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shPLCε通过YAP抑制前列腺癌细胞的丝氨酸/甘氨酸代谢和增殖 *
段李梅,杨锦潇,刘佳渝,郑永波,吴小候,罗春丽
中国生物工程杂志 ›› 2019, Vol. 39 ›› Issue (11) : 1-12.
PDF(2275 KB)
PDF(2275 KB)
shPLCε通过YAP抑制前列腺癌细胞的丝氨酸/甘氨酸代谢和增殖 *
shPLCε Inhibits Serine/Glycine Metabolism and Proliferation of Prostate Cancer via YAP Signaling Pathway
({{custom_author.role_en}}), {{javascript:window.custom_author_en_index++;}}目的 该研究旨在探讨磷脂酰肌醇特异性磷脂酶C epsilon(phospholipase C epsilon, PLCε)对前列腺癌细胞丝氨酸/甘氨酸代谢及细胞增殖的影响。方法 慢病毒及质粒转染LNCAP、PC3细胞,q-PCR、Western blot分别检测LNCAP、PC3细胞中 PLCε、Yes相关蛋白(yes associated protein,YAP)、丝氨酸/甘氨酸生成酶[包括磷酸丝氨酸转氨酶1(phosphoserine aminotransferase1,PSAT1)、磷酸丝氨酸磷酸酶(phosphoserine phosphatase,PSPH)、丝氨酸羟甲基转移酶2(serine hydroxymethyltransferase2,SHMT2)及增殖相关基因细胞周期蛋白D1(Cyclin D1)、增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)]的表达情况;克隆形成实验及MTT实验检测细胞的克隆形成率及增殖活性。结果 (1)感染LV-shPLCε可显著下调前列腺癌细胞LNCAP、PC3中的PLCε、YAP、PSAT1、PSPH、SHMT2及增殖相关基因的mRNA及蛋白质水平,同时抑制细胞的克隆形成能力和增殖活性;(2)在shPLCε组细胞中加入过表达YAP质粒后,能明显逆转YAP、PSAT1、PSPH、SHMT2及增殖相关基因的下调,但加入干扰YAP质粒后结果相反。结论 shPLCε可通过下调YAP的表达抑制前列腺癌细胞的丝氨酸/甘氨酸生成,从而抑制细胞的增殖。
Objective: To investigate the effects of phospholipase C epsilon on serine/glycine metabolism and cell proliferation in prostate cancer cells.Methods: Lentivirus and plasmid were transfected into LNCAP and PC3 cells. The expression of YAP, serine/glycine producing enzyme (PSAT1,PSPH,SHMT2) and proliferation-related genes (Cyclin D1,PCNA) were detected by q-PCR and Western blot. The cloning formation experiment and MTT assays were used to detect the clone formation rate and cell proliferation activity.Results: (1) Infection with LV-shPLCε significantly down-regulated the mRNA and protein levels of PLCε,YAP,serine/glycine producing enzymes (PSAT1,PSPH,SHMT2) and proliferation genes (Cyclin D1,PCNA) in prostate cancer cells LNCAP and PC3. At the same time, it inhibits the clone formation ability and proliferative activity of LNCAP and PC3 cells. (2) After adding over-expressing YAP plasmid to shPLCε group, YAP,serine/glycine producing enzymes and proliferation genes were significantly reversed. but the results of the interference with the down YAP plasmid were reversed.Conclusion: shPLCε inhibits the serine/glycine metabolism and proliferation in prostate cancer cells by down-regulating the expression of YAP.
前列腺癌 / 磷脂酰肌醇特异性磷脂酶C / epsilon / Yes相关蛋白 / 丝氨酸/甘氨酸代谢 / 增殖 {{custom_keyword}} /
Prostate cancer / PLCε / YAP / Serine/Glycine metabolism / Proliferation {{custom_keyword}} /
Table 1 lentivirus sequence |
| 病毒名称 | 病毒序列(5'→3') |
|---|---|
| LV-shPLCε | Sense: GGTTCTCTCCTAGAAGCAACC |
| Anti-sense: CCAAGAGAGGATCTTCGTTGG | |
| LV-shNC | Sense: TTCTCCGAACGTGTCACGT |
| Anti-sense: AAGAGGCTTGCACAGTGCA |
表2 q-PCR引物信息Table 2 The sequence of primer for q-PCR |
| 基因 | 上游引物(5'→3') | 下游引物(5'→3') |
|---|---|---|
| PLCε | GGAGAATCCTCGGTAG | GGTTGTCAGCGTATGTCC |
| YAP | TAGCCCTGCGTAGCCAGTTA | TCATGCTTAGTCCACTGTCTGT |
| PSAT1 | TGCCGCACTCAGTGTTGTTAG | GCAATTCCCGCACAAGATTCT |
| PSPH | GAGGACGCGGTGTCAGAAAT | GGTTGCTCTGCTATGAGTCTCT |
| SHMT2 | CCCTTCTGCAACCTCACGAC | TGAGCTTATAGGGCATAGACTCG |
| Cyclin D1 | GCTGGAGCCCGTGAAAAAGA | CTCCGCCTCTGGCATTTTG |
| PCNA | TCAAGAAGGTGTTGGAGGCA | CAGCGGTAGGTGTCGAAGC |
| β-actin | GGGACCTGACTGACTACCTC | ACGAGACCACCTTCAACTCCAC |
表3 质粒信息Table 3 Plasmid vector sequence |
| 质粒名称 | 质粒序列(5'→3') |
|---|---|
| pcDNA Flag Yap1 | Sense:GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATG |
| Antisense:CTGCCTAGCCCTCTAGAGGGCTAGGGGATACCAGCTGAGAGTCATGTTAGACGAGACTAC | |
| pcDNA3.2/EV | Sense:GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCC |
| Antisense:CTGTAACTAATAACTGATCAATAATTATCATTAGTTAATGCCCCAGTAATCAAGTATCGG | |
| pGPU6/GFP/Neo-YAP1 | Sense: CACCGCCACCAAGCTAGATAAAGAATTCAAGAGATTCTTTATCTAGCTTGGTGGCTTTTTTG |
| Antisense:GATCCAAAAAAGCCACCAAGCTAGATAAAGAATCTCTTGAATTCTTTATCTAGCTTGGTGGC | |
| pGPU6/GFP/Neo-shNC | Sense:CACCGTTCTCCGAACGTGTCACGTTTCAAGAGAACGTGACACGTTCGGAGAATTTTTTG |
| Antisense:GATCCAAAAAATTCTCCGAACGTGTCACGTTCTCTTGAAACGTGACACGTTCGGAGAAC |
图1 转染shPLCε慢病毒后LNCAP和PC3细胞中PLCε的mRNA和蛋白质水平Fig.1 PLCε mRNA and protein levels in LNCAP and PC3 cells infected with LV-shPLCε (a)mRNA level of PLCε in LNCAP infected with shPLCε by q-PCR (b)mRNA level of PLCε in PC3 infected with shPLCε by q-PCR (c)Western blot of PLCεin LNCAP and PC3 (d)Relative protein expression of PLCεin LNCAP and PC3 * P<0.05, ** P<0.01, *** P<0.001 vs shNC group |
图4 转染shPLCε后LNCAP和PC3细胞中PLCε、YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的mRNA水平Fig.4 PLCε,YAP,PSAT1,PSPH,SHMT2,Cyclin D1,PCNA mRNA levels in LNCAP and PC3 cells infected with shPLCε (a) mRNA levels of LNCAP infected with shPLCε (b) mRNA levels of PC3 infected with shPLCε * P<0.05, ** P<0.01, *** P<0.001 |
图5 转染shPLCε后LNCAP和PC3细胞中YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的蛋白质水平Fig.5 YAP,PSAT1,PSPH,SHMT2,Cyclin D1,PCNA protein levels in LNCAP and PC3 cells infected with shPLCε (a) Western blot of LNCAP infected with shPLCε (b)Relative protein level of LNCAP (c)Western blot of PC3 infected with shPLCε (d)Relative protein level of PC3 *P<0.05, ** P<0.01, *** P<0.001 |
Fig.6 mRNA and protein levels of YAP in LNCAP and PC3 cells infected with over-expression or knockdown of YAP plasmid(a)mRNA level of YAP in LNCAP infected with over-expression or sh-YAP plasmid by q-PCR (b)mRNA level of YAP in PC3 infected with over-expression or sh-YAP plasmid by q-PCR (c)Western blot of YAP in LNCAP (d)Relative protein level of LNCPA (e)Western blot of YAP in PC3 (f)Relative protein level of PC3 * P<0.05, ** P<0.01, *** P<0.001 |
图7 转染过表达或敲低YAP质粒后LNCAP和PC3细胞中PLCε、YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的mRNA水平Fig.7 PLCε,YAP,PSAT1,PSPH,SHMT2,Cyclin D1,PCNA mRNA levels in LNCAP and PC3 cells infected with over-expression or knockdown of YAP plasmid (a)mRNA level of LNCAP infected with plasmids (b) mRNA level of PC3 infected with plasmids * P<0.05, ** P<0.01, *** P<0.001 |
图8 转染过表达或敲低YAP质粒后LNCAP和PC3中PLCε、YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的蛋白质水平Fig.8 PLCε,YAP,PSAT1,PSPH,SHMT2,Cyclin D1,PCNA protein levels in LNCAP and PC3 cells infected with over-expression or knockdown of YAP plasmid (a)Western blot of PLCε,YAP,PSAT1,PSPH,SHMT2,Cyclin D1,PCNA in LNCAP (b)Relative protein level of LNCAP (c)Western blot of PLCε,YAP,PSAT1,PSPH,SHMT2,Cyclin D1,PCNA in PC3 (d)Relative protein level of PC3 * P<0.05, ** P<0.01, *** P<0.001 |
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Cancer constitutes an enormous burden on society in more and less economically developed countries alike. The occurrence of cancer is increasing because of the growth and aging of the population, as well as an increasing prevalence of established risk factors such as smoking, overweight, physical inactivity, and changing reproductive patterns associated with urbanization and economic development. Based on GLOBOCAN estimates, about 14.1 million new cancer cases and 8.2 million deaths occurred in 2012 worldwide. Over the years, the burden has shifted to less developed countries, which currently account for about 57% of cases and 65% of cancer deaths worldwide. Lung cancer is the leading cause of cancer death among males in both more and less developed countries, and has surpassed breast cancer as the leading cause of cancer death among females in more developed countries; breast cancer remains the leading cause of cancer death among females in less developed countries. Other leading causes of cancer death in more developed countries include colorectal cancer among males and females and prostate cancer among males. In less developed countries, liver and stomach cancer among males and cervical cancer among females are also leading causes of cancer death. Although incidence rates for all cancers combined are nearly twice as high in more developed than in less developed countries in both males and females, mortality rates are only 8% to 15% higher in more developed countries. This disparity reflects regional differences in the mix of cancers, which is affected by risk factors and detection practices, and/or the availability of treatment. Risk factors associated with the leading causes of cancer death include tobacco use (lung, colorectal, stomach, and liver cancer), overweight/obesity and physical inactivity (breast and colorectal cancer), and infection (liver, stomach, and cervical cancer). A substantial portion of cancer cases and deaths could be prevented by broadly applying effective prevention measures, such as tobacco control, vaccination, and the use of early detection tests.
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2002年全球有679 000例前列腺癌新发病例,占所有肿瘤新发病例的11.7%,位列常见肿瘤的第5位和男性肿瘤的第2位.但是前列腺癌发病率的地区分布并不均衡,在发达国家前列腺癌占肿瘤新发病例的19%,而在发展中国家仅占5.3%[1].中国是前列腺癌发病率较低的国家,2002年的标化发病率为1.6/10万,远低于美国的124.8/10万[2].
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余朝文 . 基于质谱技术的临床重要疾病代谢相关标志物的鉴定及应用研究. 重庆:重庆医科大学, 2018.
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There is a great interest in searching for diagnostic biomarkers in prostate cancer patients. The aim of the pilot study was to evaluate free amino acid profiles in their serum and urine. The presented paper shows the first comprehensive analysis of a wide panel of amino acids in two different physiological fluids obtained from the same groups of prostate cancer patients (n = 49) and healthy men (n = 40). The potential of free amino acids, both proteinogenic and non-proteinogenic, as prostate cancer biomarkers and their utility in classification of study participants have been assessed. Several metabolites, which deserve special attention in the further metabolomic investigations on searching for prostate cancer markers, were indicated. Moreover, free amino acid profiles enabled to classify samples to one of the studied groups with high sensitivity and specificity. The presented research provides a strong evidence that ethanolamine, arginine and branched-chain amino acids metabolic pathways can be a valuable source of markers for prostate cancer. The altered concentrations of the above-mentioned metabolites suggest their role in pathogenesis of prostate cancer and they should be further evaluated as clinically useful markers of prostate cancer.
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One-carbon metabolism involving the folate and methionine cycles integrates nutritional status from amino acids, glucose and vitamins, and generates diverse outputs, such as the biosynthesis of lipids, nucleotides and proteins, the maintenance of redox status and the substrates for methylation reactions. Long considered a 'housekeeping' process, this pathway has recently been shown to have additional complexity. Genetic and functional evidence suggests that hyperactivation of this pathway is a driver of oncogenesis and establishes a link to cellular epigenetic status. Given the wealth of clinically available agents that target one-carbon metabolism, these new findings could present opportunities for translation into precision cancer medicine.
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R-Ras, an atypical member of the Ras subfamily of small GTPases, enhances integrin-mediated adhesion and signaling through a poorly understood mechanism. Dynamic analysis of cell spreading by total internal reflection fluorescence (TIRF) microscopy demonstrated that active R-Ras lengthened the duration of initial membrane protrusion, and promoted the formation of a ruffling lamellipod, rich in branched actin structures and devoid of filopodia. By contrast, dominant-negative R-Ras enhanced filopodia formation. Moreover, RNA interference (RNAi) approaches demonstrated that endogenous R-Ras contributed to cell spreading. These observations suggest that R-Ras regulates membrane protrusions through organization of the actin cytoskeleton. Our results suggest that phospholipase Cepsilon (PLCepsilon) is a novel R-Ras effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion, because R-Ras was co-precipitated with PLCepsilon and increased its activity. Knockdown of PLCepsilon with siRNA reduced the formation of the ruffling lamellipod in R-Ras cells. Consistent with this pathway, inhibitors of PLC activity, or chelating intracellular Ca2+ abolished the ability of R-Ras to promote membrane protrusions and spreading. Overall, these data suggest that R-Ras signaling regulates the organization of the actin cytoskeleton to sustain membrane protrusion through the activity of PLCepsilon.
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Phosphoinositide-specific phospholipase C (PI-PLC) plays a pivotal role in regulation of intracellular signal transduction from various receptor molecules. More than 10 members of human PI-PLC isoforms have been identified and classified into three classes beta, gamma, and delta, which are regulated by distinct mechanisms. Here we report identification of a novel class of human PI-PLC, named PLCepsilon, which is characterized by the presence of a Ras-associating domain at its C terminus and a CDC25-like domain at its N terminus. The Ras-associating domain of PLCepsilon specifically binds to the GTP-bound forms of Ha-Ras and Rap1A. The dissociation constant for Ha-Ras is estimated to be approximately 40 nm, comparable with those of other Ras effectors. Co-expression of an activated Ha-Ras mutant with PLCepsilon induces its translocation from the cytosol to the plasma membrane. Upon stimulation with epidermal growth factor, similar translocation of ectopically expressed PLCepsilon is observed, which is inhibited by co-expression of dominant-negative Ha-Ras. Furthermore, using a liposome-based reconstitution assay, it is shown that the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of PLCepsilon is stimulated in vitro by Ha-Ras in a GTP-dependent manner. These results indicate that Ras directly regulates phosphoinositide breakdown through membrane targeting of PLCepsilon.
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BACKGROUND Phospholipase Cε (PLCε), a member of the plc family, has been extensively studied to reveal its role in the regulation of different cell functions, but understanding of the underlying mechanisms remains limited. In the present study, we explored the effects of PLCε on PTEN (phosphatase and tensin homolog deleted on chromosome 10) in cell proliferation in prostate cancer cells. MATERIAL AND METHODS We assessed PLCε and PTEN expression in human benign prostate tissues compared to prostate cancer tissues by immunohistochemistry. Lentivirus-shPLCε (LV-shPLCε) was designed to silence PLCε expression in DU145 and PC3 cell lines, and the effectiveness was tested by qRT-PCR and Western blotting. MTT assay and colony formation assay were conducted to observe cell proliferation. Western blotting and immunofluorescence assays were used to detect changed PTEN expression in DU145. RESULTS We observed that PLCε expression was reduced in human benign prostate tissues compared to prostate cancer tissues, while PTEN expression showed the opposite trend. Silencing of the PLCε gene significantly inhibited cell proliferation in DU145 and PC3 cell lines. DU145 is a PTEN-expressing cell, while PC3 is PTEN-deficient. After infection by LV-shPLCε, we noticed that PTEN expression was up-regulated in DU145 cells but not in PC3 cells. Furthermore, we found that PLCε gene knockdown decreased P-AKT protein levels, but AKT protein levels were not affected. Immunofluorescence assays showed that PTEN expression had an intracellular distribution change in the DU145 cell line, and Western blot analysis showed that PTEN was obviously up-regulated in cell nucleus and cytoplasm. CONCLUSIONS PLCε is an oncogene, and knockdown of expression of PLCe inhibits PCa cells proliferation via the PTEN/AKT signaling pathway.
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王晓亮, 彭志海 . 磷酯酶C家族新成员——磷酯酶CE1. 医学分子生物学杂志, 2008,4:332-335.
磷酯酶CEI是近几年发现的新的磷酯酶C同工酶,对其研究尚未深入.其分子结构中特有的CDC25和RA结构域使其功能上与小G蛋白关系密切.磷酯酶CE1激活后介导信号从细胞膜到细胞核的传递,从而调控某些基因的表达,调节细胞的生长、分化等过程.最近发现,磷酯酶CE1和肿瘤、肾病综合征等多种疾病的发生相关.
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We conducted a genome-wide association study of gastric cancer and esophageal squamous cell carcinoma (ESCC) in ethnic Chinese subjects in which we genotyped 551,152 SNPs. We report a combined analysis of 2,240 gastric cancer cases, 2,115 ESCC cases and 3,302 controls drawn from five studies. In logistic regression models adjusted for age, sex and study, multiple variants at 10q23 had genome-wide significance for gastric cancer and ESCC independently. A notable signal was rs2274223, a nonsynonymous SNP located in PLCE1, for gastric cancer (P = 8.40 x 10(-9); per-allele odds ratio (OR) = 1.31) and ESCC (P = 3.85 x 10(-9); OR = 1.34). The association with gastric cancer differed by anatomic subsite. For tumors in the cardia the association was stronger (P = 4.19 x 10(-15); OR = 1.57), and for those in the noncardia stomach it was absent (P = 0.44; OR = 1.05). Our findings at 10q23 could provide insight into the high incidence of both cancers in China.
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Receptor-initiated phospholipase C activation and generation of IP3 and DAG are important common triggers for a diversity of signal transduction processes in many cell types. Contributing to this diversity is the existence and differential cellular and subcellular distribution of distinct phospholipase C isoforms with distinct regulatory properties. The recently identified PLC epsilon enzyme is an isoform that is uniquely regulated by multiple upstream signals including ras-family GTP binding proteins as well as heterotrimeric G-proteins. In this review we will consider the well documented biochemical regulation of this isoform in the context of cell and whole animal physiology and in the context of other G protein-regulated PLC isoforms. These studies together reveal a surprisingly wide range of unexpected functions for PLC epsilon in cellular signaling, physiology and disease. (c) 2012 Elsevier Inc.
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Phospholipase Cε (PLCε) is a multifunctional enzyme implicated in inflammatory functions. There are limited data, however, on how PLCε can alter inflammatory cytokine by affecting downstream pathways. Recent studies suggest that inflammation is likely to have an important role in transitional cell carcinoma of bladder (TCCB) and cancer disease progression. Here, we showed that PLCε and p-STAT3 expression were both elevated in TCCB tissues compared to adjacent tissues, and the increase of PLCε level was associated with the increase of p-STAT3 level. Then, knockdown of PLCε using adenovirus-shPLCε significantly decreased inflammatory cytokine (IL-6, TNF-α, IL-1β) expression and inflammation-associated gene (TLR4, MyD88, p-STAT3) expression. Furthermore, we demonstrated that PLCε knockdown blocked LPS-induced inflammatory cytokine and p-STAT3 expression. Additionally, we found that combined treatment of STAT3 inhibitor S3I-201 with adenovirus-shPLCε exhibited synergistic inhibitory effects on expression of p-STAT3. Our results suggested that STAT3 phosphorylation is involved in PLCε-mediated inflammatory cytokine release. Our research is of potential importance in drug development programs using PLCε as a therapeutic target for TCCB.
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郝燕妮, 李婷, 范佳鑫 , 等. shPLCε通过下调CDC25A抑制T24细胞的瓦伯格效应. 中国生物工程杂志, 2018,38(5):33-39.
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Serine and glycine are biosynthetically linked, and together provide the essential precursors for the synthesis of proteins, nucleic acids, and lipids that are crucial to cancer cell growth. Moreover, serine/glycine biosynthesis also affects cellular antioxidative capacity, thus supporting tumour homeostasis. A crucial contribution of serine/glycine to cellular metabolism is through the glycine cleavage system,-which refuels one-carbon metabolism; a complex cyclic metabolic network based on chemical reactions of folate compounds. The importance of serine/glycine metabolism is further highlighted by genetic and functional evidence indicating that hyperactivation of the serine/glycine biosynthetic pathway drives oncogenesis. Recent developments in our understanding of these pathways provide novel translational opportunities for drug development, dietary intervention, and biomarker identification of human cancers.
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Serine metabolism is frequently dysregulated in cancers; however, the benefit that this confers to tumors remains controversial. In many cases, extracellular serine alone is sufficient to support cancer cell proliferation, whereas some cancer cells increase serine synthesis from glucose and require de novo serine synthesis even in the presence of abundant extracellular serine. Recent studies cast new light on the role of serine metabolism in cancer, suggesting that active serine synthesis might be required to facilitate amino acid transport, nucleotide synthesis, folate metabolism, and redox homeostasis in a manner that impacts cancer.
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The non-essential amino acids serine and glycine are used in multiple anabolic processes that support cancer cell growth and proliferation (reviewed in ref. 1). While some cancer cells upregulate de novo serine synthesis, many others rely on exogenous serine for optimal growth. Restriction of dietary serine and glycine can reduce tumour growth in xenograft and allograft models. Here we show that this observation translates into more clinically relevant autochthonous tumours in genetically engineered mouse models of intestinal cancer (driven by Apc inactivation) or lymphoma (driven by Myc activation). The increased survival following dietary restriction of serine and glycine in these models was further improved by antagonizing the anti-oxidant response. Disruption of mitochondrial oxidative phosphorylation (using biguanides) led to a complex response that could improve or impede the anti-tumour effect of serine and glycine starvation. Notably, Kras-driven mouse models of pancreatic and intestinal cancers were less responsive to depletion of serine and glycine, reflecting an ability of activated Kras to increase the expression of enzymes that are part of the serine synthesis pathway and thus promote de novo serine synthesis.
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Cancer cells acquire distinct metabolic adaptations to survive stress associated with tumour growth and to satisfy the anabolic demands of proliferation. The tumour suppressor protein p53 (also known as TP53) influences a range of cellular metabolic processes, including glycolysis(1,2), oxidative phosphorylation(3), glutaminolysis(4,5) and anti-oxidant response(6). In contrast to its role in promoting apoptosis during DNA-damaging stress, p53 can promote cell survival during metabolic stress(7), a function that may contribute not only to tumour suppression but also to non-cancer-associated functions of p53(8). Here we show that human cancer cells rapidly use exogenous serine and that serine deprivation triggered activation of the serine synthesis pathway and rapidly suppressed aerobic glycolysis, resulting in an increased flux to the tricarboxylic acid cycle. Transient p53-p21 (also known as CDKN1A) activation and cell-cycle arrest promoted cell survival by efficiently channelling depleted serine stores to glutathione synthesis, thus preserving cellular anti-oxidant capacity. Cells lacking p53 failed to complete the response to serine depletion, resulting in oxidative stress, reduced viability and severely impaired proliferation. The role of p53 in supporting cancer cell proliferation under serine starvation was translated to an in vivo model, indicating that serine depletion has a potential role in the treatment of p53-deficient tumours.
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Altered metabolism is a common feature of new and recurring malignancy. In this issue of Cancer Cell, Reina-Campos and colleagues report upregulation of the serine, glycine, one-carbon (SGOC) metabolic network is required for neuroendocrine prostate cancer, a castration-resistant aggressive form of the disease, and presents a targetable vulnerability.
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Increasingly effective therapies targeting the androgen receptor have paradoxically promoted the incidence of neuroendocrine prostate cancer (NEPC), the most lethal subtype of castration-resistant prostate cancer (PCa), for which there is no effective therapy. Here we report that protein kinase C (PKC)λ/ι is downregulated in de novo and during therapy-induced NEPC, which results in the upregulation of serine biosynthesis through an mTORC1/ATF4-driven pathway. This metabolic reprogramming supports cell proliferation and increases intracellular S-adenosyl methionine (SAM) levels to feed epigenetic changes that favor the development of NEPC characteristics. Altogether, we have uncovered a metabolic vulnerability triggered by PKCλ/ι deficiency in NEPC, which offers potentially actionable targets to prevent therapy resistance in PCa.
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The transcriptional regulators YAP and TAZ are the focus of intense interest given their remarkable biological properties in development, tissue homeostasis and cancer. YAP and TAZ activity is key for the growth of whole organs, for amplification of tissue-specific progenitor cells during tissue renewal and regeneration, and for cell proliferation. In tumors, YAP/TAZ can reprogram cancer cells into cancer stem cells and incite tumor initiation, progression and metastasis. As such, YAP/TAZ are appealing therapeutic targets in cancer and regenerative medicine. Just like the function of YAP/TAZ offers a molecular entry point into the mysteries of tissue biology, their regulation by upstream cues is equally captivating. YAP/TAZ are well known for being the effectors of the Hippo signaling cascade, and mouse mutants in Hippo pathway components display remarkable phenotypes of organ overgrowth, enhanced stem cell content and reduced cellular differentiation. YAP/TAZ are primary sensors of the cell's physical nature, as defined by cell structure, shape and polarity. YAP/TAZ activation also reflects the cell "social" behavior, including cell adhesion and the mechanical signals that the cell receives from tissue architecture and surrounding extracellular matrix (ECM). At the same time, YAP/TAZ entertain relationships with morphogenetic signals, such as Wnt growth factors, and are also regulated by Rho, GPCRs and mevalonate metabolism. YAP/TAZ thus appear at the centerpiece of a signaling nexus by which cells take control of their behavior according to their own shape, spatial location and growth factor context.
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| [21] |
TEAD (TEA/ATTS domain) transcription factors are the most distal effectors of the Hippo pathway. YAP (Yes-associated protein) is a coactivator protein which, upon binding to TEAD proteins, stimulates their transcriptional activity. Since the Hippo pathway is deregulated in various cancers, designing inhibitors of the YAP:TEAD interaction is an attractive therapeutic strategy for oncology. Understanding the molecular events that take place at the YAP:TEAD interface is therefore important not only to devise drug discovery approaches, but also to gain knowledge on TEAD regulation. In this report, combining single site-directed mutagenesis and double mutant analyses, we conduct a detailed analysis on the role of several residues located at the YAP:TEAD interface. Our results provide quantitative understanding of the interactions taking place at the YAP:TEAD interface and give insights into the formation of the YAP:TEAD complex and more particularly on the interaction between TEAD and the Ω-loop found in YAP.
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| [22] |
Although YAP-dependent transcription is closely associated with liver tumorigenesis, the mechanism by which YAP maintains its function is poorly understood. Here, we show that TFCP2 is required for YAP-dependent transcription and liver malignancy. Mechanistically, YAP function is stimulated by TFCP2 via a WW-PSY interaction. TFCP2 also maintains YAP stability by inhibiting βTrCP. Notably, genomic co-occupancy of YAP and TFCP2 is revealed. TFCP2 acts as a transcription co-factor that stimulates YAP transcription by facilitating YAP binding with YAP binding motif (YBF)-containing transcription factors. Interestingly, TFCP2 also stimulated the YAP-TEAD interaction and TEAD target gene expression. Finally, several genes co-regulated by YAP and TFCP2 that contribute to YAP-dependent tumorigenesis are identified and verified. Thus, we establish a model showing that TFCP2 acts as a YAP co-factor to maintain YAP-dependent transcription in liver cancer cells, suggesting that simultaneous targeting of both YAP and TFCP2 may be an effective therapeutic approach.
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| [23] |
Yes-associated protein (YAP) is a downstream effector molecule of a newly emerging tumour suppressor pathway called the Hippo pathway. YAP is a transcriptional co-activator and mis-expressed in various cancers, including colorectal cancer (CRC). Accumulating studies show that the high expression of nuclear YAP is linked with tumour progression and decreased survival. Nuclear YAP can interact with other transcription factors to promote cancer cell proliferation, apoptosis, metastasis and maintenance of stemness. Therefore, YAP has the potential to be a tumour biomarker or therapeutic target for CRC. However, recently, a number of studies have supported a contradictory role for YAP as a tumour suppressor, demonstrating inhibition of the tumorigenesis of CRC, involvement in promoting cell apoptosis, and inhibiting the maintenance of intestinal stem cells and inflammatory activity. In these studies, high expression of YAP was highly correlated with worse survival in CRC. In this review, we will comprehensively summarize and analyse these paradoxical reports, and discuss both the oncogenic and tumour suppressor functions of YAP in the differential status of CRC progression. Further investigation into the mechanisms responsible for the dual function of YAP will be of great value in the prevention, early diagnosis, and therapy of CRC.
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| [24] |
The Hippo signalling is emerging as a tumour suppressor pathway whose function is regulated by an intricate network of intracellular and extracellular cues. Defects in the signal cascade lead to the activation of the Hippo transducers TAZ and YAP. Compelling preclinical evidence showed that TAZ/YAP are often aberrantly engaged in breast cancer (BC), where their hyperactivation culminates into a variety of tumour-promoting functions such as epithelial-to-mesenchymal transition, cancer stem cell generation and therapeutic resistance. Having acquired a more thorough understanding in the biology of TAZ/YAP, and the molecular outputs they elicit, has prompted a first wave of exploratory, clinically-focused analyses aimed at providing initial hints on the prognostic/predictive significance of their expression. In this review, we discuss oncogenic activities linked with TAZ/YAP in BC, and we propose clinical strategies for investigating their role as biomarkers in the clinical setting. Finally, we address the therapeutic potential of TAZ/YAP targeting and the modalities that, in our opinion, should be pursued in order to further study the biological and clinical consequences of their inhibition.
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| [25] |
The transcriptional co-activator YES-associated protein (YAP) has been reported to act as both an oncogene and tumor suppressor in breast cancers. In this study, we evaluated YAP expression immunohistochemically in 324 breast cancer tissues and correlated the expression with clinicopathological findings and patient survival data. Additionally, we reviewed the literature to clarify the role of YAP in breast cancer. We detected YAP, estrogen receptor, progesterone receptor (PR), and human epidermal growth receptor-2 (HER2) expression and a Ki67 labeling index &gt;20% in 53.4%, 49.0%, 45.0%, 28.3%, and 57.4% of invasive ductal carcinoma tissues, respectively. YAP is mainly localized within the tumor cell nuclei, and its expression was associated with the PR status and luminal A subtype. YAP expression also inversely correlated with the HER2 and Ki67 levels and lymph node metastasis. Kaplan-Meier curves revealed associations of YAP expression with favorable disease-free survival (DFS) and overall survival in patients with luminal A breast cancer and with favorable DFS association among patients with invasive ductal carcinoma, luminal B (HER2-), and luminal B (HER2+) breast cancers. A multivariate Cox analysis revealed that YAP expression and PR status were independent favorable predictors of DFS and overall survival, respectively, among patients with breast cancer, whereas tumor-node-metastasis stage and an old age were independent predictors of a poor DFS. Our results, together with the literature review findings, suggest that YAP could be a prognostic marker in patients with breast cancer.
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| [26] |
Yes-associated protein (YAP) is an effector of the Hippo tumor suppressor pathway. The functional significance of YAP in prostate cancer has remained elusive. In this study, we first show that enhanced expression of YAP is able to transform immortalized prostate epithelial cells and promote migration and invasion in both immortalized and cancerous prostate cells. We found that YAP mRNA was upregulated in androgen-insensitive prostate cancer cells (LNCaP-C81 and LNCaP-C4-2 cells) compared to the level in androgen-sensitive LNCaP cells. Importantly, ectopic expression of YAP activated androgen receptor signaling and was sufficient to promote LNCaP cells from an androgen-sensitive state to an androgen-insensitive state in vitro, and YAP conferred castration resistance in vivo. Accordingly, YAP knockdown greatly reduced the rates of migration and invasion of LNCaP-C4-2 cells and under androgen deprivation conditions largely blocked cell division in LNCaP-C4-2 cells. Mechanistically, we found that extracellular signal-regulated kinase-ribosomal s6 kinase signaling was downstream of YAP for cell survival, migration, and invasion in androgen-insensitive cells. Finally, immunohistochemistry showed significant upregulation and hyperactivation of YAP in castration-resistant prostate tumors compared to their levels in hormone-responsive prostate tumors. Together, our results identify YAP to be a novel regulator in prostate cancer cell motility, invasion, and castration-resistant growth and as a potential therapeutic target for metastatic castration-resistant prostate cancer (CRPC).
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| [27] |
Hypoxia is a hallmark of solid tumor growth microenvironment and appropriates the major contributor for the failure and poor prognosis of clinical tumor treatment, including prostate cancer (PCa). Ectopic expression of netrin-1 is reportedly associated with the progression of several carcinomas. Here, we aimed to investigate the role of netrin-1 in hypoxic metastasis potential of prostate carcinoma. Here, hypoxia induced the up-regulation of netrin-1 mRNA and protein expression in prostate cancer cell lines PC3 and DU145. Importantly, knockdown of netrin-1 dramatically suppressed cell invasion, migration and epithelial-to-mesenchymal transition (EMT) of PC3 and DU145 cells under hypoxia. Furthermore, hypoxia treatment increased the activity of Yes-associated protein (YAP) by increasing YAP expression in the nucleus and inhibiting p-YAP levels. However, YAP activation was notably restrained following netrin-1 down-regulation. Interestingly, interrupting YAP expression attenuated hypoxia-triggered cell invasion, migration and EMT of DU145 cells. More importantly, restoring YAP expression strikingly antagonized the inhibitory effects of netrin-1 decrease on the metastatic potential of prostate cancer cells. Together, these results indicate that netrin-1 may function as a positive regulator of hypoxia-triggered malignant behavior in PCa by activating the YAP signaling. Accordingly, netrin-1 could be a promising therapeutic agent against prostate carcinoma.
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| [28] |
Aberrant up-regulation of Wnt/β-catenin signaling is associated with the development and progression of prostate cancer, but the underlying mechanism is unclear. Here we show that in the absence of androgens, the Wnt/β-catenin pathway activates AR-mediated transcription through up-regulation of the Hippo pathway effector Yes-associated protein (YAP). Wnt3a-conditioned medium (Wnt3a-CM) promotes the growth of LNCaP cells and increases AR and YAP protein levels. Moreover, Wnt3a-CM induces the nuclear translocation of YAP and the AR, but not β-catenin, thereby activating the expression of AR- and YAP-dependent genes, in an androgen-independent manner. In addition, depletion of YAP with small interfering RNA (siRNA) prevented Wnt3a-CM-mediated up-regulation of AR-dependent gene expression. Thus, our findings provide mechanistic insight into the proposed cross-talk between the Wnt/β-catenin and Hippo pathways in androgen-independent prostate cancer development.
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| [29] |
Cell growth and proliferation are tightly coupled to metabolism, and dissecting the signaling molecules which link these processes is an important step toward understanding development, regeneration, and cancer. The transcriptional regulator Yes-associated protein 1 (YAP) is a key regulator of liver size, development, and function. We now show that YAP can also suppress gluconeogenic gene expression. Yap deletion in primary hepatocytes potentiates the gluconeogenic gene response to glucagon and dexamethasone, whereas constitutively active YAP suppresses it. The effects of YAP are mediated by the transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator 1. YAP inhibits its ability to bind to and activate transcription from the promoters of its gluconeogenic targets, and the effects of YAP are blunted upon its knockdown. In vivo, constitutively active YAP lowers plasma glucose levels and increases liver size.
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| [30] |
The transcriptional regulators TAZ and YAP (TAZ/YAP) have emerged as pro-tumorigenic factors that drive many oncogenic traits, including induction of cell growth, resistance to cell death, and activation of processes that promote migration and invasion. Here, we report that TAZ/YAP reprogram cellular energetics to promote the dependence of breast cancer cell growth on exogenous glutamine. Rescue experiments with glutamine-derived metabolites suggest an essential role for glutamate and α-ketoglutarate (AKG) in TAZ/YAP-driven cell growth in the absence of glutamine. Analysis of enzymes that mediate the conversion of glutamate to AKG shows that TAZ/YAP induce glutamic-oxaloacetic transaminase (GOT1) and phosphoserine aminotransferase (PSAT1) expression and that TAZ/YAP activity positively correlates with transaminase expression in breast cancer patients. Notably, we find that the transaminase inhibitor aminooxyacetate (AOA) represses cell growth in a TAZ/YAP-dependent manner, identifying transamination as a potential vulnerable metabolic requirement for TAZ/YAP-driven breast cancer.
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| [31] |
The G protein-coupled estrogen receptor (GPER) mediates both the genomic and nongenomic effects of estrogen and has been implicated in breast cancer development. Here, we compared GPER expression in cancerous tissue and adjacent normal tissue in patients with invasive ductal carcinoma (IDC) of the breast and determined that GPER is highly upregulated in cancerous cells. Additionally, our studies revealed that GPER stimulation activates yes-associated protein 1 (YAP) and transcriptional coactivator with a PDZ-binding domain (TAZ), 2 homologous transcription coactivators and key effectors of the Hippo tumor suppressor pathway, via the Gαq-11, PLCβ/PKC, and Rho/ROCK signaling pathways. TAZ was required for GPER-induced gene transcription, breast cancer cell proliferation and migration, and tumor growth. Moreover, TAZ expression positively correlated with GPER expression in human IDC specimens. Together, our results suggest that the Hippo/YAP/TAZ pathway is a key downstream signaling branch of GPER and plays a critical role in breast tumorigenesis.
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| [32] |
Nowadays, risk factors of triple-negative breast cancer (TNBC) metastasis are not well identified. Our present study reveals that an industrial chemical, bisphenol S (BPS), can promote the migration, but not the proliferation, of TNBC cells in vitro. BPS activates YAP, a key effector of Hippo pathway, by inhibiting its phosphorylation, which promotes YAP nuclear accumulation and up-regulates its downstream genes such as CTGF and ANKRD1. Inhibition of YAP blocks the BPS-triggered cell migration and up-regulation of fibronectin (FN) and vimentin (Vim). BPS rapidly decreases the phosphorylation levels of LATS1 (Ser909) in TNBC cells, which regulates the activation and functions of YAP. Silencing LATS1/2 by siRNA increases BPS-induced dephosphorylation of YAP and extended the half-life of YAP protein. Inhibition of G protein-coupled estrogen receptor 1 (GPER) and its downstream PLCβ/PKC signals attenuate the effects of BPS-induced YAP dephosphorylation and CTGF up-regulation. Targeted inhibition of GPER/YAP inhibits BPS-induced migration of TNBC cells. Collectively, we reveal that GPER/Hippo-YAP signal is involved in BPS-induced migration of TNBC cells.
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PDF(2275 KB)
Table 1 lentivirus sequence表2 q-PCR引物信息表3 质粒信息
图1 转染shPLCε慢病毒后LNCAP和PC3细胞中PLCε的mRNA和蛋白质水平图2 LNCAP和PC3细胞的克隆形成实验图3 LNCAP和PC3细胞的MTT实验(72h)图4 转染shPLCε后LNCAP和PC3细胞中PLCε、YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的mRNA水平图5 转染shPLCε后LNCAP和PC3细胞中YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的蛋白质水平Fig.6 mRNA and protein levels of YAP in LNCAP and PC3 cells infected with over-expression or knockdown of YAP plasmid图7 转染过表达或敲低YAP质粒后LNCAP和PC3细胞中PLCε、YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的mRNA水平图8 转染过表达或敲低YAP质粒后LNCAP和PC3中PLCε、YAP、丝氨酸/甘氨酸生成酶、增殖相关基因的蛋白质水平/
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