Induction of human epithelial stem/progenitor expansion by FOXM1

Abstract

Stem cells are permanent residents of tissues and thought to be targets of cancer initiation. The frequent, and often early, upregulation of the FOXM1 transcription factor in the majority of human cancers suggests that it may participate in the initiation of human tumorigenesis. However, this hypothesis has not been tested. Herein, we show that targeting the ectopic expression of FOXM1 to the highly clonogenic cells of primary human keratinocytes with stem/progenitor cell properties, but not to differentiating cells, caused clonal expansion in vitro. We show, using a functional three-dimensional organotypic epithelial tissue regeneration system, that ectopic FOXM1 expression perturbed epithelial differentiation generating a hyperproliferative phenotype reminiscent of that seen in human epithelial hyperplasia. Furthermore, transcriptional expression analysis of a panel of 28 epithelial differentiation-specific genes reveals a role for FOXM1 in the suppression of epithelial differentiation. This study provides the first evidence that FOXM1 participates in an early oncogenic pathway that predisposes cells to tumorigenesis by expanding the stem/progenitor compartment and deregulating subsequent keratinocyte terminal differentiation. This finding reveals an important window of susceptibility to oncogenic signals in epithelial stem/progenitor cells prior to differentiation, and may provide a significant benefit to the design of cancer therapeutic interventions that target oncogenesis at its earliest incipient stage.

Figure 1

A, Immunohistochemistry staining of p75NTR (green) and FOXM1 (red) in normal human oral mucosa epithelium. B and C, Magnified views of tips of connective tissue papilla and deep-rete ridges, respectively, (dotted boxes) in A are shown here where co-expression (yellow) of p75NTR and FOXM1 are found predominantly in the suprabasal layer and also in isolated cells within the basal cell layer. D, Absolute qPCR analysis of FOXM1B and p75NTR endogenous mRNA expression levels in p75NTR-sorted primary oral keratinocytes three days after flow sorting. RS, random sorted populations. All values are representative of three independent experiments. Bars represent the mean ± SEM of triplicate samples.*P≤0.05, **P≤0.005, ***P≤0.001. E, Endogenous FOXM1 and ΔNp63α protein expression patterns in p75NTR-sorted primary oral keratinocytes following 3 (i) or 12 (ii) days in culture. β-tubulin was blotted for protein loading control. F, Clonogenecity assays for p75NTR-sorted primary oral keratinocytes. Primary human oral keratinocytes were FACS sorted according to p75NTR levels (low or high) and were plated at equal densities in 6 well plates (n=6). Cells were then allowed to clonally expand for 12 days after which time they were either trypsinised to obtain cell number values, or were stained with rhodamine B for colony visualisation and the quantification of colony growth. G, p75NTR^hi cells were transduced with either (i, ii) pSIN-EGFP or (iii, iv) pSIN-EGFP-FOXM1B and individual keratinocyte colonies were examined under bright and fluorescence (FIT-C) microscopy following 3T3-feeder removal. H, Clonogenic assays performed on p75NTR^lo and p75NTR^hi oral keratinocytes following transduction with either pSIN-EGFP or pSIN-EGFP-FOXM1B. I, Quantification of the average colony area of p75NTR^lo and p75NTR^hi oral keratinocytes transduced with either construct. Each bar represents mean ± SEM values obtained from n=9 replicates performed in two individual primary human oral keratinocyte strains. J. Graphical representation of the colony-size distribution according to surface area (mm²) that emerged 15 days after transduction. A total of 200-500 colonies were counted. *P<0.05, **P<0.01, ***P<0.001.

Figure 2

Characterisation of FOXM1B-induced hyperproliferation phenotype using 3D-Organotypic epithelial tissue regeneration model system. Haematoxylin and eosin stained organotypic cultures of human oral mucosa derived from primary human oral keratinocytes retrovirally transduced with either A, pSIN-EGFP or B, pSIN-EGFP-FOXM1B. Adjacent sections of respective organotypic tissues were stained with C and D, FOXM1, E and F, Ki-67 (arrows and arrow heads indicate expression of Ki-67 in basal and suprabasal cells, respectively; dotted line demarcates the basement membrane), G and H, cytokeratin 16 (KRT16), I and J, transglutaminase-1 (TGM1), K and L, cytokeratin 13 (KRT13), M and N, filaggrin (FLG). Nuclear DNA was stained with DAPI (blue).

Figure 3

Transcriptional regulation of keratinocyte differentiation genes in primary human oral keratinocytes transduced with varying levels of FOXM1B. Each data point represents mean ± S.E.M from triplicate determinations of 6 independent primary oral keratinocyte strains. Second-order polynomial regression analysis was performed to obtain the R² coefficient of determination value which indicates the degree of correlation between each gene with FOXM1B.

Figure 4

Absolute quantitative RT-PCR analysis of FOXM1B mRNA expression and A, cornifin (SPRR1B); B, involucrin (IVL); C, desmoglein 3 (DSG3); D, p75NTR and E, CEP55 across a cell panel consisting of 14 normal primary oral keratinocytes, 6 oral premalignant (SVpgC2a, DOK, OKF6/T, POE9n, D19 and D20) and 19 oral SCC cell lines (SCC9, SCC25, UK1, CALH2, SCC15, VB6, SqCC/Y1, CaDe12, 5PT, SCC4, H357, SVFN1-8). Second-order polynomial regression analysis was performed to obtain the R² coefficient of determination value which indicates the degree of correlation between two genes. F, Relative FOXM1B mRNA expression levels in FACS-sorted CD44^lo and CD44^hi cells from cell lines (POE9n, 5PT and CA1) and three independent primary cells extracted from oral SCC tumor tissues (T1-T3), each performed in triplicates. Inset histogram shows respective CD44^lo and CD44^hi cell populations harvested for qPCR analysis.

Figure 5

A schematic model showing the mechanism for FOXM1-induced expansion of epithelial progenitor compartment. We have previously shown that environmental factors such as nicotine (5) and ultraviolet light (6) can directly activate endogenous FOXM1 (1^st oncogenic hit) in primary human oral and skin keratinocytes, respectively. Based on multiple lines of evidence found in this study, we showed that aberrant upregulation of FOXM1 in epithelial stem cells produce progenitor cells with enhanced proliferative capacity which impacts on terminal differentiation, resulting in a hyperplastic phenotype displaying perturbed differentiation characteristics. The deregulated differentiation pathway found in hyperplastic tissues may predispose the ‘expanded’ progenitor compartment to subsequent ‘2^nd’ oncogenic hit necessary for malignant conversion. We hypothesized that FOXM1 mediates a window of cancer susceptibility in the progenitor cells whereby anticancer therapeutic intervention at this stage may prevent cancer initiation.