HUWE1 Expression Research Articles

Induced degradation of lineage-specific oncoproteins drives the therapeutic vulnerability of small cell lung cancer to PARP inhibitors.

Although BRCA1/2 mutations are not commonly found in small cell lung cancer (SCLC), a substantial fraction of SCLC shows clinically relevant response to PARP inhibitors (PARPis). However, the underlying mechanism(s) of PARPi sensitivity in SCLC is poorly understood. We performed quantitative proteomic analyses and identified proteomic changes that signify PARPi responses in SCLC cells. We found that the vulnerability of SCLC to PARPi could be explained by the degradation of lineage-specific oncoproteins (e.g., ASCL1). PARPi-induced activation of the E3 ligase HUWE1 mediated the ubiquitin-proteasome system (UPS)-dependent ASCL1 degradation. Although PARPi induced a general DNA damage response in SCLC cells, this signal generated a cell-specific response in ASCL1 degradation, leading to the identification of HUWE1 expression as a predictive biomarker for PARPi. Combining PARPi with agents targeting these pathways markedly improved therapeutic response in SCLC. The degradation of lineage-specific oncoproteins therefore represents a previously unidentified mechanism for PARPi efficacy in SCLC.

The role of KDM4A-mediated histone methylation on temozolomide resistance in glioma cells through the HUWE1/ROCK2 axis.

Temozolomide(TMZ) resistance presents a significant challenge in the treatment of gliomas. Although lysine demethylase 4A (KDM4A) has been implicated in various cancer-related processes, its role in TMZ resistance remains unclear. This study aims to elucidate the contribution of KDM4A to TMZ resistance in glioma cells and its potential implications for glioma prognosis. We assessed the expression of KDM4A in glioma cells (T98G and U251MG) using qRT-PCR and Western blot assays. To explore the role of KDM4A in TMZ resistance, we transfected siRNA targeting KDM4A into drug-resistant glioma cells. Cell viability was assessed using the CCK-8 assay and the TMZ IC50 value was determined. ChIP assays were conducted to investigate KDM4A, H3K9me3, and H3K36me3 enrichment on the promoters of ROCK2 and HUWE1. Co-immunoprecipitation confirmed the interaction between HUWE1 and ROCK2, and we examined the levels of ROCK2 ubiquitination following MG132 treatment. Notably, T98G cells exhibited greater resistance to TMZ than U251MG cells, and KDM4A displayed high expression in T98G cells. Inhibiting KDM4A resulted in decreased cell viability and a reduction in the TMZ IC50 value. Mechanistically, KDM4A promoted ROCK2 transcription by modulating H3K9me3 levels. Moreover, disruption of the interaction between HUWE1 and ROCK2 led to reduced ROCK2 ubiquitination. Inhibition of HUWE1 or overexpression of ROCK2 counteracted the sensitization effect of si-KDM4A on TMZ responsiveness in T98G cells. Our findings highlight KDM4A's role in enhancing TMZ resistance in glioma cells by modulating ROCK2 and HUWE1 transcription and expression through H3K9me3 and H3K36me3 removal.

P-021 A novel SNP in HUWE1 promoter confers increased risk of NOA by affecting the RA/RARα pathway in Chinese individuals

Abstract Study question The present study aimed to investigate the role of HUWE1 in non-obstructive azoospermia (NOA) etiology and germ cell differentiation. Summary answer An SNP mutation in HUWE1 promoter region downregulates HUWE1 expression in NOA patients, the genetic polymorphisms of HUWE1 are related to the pathogenesis of NOA. What is known already HUWE1, a ubiquitin ligase, is crucial to the development of male reproductive system and embryonic development. However, the specific role of HUWE1 in regulating germ cell differentiation is unclear, and clinical evidence for the relationship between HUWE1 and the pathogenesis of male infertility is lacking. Study design, size, duration 190 NOA Chinese patients were recruited and analyzed single nucleotide polymorphisms (SNPs) in HUWE1 gene. Participants/materials, setting, methods Regulation of HUWE1 gene expression by RARα factor was evaluated by performing ChIP assay, EMSA, and siRNA-mediated RARα knockdown. We used C18-4 cells to determine whether HUWE1 participated in RA-mediated RARα signaling and performed luciferase assay, CCK-8 assay, immunofluorescence staining, RT-qPCR, and western blot analysis. The expression level of HUWE1 and RARa was evaluated in clinical samples obtained from NOA or obstructive azoospermia (OA) patients using RT-qPCR. Main results and the role of chance We explored the mechanism by which polymorphism of the HUWE1 promoter leads to spermatogenesis disorders, and focused on the identification of mutations in the SNP site of the HUWE1 regulatory sequence among patients with NOA, which may influence HUWE1 expression. The results of in vitro experiments showed that RARα binds to the HUWE1 promoter region and regulates its expression. However, the regulatory effect of RARα weakened following introduction of the mutation into the HUWE1 regulatory sequence. The expression of spermatogonial differentiation-related gene STRA8 was impaired, and γH2AX foci accumulated following inhibition of HUWE1 gene expression using siRARα. Finally, to determine whether HUWE1 expression was important in patients with NOA, its expression was verified using testicular puncture samples collected from patients with NOA and the results revealed that HUWE1 expression was abnormal (P < 0.01). In addition, RARα expression was significantly downregulated (P < 0.001). Overall, our result support the hypothesis that HUWE1 expression is induced by the RA/RARα signaling pathway and it inhibit γH2AX foci accumulation, thereby considerably promoting sperm differentiation and meiotic prophase. Limitations, reasons for caution The effect of SNPs in HUWE1 on male infertility is further studied in a wider population. Wider implications of the findings Our study of HUWE1 and RA/RARα signaling pathway provides new information on the regulatory mechanism of spermiogenesis, suggesting that SNPs in HUWE1 promoter are etiological factors in patients with NOA and providing a rationale for further drug development to target HUWE1 in these patients. Trial registration number the National Key Research and Development Program of China (2018YFC1003603)

P16 Regulates Transcription Factor Sp1 Function to Affect Outcome in Oropharyngeal Cancer

<h3>Purpose/Objective(s)</h3> Our group has previously demonstrated that p16 activates a ubiquitin mediated pathway to repress homologous recombination via activation of HUWE1, potentially via the transcription factor Sp1. However, the specific mechanism of this interaction and its clinical significance remain to be determined. We performed <i>in vitro</i> analysis of p16 and Sp1 interaction in multiple contexts and performed correlative analysis using the head and neck TCGA clinical data, as well as analysis of HPV+ and HPV- oropharyngeal cancer (OPC) treated with chemoradiation (CRT) to evaluate the combined importance of p16 and Sp1 on outcome. We hypothesized that p16 increases the Sp1-HUWE1 interaction. <h3>Materials/Methods</h3> For clinical outcome, 100 patients with oropharyngeal cancer evenly divided between p16+ and p16- and treated with chemoradiation were evaluated for gene expression using targeted multiplex analysis platform. Additional clinical correlative analyses between HPV, p16, HUWE1 and Sp1 were performed using the TCGA Head and Neck cohort (n=530). UM-SCC-47 p16 CRISPR KO and HN5 cells harboring doxycycline-inducible p16 were used to perform <i>in vitro</i> studies. Immunoprecipitation (IP) was used to confirm the protein-protein interaction between protein p16 and transcription factor Sp1. Chromatin immunoprecipitation (ChIP) assay was used to determine the existence of the Sp1 protein-HUWE1 DNA promoter interaction. <h3>Results</h3> Sp1 (p=7.9e⁻¹⁶) and HUWE1 (p=7.9e⁻⁴) were expressed at significantly higher levels in HPV+ tumors and Sp1 and HUWE1 gene expression were highly correlated (p=5.3e10⁻⁴⁵) in the TCGA Head and Neck cohort. In addition, Sp1 expression was significantly higher in p16 positive OPC tumors in a separate patient cohort (p=3e⁻³). In OPC patents treated uniformly with CRT, the combination of p16 and Sp1 expression were associated with reduced locoregional recurrence following CRT (p=7e⁻⁶), effectively delineating good prognosis p16- patients and poor prognosis p16+ patients. IP of p16 in HN5 cells showed increased interaction with Sp1 after doxycycline-induced p16INK4a expression, and in combination with IR. ChIP assay demonstrated SP1 dependent enrichment in 2 of 5 GC box regions of the human HUWE1 promoter. More importantly, these SP1 protein-HUWE1 promoter interactions were significantly enhanced after p16 overexpression. <h3>Conclusion</h3> We show Sp1 directly interacts with HUWE1 promoter resulting in an increase of HUWE1 expression, and this interaction is mediated by p16. This increase of HUWE1 expression then leads to down-regulation of homologous recombination, sensitizing cells to DNA damage. Clinical data shows the combined expression of p16, Sp1 and HUWE1 are highly correlated and that the combination of p16 and Sp1 may be an effective biomarker in OPC.

The E3 ubiquitin ligase HUWE1 acts through the N-Myc-DLL1-NOTCH1 signaling axis to suppress glioblastoma progression.

BackgroundElucidation of the post‐transcriptional modification has led to novel strategies to treat intractable tumors, especially glioblastoma (GBM). The ubiquitin‐proteasome system (UPS) mediates a reversible, stringent and stepwise post‐translational modification which is closely associated with malignant processes of GBM. To this end, developing novel therapeutic approaches to target the UPS may contribute to the treatment of this disease. This study aimed to screen the vital and aberrantly regulated component of the UPS in GBM. Based on the molecular identification, functional characterization, and mechanism investigation, we sought to elaborate a novel therapeutic strategy to target this vital factor to combat GBM.MethodsWe combined glioma datasets and human patient samples to screen and identify aberrantly regulated E3 ubiquitin ligase. Multidimensional database analysis and molecular and functional experiments in vivo and in vitro were used to evaluate the roles of HECT, UBA and WWE domain‐containing E3 ubiquitin ligase 1 (HUWE1) in GBM. dCas9 synergistic activation mediator system and recombinant adeno‐associated virus (rAAV) were used to endogenously overexpress full‐length HUWE1 in vitro and in glioma orthotopic xenografts.ResultsLow expression of HUWE1 was closely associated with worse prognosis of GBM patients. The ubiquitination and subsequent degradation of N‐Myc mediated by HUWE1, leading to the inactivation of downstream Delta‐like 1 (DLL1)‐NOTCH1 signaling pathways, inhibited the proliferation, invasion, and migration of GBM cells in vitro and in vivo. A rAAV dual‐vector system for packaging and delivery of dCas9‐VP64 was used to augment endogenous HUWE1 expression in vivo and showed an antitumor activity in glioma orthotopic xenografts.ConclusionsThe E3 ubiquitin ligase HUWE1 acts through the N‐Myc‐DLL1‐NOTCH1 signaling axis to suppress GBM progression. Antitumor activity of rAAV dual‐vector delivering dCas9‐HUWE1 system uncovers a promising therapeutic strategy for GBM.

Ubiquitin ligase E3 HUWE1/MULE targets transferrin receptor for degradation and suppresses ferroptosis in acute liver injury.

Hepatic ischemia followed by reperfusion (I/R), a major clinical problem during liver surgical procedures, can induce liver injury with severe cell death including ferroptosis which is characterized by iron-dependent accumulation of lipid peroxidation. The HECT domain-containing ubiquitin E3 ligase HUWE1 (also known as MULE) was initially shown to promote apoptosis. However, our preliminary study demonstrates that high expression of HUWE1 in the liver donors corelates with less injury and better hepatic function after liver transplantation in patients. Thus, we investigate the role of HUWE1 in acute liver injury, and identify HUWE1 as a negative ferroptosis modulator through transferrin receptor 1(TfR1). Deficiency of Huwe1 in mice hepatocytes (HKO) exacerbated I/R and CCl4-induced liver injury with more ferroptosis occurrence. Moreover, Suppression of Huwe1 remarkably enhances cellular sensitivity to ferroptosis in primary hepatocytes and mouse embryonic fibroblasts. Mechanistically, HUWE1 specifically targets TfR1 for ubiquitination and proteasomal degradation, thereby regulates iron metabolism. Importantly, chemical and genetic inhibition of TfR1 dramatically diminishes the ferroptotic cell death in Huwe1 KO cells and Huwe1 HKO mice. Therefore, HUWE1 is a potential protective factor to antagonize both aberrant iron accumulation and ferroptosis thereby mitigating acute liver injury. These findings may provide clinical implications for patients with the high-expression Huwe1 alleles.

P53 Ubiquitination Comediated by HUWE1 and TRAF6 Contributes to White Spot Syndrome Virus Infection in Crustacean.

ABSTRACTp53, the guardian of the genome, is a short-lived protein that is tightly controlled at low levels by constant ubiquitination and proteasomal degradation in higher organisms. p53 stabilization and activation are early crucial events to cope with external stimuli in cells. However, the role of p53 ubiquitination and its relevant molecular mechanisms have not been addressed in invertebrates. In this study, our findings revealed that both HUWE1 (HECT, UBA, and WWE domain-containing E3 ubiquitin-protein ligase 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) could serve as E3 ubiquitin ligases for p53 in mud crabs (Scylla paramamosain). Moreover, the expression of HUWE1 and TRAF6 was significantly downregulated during white spot syndrome virus (WSSV) infection, and therefore the ubiquitination of p53 was interrupted, leading to the activation of apoptosis and reactive oxygen species (ROS) signals through p53 accumulation, which eventually suppressed viral invasion in the mud crabs. To the best of our knowledge, this is the first study to reveal the p53 ubiquitination simultaneously induced by two E3 ligases in arthropods, which provides a novel molecular mechanism of invertebrates for resistance to viral infection.IMPORTANCE p53, which is a well-known tumor suppressor that has been widely studied in higher animals, has been reported to be tightly controlled at low levels by ubiquitin-dependent proteasomal degradation. However, recent p53 ubiquitination-relevant research mainly involved an individual E3 ubiquitin ligase, but not whether there exist other mechanisms that need to be explored. The results of this study show that HUWE1 and TRAF6 could serve as p53 E3 ubiquitin ligases and synchronously mediate p53 ubiquitination in mud crabs (Scylla paramamosain), which confirmed the diversity of the p53 ubiquitination regulatory pathway. In addition, the effects of p53 ubiquitination are mainly focused on tumorigenesis, but a few are focused on the host immune defense in invertebrates. Our findings reveal that p53 ubiquitination could affect ROS and apoptosis signals to cope with WSSV infection in mud crabs, which is the first clarification of the immunologic functions and mechanisms of p53 ubiquitination in invertebrates.

HUWE1 Causes an Immune Imbalance in Immune Thrombocytopenic Purpura by Reducing the Number and Function of Treg Cells Through the Ubiquitination Degradation of Ets-1.

Background: Immune thrombocytopenic purpura (ITP) is an autoimmune bleeding disorder and the decreased number and immunosuppressive dysfunction of Treg cells are key promoters of ITP. However, their mechanisms in ITP development have not been fully clarified. Methods: HUWE1 mRNA and protein levels in CD4+ T cells in peripheral blood from ITP patients were assessed by quantitative real-time PCR and Western blot. HUWE1 function in ITP was estimated using flow cytometry, enzyme-linked immunosorbent assay and immunosuppression assay. Besides, the HUWE1 mechanism in reducing the number and function of Treg cells in ITP was investigated by immunoprecipitation, cycloheximide-chase assay, ubiquitin experiment and immunofluorescence assay. Results: HUWE1 expression was elevated in CD4+ T cells in peripheral blood from ITP patients and HUWE1 mRNA level was negatively correlated with platelet counts and Treg cell percentage. Moreover, the interference with HUWE1 increased the number of Treg cells and enhanced its immunosuppressive function, and the HUWE1 overexpression produced the opposite results. For the exploration of mechanism, HUWE1 interacted with E26 transformation-specific-1 (Ets-1) and this binding was dependent on the negative regulation of the phosphorylation level of Ets-1 (Thr38) and HUWE1 facilitated the ubiquitin degradation of Ets-1 protein to restrain Treg cell differentiation and weaken their immunosuppressive functions. The in vivo assay confirmed that the HUWE1 inhibitor alleviated ITP in mice. Conclusion: HUWE1 induced the immune imbalance in ITP by decreasing the number and weakening the function of Treg cells through the ubiquitination degradation of Ets-1.

The future belongs to those who prepare for it today

Hippocampal neural stem cells (NSCs) and neurogenesis decline sharply with age, though a small population remains. Two articles in this issue of Cell Stem Cell by Ibrayeva etal. (2021) and Harris etal. (2021) indicate the presence of subpopulations of NSCs whose dynamics of activation and self-renewal change over time and may be key to NSC preservation.

Targeting of the E3 ubiquitin-protein ligase HUWE1 impairs DNA repair capacity and tumor growth in preclinical multiple myeloma models

Experimental evidence suggests that ubiquitin-protein ligases regulate a number of cellular processes involved in tumorigenesis. We analysed the role of the E3 ubiquitin-protein ligase HUWE1 for pathobiology of multiple myeloma (MM), a still incurable blood cancer. mRNA expression analysis indicates an increase in HUWE1 expression levels correlated with advanced stages of myeloma. Pharmacologic as well as RNAi-mediated HUWE1 inhibition caused anti-proliferative effects in MM cell lines in vitro and in an MM1.S xenotransplantation mouse model. Cell cycle analysis upon HUWE1 inhibition revealed decreased S phase cell fractions. Analyses of potential HUWE1-dependent molecular functions did not show involvement in MYC-dependent gene regulation. However, HUWE1 depleted MM cells displayed increased DNA tail length by comet assay, as well as changes in the levels of DNA damage response mediators such as pBRCA1, DNA-polymerase β, γH2AX and Mcl-1. Our finding that HUWE1 might thus be involved in endogenous DNA repair is further supported by strongly enhanced apoptotic effects of the DNA-damaging agent melphalan in HUWE1 depleted cells in vitro and in vivo. These data suggest that HUWE1 might contribute to tumour growth by endogenous repair of DNA, and could therefore potentially be exploitable in future treatment developments.

Associations between Huwe1 and autophagy in rat cerebral neuron oxygen‑glucose deprivation and reperfusion injury.

Autophagy and the ubiquitin proteasome system (UPS) are two major protein degradation pathways involved in brain ischemia. Autophagy can compensate for UPS impairment-induced cellular dysfunction. HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1 (Huwe1), an E3 ubiquitin ligase, serves critical roles in nervous system plasticity, regeneration and disease. However, the role of Huwe1 in autophagy in brain ischemia/reperfusion (I/R) injury remains unknown. The aim of the present study was to investigate the crosstalk between autophagy and the UPS in brain ischemia. The present study established an oxygen-glucose deprivation and reperfusion (OGD/R) model in rat primary cortex neurons in vitro. Lentiviral interference was used to silence the expression of Huwe1. An autophagy promoter (rapamycin), an autophagy inhibitor (wortmannin) and a JNK pathway inhibitor (SP600125) were also used in the current study. Cellular autophagy-related proteins, including Beclin-1, autophagy related (ATG) 7, ATG5, ATG3 and microtubule associated protein 1 light chain 3 α, and apoptosis-related proteins, such as P53, cleaved caspase 3, Bax and Bcl2, were detected via western blotting and immunocytochemistry. Neuronal apoptosis was evaluated using a TUNEL assay. The results demonstrated that silencing Huwe1 increased the expression levels of autophagy-related proteins at 24 h after OGD/R. Treatment with a JNK inhibitor or cotreatment with Huwe1 shRNA significantly increased autophagy. Rapamycin increased apoptosis under OGD/R conditions. However, treatment with Huwe1 shRNA decreased the number of TUNEL-positive cells at 24 h after OGD/R. Cotreatment with Huwe1 shRNA and wortmannin alleviated neuronal apoptosis under OGD/R conditions compared with cotreatment with DMSO. Collectively, the present results suggested that silencing Huwe1 was accompanied by a compensatory induction of autophagy under OGD/R conditions. Furthermore, the JNK pathway may be a key mediator of the interaction between Huwe1 and autophagy in response to UPS impairment.

Abstract 5341: Derangements in HUWE1/c-MYC pathway confer sensitivity to the BET bromodomain inhibitor GS-626510 in uterine cervical carcinoma

Abstract Purpose: The prognosis for women with advanced or recurrent cervical cancer (CC) no longer responsive to radiation and/or chemotherapy remains poor. Whole-exome-sequencing (WES) studies have recently reported c-MYC gene amplification and HUWE1 gene mutations in a significant number of CC suggesting the HUWE1/c-MYC pathway as potential therapeutic target. We investigated HUWE1 and c-MYC expression in fresh-frozen CC and the potential activity of the novel BET bromodomain inhibitor GS-626510 (Gilead-Science-Inc.) against primary WES CC-cultures and CC-xenografts. Experimental Design: HUWE1 and c-MYC expression were evaluated by qRT-PCR in a total of 23 CC including 12 fresh frozen tumor tissues and 11 primary cell lines. c-MYC expression was also evaluated by Western-Blot (WB) and fluorescence in situ hybridization (FISH) experiments in all 11 fully sequenced primary CC cell lines. Primary tumors were evaluated for sensitivity to GS-626510 in-vitro using proliferation and viability-assays. siRNA experiments were used to evaluate the effect of HUWE1 silencing on primary CC cell line growth and sensitivity to GS-626510. Finally, the in-vivo activity of GS-626510 was studied in CC-CVX8 mouse-xenografts. Results: Fresh-frozen CC and primary CC cell lines overexpressed c-MYC when compared to normal control tissues (mean ± SEM mRNA values = 7.06 ± 1.17 vs a mean ± SEM = 2.96 ± 0.97, p = 0.01). FISH experiments demonstrated amplification of c-MYC in 9 out of 11 (82%) of the primary CC cell lines. Primary CC cell lines with derangements in the HUWE1/c-MYC pathway were highly sensitive to GS-626510, with a dose-response decrease in cell proliferation and IC50 values ranging from 3 to 57 nM. Silencing of HUWE1 by siRNA significantly increased c-MYC expression as well as CC cell proliferation and enhanced the in vitro sensitivity to GS-626510. Twice daily oral doses of GS-626510 (10 mg/kg) were well tolerated in vivo in animals and highly effective in decreasing tumor-growth (p = 0.004) and increasing survival (p value = 0.004) of mice harboring CC-CVX8 xenografts. Conclusions: Downregulation/inactivation of HUWE1 may increase c-MYC expression and proliferation in primary CC cell lines. GS-626510 may represent a novel, potentially highly effective therapeutic agent against CC overexpressing c-MYC and/or harboring HUWE1 mutations. Clinical studies with BET inhibitor in CC-patients harboring radiation/chemotherapy-resistant disease are warranted. Citation Format: Elena Bonazzoli, Stefania Bellone, Luca Zammataro, Barbara Gnutti, Adele Guglielmi, Silvia Pelligra, Paola Manara, Joan Tymon-Rosario, Burak Zeybek, Chanhee Han, Alessandro D. Santin. Derangements in HUWE1/c-MYC pathway confer sensitivity to the BET bromodomain inhibitor GS-626510 in uterine cervical carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5341.

HUWE1 cooperates with RAS activation to control leukemia cell proliferation and human hematopoietic stem cells differentiation fate.

Acute myeloid leukemia (AML) is a poor prognosis hematopoietic malignance characterized by abnormal proliferation and differentiation of hematopoietic stem cells (HSCs). Although advances in treatment have greatly improved survival rates in young patients, in the elderly population, ~70% of patients present poor prognosis. A pan-cancer analysis on the TCGA cohort showed that AML has the second higher HUWE1 expression in tumor samples among all cancer types. In addition, pathway enrichment analysis pointed to RAS signaling cascade as one of the most important pathways associated to HUWE1 expression in this particular AML cohort. In silico analysis for biological processes enrichment also revealed that HUWE1 expression is correlated with 13 genes involved in myeloid differentiation. Therefore, to understand the role of HUWE1 in human hematopoietic stem and progenitor cells (HSPC) we constitutively expressed KRASG12V oncogene concomitantly to HUWE1 knockdown in stromal co-cultures. The results showed that, in the context of KRASG12V, HUWE1 significantly reduces cell cumulative growth and changes myeloid differentiation profile of HSPCs. Overall, these observations suggest that HUWE1 might contribute to leukemic cell proliferation and impact myeloid differentiation of human HSCs, thus providing new venues for RAS-driven leukemia targeted therapy approach.

The E3 ligase HUWE1 inhibition as a therapeutic strategy to target MYC in multiple myeloma

Proteasome inhibitors have provided a significant advance in the treatment of multiple myeloma (MM). Consequently, there is increasing interest in developing strategies to target E3 ligases, de-ubiquitinases, and/or ubiquitin receptors within the ubiquitin proteasome pathway, with an aim to achieve more specificity and reduced side-effects. Previous studies have shown a role for the E3 ligase HUWE1 in modulating c-MYC, an oncogene frequently dysregulated in MM. Here we investigated HUWE1 in MM. We identified elevated expression of HUWE1 in MM compared with normal cells. Small molecule-mediated inhibition of HUWE1 resulted in growth arrest of MM cell lines without significantly effecting the growth of normal bone marrow cells, suggesting a favorable therapeutic index. Studies using a HUWE1 knockdown model showed similar growth inhibition. HUWE1 expression positively correlated with MYC expression in MM bone marrow cells and correspondingly, genetic knockdown and biochemical inhibition of HUWE1 reduced MYC expression in MM cell lines. Proteomic identification of HUWE1 substrates revealed a strong association of HUWE1 with metabolic processes in MM cells. Intracellular glutamine levels are decreased in the absence of HUWE1 and may contribute to MYC degradation. Finally, HUWE1 depletion in combination with lenalidomide resulted in synergistic anti-MM activity in both in vitro and in vivo models. Taken together, our data demonstrate an important role of HUWE1 in MM cell growth and provides preclinical rationale for therapeutic strategies targeting HUWE1 in MM.

Enhanced immortalization, HUWE1 mutations and other biological drivers of breast invasive carcinoma in Black/African American patients.

Black/African-American (B/AA) breast cancer patients tend to have more aggressive tumor biology compared to White/Caucasians. In this study, a variety of breast tumor molecular expression profiles of patients derived from the two racial groupings were investigated. Breast invasive carcinoma sample data (RNASeq version 2, Reverse Phase Protein Array, mutation, and miRSeq data) from the Cancer Genome Atlas were examined. The results affirm that B/AA patients are more likely than Caucasian patients to harbor the aggressive basal-like or the poor prognosis-associated HER2-enriched molecular subtypes of breast cancer. There is also a higher incidence of the triple-negative breast cancer (TNBC) among B/AA patients than the general population, a fact reflected in the mutation patterns of genes such as PIK3CA and TP53. Furthermore, an immortalization signature gene set, is enriched in samples from B/AA patients. Among stage III patients, TERT, DRAP1, and PQBP1, all members of the immortalization gene signature set, are among master-regulators with increased activity in B/AA patients. Master-regulators driving differences in expression profiles between the two groups include immortalization markers, senescence markers, and immune response and redox gene products. Differences in expression, between B/AA and Caucasian patients, of RB1, hsa-let-7a, E2F1, c-MYC, TERT, and other biomolecules appear to cooperate to enhance entry into the S-phase of the cell cycle in B/AA patients. Higher expression of miR-221, an oncomiR that facilitates entry into the cell cycle S-phase, is regulated by c-MYC, which is expressed more in breast cancer samples from B/AA patients. Furthermore, the cell migration- and invasion-promoting miRNA, miR-135b, has increased relative expression in B/AA patients. Knock down of the immortalization marker TERT inhibited triple-negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) cell viability and decreased expression of TERT, MYC and WNT11. For those patients with available survival data, prognosis of stage II patients 50 years of age or younger at diagnosis, was distinctly poorer in B/AA patients. Also associated with this subset of B/AA patients are missense mutations in HUWE1 and PTEN expression loss. Relative to Caucasian non-responders to endocrine therapy, B/AA non-responders show suppressed expression of a signature gene set on which biological processes including signaling by interleukins, circadian clock, regulation of lipid metabolism by PPARα, FOXO-mediated transcription, and regulation of TP53 degradation are over-represented. Thus, we identify molecular expression patterns suggesting diminished response to oxidative stress, changes in regulation of tumor suppressors/facilitators, and enhanced immortalization in B/AA patients are likely important in defining the more aggressive molecular tumor phenotype reported in B/AA patients.

HUWE1 promotes EGFR ubiquitination and degradation to protect against renal tubulointerstitial fibrosis.

Injury of renal tubular epithelial cells is a key feature of the pathogenicity associated with tubulointerstitial fibrosis and other kidney diseases. HUWE1, an E3 ubiquitin ligase, acts by participating in ubiquitination and degradation of its target proteins. However, the detailed mechanisms by which HUWE1 might regulate fibrosis in renal tubular epithelial cells have not been established. Here, the possible regulation of renal tubulointerstitial fibrosis by HUWE1 was investigated by examining the expression of HUWE1 and EGFR in unilateral ureteral obstruction (UUO) mice. Markedly consistent reciprocal changes in HUWE1 and EGFR expression were observed at the protein and mRNA levels in the kidney after UUO injury. Expression of HUWE1 inhibited TGF-β-induced injury to HK-2 cells, while HUWE1 overexpression decreased the expression of EGFR. Further analysis indicated that HUWE1 physically interacted with EGFR and promoted its ubiquitination and degradation. HUWE1 expression also showed clinical relevance in renal disease, as it notably decreased in multiple types of clinical nephropathy, while EGFR expression significantly increased when compared to the normal kidney. Therefore, this study demonstrated that HUWE1, which serves as an E3 ubiquitin ligase specific for EGFR, promotes EGFR ubiquitination and degradation, thereby regulating EGFR expression and providing protection against kidney injury.

Dysregulation of HUWE1 is associated with an increased mutational burden in Multiple Myeloma

Genomic instability is a prominent feature in the development and progression of Multiple Myeloma (MM). The E3 ligase HUWE1 contributes to the regulation of genomic stability by promoting DNA damage tolerance during replicative stress. Previous studies, by our lab and others, have shown dysregulation of HUWE1 in MM. We have demonstrated an increase in expression of HUWE1 across plasma cell dyscrasias and HUWE1 has been reported as a mutational driver in t(11;14) myeloma. The aim of this study was to investigate the role of HUWE1 in DNA replication and explore whether dysregulation of HUWE1 contributes to genomic instability in MM.

Meta-analysis of gene expression alterations and clinical significance of the HECT domain-containing ubiquitin ligase HUWE1 in cancer.

E3 ubiquitin-protein ligase (HUWE1) has previously been identified as a HECT domain-containing ubiquitin ligase (E3) that is involved in several signaling pathways, transcriptional regulation, neural differentiation, DNA damage responses and apoptosis. However, the function of HUWE1 in the various types of cancer remains unclear. A previous study indicated that HUWE1 exhibited different roles depending on the cancer type due to the ubiquitination of various substrates. The objective of the present study was to determine whether HUWE1 can be employed as a prognostic indicator in human cancer. The expression of HUWE1 was examined using the Oncomine database, and gene alterations during carcinogenesis, copy number alterations and mutations of HUWE1 were then analyzed using cBioPortal, which is the International Cancer Genome Consortium and the Tumorscape database. Furthermore, the association between HUWE1 expression and patient survival was evaluated using Kaplan-Meier plotter and the PrognoScan databases. In addition, the present study attempted to establish the functional association between HUWE1 expression and cancer phenotypes, and the results revealed that HUWE1 may serve as a diagnostic marker or therapeutic target for certain types of cancer. HUWE1 may serve an oncogenic role in breast, brain and prostate cancer, while it may serve an anti-oncogenic role in colorectal cancer and certain lung cancers. The function of HUWE1 and its mechanisms require more in-depth and extensive investigation in future studies.

Abstract 2919: p16 modulates a novel ubiquitin signaling cascade which regulates radioresponse and offers clinically relevant therapeutic targets in squamous cell carcinoma

Abstract Human papilloma virus (HPV) is associated with multiple types of squamous cell carcinoma, including that of the head and neck (HNSCC). The presence of HPV leads to increased expression of p16, to the extent that p16 is used as a surrogate marker of HPV positivity. HPV/p16 expression is associated with improved response to genotoxic therapy and improved outcome; however, the mechanism of this phenomenon is unclear. Previously, we showed that HPV/p16 expression repressed the DNA damage repair protein TRIP12 and led to radiosensitivity. In the current project we identify USP7 and HUWE1 as key links in this signaling pathway. Ubiquitin-specific-processing protease 7 (USP7) is a deubiquitinating enzyme that has previously been shown to bind to TRIP12, which we confirmed in HPV (-) HNSCC cell lines. We found that forced p16 overexpression in a panel of SCC cell lines led to decreased levels of USP7 and TRIP12 protein, while inhibition of p16 in HPV (+) HNSCC cell lines led to their increase. Forced p16 expression did not affect USP7 mRNA expression, however did lead to increased K48-linked ubiquitinylation and degradation of USP7. Chemical or shRNA mediated inhibition of USP7 in p16/HPV (-) cells led to decreased 53BP1 and BRCA1 foci and increased cell death following radiation. Expression of USP7 shRNA in a HPV (-), radioresistant cell derived xenograft (HN5) lead to a tumor growth delay (TGD) of 10 days following radiation (4 Gy x 5d), compared to a TGD of 1.4 days in control tumors (p&amp;lt;1x10-15). To determine how p16 regulates USP7 ubquitinylation and degradation, we performed immunoprecipitation/mass spectrography (IP/MS) analysis. In 3 HPV (-) and 3 HPV (+) cell lines, pulldown of USP7 identified several E3 ubquitin ligases, however only HUWE1 and TRIM21 were identified in all cell lines tested. We then examined HUWE1 expression following forced expression of p16 and found that both HUWE1 mRNA and protein were increased. Moreover, inhibition of p16 expression in HPV (+) HNSCC cell lines led to decreased HUWE1 expression. Moreover, inhibition of HUWE1 via shRNA could partially rescue the effects of p16 overexpression on expression of DNA damage repair proteins. As HUWE1 is known to be mutated in ~10% of HNSCC, we then examined the effects of HUWE1 on outcome in the TCGA HNSCC patient cohort. In HPV (+) patients, neither HUWE1 mutation nor its gene expression were associated with survival. However, in HPV (-) patients, low HUWE1 expression was associated with worse disease-free survival (DFS)(p=0.048). Additionally, truncating mutations in HUWE1 led to a median survival of 9.4 mos, compared to 67.7 mos in the remaining patients (p=0.008). In conclusion, p16-HUWE1-USP7 signaling can modulate response to radiation. These data suggest that agents under investigation to target USP7, or other members of this signaling cascade, have the potential to improve outcomes in HNSCC. Citation Format: David Molkentine, Kathleen Bridges, Aakash Sheth, David Valdecanas, Curtis Pickering, Heath D. Skinner. p16 modulates a novel ubiquitin signaling cascade which regulates radioresponse and offers clinically relevant therapeutic targets in squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2919.

Silencing Huwe1 reduces apoptosis of cortical neurons exposed to oxygen-glucose deprivation and reperfusion.

HECT, UBA and WWE domain-containing 1 (Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase (JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor (SP600125) or a p38MAPK inhibitor (SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38MAPK and of apoptosis-related proteins (p53, Gadd45a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018 (approval No. 2018013).