Synergistic activation by heterotrimeric guanine nucleotide–binding protein (G protein)-coupled receptors (GPCRs) and receptor tyrosine kinases distinguishes p110β from other class IA phosphoinositide 3-kinases (PI3Ks). Activation of p110β is specifically implicated in various physiological and pathophysiological processes, such as the growth of tumors deficient in phosphatase and tensin homolog deleted from chromosome 10 (PTEN). To determine the specific contribution of GPCR signaling to p110β-dependent functions, we identified the site in p110β that binds to the Gβγ subunit of G proteins. Mutation of this site eliminated Gβγ-dependent activation of PI3Kβ (a dimer of p110β and the p85 regulatory subunit) in vitro and in cells, without affecting basal activity or phosphotyrosine peptide–mediated activation. Disrupting the p110β-Gβγ interaction by mutation or with a cell-permeable peptide inhibitor blocked the transforming capacity of PI3Kβ in fibroblasts, and reduced proliferation, chemotaxis, and invasiveness of PTEN-null tumor cells in culture. Our data suggest that specifically targeting GPCR signaling to PI3Kβ could provide a therapeutic approach for tumors that depend on p110β for growth and metastasis.

Cancer stem cells (CSCs) play an important role during metastasis, but the dynamic behavior and induction mechanisms of CSCs are not well understood. Here, we employ high-resolution intravital microscopy using a CSC biosensor to directly observe CSCs in live mice with mammary tumors. CSCs display the slow-migratory, invadopod-rich phenotype that is the hallmark of disseminating tumor cells. CSCs are enriched near macrophages, particularly near macrophage-containing intravasation sites called Tumor Microenvironment of Metastasis (TMEM) doorways. Substantial enrichment of CSCs occurs on association with TMEM doorways, contributing to the finding that CSCs represent >60% of circulating tumor cells. Mechanistically, stemness is induced in non-stem cancer cells upon their direct contact with macrophages via Notch-Jagged signaling. In breast cancers from patients, the density of TMEM doorways correlates with the proportion of cancer cells expressing stem cell markers, indicating that in human breast cancer TMEM doorways are not only cancer cell intravasation portals but also CSC programming sites.

Invadopodia, actin-based protrusions of invasive carcinoma cells that focally activate extracellular matrix-degrading proteases, are essential for the migration and intravasation of tumor cells during dissemination from the primary tumor. We have previously shown that cortactin phosphorylation at tyrosine residues, in particular tyrosine 421, promotes actin polymerization at newly-forming invadopodia, promoting their maturation to matrix-degrading structures. However, the mechanism by which cells regulate the cortactin tyrosine phosphorylation-dephosphorylation cycle at invadopodia is unknown. Mena, an actin barbed-end capping protein antagonist, is expressed as various splice-isoforms. The MenaINV isoform is upregulated in migratory and invasive sub-populations of breast carcinoma cells, and is involved in tumor cell intravasation. Here we show that forced MenaINV expression increases invadopodium maturation to a far greater extent than equivalent expression of other Mena isoforms. MenaINV is recruited to invadopodium precursors just after their initial assembly at the plasma membrane, and promotes the phosphorylation of cortactin tyrosine 421 at invadopodia. In addition, we show that cortactin phosphorylation at tyrosine 421 is suppressed by the phosphatase PTP1B, and that PTP1B localization to the invadopodium is reduced by MenaINV expression. We conclude that MenaINV promotes invadopodium maturation by inhibiting normal dephosphorylation of cortactin at tyrosine 421 by the phosphatase PTP1B.

Activation of the Gi family of heterotrimeric guanine nucleotide-binding proteins (G proteins) releases βγ subunits, which are the major transducers of chemotactic G protein-coupled receptor (GPCR)-dependent cell migration. The small molecule 12155 binds directly to Gβγ and activates Gβγ signaling without activating the Gαi subunit in the Gi heterotrimer. We used 12155 to examine the relative roles of Gαi and Gβγ activation in the migration of neutrophils on surfaces coated with the integrin ligand intercellular adhesion molecule-1 (ICAM-1). We found that 12155 suppressed basal migration by inhibiting the polarization of neutrophils and increasing their adhesion to ICAM-1-coated surfaces. GPCR-independent activation of endogenous Gαi and Gβγ with the mastoparan analog Mas7 resulted in normal migration. Furthermore, 12155-treated cells expressing a constitutively active form of Gαi1 became polarized and migrated. The extent and duration of signaling by the second messenger cyclic adenosine monophosphate (cAMP) were enhanced by 12155. Inhibiting the activity of cAMP-dependent protein kinase (PKA) restored the polarity of 12155-treated cells but did not decrease their adhesion to ICAM-1 and failed to restore migration. Together, these data provide evidence for a direct role of activated Gαi in promoting cell polarization through a cAMP-dependent mechanism and in inhibiting adhesion through a cAMP-independent mechanism.

Background: G protein ␤␥ (G␤␥) subunits are required for chemokine-dependent directional chemotaxis. Results: A chemical activator of G␤␥ signaling activated G␤␥ signaling and induced directional chemotaxis of neutrophils. Conclusion: G␤␥ signaling is sufficient to induce directional chemotaxis of neutrophils. Significance: Demonstrates that G protein-coupled receptor signals other than G␤␥ are not required for directional migration of neutrophils in response to a gradient.