Epidermal growth factor receptor endocytic traffic perturbation by phosphatidate phosphohydrolase inhibition: new strategy against cancer

Abstract

Epidermal growth factor receptor (EGFR) exaggerated (oncogenic) function is currently targeted in cancer treatment with drugs that block receptor ligand binding or tyrosine kinase activity. Because endocytic trafficking is a crucial regulator of EGFR function, its pharmacological perturbation might provide a new anti-tumoral strategy. Inhibition of phosphatidic acid (PA) phosphohydrolase (PAP) activity has been shown to trigger PA signaling towards type 4 phosphodiesterase (PDE4) activation and protein kinase A inhibition, leading to internalization of empty/inactive EGFR. Here, we used propranolol, its L-and D-isomers and desipramine as PAP inhibitors to further explore the effects of PAP inhibition on EGFR endocytic trafficking and its consequences on EGFR-dependent cancer cell line models. PAP inhibition not only made EGFR inaccessible to stimuli but also prolonged the signaling lifetime of ligand-activated EGFR in recycling endosomes. Strikingly, such endocytic perturbations applied in acute/intermittent PAP inhibitor treatments selectively impaired cell proliferation/ viability sustained by an exaggerated EGFR function. Phospholipase D inhibition with FIPI (5-fluoro-2-indolyl des-chlorohalopemide) and PDE4 inhibition with rolipram abrogated both the anti-tumoral and endocytic effects of PAP inhibition. Prolonged treatments with a low concentration of PAP inhibitors, although without detectable endocytic effects, still counteracted cell proliferation, induced apoptosis and decreased anchorageindependent growth of cells bearing EGFR oncogenic influences. Overall, our results show that PAP inhibitors can counteract EGFR oncogenic traits, including receptor overexpression or activating mutations resistant to current tyrosine kinase inhibitors, perturbing EGFR endocytic trafficking and perhaps other as yet unknown processes, depending on treatment conditions. This puts PAP activity forward as a new suitable target against EGFR-driven malignancy.Abbreviations DAG, diacylglycerol; EEA1, early endosome antigen 1; EGF, epidermal growth factor; EGFR, EGF receptor; GFP, green fluorescent protein; LPP, lipid phosphate phosphatase; PA, phosphatidic acid; PAP, phosphatidic acid phosphohydrolase; PARP, poly ADP ribose polymerase; PDE4, type 4 phosphodiesterases; PKA, protein kinase A; PLD, phospholipase D; TfR, transferrin receptor.

Stress-Induced EGFR Trafficking: Mechanisms, Functions, and Therapeutic Implications

“…The clinically available PAP inhibitors, propranolol and desipramine, are known to cause PA accumulation and PKA inhibition, resulting in EGFR internalization. Although this mimics EGFR trafficking induced by other cellular stresses, PKA inhibition shows tumor suppression activities in EGFR-driven cancer cells [100]. Thus, PKA inhibition reagents could be further explored in combination with other therapeutic approaches such as EGFR inhibition, radiation, and autophagy suppression.…”

Repurposing Drugs in Oncology (ReDO)—Propranolol as an anti-cancer agent

“…Disrupted endocytic trafficking is implicated in the process by which tumours gain self-sufficiency in growth signals by delays in the inactivation of multiple growth factor receptors, including EGFR [163–164]. It has been proposed by Shaughnessy et al that a strategy for inhibiting EGFR function may be to interfere with the endocytic process directly rather than directly targeting receptor-ligand binding or tyrosine kinase activity [165]. Inhibition of phosphatidic acid phosphohydrolase (PAP) has been shown to cause a reversible trafficking of inactive (empty) EGFR from the cell surface to endosomes, thereby restricting receptor availability to ligands [166].…”

“…Inhibition of phosphatidic acid phosphohydrolase (PAP) has been shown to cause a reversible trafficking of inactive (empty) EGFR from the cell surface to endosomes, thereby restricting receptor availability to ligands [166]. PRO is a known inhibitor of PAP [167], and has been shown to reduce the cell viability of EGFR-dependent cancer cell lines [165]. …”

Phosphatidic Acid Increases Epidermal Growth Factor Receptor Expression by Stabilizing mRNA Decay and by Inhibiting Lysosomal and Proteasomal Degradation of the Internalized Receptor

“…Second, for inhibition of the proteasome, we used bortezomib, a specific inhibitor of proteasome-mediated intracellular proteolysis of long-lived proteins (37,44). Third, we used the PA phosphatase inhibitor D-propranolol, which induces empty or inactive EGF to be internalized and renders the receptor inaccessible to external stimuli (41). As indicated by the data shown in Fig.…”