Exploration of the Hsa-miR-1587–Protein Interaction and the Inhibition to CASK

Hsa-miR-1587 has been found to be capable of forming G-quadruplex structures and is overexpressed in multiple cancer cell lines. Here, we explored the interactions between miR-1587 and proteins. HuProt™ human proteome microarray was utilized to screen the binding proteins, and it was discovered that CASK could bind to miR-1587 on the base of the G-quadruplex structure. Moreover, reelin and p21, which are downstream of CASK, were downregulated both transcriptionally and translationally by miR-1587, uncovered by q-RT-PCR and Western blot assays. Bioinformatic analysis was performed on STRING and Panther platforms, leading to the discovery that miR-1587 may be involved in intracellular metabolic and transcriptional physiological processes. This study explores the interaction of hsa-miR-1587 with proteins and provides a new strategy for the regulation of G-rich microRNA’s function.

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The Tyrosine Kinase src Is Highly Activated in AML and Plays a Crucial Role in Leukemic Cell Proliferation.

Blood ◽

10.1182/blood.v106.11.4402.4402 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4402-4402

Author(s):

Gunter Schuch ◽

Ellen Schafer ◽

Katharina Eggert ◽

Sonja Loges ◽

Manfred Jucker ◽

...

Keyword(s):

Cell Proliferation ◽

Tyrosine Kinase ◽

Western Blot ◽

Cell Lines ◽

Crucial Role ◽

Leukemic Cell ◽

Clinical Samples ◽

Dependent Manner ◽

Coding Region ◽

Rt Pcr

Abstract The tyrosine kinase pp60src is involved in several signal transduction pathways such as signalling of hematopoetic growth factors and cytokines. The viral form v-src was the first oncogene described and mutations of regulatory tyrosine residues in cellular src (c-src) have been linked to malignant transformation. However, no mutations in the gene of c-src have been described in leukemia so far, although some data of src mutations in solid tumors have been reported. The current study was undertaken to examine the role of src in acute myeloid leukemia (AML). Blood and bone marrow specimen of patients with newly diagnosed or recurrent AML treated at our institution were sampled. AML cell lines or CD34 positive cells of healthy donors served as positive and negative controls, respectively. RNA was isolated, and RT-PCR was performed using 4 different primer pairs spanning the coding region of c-src. Protein expression and phosphorylation was studied after protein extraction and Western blot analyses using src and phospho-src specific antibodies. The effect of src inhibitors PP1 and PP2 on leukemic cell proliferation was studied in human and murine cell lines. Mutational analyses of the coding region were performed using SSCP/heteroduplex and bi-directionally sequencing. In all 60 patients analyzed expression of c-src mRNA was detected by RT-PCR. Western blot analyses confirmed strong expression of src on the protein level and revealed a robust activation of the protein as determined by tyrosine phosphorylation. Inhibition of src phosphorylation by src-specific inhibitors PP1 and PP2 was detected by Western blot using an antibody specific for phospho-src. Incubation of leukemic cells with PP1 and PP2 caused significant inhibition of proliferation in a dose dependent manner. Mutational analyses as performed by SSCP/heteroduplex and bi-directionally sequencing revealed wildtype sequence in all cell lines and 60 clinical samples. In summary, pp60src is highly expressed and activated in cell lines and clinical samples of human AML. Moreover, phosphorylation of src is essential for leukemic cell proliferation. Mutations in the coding sequence of c-src causing constitutive activation could be excluded by mutational analyses of primary AML samples. These data suggest that pp60src plays a crucial role in AML and src inhibition by targeted therapy might offer a useful new approach in the treatment of AML.

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Abnormal Expression of TRAF Adapter Proteins in Waldenstrom’s Macroglobulinemia.

Blood ◽

10.1182/blood.v108.11.4640.4640 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4640-4640

Author(s):

Xavier Leleu ◽

Lian Xu ◽

Zachary R. Hunter ◽

Anne-Sophie Moreau ◽

Xiaoying Jia ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Blot Analysis ◽

Western Blot Analysis ◽

Low Grade ◽

Adapter Proteins ◽

Rt Pcr ◽

Growth And Survival ◽

Healthy Donors ◽

Sirna Knockdown

Abstract Background: Waldenström’s Macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma with as yet unknown genetic basis for its pathogenesis. Several TNF family members (CD40L, APRIL and BAFF/BLYS) are known to regulate WM growth and survival. TRAFs are a novel family of adapter proteins that facilitate pro-apoptotic (TACI) or pro-survival/differentiation (CD40, BAFFR, BCMA) receptor signaling mediated by TNF family ligands. Therefore, understanding the TRAF system in WM may yield important clues about WM growth and survival. Methods: WM cell lines (BCWM.1 and WSU-WM), IgM secreting low-grade lymphoma cell lines (MEK1, RL, Namalwa), and primary bone marrow CD19+ selected lymphoplasmacytic cells (LPC) from 20 WM patients and 6 healthy donors were evaluated for TRAF (TRAF 2, 3, 5, 6) expression using semi quantitative RT-PCR and/or western blot analysis. Results: The TNF familiy receptors CD40, BAFFR, BCMA, and TACI were expressed in all cell lines tested as well as in CD19+ selected LPC from WM patients and healthy donors. Moreover, TRAF 2, 3, 5, 6 were expressed in all cell lines by both RT-PCR and western blot analysis. In contrast, we observed loss or abnormally low expression of both TRAF 2 and 5 in 6/20 (30%) patients, whilst TRAF 3 was absent or abnormally low in 3/30 (15%) patients. TRAF 6 was expressed in all patients. Among healthy donors, we observed expression of all TRAF adapter proteins. Conclusion: Up to one third of WM patients demonstrate loss of TRAF 2 and 5 adapter proteins which facilitate signaling through the pro-apoptotic receptor TACI. Ongoing studies including gene sequencing and siRNA knockdown models are delineating a role for TRAF loss in the pathogenesis of WM.

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NF-κB and pSTAT3 synergistically drive G6PD overexpression and facilitate sensitivity to G6PD inhibition in ccRCC

10.21203/rs.3.rs-27362/v3 ◽

2020 ◽

Author(s):

Qiao Zhang ◽

Zhe Yang ◽

Yueli Ni ◽

Honggang Bai ◽

Qiaoqiao Han ◽

...

Keyword(s):

Western Blot ◽

Real Time ◽

Signaling Pathway ◽

Protein Interaction ◽

Xenograft Model ◽

Metabolic Reprogramming ◽

Regulatory Function ◽

Rt Pcr ◽

Repressive Effect ◽

The Impact

Abstract Background: Glucose 6-phosphate dehydrogenase (G6PD) serves key roles in cancer cell metabolic reprogramming, and has been reported to be involved in certain carcinogenesis. Previous results from our laboratory demonstrated that overexpressed G6PD was a potential prognostic biomarker in clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer. G6PD could stimulate ccRCC growth and invasion through facilitating reactive oxygen species (ROS)-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) activation and ROS-MAPK-MMP2 axis pathway, respectively. However, the reasons for ectopic G6PD overexpression and the proliferation repressive effect of G6PD inhibition in ccRCC are still unclear. Methods: The impact of ROS accumulation on NF-κB signaling pathway and G6PD expression was determined by real-time RT-PCR and Western blot in ccRCC cells following treatment with ROS stimulator or scavenger. The regulatory function of NF-κB signaling pathway in G6PD transcription was analyzed by real-time RT-PCR, Western blot, luciferase and ChIP assay in ccRCC cells following treatment with NF-κB signaling activator/inhibitor or lentivirus infection. ChIP and Co-IP assay was performed to demonstrate protein-DNA and protein-protein interaction of NF-κB and pSTAT3, respectively. MTS assay, human tissue detection and xenograft model were conducted to characterize the association between NF-κB, pSTAT3, G6PD expression level and proliferation functions. Results: ROS-stimulated NF-κB and pSTAT3 signaling over-activation could activate each other, and exhibit cross-talks in G6PD aberrant transcriptional regulation. The underlying mechanism was that NF-κB signaling pathway facilitated G6PD transcription via direct DNA–protein interaction with p65 instead of p50. p65 and pSTAT3 formed a p65/pSTAT3 complex, occupied the pSTAT3-binding site on G6PD promoter, and contributed to ccRCC proliferation following facilitated G6PD overexpression. G6PD, pSTAT3, and p65 were highly expressed and positively correlated with each other in ccRCC tissues, confirming that NF-κB and pSTAT3 synergistically promote G6PD overexpression. Moreover, G6PD inhibitor exhibited tumor-suppressor activities in ccRCC and attenuated the growth of ccRCC cells both in vitro and in vivo . Conclusion: ROS-stimulated aberrations of NF-κB and pSTAT3 signaling pathway synergistically drive G6PD transcription through forming a p65/pSTAT3 complex. Moreover, G6PD activity inhibition may be a promising therapeutic strategy for ccRCC treatment.

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P169 SEXUAL HORMONE AND INSULIN RECEPTORS IN CHONDROCYTE CELL LINES C28/12 AND T/C-28a2 ANALYZED BY WESTERN BLOT AND RT-PCR

Osteoarthritis and Cartilage ◽

10.1016/s1063-4584(07)60621-9 ◽

2006 ◽

Vol 14 ◽

pp. S100

Author(s):

H. Claassen ◽

J. Brandt ◽

R. Ebersbach ◽

M. Goldring ◽

K. Reuse ◽

...

Keyword(s):

Western Blot ◽

Cell Lines ◽

Insulin Receptors ◽

Rt Pcr ◽

Sexual Hormone

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Osteonectin/SPARC Is Epigenetically Silenced in AML with MLL Gene Rearrangements and Selectively Inhibits the Growth of MLL Rearranged Cell Lines.

Blood ◽

10.1182/blood.v104.11.1106.1106 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1106-1106

Author(s):

Jorge F. DiMartino ◽

Norman J. Lacayo ◽

Marie Varadi ◽

Yaddanapudi Ravindranath ◽

Ron Yu ◽

...

Keyword(s):

Gene Expression ◽

Pancreatic Cancer ◽

Western Blot ◽

Cell Lines ◽

Chromatin Remodeling ◽

Promoter Methylation ◽

Rt Pcr ◽

Growth And Survival ◽

Mll Gene ◽

Sparc Expression

Abstract Transcriptional repression by chimeric transcription factors is emerging as a common theme in leukemogenesis and as a therapeutic target for chromatin remodeling agents. We hypothesized that rearrangements involving the MLL gene result in the inappropriate silencing of growth and survival control genes subordinate to this positive epigenetic transcriptional regulator. To identify some of these genes, we used Significance Analysis of Microarrays (SAM), a supervised learning algorithm. We found significant gene expression differences between 13 patients with MLL translocations and 12 core binding factor (CBF) rearranged patients, 2 t(8;21), 10 INV16, from a Pediatric Oncology Group AML study (POG 9421). We also analyzed gene expression data from a published study of adult AML including 8 MLL rearranged patients, 11 with t(8;21) and 15 with INV16. SAM identified 10 genes, common to both datasets, that were significantly under-expressed in the MLL rearranged patients. One of the most significant genes was osteonectin, also known as secreted protein, acidic, rich in cysteine (SPARC). This gene encodes a matricellular glycoprotein with diverse functions in cell-matrix interactions. SPARC has been identified as a target of epigenetic silencing in pancreatic cancer and addition of exogenous SPARC to cancer cell lines induces growth arrest and apoptosis. To determine if leukemia cell lines could be used as a model to study the basis for SPARC silencing and its role in cell growth and survival we measured SPARC expression in AML cell lines with rearranged and germline MLL genes. By real-time quantitative reverse transcriptase PCR (Q-RT-PCR), the cell lines THP-1 (MLL-AF-9) and ML-2 (MLL-AF6) expressed SPARC mRNA levels 40 to 1000 fold lower than Kasumi-1 (t(8;21)) and KG1a. By Western blot, SPARC was easily detectable in Kasumi-1 and KG1a but undetectable in the MLL rearranged lines. Bisulfite sequencing revealed extensive methylation of CpG dinucleotides in the promoter region and first exon of SPARC in THP-1 and ML-2 but a complete lack of methylation in KG1a. Treatment with the DNA methyltransferase inhibitor 5-aza-2′deoxycytidine (DAC) restored SPARC expression to nearly normal levels in THP-1 cells by Q-RT-PCR and Western blot, suggesting that promoter methylation is critical to the silencing of this gene. To determine if SPARC was contributing to the growth inhibitory effect of DAC, cells were cultured in varying concentrations of exogenous purified SPARC. Concentrations of SPARC that reduced the growth of ML-2 and THP-1 by 30 to 40% had no effect on the growth of KG1a cells. We conclude that SPARC, a putative tumor suppressor that is epigenetically silenced in pancreatic cancer, is also silenced by promoter methylation in AML with MLL rearrangements. These studies suggest that SPARC expression may constitute a reliable pharmacodynamic endpoint for clinical studies of chromatin remodeling agents in patients with MLL rearranged AML. Whether SPARC is a direct target of MLL and how MLL rearrangements are related to SPARC silencing are the subject of future studies.

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Prima-1Met Combined with Bortezomib Has Synergistic Anti-Myeloma Activity By Modulation of Apoptosis and Cell Cycle Regulating Genes

Blood ◽

10.1182/blood.v126.23.4213.4213 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4213-4213

Author(s):

Priya Khoral ◽

Robert J Guo ◽

Jahangir Abdi ◽

Hong Chang

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Western Blot ◽

Real Time ◽

Cell Lines ◽

Blot Analysis ◽

Drug Combination ◽

Western Blot Analysis ◽

Rt Pcr ◽

Novel Therapeutic

Abstract INTRODUCTION Multiple Myeloma (MM) is a plasma-cell malignancy characterized by dismal prognosis and a high level of relapse, thus novel therapeutic approaches are needed. PRIMA-1Met is a novel small molecule showing anti-tumour activity and currently in clinical phase I-II trials. We recently demonstrated that PRIMA-1Met has potent anti-MM activity in vitro and in vivo. Bortezomib (BTZ) is a proteasome inhibitor that has been successfully used for treating some cases of relapsed MM. The aim of the current study is to determine whether PRIMA-1Met could be used in combination with BTZ to enhance the cytotoxic effects in myeloma cells. METHODS Using three different MM cell lines (LP1, U266 and 8226), we established dose response curves for both PRIMA-1Met and BTZ, and tested drug cytotoxicity using MTT assays. We then tested drug cytotoxicity of a range of concentrations of the drugs in combination. The Chou Talay method was used to determine whether or not the drug combinations were synergistic. A gene expression array was used to investigate the mechanism of the drug combination's effects. Total RNA was isolated from MM cell pellets, then synthesized cDNAs were applied to real time RT-PCR gene expression arrays containing 84 genes of interest. The genes selected were involved in apoptotic as well as cell growth and proliferation pathways. After normalization to 4 different housekeeping genes, fold changes in gene expression were analyzed in both drug treated and control samples using the 2-ΔΔCt algorithm. Western blot analysis was used to further investigate proteins of interest. RESULTS Cell viability of 8226, LP1 and U266 cells treated with individual concentrations of PRIMA-1Met (10uM) and BTZ (10nM) was on average 65%, 45% and 72.5%, respectively. However, combination of above doses reduced viability to 20% in 8226 and LP1, and to 40% in U266. The Chou Talay method identified this drug combination as synergistic in 2 out of the three tested cell lines, with Combination Index (CI) values of 0.72 in 8226 and 0.582 in U266. The gene expression analysis in real time RT-PCR indicated that the drug combination resulted in downregulation of genes involved in cell cycle and proliferation (CCND1, CDK4, CDK6, CDK2, IGFIR), genes from the Bcl-2 family of apoptosis regulation (Bcl-2, Bcl-XL, Mcl-1), as well as MDM2 from the p53 signalling pathway, and MYC, which is involved in both apoptosis and cell cycle progression. Western blot analysis revealed up-regulation of cleaved caspase-3 and -9, implying involvement of the intrinsic apoptotic pathway in the drug combination's activity. CONCLUSION Our results reveal that PRIMA-1Met synergistically enhances the anti-MM effect of BTZ, leading to a significantly higher level of MM cell death. Real time RT-PCR gene array analysis offers some insight into the mechanism of this combination's effect, implicating apoptotic, cell cycle and growth regulating genes. Our study provides framework for further evaluation of this drug combination as a novel therapeutic strategy in MM. Disclosures No relevant conflicts of interest to declare.

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The tyrosine kinase src plays a crucial role in leukemic cell proliferation in AML

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.10531 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 10531-10531

Author(s):

G. Schuch ◽

E. Schäfer ◽

K. Eggert ◽

S. Loges ◽

M. Görn ◽

...

Keyword(s):

Cell Proliferation ◽

Western Blot ◽

Cell Lines ◽

Crucial Role ◽

Leukemic Cell ◽

Leukemic Cells ◽

Clinical Samples ◽

Dependent Manner ◽

Rt Pcr

10531 Background: Src-family tyrosine kinases are known to be involved in signal transduction pathways of growth factors and cytokines in hematopoetic cells. While the role of other src family members has been studied widely, only few data exist about c-src in leukemia. The actual study was performed to analyze src mutations in leukemic cells and to determine the role of pp60src in leukemic cell proliferation. Methods: AML cell lines and primary samples were analyzed for expression and activation of src by RT-PCR and Western blot analyses. Mutational analyses were performed by sequencing of C-terminal cDNA from 60 AML samples. The effects of src inhibition were studied by src-specific inhibitors PP1 and PP2 or by siRNA transfection. Effetcs of src inhibition were monitored in proliferation assays and analyzes of signalling through Erk1/2 and apoptosis by annexin V staining and DNA laddering. Results: In all 60 patients analyzed expression of c-src mRNA was detected by RT-PCR. Western blot analyses confirmed strong expression of src on the protein level and revealed a robust activation of the protein as determined by tyrosine phosphorylation. Incubation of leukemic cells with PP1 and PP2 caused significant inhibition of proliferation in a dose dependent manner. Similar results were observed after transfection with specific siRNAs. Src inhibition blocked phosphorylation of pp60src, Erk1/2 and induced apoptosis in leukemic cells. Mutational analyses as performed by SSCP/heteroduplex and bi-directionally sequencing revealed wildtype sequence in cell lines and 60 clinical samples. Conclusions: In summary, pp60src is highly expressed and activated in cell lines and clinical samples of human AML. Moreover, phosphorylation of src is essential for leukemic cell proliferation. Underlying mutations in the coding sequence of c-src causing constitutive activation could be excluded. These data suggest that pp60src plays a crucial role in AML and src inhibition by targeted therapy might offer a useful new approach in the treatment of AML. No significant financial relationships to disclose.

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Anticancer Activity of Platinum (II) Complex as G-Quadruplex DNA Binders

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.781-784.1130 ◽

2013 ◽

Vol 781-784 ◽

pp. 1130-1133

Author(s):

Qi Pin Qin ◽

Yu Lan Li ◽

Yan Cheng Liu ◽

Xu Jian Luo

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Normal Cell ◽

Cancer Cell Lines ◽

Cytotoxic Activities ◽

Binding Properties ◽

Quadruplex Dna ◽

Quadruplex Structure ◽

G Quadruplex ◽

Dna Binders

A platinum (II) complex has been synthesized and characterized. The complex binding properties with G-quadruplex DNA and ds26 were examined by FID and CD spectroscopic methods. The results revealed that the platinum (II) complex can induce the antiparallel G-quadruplex structure of HTG21 conformation in the absence of added K+ with selectivity over other G-quadruplex DNA and duplex DNA. The cytotoxicity of the platinum (II) was screened against four cancer cell lines and normal cells of HL-7702 in comparison to cisplatin and it showed a higher activity than cisplatin, with inhibition rates ranging from (40.06±1.65)% to (89.47±1.14)%. Furthermore, the platinum (II) complex displayed lower cytotoxic activities to HL-7702 (normal cell) compared with the cancer cell lines.

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MEK 1/2 Inhibitor U0126 Reversed Imatinib Resistance in IM-Resistant K562R

Blood ◽

10.1182/blood.v120.21.4914.4914 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4914-4914

Author(s):

Yuping Gong ◽

Juan Lin ◽

Ting Niu

Keyword(s):

Western Blot ◽

Cell Line ◽

Signaling Pathway ◽

Cell Lines ◽

Mtt Assay ◽

K562 Cell ◽

Single Agent ◽

Rt Pcr ◽

Imatinib Resistance ◽

Ic50 Values

Abstract Abstract 4914 Objective To study the anti-leukemic activity of MEK inhibitor U0126 alone or in combination with imatinib and explored the reversing mechanism to imatinib resistance in imatinib resistant K562R cell line. Methods Cytotoxicity of drug was detected by the MTT assay in the IM-sensitive cell line K562 and IM-resistant cell line K562R. Western Blot assay were employed to examine the expression of p-cAbl, p-Lyn, p-STAT5, ERK signaling pathway (p-cRaf, p-MEK, p-ERK), PI3K/AKT/mTOR signaling pathway(p-AKT, p-mTOR, p-4EBP1) and the apoptosis related proteins (Bax, Bcl2). The apoptosis rates were analyzed by Annexin V/PI double staining flow cytometry assay. The levels of lyn and erk1/2 gene were assayed by RT-PCR. Results 1. BCR-ABL-independent activation of Lyn and ERK1/2 may be the IM resistant mechanism in K562R cell line. The IC50 value of K562R cell line(1. 505±0. 459 μmol/L) inhibited by imatinib for 72 hours was higher than the values of K562(0. 159±0. 032μmol/L) and the Resistant Fold(RF)was 9. 465. Western Blot assays showed that p-Lyn, p-MEK and p-ERK of ERK pathway, p-mTOR and p-4EPB1 were over-expressed in K562R cell line relative to K562 cell line. However, the levels of p-cAbl, p-STAT5, p-Raf, p-AKT of PI3K/AKT/mTOR were similar in the K562R and K562 cell lines. The treatment of IM could reduce the expression of p-cAbl and p-MEK, but not that of p-Lyn, p-ERK, p-mTOR, p-4EBP1, and even up-regulate the p-Lyn, p-ERK, p-mTOR. The expression of Bax, Bcl2 and the apoptosis rates were the similar in both cell lines. 2. MEK1/2 inhibitor U0126 could reverse the IM resistance in K562R cell line. MTT assay showed single-agent U0126 is more sensitive to K562R than K562. The IC50 values of the two cell lines were 34. 235±5. 658 μmol/L and 85. 824±4. 474 μmol/L respectively. The combination of imatinib and U0126 markedly enhanced inhibitory effect as measured by MTT assay in K562R cell line, combination with 10μmol/L U0126, the IC50 values of IM was 0. 134±0. 059μmol/L, which reduced to 8. 9% of single IM treatment. 3. The reversing mechanism of U0126 to imatinib resistance in K562R cell line. Western Blot showed single IM up-regulated the p-Lyn and p-ERK, while U0126 reduced the expression of them, the combination of the IM and U0126 could synergisticly reduce the p-Lyn expression and neutralize the up-regulation of p-ERK caused by IM single agent. Single IM also up-regulated the p-mTOR in K562R while U0126 reduced it, single IM or U0126 had no influence on p-4EBP1 in K562R, the combination of the two drugs could synergisticly reduce the p-4EBP1 and neutralize the up-regulation of p-mTOR caused by IM single agent. IM but not U0126could reduce p-cAbl and the combination of the two was more effective than IM treatment. IM alone or combination with U0126 could not regulate p-STAT5 expression in K562R. RT-PCR showed that neither IM treatment nor its combination with U0126 could change the level of lyn and erk1, 2 gene in the cell lines. Conclusions 1. BCR-ABL-independent activation of Lyn and ERK1/2 involved in IM resistance mechanism in IM-resistant K562R cell line. Imatinib alone could up-regulated the expression of the p-Lyn, p-ERK, p-mTOR in K562R cell line. MEK1/2 inhibitor U0126 could reverse the IM resistance by reducing the expression of the p-Lyn, p-ERK, p-mTOR, p-4EBP1 of IM-resistant K562R cell line, and the combination of U0216 and Imatinib could synergisticly depress up-regulation of the p-Lyn, p-ERK, p-mTOR and p-4EBP1 caused by IM. Grant support: National Natural Science Foundation of China (No. 30770912), Foundation of the Science & Technology Department of Sichuan Province (No. 2008SZ0017). Disclosures: No relevant conflicts of interest to declare.

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NF-κB and pSTAT3 synergistically drive G6PD overexpression and facilitate sensitivity to G6PD inhibition in ccRCC

10.21203/rs.3.rs-27362/v1 ◽

2020 ◽

Author(s):

Qiao Zhang ◽

Zhe Yang ◽

Yueli Ni ◽

Honggang Bai ◽

Qiaoqiao Han ◽

...

Keyword(s):

Western Blot ◽

Binding Site ◽

Real Time ◽

Signaling Pathway ◽

Protein Interaction ◽

Metabolic Reprogramming ◽

Regulatory Function ◽

Rt Pcr ◽

Repressive Effect ◽

The Impact

Abstract Background Glucose 6-phosphate dehydrogenase (G6PD) serves key roles in cancer cell metabolic reprogramming, and has been reported to be involved in certain carcinogenesis. Previous results from our laboratory demonstrated that overexpressed G6PD was a potential prognostic biomarker in clear cell renal cell carcinoma (ccRCC), the most common subtype of kidney cancer. G6PD could stimulate ccRCC growth and invasion through facilitating reactive oxygen species (ROS)-phosphorylated signal transducer and activator of transcription 3 (pSTAT3) activation and ROS-MAPK-MMP2 axis pathway, respectively. However, the reasons for ectopic G6PD overexpression and the proliferation repressive effect of G6PD inhibition in ccRCC are still unclear. Methods The impact of ROS accumulation on NK-κB signaling pathway and G6PD expression was determined by real-time RT-PCR and Western blot in ccRCC cells following treatment with ROS stimulator or scavenger. The regulatory function of NK-κB signaling pathway in G6PD transcription was analyzed by real-time RT-PCR, Western blot, luciferase and ChIP assay in ccRCC cells following treatment with NK-κB signaling activator/inhibitor or lentivirus infection. ChIP and Co-IP assay was performed to demonstrate protein-DNA and protein-protein interaction of NK-κB and pSTAT3, respectively. MTS assay, human tissue detection and xenograft model were conducted to characterize the association between NK-κB, pSTAT3, G6PD expression level and proliferation functions. Results ROS-stimulated NF-κB and pSTAT3 signaling over-activation could activate each other, and exhibit cross-talks in G6PD aberrant transcriptional regulation. The underlying mechanism was that NF-κB signaling pathway facilitated G6PD transcription via direct DNA–protein interaction with p65 instead of p50. p65 and pSTAT3 formed a p65/pSTAT3 complex, occupied the pSTAT3-binding site on G6PD promoter, and contributed to ccRCC proliferation following facilitated G6PD overexpression. G6PD, pSTAT3, and p65 were highly expressed and positively correlated with each other in ccRCC tissues, confirming that NF-κB and pSTAT3 synergistically promote G6PD overexpression. Moreover, G6PD inhibitor exhibited tumor-suppressor activities in ccRCC and attenuated the growth of ccRCC cells both in vitro and in vivo . Conclusion ROS-stimulated aberrations of NF-κB and pSTAT3 signaling pathway synergistically drive G6PD transcription through forming a p65/pSTAT3 complex and occupy the pSTAT3-binding site on G6PD promoter. Moreover, G6PD activity inhibition may be a promising therapeutic strategy for ccRCC treatment.

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