背景:完整雌性狗中的犬乳腺肿瘤(CMT)为研究转移性人类癌症提供了自然模型。我们先前的研究发现前梯度2(AGR2)的表达升高,主要在内质网(ER)中发现的蛋白质二硫键异构酶(PDI),在CMT组织中,与CMT进展高度相关。我们进一步证明了AGR2表达的增加积极影响细胞外微环境,促进CMT细胞的趋化性。解开潜在的机制对于评估治疗靶向AGR2作为抑制促转移微环境和阻止肿瘤转移的策略的潜力至关重要。
方法:为了鉴定AGR2调节的分泌组,我们对来自异位表达AGR2的两种CMT细胞系的条件培养基(CM)进行了蛋白质组学分析,并与相应的载体表达对照进行了比较。通过异位表达验证了AGR2调节的14-3-3ε(基因:YWHAE)和α-肌动蛋白4(基因:ACTN4)的释放,击倒,并在CMT细胞中敲除AGR2基因。使用差异超速离心或尺寸排阻色谱法分离源自CMT细胞的细胞外囊泡。通过基因敲低研究了14-3-3ε和α-肌动蛋白4在AGR2调节的CM驱动的趋化性中的作用,抗体介导的干扰,和重组蛋白补充剂。此外,14-3-3ε和α-肌动蛋白4释放的临床相关性是使用CMT组织浸泡盐水和CMT患病犬的血清进行评估的。
结果:AGR2调节的分泌组的蛋白质组学分析显示14-3-3ε和α-肌动蛋白4的丰度增加。AGR2的异位表达显着增加了CM中14-3-3ε和α-肌动蛋白4的释放。相反,敲除或敲除AGR2表达显着减少其释放。沉默14-3-3ε或α-肌动蛋白4表达减少了AGR2调节的CM驱动的趋化性。此外,AGR2主要通过非囊泡途径控制14-3-3ε和α-肌动蛋白4的释放,响应内质网(ER)应激和自噬激活。AGR2的敲除导致自噬体中α-肌动蛋白4的积累增加和14-3-3ε易位受损。细胞外14-3-3ε或α-肌动蛋白4的消耗降低了AGR2调节的CM驱动的趋化性,而在CM中补充重组14-3-3ε增强了CM驱动的趋化性。值得注意的是,与配对的非肿瘤样本相比,在CMT组织浸泡盐水中观察到14-3-3ε或α-肌动蛋白4的水平升高,与健康犬相比,在CMT犬的血清中观察到。
结论:本研究阐明了AGR2在协调CMT细胞非常规分泌14-3-3ε和α-肌动蛋白4中的关键作用,从而有助于旁分泌介导的趋化性。深入了解AGR2涉及的ER压力之间的复杂相互作用,自噬,和非常规分泌为改进旨在阻止犬乳腺肿瘤和潜在人类癌症转移的策略提供了基础。
BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis.
METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs.
RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs.
CONCLUSIONS: This study elucidates AGR2\'s pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.