The cellular prion protein (PrP^C) is a cell surface glycoprotein attached to the membrane by a glycosylphosphatidylinositol (GPI)-anchor and plays a critical role in transmissible, neurodegenerative and fatal prion diseases. Alterations in membrane attachment influence PrP^C-associated signaling, and the development of prion disease, yet our knowledge of the role of the GPI-anchor in localization, processing, and function of PrP^C in vivo is limited We exchanged the PrP^C GPI-anchor signal sequence of for that of Thy-1 (PrP^CGPIThy-1) in cells and mice. We show that this modifies the GPI-anchor composition, which then lacks sialic acid, and that PrP^CGPIThy-1 is preferentially localized in axons and is less prone to proteolytic shedding when compared to PrP^C. Interestingly, after prion infection, mice expressing PrP^CGPIThy-1 show a significant delay to terminal disease, a decrease of microglia/astrocyte activation, and altered MAPK signaling when compared to wild-type mice. Our results are the first to demonstrate in vivo, that the GPI-anchor signal sequence plays a fundamental role in the GPI-anchor composition, dictating the subcellular localization of a given protein and, in the case of PrP^C, influencing the development of prion disease. The prion protein (PrP^C) is a glycoprotein attached to the neuronal surface via a GPI-anchor. When misfolded to PrP^Sc, it leads to fatal neurodegenerative diseases which propagates from host to host. PrP^Sc is the principal component of the infectious agent of prion diseases, the “prion”. Misfolding occurs at the plasma membrane, and when PrP^C lacks the GPI-anchor, neuropathology and incubation time of prion disease are strongly modified. Moreover, the composition of the PrP^C GPI-anchor impacts on the conversion process. To study the role of the GPI-anchor in the pathophysiology of prion diseases in vivo, we have generated transgenic mice where the PrP^C GPI-signal sequence (GPI-SS) is replaced for the one of Thy-1, a neuronal protein with a distinct GPI-anchor and membrane localization. We found that the resulting protein, PrP^CGPIThy-1, shows a different GPI-anchor composition, increased axonal localization, and reduced enzymatic shedding. After prion infection, disease progression is significantly delayed, and the neuropathology and cellular signaling are changed. The present work demonstrates that the GPI-SS per se determines the GPI-anchor composition and localization of a given protein and it stresses the importance of PrP^C membrane anchorage in prion disease.

The Ubiquitin-Specific Protease 22 (USP22) is a deubiquitinating subunit of the mammalian SAGA transcriptional co-activating complex. USP22 was identified as a member of the so-called “death-from-cancer” signature predicting therapy failure in cancer patients. However, the importance and functional role of USP22 in different types and subtypes of cancer remain largely unknown. In the present study, we leveraged human cell lines and genetic mouse models to investigate the role of USP22 in HER2-driven breast cancer (HER2⁺-BC) and demonstrate for the first time that USP22 is required for the tumorigenic properties in murine and human HER2⁺-BC models. To get insight into the underlying mechanisms, we performed transcriptome-wide gene expression analyses and identified the Unfolded Protein Response (UPR) as a pathway deregulated upon USP22 loss. The UPR is normally induced upon extrinsic or intrinsic stresses that can promote cell survival and recovery if shortly activated or programmed cell death if activated for an extended period. Strikingly, we found that USP22 actively suppresses UPR induction in HER2⁺-BC cells by stabilizing the major endoplasmic reticulum (ER) chaperone HSPA5. Consistently, loss of USP22 renders tumor cells more sensitive to apoptosis and significantly increases the efficiency of therapies targeting the ER folding capacity. Together, our data suggest that therapeutic strategies targeting USP22 activity may sensitize tumor cells to UPR induction and could provide a novel, effective approach to treat HER2⁺-BC.

Usp22 deficiency impairs intestinal epithelial lineage specification in vivo

Colorectal cancer (CRC) is the fourth leading cause of cancer-related deaths worldwide, and deciphering underlying molecular mechanism is essential. The loss of monoubiquitinated histone H2B (H2Bub1) was correlated with poor prognosis of CRC patients and, accordingly, H2Bub1 was suggested as a tumor-suppressive mark. Surprisingly, our previous work revealed that the H2B ubiquitin ligase RING finger protein 40 (RNF40) might exert tumor-promoting functions. Here, we investigated the effect of RNF40 loss on tumorigenic features of CRC cells and their survival in vitro. We evaluated the effects of RNF40 depletion in several human CRC cell lines in vitro. To evaluate cell cycle progression, cells were stained with propidium iodide and analyzed by flow cytometry. In addition, to assess apoptosis rates, caspase 3/7 activity was assessed in a Celigo® S-based measurement and, additionally, an Annexin V assay was performed. Genomic occupancy of H2Bub1, H3K79me3, and H3K27ac was determined by chromatin immunoprecipitation. Transcriptome-wide effects of RNF40 loss were evaluated based on mRNA-seq results, qRT-PCR, and Western blot. To rescue apoptosis-related effects, cells were treated with Z-VAD-FMK. Human CRC cell lines displayed decreased cell numbers in vitro after RNF40 depletion. While the differences in confluence were not mediated by changes in cell cycle progression, we discovered highly increased apoptosis rates after RNF40 knockdown due to elevated caspase 3/7 activity. This effect can be explained by reduced mRNA levels of anti-apoptotic and upregulation of pro-apoptotic BCL2 family members. Moreover, the direct occupancy of the RNF40-mediated H2B monoubiquitination was observed in the transcribed region of anti-apoptotic genes. Caspase inhibition by Z-VAD-FMK treatment rescued apoptosis in RNF40-depleted cells. However, knockdown cells still displayed decreased tumorigenic features despite the absence of apoptosis. Our findings reveal that RNF40 is essential for maintaining tumorigenic features of CRC cells in vitro by controlling the expression of genes encoding central apoptotic regulators.

Background: Basal-like breast cancer (BLBC) is one of the most aggressive malignant diseases in women with an increased metastatic behavior and poor prognosis compared to other molecular subtypes of breast cancer. Resistance to chemotherapy is the main cause of treatment failure in BLBC. Therefore, novel therapeutic strategies counteracting the gain of aggressiveness underlying therapy resistance are urgently needed. The epithelial-to-mesenchymal transition (EMT) has been established as one central process stimulating cancer cell migratory capacity but also acquisition of chemotherapy-resistant properties. In this study, we aimed to uncover epigenetic factors involved in the EMT-transcriptional program occurring in BLBC cells surviving conventional chemotherapy. Results: Using whole transcriptome data from a murine mammary carcinoma cell line (pG-2), we identified upregulation of Hdac4, 7 and 8 in tumor cells surviving conventional chemotherapy. Subsequent analyses of human BLBC patient datasets and cell lines established HDAC8 as the most promising factor sustaining tumor cell viability. ChIP-sequencing data analysis identified a pronounced loss of H3K27ac at regulatory regions of master transcription factors (TFs) of epithelial phenotype like Gata3, Elf5, Rora and Grhl2 upon chemotherapy. Interestingly, impairment of HDAC8 activity reverted epithelial-TFs levels. Furthermore, loss of HDAC8 activity sensitized tumor cells to chemotherapeutic treatments, even at low doses. Conclusion: The current study reveals a previously unknown transcriptional repressive function of HDAC8 exerted on a panel of transcription factors involved in the maintenance of epithelial cell phenotype, thereby supporting BLBC cell survival to conventional chemotherapy. Our data establish HDAC8 as an attractive therapeutically targetable epigenetic factor to increase the efficiency of chemotherapeutics. Graphical