PO.TB10.16 · 肿瘤生物学
调节TIMP1后与KRAS依赖性相关的血管生成谱
Angiogenic profile associated with KRAS dependency upon modulation of TIMP1
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
非小细胞肺癌(NSCLC)仍是癌症相关死亡的主要原因之一,KRAS突变赋予其癌基因成瘾性和对治疗的化疗耐药性。我们此前的工作证明,金属蛋白酶组织抑制因子-1(TIMP-1)与KRAS依赖性呈负相关,并促进上皮-间质转化(EMT)。鉴于TIMP-1在肿瘤进展中的作用,我们研究了TIMP-1调节如何影响NSCLC细胞中血管生成介质的表达。分别对KRAS依赖型(H441)和KRAS非依赖型(H460)NSCLC细胞系进行调节,使其过表达(OE)或敲低(KD)TIMP-1。使用血管生成蛋白质组分析芯片(R&D systems)对这些细胞的无血清条件培养基进行分析。我们发现,促血管生成因子VEGF1和CXCL8的水平在TIMP1过表达时升高,在敲低TIMP1时降低。另一方面,血管生成抑制剂血小板反应蛋白-1(Thrombospondin-1)在TIMP1过表达时下调,在敲低TIMP1时上调。此外,H441细胞中TIMP-1过表达增加了血管生成素(Angiogenin),并降低了内皮抑素(Endostatin)、血管抑素(Angiostatin)、Serpin E1和血小板因子4(PF4)。相反,H460细胞中TIMP-1敲低显示出相反的趋势,表明TIMP1具有促血管生成作用。对五种NSCLC细胞系的RNA-seq数据分析揭示了TIMP1:THBS1的相互表达模式,在TIMP1过表达下伴有强烈的CXCL8诱导,在H441中最为显著。TIMP1-THBS1的负相关关系支持一个以CXCL8为主导的血管生成轴。由于研究表明KRAS依赖性与c-Myc和ERK的上调相关,从而导致TSP-1水平下降,我们通过蛋白质印迹法评估了裂解物中的c-Myc水平。我们发现,c-MYC水平在KRAS依赖型亲本细胞中较高,并在TIMP-1过表达时进一步升高。然而在KRAS非依赖型细胞中,TIMP-1敲低并未显著改变c-MYC水平。正在进行的研究继续评估TIMP-1调节在常氧和缺氧条件下对血管生成反应的影响。我们的发现凸显TIMP-1为NSCLC中以KRAS依赖性特异性方式调节肿瘤血管生成的潜在调控因子,包含一个以CXCL8为主导的轴。
查看英文原文 English abstract
Non-small cell lung carcinoma (NSCLC) remains a leading cause of cancer-related deaths, with KRAS mutations conferring oncogene addiction and chemoresistance to therapy. Our previous work demonstrated that Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) inversely correlates with KRAS dependency and promotes epithelial-mesenchymal transition (EMT). Given the role of TIMP-1 in tumor progression, we investigated how TIMP-1 modulation impacts the expression of angiogenic mediators in NSCLC cells.KRAS-dependent (H441) and KRAS-independent (H460) NSCLC cell lines were modulated to overexpress (OE) or knock down (KD) TIMP-1, respectively. Serum free conditioned media from these cells was analyzed using angiogenesis proteome profiler array (R&D systems). We found that the levels of pro-angiogenic factors VEGF1 and CXCL8 increased upon TIMP1 overexpression and decreased upon knocking down TIMP1. On the other hand, Thrombospondin-1, an angiogenic inhibitor was downregulated when TIMP1 was over-expressed and upregulated when TIMP1 was knocked down. Additionally, TIMP-1 overexpression in H441 cells increased Angiogenin and decreased Endostatin, Angiostatin, Serpin E1, and Platelet Factor 4 (PF4). Conversely, TIMP-1 knockdown in H460 cells showed the opposite trend, indicating that TIMP1 is pro-angiogenic. RNA-seq data analysis across five NSCLC lines revealed a reciprocal TIMP1:THBS1 expression pattern with strong CXCL8 induction under TIMP1 overexpression, most pronounced in H441. The TIMP1-THBS1 inverse relationship supports a CXCL8-dominant angiogenic axis.As studies have shown that KRAS dependency is associated with upregulation of c-Myc and ERK, leading to a decrease in TSP-1 levels, we assessed c-Myc levels in the lysates using Western blot. We found that the levels of c-MYC were high in KRAS-dependent parental cells and further increased upon TIMP-1 overexpression. In KRAS-independent cells however, TIMP-1 knockdown did not significantly alter c-MYC levels. Ongoing studies continue to evaluate the effects of TIMP-1 modulation on angiogenic responses under normoxic and hypoxic conditions.Our findings highlight TIMP-1 as a potential regulator of tumor angiogenesis in NSCLC in a KRAS dependency specific manner, incorporating a CXCL8-dominant axis.
利益披露 Disclosure
I. M-Thirusenthilarasan, None..
P. K. Ahluwalia, None..
B. Siddiqui, None..
R. Kolhe, None..
A. M. Rojiani, None..
M. V. Rojiani, None.