c-Myc Protein Stability Research Articles

TRAF3 regulates Pim2 and c-Myc-mediated B cell survival

Abstract Tumor necrosis factor receptor associated factor 3 (TRAF3) is an adaptor protein that plays an important role in B cell homeostasis. TRAF3 deletions or inactivating mutations are common in human B cell malignancies. In human and mouse B cells, TRAF3 negatively regulates signaling of several receptors important for B cell homeostasis (including BAFFR, CD40, and IL-6R). B cell specific TRAF3-deficient (B-Traf3−/−) mice have enlarged spleens and lymph nodes and an increased frequency of B cell tumors as they age. B cells from B-Traf3−/− mice have markedly increased survival and autoantibody production. The mechanisms by which TRAF3 regulates B cell survival are not yet well defined. We found that TRAF3-deficient mouse B cells and human multiple myeloma cell lines with low expression of TRAF3 had increased RNA and protein expression of the pro-survival kinase Pim2. Additionally, TRAF3-deficient mouse B cells had increased phosphorylation of Pim2 targets BAD, p70S6K, and 4E-BP1. TRAF3-deficient B cells showed increased survival relative to TRAF3-sufficient B cells after treatment with the Pim inhibitors SGI-1776 and TP-3654. Pim2 can promote survival by phosphorylating and stabilizing the oncogenic transcription factor c-Myc. Loss of TRAF3 led to transcription-independent c-Myc protein elevation that was dependent on Pim2. K48 polyubiquitination of c-Myc was decreased in the absence of TRAF3, consistent with increased c-myc protein stability. TRAF3 deficiency correlated with B cell resistance to apoptosis mediated by the c-Myc transcription inhibitor JQ1. Our results show that TRAF3 promotes B cell survival by regulating expression of and interaction between Pim2 and c-Myc.

Disruption of the c-Myc/miR-200b-3p/PRDX2 regulatory loop enhances tumor metastasis and chemotherapeutic resistance in colorectal cancer

BackgroundMetastasis is a major threat to colorectal cancer (CRC) patients. We have reported that peroxiredoxin-2 (PRDX2) is associated with CRC invasion and metastasis. However, the mechanisms regulating PRDX2 expression remain unclear. We investigate whether microRNAs (miRNAs) regulate PRDX2 expression in CRC progression.MethodsQuantitative real-time polymerase chain reaction (qPCR) was used to measure microRNA-200b-3p (miR-200b-3p) expression. Immunohistochemistry (IHC) was performed to detect c-Myc and PRDX2 protein levels in CRC tissue samples (n = 97). Western blot was used to quantify PRDX2, c-Myc, AKT2/GSK3β pathway-associated proteins and epithelial-mesenchymal transition (EMT)-related proteins in CRC cells. Luciferase reporter assays were used to analyze the interaction between miR-200b-3p and 3′untranslated region (3′UTR) of PRDX2 mRNA and AKT2 mRNA as well as c-Myc and the miR-200b-3p promoter. Chromatin immunoprecipitation (ChIP) assay was used to evaluate binding of c-Myc to the miR-200b-3p promoter. Invasive assay and metastatic model were used to assess invasive and metastatic capacities of CRC cells in vitro and in vivo. Moreover, drug-induced apoptosis was measured by flow cytometry.ResultsWe found that miR-200b-3p was significantly downregulated, whereas c-Myc and PRDX2 were upregulated in metastatic CRC cells and CRC tissues compared to their counterparts. An inverse correlation existed between c-Myc and miR-200b-3p, and between miR-200b-3p and PRDX2. We also found that PRDX2 was a target of miR-200b-3p. Importantly, overexpression of nontargetable PRDX2 eliminated the suppressive effects of miR-200b-3p on proliferation, invasion, EMT, chemotherapeutic resistance and metastasis of CRC cells. Moreover, c-Myc bound to the promoter of miR-200b-3p and repressed its transcription. In turn, miR-200b-3p disrupted the stability of c-Myc protein by inducing c-Myc protein threonine 58 (T58) phosphorylation and serine 62 (S62) dephosphorylation via AKT2/GSK3β pathway.ConclusionsOur findings reveal that the c-Myc/miR-200b/PRDX2 loop regulates CRC progression and its disruption enhances tumor metastasis and chemotherapeutic resistance in CRC.

Hypoxic Stress Decreases c-Myc Protein Stability in Cardiac Progenitor Cells Inducing Quiescence and Compromising Their Proliferative and Vasculogenic Potential

Cardiac progenitor cells (CPCs) have been shown to promote cardiac regeneration and improve heart function. However, evidence suggests that their regenerative capacity may be limited in conditions of severe hypoxia. Elucidating the mechanisms involved in CPC protection against hypoxic stress is essential to maximize their cardioprotective and therapeutic potential. We investigated the effects of hypoxic stress on CPCs and found significant reduction in proliferation and impairment of vasculogenesis, which were associated with induction of quiescence, as indicated by accumulation of cells in the G0-phase of the cell cycle and growth recovery when cells were returned to normoxia. Induction of quiescence was associated with a decrease in the expression of c-Myc through mechanisms involving protein degradation and upregulation of p21. Inhibition of c-Myc mimicked the effects of severe hypoxia on CPC proliferation, also triggering quiescence. Surprisingly, these effects did not involve changes in p21 expression, indicating that other hypoxia-activated factors may induce p21 in CPCs. Our results suggest that hypoxic stress compromises CPC function by inducing quiescence in part through downregulation of c-Myc. In addition, we found that c-Myc is required to preserve CPC growth, suggesting that modulation of pathways downstream of it may re-activate CPC regenerative potential under ischemic conditions.

Macrocyclic peptides decrease c-Myc protein levels and reduce prostate cancer cell growth

ABSTRACTThe oncoprotein c-Myc is often overexpressed in cancer cells, and the stability of this protein has major significance in deciding the fate of a cell. Thus, targeting c-Myc levels is an attractive approach for developing therapeutic agents for cancer treatment. In this study, we report the anti-cancer activity of the macrocyclic peptides [D-Trp]CJ-15,208 (cyclo[Phe-D-Pro-Phe-D-Trp]) and the natural product CJ-15,208 (cyclo[Phe-D-Pro-Phe-Trp]). [D-Trp]CJ-15,208 reduced c-Myc protein levels in prostate cancer cells and decreased cell proliferation with IC50 values ranging from 2.0 to 16 µM in multiple PC cell lines. [D-Trp]CJ-15,208 induced early and late apoptosis in PC-3 cells following 48 hours treatment, and growth arrest in the G2 cell cycle phase following both 24 and 48 hours treatment. Down regulation of c-Myc in PC-3 cells resulted in loss of sensitivity to [D-Trp]CJ-15,208 treatment, while overexpression of c-Myc in HEK-293 cells imparted sensitivity of these cells to [D-Trp]CJ-15,208 treatment. This macrocyclic tetrapeptide also regulated PP2A by reducing the levels of its phosphorylated form which regulates the stability of cellular c-Myc protein. Thus [D-Trp]CJ-15,208 represents a new lead compound for the potential development of an effective treatment of prostate cancer.

Abstract 1335: Deubiquitinase OTUD6B isoforms are important regulators of growth and proliferation

Abstract Deubiquitinases (DUBs) are increasingly linked to the regulation of fundamental processes in normal and cancer cells, including DNA replication and repair, programmed cell death, and oncogenes and tumor suppressors signaling. Here evidence is presented that the deubiquitinase OTUD6B regulates protein synthesis in non-small cell lung cancer (NSCLC) cells, operating downstream from mTORC1. OTUD6B associates with the protein synthesis initiation complex and modifies components of the 48S preinitiation complex. The two main OTUD6B splicing isoforms seem to regulate protein synthesis in opposing fashions: the long OTUD6B-1 isoform is inhibitory, while the short OTUD6B-2 isoform stimulates protein synthesis. These properties affect NSCLC cell proliferation, since OTUD6B-1 represses DNA synthesis while OTUD6B-2 promotes it. Mutational analysis and downstream mediators suggest that the two OTUD6B isoforms modify different cellular targets. OTUD6B-2 influences the expression of cyclin D1 by promoting its translation while regulating (directly or indirectly) c-Myc protein stability. This phenomenon appears to have clinical relevance as NSCLC cells and human tumor specimens have a reduced OTUD6B-1/OTUD6B-2 mRNA ratio compared to normal samples. The global OTUD6B expression level does not change significantly between non-neoplastic and malignant tissues, suggesting that modifications of splicing factors during the process of transformation are responsible for this isoform switch. Because protein synthesis inhibition is a viable treatment strategy for NSCLC, these data indicate that OTUD6B isoform 2, being specifically linked to NSCLC growth, represents an attractive, novel therapeutic target and potential biomarker for early diagnosis of malignant NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Maurizio Bocchetta, Clodia Osipo, Anna Sobol. Deubiquitinase OTUD6B isoforms are important regulators of growth and proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1335. doi:10.1158/1538-7445.AM2017-1335

SPOP regulates prostate epithelial cell proliferation and promotes ubiquitination and turnover of c-MYC oncoprotein.

The E3 ubiquitin ligase adaptor speckle-type POZ protein (SPOP) is frequently dysregulated in prostate adenocarcinoma (PC), via either somatic mutations or mRNA downregulation, suggesting an important tumor suppressor function. To examine its physiologic role in the prostate epithelium in vivo, we generated mice with prostate-specific biallelic ablation of Spop. These mice exhibited increased prostate mass, prostate epithelial cell proliferation, and expression of c-MYC protein compared to littermate controls, and eventually developed prostatic intraepithelial neoplasia (PIN). We found that SPOPWT can physically interact with c-MYC protein and, upon exogenous expression in vitro, can promote c-MYC ubiquitination and degradation. This effect was attenuated in PC cells by introducing PC-associated SPOP mutants or upon knockdown of SPOP via short-hairpin-RNA, suggesting that SPOP inactivation directly increases c-MYC protein levels. Gene set enrichment analysis revealed enrichment of Myc-induced genes in transcriptomic signatures associated with SPOPMT. Likewise, we observed strong inverse correlation between c-MYC activity and SPOP mRNA levels in two independent PC patient cohorts. The core SPOPMT;MYCHigh transcriptomic response, defined by the overlap between the SPOPMT and c-MYC transcriptomic programs, was also associated with inferior clinical outcome in human PCs. Finally, the organoid-forming capacity of Spop-null murine prostate cells was more sensitive to c-MYC inhibition than that of Spop-WT cells, suggesting that c-MYC upregulation functionally contributes to the proliferative phenotype of Spop knock-out prostates. Taken together, our data highlight SPOP as an important regulator of luminal epithelial cell proliferation and c-MYC expression in prostate physiology, identify c-MYC as a novel bona fide SPOP substrate, and help explain the frequent inactivation of SPOP in human PC. We propose SPOPMT–induced stabilization of c-MYC protein as a novel mechanism that can increase total c-MYC levels in PC cells, in addition to amplification of c-MYC locus.

Deubiquitinase OTUD6B Isoforms Are Important Regulators of Growth and Proliferation.

Because protein synthesis inhibition is a viable treatment strategy for NSCLC, these data indicate that OTUD6B isoform 2, being specifically linked to NSCLC growth, represents an attractive, novel therapeutic target and potential biomarker for early diagnosis of malignant NSCLC. Mol Cancer Res; 15(2); 117-27. ©2016 AACR.

LncRNA-MIF, a c-Myc-activated long non-coding RNA, suppresses glycolysis by promoting Fbxw7-mediated c-Myc degradation.

The c-Myc proto-oncogene is activated in more than half of all human cancers. However, the precise regulation of c-Myc protein stability is unknown. Here, we show that the lncRNA-MIF (c-Myc inhibitory factor), a c-Myc-induced long non-coding RNA, is a competing endogenous RNA for miR-586 and attenuates the inhibitory effect of miR-586 on Fbxw7, an E3 ligase for c-Myc, leading to increased Fbxw7 expression and subsequent c-Myc degradation. Our data reveal the existence of a feedback loop between c-Myc and lncRNA-MIF, through which c-Myc protein stability is finely controlled. Additionally, we show that the lncRNA-MIF inhibits aerobic glycolysis and tumorigenesis by suppressing c-Myc and miR-586.

A natural small molecule, catechol, induces c-Myc degradation by directly targeting ERK2 in lung cancer.

Various carcinogens induce EGFR/RAS/MAPK signaling, which is critical in the development of lung cancer. In particular, constitutive activation of extracellular signal-regulated kinase 2 (ERK2) is observed in many lung cancer patients, and therefore developing compounds capable of targeting ERK2 in lung carcinogenesis could be beneficial. We examined the therapeutic effect of catechol in lung cancer treatment. Catechol suppressed anchorage-independent growth of murine KP2 and human H460 lung cancer cell lines in a dose-dependent manner. Catechol inhibited ERK2 kinase activity in vitro, and its direct binding to the ERK2 active site was confirmed by X-ray crystallography. Phosphorylation of c-Myc, a substrate of ERK2, was decreased in catechol-treated lung cancer cells and resulted in reduced protein stability and subsequent down-regulation of total c-Myc. Treatment with catechol induced G1 phase arrest in lung cancer cells and decreased protein expression related to G1-S progression. In addition, we showed that catechol inhibited the growth of both allograft and xenograft lung cancer tumors in vivo. In summary, catechol exerted inhibitory effects on the ERK2/c-Myc signaling axis to reduce lung cancer tumor growth in vitro and in vivo, including a preclinical patient-derived xenograft (PDX) model. These findings suggest that catechol, a natural small molecule, possesses potential as a novel therapeutic agent against lung carcinogenesis in future clinical approaches.

Loss of TRAF3 Promotes a Premalignant Metabolic State in B cells.

Abstract TRAF3 is a signaling protein that acts as a tumor suppressor in B cells. B cell conditional TRAF3−/− mice display a remarkable expansion of B cell compartments and parenchymal B cell infiltration, attributed to enhanced B cell survival without increased proliferation. When aged, these mice develop lymphomas, and TRAF3 mutations are common in human B cell lymphoma and multiple myeloma. The mechanism of how loss of TRAF3 promotes B cell survival phenotype is not well understood; this particular role for TRAF3 is not seen in other immune cell types. We find that loss of TRAF3 leads to increased uptake of glucose in B cells in vitro and in vivo. This is accompanied by increased oxidative phosphorylation and glycolysis. In the absence of TRAF3, expression of Glut1 and Hexokinase II, two molecules important for glucose metabolism, is increased. Treatment of B cells with Glut1 inhibitor or the competitive glycolysis inhibitor 2-DG attenuates cell survival, and B cell viability is also substantially reduced in a glucose-free environment. Mechanistically, loss of TRAF3 leads to the induction of Pim2 kinase. Pim2 in turn promotes the protein stability of the oncogenic transcription factor c-Myc, known to drive expression of glycolytic genes. Our study shows that B cell deficiency of TRAF3 is sufficient for altered metabolic programming, consistent with a premalignant abnormal survival phenotype.

C-MYC responds to glucose deprivation in a cell-type-dependent manner

Metabolic reprogramming supports cancer cells’ demands for rapid proliferation and growth. Previous work shows that oncogenes, such as MYC, hypoxia-inducible factor 1 (HIF1), have a central role in driving metabolic reprogramming. A lot of metabolic enzymes, which are deregulated in most cancer cells, are the targets of these oncogenes. However, whether metabolic change affects these oncogenes is still unclear. Here we show that glucose deprivation (GD) affects c-MYC protein levels in a cell-type-dependent manner regardless of P53 mutation status. GD dephosphorylates and then decreases c-MYC protein stability through PI3K signaling pathway in HeLa cells, but not in MDA-MB-231 cells. Role of c-MYC in sensitivity of GD also varies with cell types. c-MYC-mediated glutamine metabolism partially improves the sensitivity of GD in MDA-MB-231 cells. Our results reveal that the heterogeneity of cancer cells in response to metabolic stress should be considered in metabolic therapy for cancer.

Galectin-3 modulates the EGFR signalling-mediated regulation of Sox2 expression via c-Myc in lung cancer.

Galectin-3 is a ubiquitous lectin exerting multiple cellular functions such as RNA splicing, protein trafficking and apoptosis. Its expression is positively correlated with the poor prognosis in lung cancer patients. Galectin-3 can promote cancer progression through its effects on cell proliferation, cell survival or cancer metastasis. However, the role of galectin-3 in the regulation of cancer stem-like cells (CSCs) is still unclear. Here, we investigated the hypothesis that galectin-3 might regulate lung CSCs via the EGF receptor (EGFR) signaling pathway. In our study, galectin-3 facilitated EGFR activation and enhanced the sphere formation activity of lung cancer cells. Furthermore, galectin-3 promoted Sox2 expression in an EGFR activation-dependent manner; importantly, forced expression of Sox2 blunted the effect of galectin-3 knockdown on lung cancer sphere formation ability. These results suggest that galectin-3 promotes EGFR activation leading to the upregulation of Sox2 expression and lung CSCs properties. Moreover, we showed that the carbohydrate-binding activity of galectin-3 was important for the regulation of EGFR activation, Sox2 expression and sphere formation. We have recently reported that c-Myc is a transcriptional activator of Sox2. We further found that galectin-3 enhanced c-Myc protein stability leading to increased c-Myc binding to the Sox2 gene promoter. We also examined the effect of the stemness factors, Oct4, Nanog and Sox2 on the expression of galectin-3. We found that Oct4 enhanced galectin-3 expression. Our results together suggest that galectin-3 enhances lung cancer stemness through the EGFR/c-Myc/Sox2 axis; Oct4, in turn, promotes galectin-3 expression, forming a positive regulatory loop in lung CSCs.

SCP1 regulates c-Myc stability and functions through dephosphorylating c-Myc Ser62.

Serine 62 (Ser62) phosphorylation affects the c-Myc protein stability in cancer cells. However, the mechanism for dephosphorylating c-Myc is not well understood. In this study, we identified carboxyl-terminal domain RNA polymerase II polypeptide A small phosphatase 1 (SCP1) as a novel phosphatase specifically dephosphorylating c-Myc Ser62. Ectopically expressed SCP1 strongly dephosphorylated c-Myc Ser62, destabilized c-Myc protein and suppressed c-Myc transcriptional activity. Knockdown of SCP1 increased the c-Myc protein levels in liver cancer cells. SCP1 interacted with c-Myc both in vivo and in vitro. In addition, Ser245 at the C-terminus of SCP1 was essential for its phosphatase activity towards c-Myc. Functionally, SCP1 negatively regulated the cancer cell proliferation. Collectively, our findings indicate that SCP1 is a potential tumor suppressor for liver cancers through dephosphorylating c-Myc Ser62.

The nucleolar ubiquitin-specific protease USP36 deubiquitinates and stabilizes c-Myc

c-Myc protein stability and activity are tightly regulated by the ubiquitin-proteasome system. Aberrant stabilization of c-Myc contributes to many human cancers. c-Myc is ubiquitinated by SCF(Fbw7) (a SKP1-cullin-1-F-box complex that contains the F-box and WD repeat domain-containing 7, Fbw7, as the F-box protein) and several other ubiquitin ligases, whereas it is deubiquitinated and stabilized by ubiquitin-specific protease (USP) 28. The bulk of c-Myc degradation appears to occur in the nucleolus. However, whether c-Myc is regulated by deubiquitination in the nucleolus is not known. Here, we report that the nucleolar deubiquitinating enzyme USP36 is a novel c-Myc deubiquitinase. USP36 interacts with and deubiquitinates c-Myc in cells and in vitro, leading to the stabilization of c-Myc. This USP36 regulation of c-Myc occurs in the nucleolus. Interestingly, USP36 interacts with the nucleolar Fbw7γ but not the nucleoplasmic Fbw7α. However, it abolished c-Myc degradation mediated both by Fbw7γ and by Fbw7α. Consistently, knockdown of USP36 reduces the levels of c-Myc and suppresses cell proliferation. We further show that USP36 itself is a c-Myc target gene, suggesting that USP36 and c-Myc form a positive feedback regulatory loop. High expression levels of USP36 are found in a subset of human breast and lung cancers. Altogether, these results identified USP36 as a crucial and bono fide deubiquitinating enzyme controlling c-Myc's nucleolar degradation pathway.

Regulation of c-Myc protein stability by proteasome activator REGγ.

c-Myc is a key transcriptional factor that has a prominent role in cell growth, differentiation and tumor development. Its protein levels are tightly controlled by ubiquitin-proteasome pathway and frequently deregulated in various cancers. Here, we report that the 11S proteasomal activator REGγ is a novel regulator of c-Myc abundance in cells. We showed that overexpression of wild-type REGγ, but not inactive mutants including N151Y and G250S, significantly promoted the degradation of c-Myc. Depletion of REGγ markedly increased the protein stability of c-Myc. REGγ interacts with the C-terminal region of c-Myc and regulates c-Myc protein turnover. Functionally, REGγ negatively regulates c-Myc-mediated cell proliferation. Interestingly, depletion of the Drosophila Reg homolog (dReg) in developing wings induced the upregulation of Drosophila Myc, which contributes to cell death. Collectively, these results suggest that REGγ proteasome has a conserved role in the regulation of Myc abundance in both mammalian cells and Drosophila.

The Long Non-Coding RNA PCAT-1 Promotes Prostate Cancer Cell Proliferation through cMyc

Long non-coding RNAs (lncRNAs) represent an emerging layer of cancer biology, contributing to tumor proliferation, invasion, and metastasis. Here, we describe a role for the oncogenic lncRNA PCAT-1 in prostate cancer proliferation through cMyc. We find that PCAT-1–mediated proliferation is dependent on cMyc protein stabilization, and using expression profiling, we observed that cMyc is required for a subset of PCAT-1–induced expression changes. The PCAT-1–cMyc relationship is mediated through the post-transcriptional activity of the MYC 3′ untranslated region, and we characterize a role for PCAT-1 in the disruption of MYC-targeting microRNAs. To further elucidate a role for post-transcriptional regulation, we demonstrate that targeting PCAT-1 with miR-3667-3p, which does not target MYC, is able to reverse the stabilization of cMyc by PCAT-1. This work establishes a basis for the oncogenic role of PCAT-1 in cancer cell proliferation and is the first study to implicate lncRNAs in the regulation of cMyc in prostate cancer.

Abstract 3515: Increased TSH signaling by overexpressed thyrotrophin receptor is essential for B-RafV600E induced thyroid carcinogenesis

Abstract B-RafV600E oncogene mutation occurs most commonly in papillary thyroid cancer (PTC), and is associated with tumor initiation. However, a genetic modification by B-RafV600E in the thyrocytes results in oncogene induced senescence (OIS). In the present study, therefore, we attempted to evaluate the factors involved in the senescence overcome program in PTC. First of all, we observed downregulation of p-Erk1/2 and upregulation of dual specific phosphatase 6 (DUSP6) in the PTC with B-RafV600E mutation. In B-RafV600E expressing thyrocytes, although DUSP6 mRNA was upregulated, its protein and phosphatase activity were similar to those of normal thyrocytes. However, DUSP6 activity was reactivated when ROS was downregulated by thyroid stimulating hormone (TSH). Moreover, upregulation of MnSOD by TSH was observed in thyrocytes and PTC. Although serum TSH was not increased, its receptor was markedly upregulated in PTC with B-RafV600E. TSH together with DUSP6 inhibited B-RafV600E induced senescence in the thyrocytes, and reactivated Ras/Akt/GSK3β resulted in c-Myc protein stabilization by inhibition of its degradation. These observations led us to conclude that increased TSH signaling by overexpression of TSH receptor is essential for B-RafV600E induced papillary thyroid carcinogenesis. Citation Format: Young Hwa Kim, Yong Won Choi, Jang-Hee Kim, Tae Jun Park. Increased TSH signaling by overexpressed thyrotrophin receptor is essential for B-RafV600E induced thyroid carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3515. doi:10.1158/1538-7445.AM2014-3515

ROCK has a crucial role in regulating prostate tumor growth through interaction with c-Myc

Rho-associated kinase (ROCK) has an essential role in governing cell morphology and motility, and increased ROCK activity contributes to cancer cell invasion and metastasis. Burgeoning data suggest that ROCK is also involved in the growth regulation of tumor cells. However, thus far, the molecular mechanisms responsible for ROCK-governed tumor cell growth have not been clearly elucidated. Here we showed that inhibition of ROCK kinase activity, either by a selective ROCK inhibitor Y27632 or by specific ROCK small interfering RNA (siRNA) molecules, attenuated not only motility but also the proliferation of PC3 prostate cancer cells in vitro and in vivo. Importantly, mechanistic investigation revealed that ROCK endowed cancer cells with tumorigenic capability, mainly by targeting c-Myc. ROCK could increase the transcriptional activity of c-Myc by promoting c-Myc protein stability, and ROCK inhibition reduced c-Myc-mediated expression of mRNA targets (such as HSPC111) and microRNA targets (such as miR-17-92 cluster). We provided evidence demonstrating that ROCK1 directly interacted with and phosphorylated c-Myc, resulting in stabilization of the protein and activation of its transcriptional activity. Suppression of ROCK-c-Myc downstream molecules, such as c-Myc-regulated miR-17, also impaired tumor cell growth in vitro and in vivo. In addition, c-Myc was shown to exert a positive feedback regulation on ROCK by increasing RhoA mRNA expression. Therefore, inhibition of ROCK and its stimulated signaling might prove to be a promising strategy for restraining tumor progression in prostate cancer.

BET inhibition as a single or combined therapeutic approach in primary paediatric B-precursor acute lymphoblastic leukaemia.

Paediatric B-precursor ALL is a highly curable disease, however, treatment resistance in some patients and the long-term toxic effects of current therapies pose the need for more targeted therapeutic approaches. We addressed the cytotoxic effect of JQ1, a highly selective inhibitor against the transcriptional regulators, bromodomain and extra-terminal (BET) family of proteins, in paediatric ALL. We showed a potent in vitro cytotoxic response of a panel of primary ALL to JQ1, independent of their prognostic features but dependent on high MYC expression and coupled with transcriptional downregulation of multiple pro-survival pathways. In agreement with earlier studies, JQ1 induced cell cycle arrest. Here we show that BET inhibition also reduced c-Myc protein stability and suppressed progression of DNA replication forks in ALL cells. Consistent with c-Myc depletion and downregulation of pro-survival pathways JQ1 sensitised primary ALL samples to the classic ALL therapeutic agent dexamethasone. Finally, we demonstrated that JQ1 reduces ALL growth in ALL xenograft models, both as a single agent and in combination with dexamethasone. We conclude that targeting BET proteins should be considered as a new therapeutic strategy for the treatment of paediatric ALL and particularly those cases that exhibit suboptimal responses to standard treatment.

Abstract 510: Activation of PP2A by SET antagonism destabilizes c-Myc and inhibits breast cancer growth.

Abstract Background: Protein phosphorylation plays an important role in regulating cellular signaling and deregulation of this process can contribute to many aberrant cellular behaviors, including those that contribute to cancer. It has been demonstrated that many oncogenes and proto-oncogenes are kinases, and that uncontrolled elevated levels of their activity lead to tumorigenic transformation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed heterotrimeric Serine-Threonine (S/T) phosphatase that is responsible for 30-50% of cellular S/T protein phosphatase activity. PP2A regulates numerous signaling pathways, including stem cell self-renewal, proliferation, differentiation, migration, cell survival, and apoptosis. The PP2A holoenzyme has 3 subunits: a catalytic (C) subunit, a structural (A) subunit, and a variable regulatory (B) subunit that directs the PP2A holoenzyme to a specific substrates. PP2A loss of activity is important for cell transformation and it has been shown that B56α, B56γ, and PR72/PR130 are involved in human cell transformation. Importantly, PP2A-B56α negatively regulates the c-Myc transcription factor that is a potent oncoprotein, by decreasing its protein stability. The oncoprotein SET is an endogenous inhibitor of PP2A that is overexpressed in many cancers. The role that SET plays in the development, progression and metastasis of breast cancer has not been reported. Methods: SET expression was analyzed by qRT-PCR in human breast tumors and compared to that in matched adjacent normal. SET was knocked down in MDA-MB-231 cells and tested for xenografts experiments. SET was also stably knocked down in MDA-MB-231 cells using two different shRNAs. Clonal cell lines with reduced SET expression were assayed for cell proliferation and soft agar in vitro. PP2A activation was measured following treatment with OP449, a SET inhibitor, in breast cancer cells lines. OP449’s effect on the growth and tumorigenic potential of these cells was also measured. Lysates of cells treated with OP449 have been used to analyze the effect of SET inhibition on c-Myc protein stability and promoter binding. Results: Our data suggest that SET, is overexpressed in 50-60% of breast cancers. Treatment with OP449 and knockdown of SET reduces tumorigenic potential of some breast cancer cell lines in vitro and in vivo. OP449 treatment or SET knockdown also reduces S62-phosphorylated Myc levels and OP449 decreases Myc target gene promoter binding. OP449 also inhibits tumor growth in a Myc-driven transgenic mouse model of HER2-dependent breast cancer. Conclusion: Activating PP2A through SET inhibition reduces tumorigenic potential of some breast cancer cell lines. This can be caused in part by decreasing Myc protein levels. Because of the ubiquitous expression of PP2A in eukaryotic cells and its important role in blocking transformation, we propose that activating PP2A can be a novel approach for breast cancer therapy. Citation Format: Mahnaz Janghorban, Jessica Oddo, Tyler Risom, Amy Farrell, Michael P. Vitek, Rosalie C. Sears, Dale J. Christensen. Activation of PP2A by SET antagonism destabilizes c-Myc and inhibits breast cancer growth. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 510. doi:10.1158/1538-7445.AM2013-510