Fig 2: Treatment with DAC in combination with DEX synergistically induces the expression of miR-708 in TNBC cell lines(A–D) Expression of miR-708 in MCF-10A (A), MDA-MB-231 (B), HCC-1395 (C), and Hs578T (D) cells after treatment with 100 nM DEX, 10 μM DAC, or their combination for 6 days. The data shown here represent the normalized means ± SD (n = 3 biological replicates). ANOVA followed by Tukey’s post hoc test was used for the statistical analysis (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).(E) mRNA expression of the miR-708 downstream target genes Rap1B and CD44 in MDA-MB-231 cells after treatment with 100 nM DEX, 10 μM DAC, or their combination for 6 days. The data shown here represent the normalized means ± SD (n = 3 biological replicates). ANOVA followed by Tukey’s post hoc test was used for the statistical analysis (∗p < 0.05 and ∗∗∗p < 0.001).(F) Left panel: Western blot analysis of Rap1B and CD44 levels in MDA-MB-231 cells after treatment with 100 nM DEX, 10 μM DAC, or both drugs for 6 days. Actin was used as the internal control. Right panel: Quantitative analysis of normalized Rap1B and CD44 protein levels with actin. Histograms represent normalized means ± SEM (n = 3 biological replicates). ANOVA followed by Tukey’s post hoc test was used for the statistical analysis (∗p < 0.05).

Fig 3: miR-28-5p directly targets and downregulates Rap1b expression in glioma cells. (A,B) miR-28-5p expression is inversely correlated with Rap1b mRNA and protein expression in glioma cells. We analyzed the expression of the endogenous Rap1b after transiently transfecting cells with either (a) miR-28-5p mimics; (b) miR-28-5p inhibitor; (c) control miR-28-5p-NC (ctrl) in A172, U87MG, U373MG, and SNB19 cells. The transient transfection efficiency was determined by qRT-PCR and Western blot. As expected, Rap1b mRNA expression levels were significantly reduced by miR-28-5p mimics and significantly enhanced by miR-28-5p inhibitor in each of the four glioma cell lines (A). Similar, the Rap1b protein expressions displayed the same patterns as those of mRNA expressions in U87MG, U373MG, and SNB19 cells (B). The densitometry analysis performed using the ImageQuant program are on the right side of each Western blot gel. (C,D) miR-28-5p inhibits glioma cell proliferation and invasion by directly targeting Rap1b. To further clarify whether or not tumor suppressive roles of miR-28-5p were dependent on Rap1b expression, 100 nM of siRap1b was co-transfected with 30 nM of miR28-5p inhibitor in A172, U87MG, U373MG, and SNB19 glioma cell lines to assess glioma cells' proliferation by MTT assay and evaluate glioma cell invasion by Matrigel transwell invasion assay. The reduction of Rap1b expression by siRap1b effectively reduced the miR-28-5p inhibitor-induced glioma cell proliferation (C), and the miR-28-5p inhibitor-induced invasion (D, Photomicrographs were taken at 10x magnification) in each of the four glioma cell lines above. All data are from triplicate samples performed in two different independent experiments. (*P < 0.05, **P < 0.01).

Fig 4: Rap1b elevates glioma cell proliferation and invasion. (A) All the glioma cell lines A172, SF767, SNB19, U373MG, and U87MG expressed the Rap1b protein (upper panel). We then transfected siRap1b along with the control into the in A172, U87MG, U373MG, and SNB19 cells, the efficiency of the transfection was detected by the expression of total Rap1b protein, as examined by Western blot. Rap1b siRNA markedly inhibited Rap1b expression (lower panel). The corresponding densitometry analysis for each Western blot performed using the ImageQuant program are on the right sides correspondingly. (B,C) Rap1b elevates glioma cell proliferation and invasion. MTT and cell invasion assay were conducted to examine the effects of Rap1b on the proliferation and invasion after transfecting A172, U87MG, U373MG, and SNB19 with synthesized specific small interfering RNAs (siRap1b) targeting Rap1b mRNA. The results showed that transfection with siRap1b resulted in a significant decrease in proliferation (B) and invasion (C, Photomicrographs were taken at 10x magnification) compared with the respective siRNA control groups in the four glioma cell lines. These results suggested that Rap1b increases glioma cell line proliferation and invasion, and Rap1b may perform tumor promoting roles in glioma growth and metastasis [*P < 0.05, one-way ANOVA for proliferation assay in (B), Student t-test for invasion assay in C]. All data are from triplicate samples performed in two different independent experiments.

Fig 5: Crystal structure rendering of PIK3CAI459_T462del and expression of mTOR/AKT and MAPK signaling pathways in transduced C2C12 cell lines, OHSU-SARC001, and after molecularly targeted kinase inhibitors. (A) PIK3CAI459_T462del interfaces with the regulatory PIK3CA subunit and may disrupt the interaction between PI3K catalytic and regulatory subunits to induce catalytic activation. (B) C2C12 stable cell lines harboring wild-type and mutant cDNA suggest that PIK3CAI459_T462del modestly activates pAktT308, pAktS473, pTscT1462, p70s6kT389, pS6S235/236, p4ebp-1T37/46, and pEerkT202/Y204 compared to WT PIK3CA. Of note, 4EBP1 undergoes sequential hyperphosphorylation on Thr37 and Thr46, followed by up to six residues in the carboxyl terminus. This increase in 4EBP1 phosphorylation decreases its electrophoretic mobility. GNASR201C does not appear to activate the mTOR/Akt or MAPK pathway compared to WT GNAS. (C) OHSU-SARC001 has PTEN loss and expresses RAP1B, B-Raf, and C-Raf. (D) In OHSU-SARC001, phosphorylation of downstream mTOR/AKT signal effectors p70S6KT389 and pS6S235/236 was decreased when exposed to LY3023414, everolimus, and rapamycin. Effectors p70S6KT421/S424 were decreased in trametinib-treated cells. Trametinib treatment resulted in decreased expression of pERKT202/Y204.