SGK3 mediates INPP4B-dependent PI3K signaling in breast cancer.

Oncogenic mutations in PIK3CA, the gene encoding the catalytic subunit of phosphoinositide 3-kinase (PI3K), occur with high frequency in breast cancer. The protein kinase Akt is considered to be the primary effector of PIK3CA, although mechanisms by which PI3K mediates Akt-independent tumorigenic signals remain obscure. We show that serum and glucocorticoid-regulated kinase 3 (SGK3) is amplified in breast cancer and activated downstream of PIK3CA in a manner dependent on the phosphoinositide phosphatase INPP4B. Expression of INPP4B leads to enhanced SGK3 activation and suppression of Akt phosphorylation. Activation of SGK3 downstream of PIK3CA and INPP4B is required for 3D proliferation, invasive migration, and tumorigenesis in vivo. We further show that SGK3 targets the metastasis suppressor NDRG1 for degradation by Fbw7. We propose a model in which breast cancers harboring oncogenic PIK3CA activate SGK3 signaling while suppressing Akt, indicative of oncogenic functions for both INPP4B and SGK3 in these tumors.

Acetylation-dependent regulation of essential iPS-inducing factors: a regulatory crossroad for pluripotency and tumorigenesis.

Induced pluripotent stem (iPS) cells can be generated from somatic cells by coexpression of four transcription factors: Sox2, Oct4, Klf4, and c-Myc. However, the low efficiency in generating iPS cells and the tendency of tumorigenesis hinder the therapeutic applications for iPS cells in treatment of human diseases. To this end, it remains largely unknown how the iPS process is subjected to regulation by upstream signaling pathway(s). Here, we report that Akt regulates the iPS process by modulating posttranslational modifications of these iPS factors in both direct and indirect manners. Specifically, Akt directly phosphorylates Oct4 to modulate the Oct4/Sox2 heterodimer formation. Furthermore, Akt either facilitates the p300-mediated acetylation of Oct4, Sox2, and Klf4, or stabilizes Klf4 by inactivating GSK3, thus indirectly modulating stemness. As tumorigenesis shares possible common features and mechanisms with iPS, our study suggests that Akt inhibition might serve as a cancer therapeutic approach to target cancer stem cells.

SIRT1 phosphorylation by AMP-activated protein kinase regulates p53 acetylation.

The deacetylase SIRT1 regulates multiple biological processes including cellular metabolism and aging. Importantly, SIRT1 can also inactivate the p53 tumor suppressor via deacetylation, suggesting a role in oncogenesis. Recently, SIRT1 was shown to be released from its endogenous inhibitor DBC1 by a process requiring AMPK and the phosphorylation of SIRT1 by yet undefined kinase(s). Here we provide further evidence that AMPK directly phosphorylates SIRT1 on T344, releasing it from DBC1. Furthermore, a phospho-mimetic SIRT1 (T334E) showed decreased binding to DBC1, supporting the importance of this phosphorylation in AMPK-mediated regulation of SIRT1 activity. In addition, inhibition of AMPK by Compound C led to increased p53 acetylation, suggesting a role for the AMPK/SIRT1 pathway in regulating p53 signaling. Together, our results support a hypothesis that AMPK negatively regulates p53 acetylation via phosphorylation of SIRT1 on T344. Furthermore, our findings also define the AMPK/SIRT1 axis as a possible targetable pathway to regulate p53 function.

SCF-mediated Cdh1 degradation defines a negative feedback system that coordinates cell-cycle progression.

Proper cell-cycle transitions are driven by waves of ubiquitin-dependent degradation of key regulators by the anaphase-promoting complex (APC) and Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligase complexes. But precisely how APC and SCF activities are coordinated to regulate cell-cycle progression remains largely unclear. We previously showed that APC/Cdh1 earmarks the SCF component Skp2 for degradation. Here, we continue to report that SCF(ß-TRCP) reciprocally controls APC/Cdh1 activity by governing Cdh1 ubiquitination and subsequent degradation. Furthermore, we define both cyclin A and Plk1, two well-known Cdh1 substrates, as upstream modifying enzymes that promote Cdh1 phosphorylation to trigger Cdh1 ubiquitination and subsequent degradation by SCF(ß-TRCP). Thus, our work reveals a negative repression mechanism for SCF to control APC, thereby illustrating an elegant dual repression system between these two E3 ligase complexes to create the ordered cascade of APC and SCF activities governing timely cell-cycle transitions.

Regulation of APC(Cdh1) E3 ligase activity by the Fbw7/cyclin E signaling axis contributes to the tumor suppressor function of Fbw7.

Fbw7 and Cdh1 are substrate-recognition subunits of the SCF- and APC-type E3 ubiquitin ligases, respectively. There is emerging evidence suggesting that both Fbw7 and Cdh1 function as tumor suppressors by targeting oncoproteins for destruction. Loss of Fbw7, but not Cdh1, is frequently observed in various human tumors. However, it remains largely unknown how Fbw7 mechanistically functions as a tumor suppressor and whether there is a signaling crosstalk between Fbw7 and Cdh1. Here, we report that Fbw7-deficient cells not only display elevated expression levels of SCF(Fbw7) substrates, including cyclin E, but also have increased expression of various APC(Cdh1) substrates. We further defined cyclin E as the critical signaling link by which Fbw7 governs APC(Cdh1) activity, as depletion of cyclin E in Fbw7-deficient cells results in decreased expression of APC(Cdh1) substrates to levels comparable to those in wild-type (WT) cells. Conversely, ectopic expression of cyclin E recapitulates the aberrant APC(Cdh1) substrate expression observed in Fbw7-deficient cells. More importantly, 4A-Cdh1 that is resistant to Cdk2/cyclin E-mediated phosphorylation, but not WT-Cdh1, reversed the elevated expression of various APC(Cdh1) substrates in Fbw7-deficient cells. Overexpression of 4A-Cdh1 also resulted in retarded cell growth and decreased anchorage-independent colony formation. Altogether, we have identified a novel regulatory mechanism by which Fbw7 governs Cdh1 activity in a cyclin E-dependent manner. As a result, loss of Fbw7 can lead to aberrant increase in the expression of both SCF(Fbw7) and APC(Cdh1) substrates. Our study provides a better understanding of the tumor suppressor function of Fbw7, and suggests that Cdk2/cyclin E inhibitors could serve as effective therapeutic agents for treating Fbw7-deficient tumors.

Acetylation-dependent regulation of Skp2 function.

Aberrant Skp2 signaling has been implicated as a driving event in tumorigenesis. Although the underlying molecular mechanisms remain elusive, cytoplasmic Skp2 correlates with more aggressive forms of breast and prostate cancers. Here, we report that Skp2 is acetylated by p300 at K68 and K71, which is a process that can be antagonized by the SIRT3 deacetylase. Inactivation of SIRT3 leads to elevated Skp2 acetylation, which leads to increased Skp2 stability through impairment of the Cdh1-mediated proteolysis pathway. As a result, Skp2 oncogenic function is increased, whereby cells expressing an acetylation-mimetic mutant display enhanced cellular proliferation and tumorigenesis in vivo. Moreover, acetylation of Skp2 in the nuclear localization signal (NLS) promotes its cytoplasmic retention, and cytoplasmic Skp2 enhances cellular migration through ubiquitination and destruction of E-cadherin. Thus, our study identifies an acetylation-dependent regulatory mechanism governing Skp2 oncogenic function and provides insight into how cytoplasmic Skp2 controls cellular migration.

SCF(Fbw7) modulates the NFkB signaling pathway by targeting NFkB2 for ubiquitination and destruction.

The NFkB/Rel family of proteins play critical roles in a variety of cellular processes. Thus, their physiological activation is tightly controlled. Recently, the NFkB2/p100 precursor has been characterized as the fourth IkB type of suppressor for NFkB. However, the molecular mechanism(s) underlying regulated destruction of NFkB2 remains largely unknown. Here, we report that, unlike other IkBs, ubiquitination and destruction of NFkB2 are governed by SCF(Fbw7) in a GSK3-dependent manner. In Fbw(7-/-) cells, elevated expression of NFkB2/p100 leads to a subsequent reduction in NFkB signaling pathways and elevated sensitivity to TNFa-induced cell death. Reintroducing wild-type Fbw7, but not disease-derived mutant forms of Fbw7, rescues NFkB activity. Furthermore, T cell-specific depletion of Fbw7 also leads to reduced NFkB activity and perturbed T cell differentiation. Therefore, our work identifies Fbw7 as a physiological E3 ligase controlling NFkB20s stability. It further implicates that Fbw7 might exert its tumor-suppressor function by regulating NFkB activity.

The Fbw7 and betaTRCP E3 ubiquitin ligases and their roles in tumorigenesis.

The Ubiquitin Proteasome System (UPS) is a major regulator of protein abundance in the cell. The UPS influences the functions of multiple biological processes by targeting key regulators for destruction. E3 ubiquitin ligases are a vital component of the UPS machinery, working with E1 and E2 enzymes to bind substrates and facilitate the transfer of ubiquitin molecules onto the target protein. This poly-ubiquitination, in turn, directs the modified proteins for proteolysis by the 26S proteasome. As the UPS regulates the degradation of multiple oncogenes and tumor suppressors, the dysregulation of this pathway is known to promote various diseases including cancer. While E1 and E2 enzymes have only been minimally linked to cancer development, burgeoning amounts of evidence have implicated loss or gain of E3 function as a key factor in cancer initiation and progression. This review will examine the literature on two SCF-type E3 ligases, SCFFbw7 and SCFbeta-TRCP. In particular, we will highlight novel substrates recently identified for these two E3 ligases, and further discuss how UPS regulation of these targets may promote carcinogenesis.

Negative regulation of the stability and tumor suppressor function of Fbw7 by the Pin1 prolyl isomerase.

Fbw7 is the substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers; however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, overexpressing Pin1 reduces Fbw7 abundance and suppresses Fbw7's ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis, and Pin1 may be a promising drug target for anticancer therapy.

The two faces of FBW7 in cancer drug resistance.

Chemotherapy is an important therapeutic approach for cancer treatment. However, drug resistance is an obstacle that often impairs the successful use of chemotherapies. Therefore, overcoming drug resistance would lead to better therapeutic outcomes for cancer patients. Recently, studies by our own and other groups have demonstrated that there is an intimate correlation between the loss of the F-box and WD repeat domain-containing 7 (FBW7) tumor suppressor and the incurring drug resistance. While loss of FBW7 sensitizes cancer cells to certain drugs, FBW7-/- cells are more resistant to other types of chemotherapies. FBW7 exerts its tumor suppressor function by promoting the degradation of various oncoproteins that regulate many cellular processes, including cell cycle progression, cellular metabolism, differentiation, and apoptosis. Since loss of the FBW7 tumor suppressor is linked to drug resistance, FBW7 may represent a novel therapeutic target to increase drug sensitivity of cancer cells to conventional chemotherapeutics. This paper thus focuses on the new functional aspects of FBW7 in drug resistance.

Nodular fasciitis: a novel model of transient neoplasia induced by MYH9-USP6 gene fusion.

Nodular fasciitis (NF) is a relatively common mass-forming and self-limited subcutaneous pseudosarcomatous myofibroblastic proliferation of unknown pathogenesis. Due to its rapid growth and high mitotic activity, NF is often misdiagnosed as a sarcoma. While studying the USP6 biology in aneurysmal bone cyst and other mesenchymal tumors, we identified high expression levels of USP6 mRNA in two examples of NF. This finding led us to further examine the mechanisms underlying USP6 overexpression in these lesions. Upon subsequent investigation, genomic rearrangements of the USP6 locus were found in 92% (44 of 48) of NF. Rapid amplification of 5'-cDNA ends identified MYH9 as the translocation partner. RT-PCR and direct sequencing revealed the fusion of the MYH9 promoter region to the entire coding region of USP6. Control tumors and tissues were negative for this fusion. Xenografts of cells overexpressing USP6 in nude mice exhibited clinical and histological features similar to human NF. The identification of a sensitive and specific abnormality in NF holds the potential to be used diagnostically. Considering the self-limited nature of the lesion, NF may represent a model of 'transient neoplasia', as it is, to our knowledge, the first example of a self-limited human disease characterized by a recurrent somatic gene fusion event.

Mcl-1 ubiquitination and destruction.

Loss of the Fbw7 tumor suppressor is common in diverse human cancer types, including T-Cell Acute Lymphoblastic Leukemia (T-ALL), although the mechanistic basis of its anti-oncogenic activity remains largely unclear. We recently reported that SCFFbw7 regulates cellular apoptosis by controlling the ubiquitination and destruction of the pro-survival protein, Mcl-1, in a GSK3 phosphorylation-dependent manner. We found that human T-ALL cell lines displayed a close relationship between Fbw7 loss and Mcl-1 overexpression. More interestingly, T-ALL cell lines that are deficient in Fbw7 are particularly sensitive to sorafenib, a multi-kinase inhibitor that has been demonstrated to reduce Mcl-1 expression through an unknown mechanism. On the other hand, Fbw7-deficient T-ALL cell lines are much more resistant to the Bcl-2 antagonist, ABT-737. Furthermore, reconstitution of Fbw7 or depletion of Mcl-1 in Fbw7-deficient cells restores ABT-737 sensitivity, suggesting that elevated Mcl-1 expression is important for Fbw7-deficient cells to evade apoptosis. Therefore, our work provides a novel molecular mechanism for the tumor suppression function of Fbw7. Furthermore, it provides the rationale for targeted usage of Mcl-1 antagonists to treat Fbw7-deficient T-ALL patients.

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 SCF(FBW7) (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.

TRE17/ubiquitin-specific protease 6 (USP6) oncogene translocated in aneurysmal bone cyst blocks osteoblastic maturation via an autocrine mechanism involving bone morphogenetic protein dysregulation.

Aneurysmal bone cyst (ABC) is a pediatric osseous tumor characterized by extensive destruction of the surrounding bone. The molecular mechanisms underlying its pathogenesis are completely unknown. Recent work showed that translocation of the TRE17/USP6 locus occurs in over 60% of ABC cases resulting in TRE17 overexpression. Immature osteoblasts are presumed to be the cell type harboring translocation of TRE17 in at least a subset of ABCs. However, the effects of TRE17 overexpression on transformation and osteoblast function are unknown. TRE17 encodes a ubiquitin-specific protease (USP) and a TBC (TRE2-Bub2-Cdc16) domain that promotes activation of the Arf6 GTPase. Here we report that TRE17 potently inhibits the maturation of MC3T3 pre-osteoblasts in a USP-dependent and Arf6-independent manner. Notably, we find that TRE17 function is mediated through an autocrine mechanism. Transcriptome analysis of TRE17-expressing cells reveals dysregulation of several pathways with established roles in osteoblast maturation. In particular, signaling through the bone morphogenetic protein (BMP) pathway, a key regulator of osteogenesis, is profoundly altered. TRE17 simultaneously inhibits the expression of BMP-4 while augmenting the BMP antagonist, Gremlin-1. Osteoblastic maturation is restored in TRE17-expressing cells by the addition of exogenous BMP-4, thus establishing a functional role for BMP-4 during TRE17-induced transformation. Because bone homeostasis involves a precise balance between the activities of osteoblasts and osteoclasts, our studies raise the possibility that attenuated osteoblast maturation caused by TRE17 overexpression may contribute to the bone loss/destruction observed in ABC.

A reconfigured pattern of MLL occupancy within mitotic chromatin promotes rapid transcriptional reactivation following mitotic exit.

Mixed lineage leukemia (MLL) and its metazoan Trithorax orthologs have been linked with the epigenetic maintenance of transcriptional activity. To identify mechanisms by which MLL perpetuates active transcription in dividing cells, we investigated its role during M phase of the cell cycle. Unlike other chromatin-modifying enzymes examined, we found that MLL associates with gene promoters packaged within condensed mitotic chromosomes. Genome-wide location analysis identified a globally rearranged pattern of MLL occupancy during mitosis in a manner favoring genes that were highly transcribed during interphase. Knockdown experiments revealed that MLL retention at gene promoters during mitosis accelerates transcription reactivation following mitotic exit. MLL tethers Menin, RbBP5, and ASH2L to its occupied sites during mitosis, but is dispensable for preserving histone H3K4 methylation. These findings implicate mitotic bookmarking as a component of Trithorax-based gene regulation, which may facilitate inheritance of active gene expression states during cell division.

The adaptor complex AP-2 regulates post-endocytic trafficking through the non-clathrin Arf6-dependent endocytic pathway.

The ADP-ribosylation factor 6 (Arf6) GTPase functions as a key regulator of endocytic trafficking, participating in clathrin-independent endocytosis in most cell types. Unexpectedly, we found that siRNA-mediated depletion of clathrin or of adaptor protein 2 (AP-2)-complex subunits alters trafficking of Arf6 pathway cargo proteins, such as major histocompatibility complex class I (MHCI) and beta1 integrin. Internalization of these cargoes from the plasma membrane was not affected in cells depleted of clathrin, but was modestly delayed in cells lacking AP-2. Furthermore, depletion of clathrin or AP-2 altered the intracellular distribution of MHCI and beta1 integrin, inducing clustering in a perinuclear region. Despite this altered localization in both depleted populations, enhanced lysosomal targeting of MHCI was observed uniquely in cells that lack AP-2. Total levels of MHCI were modestly but consistently reduced in AP-2-depleted cells, and restored by the lysosomal inhibitor bafilomycin A. Furthermore, the half-life of surface-derived MHCI was reduced in AP-2-depleted cells. Consistent with enhanced degradative sorting, colocalization of Arf6 cargo with the late endosome and lysosome markers CD63 and Lamp1 was increased in cells depleted of AP-2 but not clathrin. These studies indicate a role for AP-2 in maintaining normal post-endocytic trafficking through the Arf6-regulated, non-clathrin pathway, and reveal pervasive effects of clathrin and AP-2 depletion on the endosomal and lysosomal system.

Calcium/calmodulin regulates ubiquitination of the ubiquitin-specific protease TRE17/USP6.

The TRE17 (USP6/TRE-2) oncogene induces tumorigenesis in both humans and mice. However, little is known regarding its regulation or mechanism of transformation. TRE17 encodes a TBC (Tre-2/Bub2/Cdc16)/Rab GTPase-activating protein homology domain at its N terminus and a ubiquitin-specific protease at its C terminus. In the current study, we identified the ubiquitous calcium (Ca2+)-binding protein calmodulin (CaM) as a novel binding partner for TRE17. CaM bound directly to TRE17 in a Ca2+-dependent manner both in vitro and in vivo. The CaM-binding site was mapped to two hydrophobic motifs near the C terminus of the TBC domain. Point mutations within these motifs significantly reduced the interaction of TRE17 with CaM. We further found that TRE17 is monoubiquitinated and promotes its own deubiquitination in vivo. CaM binding-deficient mutants of TRE17 exhibited significantly reduced monoubiquitination, suggesting that binding of Ca2+/CaM to TRE17 promotes this modification. Consistent with this notion, treatment of cells with the CaM inhibitor W7 reduced levels of TRE17 monoubiquitination. Interestingly, the calcium ionophore A23187 induced accumulation of a polyubiquitinated TRE17 species. The effect of A23187 was attenuated in CaM binding-deficient mutants of TRE17. Taken together, these studies indicate a role for Ca2+/CaM in regulating ubiquitination through direct interaction with TRE17.