Unknown,Transcriptomics,Genomics,Proteomics

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Expression data from highly purified MMTV-Neu Tumor Initiating Cells (TICs) and the non-TIC CD24- fraction

ABSTRACT: The cancer stem cell model maintains that tumors are organized in a hierarchy driven by tumor initiating cells (TICs), and that patient survival inversely correlates with TIC gene expression. Here we generated a prognostic signature for HER2+ breast cancer from TICs purified from MMTV-Her2/Neu mammary tumors. TICs from this model, identified as Lin-:CD24+:JAG1- at a frequency of 2-5% by serial and single cell transplantation assays, showed elevated expression of proliferation genes and low expression of differentiation genes (compared to non-TIC fraction CD24- of the same tumor). We used microarrays to detect differentially expressed genes in the TIC fraction compared to the non-TIC fraction of the same tumor Cells from each primary tumor were separated by FACS into TIC and non-TIC fractions and total RNA was extracted and hybridized on Affymetrix microarrays.

ORGANISM(S): Mus musculus

SUBMITTER: Jeff Liu

PROVIDER: E-GEOD-29590 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:To model the effect of Pten loss on breast cancer, we deleted Pten using a floxed allele and the deleter lines MMTV-Cre(NLST), which targets stem/bi-potent progenitor cells, and WAP-Cre, which targets CD24-positive, pregnancy-identified stem cells/alveolar progenitors. Mammary tumors were detected in WAP-Cre:Ptenf/f females with a latency of 15.2 months. By 18 months, nearly all mice had succumbed to cancer. MMTV-Cre:Ptenf/f mice developed mammary tumors after a longer latency of 26.4 months and reduced penetrance (70%) compared to WAP-Cre:Ptenf/f mice. Tumors from both models were heterogeneous, consisting primarily of differentiated adenocarcinoma (adenomyoepithelioma; ~70%) and adenosquamous carcinoma (20-25%). In addition, a small fraction of tumors was classified as acinar and poorly differentiated adenocarcinoma (4-7%) and adenosarcoma (3-4%). To test the consequences of combined Pten and p53 gene mutation on breast cancer, we deleted both genes via MMTV-Cre or WAP-Cre. Kaplan-Meier tumor free survival curves revealed that WAP-Cre:Ptenf/f:p53f/f and MMTV-Cre:Ptenf/f:p53f/f females developed tumors with reduced latency of 11.3 and 9.8 months, compared with 15.2, 26.4, and 16.9 months for single-mutant WAP-Cre:Ptenf/f, MMTV-Cre:Ptenf/f or MMTV-Cre:p53f/f mice, respectively. In contrast to the heterogeneity of Pten tumors and small percentage of adenosarcomas in these mice, ~70% of Pten:p53 lesions were histologically classified as adeno-sacrcomatoid-like or mesenchymal-like breast cancer, with the rest exhibiting mixed mesenchymal plus adenocarcinomas and differentiated adenocarcinomas. The adeno-sacrcomatoid-like tumors expressed the mesenchymal markers vimentin, K5, SMA, N-cadherin and desmin but not ER, as well as islands of luminal-like K18 expressing cells surrounded by a layer of K14-positive cells. We used microarrays to detect differentially expressed genes in the Pten:p53 double-knock-out vs Pten or p53 single deletions Total RNA was extracted from tumors developed by double Trizol method and hybridized on Affymetrix microarrays.

Project description:Background: cancer cells rely on glycolysis as main ATP source (Warbürg effect). Tumor-initiating cells (TICs) are the fraction of cells that give raise and repopulate tumors. TICs are exposed to prolonged periods of oxygen and glucose deprivation (OGD), as they live in a hypoxic niche and they withstand prolonged lack of blood vessels during initial tumorigenesis or metastasis formation (avascular phase). Warbürg effect is energetically inefficient; we hypothesize that TICs might have differential metabolic features. Tumor eradication requires killing TICS; finding such features would have therapeutic implications. Methodology/principal findings: 106 MDA-MB-231 breast-cancer cells (hereafter Wt) were exposed for 5 weeks to 0.2% oxygen and 0.1g/l glucose, recovering 9 clones. Both flow-cytometry (50-fold enrichment in the CD24-/CD44+/CD133+ population) and xenografts in NOD/SCID mice using 100 cells (75% vs. 16% engraftment) suggest that OGD-resistant clones are true TICs (hereafter “TIC clones”). TIC clones showed a 30-fold higher replication and viability (BRDU incorporation, colony assay) than Wt. When exposed to OGD, ATP-production dropped 5-fold in WT, but was maintained in TIC clones by increasing 5-fold the fatty acid and oxygen consumption. These properties were explained by lack of upregulation of HIF-1 alpha and PDK1, as well as an increase in ATP-synthase. Analysis with metabolic inhibitors (2-deoxyglucose, antimycin-A) confirmed glycolysis and mitochondrial respiration as main routes of metabolism for Wt and TIC clones respectively. Metabolomics revealed that glutamine catabolism generated the NADPH required to quench reactive oxygen species generated during mitochondrial respiration in TIC clones. Glutamine deprivation or mitochondrial blockers were able to abrogate the viability of TIC clones. Conclusions/significance: the TIC-fraction of a cancer cell population withstands prolonged OGD by switching from the Warbürg effect to mitochondrial respiration. Targeting this metabolic feature abrogates the survival of TICs.

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Expression data from highly purified MMTV-Neu Tumor Initiating Cells (TICs) and the non-TIC CD24- fraction

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