SMURF2 phosphorylation at Thr249 modifies glioma stemness and tumorigenicity by regulating TGF-β receptor stability

Glioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma, the most malignant form of glioma. The implication and underlying mechanisms of SMAD specific E3 ubiquitin protein ligase 2 (SMURF2) on the GSC phenotypes remain unknown. We previously demonstrated that SMURF2 phosphorylation at Thr249 (SMURF2Thr249) activates its E3 ubiquitin ligase activity. Here, we demonstrate that SMURF2Thr249 phosphorylation plays an essential role in maintaining GSC stemness and tumorigenicity. SMURF2 silencing augmented the self-renewal potential and tumorigenicity of patient-derived GSCs. The SMURF2Thr249 phosphorylation level was low in human glioblastoma pathology specimens. Introduction of the SMURF2T249A mutant resulted in increased stemness and tumorigenicity of GSCs, recapitulating the SMURF2 silencing. Moreover, the inactivation of SMURF2Thr249 phosphorylation increases TGF-β receptor (TGFBR) protein stability. Indeed, TGFBR1 knockdown markedly counteracted the GSC phenotypes by SMURF2T249A mutant. These findings highlight the importance of SMURF2Thr249 phosphorylation in maintaining GSC phenotypes, thereby demonstrating a potential target for GSC-directed therapy.

Targeting Post-Translational Regulation of p53 in Colorectal Cancer by Exploiting Vulnerabilities in the p53-MDM2 Axis

The role played by the key tumor suppressor gene p53 and the implications of p53 mutations for the development and progression of neoplasia continue to expand. This review focuses on colorectal cancer and the regulators of p53 expression and activity identified over the past decade. These newly recognized regulatory mechanisms include (1) direct regulation of mouse double minute 2 homolog (MDM2), an E3 ubiquitin-protein ligase; (2) modulation of the MDM2-p53 interaction; (3) MDM2-independent p53 degradation; and (4) inhibition of p53 nuclear translocation. We positioned these regulatory mechanisms in the context of p53 missense mutations, which not only evade canonical p53 degradation machinery but also exhibit gain-of-function phenotypes that enhance tumor survival and metastasis. Lastly, we discuss current and potential therapeutic strategies directed against p53 mutant-bearing tumors.

Human gut-microbiome-derived propionate coordinates proteasomal degradation via HECTD2 upregulation to target EHMT2 in colorectal cancer

The human microbiome plays an essential role in the human immune system, food digestion, and protection from harmful bacteria by colonizing the human intestine. Recently, although the human microbiome affects colorectal cancer (CRC) treatment, the mode of action between the microbiome and CRC remains unclear. This study showed that propionate suppressed CRC growth by promoting the proteasomal degradation of euchromatic histone-lysine N-methyltransferase 2 (EHMT2) through HECT domain E3 ubiquitin protein ligase 2 (HECTD2) upregulation. In addition, EHMT2 downregulation reduced the H3K9me2 level on the promoter region of tumor necrosis factor α-induced protein 1 (TNFAIP1) as a novel direct target of EHMT2. Subsequently, TNFAIP1 upregulation induced the apoptosis of CRC cells. Furthermore, using Bacteroides thetaiotaomicron culture medium, we confirmed EHMT2 downregulation via upregulation of HECTD2 and TNFAIP1 upregulation. Finally, we observed the synergistic effect of propionate and an EHMT2 inhibitor (BIX01294) in 3D spheroid culture models. Thus, we suggest the anticancer effects of propionate and EHMT2 as therapeutic targets for colon cancer treatment and may provide the possibility for the synergistic effects of an EHMT2 inhibitor and microbiome in CRC treatment.

Itchy E3 ubiquitin-protein ligase promotes neuroblastoma progression in vitro and in vivo

[Objective] Neuroblastoma is the most common pediatric neuroendocrine tumor. Patients with high-risk neuroblastoma have poor clinical outcomes. Understanding the mechanisms underlying neuroblastoma progression could help identify potential therapeutic targets. This study aimed to explore the roles of itchy E3 ubiquitin-protein ligase (ITCH) in neuroblastoma progression using neuroblastoma cell lines and xenograft models of neuroblastoma. [Methods] ITCH-silencing or overexpressing neuroblastoma cells were established using two different human neuroblastoma cell lines, SK-N-AS and SH-SY5Y. In vitro and in vivo experiments were carried out to determine the effects of ITCH on neuroblastoma cell behaviors. The dual-luciferase reporter assay and co-transfection experiments were applied to determine the interaction of ITCH and miR-145-5p during neuroblastoma progression. [Results] In both cell lines, ITCH overexpression significantly promotes the proliferation, migration, and invasion capacities of neuroblastoma cells, while ITCH silencing with ShITCH suppressed neuroblastoma cell proliferation and induced apoptosis. Moreover, overexpression of ITCH decreased 51% and 54% the protein expressions of large tumor suppressor kinase 1 (LATS1), and inhibited 59% and 66% the phosphorylation of Yes-associated protein (YAP), concomitant with 2.02-fold and 2.56-fold increased expressions of cell proliferation marker Ki67 and 2.51-fold and 2.26-fold elevated levels of anti-apoptosis marker Bcl2 in SK-N-AS and SH-SY5Y cells, respectively. The dual-luciferase reporter assay demonstrated that ITCH interacted with miR-145-5p. Further in vitro and xenograft experiments showed that ITCH negatively affected the tumor-suppressive effect of miR-145-5p. [Conclusion] ITCH promotes neuroblastoma cell proliferation and metastasis by inhibiting LATS1 and promoting YAP nuclear translocation.

SNPs Associated with Foliar Phylloxera Tolerance in Hybrid Grape Populations Carrying Introgression from Muscadinia

Leaf-feeding phylloxera decreases the photosynthetic activity of a grape plant, leading to decreasing number of fruit buds. In addition, phylloxera larvae emerging from the leaf galls may colonize the roots, negatively affecting the growth of the grape plant. In this study, we evaluated host tolerance of three grapevine hybrid populations obtained from crossing of the same maternal grapevine M. no. 31-77-10 with interspecific hybrids carrying introgressions from Muscadinia and other North American Vitis species against leaf-feeding grape phylloxera. Combining genotyping data of the populations obtained with 12,734 SNPs and their resistance phenotypes evaluated in the laboratory experiment, we performed an association study. As the result of GWAS, nine SNPs with the lowest significant p-values were discovered in the whole sample of 139 hybrids as associated with variation of the scores ‘the percentage of infested leaves’ and ‘intensity of gall formation’. Three of the SNPs on LG 7 were located in the same chromosome interval where a major QTL (RDV6) for root phylloxera resistance was reported from Muscadine background. Two SNPs on LG 8 were detected within the gene, encoding E3 ubiquitin-protein ligase UPL4 involved in apoptosis. SNPs detected on LG 13 and LG 18 may overlap with the previously reported QTLs for phylloxera resistance inherited from V. cinerea.

The ides of MARCH5: The E3 ligase essential for peroxisome degradation by pexophagy

A recent study by Zheng et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202103156) identifies the ubiquitin-protein ligase (E3) MARCH5 as a dual-organelle localized protein that not only targets to mitochondria but also to peroxisomes in a PEX19-mediated manner. Moreover, the authors demonstrate that the Torin1-dependent induction of pexophagy is executed by the MARCH5-catalyzed ubiquitination of the peroxisomal membrane protein PMP70.

Zfhx3 Transcription Factor Represses the Expression of SCN5A Gene and Decreases Sodium Current Density (INa)

The ZFHX3 and SCN5A genes encode the zinc finger homeobox 3 (Zfhx3) transcription factor (TF) and the human cardiac Na+ channel (Nav1.5), respectively. The effects of Zfhx3 on the expression of the Nav1.5 channel, and in cardiac excitability, are currently unknown. Additionally, we identified three Zfhx3 variants in probands diagnosed with familial atrial fibrillation (p.M1260T) and Brugada Syndrome (p.V949I and p.Q2564R). Here, we analyzed the effects of native (WT) and mutated Zfhx3 on Na+ current (INa) recorded in HL-1 cardiomyocytes. ZFHX3 mRNA can be detected in human atrial and ventricular samples. In HL-1 cardiomyocytes, transfection of Zfhx3 strongly reduced peak INa density, while the silencing of endogenous expression augmented it (from −65.9 ± 8.9 to −104.6 ± 10.8 pA/pF; n ≥ 8, p < 0.05). Zfhx3 significantly reduced the transcriptional activity of human SCN5A, PITX2, TBX5, and NKX25 minimal promoters. Consequently, the mRNA and/or protein expression levels of Nav1.5 and Tbx5 were diminished (n ≥ 6, p < 0.05). Zfhx3 also increased the expression of Nedd4-2 ubiquitin-protein ligase, enhancing Nav1.5 proteasomal degradation. p.V949I, p.M1260T, and p.Q2564R Zfhx3 produced similar effects on INa density and time- and voltage-dependent properties in WT. WT Zfhx3 inhibits INa as a result of a direct repressor effect on the SCN5A promoter, the modulation of Tbx5 increasing on the INa, and the increased expression of Nedd4-2. We propose that this TF participates in the control of cardiac excitability in human adult cardiac tissue.

660 Targeting T cell fates: converting exhaustion to memory to improve immunotherapeutic responses to cancer

BackgroundIn cancer, CD8+ T cells have the power to target and kill tumor cells with precision, but often fail due to chronic activation from the immunosuppressive tumor microenvironment (TME). T cells that experience prolonged activation in the TME differentiate into a severely dysfunctional cell state known as exhaustion. In healthy tissues, T cells differentiate into tissue-resident memory cells (TRM) in response to infection, which remain lodged in tissues to provide protection from reinfection. When TRM-like cells are found in patient tumors, they are correlated with improved outcomes and responses to immunotherapy. Understanding how to manipulate T cell fates in an effort to prevent exhaustion and favor TRM-characteristics could benefit cancer immunotherapy.MethodsTo explore differences between these cell states, we screened the core TRM gene-expression signatures for genes downregulated as T cells undergo terminal exhaustion. Targets were then overexpressed in antigen-specific T cells and adoptively transferred into tumor-bearing mice for analysis.ResultsInterestingly, many genes related to protein regulation and processing were identified, including a novel gene called Neuralized E3 Ubiquitin Protein Ligase 3 (Neurl3). Neurl3's function is not well described, however, experimentally mutating the RING domain suggests Neurl3 transfers ubiquitin to target proteins for degradation. When Neurl3 was overexpressed in tumor-specific T cells, we found tumor infiltrating lymphocytes still upregulated inhibitory receptors PD1 and Tim3, but showed enhanced anti-tumor function. Neurl3-overexpressing T cells had increased accumulation in the TME, upregulated canonical TRM markers CD69 and CD103, produced more cytokines, controlled tumor growth, and increased mouse survival in B16 melanoma.ConclusionsThese results highlight the understudied field of negative regulation of T cell function by protein degradation in T cell differentiation fate and uncover a potential gene target for immunocellular therapies to favor functional T cell fates in cancer.Ethics ApprovalThe study was approved by UCSD's Institutional Animal Care and Use Committee, protocol number S04105.