Insulin-like growth factor binding protein-6 modulates proliferative antagonism in response to progesterone in breast cancer

Abstract

Breast cancer is one of the most diagnosed cancers worldwide. The insulin-like growth factor (IGF) system promotes proliferation and survival in breast cancer cells and is regulated by 6 insulin-like growth factor binding proteins (IGFBPs). The IGFBPs sequester IGFs to prolong their half-life and attenuate binding to insulin-like growth factor 1 receptor (IGF1R). While IGFBP-6 has been studied in some cancers it has not been studied extensively in hormone receptor positive breast cancer. Survival analysis using available databases indicated that high IGFBP-6 levels improve overall survival in progesterone receptor positive breast cancers. IGFBP-6 is transcriptionally induced by progesterone in T47D breast cancer cells resulting in increased intracellular and extracellular IGFBP-6 protein. Knockdown of IGFBP-6 resulted in reduced proliferative antagonism when estradiol stimulated T47D cells were cotreated with progesterone and protein levels of both progesterone receptor isoforms (PR-A and PR-B) were decreased following knockdown of IGFBP-6. P21(Cip1/Waf1), which is progesterone responsive, was not induced in response to progesterone following knockdown of IGFBP-6. Cyclin E2, a cell cycle regulator, is induced by progesterone only when IGFBP-6 is knocked down. Stable overexpression of IGFBP-6 in MCF-7 cells resulted in an increase in Epidermal Growth Factor Receptor (EGFR) and this expression was further enhanced when cells were cotreated with progesterone and estradiol. These results indicate that IGFBP-6 is a regulator of progesterone action, and that PR is required for the observed protective effects of IGFBP-6 in breast cancer.

Keywords: breast cancer; insulin- like growth factor binding protein; progesterone; progesterone receptor; steroid hormones.

Figure 1

High IGFBP-6 expression is associated with better overall survival and recurrence free survival in R+ breast cancers. (A) Overall ival analysis using RNA-Seq data. PR positive patient data are separated by the median. (B) Overall survival analysis using GeneChip Data from GEO and EGA. PR positive patient data are again separated by the median. (C) Recurrence-free survival analysis using Gene chip data. PR positive patient data were separated by the upper quartile. (D) Overall survival analysis using RNA-seq data for ER positive and PR positive patients. Data is divided by the median. (E) Overall survival analysis using RNA-seq data for ER positive and PR negative patients. Data were divided by the median.

Figure 2

IGFBP-6 is induced by Progesterone (P4) in T47D breast cancer cells. Asterisks indicate significance relative to the vehicle. (* means p< 0.05, ** means p<0.01, and *** means p<0.001) (A) IGFBP-6 transcriptional expression with various steroid hormone and mifepristone treatments (p<0.0001). V stands for ethanol, P for progesterone, E for estradiol, and R for mifepristone (RU486). (B) IGFBP-6 protein levels follow transcriptional levels (p<0.0001). (C) Representative intracellular IGFBP-6 immunoblot. (D) IGFBP-6 secretion. Units are in fg/mL/cell. IGFBP-6 concentrations are normalized by cell count (p<0.01). (E) Relative proliferation of T47D cells in response to steroid hormones (p<0.0001).

Figure 3

Knockdown of IGFBP-6 counteracts proliferative antagonism of progesterone. Asterisks indicate significance relative to the negative control for each steroid treatment. (* means p< 0.05, ** means p<0.01). (A) IGFBP-6 transcript following siRNA mediated knockdown. All values are log base 2 transformed. For the knockdown p< 0.0001, for steroid treatments p<0.0001, and the interaction was not significant. (B) Cell proliferation after IGFBP-6 knockdown. All values are relative to cells treated with the vehicle which was set to 1 for each siRNA. For the knockdown p<0.05, for steroid treatments p< 0.0001, and for the interaction p<0.01. (C) Representative intracellular IGFBP-6 immunoblot. (D) Representative extracellular IGFBP-6 immunoblot.

Figure 4

Knockdown of IGFBP-6 reduces Progesterone Receptor levels. Asterisks indicate significance relative to the negative control for each steroid treatment. (* means p< 0.05, ** means p<0.01, and *** means p<0.001). (A) PR-A levels decrease following IGFBP-6 knockdown. Statistical analysis was performed on Rank-Transformed data. For the knockdown p<0.0001, for the steroid treatments p<0.0001, and for the interaction p<0.05. (B) PR-B levels decrease following IGFBP-6 knockdown. For the knockdown p<0.0001, for the steroid treatments p<0.0001, and interactions were nonsignificant. (C) Representative immunoblot for PR-A and PR-B.

Figure 5

Downstream Progesterone signaling is modulated by knockdown of IGFBP-6. Progesterone signaling is dysregulated upon IGFBP-6 knockdown. Asterisks indicate significance relative to the negative control for each steroid treatment. (* means p< 0.05, ** means p<0.01, and *** means p<0.001). (A) Representative P21 immunoblot. (B) Relative P21 levels following IGFBP-6 knockdown. For knockdowns p< 0.0001, steroid treatments were nonsignificant, and for the interaction p<0.001. (C) Representative Cyclin E2 Immunoblot. (D) Relative Cyclin E2 levels following IGFBP-6 knockdown. Statistical analyses were performed on Rank-transformed data. For the knockdown p<0.0001, for steroid treatments p<0.001, and for interaction p<0.05. (E) Representative EGFR immunoblot. (F) Relative EGFR levels following IGFBP-6 knockdown. For knockdowns p< 0.01, for steroid treatments p< 0.0001, and for the interaction p<0.05.

Figure 6

High IGFBP-6 levels are associated with PR levels in Hormone Receptor Positive Cells Lines. (A) Comparison of PR levels in T47D and MCF-7 cells. (B) Comparison of IGFBP-6 levels in T47D and MCF-7 Cells in response to P4 and E2.

Figure 7

Stable IGFBP-6 Over-expression increases EGFR levels in response to hormonal treatments. Asterisks indicate significance between EV and BP6 cells for each steroid treatment. (* means p< 0.05, ** means p<0.01, and *** means p<0.001). (A) Stable overexpression of IGFBP-6 increases EGFR levels in MCF-7 cells. (B) Relative EGFR levels are increased with IGFBP-6 over-expression. EGFR levels increase even higher following estradiol plus progesterone treatment. P<0.001 for each cell type, p < 0.01 for the steroid treatments, and p <0.01 for the interaction. (C) PR levels following IGFBP-6 stable overexpression in MCF-7 cells. (D) Cell Proliferation following steroid hormone treatments in stable MCF-7 cells.