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. 2021 Mar 26;12:1920. doi: 10.1038/s41467-021-22101-7

Fig. 6. AAMDC activates PI3K-AKT-mTOR signaling and promotes E2-independent tumor growth in vivo.

Fig. 6

a Activation of PI3K-AKT-mTOR signaling by various ligands increases MTHFD1L expression, as determined by immunoblot (IB). Panels 1 and 2 (from left to right): Serum-starved SUM52PE subjected to growth factor stimulation for 24 h with insulin (Ins, 300 μg/mL), fetal bovine serum (FBS, 20%), tumor necrosis factor α (TNFα, 50 ng/mL), and amino acids (4 h). Panel 3: The estrogen receptor positive (ER+) cell line SUM44PE was estrogen-starved for 72 h and then stimulated with estrogen (E2, 1 nM) for 24 h. Panel 4: Time-course of E2-mediated activation of the PI3K-AKT-mTOR-MTHFD1L axis in SUM44PE cells. Panel 5: Activation of PI3K-AKT-mTOR upon lentiviral transduction of AAMDC cDNA in MCF-7 cells compared to empty vector (EV) and untransduced wild-type (WT) cells grown either under starvation conditions (in the absence of serum, estrogen, or non-essential amino acids) or in complete medium for 24 h prior to immunoblotting with the indicated antibodies (α). Source data are provided as a Source Data file. b Left: mRNA (top) and protein expression (bottom) levels in MCF-7 cells lentivirally transduced either with AAMDC cDNA or EV grown under starvation conditions prior to their injection into nude mice. mRNA levels for the same cells grown in complete medium are shown. Middle: mRNA expression of AAMDC in the extracted tumors (top) with representative images of the tumors indicated (bottom), harvested at day 55. Right: Mean tumor volumes in nude mice injected with low growth factor (LGF) Matrigel® and with MCF-7 cells transduced with either the EV or AAMDC cDNA. Mice were injected with 1 μg of estradiol valerate (+E2, top) or with vehicle control in the absence of estrogen (-E2, bottom). The mean tumor volume of n = 8 mice is normalized to day 7 (left). Volume measurements of individual mice are plotted for the selected time-points shown (right). Statistical significance is determined using a two-tailed unpaired t-test and presented as mean values ±  SEM. For MCF-7 cells and tumors: ***p = 0.0005, ****p < 0.0001. Multiple unpaired t-test for tumor volume +E2 + LGF: *p = 0.0214, left; *p = 0.0111, right. For tumor volume −E2 + LGF (left plot): day 16 **p = 0.0086, day 20 *p = 0.0277, day 24 **p = 0.0052, day 27 *p = 0.0339, day 34 **p = 0.0026, day 38 *p = 0.0132, day 41 **p = 0.0042, day 45 ***p = 0.0009, day 48 ***p = 0.0002, day 52 ***p = 0.0004, day 55 ***p = 0.0003; (right plot): day 24 *p = 0.0175, day 55 **p = 0.0011). Source data are provided as a Source Data file. c Immunohistochemistry (IHC) analysis of two representative tumors collected at day 55 for each condition. Tumor sections were stained with hematoxylin and eosin (H&E) or the antibodies indicated and the images quantified. The error bars indicate the mean ± SEM. Statistical significance between empty vector (EV) and AAMDC cDNA conditions is determined by a two-tailed unpaired t-test (***p = 0.0002, and ****p < 0.0001). n = 3 biologically independent animals.