Neuroblastoma is a childhood solid tumor of a unique propensity to either regress
spontaneously or grow relentlessly. Emerging evidence indicated that neuroblastoma
contains heterogeneous populations of cells, and commitment of these cells to
neuronal lineage may result in aggressive progression in patients, whereas to
fibromuscular lineage may give a favorable outcome. However, mechanism(s)
controlling the lineage commitment of neuroblastoma cells remains to be identified.
Our preliminary data suggested that Kr?ppel-Like Factor 4 (KLF4) might promote
neuroblastoma regression. KLF4 is a transcription factor regulating a variety of
cellular functions, including proliferation and cell cycle progression. Recent studies
have demonstrated that KLF4 may act as both tumor suppressor and oncogene in a
cell-context dependent manner. Importantly, our preliminary data showed that low
KLF4 expression is highly associated with poor clinical outcomes of the
neuroblastoma patients. In addition, we found that overexpression of KLF4
suppresses neuroblastoma cell growth accompanied with loss of tumorigenicity.
Morphologically, KLF4 overexpressing cells changed their morphologies to become
epithelial-like, strongly substrate-adherent and expressing smooth muscle marker.
Therefore, we hypothesized that KLF4 exerts its effects through two ways, it may (i)
function to inhibit cell growth and reduce tumorigenicity; and (ii) promote
differentiation of the neuroblastoma cells to the non-tumorigenic, fibromuscular-like
cells.
RT-PCR data revealed the differential expression of KLF4 in 11 neuroblastoma cell
lines. In particular, a modest expression was found in Be(2)C, a cell line which was
formerly demonstrated to differentiate and form tumor in mice xenograft assay. It
was therefore chosen as the study model.
To assess the effects of KLF4 knockdown on tumor growth, stable knockdown clones
from Be(2)C cells were established by lentiviral transduction of KLF4-targeting
shRNA. In parallel, clones that stably expressed non-target shRNA were used as
controls. After the transduction, two stable knockdown clones showing significant
KLF4 downregulation were isolated from single colony (monoclonal stable clones)
and a pool of cells (polyclonal stable clones) respectively. The cell proliferation and
growth rate of the stable clones were then measured by 5-bromo-2’-deoxyuridine
(BrdU) proliferation assay and growth curve assay. The results have indicated that
both monoclonal and polyclonal stable KLF4 knockdown clones grow faster than the
control clones. In order to examine the tumorigenicity in vivo, the stable clones were
xenotransplanted to severe combined immunodeficient mice. The stable KLF4
knockdown clones showed a significant higher growth rate and formed a larger
tumor. The stable clones were also treated with BrdU for four weeks for
differentiation towards fibromuscular lineage. As anticipated, the control clones
showed fibromuscular features, like more flattened and epithelial-like morphology. In
contrast, the stable KLF4 knockdown clones failed to present the fibromuscular
features after treatment. In addition, immunocytochemistry staining of SMA and
quantitative analysis of the immunocytochemistry further confirmed that only the
control clones showed higher SMA expression after BrdU treatment, while there is no
change in the SMA expression in the stable KLF4 knockdown clones. These results
demonstrated that KLF4 functioned by inhibiting neuroblastoma cell proliferation
and growth, reducing the tumorigenicity, and it was required for fibromuscular
differentiation. / published_or_final_version / Surgery / Master / Master of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/208425 |
| Date | January 2011 |
| Creators | Tsoi, Lai-shan, 蔡麗珊 |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Rights | Creative Commons: Attribution 3.0 Hong Kong License, The author retains all proprietary rights, (such as patent rights) and the right to use in future works. |
| Relation | HKU Theses Online (HKUTO) |
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