Phospho-kinase profile of triple negative breast cancer and androgen receptor signaling

Abstract

Background: The androgen receptor (AR) plays a central role in the oncogenesis of different tumors, as is the case in prostate cancer. In triple negative breast cancer (TNBC) a gene expression classification has described different subgroups including a luminal androgen subtype. The AR can be controlled by several mechanisms like the activation of membrane tyrosine kinases and downstream signaling pathways. However little is known in TNBC about how the AR is modulated by these mechanisms and the potential therapeutic strategists to inhibit its expression.

Methods: We used human samples to evaluate the expression of AR by western-blot and phospho-proteomic kinase arrays that recognize membrane tyrosine kinase receptors and downstream mediators. Western-blots in human cell lines were carried out to analyze the expression and activation of individual proteins. Drugs against these kinases in different conditions were used to measure the expression of the androgen receptor. PCR experiments were performed to assess changes in the AR gene after therapeutic modulation of these pathways.

Results: AR is present in a subset of TNBC and its expression correlates with activated membrane receptor kinases-EGFR and PDGFRβ in human samples and cell lines. Inhibition of the PI3K/mTOR pathway in TNBC cell lines decreased notably the expression of the AR. Concomitant administration of the anti-androgen bicalutamide with the EGFR, PDGFRβ and Erk1/2 inhibitors, decreased the amount of AR compared to each agent given alone, and had an additive anti-proliferative effect. Administration of dihydrotestosterone augmented the expression of AR that was not modified by the inhibition of the PI3K/mTOR or Erk1/2 pathways. AR expression was posttranscriptionally regulated by PI3K or Erk1/2 inhibition.

Conclusion: Our results describe the expression of the AR in TNBC as a druggable target and further suggest the combination of bicalutamide with inhibitors of EGFR, PDGFRβ or Erk1/2 for future development.

Figures

Figure 1

Expression of AR in a panel of TNBC cell lines and in human samples. A) Eight cell lines, Hs578T, MDA-MB231, MDA-MB435, HCC 1187, HBL 100, BT 549, HCC 70 and HCC 3153 were cultured and cell lysates were analyzed by western blot for AR expression. α-tubuline was used as a loading control. Expression of the AR gene from public gene expression profile data bases. B) Human samples of triple negative breast cancer patients were processed for protein extraction and lysates were analyzed by western blot for AR expression. α-tubuline was used as a loading control.

Figure 2

Activation of TKRs and their downstream pathways in human samples and in TNBC cell lines. A) Human samples of triple negative breast cancer patients were processed for protein extraction. Lysates containing 1 mg/ml protein were analyzed for the level of tyrosine phosphorylation in a panel of 42 receptor tyrosine kinases (RTKs) and citosolic kinases using antibody array kits. The levels of phosphorylated EGFR, HER2, HER3, PDGFRβ, Akt (S473), Akt (T308) and Erk1/2 are shown (indicated by arrows). B) Relationship between pPDGFRβ and pEGFR and AR expression in human samples. Activated PDGFRβ and EGFR obtained from the antibody array kit were measured using a gray level scale in arbitrary units. AR expression measured by western blot was classified in two groups: negative or positive. Statistical difference (p < 0.05) was analyzed using a Mann–Whitney test. C) Eight cell lines Hs578T, MDA-MB231, MDA-MB435, HCC 1187, HBL 100, BT 549, HCC 70 and HCC 3153 were cultured and cell lysates were analyzed by western blot for the levels of different proteins as well as their phosphorylated forms.

Figure 3

Effect of PI3K/mTOR, Erk1/2 and EGFR/PDGFRβ inhibitors alone or in combination with bicalutamide on the AR expression and cell proliferation in Hs578T and BT549. A) Effect of drugs on AR expression in Hs578T and BT549. Cells were cultured and treated with drugs for 24 h. Cell lysates were analyzed by western blot for AR expression. α-tubuline was used as a loading control. B) Effect of drugs on cell proliferation in Hs578T and BT549. MTT metabolization was performed after 4 days to evaluate cell proliferation. Control cells were untreated. Statistical difference (* = p < 0.05, Bic versus control or drug combination versus drug alone.) was analyzed using a Test T. C) Effect of drugs on cell proliferation in Hs578T and BT549. Cell counting was performed after 4 days to evaluate cell proliferation. Control cells were untreated. Statistical difference (* = p < 0.05, Bic versus control or drug combination versus drug alone.) was analyzed using a Test T.

Figure 4

Effect of DHT and bicalutamide alone and in combination with PI3K/mTOR, Erk1/2 and EGFR/PDGFRβ inhibitors in AR expression and cell proliferation. A) Effect of DHT and bicalutamide, BEZ and PD in alone and combined on AR expression in Hs578T and BT549. Cells were cultured and treated with drugs for 24 h. Cell lysates were analyzed by western blot for AR expression. α-tubuline was used as loading control. B) In parallel, total RNA was extracted and subjected to RT-PCR. Subsequently, amplification products were resolved on agarose gel to determine AR mRNA levels. Actin was used as protein loading control and mRNA as gene control.