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The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer

ABSTRACT: BHLHE40 is a basic helix-loop-helix transcription factor that is involved in multiple cell activities including differentiation, cell cycle, and epithelial-to-mesenchymal transition. While there is growing evidence to support the functions of BHLHE40 in energy metabolism, little is known about the mechanism. In this study, we found that BHLHE40 expression was downregulated in cases of endometrial cancer of higher grade and advanced disease. Knockdown of BHLHE40 in endometrial cancer cells resulted in suppressed oxygen consumption and enhanced extracellular acidification. Suppressed pyruvate dehydrogenase (PDH) activity and enhanced lactated dehydrogenase (LDH) activity was observed in the knockdown cells. Knockdown of BHLHE40 also led to dephosphorylation of AMPKα Thr172 and enhanced phosphorylation of PDHA1 Ser293 and LDHA Tyr10. These results suggested that BHLHE40 modulates PDH and LDH activity by regulating the phosphorylation status of PDHA1 and LDHA. We found that BHLHE40 enhanced AMPKα phosphorylation by directly suppressing the transcription of an AMPKα-specific phosphatase, PPM1F. Our immunohistochemical study showed that the expression of BHLHE40, PPM1F, and phosphorylated AMPKα correlated with the prognosis of endometrial cancer patients. Because AMPK is a central regulator of energy metabolism in cancer cells, targeting the BHLHE40‒PPM1F‒AMPK axis may represent a strategy to control cancer development.

ORGANISM(S): Homo sapiens

PROVIDER: GSE241941 | GEO | 2023/09/04

REPOSITORIES: GEO

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Project description:Goal and Objectives: Here we first time report that p-PDH A1 (S293 in mouse but in Human is S264), and PKM2 can regulates the many genes upon Insulin in Human Heptocellular Carcinoma HepG2 cells Line. The goal of this study is to identify the target genes, of which the promoters are associated with p-PDHA1 and PKM2, which may regulate target genes' trascription. We performed Chromatin immunoprecipitation-sequencing (ChIP-Seq) experiments by using antibodies agaist p-PDHA1 and PKM2. Methods: HepG2 cells were cultured in DMEM media, supplemented with 10% FBS and 1% penicillin/Streptomycin antibiotics. In 100 mm (diameter) dishes, the cells were seeded (1×107 – 3×107 cells per dish) and then incubated for 24 h. The media was exchanged for DMEM without serum for 12 h and the cells were then treated with 100 nM insulin for 48 h. The cells were then washed with PBS. To crosslink proteins to DNA, we added formaldehyde drop-wise directly to PBS containing samples to a final concentration of 0.75% and incubated at room temperature for 1 h. The crosslinking was stopped by adding glycine to the samples to a final concentration of 125 mM, followed by incubation for 10 min at RT. The cells were then rinsed twice with ice cold PBS and were harvested. The cell pellets were suspended in ChIP lysis buffer with freshly added cocktail of protease/phosphatase inhibitors and incubated for 10 min on ice. Sonication of the sample was then performed with an OMNI Sonic Ruptor-400 sonicator (OMNI International, Kennesaw, GA, USA) on ice for 20 impulses of 20 sec each. This was repeated five times with a 30 sec interval for each. The sonicated sample was centrifuged at 13,000 rpm for 15 min and 200 μg of the protein–chromatin complex was used for each round of immunoprecipitation {Cap, 2020, 32046944}, performed with anti-p-PDH A1 (S293), and PKM2 antibodies overnight at 4°C. IgG antibody used as a control. The formed antibody–protein complex was captured by incubation with pre-blocked Pierce Protein A/G Beads (Pierce). After sequential washing the beads with wash buffer I, II, III and IV several times and DNA sequencing of ChIP was performed by eBiogen (Seoul, Korea). Results: From ChIP Seq data analysis, we found that the promoters of many genes are associated with both p-PDH and PKM2. p-PDHA1 and PKM2 alone or together may regulate transcription of many genes upon insulin. In our current manuscript, we sort out the common genes which may be regulated p-PDHA1/PKM2 complex common genes in hepatocellular carcinoma cell line in response to insulin stimulation. Conclusions: Our study represents the first detailed analysis of p-PDHA1/PKM2 complex regarding to transcriptional regulation through ChIP-seq. The optimized data analysis workflows reported here may provide a framework for comparative transcritional investigations of p-PDHA1 and PKM2. We conclude that ChIP-Seq data of p-PDHA1 and PKM2 would expedite genetic network analyses and permit the dissection of complex biologic functions as well as cancer biology studies.

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The BHLHE40‒PPM1F‒AMPK pathway regulates energy metabolism and is associated with the aggressiveness of endometrial cancer

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