Nupr1调控非小细胞肺癌细胞迁移、凋亡机制的研究

目的:探讨核蛋白1(Nupr1)调控非小细胞肺癌细胞迁移、凋亡机制的研究。方法:肿瘤抑制剂盐酸素(salinomycin)不同时间处理非小细胞肺癌细胞A549后采用Western Blot法检测非小细胞肺癌细胞A549中Cleaved Caspase-3、Nupr1的蛋白表达;Transwell小室检测Nupr1基因沉默后非小细胞肺癌细胞A549细胞体外迁移、侵袭能力的变化;Western Blot法检测Nupr1沉默后非小细胞肺癌细胞A549 MMP-2、TIMP-1的蛋白表达;流式细胞仪检测Nupr1沉默后非小细胞肺癌细胞A549的凋亡情况。结果:与未经肿瘤抑制剂salinomycin处理对照组相比较,salinomycin处理后的非小细胞肺癌细胞A549中Nupr1蛋白表达量下降,Cleaved Caspase-3蛋白表达量升高,并且随着作用时间呈依赖关系。Nupr1-siRNA转染组的迁移能力相比对照组未转染组下降(64.4±7.2)%,Nupr1-siRNA转染组的侵袭能力相比对照组下降(58.7±7.3)%。与未转染Nupr1-siRNA对照组相比较,转染后TIMP-1的表达明显上调,而MMP-2的表达则明显下调。流式细胞仪检测结果显示Nupr1沉默后非小细胞肺癌细胞A549出现大量凋亡。结论:Nupr1基因沉默后通过上调TIMP-1的表达,下调MMP-2的表达降低肺癌A549细胞的侵袭和迁移能力,进而促进非小细胞肺癌细胞凋亡。     

Identification of 14‐3‐3zeta associated protein networks in oral cancer

Advancements in genomics, proteomics, and bioinformatics have improved our understanding of gene/protein networks involved in intra‐ and intercellular communication and tumor–host interactions. Using proteomics integrated with bioinformatics, previously we reported overexpression of 14‐3‐3ζ in premalignant oral lesions and oral squamous cell carcinoma tissues in comparison with normal oral epithelium. 14‐3‐3ζ emerged as a novel molecular target for therapeutics and a potential prognostic marker in oral squamous cell carcinoma patients. However, the role of 14‐3‐3ζ in development and progression of oral cancer is not known yet. This study aimed to identify the 14‐3‐3ζ associated protein networks in oral cancer cell lines using IP–MS/MS and bioinformatics. A total of 287 binding partners of 14‐3‐3ζ were identified in metastatic (MDA1986) and nonmetastatic (SCC4) oral cancer cell lines including other 14‐3‐3 isoforms (2%), proteins involved in apoptosis (2%), cytoskeleton (9%), metabolism (16%), and maintenance of redox potential (2%). Our bioinformatics analysis revealed involvement of 14‐3‐3ζ in protein networks regulating cell cycle, proliferation, apoptosis, cellular trafficking, and endocytosis in oral cancer. In conclusion, our data revealed several novel protein interaction networks involving 14‐3‐3ζ in oral cancer progression and metastasis.     

Caveolin‐1 as a promoter of tumour spreading: when,how, where and why

Caveolae are non‐clathrin invaginations of the plasma membrane in most cell types; they are involved in signalling functions and molecule trafficking, thus modulating several biological functions, including cell growth, apoptosis and angiogenesis. The major structural protein in caveolae is caveolin‐1, which is known to act as a key regulator in cancer onset and progression through its role as a tumour suppressor. Caveolin‐1 can also promote cell proliferation, survival and metastasis as well as chemo‐ and radioresistance. Here, we discuss recent findings and novel concepts that support a role for caveolin‐1 in cancer development and its distant spreading. We also address the potential application of caveolin‐1 in tumour therapy and diagnosis.     

AEG‐1 induces gastric cancer metastasis by upregulation of eIF4E expression

Gastric cancer is the third leading cause of cancer‐related deaths worldwide, and patients with lymph node, peritoneal and distant metastasis have a poor prognosis. Overexpression of Astrocyte‐elevated gene‐1 (AEG‐1) has been reported to be correlated with the progression and metastasis of gastric cancer. However, its mechanisms are quite unclear. In this study, we found that elevated expression of AEG‐1 was correlated with metastasis in human gastric cancer tissues. Moreover, gain‐ or loss‐of‐function of AEG‐1, respectively, promoted or suppressed epithelial–mesenchymal transition (EMT), migration and invasion of gastric cancer cells. AEG‐1 positively regulated eIF4E, MMP‐9 and Twist expression. Manipulating eIF4E expression by transfection of overexpression constructs or siRNAs partially eliminated AEG‐1‐regulated EMT, cell migration and invasion. In addition, overexpression or knockdown of eIF4E promoted or suppressed EMT, cell migration and invasion in parallel with upregulation of MMP‐9 and Twist expression, while manipulating eIF4E expression partially abrogated AEG‐1‐induced MMP‐9 and Twist. Finally, silencing of AEG‐1 expression not only inhibited tumour growth in parallel with downregulation of eIF4E, MMP‐9 and Twist expression in a xenograft nude mouse model, but also suppressed lymph node and peritoneal metastasis of gastric cancer in an orthotopic nude mouse model. These findings suggest that AEG‐1 promotes gastric cancer metastasis through upregulation of eIF4E‐mediated MMP‐9 and Twist, which provides new diagnostic markers and therapeutic targets for cancer metastasis.     

Significance of PELP1 in ER-negative breast cancer metastasis

Breast cancer metastasis is a major clinical problem. The molecular basis of breast cancer progression to metastasis remains poorly understood. PELP1 is an estrogen receptor (ER) coregulator that has been implicated as a proto-oncogene whose expression is deregulated in metastatic breast tumors and whose expression is retained in ER-negative tumors. We examined the mechanism and significance of PELP1-mediated signaling in ER-negative breast cancer progression using two ER-negative model cells (MDA-MB-231 and 4T1 cells) that stably express PELP1-shRNA. These model cells had reduced PELP1 expression (75% of endogenous levels) and exhibited less propensity to proliferate in growth assays in vitro. PELP1 downregulation substantially affected migration of ER-negative cells in Boyden chamber and invasion assays. Using mechanistic studies, we found that PELP1 modulated expression of several genes involved in the epithelial mesenchymal transition (EMT), including MMPs, SNAIL, TWIST, and ZEB. In addition, PELP1 knockdown reduced the in vivo metastatic potential of ER-negative breast cancer cells and significantly reduced lung metastatic nodules in a xenograft assay. These results implicate PELP1 as having a role in ER-negative breast cancer metastasis, reveal novel mechanism of coregulator regulation of metastasis via promoting cell motility/EMT by modulating expression of genes, and suggest PELP1 may be a potential therapeutic target for metastatic ER-negative breast cancer.     

Tenascin C in metastasis: A view from the invasive front

The extracellular matrix protein tenascin C (TNC) is a large glycoprotein expressed in connective tissues and stem cell niches. TNC over-expression is repeatedly observed in cancer, often at the invasive tumor front, and is associated with poor clinical outcome in several malignancies. The link between TNC expression and poor survival in cancer patients suggests a role for TNC in metastatic progression, which is responsible for the majority of cancer related deaths. Indeed, functional studies using mouse models are revealing new roles of TNC in cancer progression and underscore its important contribution to the development of metastasis. TNC has a pleiotropic role in advancing metastasis by promoting migratory and invasive cell behavior, angiogenesis and cancer cell viability under stress. TNC is an essential component of the metastatic niche and modulates stem cell signaling within the niche. This may be crucial for the fitness of disseminated cancer cells confronted with a foreign environment in secondary organs, that can exert a strong selective pressure on invading cells. TNC is a compelling example of how an extracellular matrix protein can provide a molecular context that is imperative to cancer cell fitness in metastasis.     

Osteopontin contributes to hepatocyte growth factor-induced tumor growth and metastasis formation

The cytokine hepatocyte growth factor (HGF)/scatter factor-1 and its cognate receptor, Met, are involved in the etiology and progression of many types of cancer. Despite recent advances in understanding the signal transduction pathways activated by HGF, the mechanism by which HGF exerts its tumorigenic effect is not well understood. To identify proteins that may be involved in mediating HGF-induced cell motility, invasiveness, and tumorigenesis, we used two separate differential display screening methods to identify changes in gene expression that are initiated by HGF in an epithelial cell culture system. Among several known and unknown genes whose expression was modified, osteopontin (OPN), a protein previously associated with tumorigenesis, was found to be upregulated within 6 h following HGF stimulation. OPN expression was dependent on activation of the PI-3 kinase pathway. Autocrine secretion of HGF resulted in sustained expression of OPN. Downregulation of opn expression by stable antisense transfection attenuated OPN expression and repressed HGF-induced invasiveness in vitro and decreased HGF-mediated tumor growth and metastasis formation in vivo. Constitutive expression of OPN in itself exerted partial invasiveness in vitro, but its expression itself was not sufficient to initiate tumor growth or metastasis formation in vivo. Thus, together with other molecules, OPN activity contributes to HGF-induced tumor growth and invasiveness.     

Nupr1 deficiency downregulates HtrA1, enhances SMAD1 signaling,and suppresses age-related bone loss in male mice

Nuclear protein 1 (NUPR1) is a stress-induced protein activated by various stresses, such as inflammation and oxidative stress. We previously reported that Nupr1 deficiency increased bone volume by enhancing bone formation in 11-week-old mice. Analysis of differentially expressed genes between wild-type (WT) and Nupr1-knockout (Nupr1-KO) osteocytes revealed that high temperature requirement A 1 (HTRA1), a serine protease implicated in osteogenesis and transforming growth factor-β signaling was markedly downregulated in Nupr1-KO osteocytes. Nupr1 deficiency also markedly reduced HtrA1 expression, but enhanced SMAD1 signaling in in vitro-cultured primary osteoblasts. In contrast, Nupr1 overexpression enhanced HtrA1 expression in osteoblasts, suggesting that Nupr1 regulates HtrA1 expression, thereby suppressing osteoblastogenesis. Since HtrA1 is also involved in cellular senescence and age-related diseases, we analyzed aging-related bone loss in Nupr1-KO mice. Significant spine trabecular bone loss was noted in WT male and female mice during 6−19 months of age, whereas aging-related trabecular bone loss was attenuated, especially in Nupr1-KO male mice. Moreover, cellular senescence-related markers were upregulated in the osteocytes of 6−19-month-old WT male mice but markedly downregulated in the osteocytes of 19-month-old Nupr1-KO male mice. Oxidative stress-induced cellular senescence stimulated Nupr1 and HtrA1 expression in in vitro-cultured primary osteoblasts, and Nupr1 overexpression enhanced p16^ink4a expression in osteoblasts. Finally, NUPR1 expression in osteocytes isolated from the bones of patients with osteoarthritis was correlated with age. Collectively, these results indicate that Nupr1 regulates HtrA1-mediated osteoblast differentiation and senescence. Our findings unveil a novel Nupr1/HtrA1 axis, which may play pivotal roles in bone formation and age-related bone loss.     

Mass spectrometry analysis of the native protein complex containing actinin-4 in prostate cancer cells

Actinin-4 was originally identified as an actin-binding protein associated with cell motility and cancer invasion and metastasis. However, actinin-4 forms complexes with a large number of different partner proteins and is speculated to have several distinct functions depending on its partner. The level of actinin-4 expression was found to be significantly lower in prostate cancer cells than in non-cancerous basal cells, and restoration of actinin-4 expression inhibited cell proliferation by prostate cancer cell line 22RV1. Immunoprecipitation and mass spectrometry analysis revealed that actinin-4 forms native complexes with several partner proteins in 22RV1 cells, including with beta/gamma-actin, calmodulin, the clathrin heavy chain, non-muscular myosin heavy chain, heterogeneous nuclear ribonucleoprotein A1, and Ras-GTPase-activating protein SH3 domain-binding protein. Clathrin is a coat protein that covers the internalized membrane pit that forms during early endocytosis. We found that other clathrin-related and unrelated cargo proteins, including dynamin, adaptin-delta, beta subunit of neuronal adaptin-like protein, and p47A, also interact with actinin-4. Immunofluorescence microscopy revealed that dynamin and clathrin co-localized with actinin-4 at the sites of membrane ruffling, and transfection of actinin-4 cDNA facilitated the transport of transferrin into perinuclear endosomes. Endocytosis terminates signaling evoked by cell surface receptors and regulates the recycling of receptors and ligands. We identified a panel of proteins whose expression and/or subcellular localization was regulated by actinin-4 by performing organelle fractionation and ICAT-LC-MS/MS. The decreased expression of actinin-4 protein in prostate cancer cells may cause aberrations in the intracellular trafficking of various cell surface molecules and contribute to carcinogenesis.     

Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness‐induced CCN1 activates β‐catenin nuclear translocation and signaling and that this contributes to upregulate N‐cadherin levels on the surface of the endothelium, in vitro. This facilitates N‐cadherin‐dependent cancer cell–endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness‐induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis.     

Genetic inactivation of the pancreatitis-inducible gene Nupr1 impairs PanIN formation by modulating KrasG12D-induced senescence

Nuclear protein 1 (Nupr1), a small chromatin protein, has a critical role in cancer development, progression and resistance to therapy. Previously, we had demonstrated that Nupr1 cooperates with Kras^G12D to induce pancreas intraepithelial neoplasias (PanIN) formation and pancreatic ductal adenocarcinoma development in mice. However, the molecular mechanisms by which Nupr1 influences Kras-mediated preneoplastic growth remain to be fully characterized. In the current study, we report evidence supporting a role for Nupr1 as a gene modifier of Kras^G12D-induced senescence, which must be overcome to promote PanIN formation. We found that genetic inactivation of Nupr1 in mice impairs Kras-induced PanIN, leading to an increase in β-galactosidase-positive cells and an upregulation of surrogate marker genes for senescence. More importantly, both of these cellular and molecular changes are recapitulated by the results of mechanistic experiments using RNAi-based inactivation of Nupr1 in human pancreatic cancer cell models. In addition, the senescent phenotype, which results from Nupr1 inactivation, is accompanied by activation of the FoxO3a-Skp2-p27^Kip1-pRb-E2F pathway in vivo and in vitro. Thus, combined, these results show, for the first time, that Nupr1 aids oncogenic Kras to bypass senescence in a manner that cooperatively promotes PanIN formation. Besides its mechanistic importance, this new knowledge bears medical relevance as it delineates early pathobiological events that may be targeted in the future as a means to interfere with the formation of preneoplastic lesions early during pancreatic carcinogenesis.Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with <5% survival after 5 years and a median survival of <6 months after diagnosis.¹ PDAC progresses from precursor lesions named pancreas intraepithelial neoplasias (PanINs). In this regard, it has been firmly established that oncogenic mutations in KRAS behave as one of the earliest stimuli for the formation of PanINs.^{2, 3} These data are strongly supported by animal models, such as the Pdx1-Cre; LSL-Kras^G12D transgenic mice, in which the pancreas-specific expression of oncogenic Kras promotes PanIN occurrence⁴ and, at a lower frequency, pancreatic cancer. Thus, the role of Kras as an initiating cancer mutation is one of the best-established pathobiological mechanisms required for the development of pancreatic cancer. Noteworthy, however, during the initiation stage, pancreatic cells not only trigger protumoral processes but also cellular events that aim at counteracting transformation. One of these tumor-suppressive processes elicited by Kras activation is cellular senescence (oncogene-induced senescence). In the pancreas, the induction of senescence underlies the resistance of exocrine cells to oncogenic Kras-mediated transformation⁵ so as to prevent tumor promotion, which is often supported by common diseases such as chronic pancreatitis.⁶ Indeed, tissue injury, as it occurs in pancreatitis, weakens the defense mechanism posed by senescence leading to its bypass by exocrine cells, which can then readily form PanINs.⁵ Therefore, the molecular mechanism that supports the development of oncogene-induced senescence (OIS) needs to be fully elucidated, if we want to advance our understanding of pancreatic cancer development.The nuclear protein 1 (Nupr1) is a basic helix–loop–helix molecule that is strongly induced by acute pancreatitis and several other cell stresses.^{7, 8} Nupr1 is also overexpressed in several types of human cancers, including PDAC. In this regard, the expression of genes that are targets for regulation by Nupr1 has been implicated in key protumorigenic pathways, including cell cycle regulation, matrix remodeling, autophagy, cell cannibalism and apoptosis inhibition.^{9, 10, 11, 12, 13, 14, 15, 16} Moreover, the fundamental role that Nupr1 has in pancreatic tumorigenesis is underscorded by recent results, which showed that, in mice, the oncogenic form of Kras^G12D is unable to promote PanINs in the absence of this chromatin protein,¹⁷ although the mechanisms responsible for this effect remain an area of active investigation. Consequently, we designed the current study with the aim of testing the hypothesis that Nupr1 cooperates with oncogenic Kras to induce PanIN formation by modulating the expression of gene networks that are necessary for bypassing senescence. To address this question, we characterized the effects that Nupr1 inactivation has on Kras-induced senescence using genome-wide expression profiling, as well as both cellular and molecular assays for this process. As a result of these experiments, we found that, indeed, the genetic inactivation of Nupr1 induces cellular senescence in exocrine pancreatic cells and reduces Kras-induced PanIN formation. At the molecular level, we demonstrated that this phenomenon is characterized by the upregulation of gene networks, which are known mediators of this phenomenon, by regulating at the G1/S transition. Taken together, these results provide mechanistic insights into how Nupr1 cooperates with Kras to promote the development of pancreatic preneoplastic lesions by discovering and characterizing a role for this pancreatitis-inducible protein in modulating cellular senescence. Thus, the new information emerging from this study has both mechanistic and biomedical implications for a better understanding of the pathobiology of pancreatic cancer.