In-depth characterization of breast cancer tumor-promoting cell transcriptome by RNA sequencing and microarrays

Callari, Maurizio; Guffanti, Alessandro; Soldà, Giulia; Merlino, Giuseppe; Fina, Emanuela; Brini, Elena; Moles, Anna; Cappelletti, Vera; Daidone, Maria Grazia

doi:10.18632/oncotarget.5810

Cited by 8 publications

(9 citation statements)

References 49 publications

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“…Therefore, we generated oncospheres in T47D NUDT5 RES cell lines and, in order to gain a more global understanding of the gene expression changes which took place following the transition between 2D and 3D (Figure 2A), RNA-seq was carried out in NUDT5 RES grown in the two conditions (Figure S2A–C, Table S3). A significant portion of the 5850 genes, which significantly changed their expression between 2D and 3D culture conditions (Figure 2B), were found within a curated set of 480 oncosphere-regulated genes (Figure 2C and Table S4), including genes involved in EMT, oncosphere growth, signalling, adhesion, pluripotency and stem cells [21,22,23] referring to the dEMT database (). The most significant functional enrichment and expression changes between 2D and 3D were observed for genes involved in EMT (108 genes) (Figure 2C–D).…”

Section: Resultsmentioning

confidence: 99%

Expression of Oncogenic Drivers in 3D Cell Culture Depends on Nuclear ATP Synthesis by NUDT5

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Pardow

Carbonell-Caballero

et al. 2019

Cancers

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The growth of cancer cells as oncospheres in three-dimensional (3D) culture provides a robust cell model for understanding cancer progression, as well as for early drug discovery and validation. We have previously described a novel pathway in breast cancer cells, whereby ADP (Adenosine diphosphate)-ribose derived from hydrolysis of poly (ADP-Ribose) and pyrophosphate (PPi) are converted to ATP, catalysed by the enzyme NUDT5 (nucleotide diphosphate hydrolase type 5). Overexpression of the NUDT5 gene in breast and other cancer types is associated with poor prognosis, increased risk of recurrence and metastasis. In order to understand the role of NUDT5 in cancer cell growth, we performed phenotypic and global expression analysis in breast cancer cells grown as oncospheres. Comparison of two-dimensional (2D) versus 3D cancer cell cultures from different tissues of origin suggest that NUDT5 increases the aggressiveness of the disease via the modulation of several key driver genes, including ubiquitin specific peptidase 22 (USP22), RAB35B, focadhesin (FOCAD) and prostagladin E synthase (PTGES). NUDT5 functions as a master regulator of key oncogenic pathways and of genes involved in cell adhesion, cancer stem cell (CSC) maintenance and epithelial to mesenchyme transition (EMT). Inhibiting the enzymatic activities of NUDT5 prevents oncosphere formation and precludes the activation of cancer driver genes. These findings highlight NUDT5 as an upstream regulator of tumour drivers and may provide a biomarker for cancer stratification, as well as a novel target for drug discovery for combinatorial drug regimens for the treatment of aggressive cancer types and metastasis.

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Section: Resultsmentioning

confidence: 99%

Expression of Oncogenic Drivers in 3D Cell Culture Depends on Nuclear ATP Synthesis by NUDT5

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Pardow

Carbonell-Caballero

et al. 2019

Cancers

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“…Propagation of the primary mammary tumor cells in vitro in chemically defined, serum-free media as non-adherent tumorspheres preserves the high BTIC fraction found in the primary tumors, whereas culturing the tumor cells in serum-containing media as adherent cells reduced BTIC frequencies by 4–5 orders of magnitude [5]. Others have also shown that culturing cells from human breast tumors and breast tumor cell lines as tumorspheres similarly increases BTIC frequencies [6, 7].…”

Section: Introductionmentioning

confidence: 99%

Monoamine oxidase-A activity is required for clonal tumorsphere formation by human breast tumor cells

Gwynne

Shakeel

et al. 2019

Cell Mol Biol Lett

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BackgroundBreast tumor growth and recurrence are driven by an infrequent population of breast tumor-initiating cells (BTIC). We and others have reported that the frequency of BTIC is orders of magnitude higher when breast tumor cells are propagated in vitro as clonal spheres, termed tumorspheres, by comparison to adherent cells. We exploited the latter to screen > 35,000 small molecules to identify agents capable of targeting BTIC. We unexpectedly discovered that selective antagonists of serotonin signaling were among the hit compounds. To better understand the relationship between serotonin and BTIC we expanded our analysis to include monoamine oxidase-A (MAO-A), an enzyme that metabolizes serotonin.MethodsWe used the Nanostring technology and Western blotting to determine whether MAO-A is expressed in human breast tumor cell lines cultured as tumorspheres by comparison to those grown as adherent cells. We then determined whether MAO-A activity is required for tumorsphere formation, a surrogate in vitro assay for BTIC, by assessing whether selective MAO-A inhibitors affect the frequency of tumorsphere-forming cells. To learn whether MAO-A expression in breast tumor cells is associated with other reported properties of BTIC such as anticancer drug resistance or breast tumor recurrence, we performed differential gene expression analyses using publicly available transcriptomic datasets.ResultsTumorspheres derived from human breast tumor cell lines representative of every breast cancer clinical subtype displayed increased expression of MAO-A transcripts and protein by comparison to adherent cells. Surprisingly, inhibition of MAO-A activity with selective inhibitors reduced the frequency of tumorsphere-forming cells. We also found that increased MAO-A expression is a common feature of human breast tumor cell lines that have acquired anticancer drug resistance and is associated with poor recurrence-free survival (RFS) in patients that experienced high-grade, ER-negative (ER−) breast tumors.ConclusionsOur data suggests that MAO-A activity is required for tumorsphere formation and that its expression in breast tumor cells is associated with BTIC-related properties. The discovery that a selective MAO-A inhibitor targets tumorsphere-forming cells with potencies in the nanomolar range provides the first evidence of this agent’s anticancer property. These data warrant further investigation of the link between MAO-A and BTIC.

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“…The pathways of ʻsubstance metabolism and transport', including ʻsteroid metabolic process' and ʻcholesterol metabolic process' were found to be enriched in the two lists of DEGs. As steroid is an important metabolite, the dysregulation of its functions is associated with the generation of CSCs in many types of cancers and could affect progression (34)(35)(36). In this study, MVD, which is related to cholesterol biosynthesis, was found to be present in both of these two pathways: ʻsteroid biosynthetic/metabolic process' and ʻterpenoid backbone biosynthesis' in the Huh7-C sample.…”

Section: Discussionmentioning

confidence: 65%

Long non-coding RNAs and genes contributing to the generation of cancer stem cells in hepatocellular carcinoma identified by RNA sequencing analysis

et al. 2016

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Cancer stem cells (CSCs) play important roles in cancer initiation, progression and metastasis. The aim of the present study was to identify the potential targets that may contribute to the generation of hepatocellular carcinoma stem cells (HCSCs) from hepatocellular carcinoma (HCC) cells. The RNA sequencing (RNA‑Seq) dataset GSE70537 was downloaded from the Gene Expression Omnibus (GEO) database. Raw RNA sequences were mapped to the GRCh37/hg19 genome based on TopHat and assembled through Cufflinks. Cuffdiff of Cufflinks was used for the screening of differentially expressed genes (DEGs) in the two types of HCSCs (Hep3B‑C and Huh7‑C) compared with the two types of HCC cells (Hep3B and Huh7) which were satisfied by the criteria of |log2(RPKMHCSC/RPKMHCC)| >1 and p<0.05. In addition, based on the Database for Annotation, Visualization, and Integrated Discovery (DAVID), we screened the Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways which were enriched in the DEGs. For the DEGs with consistent differential expression in the two lists of DEGs, the LncRNA2Target database was used for the identification of long non‑coding RNA (lncRNA)‑gene pairs. A total of 218 and 591 DEGs were identified for the Hep3B‑C and Huh7‑C samples, respectively, and 22 overlaps were obtained. Biological processes and pathways related to steroid biosynthesis/metabolism or other substance transport were found to be enriched in the two lists of DEGs. Among the 22 overlaps, 16 were found to be consistently differentially expressed in the two HCSC samples, and the lncRNA‑gene regulatory network of these genes was constructed. Moreover, several potential biomarkers that may play important roles in the transformation of HCSCs were identified in the regulation network. Through the bioinformatics analysis of the RNA‑Seq dataset, several novel targets that were associated with the progression of HCC were obtained, and these targets may be valuable for the treatment and prognosis of HCC.

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In-depth characterization of breast cancer tumor-promoting cell transcriptome by RNA sequencing and microarrays

Cited by 8 publications

References 49 publications

Expression of Oncogenic Drivers in 3D Cell Culture Depends on Nuclear ATP Synthesis by NUDT5

Expression of Oncogenic Drivers in 3D Cell Culture Depends on Nuclear ATP Synthesis by NUDT5

Monoamine oxidase-A activity is required for clonal tumorsphere formation by human breast tumor cells

Long non-coding RNAs and genes contributing to the generation of cancer stem cells in hepatocellular carcinoma identified by RNA sequencing analysis

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