Thioredoxin-interacting protein (TXNIP) is a substrate of the NEDD4-like E3 ubiquitin-protein ligase WWP1 in cellular redox state regulation of acute myeloid leukemia cells.

The HECT-type E3 ubiquitin WWP1 (also known as NEDD4-like E3 ubiquitin-protein ligase WWP1) acts as an oncogenic factor in acute myeloid leukemia (AML) cells. WWP1 overexpression in AML confers a proliferative advantage to leukemic blasts (abnormal immature white blood cells) and counteracts apoptotic cell death and differentiation. In an effort to elucidate the molecular basis of WWP1 oncogenic activities, we identified WWP1 as a previously unknown negative regulator of thioredoxin-interacting protein (TXNIP)-mediated reactive oxygen species (ROS) production in AML cells. TXNIP inhibits the disulfide reductase enzymatic activity of thioredoxin (Trx), impairing its antioxidant function and, ultimately, leading to the disruption of cellular redox homeostasis. In addition, TXNIP restricts cell growth and survival by blocking glucose uptake and metabolism. Here, we found that WWP1 directly interacts with TXNIP, thus promoting its ubiquitin-dependent proteasomal proteolysis. As a result, accumulation of TXNIP in response to WWP1 inactivation in AML blasts reduces Trx activity and increases ROS production, hence inducing cellular oxidative stress. Increased ROS generation in WWP1-depleted cells culminates in DNA strand breaks and subsequent apoptosis. Coherently with TXNIP stabilization following WWP1 inactivation, we also observed an impairment of both glucose up-take and consumption. Hence, a contribution to the increased cell death observed in WWP1-depleted cells also possibly arises from the attenuation of glucose up-take and glycolytic flux resulting from TXNIP accumulation. Future studies are needed to establish whether TXNIP-dependent deregulation of redox homeostasis in WWP1-overexpressing blasts may affect the response of leukemic cells to chemotherapeutic drugs.

A comprehensive molecular characterization of a claudin-low luminal B breast tumor.

Breast cancer is the most common cause of death from cancer in women. Here, we present the case of a 43-year-old woman, who received a diagnosis of claudin-low luminal B breast cancer. The lesion revealed to be a poorly differentiated high-grade infiltrating ductal carcinoma, which was strongly estrogen receptor (ER)/progesterone receptor (PR) positive and human epidermal growth factor receptor (HER2) negative. Her tumor underwent in-depth chromosomal, mutational and gene expression analyses. We found a pathogenic protein truncating mutation in the TP53 gene, which is predicted to disrupt its transcriptional activity. The patient also harbors germline mutations in some mismatch repair (MMR) genes, and her tumor displays the presence of immune infiltrates, high tumor mutational burden (TMB) status and the apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3) associated signatures, which, overall, are predictive for the use of immunotherapy. Here, we propose promising prognostic indicators as well as potential therapeutic strategies based on the molecular characterization of the tumor.

Involvement of transcribed lncRNA uc.291 in hyperproliferative skin disorders.

The uc.291 transcript controls keratinocytes differentiation by physical interaction with ACTL6A and subsequent induction of transcription of the genes belonging to the epidermal differentiation complex (EDC). Uc.291 is also implicated in the dedifferentiation phenotype seen in poorly differentiated cutaneous squamous cell carcinomas. Here, we would like to investigate the contribution of uc.291 to the unbalanced differentiation state of keratinocytes observed in hyperproliferative skin disorders, e. g., psoriasis. Psoriasis is a multifactorial inflammatory disease, caused by alteration of keratinocytes homeostasis. The imbalanced differentiation state, triggered by the infiltration of immune cells, represents one of the events responsible for this pathology. In the present work, we explore the role of uc.291 and its interactor ACTL6A in psoriasis skin, using quantitative real-time PCR (RT-qPCR), immunohistochemistry and bioinformatic analysis of publicly available datasets. Our data suggest that the expression of the uc.291 and of EDC genes loricrin and filaggrin (LOR, FLG) is reduced in lesional skin compared to nonlesional skin of psoriatic patients; conversely, the mRNA and protein level of ACTL6A are up-regulated. Furthermore, we provide evidence that the expression of uc.291, FLG and LOR is reduced, while ACTL6A mRNA is up-regulated, in an in vitro psoriasis-like model obtained by treating differentiated keratinocytes with interleukin 22 (IL-22). Furthermore, analysis of a publicly available dataset of human epidermal keratinocytes treated with IL-22 (GSE7216) confirmed our in vitro results. Taken together, our data reveal a novel role of uc.291 and its functional axis with ACTL6A in psoriasis disorder and a proof of concept that biological inhibition of this molecular axis could have a potential pharmacological effect against psoriasis and, in general, in skin diseases with a suppressed differentiation programme.

p63 orchestrates serine and one carbon metabolism enzymes expression in head and neck cancer.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is characterized by high proliferation and limited differentiation. The altered expression of the p53 family members, and specifically of p63, represents a pivotal event in the pathogenesis of HNSCC. Physiologically, p63 affects metabolism through the direct transactivation of the enzyme hexokinase 2, and subsequently controls the proliferation of epithelial cells; nonetheless, its role in cancer metabolism is still largely unclear. The high energetic demand of cancer and the consequent needs of a metabolic reshape, also involve the serine and glycine catabolic and anabolic pathways, including the one carbon metabolism (OCM), to produce energetic compounds (purines) and to maintain cellular homeostasis (glutathione and S-adenosylmethionine). RESULTS: The involvement in serine/glycine starvation by other p53 family members has been reported, including HNSCC. Here, we show that in HNSCC p63 controls the expression of the enzymes regulating the serine biosynthesis and one carbon metabolism. p63 binds the promoter region of genes involved in the serine biosynthesis as well as in the one carbon metabolism. p63 silencing in a HNSCC cell line affects the mRNA and protein levels of these selected enzymes. Moreover, the higher expression of TP63 and its target enzymes, negatively impacts on the overall survival of HNSCC patients. CONCLUSION: These data indicate a direct role of p63 in the metabolic regulation of HNSCC with significant clinical effects.

p63: a crucial player in epithelial stemness regulation.

Epithelial tissue homeostasis is closely associated with the self-renewal and differentiation behaviors of epithelial stem cells (ESCs). p63, a well-known marker of ESCs, is an indispensable factor for their biological activities during epithelial development. The diversity of p63 isoforms expressed in distinct tissues allows this transcription factor to have a wide array of effects. p63 coordinates the transcription of genes involved in cell survival, stem cell self-renewal, migration, differentiation, and epithelial-to-mesenchymal transition. Through the regulation of these biological processes, p63 contributes to, not only normal epithelial development, but also epithelium-derived cancer pathogenesis. In this review, we provide an overview of the role of p63 in epithelial stemness regulation, including self-renewal, differentiation, proliferation, and senescence. We describe the differential expression of TAp63 and ΔNp63 isoforms and their distinct functional activities in normal epithelial tissues and in epithelium-derived tumors. Furthermore, we summarize the signaling cascades modulating the TAp63 and ΔNp63 isoforms as well as their downstream pathways in stemness regulation.

A BRCA2 germline mutation and high expression of immune checkpoints in a TNBC patient.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of mammary carcinoma. Here, we describe a case of an 81-year-old female diagnosed with ductal triple negative breast cancer with a germline pathogenic variant in BReast CAncer gene2 (BRCA2). Genetic testing also revealed the presence of four somatic mutations in the ephrin type-A receptor 3 (EphA3), TP53, BRCA1-associated protein (BAP1), and MYB genes. The BRCA2, TP53, and BAP1 gene mutations are highly predictive of a defective homologous recombination repair system and subsequent chromosomal instability in this patient. Coherently, the patient displayed a strong homologous recombination deficiency signature and high tumor mutational burden status, which are generally associated with increased probability of immune neoantigens formation and presentation, and with tumor immunogenicity. Analysis of immune checkpoint revealed high expression of programmed cell death ligand 1 (PD-L1), programmed cell death ligand 2 (PD-L2), programmed death 1 (PD1), and cytotoxic T-lymphocyte-associated protein 4 (CTLA 4), suggesting that the patient might likely benefit from immunotherapies. Altogether, these findings support an unveiled link between BRCA2 inactivation, HR deficiency and increased expression of immune checkpoints in TNBC. This clinical case highlights the importance of screening TNBC patients for genetic mutations and TMB biomarkers in order to predict the potential efficacy of immunotherapy.

Macrophage polarization and metabolism in atherosclerosis.

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of fatty deposits in the inner walls of vessels. These plaques restrict blood flow and lead to complications such as heart attack or stroke. The development of atherosclerosis is influenced by a variety of factors, including age, genetics, lifestyle, and underlying health conditions such as high blood pressure or diabetes. Atherosclerotic plaques in stable form are characterized by slow growth, which leads to luminal stenosis, with low embolic potential or in unstable form, which contributes to high risk for thrombotic and embolic complications with rapid clinical onset. In this complex scenario of atherosclerosis, macrophages participate in the whole process, including the initiation, growth and eventually rupture and wound healing stages of artery plaque formation. Macrophages in plaques exhibit high heterogeneity and plasticity, which affect the evolving plaque microenvironment, e.g., leading to excessive lipid accumulation, cytokine hyperactivation, hypoxia, apoptosis and necroptosis. The metabolic and functional transitions of plaque macrophages in response to plaque microenvironmental factors not only influence ongoing and imminent inflammatory responses within the lesions but also directly dictate atherosclerotic progression or regression. In this review, we discuss the origin of macrophages within plaques, their phenotypic diversity, metabolic shifts, and fate and the roles they play in the dynamic progression of atherosclerosis. It also describes how macrophages interact with other plaque cells, particularly T cells. Ultimately, targeting pathways involved in macrophage polarization may lead to innovative and promising approaches for precision medicine. Further insights into the landscape and biological features of macrophages within atherosclerotic plaques may offer valuable information for optimizing future clinical treatment for atherosclerosis by targeting macrophages.

A primary luminal/HER2 negative breast cancer patient with mismatch repair deficiency.

Here, we present the case of a 47-year-old woman diagnosed with luminal B breast cancer subtype and provide an in-depth analysis of her gene mutations, chromosomal alterations, mRNA and protein expression changes. We found a point mutation in the FGFR2 gene, which is potentially hyper-activating the receptor function, along with over-expression of its ligand FGF20 due to genomic amplification. The patient also harbors somatic and germline mutations in some mismatch repair (MMR) genes, with a strong MMR mutational signature. The patient displays high microsatellite instability (MSI) and tumor mutational burden (TMB) status and increased levels of CTLA-4 and PD-1 expression. Altogether, these data strongly implicate that aberrant FGFR signaling, and defective MMR system might be involved in the development of this breast tumor. In addition, high MSI and TMB in the context of CTLA-4 and PD-L1 positivity, suggest the potential benefit of immune checkpoint inhibitors. Accurate characterization of molecular subtypes, based on gene mutational and expression profiling analyses, will be certainly helpful for individualized treatment and targeted therapy of breast cancer patients, especially for those subtypes with adverse outcome.

Aged mesenchymal stem cells and inflammation: from pathology to potential therapeutic strategies.

Natural ageing of organisms and corresponding age-related diseases result mainly from stem cell ageing and "inflammaging". Mesenchymal stem cells (MSCs) exhibit very high immune-regulating capacity and are promising candidates for immune-related disease treatment. However, the effect of MSC application is not satisfactory for some patients, especially in elderly individuals. With ageing, MSCs undergo many changes, including altered cell population reduction and differentiation ability, reduced migratory and homing capacity and, most important, defective immunosuppression. It is necessary to explore the relationship between the "inflammaging" and aged MSCs to prevent age-related diseases and increase the therapeutic effects of MSCs. In this review, we discuss changes in naturally ageing MSCs mainly from an inflammation perspective and propose some ideas for rejuvenating aged MSCs in future treatments.

Apoptotic cell death in disease-Current understanding of the NCCD 2023.

Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.

Distinct interactors define the p63 transcriptional signature in epithelial development or cancer.

The TP63 is an indispensable transcription factor for development and homeostasis of epithelia and its derived glandular tissue. It is also involved in female germline cell quality control, muscle and thymus development. It is expressed as multiple isoforms transcribed by two independent promoters, in addition to alternative splicing occurring at the mRNA 3'-UTR. Expression of the TP63 gene, specifically the amino-deleted p63 isoform, ΔNp63, is required to regulate numerous biological activities, including lineage specification, self-renewal capacity of epithelial stem cells, proliferation/expansion of basal keratinocytes, differentiation of stratified epithelia. In cancer, ΔNp63 is implicated in squamous cancers pathogenesis of different origin including skin, head and neck and lung and in sustaining self-renewal of cancer stem cells. How this transcription factor can control such a diverse set of biological pathways is central to the understanding of the molecular mechanisms through which p63 acquires oncogenic activity, profoundly changing its down-stream transcriptional signature. Here, we highlight how different proteins interacting with p63 allow it to regulate the transcription of several central genes. The interacting proteins include transcription factors/regulators, epigenetic modifiers, and post-transcriptional modifiers. Moreover, as p63 depends on its interactome, we discuss the hypothesis to target the protein interactors to directly affect p63 oncogenic activities and p63-related diseases.

Fate mapping and scRNA sequencing reveal origin and diversity of lymph node stromal precursors.

Lymph node (LN) stromal cells play a crucial role in LN development and in supporting adaptive immune responses. However, their origin, differentiation pathways, and transcriptional programs are still elusive. Here, we used lineage-tracing approaches and single-cell transcriptome analyses to determine origin, transcriptional profile, and composition of LN stromal and endothelial progenitors. Our results showed that all major stromal cell subsets and a large proportion of blood endothelial cells originate from embryonic Hoxb6+ progenitors of the lateral plate mesoderm (LPM), whereas lymphatic endothelial cells arise from Pax3+ progenitors of the paraxial mesoderm (PXM). Single-cell RNA sequencing revealed the existence of different Cd34+ and Cxcl13+ stromal cell subsets and showed that embryonic LNs contain proliferating progenitors possibly representing the amplifying populations for terminally differentiated cells. Taken together, our work identifies the earliest embryonic sources of LN stromal and endothelial cells and demonstrates that stromal diversity begins already during LN development.

Emerging roles of the HECT-type E3 ubiquitin ligases in hematological malignancies.

Ubiquitination-mediated proteolysis or regulation of proteins, ultimately executed by E3 ubiquitin ligases, control a wide array of cellular processes, including transcription, cell cycle, autophagy and apoptotic cell death. HECT-type E3 ubiquitin ligases can be distinguished from other subfamilies of E3 ubiquitin ligases because they have a C-terminal HECT domain that directly catalyzes the covalent attachment of ubiquitin to their substrate proteins. Deregulation of HECT-type E3-mediated ubiquitination plays a prominent role in cancer development and chemoresistance. Several members of this subfamily are indeed frequently deregulated in human cancers as a result of genetic mutations and altered expression or activity. HECT-type E3s contribute to tumorigenesis by regulating the ubiquitination rate of substrates that function as either tumour suppressors or oncogenes. While the pathological roles of the HECT family members in solid tumors are quite well established, their contribution to the pathogenesis of hematological malignancies has only recently emerged. This review aims to provide a comprehensive overview of the involvement of the HECT-type E3s in leukemogenesis.

The Impact of the Ubiquitin System in the Pathogenesis of Squamous Cell Carcinomas.

The ubiquitin system is a dynamic regulatory pathway controlling the activity, subcellular localization and stability of a myriad of cellular proteins, which in turn affects cellular homeostasis through the regulation of a variety of signaling cascades. Aberrant activity of key components of the ubiquitin system has been functionally linked with numerous human diseases including the initiation and progression of human tumors. In this review, we will contextualize the importance of the two main components of the ubiquitin system, the E3 ubiquitin ligases (E3s) and deubiquitinating enzymes (DUBs), in the etiology of squamous cell carcinomas (SCCs). We will discuss the signaling pathways regulated by these enzymes, emphasizing the genetic and molecular determinants underlying their deregulation in SCCs.

DHA Affects Microtubule Dynamics Through Reduction of Phospho-TCTP Levels and Enhances the Antiproliferative Effect of T-DM1 in Trastuzumab-Resistant HER2-Positive Breast Cancer Cell Lines.

Trastuzumab emtansine (T-DM1) is an anti-human epidermal growth factor receptor 2 (HER2) antibody-drug conjugated to the microtubule-targeting agent emtansine (DM1). T-DM1 is an effective agent in the treatment of patients with HER2-positive breast cancer whose disease has progressed on the first-line trastuzumab containing chemotherapy. However, both primary and acquired tumour resistance limit its efficacy. Increased levels of the phosphorylated form of Translationally Controlled Tumour Protein (phospho-TCTP) have been shown to be associated with a poor clinical response to trastuzumab therapy in HER2-positive breast cancer. Here we show that phospho-TCTP is essential for correct mitosis in human mammary epithelial cells. Reduction of phospho-TCTP levels by dihydroartemisinin (DHA) causes mitotic aberration and increases microtubule density in the trastuzumab-resistant breast cancer cells HCC1954 and HCC1569. Combinatorial studies show that T-DM1 when combined with DHA is more effective in killing breast cells compared to the effect induced by any single agent. In an orthotopic breast cancer xenograft model (HCC1954), the growth of the tumour cells resumes after having achieved a complete response to T-DM1 treatment. Conversely, DHA and T-DM1 treatment induces a severe and irreversible cytotoxic effect, even after treatment interruption, thus, improving the long-term efficacy of T-DM1. These results suggest that DHA increases the effect of T-DM1 as poison for microtubules and supports the clinical development of the combination of DHA and T-DM1 for the treatment of aggressive HER2-overexpressing breast cancer.

HECT-Type E3 Ubiquitin Ligases in Cancer.

Ubiquitination, a post-translational modification that involves a covalent attachment of ubiquitin to a protein substrate, is essential for cellular homeostatic maintenance. At the end of a three-enzyme cascade, E3 ubiquitin ligases (E3s) recruit substrates and promote or directly catalyze ubiquitin transfer to targets. These enzymes largely determine the specificity of the ubiquitination reaction. Genetic alteration, abnormal expression, or dysfunction of E3s account for the occurrence and progression of human cancers. Indeed, excessive degradation of relevant tumor-suppressor molecules and impaired disposal of oncogenic proteins have been linked to tumorigenesis. This review focuses on the emerging roles of HECT-type E3s in tumorigenesis, and emphasizes how perturbations of these enzymes contribute to cancer pathogenesis.

Emerging roles of HECT-type E3 ubiquitin ligases in autophagy regulation.

Autophagy is a conserved self-eating process that delivers cytoplasmic material to the lysosome to allow degradation of intracellular components, including soluble, unfolded and aggregated proteins, damaged organelles, and invading microorganisms. Autophagy provides a homeostatic control mechanism and is essential for balancing sources of energy in response to nutrient stress. Autophagic dysfunction or dysregulation has been implicated in several human pathologies, including cancer and neurodegeneration, and its modulation has substantial potential as a therapeutic strategy. Given the relevant clinical and therapeutic implications of autophagy, there is emerging intense interest in the identification of the key factors regulating the components of the autophagic machinery. Various post-translational modifications, including ubiquitylation, have been implicated in autophagy control. The list of the E3 ubiquitin protein ligases involved in the regulation of several steps of the autophagic process is continuously growing. In this review, we will focus on recent advances in the understanding of the role of the homologous to the E6AP carboxyl terminus-type E3 ubiquitin ligases in autophagy control.

ΔNp63 promotes IGF1 signalling through IRS1 in squamous cell carcinoma.

Accumulating evidence has proved that deregulation of ΔNp63 expression plays an oncogenic role in head and neck squamous cell carcinomas (HNSCCs). Besides p63, the type 1-insulin-like growth factor (IGF) signalling pathway has been implicated in HNSCC development and progression. Most insulin/IGF1 signalling converges intracellularly onto the protein adaptor insulin receptor substrate-1 (IRS-1) that transmits signals from the receptor to downstream effectors, including the PI3K/AKT and the MAPK kinase pathways, which, ultimately, promote proliferation, invasion, and cell survival. Here we report that p63 directly controls IRS1 transcription and cellular abundance and fosters the PI3K/AKT and MAPK downstream signalling pathways. Inactivation of ΔNp63 expression indeed reduces tumour cell responsiveness to IGF1 stimulation, and inhibits the growth potential of HNSCC cells. In addition, a positive correlation was observed between p63 and IRS1 expression in human HNSCC tissue arrays and in publicly available gene expression data. Our findings indicate that aberrant expression of ΔNp63 in HNSSC may act as an oncogenic stimulus by altering the IGF signalling pathway.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

The p53 Family in Brain Disease.

SIGNIFICANCE: The p53 family of transcription factors, including p53, p63, and p73, plays key roles in both biological and pathological processes, including cancer and neural development. Recent Advances: In recent years, a growing body of evidence has indicated that the entire p53 family is involved in the regulation of the central nervous system (CNS) functions as well as in the pathogenesis of several neurological disorders. Mechanistically, the p53 proteins control neuronal cell fate, terminal differentiation, and survival, via a complex interplay among the family members. CRITICAL ISSUES: In this article, we discuss the involvement of the p53 family in neurobiology and in pathological conditions affecting the CNS, including neuroinflammation. FUTURE DIRECTIONS: Understanding the molecular mechanism(s) underlying the function of the p53 family could improve our general knowledge of the pathogenesis of brain disorders and potentially pave the road for new therapeutic intervention. Antioxid. Redox Signal. 29, 1-14.