Choksi Arpankumar, Parulekar Apoorva, Pant Richa, Shah Vibhuti Kumar, Nimma Ramakrishna, Firmal Priyanka, Singh Smriti, Kundu Gopal C, Shukla Sanjeev, Chattopadhyay Samit
National Centre for Cell Science, Pune, 411007, India.
Indian Institute of Science Education and Research, Bhopal, 462066, India.
Cancer Metab. 2021 Apr 16;9(1):16. doi: 10.1186/s40170-021-00252-x.
Highly proliferating cancer cells exhibit the Warburg effect by regulation of PKM alternative splicing and promoting the expression of PKM2. Majority of the alternative splicing events are known to occur in the nuclear matrix where various MARBPs actively participate in the alternative splicing events. SMAR1, being a MARBP and an important tumor suppressor, is known to regulate the splicing of various cancer-associated genes. This study focuses on the regulation of PKM alternative splicing and inhibition of the Warburg effect by SMAR1.
Immunohistochemistry was performed in breast cancer patient samples to establish the correlation between SMAR1 and PKM isoform expression. Further, expression of PKM isoforms upon modulation in SMAR1 expression in breast cancer cell lines was quantified by qRT-PCR and western blot. The acetylation status of PTBP1 was estimated by immunoprecipitation along with its enrichment on PKM pre-mRNA by CLIP in SMAR1 knockdown conditions. The role of SMAR1 in tumor metabolism and tumorigenesis was explored by in vitro enzymatic assays and functional assays upon SMAR1 knockdown. Besides, in vivo tumor formation by injecting adeno-SMAR1-transduced MDA-MB-231 cells in NOD/SCID mice was performed.
The expression profile of SMAR1 and PKM isoforms in breast cancer patients revealed that SMAR1 has an inverse correlation with PKM2 and a positive correlation with PKM1. Further quantitative PKM isoform expression upon modulation in SMAR1 expression also reflects that SMAR1 promotes the expression of PKM1 over tumorigenic isoform PKM2. SMAR1 deacetylates PTBP1 via recruitment of HDAC6 resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. SMAR1 inhibits the Warburg effect, tumorigenic potential of cancer cells, and in vivo tumor generation in a PKM2-dependent manner.
SMAR1 regulates PKM alternative splicing by causing HDAC6-dependent deacetylation of PTBP1, resulting in reduced enrichment of PTBP1 on PKM pre-mRNA. Additionally, SMAR1 suppresses glucose utilization and lactate production via repression of PKM2 expression. This suggests that tumor suppressor SMAR1 inhibits tumor cell metabolism and tumorigenic properties of cancer cells via regulation of PKM alternative splicing.
高度增殖的癌细胞通过调节丙酮酸激酶（PKM）的可变剪接和促进PKM2的表达来表现出瓦伯格效应。已知大多数可变剪接事件发生在核基质中，各种基质附着区域结合蛋白（MARBP）积极参与可变剪接事件。SMAR1作为一种MARBP和重要的肿瘤抑制因子，已知可调节多种癌症相关基因的剪接。本研究聚焦于SMAR1对PKM可变剪接的调节及对瓦伯格效应的抑制作用。
对乳腺癌患者样本进行免疫组织化学，以建立SMAR1与PKM异构体表达之间的相关性。此外，通过qRT-PCR和蛋白质免疫印迹法对乳腺癌细胞系中SMAR1表达调节后PKM异构体的表达进行定量。在SMAR1敲低条件下，通过免疫沉淀评估PTBP1的乙酰化状态，并通过CLIP评估其在PKM前体mRNA上的富集情况。通过体外酶活性测定和SMAR1敲低后的功能测定，探究SMAR1在肿瘤代谢和肿瘤发生中的作用。此外，将腺病毒转导SMAR1的MDA-MB-231细胞注射到NOD/SCID小鼠体内进行体内肿瘤形成实验。
乳腺癌患者中SMAR1和PKM异构体的表达谱显示，SMAR1与PKM2呈负相关，与PKM1呈正相关。进一步对SMAR1表达调节后的PKM异构体表达进行定量分析也表明，SMAR1促进PKM1而非致癌异构体PKM2的表达。SMAR1通过募集HDAC6使PTBP1去乙酰化，导致PTBP1在PKM前体mRNA上的富集减少。SMAR1以PKM2依赖的方式抑制瓦伯格效应、癌细胞的致癌潜能和体内肿瘤生成。
SMAR1通过引起HDAC6依赖的PTBP1去乙酰化来调节PKM可变剪接，导致PTBP1在PKM前体mRNA上的富集减少。此外，SMAR1通过抑制PKM2的表达来抑制葡萄糖利用和乳酸产生。这表明肿瘤抑制因子SMAR1通过调节PKM可变剪接来抑制肿瘤细胞代谢和癌细胞的致癌特性。
