Tumor suppressor SMAR1 regulates PKM alternative splicing by HDAC6-mediated deacetylation of PTBP1

Abstract

Background 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. Methods 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. Results 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. Conclusions 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.

RBX1 regulates PKM alternative splicing to facilitate anaplastic thyroid carcinoma metastasis and aerobic glycolysis by destroying the SMAR1/HDAC6 complex

“…Cancer cells achieve the metabolic advantage over normal cells by modulating PKM alternative splicing and facilitating the PKM2 expression [ 19 ]. To further demonstrate whether RBX1 is responsible for this phenomenon, we detected the expression levels of RBX1, PKM2, and PKM1 in ATC patient samples and then compared these levels with those in the corresponding adjacent non-cancerous tissues using western blotting.…”

“…Previous studies have proven that UPS regulated the polyubiquitination and degradation of SMAR1 [ 19 ]. As the E3 ubiquitin ligase, RBX1, plays a role in protein degradation and recycling, we initially determined the degeneration of SMAR1 protein in RBX1-knockdown cells after inhibition of protein synthesis using cycloheximide (CHX).…”

“…The cloning of dual reporter PKM minigene system and dual reporter PKM minigene assay were performed as described previously [ 19 ]. Mean ± SD fold change in eGFP/mCherry ratio was calculated for each sample and compared with the control sample.…”

“…Therefore, targeting this alternative splicing step is shown to be an efficacious strategy for the elimination of cancer cells [ 17 , 18 ]. Recently, it has been shown that tumor suppressor SMAR1 regulates PKM alternative splicing by HDAC6-mediated deacetylation of PTBP1 [ 19 ]. Therefore, identification of the factors and mechanisms underlying PKM alternative splicing, resulting in the PKM2-mediated Warburg effect, is crucial for overcoming the current challenges in cancer treatment.…”

SARS-CoV-2 Causes Lung Inflammation through Metabolic Reprogramming and RAGE

“…The last step is the conversion of phosphoenolpyruvate (PEP) into pyruvate and is performed by the pyruvate kinase enzyme, which has four isoforms including kinase muscle isoforms 1 and 2 (PKM1 and PKM2). PKM1 and PKM2 are the dominant isoforms of PKM that are found in most tissues [ 41 ]. The polypyrimidine tract-binding protein 1 (PTBP1) is important for splicing PKM into the PKM2 isoform over PKM1 [ 42 , 43 ].…”