Inositol polyphosphate-4-phosphatase type II and rucaparib treatment inhibit the growth of osteosarcoma cells dependent on phosphoinositide 3-kinase/protein kinase B pathway.

Osteosarcoma (OS) is an aggressive malignant tumor of bone, which often occurs in children and adolescents. Currently, the effective method for the treatment of OS is still limited. The study aimed to investigate the synergistic antitumor effect of inositol polyphosphate-4-phosphatase, type-II (INPP4B) and rucaparib on OS cells. The expression levels of INPP4B in OS tissues and OS cell lines were examined by quantitative real-time polymerase chain reaction and Western blot analysis. SaOS2 and U2OS cells were then transfected with overexpression vector of INPP4B or were treated with different concentrations of rucaparib, and cell viability, cell cycle, and apoptosis were determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry. Western blot assay uncovered the combined effects of INPP4B and rucaparib on cell cycle, apoptosis and phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signal pathway. Further, the tumor formation was examined in vivo. Results showed that INPP4B was low expressed in OS tissues and in OS cell lines. INPP4B overexpression significantly decreased cell viability and induced apoptosis in SaOS2 and U2OS cells. Additionally, rucaparib remarkably reduced cell viability in a dose-dependent and time-dependent manner. Meanwhile, rucaparib suppressed cell cycle progression in the S phase and promoted apoptosis in a dose-dependent manner. Further, combination of INPP4B overexpression and rucaparib declined Myc, cyclin E1 and cyclin D1 expressions, enhanced Bad, Bax, and cleaved-caspase-3 expressions, and blocked PI3K/AKT signal pathway in SaOS2 and U2OS cells. Finally, combination of INPP4B overexpression and rucaparib inhibited tumor formation in vivo. The study demonstrated that INPP4B and rucaparib exhibited synergistic antitumor effect by regulating PI3K/AKT pathway in OS cells.

MicroRNA-590-3p promotes cell proliferation and invasion by targeting inositol polyphosphate 4-phosphatase type II in human prostate cancer cells.

Inositol polyphosphate 4-phosphatase type II emerges as a tumor suppressor in prostate cancer, and its loss of expression is associated with poor prognosis for prostate cancer. However, the mechanism of downregulation of inositol polyphosphate 4-phosphatase type II in prostate cancer development has not yet been fully clarified. In this study, microRNA-590-3p was found to be upregulated in both prostate cancer tissues and cell lines. Overexpression of microRNA-590-3p by microRNA-590-3p mimics promoted prostate cancer cell proliferation and invasion and accelerated the growth of xenografted tumors, while microRNA-590-3p inhibitors contributed to inhibition of cellular proliferation and invasion as well as tumor growth. A dual-luciferase reporter assay and expression analysis further confirmed that inositol polyphosphate 4-phosphatase type II was a direct target of microRNA-590-3p. Enforced expression of microRNA-590-3p led to repression of inositol polyphosphate 4-phosphatase type II messenger RNA and protein expression, as well as upregulation of p-Akt, p-FoxO3a, and cyclin D1 and downregulation of p21 expression in prostate cancer cell lines. Overexpression of inositol polyphosphate 4-phosphatase type II could reduce microRNA-590-3p-induced cell proliferation and invasion as well as tumor growth, and decrease microRNA-590-3p-mediated upregulation of cyclin D1 and downregulation of p21 expression in prostate cancer cells. Taken together, our findings reveal that microRNA-590-3p is a potential onco-microRNA that participates in carcinogenesis of human prostate cancer by suppressing inositol polyphosphate 4-phosphatase type II expression and involving the Akt/FoxO3a pathway. MicroRNA-590-3p may represent a potential therapeutic target for prostate cancer patients.

AS1411 binds to nucleolin via its parallel structure and disrupts the exos-miRNA-27a-mediated reciprocal activation loop between glioma and astrocytes.

The interaction between glioma cells and astrocytes promotes the proliferation of gliomas. Micro-RNAs (miRNAs) carried by astrocyte exosomes (exos) may be involved in this process, but the mechanism remains unclear. The oligonucleotide AS1411, which consists of 26 bases and has a G-quadruplex structure, is an aptamer that targets nucleolin. In this study, we demonstrate exosome-miRNA-27a-mediated cross-activation between astrocytes and glioblastoma and show that AS1411 reduces astrocytes' pro-glioma activity. The enhanced affinity of AS1411 toward nucleolin is attributed to its G-quadruplex structure. After binding to nucleolin, AS1411 inhibits the entry of the NF-κB pathway transcription factor P65 into the nucleus, then downregulates the expression of miRNA-27a in astrocytes surrounding gliomas. Then, AS1411 downregulates astrocyte exosome-miRNA-27a and upregulates the expression of INPP4B, the target gene of miRNA-27a in gliomas, thereby inhibiting the PI3K/AKT pathway and inhibiting glioma proliferation. These results were verified in mouse orthotopic glioma xenografts and human glioma samples. In conclusion, the parallel structure of AS1411 allows it to bind to nucleolin and disrupt the exosome-miRNA-27a-mediated reciprocal activation loop between glioma cells and astrocytes. Our results may help in the development of a novel approach to therapeutic modulation of the glioma microenvironment.

INPP4B-mediated tumor resistance is associated with modulation of glucose metabolism via hexokinase 2 regulation in laryngeal cancer cells.

Inositol polyphosphate 4-phosphatase type II (INPP4B) was recently identified as a tumor resistance factor in laryngeal cancer cells. Herein, we show that INPP4B-mediated resistance is associated with increased glycolytic phenotype. INPP4B expression was induced by hypoxia and irradiation. Intriguingly, overexpression of INPP4B enhanced aerobic glycolysis. Of the glycolysis-regulatory genes, hexokinase 2 (HK2) was mainly regulated by INPP4B and this regulation was mediated through the Akt-mTOR pathway. Notably, codepletion of INPP4B and HK2 markedly sensitized radioresistant laryngeal cancer cells to irradiation or anticancer drug. Moreover, INPP4B was significantly associated with HK2 in human laryngeal cancer tissues. Therefore, these results suggest that INPP4B modulates aerobic glycolysis via HK2 regulation in radioresistant laryngeal cancer cells.

Determinants of the tumor suppressor INPP4B protein and lipid phosphatase activities.

The tumor suppressor INPP4B is an important regulator of phosphatidyl-inositol signaling in the cell. Reduced INPP4B expression is associated with poor outcomes for breast, prostate, and ovarian cancer patients. INPP4B contains a CX5R catalytic motif characteristic of dual-specificity phosphatases, such as PTEN. Lipid phosphatase activity of INPP4B has previously been described. In this report we show that INPP4B can dephosphorylate para-nitrophenyl phosphate (pNPP) and 6,8-difluoro-4-methylumbelliferyl (DiFMUP), synthetic phosphotyrosine analogs, suggesting that INPP4B has protein tyrosine phosphatase (PTP) activity. Using mutagenesis, we examined the functional role of specific amino acids within the INPP4B C842KSAKDR catalytic site. The K843M mutant displayed increased pNPP hydrolysis, the K846M mutant lost lipid phosphatase activity with no effect on PTP activity, and the D847E substitution ablated PTP activity and significantly reduced lipid phosphatase activity. Further, we show that INPP4B but not PTEN is able to reduce tyrosine phosphorylation of Akt1 and both the lipid and PTP activity of INPP4B likely contribute to the reduction of Akt1 phosphorylation. Taken together our data identified key residues in the INPP4B catalytic domain associated with lipid and protein phosphatase activities and found a robust downstream target regulated by INPP4B but not PTEN.

Attenuated cerebellar phenotypes in Inpp4a truncation mutants with preserved phosphatase activity.

Phosphoinositides (PIPs) act as intracellular signaling molecules that regulate various cellular processes. Abnormalities in PIP metabolism cause various pathological conditions, including neurodegenerative diseases, cancer and immune disorders. Several neurological diseases with diverse phenotypes, such as ataxia with cerebellar atrophy or intellectual disability without brain malformation, are caused by mutations in INPP4A, which encodes a phosphoinositide phosphatase. We examined two strains of Inpp4a mutant mice with distinct cerebellar phenotypes: the Inpp4aΔEx1,2 mutant exhibited striatal degeneration without cerebellar atrophy, and the Inpp4aΔEx23 mutant exhibited a severe striatal phenotype with cerebellar atrophy. Both strains exhibited reduced expression of Inpp4a mutant proteins in the cerebellum. N-terminal-truncated Inpp4a proteins were expressed from the Inpp4aΔEx1,2 allele by alternative translation initiation and had phosphatase activity for PI(3,4)P2, whereas the Inpp4a mutant protein encoded by Inpp4aΔEx23 completely lacked phosphatase activity. Our results indicate that the diverse phenotypes observed in Inpp4a-related neurological diseases could be due to the varying protein expression levels and retained phosphatase activity in different Inpp4a variants. These findings provide insights into the role of INPP4A mutations in disease pathogenesis and may help to develop personalized therapy.

Role of inositol polyphosphate-4-phosphatase type II in oncogenesis of digestive system tumors.

Inositol polyphosphate-4-phosphatase type II (INPP4B) is a newly discovered PI(3,4,5)P3 phosphatase. Many studies have revealed that INPP4B is upregulated or downregulated in tumors of the digestive system, and the abnormal expression of INPP4B may be attributed to the occurrence, development, and prognosis of tumors of the digestive system. This paper reviews studies on the correlations between INPP4B and digestive system tumors and the roles of INPP4B in the development of different tumors to provide a theoretical basis for further research on its molecular mechanism and clinical application. "INPP4B" and "tumor" were searched as key words in PubMed and in the CNKI series full text database retrieval system from January 2000 to August 2023. A total of 153 English-language studies and 30 Chinese-language studies were retrieved. The following enrollment criteria were applied: (1) Studies contained information on the biological structure and functions of INPP4B; (2) studies covered the influence of abnormal expression of INPP4B in digestive system tumors; and (3) studies covered the role of INPP4B in the diagnosis, treatment, and prognosis of digestive system tumors. After excluding the literature irrelevant to this study, 61 papers were finally included in the analysis. INPP4B expression is low in gastric cancer, colon cancer, pancreatic cancer, and liver cancer but it has high expression in esophageal cancer, colon cancer, pancreatic cancer, and gallbladder cancer. INPP4B is involved in the occurrence and development of digestive system tumors through the regulation of gene expression and signal transduction. The abnormal expression of INPP4B plays an important role in the development of digestive system tumors. Studies on INPP4B provide new molecular insights for the diagnosis, treatment, and prognosis evaluation of digestive system tumors.

The INPP4B paradox: Like PTEN, but different.

Cancer is a complex and heterogeneous disease marked by the dysregulation of cancer driver genes historically classified as oncogenes or tumour suppressors according to their ability to promote or inhibit tumour development and growth, respectively. Certain genes display both oncogenic and tumour suppressor functions depending on the biological context, and as such have been termed dual-role cancer driver genes. However, because of their context-dependent behaviour, the tumourigenic mechanism of many dual-role genes is elusive and remains a significant knowledge gap in our effort to understand and treat cancer. Inositol polyphosphate 4-phosphatase type II (INPP4B) is an emerging dual-role cancer driver gene, primarily known for its role as a negative regulator of the phosphoinositide 3-kinase (PI3K)/AKT signalling pathway. In response to growth factor stimulation, class I PI3K generates PtdIns(3,4,5)P3 at the plasma membrane. PtdIns(3,4,5)P3 can be hydrolysed by inositol polyphosphate 5-phosphatases to generate PtdIns(3,4)P2, which, together with PtdIns(3,4,5)P3, facilitates the activation of AKT to promote cell proliferation, survival, migration, and metabolism. Phosphatase and tensin homology on chromosome 10 (PTEN) and INPP4B are dual-specificity phosphatases that hydrolyse PtdIns(3,4,5)P3 and PtdIns(3,4)P2, respectively, and thus negatively regulate PI3K/AKT signalling. PTEN is a bona fide tumour suppressor that is frequently lost in human tumours. INPP4B was initially characterised as a tumour suppressor akin to PTEN, and has been implicated as such in a number of cancers, including prostate, thyroid, and basal-like breast cancers. However, evidence has since emerged revealing INPP4B as a paradoxical oncogene in several malignancies, with increased INPP4B expression reported in AML, melanoma and colon cancers among others. Although the tumour suppressive function of INPP4B has been mostly ascribed to its ability to negatively regulate PI3K/AKT signalling, its oncogenic function remains less clear, with proposed mechanisms including promotion of PtdIns(3)P-dependent SGK3 signalling, inhibition of PTEN-dependent AKT activation, and enhancing DNA repair mechanisms to confer chemoresistance. Nevertheless, research is ongoing to identify the factors that dictate the tumourigenic output of INPP4B in different human cancers. In this review we discuss the dualistic role that INPP4B plays in the context of cancer development, progression and treatment, drawing comparisons to PTEN to explore how their similarities and, importantly, their differences may account for their diverging roles in tumourigenesis.

Structural and biochemical characterization of the pleckstrin homology domain of the RhoGEF P-Rex2 and its regulation by PIP3.

P-Rex family Rho guanine-nucleotide exchange factors are important regulators of cell motility through their activation of a subset of small GTPases. Both P-Rex1 and P-Rex2 have also been implicated in the progression of certain cancers, including breast cancer and melanoma. Although these molecules display a high level of homology, differences exist in tissue distribution, physiological function, and regulation at the molecular level. Here, we sought to compare the P-Rex2 pleckstrin homology (PH) domain structure and ability to interact with PIP3 with those of P-Rex1. The 1.9 Šcrystal structure of the P-Rex2 PH domain reveals conformational differences in the loop regions, yet biochemical studies indicate that the interaction of the P-Rex2 PH domain with PIP3 is very similar to that of P-Rex1. Binding of the PH domain to PIP3 is critical for P-Rex2 activity but not membrane localization, as previously demonstrated for P-Rex1. These studies serve as a starting point in the identification of P-Rex structural features that are divergent between isoforms and could be exploited for the design of P-Rex selective compounds.

Down-regulation of Inositol Polyphosphate 4-Phosphatase Type II Expression in Colorectal Carcinoma.

BACKGROUND/AIM: Aberrant expression of survival signaling pathways causes deregulation of cellular proliferation and resistance to apoptosis, and plays a crucial role in the development, progression and metastasis of cancer. Inositol polyphosphate 4-phosphatase type II (INPP4B) negatively regulates phosphatidylinositol 3-kinase signaling and has a tumor-suppressive role in several human malignancies. MATERIALS AND METHODS: We analyzed the expression levels of INPP4B mRNA and protein in colorectal carcinoma (CRC) cell lines and tissue samples using western blot, quantitative real-time reverse-transcriptase polymerase chain reaction, and immunohistochemical staining. RESULTS: Western blot analysis revealed that the CRC cell lines HCT 116, SW620, DLD-1, and WiDr expressed significantly lower levels of INPP4B protein than the normal colonic epithelial cell lines CCD 841 CoTr and FHC. Consistent with these results, INPP4B mRNA expression in the CRC cell lines was significantly lower than in the normal colonic epithelial cells. Immunohistochemical staining revealed that normal colonic mucosa displayed uniform and strong-to-moderate INPP4B immunoreactivity, whereas 60.7% (71/117; p<0.001) and 76.5% (62/81; p<0.001) of the primary and metastatic CRC tissue samples exhibited reduced INPP4B expression, respectively. CONCLUSION: Our results indicate that INPP4B is down-regulated in CRC and that INPP4B is involved in the development and progression of CRC.

Depletion of Inositol Polyphosphate 4-Phosphatase II Suppresses Callosal Axon Formation in the Developing Mice.

The corpus callosum is a bundle of nerve fibers that connects the two cerebral hemispheres and is essential for coordinated transmission of information between them. Disruption of early stages of callosal development can cause agenesis of the corpus callosum (AgCC), including both complete and partial callosal absence, causing mild to severe cognitive impairment. Despite extensive studies, the etiology of AgCC remains to be clarified due to the complicated mechanism involved in generating AgCC. The biological function of PI3K signaling including phosphatidylinositol-3,4,5-trisphosphate is well established in diverse biochemical processes including axon and dendrite morphogenesis, but the function of the closely related phosphatidylinositol-3,4,-bisphosphate (PI(3,4)P2) signaling, particularly in the nervous system, is largely unknown. Here, we provide the first report on the role of inositol polyphosphate 4-phosphatase II (INPP4B), a PI(3,4)P2 metabolizing 4-phosphatase in the regulation of callosal axon formation. Depleting INPP4B by in utero electroporation suppressed medially directed callosal axon formation. Moreover, depletion of INPP4B significantly attenuated formation of Satb2-positive pyramidal neurons and axon polarization in cortical neurons during cortical development. Taken together, these data suggest that INPP4B plays a role in the regulating callosal axon formation by controlling axon polarization and the Satb2-positive pyramidal neuron population. Dysregulation of INPP4B during cortical development may be implicated in the generation of partial AgCC.

Qualitative and Quantitative In Vitro Analysis of Phosphatidylinositol Phosphatase Substrate Specificity.

Phosphoinositides compromise a family of eight membrane lipids which play important roles in many cellular signaling pathways. Signaling through phosphoinositides has been shown in a variety of cellular functions such cell proliferation, cell growth, apoptosis, and vesicle trafficking. Phospholipid phosphatases regulate cell signaling by modifying the concentration of phosphoinositides and their dephosphorylated products. To understand the role of individual lipid phosphatases in phosphoinositide turnover and functional signaling, it is crucial to determine the substrate specificity of the lipid phosphatase of interest. In this chapter we describe how the substrate specificity of an individual lipid phosphatase can be qualitatively and quantitatively measured in an in vitro radiometric assay. In addition, we specify the different expression systems and purification methods required to produce the necessary yield and functionality in order to further characterize these enzymes. The outstanding versatility and sensitivity of this assay system are yet unmatched and are therefore currently considered the standard of the field.

Loss of INPP4B causes a DNA repair defect through loss of BRCA1, ATM and ATR and can be targeted with PARP inhibitor treatment.

Treatment options for ovarian cancer patients remain limited and overall survival is less than 50% despite recent clinical advances. The lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma. Using microarray technology we identified a DNA repair defect in INPP4B-deficient cells, which we further characterized by comet assays and quantification of γH2AX, RAD51 and 53BP1 foci formation. INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models. Mechanistically, we discovered that INPP4B forms a protein complex with the key players of DNA repair, ATR and BRCA1, in GST pulldown and 293T overexpression assays, and INPP4B loss affects BRCA1, ATM and ATR protein stability resulting in the observed DNA repair defect. Given that INPP4B loss has been found in 40% of ovarian cancer patients, this study provides the rationale for establishing INPP4B as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients. Loss of the tumor suppressor inositol polyphosphate 4-phosphatase type II (INPP4B) results in a DNA repair defect due to concomitant loss of BRCA1, ATR and ATM and can be therapeutically targeted with PARP inhibitors.