HCPA4 - Differential complementary positional prroteomics of human carboxypeptidase 4 treated PC3-lysates
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ-Orbitrap
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Prostate Cancer Cell
SUBMITTER: 
Petra Van Damme
LAB HEAD: Petra Van Damme
PROVIDER: PRD000228 | Pride | 2010-05-19
REPOSITORIES: Pride
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Project description:BACKGROUND: Cancer stem-like cells are proposed to sustain solid tumors by virtue of their capacity for self-renewal and differentiation to cells that comprise the bulk of the tumor, and have been identified for a variety of cancers based on characteristic clonal morphologies and patterns of marker gene expression. METHODS: Single cell cloning and spheroid culture studies were used to identify a population of cancer stem-like cells in the androgen-independent human prostate cancer cell line PC3. RESULTS: We demonstrate that, under standard culture conditions, ~10% of PC3 cells form holoclones with cancer stem cell characteristics. These holoclones display high self-renewal capability in spheroid formation assays under low attachment and serum-free culture conditions, retain their holoclone morphology when passaged at high cell density, exhibit moderate drug resistance, and show high tumorigenicity in scid immunodeficient mice. PC3 holoclones readily form spheres, and PC3-derived spheres yield a high percentage of holoclones, further supporting their cancer stem cell-like nature. We identified one gene, FAM65B, whose expression is consistently up regulated in PC3 holoclones compared to paraclones, the major cell morphology in the parental PC3 cell population, and two genes, MFI2 and LEF1, that are consistently down regulated. This molecular profile, FAM65Bhigh/MFI2low/LEF1low, also characterizes spheres generated from parental PC3 cells. The PC3 holoclones did not show significant enriched expression of the putative prostate cancer stem cell markers CD44 and integrin α2β1. PC3 tumors seeded with holoclones showed dramatic down regulation of FAM65B and dramatic up regulation of MFI2 and LEF1, and unexpectedly, a marked increase in tumor vascularity compared to parental PC3 tumors, suggesting a role of cancer stem cells in tumor angiogenesis. CONCLUSIONS: These findings support the proposal that PC3 tumors are sustained by a small number of tumor-initiating cells with stem-like characteristics, including strong self-renewal and pro-angiogenic capability and marked by the expression pattern FAM65Bhigh/MFI2low/LEF1low. These markers may serve as targets for therapies designed to eliminate cancer stem cell populations associated with aggressive, androgen-independent prostate tumors such as PC3. (Mol Cancer. 2010 Dec 29;9:319. doi: 10.1186/1476-4598-9-319; PMID: 21190562; PMCID: PMC3024252).
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Project description:BACKGROUND: Cancer stem-like cells are proposed to sustain solid tumors by virtue of their capacity for self-renewal and differentiation to cells that comprise the bulk of the tumor, and have been identified for a variety of cancers based on characteristic clonal morphologies and patterns of marker gene expression. METHODS: Single cell cloning and spheroid culture studies were used to identify a population of cancer stem-like cells in the androgen-independent human prostate cancer cell line PC3. RESULTS: We demonstrate that, under standard culture conditions, ~10% of PC3 cells form holoclones with cancer stem cell characteristics. These holoclones display high self-renewal capability in spheroid formation assays under low attachment and serum-free culture conditions, retain their holoclone morphology when passaged at high cell density, exhibit moderate drug resistance, and show high tumorigenicity in scid immunodeficient mice. PC3 holoclones readily form spheres, and PC3-derived spheres yield a high percentage of holoclones, further supporting their cancer stem cell-like nature. We identified one gene, FAM65B, whose expression is consistently up regulated in PC3 holoclones compared to paraclones, the major cell morphology in the parental PC3 cell population, and two genes, MFI2 and LEF1, that are consistently down regulated. This molecular profile, FAM65Bhigh/MFI2low/LEF1low, also characterizes spheres generated from parental PC3 cells. The PC3 holoclones did not show significant enriched expression of the putative prostate cancer stem cell markers CD44 and integrin M-NM-12M-NM-21. PC3 tumors seeded with holoclones showed dramatic down regulation of FAM65B and dramatic up regulation of MFI2 and LEF1, and unexpectedly, a marked increase in tumor vascularity compared to parental PC3 tumors, suggesting a role of cancer stem cells in tumor angiogenesis. CONCLUSIONS: These findings support the proposal that PC3 tumors are sustained by a small number of tumor-initiating cells with stem-like characteristics, including strong self-renewal and pro-angiogenic capability and marked by the expression pattern FAM65Bhigh/MFI2low/LEF1low. These markers may serve as targets for therapies designed to eliminate cancer stem cell populations associated with aggressive, androgen-independent prostate tumors such as PC3. (Mol Cancer. 2010 Dec 29;9:319. doi: 10.1186/1476-4598-9-319; PMID: 21190562; PMCID: PMC3024252). Four PC3 cell-derived holoclones, selected based on their clear and unambiguous holoclone morphology and designated holoclones 2H10, 2G7, 1A8 and 5A2, were used for global transcriptome/microarray analysis in direct comparison to parental PC3 cells. Total RNA was prepared from each of four sequential cell passages for each PC3-derived holoclone, and from four passages of parental PC3 cells, 48 h after seeding the cells at 100,000 cells per well of a 6-well plate. For each sample (holoclones or parental PC3 cells), a pool of RNA was prepared by combining equal amounts of RNA from each passage to minimize the effects of passage number and inter-sample variability on gene expression profiles. All RNAs had an RNA integrity number >8.0, determined using an Agilent Bioanalyzer 2100 instrument. cDNAs transcribed from pools of RNA for each holoclone, and for parental PC3 cells, were labeled with Alexa 647 or Alexa 555 dyes in a fluorescent reverse pair (dye swap) design for competitive hybridization to Agilent Whole Human Genome Microarrays.
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