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Aldh1 Expression and Activity Increase During Tumor Evolution in Sarcoma Cancer Stem Cell Populations.

ABSTRACT: Tumors evolve from initial tumorigenic events into increasingly aggressive behaviors in a process usually driven by subpopulations of cancer stem cells (CSCs). Mesenchymal stromal/stem cells (MSCs) may act as the cell-of-origin for sarcomas, and CSCs that present MSC features have been identified in sarcomas due to their ability to grow as self-renewed floating spheres (tumorspheres). Accordingly, we previously developed sarcoma models using human MSCs transformed with relevant oncogenic events. To study the evolution/emergence of CSC subpopulations during tumor progression, we compared the tumorigenic properties of bulk adherent cultures and tumorsphere-forming subpopulations both in the sarcoma cell-of-origin models (transformed MSCs) and in their corresponding tumor xenograft-derived cells. Tumor formation assays showed that the tumorsphere cultures from xenograft-derived cells, but not from the cell-of-origin models, were enriched in CSCs, providing evidence of the emergence of bona fide CSCs subpopulations during tumor progression. Relevant CSC-related factors, such as ALDH1 and SOX2, were increasingly upregulated in CSCs during tumor progression, and importantly, the increased levels and activity of ALDH1 in these subpopulations were associated with enhanced tumorigenicity. In addition to being a CSC marker, our findings indicate that ALDH1 could also be useful for tracking the malignant potential of CSC subpopulations during sarcoma evolution.

SUBMITTER: Martinez-Cruzado L

PROVIDER: S-EPMC4904413 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Aldh1 Expression and Activity Increase During Tumor Evolution in Sarcoma Cancer Stem Cell Populations.

Martinez-Cruzado Lucia L Tornin Juan J Santos Laura L Rodriguez Aida A García-Castro Javier J Morís Francisco F Rodriguez Rene R

Scientific reports 20160613

Tumors evolve from initial tumorigenic events into increasingly aggressive behaviors in a process usually driven by subpopulations of cancer stem cells (CSCs). Mesenchymal stromal/stem cells (MSCs) may act as the cell-of-origin for sarcomas, and CSCs that present MSC features have been identified in sarcomas due to their ability to grow as self-renewed floating spheres (tumorspheres). Accordingly, we previously developed sarcoma models using human MSCs transformed with relevant oncogenic events. ...[more]

PMID: 27292183

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Project description:BackgroundAldehyde dehydrogenase 1 (ALDH1) activity has been implicated in the therapeutic drug resistance of many malignancies and has been widely used as a marker to identify stem-like cells, including in primary liver cancer. Cancer stem cells (CSCs) are thought to play a crucial role in cancer progression and metastasis. In order to clarify the validity of the rabbit VX2 liver cancer model, we questioned if it expresses ALDH1 as a potential marker of CSCs. Hepatocellular carcinoma is a common malignancy worldwide and has poor prognosis. Most of the animal models used to study hepatocellular carcinoma are rodent models which lack clinical relevance. The rabbit VX2 model is a large animal model useful for preclinical and for developing drugs targeting cancer stem cells.Materials and methodsWe used flow cytometry to identify rabbit VX2 liver tumor cells that express ALDH1A1 activity at a high level and confirmed the results with RT-PCR, immunohistochemical and western blot analyses. Further, mRNA and protein expression analysis of tumor samples also express the markers for stemness like klf4, oct3/4, CD44 and nanog as well as the differentiation marker α-fetoprotein.ResultsWe used Aldefluor flow cytometry-based assay to identify cells with high ALDH1 activity in the rabbit VX2 liver cancer model. We used the brightest 4.39 % of the total cancer cell population in our study. We performed semi-quantitative as well as real time PCR to characterize the stemness derived from VX2 tumors and tissues from normal rabbit liver. We demonstrated that VX2 tumors have higher expression of cancer stem cell markers such as AlDH1A1 and CD44 in comparison to normal rabbit liver cells. Additionally, real time PCR analysis of the same samples using syber-green demonstrated the significant change (p > 0.05) in the expression of genes. We validated the gene expression of the stemness markers by performing western blot and immunofluorescence. We showed that cancer stem cell markers (AlDH1A1, CD44) and the differentiation marker α-fetoprotein were upregulated in VX2 tumor cells. The same extent of upregulation was observed in stemness markers (klf4, oct3/4 and nanog) in VX2 tumors in comparison to normal rabbit liver.ConclusionThe overall results of this study indicate that ALDH1 is a valid CSC marker for VX2 cancer. This finding suggests that the rabbit VX2 liver cancer model is useful in studying drug resistance in hepatocellular carcinoma and may be useful for basic and preclinical studies of other types of human cancer.

Project description:Gene copy-number variation, which provides the raw material for the evolution of novel genes, is surprisingly widespread in natural populations. Experimental evolution studies have demonstrated an extremely high spontaneous rate of origin of gene duplications. When organisms are suboptimally adapted to their environment, gene duplication may compensate for reduced fitness by amplifying promiscuous activity of a gene, or increasing dosage of a suboptimal gene. The overarching goal of this study is to inverstigate whether CNVs constitute a common mechanism of adaptive genetic change during compensatory evolution and to further characterize the role of natural selection in dictating their evolutionary spread at a population-genomic level. Outcrossing populations of C. elegans with low fitness were evolved for >200 generations and the frequencies of CNVs in these populations were analyzed by oligonucleotide array comparative genome hybridization, quantitative PCR, and single-worm PCR. Multiple duplications and deletions were detected in intermediate to high frequencies and several lines of evidence suggest that the changes in frequency were adaptive. 1) Many copy-number changes reached high frequency, were near fixation, or were fixed in a short time. 2) Many independent duplications and deletions in high frequency harbor overlapping regions which likely include genes that are under selection for either higher or lower rates of expression. 3) The size spectrum of deuplications and deletions in the adaptive recovery populations is significantly larger than that of spontaneous copy-number variants in mutation accumulation experiments. This is expected if larger CNVs are more likely to encompass genes that are being selected for altered gene dosage. Out results validate the great potential borne by gene copy-number changes for compensatory evolution and adaptation. Experimental genome evolution of copy-number variants in 25 experimental lines compared to 5 ancestral control lines.

Project description:UnlabelledMicroRNAs (miRNAs) are small, ∼ 22-nucleotide-long RNAs that regulate gene expression posttranscriptionally. Kaposi's sarcoma-associated herpesvirus (KSHV) encodes 12 pre-miRNAs during latency, and the functional significance of these microRNAs during KSHV infection and their cellular targets have been emerging recently. Using a previously reported microarray profiling analysis, we identified breakpoint cluster region mRNA (Bcr) as a cellular target of the KSHV miRNA miR-K12-6-5p (miR-K6-5). Bcr protein levels were repressed in human umbilical vein endothelial cells (HUVECs) upon transfection with miR-K6-5 and during KSHV infection. Luciferase assays wherein the Bcr 3' untranslated region (UTR) was cloned downstream of a luciferase reporter showed repression in the presence of miR-K6-5, and mutation of one of the two predicted miR-K6-5 binding sites relieved this repression. Furthermore, inhibition or deletion of miR-K6-5 in KSHV-infected cells showed increased Bcr protein levels. Together, these results show that Bcr is a direct target of the KSHV miRNA miR-K6-5. To understand the functional significance of Bcr knockdown in the context of KSHV-associated disease, we hypothesized that the knockdown of Bcr, a negative regulator of Rac1, might enhance Rac1-mediated angiogenesis. We found that HUVECs transfected with miR-K6-5 had increased Rac1-GTP levels and tube formation compared to HUVECs transfected with control miRNAs. Knockdown of Bcr in latently KSHV-infected BCBL-1 cells increased the levels of viral RTA, suggesting that Bcr repression by KSHV might aid lytic reactivation. Together, our results reveal a new function for both KSHV miRNAs and Bcr in KSHV infection and suggest that KSHV miRNAs, in part, promote angiogenesis and lytic reactivation.ImportanceKaposi's sarcoma (KS)-associated herpesvirus (KSHV) infection is linked to multiple human cancers and lymphomas. KSHV encodes small nucleic acids (microRNAs) that can repress the expression of specific human genes, the biological functions of which are still emerging. This report uses a variety of approaches to show that a KSHV microRNA represses the expression of the human gene called breakpoint cluster region (Bcr). Repression of Bcr correlated with the activation of a protein previously shown to cause KS-like lesions in mice (Rac1), an increase in KS-associated phenotypes (tube formation in endothelial cells and vascular endothelial growth factor [VEGF] synthesis), and modification of the life cycle of the virus (lytic replication). Our results suggest that KSHV microRNAs suppress host proteins and contribute to KS-associated pathogenesis.

Dataset Information

Aldh1 Expression and Activity Increase During Tumor Evolution in Sarcoma Cancer Stem Cell Populations.

Publications

Aldh1 Expression and Activity Increase During Tumor Evolution in Sarcoma Cancer Stem Cell Populations.

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