Src activation by β-adrenoreceptors is a key switch for tumour metastasis

Abstract

Noradrenaline can modulate multiple cellular functions important for cancer progression; however, how this single extracellular signal regulates such a broad array of cellular processes is unknown. Here we identify Src as a key regulator of phosphoproteomic signalling networks activated in response to beta-adrenergic signalling in cancer cells. These results also identify a new mechanism of Src phosphorylation that mediates beta-adrenergic/PKA regulation of downstream networks, thereby enhancing tumour cell migration, invasion and growth. In human ovarian cancer samples, high tumoural noradrenaline levels were correlated with high pSrc(Y419) levels. Moreover, among cancer patients, the use of beta blockers was significantly associated with reduced cancer-related mortality. Collectively, these data provide a pivotal molecular target for disrupting neural signalling in the tumour microenvironment.

Figure 1. Putative phosphorylation cascade triggered by the NE-induced activation of Src

In this predicted network (see “Bioinformatics Analysis” in “Methods”), the components connecting Src to the NE-responsive proteins are shown as arrows: green, experimentally observed phosphorylation according to PhosphoELM; dark blue, predicted by NetworKIN; light blue, predicted by NetworKIN for a close homolog of the target.

Figure 2. NE-induced Src activation is mediated by an ADRB/cAMP/PKA mechanism

(a) Effect of 10 μM NE on Src^Y419 phosphorylation in HeyA8 and SKOV3ip1 cells. *P < 0.01, **P < 0.001; two-tail Student’s t-test (b) HeyA8 cells were incubated for 1h with 1 μM butoxamine (Buto, β₂ antagonist), exposed to 10 μM NE and probed for pSrc^Y419. *P < 0.001; two-tail Student’s t-test (c) Similar experiments were performed with siRNA targeted against ADRB1 (ADRB1 si) or ADRB2 (ADRB2 si) in SKOV3ip1 cells. *P < 0.001; two-tail Student’s t-test (d) Effect of 50 μM dbcAMP (PKA agonist) on pSrc^Y419 in SKOV3ip1 cells. *P < 0.01, **P <0.001; two-tail Student’s t-test (e) HeyA8 cells were exposed to 10 μM KT5720 (PKA antagonist) for 1 hr, stimulated with 10 μM NE, and probed for pSrc^Y419. *P < 0.001; two-tail Student’s t-test (f) SKOV3ip1 cells were treated with 10 μM NE, and Src was visualized by immunofluorescence (scale bar = 9.375 μm). (g) Quantification of cellular response to NE (measured as cells with increased Src expression at the focal adhesions) is shown in graphs *P < 001; two-tail Student’s t-test. In panels (a–e,), the immunoblot is shown at the top and quantification of pSrc band intensity relative to the intensity of the total Src band is shown below.

Figure 3. NE-induced Src^S17 phosphorylation on SYF cells is required for Src activation

(a) HeyA8 cells were treated with 10 μM NE and probed for pSrc^S17. *P < 0.01; **P < 0.001; two-tail Student’s t-test. SYF-null cells transfected with either WT or mutant Src (S17A) were stimulated with 10 μM NE and immunoblotted for (b) pSrc^S17 or (c) pSrc^Y419. *P < 0.01; **P < 0.001; two-tail Student’s t-test. (d) SYF-null cells transfected with either WT or mutant Src (S17A) were stimulated with 10 μM NE or PDGF (20 ng/mL) and subjected to a migration assay or Western blot analysis for pSrc^S17 expression. *P < 0.01; two-tail Student’s t-test. In panels (a–c), the immunoblot is shown at the top and quantification of pSrc band intensity relative to the intensity of the total Src band is shown below.

Figure 4. Interaction between pS17 and Src results in conformational changes that expose Y419

(a) Inactive form of the protein obtained from the protein data bank (PDB code: 2SRC). The phosphoaminophosphonic acid-adenylate ester (ANP) and all the water molecules were deleted from the PDB structure. (b) View of the three identified cavities where the peptide could be inserted. Charge distribution analysis of (c) designed model peptide, (d) Src, and (e) the model peptide docked in the proposed cavity. The red and blue regions represent negative and positive charges, respectively. (f) After the phospho-peptide was exposed to Src for 36 ns, the kinase domain moved, completely exposing Y419. (g) When Src was exposed to an unphosphorylated peptide, no significant movement was observed and Y419 was not exposed. The SH3, SH2, N-lobe, and alphaC domains are shown in red, gray, orange, and pink, respectively. The C-lobe, A-loop, C-terminus, and phosphopeptide are shown in green, yellow, purple, and brown, respectively.

Figure 5. Adrenergic-mediated Src activation leads to increased tumor invasion and growth

NE 10 μM induces HeyA8 and SKOV3ip1 (a) cell invasion and (b) migration. (c) Mice were inoculated with either HeyA8 (2.5x10⁵) or (d) SKOV3ip1 (1.0x10⁶) cells and subjected to daily restraint and treated twice a week with either control siRNA-DOPC or Src siRNA-DOPC. Treatment with Src siRNA-DOPC blocked the daily restraint mediated induction in tumor weight and number of nodules compared to control siRNA-DOPC. (e) Mice bearing SKOV3ip1 tumors undergoing daily restraint were treated daily with propranolol (2 mg/kg). Propranolol counteracts the effects of daily restraint on tumor growth. (f) Mice bearing SKOV3ip1 tumors were treated daily with either: 10 mg/kg isoproterenol, 5 mg/kg terbutaline, 1 mg/kg xamoterol, or isoproterenol plus 2 mg/kg of propranolol. Isoproterenol and terbutaline induced tumor growth, but not xamoterol. Mice treated with isoproterenol plus propranolol have tumor burden similar to control mice.

Figure 6. High pSrc^Y419 levels are associated with decreased survival and depressive symptoms

(a) Representative images of human ovarian tumors with low or high expression of Src and pSrc^Y419. Images were taken at original magnification X200 (scale bar = 50 μm). (b) Kaplan-Meier curves of disease-specific mortality for patients with epithelial ovarian carcinoma (n= 91) based on pSrc^Y419 expression. The log-rank test (two-sided) was used to compare differences between groups. (c) Percentage of ovarian cancers with high pSrc^Y419 expression based on tumoral NE levels (greater than the median value of 0.84 pg/mg versus less than 0.84 pg/mg) and CESD scores ≥ 16. (d) Effect of beta blocker usage on cancer-related mortality, as estimated based on data from the FDA Adverse Event Reporting System. Bars, mortality decrease (green) or increase (black) by cancer type; saturation representing statistical confidence. Dashed yellow line, general mortality reduction over all cancer-related cases (17%). Numbers, cases having received a beta-blocker / total number of cases with given cancer type. (e) In response to chronic stress, catecholamines are released from the sympathetic nervous system. Stress-related hormones bind and activate ADRB receptors on tumor cells, initiating a cascade of events that result in Src activation.