Open Access

Circulating fatty acid synthase: an exploratory biomarker to predict efficacy of the dual HER1/HER2 tyrosine kinase inhibitor lapatinib

  • Cristina Oliveras-Ferraros1, 2,
  • Sílvia Cufí1, 2,
  • Tamara Sauri-Nadal2, 3,
  • Sonia Del Barco2, 3,
  • Begoña Martin-Castillo2, 4,
  • Alejandro Vazquez-Martin1, 2 and
  • Javier A Menendez1, 2Email author
Breast Cancer Research201113:401

Published: 24 January 2011

In the previous issue of Breast Cancer Research, Jin and colleagues [1] demonstrated that the lipogenic enzyme fatty acid synthase (FASN) is phosphorylated when it is in complex with the tyrosine kinase receptor HER2. HER2-dependent activating phosphorylation of FASN was related to tumor progression and was sensitive to the dual HER1/HER2 tyrosine kinase inhibitor lapatinib [1]. Adding to knowledge of the therapeutic value of the phosphorylation status of FASN in HER2-driven breast cancer (BC), we now provide experimental evidence that the expression status of FASN in the extracellular milieu (that is, extracellular FASN) may function as a novel biomarker that distinctively predicts molecular functioning of lapatinib.

AMP-activated protein kinase (AMPK)-activating drugs, by mimicking an elevated AMP/ATP ratio in BC cells, drastically augment the release of extracellular FASN in HER2-positive BC cells [2]. Lapatinib-induced deprivation of tumor cell energy activates AMPK to trigger an entire cascade of metabolic events, including suppression of FASN expression and activity [1, 3]. We hypothesized that a differential ability to initiate AMPK-sensed metabolic stress responses may provide information about the efficacy of HER-targeting drugs via changes in the extracellular FASN status. Enzyme-linked immunosorbent assay (ELISA)-based quantitative analyses revealed that lapatinib treatment drastically enhanced extracellular FASN concentration (by at least 8.0-fold) (Figure 1a). Immunoblotting assessment of AMPK phosphorylation at Thr172 confirmed that lapatinib treatment induced a strong activation of AMPK (Figure 1b). A weak but detectable upregulation of PP-AMPKThr172 was observed upon treatment with gefitinib. Trastuzumab, cetuximab, and erlotinib - all of which are unable to promote FASN release - failed to activate AMPK. AMPK knockdown using short interfering RNA (siRNA) transfection [2] fully prevented lapatinib-induced FASN release (Figure S1). Immunoblotting and cell imaging analyses confirmed that FASN was depleted from the cytosol of lapatinib-treated HER2 overexpressors and accumulated in their extracellular milieu (Figure S1).
Figure 1

Lapatinib's molecular functioning involves AMPK-dependent release of extracellular fatty acid synthase (FASN). (a) HER1 or HER2 inhibitors (or both) differentially regulate extracellular expression of FASN. FASN concentration in cell culture supernatants from SKBR3 cells cultured in low-serum (0.5% fetal bovine serum)-containing Dulbecco's modified Eagle's medium in the presence or absence of cetuximab (100 μg/mL), trastuzumab (100 μg/mL), erlotinib (1 μmol/L), gefitinib (1 μmol/L), or lapatinib (1 μmol/L) for 48 hours was measured by enzyme-linked immunosorbent assay (ELISA) as described elsewhere [2]. (b) HER1 or HER2 inhibitors (or both) differentially regulate the activation status of AMPK. After the harvesting, cell cultures treated as described above were lysed and prepared for immunoblotting as described elsewhere [2]. A representative immunoblotting analysis obtained by using the AMPK Antibody Kit (Cell Signaling Technology, Inc., Danvers, MA, USA) is shown. (c) Lapatinib treatment induces extracellular release of FASN in an AMPK-dependent manner. SKBR3 cells were transiently transfected with AMPK short interfering RNA (siRNA) or control siRNA or were treated with transfection reagent alone (mock) before exposure to 1 mmol/L lapatinib for 48 hours. The FASN-detect ELISA was then used to quantitatively assess the released concentration of FASN in the extracellular milieu [2]. (d) Lapatinib-induced release of extracellular FASN is related to lapatinib efficacy in trastuzumab-refractory breast cancer cells. (e) Lapatinib-induced release of extracellular FASN is related to lapatinib's molecular functioning and involves activation of AMPK. Results in graphs are means (columns) and 95% confidence intervals (bars) of two independent experiments made in triplicate. Statistically significant differences (one-factor analysis of variance [ANOVA]) between experimental conditions and unsupplemented control cells are shown by asterisks (*P < 0.01, **P < 0.001; n.s., not statistically significant). All statistical tests were two-sided. AMPK, AMP-activated protein kinase.

Treatment with lapatinib dramatically increased FASN release (by approximately 17 times) in lapatinib-responsive SKBR3 TzbR cells (Figure 1c), which were selected for long-term outgrowth in trastuzumab-containing culture medium. Extracellular FASN remained unaltered in response to trastuzumab or lapatinib in JIMT-1 BC cells, which exhibit de novo cross-refractoriness to multiple HER1/2-targeted therapies (Figure 1c). Lapatinib treatment significantly activated AMPK and promoted an enormous accumulation of extracellular FASN in lapatinib-hypersensitive MCF-7/HER2 cells (Figure 1d). Equimolar concentrations of lapatinib failed to activate AMPK or to alter FASN release into the extracellular milieu of MCF-7/HER2 LapR cells, which were obtained by continuously subculturing parental MCF-7/HER2 cells with high-dose lapatinib.

High levels of circulating FASN can be found in peripheral blood of HER2-overexpressing BC patients [4]. The exclusive ability of lapatinib to actively promote the extracellular release of FASN via AMPK-sensed energy depletion in metabolically demanding HER2-driven BC cell growth might provide a molecular rationale to evaluate the predictive value of circulating FASN in HER2-positive BC patients receiving lapatinib.



AMP-activated protein kinase


breast cancer


fatty acid synthase.



The authors wish to dedicate this letter to the memory of Dr. Francis P Kuhajda, who passed away suddenly 10 November 2010. Dr. Kuhajda was an associate professor of pathology, oncology, and biological chemistry at Johns Hopkins University (JHU) (Baltimore, MD, USA) and director of the JHU Center for Metabolic Pathology. He pioneered the exploration of basic molecular mechanisms linking cancer cell apoptosis with inhibition of fatty acid synthase (FASN)-catalyzed endogenous fatty acid biogenesis. He was actively involved in translational research developing new inhibitors of FASN for future clinical use. He discovered that FASN, a cytosolic protein, can also be found circulating in the blood of patients with cancer. Delineating the forms of the circulating FASN antigen and understanding its role in cancer diagnosis and prognosis were among his ongoing projects.

SC is the recipient of a research fellowship (Formación de Personal Investigador, or FPI) by the Ministerio de Ciencia e Innovación (MICINN, Spain). AV-M is the recipient of a 'Sara Borrell' post-doctoral contract (CD08/00283, Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria [FIS], Spain). Work at the laboratory of JAM is supported in part by the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, FIS, Spain, grants CP05-00090, PI06-0778, and RD06-0020-0028), the Fundación Científica de la Asociación Española Contra el Cáncer (AECC, Spain), and the Ministerio de Ciencia e Innovación (SAF2009-11579, Plan Nacional de I+D+ I, MICINN, Spain).

Authors’ Affiliations

Unit of Translational Research, Catalan Institute of Oncology-Girona (ICO-Girona)
Girona Biomedical Research Institute (IdIBGi)
Medical Oncology, Catalan Institute of Oncology-Girona (ICO-Girona)
Unit of Clinical Research, Catalan Institute of Oncology-Girona (ICO-Girona)


  1. Jin Q, Yuan LXH, Boulbes DR, Baek J-M, Wang Y-N, Gomez-Cabello D, Hawke DH, Yeung S-C, Lee M-H, Hortobagyi GN, Hung M-C, Esteva FJ: Fatty acid synthase phosphorylation: a novel therapeutic target in HER2-overexpressing breast cancer cells. Breast Cancer Res. 2010, 12: R96-10.1186/bcr2777.View ArticlePubMedPubMed CentralGoogle Scholar
  2. Oliveras-Ferraros C, Vazquez-Martin A, Fernández-Real JM, Menendez JA: AMPK-sensed cellular energy state regulates the release of extracellular fatty acid synthase. Biochem Biophys Res Commun. 2009, 378: 488-493. 10.1016/j.bbrc.2008.11.067.View ArticlePubMedGoogle Scholar
  3. Shell SA, Lyass L, Trusk PB, Pry KJ, Wappel RL, Bacus SS: Activation of AMPK is necessary for killing cancer cells and sparing cardiac cells. Cell Cycle. 2008, 7: 1769-1775. 10.4161/cc.7.12.6016.View ArticlePubMedGoogle Scholar
  4. Vazquez-Martin A, Fernandez-Real JM, Oliveras-Ferraros C, Navarrete JM, Martin-Castillo B, Del Barco S, Brunet J, Menendez JA: Fatty acid synthase activity regulates HER2 extracellular domain shedding into the circulation of HER2-positive metastatic breast cancer patients. Int J Oncol. 2009, 35: 1369-1376.PubMedGoogle Scholar


© BioMed Central Ltd 2011