GPCR Signaling Mediates Tumor Metastasis via PI3Kß.

Inappropriate activation of PI3K signaling has been implicated strongly in human cancer. Although studies on the role of PI3K signaling in breast tumorigenesis and progression have focused most intensively on PI3Kα, a role for PI3Kß has begun to emerge. The PI3Kß isoform is unique among class IA PI3K enzymes in that it is activated by both receptor tyrosine kinases and G-protein-coupled receptors (GPCR). In previous work, we identified a mutation that specifically abolishes PI3Kß binding to Gßγ (p110(526KK-DD)). Expression of this mutant in p110ß-silenced breast cancer cells inhibits multiple steps of the metastatic cascade in vitro and in vivo and causes a cell autonomous defect in invadopodial matrix degradation. Our results identify a novel link between GPCRs and PI3Kß in mediating metastasis, suggesting that disruption of this link might offer a novel therapeutic target to prevent the development of metastatic disease. Cancer Res; 76(10); 2944-53. ©2016 AACR.

NRBF2 regulates macroautophagy as a component of Vps34 Complex I.

Macroautophagy is a physiological cellular response to nutrient stress, which leads to the engulfment of cytosolic contents by a double-walled membrane structure, the phagophore. Phagophores seal to become autophagosomes, which then fuse with lysosomes to deliver their contents for degradation. Macroautophagy is regulated by numerous cellular factors, including the Class III PI3K (phosphoinositide 3-kinase) Vps34 (vacuolar protein sorting 34). The autophagic functions of Vps34 require its recruitment to a complex that includes Vps15, Beclin-1 and Atg14L (autophagy-related 14-like protein) and is known as Vps34 Complex I. We have now identified NRBF2 (nuclear receptor-binding factor 2) as a new member of Vps34 Complex I. NRBF2 binds to complexes that include Vps34, Vps15, Beclin-1 and ATG-14L, but not the Vps34 Complex II component UVRAG (UV radiation resistance-associated gene). NRBF2 directly interacts with Vps15 via the Vps15 WD40 domain as well as other regions of Vps15. The formation of GFP-LC3 (light chain 3) punctae and PE (phosphatidylethanolamine)-conjugated LC3 (LC3-II) in serum-starved cells was inhibited by NRBF2 knockdown in the absence and presence of lysosomal inhibitors, and p62 levels were increased. Thus NRBF2 plays a critical role in the induction of starvation-induced autophagy as a specific member of Vps34 Complex I.

Molecular determinants of PI3Kγ-mediated activation downstream of G-protein-coupled receptors (GPCRs).

Phosphoinositide 3-kinase gamma (PI3Kγ) has profound roles downstream of G-protein-coupled receptors in inflammation, cardiac function, and tumor progression. To gain insight into how the enzyme's activity is shaped by association with its p101 adaptor subunit, lipid membranes, and Gßγ heterodimers, we mapped these regulatory interactions using hydrogen-deuterium exchange mass spectrometry. We identify residues in both the p110γ and p101 subunits that contribute critical interactions with Gßγ heterodimers, leading to PI3Kγ activation. Mutating Gßγ-interaction sites of either p110γ or p101 ablates G-protein-coupled receptor-mediated signaling to p110γ/p101 in cells and severely affects chemotaxis and cell transformation induced by PI3Kγ overexpression. Hydrogen-deuterium exchange mass spectrometry shows that association with the p101 regulatory subunit causes substantial protection of the RBD-C2 linker as well as the helical domain of p110γ. Lipid interaction massively exposes that same helical site, which is then stabilized by Gßγ. Membrane-elicited conformational change of the helical domain could help prepare the enzyme for Gßγ binding. Our studies and others identify the helical domain of the class I PI3Ks as a hub for diverse regulatory interactions that include the p101, p87 (also known as p84), and p85 adaptor subunits; Rab5 and Gßγ heterodimers; and the ß-adrenergic receptor kinase.

Mixed lineage kinase 3 is required for matrix metalloproteinase expression and invasion in ovarian cancer cells.

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells. MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells.

Mixed lineage kinase 3 negatively regulates IKK activity and enhances etoposide-induced cell death.

Mixed lineage kinase 3 (MLK3) is a mitogen activated protein kinase kinase kinase (MAP3K) that activates multiple MAPK signaling pathways. Nuclear factor kappa B (NF-kappaB) is a transcription factor that has important functions in inflammation, immunity and cell survival. We found that silencing mlk3 expression with RNA interference (RNAi) in SKOV3 human ovarian cancer epithelial cells and NIH-3T3 murine fibroblasts led to a reduction in the level of the inhibitor of kappa B alpha (IkappaBalpha) protein. In addition, we observed enhanced basal IkappaB kinase (IKK) activity in HEK293 cells transiently transfected with MLK3 siRNA and in NIH3T3 cells stably expressing MLK3 shRNA (shMLK3). Furthermore, the basal level of NF-kappaB-dependent gene transcription was elevated in shMLK3 cells. Silencing mlk3 expression conferred resistance of cells to etoposide-induced apoptotic cell death and overexpression of wild type MLK3 (MLK3-WT) or kinase-dead MLK3 (MLK3-KD) promoted apoptotic cell death and cleavage of poly (ADP-ribose) polymerase (PARP). Overexpression of MLK3-WT or MLK3-KD enhanced etoposide-induced apoptotic cell death and cleavage of PARP. These data suggest that MLK3 functions to limit IKK activity, and depleting MLK3 helps protect cells from etoposide-induced cell death through activation of IKK-dependent signaling.