Targeting SNP BRMS1v2 A273V/A273V to reduce metastases in lung adenocarcinoma

项目总结/摘要 转移性肺癌在美国每年导致16万人死亡。在早期患者中， 手术切除的肺癌10-60%会复发和远处转移。存在未满足的 需要预测发生远处转移的可能性和创新的辅助治疗， 可以降低这种风险。我们的研究小组已经表明，转移抑制基因，乳腺癌转移， 抑制因子1（BRMS 1）抑制肺腺癌转移（LUAD）。两种主要亚型 BRMS 1、v1和v2存在于人类中。BRMS 1的下一代测序揭示了一个单核苷酸 一个多态性（SNP）rs 1052566（G>A）导致BRMS 1v 2的A273 V突变。纯合子A等位基因 （BRMS 1v 2A 273 V/A273 V）存在于8%的LUAD患者中，并与LUAD患者的无进展生存率低相关。 TCGA队列中I-II期淋巴结阴性LUAD患者（N=278）。从机械上讲， BRMS 1v 2 A273 V消除BRMS 1v 2的转移抑制功能并促进稳健的细胞侵袭 和通过激活c-fos介导的基因特异性转录调节的转移。具体来说，BRMS 1v 2 A273 V增加体外细胞侵袭，并增加尾静脉注射异种移植物和LUAD中的转移 患者来源的类器官（PDO）心内注射转移体内模型。此外，我们表明， BRMS 1v 2 A273 V不能与Src相互作用，导致c-fos激活和L1 CAM增加。c-fos抑制 或L1 CAM的敲低降低BRMS 1v 2 A273 V促进的细胞侵袭。我们的总体目标是：1） 阐明c-fos在BRMS 1v 2A 273 V/A273 V LUAD转移中的作用，以及2）评估 靶向BRMS 1v 2A 273 V/A273 V，以减少转移并提高生存率。两个具体目标将考验我们的 假设：目的1）研究BRMS 1v 2 A273 V促进LUAD转移的机制。 我们将在我们的BRMS 1v 2 WT/WT和同基因BRMS 1A 273 V/A273 V细胞中进行RNA-seq，然后进行体内测序。 CRISPR/sgRNA筛选以鉴定功能性BRMS 1v 2 A273 V下游靶基因和要求 的c-fos。接下来，我们将在LUAD细胞中表达不同形式的核Src，以评估核Src/c在LUAD细胞中的作用。 BRMS 1v 2 A273 V介导的基因特异性转录调控中的fos途径。（2）理解 BRMS 1v 2A 273 V/A273 V在促进LUAD转移中的意义我们将评估 BRMS 1v 2A 273 V/A273 V通过利用我们广泛的临床注释的893个生物储存库预测转移 I-II期（淋巴结阴性）人LUAD标本。然后，我们将使用我们新开发的PDO模型， 检测1）靶向c-fos在BRMS 1v 2A 273 V/A273 V转移抑制中的治疗意义 LUAD通过重新利用c-Fos抑制剂T5224，和2）BRMS 1的主导形式，控制转移。 影响：我们的工作将提供机制和翻译证据，以突出BRMS 1v 2A 273 V/A273 V作为一种 预测早期LUAD转移的重要生殖系标志物，并为将来的研究提供依据。 靶向BRMS 1v 2A 273 V/A273 V LUAD中的c-fos以潜在地减少转移的临床试验。

PROJECT SUMMARY/ABSTRACT Metastatic lung cancer kills 160,000 people in the United States annually. In patients with early stage, surgically resected lung cancer 10-60% will develop recurrence and distant metastasis. There is an unmet need to predict the likelihood for developing distant metastases and for innovative adjuvant therapies that could decrease this risk. Our group has shown that the metastasis suppressor gene, Breast Cancer Metastasis Suppressor 1 (BRMS1), inhibits metastases in lung adenocarcinoma (LUAD). Two primary isoforms of BRMS1, v1 and v2 are present in humans. Next generation sequencing of BRMS1 reveals a single nucleotide polymorphism (SNP) rs1052566 (G>A) that causes an A273V mutation of BRMS1v2. The homozygous A allele (BRMS1v2A273V/A273V) is present in 8% of LUAD patients and correlates with a poor progression-free survival of patients with stage I-II, node-negative LUAD in the TCGA cohort (N=278). Mechanistically we show that BRMS1v2 A273V abolishes the metastasis suppressor function of BRMS1v2 and promotes robust cell invasion and metastases by activation of c-fos-mediated gene-specific transcriptional regulation. Specifically, BRMS1v2 A273V increases cell invasion in vitro and increased metastases in both tail-vein injection xenograft and LUAD patient-derived organoid (PDO) intracardiac injection metastasis in vivo models. Moreover, we show that BRMS1v2 A273V fails to interact with Src, resulting in c-fos activation and increased L1CAM. Inhibition of c-fos or knockdown of L1CAM reduces BRMS1v2 A273V-promoted cell invasion. Our overarching goals are 1) to clarify the contribution of the c-fos in BRMS1v2A273V/A273V LUAD metastases, and 2) to assess the efficacy of targeting BRMS1v2A273V/A273V to reduce metastases and improve survival. Two Specific Aims will test our hypotheses: Aim 1) Investigate the mechanisms through which BRMS1v2 A273V promotes LUAD metastasis. We will perform RNA-seq in our BRMS1v2WT/WT and isogenic BRMS1A273V/A273V cells followed with an in vivo CRISPR/sgRNAs screen to identify functional BRMS1v2 A273V downstream target genes and the requirement of c-fos. Next, we will express different forms of nuclear Src in LUAD cells to assess the role of nuclear Src/c- fos pathway in BRMS1v2 A273V-mediated gene-specific transcriptional regulation. Aim 2) Understand the significance of BRMS1v2A273V/A273V in promoting LUAD metastasis. We will assess the ability of BRMS1v2A273V/A273V to predict metastases by leveraging our extensive, clinically-annotated biorepository of 893 stage I-II (node-negative) human LUAD specimens. We then will use our newly developed PDO model to examine 1) the therapeutic significance of targeting c-fos in metastasis suppression of BRMS1v2A273V/A273V LUAD by repurposing the c-Fos inhibitor T5224, and 2) the dominant form of BRMS1 that governs metastases. Impact: Our work will provide mechanistic and translational evidence to highlight BRMS1v2A273V/A273V as an important germline marker to predict metastases in early-stage LUAD, and to provide justification for future clinical trials that target c-fos in BRMS1v2A273V/A273V LUAD to potentially decrease metastasis.