Upregulation Of c-Myc Research Articles

Abstract A123: Reversal of glucocorticoid resistance in pediatric acute lymphoblastic leukemia is dependent on restoring BIM expression

Abstract Introduction: Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Glucocorticoids (e.g. dexamethasone, prednisolone) form a critical component of chemotherapy regimens for pediatric ALL and initial resistance to glucocorticoid therapy is predictive of poor outcome. Acquired resistance to glucocorticoids is common at relapse and is disparate compared with other chemotherapeutic drugs used in the treatment of pediatric ALL. Glucocorticoids induce apoptosis in lymphoid cells which is mediated through the glucocorticoid receptor (GR). Resistance to glucocorticoids in pediatric ALL is associated with upregulation of the oncogene C-MYC and failure to induce the proapoptotic gene BIM. The purpose of this study was to identify small molecules that are able to reverse glucocorticoid resistance in pediatric ALL patient-derived xenografts (PDXs). Methods: A 40,000 compound high-throughput screening (HTS) campaign was carried out to identify compounds that potentiated the cytotoxicity of dexamethasone against a pediatric ALL PDX derived from a patient with aggressive and fatal relapse. Cytotoxicity assays were carried out by alamarBlue assay. Gene expression analysis was carried out using Illumina microarrays. mRNA and protein analysis was carried out by RT-PCR and immunoblotting, respectively. Results: The HTS campaign identified a novel glucocorticoid sensitiser, 2-((4,5-dihydro-1H-imidazol-2-yl)thio)-N-isopropyl-N-phenylacetamide (GCS-3). The sensitizing effect was specific to glucocorticoids, with synergy observed when GCS-3 was combined with dexamethasone or prednisolone, but not with other chemotherapeutic drugs. Synergy was observed in a range of dexamethasone-resistant and -sensitive xenografts representative of B-ALL, T-ALL, early T-cell precursor ALL and Philadelphia chromosome positive ALL. GCS-3 required a functional GR to sensitise ALL xenografts to dexamethasone. Gene expression analysis showed that GCS-3 in the presence of dexamethasone upregulated a previously published gene expression signature induced by glucocorticoid treatment of B-ALL patients (Rhein et al. Leukemia, 2007, 21:897-905). Moreover, GCS-3 in combination with dexamethasone significantly downregulated C-MYC and upregulated BIM expression in a glucocorticoid-resistant ALL xenograft. Treatment with the GR antagonist, RU486, significantly abrogated the cytotoxic effects of the GCS-3/dexamethasone combination. Although RU486 increased C-MYC expression in the dexamethasone treated cells, there was no increase in C-MYC expression in the GCS-3/dexamethasone combination. RU486 suppressed the marked induction of BIM following treatment with the GCS-3/dexamethasone combination, indicating that BIM upregulation is critical for the cytotoxicity of the GCS-3/dexamethasone combination. The GCS-3/dexamethasone combination significantly increased binding of the GR to an intronic binding site at the BIM locus which is associated with BIM transcription and glucocorticoid sensitivity in pediatric ALL. Conclusion: This study describes the potential of the novel glucocorticoid sensitizing agent, GCS-3, as a biological tool to understand glucocorticoid action and resistance. Citation Format: Richard B Lock, Cara E Toscan, Duohui Jing, Chelsea Mayoh. Reversal of glucocorticoid resistance in pediatric acute lymphoblastic leukemia is dependent on restoring BIM expression [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A123. doi:10.1158/1535-7163.TARG-19-A123

Cecal Tumorigenesis in Aryl Hydrocarbon Receptor–Deficient Mice Depends on Cecum-Specific Mitogen-Activated Protein Kinase Pathway Activation and Inflammation

The aryl hydrocarbon receptor (AhR) is a transcription factor known as a dioxin receptor. Recently, Ahr-/- mice were revealed to develop cecal tumors with inflammation and Wnt/β-catenin pathway activation. However, whether β-catenin degradation is AhR dependent remains unclear. To determine whether other signaling pathways function in Ahr-/- cecal tumorigenesis, we investigated histologic characteristics of the tumors and cytokine/chemokine production in tumors and Ahr-/- peritoneal macrophages. AhR expression was also assessed in human colorectal carcinomas. Of the 28 Ahr-/- mice, 10 developed cecal lesions by 50 weeks of age, an incidence significantly lower than previously reported. Cecal lesions of Ahr-/- mice developed from serrated hyperplasia to adenoma/dysplasia-like neoplasia with enhanced proliferation. Macrophage and neutrophil infiltration into the lesions was also observed early in serrated hyperplasia, although adjacent mucosa was devoid of inflammation. Il1b, Il6, Ccl2, and Cxcl5 were up-regulated at lesion sites, whereas only IL-6 production increased in Ahr-/- peritoneal macrophages after lipopolysaccharide+ATP stimulation. Neither Myc (alias c-myc) up-regulation nor β-catenin nuclear translocation was observed, unlike previously reported. Interestingly, enhanced phosphorylation of extracellular signal-regulated kinase, Src, and epidermal growth factor receptor and Amphiregulin up-regulation at Ahr-/- lesion sites were detected. In human serrated lesions, however, AhR expression in epithelial cells was up-regulated despite morphologic similarity to Ahr-/- cecal lesions. Our results suggest novel mechanisms underlying Ahr-/- cecal tumorigenesis, depending primarily on cecum-specific mitogen-activated protein kinase pathway activation and inflammation.

Gfi1 Upregulates c-Myc Expression and Promotes c-Myc-Driven Cell Proliferation

Gfi1 is a zinc-finger transcriptional repressor that plays an important role in hematopoiesis. When aberrantly activated, Gfi1 may function as a weak oncoprotein in the lymphoid system, but collaborate strongly with c-Myc in lymphomagenesis. c-Myc is a transcription factor that is frequently activated in human cancers including leukemia and lymphoma mainly due to its overexpression as a result of gene amplifications and chromosomal translocations. c-Myc overexpression may also result from stabilization of c-Myc protein, which is highly unstable and rapidly degraded through the ubiquitin-proteasome pathway. The mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis is incompletely understood. c-Myc activates gene expression by forming a heterodimeric complex with the partner protein Max, but may also repress target genes through interaction with transcription factor Miz-1. We previously showed that Gfi1 indirectly interacts with c-Myc through Miz-1 and collaborates with c-Myc to repress CDK inhibitors p21Cip1 and p15Ink4B. In this study, we show that Gfi1 augmented the level of c-Myc protein transiently expressed in Hela cells and the levels of MycER fusion protein stably expressed in the mouse pro-B Ba/F3 and myeloid 32D cells. The C-terminal ZF domains of Gfi1, but not its transcriptional repression and DNA binding activities, were required for c-Myc upregulation. Notably, although Miz-1 has been shown to stabilize c-Myc protein, the expression of c-Myc V394D mutant, which is defective in Miz-1 interaction, was still upregulated by Gfi1, suggesting that Gfi1-mediated c-Myc upregulation was independent of Miz-1 interaction. We further show that Gfi1 overexpression led to reduced polyubiquitination and increased stability of c-Myc protein. Interestingly, the levels of endogenous c-Myc mRNA and protein were augmented upon induction of Gfi1 expression in Ba/F3 and Burkitt lymphoma Ramos cells transduced with the doxycycline-inducible Gfi1 lentiviral construct, but reduced in Gfi1-knocked down human leukemic HL60 and U937 cells. Additionally, targeted deletion of Gfi1 resulted in reduced c-Myc expression in mouse lineage negative bone marrow cells, which was associated with a decline in the expression of c-Myc-activated target genes. The oncogenic potential of Myc derives from its ability to stimulate cell proliferation. Our results demonstrate that inducible expression of Gfi1 in Ba/F3 cells expressing MycER promoted Myc-driven cell cycle progression and proliferation. Thus, in addition to its role in c-Myc-mediated transcriptional repression, Gfi1 upregulates c-Myc expression at both mRNA and protein levels, leading to enhanced expression of c-Myc-activated genes and augmented cell proliferation driven by c-Myc. Together, these data may reveal a novel mechanism by which Gfi1 collaborates with c-Myc in lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

GFI1-Dependent SGPP1 Repression Promotes Growth and Survival of Myeloma Cells

Multiple myeloma (MM) remains incurable for the vast majority of patients due to emergence of drug resistant clones and mutations inducing drug resistant relapses. This is despite the fact that new therapies have greatly improved progression-free and overall survival for patients with standard risk myeloma. We recently found that the transcriptional repressor GFI1 is increased in bone marrow stromal cells of MM patients (MM-BMSC) where it causes prolonged suppression of osteoblast differentiation, and in CD138+ cells from MM patients, where GFI1 levels significantly correlate with disease progression. We also found that GFI1 overexpression (o/e) enhances MM cell growth and partially confers resistance to proteasome inhibitors in vitro as well as enhances tumor growth and osteoclastogenesis in vivo. Although the mechanisms responsible for these GFI1 effects in p53wt MM cells were p53-dependent, we found that GFI1 is also essential for MM cell survival regardless of their p53 status. The p53-independent mechanisms responsible for Gfi1 effects on MM cells growth and survival of are unknown. Sphingolipids are bioactive lipids that can control MM cell growth and survival. The balance between the levels of Sphingosine-1-phosphate (S1P) and its metabolic precursors ceramide (Cer) and sphingosine (SPH) form a rheostat that determines whether a cell proliferates or dies. We hypothesized that GFI1 represses SGPP1, the enzyme responsible for degrading S1P via salvage and recycling of sphingosine into long-chain ceramides. This repression changes the intracellular sphingolipid profile (Cer/S1P/SPH ratio) to maintain c-Myc upregulation in a protein phosphatase 2 (PP2A)-dependent manner, thus promoting growth and survival of MM cells. To test this hypothesis we measured S1P, SPH and Cer levels by mass spectrometry (LC-MS/MS). LC-MS/MS evaluation showed that bone marrow plasma of MM patients has significant higher levels of S1P when compared to normal donors. Moreover, intracellular S1P levels of MM.1S GFI1 o/e cells were also significantly higher as compared to those of MM.1S empty vector controls. Knock-down (KD) of Gfi1 in MM.1S cells strikingly increased SGPP1 and decreased SphK1 (the enzyme which catalyzes S1P production) mRNA levels, while GFI1 o/e cells had the opposite effect. We found that CD138+ cells isolated from MM patients expressed elevated levels of SphK1 mRNA compared to MGUS patients, and that SphK1 protein levels directly correlate with GFI1 levels in MM patient CD138+ cells and cell lines (r= 0.527). We also detected an indirect correlation (r= -0.961) between GFI1 and SGPP1 mRNA levels in five different MM cell lines. These results indicate a GFI1-dependent imbalance of the enzymes regulating S1P production. Further, KD GFI1 and SphK1 inhibition (5 μM SK1I) had a profound inhibitory effect on c-Myc protein levels and induced caspase 3 activation as detected by Western blotting, while GFI1 o/e cells had significant higher levels of c-Myc and were more resistant to SK1I treatment. Exogenous ceramide (10 μM Cer 16:0) treatment or SphK1 inhibition (5 μM SK1I), both treatments known to trigger intracellular ceramide production, significantly inhibited MM cell viability (measured by AlamarBlue), regardless of their p53 status (MM1.S p53 +/+ and KMS-11 p53 -/-). This inhibition of MM viability was GFI1-dependent, as GFI1 o/e cells were significantly more resistant to ceramide-induced cell death, which was PP2A dependent, as PP2A inhibition with okadaic acid (OA) restored it. MM.1S cells with KD of GFI1 exhibited significantly higher PP2A activity then control cells, supporting our observation that c-Myc modulation by GFI1 is PP2A-dependent. c-Myc protein levels were significantly decreased in MM.1S control cells treated with ceramide and rescued by OA pre-treatment; thus mimicking the effects of changing GFI1 levels and I2PP2A (the PP2A endogenous inhibitor) and confirms that PP2A mediates the effects of GFI1 on c-Myc. Taken together, our results show that GFI1 acts as a key regulator of MM growth and survival, at least partially through modulation of SGPP1. Therefore, targeting lipid metabolism to modulate the levels of specific bioactive lipid components that can modify cancer cell fate may provide a new and attractive therapeutic approach for MM. Disclosures Roodman: Amgen: Membership on an entity's Board of Directors or advisory committees.

Regulation of apoptosis in the ischemic penumbra in the first day post-stroke.

Regulation of apoptosis in the ischemic penumbra in the first day post-stroke.

The neural ELAVL protein HuB enhances endogenous proto-oncogene activation

The cytoplasmic distribution of the HuR/ELAVL1 (embryonic lethal abnormal vision 1) protein is recognized as an important prognostic factor of malignant tumors. However, the previous study suggests that exogenous over-expression of HuR is not sufficient for nuclear export. Conversely, the predominantly cytosolic distribution of neuron-specific human ELAV members, including HuB/ELAVL2, HuC/ELAVL3, and HuD/ELAVL4, has been reported. In the present study, we demonstrated the expression of HuB in several types of cancer cells, but expression of HuC and HuD was not observed. In addition, our results indicated that HuR and HuB formed a complex in the cytosolic fraction of cancer cells via the RRM3 region. Ectopic expression of HuB was capable of initiating the cytosolic translocation of HuR from the nucleus to the cytosol. Furthermore, HuB-transduced cancer cells displayed significant nuclear export of HuR, with quantitative PCR experiments revealing the simultaneous upregulation of HIF-1α, c-Fos, c-MYC, and Ets2 basal mRNA expression. Phorbol 12-myristate 13-acetate (PMA)-stimulated HuB-transduced cells demonstrated significantly enhanced activation of endogenous c-Fos and CREB dependent cascades. Finally, co-transfection of HuB with the E1 region of type 5 human adenovirus significantly enhanced E1 transformation activities but that of HuR with the E1 region did not. Collectively, our findings suggest that the neural Hu family protein HuB plays a major role in the activation of memory-related proto-oncogenes.

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.

Abstract 4436: Ubiquitin-specific peptidase 24 decreases c-Myc expression to inhibit lung cancer formation

Abstract Lung cancer is a malignant lung tumor characterized by high incidence and motility. c-Myc is a transcription factor that plays an important role in oncogenic activation to influence cellular metabolism and proliferation. Ubiquitin-specific peptidase 24 (USP24) is one of the members of USPs family to regulate protein ubiquitination. However, it is lack of evidence to uncover the role of USP24 in cancer formation. In this study, we found that cell proliferation was significantly increased by USP24 knockdown in A549 cells as accompanied by the increase of c-Myc protein. In particular, the RNA level and protein stability of c-Myc were not affected by USP24 knockdown, suggesting that USP24 affects c-Myc through regulating the translational pathway. Furthermore, c-Myc participated in USP24-knocked down-induced proliferation, not migration. To further clarify the mechanism underlying the regulation of c-Myc by USP24, we investigated whether mTOR pathway is involved in c-Myc upregulation by USP24 knockdown. Herein rapamycin, the mTOR inhibitor, prevented c-Myc upregulation caused by USP24 knockdown, suggesting that USP24 affect the protein level of c-Myc by regulating mTOR-mediated translation. We found that the downstream signaling of mTOR is activated by USP24 knockdown, including S6K and RPS6 phosphorylation. In addition, USP24 knockdown induced the binding of activated PRS6 to 5′-UTR of c-Myc, indicating that USP24 regulates c-Myc through mTOR-mediated translation. However, USP24 did not affect the phosphorylation of mTOR at Ser-2448, implying that other phosphorylation residue(s) within mTOR might be regulated by USP24. Since mTOR pathway is critical for many cancers formation, understanding the role of USP24 in regulating mTOR pathway will contribute to develop strategies to fight lung cancer. Citation Format: Jan-Jong Hung, Wen-Chang Chang. Ubiquitin-specific peptidase 24 decreases c-Myc expression to inhibit lung cancer formation [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 4436.

Abstract 3673: The BMI1/Sp1/IGF1R pathway mediates pemetrexed resistance in non-small cell lung cancer cells

Abstract Background: Lung cancer is the leading cause of cancer death in Taiwan and worldwide and non-small cell lung cancers (NSCLCs) account for more than 85% of lung cancers. Pemetrexed is approved for several steps of nonsquamous NSCLC therapy but drug resistance often occurs in long-term treatment. Cancer stem cells (CSCs) are considered as the rare subpopulation of cancer cells with the features of tumor initiation, drug resistance, and metastatic capability. The signaling pathway and potential targets contribute to the maintenance of pemetrexed-resistant cancer stem cell remain unknown. Methods: To investigate the mechanism of pemetrexed resistance in lung cancers, the pemetrexed-resistant A549 cells were established (A400). The sphere-formation assay, western blot analysis and real-time qPCR were employed to identify cancer stem cells and study their properties. Result: We first discovered that the pemetrexed-resistant A549 cells (called A400) contained more CSC activity than the parental A549 cells including the increased number of tumorspheres and the upregulation of BMI1, c-Myc, Sox2 and p-IGF1Rtyr1165/1166 expression. The treatment of PTC-209, a BMI1 inhibitor, displayed a greater inhibitory effect in cell proliferation in A400 than A549 cells and it suppressed tumorsphere formation capability of A400 cells. In addition, PTC-209 treatment downregulated the expression of IGF1R, p-IGF1Rtyr1165/1166, and Sp1, a positive regulatory transcriptional factor in IGF1R expression, in A400 cells. We also found that Sp1 was increased in A400 when compared to parental A549 cells. With the treatment of Sp1 inhibitor in A400 cells, the expression of IGF1R was suppressed without changing BMI1 level. Finally, we examined the therapeutic effect of BMI1 inhibition in vivo and resulted found that intraperitoneal administration of PTC-209 enhanced the pemetrexed efficacy in NOD/SCID mice harboring A400 xenograft tumors. Conclusion: Our data reveal that BMI1/Sp1/IGF1R pathway is upregulated in pemetrexed resistant NSCLCs and targeting BMI1 could be a therapeutic strategy in NSCLCs when pemetrexed resistance occurs. Citation Format: Wen-Ling Wang, Peng-Ju Chien, Wen-Wei Chang, Bing-Yen Wang. The BMI1/Sp1/IGF1R pathway mediates pemetrexed resistance in non-small cell lung cancer cells [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 3673.

Abstract 4307: RAGE is the key receptor for LPA in lung and mammary cancer progression and metastasis

Abstract Lung and breast cancer signaling are very complex; receptor for advanced glycation end products (RAGE) is highly expressed in various cancers and is correlated with poorer outcome in lung and other cancers. It is an immunoglobulin like membrane receptor shows binding with various ligands sharing structural likeness such as amyloid beta, advanced glycation end products (AGEs), S100B proteins, amphoterin and HMGB1 and mediates numerous inflammatory and cellular events including diseases including various cancers. LPA a RAGE ligand is a biologically active phospholipid involved in cell proliferation, migration and survival via its different G protein couple receptors (GPCRs). LPA and its signalling pathways have been implicated in different pathological conditions of lung tissue, including inflammation, fibrosis and cancer activating various signalling pathways. Here, we have identified RAGE-LPA signaling mechanisms showing AKT, STAT3 and ERK as downstream signaling pathways gets activated upon LPA treatment via RAGE receptor in both lung and breast cancer cells. RAGE knockdown with multiple independent shRNAs in lung and breast cancer cells led to decreased transwell invasion and soft agar colony formation, and proliferation upon LPA stimulation. Our RAGE silencing study in lung and breast cancer cells upon LPA treatment showed upregulation of specific oncogenes Cyclin D1, c-Myc and VEGF via RAGE receptor. RAGE association with LPA induced epithelial to mesenchymal transition (EMT) in both lung and breast cancer cells have also been observed by the RAGE attenuation studies by examining the expression of some EMT markers. The physiological significance of LPA-RAGE axis was exposed by our xenograft study by nude mice intraperitoneal injections of control and RAGE inhibited lung and mammary tumors generated by LPA doses in alternative days, results the formation of tumor foci on peritoneum wall via RAGE receptor. This study provides novel therapeutic insights as the use of RAGE blocking antibodies and antagonists or competitive inhibitors of LPA against RAGE might be useful of breast cancer treatment. This study also suggests that RAGE and LPA can be used as biomarkers for the early detection of lung and breast cancers. Citation Format: Nitish Jangde, Rashmi Ray, Vivek Rai. RAGE is the key receptor for LPA in lung and mammary cancer progression and metastasis [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 4307.

Abstract 4493: SF3B1 mutation promotes c-Myc protein stability through aberrant splicing and downregulation of PP2A B56α subunit in chronic lymphocytic leukemia

Abstract SF3B1 mutation, which occurs in 10-20% of chronic lymphocytic leukemia (CLL) patients, is associated with faster disease progression, shorter overall survival and fludarabine resistance. Mutant SF3B1 utilizes cryptic 3′ splice sites to generate aberrantly spliced mRNAs, half of which may undergo non-sense mediated mRNA decay (NMD), leading to downregulation of the affected genes. To identify potential tumor suppressor genes that are aberrantly spliced and downregulated by mutant SF3B1, we performed RNA-sequencing analysis of SF3B1-mutated primary CLL cells in the presence or absence of cycloheximide (CHX), a translation inhibitor known to inhibit NMD. Our analysis identified PPP2R5A, encoding one of the regulatory subunits of protein phosphatase-2A (PP2A B56α), as one of the key genes that were most consistently affected by an aberrantly spliced junction, and significantly downregulated (>2 fold) in CLL patient samples with various SF3B1 hotspot mutations. Splicing analyses by RT-PCR and Sanger sequencing confirmed that a 13-nucleotides sequence was added before the 5th exon of PPP2R5A via alternative splicing in CLL patients with the SF3B1 mutation. Due to this 13-nucleotides addition, three consecutive premature stop codons were created by frameshift at a position more than 55 bases upstream of an exon-exon junction, which is a canonical feature of the mRNAs degraded through NMD. The down-regulation of PPP2R5A was confirmed by quantitative PCR (p<0.05) and immunoblot at both mRNA and protein levels in CLL patients with SF3B1 mutation as compared to CLL patients without SF3B1 mutation and normal CD19+ B cell samples. Furthermore, overexpression of mutant SF3B1 in K562 and HEK 293 cells resulted in the same aberrant splicing pattern observed in CLL patients with SF3B1 mutation and downregulation of PPP2R5A (p<0.05). Treatment with CHX resulted in an increased abundance of alternatively spliced PPP2R5A transcript, suggesting that the aberrant PPP2R5A transcript was degraded by NMD. PP2A, when incorporated with PPP2R5A as the regulatory subunit, dephosphorylates c-Myc at S62, and results in the degradation of c-Myc. Here, we demonstrated that downregulation of PPP2R5A by mutant SF3B1 led to upregulation of c-Myc through promoting c-Myc protein stability in SF3B1K700E overexpressing cells when compared to SF3B1wildtype overexpressing cells. Knockdown PPP2R5A in MEC1 CLL cells by siRNA also significantly upregulated c-Myc at the protein level and upregulated mRNA expression of c-Myc downstream genes NCL and ODC1. Overall, our results demonstrate that SF3B1 mutation causes aberrant splicing and downregulation of tumor suppressor protein PP2A B56α in vitro and in vivo, leading to upregulation of c-Myc. Citation Format: Qimei Han, Libin Deng, Shuo Tu, Lirong Pei, Jeong-Hyeon Choi, Victor Jin, Huidong Shi. SF3B1 mutation promotes c-Myc protein stability through aberrant splicing and downregulation of PP2A B56α subunit in chronic lymphocytic leukemia [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 4493.

C-MYC empowers transcription and productive splicing of the oncogenic splicing factor Sam68 in cancer

The splicing factor Sam68 is upregulated in many human cancers, including prostate cancer (PCa) where it promotes cell proliferation and survival. Nevertheless, in spite of its frequent upregulation in cancer, the mechanism(s) underlying its expression are largely unknown. Herein, bioinformatics analyses identified the promoter region of the Sam68 gene (KHDRBS1) and the proto-oncogenic transcription factor c-MYC as a key regulator of Sam68 expression. Upregulation of Sam68 and c-MYC correlate in PCa patients. c-MYC directly binds to and activates the Sam68 promoter. Furthermore, c-MYC affects productive splicing of the nascent Sam68 transcript by modulating the transcriptional elongation rate within the gene. Importantly, c-MYC-dependent expression of Sam68 is under the tight control of external cues, such as androgens and/or mitogens. These findings uncover an unexpected coordination of transcription and splicing of Sam68 by c-MYC, which may represent a key step in PCa tumorigenesis.

Spalt-Like Transcription Factor 1 (SALL1) Gene Expression Inhibits Cell Proliferation and Cell Migration of Human Glioma Cells Through the Wnt/β-Catenin Signaling Pathway.

BackgroundThe spalt-like transcription factor 1 (SALL1) gene is a member of the Krüppel-associated box-containing zinc finger proteins (KRAB-ZFPs) and has been shown to modulate the onset and progression of human tumors. This study aimed to investigate the regulatory effects and mechanisms of SALL1 gene expression in human glioblastoma and glioma cells and tissue samples from patients with cerebral glioma.Material/MethodsThe human glioblastoma cell lines, LN229, U87-MG, U-251, U343, and the Hs683 glioma cell line were studied. The cell counting kit-8 (CCK-8) assay, cell cycle assay, wound-healing assay, transwell assay, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to evaluate cell proliferation, cell migration, and the cell cycle and expression of SALL1. Expression of SALL1 mRNA in 120 samples of cerebral glioma and 20 samples of normal brain were studied. Overall survival data from patients with cerebral glioma were analyzed.ResultsSALL1 expression was down-regulated in human glioblastoma and glioma cells and in cerebral glioma tissues. Down-regulation of SALL1 expression was associated with reduced overall survival. Overexpression of SALL1 was associated with inhibition of cell proliferation associated with cell cycle arrest at the G0/G1 phase. SALL1 inhibited cell migration by preventing epithelial-mesenchymal transition (EMT) and down-regulating the expression of stem cell markers. Reduced levels of β-catenin and downregulation of c-Myc and cyclin D1 and upregulation of p21and p27 expression were associated with SALL1 expression.ConclusionsIn human glioblastoma cells and cerebral glioma tissues, SALL1 acted as a tumor suppressor gene by inhibiting Wnt/β-catenin signaling.

EFFECT OF BEE AND SCORPION VENOMS ON PROSTATE CANCER IN VITRO STUDY

Prostate cancer (PCa) is the commonest diagnosed visceral malignancy among males worldwide. Recent studies have shown that bee venom target the cancer cells without effect on the normal cells by activating PC3 with oxidative substances against prostate cancer. The induction of the apoptotic cell death through several cancer cell death mechanisms, includes activation of up regulation of c-myc, and c-met genes and down regulation of Casp-7, that are important to induce anticancer. Scorpion venom is a potential bio-source and therapeutic tool to design potent drugs against variety of diseases. It has been used as medicinal and therapeutic tool since ancient times in China. Scorpion venom consists of neurotoxins, salts, low molecular weight peptides and different enzymes with high molecular activities. These activities make them novel therapeutic agents. The results revealed low cytotoxicity of bee venom and satusporin while scorpion venom showed the highest level of cytotoxicity. On the other hand, apoptosis was shown in all tested subjects at pre G1, while cell cycle arrest was different, which was at G1 by scorpion venom and at G2M by both bee venom and satusporin. Apoptosis was at late stage by scorpion venom and satusporin drug while at an early stage by bee venom as Casp-7, C-myc, C-met showed up regulation, the highest level shown in scorpion venom. This study explains that bee venom and scorpion have a potencial therapeutic effect on prostate cancer.

Connexin 43 Loss Triggers Cell Cycle Entry and Invasion in Non-Neoplastic Breast Epithelium: A Role for Noncanonical Wnt Signaling.

(1) Background: The expression of connexin 43 (Cx43) is disrupted in breast cancer, and re-expression of this protein in human breast cancer cell lines leads to decreased proliferation and invasiveness, suggesting a tumor suppressive role. This study aims to investigate the role of Cx43 in proliferation and invasion starting from non-neoplastic breast epithelium. (2) Methods: Nontumorigenic human mammary epithelial HMT-3522 S1 cells and Cx43 shRNA-transfected counterparts were cultured under 2-dimensional (2-D) and 3-D conditions. (3) Results: Silencing Cx43 induced mislocalization of β-catenin and Scrib from apicolateral membrane domains in glandular structures or acini formed in 3-D culture, suggesting the loss of apical polarity. Cell cycle entry and proliferation were enhanced, concomitantly with c-Myc and cyclin D1 upregulation, while no detectable activation of Wnt/β-catenin signaling was observed. Motility and invasion were also triggered and were associated with altered acinar morphology and activation of ERK1/2 and Rho GTPase signaling, which acts downstream of the noncanonical Wnt pathway. The invasion of Cx43-shRNA S1 cells was observed only under permissive stiffness of the extracellular matrix (ECM). (4) Conclusion: Our results suggest that Cx43 controls proliferation and invasion in the normal mammary epithelium in part by regulating noncanonical Wnt signaling.

Down-Regulation of Nfatc1 Suppresses Proliferation, Migration, Invasion, and Warburg Effect in Prostate Cancer Cells.

BackgroundProstate cancer (PCa), accounting for 28% of all male cancer cases, is the second leading cause of cancer-related death among men. NFATc1, belonging to the NFAT family, is overexpressed in PCa and is correlated with the risk of recurrence after radical prostatectomy.Material/MethodsIn the present study, the expression of NFATc, c-myc, and PKM2 in PCa cells was regulated by lentiviruses and then detected by real-time PCR and Western blot analysis. Further, proliferation, invasion, and migration assays were performed. The glucose consumption and lactate production were assessed by biochemical detection.ResultsWe found that NFATc1 down-regulation significantly suppressed the proliferation and Warburg effect of PCa cells, concurrent with a decrease of c-myc and PKM2 expression. Likewise, the abilities of migration and invasion were also inhibited in NFATc1-silenced PCa cells. In addition, NFATc1 down-regulation-induced inhibition of cell proliferation, migration, invasion, and Warburg effect were counteracted by up-regulation of c-myc or PKM2. The expression of PKM2 was positively regulated by NFATc1 and c-myc expression.ConclusionsThese results indicate that NFATc1 down-regulation can suppress the proliferation, Warburg effect, and migration and invasion abilities of PCa cells, probably by regulating c-myc and PKM2 expression. NFATc1 may be a potential therapeutic target for PCa and could be used as a diagnosis or prognosis indicator of PCa.

MiR-378 promoted cell proliferation and inhibited apoptosis by enhanced stem cell properties in chronic myeloid leukemia K562 cells

Dysregulation of miR-378 has been found in diverse types of tumors as well as in leukemia. The role of miR-378 in chronic myeloid leukemia (CML) remains unclear. The aim of the study was to reveal the potential effects of miR-378 in the pathological process and progress in CML. Our results showed general level of miR-378 was significant higher in CML patients compared to controls. Overexpression of miR-378 dramatically promoted cell proliferation and drug-resistance. Additionally, apoptosis was inhibited in cells transfected with miR-378. More and bigger stem cell sphere formation was observed in miR-378 transfected cells. Furthermore, enhanced expression of miR-378 was associated with upregulation of stem-cell makers OCT4 and c-Myc. Further study validated that miR-378 inhibited the expression of FUS1. Our research demonstrated the oncogenic nature of miR-378 in CML, and might contribute to the progress of CML.

Activation of Epithelial-Mesenchymal Transition and Altered β-Catenin Signaling in a Novel Indian Colorectal Carcinoma Cell Line.

Colorectal cancer is the third major cause of cancer-related mortality worldwide. The upward trend in incidence and mortality rates, poor sensitivity to conventional therapies and a dearth of early diagnostic parameters pose a huge challenge in the management of colorectal cancer in India. Due to the high level of genetic diversity present in the Indian population, unraveling the genetic contributions toward pathogenesis is key for understanding the etiology of colorectal cancer and in reversing this trend. We have established a novel cell line, MBC02, from an Indian colorectal cancer patient and have carried out extensive molecular characterization to unravel the pathological alterations in this cell line. In-depth molecular analysis of MBC02 revealed suppression of E-cadherin expression, concomitant with overexpression of EMT related molecules, which manifested in the form of highly migratory and invasive cells. Loss of membrane-tethered E-cadherin released β-catenin from the adherens junction resulting in its cytoplasmic and nuclear accumulation and consequently, upregulation of c-Myc. MBC02 also showed dramatic transcriptional upregulation of β-catenin. Remarkably, we observed significantly elevated proteasome activity that perhaps co-evolved to compensate for the unnaturally high mRNA level of β-catenin to regulate the increased protein load. In addition, there was substantial misregulation of other clinically relevant signaling pathways that have clinical relevance in the pathogenesis of colorectal cancer. Our findings pave the way toward understanding the molecular differences that could define pathogenesis in cancers originating in the Indian population.

An integrated analysis to predict micro-RNAs targeting both stemness and metastasis in breast cancer stem cells.

Several evidences support the idea that a small population of tumour cells representing self‐renewal potential are involved in initiation, maintenance, metastasis, and outcomes of cancer therapy. Elucidation of microRNAs/genes regulatory networks activated in cancer stem cells (CSCs) is necessary for the identification of new targets for cancer therapy. The aim of the present study was to predict the miRNAs pattern, which can target both metastasis and self‐renewal pathways using integration of literature and data mining. For this purpose, mammospheres derived from MCF‐7, MDA‐MB231, and MDA‐MB468 were used as breast CSCs model. They had higher migration, invasion, and colony formation potential, with increasing in stemness‐ and EMT‐related genes expression. Our results determined that miR‐204, ‐200c, ‐34a, and ‐10b contemporarily could target both self‐renewal and EMT pathways. This core regulatory of miRNAs could increase the survival rate of breast invasive carcinoma via up‐regulation of OCT4, SOX2, KLF4, c‐MYC, NOTCH1, SNAI1, ZEB1, and CDH2 and down‐regulation of CDH1. The majority of those target genes were involved in the regulation of pluripotency, MAPK, WNT, Hedgehog, p53, and transforming growth factor β pathways. Hence, this study provides novel insights for targeting core regulatory of miRNAs in breast CSCs to target both self‐renewal and metastasis potential and eradication of breast cancer.

Semen affects the biological behavior of HeLa cells by altering ERK signaling.

IntroductionThe aim of the study was to investigate the effects of human semen on the proliferation, survival, migration and invasion of HeLa cervical cancer cells by analyzing the extracellular regulated protein kinase (ERK) pathway.Material and methodsHeLa cells were stimulated with different concentrations of human semen. MTT assay was used to analyze the effects on cell proliferation. Apoptosis in the different experimental groups was quantified by Annexin V-FITC/PI staining. The effect of seminal plasma on in vitro invasiveness of cells was evaluated using transwell assay. Western blotting was used to evaluate ERK pathway activation.ResultsHuman semen promoted HeLa cell proliferation; ERK1/2 phosphorylation and c-myc expression also increased with increasing semen concentration. U0126 inhibited semen-induced ERK1/2 phosphorylation, c-myc upregulation and cell proliferation. Compared with the control group, semen did not significantly affect the apoptotic rate of HeLa cells (p > 0.05). The transwell assays showed that compared with the control group, the number of invading cells increased significantly with increasing semen concentration, and the difference was significant (p < 0.05) when 1 : 50 semen was added, suggesting that semen promotes the invasion of cervical cancer cells. Western blotting indicated that ERK1/2 phosphorylation began to increase when 1 : 100 semen was added; with increasing semen concentration, ERK1/2 phosphorylation was significantly up-regulated, and the expression of its downstream target gene, c-myc, was also up-regulated (p < 0.05).ConclusionsSemen promoted the proliferation of HeLa cells by activating the ERK pathway and showed increased tumorigenic potential in vivo. Human seminal plasma might be a potential factor contributing to the development of cervical cancer.