The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton

Inactivation of the phosphoinositide phosphatases Sac1p and Inp54p leads to accumulation of phosphatidylinositol 4,5-bisphosphate on vacuole membranes and vacuolar fusion defects

Phosphoinositides direct membrane trafficking, facilitating the recruitment of effectors to specific membranes. In yeast phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P-2) is proposed to regulate vacuolar fusion; however, in intact cells this phosphoinositide can only be detected at the plasma membrane. In Saccharomyces cerevisiae the 5-phosphatase, Inp54p, dephosphorylates PtdIns(4,5)P-2 forming PtdIns(4)P, a substrate for the phosphatase Sac1p, which hydrolyzes (PtdIns(4) P). We investigated the role these phosphatases in regulating PtdIns(4,5) P-2 subcellular distribution. PtdIns(4,5)P-2 bioprobes exhibited loss of plasma membrane localization and instead labeled a subset of fragmented vacuoles in Delta sac1 Delta inp54 and sac1(ts) Delta inp54 mutants. Furthermore, sac1(ts) Delta inp54 mutants exhibited vacuolar fusion defects, which were rescued by latrunculin A treatment, or by inactivation of Mss4p, a PtdIns(4)P 5-kinase that synthesizes plasma membrane PtdIns(4,5)P-2. Under these conditions PtdIns(4,5)P-2 was not detected on vacuole membranes, and vacuole morphology was normal, indicating vacuolar PtdIns(4,5)P-2 derives from Mss4p-generated plasma membrane PtdIns(4,5)P-2. Delta sac1 Delta inp54 mutants exhibited delayed carboxypeptidase Y sorting, cargo-selective secretion defects, and defects in vacuole function. These studies reveal PtdIns(4,5)P-2 hydrolysis by lipid phosphatases governs its spatial distribution, and loss of phosphatase activity may result in PtdIns(4,5)P-2 accumulation on vacuole membranes leading to vacuolar fragmentation/fusion defects

The FDA-Approved Drug Pyrvinium Selectively Targets ER⁺ Breast Cancer Cells with High INPP4B Expression

The majority of breast cancers are estrogen receptor-positive (ER+), and endocrine therapies that suppress ER signaling are the standard-of-care treatment for this subset. However, up to half of all ER+ cancers eventually relapse, highlighting a need for improved clinical therapies. The phosphoinositide phosphatase, INPP4B, is overexpressed in almost half of all ER+ breast cancers, and promotes Wnt/β-catenin signaling, cell proliferation and tumor growth. Here, using cell viability assays, we report that INPP4B overexpression does not affect the sensitivity of ER+ breast cancer cells to standard-of-care treatments including the anti-estrogen 4-hydroxytamoxifen (4-OHT) or the PI3Kα inhibitor alpelisib. Examination of four small molecule Wnt inhibitors revealed that ER+ breast cancer cells with INPP4B overexpression were more sensitive to the FDA-approved drug pyrvinium and a 4-OHT-pyrvinium combination treatment. Using 3D culture models, we demonstrated that pyrvinium selectively reduced the size of INPP4B-overexpressing ER+ breast cancer spheroids in the presence and absence of 4-OHT. These findings suggest that repurposing pyrvinium as a Wnt inhibitor may be an effective therapeutic strategy for human ER+ breast cancers with high INPP4B levels

The FDA-Approved Drug Pyrvinium Selectively Targets ER+ Breast Cancer Cells with High INPP4B Expression

The majority of breast cancers are estrogen receptor-positive (ER+), and endocrine therapies that suppress ER signaling are the standard-of-care treatment for this subset. However, up to half of all ER+ cancers eventually relapse, highlighting a need for improved clinical therapies. The phosphoinositide phosphatase, INPP4B, is overexpressed in almost half of all ER+ breast cancers, and promotes Wnt/β-catenin signaling, cell proliferation and tumor growth. Here, using cell viability assays, we report that INPP4B overexpression does not affect the sensitivity of ER+ breast cancer cells to standard-of-care treatments including the anti-estrogen 4-hydroxytamoxifen (4-OHT) or the PI3Kα inhibitor alpelisib. Examination of four small molecule Wnt inhibitors revealed that ER+ breast cancer cells with INPP4B overexpression were more sensitive to the FDA-approved drug pyrvinium and a 4-OHT-pyrvinium combination treatment. Using 3D culture models, we demonstrated that pyrvinium selectively reduced the size of INPP4B-overexpressing ER+ breast cancer spheroids in the presence and absence of 4-OHT. These findings suggest that repurposing pyrvinium as a Wnt inhibitor may be an effective therapeutic strategy for human ER+ breast cancers with high INPP4B levels

Regulation of PI3K effector signalling in cancer by the phosphoinositide phosphatases

Class I phosphoinositide 3-kinase (PI3K) generates phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) at the plasma membrane in response to growth factors, activating a signalling cascade that regulates many cellular functions including cell growth, proliferation, survival, migration and metabolism. The PI3K pathway is commonly dysregulated in human cancer, and drives tumorigenesis by promoting aberrant cell growth and transformation. PtdIns(3,4,5)P(3) facilitates the activation of many pleckstrin homology (PH) domain-containing proteins including the serine/threonine kinase AKT. There are three AKT isoforms that are frequently hyperactivated in cancer through mutation, amplification or dysregulation of upstream regulatory proteins. AKT isoforms have converging and opposing functions in tumorigenesis. PtdIns(3,4,5)P(3) signalling is degraded and terminated by phosphoinositide phosphatases such as phosphatase and tensin homologue (PTEN), proline-rich inositol polyphosphate 5-phosphatase (PIPP) (INPP5J) and inositol polyphosphate 4-phosphatase type II (INPP4B). PtdIns(3,4,5)P(3) is rapidly hydrolysed by PIPP to generate phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), which is further hydrolysed by INPP4B to form phosphatidylinositol 3-phosphate (PtdIns3P). PtdIns(3,4)P(2) and PtdIns3P are also important signalling molecules; PtdIns(3,4)P(2) together with PtdIns(3,4,5)P(3) are required for maximal AKT activation and PtdIns3P activates PI3K-dependent serum and glucocorticoid-regulated kinase (SGK3) signalling. Loss of Pten, Pipp or Inpp4b expression or function promotes tumour growth in murine cancer models through enhanced AKT isoform-specific signalling. INPP4B inhibits PtdIns(3,4)P(2)-mediated AKT activation in breast and prostate cancer; however, INPP4B expression is increased in acute myeloid leukaemia (AML), melanoma and colon cancer where it paradoxically promotes cell proliferation, transformation and/or drug resistance. This review will discuss how PTEN, PIPP and INPP4B distinctly regulate PtdIns(3,4,5)P(3) signalling downstream of PI3K and how dysregulation of these phosphatases affects cancer outcomes

A late endosome signaling hub that couples PI3Kα and WNT/β-catenin signaling in breast cancer

AKT is the central phosphoinositide 3-kinase (PI3K) signaling effector, however, PIK3CA (p110α subunit of PI3Kα)-mutant estrogen receptor-positive (ER+) breast cancers exhibit minimal AKT activation and the downstream signaling is poorly characterized. We discovered that a subset of PIK3CA-mutant ER+ breast cancers exhibit increased inositol polyphosphate 4-phosphatase type II (INPP4B) expression, which promotes late endosome formation and glycogen synthase kinase 3 beta (GSK3β) trafficking, leading to enhanced Wingless–related integration site (WNT)/catenin beta 1 (β-catenin) activation