β2-adrenoceptor signaling regulates invadopodia formation to enhance tumor cell invasion

We first confirmed expression of βAR in MDA-MB-231 and 66cl4 tumor cells, which are breast cancer cells lines that respond to stress signaling with increased metastasis from primary orthotopic mammary tumors [12, 13]. Expression analyses found higher β₂AR than β₁AR transcription in MDA-MB-231 cells, while 66cl4 expressed only β₂AR (Fig. 1a). β₃AR was not detectable in either cell line, although priming from these probes has been confirmed [26, 27]. Immunostaining confirmed that tumor cells from an archival clinical sample expressed β₂AR, suggesting that findings may be relevant to human breast cancer (Fig. 1b). To evaluate receptor functionality, we treated tumor cells with βAR ligands and quantified the effect on intracellular cAMP accumulation. In each cell line, treatment with the nonselective βAR agonist isoproterenol increased cAMP accumulation (Fig. 1c, d), consistent with previous studies [28]. This effect was blocked by the nonselective βAR antagonist propranolol or the β₂AR-selective antagonist ICI-188551. Treatment with β₁AR-selective antagonist CGP-20712A or β₃AR-selective antagonist L748337 did not block cAMP accumulation (Fig. 1c, d). These findings confirm that β₂AR is the dominant functional βAR in these breast cancer cell lines.

To investigate the role of βAR signaling in invadopodia formation, LifeAct-GFP⁺ MDA-MB-231 cells were plated on a fluorescent gelatin matrix and the effect of β-agonist isoproterenol on matrix degradation was evaluated (Fig. 2a). While actin-rich puncta were identified in the entire population, only 16 % (± 4 %) of tumor cells produced active invadopodia as defined by degradation of underlying matrix fluorescence (Fig. 2a, b). Treatment with isoproterenol led to an increase in the frequency of invadopodia-positive cells, with a maximum increase of 2.5-fold in response to 0.5 μM isoproterenol (Fig. 2b). In addition to increasing the frequency of invadopodia-positive cells in the population (Fig. 2b, c), isoproterenol also increased the average number of invadopodia per cell by 2.7-fold (Fig. 2d). Treatment with the nonselective β-blocker propranolol blocked the effect of isoproterenol on both the frequency of invadopodia-positive cells and the number of invadopodia per cell (Fig. 2c, d). This confirms that isoproterenol signals via βAR to increase invadopodia formation. To confirm the βAR subtype that mediates this effect, MDA-MB-231 cells were treated with selective antagonists and the effect on invadopodia was evaluated. β₂AR-selective antagonist ICI-118551 blocked the increase in invadopodia formation in response to isoproterenol, whereas there was no effect of β₁AR-selective antagonist CGP-20712A (Fig. 2e). To determine whether β₂AR signaling is sufficient to induce invadopodia formation, cells were treated with selective βAR agonists. β₂AR-selective agonist formoterol – but not β₁AR-selective agonist xamoterol – induced invadopodia formation (Fig. 2f, g), confirming a key role for β₂AR signaling in the formation of invadopodia in these cells.

Formation of invadopodia requires adhesion proteins that may be sequestered from focal adhesions [6]. To investigate the effect of βAR signaling on focal adhesions we treated breast cancer cells with isoproterenol and used immunostaining to quantify the number and length of paxillin-positive focal adhesions (Fig. 3a). Isoproterenol resulted in a concentration-dependent decrease in adhesion length and decreased number of focal adhesions per cell (Fig. 3a, b). Loss of focal adhesion-associated proteins has been linked to a decreased frequency of focal adhesions and reduced capacity for cell migration on two-dimensional surfaces [29]. Consistent with the change in focal adhesions having a functional effect on cell movement, isoproterenol decreased the migration of cancer cells on a two-dimensional fibronectin surface (Fig. 3c, d).

These findings suggest that β₂AR signaling induces an invasive phenotype that favors tumor cell invasion in three-dimensional environments. To investigate this, MDA-MB-231 breast cancer cells were embedded in collagen matrix and cell locomotion was tracked over time. Under control conditions, cells remained rounded with little displacement from the position of origin (Fig. 4a, b). Treatment with β₂AR-selective agonist formoterol induced cancer cells to elongate and form protrusions (Fig. 4a) and increased cell displacement over time (Fig. 4b, c), indicating that β₂AR signaling is sufficient to induce invasion of MDA-MB-231 breast cancer cells. Treatment with isoproterenol similarly induced cellular protrusion formation and these effects were blocked by the β₂AR-selective antagonist ICI-118551, confirming a role for β₂AR signaling in these effects on cell invasion through a three-dimensional collagen matrix. By identifying differential effects of βAR signaling between cell movement on two-dimensional surfaces compared with migration in three-dimensional environments, these findings may reconcile seemingly inconsistent observations for the effects of endogenous neurotransmitters and βAR-selective agonists on tumor cell migration [30‐33]. Because movement in three-dimensional environments reflects processes that are required for tumor cell intravasation and extravasation during metastasis, these findings suggest a cellular mechanism for observations that βAR signaling drives breast cancer progression [12, 13].

Src activity plays a key role in invadopodia formation [4, 6], and has been implicated in βAR regulation of ovarian cancer invasion, although the cellular mechanisms are unclear [15]. To investigate the role of Src in β₂AR regulation of invadopodia, MDA-MB-231 cells were pretreated with the Src inhibitor PP2 prior to treatment with formoterol. PP2 treatment blocked the effect of β₂AR stimulation on the frequency of invadopodia (Fig. 4d, e), as seen by loss of matrix fluorescence. Treatment with PP2 alone did not significantly change the frequency of invadopodia compared to treatment with vehicle. Treatment with PP2 also blocked the effect of formoterol on invasion of cells in three-dimensional collagen matrix (Fig. 4f), demonstrating that Src is essential for β₂AR-mediated invasion. These findings indicate that β₂AR signaling regulates invadopodia formation and tumor cell invasion through canonical Src signaling pathways. βAR activation was recently shown to phosphorylate Src at Y⁴¹⁶ in addition to S¹⁷ [15], suggesting that characterization of the activation status of Src will contribute to a more complete understanding of the molecular mechanisms involved. Additional insight may be gained from clarifying other β₂AR-induced molecular changes that may amplify effects on tumor cell dissemination. For example, norepinephrine was shown to protect cancer cells from anoikis by activating and relocalizing focal adhesion kinase [34]. Coordinated regulation of invadopodia formation and reduced anoikis in response to β₂AR-mediated neural signaling may serve to amplify the effects on tumor cell dissemination and cancer progression.

To investigate the impact of β₂AR regulation of tumor cell invasion in vivo, bioluminescence imaging was used to track the effect of β₂AR-selective agonist formoterol on metastatic progression in an orthotopic xenograft model of breast cancer. Mice were implanted with luciferase-tagged MDA-MB-231 cells into the left fourth mammary fat pad and spontaneous metastasis was tracked by bioluminescence imaging using 1-second exposure to detect signals from primary tumors and 60-second exposure to detect signals from metastases (Fig. 5a). Treatment with formoterol during tumor development accelerated the formation of metastasis (Fig. 5b). To examine whether increased metastasis was linked to increased tumor cell invasion, a subset of mammary tumors were surgically resected 9 days after tumor cell inoculation, before the onset of metastasis, and embedded in a three-dimensional collagen matrix. In contrast to tumors from vehicle-treated mice, which demonstrated minimal invasion of cells into the surrounding extracellular matrix over the 8-day imaging period, tumors from mice treated with formoterol had rapid invasion of tumor cells into the surrounding matrix (Fig. 5c). This shows that β₂AR signaling increases dissemination from explanted tumors, suggesting a possible mechanism for increased distant metastasis.

While recent studies of βAR-mediated stress biology on cancer progression have focused on the impact of neural signaling on stromal cells in the tumor microenvironment [12, 14, 35, 36], the findings presented here demonstrate that tumor cells are directly responsive to βAR signaling. By driving a shift away from focal adhesions to invadopodia formation, β₂AR signaling would favor tumor cells with enhanced capacity for invasion through the three-dimensional extracellular matrix of the tumor microenvironment. Metastasis is a highly inefficient process and requires successful completion of a series of inter-connected steps including dissemination from the primary tumor, survival in circulation, extravasation, and colonization at distant sites [37‐39]. Failure at any stage of metastasis will result in failure of the entire process [40]. By increasing both the density of invadopodia per cell and the frequency of invadopodia-positive cells in the tumor cell population, β₂AR signaling may promote metastasis by enhancing the invasive capacity of cells in the tumor.

These findings suggest that blocking tumor cell responsiveness to stress signaling may protect against tumor cell invasion and metastasis. Specifically, the effects of β₂AR signaling on invadopodia formation were reversed by β-blockade, providing a cellular mechanism for clinical observations that pharmacological β-blockade was linked to improved cancer outcomes [41‐43]. Retrospective epidemiological studies found that β-blocker treatment of comorbid hypertension was associated with reduced metastasis and improved survival in different cancer types including breast and prostate cancers and melanoma (reviewed in [44]). The current findings show that β-blockers act directly on cancer cells to decrease their invasiveness. While not excluding that β-blockade regulates cancer progression through effects on the tumor microenvironment (e.g., modulating recruitment of inflammatory cells or angiogenesis) [12, 35], these findings suggest that β-blockers may be optimally targeted to patients with high tumor cell expression of β₂AR. Recent clinical practice has preferred the use of β₁-selective blockers for treatment of hypertension, to avoid adverse effects on bronchoconstriction [45]. The findings presented here suggest that use of nonselective β-blockers such as propranolol may be required to favorably impact cancer outcomes. Additionally these findings may warrant the development of novel β₂-selective blockers that may be targeted to tumor cells to avoid adverse side effects. Finally, these findings suggest that it may be important to consider the effect of clinically used β₂AR agonists (e.g., by asthmatics for bronchodilation) on cancer progression.