Mass spectrometry to decode PTM patterns and enhance the Biomarker utility of ER

描述(由申请人提供)：雌激素受体(ER，α亚型)是第一个被临床证实为癌症治疗反应预测指标的生物标志物，并且仍然是少数几个有足够医学证据证明其在临床决策中常规使用的肿瘤生物标志物之一。虽然低表达或缺乏肿瘤ER表达(ER-)可以准确地预测内分泌治疗缺乏反应性，但ER+的肿瘤过度表达是一个很差的预测指标，其准确率平均只有50%。因此，提高目前仅测量肿瘤ER含量的ER分析的预测准确性是癌症研究中最重要的悬而未决的问题之一。由于已知任何给定蛋白质上的翻译后修饰(PTM)是决定构象、定位和细胞内功能的分子代码，我们组建了一个由翻译研究人员和蛋白质化学家组成的跨学科团队，他们开发了质谱学(MS)方法，使用多反应监测(MRM)能够检测和定量各种翻译后修饰，包括丝氨酸/苏氨酸/酪氨酸磷酸化、赖氨酸修饰(乙酰化/甲基化/泛素化)和半胱氨酸氧化内源表达的ER蛋白的六个区域。两个研究目标是基于这样一个前提，即解码ER PTM对于提高ER生物标记物的特异性和ER+乳腺癌的临床亚型至关重要。目的1研究将全面优化MRM/MS程序，以定量在一组ER+人乳腺癌细胞系中定量诱导配体依赖(雌激素)和配体非依赖性(生长因子，氧化应激)的ER PTM，以选择其抗雌激素敏感性范围。将特别强调ER铰链和DNA结合域，在这些区域中，已知特定的PTM模式改变ER功能和决定抗雌激素敏感性。还将通过引入突变的ER PTM结构并评估它们对内源性ER依赖基因表达的影响来评估新型ER PTM的功能影响。在其他目标中，AIM 1的努力将产生预测细胞系抗雌激素耐药性的候选PTM图谱，用于在AIM 2肿瘤样本中进一步评估。目的2研究将使用来自AIM 1的完全优化的MRM/MS方案来评估使用两个不同临床注释的ER+原发乳腺癌队列的肿瘤ER PTM的患病率和频谱。可用于MRM/MS分析的两个肿瘤队列：i)验证我们的目标1派生的ER PTM谱与细胞系他莫昔芬的反应性相关，以及ii)独立派生和验证肿瘤ER PTM谱，与临床对他莫昔芬的耐药性及其他与早期临床复发相关的侵袭性肿瘤特征有关。在完成这些研究目标后，定量MRM/MS分析将确定所有乳腺癌PTM的谱和流行率，并将识别和验证与更具侵袭性和抗雌激素耐药性的乳腺癌相关的PTM模式。 公共卫生相关性：雌激素受体蛋白是常规临床应用中最重要的肿瘤生物标志物之一，用于识别过度表达ER(指定为ER+)的乳腺癌，这意味着它们可能对他莫昔芬或芳香酶抑制剂的内分泌治疗有良好的反应。虽然低ER表达的肿瘤(ER-)几乎总是对这种治疗无效，但ER+肿瘤总是有效的推论是不正确的。使用我们首创的敏感和复杂的基于质谱学的方法，我们将检验这样一个假设，即解码已知或怀疑发生在各种ER氨基酸上的化学修饰(称为翻译后修饰或PTM)将能够更准确地预测ER+乳腺癌对内分泌治疗的反应，并为ER+乳腺癌与内分泌耐药相关的机制提供新的见解。

DESCRIPTION (provided by applicant): Estrogen receptor (ER, alpha isoform) was the first biomarker to be clinically validated as a predictor of cancer therapy response, and still stands as one of the few tumor biomarkers with sufficient medical evidence to justify its routine use in clinical decision making. While low or absent tumor ER expression (ER-) accurately predicts lack of responsiveness to endocrine therapy, tumor overexpression of ER (ER+) is a poor predictor of response with an accuracy averaging only 50%. Hence, improving the predictive accuracy of current ER assays that measure only tumor ER content is one of the most important unresolved issues in cancer research. Since the constellation of posttranslational modifications (PTMs) on any given protein is known to be a molecular code dictating conformation, localization, and intracellular function, we assembled an interdisciplinary team of translational investigators and protein chemists who developed mass spectrometry (MS) approaches, employing multiple reaction monitoring (MRM) capable of detecting and quantitating diverse PTMs, including Ser/Thr/Tyr phosphorylations, Lys modifications (acetylation/methylation/ubiquitination), and Cys oxidation across the six domains of endogenously expressed ER protein. Two study aims are proposed based on the premise that decoding ER PTMs is essential for improving ER biomarker specificity and the clinical subtyping of ER+ breast cancers. Aim 1 studies will fully optimize MRM/MS procedures to quantitate ligand-dependent (estrogen) and ligand-independent (growth factor, oxidative stress) induction of ER PTMs across a panel of ER+ human breast cancer cell lines selected for their range of antiestrogen sensitivities. Special emphasis will be given to the ER hinge and DNA-binding domains where specific PTM patterns are known to alter ER functionality and determine antiestrogen sensitivity. The functional impact of novel ER PTMs will also be assessed by introducing mutated ER PTM constructs and evaluating their intracellular impact on endogenous ER dependent gene expression. Among other objectives, Aim 1 efforts will generate a candidate PTM profile predictive of cell line antiestrogen resistance for further evaluation in Aim 2 tumor samples. Aim 2 studies will employ fully optimized MRM/MS protocols from Aim 1 to evaluate the prevalence and spectrum of tumor ER PTMs using two different clinically annotated cohorts of ER+ primary breast cancers. The two tumor cohorts available for MRM/MS analysis are powered to: i) validate our Aim 1 derived ER PTM profile associating with cell line tamoxifen responsiveness, and ii) independently derive and validate a tumor ER PTM profile associating with clinical resistance to adjuvant tamoxifen and other aggressive tumor features linked to early clinical relapse. On completion of these study aims, quantitative MRM/MS assays will have defined the spectrum and prevalence of all breast cancer PTMs, and a PTM pattern associated with more aggressive and antiestrogen resistant breast cancers will have been identified and validated. PUBLIC HEALTH RELEVANCE: The estrogen receptor protein represents one of the most important tumor biomarkers in routine clinical use, and is employed to identify breast cancers that overexpress ER (designated ER+), signifying that they MAY respond favorably to endocrine therapy with tamoxifen or aromatase inhibitors. Although low ER expressing tumors (ER-) are almost invariably unresponsive to such therapies, the corollary that ER+ tumors will always respond is not true. Using a sensitive and sophisticated mass spectrometry-based approach that we have pioneered, we will test the hypothesis that decoding the chemical modifications known or suspected to occur on various ER amino acids (known as post-translational modifications or PTMs) will enable more accurate prediction of ER+ breast tumors responsive to endocrine therapy, and provide new insight into ER+ breast cancer mechanisms associated with endocrine resistance.