Therapeutic targeting of the TPX2/TTK network in colorectal cancer.

BACKGROUND: While the increased screening, changes in lifestyle, and recent advances in treatment regimen have decreased colorectal cancer (CRC) mortality, metastatic disease and recurrence remains a major clinical challenge. In the era of precision medicine, the identification of actionable novel therapeutic targets could ultimately offer an alternative treatment strategy for CRC. METHODS: RNA-Seq was conducted using the illumina platform, while bioinformatics analyses were conducted using CLC genomics workbench and iDEP.951. Colony forming unit, flow cytometry, and fluorescent microscopy were used to assess cell proliferation, cell cycle distribution, and cell death, respectively. The growth potential of CRC cells under 3-dimensional (3D) conditions was assessed using Matrigel. STRING database (v11.5) and Ingenuity Pathway Analysis (IPA) tool were used for network and pathway analyses. CRISPR-Cas9 perturbational effects database was used to identify potential therapeutic targets for CRC, through integration with gene-drug interaction database. Structural modeling and molecular docking were used to assess the interaction between candidate drugs and their targets. RESULTS: In the current study, we investigated the therapeutic potential of targeting TPX2, TTK, DDX39A, and LRP8, commonly upregulated genes in CRC identified through differential expression analysis in CRC and adjacent non-cancerous tissue. Targeted depletion of TPX2 and TTK impaired CRC proliferation, cell cycle progression, and organoid formation under 3D culture conditions, while suppression of DDX39A and LRP8 had modest effects on CRC colony formation. Differential expression analysis and bioinformatics on TPX2 and TTK-deficient cells identified cell cycle regulation as the hallmark associated with loss of TPX2 and TTK. Elevated expression of TPX2 and TTK correlated with an oncogenic state in tumor tissue from patients with colon adenocarcinoma, thus corroborating an oncogenic role for the TPX2/TTK network in the pathogenesis of CRC. Gene set enrichment and pathway analysis of TPX2high/TTKhigh CRC identified numerous additional gene targets as integral components of the TPX2/TTK network. Integration of TPX2/TTK enriched network with CRISPR-Cas9 functional screen data identified numerous novel dependencies for CRC. Additionally, gene-drug interaction analysis identified several druggable gene targets enriched in the TPX2/TTK network, including AURKA, TOP2A, CDK1, BIRC5, and many others. CONCLUSIONS: Our data has implicated an essential role for TPX2 and TTK in CRC pathogenesis and identified numerous potential therapeutic targets and their drug interactions, suggesting their potential clinical use as a novel therapeutic strategy for patients with CRC. Video Abstract.

Identification of a potential DNA methyltransferase (DNMT) inhibitor.

DNA methyltransferases (DNMTs) play an important role in the epigenetic regulation of gene expression through the methylation of DNA. Since hypermethylation and consequent suppression of tumor suppressor genes are associated with cancer development and progression, DNA hypomethylating agents (HMAs) such as DNMT inhibitors have been proposed for cancer therapy. Two nucleoside analogues approved for the treatment of hematological cancers, decitabine and azacytidine, have poor pharmacokinetic properties, and hence there is a critical need for identifying novel HMAs. Virtual screening of a library of ∼40,000 compounds from the ZINC database, followed by molecular docking of 4,000 compounds having potential druggable properties with DNMT1, DNMT3A and DNMT3B were performed. One unique inhibitor (ZINC167686681) was identified that successfully passed through the Lipinski Rule of 5, geometry constraints as well as ADME/Tox filters and having strong binding energy for DNMTs. Further, molecular dynamics simulations of the docked complexes showed detailed structural features critical for its binding with the DNMTs and the stability of their interaction. Our study identified a compound with potential druggable properties and predicted to bind and inhibit DNMTs. Further investigations involving cellular and animal models of ZINC167686681 will help in potentially taking it into clinical trials for the treatment of cancers.Communicated by Ramaswamy H. Sarma.

Artificial Intelligence for the Prediction and Early Diagnosis of Pancreatic Cancer: Scoping Review.

BACKGROUND: Pancreatic cancer is the 12th most common cancer worldwide, with an overall survival rate of 4.9%. Early diagnosis of pancreatic cancer is essential for timely treatment and survival. Artificial intelligence (AI) provides advanced models and algorithms for better diagnosis of pancreatic cancer. OBJECTIVE: This study aims to explore AI models used for the prediction and early diagnosis of pancreatic cancers as reported in the literature. METHODS: A scoping review was conducted and reported in line with the PRISMA-ScR (Preferred Reporting Items for Systematic Reviews and Meta-Analyses Extension for Scoping Reviews) guidelines. PubMed, Google Scholar, Science Direct, BioRXiv, and MedRxiv were explored to identify relevant articles. Study selection and data extraction were independently conducted by 2 reviewers. Data extracted from the included studies were synthesized narratively. RESULTS: Of the 1185 publications, 30 studies were included in the scoping review. The included articles reported the use of AI for 6 different purposes. Of these included articles, AI techniques were mostly used for the diagnosis of pancreatic cancer (14/30, 47%). Radiological images (14/30, 47%) were the most frequently used data in the included articles. Most of the included articles used data sets with a size of <1000 samples (11/30, 37%). Deep learning models were the most prominent branch of AI used for pancreatic cancer diagnosis in the studies, and the convolutional neural network was the most used algorithm (18/30, 60%). Six validation approaches were used in the included studies, of which the most frequently used approaches were k-fold cross-validation (10/30, 33%) and external validation (10/30, 33%). A higher level of accuracy (99%) was found in studies that used support vector machine, decision trees, and k-means clustering algorithms. CONCLUSIONS: This review presents an overview of studies based on AI models and algorithms used to predict and diagnose pancreatic cancer patients. AI is expected to play a vital role in advancing pancreatic cancer prediction and diagnosis. Further research is required to provide data that support clinical decisions in health care.

First Whole Transcriptome RNAseq on CHD8 Haploinsufficient Patient and Meta-Analyses Across Cellular Models Uncovers Likely Key Pathophysiological Target Genes.

In 2019, we confirmed that the haploinsufficiency of CHD8 does indeed cause the novel syndromic neurodevelopmental disease we first discovered a dozen years before. Here, we report the first whole transcriptome RNAseq gene expression profiling for a patient with this new syndrome, as a preliminary exploration of potential pathophysiological mechanisms. We compared our patient transcriptome profile with that of all publicly available RNAseq datasets from human cellular models including neuronal progenitor cells, neurons and organoids. We compared differential gene expression profiles overall and conducted phenotype-informed data filtration based on the characteristic syndrome presentation. We found that concordance among differential gene expression profiles was poor across all datasets. Nevertheless, remarkably, we show that the patient blood differential gene expression profile most resembled that of the neuronal cell model, a finding that encourages further transcriptome profiling using patient blood samples. In addition, our custom phenotype-informed analyses yielded important, differentially expressed syndrome pathophysiology target genes. Finally, we note that genes dysregulated due to CHD8 heterozygous deletion are linked to known neurological as well as oncological pathways.

The clinical and genetic characteristics of permanent neonatal diabetes (PNDM) in the state of Qatar.

BACKGROUND: Neonatal diabetes mellitus (NDM) is a rare condition that occurs within the first six months of life. Permanent NDM (PNDM) is caused by mutations in specific genes that are known for their expression at early and/or late stages of pancreatic beta- cell development, and are either involved in beta-cell survival, insulin processing, regulation, and release. The native population in Qatar continues to practice consanguineous marriages that lead to a high level of homozygosity. To our knowledge, there is no previous report on the genomics of NDM among the Qatari population. The aims of the current study are to identify patients with NDM diagnosed between 2001 and 2016, and examine their clinical and genetic characteristics. METHODS: To calculate the incidence of PNDM, all patients with PNDM diagnosed between 2001 and 2016 were compared to the total number of live births over the 16-year-period. Whole Genome Sequencing (WGS) was used to investigate the genetic etiology in the PNDM cohort. RESULTS: PNDM was diagnosed in nine (n = 9) patients with an estimated incidence rate of 1:22,938 live births among the indigenous Qatari. Seven different mutations in six genes (PTF1A, GCK, SLC2A2, EIF2AK3, INS, and HNF1B) were identified. In the majority of cases, the genetic etiology was part of a previously identified autosomal recessive disorder. Two novel de novo mutations were identified in INS and HNF1B. CONCLUSION: Qatar has the second highest reported incidence of PNDM worldwide. A majority of PNDM cases present as rare familial autosomal recessive disorders. Pancreas associated transcription factor 1a (PTF1A) enhancer deletions are the most common cause of PNDM in Qatar, with only a few previous cases reported in the literature.

Monoallelic expression in melanoma.

BACKGROUND: Monoallelic expression (MAE) is a frequent genomic phenomenon in normal tissues, however its role in cancer is yet to be fully understood. MAE is defined as the expression of a gene that is restricted to one allele in the presence of a diploid heterozygous genome. Constitutive MAE occurs for imprinted genes, odorant receptors and random X inactivation. Several studies in normal tissues have showed MAE in approximately 5-20% of the cases. However, little information exists on the MAE rate in cancer. In this study we assessed the presence and rate of MAE in melanoma. The genetic basis of melanoma has been studied in depth over the past decades, leading to the identification of mutations/genetic alterations responsible for melanoma development. METHODS: To examine the role of MAE in melanoma we used 15 melanoma cell lines and compared their RNA-seq data with genotyping data obtained by the parental TIL (tumor infiltrating lymphocytes). Genotyping was performed using the Illumina HumanOmni1 beadchip. The RNA-seq library preparation and sequencing was performed using the Illumina TruSeq Stranded Total RNA Human Kit and subsequently sequenced using a HiSeq 2500 according to manufacturer's guidelines. By comparing genotyping data with RNA-seq data, we identified SNPs in which DNA genotypes were heterozygous and corresponding RNA genotypes were homozygous. All homozygous DNA genotypes were removed prior to the analysis. To confirm the validity to detect MAE, we examined heterozygous DNA genotypes from X chromosome of female samples as well as for imprinted and olfactory receptor genes and confirmed MAE. RESULTS: MAE was detected in all 15 cell lines although to a different rate. When looking at the B-allele frequencies we found a preferential pattern of complete monoallelic expression rather then differential monoallelic expression across the 15 melanoma cell lines. As some samples showed high differences in the homozygous and heterozygous call rate, we looked at the single chromosomes and showed that MAE may be explained by underlying large copy number imbalances in some instances. Interestingly these regions included genes known to play a role in melanoma initiation and progression. Nevertheless, some chromosome regions showed MAE without CN imbalances suggesting that additional mechanisms (including epigenetic silencing) may explain MAE in melanoma. CONCLUSION: The biological implications of MAE are yet to be realized. Nevertheless, our findings suggest that MAE is a common phenomenon in melanoma cell lines. Further analyses are currently being undertaken to evaluate whether MAE is gene/pathway specific and to understand whether MAE can be employed by cancers to achieve a more aggressive phenotype.

Integrated omics profiling reveals novel patterns of epigenetic programming in cancer-associated myofibroblasts.

There is increasing evidence that stromal myofibroblasts play a key role in the tumour development however, the mechanisms by which they become reprogrammed to assist in cancer progression remain unclear. As cultured cancer-associated myofibroblasts (CAMs) retain an ability to enhance the proliferation and migration of cancer cells in vitro, it is possible that epigenetic reprogramming of CAMs within the tumour microenvironment may confer long-term pro-tumourigenic changes in gene expression. This study reports the first comparative multi-omics analysis of cancer-related changes in gene expression and DNA methylation in primary myofibroblasts derived from gastric and oesophageal tumours. In addition, we identify novel CAM-specific DNA methylation signatures, which are not observed in patient-matched adjacent tissue-derived myofibroblasts, or corresponding normal tissue-derived myofibroblasts. Analysis of correlated changes in DNA methylation and gene expression shows that different patterns of gene-specific DNA methylation have the potential to confer pro-tumourigenic changes in metabolism, cell signalling and differential responses to hypoxia. These molecular signatures provide new insights into potential mechanisms of stromal reprogramming in gastric and oesophageal cancer, while also providing a new resource to facilitate biomarker identification and future hypothesis-driven studies into mechanisms of stromal reprogramming and tumour progression in solid tumours.

The Unfolded Protein Response: A Novel Therapeutic Target for Poor Prognostic BRAF Mutant Colorectal Cancer.

BRAFV600E mutations occur in ∼10% of colorectal cancer cases, are associated with poor survival, and have limited responses to BRAF/MEK inhibition with or without EGFR inhibition. There is an unmet need to understand the biology of poor prognostic BRAFMT colorectal cancer. We have used differential gene expression and pathway analyses of untreated stage II and stage III BRAFMT (discovery set: n = 31; validation set: n = 26) colorectal cancer, and an siRNA screen to characterize the biology underpinning the BRAFMT subgroup with poorest outcome. These analyses identified the unfolded protein response (UPR) as a novel and druggable pathway associated with the BRAFMT colorectal cancer subgroup with poorest outcome. We also found that oncogenic BRAF drives endoplasmic reticulum (ER) stress and UPR pathway activation through MEK/ERK. Furthermore, inhibition of GRP78, the master regulator of the UPR, using siRNA or small molecule inhibition, resulted in acute ER stress and apoptosis, in particular in BRAFMT colorectal cancer cells. In addition, dual targeting of protein degradation using combined Carfilzomib (proteasome inhibitor) and ACY-1215 (HDAC6-selective inhibitor) treatment resulted in marked accumulation of protein aggregates, acute ER stress, apoptosis, and therapeutic efficacy in BRAFMT in vitro and xenograft models. Mechanistically, we found that the apoptosis following combined Carfilzomib/ACY-1215 treatment is mediated through increased CHOP expression. Taken together, our findings indicate that oncogenic BRAF induces chronic ER stress and that inducers of acute ER stress could be a novel treatment strategy for poor prognostic BRAFMT colorectal cancer. Mol Cancer Ther; 17(6); 1280-90. ©2018 AACR.

Gene expression profiling in bladder cancer identifies potential therapeutic targets.

Despite advances in management, bladder cancer remains a major cause of cancer related complications. Characterisation of gene expression patterns in bladder cancer allows the identification of pathways involved in its pathogenesis, and may stimulate the development of novel therapies targeting these pathways. Between 2004 and 2005, cystoscopic bladder biopsies were obtained from 19 patients and 11 controls. These were subjected to whole transcript-based microarray analysis. Unsupervised hierarchical clustering was used to identify samples with similar expression profiles. Hypergeometric analysis was used to identify canonical pathways and curated networks having statistically significant enrichment of differentially expressed genes. Osteopontin (OPN) expression was validated by immunohistochemistry. Hierarchical clustering defined signatures, which differentiated between cancer and healthy tissue, muscle-invasive or non-muscle invasive cancer and healthy tissue, grade 1 and grade 3. Pathways associated with cell cycle and proliferation were markedly upregulated in muscle-invasive and grade 3 cancers. Genes associated with the classical complement pathway were downregulated in non-muscle invasive cancer. Osteopontin was markedly overexpressed in invasive cancer compared to healthy tissue. The present study contributes to a growing body of work on gene expression signatures in bladder cancer. The data support an important role for osteopontin in bladder cancer, and identify several pathways worthy of further investigation.

Diagnosis implications of the whole genome sequencing in a large Lebanese family with hyaline fibromatosis syndrome.

BACKGROUND: Hyaline fibromatosis syndrome (HFS) is a recently introduced alternative term for two disorders that were previously known as juvenile hyaline fibromatosis (JHF) and infantile systemic hyalinosis (ISH). These two variants are secondary to mutations in the anthrax toxin receptor 2 gene (ANTXR2) located on chromosome 4q21. The main clinical features of both entities include papular and/or nodular skin lesions, gingival hyperplasia, joint contractures and osteolytic bone lesions that appear in the first few years of life, and the syndrome typically progresses with the appearance of new lesions. METHODS: We describe five Lebanese patients from one family, aged between 28 and 58 years, and presenting with nodular and papular skin lesions, gingival hyperplasia, joint contractures and bone lesions. Because of the particular clinical features and the absence of a clinical diagnosis, Whole Genome Sequencing (WGS) was carried out on DNA samples from the proband and his parents. RESULTS: A mutation in ANTXR2 (p. Gly116Val) that yielded a diagnosis of HFS was noted. CONCLUSIONS: The main goal of this paper is to add to the knowledge related to the clinical and radiographic aspects of HFS in adulthood and to show the importance of Next-Generation Sequencing (NGS) techniques in resolving such puzzling cases.

A collection of annotated and harmonized human breast cancer transcriptome datasets, including immunologic classification.

The increased application of high-throughput approaches in translational research has expanded the number of publicly available data repositories. Gathering additional valuable information contained in the datasets represents a crucial opportunity in the biomedical field. To facilitate and stimulate utilization of these datasets, we have recently developed an interactive data browsing and visualization web application, the Gene Expression Browser (GXB). In this note, we describe a curated compendium of 13 public datasets on human breast cancer, representing a total of 2142 transcriptome profiles. We classified the samples according to different immune based classification systems and integrated this information into the datasets. Annotated and harmonized datasets were uploaded to GXB. Study samples were categorized in different groups based on their immunologic tumor response profiles, intrinsic molecular subtypes and multiple clinical parameters. Ranked gene lists were generated based on relevant group comparisons. In this data note, we demonstrate the utility of GXB to evaluate the expression of a gene of interest, find differential gene expression between groups and investigate potential associations between variables with a specific focus on immunologic classification in breast cancer. This interactive resource is publicly available online at: http://breastcancer.gxbsidra.org/dm3/geneBrowser/list.

AKT1 has dual actions on the glucocorticoid receptor by cooperating with 14-3-3.

Glucocorticoids are important therapeutic compounds for acute lymphoblastic leukemia (ALL). AKT1 or the protein kinase B is frequently activated in ALL, and contributes to the development of glucocorticoid resistance. We examined impact of AKT1 on glucocorticoid receptor (GR)-induced transcriptional activity in cooperation with phospho-serine/threonine-binding protein 14-3-3. AKT1 has two distinct actions on GR transcriptional activity, one through segregation of GR in the cytoplasm by phosphorylating GR at Ser-134 and subsequent association of 14-3-3, and the other through direct modulation of GR transcriptional activity in the nucleus. For the latter, AKT1 and 14-3-3 are attracted to DNA-bound GR, accompanied by AKT1-dependent p300 phosphorylation, H3S10 phosphorylation and H3K14 acetylation at the DNA site. These two actions of AKT1 regulate distinct sets of glucocorticoid-responsive genes. Our results suggest that specific inhibition of the AKT1/14-3-3 activity on the cytoplasmic retention of GR may be a promising target for treating glucocorticoid resistance observed in ALL.

Ran GTPase promotes cancer progression via Met recepto-rmediated downstream signaling.

It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival.Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reduction-mediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a pre-requisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator.

KDM6B histone demethylase is an epigenetic regulator of estrogen receptor ß expression in human pleural mesothelioma.

AIM: To assess the correlation between KDM6B and estrogen receptor ß (ERß) expression in malignant pleural mesothelioma (MPM). MATERIALS & METHODS: We evaluated gene expression by in silico analysis of microarray data, real-time PCR and western blot in MPM tumors and cell lines. RESULTS & CONCLUSION: We report a strong positive correlation between the expression of KDM6B and ERß in MPM tumors and cell lines. We describe that, in hypoxia, the HIF2α-KDM6B axis induces an epithelioid morphology and ERß expression in biphasic MPM cells with estrogen receptor-negative phenotype. Reduced histone H3K27 tri-methylation confirms KDM6B activity under hypoxic conditions. Importantly, cells treated during reoxygenation with the selective ERß agonist, KB9520, maintain ERß expression and the less aggressive phenotype acquired in hypoxia.

Temple-Baraitser Syndrome and Zimmermann-Laband Syndrome: one clinical entity?

BACKGROUND: KCNH1 encodes a voltage-gated potassium channel that is predominantly expressed in the central nervous system. Mutations in this gene were recently found to be responsible for Temple-Baraitser Syndrome (TMBTS) and Zimmermann-Laband syndrome (ZLS). METHODS: Here, we report a new case of TMBTS diagnosed in a Lebanese child. Whole genome sequencing was carried out on DNA samples of the proband and his parents to identify mutations associated with this disease. Sanger sequencing was performed to confirm the presence of detected variants. RESULTS: Whole genome sequencing revealed three missense mutations in TMBTS patient: c.1042G > A in KCNH1, c.2131 T > C in STK36, and c.726C > A in ZNF517. According to all predictors, mutation in KCNH1 is damaging de novo mutation that results in substitution of Glycine by Arginine, i.e., p.(Gly348Arg). This mutation was already reported in a patient with ZLS that could affect the connecting loop between helices S4-S5 of KCNH1 with a gain of function effect. CONCLUSIONS: Our findings demonstrate that KCNH1 mutations cause TMBTS and expand the mutational spectrum of KCNH1 in TMBTS. In addition, all cases of TMBTS were reviewed and compared to ZLS. We suggest that the two syndromes are a continuum and that the variability in the phenotypes is the result of the involvement of genetic modifiers.

Analysis of Gene Expression in 3D Spheroids Highlights a Survival Role for ASS1 in Mesothelioma.

To investigate the underlying causes of chemoresistance in malignant pleural mesothelioma, we have studied mesothelioma cell lines as 3D spheroids, which acquire increased chemoresistance compared to 2D monolayers. We asked whether the gene expression of 3D spheroids would reveal mechanisms of resistance. To address this, we measured gene expression of three mesothelioma cell lines, M28, REN and VAMT, grown as 2D monolayers and 3D spheroids. A total of 209 genes were differentially expressed in common by the three cell lines in 3D (138 upregulated and 71 downregulated), although a clear resistance pathway was not apparent. We then compared the list of 3D genes with two publicly available datasets of gene expression of 56 pleural mesotheliomas compared to normal tissues. Interestingly, only three genes were increased in both 3D spheroids and human tumors: argininosuccinate synthase 1 (ASS1), annexin A4 (ANXA4) and major vault protein (MVP); of these, ASS1 was the only consistently upregulated of the three genes by qRT-PCR. To measure ASS1 protein expression, we stained 2 sets of tissue microarrays (TMA): one with 88 pleural mesothelioma samples and the other with additional 88 pleural mesotheliomas paired with matched normal tissues. Of the 176 tumors represented on the two TMAs, ASS1 was expressed in 87 (50%; staining greater than 1 up to 3+). For the paired samples, ASS1 expression in mesothelioma was significantly greater than in the normal tissues. Reduction of ASS1 expression by siRNA significantly sensitized mesothelioma spheroids to the pro-apoptotic effects of bortezomib and of cisplatin plus pemetrexed. Although mesothelioma is considered by many to be an ASS1-deficient tumor, our results show that ASS1 is elevated at the mRNA and protein levels in mesothelioma 3D spheroids and in human pleural mesotheliomas. We also have uncovered a survival role for ASS1, which may be amenable to targeting to undermine mesothelioma multicellular resistance.

Distinct miRNA profiles in normal and gastric cancer myofibroblasts and significance in Wnt signaling.

Stromal cells influence epithelial function in both health and disease. Myofibroblasts are abundant stromal cells that influence the cellular microenvironment by release of extracellular matrix (ECM) proteins, growth factors, proteases, cytokines, and chemokines. Cancer-associated myofibroblasts (CAMs) differ from adjacent tissue (ATMs) and normal tissue myofibroblasts (NTMs), but the basis of this is incompletely understood. We report now the differential expression of miRNAs in gastric cancer CAMs. MicroRNA arrays identified differences in the miRNA profile in gastric and esophageal NTMs and in CAMs from stomach compared with NTMs. miR-181d was upregulated in gastric CAMs. Analysis of differentially regulated miRNAs indicated an involvement in Wnt signaling. Examination of a microarray data set then identified Wnt5a as the only consistently upregulated Wnt ligand in gastric CAMs. Wnt5a stimulated miR-181d expression, and knockdown of miR-181d inhibited Wnt5a stimulation of CAM proliferation and migration. Analysis of miR-181d targets suggested a role in chemotaxis. Conditioned medium from CAMs stimulated gastric cancer cell (AGS) migration more than that from ATMs, and miR-181d knockdown reduced the effect of CAM-CM on AGS cell migration but had no effect on AGS cell responses to ATM conditioned media. The data suggest that dysregulation of miRNA expression in gastric CAMs, secondary to Wnt5a signaling, accounts at least in part for the effect of CAMs in promoting cancer cell migration.

SIRT1 at the crossroads of AKT1 and ERß in malignant pleural mesothelioma cells.

In this report, we show that malignant pleural mesothelioma (MPM) patients whose tumors express high levels of AKT1 exhibit a significantly worse prognosis, whereas no significant correlation with AKT3 expression is observed. We provide data that establish a phosphorylation independent role of AKT1 in affecting MPM cell shape and anchorage independent cell growth in vitro and highlight the AKT1 isoform-specific nature of these effects.We describe that AKT1 activity is inhibited by the loss of SIRT1-mediated deacetylation and identify, by mass spectrometry, 11 unique proteins that interact with acetylated AKT1.Our data demonstrate a role of the AKT1/SIRT1/FOXM1 axis in the expression of the tumor suppressor ERß. We further demonstrate an inhibitory feedback loop by ERß, activated by the selective agonist KB9520, on this axis both in vitro and in vivo.Our data broaden the current knowledge of ERß and AKT isoform-specific functions that could be valuable in the design of novel and effective therapeutic strategies for MPM.

Total proteome analysis identifies migration defects as a major pathogenetic factor in immunoglobulin heavy chain variable region (IGHV)-unmutated chronic lymphocytic leukemia.

The mutational status of the immunoglobulin heavy chain variable region defines two clinically distinct forms of chronic lymphocytic leukemia (CLL) known as mutated (M-CLL) and unmutated (UM-CLL). To elucidate the molecular mechanisms underlying the adverse clinical outcome associated with UM-CLL, total proteomes from nine UM-CLL and nine M-CLL samples were analyzed by isobaric tags for relative and absolute quantification (iTRAQ)-based mass spectrometry. Based on the expression of 3521 identified proteins, principal component analysis separated CLL samples into two groups corresponding to immunoglobulin heavy chain variable region mutational status. Computational analysis showed that 43 cell migration/adhesion pathways were significantly enriched by 39 differentially expressed proteins, 35 of which were expressed at significantly lower levels in UM-CLL samples. Furthermore, UM-CLL cells underexpressed proteins associated with cytoskeletal remodeling and overexpressed proteins associated with transcriptional and translational activity. Taken together, our findings indicate that UM-CLL cells are less migratory and more adhesive than M-CLL cells, resulting in their retention in lymph nodes, where they are exposed to proliferative stimuli. In keeping with this hypothesis, analysis of an extended cohort of 120 CLL patients revealed a strong and specific association between UM-CLL and lymphadenopathy. Our study illustrates the potential of total proteome analysis to elucidate pathogenetic mechanisms in cancer.