Abstract Background: Hepatocellular carcinoma (HCC), the most common type of liver cancer, is an aggressive malignancy with limited therapeutic options and poor prognosis, underscoring the need for novel therapeutic targets. Emerging evidence implicates dysregulated creatine metabolism in HCC pathogenesis. Serum creatine levels inversely correlate with HCC progression and mitochondrial creatine kinase (MtCK), encoded by CKMT1, has been identified as a potential diagnostic biomarker. MtCK catalyzes the reversible transfer of a phosphate group from ATP to creatine to generate phosphocreatine, a key energy buffer that supports cellular bioenergetics. However, the functional role of CKMT1 in HCC progression remains poorly understood. Methods: We investigated the role of CKMT1 in HCC progression using both human (Hep3B) and porcine (Oncopig A272) HCC cells. CKMT1 overexpression was achieved by lentiviral transduction of human CKMT1 followed by antibiotic selection, and CKMT1 knockout (KO) clones were generated by CRISPR/Cas9 and single-cell clone isolation. Altered CKMT1 expression was confirmed by Western blotting. Cell proliferation and migration were assessed by MTS and wound-healing assays, respectively. Intracellular creatine levels were quantified using colorimetric method, and in vivo tumor growth was evaluated by subcutaneous injection into both flanks of SCID mice (n=6 mice/group). Results: CKMT1 overexpression significantly decreased intracellular creatine levels and increased Hep3B cell proliferation and migration. A272 cells, which exhibit higher endogenous CKMT1 expression compared to Hep3b, did not show further increase in proliferation or migration upon overexpression. Conversely, A272 CKMT1 KO clones exhibited significantly elevated intracellular creatine levels and reduced proliferation and migration. In vivo, xenografts derived from CKMT1-overexpressing A272 cells exhibited significantly increased tumor growth rate compared to parental controls (p 0.001), whereas CKMT1 KO tumors grew at rates comparable to the parental line. Conclusions: These findings identify CKMT1 as a promoter of HCC progression, linking altered creatine metabolism to HCC growth. Targeting mitochondrial creatine kinase or its metabolic network may represent a promising therapeutic strategy. Ongoing studies focus on developing porcine HCC models with modulated creatine metabolism to enable integrated metabolic and molecular imaging approaches for improved diagnosis, risk stratification, and treatment monitoring in a physiologically relevant large-animal model. Citation Format: Nusrat Israr Khan, Luke Jordan, Olayinka David, Shreya Pattisapu, Kyle Schachtschneider, Kejia Cai, Ron Gaba, Lobna Elkhadragy, . Mitochondrial creatine kinase promotes hepatocellular carcinoma progression abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7334.
Khan et al. (Fri,) studied this question.