Hydroxysteroid (17β) dehydrogenase type 12 (HSD17B12) is a multifunctional isoenzyme functional in the conversion of estrone to estradiol (E2), and elongation of long-chain fatty acids, in particular the conversion of palmitic to archadonic (AA) acid, the precursor of sterols and the inflammatory mediator, prostaglandin E(2). Its overexpression together with that of COX-2 in breast carcinoma is associated with a poor prognosis. We have identified the HSD17B12(114-122) peptide (IYDKIKTGL) as a naturally presented HLA-A*0201 (HLA-A2)-restricted CD8(+) T-cell-defined epitope. The HSD17B12(114-122) peptide, however, is poorly immunogenic in its in vitro ability to induce peptide-specific CD8(+) T cells. Acting as an "optimized peptide", a peptide (TYDKIKTGL), which is identical to the HSD17B12(114-122) peptide except for threonine at residue 1, was required for inducing in vitro the expansion of CD8(+) T-cell effectors cross-reactive against the HSD17B12(114-122) peptide. In IFN-γ ELISPOT assays, these effector cells recognize HSD17B12(114-122) peptide-pulsed target cells, as well as HLA-A2(+) squamous cell carcinoma of the head and neck (SCCHN) and breast carcinoma cell lines overexpressing HSD17B12 and naturally presenting the epitope. Whereas growth inhibition of a breast carcinoma cell line induced by HSD17B12 knockdown was only reversed by AA, in a similar manner, the growth inhibition of the SCCHN PCI-13 cell line by HSD17B12 knockdown was reversed by E2 and AA. Our findings provide the basis for future studies aimed at developing cancer vaccines for targeting HSD17B12, which apparently can be functional in critical metabolic pathways involved in inflammation and cancer.