抗 HER2-hTNF-α新型免疫毒素的制备及功能研究

HER2/neu 基因在肿瘤中的过度表达使其成为许多肿瘤的标志分子 . 为了增加过度表达 HER2/neu 的肿瘤细胞对肿瘤坏死因子 (TNF) 的敏感性和提高 HER2/neu 抗体的肿瘤杀伤效应,将抗 HER2/neu 单链抗体 C6.5 与人肿瘤坏死因子 hTNF-α融合,构建了 scFvC6.5-hTNF-α融合蛋白,完成了重组蛋白在大肠杆菌中的表达,产率为 400 μg/L 菌液 . 经过亲和层析和柱复性,融合蛋白的纯度达 95％以上 . ELISA 试验表明, scFvC6.5-hTNF-α能够特异结合 HER2/neu 阳性卵巢癌细胞 SKOV-3 和乳腺癌细胞 MCF-7 ,而不结合 HER2/neu 阴性的黑色素瘤细胞 A375. MTT 试验表明, scFvC6.5-hTNF-α能够选择性地杀伤 SKOV-3 和 MCF-7 细胞,而不影响 A375 细胞的生长 . 这种肿瘤细胞特异性杀伤作用提示该免疫毒素具有肿瘤靶向治疗的潜在应用价值 .     

胃癌HER2蛋白表达及基因拷贝分析

目的：探讨胃癌组织HER2蛋白表达和基因拷贝数增加频率,以及蛋白表达与基因拷贝数之间的相关性。方法：分别采用免疫组织化学（1HC）和显色原位杂交（ClSH）方法,检测80例青岛人胃癌组织中HER2蛋白表达和基因扩增情况。结果：HER2免疫组织化学0者51例、1＋者12例、2＋者12例、3＋者5例。通过CISH分析：这组患者中HER2基因拷贝数扩增者共7例（8．8％）,其中包括基因临界扩增3例（3．8％）。HER2的蛋白表达与基因拷贝数增加相关（P〈0．05）,这两个指标与肿瘤分化程度相关。HER2免疫组织化学3＋和基因扩增结果一致,与乳腺癌相似。结论：这组患者中,具有较高的HER2蛋白表达和基因拷贝数增加的比率,基因扩增可能是其蛋白过表达的主要分子机制。     

慢病毒介导的RNA干涉对乳腺癌SKBR3细胞HER2受体的下调及生长抑制

大约30％的乳腺癌中有表皮生长因子受体家族蛋白HER2的过表达,此类癌症的预后差,恶性程度高。RNA干涉（RNAi）是最近发展起来能特异性抑制哺乳动物细胞中基因表达的新技术。本文在以往获得的能够产生良好基因沉默效应的小干涉RNA（siRNA）的基础上,构建了U6和H1双启动子siRNA表达载体,并转染HER2高表达乳腺癌SKBR3细胞定量测定了其HER2下调效应。随后,siRNA表达盒经LR重组反应被克隆入慢病毒载体中,在成功包装成病毒后,感染SKBR3并经荧光定量PCR、蛋白印迹杂交和流式细胞仪一系列实验证明慢病毒介导的RNAi确实能有效地下调肿瘤抗原HER2的表达。细胞长期增殖实验表明经慢病毒处理后细胞生长得到抑制。我们的研究为进一步阐明HER2与癌症恶化的关系以及发展新的基因治疗药物提供了工具和可能。     

microRNA调控网络与肿瘤

microRNA(miRNA)是一类长度约为22nt的小分子非编码RNA,对基因表达进行转录后调控。基因调控网络由各种回路,如前馈和反馈回路组成。它通过将外界刺激整合成合适的输出信号,控制细胞从增殖到死亡几乎所有的生命活动。已知基因调控网络的失调与肿瘤的发生发展密切相关。新近的实验证据表明,miRNA广泛参与基因调控网络中的反馈和前馈回路。该文主要介绍miRNA在基因表达调控网络中的作用及其与肿瘤发生发展的关系。     

HER-2/neu基因高表达及靶向治疗

HER-2/neu癌基因在许多肿瘤,如乳腺癌、卵巢癌、非小细胞肺癌等肿瘤中高表达,在肿瘤的发生与发展中起重要作用,与肿瘤的转化、转移、复发、预后差、患者生存期缩短有关。HER-2/neu在乳腺癌过度表达率约为20%～30%,编码蛋白P185HER2属生长因子受体家族,抗P185HER2单克隆抗体(Herceptin)作为靶向药物已临床应用治疗HER2/neu高表达乳腺癌。     

果蝇发育中细胞决定和分化与基因表达环境

胚胎发育是个程序化的,复杂而有趣的生命现象。在胚胎发育中,不同细胞的分化和其 功能由基因决定,受到核内遗传物质的控制。而细胞的决定和分化则是在不同的细胞质对细胞核的不断作用下,才能逐步进行。核质之间的相互作用先建立特定的基因表达状态,从而选择性表达发育调控基因或分化基因。发育调控基因产物一旦进入胞质,就可改变原来的基因表达环境,使细胞核进入新的基因表达状态,选择表达新的发育调控基因。如果新的发育调控基因的产物再影响细胞核,改变原来的基因表达状态,其它的发育调控基因的表达就可使胚胎细胞进一步分化。在发育过程中,细胞质和细胞核的这个相互作用不断进行,使控制发育程序的不同基因群在特定的时空中表达,受精卵分裂产生的子细胞才能不断决定和逐步分化,最后形成组成个体所必须的各种细胞类型。     

胃癌组织中HER2 基因及p53蛋白表达及其临床意义

目的:研究胃癌组织中HER2基因及P53蛋白表达情况,分析其对临床诊疗的意义与价值。方法:选取2013年7月到2015年7月我院确诊的胃癌患者110例,检测患者胃癌组织中的HER2蛋白表达与基因扩增,及P53蛋白的表达情况,分析HER2基因扩增与P53蛋白表达与病理关系间的关系。结果:HER2基因扩增率为17.3%(19/110),HER2蛋白表达率为42.7%(47/110),P53蛋白表达率为58.2%(64/110),其中HER2蛋白表达3+、2+者HER2基因的扩增比例分别为3/4、6/9与1+表达者的3/16比较具有统计学意义(P0.05);HER2基因扩增和P53蛋白表达与胃癌淋巴结转移以及浸润程度有关(P0.05);相关性分析显示:HER2基因扩增与P53蛋白表达具有正相关关系(P0.05)。结论:胃癌组织中HER2基因扩增和P53蛋白协同表达,促进胃癌浸润和淋巴结转移,对胃癌早期诊断具有重要参考价值。     

脱落酸应答基因的结构,表达调控及信号转导

许多脱落酸应答的基因已被克隆,并已研究了一些基因的调控。其启动子有G－box和ABARE顺式元件。ABARE与Leuzippier（亮氨酸拉链蛋白）互相作用,ABA与某些基因产物结合以及蛋白质磷酸化在基因表达调控中起重要作用。ABA涉及的信号转导是多途径或单途径的。文中讨论了ABA应答基因的结构、表达调控及信号转导的研究方向。     

报告基因

报告基因(ｒｅｐｏｒｔｅｒｇｅｎｅ)是一种编码可被检测的蛋白质或酶的基因,也就是说,是一个其表达产物非常容易被鉴定的基因。把它的编码序列和基因表达调节序列相融合形成嵌合基因,或与其它目的基因相融合,在调控序列控制下进行表达,从而利用它的表达产物来标定目的基因的表达调控,筛选得到转化体。作为报告基因,在遗传选择和筛选检测方面必须具有以下几个条件:(1)已被克隆和全序列已测定;(2)表达产物在受体细胞中不存在,即无背景,在被转染的细胞中无相似的内源性表达产物;(3)其表达产物能进行定量测定。在植物基因工程研究领域,已使用的…     

HER2与肿瘤浸润转移

原癌基因HER2/neu编码的2型人类表皮生长因子受体（human epidermal growth factor receptor type2,HER2）在许多肿瘤中有不同程度的表达。HER2被激活后可通过多种途径增强肿瘤细胞的浸润、转移能力,比如：促进肿瘤细胞增殖、抑制其凋亡、增加基质金属蛋白酶（matrix metalloproteinase,MMP）和血管内皮生长因子（vascular endot-helial growth factor,VEGF）等的表达。阐明HER2与肿瘤浸润转移的关系,将有可能为延长患者生存期,减少肿瘤复发、转移的针对性治疗提供理论依据。     

HER2/HER3 signaling regulates NK cell-mediated cytotoxicity via MHC class I chain-related molecule A and B expression in human breast cancer cell lines

Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understanding of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we demonstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1β promoted the expression in a process that was antagonized by pharmacological and genetic interference with HER3 but not by the ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related protein kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B-dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells.     

Negative/low HER2 expression alone or combined with E-cadherin positivity is predictive of better prognosis in patients with breast carcinoma

The loss of E-cadherin expression leads to absence of tissue integrity, an essential step in tumor progression. Methylation of CpG islands in the promoter region of the CDH1 gene coding E-cadherin might be an alternative for gene silencing. In the present study, we investigate the expression of E-cadherin and hormone receptors in invasive ductal breast carcinoma (IDCs). Protein expression was analysed immunohistochemically in 87 cases, including 26 familial tumors. The most interesting results revealed a significantly reduced E-cadherin expression in cases with familial history compared to sporadic tumors (p=0.009), as well as with tumors ≤5 cm (p=0.022). Moreover, HER2 over-expression was associated with distant metastasis (p=0.011) and overall survival (p log rank=0.028). Tumors displaying negative/low HER2 expression combined with E-cadherin positivity confer better patient survival (p=0.052). Triple Negative tumors (TN) were more frequently found in patients with advanced grade (GIII) (p=0.001) and TNM (III+IV) (p=0.018) which supports the aggressive behavior of TN tumors. On the other hand, hypermethylation of CDH1 gene promoter was observed in 46% of hereditary cases and strongly associated with loss of E-cadherin expression (p=0.002). Furthermore, patients with unmethylated CDH1 pattern have a better 5-year disease free survival (p=0.021). In conclusion, in patients with hereditary breast cancer, the CpG methylation event contributes to the loss of E-cadherin expression. On the other hand, HER2 over-expression is predictive of worse prognosis, either alone or combined with loss of E-cadherin expression in Tunisian patients with breast cancer.     

Role of HER2 gene overexpression in breast carcinoma

The HER2 proto-oncogene encodes a transmembrane glycoprotein of 185 kDa (p185(HER2)) with intrinsic tyrosine kinase activity. Amplification of the HER2 gene and overexpression of its product induce cell transformation. Numerous studies have demonstrated the prognostic relevance of p185(HER2), which is overexpressed in 10% to 40% of human breast tumors. Recent data suggest that p185(HER2) is a ligand orphan receptor that amplifies the signal provided by other receptors of the HER family by heterodimerizing with them. Ligand-dependent activation of HER1, HER3, and HER4 by EGF or heregulin results in heterodimerization and, thereby, HER2 activation. HER2 overexpression is associated with breast cancer patient responsiveness to doxorubicin, to cyclophosphamide, methotrexate, and fluorouracil (CMF), and to paclitaxel, whereas tamoxifen was found to be ineffective and even detrimental in patients with HER2-positive tumors. In vitro analyses have shown that the role of HER2 overexpression in determining the sensitivity of cancer cells to drugs is complex, and molecules involved in its signaling pathway are probably the actual protagonists of the sensitivity to drugs. The association of HER2 overexpression with human tumors, its extracellular accessibility, as well as its involvement in tumor aggressiveness are all factors that make this receptor an appropriate target for tumor-specific therapies. A number of approaches are being investigated as possible therapeutic strategies that target HER2: (1) growth inhibitory antibodies, which can be used alone or in combination with standard chemotherapeutics; (2) tyrosine kinase inhibitors (TKI), which have been developed in an effort to block receptor activity because phosphorylation is the key event leading to activation and initiation of the signaling pathway; and (3) active immunotherapy, because the HER2 oncoprotein is immunogenic in some breast carcinoma patients.     

Co-Targeting of JNK and HUNK in Resistant HER2-Positive Breast Cancer

Strategies for successful primary treatment of HER2-positive breast cancer include use of the HER2 inhibitors trastuzumab or lapatinib in combination with standard chemotherapy. While successful, many patients develop resistance to these HER2 inhibitors indicating an unmet need. Consequently, current research efforts are geared toward understanding mechanisms of resistance and the signaling modalities that regulate these mechanisms. We have undertaken a study to examine whether signaling molecules downstream of epidermal growth factor receptor, which often act as compensatory signaling outlets to circumvent HER2 inhibition, can be co-targeted to overcome resistance. We identified JNK signaling as a potential area of intervention and now show that inhibiting JNK using the pan-JNK inhibitor, SP600125, is effective in the HER2-positive, resistant JIMT-1 xenograft mammary tumor model. We also investigate potential combination strategies to bolster the effects of JNK inhibition and find that co-targeting of JNK and the protein kinase HUNK can prohibit tumor growth of resistant HER2-positive mammary tumors in vivo.     

Patterns of HER2 Gene Amplification and Response to Anti-HER2 Therapies

A chromosomal region that includes the gene encoding HER2, a receptor tyrosine kinase (RTK), is amplified in 20% of breast cancers. Although these tumors tend to respond to drugs directed against HER2, they frequently become resistant and resume their malignant progression. Gene amplification in double minutes (DMs), which are extrachromosomal entities whose number can be dynamically regulated, has been suggested to facilitate the acquisition of resistance to therapies targeting RTKs. Here we show that ~30% of HER2-positive tumors show amplification in DMs. However, these tumors respond to trastuzumab in a similar fashion than those with amplification of the HER2 gene within chromosomes. Furthermore, in different models of resistance to anti-HER2 therapies, the number of DMs containing HER2 is maintained, even when the acquisition of resistance is concomitant with loss of HER2 protein expression. Thus, both clinical and preclinical data show that, despite expectations, loss of HER2 protein expression due to loss of DMs containing HER2 is not a likely mechanism of resistance to anti-HER2 therapies.     

Prognostic and Predictive Significance of MYC and KRAS Alterations in Breast Cancer from Women Treated with Neoadjuvant Chemotherapy

Breast cancer is a complex disease, with heterogeneous clinical evolution. Several analyses have been performed to identify the risk factors for breast cancer progression and the patients who respond best to a specific treatment. We aimed to evaluate whether the hormone receptor expression, HER2 and MYC genes and their protein status, and KRAS codon 12 mutations may be prognostic or predictive biomarkers of breast cancer. Protein, gene and mutation status were concomitantly evaluated in 116 breast tumors from women who underwent neoadjuvant chemotherapy with doxorubicin plus cyclophosphamide. We observed that MYC expression was associated with luminal B and HER2 overexpression phenotypes compared to luminal A (p<0.05). The presence of MYC duplication or polysomy 8, as well as KRAS mutation, were also associated with the HER2 overexpression subtype (p<0.05). MYC expression and MYC gain were more frequently observed in early-onset compared to late-onset tumors (p<0.05). KRAS mutation was a risk factor of grade 3 tumors (p<0.05). A multivariate logistic regression demonstrated that MYC amplification defined as MYC/nucleus ratio of ≥2.5 was a protective factor for chemotherapy resistance. On the other hand, age and grade 2 tumors were a risk factor. Additionally, luminal B, HER2 overexpression, and triple-negative tumors presented increased odds of being resistant to chemotherapy relative to luminal A tumors. Thus, breast tumors with KRAS codon 12 mutations seem to present a worse prognosis. Additionally, MYC amplification may help in the identification of tumors that are sensitive to doxorubicin plus cyclophosphamide treatment. If confirmed in a large set of samples, these markers may be useful for clinical stratification and prognosis.     

In vivo properties of three human HER2/neu-expressing murine cell lines in immunocompetent mice.

BACKGROUND AND PURPOSE: Expression of the HER2/neu proto-oncogene, a receptor-like transmembrane protein expressed at low levels on some normal cells, is markedly increased in a subset of human breast, colon, lung, and ovarian cancers. A humanized HER2/neu antibody has been tested as a therapeutic agent in several clinical trials, with promising results. We have developed a family of anti-HER2/neu fusion proteins. To evaluate the immunologic efficacy of these proteins, it is critical that tumors expressing the target antigen can grow in immunologically intact mice. METHOD: To produce murine tumors expressing human HER2/neu on the surface, CT26, MC38, and EL4 murine cell lines were transduced by use of a retroviral construct containing the cDNA encoding the human HER2/neu gene. RESULTS: Histologic features and kinetics of tumor growth in subcutaneous space of the human HER2/neu-expressing cells were similar to those of the respective parental cell lines. Intravenous inoculation with these cells induced disseminated malignant disease. Flow cytometric and immmunohistochemical analyses of freshly isolated tumors revealed in vivo expression of human HER2/neu. Secretion of antigen was not detected by use of an ELISA. CONCLUSION: Although an antibody response against the human HER2/neu antigen was observed, this response does not affect the growth rate of the HER2/neu-expressing cells. These murine models may be useful tools for evaluation of anti-cancer therapeutic approaches that target human HER2/neu.