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The Kluyveromyces lactis superoxide dismutase 1(KlSOD1) and K. lactis CCS1 (KlCCS1) genes are isolated and the effect of their increased dosage on the secretion of heterologous proteins is evaluated. The results have shown that the production of some heterologous proteins is improved by manipulating genes involved in general stress responses.

Background Hailey–Hailey disease (HHD), also known as familial benign chronic pemphigus, is a rare, chronic and recurrent blistering disorder, histologically characterized by suprabasal acantholysis. HHD has been linked to mutations in ATP2C1 , the gene encoding the human adenosine triphosphate (ATP)-powered calcium channel pump. Methods In this work, the genetically tractable yeast Kluyveromyces lactis has been used to study the molecular basis of Hailey-Hailey disease. The K. lactis strain depleted of PMR1 , the orthologue of the human ATP2C1 gene, was used to screen a Madin-Darby canine kidney (MDCK) cDNA library to identify genetic interactors able to suppress the oxidative stress occurring in those cells. Results We have identified the Glutathione S-transferase ϴ-subunit (GST), an important detoxifying enzyme, which restores many of the defects associated with the pmr1Δ mutant. GST overexpression in those cells suppressed the sensitivity to calcium chelating agents and partially re-established calcium (Ca 2 + ) homeostasis by decreasing the high cytosolic Ca 2 + levels in pmr1Δ strain. Moreover, we found that in the K. lactis mutant the mitochondrial dysfunction was suppressed by GST overexpression independently from calcineurin. In agreement with yeast results, a decreased expression of the human GST counterpart ( GSTT1/M1 ) was observed in lesion-derived keratinocytes from HHD patients. Conclusions These data highlighted the Glutathione S-transferase as a candidate gene associated with Hailey-Hailey disease. General significance Kluyveromyces lactis can be considered a good model to study the molecular basis of this pathology. [ABSTRACT FROM AUTHOR]

Hailey-Hailey disease (HHD) is a rare autosomal dominantly inherited disorder caused by mutations in the ATP2C1 gene that encodes an adenosine triphosphate (ATP)-powered calcium channel pump. HHD is characterized by impaired epidermal cell-to-cell adhesion and defective keratinocyte growth/differentiation. The mechanism by which mutant ATP2C1 causes HHD is unknown and current treatments for affected individuals do not address the underlying defects and are ineffective. Notch signalling is a direct determinant of keratinocyte growth and differentiation. We found that loss of ATP2C1 leads to impaired Notch1 signalling, thus deregulation of the Notch signalling response is therefore likely to contribute to HHD manifestation. NOTCH1 is a transmembrane receptor and upon ligand binding, the intracellular domain (NICD) translocates to the nucleus activating its target genes. In the context of HHD, we found that loss of ATP2C1 function promotes upregulation of the active NOTCH1 protein (NICD-Val1744). Here, deeply exploring this aspect, we observed that NOTCH1 activation is not associated with the transcriptional enhancement of its targets. Moreover, in agreement with these results, we found a cytoplasmic localization of NICD-Val1744. We have also observed that ATP2C1-loss is associated with the degradation of NICD-Val1744 through the lysosomal/proteasome pathway. These results show that ATP2C1-loss could promote a mechanism by which NOTCH1 is endocytosed and degraded by the cell membrane. The deregulation of this phenomenon, finely regulated in physiological conditions, could in HHD lead to the deregulation of NOTCH1 with alteration of skin homeostasis and disease manifestation., (© 2023 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.)

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy considered curable by modern clinical management. Nevertheless, the prognosis for T-ALL high-risk cases or patients with relapsed and refractory disease is still dismal. Therefore, there is a keen interest in developing more efficient and less toxic therapeutic approaches. T-ALL pathogenesis is associated with Notch signaling alterations, making this pathway a highly promising target in the fight against T-ALL. Here, by exploring the anti-leukemic capacity of the natural polyphenol curcumin and its derivatives, we found that curcumin exposure impacts T-ALL cell line viability and decreases Notch signaling in a dose- and time-dependent fashion. However, our findings indicated that curcumin-mediated cell outcomes did not depend exclusively on Notch signaling inhibition, but might be mainly related to compound-induced DNA-damage-associated cell death. Furthermore, we identified a novel curcumin-based compound named CD2066, endowed with potentiated anti-proliferative activity in T-ALL compared to the parent molecule curcumin. At nanomolar concentrations, CD2066 antagonized Notch signaling, favored DNA damage, and acted synergistically with the CDK1 inhibitor Ro3306 in T-ALL cells, thus representing a promising novel candidate for developing therapeutic agents against Notch-dependent T-ALL.

The cross-talk between axon and glial cells during development and in adulthood is mediated by several molecules. Among them are neurotransmitters and their receptors, which are involved in the control of myelinating and non-myelinating glial cell development and physiology. Our previous studies largely demonstrate the functional expression of cholinergic muscarinic receptors in Schwann cells. In particular, the M2 muscarinic receptor subtype, the most abundant cholinergic receptor expressed in Schwann cells, inhibits cell proliferation downregulating proteins expressed in the immature phenotype and triggers promyelinating differentiation genes. In this study, we analysed the in vitro modulation of the Neuregulin-1 (NRG1)/erbB pathway, mediated by the M2 receptor activation, through the selective agonist arecaidine propargyl ester (APE). M2 agonist treatment significantly downregulates NRG1 and erbB receptors expression, both at transcriptional and protein level, and causes the internalization and intracellular accumulation of the erbB2 receptor. Additionally, starting from our previous results concerning the negative modulation of Notch-active fragment NICD by M2 receptor activation, in this work, we clearly demonstrate that the M2 receptor subtype inhibits erbB2 receptors by Notch-1/NICD downregulation. Our data, together with our previous results, demonstrate the existence of a cross-interaction between the M2 receptor and NRG1/erbB pathway-Notch1 mediated, and that it is responsible for the modulation of Schwann cell proliferation/differentiation.

Notch signaling guides cell fate decisions by affecting proliferation, apoptosis, stem cell self-renewal, and differentiation depending on cell and tissue context. Given its multifaceted function during tissue development, both overactivation and loss of Notch signaling have been linked to tumorigenesis in ways that are either oncogenic or oncosuppressive, but always context-dependent. Notch signaling is critical for several mechanisms of chemoresistance including cancer stem cell maintenance, epithelial-mesenchymal transition, tumor-stroma interaction, and malignant neovascularization that makes its targeting an appealing strategy against tumor growth and recurrence. During the last decades, numerous Notch-interfering agents have been developed, and the abundant preclinical evidence has been transformed in orphan drug approval for few rare diseases. However, the majority of Notch-dependent malignancies remain untargeted, even if the application of Notch inhibitors alone or in combination with common chemotherapeutic drugs is being evaluated in clinical trials. The modest clinical success of current Notch-targeting strategies is mostly due to their limited efficacy and severe on-target toxicity in Notch-controlled healthy tissues. Here, we review the available preclinical and clinical evidence on combinatorial treatment between different Notch signaling inhibitors and existent chemotherapeutic drugs, providing a comprehensive picture of molecular mechanisms explaining the potential or lacking success of these combinations.

In the physiopathology of cystic fibrosis (CF), oxidative stress implications are recognized and widely accepted. The cystic fibrosis transmembrane conductance regulator (CFTR) defects disrupt the intracellular redox balance causing CF pathological hallmarks. Therefore, oxidative stress together with aberrant expression levels of detoxification genes and microRNAs (miRNAs/miRs) may be associated with clinical outcome. Using total RNA extracted from epithelial nasal cells, the present study analyzed the expression levels of oxidative stress genes and one miRNA using quantitative PCR in a representative number of patients with CF compared with in healthy individuals. The present pilot study revealed the existence of an association among CFTR, genes involved in the oxidative stress response and miR-125b. The observed downregulation of CFTR gene expression was accompanied by increased expression levels of Nuclear factor erythroid derived-2 like2 and its targets NAD(P)H:Quinone Oxidoreductase and glutathione S-transferase 1. Moreover, the expression levels of heme oxygenase-1 (HO-1) and miR-125b were positively correlated with a forced expiratory volume in 1 sec (FEV1) >60% in patients with CF with chronic Pseudomonas aeruginosa lung infection (r=0.74; P<0.001 and r=0.57; P<0.001, respectively). The present study revealed the activation of an inducible, but not fully functional, oxidative stress response to protect airway cells against reactive oxygen species-dependent injury in CF disease. Additionally, the correlations of HO-1 and miR-125b expression with an improved FEV1 value suggested that these factors may synergistically protect the airway cells from oxidative stress damage, inflammation and apoptosis. Furthermore, HO-1 and miR-125b may be used as prognostic markers explaining the wide CF phenotypic variability as an additional control level over the CFTR gene mutations., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021, Spandidos Publications.)

Both CDKN1A (p21 Waf1/Cip1 ) and Apoptosis signal-regulating kinase 1 (ASK1) play important roles in tumorigenesis. The role of p21 Waf1/Cip1 in attenuating ASK1-induced apoptosis by various stress conditions is well established. However, how ASK1 and p21 Waf1/Cip1 functionally interact during tumorigenesis is still unclear. To address this aspect, we crossed ASK1 knockout (ASK1KO) mice with p21 Waf1/Cip1 knockout (p21KO) mice to compare single and double-mutant mice. We observed that deletion of p21 Waf1/Cip1 leads to increased keratinocyte proliferation but also increased cell death. This is mechanistically linked to the ASK1 axis-induced apoptosis, including p38 and PARP. Indeed, deletion of ASK1 does not alter the proliferation but decreases the apoptosis of p21KO keratinocytes. To analyze as this interaction might affect skin carcinogenesis, we investigated the response of ASK1KO and p21KO mice to DMBA/TPA-induced tumorigenesis. Here we show that while endogenous ASK1 is dispensable for skin homeostasis, ASK1KO mice are resistant to DMBA/TPA-induced tumorigenesis. However, we found that epidermis lacking both p21 and ASK1 reacquires increased sensitivity to DMBA/TPA-induced tumorigenesis. We demonstrate that apoptosis and cell-cycle progression in p21KO keratinocytes are uncoupled in the absence of ASK1. These data support the model that a critical event ensuring the balance between cell death, cell-cycle arrest, and successful divisions in keratinocytes during stress conditions is the p21-dependent ASK1 inactivation.

SPONASTRIME dysplasia is an ultrarare spondyloepimetaphyseal dysplasia featuring short stature and short limbs, platyspondyly, depressed nasal bridge with midface hypoplasia and striated metaphyses. In 2019, an autosomal recessive inheritance was demonstrated by the identification of bi-allelic hypomorphic alleles in TONSL. The encoded protein has a critical role in maintaining genome integrity by promoting the homologous recombination required for repairing spontaneous replication-associated DNA lesions at collapsed replication forks. We report a 9-year-old girl with typical SPONASTRIME dysplasia and resulted in carrier of the novel missense p.(Gln430Arg) and p.(Leu1090Arg) variants in TONSL at whole-exome sequencing. In silico analysis predicted that these variants induced thermodynamic changes with a pathogenic impact on protein function. To support the pathogenicity of the identified variants, cytogenetic analysis and microscopy assays showed that patient-derived fibroblasts exhibited spontaneous chromosomal breaks and flow cytometry demonstrated defects in cell proliferation and enhanced apoptosis. These findings contribute to our understanding of the molecular pathogenesis of SPONASTRIME dysplasia and might open the way to novel therapeutic approaches., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Unfolded protein response (UPR) is a conserved adaptive response that tries to restore protein homeostasis after endoplasmic reticulum (ER) stress. Recent studies highlighted the role of UPR in acute leukemias and UPR targeting has been suggested as a therapeutic approach. Aberrant Notch signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL), as downregulation of Notch activity negatively affects T-ALL cell survival, leading to the employment of Notch inhibitors in T-ALL therapy. Here we demonstrate that Notch3 is able to sustain UPR in T-ALL cells, as Notch3 silencing favored a Bip-dependent IRE1α inactivation under ER stress conditions, leading to increased apoptosis via upregulation of the ER stress cell death mediator CHOP. By using Juglone, a naturally occurring naphthoquinone acting as an anticancer agent, to decrease Notch3 expression and induce ER stress, we observed an increased ER stress-associated apoptosis. Altogether our results suggest that Notch3 inhibition may prevent leukemia cells from engaging a functional UPR needed to compensate the Juglone-mediated ER proteotoxic stress. Notably, in vivo administration of Juglone to human T-ALL xenotransplant models significantly reduced tumor growth, finally fostering the exploitation of Juglone-dependent Notch3 inhibition to perturb the ER stress/UPR signaling in Notch3-dependent T-ALL subsets.

The eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for the viability of the cells whose proposed function is to prevent the stalling of the ribosomes during translation elongation. eIF5A activity requires a unique and functionally essential post-translational modification, the change of a lysine to hypusine. eIF5A is recognized as a promoter of cell proliferation, but it has also been suggested to induce apoptosis. To date, the precise molecular mechanism through which eIF5A affects these processes remains elusive. In the present study, we explored whether eIF5A is involved in controlling the stress-induced expression of the key cellular regulator p53. Our results show that treatment of HCT-116 colon cancer cells with the deoxyhypusine (DHS) inhibitor N1-guanyl-1,7-diamineheptane (GC7) caused both inhibition of eIF5A hypusination and a significant reduction of p53 expression in UV-treated cells, and that eIF5A controls p53 expression at the level of protein synthesis. Furthermore, we show that treatment with GC7 followed by UV-induced stress counteracts the pro-apoptotic process triggered by p53 up-regulation. More in general, the importance of eIF5A in the cellular stress response is illustrated by the finding that exposure to UV light promotes the binding of eIF5A to the ribosomes, whereas UV treatment complemented by the presence of GC7 inhibits such binding, allowing a decrease of de novo synthesis of p53 protein.

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.

Hailey-Hailey disease (HHD) is a rare, chronic and recurrent blistering disorder, characterized by erosions occurring primarily in intertriginous regions and histologically by suprabasal acantholysis. Mutation of the Golgi Ca 2+ -ATPase ATP2C1 has been identified as having a causative role in Hailey-Hailey disease. HHD-derived keratinocytes have increased oxidative-stress that is associated with impaired proliferation and differentiation. Additionally, HHD is characterized by skin lesions that do not heal and by recurrent skin infections, indicating that HHD keratinocytes might not respond well to challenges such as wounding or infection. Hypochlorous acid has been demonstrated in vitro and in vivo to possess properties that rescue both oxidative stress and altered wound repair process. Thus, we investigated the potential effects of a stabilized form of hypochlorous acid (APR-TD012) in an in vitro model of HHD. We found that treatment of ATP2C1-defective keratinocytes with APR-TD012 contributed to upregulation of Nrf2 (nuclear factor (erythroid-derived 2)-like 2). Additionally, APR TD012-treatment restored the defective proliferative capability of siATP2C1-treated keratinocytes. We also found that the APR-TD012 treatment might support wound healing process, due to its ability to modulate the expression of wound healing associated cytokines. These observations suggested that the APR-TD012 might be a potential therapeutic agent for HHD-lesions.

Calcium signaling can elicit different pathways involved in an extreme variety of biological processes. Calcium levels must be tightly regulated in a spatial and temporal manner in order to be efficiently and properly utilized in the host physiology. The Ca 2+ -ATPase, encoded by pmr-1 gene, was first identified in yeast and localized to the Golgi and it appears to be involved in calcium homeostasis. PMR-1 function is evolutionary conserved from yeast to human, where mutations in the orthologous gene ATP2C1 cause Hailey-Hailey disease. In this work, we used the Caenorhabditis elegans model system to gain insight into the downstream response elicited by the loss of pmr-1 gene. We found that pmr-1 knocked down animals not only showed defects in the oligosaccharide structure of glycoproteins at the cell surface but also were characterized by reduced susceptibility to bacterial infection. Although increased resistance to the infection might be related to lack of regular recognition of C. elegans surface glycoproteins by microbial agents, we provide genetic evidence that pmr-1 interfered nematodes mounted a stronger innate immune response to Gram-positive bacterial infection. Thus, our observations indicate pmr-1 as a candidate gene implicated in mediating the worm's innate immune response.

Notch signaling plays a complex role in carcinogenesis, and its signaling pathway has both tumor suppressor and oncogenic components. To identify regulators that might control this dual activity of NOTCH1, we screened a chemical library targeting kinases and identified Polo-like kinase 1 (PLK1) as one of the kinases involved in arsenite-induced NOTCH1 down-modulation. As PLK1 activity drives mitotic entry but also is inhibited after DNA damage, we investigated the PLK1-NOTCH1 interplay in the G 2 phase of the cell cycle and in response to DNA damage. Here, we found that PLK1 regulates NOTCH1 expression at G 2 /M transition. However, when cells in G 2 phase are challenged with DNA damage, PLK1 is inhibited to prevent entry into mitosis. Interestingly, we found that the interaction between NOTCH1 and PLK1 is functionally important during the DNA damage response, as we found that whereas PLK1 activity is inhibited, NOTCH1 expression is maintained during DNA damage response. During genotoxic stress, cellular transformation requires that promitotic activity must override DNA damage checkpoint signaling to drive proliferation. Interestingly, we found that arsenite-induced genotoxic stress causes a PLK1-dependent signaling response that antagonizes the involvement of NOTCH1 in the DNA damage checkpoint. Taken together, our data provide evidence that Notch signaling is altered but not abolished in SCC cells. Thus, it is also important to recognize that Notch plasticity might be modulated and could represent a key determinant to switch on/off either the oncogenic or tumor suppressor function of Notch signaling in a single type of tumor., (© 2019 De Blasio et al.)

Colorectal cancer is characterized by well-known genetic defects and approximately 50% of cases harbor oncogenic Ras mutations. Increased expression of Notch ligand Jagged1 occurs in several human malignancies, including colorectal cancer, and correlates with cancer progression, poor prognosis, and recurrence. Herein, we demonstrated that Jagged1 was constitutively processed in colorectal cancer tumors with mutant Kras, which ultimately triggered intrinsic reverse signaling via its nuclear-targeted intracellular domain Jag1-ICD. This process occurred when Kras/Erk/ADAM17 signaling was switched on, demonstrating that Jagged1 is a novel target of the Kras signaling pathway. Notably, Jag1-ICD promoted tumor growth and epithelial-mesenchymal transition, enhancing colorectal cancer progression and chemoresistance both in vitro and in vivo . These data highlight a novel role for Jagged1 in colorectal cancer tumor biology that may go beyond its effect on canonical Notch activation and suggest that Jag1-ICD may behave as an oncogenic driver that is able to sustain tumor pathogenesis and to confer chemoresistance through a noncanonical mechanism. SIGNIFICANCE: These findings present a novel role of the transcriptionally active Jag1-ICD fragment to confer and mediate some of the activity of oncogenic KRAS., (©2019 American Association for Cancer Research.)

Light influences a wide range of physiological processes from prokaryotes to mammals. Neurospora crassa represents an important model system used for studying this signal pathway. At molecular levels, the WHITE COLLAR Complex (WCC), a heterodimer formed by WC-1 (the blue light photo-sensor) and WC-2 (the transcriptional activator), is the critical positive regulator of light-dependent gene expression. GATN (N indicates any other nucleotide) repeats are consensus sequences within the promoters of light-dependent genes recognized by the WCC. The distal GATN is also known as C-box since it is involved in the circadian clock. However, we know very little about the role of the proximal GATN, and the molecular mechanism that controls the transcription of light-induced genes during the dark/light transition it is still unclear. Here we showed a first indication that mutagenesis of the proximal GATA sequence within the target promoter of the albino-3 gene or deletion of the WC-1 zinc finger domain led to a rise in expression of light-dependent genes already in the dark, effectively decoupling light stimuli and transcriptional activation. This is the first observation of cis -/ trans -acting repressive machinery, which is not consistent with the light-dependent regulatory mechanism observed in the eukaryotic world so far.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer caused by the deregulation of key T-cell developmental pathways, including Notch signaling. Aberrant Notch signaling in T-ALL occurs by NOTCH1 gain-of-function mutations and by NOTCH3 overexpression. Although NOTCH3 is assumed as a Notch1 target, machinery driving its transcription in T-ALL is undefined in leukemia subsets lacking Notch1 activation. Here, we found that the binding of the intracellular Notch3 domain, as well as of the activated Notch1 fragment, to the NOTCH3 gene locus led to the recruitment of the H3K27 modifiers JMJD3 and p300, and it was required to preserve transcriptional permissive/active H3K27 marks and to sustain NOTCH3 gene expression levels. Consistently, pharmacological inhibition of JMJD3 by GSKJ4 treatment or of p300 by A-485 decreased the levels of expression of NOTCH3, NOTCH1 and of the Notch target genes DELTEX1 and c-Myc and abrogated cell viability in both Notch1- and Notch3-dependent T-cell contexts. Notably, re-introduction of exogenous Notch1, Notch3 as well as c-Myc partially rescued cells from anti-growth effects induced by either treatment. Overall our findings indicate JMJD3 and p300 as general Notch1 and Notch3 signaling co-activators in T-ALL and suggest further investigation on the potential therapeutic anti-leukemic efficacy of their enzymatic inhibition in Notch/c-Myc axis-related cancers and diseases.

DNA is an entity shielded by mechanisms that maintain genomic stability and are essential for living cells; however, DNA is constantly subject to assaults from the environment throughout the cellular life span, making the genome susceptible to mutation and irreparable damage. Cells are prepared to mend such events through cell death as an extrema ratio to solve those threats from a multicellular perspective. However, in cells under various stress conditions, checkpoint mechanisms are activated to allow cells to have enough time to repair the damaged DNA. In yeast, entry into the cell cycle when damage is not completely repaired represents an adaptive mechanism to cope with stressful conditions. In multicellular organisms, entry into cell cycle with damaged DNA is strictly forbidden. However, in cancer development, individual cells undergo checkpoint adaptation, in which most cells die, but some survive acquiring advantageous mutations and selfishly evolve a conflictual behavior. In this review, we focus on how, in cancer development, cells rely on checkpoint adaptation to escape DNA stress and ultimately to cell death.

Autophagy is a not well-understood conserved mechanism activated during nutritional deprivation in order to maintain cellular homeostasis. In the present study, we investigated the correlations between autophagy, apoptosis and the MAPK pathways in melanoma cell lines. We demonstrated that during starvation the EGF receptor mediated signaling activates many proteins involved in the MAPK pathway. Our data also suggest a previously unidentified link between the EGFR and Beclin-1 in melanoma cell line. We demonstrated that, following starvation, EGFR binds and tyrosine-phosphorylates Beclin-1, suggesting that it may play a key inhibitory role in the early stage of starvation, possibly through the Beclin-1 sequestration. Furthermore, EGFR releases Beclin-1 and allows initiating steps of the autophagic process. Interestingly enough, when the EGFR pathway was blocked by anti-EGF antibodies, immunoprecipitated Beclin-1 did not bind the phospho-EGFR. In addition, an extended binding of p-Bcl2 either with Beclin-1 or with Bax was observed with a decreased activation of the stress-induced JNK kinase, thus avoiding the transduction pathways that activate autophagy and apoptosis, respectively. For this reason, we advance the hypothesis that the activation of the EGFR is a necessary event that allows the ignition and progression of the autophagic process, at least in melanoma cells., Competing Interests: CONFLICTS OF INTEREST The authors report no conflicts of interest.

The term orthodisease defines human disorders in which the pathogenic gene has orthologs in model organism genomes. Yeasts have been instrumental for gaining insights into the molecular basis of many human disorders, particularly those resulting from impaired cellular metabolism. We and others have used yeasts as a model system to study the molecular basis of Hailey-Hailey disease (HHD), a human blistering skin disorder caused by haploinsufficiency of the gene ATP2C1 the orthologous of the yeast gene PMR1 . We observed that K. lactis cells defective for PMR1 gene share several biological similarities with HHD derived keratinocytes. Based on the conservation of ATP2C1/PMR1 function from yeast to human, here we used a yeast-based assay to screen for molecules able to influence the pleiotropy associated with PMR1 deletion. We identified six compounds, Kaempferol, Indirubin, Lappaconite, Cyclocytidine, Azomycin and Nalidixic Acid that induced different major shape phenotypes in K. lactis . These include mitochondrial and the cell-wall morphology-related phenotypes. Interestingly, a secondary assay in mammalian cells confirmed activity for Kaempferol. Indeed, this compound was also active on human keratinocytes depleted of ATP2C1 function by siRNA-treatment used as an in-vitro model of HHD. We found that Kaempferol was a potent NRF2 regulator, strongly inducing its expression and its downstream target NQO1 . In addition, Kaempferol could decrease oxidative stress of ATP2C1 defective keratinocytes, characterized by reduced NRF2-expression. Our results indicated that the activation of these pathways might provide protection to the HHD-skin cells. As oxidative stress plays pivotal roles in promoting the skin lesions of Hailey-Hailey, the NRF2 pathway could be a viable therapeutic target for HHD.

Glioblastoma (GBM) is the most aggressive human brain tumor. The high growth potential and decreased susceptibility to apoptosis of the glioma cells is mainly dependent on genetic amplifications or mutations of oncogenic or pro-apoptotic genes, respectively. We have previously shown that the activation of the M2 acetylcholine muscarinic receptors inhibited cell proliferation and induced apoptosis in two GBM cell lines and cancer stem cells. The aim of this study was to delve into the molecular mechanisms underlying the M2-mediated cell proliferation arrest. Exploiting U87MG and U251MG cell lines as model systems, we evaluated the ability of M2 receptors to interfere with Notch-1 and EGFR pathways, whose activation promotes GBM proliferation. We demonstrated that the activation of M2 receptors, by agonist treatment, counteracted Notch and EGFR signaling, through different regulatory cascades depending, at least in part, on p53 status. Only in U87MG cells, which mimic p53-wild type GBMs, did M2 activation trigger a molecular circuitry involving p53, Notch-1, and the tumor suppressor mir-34a-5p. This regulatory module negatively controls Notch-1, which affects cell proliferation mainly through the Notch-1/EGFR axis. Our data highlighted, for the first time, a molecular circuitry that is deregulated in the p53 wild type GBM, based on the cross-talk between M2 receptor and the Notch-1/EGFR pathways, mediated by mir-34a-5p.

Notch dysregulation has been implicated in numerous tumors, including triple-negative breast cancer (TNBC), which is the breast cancer subtype with the worst clinical outcome. However, the importance of individual receptors in TNBC and their specific mechanism of action remain to be elucidated, even if recent findings suggested a specific role of activated-Notch3 in a subset of TNBCs. Epidermal growth factor receptor (EGFR) is overexpressed in TNBCs but the use of anti-EGFR agents (including tyrosine kinase inhibitors, TKIs) has not been approved for the treatment of these patients, as clinical trials have shown disappointing results. Resistance to EGFR blockers is commonly reported. Here we show that Notch3-specific inhibition increases TNBC sensitivity to the TKI-gefitinib in TNBC-resistant cells. Mechanistically, we demonstrate that Notch3 is able to regulate the activated EGFR membrane localization into lipid rafts microdomains, as Notch3 inhibition, such as rafts depletion, induces the EGFR internalization and its intracellular arrest, without involving receptor degradation. Interestingly, these events are associated with the EGFR tyrosine dephosphorylation at Y1173 residue (but not at Y1068) by the protein tyrosine phosphatase H1 (PTPH1), thus suggesting its possible involvement in the observed Notch3-dependent TNBC sensitivity response to gefitinib. Consistent with this notion, a nuclear localization defect of phospho-EGFR is observed after combined blockade of EGFR and Notch3, which results in a decreased TNBC cell survival. Notably, we observed a significant correlation between EGFR and NOTCH3 expression levels by in silico gene expression and immunohistochemical analysis of human TNBC primary samples. Our findings strongly suggest that combined therapies of TKI-gefitinib with Notch3-specific suppression may be exploited as a drug combination advantage in TNBC treatment.

Sonic hedgehog (Shh) signaling is essential for proliferation of cerebellar granule cell progenitors (GCPs) and its misregulation is linked to various disorders, including cerebellar cancer medulloblastoma. The effects of Shh pathway are mediated by the Gli family of transcription factors, which controls the expression of a number of target genes, including Gli1. Here, we identify Mastermind-like 1 (Maml1) as a novel regulator of the Shh signaling since it interacts with Gli proteins, working as a potent transcriptional coactivator. Notably, Maml1 silencing results in a significant reduction of Gli target genes expression, with a negative impact on cell growth of NIH3T3 and Patched1 -/- mouse embryonic fibroblasts (MEFs), bearing a constitutively active Shh signaling. Remarkably, Shh pathway activity results severely compromised both in MEFs and GCPs deriving from Maml1 -/- mice with an impairment of GCPs proliferation and cerebellum development. Therefore Maml1 -/- phenotype mimics aspects of Shh pathway deficiency, suggesting an intrinsic requirement for Maml1 in cerebellum development. The present study shows a new role for Maml1 as a component of Shh signaling, which plays a crucial role in both development and tumorigenesis.

Notch signaling is considered a rational target in the therapy of several cancers, particularly those harbouring Notch gain of function mutations, including T-cell acute lymphoblastic leukemia (T-ALL). Although currently available Notch-blocking agents are showing anti-tumor activity in preclinical studies, they are not effective in all the patients and often cause severe side-effects, limiting their widespread therapeutic use. Here, by functional and biological analysis of the most representative molecules of an in house library of natural products, we have designed and synthetized the chalcone-derivative 8 possessing Notch inhibitory activity at low micro molar concentration in T-ALL cell lines. Structure-activity relationships were afforded for the chalcone scaffold. Short term treatments with compound 8 resulted in a dose-dependent decrease of Notch signaling activity, halted cell cycle progression and induced apoptosis, thus affecting leukemia cell growth. Taken together, our data indicate that 8 is a novel Notch inhibitor, candidate for further investigation and development as an additional therapeutic option against Notch-dependent cancers.