2018; 167:731C740. models of varying PTEN status and tumor types. RESULTS AZD8186 has antitumor efficacy in TNBC cells with PTEN loss mutation that did not result in loss of PTEN protein. Open in a separate window Figure 1 Effects of AZD8186 on cell proliferation studies [12], we defined that the cell lines with IC50 less than 1 M are sensitive to AZD8186. Cell lines were treated for 72 hours with DMSO or AZD8186. Cell growth was measured using SRB colorimetric assay. IC50 calculated by dose-response isobologram curves was used to evaluate sensitivity. The results showed that out of ten TNBC cell lines, three (BT-549, MDA-MB-468, and MDA-MB-436) were sensitive to AZD8186 with IC50 of 31, 358, and 899 nM, respectively (Figure 1B, Supplementary Table 1). Other seven cell lines were not Tulobuterol responding to AZD8186 treatment as their IC50s were all over 2 M (Figure 1B, Supplementary Table 1). Using colony formation assays, we confirmed the sensitivity of the three PTEN-deficient cell lines, MDA-MB-436, MDA-MB-468, and BT-549, to AZD8186. Cells were treated with DMSO or AZD8186 at 1 M every other 3-4 days for two weeks. Colony staining showed that AZD8186 treatment substantially reduced the capability of all these cell lines to form colonies, compared to the vehicle controls (Figure 2A). Quantitation results demonstrated that cells with AZD8186 treatment had significantly less total colony area than control in all the three cell lines (0.01) (Figure 2B). Together with the results of cell proliferation assay Tulobuterol above, these findings suggest that AZD8186 is capable of inhibiting cell growth of TNBC cells that are deficient in PTEN. Open in a separate window Figure 2 Effect of AZD8186 on colony formation ability.(A) Colony formation assay. TNBC MDA-MB-436, MDA-MB-468, and BT-549 cells were seeded in 6-well plates in triplicate for each treatment group. Cells were treated with DMSO or AZD8186 at 1 M for 2 weeks, with refreshing drug every 3-4 days. Crystal violet-stained colonies were imaged and scanned. (B) Colony quantitation. Total colony area was quantitated using Image J v.1.48 software. Values presented as mean SD are obtained from triplicate wells from single experiment. * values for AZD8186 vs DMSO control (< 0.01 for all three cell lines). AZD8186 inhibits Akt signaling To evaluate the mechanism of action of AZD8186 in PTEN-deficient tumors, we assessed its effect on PI3K signaling. Four TNBC cell lines, including PTEN loss MDA-MB-436, MDA-MB-468 cell lines and non-PTEN loss Sum-159, MFM-223 cell lines, were evaluated. The TNBC cells were treated with AZD8186 at 2 M, or DMSO for 2 hours. Immunoblotting showed that compared to the vehicle control, treatment with AZD8186 moderately but significantly decreased levels of phospho-AKT in both PTEN loss cell lines, normalized by -actin (0.037 and 0.0002, respectively) (Figure 3A, 3B-1). On the other hand, in both non-PTEN loss cell lines, AZD8186 did not reduce phospho-AKT levels (Figure 3A, 3B-1). These phospho-AKT changes were also seen by normalization with total AKT levels (Figure 3B-2). Normalization with total proteins also showed that AZD8186 inhibited phosphorylation of S6K and PRAS40 which are downstream targets of AKT in the PTEN loss cell lines (Figure 3A, 3B-3, 3B-4). In non-PTEN loss cell lines, except phospho-S6K in Sum-159 cells, AZD8186 did not exhibit an inhibitory effect on phosphorylation of S6K and PRAS40 (Figure 3A, 3B-3, 3B-4). In order to validate drug efficacy and immunoblotting, we compared AZD8186 with AKT inhibitor AZD5363. We found that AZD5363 enhanced phospho-AKT levels in all the four cell lines, particularly striking in non-PTEN loss cells (Figure 3A, 3B-1, 3B-2), but AZD5363 decreased phospho-S6K and phospho-PRAS40 levels in some of these cell lines (Figure 3A, 3B-3, 3B-4). Open in a separate window Figure 3 Effects of AZD8186 Tulobuterol on cell signaling and cell cycle.(A) Immunoblotting of PI3K pathway. PTEN-loss MDA-MB-436 and MDA-MB-468 cells and PTEN-wild-type Sum-159 and MFM-223 cells were treated with DMSO or AZD8186 at 2 M for 2 hours. Cell lysates were loaded for SDS-PAGE and blotted with the indicated antibodies. Top -actin panel is a loading control for phosphoproteins, and bottom -actin panel is a loading TMPRSS2 control for non-phosphoproteins. (B) Quantitation of immunoblotting..