Abstract
BackgroundLigand-dependent activation of the estrogen receptor (ER) as well as of the insulin-like growth factor type 1 (IGF1R) induces the proliferation of luminal breast cancer cells. These two pathways cooperate and are interdependent. We addressed the question of the mechanisms of crosstalk between the ER and IGF1R.MethodsWe evaluated the mitogenic effects of estradiol (E2; agonist ligand of ER) and of insulin (a ligand of IGF1R) in the MCF-7 cells by flow cytometry and by analyzing the cell levels of cell cycle-related proteins (immunoblotting) and mRNA (RT-QPCR). To verify the requirement for the kinase activity of Akt (a downstream target of IGF1R) in the mitogenic action of estradiol, we used shRNA strategy and shRNA-resistant expression vectors.ResultsThe activation of the ER by E2 is unable to induce the cell cycle progression when the phosphatidyl inositol-3 kinase (PI3K)/Akt signaling is blocked by a chemical inhibitor (LY 294002) or by shRNA targeting Akt1 and Akt2. shRNA-resistant Akt wild-type constructs efficiently complemented the mitogenic signaling activity of E2 whereas constructs with inactivated kinase function did not. In growth factor-starved cells, the residual PI3K/Akt activity is sufficient to complement the mitogenic action of E2. Conversely, when ER function is blocked by the antiestrogen ICI 182780, IGF1R signaling is intact but does not lead to efficient reinitiation of the cell cycle in quiescent, growth factor-starved MCF-7 cells. The basal transcription-promoting activity of ligand-free ER in growth factor-starved cells is sufficient to complement the mitogenic action of the IGF1R-dependent signaling.ConclusionsThe basal ER activity in the absence of ligand is sufficient to allow efficient mitogenic action of IGF1R agonists and needs to be blocked to prevent the cell cycle progression.
Highlights
Ligand-dependent activation of the estrogen receptor (ER) as well as of the insulin-like growth factor type 1 (IGF1R) induces the proliferation of luminal breast cancer cells
It has been reported that the mitogenic activity of Insulin-like growth factor type 1 receptor (IGF1R) is blocked by ICI 182780 [11,12]; this anti-estrogen belongs to the category of selective estrogen receptor down-regulators (SERD) since its presence in the cell culture medium leads to a substantial decrease in the content of ER [13]
In our previous work we showed that depletion of Akt1 and 2 prevented the mitogenic signaling by E2 in the MCF-7 cells
Summary
Cell culture Breast cancer-derived cell lines (MCF-7, MELN) were propagated in DMEM supplemented with 10% fetal bovine serum (FBS). It contains a sequence (cloned under pol III promoter in a U6 vector) common to isoforms of Akt and Akt2 [14]. Transfection experiments Cells were transfected with expression vectors containing: shRNA sequence complementary to Akt and Akt mRNA (shAkt1 + 2); shRNA-resistant Akt or Akt (Akt1R and Akt2R); shRNA-kinase dead Akt and Akt (Akt1R/KD and Akt2R/KD); cyclin A-luciferase (indicator of late G1 phase; β-galactosidase (indicator of transfection efficiency). After 3 h incubation with the DNA-containing liposomes, the cells were rinsed and incubated 40 h in serum-free, phenol red-free DMEM with 10 nM ICI 182780 prior to stimulation with E2 for additional 24 h. One microgram of total RNA was reverse transcribed with 200 ng random primers (Invitrogen) and ImProm-II reverse transcriptase (Promega) for 60 min at 42°C, in 20 μl final volume. The mRNA contents were evaluated based on the comparative ΔCT method and normalized to the housekeeping gene 36B4 as described previously [18]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
