Identification of Histone Lysine Acetoacetylation as a Dynamic Post-Translational Modification Regulated by HBO1.

Ketone bodies have long been known as a group of lipid-derived alternative energy sources during glucose shortages. Nevertheless, the molecular mechanisms underlying their non-metabolic functions remain largely elusive. This study identified acetoacetate as the precursor for lysine acetoacetylation (Kacac), a previously uncharacterized and evolutionarily conserved histone post-translational modification. This protein modification is comprehensively validated using chemical and biochemical approaches, including HPLC co-elution and MS/MS analysis using synthetic peptides, Western blot, and isotopic labeling. Histone Kacac can be dynamically regulated by acetoacetate concentration, possibly via acetoacetyl-CoA. Biochemical studies show that HBO1, traditionally known as an acetyltransferase, can also serve as an acetoacetyltransferase. In addition, 33 Kacac sites are identified on mammalian histones, depicting the landscape of histone Kacac marks across species and organs. In summary, this study thus discovers a physiologically relevant and enzymatically regulated histone mark that sheds light on the non-metabolic functions of ketone bodies.

HBO1 catalyzes lysine benzoylation in mammalian cells.

Lysine benzoylation (Kbz) is a newly discovered protein post-translational modification (PTM). This PTM can be stimulated by benzoate and contributes to gene expression. However, its regulatory enzymes and substrate proteins remain largely unknown, hindering further functional studies. Here we identified and validated the lysine acetyltransferase (KAT) HBO1 as a "writer" of Kbz in mammalian cells. In addition, we report the benzoylome in mammalian cells, identifying 1747 Kbz sites; among them at least 77 are the HBO1-targeted Kbz substrates. Bioinformatics analysis showed that HBO1-targeted Kbz sites were involved in multiple processes, including chromatin remodeling, transcription regulation, immune regulation, and tumor growth. Our results thus identify the regulatory elements of the Kbz pathway and reveal the non-canonical enzymatic activity and functions of HBO1 in cellular physiology.

Let-7b-5p inhibits proliferation and motility in squamous cell carcinoma cells through negative modulation of KIAA1377.

KIAA1377 has been found to be linked with lymph node metastasis in esophageal squamous cell carcinoma (SCC) in our previous study; however, the regulation of KIAA1377 remains far from understood. Herein, to understand the regulation of KIAA1377 from the angle of microRNA (miRNA)-messenger RNA (mRNA) modulation in the setting of SCC cells, the basal level of KIAA1377 was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis in KYSE-150 and HeLa cells; biological roles of KIAA1377 contributing in the proliferation, migration, and invasion were evaluated using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), wound-healing and Transwell assays, respectively, after KIAA1377 was knocked out mediated by the CRISPR-Cas9 system. Bioinformatic prediction revealed that let-7b-5p was a putative miRNA regulating KIAA1377, which was ensuingly validated by the luciferase reporter assay; after which, variation of KIAA1377 expression was further verified by qRT-PCR and western blot analysis. Moreover, the biological roles of let-7b-5p in proliferation, migration, and invasion of KYSE-150 and HeLa cells were also evaluated. It was exhibited that KIAA1377 was able to promote the proliferation and motility of both KYSE-150 and HeLa cells, which can be reverted by re-expression of let-7b-5p. The luciferase reporter assay verified that let-7b-5p can diametrically target KIAA1377. Collectively, our data demonstrated that let-7b-5p can directly but negatively regulate KIAA1377 in SCC cell lines, Ecal109, and HeLa cells.

PKM2-regulated STAT3 promotes esophageal squamous cell carcinoma progression via TGF-ß1-induced EMT.

Recent studies have demonstrated pleiotropic roles of pyruvate kinase isoenzyme type M2 (PKM2) in tumor progression. However, the precise mechanisms underlying the effects of PKM2 on esophageal squamous cell carcinoma (ESCC) metastasis and transforming growth factor ß1 (TGF-ß1)-induced epithelial-mesenchymal transition (EMT) remain to be established. In this study, we observed upregulation of PKM2 in ESCC tissues that was markedly associated with lymph node metastasis and poor prognosis. High PKM2 expression in tumor tissues frequently coincided with the high pSTAT3Tyr705 expression and low E-cadherin expression. Furthermore, altered PKM2 expression was significantly associated with proliferation, migration, and invasion of ESCC cells, in addition to expression patterns of EMT markers (Snail, E-cadherin, and vimentin) and pSTAT3Tyr705 /STAT3 ratio. Overexpression of STAT3 significantly attenuated the effects of PKM2 knockdown on cell proliferation and motility as well as expression of pSTAT3 Tyr705 and EMT markers. Consistently, stable short hairpin RNA (shRNA)-mediated silencing of PKM2 reversed the effects of TGF-ß1 treatment, specifically, upregulation of PKM2, phosphorylation of STAT3 at Tyr705, and increased EMT, migration, and invasion. We propose that PKM2 regulates cell proliferation, migration, and invasion via phosphorylation of STAT3 through TGF-ß1-induced EMT. Our findings collectively provide mechanistic insights into the tumor-promoting role of PKM2, supporting its prognostic value and the therapeutic utility of PKM2 inhibitors as potential antitumor agents in ESCC.

Infiltrated M2 tumour-associated macrophages in the stroma promote metastasis and poor survival in oesophageal squamous cell carcinoma.

Although M2 tumour-associated macrophages (M2 TAMs) have been shown to be associated with the progression and metastasis of breast cancer, their role in oesophageal squamous cell carcinoma (ESCC) remains less well understood. Therefore, to understand the clinicopathological significance of infiltrated M2 TAMs in ESCC, statistical analysis was performed after immunohistochemical evaluation of CD163 expression, a well-accepted surface marker of M2 TAMs in ESCC. To gain insight into the effect of M2 TAMs, ESCC cell lines Eca109 and KYSE150 cells were co-cultured with M2 TAMs artificially induced from THP-1 cells. The variations in the proliferation, migration and invasion were assessed using the MTT, wound-healing and Transwell assays, respectively. The variation in the typical biomarkers of the epithelial-mesenchymal transition (EMT) was evaluated using western blotting. Infiltrated M2 TAMs were confirmed to predominate in the stroma of ESCC relative to normal controls. Moreover, it turned out that M2 TAMs were shown to promote the migration and invasion of ESCC cells but not proliferation. Furthermore, M2 TAMs were observed to induce EMT in ESCC cells. Together, our results showed that infiltrated M2 TAMs in the stroma is a feature accompanying ESCC metastasis and that M2 TAMs can promote the migration and invasion, but not proliferation, of ESCC cells, thereby inducing EMT. Thus, M2 TAMs could be an alternative therapeutic target in ESCC.

IL-1ß from M2 macrophages promotes migration and invasion of ESCC cells enhancing epithelial-mesenchymal transition and activating NF-κB signaling pathway.

Despite the phenotype has been established that M2 macrophages promotes the metastasis of ESCC, question still remains as to how the M2 macrophages facilitated metastasis of ESCC cells. To begin with, immunohistochemistry was performed to detect the expression of CD163, one typical surface marker of M2 macrophages in 90 paired ESCC and its normal controls after meta-analyzing the relevant studies regarding M2 macrophages in ESCC, confirming that infiltration of M2 macrophages was significantly linked with lymph node metastasis, T classification, and inferior overall survival of ESCC. To explore the mechanism behind, protein factors secreted by M2 macrophages were identified using antibody microarray. Six different significantly differential protein factors were screened out, including IL-1ß, TIMP1, IL-1α, MDC, TGF-ß1, and TGF-ß2. Among which, IL-1ß was picked up as cytokine as interest based on previous reports and its absolute fold. Functional analysis of IL-1ß showed that IL-1ß was able to promote migration and invasion of ESCC cells, enhancing epithelial-mesenchymal transition, and activating NF-κB pathway. Our study supports the promoting role of M2 macrophages in metastasis of ESCC cells, enriching the profile of protein factors released from M2 macrophages.

MCP2 activates NF-κB signaling pathway promoting the migration and invasion of ESCC cells.

MCP2, aliased CCL8, has been suggested to be implicated in the metastasis of cancer cells; however, no direct evidence has been established in esophageal squamous cell carcinoma (ESCC). In our present study, to investigate the role MCP2 played in the metastasis of ESCC cells; in vitro cell co-culture system was established. Wound-healing and Transwell assays were used to evaluate the migratory and invasive variation of ESCC cells before and after treatment with recombinant human MCP2. It was shown that MCP2 was able to activate the NF-κB signaling pathway inducing the epithelial-mesenchymal transition (EMT) and promoting the migration and invasion of ESCC cells in vitro. Our study provides an alternative working mechanism for M2 macrophage mediated the metastasis in tumor microenvironment in ESCC.