Objective

Carbonic anhydrase IX (CA9) is important in the regulation of intra- and extracellular pH in solid tumors, contributing to cell growth and invasion. In urothelial carcinoma (UC), CA9 has been identified as a urinary marker for disease detection, but its biologic role is unknown. To date, differential gene expression patterns of CA9 in various molecular subtypes and potential effects of CA9 inhibition in UC cells are unknown. We aimed to investigate the function of CA9 and the effects of CA9 inhibition in invasive UC.

Methods

Immunohistochemistry was used to assess CA9 expression in a cohort of 153 patients undergoing radical cystectomy. CA9 expression was correlated with molecular subtype by analysis of the TCGA data and of our own cohort of 223 patients with invasive UC receiving neoadjuvant chemotherapy. CA9 expression was assessed in a panel of 12 UC cell lines by Western Blot and qPCR, and multiple siRNAs were used to silence CA9 in 2 cell lines. Effects of CA9 silencing on cell growth, migration, and invasion were assessed. We also used the small molecule inhibitor U-104 to inhibit CA9 in vitro and in an orthotopic xenograft model.

Results

CA9 expression was higher in cancer tissue compared to benign urothelium and was particularly highly expressed in luminal papillary and basal squamous tumors. CA9 expression did not correlate with outcome after neoadjuvant chemotherapy and/or radical cystectomy. Silencing of CA9 by siRNA diminished invasion but did not induce a consistent change of cell growth and migration. Treatment with U-104 led to cell growth reduction only at high concentrations in vitro and failed to have a significant effect on tumor growth in vivo.

Conclusions

The present study confirms over-expression of CA9 in UC and for the first time shows a correlation with molecular subtypes. However, CA9 expression showed no association with the outcome of patients with muscle invasive bladder cancer and inhibition of CA9 did not lead to a consistent inhibition of tumor growth. Based on these data, CA9 exhibits a role neither as a predictive or prognostic marker nor as a therapeutic target in invasive UC.

中文翻译：

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碳酸酐酶 IX 作为尿路上皮癌潜在治疗靶点的评估

客观的

碳酸酐酶 IX (CA9) 在调节实体瘤的细胞内和细胞外 pH 值中很重要，有助于细胞生长和侵袭。在尿路上皮癌 (UC) 中，CA9 已被确定为疾病检测的泌尿标志物，但其生物学作用尚不清楚。迄今为止，CA9 在各种分子亚型中的差异基因表达模式以及 CA9 抑制在 UC 细胞中的潜在影响尚不清楚。我们旨在研究 CA9 的功能和 CA9 抑制在侵袭性 UC 中的作用。

方法

免疫组织化学用于评估一组 153 名接受根治性膀胱切除术的患者的 CA9 表达。通过分析 TCGA 数据和我们自己的 223 名接受新辅助化疗的侵袭性 UC 患者队列，CA9 表达与分子亚型相关。通过蛋白质印迹和 qPCR 在一组 12 个 UC 细胞系中评估 CA9 表达，并使用多种 siRNA 使 2 个细胞系中的 CA9 沉默。评估了 CA9 沉默对细胞生长、迁移和侵袭的影响。我们还使用小分子抑制剂 U-104 在体外和原位异种移植模型中抑制 CA9。

结果

与良性尿路上皮相比，癌组织中的 CA9 表达更高，并且在管腔乳头状和基底鳞状肿瘤中表达特别高。CA9 表达与新辅助化疗和/或根治性膀胱切除术后的结果无关。siRNA 对 CA9 的沉默减少了侵袭，但不会引起细胞生长和迁移的一致变化。用 U-104 处理仅在体外高浓度下导致细胞生长减少，而未能对体内肿瘤生长产生显着影响。

结论

本研究证实了 CA9 在 UC 中的过度表达，并首次显示出与分子亚型的相关性。然而，CA9 表达与肌肉浸润性膀胱癌患者的结果没有关联，并且 CA9 的抑制不会导致对肿瘤生长的一致抑制。基于这些数据，CA9 在侵袭性 UC 中既不能作为预测或预后标志物，也不能作为治疗靶点。