Stemness Gene Expression Profiles in Cancer Stem Cell Progenies Derived from a Cell Line Panel +/- BRAF Mutation
Gary Sommerville, Paul Smyth, John J O'Leary, Orla Sheils. Trinity College Dublin, Dublin, Ireland
Background: BRAF mutation is implicated in up to 8% of all solid tumours and so represents an important target for therapeutic intervention. The V600E BRAF mutation confers constitutive kinase activity, independent of mitogen initiation, within the MAPK-ERK pathway leading to aberrant cell signalling.
Cancer stem cells are reputed to exist in many human cancers. They possess characteristics similar to normal human stem cells and are believed to contribute to disease aggressiveness, metastasis and chemoresistance through their capacity for self-renewal and differentiation.
This project sought to derive stem cell holoclones from various different cancer cell lines, (BRAF WT & MUT), to determine if they have stemness characteristics and to compare their expression profiles.
Design: Established melanoma, thyroid, ovarian and colon cancer cell lines were used in this study. BRAF status was determined using a modified SNP-allelic discrimination TaqMan assay. Parental cells were plated at low density onto high salt agar to positively select for holoclone formation. RNA was extracted from parental cells and derived holoclones. TaqMan RT-PCR was employed to investigate stemness gene expression profiles of holoclones relative to parental cells. The targets examined were:
NANOG -Stem cell proliferation and self-renewal
Oct4 -Stem cell pluripotency
SNAIL2 -Epithelial Mesenchymal Transition
Sonic hedgehog homolog (Shh) -Stem cell proliferation control
TGF-β -TGF pathway ligand
CDKNIB -Endogenous control
Expression of NANOG protein was assessed by immunoflouresence with Confocal Microscopy.
Results: Each holoclone displayed upregulation of at least 2 stemness genes compared to parent cells. NANOG; involved in self-renewal, was the most consistently upregulated marker of all holoclones analyzed. Melanoma cell lines (WT & MUT) had upregulation of each target analysed.
NANOG protein expression was significantly increased in holoclones compared to parental cells, was localised to the apical side and cell surface -a pattern also seen in ES cells and putative cancer stem cells.
After holoclone harvesting, plates were maintained. New holoclones consistently grew at the precise locations previously harvested, implying a strong capacity for self-renewal.
Conclusions: We present evidence that cancer stem cells can be consistently derived from established cell lines. These cells possess characteristics similar to normal human stem cells as displayed by gene expression profiling and immunofluoresence. We also observed capacity for self-renewal in these cancer cell supopulations.
Category: Special Category - Pan-genomic/Pan-proteomic approaches to Cancer
Tuesday, March 20, 2012 9:30 AM
Poster Session III # 281, Tuesday Morning