A novel activating role of SRC and STAT3 on HGF transcription in human breast cancer cells

Abstract

We have previously determined that the HGF promoter can be transactivated by a combination of activated Src and wild-type Stat3 in the mouse breast cell lines HC11 and SP1. To determine if this pathway is of relevance for the human disease, a series of human breast and other human cells lines were examined, and the status of key proteins in these cells determined. All of the human breast cell lines exhibited strong transactivation by a combination of activated Src and Stat3. This activation was dependent on a Stat3 recognition element present at nt-95. The exception was the ErbB2 over-expressing cell line SK-BR-3 where Stat3 alone could transactivate HGF though Src augmented this effect. Increased phosphorylation of Stat3 tyrosine 705 was also observed in this line. Analysis of three ovarian cell lines revealed that Src/Stat3 expression was not able to activate the HGF promoter in two of these lines (SKOV3 and IOSE-80PC). Src/Stat3 expression did activate HGF transcription in OVCAR3 cells, but this effect was not mediated by the Stat3 site at nt-95. Stat3 phosphorylation at tyrosine 705 was observed in IOSE-80PC cells, but was insufficient to allow for activation of the HGF promoter. Human kidney (HEK293) and cervical carcinoma (HeLa) cells were also not Src/Stat3 permissive, despite high levels of Stat3 phospho-Y705. These results suggest that human breast cells are a uniquely permissive environment for HGF transactivation by Src/Stat3 which may allow for the inappropriate activation of HGF transcription during the early stages of breast transformation. This could lead to paracrine or autocrine activation of the Met receptor in breast carcinoma cells.

Figure 1

Src/Stat3 mediated HGF promoter transactivation in mouse epithelial breast cell lines. A DNA sequence alignment between the mouse HGF proximal promoter spanning from nt-274 to +29 is shown with the comparable region of the human gene (A). The Stat3 site at nt-95 is boxed and the transcriptional start site is indicated by an arrow. The 0.3 HGF-Renilla or -95 M 0.3 HGF-Renilla mouse proximal promoters were co-transfected with either activated Src or Stat3 alone, or in combination in: mouse mammary carcinoma SP1 cells (B), non-tumorigenic mouse mammary HC11 cells (C), and non-tumorigenic mouse mammary EPH4 cells (D). 48 hours post-transfection, cells were lysed and assayed for dual-luciferase activity. Values were normalized using a CMV-Luc internal control and are presented as fold induction relative to the empty vector (EV) for 0.3 HGF-Renilla. Asterisks indicate a significant increase in HGF promoter activity compared to the 0.3 HGF-Renilla empty vector control (P = 0.001*, P = 0.0005** using a Student's T-test). Transfections were repeated three times and values represent average results of triplicate samples +/- SD.

Figure 2

Endogenous expression and activity of HGF/Met signaling proteins in mouse and human epithelial cell lines. Endogenous protein expression and activation levels of HGF, Met, Src and Stat3 were characterized in mouse and human epithelial cell lines by western blot analysis. Whole cell lysates collected from the indicated cell lines were normalized and resolved on a reducing SDS-PAGE gel. Western blot analysis was performed using antibodies probing for the indicated proteins (arrows point to relevant band). γ-Tubulin was used as a loading control to normalize for overall protein concentration, and recombinant HGF (rHGF) served as a control for HGFα migration.

Figure 3

Src/Stat3 mediated HGF promoter transactivation in human epithelial breast cell lines. The 0.3 HGF-Renilla or -95 M 0.3 HGF-Renilla mouse proximal promoters were co-transfected with either activated Src or Stat3 alone, or in combination in: human non-tumorigenic 184-hTERT (A) and MCF10a breast cells (B), human MCF-7 adenocarcinoma breast cells (C), human T47-D ductal carcinoma breast cells (D), and human SK-BR-3 adenocarinoma breast cells (E). 48 hours post-transfection, cells were lysed and assayed for dual-luciferase activity. Values were normalized using a CMV-Luc internal control and are presented as fold induction relative to the empty vector (EV) for 0.3 HGF-Renilla. Asterisks indicate a significant increase in HGF promoter activity compared to the 0.3 HGF-Renilla empty vector control (P = 0.001*, 0.0003**, 0.0005*** using a Student's T-test). Transfections were repeated three times and values represent average results of triplicate samples +/- SD.

Figure 4

Src/Stat3 mediated HGF promoter transactivation in human non-breast epithelial cell lines. The 0.3 HGF-Renilla or -95 M 0.3 HGF-Renilla mouse proximal promoters were co-transfected with either activated Src or Stat3 alone, or in combination in: human ovarian tumorigenic OVCAR3 (A) and SKOV3 cells (B), human ovarian non-tumorigenic ovarian IOSE-80PC cells (C), human embryonic kidney HEK293 adenocarcinoma cells (D), and human cervical carcinoma HeLa cells (E). 48 hours post-transfection, cells were lysed and assayed for dual-luciferase activity. Values were normalized using a CMV-Luc internal control and are presented as fold induction relative to the empty vector (EV) for 0.3 HGF-Renilla. Asterisks indicate a significant increase in HGF promoter activity compared to the 0.3 HGF-Renilla empty vector control (P = 0.0005* using a Student's T-test). Transfections were repeated three times and values represent average results of triplicate samples +/- SD.