GRP78-targeted nanotherapy against castrate-resistant prostate cancer cells expressing membrane GRP78

Glucose-regulated protein 78, GRP78, is a chaperone protein mainly located in the endoplasmic reticulum (ER) of normal cells. In stress conditions, GRP78 is overexpressed and in different cancer cell types, it is expressed at the cell surface, whereas it stays intracellular in non-cancerous cells. Therefore, it appears as a strategic target to recognize malignant cells. Prostate cancer is one of the most diagnosed cancers in men. The development of castrate resistant tumors and the resistance to chemotherapy frequently occur. The carboxy-terminal ER retention domain is defined by the KDEL amino acid sequence. We developed anti-KDEL functionalized polymeric nanoparticles (NPs) loaded with paclitaxel (Tx) to specifically target prostate cancer cells expressing GRP78. The sensitivity to Tx in different formulations was compared in three prostate cell lines: PNT1B, a normal cell line, PC3, a cancer cell line faintly expressing GRP78 at its surface, and DU145, a cancer cell line expressing GRP78 at its cell surface. Our results show that the targeted formulation significantly increases Tx sensitivity of cell line expressing GRP78 at its surface compared to other treatments suggesting the added value of GRP78 targeted therapy for castrate resistant tumor which expresses GRP78 at its cell surface.     

sGRP78在恶性肿瘤中的研究进展

随着对葡萄糖调节蛋白78（GRP78）的不断研究,发现GRP78不仅存在于内质网,还存在于多种肿瘤细胞的表面,而细胞表面GRP78(sGRP78)作为一种多功能蛋白质,能够与组织型纤溶酶原激活剂（t-PA）、纤溶蛋白酶原（PG）、α2-巨球蛋白、PI3K的调节亚基p85等多种配体结合,参与多种恶性肿瘤细胞的生长、侵袭、转移及耐药等多种生物学特性的调节。越来越多的研究表明,sGRP78可作为恶性肿瘤的生物标志物和治疗靶点。全文就sGRP78在恶性肿瘤中的研究进展作一综述。     

GRP78 as a novel predictor of responsiveness to chemotherapy in breast cancer

The discovery of predictive factors for chemoresistance is critical for improving adjuvant therapy for cancer patients. The 78-kDa glucose-regulated protein (GRP78), widely used as an indicator of the unfolded protein response (UPR), is induced in the tumor microenvironment. In vitro studies suggest that GRP78 confers chemoresistance to topoisomerase inhibitors, such as Adriamycin (doxorubicin). Here, we report on a retrospective cohort study of 127 stage II and III breast cancer patients who were treated with Adriamycin-based chemotherapy. Archival tumor specimens were available for analysis and the relationship of GRP78 expression level to "time to recurrence" (TTR), used as a surrogate marker for drug resistance, was examined. Our data show that 67% of the study subjects expressed high level of GRP78 in their tumors before the initiation of chemotherapy and suggest an association between GRP78 positivity and shorter TTR [hazard ratio (HR), 1.78; P = 0.16]. Interestingly, subgroup analysis reveals that the HR for the GRP78-positive group increased significantly among patients who did not receive further taxane treatment (HR, 3.00; P = 0.022) and among mastectomy patients (HR, 3.33; P = 0.027). The HR was even stronger among mastectomy patients who did not receive further taxane treatment (HR, 4.82; P = 0.010). The use of GRP78 as a predictor for chemoresponsiveness and the potential interaction of GRP78 and/or the UPR pathways with taxanes warrant larger studies.     

GRP78 as potential predictor for breast cancer response to adjuvant taxane therapy

Few predictive markers exist for response to adjuvant chemotherapy in breast cancer. The 78‐kDa glucose‐regulated protein (GRP78) is a potent antiapoptotic factor, conferring drug resistance. Recently, we reported that high GRP78 expression in breast cancer specimens predicts a shorter recurrence‐free survival in patients who received doxorubicin‐based adjuvant chemotherapy. Interestingly, the opposite effect was observed in 25 patients who additionally received a taxane. To confirm this potentially paradigm shifting finding, we investigated whether GRP78 is associated with recurrence‐free survival in an independent cohort of taxane‐treated breast cancer patients. Immunohistochemical staining of GRP78 was performed on archival paraffin‐embedded formalin‐fixed tumor specimens obtained from 48 female breast cancer patients before chemotherapy treatment. These patients received doxorubicin and cyclophosphamide, followed by paclitaxel or docetaxel on a clinical trial. GRP78 expression level was evaluated by a pathologist, masked to all clinical and outcome data. Association between GRP78 expression and recurrence‐free survival was evaluated. GRP78 positivity predicts a better recurrence‐free survival, independent of other prognostic factors [hazard ratio (HR) for moderate positivity: 0.40 (95% confidence interval (CI): 0.087–1.83); HR for strong positivity: 0.16 (95% CI: 0.018–1.50); p_trend = 0.053]. In a pooled analysis with the previous 25 patients, almost identical HRs were obtained with p_trend = 0.024. This provides further evidence that GRP78 is a potential independent predictor for response to taxane‐based adjuvant chemotherapy in breast cancer.     

IGFBP-3 sensitizes antiestrogen-resistant breast cancer cells through interaction with GRP78

IGFBP-3 is known to possess intrinsic biological activities such as anti-tumor property in addition to its IGF/IGF-R axis-dependent actions in a variety of human cancers including breast cancer. To investigate the molecular mechanisms underlying the intrinsic biological actions of IGFBP-3 on breast cancer cells, we performed yeast two-hybrid screening and found GRP78, known to cause drug-resistance, as a binding partner of IGFBP-3. Overexpression of IGFBP-3 in antiestrogen-resistant LCC9 cells showed that IGFBP-3 interacted with GRP78, resulting in disruption of the GRP78-caspase-7 complex, thereby activating caspase-7, and further inducing apoptosis. Combination of overexpression of IGFBP-3 and application of siRNAs against GRP78 led to decrease in cell viability upon ICI 182,780 treatment. These data suggest that IGFBP-3 could sensitize antiestrogen-resistant breast cancer cells to ICI 182,780 by preventing the anti-apoptotic function of GRP78.     

胃癌细胞表面标志CD44表达生物学意义的研究进展

目的:总结CD44分子与胃癌临床病理特征及预后的关系,探讨CD44基因在胃癌发生发展中的作用.方法:应用PubMed及CNKI全文数据库检索系统,以“CD44、胃癌和肿瘤干细胞”等为关键词,检索2003-01-2012-07的相关文献,共检索到45篇文献,其中英文41篇,中文4篇.文献纳入标准:1)CD44分子的生物学特性;2)CD44与肿瘤干细胞的关系;3)CD44与胃癌发生、临床病理特征及患者预后的关系.根据纳入标准,符合分析的文献33篇.结果:CD44+细胞具有自我更新、多向分化、克隆球形成及体内成瘤等肿瘤干细胞/肿瘤起始细胞特性.CD44分子在胃癌组织的表达量显著高于癌旁正常组织,其表达水平越高,癌肿浸润越深,转移概率越大,患者预后越差,并与胃癌的组织类型具有相关性.结论:CD44能够介导胃癌的发生,促进癌细胞的侵袭和转移,其阳性表达影响患者预后,可能会为胃癌的早期诊断和靶向治疗提供新的科学依据.     

Retrovirus-mediated transduction of a short hairpin RNA gene for GRP78 fails to downregulate GRP78 expression but leads to cisplatin sensitization in HeLa cells

Glucose-regulated protein 78 (GRP78) is expressed abundantly in various types of cancer cells and is believed to contribute to chemotherapeutic resistance. In this study, we investigated the effect of a continuous approach for the expression of a short hairpin RNA (shRNA) targeted to GRP78 with retrovirus transduction on the sensitivity to the anticancer drugs VP-16 and cisplatin. The reduction of GRP78 expression failed, and the expression of GRP94 and P5 chaperon mRNA increased; this increase was associated with a mild activation of the unfolded protein response in HeLa cells, which were stably transduced with GRP78 shRNA gene. The transduced cells exhibited similar sensitivity to VP-16-induced cell death when compared to control GFP shRNA gene-transduced cells. However, sensitivity to cisplatin-induced cell death was higher in GRP78 shRNA gene-transduced cells compared to control cells. These results demonstrate that the continuous or prolonged approach targeting GRP78 confers sensitization of HeLa cells to cisplatin independently of the down-regulation of GRP78 expression. The role of the unfolded protein response in sensitization to cisplatin is discussed.     

Nestin as a marker of cancer stem cells

The crucial role of cancer stem cells (CSCs) in the pathology of malignant diseases has been extensively studied during the last decade. Nestin, a class VI intermediate filament protein, was originally detected in neural stem cells during development. Its expression has also been reported in different tissues under various pathological conditions. Specifically, nestin has been shown to be expressed in transformed cells of various human malignancies, and a correlation between its expression and the clinical course of some diseases has been proved. Furthermore, the coexpression of nestin with other stem cell markers was described as a CSC phenotype that was subsequently verified using tumorigenicity assays. The primary aim of this review is to summarize the recent findings regarding nestin expression in CSCs, its possible role in CSC phenotypes, particularly with respect to capacity for self‐renewal, and its utility as a putative marker of CSCs.     

Purified autoantibodies against glucose-regulated protein 78 (GRP78) promote apoptosis and decrease invasiveness of ovarian cancer cells

Circulating glucose-regulated protein 78 (GRP78) autoantibodies represent potential biomarker of epithelial ovarian cancer. However there is relatively limited identification of these antibodies in response to ovarian cancer. Here, we were interested in characterization of these antibodies and into their role in tumor development. We first purified GRP78 autoantibodies from sera of patients with ovarian cancer, and then tested them on SKOV-3 ovarian cancer cell line. A decrease of invasion and an increase of H(2)O(2)-induced apoptosis of SKOV-3 cells were observed, suggesting their protective role against ovarian cancer cells.     

Phage display derived human monoclonal antibodies isolated by binding to the surface of live primary breast cancer cells recognize GRP78

Clinical trials using monoclonal antibodies (mAb) against cell-surface markers have yielded encouraging therapeutic results in several cancer types. Generally, however, anticancer antibodies are only efficient against a subpopulation of cancers, and there is a strong need for identification of novel targets and human antibodies against them. We have isolated single-chain human mAbs from a large na?ve antibody phage display library by panning on a single-cell suspension of freshly isolated live cancer cells from a human breast cancer specimen, and these antibodies were shown to specifically recognize cancer-associated cell-surface proteins. One of the isolated human antibody fragments, Ab39, recognizes a cell-surface antigen expressed on a subpopulation of cancer cell lines of different origins. Immunohistochemical analysis of a large panel of cancerous and normal tissues showed that Ab39 bound strongly to several cancers, including 45% breast carcinomas, 35% lung cancers, and 86% melanomas, but showed no or weak binding to normal tissues. A yeast two-hybrid screen of a large human testis cDNA library identified the glucose-regulated protein of 78 kDa (GRP78) as the antigen recognized by Ab39. The interaction was confirmed by colocalization studies and antibody competition experiments that also mapped the epitope recognized by Ab39 to the COOH terminus of GRP78. The expression of GRP78 on the surface of cancer cells, but not normal cells, makes it an attractive target for cancer therapies including mAb-based immunotherapy. Our results suggest that the human antibody Ab39 may be a useful starting point for further genetic optimization that could render it a useful diagnostic and therapeutic reagent for a variety of cancers.     

GRP78在蛋白酶体抑制剂诱导甲状腺癌细胞凋亡中的作用

目的:探讨葡萄糖调节蛋白78(GRP78)在蛋白酶体抑制剂诱导甲状腺癌细胞凋亡中的作用.方法:对2种人甲状腺未分化癌细胞系FRO、AR0分别设空白对照组和蛋白酶体抑制荆处理组;及siGRP78、随机序列核酸siRNA和错位型siGRP78组.利用蛋白酶体抑制荆MG132(1μtmol/L)、PSI(10 nmol/L)和EPOX(5 nmol/L)作用2种细胞系,实时定量RT-PCR和蛋白质印迹法检测各组细胞GRP78 mRNA、蛋白表达.流式细胞仪检测细胞凋亡.结果:蛋白酶体抑制剂处理24 h后,2种细胞系中GRP78mRNA及蛋白水平保持相似增长高峰,为空白对照组2～3倍(P<0.01);siGRP78转染24 h,细胞总GRP78 mRNA丢失>80%,转染48 h,GRP78蛋白总量丢失>75%.蛋白酶体抑制剂MG132作用后,ARO、FRO细胞凋亡率分别为8.3%和78.3%;当siGRP78靶向沉默GRP78后,蛋白酶体抑制剂诱导2种细胞凋亡率显著增加,AR0细胞凋亡率提高5倍左右(P<0.01).结论:2种甲状腺癌细胞系存在GRP78基因表达,不同程度干扰蛋白酶体抑制荆凋亡诱导作用;沉默GRP78基因表达可提高蛋白酶体抑制荆对肿瘤细胞杀伤率.     

Regeneration of irradiated salivary glands with stem cell marker expressing cells

Background

Stem cell therapy could be a potential way for reducing radiation-induced hyposalivation and improving the patient’s quality of life. However, the identification and purification of salivary gland stem cells have not been accomplished. This study aims to better characterize the stem/progenitor cell population with regenerative potential residing in the mouse salivary gland.

Methods

Mouse submandibular gland tissue, isolated cells and cultured 3 day old salispheres were tested for their expression of stem cell markers c-Kit, CD133, CD49f, and CD24 using immunohistochemistry for tissue and flow cytometry for cells. Mice were locally irradiated with a single dose of 15 Gy and transplanted with cells expressing defined markers.

Results

Cells expressing known stem cell markers are localized in the larger ducts of the mouse salivary gland. Isolated cells and cells from day 3 salispheres also express these markers: c-Kit (0.058% vs. 0.65%), CD133 (6% vs. 5%), CD49f (78% vs. 51%), and CD24 (60% vs. 60%, respectively). Intraglandular transplantation of these cells into irradiated salivary glands of mice resulted in stem cell marker-specific recovery of salivary gland function.

Conclusions

Different stem cell-associated markers are expressed in mouse salivary gland cells, which upon transplantation are able to regenerate the irradiation damaged salivary gland.     

GRP78 promoter polymorphism rs391957 as potential predictor for clinical outcome in gastric and colorectal cancer patients

BackgroundRecently, the analysis of gastric and colorectal tumor specimens determined that 78-kiloDalton glucose-regulated protein (GRP78), an endoplasmic reticulum chaperone, up-regulation serves as an efficient mechanism protecting cells against apoptosis and can confer drug resistance. We tested whether functional polymorphisms within the GRP78 gene are related to clinical outcome in gastric and colorectal cancer (CRC) patients.Patients and methodsBlood samples of 234 stage II/III CRC patients at the University of Southern California (USC) and formalin-fixed paraffin-embedded tissues of 137 patients with localized gastric adenocarcinoma (GA) at USC and Memorial Sloan-Kettering Cancer Centers were obtained. GRP78 polymorphisms analyzed on germline DNA were correlated with clinical outcome using univariate and multivariate analyses.ResultsGA patients with the combined GRP78 rs391957 C/T and T/T genotype were at higher risk for tumor recurrence and death [hazard ratio (HR) 2.61; P < 0.001 and HR 3.17; P < 0.001, respectively], than those with C/C. These findings were subsequently tested in a CRC cohort where patients with the homozygous T/T genotype were at highest risk for tumor recurrence (HR 2.61; P = 0.015). The results remained significant after adjusting for clinicopathologic determinants.ConclusionThese data provide the first evidence that the GRP78 rs391957 polymorphism can predict clinical outcome in localized GA and locally advanced CRC patients.     

非小细胞肺癌体外化疗药物敏感性与GRP78表达的相关性

背景和目的 糖调节蛋白78（GRP78）在乳腺癌细胞中表达增高并与其对化疗药物的耐药性有关，而非小细胞肺癌（NSCLC）中GRP78的表达与其对化疗药物的耐药性是否相关，研究较少。本研究拟探讨NSCLC对8种化疗药物的体外敏感性与GRP78表达的相关性。方法 采用四噻唑蓝（MTT）法对52例手术切除的新鲜NSCLC组织进行8种化疗药物体外敏感性检测；采用免疫组织化学方法检测其GRP78的表达水平。采用Spearman相关分析对NSCLC的耐药性与GRP78表达的相关性进行分析。结果 52例NSCLC对紫杉醇（PTX）、阿霉素（ADM）、卡铂（CBP）、拓扑替康（TPT）、长春瑞滨（NVB）、长春新碱（VCR）、顺铂（DDP）和鬼臼乙叉苷（VP-16）8种化疗药物的耐药率分别为42．31％、57．69％、63．46％、65．38％、67．31％、73．08％、78．85％、90．38％，其中14例表现为完全耐药。GRP78的表达水平随着肺癌恶性程度的提高而增加，且其高表达与肺癌细胞对VP-16、ADM、VCR和TPT的耐药具有相关性（P〈0．05）。结论 MTT法体外化疗药物敏感性实验对NSCLC的治疗具有一定的指导作用，GRP78对判定NSCLC的恶性程度及其耐药性具有一定的参考意义。     

Down-regulation of GRP78 is associated with the sensitivity of chemotherapy to VP-16 in small cell lung cancer NCI-H446 cells

Background  

Chemotherapy resistance remains a major obstacle for the treatment of small cell lung cancer (SCLC). Glucose-regulated protein 78 (GRP78), an endoplasmic reticulum chaperone, plays a critical role in chemotherapy resistance in some cancers. However, whether the suppression of the chaperone can enhance the sensitivity of chemotherapy in SCLC is still unclear.     

Evaluation of squamous cell carcinoma antigen as a new marker for lung cancer

Carcinoembryonic antigen (CEA) is the only tumor marker of proven, although limited, value for the management of patients with non-small cell lung cancer (NSCLC). The authors have prospectively assessed the potential value of a new tumor marker, squamous cell carcinoma antigen (SCC Ag), in a large series of patients with advanced lung cancer (LC). Squamous cell carcinoma antigen and CEA levels were measured in 382 healthy persons (N1 group), 90 patients with benign pulmonary diseases, and 291 patients with LC (129 with SCLC and 162 with NSCLC, including 96 with squamous LC). Carcinoembryonic antigen levels were higher in smokers than in nonsmokers, but smoking habits did not influence the serum concentrations of SCC Ag. Elevated values (above the 95th percentiles of N1, i.e., 7.5 ng/ml for CEA and 3.0 ng/ml for SCC Ag) were observed in 11.1% of patients with benign pulmonary diseases for both markers. Carcinoembryonic antigen was more sensitive than SCC Ag, even for squamous LC (56% versus 35% of elevated values, P less than 0.01). The specificity toward squamous LC was better, however, for SCC Ag, for which levels were elevated in only 8.5% of SCLC and in 18% of other forms of NSCLC, compared with 49% and 55%, respectively, for CEA. Moreover, measurement of SCC Ag and CEA levels did not give redundant information: thus, in squamous LC and SCC Ag level was elevated in 32% of the patients with a normal CEA level, increasing from 57% to 71% the proportion of patients with at least one elevated marker. Lastly, elevation of CEA or SCC Ag levels was an adverse prognostic factor in squamous LC (P = 0.05 for CEA; P = 0.07 for SCC Ag). In conclusion, SCC Ag appears to be worthwhile of further investigation in squamous LC. The authors found that this new marker provided additional information on CEA and that it was more specific for squamous LC than CEA.     

Inhibin as a diagnostic marker for ovarian cancer

The inhibins are a family of growth factors comprised of several different species that are secreted in the female principally from the ovarian follicle. The inhibins perform best as markers of ovarian cancer when measured collectively (total inhibin) by immunoassays targeted to common epitopes. After menopause with the depletion of ovarian follicles, the circulating level of total inhibin becomes undetectable. In contrast, serum total inhibin levels are elevated in women with ovarian cancer, in particular those with granulosa cell tumours and those with the mucinous subtype of epithelial carcinoma. Investigations into the clinical utility of inhibin to detect ovarian cancer have shown that it complements CA125, an established marker of epithelial ovarian cancer, in that each performs best in detecting different subtypes of ovarian cancer. In some published studies, the two markers together have detected up to 95% of ovarian cancers with 95% specificity.     

The unfolded protein response regulator GRP78 is a novel predictive biomarker in colorectal cancer

Adjuvant fluoropyrimidine‐based (5‐FU) chemotherapy is a mainstay of treatment for colorectal cancer (CRC), but only provides benefit for a subset of patients. To improve stratification we examined (for the first time in CRC), whether analysis of GRP78 expression provides a predictive biomarker and performed functional studies to examine the role of GRP78 in sensitivity to 5‐FU. 396 CRC patient samples were collected in a prospective uniform manner and GRP78 expression was determined by immunohistochemistry on tissue microarrays using a well‐validated antibody. Expression was correlated with clinicopathological parameters and survival. The role of GRP78 in 5‐FU sensitivity was examined in CRC cells using siRNA, drug inhibition and flow cytometry. GRP78 expression was significantly elevated in cancer tissue (p < 0.0001), and correlated with depth of invasion (p = 0.029) and stage (p = 0.032). Increased overall 5‐year survival was associated with high GRP78 expression (p = 0.036). Patients with stage II cancers treated by surgery alone, with high GRP78 also had improved survival (71% v 50%; p = 0.032). Stage III patients with high GRP78 showed significant benefit from adjuvant chemotherapy (52% vs. 28%; p = 0.026), whereas patients with low GRP78 failed to benefit (28% vs. 32%; p = 0.805). Low GRP78 was an independent prognostic indicator of reduced overall 5‐year survival (p = 0.004; HR = 1.551; 95%CI 1.155–2.082). In vitro, inhibition of GRP78 reduces apoptosis in response to 5‐FU in p53 wild‐type cells. GRP78 expression may provide a simple additional risk stratification to inform the adjuvant treatment of CRC and future studies should combine analysis with determination of p53 status.