Enhancement of colorectal cancer therapy through interruption of the HSF1-HSP90 axis by p53 activation or cell cycle inhibition.

The stress-associated molecular chaperone system is an actionable target in cancer therapies. It is ubiquitously upregulated in cancer tissues and enables tumorigenicity by stabilizing hundreds of oncoproteins and disturbing the stoichiometry of protein complexes. Most inhibitors target the key component heat-shock protein 90 (HSP90). However, although classical HSP90 inhibitors are highly tumor-selective, they fail in phase 3 clinical oncology trials. These failures are at least partly due to an interference with a negative feedback loop by HSP90 inhibition, known as heat-shock response (HSR): in response to HSP90 inhibition there is compensatory synthesis of stress-inducible chaperones, mediated by the transcription factor heat-shock factor 1 (HSF1). We recently identified that wildtype p53 (p53) actively reduces the HSR by repressing HSF1 via a p21-CDK4/6-MAPK-HSF1 axis. Here we test the hypothesis that in HSP90-based therapies simultaneous p53 activation or direct cell cycle inhibition interrupts the deleterious HSF1-HSR axis and improves the efficiency of HSP90 inhibitors. Indeed, we find that the clinically relevant p53 activator Idasanutlin suppresses the HSF1-HSR activity in HSP90 inhibitor-based therapies. This combination synergistically reduces cell viability and accelerates cell death in p53-proficient colorectal cancer (CRC) cells, murine tumor-derived organoids and patient-derived organoids (PDOs). Mechanistically, upon combination therapy human CRC cells strongly upregulate p53-associated pathways, apoptosis, and inflammatory immune pathways. Likewise, in the chemical AOM/DSS CRC model in mice, dual HSF1-HSP90 inhibition strongly represses tumor growth and remodels immune cell composition, yet displays only minor toxicities in mice and normal mucosa-derived organoids. Importantly, inhibition of the cyclin dependent kinases 4 and 6 (CDK4/6) under HSP90 inhibition phenocopies synergistic repression of the HSR in p53-proficient CRC cells. Even more important, in p53-deficient (mutp53-harboring) CRC cells, an HSP90 inhibition in combination with CDK4/6 inhibitors similarly suppresses the HSF1-HSR system and reduces cancer growth. Likewise, p53-mutated PDOs strongly respond to dual HSF1-HSP90 pathway inhibition and thus, providing a strategy to target CRC independent of the p53 status. In sum, activating p53 (in p53-proficient cancer cells) or inhibiting CDK4/6 (independent of the p53 status) provide new options to improve the clinical outcome of HSP90-based therapies and to enhance colorectal cancer therapy.

USP22 supports the aggressive behavior of basal-like breast cancer by stimulating cellular respiration.

BACKGROUND: Breast cancer (BC) is the most frequent tumor entity in women worldwide with a high chance of therapeutic response in early- and non-metastatic disease stages. Among all BC subtypes, triple-negative BC (TNBC) is the most challenging cancer subtype lacking effective molecular targets due to the particular enrichment of cancer stem cells (CSCs), frequently leading to a chemoresistant phenotype and metastasis. The Ubiquitin Specific Peptidase 22 (USP22) is a deubiquitinase that has been frequently associated with a CSC-promoting function and intimately implicated in resistance to conventional therapies, tumor relapse, metastasis and overall poor survival in a broad range of cancer entities, including BC. To date, though, the role of USP22 in TNBC has been only superficially addressed. METHODS: The current study utilized the MMTV-cre, Usp22fl/fl transgenic mouse model to study the involvement of USP22 in the stem cell-like properties of the growing mammary tissue. Additionally, we combined high-throughput transcriptomic analyses with publicly available patient transcriptomic data and utilized TNBC culture models to decipher the functional role of USP22 in the CSC characteristics of this disease. RESULTS: Interestingly, we identified that USP22 promotes CSC properties and drug tolerance by supporting the oxidative phosphorylation program, known to be largely responsible for the poor response to conventional therapies in this particularly aggressive BC subtype. CONCLUSIONS: This study suggests a novel tumor-supportive role of USP22 in sustaining cellular respiration to facilitate the drug-tolerant behavior of HER2+-BC and TNBC cells. Therefore, we posit USP22 as a promising therapeutic target to optimize standard therapies and combat the aggressiveness of these malignancies. Video Abstract.

RNF40 epigenetically modulates glycolysis to support the aggressiveness of basal-like breast cancer.

Triple-negative breast cancer (TNBC) is the most difficult breast cancer subtype to treat due to the lack of targeted therapies. Cancer stem cells (CSCs) are strongly enriched in TNBC lesions and are responsible for the rapid development of chemotherapy resistance and metastasis. Ubiquitin-based epigenetic circuits are heavily exploited by CSCs to regulate gene transcription and ultimately sustain their aggressive behavior. Therefore, therapeutic targeting of these ubiquitin-driven dependencies may reprogram the transcription of CSC and render them more sensitive to standard therapies. In this work, we identified the Ring Finger Protein 40 (RNF40) monoubiquitinating histone 2B at lysine 120 (H2Bub1) as an indispensable E3 ligase for sustaining the stem-cell-like features of the growing mammary gland. In addition, we found that the RNF40/H2Bub1-axis promotes the CSC properties and drug-tolerant state by supporting the glycolytic program and promoting pro-tumorigenic YAP1-signaling in TNBC. Collectively, this study unveils a novel tumor-supportive role of RNF40 and underpins its high therapeutic value to combat the malignant behavior of TNBC.

Identification of a ΔNp63-Dependent Basal-Like A Subtype-Specific Transcribed Enhancer Program (B-STEP) in Aggressive Pancreatic Ductal Adenocarcinoma.

A major hurdle to the application of precision oncology in pancreatic cancer is the lack of molecular stratification approaches and targeted therapy for defined molecular subtypes. In this work, we sought to gain further insight and identify molecular and epigenetic signatures of the Basal-like A pancreatic ductal adenocarcinoma (PDAC) subgroup that can be applied to clinical samples for patient stratification and/or therapy monitoring. We generated and integrated global gene expression and epigenome mapping data from patient-derived xenograft models to identify subtype-specific enhancer regions that were validated in patient-derived samples. In addition, complementary nascent transcription and chromatin topology (HiChIP) analyses revealed a Basal-like A subtype-specific transcribed enhancer program in PDAC characterized by enhancer RNA (eRNA) production that is associated with more frequent chromatin interactions and subtype-specific gene activation. Importantly, we successfully confirmed the validity of eRNA detection as a possible histologic approach for PDAC patient stratification by performing RNA-ISH analyses for subtype-specific eRNAs on pathologic tissue samples. Thus, this study provides proof-of-concept that subtype-specific epigenetic changes relevant for PDAC progression can be detected at a single-cell level in complex, heterogeneous, primary tumor material. IMPLICATIONS: Subtype-specific enhancer activity analysis via detection of eRNAs on a single-cell level in patient material can be used as a potential tool for treatment stratification.

Basal-like mammary carcinomas stimulate cancer stem cell properties through AXL-signaling to induce chemotherapy resistance.

Basal-like breast cancer (BLBC) is the most aggressive and heterogeneous breast cancer (BC) subtype. Conventional chemotherapies represent next to surgery the most frequently employed treatment options. Unfortunately, resistant tumor phenotypes often develop, resulting in therapeutic failure. To identify the early events occurring upon the first drug application and initiating chemotherapy resistance in BLBC, we leveraged the WAP-T syngeneic mammary carcinoma mouse model and we developed a strategy combining magnetic-activated cell sorting (MACS)-based tumor cell enrichment with high-throughput transcriptome analyses. We discovered that chemotherapy induced a massive gene expression reprogramming toward stemness acquisition to tolerate and survive the cytotoxic treatment in vitro and in vivo. Retransplantation experiments revealed that one single cycle of cytotoxic drug combination therapy (Cyclophosphamide, Adriamycin and 5-Fluorouracil) suffices to induce resistant tumor cell phenotypes in vivo. We identified Axl and its ligand Pros1 as highly induced genes driving cancer stem cell (CSC) properties upon chemotherapy in vivo and in vitro. Furthermore, from our analysis of BLBC patient datasets, we found that AXL expression is also strongly correlated with CSC-gene signatures, a poor response to conventional therapies and worse survival outcomes in those patients. Finally, we demonstrate that AXL inhibition sensitized BLBC-cells to cytotoxic treatment in vitro. Together, our data support AXL as a promising therapeutic target to optimize the efficiency of conventional cytotoxic therapies in BLBC.

ROBO3s: a novel ROBO3 short isoform promoting breast cancer aggressiveness.

Basal-like breast cancer (BLBC) is a highly aggressive breast cancer subtype frequently associated with poor prognosis. Due to the scarcity of targeted treatment options, conventional cytotoxic chemotherapies frequently remain the standard of care. Unfortunately, their efficacy is limited as BLBC malignancies rapidly develop resistant phenotypes. Using transcriptomic and proteomic approaches in human and murine BLBC cells, we aimed to elucidate the molecular mechanisms underlying the acquisition of aggressive and chemotherapy-resistant phenotypes in these mammary tumors. Specifically, we identified and characterized a novel short isoform of Roundabout Guidance Receptor 3 (ROBO3s), upregulated in BLBC in response to chemotherapy and encoding for a protein variant lacking the transmembrane domain. We established an important role for the ROBO3s isoform, mediating cancer stem cell properties by stimulating the Hippo-YAP signaling pathway, and thus driving resistance of BLBC cells to cytotoxic drugs. By uncovering the conservation of ROBO3s expression across multiple cancer types, as well as its association with reduced BLBC-patient survival, we emphasize its potential as a prognostic marker and identify a novel attractive target for anti-cancer drug development.

Axon guidance receptor ROBO3 modulates subtype identity and prognosis via AXL-associated inflammatory network in pancreatic cancer.

Metastatic pancreatic cancer (PDAC) has a poor clinical outcome with a 5-year survival rate below 3%. Recent transcriptome profiling of PDAC biopsies has identified 2 clinically distinct subtypes - the "basal-like" (BL) subtype with poor prognosis and therapy resistance compared with the less aggressive and drug-susceptible "classical" (CLA) subtype. However, the mechanistic events and environmental factors that promote the BL subtype identity are not very clear. Using preclinical models, patient-derived xenografts, and FACS-sorted PDAC patient biopsies, we report here that the axon guidance receptor, roundabout guidance receptor 3 (ROBO3), promotes the BL metastatic program via a potentially unique AXL/IL-6/phosphorylated STAT3 (p-STAT3) regulatory axis. RNA-Seq identified a ROBO3-mediated BL-specific gene program, while tyrosine kinase profiling revealed AXL as the key mediator of the p-STAT3 activation. CRISPR/dCas9-based ROBO3 silencing disrupted the AXL/p-STAT3 signaling axis, thereby halting metastasis and enhancing therapy sensitivity. Transcriptome analysis of resected patient tumors revealed that AXLhi neoplastic cells associated with the inflammatory stromal program. Combining AXL inhibitor and chemotherapy substantially restored a CLA phenotypic state and reduced disease aggressiveness. Thus, we conclude that a ROBO3-driven hierarchical network determines the inflammatory and prometastatic programs in a specific PDAC subtype.

Osmotic Stress Interferes with DNA Damage Response and H2AX Phosphorylation in Human Keratinocytes.

The human skin and in particular its outermost layer, the epidermis, protects the body from potentially harmful substances, radiation as well as excessive water loss. However, the interference between the various stress responses of the epidermal keratinocytes, which often occur simultaneously, is largely unknown. The focus of this study was to investigate the interference between osmotic stress and DNA damage response. In addition to revealing the already well-described regulation of diverse gene sets, for example, cellular processes such as transcription, translation, and metabolic pathways (e.g., the KEGG citrate cycle and Reactome G2/M checkpoints), gene expression analysis of osmotically stressed keratinocytes revealed an influence on the transcription of genes also related to UV-induced DNA damage response. A gene network regulating the H2AX phosphorylation was identified to be regulated by osmotic stress. To analyze and test the interference between osmotic stress and DNA damage response, which can be triggered by UV stress on the one hand and oxidative stress on the other, in more detail, primary human keratinocytes were cultured under osmotic stress conditions and subsequently exposed to UV light and H2O2, respectively. γH2AX measurements revealed lower γH2AX levels in cells previously cultured under osmotic stress conditions.

HDAC8 suppresses the epithelial phenotype and promotes EMT in chemotherapy-treated basal-like breast cancer.

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.

Decreased PRC2 activity supports the survival of basal-like breast cancer cells to cytotoxic treatments.

Breast cancer (BC) is the most common cancer occurring in women but also rarely develops in men. Recent advances in early diagnosis and development of targeted therapies have greatly improved the survival rate of BC patients. However, the basal-like BC subtype (BLBC), largely overlapping with the triple-negative BC subtype (TNBC), lacks such drug targets and conventional cytotoxic chemotherapies often remain the only treatment option. Thus, the development of resistance to cytotoxic therapies has fatal consequences. To assess the involvement of epigenetic mechanisms and their therapeutic potential increasing cytotoxic drug efficiency, we combined high-throughput RNA- and ChIP-sequencing analyses in BLBC cells. Tumor cells surviving chemotherapy upregulated transcriptional programs of epithelial-to-mesenchymal transition (EMT) and stemness. To our surprise, the same cells showed a pronounced reduction of polycomb repressive complex 2 (PRC2) activity via downregulation of its subunits Ezh2, Suz12, Rbbp7 and Mtf2. Mechanistically, loss of PRC2 activity leads to the de-repression of a set of genes through an epigenetic switch from repressive H3K27me3 to activating H3K27ac mark at regulatory regions. We identified Nfatc1 as an upregulated gene upon loss of PRC2 activity and directly implicated in the transcriptional changes happening upon survival to chemotherapy. Blocking NFATc1 activation reduced epithelial-to-mesenchymal transition, aggressiveness, and therapy resistance of BLBC cells. Our data demonstrate a previously unknown function of PRC2 maintaining low Nfatc1 expression levels and thereby repressing aggressiveness and therapy resistance in BLBC.

RNF20 and RNF40 regulate vitamin D receptor-dependent signaling in inflammatory bowel disease.

Despite the identification of several genetic factors linked to increased susceptibility to inflammatory bowel disease (IBD), underlying molecular mechanisms remain to be elucidated in detail. The ubiquitin ligases RNF20 and RNF40 mediate the monoubiquitination of histone H2B at lysine 120 (H2Bub1) and were shown to play context-dependent roles in the development of inflammation. Here, we aimed to examine the function of the RNF20/RNF40/H2Bub1 axis in intestinal inflammation in IBD patients and mouse models. For this purpose, intestinal sections from IBD patients were immunohistochemically stained for H2Bub1. Rnf20 or Rnf40 were conditionally deleted in the mouse intestine and mice were monitored for inflammation-associated symptoms. Using mRNA-seq and chromatin immunoprecipitation (ChIP)-seq, we analyzed underlying molecular pathways in primary intestinal epithelial cells (IECs) isolated from these animals and confirmed these findings in IBD resection specimens using ChIP-seq.The majority (80%) of IBD patients displayed a loss of H2Bub1 levels in inflamed areas and the intestine-specific deletion of Rnf20 or Rnf40 resulted in spontaneous colorectal inflammation in mice. Consistently, deletion of Rnf20 or Rnf40 promoted IBD-associated gene expression programs, including deregulation of various IBD risk genes in these animals. Further analysis of murine IECs revealed that H3K4me3 occupancy and transcription of the Vitamin D Receptor (Vdr) gene and VDR target genes is RNF20/40-dependent. Finally, these effects were confirmed in a subgroup of Crohn's disease patients which displayed epigenetic and expression changes in RNF20/40-dependent gene signatures. Our findings reveal that loss of H2B monoubiquitination promotes intestinal inflammation via decreased VDR activity thereby identifying RNF20 and RNF40 as critical regulators of IBD.

Suppression of HSF1 activity by wildtype p53 creates a driving force for p53 loss-of-heterozygosity.

The vast majority of human tumors with p53 mutations undergo loss of the remaining wildtype p53 allele (loss-of-heterozygosity, p53LOH). p53LOH has watershed significance in promoting tumor progression. However, driving forces for p53LOH are poorly understood. Here we identify the repressive WTp53-HSF1 axis as one driver of p53LOH. We find that the WTp53 allele in AOM/DSS chemically-induced colorectal tumors (CRC) of p53R248Q/+ mice retains partial activity and represses heat-shock factor 1 (HSF1), the master regulator of the proteotoxic stress response (HSR) that is ubiquitously activated in cancer. HSR is critical for stabilizing oncogenic proteins including mutp53. WTp53-retaining CRC tumors, tumor-derived organoids and human CRC cells all suppress the tumor-promoting HSF1 program. Mechanistically, retained WTp53 activates CDKN1A/p21, causing cell cycle inhibition and suppression of E2F target MLK3. MLK3 links cell cycle with the MAPK stress pathway to activate the HSR response. In p53R248Q/+ tumors WTp53 activation by constitutive stress represses MLK3, thereby weakening the MAPK-HSF1 response necessary for tumor survival. This creates selection pressure for p53LOH which eliminates the repressive WTp53-MAPK-HSF1 axis and unleashes tumor-promoting HSF1 functions, inducing mutp53 stabilization enabling invasion.

USP22 promotes HER2-driven mammary carcinoma aggressiveness by suppressing the unfolded protein response.

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.

TNF-α-producing macrophages determine subtype identity and prognosis via AP1 enhancer reprogramming in pancreatic cancer.

Large-scale genomic profiling of pancreatic cancer (PDAC) has revealed two distinct subtypes: 'classical' and 'basal-like'. Their variable coexistence within the stromal immune microenvironment is linked to differential prognosis; however, the extent to which these neoplastic subtypes shape the stromal immune landscape and impact clinical outcome remains unclear. By combining preclinical models, patient-derived xenografts, as well as FACS-sorted PDAC patient biopsies, we show that the basal-like neoplastic state is sustained via BRD4-mediated cJUN/AP1 expression, which induces CCL2 to recruit tumor necrosis factor (TNF)-α-secreting macrophages. TNF-α+ macrophages force classical neoplastic cells into an aggressive phenotypic state via lineage reprogramming. Integration of ATAC-, ChIP- and RNA-seq data revealed distinct JUNB/AP1 (classical) and cJUN/AP1 (basal-like)-driven regulation of PDAC subtype identity. Pharmacological inhibition of BRD4 led to suppression of the BRD4-cJUN-CCL2-TNF-α axis, restoration of classical subtype identity and a favorable prognosis. Hence, patient-tailored therapy for a cJUNhigh/TNF-αhigh subtype is paramount in overcoming highly inflamed and aggressive PDAC states.

RNF40 exerts stage-dependent functions in differentiating osteoblasts and is essential for bone cell crosstalk.

The role of histone ubiquitination in directing cell lineage specification is only poorly understood. Our previous work indicated a role of the histone 2B ubiquitin ligase RNF40 in controlling osteoblast differentiation in vitro. Here, we demonstrate that RNF40 has a stage-dependent function in controlling osteoblast differentiation in vivo. RNF40 expression is essential for early stages of lineage specification, but is dispensable in mature osteoblasts. Paradoxically, while osteoblast-specific RNF40 deletion led to impaired bone formation, it also resulted in increased bone mass due to impaired bone cell crosstalk. Loss of RNF40 resulted in decreased osteoclast number and function through modulation of RANKL expression in OBs. Mechanistically, we demonstrate that Tnfsf11 (encoding RANKL) is an important target gene of H2B monoubiquitination. These data reveal an important role of RNF40-mediated H2B monoubiquitination in bone formation and remodeling and provide a basis for exploring this pathway for the treatment of conditions such as osteoporosis or cancer-associated osteolysis.

Influence of ARHGAP29 on the Invasion of Mesenchymal-Transformed Breast Cancer Cells.

Aggressive and mesenchymal-transformed breast cancer cells show high expression levels of Rho GTPase activating protein 29 (ARHGAP29), a negative regulator of RhoA. ARHGAP29 was the only one of 32 GTPase-activating enzymes whose expression significantly increased after the induction of mesenchymal transformation in breast cancer cells. Therefore, we investigated the influence of ARHGAP29 on the invasiveness of aggressive and mesenchymal-transformed breast cancer cells. After knock-down of ARHGAP29 using siRNA, invasion of HCC1806, MCF-7-EMT, and T-47D-EMT breast cancer cells was significantly reduced. This could be explained by reduced inhibition of RhoA and a consequent increase in stress fiber formation. Proliferation of the breast cancer cell line T-47D-EMT was slightly increased by reduced expression of ARHGAP29, whereas that of HCC1806 and MCF-7-EMT significantly increased. Using interaction analyses we found that AKT1 is a possible interaction partner of ARHGAP29. Therefore, the expression of AKT1 after siRNA knock-down of ARHGAP29 was tested. Reduced ARHGAP29 expression was accompanied by significantly reduced AKT1 expression. However, the ratio of active pAKT1 to total AKT1 remained unchanged or was significantly increased after ARHGAP29 knock-down. Our results show that ARHGAP29 could be an important factor in the invasion of aggressive and mesenchymal-transformed breast cancer cells. Further research is required to fully understand the underlying mechanisms.

The histone H2B ubiquitin ligase RNF40 is required for HER2-driven mammary tumorigenesis.

The HER2-positive breast cancer subtype (HER2+-BC) displays a particularly aggressive behavior. Anti-HER2 therapies have significantly improved the survival of patients with HER2+-BC. However, a large number of patients become refractory to current targeted therapies, necessitating the development of new treatment strategies. Epigenetic regulators are commonly misregulated in cancer and represent attractive molecular therapeutic targets. Monoubiquitination of histone 2B (H2Bub1) by the heterodimeric ubiquitin ligase complex RNF20/RNF40 has been described to have tumor suppressor functions and loss of H2Bub1 has been associated with cancer progression. In this study, we utilized human tumor samples, cell culture models, and a mammary carcinoma mouse model with tissue-specific Rnf40 deletion and identified an unexpected tumor-supportive role of RNF40 in HER2+-BC. We demonstrate that RNF40-driven H2B monoubiquitination is essential for transcriptional activation of RHO/ROCK/LIMK pathway components and proper actin-cytoskeleton dynamics through a trans-histone crosstalk with histone 3 lysine 4 trimethylation (H3K4me3). Collectively, this work demonstrates a previously unknown essential role of RNF40 in HER2+-BC, revealing the H2B monoubiquitination axis as a possible tumor context-dependent therapeutic target in breast cancer.

Egr2-guided histone H2B monoubiquitination is required for peripheral nervous system myelination.

Schwann cells are the nerve ensheathing cells of the peripheral nervous system. Absence, loss and malfunction of Schwann cells or their myelin sheaths lead to peripheral neuropathies such as Charcot-Marie-Tooth disease in humans. During Schwann cell development and myelination chromatin is dramatically modified. However, impact and functional relevance of these modifications are poorly understood. Here, we analyzed histone H2B monoubiquitination as one such chromatin modification by conditionally deleting the Rnf40 subunit of the responsible E3 ligase in mice. Rnf40-deficient Schwann cells were arrested immediately before myelination or generated abnormally thin, unstable myelin, resulting in a peripheral neuropathy characterized by hypomyelination and progressive axonal degeneration. By combining sequencing techniques with functional studies we show that H2B monoubiquitination does not influence global gene expression patterns, but instead ensures selective high expression of myelin and lipid biosynthesis genes and proper repression of immaturity genes. This requires the specific recruitment of the Rnf40-containing E3 ligase by Egr2, the central transcriptional regulator of peripheral myelination, to its target genes. Our study identifies histone ubiquitination as essential for Schwann cell myelination and unravels new disease-relevant links between chromatin modifications and transcription factors in the underlying regulatory network.

USP22 exerts tumor-suppressive functions in colorectal cancer by decreasing mTOR activity.

USP22, the deubiquitinating subunit of the SAGA transcriptional cofactor complex, is a member of an 11-gene "death-from-cancer" signature. USP22 has been considered an attractive therapeutic target since high levels of its expression were associated with distant metastasis, poor survival, and high recurrence rates in a wide variety of solid tumors, including colorectal cancer (CRC). We sought to investigate the role of Usp22 during tumorigenesis in vivo using a mouse model for intestinal carcinogenesis with a tissue-specific Usp22 ablation. In addition, we assessed the effects of USP22 depletion in human CRC cells on tumorigenic potential and identified underlying molecular mechanisms. For the first time, we report that USP22 has an unexpected tumor-suppressive function in vivo. Intriguingly, intestine-specific Usp22 deletion exacerbated the tumor phenotype caused by Apc mutation, resulting in significantly decreased survival and higher intestinal tumor incidence. Accordingly, human CRC cells showed increased tumorigenic properties upon USP22 reduction in vitro and in vivo and induced gene expression signatures associated with an unfavorable outcome in CRC patients. Notably, USP22 loss resulted in increased mTOR activity with the tumorigenic properties elicited by the loss of USP22 being reversible by mTOR inhibitor treatment in vitro and in vivo. Here, we demonstrate that USP22 can exert tumor-suppressive functions in CRC where its loss increases CRC burden by modulating mTOR activity. Importantly, our data uncover a tumor- and context-specific role of USP22, suggesting that USP22 expression could serve as a marker for therapeutic stratification of cancer patients.

USP22-dependent HSP90AB1 expression promotes resistance to HSP90 inhibition in mammary and colorectal cancer.

As a member of the 11-gene "death-from-cancer" gene expression signature, overexpression of the Ubiquitin-Specific Protease 22 (USP22) was associated with poor prognosis in various human malignancies. To investigate the function of USP22 in cancer development and progression, we sought to detect common USP22-dependent molecular mechanisms in human colorectal and breast cancer cell lines. We performed mRNA-seq to compare gene expression profiles of various colorectal (SW837, SW480, HCT116) and mammary (HCC1954 and MCF10A) cell lines upon siRNA-mediated knockdown of USP22. Intriguingly, while USP22 depletion had highly heterogeneous effects across the cell lines, all cell lines displayed a common reduction in the expression of Heat Shock Protein 90 Alpha Family Class B Member 1 (HSP90AB1). The downregulation of HSP90AB1 was confirmed at the protein level in these cell lines as well as in colorectal and mammary tumors in mice with tissue-specific Usp22 deletions. Mechanistically, we detected a significant reduction of H3K9ac on the HSP90AB1 gene in USP22-deficient cells. Interestingly, USP22-deficient cells displayed a high dependence on HSP90AB1 expression and diminishing HSP90 activity further using the HSP90 inhibitor Ganetespib resulted in increased therapeutic vulnerability in both colorectal and breast cancer cells in vitro. Accordingly, subcutaneously transplanted CRC cells deficient in USP22 expression displayed increased sensitivity towards Ganetespib treatment in vivo. Together, we discovered that HSP90AB1 is USP22-dependent and that cooperative targeting of USP22 and HSP90 may provide an effective approach to the treatment of colorectal and breast cancer.