Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta University, Augusta, Georgia.
Sheila and David Fuente Graduate Program in Cancer Biology, University of Miami-Miller School of Medicine, Miami, Florida.
Clin Cancer Res. 2020 Jul 1;26(13):3455-3467. doi: 10.1158/1078-0432.CCR-19-2912. Epub 2020 Feb 24.
Poor prognosis of patients with muscle-invasive bladder cancer that often metastasizes drives the need for discovery of molecular determinants of bladder cancer progression. Chondroitin sulfate proteoglycans, including CD44, regulate cancer progression; however, the identity of a chondroitinase (Chase) that cleaves chondroitin sulfate from proteoglycans is unknown. HYAL-4 is an understudied gene suspected to encode a Chase, with no known biological function. We evaluated HYAL-4 expression and its role in bladder cancer.
In clinical specimens, HYAL-4 wild-type (Wt) and V1 expression was evaluated by RT-qPCR, IHC, and/or immunoblotting; a novel assay measured Chase activity. Wt and V1 were stably expressed or silenced in normal urothelial and three bladder cancer cell lines. Transfectants were analyzed for stem cell phenotype, invasive signature and tumorigenesis, and metastasis in four xenograft models, including orthotopic bladder.
HYAL-4 expression, specifically a novel splice variant (V1), was elevated in bladder tumors; Wt expression was barely detectable. V1 encoded a truncated 349 amino acid protein that was secreted. In bladder cancer tissues, V1 levels associated with metastasis and cancer-specific survival with high efficacy and encoded Chase activity. V1 cleaved chondroitin-6-sulfate from CD44, increasing CD44 secretion. V1 induced stem cell phenotype, motility/invasion, and an invasive signature. CD44 knockdown abrogated these phenotypes. V1-expressing urothelial cells developed angiogenic, muscle-invasive tumors. V1-expressing bladder cancer cells formed tumors at low density and formed metastatic bladder tumors when implanted orthotopically.
Our study discovered the first naturally-occurring eukaryotic/human Chase and connected it to disease pathology, specifically cancer. V1-Chase is a driver of malignant bladder cancer and potential predictor of outcome in patients with bladder cancer.
肌层浸润性膀胱癌患者预后不良，常发生转移，这促使我们需要发现膀胱癌进展的分子决定因素。软骨素硫酸蛋白聚糖，包括 CD44，可调节癌症进展；然而，能从蛋白聚糖上切割软骨素硫酸的软骨素酶（Chase）的身份尚不清楚。HYAL-4 是一个研究较少的基因，推测其编码一种 Chase，但目前尚不清楚其生物学功能。我们评估了 HYAL-4 的表达及其在膀胱癌中的作用。
在临床标本中，通过 RT-qPCR、免疫组化和/或免疫印迹评估 HYAL-4 野生型（Wt）和 V1 的表达；一种新的测定方法测量了 Chase 活性。在正常尿路上皮和三种膀胱癌细胞系中，稳定表达或沉默 Wt 和 V1。转染子分析了在四个异种移植模型（包括原位膀胱癌）中的干细胞表型、侵袭特征和肿瘤发生和转移。
HYAL-4 表达，特别是一种新的剪接变体（V1），在膀胱癌中升高；Wt 表达几乎检测不到。V1 编码一个截断的 349 个氨基酸的蛋白质，该蛋白质被分泌。在膀胱癌组织中，V1 水平与转移和癌症特异性生存相关，具有高效性，并编码 Chase 活性。V1 从 CD44 上切割软骨素-6-硫酸，增加 CD44 的分泌。V1 诱导了干细胞表型、运动/侵袭和侵袭特征。CD44 敲低可消除这些表型。表达 V1 的尿路上皮细胞形成血管生成、肌肉浸润性肿瘤。表达 V1 的膀胱癌细胞以低密度形成肿瘤，并在原位植入时形成转移性膀胱癌肿瘤。
我们的研究发现了第一个天然存在的真核/人源 Chase，并将其与疾病病理，特别是癌症联系起来。V1-Chase 是恶性膀胱癌的驱动因素，也是膀胱癌患者预后的潜在预测因子。
