Potential Tumor Suppressor Protein Research Articles

Disable 2, A Versatile Tissue Matrix Multifunctional Scaffold Protein with Multifaceted Signaling: Unveiling Role in Breast Cancer for Therapeutic Revolution.

The Disabled-2 (DAB2) protein, found in 80-90% of various tumors, including breast cancer, has been identified as a potential tumor suppressor protein. On the contrary, some hypothesis suggests that DAB2 is associated with the modulation of the Ras/MAPK pathway by endocytosing the Grb/Sos1 signaling complex, which produces oncogenes and chemoresistance to anticancer drugs, leading to increased tumor growth and metastasis. DAB2 has multiple functions in several disorders and is typically under-regulated in several cancers, making it a potential target for treatment of cancer therapy. The primary function of DAB2 is the modulation of transforming growth factor- β (TGF-β) mediated endocytosis, which is involved in several mechanisms of cancer development, including tumor suppression through promoting apoptosis and suppressing cell proliferation. In this review, we will discuss in detail the mechanisms through which DAB2 leads to breast cancer and various advancements in employing DAB2 in the treatment of breast cancer. Additionally, we outlined its role in other diseases. We propose that upregulating DAB2 could be a novel approach to the therapeutics of breast cancer.

RBM10 recruits METTL3 to induce N6-methyladenosine-MALAT1-dependent modification, inhibiting the invasion and migration of NSCLC

AimsPrevious studies have shown that RNA binding motif 10 (RBM10) is a potential tumor suppressor protein that can inhibit proliferation and promote apoptosis of non-small cell lung cancer (NSCLC). Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) plays an important role in promoting the development of lung cancer. Inhibiting its m6A methylation can effectively inhibit the invasion and metastasis of lung cancer. There is concern that RBM10 could affect MALAT1 m6A methylation for the invasion and migration of NSCLC. Main methods and findingsTranswell and wound healing assays showed that RBM10 significantly inhibited the invasion and migration of NSCLC. CLIP-Seq showed that among all RBM10 binding RNAs, MALAT1 had the highest binding peak among all non-coding RNAs. RNA immunoprecipitation verified the direct combination of RBM10 and MALAT1. The rescue experiment confirmed that RBM10 affected the phosphorylation of the PI3K/AKT/mTOR pathway protein as well as the invasion and migration ability by regulating MALAT1. MeRIP-qPCR confirmed that RBM10 could inhibit the MALAT1 m6A methylation level by recruiting Methyltransferase Like 3 (METTL3). SignificanceThe study suggests that RBM10, as an RNA-binding protein, may inhibit the m6A methylation of MALAT1 by recruiting METTL3 and affecting phosphorylation of the downstream PI3K/AKT/mTOR pathway by binding and regulating MALAT1, ultimately affecting the invasion and migration of NSCLC.

Par-4 activation restrains EMT-induced chemoresistance in PDAC by attenuating MDM-2

BackgroundWe recently reported prostate apoptosis response 4 (Par-4), a potential tumor suppressor protein restrains epithelial-mesenchymal transition (EMT) properties and promotes mesenchymal-epithelial transition (MET) in invasive cancer cells by repressing Twist-1 promoter activity. Here, we demonstrate that genetic as well as pharmacological modulation of Par-4 by NGD16 (a small molecule antimetastatic agent), limits EMT-induced chemoresistance in aggressive cancer cells by suppressing MDM-2, a downstream effector of Twist-1. MethodsMatrigel invasion assay, gelatin degradation assay, cell scattering assay, MTT assay and colony formation assay were used to study the proliferation and migration abilities of invasive cancer cells. Immunoblotting, immunocytochemistry, and immunoprecipitation analysis were utilized for determining protein expression and protein-protein interaction. 4T1 aggressive mouse carcinoma model was employed to evaluate tumor growth and lung metastasis. ResultsTreatment of gemcitabine (nucleoside analogue anticancer agent) to pancreatic cancer (Panc-1, MiaPaca-2) and breast cancer (MDA-MB-231) cells amplified MDM-2 expression along with increase in EMT properties. Conversely, NGD16 boosted expression of tumor suppressor Par-4 and inhibited invasion and migration abilities of these cells. Moreover, induction of Par-4 effectively diminished MDM-2 along with pro-EMT markers, whereas, augmented the expression of epithelial markers. Furthermore, siRNA-mediated silencing of Par-4 divulged that NGD16 exerts its EMT inhibitory effects in a Par-4-dependent manner. Mechanistically, Par-4 activation provokes p53 by disrupting MDM-2-p53 interaction, which restored epithelial characteristics in cancer cells. Additionally, partial knockdown of MDM-2 through siRNA pronounced the anti-proliferative and anti-invasive effects of NGD16. Finally, NGD16 efficiently inhibited tumor growth and lung metastasis in mouse mammary carcinoma model without showing any undesirable effects. ConclusionOur findings unveil Par-4 as a key therapeutic target and NGD16 (the pharmacological modulator of Par-4) are potential tools to suppress EMT and associated chemoresistance, which could be exploited clinically for the treatment of aggressive cancers.

PAQR3 protects against oxygen-glucose deprivation/reperfusion-induced injury through the ERK signaling pathway in N2A cells.

Cerebral ischemia-reperfusion injury is pivotal in the development of multiple-subcellular organelle and tissue injury after acute ischemic stroke. Recently, the Golgi apparatus (GA) has been shown to be a key subcellular organelle that plays an important role in neuroprotection against oxygen-glucose deprivation/reperfusion (OGD/R) injury. PAQR3, a scaffold protein exclusively localized in the GA, was originally discovered as a potential tumor suppressor protein. PAQR3 acts as a spatial regulator of Raf-1 that binds Raf-1 and sequesters it to the GA, where it negatively modulates the Ras/Raf/MEK/ERK signaling pathway in tumor models. Studies suggest that suppression of the ERK pathway can alleviate OGD/R-induced cell apoptosis. However, whether PAQR3 has potential effects on ischemic stroke and the underlying mechanism(s) remain unexplored. The current study is the first to show that PAQR3 was significantly downregulated in mouse neuroblastoma (N2A) cells upon OGD/R exposure, both at the mRNA and protein levels. Compared to that in controls, the mRNA level of PAQR3 began to decline at 0h (0h) after reperfusion, while the protein level began to decline at 4h. Furthermore, overexpression of PAQR3 reduced OGD/R-induced apoptosis. The mRNA and protein levels of total ERK1 and ERK2 were unaltered, while activated p-ERK1 and p-ERK2 were decreased in N2A cells transfected with a PAQR3 expression vector after OGD for 4h plus 24h of reperfusion. Collectively, these data indicated that increased PAQR3 expression protected against OGD/R-induced apoptosis possibly by inhibiting the ERK signaling pathway. Therefore, PAQR3 might be a new attractive target in the treatment of OGD/R insult, and the underlying mechanism will pave the way for its potential experimental and clinical application.

Combined Chibby and β-Catenin Predicts Clinical Outcomes in Patients with Hepatocellular Carcinoma.

Chibby is an antagonist of β-catenin and is considered a potential tumor suppressor protein, but the role of Chibby in hepatocellular carcinoma (HCC) has not been characterized. The expression patterns of Chibby and β-catenin in HCC specimens and paired adjacent noncancerous tissues were measured by Western blotting and immunohistochemistry. The correlations between Chibby expression and clinicopathological parameters were analyzed. Then the biological functions of Chibby were analyzed in vitro. The Chibby protein was significantly downexpressed in human primary HCC tissues compared to that in matched adjacent normal liver tissue and is a risk factor for HCC recurrence and shorter survival. Furthermore, we found that in HCC tissues the high expression of β-catenin with low expression of Chibby in the nuclei was an independent predictor for disease-free survival (DFS) (p = 0.012) and overall survival (OS) (p = 0.005). Subsequent genetic manipulation in vitro studies revealed that Chibby knockdown induced the expression of β-catenin and C-myc, cyclin D1 protein, which promoted cell proliferation and invasiveness. In contrast, overexpression of Chibby decreased β-catenin expression and inhibited the cell proliferation and invasiveness. Our results suggest that low expression of Chibby was associated with advanced tumor-node-metastasis (TNM) stage and poor differentiation. Furthermore, the combination of Chibby and β-catenin can predict poor prognosis in patients with HCC. Chibby inhibited HCC progression by blocking β-catenin signaling in vitro. Chibby is a biomarker and may be a potential therapeutic target for HCC.

UBIAD1 protects against oxygen-glucose deprivation/reperfusion-induced multiple subcellular organelles injury through PI3K/AKT pathway in N2A cells.

Cerebral ischemia/reperfusion-induced injury plays a significant role in the development of multi-subcellular organelles injury after ischemic stroke. UBIAD1 was discovered originally as a potential tumor suppressor protein. Recently, analysis of UBIAD1 has indicated it is a prenyltransferase enzyme for both non-mitochondrial CoQ10 and vitamin K2 production. Further, UBIAD1 has been localized to multiple subcellular organelles. Particularly, UBIAD1 plays an important role in the regulation of oxidative stress, apoptosis and cell proliferation, cholesterol and lipid metabolism, which was closely associated with the cerebral ischemic/reperfusion mechanism. However, the mechanism underlying effects of UBIAD1 on cerebral ischemia/reperfusion-induced injury remains largely unknown. We aimed to investigate the effects of UBIAD1 on ischemia/reperfusion-induced multiple subcellular organelles injury in vitro, mouse N2A cells were subjected to a classical oxygen-glucose deprivation and reperfusion (OGD/R) insult. The expression of UBIAD1 was reduced in mouse N2A cells after OGD/R. UBIAD1 exhibits multi-subcellular organelles co-localization in N2a cells, including in the mitochondria, endoplasmic reticulum, and Golgi apparatus. The over-expression of UBIAD1 significantly protects against OGD/R-induced cell death. UBIAD1 over-expression also attenuated OGD/R-induced mitochondrial fragmentation and dysfunction and mediated the level of apoptosis-associated protein. Moreover, we observed that the over-expression of UBIAD1 ameliorated OGD/R-induced fragmentation and reduced the level of oxidative stress-related protein expression in both the endoplasmic reticulum and Golgi apparatus. Besides, the neuroprotective effect of UBIAD1 was correlated with the PI3K/AKT pathway, which was demonstrated using the PI3K inhibitor LY294002 and perifosion. Collectively, these findings identified that UBIAD1 protects against OGD/R-induced multiple subcellular organelles injury through PI3K/AKT Pathway.

S6K1 phosphorylation-dependent degradation of Mxi1 by β-Trcp ubiquitin ligase promotes Myc activation and radioresistance in lung cancer.

Rationale: Mxi1 is regarded as a potential tumor suppressor protein that antagonizes the transcriptional activity of proto-oncogene Myc. However, the clinical significances and underlying mechanisms by which Mxi1 is regulated in lung cancer remain poorly understood.Methods: Mass spectrometry analysis and immunoprecipitation assay were utilized to detect the protein-protein interaction. The phosphorylation of Mxi1 was evaluated by in vitro kinase assays. Poly-ubiquitination of Mxi1 was examined by in vivo ubiquitination assay. Lung cancer cells stably expressing wild-type Mxi1 or Mxi1-S160A were used for functional analyses. The expression levels of Mxi1 and S6K1 were determined by immunohistochemistry in lung cancer tissues and adjacent normal lung tissues.Results: We found that Mxi1 is downregulated and correlated with poor prognosis in lung cancer. Using tandem affinity purification technology, we provided evidence that β-Trcp E3 ubiquitin ligase interacts with and promotes the ubiquitination and degradation of Mxi1. Furthermore, we demonstrated that Mxi1 is phosphorylated at S160 site by the protein kinase S6K1 and subsequently degraded via the ubiquitin ligase β-Trcp. Moreover, a phosphorylation mutant form of Mxi1 (Mxi1-S160A), which cannot be degraded by S6K1 and β-Trcp, is much more stable and efficient in suppressing the transcriptional activity of Myc and radioresistance in lung cancer cells. More importantly, a strong inverse correlation between S6K1 and Mxi1 expression was observed in human lung cancer tissues.Conclusion: Our findings not only establish a crosstalk between the mTOR/S6K1 signaling pathway and Myc activation, but also suggest that targeting S6K1/Mxi1 pathway is a promising therapeutic strategy for the treatment of lung cancer.

The PTPN14 Tumor Suppressor Is a Degradation Target of Human Papillomavirus E7.

Activation of signaling pathways ensuring cell growth is essential for the proliferative competence of human papillomavirus (HPV)-infected cells. Tyrosine kinases and phosphatases are key regulators of cellular growth control pathways. A recently identified potential cellular target of HPV E7 is the cytoplasmic protein tyrosine phosphatase PTPN14, which is a potential tumor suppressor and is linked to the control of the Hippo and Wnt/beta-catenin signaling pathways. In this study, we show that the E7 proteins of both high-risk and low-risk mucosal HPV types can interact with PTPN14. This interaction is independent of retinoblastoma protein (pRb) and involves residues in the carboxy-terminal region of E7. We also show that high-risk E7 induces proteasome-mediated degradation of PTPN14 in cells derived from cervical tumors. This degradation appears to be independent of cullin-1 or cullin-2 but most likely involves the UBR4/p600 ubiquitin ligase. The degree to which E7 downregulates PTPN14 would suggest that this interaction is important for the viral life cycle and potentially also for the development of malignancy. In support of this we find that overexpression of PTPN14 decreases the ability of HPV-16 E7 to cooperate with activated EJ-ras in primary cell transformation assays.IMPORTANCE This study links HPV E7 to the deregulation of protein tyrosine phosphatase signaling pathways. PTPN14 is classified as a potential tumor suppressor protein, and here we show that it is very susceptible to HPV E7-induced proteasome-mediated degradation. Intriguingly, this appears to use a mechanism that is different from that employed by E7 to target pRb. Therefore, this study has important implications for our understanding of the molecular basis for E7 function and also sheds important light on the potential role of PTPN14 as a tumor suppressor.

Molecular characterization and function of the Prohibitin2 gene in Litopenaeus vannamei responses to Vibrio alginolyticus

Prohibitin2 (PHB2), a potential tumor suppressor protein, plays important roles in inhibition of cell cycle progression, transcriptional regulation, apoptosis and the mitochondrial respiratory chain. To explore its potential roles in crustaceans' immune responses we have identified and characterized LvPHB2, a 891 bp gene encoding a 297 amino acids protein in the shrimp Litopenaeus vannamei. Expression analyses showed that LvPHB2 is expressed in all examined tissues, and largely present in cytoplasm, correlating with its known anti-oxidation function in mitochondria. Luciferase reporter assays showed that over-expression of LvPHB2 could activate the p53 pathway, indicating that it might participate in apoptosis regulation. Quantitative real-time PCR revealed that infection with Vibrio alginolyticus induces its up-regulation in hepatopancreas. Moreover, RNAi knock-down of LvPHB2 in vivo raises mortality rates of L. vannamei infected by V. alginolyticus, and affects expression of STAT3, Caspase3 and p53 genes. We found significantly higher reactive oxygen species production, DNA damage and apoptosis rates in LvPHB2-silenced shrimp challenged with V. alginolyticus than in controls injected with a Green Fluorescent Protein-silencing construct. Our results suggest that LvPHB2 plays a vital role in shrimp responses to V. alginolyticus infection through its participation in regulation of oxidants and apoptosis.

The interaction of botanicals and AMPK signaling in the inhibition of castrate-resistant prostate cancer

Materials and methods CWR22Rv1 cells (a castrate-resistant human PCa cell line) were treated with a polyherbal mixture of botanicals (PHBs) consisting of 10 herbal extracts [1] and we investigated whether inhibition of cell proliferation was linked to the activity of 5’adenosine monophosphate (AMP)activated protein kinase (AMPK), a potential tumor suppressor protein that functions as a central energy sensor within the cell. In addition, we investigated whether this inhibition was also the result of synergy among the bioactives as determined by the Chou-Talalay method [2].

The TERE1 protein interacts with mitochondrial TBL2: Regulation of trans‐membrane potential, ROS/RNS and SXR target genes

We originally discovered TERE1 as a potential tumor suppressor protein based upon reduced expression in bladder and prostate cancer specimens and growth inhibition of tumor cell lines/xenografts upon ectopic expression. Analysis of TERE1 (aka UBIAD1) has shown it is a prenyltransferase enzyme in the natural bio-synthetic pathways for both vitamin K-2 and COQ10 production and exhibits multiple subcellular localizations including mitochondria, endoplasmic reticulum, and golgi. Vitamin K-2 is involved in mitochondrial electron transport, SXR nuclear hormone receptor signaling and redox cycling: together these functions may form the basis for tumor suppressor function. To gain further insight into mechanisms of growth suppression and enzymatic regulation of TERE1 we isolated TERE1 associated proteins and identified the WD40 repeat, mitochondrial protein TBL2. We examined whether disease specific mutations in TERE1 affected interactions with TBL2 and the role of each protein in altering mitochondrial function, ROS/RNS production and SXR target gene regulation. Biochemical binding assays demonstrated a direct, high affinity interaction between TERE1 and TBL2 proteins; TERE1 was localized to both mitochondrial and non-mitochondrial membranes whereas TBL2 was predominantly mitochondrial; multiple independent single amino acid substitutions in TERE1 which cause a human hereditary corneal disease reduced binding to TBL2 strongly suggesting the relevance of this interaction. Ectopic TERE1 expression elevated mitochondrial trans-membrane potential, oxidative stress, NO production, and activated SXR targets. A TERE1-TBL2 complex likely functions in oxidative/nitrosative stress, lipid metabolism, and SXR signaling pathways in its role as a tumor suppressor.

A Novel Mathematical Model of Targeted Cancer Therapy along p53 Proteasomal Degradation Pathways

Overzealous MDM2-mediated ubiquitination of p53 characterizesand sustains over 50% of all human cancers. Successful targeted cancer therapyhinges on a thorough understanding of the ubiquitination process.Unfortunately, current steady-state enzyme kinetic models fail to accuratelydescribe the ubiquitin-proteasome system, due to the assumption that the enzyme(E3 ligase) is expressed at infinitesimally smaller concentrations than thesubstrates (E2 conjugase and target protein). This limitation of the quasi-steadystate assumption prohibits the use of steady-state models when analyzing thecancerous consequences of extreme ubiquitin-ligase overexpression. This paperderives a novel non-steady-state mathematical model of ternary complex enzymekinetics, which can be used to simulate the behavioral response of theubiquitin-proteasome system to specific variations in the cellularconcentrations of targeted the E2 conjugase, E3 ligase, and target protein.Computer simulations of the model were used to study the effects of E2D3 andMDM2 concentrations on the rates of p53 ubiquitination at differenttemperatures. At each temperature, it was observed that the ubiquitin-ligaseMDM2 accelerates the dangerous degradation process, while the ubiquitinconjugating-enzyme E2D3 inhibits it. It was also discovered that E2D3 is a moreeffective inhibitor of p53 ubiquitination at higher body temperatures, whereas MDM2 is then a less-effective catalyst. The derived model therefore suggests MDM2 as a prospective target for cancer therapy. In addition, the findings of this project propose recombinant E2D3 as a new promising protein-basedanticancer drug and a potential tumor suppressor protein. The mathematical model successfully reproduced the experimentally observed p53-MDM2 interaction. Further in vivo experimentation is necessary for additional validation of thecomputational results. The derived model can suggest new therapeutic solutions for decreasing the harmful effects of many human cancers, bacterial infections,and inflammatory diseases (such as rheumatoid arthritis) characterized by an overzealous ubiquitin-proteasome system.

Botanicals inhibit castrate‐resistant prostate cancer via mechanisms involving AMPK, inhibition of lipogenesis, mTOR‐mediated proliferation, and histone modification

Reprogramming of metabolic pathways and abnormal acetylation of DNA are two common features of cancer cells, including prostate cancer (PCa). Our results show that polyherbal mixtures of botanicals, like Zyflamend®, can modify lipogenesis in CWR22Rv1 cells (a castrate‐resistant human PCa cell line) by enhancing the activity of 5'adenosine monophosphate (AMP)‐ activated protein kinase (AMPK), a potential tumor suppressor protein that functions as a central energy sensor within the cell. By increasing the phosphorylation of AMPK, Zyflamend® decreases de novo lipid biosynthesis by decreasing the activity of acetyl‐CoA carboxylase (ACC) and the expression of FASN and SREBP1c. Also via AMPK, Zyflamend® inhibits the activity of mTORC1 complex by directly phosphorylating Raptor, decreasing the activity of P70 (S6K, the marker for protein synthesis). All of these effects can be mimicked by treatment of AICAR (activator of AMPK). Furthermore, Zyflamend® increases the acetylation of DNA via mechanisms that appear to involve ATP‐citrate lyase (ACLY). Thus, our results suggest that botanicals, via AMPK, inhibit castrate‐resistant prostate cancer via mechanisms that involve lipid metabolism that may reshuttle acetyl CoAs for histone acetylation, and inhibition of mTOR mediated cell proliferation.Grant Funding Source: This research was supported in part by Tennessee Agricultural Experiment Station

Medicinal herbs inhibit castrate‐resistant prostate cancer via mechanisms involving AMPK

Prostate cancer (PCa) is the second most common type of cancer among males in the USA. Preliminary data has shown that Zyflamend®, a mixture containing extracts of 10 medicinal herbs, inhibited growth of CWR22Rv1 cells (a castrate‐resistant human PCa cell line), by down‐regulating the expression of key mediators of lipogenesis, including SREBP‐1c, fatty acid synthase and HMG CoA reductase. Down‐regulation of lipogenic genes is associated with tumor growth inhibition. These same pathways are negatively regulated by 5’adenosine monophosphate (AMP)‐activated protein kinase (AMPK), a potential tumor suppressor protein that functions as a central energy sensor within the cell. Proliferation of CWR22Rv1 cells was enhanced following chemical inhibition of AMPK by Compound C and inhibited with constitutive over‐expression of AMPK following adenoviral infection. These effects are believed to be mediated by AMPKs phosphorylation of mTOR (mammalian target of rapamycin), a known activator of cell proliferation and a downstream target of AMPK. Similarly, Zyflamend® up‐regulates AMPK activation, inhibits cell growth, and coordinately increases the phosphorylation of down‐stream targets of AMPK, mTOR and acetyl CoA carboxylase (negatively effecting fatty acid synthesis). Thus, our results suggest that the polyherbal mixture Zyflamend® inhibits castrate‐resistant PCa through mechanisms involving AMPK.

Reduced Expression of RNA Binding Protein CELF2, a Putative Tumor Suppressor Gene in Colon Cancer

Colon cancer is the third leading cause of cancer death in both men and women in the United States. Every year, 160000 cases of colorectal cancer are diagnosed, and 57000 patients die. CUGBP, Elav-like family member 2 (CELF2) is an RNA binding protein that modulates various posttranscriptional events including RNA splicing, shuttling, editing, stability and translation. Previous studies have demonstrated that CELF2 expression is low in colon cancer cells. Furthermore, ectopic overexpression of CELF2 induces cells to undergo death by mitotic catastrophe. Based on the above observations, we hypothesized that CELF2 expression might be reduced during neoplastic transformation of colon cells. Forty human colon cancer tissues along with 10 uninvolved normal colon tissues from cancer patients were utilized for immunohistochemical analysis of CELF2 expression. We have observed that CELF2 levels are reduced in colon tumor tissues when compared to the normal intestinal tissues. The data set suggests that RNA binding protein CELF2 could be a potential tumor suppressor protein. CELF2 was predominantly nuclear in normal cells, while the cancer tissues had diffused cytoplasmic staining. CELF2 expression is consistently reduced during neoplastic transformation suggesting that it might play a crucial role in tumor initiation and progression.

DRO1 sensitizes colorectal cancer cells to receptor-mediated apoptosis

The molecule DRO1 (down‑regulated by oncogenes1) is a potential tumor suppressor protein that is frequently down‑regulated in primary colorectal cancers and colorectal cancer cell lines. Although the mechanism of DRO1 action has yet to be elucidated, previous data have suggested that DRO1 interferes with tumor growth by sensitizing cells to apoptosis. The effect of DRO1 expression on receptor-, mitochondrial- and endoplasmic reticulum‑mediated apoptosis in colorectal cancer cell lines was analyzed in this study, following the generation of DLD1/DRO1 and HCT116/DRO1 cell lines. Cells were cultured, and then analyzed using flow cytometry. DRO1 was found to sensitize cells to receptor-mediated apoptosis by promoting the activation of components of the death-inducing signaling complex (DISC).

Disabled-2 and Axin are concurrently colocalized and underexpressed in lung cancers

Disabled-2 expression is reduced in many cancers, suggesting that it is a potential tumor suppressor protein. To elucidate the role of Disabled-2 in lung cancer, we examined the expression of Disabled-2, the Disabled-2-binding protein Axin, and DNA methyltransferase-1 in lung cancer tissues and corresponding normal lung tissues using immunohistochemistry and Western blots. We also determined the subcellular localization of Axin and Disabled-2 in A549 cells using confocal immunofluorescence. Disabled-2 expression was significantly reduced in lung cancers and was colocalized and coexpressed with Axin (correlation coefficient = 0.321, P < .001 for cytoplasmic expression; correlation coefficient = 0.393, P < .001 for nuclear expression). Reduced nuclear Disabled-2 expression was correlated with the differentiation (P = .048) and TNM stage (P = .048) of the tumor. The cytoplasmic expression of Axin was also correlated with differentiation (P = .042), whereas the nuclear expression of Axin was correlated with both histologic type (P = .001) and TNM stage (P < .001) of lung cancers. Expression of DNA methyltransferase-1 was negatively correlated with the cytoplasmic expression of Axin (correlation coefficient = -0.244, P = .012) but positively correlated with thehistologic type (P = .004), differentiation (P = .036), TNM stage (P = .044), and lymphatic metastasis (P = .011). Expressions of Disabled-2 and Axin were concurrently reduced and correlated with the malignant phenotype of lung cancers. Enhanced expression of DNA methyltransferase-1 correlated with the reduced expression of Axin and could be a marker for lung cancer development and progression.

Identification of nuclear substrates of Akt/PKB by functional proteomics: prohibitin 2 is a target of Akt phosphorylation in human promyelocytic leukemia cells

The serine/threonine protein kinase Akt is a major signal transducer of the phosphoinositide 3-kinase (PI 3-K) pathway in all cells and tissues and plays a pivotal role in the maintenance of cellular processes including cell growth, proliferation, survival, metabolism and development of many malignancies including acute myeloid leukemia. The frequent aberrant activation of the PI 3-K/Akt pathway in human cancer has made it an attractive therapeutic target. Therefore, the study of effector proteins downstream of Akt could clarify the role of Akt in the development of myeloid leukemia. Although both localization and activity of Akt in the nuclear compartment are well documented, most Akt substrates identified so far are located in the cytoplasm, while nuclear substrates have remained elusive. In this study, we applied a proteomic approach to identify novel Akt substrates by using an antibody that recognized a consensus motif phosphorylated by Akt (K/RXK/RXXS/T) when phosphorylated on S/T (anti-phospho-Akt substrate antibody). NB4 cells were treated with ATRA, and the putative Akt substrate proteins were isolated by immunoprecipitation with the anti-phospho-Akt substrate antibody. The proteins were separated on SDS-PAGE and analyzed by ESI-Q-TOF mass spectrometry. This analysis indicated prohibitin 2, a potential tumor suppressor protein with potent transcriptional functions in the nucleus, as a putative substrate of Akt in the nucleus of NB4 cells. The putative Akt-Prohibitin 2 interaction was validated by reverse in vivo immunoprecipitation from nuclear protein of NB4 cells. In vitro phosphorylation of endogenous prohibitin 2 by recombinant Akt further validated this result. Vandermoere F, El Yazidi-Belkoura I, Demont Y, Slomianny C, Antol J, Lemoine J, Hondermarck H. Proteomics exploration reveals that actin is a signaling target of the kinase Akt. Mol Cell Proteomics. 2007 Jan;6(1):114-24. Kasashima K, Ohta E, Kagawa Y, Endo H. Mitochondrial functions and estrogen receptor-dependent nuclear translocation of pleiotropic human prohibitin 2. J Biol Chem. 2006 Nov 24;281(47):36401-10.

Abstract 4071: Prohibitin peptides induce apoptosis in cancer cells and inhibit angiogenesis

Abstract Prohibitin is a potential tumor suppressor protein that can repress E2F-mediated transcription and arrest cell proliferation. E2F1 binding domain of prohibitin is a coiled-coil structure and prohibitin functions through this domain to repress E2F1 transcription activity by recruiting HDAC1. The coiled- coil domain (175-217aa) by itself exhibited growth suppressive properties and showed a reduction in colony formation when transfected into breast cancer cells, T47D cells. It was shown that a synthetic peptide (42aa), designated as H1-6, corresponding to the coiled- coil domain induced apoptosis in four different human cell lines; MCF7, HAEC, BL18 and WI38 cells. Hence we decided to synthesize peptides of 14-mer (H1-2, H3-4, H5-6) and 7-mer (H2, H5) from the coiled coil domain of prohibitin and study their effect on cells. We found that each one of the prohibitin peptides (20μM) induced a marked increase in apoptosis in a panel of breast, lung and prostate cancer cell lines when compared to the control peptide (CP1). Interestingly, the peptides had no effect on breast epithelial cells, MCF10A. Treatment of MDA-MB-231 cells with 20μM of each prohibitin peptide individually reduced the invasive capability of these cells when compared to the control peptide. We also investigated their effect on angiogenesis in HUVEC cells and found that these peptides could significantly inhibit angiogenesis in vitro. In conclusion, these results suggest that these peptides could be of significant therapeutic value. 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 4071.

Plexin C1, A Receptor for Semaphorin 7A, Inactivates Cofilin and Is a Potential Tumor Suppressor for Melanoma Progression

Melanocytes are progenitor cells for melanoma, which arises through step-wise progression from dysplastic to invasive, to metastatic tumor. Our previous data showed that semaphorin 7A (Sema7A), a protein involved in axon guidance, stimulates melanocyte adhesion and dendricity through opposing actions of beta1-integrin and Plexin C1 receptors. We now show that Plexin C1 is diminished or absent in human melanoma cell lines; analysis of tissue microarrays of nevi, melanoma, and metastatic melanoma showed a decrease in Plexin C1 expression in metastatic melanoma, and an inverse correlation of Plexin C1 expression with depth of invasion. We examined the signaling intermediates of Sema7A and downstream targets of Plexin C1 in human melanocytes. Sema7A activated mitogen-activated protein kinase and inactivated cofilin, an actin-binding protein involved in cell migration. When Plexin C1 expression was silenced, Sema7A failed to phosphorylate cofilin, indicating that cofilin is downstream of Plexin C1. Further, Lim kinase II, a protein that phosphorylates cofilin, is upregulated by Sema7A in a Plexin C1-dependent manner. These data identify Plexin C1 as a potential tumor suppressor protein in melanoma progression, and suggest that loss of Plexin C1 expression may promote melanoma invasion and metastasis through loss of inhibitory signaling on cofilin activation.