Keratinocyte Growth Factor Receptor Restores 5-Fluorouracil and Tamoxifen Efficacy on Resp

Silencing of Keratinocyte Growth Factor Receptor Restores 5-Fluorouracil and Tamoxifen Efficacy on Responsive Cancer Cells.Sabrina Rotolo 1, Simona Ceccarelli 1, Ferdinando Romano 1, Luigi Frati 1,2, Cinzia Marchese 1, Antonio Angeloni 1* 1 Dipartimento di Medicina Sperimentale, Universita` Sapienza di Roma, Roma, Italy, 2 Istituto Neurologico Mediterraneo ‘‘Neuromed, Pozzilli, ItalyAbstract : BackgroundKeratinocyte 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.Abstract : Methodology/Principal FindingsSmall 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).Abstract : Conclusions/SignificanceThese 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.

Keratinocyte growth factor receptor (KGFR/FGFR2-IIIb) is a tyrosine kinase protein that belongs to the family of the fibroblast growth factor receptors (FGFRs). KGFR represents a splicing transcript variant of FGFR2 gene and is expressed on epithelial cells of different organs. The alternatively spliced isoform, known as FGFR2-IIIc, is found in cells of mesenchymal lineages [1], [2]. KGFR plays a key role in the control of epithelial growth and differentiation, carrying out its biological effects in a paracrine way [3] through high affinity binding to its specific ligands, namely keratinocyte growth factor (KGF/FGF7), FGF10 and FGF22 [4]. Among them, KGF acts not only as a potent mitogen for primary human keratinocytes, but also promoting their differentiation program [5] and protecting them against apoptosis induction [6], [7]. Furthermore, KGF is involved in both experimental [8], [9] and in vivo [10] wound healing models, stimulating migration of keratinocytes [11], [12] and inducing reorganization of actin cytoskeleton, therefore increasing epithelial cell motility [13].Recently, there has been growing interest about the potential role of alterations of KGF/KGFR signaling in epithelial tumorigenesis. Increased KGFR mRNA expression has been detected in a wide range of tumors of epithelial origin, such as lung, colon, gastric, pancreas and prostate cancers. In some cases, such increased expression seems to be associated with cell transformation and, perhaps, malignant progression [14]. Moreover, KGF administration has been shown to increase cell motility in estrogen receptor (ER)-positive breast tumor cells [15], [16], to be potentially involved in breast cancer progression and metastasis [17] and to enhance the invasive potential of gastric carcinoma derived cell lines overexpressing KGFR [18].Other studies led to hypothesize that KGF may exert antiapoptotic activity on certain cancer cells as well as inhibition of apoptosis induced by the chemotherapeutic drug 5-fluorouracil (5-FU) [19]–[22]. The development by cancer cells of resistance to traditional chemotherapeutic agents, such as 5-FU, is frequently observed and remains a major obstacle to a successful treatment of cancer and a prominent cause of tumor recurrence after chemotherapy [23].Furthermore, it has been suggested that alterations of the pathways involving FGFs and cognate receptors might represent one of the mechanisms of resistance to tamoxifen that finally develops in many ER-positive breast cancers [24]–[27]. However, this hypothesis is not completely ascertained and the specific role played by the large family of the FGFs is far from being understood.The approach based on selective downregulation of proteins involved in cellular processes correlated to tumor progression represents a promising frontier for cancer treatment. Transfection of specific small interfering RNAs (siRNAs) is a powerful tool to achieve a gene-specific knockdown and represents a potent therapeutic strategy for the treatment of several diseases, such as viral infections, neurological disorders and cancers [28].The exclusive target specificity of siRNA against disease-relevant mRNAs is an essential prerequisite for utilization of this technology. In this study, we selectively downregulated KGFR mRNA and protein expression in three epithelial cell lines, HaCaT keratinocytes, MCF-7 breast cancer cell and primary cultured keratinocytes (KCs), by a new approach of siRNA design, based on the utilization of DICER endonuclease substrate 27-mer dsRNAs to trigger RNA interference (RNAi). This technique provides enhanced efficacy and longer duration of RNAi as compared to traditional 21-mer siRNAs, allowing usage of lower concentrations of RNAi in target cells, which greatly reduces the side effects. Furthermore, it allows the targeting of sites that are refractory to suppression with 21-mer siRNAs [29].We analyzed the effects of KGFR siRNA on characteristics bio-parameters such as cell viability, proliferation, apoptosis and migration of the tested cell lines. Finally, we evaluated whether the downregulation of KGFR expression is able to inhibit 5-FU and tamoxifen resistance induced by KGF in cell cultures.
Citation: Rotolo S, Ceccarelli S, Romano F, Frati L, Marchese C, et al. (2008) Silencing of Keratinocyte Growth Factor Receptor Restores 5-Fluorouracil and Tamoxifen Efficacy on Responsive Cancer Cells. PLoS ONE 3(6):e2528.doi:10.1371/journal.pone.0002528
Editor: Stefan Maas, Lehigh University, United States of America
Copyright: © 2008 Rotolo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was partially supported by grants from the MIUR, Cofin 2005, and from Regione Lazio grant issued for Progetto di Ricerca Finalizzata 2005. Funders were not directly nor indirectly involved in designing or conducting the study in any part.
Competing interests: The authors have declared that no competing interests exist.

E-mail: antonio.angeloni@uniroma1.it

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