Figure 1.
Leptin induces the expression of Notch and its molecular targets in mammary cancer cells.
Representative results of leptin-dose effects on increase in protein levels of Notch (ligands and receptors) and molecular targets as determined by immunocytochemistry in 4T1 cells (A) and western blot (WB) in 4T1 (B and C), EMT6 (D and E) and MMT (F and G) cells. Cells were cultured for 24 h and leptin dose-induced (0, 0.6, 1.2 and 6.2 nM) effects were determined as described (See M &M). The WB results were normalized to β-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software. (a) P<0.05 when comparing levels of protein to control (basal). Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments. Bar = 200 µm.
Figure 2.
Leptin-mediated upregulation of protein and mRNA levels of Notch receptors and targets in mammary cancer cells are abrogated by a γ-secretase inhibitor (DAPT).
Leptin-induced transcriptional expression of Notch receptors and targeted genes was abrogated by DAPT in 4T1 cells. Levels of Notch receptors mRNA (A, Notch1; B, Notch2 and C, Notch3) and targeted genes (D, Hey2 and E, survivin) as determined by real-time RT-PCR. GAPDH was used as internal control. (F–M) Representative results of leptin upregulation of protein levels of Notch1–3 and activated NICD1 as well as Notch targeted genes, Hey2 and survivin in 4T1 cells as determined by western blot (WB). The WB results were normalized to β-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software for (G) NICD1; (H) Notch1; (I) Notch 2; (J) Notch 3; (K) Hey2 and (M) survivin. 4T1 cells were cultured in a medium containing 0 or 1.2 nM leptin for 24 h. (a) P<0.05 when comparing levels of mRNA or protein to control (basal). Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.
Figure 3.
Leptin-induced expression of Notch receptors and targeted genes in mammary cancer cells are abrogated by CSL-RNA knockdown.
(A) Representative results of CSL-siRNA inhibition on leptin upregulation of protein levels of Notch 1-3, activated NICD1 and Notch targeted genes, CSL, survivin and Hey2 in 4T1 cells as determined by western blot (WB). The WB results were normalized to β-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software for (B) CSL; (C) Notch1; (D) NICD1; (E) Notch 2; (F) Notch 3; (G) Hey2 and (H) survivin. Cells cotransfected with siRNA oligonucleotides (CSL-siRNA or control-siRNA) were treated with leptin (0 and 1.2 nM) for 24 h. (a) P<0.05 when comparing protein levels to cells treated with control-siRNA (basal) or CSL-siRNA. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.
Figure 4.
Leptin upregulates the CSL gene in mammary cancer cells.
Leptin transcriptional activation of CBF (or CSL) promoter is linked to several leptin-induced signaling pathways. (A) 4T1 cells were transiently transfected with a CBF-Luc reporter construct and incubated with leptin (0 and 1.2 nM) alone or plus siRNA oligonucleotides (SignalSilence control-siRNA and STAT3, MEK1, AKT1, mTOR, JNK, p38-siRNA). (B) Protein levels of kinases after siRNA treatment were determined by WB analysis using β-actin as loading control. (C) 4T1 cells transfected with CBF-Luc reporter were incubated with leptin alone or plus pharmacological inhibitors of JAK2/STAT3 (AG490, 30 µM), MEK/MAPK/ERK1/2 (PD98059, 30 µΜ), PI-3K/AKT1 (Wortmannin, 50 nM), PKC-Ca dependant (Gö6976, 30 µΜ), p38 kinase (SB203580, 2 µΜ), JNK (SP600125, 30 µM) and mTOR (Rapamycin, 20 nM) signaling pathways for 24 h. Luciferase activity was determined as described (see M &M) and expressed as a percent of basal or leptin-treated cells. (a) P<0.05 when comparing levels of luciferase activity to control (basal) or leptin-treated cells. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.
Figure 5.
Leptin-induced 4T1 cell migration and proliferation was abrogated by Notch inhibition.
A) Representative results from immunocytochemistry (ICC, hematoxylin staining) of leptin and DAPT effects on migration of 4T1 cells as compared to basal conditions. B) Quantitative assessment of cell migration under the effects of leptin and DAPT. C) Representative results from ICC after addition of control-siRNA (Ctr-siRNA), CLS-siRNA and leptin. D) Quantitative assessment of cell migration under the effects of leptin, CSL-siRNA and Crt-siRNA. E) Effects of leptin and DAPT on 4T1 cell proliferation. F) Effects of leptin, Ctr-siRNA and CSL-siRNA on 4T1 cell proliferation. Results from cell migration (Boyden chamber cell migration assay) and proliferation (MTT cell proliferation assay) were obtained after 24 h and normalized to basal conditions (see Material and Methods). Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.
Figure 6.
Leptin-Notch crosstalk regulates leptin-induced levels of pro-angiogenic and pro-inflammatory factors in mammary cancer cells.
(A) DAPT inhibition of γ-secretase abrogated leptin transcriptional induction of VEGF (A); VEGFR-2 (B) and IL-1α mRNA (C) as determined by real-time RT-PCR and normalized to the GAPDH expression. CSL-siRNA also inhibited leptin-induced effects on protein levels of VEGF (D); VEGFR-2 (E) and IL-1α (F) mRNA. Representative results of DAPT (G) and CSL-siRNA (M) inhibition of leptin upregulation of protein levels of VEGF, VEGFR-2, IL-1α, ERα and OB-R in 4T1 cells as determined by western blot (WB). The WB results were normalized to β-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software for VEGF (H and N); VEGFR-2 (I and O); IL-1α (J and P); ERα (K and Q) and OB-R (L and R) in cells treated with leptin (0 and 1.2 nM) and DAPT for 24 h or cotransfected with CSL-siRNA and control (Ctr)-SiRNA, respectively. (a) P<0.05 when comparing levels of antigens to basal conditions and control-siRNA. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.
Figure 7.
Inhibition of IL-1R tI signaling negatively impacts on leptin-mediated upregulation of Notch receptors and target genes in mammary cancer cells.
Anti-IL-1R tI antibodies abrogated leptin transcriptional induction of Notch1 (A); Notch2 (B); Notch3 (C) and targeted genes, Hey2 (D) and survivin (E) in 4T1 cells as determined by real-time RT-PCR and normalized to the GAPDH expression. Blockade of IL-1R tI with antibodies also inhibited leptin-induced effects on protein levels of Notch and targeted genes. Representative results of IL-1R tI blockade on leptin upregulation of protein levels of Notch1, NICD1, Notch2, Notch3, Hey2) and survivin (F) as determined by western blot (WB). The WB results were normalized to β\-actin as loading control and densitometric analysis of bands was carried-out with the imageJ software for Notch1 (G); NICD1 (H); Notch2 (I); Notch3 (J), Hey2 (K) and survivin (M). 4T1 cells treated were treated with leptin (0 and 1.2 nM) and IL-1R tI antibodies or nonspecific species-matched IgG (Control Ab) for 24 h. (a) P<0.05 when comparing Basal to Leptin+Control Ab and, (b) comparing Leptin+IL-1R tI Ab to Leptin+Control Ab. Data (mean ± standard error) representative results derived from a minimum of 3 independent experiments.
Figure 8.
Notch, IL-1 and leptin crosstalk outcome (NILCO) upregulates VEGF/VEGFR-2 and mediates leptin-induced breast cancer cell proliferation/migration.
Leptin up-regulates Notch and IL-1 in breast cancer cells [18]. Leptin can directly [27], or through IL-1 [18] and Notch, induce VEGF/VEGFR-2 up-regulation. VEGF signaling could also upregulate Notch [45]. Leptin-induced proliferation and migration of breast cancer cells was related to an intact Notch signaling axis. NILCO could be a master process for the regulation of breast cancer angiogenesis.