Abstract. Objectives: More than 50 years ago, we learned that breast cancer cells (and those of many other types of tumour) endogenously synthesize 95% of fatty acids (FAs) de novo, despite having adequate nutritional lipid supply. Today, we know that breast cancer cells benefit from this phenomenon in terms of enhanced cell proliferation, survival, chemoresistance and metastasis. However, the exact role of the major lipogenic enzyme fatty acid synthase (FASN) as cause, correlate or facilitator of breast cancer remains unidentified. Materials and methods: To evaluate a causal effect of FASN-catalysed endogenous FA biosynthesis in the natural history of breast cancer disease, HBL100 cells (an SV40-transformed in vitro model for near-normal gene expression in the breast epithelium), and MCF10A cells (a non-transformed, near diploid, spontaneously immortalized human mammary epithelial cell line) were acutely forced to overexpress the human FASN gene. Results: Following transient transfection with plasmid pCMV6-XL4 carrying full-length human FASN cDNA (gi: NM 004104), HBL100 cells enhanced their endogenous lipid synthesis while acquiring canonical oncogenic properties such as increased size and number of colonies in semisolid (i.e. soft-agar) anchorage-independent cultures. Anchorage-dependent cell proliferation assays in low serum (0.1% foetal bovine serum), MTT-based assessment of cell metabolic status and cell death ELISA-based detection of apoptosis-induced DNA-histone fragmentation, together revealed that sole activation of endogenous FA biosynthesis was sufficient to significantly enhance breast epithelial cell proliferation and survival. When analysing molecular mechanisms by which acute activation of de novo FA biosynthesis triggered a transformed phenotype, HBL100 cells, transiently transfected with pCMV6-XL4/FASN, were found to exhibit a dramatic increase in the number of phosphor-tyrosine (Tyr)-containing proteins, as detected by 4G10 antiphosphor-Tyr monoclonal antibody. Phosphor-Tyr-specific antibodies recognizing the phosphorylation status of either the 1173 Tyr residue of epidermal growth factor receptor (HER1) or the 1248 Tyr residue of HER2, further revealed that FASN-induced Tyr-phosphorylation at ∼180 kDa region mainly represented that of these key members of the HER (erbB) network, which remained switched-off in mock-transfected HBL100 cells. ELISA and immunoblotting procedures demonstrated that FASN overactivation significantly increased (> 200%) expression levels of epidermal growth factor receptor and HER2 proteins in HBL100 cells. Proteome Profiler™ antibody arrays capable of simultaneously detecting relative levels of phosphorylation of 42 phospho-receptor Tyr-kinases (RTKs) confirmed that acute activation of endogenous FA biosynthesis specifically promoted hyper-Tyr-phosphorylation of HER1 and HER2 in MCF10A cells. This FASN-triggered HER1/HER2-breast cancer-like phenotype was specifically inhibitable either by FASN inhibitor C75 or by Tyr-kinase inhibitors (TKIs) gefitinib (Iressa™) and lapatinib (Tykerb™) but not by chemotherapeutic agents such as cisplatin. Transient overexpression of FASN dramatically increased HBL100 breast epithelial cells’ sensitivity to cytotoxic effects of C75, gefitinib and lapatinib (∼8, 10 and > 15 times, respectively), while significantly decreasing (∼3 times) cisplatin efficacy. Conclusions: Although we cannot definitely establish FASN as a novel oncogene in breast cancer, this study reveals for the first time that exacerbated endogenous FA biosynthesis in non-cancerous epithelial cells is sufficient to induce a cancer-like phenotype functionally dependent on the HER1/HER2 duo. These findings may perhaps radically amend our current perspective of endogenously synthesized fat, as on its own, it appears to actively increase signal-to-noise ratio in the HER1/HER2-driven progression of human breast epithelial cells towards malignancy.