Notch and mTOR Signaling Pathways Promote Human Gastric Cancer Cell Proliferation - PubMed (original) (raw)

Notch and mTOR Signaling Pathways Promote Human Gastric Cancer Cell Proliferation

Elise S Hibdon et al. Neoplasia. 2019 Jul.

Abstract

Notch pathway signaling is known to promote gastric stem cell proliferation, and constitutive pathway activation induces gastric tumors via mTORC1 activation in mouse genetic models. The purpose of this study was to determine whether human gastric adenocarcinomas are similarly dependent on Notch and mTORC1 signaling for growth. Gene expression profiling of 415 human gastric adenocarcinomas in The Cancer Genome Atlas, and a small set of locally obtained gastric cancers showed enhanced expression of Notch pathway components, including Notch ligands, receptors and downstream target genes. Human gastric adenocarcinoma tissues and chemically induced mouse gastric tumors both exhibited heightened Notch and mTORC1 pathway signaling activity, as evidenced by increased expression of the NOTCH1 receptor signaling fragment NICD, the Notch target HES1, and the mTORC1 target phosphorylated S6 ribosomal protein. Pharmacologic inhibition of either Notch or mTORC1 signaling reduced growth of human gastric cancer cell lines, with combined pathway inhibition causing a further reduction in growth, suggesting that both pathways are activated to promote gastric cancer cell proliferation. Further, mTORC1 signaling was reduced after Notch inhibition suggesting that mTOR is downstream of Notch in gastric cancer cells. Analysis of human gastric organoids derived from paired control and gastric cancer tissues also exhibited reduced growth in culture after Notch or mTOR inhibition. Thus, our studies demonstrate that Notch and mTOR signaling pathways are commonly activated in human gastric cancer to promote cellular proliferation. Targeting these pathways in combination might be an effective therapeutic strategy for gastric cancer treatment.

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Figures

Figure 1

Notch and mTOR pathway activation in mouse gastric tumors induced by N-nitroso-N-methylurea (MNU). (A-B) Gross morphology of stomachs isolated from control (Veh) or MNU-treated mice. (C) Stomach wet weight in control and MNU-treated mice 9–12 months post-treatment. (D&E) Histology of control antrum and an MNU-induced antral tumor assessed by H&E staining. (F&G) Epithelial cell proliferation was assessed by EdU incorporation (red), with DAPI nuclear stain (blue). (H-K) mTOR and Notch pathway activity in MNU-induced tumors was determined via immunostaining for pS6 (H), NICD (I) or Hes1 (J&K). Examples of epithelial and stromal Notch activity are indicated by the dotted outlines or arrowheads, respectively. DAPI or hematoxylin was used as nuclear counterstain. Data are presented as mean ± SEM (n = 6–7 mice/group). **P < .01 vs. control. Scale bars: 100 (D-H, J) or 50 μm (I).

Figure 2

Increased Notch pathway component expression in human gastric adenocarcinoma. (A-E) Normalized RNAseq data from The Cancer Genome Atlas (TCGA) database examining 35 normal stomach and 415 gastric adenocarcinoma tissue samples. Gene expression analysis of (A) Notch receptors, (B&C) Notch ligands and (D&E) Notch target genes in normal (white circles) and gastric adenocarcinoma (black circles) samples. Analysis of the 4 molecular subtypes of gastric adenocarcinomas is presented in Supplementary Figure 1. (F&G) RT-qPCR analysis of Notch receptors (F) and Notch ligands (G) in paired non-cancer (white circles) and gastric cancer (gray circles) full-thickness tissue obtained from Michigan Medicine patients. See Supplementary Table 1 for information related to patient samples. Data are presented as mean ± SEM. *P < .05, **P < .01, ****P < .0001 vs. normal or non-cancer.

Figure 3

Notch and mTOR pathway activation in human gastric adenocarcinoma tissue. (A-E) Histology of 5 primary gastric adenocarcinomas was assessed by H&E staining. Comparative histology of adjacent non-cancer tissue from the same patients is presented in Supplementary Figure 2. (F-J) mTOR pathway activity was detected via pS6 immunohistochemistry (brown). (K-T) Notch pathway activity was detected by immunostaining for (K-O) Hes1 (brown) or (P–T) NICD (green). (A-O) Hematoxylin or (P-T) DAPI was used as nuclear counterstains. Epithelial and stromal Notch activity are indicated by the dotted lines or arrowheads, respectively. Scale bars: (A-J) 100 or (K-T) 50 μm.

Figure 4

Notch and mTOR pathway signaling are both required for human gastric cancer cell growth. (A-C) Gene expression analysis of Notch pathway components in AGS (A), MKN45 (B), and NCI-N87 (C) human gastric cancer cell lines by RT-qPCR. (D-F) Cell growth was measured using a colorimetric assay kit in human gastric cancer cell lines AGS (D), MKN45 (E) and NCI-N87(F), after treatment with vehicle (Veh; white circles), the Notch inhibitor DAPT (30 μM; black circles), the mTORC1 inhibitor rapamycin (Rapa; 250 nM; white triangles), or a combination of both inhibitors (black triangles). Data are presented as mean ± SEM, with n = 6–12 technical replicates (A-C) or n = 4 technical replicates (D-F) for each cell line. *P < .05, ****P < .0001 vs. Day 5 vehicle, or as indicated on graph. ND = not detected.

Figure 5

mTORC1 activity is dependent on Notch signaling in gastric cancer cells. MKN45 cells were treated daily with DAPT (30 μM) or vehicle for 5 days and cells were collected for protein extraction. (A) Western blot analysis for mTORC1 pathway targets, measuring phosphorylated (p) or total (t) protein, with β-actin loading control. (B-D) Quantification of phospho-AKT/total-AKT (B), phosphor-S6/total S6 (C) and phospho-4EBP/total-4EBP. Data are presented as mean ± SEM, with 3 independent cultures. *P < .05 vs. vehicle by student's t-test.

Figure 6

Notch regulates growth of human gastric cancer-derived organoids. Human gastric organoids from paired non-cancer (H46) and gastric cancer (H47) tissue isolated from the same patient were treated with Veh or DAPT (10 μM), renewed every other day for 5 days. Morphology of non-cancer (A, E) and gastric cancer (C, G)-derived organoids after 5 days of treatment. Cell proliferation was assessed by EdU incorporation (green) in non-cancer (B,F) and gastric cancer (D,H) organoids. (I) Morphometric analysis of organoid size in Veh-treated H46 non-cancer and H47 gastric cancer organoids. (J,K) Dose response of non-cancer and gastric cancer organoid growth in various doses of DAPT, as indicated. Data are presented as mean ± SEM, with n = 3 technical replicates for each organoid line. *P < .05, **P < .01, ***P < .001, ****P < .0001 vs. vehicle. Scale bars: (A,C,E,G) 500 μm or (B,D,F,H) 50 μm.

Figure 7

mTOR signaling is required for human gastric cancer organoid growth. (A-G) Human gastric organoids from paired (A-C) non-cancer (H46) and (D-F) gastric cancer (H47) tissues were treated with (A&D) vehicle, (B&E) the mTORC1 inhibitor rapamycin (250 nM), or (C&F) 10 μM DAPT +250 nM rapamycin, renewed every other day for 5 days. (G) Morphometric analysis of organoid size after 5 days of inhibitor treatment. (H-N) Paired non-cancer (H-J) and gastric cancer (K-M) tissues were treated with (H&K) vehicle, (I&L) the pan-mTOR inhibitor Torin1 (250 nM), or (J&M) 10 μM DAPT +250 nM Torin1, renewed every other day for 5 days. (N) Morphometric analysis of organoid size after 5 days of inhibitor treatment. Data are presented as mean ± SEM, with n = 3–6 technical replicates for each organoid line. **P < .01, ***P < .001, ****P < .0001 vs. vehicle or as indicated on graph. Scale bars: 500 μm.

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Notch and mTOR Signaling Pathways Promote Human Gastric Cancer Cell Proliferation - PubMed (original) (raw)