PTEN-deficient Cells Research Articles

Abstract 5510: ABT-737 synergizes with Vorinostat to induce apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines

Abstract We have previously observed that glioma cells are differentially sensitive to ABT-737 and, when used as a single-agent, this drug failed to induce apopotsis. Identification of therapeutic strategies to enhance the efficacy of the Bcl-2 inhibitor ABT-737 in human glioma is of interest. Histone deacetylation inhibitors (HDACi) are currently being assessed clinically in patients with glioma, as regulation of epigenetic abnormalities is expected to produce pro-apoptotic effects. We hypothesized that cotreatment of glioma with a BH3-mimetic and HDACi may synergistically produce cell death. We assessed the combination of the Bcl-2 family inhibitor ABT-737 and the HDACi vorinostat in established and primary cultured glioma cells. We found that combined treatment with these agents led to significant cellular death when compared to either drug as a single agent and demonstrated activation of the caspase cascade. The enhanced apoptosis observed with the combination also appears dependent upon the loss of mitochondrial membrane potential, release of cytochrome c, smac/DIABLO and apoptosis-inducing factor to the cytosol. We observed that the upregulation of Noxa, truncation of Bid, and the activation of Bax caused by this combination were important factors for synergistic cell death. We demonstrated that Noxa functioned to occupy Mcl-1 in cells exposed to the combination, yet, contrary to studies in other malignancies, this combination in glioma occurred without the release of proapoptotic proteins Bim and Bak from Mcl-1. We showed this combination to be less effective in PTEN-deficient glioma cells. Both genetic manipulation of PTEN status and pharmacologic inhibition of PI3 kinase sensitized PTEN-deleted glioma cells to the combination. This study demonstrates that antagonizing apoptosis-resistance pathways, such as the Bcl-2 family, and regulating epigenetic aberrancies, such as histone regulation, may induce cell death. Moreover, we reiterate that therapies to target the PI3-kinase/Akt/mTor pathway may be additionally necessary in cancers lacking PTEN functionality. Citation Format: Kimberly A. Foster, Daniel R. Premkumar, Esther P. Jane, Alejandro Morales, Ian F. Pollack. ABT-737 synergizes with Vorinostat to induce apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5510. doi:10.1158/1538-7445.AM2014-5510

Abstract 2254: Pro-angiogenic CXCL8 signalling underpins microenvironment-induced relapse to anti-androgen therapy of prostate cancer

Abstract Introduction and Objectives: Bicalutamide induces a rapid and sustained hypoxia in growing LNCaP prostatic tumors. This treatment-induced hypoxia (>14 days) increases synthesis of the CXC-chemokine Interleukin-8(CXCL8) co-incident with increasing oxygen tension and the restoration of tumor vascularisation1. Consequently, our current hypothesis is that hypoxia induced CXCL8 signalling underpins resistance to bicalutamide-treated tumors and facilitates the adoption of more aggressive cancer cells. Results: Culturing of LNCaP (PTEN null) and 22Rv1 (PTEN wt) cells under hypoxic conditions (mimicking a bicalutamide-treated tumor microenvironment) increased transcriptional activity of the androgen receptor (AR), Hypoxia-Inducible Factor (HIF-1) and NF-kB, inducing expression of downstream target genes associated with survival, metabolic adaptation and angiogenesis. The magnitude and duration of these responses was greater in PTEN-null LNCaP cells. Experiments were conducted to determine how bicalutamide-mediated inhibition of AR signalling would affect hypoxia-induced potentiation of AR, HIF and NF-kB transcription. Bicalutamide or an AR-targeted siRNA failed to reduce expression of pro-angiogenic, pro-survival and metabolism-associated genes. However, concurrent knockdown of both HIF-1 and NF-kB activity using gene-targeted siRNA's repressed expression of these hypoxia-induced disease-progressing genes. Amongst the genes up-regulated in hypoxic and PTEN-deficient LNCaP cells was expression of CXCL8 and its receptors, CXCR1 and CXCR2, which we have previously shown to be regulated by HIF-1 and NF-kB activity. Potentiation of CXCL8 signalling under hypoxia was found to sustain AR, HIF and NF-kB transcription. Combination of bicalutamide with the proteasome inhibitor bortezomib attenuated hypoxia-induced AR expression and transcriptional activity and decreased HIF-1 activity but failed to reduce NF-kB activity. In contrast, only direct inhibition of CXCL8 signalling was shown to repress activity of AR, HIF-1 and NF-kB in hypoxic LNCaP cells and increased LNCaP cells sensitivity to bicalutamide. Conclusion: Our data suggests that targeting CXCL8 signalling may therefore be a relevant strategy to enhance tumor response to anti-androgen therapies. Inhibiting signalling of this chemokine attenuates key transcriptional approaches induced in the hypoxic microenvironment of bicalutamide-treated tumors. Moreover, inhibition of CXCL8 signalling may prevent the re-vascularization previously observed in hypoxic LNCaP tumors. Citation Format: Melanie McKechnie, Pamela J. Maxwell, David J. J. Waugh. Pro-angiogenic CXCL8 signalling underpins microenvironment-induced relapse to anti-androgen therapy of prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2254. doi:10.1158/1538-7445.AM2014-2254

Abstract 5253: PTEN represses oncogene expression by regulating Daxx-H3.3 deposition in the chromatin

Abstract One of the most mutated and deleted gene in cancer is the tumor suppressor gene pten (phosphatase and tensin homology, deleted on chromosome ten). To further understand how PTEN inactivation contributes to cancer predisposition, we investigated how PTEN regulates gene expression. We found that the histone chaperon protein Daxx (death domain-associated protein) associates with PTEN, in vivo and in vitro, and regulates PTEN stability through acetylation and ubiquitination. We observed that PTEN regulates Histone 3.3 (H3.3 variant) loading in the chromatin by limiting Daxx interactions, and thereby controls expression of several tumor driver genes. Furthermore, when Daxx expression is disrupted in pten-deficient glioma stem cells, we observed a dramatic diminution of pluripotency transcription factors, a decrease in differentiation and sphere-forming capacity, and an increase in radiation sensitivity. In conclusion, this work supports a novel chromatin-associated function for PTEN that regulates H3.3 deposition, and gene expression, through Daxx interaction. This nominates Daxx as a therapeutic target to treat patients with gliomas and other types of cancers in which PTEN is mutated. Citation Format: Jorge A. Benitez, Webster K. Cavenee, Frank F. Furnari. PTEN represses oncogene expression by regulating Daxx-H3.3 deposition in the chromatin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5253. doi:10.1158/1538-7445.AM2014-5253

Differential SKIP expression in PTEN-deficient glioblastoma regulates cellular proliferation and migration.

Glioblastoma is the most common and lethal primary malignant brain tumor in adults. The tumor suppressor gene PTEN is deleted, mutated or hypermethylated in more than 60% of glioblastoma cases resulting in hyperactivation of the phosphoinositide 3-kinase pathway, which leads to sustained PI(3,4,5)P3 signaling, and thereby hyperactivation of Akt and other effectors. PI(3,4,5)P3 is also hydrolyzed to PI(3,4)P2 by inositol polyphosphate 5-phosphatases such as SKIP, but the role this pathway has in glioblastoma is unknown. Microarray expression profiling of SKIP in human glioblastoma has revealed both increased and decreased SKIP gene expression. Here we have screened PTEN-deficient glioblastoma for SKIP protein expression by immunohistochemistry and report that SKIP expression is increased in some cases or decreased relative to normal brain. Using the U-87MG PTEN-deficient cell line we show that SKIP knockdown did not further enhance cell proliferation or survival. However, SKIP overexpression in U-87MG cells suppressed anchorage-independent cell growth and growth factor-induced PI(3,4,5)P3/Akt signaling. Although, SKIP knockdown did not affect cell proliferation or survival, cell migration was significantly retarded, associated with significantly increased PI(4,5)P2 signals, and decreased phosphorylation of the actin-regulatory protein cofilin, a PI(4,5)P2-binding protein. Notably, overexpression of SKIP also inhibited migration of U-87MG cells to a similar degree as observed with PTEN reconstitution, however, via distinct mechanisms. PTEN reconstitution promoted sustained lamellipodia generation and focal adhesion formation. In contrast, SKIP overexpression reduced sustained lamellipodia formation, talin incorporation into focal adhesions and recruitment of PI(4,5)P2-binding proteins to the plasma membrane. Notably, analysis of two independent ONCOMINE microarray data sets revealed a significant correlation between increased SKIP mRNA expression in glioblastoma and improved long-term survival. Therefore, SKIP expression in glioblastoma may affect the local invasion of PTEN-deficient tumors.

Phosphorylation of PTEN at STT motif is associated with DNA damage response

Phosphatase and tensin homolog deleted on chromosome Ten (PTEN), a tumor suppressor protein participates in multiple cellular activities including DNA repair. In this work we found a relationship between phosphorylation of carboxy (C)-terminal STT motif of PTEN and DNA damage response. Ectopic expression of C-terminal phospho-mutants of PTEN, in PTEN deficient human glioblastoma cells, U87MG, resulted in reduced viability and DNA repair after etoposide induced DNA damage compared to cells expressing wild type PTEN. Also, after etoposide treatment phosphorylation of PTEN increased at C-terminal serine 380 and threonine 382/383 residues in PTEN positive HEK293T cells and wild type PTEN transfected U87MG cells. One-step further, DNA damage induced phosphorylation of PTEN was confirmed by immunoprecipitation of total PTEN from cellular extract followed by immunobloting with phospho-specific PTEN antibodies. Additionally, phospho-PTEN translocated to nucleus after etoposide treatment as revealed by indirect immunolabeling. Further, phosphorylation dependent nuclear foci formation of PTEN was observed after ionizing radiation or etoposide treatment which colocalized with γH2AX. Additionally, etoposide induced γH2AX, Mre11 and Ku70 foci persisted for a longer period of times in U87MG cells after ectopic expression of PTEN C-terminal phospho-mutant constructs compared to wild type PTEN expressing cells. Thus, our findings strongly suggest that DNA damage induced phosphorylation of C-terminal STT motif of PTEN is necessary for DNA repair.

Bidirectional molecular transport shapes cell polarization in a two-dimensional model of eukaryotic chemotaxis

Chemotacting eukaryotic cells can sense shallow gradients of chemoattractants and respond by assuming an asymmetric shape with well-defined front and back regions. Such a striking polarization phenomenon is produced largely through the interconversions and interactions between several cellular components, including Rac GTPase (Rac), phosphoinositide 3-kinase (PI3K), tensin homology protein (PTEN), phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) and phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2). Here, we developed a mathematical model of cell polarization by exploring bidirectional molecular transport that arose from phosphoinositides (PIs) and Rac-mediated feedback loops. We assumed a static gradient of activated Rac derived from an external signal field as the internal trigger signal. The evolution of PI(3,4,5)P3 and PI(4,5)P2 along with PI3K and PTEN that act as activator and inhibitor, respectively, were described by a pair of coupled transient reaction-diffusion equations. The entire system was solved using a Lattice–Boltzmann method with an embedded Monte-Carlo method to track the stochastic translocation behaviors of discrete PI3K/PTEN molecules. We first showed that, upon a graded external stimulus, the Rac to PI(3,4,5)P3 cascade exhibited a short range positive-feedback loop, while the PTEN to PI(4,5)P2 cascade contributed another long range negative-feedback loop, which dominated the “forward” and “backward” molecular transport, respectively. Second, polarization was governed by the ratio of [PI3K] to [PTEN], and manifested a switch-like behavior. Third, with a uniform stimulus, spontaneous polarization could occur in PTEN-deficient cells.

Co-administration of ABT-737 and SAHA induces apoptosis, mediated by Noxa upregulation, Bax activation and mitochondrial dysfunction in PTEN-intact malignant human glioma cell lines.

We previously observed that glioma cells are differentially sensitive to ABT-737 and, when used as a single-agent, this drug failed to induce apoptosis. Identification of therapeutic strategies to enhance the efficacy of the Bcl-2 inhibitor ABT-737 in human glioma is of interest. Histone deacetylation inhibitors (HDACI) are currently being assessed clinically in patients with glioma, as regulation of epigenetic abnormalities is expected to produce pro-apoptotic effects. We hypothesized that co-treatment of glioma with a BH3-mimetic and HDACI may induce cellular death. We assessed the combination of ABT-737 and HDACI SAHA in established and primary cultured glioma cells. We found combination treatment led to significant cellular death when compared to either drug as single agent and demonstrated activation of the caspase cascade. This enhanced apoptosis also appears dependent upon the loss of mitochondrial membrane potential and the release of cytochrome c and AIF into the cytosol. The upregulation of Noxa, truncation of Bid, and activation of Bax caused by this combination were important factors for cell death and the increased levels of Noxa functioned to sequester Mcl-1. This combination was less effective in PTEN-deficient glioma cells. Both genetic and pharmacologic inactivation of the PI3K/Akt signaling pathway sensitized PTEN-deleted glioma cells to the combination. This study demonstrates that antagonizing apoptosis-resistance pathways, such as targeting the Bcl-2 family in combination with epigenetic modifiers, may induce cell death. These findings extend our previous observations that targeting the PI3K/Akt pathway may be additionally necessary to promote apoptosis in cancers lacking PTEN functionality.

MDM2 Regulates Hypoxic Hypoxia-inducible Factor 1α Stability in an E3 Ligase, Proteasome, and PTEN-Phosphatidylinositol 3-Kinase-AKT-dependent Manner

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α-HIF1β heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics.

The PTEN/NRF2 Axis Promotes Human Carcinogenesis

A recent study conducted in mice reported that liver-specific knockout of tumor suppressor Pten augments nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcriptional activity. Here, we further investigated how phosphatase and tensin homolog deleted on chromosome 10 (PTEN) controls NRF2 and the relevance of this pathway in human carcin ogenesis. Drug and genetic targeting to PTEN and phosphoproteomics approaches indicated that PTEN leads to glycogen synthase kinase-3 (GSK-3)-mediated phosphorylation of NRF2 at residues Ser(335) and Ser(338) and subsequent beta-transducin repeat containing protein (β-TrCP)-dependent but Kelch-like ECH-associated protein 1 (KEAP1)-independent degradation. Rescue experiments in PTEN-deficient cells and xerographs in athymic mice indicated that loss of PTEN leads to increased NRF2 signature which provides a proliferating and tumorigenic advantage. Tissue microarrays from endometrioid carcinomas showed that 80% of PTEN-negative tumors expressed high levels of NRF2 or its target heme oxygenase-1 (HO-1). These results uncover a new mechanism of oncogenic activation of NRF2 by loss of its negative regulation by PTEN/GSK-3/β-TrCP that may be relevant to a large number of tumors, including endometrioid carcinomas. Increased activity of NRF2 due to loss of PTEN is instrumental in human carcinogenesis and represents a novel therapeutic target.

Tumor-derived CXCL8 signaling augments stroma-derived CCL2-promoted proliferation and CXCL12-mediated invasion of PTEN-deficient prostate cancer cells.

Impaired PTEN function is a genetic hallmark of aggressive prostate cancers (CaP) and is associated with increased CXCL8 expression and signaling. The current aim was to further characterize biological responses and mechanisms underpinning CXCL8-promoted progression of PTEN-depleted prostate cancer, focusing on characterizing the potential interplay between CXCL8 and other disease-promoting chemokines resident within the prostate tumor microenvironment. Autocrine CXCL8-stimulation (i) increased expression of CXCR1 and CXCR2 in PTEN-deficient CaP cells suggesting a self-potentiating signaling axis and (ii) induced expression of CXCR4 and CCR2 in PTEN-wild-type and PTEN-depleted CaP cells. In contrast, paracrine CXCL8 signaling induced expression and secretion of the chemokines CCL2 and CXCL12 from prostate stromal WPMY-1 fibroblasts and monocytic macrophage-like THP-1 cells. In vitro studies demonstrated functional co-operation of tumor-derived CXCL8 with stromal-derived chemokines. CXCL12-induced migration of PC3 cells and CCL2-induced proliferation of prostate cancer cells were dependent upon intrinsic CXCL8 signaling within the prostate cancer cells. For example, in co-culture experiments, CXCL12/CXCR4 signaling but not CCL2/CCR2 signaling supported fibroblast-mediated migration of PC3 cells while CXCL12/CXCR4 and CCL2/CCR2 signaling underpinned monocyte-enhanced migration of PC3 cells. Combined inhibition of both CXCL8 and CXCL12 signaling was more effective in inhibiting fibroblast-promoted cell motility while repression of CXCL8 attenuated CCL2-promoted proliferation of prostate cancer cells. We conclude that tumor-derived CXCL8 signaling from PTEN-deficient tumor cells increases the sensitivity and responsiveness of CaP cells to stromal chemokines by concurrently upregulating receptor expression in cancer cells and inducing stromal chemokine synthesis. Combined chemokine targeting may be required to inhibit their multi-faceted actions in promoting the invasion and proliferation of aggressive CaP.

MCT-1 expression and PTEN deficiency synergistically promote neoplastic multinucleation through the Src/p190B signaling activation.

Multinucleation is associated with malignant neoplasms; however, the molecular mechanism underlying the nuclear abnormality remains unclear. Loss or mutation of PTEN promotes the development of malignant tumors. We now demonstrate that increased expression of the oncogene MCT-1 (multiple copies in T-cell malignancy 1) antagonizes PTEN gene presentation, PTEN protein stability and PTEN functional activity, thereby further promoting phosphoinositide 3 kinase/AKT signaling, survival rate and malignancies of the PTEN-deficient cells. In the PTEN-null cancer cells, MCT-1 interacts with p190B and Src in vivo, supporting that they are in proximity of the signaling complexes. MCT-1 overexpression and PTEN loss synergistically augments the Src/p190B signaling function that leads to inhibition of RhoA activity. Under such a condition, the incidence of mitotic catastrophes including spindle multipolarity and cytokinesis failure is enhanced, driving an Src/p190B/RhoA-dependent neoplastic multinucleation. Targeting MCT-1 by the short hairpin RNA markedly represses the Src/p190B function, improves nuclear structures and suppresses xenograft tumorigenicity of the PTEN-null breast cancer cells. Consistent with the oncogenic effects in vitro, clinical evidence has confirmed that MCT-1 gene stimulation is correlated with p190B gene promotion and PTEN gene suppression in human breast cancer. Accordingly, MCT-1 gene induction is recognized as a potential biomarker of breast tumor development. Abrogating MCT-1 function may be a promising stratagem for management of breast cancer involving Src hyperactivation and/or PTEN dysfunction.

Emergence, Involution, and Progression to Carcinoma of Mutant Clones in Normal Endometrial Tissues

Sporadic somatic inactivation of genes such as PTEN within histologically normal endometrium (latent precancers) is an early step in endometrial carcinogenesis. We have used clone-specific mutations of PTEN to determine the fate of latent precancers over time in women who do (high risk) and do not (low risk) develop endometrial neoplasia. PTEN immunohistochemistry was performed on 45 occurrences of endometrial neoplasia and their paired antecedent benign biopsies, along with age matched sample pairs from 167 patients who did not develop a neoplasm. When PTEN-deficient cells were present at both time points, DNA sequencing was performed to determine whether they were single or multiple independent events. Loss of PTEN protein in isolated glands was common in the initial normal biopsies of high- and low-risk groups (42% and 27%, respectively, P = 0.066). Protein-deficient glands have a tendency to disappear over time in low-risk women (P = 0.047) and, even when "persistent," are infrequently (19%, 3/16) confirmed to be the same clone. Similarly, only a small proportion (6.7%, 1/15) of latent precancers seen in high-risk women are the direct progenitors of subsequent neoplasia. There is a high rate of latent precancer turnover in both low- and high-risk patients, with rare long-term persistence of unique clones, which may or may not progress to a histologic lesion. The temporal dynamics of clonal emergence, persistence, and involution are sufficiently complex that in the individual patient, the presence of a latent precancer has an unknown contribution to long-term cancer risk.

A novel PI3K inhibitor displays potent preclinical activity against an androgen-independent and PTEN-deficient prostate cancer model established from the cell line PC3

Recent studies demonstrated that targeting the phosphatidylinositide 3-kinase (PI3K)/AKT signaling pathway is a major strategy for the treatment of androgen-independent prostate cancer. In the present study, we developed an analog BENC-511 from a recently reported PI3K inhibitor S14161 by structural optimization. Using PC3 and DU145 as the model cell lines, we found PTEN-deficient PC3 cells were more sensitive than PTEN-expressing DU145 ones in terms of cell proliferation, apoptosis, and caspase-3 activation. These findings were consistent with the inhibition on PI3K/AKT signals. BENC-511 preferably suppressed AKT activation in PC3 over DU145 cells. Notably, PTEN restoration attenuated BENC-511 induced apoptosis. Moreover, BENC-511 displayed great therapeutic efficacy in a PC3-derived prostate cancer model in nude mice. With an oral dosage of 50mg/kg, BENC-511 decreased tumor growth more than 50% in 27 days, which was accompanied with PARP cleavage, but did not show overt toxicity. This study lays a solid rationale for the development of BENC-511 as a drug for the treatment of PTEN-deficient and androgen-independent prostate cancers.

PTEN regulates natural killer cell trafficking and effector functions (IRM7P.484)

Abstract Phosphatase and tensin homolog (PTEN) is a critical negative regulator of the PI3 kinase pathway, members of which are essential for regulating natural killer (NK) cell signaling and activation. To evaluate whether PTEN was required for normal NK cell functions, we utilized a mouse model of NK specific PTEN deficiency (Ncr1iCre knockin x PTENflox). PTEN deletion resulted in significant loss of NK cells in the bone marrow and other lymphoid tissues, but markedly increased numbers within the peripheral blood and lung. Surprisingly, we observed near equivalent NK cell maturation within peripheral organs regardless of PTEN expression, suggesting that PTEN operates to re-distribute NK cells during development. Similarly, despite impaired migration to chemoattractants in vitro, PTEN-deficient NK cells egress more efficiently from the bone marrow and preferentially reside in sinusoidal compartments, but are specifically retained in the lung and peripheral vasculature. Given the inappropriate localization in the absence of PTEN, we further evaluated whether cytotoxic functions were altered. While PTEN deficient NK cells have enhanced cytotoxicity against co-localized RMAS tumors, they are unable to migrate to distal tumor sites. These data suggest that PTEN is required for the trafficking of NK cells during both homeostasis and in the presence of malignancy, during which it represses cytotoxic functions.

Targeting human apurinic/apyrimidinic endonuclease 1 (APE1) in phosphatase and tensin homolog (PTEN) deficient melanoma cells for personalized therapy.

Phosphatase and tensin homolog (PTEN) loss is associated with genomic instability. APE1 is a key player in DNA base excision repair (BER) and an emerging drug target in cancer. We have developed small molecule inhibitors against APE1 repair nuclease activity. In the current study we explored a synthetic lethal relationship between PTEN and APE1 in melanoma. Clinicopathological significance of PTEN mRNA and APE1 mRNA expression was investigated in 191 human melanomas. Preclinically, PTEN-deficient BRAF-mutated (UACC62, HT144, and SKMel28), PTEN-proficient BRAF-wildtype (MeWo), and doxycycline-inducible PTEN-knockout BRAF-wildtype MeWo melanoma cells were DNA repair expression profiled and investigated for synthetic lethality using a panel of four prototypical APE1 inhibitors. In human tumours, low PTEN mRNA and high APE1 mRNA was significantly associated with reduced relapse free and overall survival. Pre-clinically, compared to PTEN-proficient cells, PTEN-deficient cells displayed impaired expression of genes involved in DNA double strand break (DSB) repair. Synthetic lethality in PTEN-deficient cells was evidenced by increased sensitivity, accumulation of DSBs and induction of apoptosis following treatment with APE1 inhibitors. We conclude that PTEN deficiency is not only a promising biomarker in melanoma, but can also be targeted by a synthetic lethality strategy using inhibitors of BER, such as those targeting APE1.

KAP regulates ROCK2 and Cdk2 in an RNA-activated glioblastoma invasion pathway

Aberrant splicing of the cyclin-dependent kinase-associated phosphatase, KAP, promotes glioblastoma invasion in a Cdc2-dependent manner. However, the mechanism by which this occurs is unknown. Here we show that miR-26a, which is often amplified in glioblastoma, promotes invasion in phosphatase and tensin homolog (PTEN)-competent and PTEN-deficient glioblastoma cells by directly downregulating KAP expression. Mechanistically, we find that KAP binds and activates ROCK2. Thus, RNA-mediated downregulation of KAP leads to decreased ROCK2 activity and this, in turn, increases Rac1-mediated invasion. In addition, the decrease in KAP expression activates the cyclin-dependent kinase, Cdk2, and this directly promotes invasion by increasing retinoblastoma phosphorylation, E2F-dependent Cdc2 expression and Cdc2-mediated inactivation of the actomyosin inhibitor, caldesmon. Importantly, glioblastoma cell invasion mediated by this pathway can be antagonized by Cdk2/Cdc2 inhibitors in vitro and in vivo. Thus, two distinct RNA-based mechanisms activate this novel KAP/ROCK2/Cdk2-dependent invasion pathway in glioblastoma.

Reciprocal positive regulation between TRPV6 and NUMB in PTEN-deficient prostate cancer cells

Calcium acts as a second messenger and plays a crucial role in signaling pathways involved in cell proliferation. Recently, calcium channels related to calcium influx into the cytosol of epithelial cells have attracted attention as a cancer therapy target. Of these calcium channels, TRPV6 is overexpressed in prostate cancer and is considered an important molecule in the process of metastasis. However, its exact role and mechanism is unclear.NUMB, well-known tumor suppressor gene, is a novel interacting partner of TRPV6. We show that NUMB and TRPV6 have a reciprocal positive regulatory relationship in PC-3 cells. We repeated this experiment in two other prostate cancer cell lines, DU145 and LNCaP. Interestingly, there were no significant changes in TRPV6 expression following NUMB knockdown in DU145. We revealed that the presence or absence of PTEN was the cause of NUMB–TRPV6 function. Loss of PTEN caused a positive correlation of TRPV6–NUMB expression. Collectively, we determined that PTEN is a novel interacting partner of TRPV6 and NUMB. These results demonstrated a novel relationship of NUMB–TRPV6 in prostate cancer cells, and show that PTEN is a novel regulator of this complex.

Pten regulates homeostasis and inflammation-induced migration of myelocytes in zebrafish

BackgroundLoss of the tumor suppressor phosphatase and tensin homolog (PTEN) is frequently observed in hematopoietic malignancies. Although PTEN has been implicated in maintaining the quiescence of hematopoietic stem cells (HSCs), its role in hematopoiesis during ontogeny remains largely unexplored.MethodsThe expression of hematopoietic marker genes was analyzed via whole mount in situ hybridization assay in ptena and ptenb double mutant zebrafish. The embryonic myelopoiesis was characterized by living imaging and whole mount in situ immunofluorescence with confocal microscopy, as well as cell-specific chemical staining for neutrophils and macrophages. Analyses of the involved signaling pathway were carried out by inhibitor treatment and mRNA injection.ResultsPten-deficient zebrafish embryos exhibited a strikingly increased number of myeloid cells, which were further characterized as being immune deficient. In accordance with this finding, the inhibition of phosphoinositide 3-kinase (PI3K) or the mechanistic target of rapamycin (mTOR) corrected the expansive myelopoiesis in the pten-deficient embryos. Further mechanistic studies revealed that the expression of cebpa, a critical transcription factor in myeloid precursor cells, was downregulated in the pten-deficient myeloid cells, whereas the injection of cebpa mRNA markedly ameliorated the dysmyelopoiesis induced by the loss of pten.ConclusionsOur data provide in vivo evidence that definitive myelopoiesis in zebrafish is critically regulated by pten via the elevation of cebpa expression.

Discovery and Optimization of Pyrimidone Indoline Amide PI3Kβ Inhibitors for the Treatment of Phosphatase and Tensin Homologue (PTEN)-Deficient Cancers

Compelling molecular biology publications have reported the implication of phosphoinositide kinase PI3Kβ in PTEN-deficient cell line growth and proliferation. These findings supported a scientific rationale for the development of PI3Kβ-specific inhibitors for the treatment of PTEN-deficient cancers. This paper describes the discovery of 2-[2-(2,3-dihydro-indol-1-yl)-2-oxo-ethyl]-6-morpholin-4-yl-3H-pyrimidin-4-one (7) and the optimization of this new series of active and selective pyrimidone indoline amide PI3Kβ inhibitors. 2-[2-(2-Methyl-2,3-dihydro-indol-1-yl)-2-oxo-ethyl]-6-morpholin-4-yl-3H-pyrimidin-4-one (28), identified following a carefully designed methyl scan, displayed improved physicochemical and in vitro pharmacokinetic properties. Structural biology efforts enabled the acquisition of the first X-ray cocrystal structure of p110β with the selective inhibitor compound 28 bound to the ATP site. The nonplanar binding mode described herein is consistent with observed structure-activity relationship for the series. Compound 28 demonstrated significant in vivo activity in a UACC-62 xenograft model in mice, warranting further preclinical investigation. Following successful development, compound 28 entered phase I/Ib clinical trial in patients with advanced cancer.

Rosiglitazone regulates anti-inflammation and growth inhibition via PTEN.

Peroxisome proliferator-activated receptor gamma (PPARγ) agonist has anti-inflammatory and anticancer properties. However, the mechanisms by which PPARγ agonist rosiglitazone interferes with inflammation and cancer via phosphatase and tensin homolog-(PTEN)-dependent pathway remain unclear. We found that lower doses (<25 μM) of rosiglitazone significantly inhibited lipopolysaccharide-(LPS)-induced nitric oxide (NO) release (via inducible nitric oxide synthase, iNOS), prostaglandin E2 (PGE2) production (via cyclooxygenase-2, COX-2), and activation of Akt in RAW 264.7 murine macrophages. However, rosiglitazone did not inhibit the production of reactive oxygen species (ROS). In PTEN knockdown (shPTEN) cells exposed to LPS, rosiglitazone did not inhibit NO release, PGE2 production, and activation of Akt. These cells had elevated basal levels of iNOS, COX-2, and ROS. However, higher doses (25–100 μM) of rosiglitazone, without LPS stimulation, did not block NO release and PGE2 productions, but they inhibited p38 MAPK phosphorylation and blocked ROS generation in shPTEN cells. In addition, rosiglitazone caused G1 arrest and reduced the number of cells in S + G2/M phase, leading to growth inhibition. These results indicate that the anti-inflammatory property of rosiglitazone is related to regulation of PTEN independent of inhibition on ROS production. However, rosiglitazone affected the dependence of PTEN-deficient cell growth on ROS.