Hanbing An

PPM1A is a RelA phosphatase with tumor suppressor-like activity

Nuclear factor-κB (NF-κB) signaling contributes to human disease processes, notably inflammatory diseases and cancer. NF-κB has a role in tumorigenesis and tumor growth, as well as promotion of metastases. Mechanisms responsible for abnormal NF-κB activation are not fully elucidated; however, RelA phosphorylation, particularly at serine residues S536 and S276, is critical for RelA function. Kinases that phosphorylate RelA promote oncogenic behaviors, suggesting that phosphatases targeting RelA could have tumor-inhibiting activities; however, few RelA phosphatases have been identified. Here, we identified tumor inhibitory and RelA phosphatase activities of the protein phosphatase 2C (PP2C) phosphatase family member, PPM1A. We show that PPM1A directly dephosphorylated RelA at residues S536 and S276 and selectively inhibited NF-κB transcriptional activity, resulting in decreased expression of monocyte chemotactic protein-1/chemokine (C–C motif) ligand 2 and interleukin-6, cytokines implicated in cancer metastasis. PPM1A depletion enhanced NF-κB-dependent cell invasion, whereas PPM1A expression inhibited invasion. Analyses of human expression data revealed that metastatic prostate cancer deposits had lower PPM1A expression compared with primary tumors without distant metastases. A hematogenous metastasis mouse model revealed that PPM1A expression inhibited bony metastases of prostate cancer cells after vascular injection. In summary, our findings suggest that PPM1A is a RelA phosphatase that regulates NF-κB activity and that PPM1A has tumor suppressor-like activity. Our analyses also suggest that PPM1A inhibits prostate cancer metastases and as neither gene deletions nor inactivating mutations of PPM1A have been described, increasing PPM1A activity in tumors represents a potential therapeutic strategy to inhibit NF-κB signaling or bony metastases in human cancer.

LZAP Inhibits p38 MAPK (p38) Phosphorylation and Activity by Facilitating p38 Association with the Wild-Type p53 Induced Phosphatase 1 (WIP1)

LZAP (Cdk5rap3, C53) is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation.

LZAP is a novel Wip1 binding partner and positive regulator of its phosphatase activity in vitro

ABSTRACT The phosphatase Wip1 attenuates the DNA damage response (DDR) by removing phosphorylation marks from a number of DDR proteins (p53, MDM2, Chk1/2, p38). Wip1 also dephosphorylates and inactivates RelA. Notably, LZAP, a putative tumor suppressor, has been linked to dephosphorylation of several of these substrates, including RelA, p38, Chk1, and Chk2. LZAP has no known catalytic activity or functional motifs, suggesting that it exerts its effects through interaction with other proteins. Here we show that LZAP binds Wip1 and stimulates its phosphatase activity. LZAP had been previously shown to bind many Wip1 substrates (RelA, p38, Chk1/2), and our results show that LZAP also binds the previously identified Wip1 substrate, MDM2. This work identifies 2 novel Wip1 substrates, ERK1 and HuR, and demonstrates that HuR is a binding partner of LZAP. Pleasingly, LZAP potentiated Wip1 catalytic activity toward each substrate tested, regardless of whether full-length substrates or phosphopeptides were utilized. Since this effect was observed on ERK1, which does not bind LZAP, as well as for each of 7 peptides tested, we hypothesize that LZAP binding to the substrate is not required for this effect and that LZAP directly binds Wip1 to augment its phosphatase activity.

Isoxazole compound ML327 blocks MYC expression and tumor formation in neuroblastoma

Neuroblastomas are the most common extracranial solid tumors in children and arise from the embryonic neural crest. MYCN-amplification is a feature of ∼30% of neuroblastoma tumors and portends a poor prognosis. Neural crest precursors undergo epithelial-to-mesenchymal transition (EMT) to gain migratory potential and populate the sympathoadrenal axis. Neuroblastomas are posited to arise due to a blockade of neural crest differentiation. We have recently reported effects of a novel MET inducing compound ML327 (N-(3-(2-hydroxynicotinamido) propyl)-5-phenylisoxazole-3-carboxamide) in colon cancer cells. Herein, we hypothesized that forced epithelial differentiation using ML327 would promote neuroblastoma differentiation. In this study, we demonstrate that ML327 in neuroblastoma cells induces a gene signature consistent with both epithelial and neuronal differentiation features with adaptation of an elongated phenotype. These features accompany induction of cell death and G1 cell cycle arrest with blockage of anchorage-independent growth and neurosphere formation. Furthermore, pretreatment with ML327 results in persistent defects in proliferative potential and tumor-initiating capacity, validating the pro-differentiating effects of our compound. Intriguingly, we have identified destabilization of MYC signaling as an early and consistent feature of ML327 treatment that is observed in both MYCN-amplified and MYCN-single copy neuroblastoma cell lines. Moreover, ML327 blocked MYCN mRNA levels and tumor progression in established MYCN-amplified xenografts. As such, ML327 may have potential efficacy, alone or in conjunction with existing therapeutic strategies against neuroblastoma. Future identification of the specific intracellular target of ML327 may inform future drug discovery efforts and enhance our understanding of MYC regulation.Neuroblastomas are the most common extracranial solid tumors in children and arise from the embryonic neural crest. MYCN-amplification is a feature of ~30% of neuroblastoma tumors and portends a poor prognosis. Neural crest precursors undergo epithelial-to-mesenchymal transition (EMT) to gain migratory potential and populate the sympathoadrenal axis. Neuroblastomas are posited to arise due to a blockade of neural crest differentiation. We have recently reported effects of a novel MET inducing compound ML327 (N-(3-(2-hydroxynicotinamido) propyl)-5-phenylisoxazole-3-carboxamide) in colon cancer cells. Herein, we hypothesized that forced epithelial differentiation using ML327 would promote neuroblastoma differentiation. In this study, we demonstrate that ML327 in neuroblastoma cells induces a gene signature consistent with both epithelial and neuronal differentiation features with adaptation of an elongated phenotype. These features accompany induction of cell death and G1 cell cycle arrest with blockage of anchorage-independent growth and neurosphere formation. Furthermore, pretreatment with ML327 results in persistent defects in proliferative potential and tumor-initiating capacity, validating the pro-differentiating effects of our compound. Intriguingly, we have identified destabilization of MYC signaling as an early and consistent feature of ML327 treatment that is observed in both MYCN-amplified and MYCN-single copy neuroblastoma cell lines. Moreover, ML327 blocked MYCN mRNA levels and tumor progression in established MYCN-amplified xenografts. As such, ML327 may have potential efficacy, alone or in conjunction with existing therapeutic strategies against neuroblastoma. Future identification of the specific intracellular target of ML327 may inform future drug discovery efforts and enhance our understanding of MYC regulation.

cFLIP critically modulates apoptotic resistance in epithelial-to-mesenchymal transition

Epithelial cancers (carcinomas) comprise the top four causes of cancer-related deaths in the United States. While overall survival has been steadily improving, therapy-resistant disease continues to present a major therapeutic challenge. Carcinomas often exploit the normal developmental program, epithelial-to-mesenchymal transition (EMT), to gain a mesenchymal phenotype associated with increased invasiveness and resistance to apoptosis. We have previously shown that an isoxazole-based small molecule, ML327, partially reverses TGF-β-induced EMT in an immortalized mouse mammary epithelial cell line. Herein, we demonstrate that ML327 reverses much of the EMT gene expression program in cultured carcinoma cell lines. The reversal of EMT sensitizes these cancer cells to the apoptosis-inducing ligand TRAIL. This sensitization is independent of E-cadherin expression and rather relies on the downregulation of a major anti-apoptotic protein, cFLIPS. Loss of cFLIPS is sufficient to overcome resistance to TRAIL and exogenous overexpression of cFLIPS restores resistance to TRAIL-induced apoptosis despite EMT reversal with ML327. In summary, we have utilized an isoxazole-based small molecule that partially reverses EMT in carcinoma cells to demonstrate that cFLIPS critically regulates the apoptosis resistance phenotype associated with EMT.

Abstract 4097: Small molecule mediated transcriptional derepression of E-cadherin and inhibition of epithelial to mesenchymal transition

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA BACKGROUND: Transcriptional repression of E-Cadherin is a hallmark of Epithelial-to-Mesenchymal Transition (EMT) and is associated with metastasis in cancer. Understanding mechanisms behind E-cadherin repression may provide important insights into the regulation of EMT and novel therapeutic strategies for cancer. In this report, we applied a medicinal chemistry approach to generate a novel EMT-reversing compound, ML327. We tested the hypothesis that ML327 activity impinges directly on the mechanism of EMT in cancer and represents a potential therapeutic agent to block or reverse EMT in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We used cell based biochemical approaches to show that ML327 increases E-cadherin expression in 5 cancer cell lines and 2 non-transformed cell lines. ML327 was shown to induce global changes in histone modifications, but through a distinct mechanism than similar chemical agents such as Histone De-Acetylase (HDAC) inhibitors. Cell based assays were used to show that ML327 inhibits cell invasion, partially reverses EMT, and inhibits metastasis in vivo. We examined gene expression changes induced by ML327 within 3 hrs in the presence of cycloheximide by RNA-seq and determined a mechanistic requirement for Hepatocyte Nuclear Factor 4α (HNF4α) in ML327 activity on E-cadherin gene expression. Through direct testing, we have determined the ML327 is not a promiscuous chemical probe and that it lacks direct HDAC inhibitory and kinase inhibitory activity, however, our study is limited by the lack of identification of the direct cellular target. Significant ongoing studies are designed to address this limitation. CONCLUSIONS/SIGNIFICANCE: ML327 increases E-cadherin in cells and translate to an important biological behavior in vivo that may be exploited for therapeutic potential in cancer. Furthermore, ML327 represents a novel chemical structure for continued pharmacological optimization toward the development of novel therapeutics. ACKNOWLEDGEMENTS: This work was supported by NIH through the Molecular Library Probe Center Network (MPLCN) under grant U54MH084659 to CWL. DISCLOSURE of POTENTIAL CONFLICT of INTEREST: No potential conflicts of interest were disclosed. DISCLOSURE of CHEMICAL STRUCTURES: Chemical compounds were used in this scientific work to generate the data, and the structures of the compounds will be disclosed at the time of presentation at the meeting. Citation Format: Hanbing An, Natasha Deane, Sydney Stoops, Jing Zhu, Alex Waterson, Andries Zijlstra, Craig Lindsley, Robert Daniel Beauchamp. Small molecule mediated transcriptional derepression of E-cadherin and inhibition of epithelial to mesenchymal transition. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4097. doi:10.1158/1538-7445.AM2015-4097

Abstract 1078: PPM1A acts as a novel RelA phosphatase to negatively regulate NF-κB signaling

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Phosphorylation of RelA is required to fully activate NF-κB transcription activity and dephosphorylation of RelA can inhibit its activity. To date, Wip1 (PPM1D) and PP2A have been described as RelA phosphatases. The purpose of this study was to identify novel RelA phosphatase(s) and determine if they play a role in regulation of NF-κB activity related to tumorigenesis. PPM1A belongs to PP2C protein phosphatase family, the same family of Wip1. To begin exploring PPM1A activity toward RelA, serine 536 (S536) phosphorylation of RelA was determined with and without expression or knockdown of PPM1A. Expression of PPM1A in U2OS cells decreased RelA phosphorylation, whereas siRNA-mediated loss of PPM1A increased S536RelA phosphorylation. Luciferase assay using a κB responsive reporter with expression of either PPM1A or phosphatase-dead PPM1A (PPM1A-PD) revealed that wild-type, but not PPM1A-PD, inhibited RelA transcription. Furthermore, in vitro phosphatase assays using bacterially expressed PPM1A with substrate of either full length RelA or phosphorylated RelA peptides, suggest that PPM1A can function as a direct RelA phosphatase at residuesS536 and S276. We could not identify reports of additional phosphatases with activity toward the S276 residue of RelA. Using liquid chromatography and tandem mass spectroscopy (LC-MS/MS) LZAP-associated proteins (LAPs) were sought with PPM1A being identified. LZAP (C53, CDK5RAP3) has tumor suppressor-like activity that is mediated at least partially through inhibition of RelA S536 phosphorylation and activity. PPM1As ability to decrease RelA phosphorylation at S536 site was largely inhibited in cells with siRNA-mediated loss of LZAP. To determine if PPM1A has tumor suppressor activity, HeLa cells were infected with retrovirus directing shRNA-mediated knockdown of PPM1A and tumorigenic properties of these cells were determined. Preliminary xenograft tumor data suggest PPM1A has a negative effect on tumor initiation and proliferation and studies are underway to determine if PPM1A activity toward NF-κB plays a role. In summary, our data suggest that PPM1A is a novel RelA phosphatase with activity to dephosphorylate RelA at serines 536 and 276. PPM1A inhibits NF-κB transcriptional activity and that PPM1A activity toward RelA is at least partially dependent on LZAP. Our preliminary data also suggest PPM1A might have tumor suppressor-like activity that could be dependent on LZAP or RelA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1078. doi:10.1158/1538-7445.AM2011-1078

Abstract 3063: Novel putative tumor suppressor LZAP binds and regulates Wip1

Our group has shown that LZAP is lost in ∼30% of head and neck squamous cell carcinomas and that inhibition of LZAP expression is associated cellular transformation and xenograft tumor growth. LZAP increases invasion and inhibits cellular proliferation and clonogenic growth mediated correlating with inhibition of NF-kB and activation of p53. Other researchers have found that LZAP promotes cell death in response to cytotoxic therapy at least partially dependent on activation of Cdk1 through Chk1/2 inhibition. We have also found that LZAP binds and inhibits p38 and this inhibition does not depend on upstream regulators MKK3 and MKK6. The activity of MAPKs reflects a balance between the upstream activating kinases and inactivating protein phosphatases. Since LZAP inhibition of p38 is not through upstream kinases, we determined if phosphatase activity targeting p38 was regulated by LZAP. Wip1 is a major phosphatase regulating p38 phosphorylation and activation. Wip1 is a member of the PP2C family of serine/threonine phosphatases and was identified in a screen to detect p53 responsive genes. We noted that LZAP and Wip1 had many binding partners/targets including RelA, p38, and Chk1/Chk2 in common. Mechanisms of LZAP inhibition of RelA, p38 and Chk1/Chk2 are not well described, but expression of LZAP is associated with decreased phosphorylation of all three target proteins. To determine if LZAP could regulate Wip1 activity, we first evaluated LZAP and Wip1 interaction through immuno- or affinity precipitation. LZAP and Wip1 co-precipitated after expression in mammalian cells and bacterially expressed LZAP pulled down Wip1 generated through in vitro transcription and translation. After transient expression in mammalian cells, Wip1 and LZAP co-localized by indirect immunofluorescence staining. When expressed singly, Flag-tagged Wip1 localized to the nucleus and LZAP was excluded from the nucleolus, but localized to both cytoplasm and nucleus. When Wip1 and LZAP were co-expressed LZAP localization was altered so that nucleolar LZAP was observed. These findings indicate that co-expression of LZAP and Wip1 results in alternation of LZAP subcellular localization with resultant co-localization of these two proteins to nucleus. Functionally, co-expression of LZAP with Wip1 increased Wip1 content within p38 immunocomplexes which was associated with decreased p38 phosphorylation. Together, our findings indicate LZAP binds and co-localizes with Wip1 and increases activity of Wip1 towards p38. Wip1 may play a common mediator for LZAP activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3063.