Lorenzo K, Ton P, Clark J L, Coulibaly S, Mach L
Department of Biochemistry, University of Western Australia, Nedlands, Australia.
Cancer Res. 2000 Aug 1;60(15):4070-6.
Penetration of basement membrane layers is a hallmark feature of metastatic tumor cells. The invasive propensity of murine SCC-VII squamous carcinoma cells is in part attributable to the extracellular action of the lysosomal cysteine proteinase cathepsin B. Although most noncancerous cells store this enzyme in the lysosomes, we found that SCC-VII cells release a large fraction (42%) of their newly synthesized procathepsin B into the culture medium. Procathepsins D and L, the precursors of other major lysosomal proteinases, are also secreted in significant amounts (24 and 75%, respectively). In contrast, normal murine 3T3-L1 fibroblasts exocytose only minor amounts of their newly synthesized procathepsins B (10%), D (<1%), and L (16%). Western blotting analysis revealed that SCC-VII cells are deficient in the 300 kDa mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R), a tumor suppressor with a central role in the intracellular transport of lysosomal enzymes. Consistent with the absence of M6P/IGF2R, SCC-VII cells lack dense lysosomes, with the bulk of intracellular acid hydrolases residing in late endosomes/ prelysosomes. On the other hand, the synthesis of the M6P recognition marker on lysosomal enzymes is not impaired in SCC-VII cells, because [33P]Pi is incorporated into the carbohydrate moieties of procathepsins B, D, and L. Furthermore, 69% of the phosphorylated N-linked oligosaccharides synthesized by SCC-VII cells carry phosphomonoester groups and as such constitute high-affinity ligands for M6P receptors. SCC-VII cells express the 46 kDa cation-dependent M6P receptor (MPR46), but intracellular retention of procathepsins B, D, and L is not affected by ammonium chloride and chloroquine, agents known to perturb the M6P receptor system. Our results indicate that failure to express the 300 kDa M6P/IGF2R may enhance the metastatic capacity of tumor cells by inducing the secretion of procathepsins B, D, and L.
穿透基底膜层是转移性肿瘤细胞的一个标志性特征。小鼠SCC - VII鳞状癌细胞的侵袭倾向部分归因于溶酶体半胱氨酸蛋白酶组织蛋白酶B的细胞外作用。尽管大多数非癌细胞将这种酶储存在溶酶体中，但我们发现SCC - VII细胞将其新合成的组织蛋白酶B的很大一部分（42%）释放到培养基中。其他主要溶酶体蛋白酶的前体，组织蛋白酶D和L，也大量分泌（分别为24%和75%）。相比之下，正常小鼠3T3 - L1成纤维细胞仅少量胞吐其新合成的组织蛋白酶B（10%）、D（<1%）和L（16%）。蛋白质印迹分析表明，SCC - VII细胞缺乏300 kDa的甘露糖6 - 磷酸/胰岛素样生长因子II受体（M6P/IGF2R），这是一种在溶酶体酶的细胞内运输中起核心作用的肿瘤抑制因子。与缺乏M6P/IGF2R一致，SCC - VII细胞缺乏致密的溶酶体，大部分细胞内酸性水解酶存在于晚期内体/前溶酶体中。另一方面，SCC - VII细胞中溶酶体酶上M6P识别标记的合成并未受损，因为[33P]Pi被掺入到组织蛋白酶B、D和L的碳水化合物部分。此外，SCC - VII细胞合成的69%的磷酸化N - 连接寡糖带有磷酸单酯基团，因此构成M6P受体的高亲和力配体。SCC - VII细胞表达46 kDa的阳离子依赖性M6P受体（MPR46），但氯化铵和氯喹（已知会干扰M6P受体系统的试剂）对组织蛋白酶B、D和L的细胞内滞留没有影响。我们的结果表明，未能表达300 kDa的M6P/IGF2R可能通过诱导组织蛋白酶B、D和L的分泌来增强肿瘤细胞的转移能力。
