Silencing of Keratinocyte Growth Factor Receptor Restores 5-Fluorouracil and Tamoxifen Efficacy on Responsive Cancer Cells

BACKGROUND: Keratinocyte growth factor receptor (KGFR) is a splice variant of the FGFR2 gene expressed in epithelial cells. Activation of KGFR is a key factor in the regulation of physiological processes in epithelial cells such as proliferation, differentiation and wound healing. Alterations of KGFR signaling have been linked to the pathogenesis of different epithelial tumors. It has been also hypothesized that its specific ligand, KGF, might contribute to the development of resistance to 5-fluorouracil (5-FU) in epithelial cancers and tamoxifen in estrogen-positive breast cancers. METHODOLOGY/PRINCIPAL FINDINGS: Small interfering RNA was transfected into a human keratinocyte cell line (HaCaT), a breast cancer derived cell line (MCF-7) and a keratinocyte primary culture (KCs) to induce selective downregulation of KGFR expression. A strong and highly specific reduction of KGFR expression was observed at both RNA (reduction = 75.7%, P = 0.009) and protein level. KGFR silenced cells showed a reduced responsiveness to KGF treatment as assessed by measuring proliferation rate (14.2% versus 39.0% of the control cells, P<0.001) and cell migration (24.6% versus 96.4% of the control cells, P = 0.009). In mock-transfected MCF-7 cells, KGF counteracts the capacity of 5-FU to inhibit cell proliferation, whereas in KGFR silenced cells KGF weakly interferes with 5-FU antiproliferative effect (11.2% versus 28.4% of the control cells, P = 0.002). The capacity of 5-FU to induce cell death is abrogated by co-treatment with KGF, whereas in KGFR silenced cells 5-FU efficiently induces cell death even combined to KGF, as determined by evaluating cell viability. Similarly, the capacity of tamoxifen to inhibit MCF-7 and KCs proliferation is highly reduced by KGF treatment and is completely restored in KGFR silenced cells (12.3% versus 45.5% of the control cells, P<0.001). CONCLUSIONS/SIGNIFICANCE: These findings suggest that selective inhibition of the KGF/KGFR pathway may provide a useful tool to ameliorate the efficacy of the therapeutic strategies for certain epithelial tumors

Analysis of Endogenous LRP6 Function Reveals a Novel Feedback Mechanism by Which Wnt Negatively Regulates Its Receptor▿

The canonical Wnt pathway plays a crucial role in embryonic development, and its deregulation is involved in human diseases. The LRP6 single-span transmembrane coreceptor is essential for transmission of canonical Wnt signaling. However, due to the lack of immunological reagents, our understanding of LRP6 structure and function has relied on studies involving its overexpression, and regulation of the endogenous receptor by the Wnt ligand has remained unexplored. Using a highly sensitive and specific antibody to LRP6, we demonstrate that the endogenous receptor is modified by N-glycosylation and is phosphorylated in response to Wnt stimulation in a sustained yet ligand-dependent manner. Moreover, following triggering by Wnt, endogenous LRP6 is internalized and recycled back to the cellular membrane within hours of the initial stimulus. Finally, we have identified a novel feedback mechanism by which Wnt, acting through β-catenin, negatively regulates LRP6 at the mRNA level. Together, these findings contribute significantly to our understanding of LRP6 function and uncover a new level of regulation of Wnt signaling. In light of the direct role that the Wnt pathway plays in human bone diseases and malignancies, our findings may support the development of novel therapeutic approaches that target Wnt signaling through LRP6

AKT and MAPK signaling in KGF-Treated and UVB-Exposed human epidermal cells

Regulation of proliferation and differentiation in keratinocyte is a complex and dynamic process that involves activation of multiple signaling pathways triggered by different growth factors. Keratinocyte growth factor (KGF) is not only a potent mitogen, but differently from other growth factors, is a potent inducer of differentiation. The MAP kinase and AKT pathways are involved in proliferation and differentiation of many cell types including keratinocytes. We investigated here the role of KGF in modulating AKT and MAPK activity during differentiation of human keratinocytes. Our results show that the mechanisms of action of KGF are dose-dependent and that a sustained activation of the MAPK signaling cascade causes a negative regulation of AKT. We also demostrated increasing expression of KGFR substrates, such as PAK4 during keratinocyte differentiation parallel to the receptor upregulation

The clinical application of autologous bioengineered skin based on a hyaluronic acid scaffold

The aim of this work was to generate an in vitro skin substitute harbouring autologous fibroblasts, keratinocytes and melanocytes, to establish a new one-step clinical method in problems associated with skin disorders. Here we present a case of a nine-year-old girl with a congenital giant nevus treated by surgical approach, with primary co-cultures of keratinocytes, melanocytes and fibroblasts obtained from autologous skin biopsy. Generally these lesions need to be removed to avoid the risk of transformation into malignant melanoma. With this purpose we analyzed the melanocytes contained in the new skin substitute for the presence of genetic alterations correlated to increased risk for melanoma. The organotypical cultures were designed including an engineered scaffold of a non-woven mesh of hyaluronic acid (HYAFF (R) 11). This biomaterial has been previously demonstrated to be the most suitable to maintain polarity and to support the in vitro constructs. Six dermal-epidermal skin substitutes were transplanted and 14 days after surgery the re-epithelialized area was about 90%. Our results suggest that this new dermal-epidermal construct not only reduces hospitalization time and ameliorates scar retraction, but might also represent a solution for the high risk of developing a tumour derived from the original nevus. (c) 2007 Elsevier Ltd. All fights reserved

Modulation of the expression of the FGFR2-IIIb and FGFR2-IIIc molecules in dermatofibroma

[No abstract available

Hair Regeneration from Transected Follicles in Duplicative Surgery: Rate of Success and Cell Populations Involved

BACKGROUND The use of bisected hair follicles in hair transplantation has been previously reported, but the capacity of each half to regenerate the entire hair has not been clarified. OBJECTIVE To evaluate duplicative surgery rate of success and to analyze the cell populations involved in hair regeneration. METHODS We screened 28 patients undergoing duplicative surgery. Approximately 100 hair follicles from each patient were horizontally bisected and implanted. Upper and lower portions were stained for the known epithelial stem cell markers CD200, p63, beta 1-integrin, CD34, and K19. RESULTS Similar percentages of hair regrowth after 12 months were observed when implanting the upper (72.7 +/- 70.4%) and lower (69.2 +/- 71.1%) portions. Expression of CD200, p63, and beta 1-integrin was detected in both portions, whereas K19 and CD34 stained different cell populations in the upper and lower fragment, respectively. CONCLUSION Duplicative surgery might represent a successful alternative for hair transplantation, because both portions are capable of regenerating a healthy hair. Moreover, our results suggest the possible presence of stem cells in both halves of the follicle

Effect of siKGFR transfection on KGFR mRNA and protein in cells treated with 5-FU.

<p>(A) MCF-7 cells were mock transfected or transfected with 5 nM siKGFR. 48 h after transfection, cells were treated with 20 ng/ml KGF, 25 µg/ml 5-FU or KGF plus 5-FU for 24 h. The levels of KGFR mRNA expression were determined by Q-RT-PCR, and normalized to β-actin mRNA levels. The amount of KGFR mRNA in siKGFR-transfected cells was expressed as fold of the level of KGFR mRNA with respect to untreated mock-transfected cells. For each treatment, the graph shows the interquartile range of three independent experiments (boxes), their mean (horizontal dotted bars), and 95% CI (whiskers). The accompanying table reports mean expression level, Standard Error (S.E.), and 95% CI for each assayed sample. <i>P</i> values were determined using the Student's <i>t</i> test: * <i>P</i> = 0.039 versus untreated mock-transfected cells. (B) MCF-7 cells were transfected and treated as in (A), and the amount of KGFR protein was evaluated by Western blot analysis with an anti-bek polyclonal antibody. Tubulin served as a loading control. The amount of KGFR protein was evaluated by densitometric analysis: the values from a representative experiment were standardized to tubulin levels, expressed as fold of KGFR level with respect to untreated mock-transfected cells and reported as a graph.</p

Effect of siKGFR transfection on KGFR mRNA and protein expression levels.

<p>(A) HaCaT cells were mock transfected or transfected with 5 nM siKGFR, and total RNA was extracted from the transfected cells 6, 24, 48, 72 and 96 h later. The levels of KGFR mRNA expression were determined by Q-RT-PCR, and normalized to β-actin mRNA levels. The amount of KGFR mRNA at each time point was expressed as fold of KGFR mRNA level with respect to the 6 h time point. For each time point, the graph shows the interquartile range of three independent experiments (boxes), their mean (horizontal dotted bars) and 95% CI (whiskers). The accompanying table reports mean expression level, Standard Error (S.E.), and 95% CI for each assayed sample. Two-sided Student's <i>t</i> test was used to compare siKGFR-transfected versus mock-transfected cells: * <i>P</i><0.001 (24 h); ** <i>P</i><0.001 (48 h); † <i>P</i><0.001 (72 h); ‡ <i>P</i> = 0.017 (96 h). (B) HaCaT cells were mock transfected or transfected with 5 nM siKGFR. 24 h after transfection, cells were treated with 20 ng/ml KGF for 48 h. The levels of KGFR mRNA expression were determined by Q-RT-PCR, and normalized to β-actin mRNA levels. The amount of KGFR mRNA was expressed as fold of KGFR mRNA levels with respect to untreated mock-transfected cells. Graph and table report the same sets of data as in (A) for each assayed sample. <i>P</i> values were determined using two-sided Student's <i>t</i> test: * <i>P</i> = 0.003 versus untreated mock-transfected cells; ** <i>P</i> = 0.018 versus KGF-treated mock-transfected cells. (C) HaCaT cells were transfected and treated as in (B), and the levels of KGFR protein expression were determined by Western blot analysis using a polyclonal anti-bek antibody. The same blot was probed for tubulin as control for equal loading. The amount of KGFR protein was evaluated by densitometric analysis; the values from a representative experiment were standardized to tubulin levels, expressed as fold of KGFR protein with respect to untreated mock-transfected cells and reported as a graph.</p

Effect of siKGFR transfection on KGF-induced inhibition of 5-FU antiproliferative activity.

<p>(A) MCF-7 cells, grown on coverslips, were mock transfected or transfected with 5 nM siKGFR and 48 h later they were treated or not with 20 ng/ml KGF, 25 µg/ml 5-FU or KGF plus 5-FU. After 24 h, cells were fixed and subjected to immunofluorescence analysis with an anti-Ki67 polyclonal antibody. Nuclei were visualized using 4′, 6-diamido-2-phenylindole dihydrochloride (DAPI). Cell proliferation was evaluated as percentage of Ki67-positive nuclei versus total number of nuclei in ten different areas randomly taken from three different experiments, expressed as mean value±95% CI and reported as a graph. <i>P</i> values were determined using the Student's <i>t</i> test: * <i>P</i> = 0.004 and ** <i>P</i><0.001 versus untreated mock-transfected cells; *** <i>P</i><0.001 versus 5-FU-treated mock-transfected cells; † <i>P</i><0.001 versus KGF-treated mock-transfected cells; ‡ <i>P</i> = 0.002 versus KGF plus 5-FU-treated mock-transfected cells. (B) MCF-7 cells were transfected and treated as in (A), and Western blot analysis of the phosphorylation status of ERK was carried out using a phospho-specific ERK monoclonal antibody (p-ERK). Levels of total ERK were assessed by blotting with an ERK2-specific antibody. The amount of activated ERK was evaluated by densitometric analysis: the values from a representative experiment were standardized to total ERK levels, expressed as fold of p-ERK expression with respect to untreated mock-transfected cells and reported as a graph.</p

Effect of siKGFR transfection on KGF-induced inhibition of Tamoxifen antiproliferative activity in MCF-7 cells.

<p>(A) MCF-7 cells were mock transfected or transfected with 5 nM siKGFR. 48 h after transfection, cells were treated with 20 ng/ml KGF, 20 ng/ml E₂, 100 nM tamoxifen (Tam), KGF plus Tam or E₂ plus Tam for 24 h. The levels of KGFR mRNA expression were determined by Q-RT-PCR, and normalized to β-actin mRNA levels. The amount of KGFR mRNA in siKGFR-transfected cells was expressed as fold of the level of KGFR mRNA with respect to untreated mock-transfected cells. For each treatment, the graph shows the interquartile range of three independent experiments (boxes), their mean (horizontal dotted bars), and 95% CI (whiskers). The accompanying table reports mean expression level, Standard Error (S.E.), and 95% CI for each assayed sample. <i>P</i> values were determined using the Student's <i>t</i> test: * <i>P</i><0.001 versus untreated mock-transfected cells. (B) MCF-7 cells were transfected and treated as in (A), and the amount of KGFR protein was evaluated by Western blot analysis with an anti-bek polyclonal antibody. Tubulin served as a loading control. (C) MCF-7 cells, grown on coverslips, were transfected and treated as in (A). After 24 h, cells were fixed and subjected to immunofluorescence analysis with an anti-Ki67 polyclonal antibody. Nuclei were visualized using 4′, 6-diamido-2-phenylindole dihydrochloride (DAPI). Cell proliferation was evaluated as percentage of Ki67-positive nuclei versus total number of nuclei in ten different areas randomly taken from three different experiments, expressed as mean value±95% CI and reported as a graph. <i>P</i> values were determined using the Student's <i>t</i> test: * <i>P</i><0.001 and ** <i>P</i> = 0.001 versus untreated mock-transfected cells; *** <i>P</i><0.001 versus Tam-treated mock-transfected cells; † <i>P</i><0.001 versus KGF-treated mock-transfected cells; ‡ <i>P</i><0.001 versus KGF plus Tam-treated mock-transfected cells.</p