Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells - PubMed (original) (raw)

Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells

A Vazquez-Martin et al. Cell Prolif. 2008 Feb.

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 approximately 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 Profilertrade mark 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 (approximately 8, 10 and > 15 times, respectively), while significantly decreasing (approximately 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.

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Figures

Figure 1

Development of noncancerous breast epithelial cells acutely overexpressing FASN. Normal breast epithelial cells were acutely forced to activate FASN‐driven endogenous lipogenesis upon transient transfection with the plasmid pCMV6‐XL4 carrying the full‐length human FASN cDNA (gi: NM 004104). FASN expression was characterized by immunoblotting procedures and FASN enzymatic activity was evaluated by recording spectrophotometrically the linear increase in NADPH oxidation following the addition of the FASN substrate malonyl‐CoA (the method by Dils & Carey). Lipogenic activity was assessed by measuring incorporation of 2‐14C‐acetate into lipids. Changes in the cellular pattern of tyrosine (Tyr) phosphorylation were detected either by immunoblotting procedures using the 4G10 antiphosphor‐Tyr monoclonal antibody or by antibody arrays‐based Proteome Profiling of Human Phospho‐RTKs. The expression level and the activation status of EGFR (HER1) and HER2 (_erb_B‐2) receptors was assessed by ELISA and immunoblotting procedures, respectively. Semiliquid colony formation (i.e. soft agar), anchorage‐dependent cell growth in low serum, MTT and cell death ELISA assays were used to evaluate the effects of FASN overexpression on cell proliferation and survival.

Figure 2

Acute activation of FASN‐catalysed endogenous FA biogenesis promotes the appearance of oncogenic canonical properties in breast epithelial cells. (a) Top. FASN‐specific activity was assayed spectrophotometrically in fresh 14 000 g supernatants from pCMV6‐XL4/FASN‐transfected, pCMV6‐XL4 (mock)‐transfected and parental (non‐transfected) HBL100 cells by measuring the rate of disappearance of NADPH at 340 nm. Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. One‐factor

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was used to analyse differences FASN activity between each experimental condition (P < 0.001 for transfection with pCMV6‐XL4/FASN cells versus all other conditions; one‐factor analysis of variance). All statistical tests were two‐sided. Bottom. Five micrograms of total protein from cultures of HBL100 cells engineered to transiently express either the pCMV6‐XL4 plasmid vector carrying the full‐length human FASN cDNA (pCMV6‐XL4/FASN) or the empty vector pCMV6‐XL4 were resolved by 3–8% Tris‐Acetate NuPAGE and subjected to immunoblotting analyses for FASN using an anti‐FASN monoclonal antibody (clone 23) as described in ‘Materials and methods’. Blots were re‐probed for β‐actin expression to control for protein loading and transfer. Figure shows a representative immunoblotting analysis. Equivalent results were obtained in three independent experiments. (b) HBL100 cells grown in 60‐mm plates were transfected either with the pCMV6‐XL4 plasmid vector carrying the full‐length human FASN cDNA (pCMV6‐XL4/FASN) or the empty vector pCMV6‐XL4. Two days later, medium was changes, and fresh medium was added together with [2‐14C] acetate (57 mCi/mmol; 2 µCi/dish) and incubation was continued for another 4 h. Cells were washed with PBS and trypsinized. Lipids were extracted as described in ‘Materials and methods’, and the radioactivity of aliquots was measured by scintillation counting. Results shown represent mean d.p.m. × 10−3 (columns) and 95% confidence intervals (bars) of incubations performed in triplicate following normalization by protein content in cell lysates. One‐factor

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was used to analyse differences in the lipogenic activity between each experimental condition (P < 0.001 for HBL100‐pCMV6‐XL4/FASN cells versus HBL100‐pCMV6‐XL4 matched control cells; one‐factor analysis of variance).

Figure 2

. (c) HBL100 cells engineered to transiently express either the pCMV6‐XL4 plasmid vector carrying the full‐length human FASN cDNA (pCMV6‐XL4/FASN) or the empty vector pCMV6‐XL4 were seeded (10 000 per well) in six‐well plates in culture medium containing 0.35% low‐melting agarose over a 0.7% agarose base layer and incubated for 10 days. Colonies were then stained with p‐iodonitrotetrazolium violet (1 mg/mL stock diluted at 1 : 500) for 18 h, and colonies larger than 50 µm in diameter were counted. Each experimental value on the graph (right panel) represents the mean colony number (columns) and 95% confidence intervals (bars) from three separate experiments in which triplicate dishes were counted. One‐factor anova was used to analyse differences in the number of colonies between each experimental condition (P < 0.001 for HBL100‐pCMV6‐XL4/FASN cells versus HBL100‐pCMV6‐XL4 matched control cells; one‐factor analysis of variance). Representative microphotographs of soft agar assays are shown (200‐fold magnification). (d) Top. Following 4 h incubation with [2‐14C] acetate (57 mCi/mmol; 2 µCi/dish), lipids from cultures of HBL100 cells at different passage levels were extracted as described in ‘Materials and methods’, and the radioactivity of aliquots was measured by scintillation counting. Results shown represent mean d.p.m. × 10–3 (columns) and 95% confidence intervals (bars) of incubations performed in triplicate following normalization by protein content in cell lysates. One‐factor anova was used to analyse differences in the lipogenic activity between high passage level (HPL) and the low passage level (LPL) control group. Bottom. Five micrograms of total protein from HBL100 cultures were resolved by 3–8% Tris‐Acetate NuPAGE and subjected to immunoblotting analyses for FASN using an anti‐FASN monoclonal antibody (clone 23) as described in ‘Materials and methods’. Blots were re‐probed for β‐actin expression to control for protein loading and transfer. Figure shows a representative immunoblotting analysis. Equivalent results were obtained in three independent experiments.

Figure 2

. (e) Left. HBL100 cells engineered to transiently express either the pCMV6‐XL4 plasmid vector carrying the full‐length human FASN cDNA (pCMV6‐XL4/FASN) or the empty vector pCMV6‐XL4 were seeded (10 000 per well) in 24‐well plates. Cells were plated in the presence of 10% FBS, changed to serum‐free conditions overnight, and processed further in culture medium supplemented with 0.1% FBS. For each experimental condition and time point cell numbers were determined using a trypan blue exclusion assay as described in ‘Materials and methods’. The data presented are mean of cell numbers × 104 (circles) and 95% confidence intervals (bars) from three separate experiments in which triplicate wells were counted. All assays were performed at least three times in triplicate. One‐factor anova was used to analyse differences in the number of cells between each experimental condition (P < 0.01, 48 and 72 h; and P < 0.001, 96 and 120 h for transfection with pCMV6‐XL4/FASN cells versus all other conditions; one‐factor analysis of variance). All statistical tests were two‐sided. Right. Representative microphotographs of anchorage‐dependent HBL100 cultures (at day 4 after transfection) are shown. (f) Left. NIH‐3T3 cells engineered to transiently express either the pCMV6‐XL4 plasmid vector carrying the full‐length human FASN cDNA (pCMV6‐XL4/FASN) or the empty vector pCMV6‐XL4 were seeded (10 000 per well) in 24‐well plates. Cells were plated and processed further in culture medium supplemented with 10% FBS. For each experimental condition, cell numbers were determined using a trypan blue exclusion assay as described in ‘Materials and methods’. The data presented are mean of cell numbers × 104 (circles) and 95% confidence intervals (bars) from three separate experiments in which triplicate wells were counted at day 4 after transfection. All assays were performed at least three times in triplicate. One‐factor anova was used to analyse differences in the number of cells between each experimental condition (P < 0.001 for transfection with pCMV6‐XL4/FASN cells versus all other conditions; one‐factor analysis of variance). All statistical tests were two‐sided. Right. Representative microphotographs of anchorage‐dependent NIH‐3T3 cultures (at day 4 after transfection) are shown.

Figure 3

Acute activation of FASN‐catalysed endogenous FA biogenesis enhances breast epithelial cell viability and survival. (a) HBL100 cells transiently transfected with pCMV6‐XL4/FASN or the empty vector pCMV6‐XL4 for 48 h were incubated in the absence or presence of the specified concentrations of the FASN inhibitor C75 for 5 days. Cell growth, measured using MTT assay, was expressed as percentage of untreated cells (100% cell viability). Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. One‐factor

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was used to analyse differences in the levels of sensitivity to C75 by comparing MTT‐based determination of optical densities at 570 nm in C75‐treated and control‐untreated groups; NS, not statistically significant, one‐factor analysis of variance). All statistical tests were two‐sided. (b) Quantification of apoptosis‐related cell death upon treatment with the FASN inhibitor C75 was determined by cell death ELISA, which measures cytoplasmic histone‐DNA fragments produced during apoptosis. The enrichment of histone‐DNA fragments in treated cells was expressed as fold increase in absorbance by comparing with control (vehicle‐treated) cells using the following formula: [A405–A490]TREATED/[A405–A490]UNTREATED. Data are the mean (columns) and 95% confidence intervals (bars) of three independent experiments performed in duplicate. One‐factor

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was used to analyse differences in the percentage of apoptosis between the C75 treatment group and the control‐untreated group. NS, not statistically significant (one‐factor analysis of variance). All statistical tests were two‐sided.

Figure 4

(a,b) Acute activation of FASN‐catalysed endogenous FA biogenesis alters both the activation (phosphorylation) and the expression status of HER‐ axis in normal breast epithelial cells. Overnight serum‐starved HBL100 cells were transiently transfected with 5 µg of either the pCMV6‐XL4 empty plasmid vector or the pCMV6‐XL4 plasmid vector carrying the full‐length human FASN cDNA in DMEM, 0.1% FBS for 0, 12, 24 or 48 h in the absence or presence of 5 µg/mL C75 as specified. Total content in phosphotyrosine (P)‐Tyr‐proteins and activation status of HER1 and HER2 receptors were tested using P‐Tyr (4G10), P‐Tyr1173‐HER1 and P‐Tyr1248‐HER2 immunoblotting procedures as described in ‘Materials and methods’. Figure shows a representative immunoblotting analysis. Equivalent results were obtained in, at least, three independent experiments.

Figure 4

(c) Five hundred micrograms of total cell lysates from MCF10A transiently transfected with 5 µg of the pCMV6‐XL4 empty plasmid (48 h), with 5 µg of the pCMV6‐XL4 vector plasmid carrying the full‐length human FASN cDNA (48 h), or with 5 µg of pCMV6‐XL4/FASN (48 h) prior treatment with 5 µg/mL C75 (24 h) were incubated with membranes of the Human Phospho‐RTK Array Kit (Proteome Profiler™; R&D Systems) as per manufacturer's instructions. Phospho‐RTK array data were developed on X‐ray films following exposure to chemiluminescent reagents. Figure shows a representative phosphor‐proteome analysis. Equivalent results were obtained in three independent experiments. Representative microphotographs of anchorage‐dependent cultures (at day 2 after transfection) are shown. (d) Left. MCF10A cells engineered to transiently express either the pCMV6‐XL4 plasmid vector carrying the full‐length human FASN cDNA (pCMV6‐XL4/FASN) or the empty vector pCMV6‐XL4 were seeded (10 000 per well) in six‐well plates in culture medium containing 0.35% low‐melting agarose over a 0.7% agarose base layer and incubated for 14 days. Colonies were then stained with _p_‐iodonitrotetrazolium violet (1 mg/mL stock diluted at 1 : 500) for 18 h, and colonies larger than 50 µm in diameter were counted. Each experimental value on the graph (right panel) represents the mean colony number (columns) and 95% confidence intervals (bars) from three separate experiments in which triplicate dishes were counted. One‐factor

anova

was used to analyse differences in the number of colonies between each experimental condition (P < 0.001 for MCF10A‐pCMV6‐XL4/FASN cells versus MCF10A‐pCMV6‐XL4 matched control cells; one‐factor analysis of variance). Representative microphotographs of soft agar assays are shown (200‐fold magnification). Right. Fifty micrograms (for P‐HER2) or 5 µg (for FASN) of total protein from MCF10A, MCF10AT and MCF10CA1 cultures were resolved by 3–8% Tris‐Acetate NuPAGE and subjected to immunoblotting analyses for P‐HER2 and FASN using anti‐P‐HER2 and anti‐FASN monoclonal antibodies (clone PN2A and clone 23, respectively) as described in ‘Materials and methods’. Blots were re‐probed for β‐actin expression to control for protein loading and transfer. Figure shows a representative immunoblotting analysis. Equivalent results were obtained in three independent experiments. (e) Left. The Oncogene Science HER1 and HER2 microtiter ELISAs were used according to the manufacturer's instructions to compare HER1 and HER2 concentrations in HBL100 cells transfected as described above. Results are means (columns) and 95% confidence intervals (bars) of three independent experiments made in triplicate. One‐factor

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was used to analyse differences in the amounts of HER1 or HER2 24 and 48 h after transfection with pCMV6‐XL4/FASN (one‐factor analysis of variance). All statistical tests were two‐sided. No changes were observed when HBL100 cells were mock‐transfected with 5 µg of the empty vector pCMV6‐XL4 (data not shown). Right. Fifty micrograms of total protein from HBL100 cells transfected as described above were subjected to Western blot analyses with specific antibodies against HER1 and HER2. Blots were re‐probed for β‐actin expression to control for protein loading and transfer. A representative immunoblotting analysis is shown. Equivalent results were obtained in three independent experiments.

Figure 4

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Figure 5

Cell viability of breast epithelial cells engineered to overexpress FASN depends on the function of the HER1/HER2 duo. Figure shows dose–response curves of HBL100 cells engineered to transiently express increasing amounts of the pCMV6‐XL4 plasmid vector carrying the full‐length human FASN cDNA (i.e. 2.5 and 5 µg of pCMV6‐XL4/FASN) or 5 µg of the empty vector pCMV6‐XL4 to gefitinib (a), lapatinib (b) and cisplatin (c). Cells seeded in 96‐well plates (2000–3000 per well) were cultured in triplicate in the absence or presence of graded concentrations of the agents, which were not renewed during the entire period of cell exposure. Once control‐untreated wells reached confluency, cells were exposed to MTT reagent and optical density at 570 nm was measured in a microplate reader. The cell viability effects from exposure of cells to gefitinib, lapatinib and cisplatin were analysed by generating concentration‐effect curves as a plot of the fraction of unaffected (surviving) cells versus drug concentration. Dose–response curves were plotted as percentages of the control cells’ absorbance (= 100%), which was obtained from wells treated with appropriate concentrations (DMSO, v/v) of agents vehicle that were processed simultaneously. IC50 values were designated for the concentrations of the agents (µ

) decreasing absorbance values at 570 nm by 50%, as determined by interpolation using the MTT‐based colorimetric growth viability assay (see ‘Materials and methods’). Values are means of three independent experiments carried out in triplicate. Sensitization/resistance factors were obtained by dividing the IC50 values of gefitinib, lapatinib and cisplatin from HBL100 cells transfected with the empty vector pCMV6‐XL4 by those obtained when cells were transfected with pCMV6‐XL4/FASN.

Figure 5

Figure 6

A new mode of action for endogenous lipogenesis in breast cancer disease. It is widely accepted that tumour‐associated FASN represents a growth factors/growth factor receptors‐driven change in the genetic program controlling lipogenesis through sterol regulatory element‐binding proteins (SREBPs), a family of transcription factors that coordinately activate genes involved in the synthesis of cholesterol and FAs. In this classic ‘out‐to‐in_’_model (left), changes in up‐stream regulatory circuits (e.g. activation of the HER network by either gene amplification of HER members or by overproduction of HER ligands ‐1‐) lead to down‐stream activation of the transduction cascades Ras/Raf/MEK/ERK and PI‐3′K/AKT ‐2‐, and ultimately to SREBP‐dependent up‐regulation of FASN gene expression ‐3‐. We here propose an alternative ‘in‐to‐out_’_scenario (right) in which, de novo FA biogenesis, directly or indirectly, significantly affects the formation of HER1/HER2 signal transduction complexes at the membrane, changing their state from ‘_off_’ to ‘_on_’ ‐1‐. FASN‐induced activation of the HER network would indirectly up‐regulate SREBP‐driven FASN gene expression ‐2‐ and therefore FASN hyperactivity may create a feed‐back able to continuously maintain high levels of FASN in breast cancer cells ‐3‐.

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Overexpression of fatty acid synthase gene activates HER1/HER2 tyrosine kinase receptors in human breast epithelial cells - PubMed (original) (raw)