Keratinocyte growth factor (KGF) induces tamoxifen (Tam) resistance in human breast cancer MCF-7 cells

Both estrogen receptor-alpha (ER-alpha) and progesterone receptor (PR) are good prognostic factors and indicators of benefit from endocrine therapy in breast cancer patients. The relationship of the ER-alpha and PR status and the difference in clinical benefit from endocrine therapy in breast cancer is currently unclear. It has been suggested that keratinocyte growth factors (KGFs) are important regulatory factors in breast cancer. Our laboratory has demonstrated that KGF can act as an estromedin for the stimulation of breast cancer cell growth. Also, KGF stimulates aromatase activity in primary cultured human breast cells. This enzyme is a key to the conversion of androgens to estrogens. In the present study, ER-alpha, two estrogen-regulated genes, PR and PTPgamma, KGF and their relationship with endocrine resistance in human breast cancer cells were investigated.

MCF-7 cells were treated with KGF (1, 5, 10, 20 ng/ml), KGF-13 (0.1, 1, 10 microM) or vehicles as control for 24 hours. KGF-13 is a potential receptor-binding pentapeptide constructed using the KGF peptide residues 101-105 (RTVAV) as a template, located within the beta 4--beta 5 loop. Total RNA were isolated and real-time PCR was employed to identify ER-alpha, PR and PTPgamma gene expressions in response to KGF and KGF-13. Western blot analysis was used to verify the levels of ER-alpha and PR protein, whereas immunohistochemical staining was used to detect PTPgamma expression in MCF-7 cells. To determine the response of MCF-7 cells to endocrine therapy, MCF-7 was treated with either 20 ng/ml KGF or 10 microM KGF-13 combined with 1, 3 and 5 microM of 4-hydroxytamoxifen (4OH-Tam). A non-radioactive cell proliferation assay was applied to determine the growth rate of MCF-7 cells. The results of real-time PCR and the cell proliferation assay were analyzed by Student's t-test and p-values of less than 0.05 were considered statistically significant.

Our data showed that KGF significantly suppressed ER-alpha, PR and PTPgamma expression in MCF-7 cells. KGF suppressed ER-alpha, PR and PTPgamma mRNA to a maximal inhibition at 20 ng/ml by 88%, 57% and 61%, respectively. Western blot analysis and immunohistochemical staining confirmed the down-regulation of ER-alpha, PR and PTPgamma by KGF in protein levels. Ten microM KGF-13 also decreased ER-alpha expression. Ten microM KGF-13 significantly decreased ER-alpha, PR and PTPgamma mRNA expressions by 51%, 57% and 67%, respectively. These KGF-13-mediated mRNA down-regulations were also observed in protein levels. In a cell proliferation assay, 4OH-Tam (3, 5 microM) induced MCF-7 cell death. KGF and KGF-13 alone did not stimulate MCF-7 cell growth. KGF significantly disrupted 4OH-Tam cell killing effects by 1.2- and 1.3-fold at 4OH-Tam concentrations of 3 microM and 5 microM, respectively. KGF-13 significantly disrupted 4OH-Tam cell killing effects by 1.2- and 1.7-fold at 4OH-Tam concentrations of 3 microM and 5 microM, respectively.

Our results suggested that not only ER-alpha and PR but also PTPgamma could be potential bio-makers for growth factor-induced endocrine resistant in breast cancer. KGF might increase the endocrine resistance via decreasing ER-alpha, PR and PTPgamma as well. Moreover, the functional analysis of KGF-13 implied possible applications of using short receptor-binding peptides derived from intact KGF as breast cancer therapeutic agents. Thus, our experimental data provided evidence of KGF-induced anti-hormone resistance in human breast cancer and suggested novel strategies for breast cancer via interference with KGF signaling.