Metabolic reprogramming during TGFβ1-induced epithelial-to-mesenchymal transition

Abstract

Metastatic progression, including extravasation and micrometastatic outgrowth, is the main cause of cancer patient death. Recent studies suggest that cancer cells reprogram their metabolism to support increased proliferation through increased glycolysis and biosynthetic activities, including lipogenesis pathways. However, metabolic changes during metastatic progression, including alterations in regulatory gene expression, remain undefined. We show that transforming growth factor beta 1 (TGFβ1)-induced epithelial-to-mesenchymal transition (EMT) is accompanied by coordinately reduced enzyme expression required to convert glucose into fatty acids, and concomitant enhanced respiration. Overexpressed Snail1, a transcription factor mediating TGFβ1-induced EMT, was sufficient to suppress carbohydrate-responsive-element-binding protein (ChREBP, a master lipogenic regulator), and fatty acid synthase (FASN), its effector lipogenic gene. Stable FASN knockdown was sufficient to induce EMT, stimulate migration and extravasation in vitro. FASN silencing enhanced lung metastasis and death in vivo. These data suggest that a metabolic transition that suppresses lipogenesis and favors energy production is an essential component of TGFβ1-induced EMT and metastasis.

Figure 1. Changes of de novo lipogenesis during TGFβ1 induced EMT

A549 cells were treated with 2ng/ml TGFβ1, with or without 400nM TGF inhibitor for 48h. (a) EMT functional proteins E-cadherin, N-cadherin and Snail1, were analyzed by real-time PCR (RT-PCR). (b) Transwell assays of A549 cells were treated with TGFβ1, with or without TGF inhibitor. (c) mRNA expression levels of FASN and ChREBP were analyzed by RT-PCR as in panels A. (d) Labeling of palmitate in A549, after culture in medium containing [U-¹³C] glucose for 24 hours, and calculated glucose contributed lipogenic acetyl-CoA. (e) Intra-cellular ATP levels and OCRs were measured in TGFβ1 treated cells. (*P<0.05 comparing to BCA control.) (Data are represented as mean ± SEM.)

Figure 2. TGFβ1 induced reversible EMT responses in A549 cells

A549 cells were treated with TGFβ1 (2 ng/ml) for the indicated days (marked TGFβ1 treatment). After 12 days, TGFβ1-treated A549 cells were washed with PBS and subsequently cultured in normal growth medium without TGFβ1 for 3, 6, or 9 days. (a) Protein levels of key EMT functional proteins and metabolic enzymes were analyzed by western-blotting. (b) mRNA levels of ChREBP were monitored by RT-PCR. (c) mRNA levels of EMT functional proteins (E-cadherin, N-cadherin, and Snail1) and (d) lipogenic transcriptional regulatory factors (PPAR-gamma, SREBP1, and SREBP2) were monitored by RT-PCR. (*P<0.05 comparing to day0. ^#P<0.05 comparing to day12 TGFβ1 treatment.) (Data are represented as mean ± SEM.)

Figure 3. Snail1 involved in TGFβ1 regulated lipogenesis

(a) Protein levels of E-cadherin, Vimentin, FASN and ACC were analyzed by western-blotting in stable Snail1 over-expressed A549 cells. (b) The mRNA expression levels of Vimentin and E-cadherin were analyzed by RT-PCR. (c) Transwell assay and scratch assay of control and Snail1 over-expressed A549 cells. (d) Intracellular ATP content and OCRs in Snail1 over-expressed cells with or without TGFβ1 treatment. (e) mRNA levels of lipogenic genes FASN and ACC were analyzed by RT-PCR. (f) mRNA levels of ChREBP were shown by RT-PCR. (*P<0.05 comparing to control A549 cells.) (Data are represented as mean ± SEM.)

Figure 4. Fatty acid synthase knockdown mimic TGFβ1 induced EMT

(a) Whole A549cell lysates were harvested 48h after siRNA transfection, and protein levels of FASN and E-cadherin were shown by western-blotting. (b) mRNA levels of FASN were monitored by RT-PCR in three FASN knockdown cell populations. (c) Protein levels of FASN and EMT functional proteins were shown by western-blotting in FASN knockdown cell lines. (d) Labeling of palmitate in control or FASN-silenced cell lines cultured in medium containing [U-¹³C] glucose for 24 hours, and calculated glucose contributed lipogenic acetyl-CoA. (e) mRNA levels of EMT functional proteins E-cadherin, N-cadherin and Vimentin were analyzed by RT-PCR. (f) Cell mobility was measured by transwell migration assays. (g) Intracellular ATP content and ORCs were monitored for stable shFASN knockdown and shNT A549 cells. (*P<0.05 comparing to shNT A549 cells.) (Data are represented as mean ± SEM.)

Figure 5. Stable shFASN knockdown increases metastasis in A549 NSCLC cells

(a) Growth curve of A549 shFASN knockdown cells in vitro. (b) Stable knockdown shNT (top) or shFASN (bottom) A549 cells (1×10⁶) were injected into the tail veins of anesthetized NOD/SCID female mice, and cell deposition into the lungs were analyzed 4h post-injection. Note that identical viable cell numbers (measured by BLI) deposited into the lungs. (c) Representative images of mice injected with stable shNT or shFASN A549 cells as in ‘A’, but analyzed 20 days later. (d) Relative tumor volumes were measured by BLI intensities at the indicated times. (e) Kaplan-Meier survival for mice bearing stable shNT vs shFASN knockdown A549 lung xenografts. (Data are represented as mean ± SEM.)