Comprehensive Molecular Profiling of NPM1-Mutated Acute Myeloid Leukemia Using RNAseq Approach.

Acute myeloid leukemia (AML) is a complex hematologic malignancy with high morbidity and mortality. Nucleophosmin 1 (NPM1) mutations occur in approximately 30% of AML cases, and NPM1-mutated AML is classified as a distinct entity. NPM1-mutated AML patients without additional genetic abnormalities have a favorable prognosis. Despite this, 30-50% of them experience relapse. This study aimed to investigate the potential of total RNAseq in improving the characterization of NPM1-mutated AML patients. We explored genetic variations independently of myeloid stratification, revealing a complex molecular scenario. We showed that total RNAseq enables the uncovering of different genetic alterations and clonal subtypes, allowing for a comprehensive evaluation of the real expression of exome transcripts in leukemic clones and the identification of aberrant fusion transcripts. This characterization may enhance understanding and guide improved treatment strategies for NPM1mut AML patients, contributing to better outcomes. Our findings underscore the complexity of NPM1-mutated AML, supporting the incorporation of advanced technologies for precise risk stratification and personalized therapeutic strategies. The study provides a foundation for future investigations into the clinical implications of identified genetic variations and highlights the importance of evolving diagnostic approaches in leukemia management.

Albumin/Mitotane Interaction Affects Drug Activity in Adrenocortical Carcinoma Cells: Smoke and Mirrors on Mitotane Effect with Possible Implications for Patients' Management.

BACKGROUND: Mitotane is the only drug approved for the treatment of adrenocortical carcinoma (ACC). Although it has been used for many years, its mechanism of action remains elusive. H295R cells are, in ACC, an essential tool to evaluate drug mechanisms, although they often lead to conflicting results. METHODS: Using different in vitro biomolecular technologies and biochemical/biophysical experiments, we evaluated how the presence of "confounding factors" in culture media and patient sera could reduce the pharmacological effect of mitotane and its metabolites. RESULTS: We discovered that albumin, the most abundant protein in the blood, was able to bind mitotane. This interaction altered the effect of the drug by blocking its biological activity. This blocking effect was independent of the albumin source or methodology used and altered the assessment of drug sensitivity of the cell lines. CONCLUSIONS: In conclusion, we have for the first time demonstrated that albumin does not only act as an inert drug carrier when mitotane or its metabolites are present. Indeed, our experiments clearly indicated that both albumin and human serum were able to suppress the pharmacological effect of mitotane in vitro. These experiments could represent a first step towards the individualization of mitotane treatment in this rare tumor.

Apelin-13 Increases Functional Connexin-43 through Autophagy Inhibition via AKT/mTOR Pathway in the Non-Myocytic Cell Population of the Heart.

Studies have shown a link between the downregulation of connexin 43 (Cx43), the predominant isoform in cardiac gap junctions, and high susceptibility to cardiac arrhythmias and cardiomyocyte death. Non-myocytic cells (NMCs), the most abundant component of the heart, exert multiple cardiac functions and represent an important therapeutic target for diseased cardiac tissue. A few studies have investigated the effect of Apelin-13, an endogenous peptide with a key role in various cardiovascular functions, on Cx43 expression in cardiomyocytes. However, it remained unknown whether Apelin-13 influences Cx43 expression in NMCs. Here, we found that in NMCs, Cx43 protein expression increased after Apelin-13 treatment (100 nM for 48 h). Furthermore, dye transfer assays proved that Apelin-13-treated NMCs had a greater ability to communicate with surrounding cardiomyocytes, and this effect was abrogated by carbenoxolone, a gap junction inhibitor. Interestingly, we showed that Apelin-13 increased Cx43 through autophagy inhibition, as proved by the upregulation of p62 and LC3I, acting as 3-MA, a well-known autophagy inhibitor. In addition, Apelin-13-induced AKT and mTOR phosphorylation was abolished by LY294002 and rapamycin inhibitors resulting in Cx43 increased suppression. These results open the possibility of targeting gap junctions in NMCs with Apelin-13 as an exciting therapeutic approach with great potential.

Two conserved glycine residues in mammalian and Dictyostelium Rictor are required for mTORC2 activity and integrity.

Mammalian, or mechanistic, target of rapamycin complex 2 (mTORC2) regulates a variety of vital cellular processes, and its aberrant functioning is often associated with various diseases. Rictor is a peculiar and distinguishing mTORC2 component playing a pivotal role in controlling its assembly and activity. Among extant organisms, Rictor is conserved from unicellular eukaryotes to metazoans. We replaced two distinct, but conserved, glycine residues in both the Dictyostelium piaA gene and its human ortholog, RICTOR The two conserved residues are spaced ∼50 amino acids apart, and both are embedded within a conserved region falling in between the Ras-GEFN2 and Rictor-_V domains. The effects of point mutations on the mTORC2 activity and integrity were assessed by biochemical and functional assays. In both cases, these equivalent point mutations in the mammalian RICTOR and DictyosteliumpiaA gene impaired mTORC2 activity and integrity. Our data indicate that the two glycine residues are essential for the maintenance of mTORC2 activity and integrity in organisms that appear to be distantly related, suggesting that they have a evolutionarily conserved role in the assembly and proper mTORC2 functioning.

Bcl-xL represents a therapeutic target in Philadelphia negative myeloproliferative neoplasms.

Myeloproliferative neoplasms are divided into essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). Although ruxolitinib was proven to be effective in reducing symptoms, patients rarely achieve complete molecular remission. Therefore, it is relevant to identify new therapeutic targets to improve the clinical outcome of patients. Bcl-xL protein, the long isoform encoded by alternative splicing of the Bcl-x gene, acts as an anti-apoptotic regulator. Our study investigated the role of Bcl-xL as a marker of severity of MPN and the possibility to target Bcl-xL in patients. 129 MPN patients and 21 healthy patients were enrolled in the study. We analysed Bcl-xL expression in leucocytes and in enriched CD34+ and CD235a+ cells. Furthermore, ABT-737, a Bcl-xL inhibitor, was tested in HEL cells and in leucocytes from MPN patients. Bcl-xL was found progressively over-expressed in cells from ET, PV and PMF patients, independently by JAK2 mutational status. Moreover, our data indicated that the combination of ABT-737 and ruxolitinib resulted in a significantly higher apoptotic rate than the individual drug. Our study suggests that Bcl-xL plays an important role in MPN independently from JAK2 V617F mutation. Furthermore, data demonstrate that targeting simultaneously JAK2 and Bcl-xL might represent an interesting new approach.

Curcumin induces apoptosis in JAK2-mutated cells by the inhibition of JAK2/STAT and mTORC1 pathways.

Myeloproliferative neoplasms are chronic myeloid cancers divided in Philadelphia positive and negative. The JAK2 V617F is the most common mutation in Philadelphia negative patients and results in a constitutive activation of the JAK/STAT pathway, conferring a proliferative advantage and apoptosis inhibition. Recent studies identified a functional crosstalk between the JAK/STAT and mTOR pathways. The identification of an effective therapy is often difficult, so the availability of new therapeutic approaches might be attractive. Previous studies showed that curcumin, the active principle of the Curcuma longa, can suppress JAK2/STAT pathways in different type of cancer and injuries. In this study, we investigated the anti-proliferative and pro-apoptotic effects of curcumin in JAK2 V617F-mutated cells. HEL cell line and cells from patients JAK2 V617F mutated have been incubated with increasing concentrations of curcumin for different time. Apoptosis and proliferation were evaluated. Subsequently, JAK2/STAT and AKT/mTOR pathways were investigated at both RNA and protein levels. We found that curcumin induces apoptosis and inhibition of proliferation in HEL cells. Furthermore, we showed that curcumin inhibits JAK2/STAT and mTORC1 pathways in JAK2 V617F-mutated cells. This inhibition suggests that curcumin could represent an alternative strategy to be explored for the treatment of patients with myeloproliferative neoplasms.

Persistent DNA damage-induced premature senescence alters the functional features of human bone marrow mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) are adult multipotent stem cells located in various tissues, including the bone marrow. In contrast to terminally differentiated somatic cells, adult stem cells must persist and function throughout life to ensure tissue homeostasis and repair. For this reason, they must be equipped with DNA damage responses able to maintain genomic integrity while ensuring their lifelong persistence. Evaluation of hMSC response to genotoxic insults is of great interest considering both their therapeutic potential and their physiological functions. This study aimed to investigate the response of human bone marrow MSCs to the genotoxic agent Actinomycin D (ActD), a well-known anti-tumour drug. We report that hMSCs react by undergoing premature senescence driven by a persistent DNA damage response activation, as hallmarked by inhibition of DNA synthesis, p21 and p16 protein expression, marked Senescent Associated ß-galactosidase activity and enlarged γH2AX foci co-localizing with 53BP1 protein. Senescent hMSCs overexpress several senescence-associated secretory phenotype (SASP) genes and promote motility of lung tumour and osteosarcoma cell lines in vitro. Our findings disclose a multifaceted consequence of ActD treatment on hMSCs that on the one hand helps to preserve this stem cell pool and prevents damaged cells from undergoing neoplastic transformation, and on the other hand alters their functional effects on the surrounding tissue microenvironment in a way that might worsen their tumour-promoting behaviour.

ATF2 contributes to cisplatin resistance in non-small cell lung cancer and celastrol induces cisplatin resensitization through inhibition of JNK/ATF2 pathway.

ATF2 is a transcription factor involved in stress and DNA damage. A correlation between ATF2 JNK-mediated activation and resistance to damaging agents has already been reported. The purpose of the present study was to investigate whether ATF2 may have a role in acquired resistance to cisplatin in non-small cell lung cancer (NSCLC). mRNA and protein analysis on matched cancer and corresponding normal tissues from surgically resected NSCLC have been performed. Furthermore, in NSCLC cell lines, ATF2 expression levels were evaluated and correlated to platinum (CDDP) resistance. Celastrol-mediated ATF2/cJUN activity was measured. High expression levels of both ATF2 transcript and proteins were observed in lung cancer specimens (p << 0.01, Log2 (FC) = +4.7). CDDP-resistant NSCLC cell lines expressed high levels of ATF2 protein. By contrast, Celastrol-mediated ATF2/cJUN functional inhibition restored the response to CDDP. Moreover, ATF2 protein activation correlates with worse outcome in advanced CDDP-treated patients. For the first time, it has been shown NSCLC ATF2 upregulation at both mRNA/protein levels in NSCLC. In addition, we reported that in NSCLC cell lines a correlation between ATF2 protein expression and CDDP resistance occurs. Altogether, our results indicate a potential increase in CDDP sensitivity, on Celastrol-mediated ATF2/cJUN inhibition. These data suggest a possible involvement of ATF2 in NSCLC CDDP-resistance.

Direct Reprogramming of Resident Non-Myocyte Cells and Its Potential for In Vivo Cardiac Regeneration.

Cardiac diseases are the foremost cause of morbidity and mortality worldwide. The heart has limited regenerative potential; therefore, lost cardiac tissue cannot be replenished after cardiac injury. Conventional therapies are unable to restore functional cardiac tissue. In recent decades, much attention has been paid to regenerative medicine to overcome this issue. Direct reprogramming is a promising therapeutic approach in regenerative cardiac medicine that has the potential to provide in situ cardiac regeneration. It consists of direct cell fate conversion of one cell type into another, avoiding transition through an intermediary pluripotent state. In injured cardiac tissue, this strategy directs transdifferentiation of resident non-myocyte cells (NMCs) into mature functional cardiac cells that help to restore the native tissue. Over the years, developments in reprogramming methods have suggested that regulation of several intrinsic factors in NMCs can help to achieve in situ direct cardiac reprogramming. Among NMCs, endogenous cardiac fibroblasts have been studied for their potential to be directly reprogrammed into both induced cardiomyocytes and induced cardiac progenitor cells, while pericytes can transdifferentiate towards endothelial cells and smooth muscle cells. This strategy has been indicated to improve heart function and reduce fibrosis after cardiac injury in preclinical models. This review summarizes the recent updates and progress in direct cardiac reprogramming of resident NMCs for in situ cardiac regeneration.

APJ as Promising Therapeutic Target of Peptide Analogues in Myocardial Infarction- and Hypertension-Induced Heart Failure.

The widely expressed G protein-coupled apelin receptor (APJ) is activated by two bioactive endogenous peptides, apelin and ELABELA (ELA). The apelin/ELA-APJ-related pathway has been found involved in the regulation of many physiological and pathological cardiovascular processes. Increasing studies are deepening the role of the APJ pathway in limiting hypertension and myocardial ischaemia, thus reducing cardiac fibrosis and adverse tissue remodelling, outlining APJ regulation as a potential therapeutic target for heart failure prevention. However, the low plasma half-life of native apelin and ELABELA isoforms lowered their potential for pharmacological applications. In recent years, many research groups focused their attention on studying how APJ ligand modifications could affect receptor structure and dynamics as well as its downstream signalling. This review summarises the novel insights regarding the role of APJ-related pathways in myocardial infarction and hypertension. Furthermore, recent progress in designing synthetic compounds or analogues of APJ ligands able to fully activate the apelinergic pathway is reported. Determining how to exogenously regulate the APJ activation could help to outline a promising therapy for cardiac diseases.

Expression and pharmacological inhibition of thymidylate synthase and Src kinase in nonsmall cell lung cancer.

The combination of cytotoxic chemotherapy with signaling pathway inhibitors represents a potential strategy to improve the treatment of nonsmall cell lung cancer (NSCLC). Thymidylate synthase (TS) is an enzyme essential for DNA synthesis, and its overexpression has been associated with the reduced sensitivity to antifolate agents. Src is a tyrosine kinase that modulates the cytotoxicity of cancer cells after drug treatment, and in vitro data indicate that its inhibition could revert the resistance to TS-inhibiting drugs. Our study investigated the significance of TS and Src expression in NSCLC tissues, and the effects of their pharmacological inhibition in cell lines. In tumor and normal tissues from 94 resected NSCLC patients, TS and Src transcript levels were found positively correlated (R(S) = 0.66), associated with patients smoking history and overall survival. At multivariate analysis, TS gene expression was an independent prognostic factor (relative risk (RR) = 1.78, from 1.16 to 2.72; p < 0.01). Immunohistochemical detection in tumor specimens confirmed that Src kinase activation, evaluated by phospho-specific antibody, was associated to a higher TS expression. In cell lines, dasatinib, a Src-inhibiting agent, synergistically enhanced pemetrexed-cytotoxicity of A549 cells, as evaluated by MTT and apoptosis assays. The biological explanation for this interaction was based on the upregulation of TS messenger RNA and protein levels induced by pemetrexed, which was significantly prevented by dasatinib cotreatment. The data of our study suggest that TS and Src may belong to a common pathway that bears prognostic significance in NSCLC, and that Src represents a potential target to improve the efficacy of TS-inhibiting agents.

Role of Chaperone-Mediated Autophagy in Ageing Biology and Rejuvenation of Stem Cells.

What lies at the basis of the mechanisms that regulate the maintenance and self-renewal of pluripotent stem cells is still an open question. The control of stemness derives from a fine regulation between transcriptional and metabolic factors. In the last years, an emerging topic has concerned the involvement of Chaperone-Mediated Autophagy (CMA) as a key mechanism in stem cell pluripotency control acting as a bridge between epigenetic, transcriptional and differentiation regulation. This review aims to clarify this new and not yet well-explored horizon discussing the recent studies regarding the CMA impact on embryonic, mesenchymal, and haematopoietic stem cells. The review will discuss how CMA influences embryonic stem cell activity promoting self-renewal or differentiation, its involvement in maintaining haematopoietic stem cell function by increasing their functionality during the normal ageing process and its effects on mesenchymal stem cells, in which modulation of CMA regulates immunosuppressive and differentiation properties. Finally, the importance of these new discoveries and their relevance for regenerative medicine applications, from transplantation to cell rejuvenation, will be addressed.

Silica Nanoparticle Internalization Improves Chemotactic Behaviour of Human Mesenchymal Stem Cells Acting on the SDF1α/CXCR4 Axis.

Human mesenchymal stem cell (hMSC)-based therapy is an emerging resource in regenerative medicine. Despite the innate ability of hMSCs to migrate to sites of injury, homing of infused hMSCs to the target tissue is inefficient. It was shown that silica nanoparticles (SiO2-NPs), previously developed to track the stem cells after transplantation, accumulated in lysosomes leading to a transient blockage of the autophagic flux. Since CXCR4 turnover is mainly regulated by autophagy, we tested the effect of SiO2-NPs on chemotactic migration of hMSCs along the SDF1α/CXCR4 axis that plays a pivotal role in directing MSC homing to sites of injury. Our results showed that SiO2-NP internalization augmented CXCR4 surface levels. We demonstrated that SiO2-NP-dependent CXCR4 increase was transient, and it reversed at the same time as lysosomal compartment normalization. Furthermore, the autophagy inhibitor Bafilomycin-A1 reproduced CXCR4 overexpression in control hMSCs confirming the direct effect of the autophagic degradation blockage on CXCR4 expression. Chemotaxis assays showed that SiO2-NPs increased hMSC migration toward SDF1α. In contrast, migration improvement was not observed in TNFα/TNFR axis, due to the proteasome-dependent TNFR regulation. Overall, our findings demonstrated that SiO2-NP internalization increases the chemotactic behaviour of hMSCs acting on the SDF1α/CXCR4 axis, unmasking a high potential to improve hMSC migration to sites of injury and therapeutic efficacy upon cell injection in vivo.

Detection of SF3B1 p.Lys700Glu Mutation by PNA-PCR Clamping in Myelodysplastic Syndromes and Myeloproliferative Neoplasms.

Mutations in SF3B1 are found in 20% of myelodysplastic syndromes and 5-10% of myeloproliferative neoplasms, where they are considered important for diagnosis and therapy decisions. Sanger sequencing and NGS are the currently available methods to identify SF3B1 mutations, but both are time-consuming and expensive techniques that are not practicable in most small-/medium-sized laboratories. To identify the most frequent SF3B1 mutation, p.Lys700Glu, we developed a novel fast and cheap assay based on PNA-PCR clamping. After setting the optimal PCR conditions, the limit of detection of PNA-PCR clamping was evaluated, and the method allowed up to 0.1% of mutated SF3B1 to be identified. Successively, PNA-PCR clamping and Sanger sequencing were used to blind test 90 DNA from patients affected by myelodysplastic syndromes and myeloproliferative neoplasms for the SF3B1 p.Lys700Glu mutation. PNA-PCR clamping and Sanger sequencing congruently identified 75 negative and 13 positive patients. Two patients identified as positive by PNA-PCR clamping were missed by Sanger analysis. The discordant samples were analyzed by NGS, which confirmed the PNA-PCR clamping result, indicating that these samples contained the SF3B1 p.Lys700Glu mutation. This approach could easily increase the characterization of myelodysplastic syndromes and myeloproliferative neoplasms in small-/medium-sized laboratories, and guide patients towards more appropriate therapy.

ApoE3 vs. ApoE4 Astrocytes: A Detailed Analysis Provides New Insights into Differences in Cholesterol Homeostasis.

The strongest genetic risk factor for sporadic Alzheimer's disease (AD) is the presence of the ε4 allele of the apolipoprotein E (ApoE) gene, the major apolipoprotein involved in brain cholesterol homeostasis. Being astrocytes the main producers of cholesterol and ApoE in the brain, we investigated the impact of the ApoE genotype on astrocyte cholesterol homeostasis. Two mouse astrocytic cell lines expressing the human ApoE3 or ApoE4 isoform were employed. Gas chromatography-mass spectrometry (GC-MS) analysis pointed out that the levels of total cholesterol, cholesterol precursors, and various oxysterols are altered in ApoE4 astrocytes. Moreover, the gene expression analysis of more than 40 lipid-related genes by qRT-PCR showed that certain genes are up-regulated (e.g., CYP27A1) and others down-regulated (e.g., PPARγ, LXRα) in ApoE4, compared to ApoE3 astrocytes. Beyond confirming the significant reduction in the levels of PPARγ, a key transcription factor involved in the maintenance of lipid homeostasis, Western blotting showed that both intracellular and secreted ApoE levels are altered in ApoE4 astrocytes, as well as the levels of receptors and transporters involved in lipid uptake/efflux (ABCA1, LDLR, LRP1, and ApoER2). Data showed that the ApoE genotype clearly affects astrocytic cholesterol homeostasis; however, further investigation is needed to clarify the mechanisms underlying these differences and the consequences on neighboring cells. Indeed, drug development aimed at restoring cholesterol homeostasis could be a potential strategy to counteract AD.

Aurora Kinase A expression is associated with lung cancer histological-subtypes and with tumor de-differentiation.

BACKGROUND: Aurora kinase A (AURKA) is a member of serine/threonine kinase family. Several kinases belonging to this family are activated in the G2/M phase of the cell cycle being involved in mitotic chromosomal segregation. AURKA overexpression is significantly associated with neoplastic transformation in several tumors and deregulated Aurora Kinases expression leads to chromosome instability, thus contributing to cancer progression. The purpose of the present study was to investigate the expression of AURKA in non small cell lung cancer (NSCLC) specimens and to correlate its mRNA or protein expression with patients' clinico-pathological features. MATERIALS AND METHODS: Quantitative real-time PCR and immunohistochemistry analysis on matched cancer and corresponding normal tissues from surgically resected non-small cell lung cancers (NSCLC) have been performed aiming to explore the expression levels of AURKA gene. RESULTS: AURKA expression was significantly up-modulated in tumor samples compared to matched lung tissue (p<0.01, mean log2(FC)=1.5). Moreover, AURKA was principally up-modulated in moderately and poorly differentiated lung cancers (p<0.01), as well as in squamous and adenocarcinomas compared to the non-invasive bronchioloalveolar histotype (p=0.029). No correlation with survival was observed. CONCLUSION: These results indicate that in NSCLC AURKA over-expression is restricted to specific subtypes and poorly differentiated tumors.

Molecular Mechanisms of Mitotane Action in Adrenocortical Cancer Based on In Vitro Studies.

Mitotane is the only approved drug for the treatment of advanced adrenocortical carcinoma and is increasingly used for postoperative adjuvant therapy. Mitotane action involves the deregulation of cytochromes P450 enzymes, depolarization of mitochondrial membranes, and accumulation of free cholesterol, leading to cell death. Although it is known that mitotane destroys the adrenal cortex and impairs steroidogenesis, its exact mechanism of action is still unclear. The most used cell models are H295-derived cell strains and SW13 cell lines. The diverging results obtained in presumably identical cell lines highlight the need for a stable in vitro model and/or a standard methodology to perform experiments on H295 strains. The presence of several enzymatic targets responsive to mitotane in mitochondria and mitochondria-associated membranes causes progressive alteration in mitochondrial structure when cells were exposed to mitotane. Confounding factors of culture affecting in vitro experiments could reduce the significance of any molecular mechanism identified in vitro. To ensure experimental reproducibility, particular care should be taken in the choice of culture conditions: aspects such as cell strains, culture serum, lipoproteins concentration, and culture passages should be carefully considered and explicated in the presentation of results. We aimed to review in vitro studies on mitotane effects, highlighting how different experimental conditions might contribute to the controversial findings. If the concerns pointed out in this review will be overcome, the new insights into mitotane mechanism of action observed in-vitro could allow the identification of novel pharmacological molecular pathways to be used to implement personalized therapy.

Genetic Screening for Potential New Targets in Chronic Myeloid Leukemia Based on Drosophila Transgenic for Human BCR-ABL1.

Chronic myeloid leukemia is a myeloproliferative neoplasm characterized by the presence of the Philadelphia chromosome that originates from the reciprocal translocation t(9;22)(q34;q11.2) and encodes for the constitutively active tyrosine kinase protein BCR-ABL1 from the Breakpoint Cluster Region (BCR) sequence and the Abelson (ABL1) gene. Despite BCR-ABL1 being one of the most studied oncogenic proteins, some molecular mechanisms remain enigmatic, and several of the proteins, acting either as positive or negative BCR-ABL1 regulators, are still unknown. The Drosophila melanogaster represents a powerful tool for genetic investigations and a promising model to study the BCR-ABL1 signaling pathway. To identify new components involved in BCR-ABL1 transforming activity, we conducted an extensive genetic screening using different Drosophila mutant strains carrying specific small deletions within the chromosomes 2 and 3 and the gmrGal4,UAS-BCR-ABL1 4M/TM3 transgenic Drosophila as the background. From the screening, we identified several putative candidate genes that may be involved either in sustaining chronic myeloid leukemia (CML) or in its progression. We also identified, for the first time, a tight connection between the BCR-ABL1 protein and Rab family members, and this correlation was also validated in CML patients. In conclusion, our data identified many genes that, by interacting with BCR-ABL1, regulate several important biological pathways and could promote disease onset and progression.

Overexpression of YAP1 induces immortalization of normal human keratinocytes by blocking clonal evolution.

YAP1 is a transcriptional co-activator able to bind several transcription factors. YAP1 was termed a candidate oncogene after it was shown to be in human chromosome 11q22 amplicon; besides the genomic amplification, several experiments indicated that it has oncogenic function. However, YAP1 was also reported to be a tumor suppressor as its gene locus is deleted in some breast cancers. To clarify the role of this protein in the physiology of rapidly renewal cells, we investigated YAP1 in human keratinocytes. Here, we show that YAP1 overexpression in primary human keratinocytes blocks clonal evolution and induces cell immortalization, but not malignant transformation. YAP1 overexpression led to an increase in cell proliferation, colony forming efficiency and holoclone percentage. Cells escaped from senescence, immortalized but still remained unable to grow in soft agar or express mesenchymal markers, suggesting that YAP1 overexpression is not sufficient to promote a complete epithelial-mesenchymal transition and tumorigenic transformation. Protein analysis showed an increase in epithelial proliferation markers and a decrease in epithelial differentiation markers. The expression of LEKTI, a late differentiation marker, dramatically dropped to undetectable levels. Taken together, these data suggest that YAP1-overexpressing keratinocytes are maintained in the proliferative compartment.