Nucleolar HEAT Repeat Containing 1 Up-regulated by the Mechanistic Target of Rapamycin Complex 1 Signaling Promotes Hepatocellular Carcinoma Growth by Dominating Ribosome Biogenesis and Proteome Homeostasis.

BACKGROUND & AIMS: Hyperactivation of ribosome biogenesis leads to hepatocyte transformation and plays pivotal roles in hepatocellular carcinoma (HCC) development. We aimed to identify critical ribosome biogenesis proteins that are overexpressed and crucial in HCC progression. METHODS: HEAT repeat containing 1 (HEATR1) expression and clinical correlations were analyzed using The Cancer Genome Atlas and Gene Expression Omnibus databases and further evaluated by immunohistochemical analysis of an HCC tissue microarray. Gene expression was knocked down by small interfering RNA. HEATR1-knockdown cells were subjected to viability, cell cycle, and apoptosis assays and used to establish subcutaneous and orthotopic tumor models. Chromatin immunoprecipitation and quantitative polymerase chain reaction were performed to detect the association of candidate proteins with specific DNA sequences. Endogenous coimmunoprecipitation combined with mass spectrometry was used to identify protein interactions. We performed immunoblot and immunofluorescence assays to detect and localize proteins in cells. The nucleolus ultrastructure was detected by transmission electron microscopy. Click-iT (Thermo Fisher Scientific) RNA imaging and puromycin incorporation assays were used to measure nascent ribosomal RNA and protein synthesis, respectively. Proteasome activity, 20S proteasome foci formation, and protein stability were evaluated in HEATR1-knockdown HCC cells. RESULTS: HEATR1 was the most up-regulated gene in a set of ribosome biogenesis mediators in HCC samples. High expression of HEATR1 was associated with poor survival and malignant clinicopathologic features in patients with HCC and contributed to HCC growth in vitro and in vivo. HEATR1 expression was regulated by the transcription factor specificity protein 1, which can be activated by insulin-like growth factor 1-mammalian target of rapamycin complex 1 signaling in HCC cells. HEATR1 localized predominantly in the nucleolus, bound to ribosomal DNA, and was associated with RNA polymerase I transcription/processing factors. Knockdown of HEATR1 disrupted ribosomal RNA biogenesis and impaired nascent protein synthesis, leading to reduced cytoplasmic proteasome activity and inhibitory-κB/nuclear factor-κB signaling. Moreover, HEATR1 knockdown induced nucleolar stress with increased nuclear proteasome activity and inactivation of the nucleophosmin 1-MYC axis. CONCLUSIONS: Our study revealed that HEATR1 is up-regulated by insulin-like growth factor 1-mammalian target of rapamycin complex 1-specificity protein 1 signaling in HCC and functions as a crucial regulator of ribosome biogenesis and proteome homeostasis to promote HCC development.

Identification of a novel m6A-related lncRNAs signature and immunotherapeutic drug sensitivity in pancreatic adenocarcinoma.

BACKGROUND: Pancreatic adenocarcinoma (PDAC) ranks as the fourth leading cause for cancer-related deaths worldwide. N6-methyladenosine (m6A) and long non-coding RNAs (lncRNAs) are closely related with poor prognosis and immunotherapeutic effect in PDAC. The aim of this study is to construct and validate a m6A-related lncRNAs signature and assess immunotherapeutic drug sensitivity in PDAC. METHODS: RNA-seq data for 178 cases of PDAC patients and 167 cases of normal pancreatic tissue were obtained from TCGA and GTEx databases, respectively. A set of 21 m6A-related genes were downloaded based on the previous report. Co-expression network was conducted to identify m6A-related lncRNAs in PDAC. Cox analyses and least absolute shrinkage and selection operator (Lasso) regression model were used to construct a risk prognosis model. The relationship between signature genes and immune function was explored by single-sample GSEA (ssGSEA). The tumor immune dysfunction and exclusion (TIDE) score and tumor mutation burden (TMB) were utilized to evaluate the response to immunotherapy. Furthermore, the expression levels of 4 m6A-related lncRNAs on PDAC cell lines were measured by the quantitative real-time PCR (qPCR). The drug sensitivity between the high- and low-risk groups was validated using PDAC cell lines by Cell-Counting Kit 8 (CCK8). RESULTS: The risk prognosis model was successfully constructed based on 4 m6A-related lncRNAs, and PDAC patients were divided into the high- and low-risk groups. The overall survival (OS) of the high-risk groups was more unfavorable compared with the low-risk groups. Receiver operating characteristic (ROC) curves demonstrated that the risk prognosis model reasonably predicted the 2-, 3- and 5-year OS of PDAC patients. qPCR analysis confirmed the decreased expression levels of 4 m6A-related lncRNAs in PDAC cells compared to the normal pancreatic cells. Furthermore, CCK8 assay revealed that Phenformin exhibited higher sensitivity in the high-risk groups, while Pyrimethamine exhibited higher sensitivity in the low-risk groups. CONCLUSION: The prognosis of patients with PDAC were well predicted in the risk prognosis model based on m6A-related lncRNAs, and selected immunotherapy drugs have potential values for the treatment of pancreatic cancer.