Fig. 9: PHLDA1/2 mediate cancer-specific ERK-AKT crosstalk and impact on the drug-sensitivity of tumor cells. | Nature Communications

Fig. 9: PHLDA1/2 mediate cancer-specific ERK-AKT crosstalk and impact on the drug-sensitivity of tumor cells.

From: Qualitative differences in disease-associated MEK mutants reveal molecular signatures and aberrant signaling-crosstalk in cancer

Fig. 9

a Simultaneous expression of PHLDA1/2 is critical for efficient inhibition of AKT. HEK293 cells were transfected with inactive HA-AKT1(K/M), together with Myc-PHLDA1 and/or 2, or 3 as indicated. The cells were treated with insulin for 10 min, and the cell lysates were probed for AKT1 phosphorylated at T308 and S473 and for protein levels. b H1299 and HCT116 cells were transfected with siRNAs targeting PHLDA1 and/or PHLDA2. Expression levels of PHLDA1/2, the phosphorylation states of AKT and ERK, and their protein levels were analyzed by immunoblotting. c, d Trametinib-mediated PHLDA1/2 downregulation activates AKT. H1299 cells (c), and those stably expressing Myc-PHLDA1/2 or empty vector (control) (d) were treated with trametinib for the indicated times. Immunoblotting was performed as in b. In (d), apoptosis was monitored by immunoblotting of cleaved-caspase 3 and PARP. e The indicated cells were treated with trametinib (10 μM) for 72 h, and apoptosis was detected by annexin V staining. f The indicated cells were cultured with etoposide (1 μM) with or without trametinib (10 μM) for 4 days. The cell survival rate was determined by CCK8 assay. e, f Data are mean ± SEM (n = 3). P-values were assessed using one-way ANOVA followed by Tukey’s multiple comparisons test. ns, not significant. g–i H1299 control cells (black arrowheads) and H1299-PHLDA1/2 cells (red arrowheads) were transplanted into the left and right flanks of mice, respectively. Mice were then treated with vehicle or trametinib (3 mg/kg, 3 times a week) by oral gavage. g Representative images of the mice on day 16. h The growth curve of the xenograft tumors. i The weight of excised xenograft tumors on day 16 (vehicle, n = 7; trametinib, n = 6). Data are mean ± SEM. P-values were assessed using two-tailed Student t-test (h) or using one-way ANOVA followed by Tukey’s multiple comparisons test (i). In (h), *p < 0.05; **p < 0.01. Source data are provided as a Source Data file.

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