Cufí Sílvia, Vazquez-Martin Alejandro, Oliveras-Ferraros Cristina, Corominas-Faja Bruna, Urruticoechea Ander, Martin-Castillo Begoña, Menendez Javier A
Metabolism and Cancer Group, Translational Research Laboratory, Catalan Institute of Oncology-Girona, ICO-Girona.
Oncotarget. 2012 Dec;3(12):1600-14. doi: 10.18632/oncotarget.742.
The autophagic process, which can facilitate breast cancer resistance to endocrine, cytotoxic, and molecularly targeted agents, is mainly regulated at the post-translational level. Although recent studies have suggested a possible transcriptome regulation of the autophagic genes, little is known about either the analysis tools that can be applied or the functional importance of putative candidate genes emerging from autophagy-dedicated transcriptome studies. In this context, we evaluated whether the constitutive activation of the autophagy machinery, as revealed by a transcriptome analysis using an autophagy-focused polymerase chain reaction (PCR) array, might allow for the identification of novel autophagy-specific biomarkers for intrinsic (primary) resistance to HER2-targeted therapies. Quantitative real-time PCR (qRT-PCR)-based profiling of 84 genes involved in autophagy revealed that, when compared to trastuzumab-sensitive SKBR3 cells, the positive regulator of autophagic vesicle formation ATG12 (autophagy-related gene 12) was the most differentially up-regulated gene in JIMT1 cells, a model of intrinsic cross-resistance to trastuzumab and other HER1/2-targeting drugs. An analysis of the transcriptional status of ATG12 in > 50 breast cancer cell lines suggested that the ATG12 transcript is commonly upregulated in trastuzumab-unresponsive HER2-overexpressing breast cancer cells. A lentiviral-delivered small hairpin RNA stable knockdown of the ATG12 gene fully suppressed the refractoriness of JIMT1 cells to trastuzumab, erlotinib, gefitinib, and lapatinib in vitro. ATG12 silencing significantly reduced JIMT1 tumor growth induced by subcutaneous injection in nude mice. Remarkably, the outgrowth of trastuzumab-unresponsive tumors was prevented completely when trastuzumab treatment was administered in an ATG12-silenced genetic background. We demonstrate for the first time the usefulness of low-density, autophagy-dedicated qRT-PCR-based platforms for monitoring primary resistance to HER2-targeted therapies by transcriptionally screening the autophagy interactome. The degree of predictive accuracy warrants further investigation in the clinical situation.
自噬过程可促进乳腺癌对内分泌、细胞毒性和分子靶向药物产生抗性，该过程主要在翻译后水平受到调控。尽管最近的研究表明自噬基因可能存在转录组调控，但对于可应用的分析工具以及自噬特异性转录组研究中出现的假定候选基因的功能重要性却知之甚少。在此背景下，我们评估了使用聚焦自噬的聚合酶链反应（PCR）阵列进行转录组分析所揭示的自噬机制的组成性激活，是否有助于识别针对HER2靶向治疗的内在（原发性）抗性的新型自噬特异性生物标志物。基于定量实时PCR（qRT-PCR）对84个参与自噬的基因进行分析，结果显示，与曲妥珠单抗敏感的SKBR3细胞相比，自噬小泡形成的正调控因子ATG12（自噬相关基因12）是JIMT1细胞中差异上调最明显的基因，JIMT1细胞是对曲妥珠单抗和其他HER1/2靶向药物具有内在交叉抗性的模型。对50多种乳腺癌细胞系中ATG12转录状态的分析表明，在曲妥珠单抗无反应的HER2过表达乳腺癌细胞中，ATG12转录本通常上调。通过慢病毒传递的小发夹RNA稳定敲低ATG12基因，在体外完全抑制了JIMT1细胞对曲妥珠单抗、厄洛替尼、吉非替尼和拉帕替尼的耐药性。ATG12沉默显著降低了裸鼠皮下注射诱导的JIMT1肿瘤生长。值得注意的是，在ATG12沉默的基因背景下给予曲妥珠单抗治疗时，曲妥珠单抗无反应肿瘤的生长被完全阻止。我们首次证明了基于低密度、聚焦自噬的qRT-PCR平台通过转录筛选自噬相互作用组来监测对HER2靶向治疗的原发性抗性的有用性。预测准确性的程度值得在临床情况下进一步研究。
