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

项目摘要/摘要 在美国，转移性肺癌每年导致16万人死亡。在早期患者中， 手术切除的肺癌有10%-60%会发生复发和远处转移。有一个未满足的人 需要预测发生远处转移的可能性和创新的辅助疗法 可以降低这种风险。我们课题组已经证明转移抑制基因乳腺癌转移 抑制因子1(BRMS1)，抑制肺腺癌(LUAD)的转移。两种主要的亚型 BRMS1、v1和v2存在于人类体内。BRMS1的下一代测序揭示了一个单核苷酸 导致BRMS1v2的A273V突变的多态(SNP)rs1052566(G&gt；A)。纯合子A等位基因 (BRMS1v2A273V/A273V)在8%的LUAD患者中存在，并与LUAD患者较差的无进展生存率相关 TCGA队列中I-II期、结节阴性的LUAD患者(N=278)。从机制上讲，我们表明 BRMS1v2 A273V取消BRMS1v2的转移抑制功能并促进细胞侵袭 通过激活c-fos介导的基因特异性转录调控而发生转移。具体而言，BRMS1v2 A273V在体外增加细胞侵袭力并增加尾静脉注射异种移植瘤和LUAD的转移 患者来源的有机化合物(PDO)心内注射转移的体内模型。此外，我们证明了 BRMS1v2 A273V不能与Src相互作用，导致c-fos激活和L1CAM增加。C-fos的抑制作用 或敲除L1CAM可减少BRMS1v2 A273V促进的细胞侵袭。我们的首要目标是：1) 阐明c-fos在BRMS1v2A273V/A273V LUAD转移中的作用，以及2)评估 靶向BRMS1v2A273V/A273V以减少转移和提高存活率。两个具体的目标将考验我们的 假说：目的1)探讨BRMS1v2 A273V促进LUAD转移的机制。 我们将在我们的BRMS1v2WT/WT和等基因BRMS1A273V/A273V细胞中进行RNA-SEQ，然后在体内进行 CRISPR/sgRNAs筛选鉴定BRMS1v2 A273V下游功能靶基因及其要求 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- BRMS1v2 A273V介导的基因特异性转录调控中的FOS通路。目标2)了解 BRMS1v2A273V/A273V在促进LUAD转移中的意义我们将评估他们的能力 BRMS1v2A273V/A273V通过利用我们广泛的、经过临床注释的893生物信息库来预测转移 I-II期(结节阴性)人LUAD标本。然后我们将使用我们新开发的PDO模型来 1)靶向c-fos基因在抑制BRMS1v2A273V/A273V细胞转移中的治疗意义 LUAD通过改变c-Fos抑制剂T5224的用途，以及2)主导转移的BRMS1形式。 影响：我们的工作将提供机械和翻译证据，以突出BRMS1v2A273V/A273V作为 预测LUAD早期转移的重要种系标记，并为未来提供依据 以BRMS1v2A273V/A273V LUAD中的c-fos为靶点的临床试验可能会减少转移。

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.