Ichiro Onoyama

SCF(FBW7) regulates cellular apoptosis by targeting MCL1 for ubiquitylation and destruction.

The effective use of targeted therapy is highly dependent on the identification of responder patient populations. Loss of FBW7, which encodes a tumour-suppressor protein, is frequently found in various types of human cancer, including breast cancer, colon cancer and T-cell acute lymphoblastic leukaemia (T-ALL). In line with these genomic data, engineered deletion of Fbw7 in mouse T cells results in T-ALL, validating FBW7 as a T-ALL tumour suppressor. Determining the precise molecular mechanisms by which FBW7 exerts antitumour activity is an area of intensive investigation. These mechanisms are thought to relate in part to FBW7-mediated destruction of key proteins relevant to cancer, including Jun, Myc, cyclin E and notch 1 (ref. 9), all of which have oncoprotein activity and are overexpressed in various human cancers, including leukaemia. In addition to accelerating cell growth, overexpression of Jun, Myc or notch 1 can also induce programmed cell death. Thus, considerable uncertainty surrounds how FBW7-deficient cells evade cell death in the setting of upregulated Jun, Myc and/or notch 1. Here we show that the E3 ubiquitin ligase SCFFBW7 (a SKP1–cullin-1–F-box complex that contains FBW7 as the F-box protein) governs cellular apoptosis by targeting MCL1, a pro-survival BCL2 family member, for ubiquitylation and destruction in a manner that depends on phosphorylation by glycogen synthase kinase 3. Human T-ALL cell lines showed a close relationship between FBW7 loss and MCL1 overexpression. Correspondingly, T-ALL cell lines with defective FBW7 are particularly sensitive to the multi-kinase inhibitor sorafenib but resistant to the BCL2 antagonist ABT-737. On the genetic level, FBW7 reconstitution or MCL1 depletion restores sensitivity to ABT-737, establishing MCL1 as a therapeutically relevant bypass survival mechanism that enables FBW7-deficient cells to evade apoptosis. Therefore, our work provides insight into the molecular mechanism of direct tumour suppression by FBW7 and has implications for the targeted treatment of patients with FBW7-deficient T-ALL.

P57 Is required for quiescence and maintenance of adult hematopoietic stem cells

Quiescence is required for the maintenance of hematopoietic stem cells (HSCs). Members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) have been implicated in HSC quiescence, but loss of p21 or p27 in mice affects HSC quiescence or functionality only under conditions of stress. Although p57 is the most abundant family member in quiescent HSCs, its role has remained uncharacterized. Here we show a severe defect in the self-renewal capacity of p57-deficient HSCs and a reduction of the proportion of the cells in G(0) phase. Additional ablation of p21 in a p57-null background resulted in a further decrease in the colony-forming activity of HSCs. Moreover, the HSC abnormalities of p57-deficient mice were corrected by knocking in the p27 gene at the p57 locus. Our results therefore suggest that, among Cip/Kip family CDK inhibitors, p57 plays a predominant role in the quiescence and maintenance of adult HSCs.

p53-Altered FBXW7 Expression Determines Poor Prognosis in Gastric Cancer Cases

A molecular target associated with the progression of gastric cancer has not yet been uncovered. FBXW7 is a tumor suppressor gene transcriptionally controlled by p53 that plays a role in the regulation of cell cycle exit and reentry via c-Myc degradation. Few studies have addressed the clinical significance of FBXW7 expression in gastric cancer. Therefore, we examined FBXW7 mRNA expression to determine its clinicopathologic significance in 100 cases of gastric cancer. Low expression levels of FBXW7 in primary gastric cancer contributed to malignant potential, such as lymph node metastasis (P = 0.0012), tumor size (P = 0.0003), and poor prognosis (P = 0.018). In comparison with 52 cases of gastric cancer without the p53 mutation, 29 cases with the mutation exhibited lower expression levels of FBXW7 (P = 0.0034), revealing a significant relationship between p53 mutation and FBXW7 expression. Furthermore, we found that gastric cancer patients who had low FBXW7 expression levels and p53 mutation had a distinctively poor prognosis in comparison with other subgroups (P = 0.0033). In conclusion, we showed a role for p53 in the transcriptional regulation of FBXW7 expression in clinical gastric cancer cases and showed that disruption of both p53 and FBXW7 contributes to poor prognosis.

Ablation of Fbxw7 Eliminates Leukemia-Initiating Cells by Preventing Quiescence

Imatinib eradicates dividing progenitor cells of chronic myeloid leukemia (CML) but does not effectively target nondividing leukemia-initiating cells (LICs); thus, the disease often relapse after its discontinuation. We now show that Fbxw7 plays a pivotal role in maintenance of quiescence in LICs of CML by reducing the level of c-Myc. Abrogation of quiescence in LICs by Fbxw7 ablation increased their sensitivity to imatinib, and the combination of Fbxw7 ablation with imatinib treatment resulted in a greater depletion of LICs than of normal hematopoietic stem cells in mice. Purging of LICs by targeting Fbxw7 to interrupt their quiescence and subsequent treatment with imatinib may thus provide the basis for a promising therapeutic approach to CML.

Loss of FBXW7, a cell cycle regulating gene, in colorectal cancer: Clinical significance

This study focused on a cell cycle regulatory gene, FBXW7, which ubiquitinates c‐Myc and cyclin E and promotes exit from the cell cycle. We determined the expression level of FBXW7 in colorectal cancer (CRC) cases, correlated those values with clinicopathologic features, and characterized the molecular mechanism of reduced expression of FBXW7 in CRC cells in vitro. FBXW7 mRNA and protein expression were evaluated in 93 CRC cases. Using CGH array, the copy number aberrations of the flanking region of FBXW7 were evaluated in another 130 CRC specimens. In vitro analysis of FBXW7 gene silencing in CRC cells was conducted. FBXW7 mRNA expression was significantly lower in tumor tissues than the corresponding normal tissues. The low FBXW7 expression group showed a significantly poorer prognosis than patients in the high expression group. A concordant relationship was observed between the incidence of FBXW7 repression and the genetic alteration. The incidence of genetic alteration was associated with the stage of disease progression. In vitro, FBXW7‐specific siRNA enhanced expression of c‐MYC and cyclin E proteins and up‐regulated cell proliferation. Genetic alterations in tumors led to the loss of FBXW7 expression and increased cell proliferation. FBXW7 expression provides a prognostic factor for patients with CRC.

Expression of lysophosphatidic acid receptors and vascular endothelial growth factor mediating lysophosphatidic acid in the development of human ovarian cancer

Lysophosphatidic acid (LPA) receptors including LPA(1), LPA(2), and LPA(3) mediate lysophosphatidic acid signals. We analyzed the expression of LPA receptors, vascular endothelial growth factor (VEGF), and interleukin-8 in 97 patients from normal ovary to ovarian cancer, using reverse transcription polymerase chain reaction. LPA(2), LPA(3), and VEGF expression ratios significantly increased in cancer, compared to those in non-cancerous state (P<0.05). A significant correlation in the expression ratios between LPA(2) or LPA(3) and VEGF was found (gamma=0.617, P<0.0001; gamma=0.431, P<0.001) in patients with cancer. These results suggested that LPA(2) and LPA(3) may be involved in VEGF expression mediated by LPA signals in human ovarian oncogenesis.

Fbxw7-dependent degradation of Notch is required for control of stemness and neuronal-glial differentiation in neural stem cells

Control of the growth and differentiation of neural stem cells is fundamental to brain development and is largely dependent on the Notch signaling pathway. The mechanism by which the activity of Notch is regulated during brain development has remained unclear, however. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members. We now show that mice with brain-specific deletion of Fbxw7 (Nestin-Cre/Fbxw7F/F mice) die shortly after birth with morphological abnormalities of the brain and the absence of suckling behavior. The maintenance of neural stem cells was sustained in association with the accumulation of Notch1 and Notch3, as well as up-regulation of Notch target genes in the mutant mice. Astrogenesis was also enhanced in the mutant mice in vivo, and the differentiation of neural progenitor cells was skewed toward astrocytes rather than neurons in vitro, with the latter effect being reversed by treatment of the cells with a pharmacological inhibitor of the Notch signaling pathway. Our results thus implicate Fbxw7 as a key regulator of the maintenance and differentiation of neural stem cells in the brain.

Fbxw7 regulates lipid metabolism and cell fate decisions in the mouse liver

E3 ubiquitin ligase complexes of the SCF type consist of ring-box 1 (Rbx1), cullin 1 (Cul1), S-phase kinase-associated protein 1 (Skp1), and a member of the F-box family of proteins. The identity of the F-box protein determines the substrate specificity of the complex. The F-box family member F-box- and WD repeat domain-containing 7 (Fbxw7; also known as Fbw7, SEL-10, hCdc4, and hAgo) targets for degradation proteins with wide-ranging functions, and uncovering its in vivo role has been difficult, because Fbxw7-/- embryos die in utero. Using two different Cre-loxP systems (Mx1-Cre and Alb-Cre), we generated mice with liver-specific null mutations of Fbxw7. Hepatic ablation of Fbxw7 resulted in hepatomegaly and steatohepatitis, with massive deposition of triglyceride, a phenotype similar to that observed in humans with nonalcoholic steatohepatitis. Both cell proliferation and the abundance of Fbxw7 substrates were increased in the Fbxw7-deficient liver. Long-term Fbxw7 deficiency resulted in marked proliferation of the biliary system and the development of hamartomas. Fbxw7 deficiency also skewed the differentiation of liver stem cells toward the cholangiocyte lineage rather than the hepatocyte lineage in vitro. This bias was corrected by additional loss of the Notch cofactor RBP-J, suggesting that Notch accumulation triggered the abnormal proliferation of the biliary system. Together, our results suggest that Fbxw7 plays key roles, regulating lipogenesis and cell proliferation and differentiation in the liver.

Role of the UBL-UBA protein KPC2 in degradation of p27 at G1 phase of the cell cycle

ABSTRACT KPC2 (Kip1 ubiquitylation-promoting complex 2) together with KPC1 forms the ubiquitin ligase KPC, which regulates degradation of the cyclin-dependent kinase inhibitor p27 at the G1 phase of the cell cycle. KPC2 contains a ubiquitin-like (UBL) domain, two ubiquitin-associated (UBA) domains, and a heat shock chaperonin-binding (STI1) domain. We now show that KPC2 interacts with KPC1 through its UBL domain, with the 26S proteasome through its UBL and NH2-terminal UBA domains, and with polyubiquitylated proteins through its UBA domains. The association of KPC2 with KPC1 was found to stabilize KPC1 in a manner dependent on the STI1 domain of KPC2. KPC2 mutants that lacked either the NH2-terminal or the COOH-terminal UBA domain supported the polyubiquitylation of p27 in vitro, whereas a KPC2 derivative lacking the STI1 domain was greatly impaired in this regard. Depletion of KPC2 by RNA interference resulted in inhibition of p27 degradation at the G1 phase, and introduction of KPC2 derivatives into the KPC2-depleted cells revealed that the NH2-terminal UBA domain of KPC2 is essential for p27 degradation. These observations suggest that KPC2 cooperatively regulates p27 degradation with KPC1 and that the STI1 domain as well as the UBL and UBA domains of KPC2 are indispensable for its function.

F-box protein FBXW7 inhibits cancer metastasis in a non-cell-autonomous manner

The gene encoding F-box protein FBXW7 is frequently mutated in many human cancers. Although most previous studies have focused on the tumor-suppressive capacity of FBXW7 in tumor cells themselves, we determined that FBXW7 in the host microenvironment also suppresses cancer metastasis. Deletion of Fbxw7 in murine BM-derived stromal cells induced accumulation of NOTCH and consequent transcriptional activation of Ccl2. FBXW7-deficient mice exhibited increased serum levels of the chemokine CCL2, which resulted in the recruitment of both monocytic myeloid-derived suppressor cells and macrophages, thereby promoting metastatic tumor growth. Administration of a CCL2 receptor antagonist blocked the enhancement of metastasis in FBXW7-deficient mice. Furthermore, in human breast cancer patients, FBXW7 expression in peripheral blood was associated with serum CCL2 concentration and disease prognosis. Together, these results suggest that FBXW7 antagonizes cancer development in not only a cell-autonomous manner, but also a non-cell-autonomous manner, and that modulation of the FBXW7/NOTCH/CCL2 axis may provide a potential approach to suppression of cancer metastasis.