Fibroblast-Induced Paradoxical PI3K Pathway Activation in PTEN-Competent Colorectal Cancer: Implications for Therapeutic PI3K/mTOR Inhibition

Abstract

PurposeTumor-microenvironment interactions are important determinants of drug resistance in colorectal cancer (CRC). We, therefore, set out to understand how interactions between genetically characterized CRC cells and stromal fibroblasts might influence response to molecularly targeted inhibitors.TechniquesSensitivity to PI3K/AKT/mTOR pathway inhibitors of CRC cell lines, with known genetic background, was investigated under different culture conditions [serum-free medium, fibroblasts’ conditioned medium (CM), direct co-culture]. Molecular pathway activation was monitored using Western Blot analysis. Immunoprecipitation was used to detect specific mTOR complex activation. Immunofluorescence was used to analyze cellular PTEN distribution, while different mutant PTEN plasmids were used to map the observed function to specific PTEN protein domains.ResultsExposure to fibroblast-CM resulted in increased growth-inhibitory response to double PI3K/mTOR inhibitors in PTEN-competent CRC cell lines harboring KRAS and PI3K mutations. Such functional effect was attributable to fibroblast-CM induced paradoxical PI3K/mTORC1 pathway activation, occurring in the presence of a functional PTEN protein. At a molecular level, fibroblast-CM induced C-tail phosphorylation and cytoplasmic redistribution of the PTEN protein, thereby impairing its lipid phosphatase function and favored the formation of active, RAPTOR-containing, mTORC1 complexes. However, PTEN’s lipid phosphatase function appeared to be dispensable, while complex protein-protein interactions, also involving PTEN/mTOR co-localization and subcellular distribution, were crucial for both mTORC1 activation and sensitivity to double PI3K/mTOR inhibitors.Data InterpretationMicroenvironmental cues, in particular soluble factors produced by stromal fibroblasts, profoundly influence PI3K pathway signaling and functional response to specific inhibitors in CRC cells, depending on their mutational background and PTEN status.

Exosomes Derived from Hypoxic Glioma Cells Reduce the Sensitivity of Glioma Cells to Temozolomide Through Carrying miR-106a-5p

“…36,37 In addition, PTEN was confirmed to inhibit the cell growth via inactivation of Akt signaling. 38,39 Consistently, this research found exosomes derived from hypoxic glioma cells could 40 and our study was similar to this previous research. Smad7 was known to be an inhibitor of cancer cell invasion, and its overexpression could inhibit the malignant behavior of cancer cells.…”

“… 36 , 37 In addition, PTEN was confirmed to inhibit the cell growth via inactivation of Akt signaling. 38 , 39 Consistently, this research found exosomes derived from hypoxic glioma cells could reduce the expression of PTEN and upregulate the level of p-Akt in TMZ-treated glioma cells. Meanwhile, Zhu et al found miR-106a-5p could promote the tumorigenesis of gastric cancer through targeting Smad7, 40 and our study was similar to this previous research.…”

Bcl-2 dependent modulation of Hippo pathway in cancer cells

“…Human melanoma A375 and M14, non-small cell lung cancer (NSCLC) H460, and breast adenocarcinoma MDA-MB-231 cell lines were maintained in RPMI-1640 complete medium (Euroclone, Milan, IT) containing 10% inactivated fetal bovine serum (FBS) (GIBCO, ThermoFisher Scientific, Waltham, MA, USA), 1% L-glutamine (Euroclone) and 100 µg/ml penicillin/streptomycin (Euroclone). Human foreskin fibroblasts (HFF), purchased by ATCC and green fluorescent protein (GFP)-labeled HFF, kindly provided by Dr Fabiana Conciatori [ 26 ] were maintained in DMEM complete medium (Euroclone) supplemented with 10% FBS, 1% L-glutamine and 100 µg/ml penicillin/streptomycin. Cell lines were routinely tested for mycoplasma contamination and authenticated.…”