Tumor suppressive functions of Krüppel-like factor 4 (KLF 4) in neuroblastoma

Abstract

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.