Telomerase deficiency and dysfunctional telomeres in the lung tumor microenvironment impair tumor progression in NSCLC mouse models and patient-derived xenografts

Abstract

Non-small cell lung cancer (NSCLC) is a leading cause of cancer death. Tumor progression depends on interactions of cancer cells with the tumor microenvironment. Here, we find increased copy number and mRNA expression of the catalytic subunit of telomerase, TERT, in tumors from NSCLC patients, contributing to a lower survival. Moreover, TERT expression in NSCLC patients from the TCGA cohort is mainly associated to the reduced infiltration of CD8+ T lymphocytes, as well as to increased infiltration of myeloid-derived suppressor cells (MDSCs). We also show that TERT deficiency and dysfunctional telomeres induced by 6-thio-dG treatment in mice reduced lung tumor implantation and vascularization, increased DNA damage response, cell cycle arrest and apoptosis, as well as reduced proliferation, inflammation, lung tumor immunosupression and invasion upon induction of a Lewis lung carcinoma (LLC). Furthermore, 6-thio-dG-treated human NSCLC xenografts exhibited increased telomere damage, cell cycle arrest and apoptosis, as well as reduced proliferation, resulting in a reduced tumor growth. Our results show that targeting telomeres might be an effective therapeutic strategy in NSCLC.

Differential contribution for ERK1 and ERK2 kinases in BRAFV600E-triggered phenotypes in adult mouse models

“…For histological examination, sections were stained with hematoxylin and eosin, according to standard procedures as previously described [ 59 – 62 ]. CC3 Asp175 (Cell Signaling Technology 9661), CC10 (Santa Cruz Biotechnology sc-9772), CD4 [ 15 , 60 , 63 ] (1:50, Clone D7D2Z, 25229, Cell Signaling Technology), CD8 [ 60 , 62 ] (1:200, Clone 94A, CNIO Monoclonal Antibodies Core Unit handmade), prosurfactant protein C (millipore AB3786), p21 (291 H/B5, CNIO Monoclonal antibodies facility homemade), γH2AX Ser 139 (Millipore 05-636), PPERK Thr202/Tyr204 (Cell Signaling Tehcnology 9101), Ki67 (Cell Signaling 12202), total ERK1/2 (Abcam ab54230, ERK-7D8), F4-80 (ABD Serotec MCA497), CD3 [ 64 ] (undiluted, Roche, 2GV6, ref.790–4341); CD45R/B220 [ 65 ] (1:150, BD biosciences, 557390, RA3-6B2); FOXP3 [ 62 , 66 ] (1:50, 221D, CNIO Monoclonal antibodies facility homemade), p53 (POE316A, CNIO Monoclonal antibodies facility homemade), antibodies were used for immunohistochemistry in tissue sections. The antibodies used to identify the different lymphocytes subpopulations were previously tested, validated, and used by us [ 15 , 60 , 62 ] after the proper testing and validation performed by the corresponding manufacturers [ 63 – 66 ].…”

“…CC3 Asp175 (Cell Signaling Technology 9661), CC10 (Santa Cruz Biotechnology sc-9772), CD4 [ 15 , 60 , 63 ] (1:50, Clone D7D2Z, 25229, Cell Signaling Technology), CD8 [ 60 , 62 ] (1:200, Clone 94A, CNIO Monoclonal Antibodies Core Unit handmade), prosurfactant protein C (millipore AB3786), p21 (291 H/B5, CNIO Monoclonal antibodies facility homemade), γH2AX Ser 139 (Millipore 05-636), PPERK Thr202/Tyr204 (Cell Signaling Tehcnology 9101), Ki67 (Cell Signaling 12202), total ERK1/2 (Abcam ab54230, ERK-7D8), F4-80 (ABD Serotec MCA497), CD3 [ 64 ] (undiluted, Roche, 2GV6, ref.790–4341); CD45R/B220 [ 65 ] (1:150, BD biosciences, 557390, RA3-6B2); FOXP3 [ 62 , 66 ] (1:50, 221D, CNIO Monoclonal antibodies facility homemade), p53 (POE316A, CNIO Monoclonal antibodies facility homemade), antibodies were used for immunohistochemistry in tissue sections. The antibodies used to identify the different lymphocytes subpopulations were previously tested, validated, and used by us [ 15 , 60 , 62 ] after the proper testing and validation performed by the corresponding manufacturers [ 63 – 66 ]. Pictures were taken using Olympus AX70 microscope.…”

“…For the quantification, protein‐band intensities were quantified by densitometric analysis with ImageLab software (Biorad). The total levels of each protein analyzed have been normalized versus actin and the mean of the specific protein/actin ratio deriving from at least 3 different replicates has been used to generate the chart as previously described [ 62 , 67 , 68 ].…”

Combinational Pulsing of TAAs Enforces Dendritic Cell-Based Immunotherapy through T-Cell Proliferation and Interferon-γ Secretion in LLC1 Mouse Model

“…For instance, WT-1, widely used as an antigen for anti-cancer immunotherapy, is expressed not only in various cancer types but also in cancer-surrounding endothelial cells, stimulating angiogenesis [19,20]. MUC-1 provides a sustained survival signal to tumors [21]; BIRC5 is known to be involved in drug resistance [22]; and TERT is reactivated in approximately 90% of primary tumors, including NSCLC [23,24].…”

Dual-Stimuli Regulation of DNAzyme Cleavage Reaction by Coordination-Driven Nanoprobes for Cancer Cell Imaging

“…GSH and TE were selected as activators of interest due to their close relationship. First, the simultaneous elevation of both markers is more uniquely characteristic of cancer cells, associated with rapid tumor proliferation. , TE, a ribonucleoprotein enzyme that is commonly overexpressed in tumor cells, − leads to enhanced TE activity. This increase, in turn, causes the accumulation of oxidative stress, resulting in a significant surge in both the concentration and activity of GSH. − Second, employing only GSH might lead to false positives in conditions of oxidative stress in normal cells .…”