Uncontrolled RHOA signaling promotes cancer progression, but the mechanisms governing its activation beyond direct gene alterations remain poorly understood. Lactylation, a metabolic modification derived from lactate, connects altered cellular metabolism to cancer development. However, whether this modification can imitate the effects of cancer-causing mutations has not been examined. The present research explores lactylation of RHOA at key mutation-prone sites and its function as a reversible “epi-mutation” mechanism. RHOA lactylation sites were detected through mass spectrometry using a pan-anti-lactyllysine (Kla) antibody. Site-specific lactylation was introduced via an orthogonal Mb-Pyl Kla-RS/Pyl-tRNA system to add lactyl-lysine at positions K118 and K162 in recombinant RHOA; incorporation was confirmed by immunoblotting and fluorescence methods. Molecular dynamics simulations (using AlphaFold 3 and BIOVIA DS) evaluated impacts on GTPase function and hydrogen-bonding patterns. ROCK2-RBD pull-down assays and GTPase activity tests measured RHOA activation status. Protein ubiquitination and stability were analyzed with cycloheximide chase experiments and K48/K63-ubiquitin variants. In vitro lactylation and delactylation reactions with PCAF and HDAC3 established enzyme preferences. Functional effects were assessed in cell migration/invasion assays and in xenograft tumor studies. Clinical significance was assessed in breast cancer specimens and patient survival datasets. Lactylation of RHOA occurs at oncogenic mutation hotspots K118 and K162, controlled by the lactate-PCAF/HDAC3 regulatory axis. Mechanistically, lactylation at K118 leads to persistent RHOA activation by reducing its intrinsic GTPase activity, while modification at K162 enhances RHOA protein stability by blocking ubiquitination; USP9X further supports this stability by deubiquitinating RHOA. These modifications drive increased tumor cell motility, invasiveness, and metastatic spread. In clinical samples, RHOA lactylation levels are higher in breast tumors compared with nearby normal tissue. Combined inhibition of lactate generation (using the LDHA inhibitor sodium oxamate) and RHOA signaling (with the ROCK inhibitor Y-27632) effectively restrained tumor growth. Through a site-specific lactylation approach, we demonstrate that this modification replicates the functional consequences of oncogenic mutations by boosting both RHOA activity and longevity, thereby defining lactylation at mutation-sensitive residues as a dynamic, reversible “epi-mutation” pathway that mirrors the outcomes of genetic mutations. Lactylation of RHOA at K118 (promoting activation) and K162 (promoting stabilization), orchestrated by the PCAF/HDAC3 enzyme pair, sustains continuous oncogenic signaling and accelerates tumor development. Importantly, this work establishes that lactylation at mutation hotspots operates as a reversible “epi-mutation” system in which metabolic modification functionally reproduces the effects of genetic mutations. This concept blurs traditional distinctions between genetic and epigenetic drivers in cancer and highlights combined inhibition of lactylation and standard RHOA pathways as a promising therapeutic avenue.
