RASSF1A Controls Tissue Stiffness and Cancer Stem-like Cells in Lung Adenocarcinoma.
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ABSTRACT:
SUBMITTER: Dr. Daniela Pankova
PROVIDER: S-SCDT-EMBOJ-2018-100532 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Lung cancer remains the leading cause of cancer-related death due to poor treatment responses and resistance arising from tumour heterogeneity. Here, we show that adverse prognosis associated with epigenetic silencing of the tumour suppressor RASSF1A is due to increased deposition of extracellular matrix (ECM), tumour stiffness and metastatic dissemination in vitro and in vivo. We find that lung cancer cells with RASSF1A promoter methylation display constitutive nuclear YAP1 accumulation and expression of prolyl 4-hydroxylase alpha-2 (P4HA2) which increases collagen deposition. Furthermore, we identify that elevated collagen creates a stiff ECM which in turn triggers cancer stem-like programming and metastatic dissemination in vivo. Re-expression of RASSF1A or inhibition of P4HA2 activity reverses these effects and increases markers of lung differentiation (TTF-1 and Mucin 5B). Our study identifies RASSF1A as a clinical biomarker associated with mechanical properties of ECM which increases the levels of cancer stemness and risk of metastatic progression in lung adenocarcinoma. Moreover, we highlight P4HA2 as a potential target for uncoupling ECM signals that support cancer stemness.
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Project description:Lung cancer remains the leading cause of cancer-related death due to poor treatment responses and resistance arising from tumor heterogeneity. Here we show that adverse prognosis associated with epigenetic silencing of the tumour suppressor RASSF1A is due to increased deposition of extracellular matrix (ECM), tumour stiffness and metastatic dissemination in vitro and in vivo. We find that lung cancer cells with RASSF1A promoter methylation display constitutive nuclear YAP1 and expression of collagen prolylhydroxylase (P4HA2) which increases collagen deposition. Furthermore, we identify that elevated collagen creates a stiff-ECM which in turn triggers cancer stem-like programming and metastatic dissemination in vivo. Re-expression of RASSF1A or inhibition of P4HA2 activity reverse these effects and increase markers of lung differentiation (TTF-1, Mucin5B). Our study identifies RASSF1A as a clinical biomarker associated with mechanical properties of ECM which increases levels of cancer stemness and risk of metastatic progression in lung adenocarcinoma. Moreover, we highlight P4HA2 as a potential target for uncoupling ECM signals that support cancer stemness.
2019-05-20 | PXD012694 | Pride
Project description:Aim: CC chemokine receptor 9 (CCR9) interacts with its exclusive ligand CCL25, resulting in promoting tumor progression and metastasis. However, the effect and mechanisms of CCR9 on lung adenocarcinoma distant metastasis remain largely unknown. To preliminary clarify the underlying mechanisms, we investigate the correlation between CCR9 and ALDH1A1+cancer stem cells (CSCs), as well as the effect of CCR9 on the migration and invasion of CSCs. Methods: Immunohistochemistry was performed to detect the expression of CCR9 in lung adenocarcinoma tissues. The correlations of CCR9 with distant metastasis and overall survival were investigated. Serial paraffin-embedded tissue blocks were used to detect ALDH1A1+CSCs expression. The correlations between CCR9 expression and ALDH1A1+CSCs were evaluated. We further studied the effect of CCR9/CCL25 on the migration and invasion of CSCs using transwell assays. Results: There were positive correlations between CCR9 expression and distant metastasis, as well as poor overall survival. Patients with high CCR9 expression were more likely to develop distant metastasis and demonstrated poorer overall survival than patients with low CCR9 expression. In addition, there was positive correlation between the expression of CCR9 and ALDH1A1 in the same tumor microenvironment. ALDHhigh CSCs demonstrated enhanced expression of CCR9 than ALDHlow cells. Further transwell assays demonstrated that the numbers of CSCs migrated or invaded in response to CCL25 were more than that without CCL25 stimulation. Additional application of anti-CCR9 antibody reversed the CCL25-induced migration and invasion of CSCs. Conclusions: In summary, our study demonstrated that CCR9/CCL25 promoted the migration and invasion of CSCs, which might contribute to distant metastasis and poor overall survival. Our findings provided evidence that CCR9/CCL25 could be used as novel therapeutic targets for lung adenocarcinoma.
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Project description:IntroductionThe growth and expression of cancer stem cells (CSCs) depend on many factors in the tumor microenvironment. The objective of this work was to investigate the effect of cancer cells' tissue origin on the optimum matrix stiffness for CSC growth and marker expression in a model polyethylene glycol diacrylate (PEGDA) hydrogel without the interference of other factors in the microenvironment.MethodsHuman MCF7 and MDA-MB-231 breast carcinoma, HCT116 colorectal and AGS gastric carcinoma, and U2OS osteosarcoma cells were used. The cells were encapsulated in PEGDA gels with compressive moduli in the 2-70 kPa range and optimized cell seeding density of 0.6x106 cells/mL. Micropatterning was used to optimize the growth of encapsulated cells with respect to average tumorsphere size. The CSC sub-population of the encapsulated cells was characterized by cell number, tumorsphere size and number density, and mRNA expression of CSC markers.ResultsThe optimum matrix stiffness for growth and marker expression of CSC sub-population of cancer cells was 5 kPa for breast MCF7 and MDA231, 25 kPa for colorectal HCT116 and gastric AGS, and 50 kPa for bone U2OS cells. Conjugation of a CD44 binding peptide to the gel stopped tumorsphere formation by cancer cells from different tissue origin. The expression of YAP/TAZ transcription factors by the encapsulated cancer cells was highest at the optimum stiffness indicating a link between the Hippo transducers and CSC growth. The optimum average tumorsphere size for CSC growth and marker expression was 50 μm.ConclusionThe marker expression results suggest that the CSC sub-population of cancer cells resides within a niche with optimum stiffness which depends on the cancer cells' tissue origin.
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Project description:The acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is the major reason for the failure of target therapy in advanced non small cell lung cancer (NSCLC) patients, the mechanism of which has not been fully elucidated yet. The present study aimed to investigate the different DNA methylation profile before and after acquired EGFR-TKI resistance, and explore the influence of the DNA demethylater, decitabine, on EGFR-TKI resistance. The DNA methylation chip was used to screen the genes whose DNA methylation status were changed in the EGFR-TKI sensitive human NSCLC cell line PC9, and the induced EGFR-TKI resistant NSCLC cell line PC9/GR (harboring T790M mutation). According to the results and literature reports, the tumor suppressor genes, RASSF1A and GADD45? were selected for further research. Methylation specific PCR (MSP) and western blot further confirmed that the promoters of these two genes were methylated, and the protein expressions were significantly inhibited in PC9/GR cells. Additionally, decitabine, the DNA methyl transferase inhibitor, could reverse the methylation status of RASSF1A and GADD45? promoters, elevate protein expression, and partially restore the sensitivity of PC9/GR cells to EGFR-TKI. To conclude, our results suggested that the DNA methylation of RASSF1 and GADD45? may play a role in EGFR-TKI resistance, and epigenetic intervention might be an effective strategy to reverse EGFR-TKI resistance, suggesting further study.
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Project description:S-palmitoylation, catalyzed by a family of 23 zinc finger Asp-His-His-Cys (DHHC) domain-containing (ZDHHC) protein acyltransferases localized on the cell membrane. However, stemness genes modulated by ZDHHCs in lung adenocarcinoma (LUAD) remain to be defined. Previously, we have constructed a network of cancer stem cell genes, including INCENP, based on mRNA stemness indices (mRNAsi) of LUAD. INCENP has the function of a chromosomal passenger complex locating to centromeres, which is performed by the conserved region of its N-terminal domain. INCENP protein with a deletion of the first non-conserved 26 amino acid sequence failed to target centromeres. However, the exact function of the deleted sequence has not been elucidated. To identify novel cancer stem cell-relevant palmitoylated proteins and responsible ZDHHC enzymes in LUAD, we analyzed multi-omics data obtained from the database of The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and the Human Protein Atlas (HPA). ZDHHC5 is distinguished from the ZDHHC family for being up-regulated in mRNA and protein levels and associated with malignant prognosis. ZDHHC5 was positively associated with INCENP, and the correlation score increased with LUAD stages. CSS-Palm results showed Cys15 was the S-palmitoylation site of INCENP. Interestingly, Cys15 locates in the 1-26 aa sequence of INCENP, and is a conserved site across species. As INCENP is a nuclear protein, we predicted that the nuclear localization signal of ZDHHC5 was specific to the importin αβ pathway, and the result of immunofluorescence proves that ZDHHC5 is located in the nucleoplasm, in addition to the plasma membrane. Therefore, our study indicates the S-palmitoylation of INCENP mediated by ZDHHC5 as a potential mechanism of S-palmitoylation to modulate CSCs in LUAD.
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Project description:BACKGROUND:Human mesenchymal stem cell (hMSC) differentiation into osteoblasts has important clinical significance in treating bone injury, and the stiffness of the extracellular matrix (ECM) has been shown to be an important regulatory factor for hMSC differentiation. The aim of this study was to further delineate how matrix stiffness affects intracellular signaling through integrin ?5/?1, FAK, and Wnt signaling, subsequently regulating the osteogenic phenotype of hMSCs. METHODS:hMSCs were cultured on tunable polyacrylamide hydrogels coated with fibronectin with stiffness corresponding to a Young's modulus of 13-16 kPa and 62-68 kPa. After hMSCs were cultured on gels for 1 week, gene expression of alpha-1 type I collagen, BGLAP, and RUNX2 were evaluated by real-time PCR. After hMSCs were cultured on gels for 24 h, signaling molecules relating to integrin ?5 (FAK, ERK, p-ERK, Akt, p-Akt, GSK-3?, p-GSK-3?, and ?-catenin) were evaluated by western blot analysis. RESULTS:Osteogenic differentiation was increased on 62-68 kPa ECM, as evidenced by alpha-1 type I collagen, BGLAP, and RUNX2 gene expression, calcium deposition, and ALP staining. In the process of differentiation, gene and protein expression of integrin ?5/?1 increased, together with protein expression of the downstream signaling molecules FAK, p-ERK, p-Akt, GSK-3?, p-GSK-3?, and ?-catenin, indicating that these molecules can affect the osteogenic differentiation of hMSCs. An antibody blocking integrin ?5 suppressed the stiffness-induced expression of all osteoblast markers examined. In particular, alpha-1 type I collagen, RUNX2, and BGLAP were significantly downregulated, indicating that integrin ?5 regulates hMSC osteogenic differentiation. Downstream expression of FAK, ERK, p-ERK, and ?-catenin protein was unchanged, whereas Akt, p-Akt, GSK-3?, and p-GSK-3? were upregulated. Moreover, expression of Akt and p-Akt was upregulated with anti-integrin ?5 antibody, but no difference was observed for FAK, ERK, and p-ERK between the with or without anti-integrin ?5 antibody groups. At the same time, expression of GSK-3? and p-GSK-3? was upregulated and ?-catenin levels showed no difference between the groups with or without anti-integrin ?5 antibody. Since Akt, p-Akt, GSK-3?, and p-GSK-3? displayed the same changes between the groups with or without anti-integrin ?5 antibody, we then detected the links among them. Expression of p-Akt and p-GSK-3? was reduced effectively in the presence of the Akt inhibitor Triciribine. However, Akt, GSK-3?, and ?-catenin were unchanged. These results suggested that expression of p-GSK-3? was regulated by p-Akt on 62-68 kPa ECM. CONCLUSIONS:Taken together, our results provide evidence that matrix stiffness (62-68 kPa) affects the osteogenic outcome of hMSCs through mechanotransduction events that are mediated by integrin ?5.
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