Targeting of GPCR and EGF receptor overcomes PI3K inhibitor resistance in PTEN-null breast cancer (2020)
Figure 6

Figure 6. PAR1 signals through GNB2 to sustain the activation of PI3K pathway in the presence of PI3K or HER inhibitors.

(A) Schematic of the results from the drug screening with compounds targeting GPCR signaling. MDA-MB-468 cells were treated with the compounds of the GPCR-targeted library at three different concentrations (0.1, 1 or 10 μM) in combination with vehicle, GDC0941 450 nM or lapatinib 1 μM. The pS6 signal was measured by IF after 24 hours of treatment and normalized to DAPI. Z scores of the normalized fluorescence values for each drug measured in the presence of vehicle, GDC0941 or lapatinib are reported on the y axis or on the left or right x axis of the dot plot, respectively. Readings acquired following treatment with the three different concentrations of the GPCR-targeted drugs are reported in 3 different colours. Dots corresponding to vorapaxar treatments at the three different concentrations are highlighted in the plot. The values reported are mean of a biological triplicate of the experiment.

(B) MDA-MB-468 were treated for 24 h with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The cell lysates were probed with the indicated antibodies.

(C) MDA-MB-468 parental cells or GNB2 KO clones were starved and treated with scramble or PAR1 activating peptide for 5 minutes, alone or in combination with vorapaxar. The cell lysates were probed with the indicated antibodies. Phospho-AKT, pan-AKT, phospho-ERK1/2 and pan-ERK1/2 bands were quantified by the use of ImageLite software: the ratio of phospho-AKT to pan-AKT and phospho-ERK1/2 to pan-ERK1/2 normalised to the control peptide-treated conditions for WT and GNB2 KO cells (first and forth lanes, respectively) is shown.

(D) p-S6/loading control signal from western blots experiments in which a panel of six TNBC PTEN-null cell lines and HCC70 cells that acquired resistance to AZD8186 or MK2206 were treated with vehicle, vorapaxar (10 μM), GDC0941 (1 μM) or lapatinib (1 μM) alone or in the indicated combinations. The values were normalised to vehicle treatment for all cell lines. Mean of 2 independent experiments ± SD. P values calculated by two-tailed paired student t-test *P=0.0235 and **P=0.002. Cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395, BT-549, HCC70 AZD8186-resistant and HCC70 MK2206-resistant.

(E) Long-term proliferation assay of MDA-MB-468 cells treated with vorapaxar (5 or 2.5 μM), GDC0941 (1 μM) or lapatinib (1 μM), alone or in the indicated combinations. Cells were treated for two weeks and stained by crystal violet. One representative experiment of three is shown.

(F) Schematic of the network controlling the phosphorylation of AKT and S6 in PTEN-null TNBC cells. EGFR and PAR1-βγ subunits of G protein signal in parallel to p110β and AKT. The two arms of the pathways are inter-connected by feedback compensation mechanisms that act when any of the two signals is perturbed. Combined targeting of both the signaling arms results in a sustained inhibition of the downstream pathway. Erlotinib (EGFR inhibitor) and AZD8186 (p110β inhibitor) are highlighted in the figure as these drugs proved effective in in vivo experiments.