Immuno-genomic classification of colorectal cancer organoids reveals cancer cells with intrinsic immunogenic properties associated with patient survival.

BACKGROUND: The intrinsic immuno-ge7nomic characteristics of colorectal cancer cells that affect tumor biology and shape the tumor immune microenvironment (TIM) are unclear. METHODS: We developed a patient-derived colorectal cancer organoid (CCO) model and performed pairwise analysis of 87 CCOs and their matched primary tumors. The TIM type of the primary tumor was classified as immuno-active, immuno-exhausted, or immuno-desert. RESULTS: The gene expression profiles, signaling pathways, major oncogenic mutations, and histology of the CCOs recapitulated those of the primary tumors, but not the TIM of primary tumors. Two distinct intrinsic molecular subgroups of highly proliferative and mesenchymal phenotypes with clinical significance were identified in CCOs with various cancer signaling pathways. CCOs showed variable expression of cancer-specific immune-related genes such as those encoding HLA-I and HLA-II, and molecules involved in immune checkpoint activation/inhibition. Among these genes, the expression of HLA-II in CCOs was associated with favorable patient survival. K-means clustering analysis based on HLA-II expression in CCOs revealed a subgroup of patients, in whom cancer cells exhibited Intrinsically Immunogenic Properties (Ca-IIP), and were characterized by high expression of signatures associated with HLA-I, HLA-II, antigen presentation, and immune stimulation. Patients with the Ca-IIP phenotype had an excellent prognosis, irrespective of age, disease stage, intrinsic molecular type, or TIM status. Ca-IIP was negatively correlated with intrinsic E2F/MYC signaling. Analysis of the correlation between CCO immuno-genotype and TIM phenotype revealed that the TIM phenotype was associated with microsatellite instability, Wnt/ß-catenin signaling, APC/KRAS mutations, and the unfolded protein response pathway linked to the FBXW7 mutation in cancer cells. However, Ca-IIP was not associated with the TIM phenotype. CONCLUSIONS: We identified a Ca-IIP phenotype from a large set of CCOs. Our findings may provide an unprecedented opportunity to develop new strategies for optimal patient stratification in this era of immunotherapy.

LNX1 contributes to tumor growth by down-regulating p53 stability.

The well-known tumor suppressor p53 inhibits the formation of various cancers by inducing cell cycle arrest and apoptosis. Although p53 mutations are commonly found in many cancers, p53 is functionally inactivated in tumor cells that retain wild-type p53. Here, we show that the ligand of numb protein X1 (LNX1) inhibited p53-dependent transcription by decreasing the half-life of p53. We generated LNX1 knockout (KO) cells in p53 wild-type cancer cells (A549, HCT116, and MCF7) using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 gene-editing system. LNX1 KO activated p53-dependent transcription by increasing the stability of p53. Moreover, lentivirus-mediated overexpression of LNX1 decreased p53 protein levels and inhibited p53-dependent transcription. LNX1 interacted with p53 and mouse double minute 2 (MDM2) and increased the ubiquitination of p53 in an MDM2-dependent manner. Finally, we demonstrated that LNX1 was required for efficient tumor growth both in cell culture and in a mouse tumor xenograft model. These results collectively indicated that LNX1 contributed to tumor growth by inhibiting p53-dependent signaling in p53 wild-type cancer cells.-Park, R., Kim, H., Jang, M., Jo, D., Park, Y.-I., Namkoong, S., Lee, J. I., Jang, I.-S., Park, J. LNX1 contributes to tumor growth by down-regulating p53 stability.

AMP-activated protein kinase regulates the expression of human telomerase reverse transcriptase.

The expression of hTERT in tumor cells contributes to oncogenic transformation by promoting immortalization. For this reason, hTERT is one of the major targets for cancer therapy, and an efficient method to downregulate hTERT expression is required for treatment of hTERT-positive cancer. In this report, we demonstrated that inhibition of AMP-activated protein kinase (AMPK) downregulates the expression of hTERT. We screened cell signaling pathways in AMPK α1 knockout cells and found that AMPKα1 is required for activity of the hTERT promoter. AMPKα1 knockout cells showed decreased expression of hTERT mRNA and protein. We also demonstrated that compound C, a reversible AMPK inhibitor, suppressed the expression of hTERT. However, AMPK activators, including AICAR and metformin, did not increase the level of hTERT protein. Finally, we showed that tumor cells stably expressing hTERT are resistant to compound C treatment. These results indicate that AMPK activity is required for tumor progression.

AMPK contributes to autophagosome maturation and lysosomal fusion.

AMP-activated protein kinase (AMPK) regulates autophagy initiation when intracellular ATP level decreases. However, the role of AMPK during autophagosome maturation is not fully understood. Here, we report that AMPK contributes to efficient autophagosome maturation and lysosomal fusion. Using CRISPR-Cas9 gene editing, we generated AMPK α1 knockout HEK293T cell lines, in which starvation-induced autophagy is impaired. Compound C, an AMPK-independent autophagy inducer, and trehalose, an mTOR-independent autophagy inducer were used to examine the role of AMPK in autophagosome maturation and lysosomal fusion. While the treatment of control cells with either compound C or trehalose induces activation of autophagosomes as well as autolysosomes, the treatment of AMPK α1 knockout cells with compound C or trehalose induces mainly activation of autophagosomes, but not autolysosomes. We demonstrate that this effect is due to interference with the fusion of autophagosomes with lysosomes in AMPK α1 knockout cells. The transient expression of AMPK α1 can rescue autophagosome maturation. These results indicate that AMPK α1 is required for efficient autophagosome maturation and lysosomal fusion.

Conessine Treatment Reduces Dexamethasone-Induced Muscle Atrophy by Regulating MuRF1 and Atrogin-1 Expression.

Conessine, a steroidal alkaloid, is a potent histamine H3 antagonist with antimalarial activity. We recently reported that conessine treatment interferes with H2O2-induced cell death by regulating autophagy. However, the cellular signaling pathways involved in conessine treatment are not fully understood. Here, we report that conessine reduces muscle atrophy by interfering with the expression of atrophy-related ubiquitin ligases MuRF-1 and atrogin-1. Promoter reporter assay revealed that conessine treatment inhibits FoxO3a-dependent transcription, NF-κB-dependent transcription, and p53-dependent transcription. We also showed by quantitative RT-PCR and western blot assays that conessine treatment reduced dexamethasone-induced expression of MuRF1 and atrogin-1. Finally, we demonstrated that conessine treatment reduced dexamethasone-induced muscle atrophy using differentiated C2C12 cells. These results collectively suggest that conessine is potentially useful in the treatment of muscle atrophy.

Conessine treatment reduces dexamethasone-induced muscle atrophy by regulating MuRF1 and atrogin-1 expression.

Conessine, a steroidal alkaloid, is a potent histamine H3 antagonist with anti-malarial activity. We recently reported that conessine treatment interferes with H2O2-induced cell death by regulating autophagy. However, the cellular signaling pathways involved in conessine treatment are not fully understood. Here, we report that conessine reduces muscle atrophy by interfering with the expression of atrophy-related ubiquitin ligases MuRF-1 and atrogin-1. Promoter reporter assay revealed that conessine treatment inhibits FoxO3a-dependent transcription, NF-kappaB-dependent transcription and p53-dependent transcription. We also showed that conessine treatment reduced dexamethasone-induced expression of MuRF1 and atrogin-1 by the quantitative RT-PCR and Western blot. Finally, we demonstrated that conessine treatment reduced dexamethasone-induced muscle atrophy using differentiated C2C12 cells. These results collectively suggest that conessine is potentially useful in the treatment of muscle atrophy.

2,2'-Methylenebis (6-tert-butyl 4-methylphenol) enhances the antitumor efficacy of belotecan, a derivative of camptothecin, by inducing autophagy.

Autophagy regulation is important for tumor cell survival. Activation and inhibition of autophagy can sensitize tumor cells to anticancer drugs. However, few autophagy-regulating small molecules are available to increase the efficacy of anticancer drugs. Here, we report that 2,2'-methylenebis (6-tert-butyl 4-methylphenol), hereafter referred to as methylenebis, is a novel autophagy-regulating small molecule that sensitizes tumor cells to belotecan, which is a derivative of camptothecin, a topoisomerase I inhibitor. Methylenebis activates autophagic flux by increasing the level of LC3-II and forming autolysosome puncta. Moreover, methylenebis enhances the antitumor efficacy of belotecan by activating both autophagy and apoptosis. Interestingly, methylenebis increased the level of LC3-II and belotecan independently decreased the level of p62, suggesting that methylenebis and belotecan target different steps of autophagy. Finally, we searched for compounds that are structurally similar to methylenebis. Our results imply that the specific structure of methylenebis contributes to its ability to activate autophagy.