MiRNA-27a controls FBW7/hCDC4-dependent cyclin E degradation and cell cycle progression.

The F-box protein FBW7/hCDC4 is a tumor suppressor that acts as the substrate recognition component of an SCF ubiquitin ligase that targets numerous oncoproteins for proteasomal degradation. In this study, we investigated whether FBW7 is regulated by microRNAs, using a screen combining bioinformatic analysis, luciferase reporters and microRNA libraries. The ubiquitous miR-27a was identified as a major suppressor of FBW7 and in line with this, miR-27a prohibited ubiquitylation and turnover of the key FBW7 substrate cyclin E. Notably, we found that miR-27a only suppresses FBW7 during specific cell cycle phases, relieving its negative impact at the G1 to S-phase transition, prior to cyclin E protein degradation. We also demonstrate that attenuation of FBW7 by miR-27a overexpression leads to improper cell cycle progression and DNA replication stress, consistent with dysregulation of cyclin E expression. Finally, in the context of human cancer, miR-27a was discovered to be generally overexpressed in pediatric B-ALL and its expression to be inversely correlated with that of FBW7 in hyperdiploid cases of B-ALL. These data provide evidence for microRNA-mediated regulation of FBW7, and highlight the role of miR-27a as a novel factor fine-tuning the periodic events regulating cell cycle progression.

Inactivation of FBXW7/hCDC4-ß expression by promoter hypermethylation is associated with favorable prognosis in primary breast cancer.

INTRODUCTION: Mutational inactivation of the FBXW7/hCDC4 tumor suppressor gene (TSG) is common in many cancer types, but infrequent in breast cancers. This study investigates the presence and impact of FBXW7/hCDC4 promoter methylation in breast cancer. METHODS: FBXW7/hCDC4-ß expression and promoter methylation was assessed in 161 tumors from two independent breast cancer cohorts. Associations between methylation status and clinicopathologic characteristics were assessed by Fisher's exact test. Survival was analyzed using the Kaplan-Meier method in addition to modeling the risk by use of a multivariate proportional hazard (Cox) model adjusting for possible confounders of survival. RESULTS: Methylation of the promoter and loss of mRNA expression was found both in cell lines and primary tumors (43% and 51%, respectively). Using Cox modeling, a trend was found towards decreased hazard ratio (HR) for death in women with methylation of FBXW7/hCDC4-ß in both cohorts (HR 0.53 (95% CI 0.23 to 1.23) and HR 0.50 (95% CI 0.23 to 1.08), respectively), despite an association between methylation and high-grade tumors (P = 0.017). Interestingly, in subgroups of patients whose tumors are p53 mutated or lymph-node positive, promoter methylation identified patients with significantly improved survival (P = 0.048 and P = 0.017, respectively). CONCLUSIONS: We demonstrate an alternative mechanism for inactivation of the TSG FBXW7/hCDC4, namely promoter specific methylation. Importantly, in breast cancer, methylation of FBXW7/hCDC4-ß is related to favorable prognosis despite its association with poorly differentiated tumors. Future work may define whether FBXW7/hCDC4 methylation is a biomarker of the response to chemotherapy and a target for epigenetic modulation therapy.

FBXW7/hCDC4 is a general tumor suppressor in human cancer.

The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skp1-Cdc53/Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin E1, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of approximately 6%. The highest mutation frequencies were found in tumors of the bile duct (cholangiocarcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational "hotspots," which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer.

The tumor suppressor gene hCDC4 is frequently mutated in human T-cell acute lymphoblastic leukemia with functional consequences for Notch signaling.

Notch signaling is of crucial importance in normal T-cell development and Notch 1 is frequently mutated in T-cell acute lymphoblastic leukemias (T-ALL), leading to aberrantly high Notch signaling. In this report, we determine whether T-ALL mutations occur not only in Notch1 but also in the F-box protein hCdc4 (Sel-10, Ago, or Fbxw7), a negative regulator of Notch1. We show that the hCDC4 gene is mutated in leukemic cells from more than 30% of patients with pediatric T-ALL and derived cell lines. Most hCDC4 mutations found were missense substitutions at critical arginine residues (Arg(465), Arg(479), and Arg(505)) localized in the substrate-binding region of hCdc4. Cells inactivated for hCdc4 and T-ALL cells containing hCDC4 mutations exhibited an increased Notch1 protein half-life, consistent with the proposed role of hCdc4 in ubiquitin-dependent proteolysis of Notch1. Furthermore, restoration of wild-type but not mutant hCdc4 in HCT 116 hCDC4-negative cells led to an increased Notch1 ubiquitylation and decreased Notch1 signaling. These results show that hCdc4 mutations interfere with normal Notch1 regulation in vivo. Finally, we found that mutations in hCDC4 and NOTCH1 can occur in the same cancers and that patients carrying hCDC4 and/or NOTCH1 mutations have a favorable overall survival. Collectively, these data show that mutation of hCDC4 is a frequent event in T-ALL and suggest that hCDC4 mutations and gain-of-function mutations in NOTCH1 might synergize in contributing to the development of pediatric T-ALL leukemogenesis.