Campanella Nathália Cristina, Gomes Izabela Natalia Faria, Alves Ana Laura Vieira, Leal Leticia Ferro, Evangelista Adriane Feijó, Rosa Marcela Nunes, Melendez Matias Eliseo, Silva Viviane Aline Oliveira, Dias Richard Lucas Konichi, Abrahão-Machado Lucas Faria, Santana Iara, Martinho Olga, Guimarães Denise Peixoto, Faça Vitor Marcel, Reis Rui Manuel
Molecular Oncology Research Center, Barretos Cancer Hospital, Rua Antenor Duarte Villela, 1331, CEP 14784 400, Barretos, S. Paulo, 14784-400, Brazil.
School of Health Sciences Dr. Paulo Prata (FACISB), Barretos, 14785-002, Brazil.
Cancer Cell Int. 2023 Oct 31;23(1):256. doi: 10.1186/s12935-023-03102-6.
Gastrointestinal stromal tumors (GIST) represent a significant clinical challenge due to their metastatic potential and limited treatment options. Raf kinase inhibitor protein (RKIP), a suppressor of the MAPK signaling pathway, is downregulated in various cancers and acts as a metastasis suppressor. Our previous studies demonstrated low RKIP expression in GIST and its association with poor outcomes. This study aimed to expand on the previous findings and investigate the biological and therapeutic implications of RKIP loss on GIST.
To validate the RKIP prognostic significance, its expression was evaluated by immunohistochemistry in 142 bona fide GIST cases. The functional role of RKIP was evaluated in vitro, using the GIST-T1 cell line, which was knocked out for RKIP. The biological and therapeutic implications of RKIP were evaluated by invasion, migration, apoptosis, and 2D / 3D viability assays. Additionally, the transcriptome and proteome of RKIP knockout cells were determined by NanoString and mass spectrometry, respectively.
Immunohistochemical analysis revealed the absence of RKIP in 25.3% of GIST cases, correlating with a tendency toward poor prognosis. Functional assays demonstrated that RKIP knockout increased GIST cells' invasion and migration potential by nearly 60%. Moreover, we found that RKIP knockout cells exhibited reduced responsiveness to Imatinib treatment and higher cellular viability in 2D and 3D in vitro models, as assessed by apoptosis-related protein expression. Through comprehensive genetic and proteomic profiling of RKIP knockout cells, we identified several putative RKIP-regulated proteins in GIST, such as COL3A1.
Using a multidimensional integrative analysis, we identified, for the first time in GIST, molecules and pathways modulated by RKIP that may potentially drive metastasis and, consequently, poor prognosis in this disease.
胃肠道间质瘤（GIST）因其转移潜能和有限的治疗选择而构成重大临床挑战。Raf激酶抑制蛋白（RKIP）是丝裂原活化蛋白激酶（MAPK）信号通路的一种抑制因子，在多种癌症中表达下调，并作为一种转移抑制因子发挥作用。我们之前的研究表明，RKIP在GIST中表达较低，且与不良预后相关。本研究旨在扩展先前的发现，并探讨RKIP缺失对GIST的生物学和治疗意义。
为验证RKIP的预后意义，采用免疫组织化学方法对142例确诊的GIST病例进行其表达评估。利用RKIP基因敲除的GIST-T1细胞系在体外评估RKIP的功能作用。通过侵袭、迁移、凋亡和二维/三维活力测定评估RKIP的生物学和治疗意义。此外，分别通过NanoString和质谱法测定RKIP基因敲除细胞的转录组和蛋白质组。
免疫组织化学分析显示，25.3%的GIST病例中不存在RKIP，这与预后不良的趋势相关。功能试验表明，RKIP基因敲除使GIST细胞的侵袭和迁移潜能增加了近60%。此外，通过凋亡相关蛋白表达评估，我们发现RKIP基因敲除细胞在二维和三维体外模型中对伊马替尼治疗的反应性降低，细胞活力更高。通过对RKIP基因敲除细胞进行全面的基因和蛋白质组分析，我们在GIST中鉴定出几种推测受RKIP调控的蛋白，如Ⅲ型胶原蛋白α1（COL3A1）。
通过多维综合分析，我们首次在GIST中鉴定出受RKIP调控的分子和通路，这些分子和通路可能潜在地驱动转移，进而导致该疾病预后不良。
