Joshi Bharat H, Leland Pamela, Lababidi Samir, Varrichio Frederick, Puri Raj K
Tumor Vaccines and Biotechnology Branch, Division of Cellular and Gene Therapies, Office of Cellular, Tissue and Gene Therapy, Center for Biologics Evaluation and Research, NIH Building 29B, Room 2E1229 Lincoln Drive, Bethesda, 20892, Maryland.
Cancer Med. 2014 Dec;3(6):1615-28. doi: 10.1002/cam4.330. Epub 2014 Sep 10.
Previously, we have demonstrated that interleukin-4 receptor α (IL-4Rα) is overexpressed on a variety of human cancers and can serve as target for IL-4 immunotoxin comprised of IL-4 and a mutated Pseudomonas exotoxin. However, its expression and association with grade and clinical stage of bladder cancer has not been studied. IL-4Rα expression was examined in human bladder cancer cell lines, mouse xenografts, and biopsy specimens at mRNA and protein levels by real-time RT-PCR and IHC/ISH techniques. We also examined the effect of IL-4 on proliferation and invasion of bladder carcinoma cell lines. For tissue microarray (TMA) results, we analyzed the precision data using exact binomial proportion with exact two-sided P-values. We used Cochran-Armitage Statistics with exact two-sided P-values to examine the trend analysis of IL-4Rα over grade or stage of the bladder cancer specimens. The influence of age and gender covariates was also analyzed using multiple logistic regression models. IL-4Rα is overexpressed in five bladder cancer cell lines, while normal bladder and human umbilical vein cell lines (HUVEC) expressed at low levels. Two other chains of IL-4 receptor complex, IL-2RγC and IL-13Rα1, were absent or weakly expressed. IL-4 modestly inhibited the cell proliferation, but enhanced cell invasion of bladder cancer cell lines in a concentration-dependent manner. Bladder cancer xenografts in immunodeficient mice also maintained IL-4Rα overexpression in vivo. Analysis of tumor biopsy specimens in TMAs revealed significantly higher IL-4Rα immunostaining (≥ 2+) in Grade 2 (85%) and Grade 3 (97%) compared to Grade 1 tumors (0%) (P ≤ 0.0001). Similarly, 9% stage I tumors were positive for IL-4Rα (≥ 2+) compared to 84% stage II (P ≤ 0.0001) and 100% stages III-IV tumors (P ≤ 0.0001). IL-13Rα1 was also expressed in tumor tissues but at low levels and it did not show any correlation with the grade and stage of disease. However, the IL-2RγC was not expressed. Ten normal bladder specimens demonstrated ≤ 1+ staining for IL-4Rα and IL-13Rα1 and no staining for IL-2RγC. These results demonstrate that IL-4Rα is overexpressed in human bladder cancer, which correlates with advanced grade and stage of the disease. Thus, IL-4Rα may be a bladder tumor-associated protein and a prognostic biomarker.
此前，我们已经证明白细胞介素-4受体α（IL-4Rα）在多种人类癌症中过表达，并且可以作为由IL-4和突变的铜绿假单胞菌外毒素组成的IL-4免疫毒素的靶点。然而，其在膀胱癌中的表达以及与膀胱癌分级和临床分期的关系尚未得到研究。通过实时RT-PCR和免疫组化/原位杂交技术，在mRNA和蛋白质水平上检测了人膀胱癌细胞系、小鼠异种移植瘤和活检标本中的IL-4Rα表达。我们还研究了IL-4对膀胱癌细胞系增殖和侵袭的影响。对于组织微阵列（TMA）结果，我们使用精确二项式比例和精确双侧P值分析精确数据。我们使用具有精确双侧P值的 Cochr an-Armitage统计量来检验IL-4Rα在膀胱癌标本分级或分期上的趋势分析。还使用多元逻辑回归模型分析了年龄和性别协变量的影响。IL-4Rα在五种膀胱癌细胞系中过表达，而正常膀胱和人脐静脉细胞系（HUVEC）表达水平较低。IL-4受体复合物的另外两条链，IL-2RγC和IL-13Rα1，不存在或弱表达。IL-4适度抑制细胞增殖，但以浓度依赖的方式增强膀胱癌细胞系的细胞侵袭。免疫缺陷小鼠中的膀胱癌异种移植瘤在体内也维持IL-4Rα过表达。TMA中肿瘤活检标本的分析显示，与1级肿瘤（0%）相比，2级（85%）和3级（97%）肿瘤中IL-4Rα免疫染色（≥2+）显著更高（P≤0.0001）。同样，I期肿瘤中9%的IL-4Rα呈阳性（≥2+），而II期为84%（P≤0.0001），III-IV期为100%（P≤0.0001）。IL-13Rα1也在肿瘤组织中表达，但水平较低，并且与疾病的分级和分期没有任何相关性。然而，IL-2RγC未表达。十个正常膀胱标本中IL-4Rα和IL-13Rα1的染色≤1+，IL-2RγC无染色。这些结果表明，IL-4Rα在人膀胱癌中过表达，这与疾病的高级别和晚期相关。因此，IL-4Rα可能是一种膀胱肿瘤相关蛋白和预后生物标志物。
