First, as shown in Fig. 2A, in SNU-1 and SNU-601 cells along with PI3K inhibition by NVP-BKM120 single treatment, AKT is activated through activated mTOR/S6K/IRS-1 negative feedback mechanism. This is in accordance with recent studies that mTOR or AKT inhibition increases PI3K activity and enhances AKT-independent PI3K pathway (23,26,27). Second, our data showed ERK or STAT3 any other enquiries activation after PI3K inhibition alone in KRAS mutant cancer cell lines as a previous study showed that inhibition of mTORC1 induces RAS pathway as well (28). This compensatory activation of other pro-survival pathways due to inhibition of the PI3K pathway has been reported as prominent between the PI3K/AKT and RAS/MAPK pathways. The PI3K/AKT and RAS/MAPK signaling pathways influence each other rather than function independently, resulting in active and complex crosstalk.

For instance, it has been reported that the interaction of RAS with p110�� is required for RAS-driven oncogenic transformation (29). In this respect, in cancers, RAS activation may limit the activity of single-agent PI3K inhibitors. One of the most typical resistant mechanisms to PI3K inhibition is activating mutations in the RAS/MAPK pathway (14,15,24,30,31). Therefore, to combine inhibition of the PI3K/AKT pathway with inhibition of the RAS/MAPK pathway is a possible approach in order to overcome a compensatory interaction between these pathways (14,24,32). In contrast to the relation between PI3K and RAS pathways, there has been only a few reports on interaction between PI3K and STAT pathways.

STAT3 is a latent cytoplasmic transcription factor and its pathway is one of the key signaling pathways of which deregulation drives tumorigenesis. STAT3 transmits signals to the nucleus where STAT3 binds to specific DNA promoter and regulates gene expression (17,33). In many human cancers, STAT3 is constitutively activated and has been described as a novel molecular target for cancer drug discovery. According to previous studies, mTOR as a serine kinase positively activates STAT3 (34,35). For RAS-dependent malignant transformation, activated STAT3 is essential (16). In addition, activated RAS/MAPK signaling can directly regulate mTOR via p90 ribosomal S6 kinase (RSK)-mediated phosphorylation of Raptor independently of the PI3K/AKT pathway (36).

Taken together, although a PI3K inhibitor suppresses the PI3K/AKT/mTOR signaling pathway, oncogenic RAS activates RAS/mTOR/STAT3 signaling (Fig. 6). Figure Brefeldin_A 6 Schematic representation of RAS/PI3K/mTOR/STAT3 signaling. Constitutively active RAS can regulate the STAT3 pathway in dependent of the canonical PI3K/AKT pathway. In addition, RAS can directly activate mTOR via RSK-mediated phosphorylation of Raptor. … Therefore, we hypothesized that in KRAS mutant gastric cancer cells, the blockade of both PI3K/AKT/mTOR and KRAS/mTOR/STAT pathways using NVP-BKM120 and AG490 would be synergistic.