c-Myc Phosphorylation Research Articles

Peroxynitrite promotes serine-62 phosphorylation-dependent stabilization of the oncoprotein c-Myc

Stabilization of c-Myc oncoprotein is dependent on post-translational modifications, especially its phosphorylation at serine-62 (S62), which enhances its tumorigenic potential. Herein we report that increase in intracellular superoxide induces phospho-stabilization and activation of c-Myc in cancer cells. Importantly, sustained phospho-S62 c-Myc was necessary for promoting superoxide dependent chemoresistance as non-phosphorylatable S62A c-Myc was insensitive to the redox impact when subjected to chemotherapeutic insults. This redox-dependent sustained S62 phosphorylation occurs through nitrative inhibition of phosphatase, PP2A, brought about by peroxynitrite, a reaction product of superoxide and nitric oxide. We identified a conserved tyrosine residue (Y238) in the c-Myc targeting subunit B56α of PP2A, which is selectively amenable to nitrative inhibition, further preventing holoenzyme assembly. In summary, we have established a novel mechanism wherein the pro-oxidant microenvironment stimulates a pro-survival milieu and reinforces tumor maintenance as a functional consequence of c-Myc activation through its sustained S62 phosphorylation via inhibition of phosphatase PP2A. Significance statementIncreased peroxynitrite signaling in tumors causes sustained S62 c-Myc phosphorylation by PP2A inhibition. This is critical to promoting c-Myc stabilization and activation which promotes chemoresistance and provides significant proliferative and growth advantages to osteosarcomas.

Polyphyllin I attenuates pressure over-load induced cardiac hypertrophy via inhibition of Wnt/β-catenin signaling pathway

AimsCardiac hypertrophy is one of most important risk factors for cardiovascular mortality. Activation of Wnt/β-catenin signaling pathway is acknowledged to be an important mechanism for pathogenesis of cardiac hypertrophy. Polyphyllin I (PPI), a component in the traditional Chinese medicinal herb, has shown anticancer effect partially via interruption of Wnt/β-catenin signaling pathway. Our aim was to test whether PPI attenuates cardiac hypertrophy. Materials and methodsAdult male C57BL/6J mice were subjected to either pressure overload generated by transverse aortic constriction (TAC) or sham surgery (control group). Angiotensin-II (Ang-II) was used to induce cardiomyocyte hypertrophy in vitro. PPI was intraperitoneally administrated daily for 4 weeks after TAC surgery and then cardiac function was determined by echocardiography and histological analysis was performed. Key findingsPPI significantly ameliorated cardiac dysfunction of mice subjected to TAC. Meanwhile, PPI attenuated TAC induced cardiac hypertrophy indicated by blunted increase in heart mass, cross section area of cardiomyocyte, cardiac fibrosis and expression of hypertrophic biomarkers ANP, BNP and β-MHC. In addition, PPI also ameliorated Ang-II induced cardiomyocyte hypertrophy in vitro. Importantly, PPI decreased protein expression of active β-catenin/total β-catenin, phosphorylation of GSK3β and Wnt target genes c-myc, c-jun, c-fos and cyclin D1 and its anti-hypertrophic effect was blunted by supplementation of Wnt 3a. SignificanceOur results suggest that PPI attenuates cardiac dysfunction and attenuate development of pressure over-load induced cardiac hypertrophic via suppressing Wnt/β-catenin signaling pathway. PPI might be a candidate drug for treatment of cardiac hypertrophy.

Protein Kinase D3 promotes the cell proliferation by activating the ERK1/c-MYC axis in breast cancer.

Breast cancer is the second leading death cause of cancer death for all women. Previous study suggested that Protein Kinase D3 (PRKD3) was involved in breast cancer progression. In addition, the protein level of PRKD3 in triple‐negative breast adenocarcinoma was higher than that in normal breast tissue. However, the oncogenic mechanisms of PRKD3 in breast cancer is not fully investigated. Multi‐omic data showed that ERK1/c‐MYC axis was identified as a major pivot in PRKD3‐mediated downstream pathways. Our study provided the evidence to support that the PRKD3/ERK1/c‐MYC pathway play an important role in breast cancer progression. We found that knocking out PRKD3 by performing CRISPR/Cas9 genome engineering technology suppressed phosphorylation of both ERK1 and c‐MYC but did not down‐regulate ERK1/2 expression or phosphorylation of ERK2. The inhibition of ERK1 and c‐MYC phosphorylation further led to the lower protein level of c‐MYC and then reduced the expression of the c‐MYC target genes in breast cancer cells. We also found that loss of PRKD3 reduced the rate of the cell proliferation in vitro and tumour growth in vivo, whereas ectopic (over)expression of PRKD3, ERK1 or c‐MYC in the PRKD3‐knockout breast cells reverse the suppression of the cell proliferation and tumour growth. Collectively, our data strongly suggested that PRKD3 likely promote the cell proliferation in the breast cancer cells by activating ERK1‐c‐MYC axis.

Phosphoenolpyruvate from Glycolysis and PEPCK Regulate Cancer Cell Fate by Altering Cytosolic Ca2.

Changes in phosphoenolpyruvate (PEP) concentrations secondary to variations in glucose availability can regulate calcium signaling in T cells as this metabolite potently inhibits the sarcoplasmic reticulum Ca2+/ATPase pump (SERCA). This regulation is critical to assert immune activation in the tumor as T cells and cancer cells compete for available nutrients. We examined here whether cytosolic calcium and the activation of downstream effector pathways important for tumor biology are influenced by the presence of glucose and/or cataplerosis through the phosphoenolpyruvate carboxykinase (PEPCK) pathway, as both are hypothesized to feed the PEP pool. Our data demonstrate that cellular PEP parallels extracellular glucose in two human colon carcinoma cell lines, HCT-116 and SW480. PEP correlated with cytosolic calcium and NFAT activity, together with transcriptional up-regulation of canonical targets PTGS2 and IL6 that was fully prevented by CsA pre-treatment. Similarly, loading the metabolite directly into the cell increased cytosolic calcium and NFAT activity. PEP-stirred cytosolic calcium was also responsible for the calmodulin (CaM) dependent phosphorylation of c-Myc at Ser62, resulting in increased activity, probably through enhanced stabilization of the protein. Protein expression of several c-Myc targets also correlated with PEP levels. Finally, the participation of PEPCK in this axis was interrogated as it should directly contribute to PEP through cataplerosis from TCA cycle intermediates, especially in glucose starvation conditions. Inhibition of PEPCK activity showed the expected regulation of PEP and calcium levels and consequential downstream modulation of NFAT and c-Myc activities. Collectively, these results suggest that glucose and PEPCK can regulate NFAT and c-Myc activities through their influence on the PEP/Ca2+ axis, advancing a role for PEP as a second messenger communicating metabolism, calcium cell signaling, and tumor biology.

Histone Demethylase KDM4C Stimulates the Proliferation of Prostate Cancer Cells via Activation of AKT and c-Myc.

Our three-dimensional organotypic culture revealed that human histone demethylase (KDM) 4C, a histone lysine demethylase, hindered the acini morphogenesis of RWPE-1 prostate cells, suggesting its potential oncogenic role. Knockdown (KD) of KDM4C suppressed cell proliferation, soft agar colony formation, and androgen receptor (AR) transcriptional activity in PCa cells as well as reduced tumor growth of human PCa cells in zebrafish xenotransplantation assay. Micro-Western array (MWA) analysis indicated that KD of KDM4C protein decreased the phosphorylation of AKT, c-Myc, AR, mTOR, PDK1, phospho-PDK1 S241, KDM8, and proteins involved in cell cycle regulators, while it increased the expression of PTEN. Fluorescent microscopy revealed that KDM4C co-localized with AR and c-Myc in the nuclei of PCa cells. Overexpression of either AKT or c-Myc rescued the suppressive effect of KDM4C KD on PCa cell proliferation. Echoing the above findings, the mRNA and protein expression of KDM4C was higher in human prostate tumor tissues as compared to adjacent normal prostate tissues, and higher KDM4C protein expression in prostate tumors correlated to higher protein expression level of AKT and c-Myc. In conclusion, KDM4C promotes the proliferation of PCa cells via activation of c-Myc and AKT.

CDK5 neutralizes the tumor suppressing effect of BIN1 via mediating phosphorylation of c-MYC at Ser-62 site in NSCLC

BackgroundBridging integrator 1 (BIN1) has showed outstanding tumor-suppressive potential via inhibiting c-MYC-mediated tumorigenesis. However, a frequent phosphorylation of c-MYC at Ser-62 site could block the BIN1/c-MYC interaction and limits the tumor-suppressive effect of BIN1. Cyclin-dependent kinase 5 (CDK5), a generally dysregulated protein in various carcinomas, can mediate c-MYC phosphorylation at Ser-62 site. However, whether the existence of CDK5 could block the BIN1/c-MYC interaction remains unclear.Materials and methodsThe expression of CDK5 and BIN1 in non-small cell lung cancer (NSCLC) cell lines were measured. CDK5 was knocked down and overexpressed in H460 and PC9 cells, respectively. CCK-8, wound healing and transwell were used to detect the proliferation, migration and invasion ability of NSCLC cells. Tumor-bearing nude mouse model was built with H460 cells. Dinaciclib was added to realize the effect of CDK5 inhibition in vivo. NSCLC and matched para-carcinoma specimens were collected from 153 patients who underwent radical operation. IHC was performed to determine the expression of CDK5 in the specimens. Kaplan–Meier analysis was used to analyze the correlation between the postoperative survival and CDK5 expression.ResultsCDK5 was highly expressed in H460 cells, and knockdown of CDK5 could restore the BIN1/c-MYC interaction. Meanwhile, low expression of CDK5 was observed in PC9 cells, and overexpression of CDK5 blocked the BIN1/c-MYC interaction. Consequently, the growth, migration, invasion and epithelial mesenchymal transition (EMT) ability of H460 and PC9 cells could be facilitated by CDK5. The addition of CDK5 inhibitor Dinaciclib significantly suppressed the tumorigenesis ability of NSCLC cells in tumor-bearing mouse model. Furthermore, high expression of CDK5, along with low expression of BIN1, could predict poor postoperative prognosis of NSCLC patients. The patients with high expression of CDK5 and low expression of BIN1 showed similar prognosis, indicating that CDK5 could neutralize the tumor suppressing effect of BIN1 in clinical situation.ConclusionsCDK5 blocked the interaction of BIN1 and c-MYC via promoting phosphorylation of c-MYC at ser-62 site, ultimately facilitated the progression of NSCLC.

Abstract 1310: Abrogation of KRas-addicted tumors by GSK3 suppression-mediated upregulation of β-catenin and c-myc

Abstract The significant involvement of mutant KRas in human cancer development underscores the need to develop approaches that disable mutant KRas-driven tumors. Targeting KRas directly is challenging, and such identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach. In this study, we discovered that glycogen synthase kinase 3 (GSK3) is required for the in vitro and in vivo growth and survival of human mutant KRas-dependent tumors but not for mutant KRas-independent tumors. Pharmacological inhibition with the GSK3 inhibitor SB as well as siRNA depletion of GSK3 lead to tumor suppression that is mediated at least in part by inhibition of the phosphorylation of the GSK3 substrates c-Myc on T58 and β-catenin on S33/S37/T41. CRISPR/Cas9 targeted knock out studies demonstrated that c-Myc and β-catenin upregulation mediate the antitumor activity of SB. Importantly, GSK3 blockade inhibits the in vivo growth of G12D, G12V, and G12C mutant KRas primary and metastatic patient-derived xenografts from pancreatic cancer patients who progressed on chemo- and radiation therapies. This discovery warrants advanced pre-clinical and clinical investigations of GSK3 inhibitors in mutant KRas-dependent cancers. Note: This abstract was not presented at the meeting. Citation Format: Aslamuzzaman Kazi, Shengyan Xiang, Hua Yang, Daniel Delitto, Jose Trevino, Rays H. Jiang, Muhammad Ayaz, Harshani Lawrence, Perry Kennedy, Said M. Sebti. Abrogation of KRas-addicted tumors by GSK3 suppression-mediated upregulation of β-catenin and c-myc [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1310.

Loss of FOXP3 and TSC1 Accelerates Prostate Cancer Progression through Synergistic Transcriptional and Posttranslational Regulation of c-MYC.

Although c-MYC and mTOR are frequently activated proteins in prostate cancer, any interaction between the two is largely untested. Here, we characterize the functional cross-talk between FOXP3-c-MYC and TSC1-mTOR signaling during tumor progression. Deletion of Tsc1 in mouse embryonic fibroblasts (MEF) decreased phosphorylation of c-MYC at threonine 58 (pT58) and increased phosphorylation at serine 62 (pS62), an observation validated in prostate cancer cells. Conversely, inhibition of mTOR increased pT58 but decreased pS62. Loss of both FOXP3 and TSC1 in prostate cancer cells synergistically enhanced c-MYC expression via regulation of c-Myc transcription and protein phosphorylation. This crosstalk between FOXP3 and TSC1 appeared to be mediated by both the mTOR-4EBP1-c-MYC and FOXP3-c-MYC pathways. In mice, Tsc1 and Foxp3 double deletions in the prostate led to prostate carcinomas at an early age; this did not occur in these mice with an added c-Myc deletion. In addition, we observed synergistic antitumor effects of cotreating mice with inhibitors of mTOR and c-MYC in prostate cancer cells and in Foxp3 and Tsc1 double-mutant mice. In human prostate cancer, loss of nuclear FOXP3 is often accompanied by low expression of TSC1. Because loss of FOXP3 transcriptionally induces c-Myc expression and loss of TSC1 activates mTOR signaling, these data suggest cross-talk between FOXP3-c-MYC and TSC1-mTOR signaling that converges on c-MYC to regulate tumor progression. Coadministration of c-MYC and mTOR inhibitors may overcome the resistance to mTOR inhibition commonly observed in prostate cancer cells. SIGNIFICANCE: These results establish the principle of a synergistic action of TSC1 and FOXP3 during prostate cancer progression and provide new therapeutic targets for patients who have prostate cancer with two signaling defects.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1413/F1.large.jpg.

GSK3 suppression upregulates \u03b2-catenin and c-Myc to abrogate KRas-dependent tumors

Mutant KRas is a significant driver of human oncogenesis and confers resistance to therapy, underscoring the need to develop approaches that disable mutant KRas-driven tumors. Because targeting KRas directly has proven difficult, identifying vulnerabilities specific for mutant KRas tumors is an important alternative approach. Here we show that glycogen synthase kinase 3 (GSK3) is required for the in vitro and in vivo growth and survival of human mutant KRas-dependent tumors but is dispensable for mutant KRas-independent tumors. Further, inhibiting phosphorylation of GSK3 substrates c-Myc on T58 and β-catenin on S33/S37/T41 and their subsequent upregulation contribute to the antitumor activity of GSK3 inhibition. Importantly, GSK3 blockade inhibits the in vivo growth of G12D, G12V, and G12C mutant KRas primary and metastatic patient-derived xenografts from pancreatic cancer patients who progressed on chemo- and radiation therapies. This discovery opens new avenues to target mutant KRas-dependent cancers.

Metabolic Reprogramming by Dual AKT/ERK Inhibition through Imipridones Elicits Unique Vulnerabilities in Glioblastoma.

Purpose: The goal of this study is to enhance the efficacy of imipridones, a novel class of AKT/ERK inhibitors that displayed limited therapeutic efficacy against glioblastoma (GBM).Experimental Design: Gene set enrichment, LC/MS, and extracellular flux analyses were used to determine the mechanism of action of novel imipridone compounds, ONC206 and ONC212. Orthotopic patient-derived xenografts were utilized to evaluate therapeutic potency.Results: Imipridones reduce the proliferation of patient-derived xenograft and stem-like glioblastoma cell cultures in vitro and in multiple xenograft models in vivo ONC212 displayed the highest potency. High levels of c-myc predict susceptibility to growth inhibition and apoptosis induction by imipridones and increased host survival in orthotopic patient-derived xenografts. As early as 1 hour, imipridones elicit on-target inhibition, followed by dephosphorylation of GSK3β at serine 9. GSK3β promotes phosphorylation of c-myc at threonine 58 and enhances its proteasomal degradation. Moreover, inhibition of c-myc by BRD4 antagonists sensitizes for imipridone-induced apoptosis in stem-like GBM cells in vitro and in vivo Imipridones affect energy metabolism by suppressing both glycolysis and oxidative phosphorylation, which is accompanied by a compensatory activation of the serine-one carbon-glycine (SOG) pathway, involving the transcription factor ATF4. Interference with the SOG pathway through novel inhibitors of PHGDH results in synergistic cell death induction in vitro and in vivo Conclusions: These results suggest that c-myc expression predicts therapeutic responses to imipridones and that imipridones lead to suppression of tumor cell energy metabolism, eliciting unique metabolic vulnerabilities that can be exploited for clinical relevant drug combination therapies. Clin Cancer Res; 24(21); 5392-406. ©2018 AACR.

Pim1 promotes cell proliferation and regulates glycolysis via interaction with MYC in ovarian cancer.

BackgroundOvarian cancer (OC) is the leading cause of death among women with gynecologic malignancies. Recent studies have highlighted the role of Pim1, which belongs to a group of constitutively activated serine/threonine kinases, in cancer development. However, the effect of Pim1 in OC is largely unclear.MethodsOC cell lines with Pim1 overexpression or knockdown were constructed with len-tivirus transduction. Cell Counting Kit-8, colony formation, glycolysis stress test and in vivo mice models were carried out to assess the effect of Pim1 on OC biological functions. Co-immunoprecipitation assay coupled with western blot were performed to explore the intrinsic mechanisms of Pim1 in OC. Bioinformatic analysis was then performed to evaluate the expression and prognostic value of Pim1.ResultsWe present the first evidence that silencing or overexpressing Pim1 can suppress or promote, respectively, OC cell proliferation. Furthermore, we demonstrated that Pim1 can significantly enhance glycolysis in OC cells. In vivo experiments further confirmed that knockdown of Pim1 inhibits the growth of tumors derived from the SKOV3 cell line. To search for the underlying molecular mechanism, we examined the effect of Pim1 on MYC, a pivotal gene in glycolysis, and observed that Pim1-mediated phosphorylation of c-Myc activated the expression of glycolysis-associated key genes such as PGK1 and LDHA. Moreover, we found that the Pim1 inhibitor SMI4a induced chemosensitization to cisplatin. Clinically, Pim1 was also overexpressed in OC and correlated with poor overall survival by bioinformatics analysis.ConclusionTogether, these results suggest that Pim1 contributes to proliferation and gly-colysis in OC via interaction with MYC and may serve as a potential target in the treatment of OC patients.

Fluorescent resonance energy transfer -based biosensor for detecting conformational changes of Pin1

Pin1, a peptidyl prolyl cis/trans isomerase (PPIase), regulates the activity and stability of various phosphorylated proteins. Pin1 consists of a PPIase domain and WW domain, both of which are required for the function of Pin1. However, how the behavior of these domains changes upon binding to phosphorylated proteins has not been analyzed. We created a Fluorescent Resonance Energy Transfer (FRET)-based biosensor “CPinY”, which is composed of Pin1 flanked by CFP and YFP, and analyzed the interaction between Pin1 and c-Myc. Our results indicated that the dual phosphorylation of c-Myc at Thr58 and Ser62 is essential for tight interaction with Pin1. Additionally, this interaction caused a significant conformational change in Pin1. Our CPinY biosensor also detected a novel type of inhibitor of Pin1 function. We believe that his biosensor will be a novel drug screening technology targeting Pin1.

Dihydroartemisinin triggers c-Myc proteolysis and inhibits protein kinase B/glycogen synthase kinase 3β pathway in T-cell lymphoma cells.

Recent studies have revealed a positive therapeutic effect of dihydroartemisinin (DHA) on tumor cells. However, the underlying mechanism of this has not yet been elucidated. The present study examined the potential therapeutic role and mechanism of DHA in T-cell lymphoma cells. It was revealed that DHA inhibited the proliferation of Jurkat and HuT-78 T-cell lymphoma cells in a concentration- and time-dependent manner. Furthermore, DHA reduced c-Myc protein expression at the transcriptional level, and induced the phosphorylation of c-Myc and the degradation of c-Myc oncoprotein levels. DHA treatment resulted in decreased phosphorylation of protein kinase B (Akt) and glycogen synthase 3β (GSK3β) in T-cell lymphoma cells. In addition, DHA treatment induced cell apoptosis, which was accompanied by an increased ratio of Bax/Bcl-2. Taken together, the results of the present study suggested that DHA may exert its antitumor role by accelerating c-Myc proteolysis and inhibiting the Akt/GSK3β pathway in T-cell lymphoma cells.

P-32 - The tumor environment stabilizes c-Myc oncoprotein through its sustained phosphorylation at serine 62, which promotes its oncogenic activity

We have previously demonstrated that redox homeostasis is governed by two key ROS – superoxide anion (O2•-) and hydrogen peroxide (H2O2). Tilting the balance to O2•- contributes to engaging cancers onto a pro-survival trajectory. Here, we present a singular event wherein redox modulation of c-Myc attributes cancer cells with survival advantages and elevates their chemoresistance potential. Phosphorylation of c-Myc correlates with its stabilization in cancers. While S62 phosphorylation increases c-Myc stability, T58 phosphorylation destabilizes it. Of particular interest is phosphatase PP2A, as we have shown that it is amenable to redox insults. We have now elucidated that B56α regulatory subunit of PP2A is amenable to redox modification preventing interaction of c-Myc bound B56α with PP2A-AC core enzyme. As assembly of PP2A holoenzyme is disrupted, c-Myc remains phosphorylated at S62. Hyperphosphorylated c-Myc cannot be recognized and engaged by the proteasome leading to increased protein stability and a moderate rise in activation of c-Myc targets. Thus, we have established a novel mechanism wherein the balance between O2•-:H2O2 ratio stimulates a pro-survival milieu and reinforces chemoresistance through redox modification of PP2A-B56α, consequentially resulting in stabilization of c-Myc.

Zinc finger protein 746 promotes colorectal cancer progression via c-Myc stability mediated by glycogen synthase kinase 3β and F-box and WD repeat domain-containing 7.

To elucidate the underlying oncogenic mechanism of zinc finger protein 746 (ZNF746), current study was conducted in colorectal cancers (CRCs). Herein, ZNF746 was overexpressed in HCT116, SW620, and SW480 cells, which was supported by CRC tissue microarray and TCGA analysis. Also, DNA microarray revealed the differentially expressed gene profile particularly related to cell cycle genes and c-Myc in ZNF746 depleted HCT116 cells. Furthermore, ZNF746 enhanced the stability of c-Myc via their direct binding through nuclear colocalization by immunoprecipitation and immunofluorescence, while ZNF746 and c-Myc exist mainly in nucleoplasm. Conversely, ZNF746 depletion attenuated phosphorylation of c-Myc (S62) and glycogen synthase kinase 3β (GSK3β) (S9) and also activated p-c-Myc (T58), which was reversed by GSK3 inhibitors such as SB-216763 and Enza. Also, c-Myc degradation by ZNF746 depletion was blocked by knockdown of F-box/WD repeat-containing protein 7 (FBW7) ubiquitin ligase or proteosomal inhibitor MG132. Additionally, the growth of ZNF746 depleted HCT116 cancer cells was retarded with decreased expression of ZNF746 and c-Myc. Overall, these findings suggest that ZNF746 promotes CRC progression via c-Myc stability mediated by GSK3 and FBW7.

LIN28B enhanced tumorigenesis in an autochthonous KRASG12V-driven lung carcinoma mouse model.

LIN28B is a RNA-binding protein regulating predominantly let-7 microRNAs with essential functions in inflammation, wound healing, embryonic stem cells, and cancer. LIN28B expression is associated with tumor initiation, progression, resistance, and poor outcome in several solid cancers, including lung cancer. However, the functional role of LIN28B, especially in non-small cell lung adenocarcinomas, remains elusive. Here, we investigated the effects of LIN28B expression on lung tumorigenesis using LIN28B transgenic overexpression in an autochthonous KRASG12V-driven mouse model. We found that LIN28B overexpression significantly increased the number of CD44+/CD326+ tumor cells, upregulated VEGF-A and miR-21 and promoted tumor angiogenesis and epithelial-to-mesenchymal transition (EMT) accompanied by enhanced AKT phosphorylation and nuclear translocation of c-MYC. Moreover, LIN28B accelerated tumor initiation and enhanced proliferation which led to a shortened overall survival. In addition, we analyzed lung adenocarcinomas of the Cancer Genome Atlas (TCGA) and found LIN28B expression in 24% of KRAS-mutated cases, which underscore the relevance of our model.

PIM1 mediates epithelial-mesenchymal transition by targeting Smads and c-Myc in the nucleus and potentiates clear-cell renal-cell carcinoma oncogenesis

Emerging evidence has shown that the PIM serine/threonine kinase family, including PIM1, PIM2 and PIM3, is associated with tumour progression towards metastasis. PIM1, an attractive molecular target, has been identified as a potential prognostic biomarker for haematological and epithelial malignancies. However, to date, the potential regulatory roles and molecular mechanisms by which PIM1 affects the development and progression of cancers, including clear-cell renal-cell carcinoma (ccRCC), remain largely unknown. Herein, we present the first evidence that PIM1 is aberrantly overexpressed in human ccRCC tissues and cell lines and positively correlated with human ccRCC progression. In our study, depletion of PIM1 attenuated ccRCC cell proliferation, colony formation, migration, invasion and angiogenesis, suggesting that PIM1 expression may be a cancer-promoting event in ccRCC. Mechanistically, we observed that PIM1 could interact with Smad2 or Smad3 in the nucleus and subsequently phosphorylate Smad2 and Smad3 to induce the expression of transcription factors, including ZEB1, ZEB2, Snail1, Snail2 and Twist, to promote epithelial-mesenchymal transition (EMT). In addition, PIM1-mediated phosphorylation of c-Myc activates the expression of the above transcription factors to synergistically promote EMT but does not activate Smads. Collectively, our results demonstrate that aberrant expression of PIM1 contributes to ccRCC development and progression. Moreover, our data reveal a potential molecular mechanism in which PIM1 mediates crosstalk between signalling pathways, including different Smad proteins and c-Myc, which target downstream transcription factors (ZEB1, ZEB2, Snail1, Snail2 and Twist) to trigger EMT. Together, our data suggest that PIM1 may be a potential therapeutic target for ccRCC patients.

PP2A Inactivation Mediated by PPP2R4 Haploinsufficiency Promotes Cancer Development.

Protein phosphatase 2A (PP2A) complexes counteract many oncogenic kinase pathways. In cancer cells, PP2A function can be compromised by several mechanisms, including sporadic mutations in its scaffolding A and regulatory B subunits or more frequently through overexpression of cellular PP2A inhibitors. Here, we identify a novel genetic mechanism by which PP2A function is recurrently affected in human cancer, involving haploinsufficiency of PPP2R4, a gene encoding the cellular PP2A activator PTPA. Notably, up to 70% of cancer patients showed a heterozygous deletion or missense mutations in PPP2R4 Cancer-associated PTPA mutants exhibited decreased abilities to bind the PP2A-C subunit or activate PP2A and failed to reverse the tumorigenic phenotype induced by PTPA suppression, indicating they function as null alleles. In Ppp2r4 gene-trapped (gt) mice showing residual PTPA expression, total PP2A activity and methylation were reduced, selectively affecting specific PP2A holoenzymes. Both PTPAgt/gt and PTPA+/gt mice showed higher rates of spontaneous tumors, mainly hematologic malignancies and hepatocellular adenomas and carcinomas. These tumors exhibited increased c-Myc phosphorylation and increased Wnt or Hedgehog signaling. We observed a significant reduction in lifespan in PTPA+/gt mice compared with wild-type mice. In addition, chemical-induced skin carcinogenesis was accelerated in PTPA+/gt compared with wild-type mice. Our results provide evidence for PPP2R4 as a haploinsufficient tumor suppressor gene, defining a high-penetrance genetic mechanism for PP2A inhibition in human cancer. Cancer Res; 77(24); 6825-37. ©2017 AACR.

Polo-like-kinase 1 (PLK-1) and c-myc inhibition with the dual kinase-bromodomain inhibitor volasertib in aggressive lymphomas.

Survival following anthracycline-based chemotherapy remains poor among patients with most T-cell lymphoproliferative disorders. This may be attributed, at least in part, to cell-autonomous mechanisms of chemotherapy resistance observed in these lymphomas, including the loss of important tumor suppressors and the activation of signaling cascades that culminate in the expression and activation of transcription factors promoting cell growth and survival. Therefore, the identification of novel therapeutic targets is needed. In an effort to identify novel tumor dependencies, we performed a loss-of-function screen targeting ≈500 kinases and identified polo-like kinase 1 (PLK-1). This kinase has been implicated in the molecular cross-talk with important oncogenes, including c-Myc, which is itself an attractive therapeutic target in subsets of T-cell lymphomas and in high-grade (“double hit”) diffuse large B-cell lymphomas. We demonstrate that PLK-1 expression is prevalent among these aggressive lymphomas and associated with c-myc expression. Importantly, PLK-1 inhibtion with the PLK-1 inhibitor volasertib significantly reduced downstream c-myc phosphorylation and impaired BRD4 binding to the c-myc gene, thus inhibiting c-myc transcription. Therefore, volasertib led to a nearly complete loss of c-myc expression in cell lines and tumor xenografts, induced apoptosis, and thus warrants further investigation in these aggressive lymphomas.

The tumor suppressor phosphatase PP2A-B56α regulates stemness and promotes the initiation of malignancies in a novel murine model.

Protein phosphatase 2A (PP2A) is a ubiquitously expressed Serine-Threonine phosphatase mediating 30–50% of protein phosphatase activity. PP2A functions as a heterotrimeric complex, with the B subunits directing target specificity to regulate the activity of many key pathways that control cellular phenotypes. PP2A-B56α has been shown to play a tumor suppressor role and to negatively control c-MYC stability and activity. Loss of B56α promotes cellular transformation, likely at least in part through its regulation of c-MYC. Here we report generation of a B56α hypomorph mouse with very low B56α expression that we used to study the physiologic activity of the PP2A-B56α phosphatase. The predominant phenotype we observed in mice with B56α deficiency in the whole body was spontaneous skin lesion formation with hyperproliferation of the epidermis, hair follicles and sebaceous glands. Increased levels of c-MYC phosphorylation on Serine62 and c-MYC activity were observed in the skin lesions of the B56αhm/hm mice. B56α deficiency was found to increase the number of skin stem cells, and consistent with this, papilloma initiation was accelerated in a carcinogenesis model. Further analysis of additional tissues revealed increased inflammation in spleen, liver, lung, and intestinal lymph nodes as well as in the skin lesions, resembling elevated extramedullary hematopoiesis phenotypes in the B56αhm/hm mice. We also observed an increase in the clonogenicity of bone marrow stem cells in B56αhm/hm mice. Overall, this model suggests that B56α is important for stem cells to maintain homeostasis and that B56α loss leading to increased activity of important oncogenes, including c-MYC, can result in aberrant cell growth and increased stem cells that can contribute to the initiation of malignancy.