Proposed model of EGFR and HER2 transactivation by SP.

<p>NK-1R induces signal transduction through the activation of G proteins. Heterotrimeric G proteins consist of three different subunits: the Gα subunit that binds GDP/GTP, and the Gβ and Gγ subunits that form the Gβγ complex. The binding of an agonist (in this case Substance P) to NK-1R induces the activation of G proteins which in turn induce their own signaling cascade, such as the activation of the MAPK pathway or the phosphorylation of c-Src. The activation of the MAPK pathway in turn contributes to raising MMP secretion, which increases the cleavage of membrane-anchored ligands that in turn will bind the EGFR receptor. On the other hand, c-Src directly phosphorylates the cytoplasmic tails of both EGFR and HER2, allowing the binding of scaffold proteins that will further activate signal transduction. Based on our results, we propose that this direct phosphorylation of the cytoplasmic tails of EGFR and HER2 by c-Src may overcome the effects of tyrosine kinase inhibitors or antibodies or molecules against extracellular domains [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129661#pone.0129661.ref017" target="_blank">17</a>], since c-Src may act independently of EGFR and HER2 tyrosine kinase activity. <i>This figure has been made using Servier Medical Art</i> collection (<a href="http://creativecommons.org/licenses/by/3.0" target="_blank">http://creativecommons.org/licenses/by/3.0</a>).</p

Blockade of c-Src, MMPs or NK-1R inhibits tumor cell viability and migration in SK-BR-3 and MDA-MB-468 cells.

<p>(A) Cell viability quantification of SK-BR-3 cells and MDA-MB-468 cells treated for 24h with IC50 values of MMP inhibitor, c-Src inhibitor, L-733,060 antagonist or with the combinations of drugs: MMP inhibitor + Src inhibitor, MMP inhibitor + L-733,060), Src inhibitor + L-733,060 and MMP inhibitor + Src inhibitor + L-733,060 in serum free medium. After 24h, the cells were treated with calcein (2 μM) for 45 min and calcein AM fluorescence was measured to determine cell viability. (B) Migration rate quantification of SK-BR-3 cells and MDA-MB-468 cells treated for 24h with IC50 values of MMP inhibitor, c-Src inhibitor, L-733,060 antagonist or with the combination of MMP inhibitor + Src inhibitor in serum-free medium. After 24h, detection of cell migration was quantified using calcein AM. Results are represented as mean of % viability or % migration ± SD. All the quantitative data are for a minimum of 6 replicates. Significant differences by ANOVA with Tukey Multiple Comparison post-hoc test are indicated as * P<0.05, ** P<0.01 and *** P<0.001.</p

SP transmodulates HER2 by c-Src and MMP-dependent mechanisms in SKBR3 cell line.

<p>(A) Representative images of Western blots evaluating the effects of the single or combined inhibition of c-Src (Y416) with 4-(4′-phenoxyanilino)-6,7-dimethoxyquinazoline (1μM) and MMPs with 1–10, phenanthroline monohydrate (7μM) on the activation of HER2 and p42/44 MAPK triggered by SP 100 nM for 6, 10 and 15 minutes. The plots show the densitometric quantification of the Western blots on phosphorylated (denoted by p-) HER2 (B) and p42/44 MAPK (C) relative to the expression of tubulin, which was used to ensure equal protein loading. Western blots are representative of at least two independent experiments.</p

c-Src phosphorylation levels at baseline or under SP treatment.

<p>Representative images of Western blots corresponding to experiments showing (A) the basal levels of c-Src Y416 phosphorylation in BC cell lines and (B) c-Src (Y416) phosphorylation at 0, 1, 2, 4, 6, 8, 10, 15 and 30 minutes after SP 100 nM stimulation in different BC cell lines. The blot was standardized to c-Src levels. The plots accompanying each panel show the densitometric quantification of Western blots (the ratio of intensities of the bands corresponding to phospho-Y416 and total c-Src) relative to the expression of tubulin or actin, which was used to ensure equal protein loading.</p

NK-1R contributes to c-Src activation in BC cell lines.

<p>(A) The contribution of NK-1R to the activation of c-Src Y416 phosphorilation protein in the MDA-MB-231 cell line transfected with pcDNA3.1(+)-<i>TACR1</i> or empty vector and treated for 6 and 10 minutes with SP 100 nM; (B) the effects of single NK-1R inhibition during 24h with L-733,060 (20 μM (SKBR3 and BT-474), 30 μM (MDA-MB-453)) or (C) combined NK-1R, NK-2R and NK-R3 inhibition during 24h with L-733,060 (20 μM), MEN 10376 (30 μM) and SB218795 (20 μM), respectively on c-Src (Y416). The blot was standardized to c-Src levels. All quantitative data are generated from a minimum of 3 replicates and are presented as mean + S.D. and compared by <i>t</i>-test (two-tailed) as * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001.</p

Inhibition of MMP7 by siRNA or addition of rhMMP7 modulates cell response to oxaliplatin-induced apoptosis.

<p>A) MMP7 expression was inhibited by siRNA and then treated with different doses of oxaliplatin in order to analyze the effects of MMP7 inhibition in the response to oxaliplatin treatment. Cell viability was then assessed by MTS assay. To analyze the protective effects of MMP7 cells were treated with rhMMP7 (5 ng/ml) and with different doses of oxaliplatin. The effects on cell viability were determined by B) MTS or C) by annexin V/IP staining. D) The effects of MMPs inhibition on cell survival were determined in HT29 and RHT29 cells treated with 1,10-phenantroline monohydrate and then with different doses of oxaliplatin. It can be observed that cells response equally to oxaliplatin when MMPs are inhibited. Results are the mean±SEM for 3 samples per group. Values that are significantly different between control and treated group by ANOVA analysis are indicated by ***p<0.0001. <i>OXA</i>: oxaliplatin.</p

MMP7 shows a higher expression in the oxaliplatin resistant cell lines.

<p>A) Immunofluorescence (IF) staining of MMP7 in the non-resistant (HT29, HCT116 p53^+/+, HCT116 p53^−/−) and resistant cells (RHT29, RHCT116 p53^+/+, RHCT116 p53^−/−). MMP7 is strongly upregulated in the RHT29 and RHCT116 p53^−/− cell lines and its expression is also increased by oxaliplatin treatment. Levels of MMP7 were also analyzed by qPCR both <i>in vitro</i> (B) and <i>in vivo</i> (C) obtaining a similar pattern than in the IF assay. Results represent the mean±SEM for 6 samples in each group. Values that are significantly different between control and treated group by ANOVA analysis are indicated by *p<0.05,**p<0.001,***p<0.0001, and between different cell lines †††p<0.0001. <i>OXA</i>: oxaliplatin.</p

Modulation of FasL expression in HT29 and RHT29 cells lines.

<p>FasL expression was detected in HT29 and RHT29 cell lines treated or not with oxaliplatin 10 µM by A) FACS analysis of its surface expression or B) qPCR of its mRNA levels. C) NF-κB p65 was detected by Western Blot by the use of an antibody that recognizes the nuclear localization signal (NLS) epitope. Tubulin expression was used as endogenous control. D) Sensitivity of both cell lines to the inhibition of the NF-κB pathway was measured by an MTS assay in which cells were treated with different doses of BAY117085, a known inhibitor of IKK activation. Values that are significantly different between control and treated group by ANOVA analysis are indicated by ***p<0.0001. <i>OXA</i>: oxaliplatin.</p

Mechanism of acquisition of resistance to oxaliplatin-induced cell death.

<p>A) In a tumor with <i>low levels of MMP7</i> oxaliplatin is able to induce an upregulation of Fas receptor at the plasma membrane (1), allowing its activation by cells expressing FasL, such as cytotoxic cells or the surrender cells of the tumor (2). B) On the other hand, in cells with <i>higher levels of MMP7</i>, such as cells that have acquired resistance to oxaliplatin, the activation of Fas receptor is different. Oxaliplatin in these cells induce a stronger upregulation of FasL (1) that is shaded by MMP7 to generate sFasL (2). This sFasL binds to Fas probably inducing its activation (3). Fas receptor could, in turn, be shaded by MMP7, decreasing the availability of Fas at the cell surface (4), although the remaining Fas receptor could be activated by its ligand inducing pathways related to cell survival.</p

Analysis of HT29 and RHT29 growth rate.

<p>A) HT29 and RHT29 cells were subcutaneously inoculated into nude mice. Two weeks later, animals were treated with oxaliplatin 10 mg/kg once a week for 27 days. Results illustrate tumor volume and are represented as mean±SEM for 10 animals per group. The lower panel is a representative image of tumors obtained from animals treated or not with oxaliplatin. B) Growth kinetics was studied by colony formation assay. This representative image shows that HT29 cells are not able to grow under oxaliplatin treatment compared to RHT29 cell line. However, HT29 cells have a higher clonogenic capacity than RHT29 after 21 days of growth. <i>OXA</i>: oxaliplatin.</p