Expression of HER2 and estrogen receptor alpha depends upon nuclear localization of Y-box binding protein-1 in human breast cancers

In our present study, we examined whether nuclear localization of Y-box binding protein-1 (YB-1) is associated with the expression of epidermal growth factor receptors (EGFR), hormone receptors, and other molecules affecting breast cancer prognosis. The expression of nuclear YB-1, clinicopathologic findings, and molecular markers [EGFR, HER2, estrogen receptor (ER)alpha, ER beta, progesterone receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), phosphorylated Akt, and major vault protein/lung resistance protein] were immunohistochemically analyzed. The association of the expression of nuclear YB-1 and the molecular markers was examined in breast cancer cell lines using microarrays, quantitative real-time PCR, and Western blot analyses. Knockdown of YB-1 with siRNA significantly reduced EGFR, HER2, and ER alpha expression in ER alpha-positive, but not ER alpha-negative, breast cancer cell lines. Nuclear YB-1 expression was positively correlated with HER2 (P = 0.0153) and negatively correlated with ER alpha (P = 0.0122) and CXCR4 (P = 0.0166) in human breast cancer clinical specimens but was not correlated with EGFR expression. Nuclear YB-1 expression was an independent prognostic factor for overall (P = 0.0139) and progression-free (P = 0.0280) survival. In conclusion, nuclear YB-1 expression might be essential for the acquisition of malignant characteristics via HER2-Akt-dependent pathways in breast cancer patients. The nuclear localization of YB-1 could be an important therapeutic target against not only multidrug resistance but also tumor growth dependent on HER2 and ER alpha.     

慢病毒导入的CDC2582 siRNA对胰腺癌细胞CFPAC-1生物学行为的影响

目的利用已构建成功的、在人胰腺癌细胞株CFPAC-1中稳定抑制细胞分裂周期25同源体B(CDC2582)的重组慢病毒,建立CDC2582表达降低的细胞株,以研究该基因的作用.方法将产生CDC2582小干扰RNA(siRNA)分子的、携带绿色荧光蛋白的重组慢病毒感染CFPAC-1细胞后,用流式细胞仪筛选稳定表达的细胞株.应用RT-PCR和Western blot分别对细胞中CDC2582的mRNA和蛋白表达水平进行分析.应用MTT法、流式细胞仪、平板克隆形成和Transwell小室法分别检测其对细胞增殖、周期、细胞克隆形成能力和迁移侵袭能力的影响.结果 CDC2582 siRNA显著下调CFPAC-1细胞中CDC2582的表达,所构建的重组慢病毒导入的siRNA有较高的沉默效率,CFPAC-1细胞的增殖能力、克隆形成能力以及迁移侵袭能力显著增强,细胞周期无明显改变.结论 CDC2582基因可显著影响胰腺癌CFPAC-1细胞的增殖、克隆形成和迁移能力,为利用CDC2582所介导的信号通路进行肿瘤基因治疗提供了实验依据.     

Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression

Summary We have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1–S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.     

Akt-dependent nuclear localization of Y-box-binding protein 1 in acquisition of malignant characteristics by human ovarian cancer cells

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.     

弥漫性大B细胞淋巴瘤中YB-1核表达的临床意义

 目的　探讨Y-盒结合蛋白（YB-1）在弥漫性大B细胞淋巴瘤(DLBCL)中表达及其临床意义。方法　采用Envision两步法检测50例DLBCL和10例反应性淋巴结增生（RLH）患者石蜡标本中YB-1与增生细胞核抗原（PCNA）的表达,分析YB-1表达与临床参数及PCNA表达的关系。结果　DLBCL组与RLH组的YB-1胞质阳性表达率差异无统计学意义（P＞0.05）,而DLBCL组的YB-1胞核阳性表达率明显高于RLH组（P＜0.05）;DLBCL临床Ⅲ~Ⅳ期患者的YB-1胞核阳性表达率明显高于Ⅰ~Ⅱ期（P＜0.05）;结外淋巴组织侵犯组的YB-1胞核阳性表达率明显高于结内病变组（P＜0.05）;骨髓浸润组的YB-1胞核阳性表达率显著高于无骨髓浸润组（P＜0.05）;YB-1的胞核阳性表达与PCNA积分呈正相关（P＜0.05）;化疗无效组的YB-1核阳性表达率及PCNA积分均明显高于化疗有效组（P＜0.05）。结论　YB-1的核阳性表达可能参与了肿瘤的发生;YB-1核阳性表达与肿瘤的高侵袭力及肿瘤细胞的高增生能力密切相关,提示患者预后不良。     

Alteration of Y-box binding protein-1 expression modifies the response to endocrine therapy in estrogen receptor-positive breast cancer

Y-box binding protein-1 (YB-1) plays an important role in tumor progression and drug resistance. This study examined whether YB-1 is involved in the alteration of response to endocrine therapy in estrogen receptor (ER)-positive breast cancer cells. MCF7 cells that stably expressed YB-1 (MCF7-YB-1) and vector control cells (MCF7-vector) were established. These cells were used to analyze the expression of the factors related to ER and growth factor receptor signaling pathways and responses to antiestrogens (tamoxifen and fulvestrant) and estrogen responsive element (ERE) activity. The effect of knocking down endogenous YB-1 expression was tested in wild-type MCF7 cells. In addition, the expression of YB-1 and the factors related to ER and growth factor receptor signaling pathways were evaluated in clinical breast cancers treated with preoperative chemotherapy. The expression of HER2, AIB1, p-Erk, and c-Myc was increased in MCF7-YB-1 cells. In contrast, knocking down of YB-1 decreased the expression of these factors but increased the expression of ERα in wild-type MCF7 cells. Furthermore, sensitivity to antiestrogens was decreased in the MCF7-YB-1 in comparison to that in MCF7-vector cells. The introduction of YB-1 into MCF7 cells inhibited apoptosis and cell cycle arrest at G1 phase induced by antiestrogens. In MCF7-YB-1 cells, the expression levels of p-Erk and c-Myc were continuously upregulated when cells were treated with either tamoxifen or fulvestrant. The ERE activity was reduced in MCF7-YB-1 cells in comparison to MCF7-vector cells, and the ERE activity in MCF7-YB-1 cells was inhibited by fulvestrant at a lower concentration than that which inhibited the ERE activity in MCF7-vector cells. In ER-positive clinical breast cancers treated with preoperative chemotherapy, significantly more number of specimens that showed increased or positive YB-1 expression after chemotherapy was positive for HER2 expression. These data suggest that alteration of YB-1 may modify the crosstalk between the ER pathway and HER2 pathway in ER-positive breast cancer cells, and consequently, may alter the response to endocrine therapy in ER-positive breast cancer cells.     

YB-1 bridges neural stem cells and brain tumor-initiating cells via its roles in differentiation and cell growth

The Y-box binding protein 1 (YB-1) is upregulated in many human malignancies including glioblastoma (GBM). It is also essential for normal brain development, suggesting that YB-1 is part of a neural stem cell (NSC) network. Here, we show that YB-1 was highly expressed in the subventricular zone (SVZ) of mouse fetal brain tissues but not in terminally differentiated primary astrocytes. Conversely, YB-1 knockout mice had reduced Sox-2, nestin, and musashi-1 expression in the SVZ. Although primary murine neurospheres were rich in YB-1, its expression was lost during glial differentiation. Glial tumors often express NSC markers and tend to loose the cellular control that governs differentiation; therefore, we addressed whether YB-1 served a similar role in cancer cells. YB-1, Sox-2, musashi-1, Bmi-1, and nestin are coordinately expressed in SF188 cells and 9/9 GBM patient-derived primary brain tumor-initiating cells (BTIC). Silencing YB-1 with siRNA attenuated the expression of these NSC markers, reduced neurosphere growth, and triggered differentiation via coordinate loss of GSK3-β. Furthermore, differentiation of BTIC with 1% serum or bone morphogenetic protein-4 suppressed YB-1 protein expression. Likewise, YB-1 expression was lost during differentiation of normal human NSCs. Consistent with these observations, YB-1 expression increased with tumor grade (n = 49 cases). YB-1 was also coexpressed with Bmi-1 (Spearmans 0.80, P > 0.001) and Sox-2 (Spearmans 0.66, P > 0.001) based on the analysis of 282 cases of high-grade gliomas. These proteins were highly expressed in 10/15 (67%) of GBM patients that subsequently relapsed. In conclusion, YB-1 correlatively expresses with NSC markers where it functions to promote cell growth and inhibit differentiation.     

Targeting of cytosolic phospholipase A2α impedes cell cycle re-entry of quiescent prostate cancer cells

Cell cycle re-entry of quiescent cancer cells has been proposed to be involved in cancer progression and recurrence. Cytosolic phospholipase A₂α (cPLA₂α) is an enzyme that hydrolyzes membrane glycerophospholipids to release arachidonic acid and lysophospholipids that are implicated in cancer cell proliferation. The aim of this study was to determine the role of cPLA₂α in cell cycle re-entry of quiescent prostate cancer cells. When PC-3 and LNCaP cells were rendered to a quiescent state, the active form of cPLA₂α with a phosphorylation at Ser⁵⁰⁵ was lower compared to their proliferating state. Conversely, the phospho-cPLA₂α levels were resurgent during the induction of cell cycle re-entry. Pharmacological inhibition of cPLA₂α with Efipladib upon induction of cell cycle re-entry inhibited the re-entry process, as manifested by refrained DNA synthesis, persistent high proportion of cells in G₀/G₁ and low percentage of cells in S and G₂/M phases, together with a stagnant recovery of Ki-67 expression. Simultaneously, Efipladib prohibited the emergence of Skp2 while maintained p27 at a high level in the nuclear compartment during cell cycle re-entry. Inhibition of cPLA₂α also prevented an accumulation of cyclin D1/CDK4, cyclin E/CDK2, phospho-pRb, pre-replicative complex proteins CDC6, MCM7, ORC6 and DNA synthesis-related protein PCNA during induction of cell cycle re-entry. Moreover, a pre-treatment of the prostate cancer cells with Efipladib during induction of cell cycle re-entry subsequently compromised their tumorigenic capacity in vivo. Hence, cPLA₂α plays an important role in cell cycle re-entry by quiescent prostate cancer cells.     

Y-box factor YB-1 predicts drug resistance and patient outcome in breast cancer independent of clinically relevant tumor biologic factors HER2, uPA and PAI-1.

Intrinsic or acquired resistance to chemotherapy is responsible for failure of current treatment regimens in breast cancer patients. The Y-box protein YB-1 regulates expression of the P-glycoprotein gene mdr1, which plays a major role in the development of a multidrug-resistant tumor phenotype. In human breast cancer, overexpression and nuclear localization of YB-1 is associated with upregulation of P-glycoprotein. In our pilot study, we analyzed the clinical relevance of YB-1 expression in breast cancer (n = 83) after a median follow-up of 61 months and compared it with tumor-biologic factors already used for clinical risk-group discrimination, i.e., HER2, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1). High YB-1 expression in tumor tissue and surrounding benign breast epithelial cells was significantly associated with poor patient outcome. In patients who received postoperative chemotherapy, the 5-year relapse rate was 66% in patients with high YB-1 expression. In contrast, in patients with low YB-1 expressions, no relapse has been observed so far. YB-1 expression thus indicates clinical drug resistance in breast cancer. Moreover, YB-1 correlates with breast cancer aggressiveness: in patients not treated with postoperative chemotherapy, those with low YB-1 expression are still free of disease, whereas the 5-year relapse rate in those with high YB-1 was 30%. There was no significant correlation between YB-1 expression and either HER2 expression or uPA and PAI-1 levels. Risk-group assessment achieved by YB-1 differed significantly from that by HER2 or uPA/PAI-1. In conclusion, YB-1 demonstrated prognostic and predictive significance in breast cancer by identifying high-risk patients in both the presence and absence of postoperative chemotherapy, independent of tumor-biologic factors currently available for clinical decision making.     

miR-147a通过抑制CCND1和CDK4的表达对膀胱癌细胞周期和增殖的影响

目的:探讨miR-147a在膀胱癌组织、细胞中的表达及对膀胱癌细胞增殖和细胞周期的影响,研究miR-147a靶向负性调控CCND1和CDK4的表达对膀胱癌细胞周期和增殖的影响。方法:通过qRT-PCR检测miR-147a在正常膀胱组织和膀胱癌组织及正常膀胱上皮细胞、膀胱癌细胞系中的表达;使用miR-147a mimics转染观察细胞特性变化;CCK-8检测方法观察细胞增殖能力变化;流式细胞术观察细胞周期变化;生物信息学方法分析miR-147a的靶基因,并将靶基因进行GO分类和KEGG分析,找到与增殖和周期相关的基因;通过western blot方法验证miR-147a的靶基因,使用双萤光素酶报告基因系统检测miR-147a对CCND1和CDK4的转录活性影响;使用CCND1和CDK4特异性siRNA观察它们对细胞周期的影响,使用miR-147a inhibitor进行回补实验。结果:同正常组织/细胞相比,miR-147a在膀胱癌组织/膀胱癌细胞系中表达水平显著降低（P＜0.05）,miR-147a能够使膀胱癌细胞发生G1/S期阻滞,细胞增殖能力降低;通过一系列生信分析找到两个与细胞增殖和周期相关的miR-147a的靶基因CCND1和CDK4;通过间接和直接实验验证CCND1和CDK4是miR-147a的靶基因;下调CCND1和CDK4能够使膀胱癌细胞发生G1/S期阻滞,同时抑制miR-147a的表达能够解除G1/S期阻滞。结论:miR-147a作为一个抑癌因子,可作为诊断膀胱癌的生物标志物;miR-147a通过抑制CCND1和CDK4的表达抑制膀胱癌细胞周期G1/S期进程并抑制膀胱癌细胞增殖。     

CDK1、CDK2 siRNA干扰对肿瘤细胞凋亡和细胞周期的影响

Objective: We investigated the influence of CDK1 and CDK2 expression inhibited by cotransfection of CDK1 and CDK2 siRNA on cell cycle and apoptosis, explored the exact role of cell cycle master regulator in tumor cell apoptosis process. Methods: The siRNA targeting the CDK1 and CDK2 genes were synthesized and simultaneously cotransfected into Hela cells by lipofectamine 2000.48 or 60 h after the cotransfection, CDK1 and CDK2 protein expressions were examined by Western blot. Cell cycle distribution was analyzed by flow cytometry. Cell apoptosis was detected by the Annexin V/PI method. The changes of the transfected cell morphological under a microscope after Wright-Giemsa Staining were studied. Results: CDK1 and CDK2 protein expression was decreased at 48 or 60 h after cotransfection. The accumulation of the G2/M and S phase population in cell cycle of the cotrensfected cells at 48 or 60 h after transfection was enhanced obviously compared with control. The ratio of apoptotic cell of cotransfected cells at 48 or 60 h after transfection was increased significantly compared with control. More binucleate or multinucleate cells among cotransfected cells were observed under the microscope. Conclu- sion: The decreased expression of CDK1 and CDK2 by cotransfection of CDK1 and CDK2 siRNA not only leads to tumor cell cycle arrest in S phase and G2/M phase, but also induces tumor cell apoptosis.