The Oncogenic MicroRNA-21 Inhibits the Tumor Suppressive Activity of FBXO11 to Promote Tumorigenesis

The microRNA miR-21 is overexpressed in most human cancers and accumulating evidence indicates that it functions as an oncogene. Since miRNAs suppress the expression of their target genes, we hypothesized that some miR-21 targets may act as tumor suppressors, and thus their expression would be anticipated to be reduced by the high miR-21 levels observed in various human cancers. By microarray analysis and quantitative PCR we identified and validated FBXO11 (a member of the F-box subfamily lacking a distinct unifying domain) as a miR-21 target gene. FBXO11 is a component of the SKP1-CUL1-F-box ubiquitin ligase complex that targets proteins for ubiquitination and proteosomal degradation. By loss of function and gain of function studies, we show that FBXO11 acts as a tumor suppressor, promotes apoptosis and mediates the degradation of the oncogenic protein BCL6. The critical role that FBXO11 plays in miR-21-mediated tumorigenesis was demonstrated by a rescue experiment, in which silencing FBXO11 in miR-21KD cancer cells restored their high tumorigenicity. Expression of miR-21 and FBXO11 are inversely correlated in tumor tissue, and their expression correlates with patient survival and tumor grade. High FBXO11 expression correlates with better patient survival and lower tumor grade consistent with its tumor suppressor activity. In contrast high miR-21 expression, which correlates with poor patient survival and higher tumor grade, is consistent with its oncogenic activity. Our results identify FBXO11 as a novel miR-21 target gene, and demonstrate that the oncogenic miRNA miR-21 decreases the expression of FBXO11, which normally acts as a tumor suppressor, and thereby promotes tumorigenesis.     

Deubiquitinating c-Myc: USP36 steps up in the nucleolus

Ubiquitination plays a key and complex role in the regulation of c-Myc stability, transactivation, and oncogenic activity. c-Myc is ubiquitinated by a number of ubiquitin ligases (E3s), such as SCF^Fbw7 and SCF^Skp2. Depending on the E3s, ubiquitination can either positively or negatively regulate c-Myc levels and activity. Meanwhile, c-Myc ubiquitination can be reversed by deubiquitination. An early study showed that USP28 deubiquitinates c-Myc via interacting with Fbw7α whereas a recent study reveals that USP37 deubiquitinates c-Myc independently of Fbw7 and c-Myc phosphorylation. Consequently, both USP28 and USP37 stabilize c-Myc and enhance its activity. We recently found the nucleolar USP36 as a novel c-Myc deubiquitinase that controls the end-point of c-Myc degradation pathway in the nucleolus. Here we briefly review the current understanding of ubiquitination and deubiquitination regulation of c-Myc and further discuss the USP36-c-Myc regulatory pathway.     

Rictor regulates FBXW7-dependent c-Myc and cyclin E degradation in colorectal cancer cells

Rictor (Rapamycin-insensitive companion of mTOR) forms a complex with mTOR and phosphorylates and activates Akt. Activation of Akt induces expression of c-Myc and cyclin E, which are overexpressed in colorectal cancer and play an important role in colorectal cancer cell proliferation. Here, we show that rictor associates with FBXW7 to form an E3 complex participating in the regulation of c-Myc and cyclin E degradation. The Rictor-FBXW7 complex is biochemically distinct from the previously reported mTORC2 and can be immunoprecipitated independently of mTORC2. Moreover, knocking down of rictor in serum-deprived colorectal cancer cells results in the decreased ubiquitination and increased protein levels of c-Myc and cyclin E while overexpression of rictor induces the degradation of c-Myc and cyclin E proteins. Genetic knockout of FBXW7 blunts the effects of rictor, suggesting that rictor regulation of c-Myc and cyclin E requires FBXW7. Our findings identify rictor as an important component of FBXW7 E3 ligase complex participating in the regulation of c-Myc and cyclin E protein ubiquitination and degradation. Importantly, our results suggest that elevated growth factor signaling may contribute to decrease rictor/FBXW7-mediated ubiquitination of c-Myc and cyclin E, thus leading to accumulation of cyclin E and c-Myc in colorectal cancer cells.     

A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells

The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.     

Recombinant Human Adenovirus-p53 Injection Induced Apoptosis in Hepatocellular Carcinoma Cell Lines Mediated by p53-Fbxw7 Pathway,Which Controls c-Myc and Cyclin E

F-box and WD repeat domain-containing 7 (Fbxw7/hAgo/hCdc4/Fbw7) is a p53-dependent tumor suppressor and leads to ubiquitination-mediated suppression of several oncoproteins including c-Myc, cyclin E, Notch, c-Jun and others. Our previous study has indicated that low expression of Fbxw7 was negatively correlated with c-Myc, cyclin E and mutant-p53 in hepatocellular carcinoma (HCC) tissues. But the role and mechanisms of Fbxw7 in HCC are still unknown. Here, we investigated the function of Fbxw7 in HCC cell lines and the anti-tumor activity of recombinant human adenovirus-p53 injection (rAd-p53, Gendicine) administration in vitro and in vivo. Fbxw7-specific siRNA enhanced expression of c-Myc and cyclin E proteins and increased proliferation in cell culture. rAd-p53 inhibited tumor cell growth with Fbxw7 upregulation and c-Myc and cyclin E downregulation in vitro and a murine HCC model. This effect could be partially reverted using Fbxw7-specific siRNA. Here, we suggest that the activation of Fbxw7 by adenoviral delivery of p53 leads to increased proteasomal degradation of c-Myc and cyclin E enabling growth arrest and apoptosis. Addressing this pathway, we identified that rAd-p53 could be a potential therapeutic agent for HCC.     

c-Jun-NH2 kinase (JNK) contributes to the regulation of c-Myc protein stability

In accord with the central role c-Myc plays in control of cell growth and death, the stability of this protein is tightly regulated. Although the NH2-terminal domain of c-Myc has been implicated in the regulation of its stability, c-Myc-S, which lacks this domain, is equally unstable, pointing to the role of additional domains in the regulation of c-Myc stability. Our former studies revealed that amino acids (aa) 127-189 of c-Myc are responsible for stress-induced stability of the c-Myc protein. This region of c-Myc shares homology with the delta domain of c-Jun, which is required for JNK association and subsequent targeting of c-Jun for ubiquitination under non-stressed growth conditions. Here we demonstrate that JNK associates with, and mediates, c-Myc ubiquitination and degradation. Addition of JNK increased the degree of c-Myc ubiquitination in in vitro ubiquitination reactions. Increased c-Myc stability following MEKK1/JNK stimuli is abolished upon mutation within the delta-like domain of c-Myc (aa 166-181), as well as deletion of aa 127-189. Significantly, inhibition of JNK expression via small interfering RNA increased c-Myc protein expression. Similarly, squelching JNK association with c-Myc by overexpression of a peptide corresponding to aa 127-189 of c-Myc increased endogenous c-Myc stability and elevated the fraction of cells within the G2/M phase of the cell cycle. In all, these findings point to the contribution of JNK to the regulation of c-Myc protein stability under normal growth conditions.     

The F-box Protein FBXO25 Promotes the Proteasome-dependent Degradation of ELK-1 Protein

FBXO25 is one of the 69 known human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of SKP1, Rbx1, Cullin1, and F-box protein (SCF1) that are involved in targeting proteins for degradation across the ubiquitin proteasome system. However, the substrates of most SCF E3 ligases remain unknown. Here, we applied an in chip ubiquitination screen using a human protein microarray to uncover putative substrates for the FBXO25 protein. Among several novel putative targets identified, the c-fos protooncogene regulator ELK-1 was characterized as the first endogenous substrate for SCF1(FBXO25) E3 ligase. FBXO25 interacted with and mediated the ubiquitination and proteasomal degradation of ELK-1 in HEK293T cells. In addition, FBXO25 overexpression suppressed induction of two ELK-1 target genes, c-fos and egr-1, in response to phorbol 12-myristate 13-acetate. Together, our findings show that FBXO25 mediates ELK-1 degradation through the ubiquitin proteasome system and thereby plays a role in regulating the activation of ELK-1 pathway in response to mitogens.     

SIRT1 and c-Myc Promote Liver Tumor Cell Survival and Predict Poor Survival of Human Hepatocellular Carcinomas

The increased expression of SIRT1 has recently been identified in numerous human tumors and a possible correlation with c-Myc oncogene has been proposed. However, it remains unclear whether SIRT1 functions as an oncogene or tumor suppressor. We sought to elucidate the role of SIRT1 in liver cancer under the influence of c-Myc and to determine the prognostic significance of SIRT1 and c-Myc expression in human hepatocellular carcinoma. The effect of either over-expression or knock down of SIRT1 on cell proliferation and survival was evaluated in both mouse and human liver cancer cells. Nicotinamide, an inhibitor of SIRT1, was also evaluated for its effects on liver tumorigenesis. The prognostic significance of the immunohistochemical detection of SIRT1 and c-Myc was evaluated in 154 hepatocellular carcinoma patients. SIRT1 and c-Myc regulate each other via a positive feedback loop and act synergistically to promote hepatocellular proliferation in both mice and human liver tumor cells. Tumor growth was significantly inhibited by nicotinamide in vivo and in vitro. In human hepatocellular carcinoma, SIRT1 expression positively correlated with c-Myc, Ki67 and p53 expression, as well as high á-fetoprotein level. Moreover, the expression of SIRT1, c-Myc and p53 were independent prognostic indicators of hepatocellular carcinoma. In conclusion, this study demonstrates that SIRT1 expression supports liver tumorigenesis and is closely correlated with oncogenic c-MYC expression. In addition, both SIRT1 and c-Myc may be useful prognostic indicators of hepatocellular carcinoma and SIRT1 targeted therapy may be beneficial in the treatment of hepatocellular carcinoma.     

ASK1-signaling promotes c-Myc protein stability during apoptosis

We previously reported that JNK is involved in the regulation of c-Myc-mediated apoptosis triggered by UV irradiation and anticancer drug treatment. Here we show that ASK1 is an upstream regulator for c-Myc-mediated apoptosis triggered by UV, and we found a direct role for Ser-62 and Ser-71 in the regulation of protein stability and function of c-Myc. The ASK1-JNK pathway enhanced the protein stability of c-Myc through phosphorylation at Ser-62 and Ser-71, which was required for c-Myc-dependent apoptosis by ASK1-signaling. Interestingly, ASK1-signaling attenuated the degradation of ubiquitinated c-Myc without affecting the ubiquitination process. Together, these findings indicate that the ASK1-JNK pathway promotes the proapoptotic activity of c-Myc by modulating c-Myc protein stability through phosphorylation at Ser-62 and Ser-71.