Mark A. Gregory

c-Myc Proteolysis by the Ubiquitin-Proteasome Pathway: Stabilization of c-Myc in Burkitt's Lymphoma Cells

ABSTRACT The c-Myc oncoprotein is a transcription factor which is a critical regulator of cellular proliferation. Deregulated expression of c-Myc is associated with many human cancers, including Burkitts lymphoma. The c-Myc protein is normally degraded very rapidly with a half-life of 20 to 30 min. Here we demonstrate that proteolysis of c-Myc in vivo is mediated by the ubiquitin-proteasome pathway. Inhibition of proteasome activity blocks c-Myc degradation, and c-Myc is a substrate for ubiquitination in vivo. Furthermore, an increase in c-Myc stability occurs in mitotic cells and is associated with inhibited c-Myc ubiquitination. Deletion analysis was used to identify regions of the c-Myc protein which are required for rapid proteolysis. We found that a centrally located PEST sequence, amino acids 226 to 270, is necessary for rapid c-Myc degradation, but not for ubiquitination. Also, N-terminal sequences, located within the first 158 amino acids of c-Myc, are necessary for both efficient c-Myc ubiquitination and subsequent degradation. We found that c-Myc is significantly stabilized (two- to sixfold) in many Burkitts lymphoma-derived cell lines, suggesting that aberrant c-Myc proteolysis may play a role in the pathogenesis of Burkitts lymphoma. Finally, mutation of Thr-58, a major phosphorylation site in c-Myc and a mutational hot spot in Burkitts lymphoma, increases c-Myc stability; however, mutation of c-Myc is not essential for stabilization in Burkitts lymphoma cells.

Phosphorylation by Glycogen Synthase Kinase-3 Controls c-Myc Proteolysis and Subnuclear Localization

The c-Myc protein is a transcription factor that is a central regulator of cell growth and proliferation. Thr-58 is a major phosphorylation site in c-Myc and is a mutational hotspot in Burkitts and other aggressive human lymphomas, indicating that Thr-58 phosphorylation restricts the oncogenic potential of c-Myc. Mutation of Thr-58 is also associated with increased c-Myc protein stability. Here we show that inhibition of glycogen synthase kinase-3 (GSK-3) activity with lithium increases c-Myc stability and inhibits phosphorylation of c-Myc specifically at Thr-58 in vivo. Conversely, overexpression of GSK-3α or GSK-3β enhances Thr-58 phosphorylation and ubiquitination of c-Myc. Together, these observations suggest that phosphorylation of Thr-58 mediated by GSK-3 facilitates c-Myc rapid proteolysis by the ubiquitin pathway. Furthermore, we demonstrate that GSK-3 binds c-Myc in vivo and in vitro and that GSK-3 colocalizes with c-Myc in the nucleus, strongly arguing that GSK-3 is the c-Myc Thr-58 kinase. We found that c-MycS, which lacks the N-terminal 100 amino acids of c-Myc, is unable to bind GSK-3; however, mutation of Ser-62, the priming phosphorylation site necessary for Thr-58 phosphorylation, does not disrupt GSK-3 binding. Finally, we show that Thr-58 phosphorylation alters the subnuclear localization of c-Myc, enhancing its localization to discrete nuclear bodies together with GSK-3.

MCL1 is phosphorylated in the PEST region and stabilized upon ERK activation in viable cells, and at additional sites with cytotoxic okadaic acid or taxol

BCL2 family members are subject to regulation at multiple levels, providing checks on their ability to contribute to tumorigenesis. However, findings on post-translational BCL2 phosphorylation in different systems have been difficult to integrate. Another antiapoptotic family member, MCL1, exhibits a difference in electrophoretic mobility upon phosphorylation induced by an activator of PKC (12-O-tetradecanoylphorbol 13-acetate; TPA) versus agents that act on microtubules or protein phosphatases 1/2A. A multiple pathway model is now presented, which demonstrates that MCL1 can undergo distinct phosphorylation events – mediated through separate signaling processes and involving different target sites – in cells that remain viable in the presence of TPA versus cells destined to die upon exposure to taxol or okadaic acid. Specifically, TPA induces phosphorylation at a conserved extracellular signal-regulated kinase (ERK) site in the PEST region (Thr 163) and slows turnover of the normally rapidly degraded MCL1 protein; however, okadaic acid and taxol induce ERK-independent MCL1 phosphorylation at additional discrete sites. These findings add a new dimension to our understanding of the complex regulation of antiapoptotic BCL2 family members by demonstrating that, in addition to transcriptional and post-transcriptional regulation, MCL1 is subject to multiple, separate, post-translational phosphorylation events, produced in living versus dying cells at ERK-inducible versus ERK-independent sites.

p19ARF directly and differentially controls the functions of c-Myc independently of p53

Increased expression of the oncogenic transcription factor c-Myc causes unregulated cell cycle progression. c-Myc can also cause apoptosis, but it is not known whether the activation and/or repression of c-Myc target genes mediates these diverse functions of c-Myc. Because unchecked cell cycle progression leads to hyperproliferation and tumorigenesis, it is essential for tumour suppressors, such as p53 and p19ARF (ARF), to curb cell cycle progression in response to increased c-Myc (refs 2, 3). Increased c-Myc has previously been shown to induce ARF expression, which leads to cell cycle arrest or apoptosis through the activation of p53 (ref. 4). Here we show that ARF can inhibit c-Myc by a unique and direct mechanism that is independent of p53. When c-Myc increases, ARF binds with c-Myc and dramatically blocks c-Mycs ability to activate transcription and induce hyperproliferation and transformation. In contrast, c-Mycs ability to repress transcription is unaffected by ARF and c-Myc-mediated apoptosis is enhanced. These differential effects of ARF on c-Myc function suggest that separate molecular mechanisms mediate c-Myc-induced hyperproliferation and apoptosis. This direct feedback mechanism represents a p53-independent checkpoint to prevent c-Myc-mediated tumorigenesis.

Disruption of Myc-Tubulin Interaction by Hyperphosphorylation of c-Myc during Mitosis or by Constitutive Hyperphosphorylation of Mutant c-Myc in Burkitt's Lymphoma

ABSTRACT Somatic mutations at Thr-58 of c-Myc have been detected in Burkitts lymphoma (BL) tumors and have been shown to affect the transforming potential of the Myc oncoprotein. In addition, the N-terminal domain of c-Myc has been shown to interact with microtubules in vivo, and the binding of c-Myc to α-tubulin was localized to amino acids 48 to 135 within the c-Myc protein. We demonstrate that c-Myc proteins harboring a naturally occurring mutation at Thr-58 from BL cell lines have increased stability and are constitutively hyperphosphorylated, which disrupts the in vivo interaction of c-Myc with α-tubulin. In addition, we show that wild-type c-Myc–α-tubulin interactions are also disrupted during a transient mitosis-specific hyperphosphorylation of c-Myc, which resembles the constitutive hyperphosphorylation pattern of Thr-58 in BL cells.

Rational Combination of a MEK Inhibitor, Selumetinib, and the Wnt/Calcium Pathway Modulator, Cyclosporin A, in Preclinical Models of Colorectal Cancer

Purpose: The mitogen-activated protein kinase (MAPK) pathway is a crucial regulator of cell proliferation, survival, and resistance to apoptosis. MEK inhibitors are being explored as a treatment option for patients with KRAS-mutant colorectal cancer who are not candidates for EGFR-directed therapies. Initial clinical results of MEK inhibitors have yielded limited single-agent activity in colorectal cancer, indicating that rational combination strategies are needed. Experimental Design: In this study, we conducted unbiased gene set enrichment analysis and synthetic lethality screens with selumetinib, which identified the noncanonical Wnt/Ca++ signaling pathway as a potential mediator of resistance to the MEK1/2 inhibitor selumetinib. To test this, we used shRNA constructs against relevant WNT receptors and ligands resulting in increased responsiveness to selumetinib in colorectal cancer cell lines. Further, we evaluated the rational combination of selumetinib and WNT pathway modulators and showed synergistic antiproliferative effects in in vitro and in vivo models of colorectal cancer. Results: Importantly, this combination not only showed tumor growth inhibition but also tumor regression in the more clinically relevant patient-derived tumor explant (PDTX) models of colorectal cancer. In mechanistic studies, we observed a trend toward increased markers of apoptosis in response to the combination of MEK and WntCa++ inhibitors, which may explain the observed synergistic antitumor effects. Conclusions: These results strengthen the hypothesis that targeting both the MEK and Wnt pathways may be a clinically effective rational combination strategy for patients with metastatic colorectal cancer. Clin Cancer Res; 19(15); 4149–62. ©2013 AACR.

Thr 163 Phosphorylation Causes Mcl-1 Stabilization when Degradation Is Independent of the Adjacent GSK3-Targeted Phosphodegron, Promoting Drug Resistance in Cancer

The antiapoptotic Bcl-2 family member Mcl-1 is a PEST protein (containing sequences enriched in proline, glutamic acid, serine, and threonine) and is subject to rapid degradation via multiple pathways. Impaired degradation leading to the maintenance of Mcl-1 expression is an important determinant of drug resistance in cancer. Phosphorylation at Thr 163 in the PEST region, stimulated by 12-O-tetradecanoylphorbol acetic acid (TPA)-induced activation of extracellular signal-regulated kinase (ERK), is associated with Mcl-1 stabilization in BL41-3 Burkitt lymphoma cells. This contrasts with the observation that Thr 163 phosphorylation in normal fibroblasts primes glycogen synthase kinase (GSK3)-induced phosphorylation at Ser 159, producing a phosphodegron that targets Mcl-1 for degradation. In the present follow-up studies in BL41-3 cells, Mcl-1 degradation was found to be independent of the GSK3-mediated pathway, providing a parallel to emerging findings showing that Mcl-1 degradation through this pathway is lost in many different types of cancer. Findings in Mcl-1-transfected CHO cells corroborated those in BL41-3 cells in that the GSK3-targeted phosphodegron did not play a major role in Mcl-1 degradation, and a phosphomimetic T163E mutation resulted in marked Mcl-1 stabilization. TPA-treated BL41-3 cells, in addition to exhibiting Thr 163 phosphorylation and Mcl-1 stabilization, exhibited an ∼10-fold increase in resistance to multiple chemotherapeutic agents, including Ara-C, etoposide, vinblastine, or cisplatin. In these cancer cells in which Mcl-1 degradation is not dependent on the GSK3/phosphodegron-targeted pathway, ERK activation and Thr 163 phosphorylation are associated with pronounced Mcl-1 stabilization and drug resistance – effects that can be suppressed by inhibition of ERK activation.

Domain-specific c-Myc ubiquitylation controls c-Myc transcriptional and apoptotic activity

The oncogenic transcription factor c-Myc causes transformation and tumorigenesis, but it can also induce apoptotic cell death. Although tumor suppressors are necessary for c-Myc to induce apoptosis, the pathways and mechanisms are unclear. To further understand how c-Myc switches from an oncogenic protein to an apoptotic protein, we examined the mechanism of p53-independent c-Myc–induced apoptosis. We show that the tumor suppressor protein ARF mediates this switch by inhibiting ubiquitylation of the c-Myc transcriptional domain (TD). Whereas TD ubiquitylation is critical for c-Myc canonical transcriptional activity and transformation, inhibition of ubiquitylation leads to the induction of the noncanonical c-Myc target gene, Egr1, which is essential for efficient c-Myc–induced p53-independent apoptosis. ARF inhibits the interaction of c-Myc with the E3 ubiquitin ligase Skp2. Overexpression of Skp2, which occurs in many human tumors, inhibits the recruitment of ARF to the Egr1 promoter, leading to inhibition of c-Myc–induced apoptosis. Therapeutic strategies could be developed to activate this intrinsic apoptotic activity of c-Myc to inhibit tumorigenesis.

The ARF Tumor Suppressor Keeping Myc on a Leash

The ARF tumor suppressor protein acts in a checkpoint that guards against unscheduled cellular proliferation in response to oncogenic signaling. Deregulated expression of c-Myc induces ARF expression and apoptosis through the ARF-Mdm2-p53 axis. Our recent study reveals a new direct role for ARF in controlling c-Myc’s oncogenic activity that is independent of p53. ARF binds to and selectively impairs the transactivation ability of c-Myc while leaving its transrepression ability intact. Biologically, ARF prevents hyperproliferation and transformation caused by c-Myc and enhances c-Myc-induced apoptosis independently of p53. These new findings may be especially relevant for therapeutic strategies targeting c-Myc-induced cancers.

B-Myc is preferentially expressed in hormonally-controlled tissues and inhibits cellular proliferation

The myc family of genes plays an important role in several cellular processes including proliferation, apoptosis, differentiation, and transformation. B-myc, a relatively new and largely unstudied member of the myc family, encodes a protein that is highly homologous to the N-terminal transcriptional regulatory domain of c-Myc. Here, we show that high level B-myc expression is restricted to specific mouse tissues, primarily hormonally-controlled tissues, with the highest level of expression in the epididymis. We also report the identification of the endogenous B-Myc protein from mouse tissues. Like other Myc family proteins, B-Myc is a short-lived nuclear protein which is phosphorylated on residues Ser-60 and Ser-68. Rapid proteolysis of B-Myc occurs via the ubiquitin-proteasome pathway. Finally, we found that overexpression of B-Myc significantly slows the growth of Rat 1a fibroblasts and COS cells suggesting B-Myc functions as an inhibitor of cellular proliferation.