Autophagy related RHEB-CSF1R complex promotes tumor metastasis via advancing phosphorylation levels of PI3K, AKT, mTOR in pancreatic cancer

Figure 7 Ras homolog enriched in brain-colony stimulating factor 1 receptor complex advances phosphorylation levels of phosphatidylinositol 3-kinase, AKT serine/threonine kinase 1, and mammalian target of rapamycin in pancreatic cancer cells. A and B: Western blot analyses of the phosphatidylinositol 3-kinase (PI3K)/AKT serine/threonine kinase 1 (AKT)/mammalian target of rapamycin (mTOR) signaling pathway biomarkers, PI3K, p-PI3K, AKT, p-AKT, mTOR and p-mTOR (p-PI3K corresponds to the Tyr524 phosphorylation site of PI3K; p-AKT corresponds to the Ser473 phosphorylation site of AKT); the autophagy biomarkers, Beclin 1, autophagy related 5 and microtubule-associated protein 1 light chain 3; and the epithelial-mesenchymal transformation biomarkers, E-cadherin, N-cadherin and vimentin, and Ras homolog enriched in brain (RHEB); C and D: Western blot analyses of p-PI3K, p-AKT, and p-mTOR, and the autophagy biomarkers, Beclin 1, autophagy related 5 and microtubule-associated protein 1 light chain 3, and the epithelial-mesenchymal transformation biomarkers, E-cadherin, N-cadherin and vimentin, and RHEB in the Capan-1 and CFPAC-1 cells stably expressing the small interfering negative control and the small interfering RHEB were transfected transiently with the vector or the colony stimulating factor 1 receptor, Colo357 cells stably expressing vector and RHEB transfected with short hairpin negative control and short hairpin colony stimulating factor 1 receptor; E and F: Representative images and quantitative analyses of Transwell assay in the small interfering negative control and the small interfering RHEB transfected the Capan-1 and CFPAC-1 cells with PI3K activator 740 Y-P, and the vector and the RHEB transfected the Colo357 cells with PI3K inhibitor LY294002. DMSO was used as controls. ^aP < 0.05, ^bP < 0.01, ^cP value < 0.001, and ^dP value < 0.0001. RHEB: Ras homolog enriched in brain; PC: Pancreatic cancer; PI3K: Phosphatidylinositol 3-kinase; AKT: AKT serine/threonine kinase 1; mTOR: Mammalian target of rapamycin; shNC: Short hairpin negative control; shRHEB: Short hairpin Ras homolog enriched in brain; ATG5: Autophagy related 5; LC3: Microtubule-associated protein 1 light chain 3; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; CSF1R: Colony stimulating factor 1 receptor; siNC: Small interfering negative control; siRHEB: Small interfering Ras homolog enriched in brain.