Synthesis and Structure of Novel Phenothiazine Derivatives, and Compound Prioritization via In Silico Target Search and Screening for Cytotoxic and Cholinesterase Modulatory Activities in Liver Cancer Cells and In Vivo in Zebrafish.

Phenothiazines (PTZ) are antipsychotics known to modulate a variety of neurotransmitter activities that include dopaminergic and cholinergic signaling and have been identified as potential anticancer agents in vitro. However, it is important to also test whether a highly cytotoxic, repurposed, or novel PTZ has low toxicity and neuromodulatory activity in vivo using vertebrate model organisms, such as zebrafish. In this study, we synthesized novel phenothiazines and screened them in vitro in liver cancer and in vivo in zebrafish embryos/larvae. The syntheses of several intermediate PTZ 10-yl acyl chlorides were followed by elemental analysis and determination of 1H NMR and 13C NMR mass (ESI+) spectra of a large number of novel PTZ 10-carboxamides. Cytotoxicities of 28 PTZ derivatives (1-28) screened against Hep3B and SkHep1 liver cancer cell lines revealed five intermediate and five novel leads along with trifluoperazine (TFP), prochlorperazine (PCP), and perphenazine, which are relatively more cytotoxic than the basic PTZ core. Overall, the derivatives were more cytotoxic to Hep3B than SkHep1 cells. Moreover, in silico target screening identified cholinesterases as some of the commonest targets of the screened phenothiazines. Interestingly, molecular docking studies with acetylcholinesterase (AChE) and butyrylcholinesterase proteins showed that the most cytotoxic compounds 1, 3, PCP, and TFP behaved similar to Huprin W in their amino acid interactions with the AChE protein. The highly cytotoxic intermediate PTZ derivative 1 exhibited a relatively lower toxicity profile than those of 2 and 3 during the zebrafish development. It also modulated in vivo the cholinesterase activity in a dose-dependent manner while significantly increasing the total cholinesterase activity and/or ACHE mRNA levels, independent of the liver cancer cell type. Our screen also identified novel phenothiazines, i.e., 8 and 10, with significant cytotoxic and cholinesterase modulatory effects in liver cancer cells; yet both compounds had low levels of toxicity in zebrafish. Moreover, they modulated the cholinesterase activity or expression of ACHE in a cancer cell line-specific manner, and compound 10 significantly inhibited the cholinesterase activity in zebrafish. Accordingly, using a successful combination of in silico, in vitro, and in vivo approaches, we identified several lead anticancer and cholinesterase modulatory PTZ derivatives for future research.

CAP-RNAseq: an integrated pipeline for functional annotation and prioritization of co-expression clusters.

Cluster analysis is one of the most widely used exploratory methods for visualization and grouping of gene expression patterns across multiple samples or treatment groups. Although several existing online tools can annotate clusters with functional terms, there is no all-in-one webserver to effectively prioritize genes/clusters using gene essentiality as well as congruency of mRNA-protein expression. Hence, we developed CAP-RNAseq that makes possible (1) upload and clustering of bulk RNA-seq data followed by identification, annotation and network visualization of all or selected clusters; and (2) prioritization using DepMap gene essentiality and/or dependency scores as well as the degree of correlation between mRNA and protein levels of genes within an expression cluster. In addition, CAP-RNAseq has an integrated primer design tool for the prioritized genes. Herein, we showed using comparisons with the existing tools and multiple case studies that CAP-RNAseq can uniquely aid in the discovery of co-expression clusters enriched with essential genes and prioritization of novel biomarker genes that exhibit high correlations between their mRNA and protein expression levels. CAP-RNAseq is applicable to RNA-seq data from different contexts including cancer and available at http://konulabapps.bilkent.edu.tr:3838/CAPRNAseq/ and the docker image is downloadable from https://hub.docker.com/r/konulab/caprnaseq.

Doxorubicin induces prolonged DNA damage signal in cells overexpressing DEK isoform-2.

DEK has a short isoform (DEK isoform-2; DEK2) that lacks amino acid residues between 49-82. The full-length DEK (DEK isoform-1; DEK1) is ubiquitously expressed and plays a role in different cellular processes but whether DEK2 is involved in these processes remains elusive. We stably overexpressed DEK2 in human bone marrow stromal cell line HS-27A, in which endogenous DEKs were intact or suppressed via short hairpin RNA (sh-RNA). We have found that contrary to ectopic DEK1, DEK2 locates in the nucleus and nucleolus, causes persistent γH2AX signal upon doxorubicin treatment, and couldn't functionally compensate for the loss of DEK1. In addition, DEK2 overexpressing cells were more sensitive to doxorubicin than DEK1-cells. Expressions of DEK1 and DEK2 in cell lines and primary tumors exhibit tissue specificity. DEK1 is upregulated in cancers of the colon, liver, and lung compared to normal tissues while both DEK1 and DEK2 are downregulated in subsets of kidney, prostate, and thyroid carcinomas. Interestingly, only DEK2 was downregulated in a subset of breast tumors suggesting that DEK2 can be modulated differently than DEK1 in specific cancers. In summary, our findings show distinct expression patterns and subcellular location and suggest non-overlapping functions between the two DEK isoforms.

Global miRNA expression of bone marrow mesenchymal stem/stromal cells derived from Fanconi anemia patients.

Fanconi anemia (FA) is a rare genetic disorder characterized by genomic instability, developmental defects, and bone marrow (BM) failure. Hematopoietic stem cells (HSCs) in BM interact with the mesenchymal stem/stromal cells (MSCs); and this partly sustains the tissue homeostasis. MicroRNAs (miRNAs) can play a critical role during these interactions possibly via paracrine mechanisms. This is the first study addressing the miRNA profile of FA BM-MSCs obtained before and after BM transplantation (preBMT and postBMT, respectively). Non-coding RNA expression profiling and quality control analyses were performed in Donors (n = 13), FA preBMT (n = 11), and FA postBMT (n = 6) BM-MSCs using GeneChip miRNA 2.0 Array. Six Donor-FA preBMT pairs were used to identify a differentially expressed miRNA expression signature containing 50 miRNAs, which exhibited a strong correlation with the signature obtained from unpaired samples. Five miRNAs (hsa-miR-146a-5p, hsa-miR-148b-3p, hsa-miR-187-3p, hsa-miR-196b-5p, and hsa-miR-25-3p) significantly downregulated in both the paired and unpaired analyses were used to generate the BM-MSCs' miRNA-BM mononuclear mRNA networks upon integration of a public dataset (GSE16334; studying Donor versus FA samples). Functionally enriched KEGG pathways included cellular senescence, miRNAs, and pathways in cancer. Here, we showed that hsa-miR-146a-5p and hsa-miR-874-3p were rescued upon BMT (n = 3 triplets). The decrease in miR-146a-5p was also validated using RT-qPCR and emerged as a strong candidate as a modulator of BM mRNAs in FA patients.

Discovery of Cancer-Specific and Independent Prognostic Gene Subsets of the Slit-Robo Family Using TCGA-PANCAN Datasets.

The Slit-Robo family of axon guidance molecules works in concert, playing important roles in organ development and cancer. Expressions of individual Slit-Robo genes have been used in calculating univariable hazard ratios (HRuni) for predicting cancer prognosis in the literature. However, Slit-Robo members do not act independently; hence, hazard ratios from multivariable Cox regression (HRmulti) on the whole gene set can further lead to identification of cancer-specific, novel, and independent prognostic gene pairs or modules. Herein, we obtained mRNA expressions of the Slit-Robo family consisting of four Robos (ROBO1/2/3/4) and three Slits (SLIT1/2/3), along with four types of survival outcome across cancers found in the Cancer Genome Atlas (TCGA). We used cluster heat maps to visualize closely associated pairs/modules of prognostic genes across 33 different cancers. We found a smaller number of significant genes in HRmulti than in HRuni, suggesting that the former analysis was less redundant. High ROBO4 expression emerged as relatively protective within the family, in both types of HR analyses. Multivariable Cox regression, on the other hand, revealed significantly more HR signatures containing Slit-Robo pairs acting in opposing directions than those containing Slit-Slit or Robo-Robo pairs for disease-specific survival. Furthermore, we discovered, through the online app SmulTCan's lasso regression, Slit-Robo gene subsets that significantly differentiated between high- versus low-risk prognosis patient groups, particularly for renal cancers and low-grade glioma. The statistical pipeline reported herein can help test independent and significant pairs/modules within a codependent gene family for cancer prognostication, and thus should also prove useful in personalized/precision medicine research.

SmulTCan: A Shiny application for multivariable survival analysis of TCGA data with gene sets.

BACKGROUND: Survival analysis is widely used in cancer research, and although several methods exist in R, there is the need for a more interactive, flexible, yet comprehensive online tool to analyze gene sets using Cox proportional hazards (CPH) models. The web-based Shiny application (app) SmulTCan extends existing tools to multivariable CPH models of gene sets-as exemplified using the netrins and their receptors (netrins-receptors). It can be used to identify survival gene signatures (GSs) and select the best subsets of input gene, microRNA, methylation level, and copy number variation sets from the Cancer Genome Atlas (TCGA). OBJECTIVES: To create a tool for CPH model building and best subset selection, using survival data from TCGA with input gene expression files from UCSC Xena. Furthermore, we aim to analyze the input TSV file of netrins-receptors in SmulTCan and discuss our findings. METHODS: SmulTCan uses Shiny's reactivity with built-in R functions from packages for CPH model analysis and best subset selection including "survminer", "riskRegression", "rms", "glmnet", and "BeSS". RESULTS: Results from the SmulTCan app with the netrins-receptors gene set indicated unique hazard ratio GSs in certain renal and neural cancers, while the best subsets for this gene set, obtained via the app, could differentiate between prognostic outcomes in these cancers. AVAILABILITY: SmulTCan is available at http://konulabapps.bilkent.edu.tr:3838/SmulTCan/. The input file for netrins-receptors is available in the online version of this paper. TCGA dataset folders containing survival files are available through https://github.com/aozh7/SmulTCan/. SUPPLEMENTARY INFORMATION: The supplementary information (SI) accompanies the online version of this article.

Transcriptome profiles associated with selenium-deficiency-dependent oxidative stress identify potential diagnostic and therapeutic targets in liver cancer cells.

Hepatocellular carcinoma (HCC) is one of the most common cancer types with high mortality rates and displays increased resistance to various stress conditions such as oxidative stress. Conventional therapies have low efficacies due to resistance and off-target effects in HCC. Here we aimed to analyze oxidative stress-related gene expression profiles of HCC cells and identify genes that could be crucial for novel diagnostic and therapeutic strategies. To identify important genes that cause resistance to reactive oxygen species (ROS), a model of oxidative stress upon selenium (Se) deficiency was utilized. The results of transcriptome-wide gene expression data were analyzed in which the differentially expressed genes (DEGs) were identified between HCC cell lines that are either resistant or sensitive to Se-deficiency-dependent oxidative stress. These DEGs were further investigated for their importance in oxidative stress resistance by network analysis methods, and 27 genes were defined to have key roles; 16 of which were previously shown to have impact on liver cancer patient survival. These genes might have Se-deficiency-dependent roles in hepatocarcinogenesis and could be further exploited for their potentials as novel targets for diagnostic and therapeutic approaches.

CHRNA5 belongs to the secondary estrogen signaling network exhibiting prognostic significance in breast cancer.

PURPOSE: Cholinergic signals can be important modulators of cellular signaling in cancer. We recently have shown that knockdown of nicotinic acetylcholine receptor subunit alpha 5, CHRNA5, diminishes the proliferative potential of breast cancer cells. However, modulation of CHRNA5 expression in the context of estrogen signaling and its prognostic implications in breast cancer remained unexplored. METHODS: Meta-analyses of large breast cancer microarray cohorts were used to evaluate the association of CHRNA5 expression with estrogen (E2) treatment, estrogen receptor (ER) status and patient prognosis. The results were validated through RT-qPCR analyses of multiple E2 treated cell lines, CHRNA5 depleted MCF7 cells and across a breast cancer patient cDNA panel. We also calculated a predicted secondary (PS) score representing direct/indirect induction of gene expression by E2 based on a public dataset (GSE8597). Co-expression analysis was performed using a weighted gene co-expression network analysis (WGCNA) pipeline. Multiple other publicly available datasets such as CCLE, COSMIC and TCGA were also analyzed. RESULTS: Herein we found that CHRNA5 expression was induced by E2 in a dose- and time-dependent manner in breast cancer cell lines. ER- breast tumors exhibited higher CHRNA5 expression levels than ER+ tumors. Independent meta-analysis for survival outcome revealed that higher CHRNA5 expression was associated with a worse prognosis in untreated breast cancer patients. Furthermore, CHRNA5 and its co-expressed gene network emerged as secondarily induced targets of E2 stimulation. These targets were largely downregulated by exposure to CHRNA5 siRNA in MCF7 cells while the response of primary ESR1 targets was dependent on the direction of the PS-score. Moreover, primary and secondary target genes were uncoupled and clustered distinctly based on multiple public datasets. CONCLUSION: Our findings strongly associate increased expression of CHRNA5 and its co-expression network with secondary E2 signaling and a worse prognosis in breast cancer.

Zebrafish Xenotransplantation Models for Studying Gene Function and Drug Treatment in Hepatocellular Carcinoma.

INTRODUCTION: Zebrafish is a promising model organism for human disease including hepatocellular cancer (HCC). Recently, zebrafish has emerged also as a host for xenograft studies of liver cancer cell lines and patient derived tumors of HCC. Zebrafish embryos enable drug screening and gene function studies of xenografted cells via ease of microinjection and visualization of tumor growth and metastasis. OBJECTIVES: In this review, we aimed to overview zebrafish HCC and liver cancer xenotransplantation studies focusing on 'gene functional analysis' and 'drug/chemical screening'. METHODS: Herein, a comprehensive literature search was performed for liver and HCC xenografts in zebrafish on PubMed using different key words and filters for molecular modifications or drug exposure. RESULTS: Our literature search revealed around 250 studies which were filtered and summarized in a table (Table 1) revealing comprehensive collection of experimental and technical details on microinjection, injected cell lines, molecular modifications of injected cells, types and doses of drug treatments as well as biological assessments. CONCLUSION: This review provides a platform for HCC and liver xenografts and highlights studies performed to understand gene functionality and drug efficacy in vivo in zebrafish.

ZenoFishDb v1.1: A Database for Xenotransplantation Studies in Zebrafish.

Rapidly accumulating literature has proven feasibility of the zebrafish xenograft models in cancer research. Nevertheless, online databases for searching the current zebrafish xenograft literature are in great demand. Herein, we have developed a manually curated database, called ZenoFishDb v1.1 (https://konulab.shinyapps.io/zenofishdb), based on R Shiny platform aiming to provide searchable information on ever increasing collection of zebrafish studies for cancer cell line transplantation and patient-derived xenografts (PDXs). ZenoFishDb v1.1 user interface contains four modules: DataTable, Visualization, PDX Details, and PDX Charts. The DataTable and Visualization pages represent xenograft study details, including injected cell lines, PDX injections, molecular modifications of cell lines, zebrafish strains, as well as technical aspects of the xenotransplantation procedures in table, bar, and/or pie chart formats. The PDX Details module provides comprehensive information on the patient details in table format and can be searched and visualized. Overall, ZenoFishDb v1.1 enables researchers to effectively search, list, and visualize different technical and biological attributes of zebrafish xenotransplantation studies particularly focusing on the new trends that make use of reporters, RNA interference, overexpression, or mutant gene constructs of transplanted cancer cells, stem cells, and PDXs, as well as distinguished host modifications.

Design, synthesis and anticancer/antiestrogenic activities of novel indole-benzimidazoles.

Indole-benzimidazoles have recently gained attention due to their antiproliferative and antiestrogenic effects. However, their structural similarities and molecular mechanisms shared with selective estrogen receptor modulators (SERMs) have not yet been investigated. In this study, we synthesized novel ethylsulfonyl indole-benzimidazole derivatives by substituting the first (R1) and fifth (R2) positions of benzimidazole and indole groups, respectively. Subsequently, we performed 1H NMR, 13C NMR, and Mass spectral and in silico docking analyses, and anticancer activity screening studies of these novel indole-benzimidazoles. The antiproliferative effects of indole-benzimidazoles were found to be more similar between the estrogen (E2) responsive cell lines MCF-7 and HEPG2 in comparison to the Estrogen Receptor negative (ER-) cell line MDA-MB-231. R1:p-fluorobenzyl group members were selected as lead compounds for their potent anticancer effects and moderate structural affinity to ER. Microarray expression profiling and gene enrichment analyses (GSEA) of the selected compounds (R1:p-fluorobenzyl: 48, 49, 50, 51; R1:3,4-difluorobenzyl: 53) helped determine the similarly modulated cellular signaling pathways among derivatives. Moreover, we identified known compounds that have significantly similar gene signatures to that of 51 via queries performed in LINCS database; and further transcriptomics comparisons were made using public GEO datasets (GSE35428, GSE7765, GSE62673). Our results strongly demonstrate that these novel indole-benzimidazoles can modulate ER target gene expression as well as dioxin-mediated aryl hydrocarbon receptor and amino acid deprivation-mediated integrated stress response signaling in a dose-dependent manner.

The Transcription Factor Elf3 Is Essential for a Successful Mesenchymal to Epithelial Transition.

The epithelial to mesenchymal transition (EMT) and the mesenchymal to epithelial transition (MET) are two critical biological processes that are involved in both physiological events such as embryogenesis and development and also pathological events such as tumorigenesis. They present with dramatic changes in cellular morphology and gene expression exhibiting acute changes in E-cadherin expression. Despite the comprehensive understanding of EMT, the regulation of MET is far from being understood. To find novel regulators of MET, we hypothesized that such factors would correlate with Cdh1 expression. Bioinformatics examination of several expression profiles suggested Elf3 as a strong candidate. Depletion of Elf3 at the onset of MET severely impaired the progression to the epithelial state. This MET defect was explained, in part, by the absence of E-cadherin at the plasma membrane. Moreover, during MET, ELF3 interacts with the Grhl3 promoter and activates its expression. Our findings present novel insights into the regulation of MET and reveal ELF3 as an indispensable guardian of the epithelial state. A better understanding of MET will, eventually, lead to better management of metastatic cancers.

Lower connectivity of tumor coexpression networks is not specific to cancer.

Global level network analysis of molecular links is necessary for systems level view of complex diseases like cancer. Using genome-wide expression datasets, we constructed and compared gene co-expression based specific networks of pre-cancerous tumors (adenoma) and cancerous tumors (carcinoma) with paired normal networks to assess for any possible changes in network connectivity. Previously, loss of connectivity was reported as a characteristics of cancer samples. Here, we observed that pre-cancerous conditions also had significantly less connections than paired normal samples. We observed a loss of connectivity trend for colorectal adenoma, aldosterone producing adenoma and uterine leiomyoma. We also showed that the loss of connectivity trend is not specific to positive or negative correlation based networks. Differential hub genes, which were the most highly differentially less connected genes in tumor, were mostly different between different datasets. No common gene list could be defined which underlies the lower connectivity of tumor specific networks. Connectivity of colorectal cancer methylation targets was different from other genes. Extracellular space related terms were enriched in negative correlation based differential hubs and common methylation targets of colorectal carcinoma. Our results indicate a systems level change of lower connectivity as cells transform to not only cancer but also pre-cancerous conditions. This systems level behavior could not be attributed to a group of genes.

PKNOX2 expression and regulation in the bone marrow mesenchymal stem cells of Fanconi anemia patients and healthy donors.

HOX and TALE transcription factors are important regulators of development and homeostasis in determining cellular identity. Deregulation of this process may drive cancer progression. The aim of this study was to investigate the expression of these transcription factors in the bone marrow derived mesenchymal stem cells (BM-MSCs) of Fanconi anemia (FA) patients, which is a cancer-predisposing disease. Expression levels of HOX and TALE genes in BM-MSCs were obtained from FA patients and healthy donors by RT-qPCR and highly conserved expression levels were observed between patient and donor cells, except PKNOX2, which is a member of TALE class. PKNOX2 was significantly downregulated in FA cells compared to donors (P < 0.05). PKNOX2 expression levels did not change with diepoxybutane (DEB), a DNA crosslinking agent, in either donor or FA cells except one patient's with a truncation mutation of FANCA. A difference of PKNOX2 protein level was not obtained between FA patient and donor BM-MSCs by western blot analysis. When human TGF-ß1 (rTGF-ß1) recombinant protein was provided to the cultures, PKNOX2 as well as TGF-ß1 expression increased both in FA and donor BM-MSCs in a dose dependent manner. 5 ng/mL rTGF-ß stimulation had more dominant effect on the gene expression of donor BM-MSCs compared to FA cells. Decreased PKNOX2 expression in FA BM-MSCs may provide new insights into the molecular pathophysiology of the disease and TGF-ß1 levels of the microenvironment may be the cause of PKNOX2 downregulation.

Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) RNAi is associated with cell cycle inhibition, apoptosis, DNA damage response and drug sensitivity in breast cancer.

Cholinergic Receptor Nicotinic Alpha 5 (CHRNA5) is an important susceptibility locus for nicotine addiction and lung cancer. Depletion of CHRNA5 has been associated with reduced cell viability, increased apoptosis and alterations in cellular motility in different cancers yet not in breast cancer. Herein we first showed the expression of CHRNA5 was variable and positively correlated with the fraction of total genomic alterations in breast cancer cell lines and tumors indicating its potential role in DNA damage response (DDR). Next, we demonstrated that silencing of CHRNA5 expression in MCF7 breast cancer cell line by RNAi affected expression of genes involved in cytoskeleton, TP53 signaling, DNA synthesis and repair, cell cycle, and apoptosis. The transcription profile of CHRNA5 depleted MCF7 cells showed a significant positive correlation with that of A549 lung cancer cell line while exhibiting a negative association with the CHRNA5 co-expression profile obtained from Cancer Cell Line Encylopedia (CCLE). Moreover, it exhibited high similarities with published MCF7 expression profiles obtained from exposure to TP53 inducer nutlin-3a and topoisomerase inhibitors. We then demonstrated that CHRNA5 siRNA treatment reduced cell viability and DNA synthesis indicating G1 arrest while it significantly increased apoptotic sub-G1 cell population. Accordingly, we observed lower levels of phosphorylated RB (Ser807/811) and an increased BAX/BCL2 ratio in RNAi treated MCF7 cells. We also showed that CHRNA5 RNAi transcriptome correlated negatively with DDR relevant gene expression profile in breast cancer gene expression datasets while the coexposure to topoisomerase inhibitors in the presence of CHRNA5 RNAi enhanced chemosensitivity potentially due to reduced DDR. CHRNA5 RNAi consistently lowered total CHEK1 mRNA and protein levels as well as phosphorylated CHEK1 (Ser345) in MCF7 cells. We also detected a significant positive correlation between the expression levels of CHRNA5 and CHEK1 in CCLE, TCGA and METABRIC breast cancer datasets. Our study suggests CHRNA5 RNAi is associated with cell cycle inhibition, apoptosis as well as reduced DDR and increased drug sensitivity in breast cancer yet future studies are warranted since dose- and cell line-specific differences exist in response to CHRNA5 depletion. Gene expression microarray data can be accessed from GEO database under the accession number GSE89333.

Development of a novel zebrafish xenograft model in ache mutants using liver cancer cell lines.

Acetylcholinesterase (AChE), an enzyme responsible for degradation of acetylcholine, has been identified as a prognostic marker in liver cancer. Although in vivo Ache tumorigenicity assays in mouse are present, no established liver cancer xenograft model in zebrafish using an ache mutant background exists. Herein, we developed an embryonic zebrafish xenograft model using epithelial (Hep3B) and mesenchymal (SKHep1) liver cancer cell lines in wild-type and ache sb55 sibling mutant larvae after characterization of cholinesterase expression and activity in cell lines and zebrafish larvae. The comparison of fluorescent signal reflecting tumor size at 3-days post-injection (dpi) revealed an enhanced tumorigenic potential and a reduced migration capacity in cancer cells injected into homozygous ache sb55 mutants when compared with the wild-type. Increased tumor load was confirmed using an ALU based tumor DNA quantification method modified for use in genotyped xenotransplanted zebrafish embryos. Confocal microscopy using the Huh7 cells stably expressing GFP helped identify the distribution of tumor cells in larvae. Our results imply that acetylcholine accumulation in the microenvironment directly or indirectly supports tumor growth in liver cancer. Use of this model system for drug screening studies holds potential in discovering new cholinergic targets for treatment of liver cancers.

15-Lipoxygenase-1 re-expression in colorectal cancer alters endothelial cell features through enhanced expression of TSP-1 and ICAM-1.

15-lipoxygenase-1 (15-LOX-1) oxygenates linoleic acid to 13(S)-hydroxyoctadecadienoic acid (HODE). The enzyme is widely suppressed in different cancers and its re-expression has tumor suppressive effects. 15-LOX-1 has been shown to inhibit neoangiogenesis in colorectal cancer (CRC); in the present study we confirm this phenomenon and describe the mechanistic basis. We show that re-expression of 15-LOX-1 in CRC cell lines resulted in decreased transcriptional activity of HIF1α and reduced the expression and secretion of VEGF in both normoxic and hypoxic conditions. Conditioned medium (CM) was obtained from CRC or prostate cancer cell lines re-expressing 15-LOX-1 (15-LOX-1CM). 15-LOX-1CM treated aortic rings from 6-week old C57BL/6 mice showed significantly less vessel sprouting and more organized structure of vascular network. Human umbilical vein endothelial cells (HUVECs) incubated with 15-LOX-1CM showed reduced motility, enhanced expression of intercellular cell adhesion molecule (ICAM-1) and reduced tube formation but no change in proliferation or cell-cycle distribution. HUVECs incubated with 13(S)-HODE partially phenocopied the effects of 15-LOX-1CM, i.e., showed reduced motility and enhanced expression of ICAM-1, but did not reduce tube formation, implying the importance of additional factors. Therefore, a Proteome Profiler Angiogenesis Array was carried out, which showed that Thrombospondin-1 (TSP-1), a matrix glycoprotein known to strongly inhibit neovascularization, was expressed significantly more in HUVECs incubated with 15-LOX-1CM. TSP-1 blockage in HUVECs reduced the expression of ICAM-1 and enhanced cell motility, thereby providing a mechanism for reduced angiogenesis. The anti-angiogenic effects of 15-LOX-1 through enhanced expressions of ICAM-1 and TSP-1 are novel findings and should be explored further to develop therapeutic options.

A Combined ULBP2 and SEMA5A Expression Signature as a Prognostic and Predictive Biomarker for Colon Cancer.

Background: Prognostic biomarkers for cancer have the power to change the course of disease if they add value beyond known prognostic factors, if they can help shape treatment protocols, and if they are reliable. The aim of this study was to identify such biomarkers for colon cancer and to understand the molecular mechanisms leading to prognostic stratifications based on these biomarkers. Methods and Findings: We used an in house R based script (SSAT) for the in silico discovery of stage-independent prognostic biomarkers using two cohorts, GSE17536 and GSE17537, that include 177 and 55 colon cancer patients, respectively. This identified 2 genes, ULBP2 and SEMA5A, which when used jointly, could distinguish patients with distinct prognosis. We validated our findings using a third cohort of 48 patients ex vivo. We find that in all cohorts, a combined ULBP2/SEMA5A classification (SU-GIB) can stratify distinct prognostic sub-groups with hazard ratios that range from 2.4 to 4.5 (p≤0.01) when overall- or cancer-specific survival is used as an end-measure, independent of confounding prognostic parameters. In addition, our preliminary analyses suggest SU-GIB is comparable to Oncotype DX colon(®) in predicting recurrence in two different cohorts (HR: 1.5-2; p≤0.02). SU-GIB has potential as a companion diagnostic for several drugs including the PI3K/mTOR inhibitor BEZ235, which are suitable for the treatment of patients within the bad prognosis group. We show that tumors from patients with worse prognosis have low EGFR autophosphorylation rates, but high caspase 7 activity, and show upregulation of pro-inflammatory cytokines that relate to a relatively mesenchymal phenotype. Conclusions: We describe two novel genes that can be used to prognosticate colon cancer and suggest approaches by which such tumors can be treated. We also describe molecular characteristics of tumors stratified by the SU-GIB signature.

miR-564 acts as a dual inhibitor of PI3K and MAPK signaling networks and inhibits proliferation and invasion in breast cancer.

Dysregulation of PI3K and MAPK pathways promotes uncontrolled cell proliferation, apoptotic inhibition and metastasis. Individual targeting of these pathways using kinase inhibitors has largely been insufficient due to the existence of cross-talks between these parallel cascades. MicroRNAs are small non-coding RNAs targeting several genes simultaneously and controlling cancer-related processes. To identify miRNAs repressing both PI3K and MAPK pathways in breast cancer, we re-analyzed our previous miRNA mimic screen data with reverse phase protein array (RPPA) output, and identified miR-564 inhibiting both PI3K and MAPK pathways causing markedly decreased cell proliferation through G1 arrest. Moreover, ectopic expression of miR-564 blocks epithelial-mesenchymal transition (EMT) and reduces migration and invasion of aggressive breast cancer cells. Mechanistically, miR-564 directly targets a network of genes comprising AKT2, GNA12, GYS1 and SRF, thereby facilitating simultaneous repression of PI3K and MAPK pathways. Notably, combinatorial knockdown of these target genes using a cocktail of siRNAs mimics the phenotypes exerted upon miR-564 expression. Importantly, high miR-564 expression or low expression of target genes in combination is significantly correlated with better distant relapse-free survival of patients. Overall, miR-564 is a potential dual inhibitor of PI3K and MAPK pathways, and may be an attractive target and prognostic marker for breast cancer.