Inflammation plays a crucial role in tumorigenesis, primarily mediated by NF-κB. RhoA GTPases are instrumental in regulating the activation of NF-κB. Specifically, the phosphorylation of Tyrosine 42 on RhoA ensures the activation of NF-κB by directly activating the IKKβ associated with IKKγ (NEMO). This study aimed to uncover the molecular mechanism through which p-Tyrosine 42 RhoA, in conjunction with NF-κB, promotes tumorigenesis. Notably, we observed that p-Tyrosine 42 RhoA co-immunoprecipitated with the p-Ser 536 p65/RelA subunit in NF-κB in response to LPS. Moreover, both p-Tyrosine 42 RhoA and p-p65/RelA translocated to the nucleus, where they formed a protein complex associated with the promoter of phosphoglycerate kinase 1 (PGK1) and regulated the expression of PGK1. In addition, p-p65/RelA and p-Tyr42 RhoA co-immunoprecipitated with p300 histone acetyltransferase. Intriguingly, PGK1 exhibited an interaction with β-catenin, PKM1 and PKM2. Of particular interest, si-PGK1 led to a reduction in the levels of β-catenin and phosphorylated pyruvate dehydrogenase A1 (p-PDHA1). We also found that PGK1 phosphorylated β-catenin at the Thr551 and Ser552 residues. These findings discovered that PGK1 may play a role in transcriptional regulation, alongside other transcription factors.

Insulin promotes HepG2 cell proliferation by inducing phosphorylation of the pyruvate dehydrogenase E1α (PDHA1) subunit at Ser293, a mechanism distinct from normal liver tissue. This study investigates how phosphorylated PDHA1 drives hepatocellular carcinoma cell proliferation. We identified eukaryotic elongation factor-1γ (eEF-1γ) as a key binding protein interacting with p-PDHA1 in response to insulin, facilitating their nuclear translocation. Silencing eEF-1γ (si-eEF-1γ) significantly reduced p-PDHA1 and PKM2 levels, highlighting eEF-1γ’s role in stabilizing these proteins. Additionally, eEF-1γ interacts with ATP-citrate lyase (ACL) and p300 acetyltransferase, and its knockdown decreased histone acetylation at H3K9/14, H3K18, and H3K27, along with RBP4 expression. Chromatin immunoprecipitation PCR (ChIP-PCR) confirmed eEF-1γ association with RBP4 promoter. Functionally, si-eEF-1γ reduced cell proliferation and deceased c-Myc and cyclin D1 protein levels. It also suppressed migration, and altered epithelial-mesenchymal transition (EMT) markers, increasing E-cadherin while reducing ZEB1, snail1, vimentin, and N-cadherin levels. Similarly, RBP4 knockdown with siRNA diminished cell proliferation and migration. In vivo, eEF-1γ knockdown in 4T1 xenografts using siRNA led to reduced tumor mass. These findings highlight eEF-1γ as a crucial driver of insulin-induced tumor progression and suggest its potential as a therapeutic target in hepatocellular carcinoma.

Cell migration is a crucial contributor to metastasis, a critical process associated with the mortality of cancer patients. The initiation of metastasis is triggered by epithelial–mesenchymal transition (EMT), along with the changes in the expression of EMT marker proteins. Inflammation plays a significant role in carcinogenesis and metastasis. Lipopolysaccharide (LPS), a typical inflammatory agent, promoted the generation of superoxide through the activation of p-Tyr42 RhoA, Rho-dependent kinase 2 (ROCK2), and the phosphorylation of p47phox. In addition, p-Tyr42 RhoA activated phospholipase D1 (PLD1), with PLD1 and phosphatidic acid (PA) being involved in superoxide production. PA also regulated the expression of EMT proteins. Consequently, we have identified MHY9 (Myosin IIA, NMIIA) as a PA-binding protein in response to LPS. MYH9 also contributed to cell migration and the alteration in the expression of EMT marker proteins. Co-immunoprecipitation revealed the formation of a complex involving p-Tyr42 RhoA, PLD1, and MYH9. These proteins were found to be distributed in both the cytosol and nucleus. In addition, we have found that p-Tyr42 RhoA PLD1 and MYH9 associate with the ZEB1 promoter. The suppression of ZEB1 mRNA levels was achieved through the knockdown of RhoA, PLD1, and MYH9 using si-RNAs. Taken together, we propose that p-Tyr42 RhoA and PLD1, responsible for producing PA, and PA-bound MYH9 are involved in the regulation of ZEB1 expression, thereby promoting cell migration.

Tumor-secreted proteins represent valuable molecular targets for cancer diagnosis and therapy. Moesin (Msn) has been reported to be secreted into the tumor microenviron-ment (TME) and to promote malignant progression; however, its extracellular regula-tory mechanisms remain poorly understood. In this study, we identified extracellular moesin (exMsn) as a common secretory protein derived from 4T1 breast cancer cells and RAW264.7 macrophages using mass spectrometry, which was further validated by western blotting. Functional analyses demonstrated that exMsn enhanced cancer cell migration through activation of the RhoA/ROCK and ZEB1 signaling axes. Neutraliza-tion of exMsn using a specific antibody significantly reduced reactive oxygen species production and cell migration. We further observed that extracellular pyruvate kinase M2 (exPKM2) phosphorylated extracellular proteins in the TME, among which exMsn was identified by peptide mass fingerprinting (PMF) and confirmed by in vitro kinase assays. Mechanistically, exPKM2 phosphorylated exMsn in a phosphoenolpyruvate (PEP)-dependent manner, and site-directed mutagenesis revealed Thr413 as the pri-mary phosphorylation site. This phosphorylation may protect exMsn from extracellu-lar degradation. Additionally, exPKM2 physically interacted with exMsn, with this in-teraction enhanced during cancer–macrophage co-culture. Collectively, our findings un-cover a novel extracellular exPKM2–exMsn signaling mechanism that promotes tumor progression and may provide new therapeutic targets.