Regulation of cancer testis antigens by unfolded protein response and their function in breast cancer

Stressful conditions of the tumour microenvironment, which overwhelm the folding capacity of the endoplasmic reticulum (EnR), activate an evolutionarily conserved signalling pathway known as the unfolded protein response (UPR). UPR is regulated by three EnR transmembrane sensors—ATF6, PERK and IRE1—each playing a distinct role in restoring EnR homeostasis. Through the coordinated actions of these sensors, the UPR aims to restore EnR homeostasis by reducing the influx of client proteins, enhancing the cell’s ability to fold misfolded proteins and removing unfolded proteins via the EnR-associated degradation (ERAD) pathway. The degradation of unfolded proteins and the enhancement of protein folding capacity, mediated by the coordinated efforts of the UPR, attempts to restore protein homeostasis. UPR is an adaptive reaction that reduces unfolded protein load to maintain cell viability and function. UPR can exert both pro-survival and deleterious effects on the survival of cancer cells. In addition to promoting cellular survival, UPR can initiate apoptosis under conditions of chronic stress. Chronic EnR stress has been linked to the occurrence of many diseases, including cancer, neurodegeneration, ischemia and diabetes. Accordingly, a comprehensive mechanistic understanding of UPR signalling is very critical for the evaluation of its biological effects in normal and/or disease conditions, as well as for developing preventive and therapeutic measures against UPR-associated diseases. Cancer/testis antigens (CTAs) are a group of proteins that are restrictively expressed in testes but are either absent or expressed at very low levels in somatic cells and normal tissues. CTAs are highly expressed during embryonic development to promote cell proliferation, migration and survival, playing a crucial role in embryonic development. After birth, most CTAs, especially the CTA genes located at the X-chromosome, are restricted to expression only in male testis, with no expression in somatic cells, mainly due to the hyper-methylation of genomic DNA in the promoter and enhancer regions of CTAs. Their expression is tightly controlled by DNA methylation and histone modifications, involving epigenetic modulatory proteins such as the germ-cell specific CCCTC-binding factor (CTCF) and the brother of regulator of imprinted sites (BORIS). While the expression of CTAs is a recurrent observation in tumours, the extent of their expression differs between cancers and even among tumours of the same cancer type. CTAs promote tumour growth, metastasis and drug resistance, and are closely associated with poor prognoses in various types of cancer patients. Given their highly tumour specific expression and pro-tumourigenic functions, CTA-based therapy provides novel opportunities for potent treatment responses with minimal adverse effects. Despite the wealth of information about their function in cancer, the regulation of their expression in cancer remains poorly understood. In this study, we performed transcriptomic analysis using total RNA from parental T47D breast cancer cells treated with the UPR activators, thapsigargin (TG) and brefeldin A (BFA). Through bioinformatic analysis of the RNA-Seq data, combined with a comprehensive literature review, we identified eight candidate CTA genes, including MAGEA11, DMRT1, SPATA17, LYPD4, MAGEB18, Calmegin (CLGN), PLAC1 and ZNF165. The RNA-Seq and qPCR results showed that CLGN expression was significantly upregulated in breast cancer cells under UPR conditions. CLGN protein is localized within the EnR membrane, where it plays an important role in maintaining EnR homeostasis. Pan-cancer analysis suggests that CLGN expression was significantly upregulated across various cancers. Further bioinformatic analysis revealed that CLGN expression was significantly upregulated in primary tumour samples of breast cancer, and the higher expression of CLGN indicated poorer survival. Our results show that CLGN expression was upregulated in a PERK-dependent manner during UPR. This study also demonstrated that CLGN knockdown reduced the proliferation, migration and colony formation abilities of breast cancer cells. Furthermore, CLGN knockdown sensitized 293T cells to UPR-induced cell death and the chemotherapeutic drug cisplatin. EnR is the major cellular compartment where the folding and maturation of secretory and membrane proteins take place. When protein folding demands exceed EnR capacity, the UPR pathway reduces the client protein load in the EnR by inhibiting protein translation and increases protein handling capacity by upregulating genes encoding EnR-resident molecular chaperones. The main pathway for translational repression in response to EnR stress has been the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) by PERK. In addition, PERK-dependent phosphorylation of eIF2α plays a critical role in the inactivation of RRN3/TIF-IA, leading to the downregulation of ribosomal RNA (rRNA) synthesis. Downregulation of rRNA transcription occurs simultaneously or slightly prior to eIF2α phosphorylation-induced translation repression. The EnR transmembrane kinase/ribonuclease IRE1β induces 28S rRNA cleavage and represses protein synthesis in response to EnR stress. It is an efficient strategy that IRE1β directly cleaves 28S rRNA on the rough EnR. rRNA methylation and pseudouridylation can fine-tune protein synthesis. Small nucleolar RNAs (SnoRNAs) are a family of small non-coding RNAs located in the nucleolus and guide the post-transcriptional modifications of rRNAs. SnoRNPs are mainly separated into two subtypes: C/D box and H/ACA box. C/D box SnoRNP is a stable assembly of C/D box SnoRNA and core proteins (NHP2L1, NOP56, NOP58 and FBL) and guides rRNA 2'-O-methylation. H/ACA SnoRNP consists of H/ACA box SnoRNA and core proteins (NHP2, NOP10, GAR1 and DKC1) and catalyses the pseudouridylation modifications of rRNAs. We hypothesize that the UPR may regulate translational fidelity by regulating the expression of core proteins of C/D and H/ACA box SnoRNPs. The bioinformatics results suggest that these core proteins were upregulated, except for FBL, in breast cancer, and higher expression levels of NOP56, NOP58, NHP2, NOP10 and DKC1 indicated poorer survival. Here, we evaluated the expression of core proteins belonging to C/D and H/ACA box SnoRNPs during EnR stress. This study also suggests that NHP2L1, NOP56 and FBL were significantly downregulated by UPR. In addition, FBL knockdown reduced cell proliferation and colony formation. Furthermore, we show the effects of UPR and FBL knockdown on rRNA methylation and translation fidelity, including alterations in nonsense suppression, frameshifts, ribosome pausing and translation initiation by internal ribosome entry site (IRES). In conclusion, this study reveals the regulation of CLGN expression by UPR and its function in breast cancer, as well as the regulation of translational fidelity during UPR by mediating the downregulation of C/D box SnoRNP, leading to change in rRNA methylation.

Funder

China Scholarship Council 202006370066

Publisher

University of Galway

Rights

CC BY-NC-ND

cbn

Collections

University of Galway Theses (PhD Theses)