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The class IA PI3K signaling pathway is activated by growth factor stimulation and regulates a signaling cascade that promotes diverse events including cell growth, proliferation, migration and metabolism. PI3K signaling is one of the most commonly hyperactivated pathways in breast cancer, leading to increased tumor growth and progression. PI3K hyperactivation occurs via a number of genetic and epigenetic mechanisms including mutation or amplification of PIK3CA, the gene encoding the p110α subunit of PI3Kα, as well as via dysregulation of the upstream growth factor receptors or downstream signaling effectors. Over the past decade, extensive efforts to develop therapeutics that suppress oncogenic PI3K signaling have been undertaken. Although FDA-approved PI3K inhibitors are now emerging, their clinical success remains limited due to adverse effects and negative feedback mechanisms which contribute to their reduced efficacy. There is an emerging body of evidence demonstrating crosstalk between the PI3K and Wnt/βcatenin pathways in breast cancer. However, PI3K exhibits opposing effects on Wnt/βcatenin signaling in distinct tumor subsets, whereby PI3K promotes Wnt/β-catenin activation in ER+ cancers, but paradoxically suppresses this pathway in ER− breast cancers. This review discusses the molecular mechanisms for PI3K–Wnt crosstalk in breast cancer, and how Wnt-targeted therapies have the potential to contribute to treatment regimens for breast cancers with PI3K dysregulation. [ABSTRACT FROM AUTHOR]

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research. [ABSTRACT FROM AUTHOR]

Simple Summary: Wnt/β-catenin signaling is hyperactivated in many human cancers including up to 50% of breast cancers. Although there has been significant progress in developing therapeutics that suppress Wnt/β-catenin signaling, particularly in colon cancer, the repurposing of FDA-approved therapeutics is likely to be a faster and more cost-effective method to target this pathway in human disease. Pyrvinium is an FDA-approved anthelmintic drug used to treat pinworms, which also suppresses Wnt/β-catenin signaling by activating the β-catenin destruction complex protein, CK1α. Here, we demonstrate that breast cancer cells with increased expression of the oncogene INPP4B, a PI3K regulator that promotes Wnt/β-catenin activation, are selectively sensitive to pyrvinium treatment in 2D and 3D culture. Therefore, Wnt inhibition using pyrvinium may be an effective strategy for treating human breast cancers 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. [ABSTRACT FROM AUTHOR]

INPP4B suppresses PI3K/AKT signaling by converting PI(3,4)P2 to PI(3)P and INPP4B inactivation is common in triple-negative breast cancer. Paradoxically, INPP4B is also a reported oncogene in other cancers. How these opposing INPP4B roles relate to PI3K regulation is unclear. We report PIK3CA-mutant ER+ breast cancers exhibit increased INPP4B mRNA and protein expression and INPP4B increased the proliferation and tumor growth of PIK3CA-mutant ER+ breast cancer cells, despite suppression of AKT signaling. We used integrated proteomics, transcriptomics and imaging to demonstrate INPP4B localized to late endosomes via interaction with Rab7, which increased endosomal PI3Kα-dependent PI(3,4)P2 to PI(3)P conversion, late endosome/lysosome number and cargo trafficking, resulting in enhanced GSK3β lysosomal degradation and activation of Wnt/β-catenin signaling. Mechanistically, Wnt inhibition or depletion of the PI(3)P-effector, Hrs, reduced INPP4B-mediated cell proliferation and tumor growth. Therefore, INPP4B facilitates PI3Kα crosstalk with Wnt signaling in ER+ breast cancer via PI(3,4)P2 to PI(3)P conversion on late endosomes, suggesting these tumors may be targeted with combined PI3K and Wnt/β-catenin therapies. The PI(3,4)P2 4-phosphatase, INPP4B, functions as a tumour suppressor in triple negative breast cancer. Here, the authors show that INPP4B enhances proliferation and growth of PIK3CA-mutant ER+ breast cancers by promoting PI3Kα-dependent late endosome formation and trafficking that leads to the activation of Wnt/β-catenin signalling. [ABSTRACT FROM AUTHOR]

The Rac-GEF, P-Rex1, activates Rac1 signaling downstream of G protein-coupled receptors and PI3K. Increased P-Rex1 expression promotes melanoma progression; however, its role in breast cancer is complex, with differing reports of the effect of its expression on disease outcome. To address this we analyzed human databases, undertook gene array expression analysis, and generated unique murine models of P-Rex1 gain or loss of function. Analysis of PREX1 mRNA expression in breast cancer cDNA arrays and a METABRIC cohort revealed that higher PREX1 mRNA in ER+ve/luminal tumors was associated with poor outcome in luminal B cancers. Prex1 deletion in MMTV-neu or MMTV-PyMT mice reduced Rac1 activation in vivo and improved survival. High level MMTV-driven transgenic PREX1 expression resulted in apicobasal polarity defects and increased mammary epithelial cell proliferation associated with hyperplasia and development of de novo mammary tumors. MMTV-PREX1 expression in MMTV-neu mice increased tumor initiation and enhanced metastasis in vivo, but had no effect on primary tumor growth. Pharmacological inhibition of Rac1 or MEK1/2 reduced P-Rex1-driven tumoroid formation and cell invasion. Therefore, P-Rex1 can act as an oncogene and cooperate with HER2/neu to enhance breast cancer initiation and metastasis, despite having no effect on primary tumor growth. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger (P-Rex) proteins are RacGEFs that are synergistically activated by phosphatidylinositol 3,4,5-trisphosphate and Gβγ subunits of G-protein-coupled receptors. P-Rex1 and P-Rex2 share similar amino acid sequence homology, domain structure, and catalytic function. Recent evidence suggests that both P-Rex proteins may play oncogenic roles in human cancers. P-Rex1 and P-Rex2 are altered predominantly via overexpression and mutation, respectively, in various cancer types, including breast cancer, prostate cancer, and melanoma. This review compares the similarities and differences between P-Rex1 and P-Rex2 functions in human cancers in terms of cellular effects and signalling mechanisms. Emerging clinical data predict that changes in expression or mutation of P-Rex1 and P-Rex2 may lead to changes in tumour outcome, particularly in breast cancer and melanoma. [ABSTRACT FROM AUTHOR]

Class I phosphoinositide 3-kinase (PI3K) generates phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) 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)P3 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)P3 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)P3 is rapidly hydrolysed by PIPP to generate phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), which is further hydrolysed by INPP4B to form phosphatidylinositol 3-phosphate (PtdIns3P). PtdIns(3,4)P2 and PtdIns3P are also important signalling molecules; PtdIns(3,4)P2 together with PtdIns(3,4,5)P3 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)P2-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)P3 signalling downstream of PI3K and how dysregulation of these phosphatases affects cancer outcomes. [ABSTRACT FROM AUTHOR]

Background: Hearing impairment is the most common sensory impairment in humans, affecting 1:1,000 births. We have identified an ENU generated mouse mutant, Mozart, with recessively inherited, non-syndromic progressive hearing loss caused by a mutation in the synaptojanin 2 (Synj2), a central regulatory enzyme in the phosphoinositide-signaling cascade. Methodology/Principal Findings: The hearing loss in Mozart is caused by a p.Asn538Lys mutation in the catalytic domain of the inositol polyphosphate 5-phosphatase synaptojanin 2. Within the cochlea, Synj2 mRNA expression was detected in the inner and outer hair cells but not in the spiral ganglion. Synj2N538K mutant protein showed loss of lipid phosphatase activity, and was unable to degrade phosphoinositide signaling molecules. Mutant Mozart mice (Synj2N538K/N538K) exhibited progressive hearing loss and showed signs of hair cell degeneration as early as two weeks of age, with fusion of stereocilia followed by complete loss of hair bundles and ultimately loss of hair cells. No changes in vestibular or neurological function, or other clinical or behavioral manifestations were apparent. Conclusions/Significance: Phosphoinositides are membrane associated signaling molecules that regulate many cellular processes including cell death, proliferation, actin polymerization and ion channel activity. These results reveal Synj2 as a critical regulator of hair cell survival that is essential for hair cell maintenance and hearing function. [ABSTRACT FROM AUTHOR]

Inositol polyphosphate 4-phosphatase-Il (INPP4B) is a regulator of the phosphoinositide 3-kinase (P13K) signaling pathway and is implicated as a tumor suppressor in epithelial carcinomas. INPP4B loss of heterozygosity (LOH) is detected in some human breast cancers; however, the expression of INPP4B protein in breast cancer subtypes and the normal breast is unknown. We report here that INPP4B is expressed in nonproliferative estrogen receptor (ER)-positive cells in the normal breast, and in ER-positive, but not negative, breast cancer cell lines. INPP4B knockdown in ER-positive breast cancer cells increased Akt activation, cell proliferation, and xenograft tumor growth. Conversely, reconstitution of INPP4B ex- pression in ER-negative, INPP4B-null human breast cancer cells reduced Akt activation and anchorage-independent growth. INPP4B protein expression was frequently lost in primary human breast carcinomas, associated with high clinical grade and tumor size and loss of hormone receptors and was lost most commonly in aggressive basal-like breast carcinomas. INPP4B protein loss was also frequently observed in phosphatase and tensin homolog (PTEN)-null tumors. These studies provide evidence that INPP4B functions as a tumor suppressor by negatively regulating normal and malignant mammary epithelial cell proliferation through regulation of the PI3K/Akt signaling pathway, and that loss of INPP4B protein is a marker of aggressive basal-like breast carcinomas. [ABSTRACT FROM AUTHOR]

Identifies the cellular functions of inositol polyphosphate 5-phosphatases. Regulation of hemapoietic cell proliferation and activation; Recruitment of various effector proteins to membrane; Distinction of subcellular localization of various isoforms.

The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is hyperactivated in ~70% of breast cancers. Class I PI3K generates PtdIns(3,4,5)P3 at the plasma membrane in response to growth factor stimulation, leading to AKT activation to drive cell proliferation, survival and migration. PTEN negatively regulates PI3K/AKT signalling by dephosphorylating PtdIns(3,4,5)P3 to form PtdIns(4,5)P2. PtdIns(3,4,5)P3 can also be hydrolysed by the inositol polyphosphate 5-phosphatases (5-phosphatases) to produce PtdIns(3,4)P2. Interestingly, while PTEN is a bona fide tumour suppressor and is frequently mutated/lost in breast cancer, 5-phosphatases such as PIPP, SHIP2 and SYNJ2, have demonstrated more diverse roles in regulating mammary tumourigenesis. Reduced PIPP expression is associated with triple negative breast cancers and reduced relapse-free and overall survival. Although PIPP depletion enhances AKT phosphorylation and supports tumour growth, this also inhibits cell migration and metastasis in vivo, in a breast cancer oncogene-driven murine model. Paradoxically, SHIP2 and SYNJ2 are increased in primary breast tumours, which correlates with invasive disease and reduced survival. SHIP2 or SYNJ2 overexpression promotes breast tumourigenesis via AKT-dependent and independent mechanisms. This review will discuss how PTEN, PIPP, SHIP2 and SYNJ2 distinctly regulate multiple functional targets, and the mechanisms by which dysregulation of these distinct phosphoinositide phosphatases differentially affect breast cancer progression. [ABSTRACT FROM AUTHOR]

The Rac-GEF P-Rex1 promotes membrane ruffling and cell migration in response to Rac activation, but its role in neuritogenesis is unknown. Rac1 promotes neurite differentiation; Rac3, however, may play an opposing role. Here we report that in nerve growth factor (NGF)-differentiated rat PC12 cells, P-Rex1 localised to the distal tips of developing neurites and to the axonal shaft and growth cone of differentiating hippocampal neurons. P-Rex1 expression inhibited NGF-stimulated PC12 neurite differentiation and this was dependent on the Rac-GEF activity of P-Rex1. P-Rex1 inhibition of neurite outgrowth was rescued by low-dose cytochalasin D treatment, which prevents actin polymerisation. P-Rex1 activated Rac3 GTPase activity when coexpressed in PC12 cells. In the absence of NGF stimulation, targeted depletion of P-Rex1 in PC12 cells by RNA interference induced the spontaneous formation of β-tubulin-enriched projections. Following NGF stimulation, enhanced neurite differentiation, with neurite hyper-elongation correlating with decreased F-actin at the growth cone, was demonstrated in P-Rex1 knockdown cells. Interestingly, P-Rex1-depleted PC12 cells exhibited reduced Rac3 and Rac1 GTPase activity. This study has identified P-Rex1 as a Rac3-GEF in neuronal cells that localises to, and regulates, actin cytoskeletal dynamics at the axonal growth cone to in turn regulate neurite differentiation. [ABSTRACT FROM AUTHOR]