Protein disulphide isomerase A3 (PDIA3) is an endoplasmic reticulum (ER)-resident disulphide isomerase and oxidoreductase with known substrates that include some extracellular matrix (ECM) proteins. PDIA3 is up-regulated in invasive breast cancers and correlates in a mouse orthotopic xenograft model with breast cancer metastasis to bone. However, the underlying cellular mechanisms remain unclear. Here we investigated the function of protein disulphide isomerases in attachment, spreading and migration of three human breast cancer lines representative of luminal (MCF-7) or basal (MDA-MB-231 and HCC1937) tumour phenotypes. Pharmacological inhibition by 16F16 decreased initial cell spreading more effectively than inhibition by PACMA-31. Cells displayed diminished cortical F-actin projections, stress fibres and focal adhesions. Cell migration was reduced in a quantified 'scratch wound' assay. To examine whether these effects might result from alterations to secreted proteins in the absence of functional PDIA3, adhesion and migration were quantified in the above cells exposed to media conditioned by wildtype (WT) or Pdia3-/- mouse embryonic fibroblasts (MEFs). The conditioned medium (CM) of Pdia3-/- MEFs was less effective in promoting cell spreading and F-actin organisation or supporting 'scratch wound' closure. Similarly, ECM prepared from HCC1937 cells after 16F16 inhibition was less effective than control ECM to support spreading of untreated HCC1937 cells. Overall, these results advance the concept that protein disulphide isomerases including PDIA3 drive the production of secreted proteins that promote a microenvironment favourable to breast cancer cell adhesion and motility, characteristics that are integral to tumour invasion and metastasis. Inhibition of PDIA3 or related isomerases may have potential for anti-metastatic therapies.

中文翻译：

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通过抑制三种乳腺癌细胞系中的蛋白质二硫键异构酶损害细胞粘附和迁移。

蛋白二硫键异构酶A3（PDIA3）是内质网（ER）驻留的二硫键异构酶和氧化还原酶，具有已知的底物，包括一些细胞外基质（ECM）蛋白。PDIA3在浸润性乳腺癌中上调，在小鼠原位异种移植模型中与乳腺癌转移至骨相关。但是，潜在的细胞机制仍不清楚。在这里，我们研究了蛋白质二硫键异构酶在三种代表腔（MCF-7）或基础（MDA-MB-231和HCC1937）肿瘤表型的人类乳腺癌细胞系的附着，扩散和迁移中的功能。16F16的药理抑制作用比PACMA-31的抑制作用更有效地降低了初始细胞扩散。细胞显示皮质F-肌动蛋白投射，应力纤维和粘着斑减少。在定量的“划痕伤口”测定中减少了细胞迁移。为了检查这些作用是否可能是由于在没有功能性PDIA3的情况下分泌蛋白的改变而引起的，在暴露于野生型（WT）或Pdia3-/-小鼠胚胎成纤维细胞（MEFs）的培养基中，对上述细胞中的粘附和迁移进行了定量。Pdia3-/-MEF的条件培养基（CM）在促进细胞扩散和F-肌动蛋白组织或支持“从头开始”闭合方面效果较差。同样，抑制16F16后由HCC1937细胞制备的ECM在支持未处理的HCC1937细胞扩散方面不如对照ECM有效。总体，这些结果推动了包括PDIA3在内的蛋白质二硫键异构酶驱动分泌的蛋白质产生的构想，这些分泌的蛋白质促进了有利于乳腺癌细胞粘附和运动的微环境，这是肿瘤侵袭和转移所必需的。抑制PDIA3或相关异构酶可能具有抗转移疗法的潜力。