Oncogenic Met Signaling in Urologic Malignancies

泌尿系统恶性肿瘤中的致癌 Met 信号转导

• 批准号: 10014543
• 负责人: Donald Bottaro
• 金额: $ 52.25万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014543
• 关键词: AF2; Adult; Affinity; Androgen Receptor; Androgens; Attenuated; Autocrine Communication; Automobile Driving; Binding; Biological Assay; Biological Markers; Biological Models; C-terminal; CSF1 gene; CSF1R gene; Carcinoma; Cell Cycle Progression; Cell Line; Cell Nucleus; Cell Proliferation; Cell Survival; Cell model; Cells; Cessation of life; Characteristics; Clinical; Clinical Trials; Colorectal Cancer; Conventional (Clear Cell) Renal Cell Carcinoma; Coupled; Cystoscopy; DDR1 gene; DDR2 gene; DNA Binding Domain; DNA Sequence Alteration; Data; Data Set; Databases; Development; Diagnosis; Disease; Disease Progression; Dissociation; Distant Metastasis; Down-Regulation; Drug Combinations; Drug resistance; Embryonic Development; Epithelial; Epithelial Cells; Estrogen Receptors; Event; Exons; Extracellular Matrix; FLT3 gene; Frequencies; Gene Dosage; Gene Mutation; Genes; Growth; HGF gene; Head and Neck Cancer; Heart Injuries; Homeostasis; Immunosuppression; Individual; Inherited; Kidney; Lead; Lethal Genes; Ligand Binding Domain; Ligands; Lung Adenocarcinoma; Lymph Node Involvement; MERTK gene; MET Gene Mutation; MET gene; MST1R gene; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Malignant neoplasm of prostate; Malignant neoplasm of urinary bladder; Mediating; Mesenchymal; Messenger RNA; Modeling; Molecular; Morphogenesis; Mucous Membrane; Muscle; Mutation; N-terminal; Natural regeneration; Neoplasm Metastasis; Newly Diagnosed Disease; Nuclear Receptors; Oncogenic; Operative Surgical Procedures; Organogenesis; Oxidative Phosphorylation; Oxytocin Receptor; Papillary; Pathogenesis; Pathway interactions; Patient Selection; Patients; Pattern; Peptide Hydrolases; Peroxisome Proliferator-Activated Receptors; Pharmacodynamics; Phase; Phenotype; Platinum; Principal Investigator; Production; Progression-Free Survivals; Prostatic Tissue; Protein Dephosphorylation; Protein Tyrosine Kinase; Proteins; Proto-Oncogene Proteins c-akt; ROS1 gene; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Recurrence; Regulation; Renal Cell Carcinoma; Renal carcinoma; Reporting; Role; Signal Transduction; Small Interfering RNA; Stanolone; Steroids; Structure; Suggestion; Survival Rate; TWIST1 gene; TYRO3 gene; Testosterone; The Cancer Genome Atlas; Therapeutic; Thyroid Hormone Receptor; Tissues; Transcription Coactivator; Transforming Growth Factor beta; Transitional Cell Carcinoma; Tyrosine Kinase Inhibitor; Up-Regulation; Variant; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-3; Work; adverse outcome; autocrine; base; bladder Carcinoma; cell motility; cell type; chemoradiation; chemotherapy; epithelial to mesenchymal transition; extracellular; improved; in vivo; in vivo imaging; inhibitor/antagonist; liver injury; lung small cell carcinoma; malignant breast neoplasm; melanoma; migration; molecular imaging; muscle invasive bladder cancer; mutant; nano-string; neoplastic cell; novel therapeutics; overexpression; paracrine; patient biomarkers; phase 2 study; programs; receptor; receptor expression; receptor internalization; reconstitution; repaired; response; standard of care; targeted treatment; therapeutic development; therapeutic target; transcriptome sequencing; tumor; tumor microenvironment; tumorigenic; urologic

Aim 1 and 2: In 2018, 81,190 new cases of bladder cancer (BCa; urothelial (transitional) cell carcinoma of the bladder) and 17,240 bladder cancer-related deaths were estimated in the U.S. alone. Although 70% of newly diagnosed disease is confined to the mucosa, recurrence and progression are frequent and long-term surveillance by cystoscopy is required. The remaining 30% of new cases are more advanced, with muscle invasion, lymph node involvement or distant metastases. Half of those individuals with muscle-invasive bladder cancer fail definitive therapy (surgery or chemoradiation) within 5 years and succumb to the disease. The 5- and 10-year survival rates for patients with lymph node involvement are 31 and 23%, respectively. Standard of care combination platinum-based chemotherapy for patients with metastatic disease provides a median survival of only 15 months and a 5-year survival rate of 15%. These circumstances underscore the urgent need to identify oncogenic pathways and therapeutic targets for this disease. Interrogating BCa datasets in The Cancer Genome Atlas (TCGA) project revealed potentially oncogenic alterations in MET or the gene encoding its cognate ligand, HGF in 15% of cases in the TCGA Provisional dataset. In addition to genetic alterations, overproduction of wild type RTKs and/or their cognate ligands, by tumor cells or by the tumor microenvironment (TME), can contribute profoundly to tumor cell survival, proliferation, immunosuppression and metastasis. In particular, overproduction of HGF in the TME has been found in many cases where genetic alterations in MET are absent: HGF overproduction was found in 50% head and neck cancers and high levels were correlated with metastasis, and with concerning frequency in cases of drug resistance in melanoma, colorectal, pancreatic, breast and lung cancers. Evidence of HGF/Met pathway involvement in BCa has been found in model systems and in vivo. Positive interim results from a phase II NCI clinical trial of the multikinase inhibitor cabozantinib for patients with advanced BCa (see NCT01688999/NCI-12-C-0205-N: "A Phase 2 Study of Cabozantinib (XL184) in Patients With Advanced /Metastatic Urothelial Carcinoma, Principal Investigator: AB Apolo, GMB, NCI) also implicate RTKs in disease progression. Known cabozantinib targets include the RTKs encoded by MET, KDR, RET, VEGFR1, VEGFR3, KIT, FLT3, ROS1, and AXL. Our unpublished results newly identify those encoded by MERTK, TYRO3, CSF1R, DDR1, and DDR2 as additional high affinity targets. Potentially oncogenic gene alterations of these 18 RTKs occur in a combined 68% of cases in the BCa TCGA Provisional database. Moreover, 6 of these BCa-associated RTKs (BCaRTKs) show significant co-occurrence of 2-fold expression for their cognate ligands in 34% of TCGA BCa cases combined, suggestive of oncogenic autocrine activation. Identifying the BCaRTKs that are likely to mediate clinical responses to existing TKIs will provide a basis for improving RTK inhibitor-based therapies by informing drug combination strategies, driving the development of inhibitors with relevant target selectivity, and guiding the development of needed biomarkers for patient selection. This information will also further define BCa molecular pathogenesis and diversity. Quantitative mRNA analysis of RTKs and their cognate ligands in BCa-derived cell lines revealed robust co-expression of MST1R/MST1, AXL/GAS6 and CSF1R/CSF1 in 13/13, 10/13 and 8/13 BCa cell lines, respectively. In the same cell lines we found mRNA overexpression of 11 common downstream transcriptional activators (TAs) known to promote cell cycle progression and/or contribute to epithelial to mesenchymal transition (EMT): ZEB1, ZEB2, RUNX2, SOX2, SOX9, POU5F1, NANOG, MYC, SNAI1, SNAI2 and TWIST1. These genes were also queried for overexpression (mRNA z-score 2) in the BCa Provisional TCGA dataset. Overexpression was noted exclusively, i.e. no cases of reduced expression were present, and occurred at individual frequencies from 3 to 8%, yielding a combined frequency of 44%. Statistically significant frequency of co-occurrence was noted among several TAs (ZEB1/2, SOX9/MYC, SNAI2/MYC, SNA1/TWIST1 and SOX9/TWIST1) and 24 unique BCaRTK/TA pairs. Functional evidence of autocrine signaling by BCaRTKs in BCa cells was obtained using siRNA suppression. Suppressing GAS6 in J82 cells significantly decreased basal AXL activation and AKT activation. siRNA suppression of AXL in J82 attenuated the motogenic response to exogenous GAS6 and mimicked inhibition of basal migration by cabozantinib. Similar results were observed in cell proliferation assays, where siRNA suppression of either GAS6 or AXL mimicked the effect of cabozantinib. siRNA suppression of MST1R (RON) in FL3 cells attenuated the motogenic response to exogenous MST1 and significantly reduced basal motility, and suppression of either ligand or receptor significantly reduced FL3 cell proliferation. These results: (1) define a large group of RTKs as potential drivers BCa progression; (2) reveal patterns of their overexpression in BCa cell models and subpopulations of BCa patients, and (3) reveal frequent co-overexpression of cognate ligands and shared TAs that are known to drive tumor cell proliferation, invasion and EMT. They provide a framework for an efficient, multilevel approach to better define the functional relevance of BCaRTKs in disease progression, to develop biomarkers that indicate their criticality in individual BCa patients, and in turn, to identify those in whom relevant RTK-targeted therapies will be most effective. Aim 3: Renal cancer (RCC) is comprised of multiple subtypes of which clear cell renal carcinoma (ccRCC) is the most common. The Cancer Genome Atlas (TCGA) provisional ccRCC data indicates that MET mutation, copy number alteration, and amplification/over-expression occur in 12% of 446 total cases. In prostate cancer, androgen receptor (AR) represses MET expression and hence negatively regulates Met signaling. AR, a steroid nuclear receptor, is activated by androgens testosterone/dihydrotestosterone (T/DHT) leading to AR translocation into the nucleus and regulation of AR dependent genes. AR structure contains a N-terminal activation function (AF1-Ligand independent) domain, DNA binding domain, hinge region, and C-terminal ligand binding domain (LBD/AF2) domain. Deletion of exons 4-8 of AR removes the LBD resulting in a truncated constitutively activated mutant (ARv7/ARv3). ARv7 is expressed in prostatic tissues and associated with androgen independent growth of PCa cells. Intriguingly, AR over-expression occurs in 6% of 534 ccRCC TCGA cases and was associated with significantly improved over-all (OS) and progression-free survival (PFS). We found that reconstitution of AR in ccRCC cell lines with robust MET expression reduced Met protein content, implying that an additional negative regulatory mechanism by AR may exist. To gain a more global view of how AR expression might potentially benefit ccRCC patients and identify potentially novel therapeutic strategies, we performed RNA-Seq and Nanostring analyses on ccRCC cell lines over-expressing wild-type AR or the constitutively active variant ARv7 in the presence or absence of the AR ligand dihydrotestosterone (DHT). This work has identified significant alterations in steroid pathways (e.g. PPAR, thyroid hormone, oxytocin and estrogen receptor signaling) and RCC relevant pathways (e.g. Hippo, TGF-beta, Wnt, FOXO, oxidative phosphorylation pathways), including those of therapeutically targetable proteins.

AIM 1和2：在2018年，仅在美国，仅在美国就估计了81,190例新的膀胱癌病例（BCA；膀胱尿路上皮（过渡）细胞癌）和17,240例与膀胱癌相关的死亡。尽管有70％的新诊断疾病仅限于粘膜，但经常复发和进展是经常进行的，并且需要通过膀胱镜检查进行长期监测。其余30％的新病例更为先进，肌肉浸润，淋巴结受累或远处转移。在5年内，有一半的患有肌肉侵入性膀胱癌的人失败了（手术或化学放疗），并屈服于该疾病。淋巴结受累患者的5年和10年生存率分别为31％和23％。针对转移性疾病患者的基于铂金的化学疗法的护理标准，可提供仅15个月的中位生存期，而5年生存率为15％。这些情况强调了迫切需要确定该疾病的致癌途径和治疗靶标。在TCGA临时数据集中，在癌症基因组图集（TCGA）项目中询问BCA数据集（TCGA）项目中有可能在15％的病例中编码其同源配体（HGF）的基因。除了遗传改变外，野生型RTK和/或其同源配体，肿瘤细胞或通过肿瘤微环境（TME）的生产过多，可以对肿瘤细胞的存活，增殖，免疫抑制和转移产生深远的贡献。特别是，在许多情况下，在50％的头颈癌中发现了HGF的HGF过量，在许多情况下发现了HGF的过量生产，并且高水平与转移相关，并且与黑色素瘤，大乳瘤，结婚，肉质，乳肉，乳腺癌，胸部和lung癌症的耐药性频率相关。在模型系统和体内发现了HGF/MET途径参与BCA的证据。积极的临时临时是由多次酶抑制剂Cabozantinib对晚期BCA患者的II期NCI临床试验产生的（请参阅NCT01688999 /NCI-12-C-0205-N：“ Cabozantinib（XL184）的2期研究（XL184）患者对晚期 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性 /转移性。疾病的进展。在BCA TCGA临时数据库中，有68％的病例。此外，这些BCA相关的RTK（BCARTKS）中有6例表现出34％的TCGA BCA案例的同源配体的显着相关表达。基于RTK抑制剂的疗法通过告知药物组合策略，推动具有相关靶标选择性的抑制剂的开发以及指导所需的生物标志物进行患者选择的开发。该信息还将进一步定义BCA分子发病机理和多样性。在BCA衍生的细胞系中RTK及其同源配体的定量mRNA分析显示，分别在13/13、10/13和8/13 BCA细胞系中MST1R/MST1，AXL/GAS6和CSF1R/CSF1的MST1R/MST1，AXL/GAS6和CSF1R/CSF1的强劲表达。在相同的细胞系中，我们发现已知的11个常见下游转录活化剂（TA）的mRNA过表达，可促进细胞周期进展和/或有助于上皮层次上质膜过渡（EMT）：ZEB1，ZEB2，RUNX2，RUNX2，RUNX2，SOX2，SOX9，SOX9，SOX9，SOX9，POU5F1，NANOG，NANOG，NANOG，MYC，MYC，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI1，SNAI2和TWIST1和TWIST1。这些基因也被查询是否在BCA临时TCGA数据集中查询过表达（mRNA z得分2）。仅注意到过表达，即不存在表达降低的病例，并以3％至8％的个人频率发生，产生44％的综合频率。在几个TAS（Zeb1/2，Sox9/Myc，Snai2/Myc，Sna1/Twist1和Sox9/Twist1）和24个独特的BCARTK/TA对中，同时存在的统计显着频率已注意到。使用siRNA抑制获得了BCARTKS自分泌信号传导的功能证据。在J82细胞中抑制GAS6可显着降低基础AXL激活和AKT激活。 J82中AXL抑制AXL的siRNA抑制了对外源性GAS6的运动反应，并模仿了Cabozantinib对基底迁移的抑制。在细胞增殖测定中观察到了相似的结果，在细胞增殖分析中，siRNA抑制GAS6或AXL模仿了Cabozantinib的作用。 FL3细胞中MST1R（RON）的siRNA抑制减弱了对外源MST1的运动反应，并显着降低了基础运动，并且对配体或受体的抑制显着降低了FL3细胞增殖。这些结果：（1）将大量RTK定义为潜在的驱动因素BCA进展； （2）揭示了其在BCA细胞模型和BCA患者亚群中的过表达模式，（3）揭示了频繁的同源配体和共享TA的共同表达，这些TA已知可驱动肿瘤细胞的增殖，侵袭和EMT。它们为一种有效的多级方法提供了一个框架，以更好地定义BCARTKS在疾病进展中的功能相关性，开发出生物标志物，以表明其在个别BCA患者中的关键性，并且可以确定那些相关的RTK靶向疗法的功能相关性。 AIM 3：肾癌（RCC）由多种亚型组成，其中透明细胞肾癌（CCRCC）是最常见的。癌症基因组图集（TCGA）临时CCRCC数据表明，在446例总病例中，有12％发生MET突变，拷贝数改变和扩增/过表达。在前列腺癌中，雄激素受体（AR）抑制MET表达，因此负调节MET信号传导。 AR是一种类固醇受体，被雄激素睾丸激素/二氢睾丸激素（T/DHT）激活，导致AR转移到核中，并调节AR依赖性基因。 AR结构包含N末端激活函数（AF1配体独立）结构域，DNA结合结构域，铰链区域和C末端配体结合结构域（LBD/AF2）域。 AR 4-8外显子的缺失去除LBD，从而导致组成型激活的突变体（ARV7/ARV3）。 ARV7在前列腺组织中表达，与PCA细胞的雄激素无关生长有关。有趣的是，AR过表达发生在534个CCRCC TCGA病例中的6％，并且与超过（OS）和无进展生存期（PFS）显着改善。我们发现，在具有稳健MET表达的CCRCC细胞系中，AR的重建降低了MET蛋白含量，这意味着可能存在AR的额外负调节机制。为了对AR表达如何使CCRCC患者有可能受益并确定潜在的新型治疗策略的更全球性观点，我们对CCRCC细胞系进行了RNA-Seq和纳米静止分析，过表达野生型AR或在存在或不存在AR aR ligandand-ligandand-ligandand-ligandand-ligandand-ligandand-ligand dihydrototeperone（dhydrotasterone）的情况下（dht）。这项工作已经确定了类固醇途径（例如PPAR，甲状腺激素，催产素和雌激素受体信号传导）和RCC相关途径的重大变化，包括河马，TGF-BETA，WNT，FOXO，FOXO，氧化磷酸化途径），包括治疗蛋白质的蛋白质。