip1 connote poor prognosis in several human malignancies. p27Kip1 levels are predominately regulated by ubiquitin-mediated turnover of the protein, which is marked for destruction by the E3 ubiquitin ligase SCFSkp2 complex following its phosphorylation by the cyclin E–cyclin-dependent kinase 2 complex. Binding of phospho-p27Kip1 is directed by the Skp2 F-box protein, and this is greatly augmented by its allosteric regulator Cks1. We have established that programmed expression of c-Myc in the B cells of Eμ-Myc transgenic mice triggers p27Kip1 destruction by inducing Cks1, that this response controls Myc-driven proliferation, and that loss of Cks1 markedly delays Myc-induced lymphomagenesis and cancels the dissemination of these tumors. Here, we report that elevated levels of Skp2 are a characteristic of Eμ-Myc lymphomas and of human Burkitt lymphoma that bear MYC/Immunoglobulin chromosomal translocations. As expected, Myc-mediated suppression of p27Kip1 was abolished in Skp2-null Eμ-Myc B cells. However, the effect of Skp2 loss on Myc-driven proliferation and lymphomagenesis was surprisingly modest compared with the effects of Cks1 loss. Collectively, these findings suggest that Cks1 targets, in addition to p27Kip1, are critical for Myc-driven proliferation and tumorigenesis. Mol Cancer Res; 8(3); 353–62

Abstract 1328 Cks1 is an activator of the SCFSkp2 ubiquitin ligase complex that targets the cell cycle inhibitor p27Kip1 for degradation. Loss of Cks1 results in p27Kip1 accumulation and decreased proliferation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. Analysis of Cks1−/− murine embryonic fibroblasts (MEFs) unveiled a significant growth defect and comparison with double knock out Cks1−/−;p27Kip1−/− MEFs revealed a p27Kip1 independent defect at G1/S transition. These effects were associated with suppressed Cdk1 protein and Cdk2 activity in Cks1−/− cells. We hypothesized that Cks1 recruits further cell cycle regulators or facilitators of proliferation, which may explain the dramatic growth defect and the prolonged latency of lymphomagenesis upon loss of Cks1. Using Strep-Flag tagged Cks1 and tandem affinity purification followed by mass spectrometry we identified Geminin as a new Cks1 binding partner. Geminin is an inhibitor of rereplication and loss of Geminin leads to recurrent origin firing in S phase. The physical interaction of Cks1 and Geminin was present in cells of human and murine origin and was associated with Cdk2 binding to Geminin. Detailed analysis proved Cks1 dispensibel for cell cycle controlled Geminin degradation or phosphorylation. Surprisingly when mining public databases and RNA microarray data we found Geminin overexpressend in aggressive human lymphoma subtypes associated with high Ki-67 expression, and in murine Myc-induced lymphoma. Real time PCR revealed increased expression of Geminin during lymphomagenesis in Eμ-Myc mice, which correlated with protein expression. In conclusion, we established the physical Geminin-Cks1 protein interaction, present in human and murine cells, which could act in control of rereplication and genomic stability. Geminin is overexpressed in human and murine lymphoma revealing a potential role in lymphomagenesis. Disclosures: No relevant conflicts of interest to declare.