Sustained proliferation in cancer: mechanisms and novel therapeutic targets

Proliferation is an important part of cancer development and progression. This is manifest by altered expression and/or activity of cell cycle related proteins. Constitutive activation of many signal transduction pathways also stimulates cell growth. Early steps in tumor development are associated with a fibrogenic response and the development of a hypoxic environment which favors the survival and proliferation of cancer stem cells. Part of the survival strategy of cancer stem cells may manifested by alterations in cell metabolism. Once tumors appear, growth and metastasis may be supported by overproduction of appropriate hormones (in hormonally dependent cancers), by promoting angiogenesis, by undergoing epithelial to mesenchymal transition, by triggering autophagy, and by taking cues from surrounding stromal cells. A number of natural compounds (e.g., curcumin, resveratrol, indole-3-carbinol, brassinin, sulforaphane, epigallocatechin-3-gallate, genistein, ellagitannins, lycopene and quercetin) have been found to inhibit one or more pathways that contribute to proliferation (e.g., hypoxia inducible factor 1, nuclear factor kappa B, phosphoinositide 3 kinase/Akt, insulin-like growth factor receptor 1, Wnt, cell cycle associated proteins, as well as androgen and estrogen receptor signaling). These data, in combination with bioinformatics analyses, will be very important for identifying signaling pathways and molecular targets that may provide early diagnostic markers and/or critical targets for the development of new drugs or drug combinations that block tumor formation and progression

Notch, IL-1 and leptin crosstalk outcome (NILCO) is critical for leptin-induced proliferation, migration and VEGF/VEGFR-2 expression in breast cancer.

High levels of pro-angiogenic factors, leptin, IL-1, Notch and VEGF (ligands and receptors), are found in breast cancer, which is commonly correlated with metastasis and lower survival of patients. We have previously reported that leptin induces the growth of breast cancer and the expression of VEGF/VEGFR-2 and IL-1 system. We hypothesized that Notch, IL-1 and leptin crosstalk outcome (NILCO) plays an essential role in the regulation of leptin-mediated induction of proliferation/migration and expression of pro-angiogenic molecules in breast cancer. To test this hypothesis, leptin's effects on the expression and activation of Notch signaling pathway and VEGF/VEGFR-2/IL-1 were determined in mouse (4T1, EMT6 and MMT) breast cancer cells. Remarkably, leptin up-regulated Notch1-4/JAG1/Dll-4, Notch target genes: Hey2 and survivin, together with IL-1 and VEGF/VEGFR-2. RNA knockdown and pharmacological inhibitors of leptin signaling significantly abrogated activity of reporter gene-luciferase CSL (RBP-Jk) promoter, showing that it was linked to leptin-activated JAK2/STAT3, MAPK, PI-3K/mTOR, p38 and JNK signaling pathways. Interestingly, leptin upregulatory effects on cell proliferation/migration and pro-angiogenic factors Notch, IL-1 and VEGF/VEGFR-2 were abrogated by a γ-secretase inhibitor, DAPT, as well as siRNA against CSL. In addition, blockade of IL-1R tI inhibited leptin-induced Notch, Hey2 and survivin as well as VEGF/VEGFR-2 expression. These data suggest leptin is an inducer of Notch (expression/activation) and IL-1 signaling modulates leptin effects on Notch and VEGF/VEGFR-2. We show for the first time that a novel unveiled crosstalk between Notch, IL-1 and leptin (NILCO) occurs in breast cancer. Leptin induction of proliferation/migration and upregulation of VEGF/VEGFR-2 in breast cancer cells were related to an intact Notch signaling axis. NILCO could represent the integration of developmental, pro-inflammatory and pro-angiogenic signals critical for leptin-induced cell proliferation/migration and regulation of VEGF/VEGFR-2 in breast cancer. Targeting NILCO might help to design new pharmacological strategies aimed at controlling breast cancer growth and angiogenesis

Leptin-Notch crosstalk regulates leptin-induced levels of pro-angiogenic and pro-inflammatory factors in mammary cancer cells.

<p>(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) <i>P<0.05</i> 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.</p

Leptin upregulates the CSL gene in mammary cancer cells.

<p>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) <i>P<0.05</i> 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.</p

Notch, IL-1 and leptin crosstalk outcome (NILCO) upregulates VEGF/VEGFR-2 and mediates leptin-induced breast cancer cell proliferation/migration.

<p>Leptin up-regulates Notch and IL-1 in breast cancer cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021467#pone.0021467-Zhou1" target="_blank">[18]</a>. Leptin can directly <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021467#pone.0021467-GonzalezPerez1" target="_blank">[27]</a>, or through IL-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021467#pone.0021467-Zhou1" target="_blank">[18]</a> and Notch, induce VEGF/VEGFR-2 up-regulation. VEGF signaling could also upregulate Notch <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021467#pone.0021467-Hainaud1" target="_blank">[45]</a>. 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.</p

Leptin-induced 4T1 cell migration and proliferation was abrogated by Notch inhibition.

<p>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.</p