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

Figure 1 Flow chart of the overall study design. RHEB: Ras homolog enriched in brain.

Figure 2 Expression of Ras homolog enriched in brain in pancreatic cancer. A and B: The mRNA expression levels of Ras homolog enriched in brain (RHEB) in five pancreatic cancer (PC) cell lines and normal pancreas line; C: The protein expression levels of RHEB in normal pancreas and PC lines; D: The mRNA expression levels of RHEB in five paired PC and non-tumor tissues; E and F: Scores and representative images of RHEB immunohistochemical staining in the non-paired normal pancreas (n = 15), primary PC (n = 20), distant liver metastasis (n = 18), and lymph node metastasis tissues (n = 6). ^aP < 0.05, ^bP < 0.01, ^cP value < 0.001, and ^dP < 0.0001. PAAD: Pancreatic adenocarcinoma; RT-PCR: Reverse transcription-polymerase chain reaction; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; RHEB: Ras homolog enriched in brain.

Figure 3 High Ras homolog enriched in brain expression indicated poor prognosis and poor sensitivity to chemotherapy in pancreatic cancer. A: Kaplan-Meier analysis of external validation patients’ high-Ras homolog enriched in brain (RHEB) and low-RHEB groups (n = 44); B: Kaplan-Meier analysis of The Cancer Genome Atlas’ high-RHEB and low-RHEB groups (n = 177); C: Univariate and multivariate analyses of prognostic factors; D: Multivariate Cox regression analyses of prognostic factors in The Cancer Genome Atlas; E: A constructed nomogram for prognostic prediction of pancreatic cancer patients; F: Calibration curves of the prognostic model in predicting the overall survival probability at 1/2/3 years; G: Receiver operating characteristic curves of the model of pancreatic adenocarcinoma patients at 1/2/3 years; H: Receiver operating characteristic curves of RHEB of pancreatic adenocarcinoma patients at 1/2/3 years. AUC: Area under the curve; HR: Hazard ratio; RHEB: Ras homolog enriched in brain; CI: Confidence interval; TPR: True positive rate; FPR: False positive rate.

Figure 4 Ras homolog enriched in brain promotes proliferation and metastasis in pancreatic cell lines. A-C: Measurement of cell proliferation of the Capan-1 and CFPAC-1 cells transfected with the control small interfering RNA (siRNA) or the Ras homolog enriched in brain (RHEB) silencing by siRNA, and in Colo357 cells transfected with the control vector or the RHEB overexpression vector by cell counting kit-8 assay; D-I: Representative images and quantitative analyses of colony formation assay in the Capan-1 and CFPAC-1 cells transfected with the control siRNA or the RHEB silencing by siRNA, and in Colo357 cells transfected with the control vector or the RHEB overexpression vector; J-L: Representative images and quantitative analyses of Transwell assay in the Capan-1 and CFPAC-1 cells transfected with the control siRNA or the RHEB silencing by siRNA, and in Colo357 cells transfected with the control vector or the RHEB overexpression vector. ^aP < 0.05, ^bP < 0.01, and ^cP value < 0.001. RHEB: Ras homolog enriched in brain; CCK-8: Cell counting kit-8; siNC: Small interfering negative control; siRHEB: Small interfering Ras homolog enriched in brain.

Figure 5 Ras homolog enriched in brain promotes proliferation and metastasis in vivo. A-D: Tumor volumes and weights in the short hairpin negative control (shNC) and short hairpin Ras homolog enriched in brain (shRHEB) groups implanted with Capan-1 cells in subcutaneous tumors models; E and F: Representative images and scores of immunohistochemical staining in the shNC and shRHEB groups implanted with Capan-1 cells; G and H: Spleen primary tumors and liver metastases in the shNC and shRHEB groups implanted with Capan-1 cells in liver metastasis models; I: Hematoxylin-eosin staining of harvested tumor. ^aP < 0.05, and ^bP < 0.01. RHEB: Ras homolog enriched in brain; shNC: Short hairpin negative control; shRHEB: Short hairpin Ras homolog enriched in brain; HE: Hematoxylin-eosin staining.

Figure 6 Ras homolog enriched in brain upregulates and interacts with colony stimulating factor 1 receptor in pancreatic cell cells. A-C: The expression of Ras homolog enriched in brain (RHEB) and colony stimulating factor 1 receptor (CSF1R) was confirmed by western blot analyses in the Capan-1 and CFPAC-1 cells transfected with the short hairpin (sh) RNA or the shRHEB, in Colo357 cells transfected with the vector or the RHEB, co-immunoprecipitation assay demonstrated that RHEB directly interacts with CSF1R; D-I: Representative images and quantitative analyses of Transwell assay 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 CSF1R, Colo357 cells stably expressing vector and RHEB transfected with sh-negative control and shCSF1R. ^aP < 0.05, ^bP < 0.01, and ^cP value < 0.001. RHEB: Ras homolog enriched in brain; shNC: Short hairpin negative control; shRHEB: Short hairpin Ras homolog enriched in brain; CSF1R: Colony stimulating factor 1 receptor; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; siNC: Small interfering negative control; siRHEB: Small interfering Ras homolog enriched in brain; shNC: Short hairpin negative control; shRHEB: Short hairpin Ras homolog enriched in brain.

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.

Figure 8 Ras homolog enriched in brain-colony stimulating factor 1 receptor complex may inhibit autophagy by advancing phosphorylation levels of phosphatidylinositol 3-kinase, AKT serine/threonine kinase 1, and mammalian target of rapamycin. A and B: Representative images and quantitative analyses of autophagic vacuoles (autophagosomes and autolysosomes) in subsets of 3 randomly-selected cells of the Capan-1 and CFPAC-1 cells transfected with the short hairpin negative control or the short hairpin Ras homolog enriched in brain (RHEB); in Colo357 cells transfected with the vector or the RHEB by using transmission electron microscopy; C-F: Immunofluorescence staining of RHEB with microtubule-associated protein 1 light chain 3 in Capan-1, CFPAC-1, and Colo357 cells; G-J: Western blot analyses of phosphatidylinositol 3-kinase (PI3K), p-PI3K, AKT serine/threonine kinase 1 (AKT), p-AKT (p-PI3K corresponds to the Tyr524 phosphorylation site of PI3K; p-AKT corresponds to the Ser473 phosphorylation site of AKT), mammalian target of rapamycin and p-mammalian target of rapamycin, and the autophagy biomarkers, Beclin 1, autophagy related 5 and microtubule-associated protein 1 light chain 3 in the short hairpin negative control and the short hairpin RHEB transfected the Capan-1 and CFPAC-1 cells with PI3K activator 740 Y-P, and the control vector and the RHEB overexpression vector transfected the Colo357 cells with PI3K inhibitor LY294002. DMSO was used as controls. ^aP < 0.05, ^bP < 0.01, and ^cP value < 0.001. shNC: Short hairpin negative control; shRHEB: Short hairpin Ras homolog enriched in brain; LC3: Microtubule-associated protein 1 light chain 3; RHEB: Ras homolog enriched in brain; PI3K: Phosphatidylinositol 3-kinase; AKT: AKT serine/threonine kinase 1; mTOR: Mammalian target of rapamycin; ATG5: Autophagy related 5; LC3: Microtubule-associated protein 1 light chain 3; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; shNC: Short hairpin negative control; shRHEB: Short hairpin Ras homolog enriched in brain; ns: No significance.

Figure 9 Ras homolog enriched in brain-colony stimulating factor 1 receptor complex promotes epithelial-mesenchymal transformation markers expression levels by autophagy inhibition. A-C: Western blot and quantitative analyses of 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) in the short hairpin negative control and the short hairpin RHEB transfected the Capan-1 and CFPAC-1 cells with chloroquine. DMSO was used as controls; D-G: Representative images and quantitative analyses of the Transwell assay in the small interfering negative control and the small interfering Ras homolog enriched in brain transfected the Capan-1 and CFPAC-1 cells with chloroquine. DMSO was used as controls. ^aP < 0.05, ^bP < 0.01, ^cP value < 0.001, and ^dP < 0.0001. RHEB: Ras homolog enriched in brain; 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; siNC: Small interfering negative control; siRHEB: Small interfering Ras homolog enriched in brain.

Figure 10  Ras homolog enriched in brain silencing inhibited colony stimulating factor 1 receptor, phosphorylate of phosphatidylinositol 3-kinase, AKT serine/threonine kinase 1, mammalian target of rapamycin, and promoted microtubule-associated protein 1 light chain 3 and E-cadherin in vivo. A and B: Representative images and scores of Ras homolog enriched in brain (RHEB), colony stimulating factor 1 receptor, p-phosphorylate of phosphatidylinositol 3-kinase, p-AKT serine/threonine kinase 1, p-mammalian target of rapamycin. Microtubule-associated protein 1 light chain 3 and E-cadherin immunohistochemical staining in the short hairpin negative control and short hairpin Ras homolog enriched in brain groups. ^aP < 0.05, and ^bP < 0.01. RHEB: Ras homolog enriched in brain; CSF1R: Colony stimulating factor 1 receptor; PI3K: Phosphatidylinositol 3-kinase; AKT: AKT serine/threonine kinase 1; mTOR: Mammalian target of rapamycin; LC3: Microtubule-associated protein 1 light chain 3; shNC: Short hairpin negative control; shRHEB: Short hairpin Ras homolog enriched in brain; IRS: Immunoreactivity score.

Figure 11  Proposed mechanism of Ras homolog enriched in brain-mediated cell metastasis in pancreatic cancer. A summary of significant results shows the following: (1) Ras homolog enriched in brain (RHEB) expression is upregulated in pancreatic cancer in vivo and in vitro experiments. It is associated with poor prognosis in our clinical samples and the The Cancer Genome Atlas database; (2) RHEB forms a complex with colony stimulating factor 1 receptor and inhibits autophagy by advancing phosphorylation levels of phosphatidylinositol 3-kinase, AKT serine/threonine kinase 1, and mammalian target of rapamycin; and (3) RHEB-colony stimulating factor 1 receptor complex may promote pancreatic cancer metastasis by upregulating the expression of epithelial-mesenchymal transformation markers N-cadherin and vimentin and downregulating E-cadherin expression via autophagy inhibitors. RHEB: Ras homolog enriched in brain; CSF1R: Colony stimulating factor 1 receptor; PI3K: Phosphatidylinositol 3-kinase; AKT: AKT serine/threonine kinase 1; mTOR: Mammalian target of rapamycin; E-cad: E-cadherin; N-cad: N-cadherin.