Molecular mechanisms underlying the role of GRK2 in Breast Cancer progression

Abstract

Cancer is a highly complex process often driven by oncogenic mutations and/or altered signaling nodes, the so-called onco-modulators that cooperate with oncogene-governed pathways to strengthen tumor hallmarks. G protein-coupled receptor kinase 2 (GRK2) has recently emerged as a relevant oncomodulator by modulating G protein-coupled receptors (GPCR) and Growth Factor-Receptor Tyrosine Kinases (GF-RTKs), as well as non-receptor partners/substrates in a cell type-specific manner. In this work, we further explore the role of increased GRK2 protein levels in breast cancer (BC) growth and metastasis. Our results show that GRK2 fosters mitogenic signaling by cross-modulation of chemokine GPCRs CXCR4/ACKR3 and EGF-responding GF-RTKs (EGFR and HER2/3), often dysregulated in most types of BC. Our data shows that GRK2 enhances GFtransducing molecules Ras and Pin1 and MAPK activation in the luminal-A subtype BC cell line MCF7, leading to increased primary tumor growth in vivo. Furthermore, GRK2 reshapes the molecular profile of MCF7 or triple-negative MDA-MB-231 cells, intensifying mesenchymal features and functionality of invadopodia and actin regulators, which ultimately fosters pulmonary metastasis occurrence. We identify the actin cytoskeleton modulator Cofilin-1 (CFL1) as new target of the BC over-activated GRK2-Histone Deacetylase-6 (HDAC6) axis in which mitogenic stimuli converge. Our data indicate that MAPK-mediated phosphorylation of GRK2 and subsequent modulation of HDAC6 would have opposite effects in the acetylation status of HDAC6 substrates such as tubulin or CFL1, altogether favoring cell migration and invasion. We also show that both GRK2 and EGFR can alter CXCR4-mediated Gi1 activation in model HEK-293 cells, while more complex interactions between these receptors and modulation by GRK2 occur in BC cell lines. In luminal-A, luminal-B or triple negative BC cell models, we find an unanticipated heterogeneity in expression profiles of GRK2 and CXCR4/ACKR3, EGFR and HER2/3 receptors, subcellular distribution of CXCR4/ACKR3 receptors and ability of CXCL12 to signal to downstream receptor cascades. Of note, GRK2 mediates cooperation of CXCR4/ACKR3 and EGFRHER2 receptors towards ERK1/2 activation in Her2-enriched BC cell line MDA-MB-361, with the involvement of Gi and tyrosine kinase Lyn proteins. Moreover, we identify GRK2 as a potential target of Lyn, and the inhibition of either GRK2 or Lyn decreases CXCL12-directed migration in MD-MB-231 cells, suggesting both kinases act as a functional tandem linking CXCR4/ACKR3 and GF-RTKs in oncogenic contexts. Overall, these results confirm an integrative role of GRK2 in GF-RTK and CXCR4/ACKR3 cooperation and downstream signaling, promoting growth of BC cells and changes in the acetylome profile of cytoskeleton components and regulators for enhanced invasive motility, thus pointing out GRK2 as a new drug target for the treatment of breast cancer subtypes with different molecular signatures