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Simple Summary: Protein arginine methyltransferase 5 (PRMT5) is known to be oncogenic in many cancers, including squamous cell carcinoma (SCC). Our analyses of multiple public databases revealed that PRMT5 overexpression correlates with poor survival in SCC patients and is essential to the survival of SCC cell lines. This study focused on understanding how PRMT5 and its binding partner, WDR77 (WD repeat domain 77), regulate SCC cell growth, particularly through the p63 ΔNp63α isoform, a key factor in SCC. Furthermore, PRMT5 depletion inhibited SCC proliferation by inducing cell cycle arrest in the G1 phase. Additionally, we showed that PRMT5 and WDR77 stabilized ΔNp63α protein expression, which in turn inhibited p21 (cyclin-dependent kinase inhibitor 1). These findings provide new insights into the potential of targeting PRMT5 as a therapeutic strategy for SCC. Protein arginine methyltransferase 5 (PRMT5) is a critical oncogenic factor in various cancers, and its inhibition has shown promise in suppressing tumor growth. However, the role of PRMT5 in squamous cell carcinoma (SCC) remains largely unexplored. In this study, we analyzed SCC patient data from The Cancer Genome Atlas (TCGA) and the Cancer Dependency Map (DepMap) to investigate the relationship between PRMT5 and SCC proliferation. We employed competition-based cell proliferation assays, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assays, flow cytometry, and in vivo mouse modeling to examine the regulatory roles of PRMT5 and its binding partner WDR77 (WD repeat domain 77). We identified downstream targets, including the p63 isoform ΔNp63α and the cyclin-dependent kinase inhibitor p21, through single-cell RNA-seq, RT-qPCR, and Western blot analyses. Our findings demonstrate that upregulation of PRMT5 and WDR77 correlates with the poor survival of head and neck squamous cell carcinoma (HNSCC) patients. PRMT5/WDR77 regulates the HNSCC-specific transcriptome and facilitates SCC proliferation by promoting cell cycle progression. The PRMT5 and WDR77 stabilize the ΔNp63α Protein, which in turn, inhibits p21. Moreover, depletion of PRMT5 and WDR77 repress SCC in vivo. This study reveals for the first time that PRMT5 and WDR77 synergize to promote SCC proliferation via the ΔNp63α-p21 axis, highlighting a novel therapeutic target for SCC. [ABSTRACT FROM AUTHOR]

COVID-19 pandemic is caused by the SARS-CoV-2 virus, whose internalization and infection are mediated by the angiotensin-converting enzyme 2 (ACE2). The identification of novel approaches to tackle this step is instrumental for the development of therapies for the management of COVID-19 and other diseases with a similar mechanism of infection. Thalidomide, a drug sadly known for its teratogenic effects, has potent immunomodulatory and anti-inflammatory properties. Treatment with this drug has been shown to improve the immune functions of COVID-19 patients and proposed for the management of COVID-19 in clinical practice through drug repositioning. Here, we investigated the molecular details linking thalidomide to ACE2 and COVID-19, showing that in conditions mimicking SARS-CoV-2-associated cytokine storm, the transcription factor ΔNp63α and ACE2 are stabilized, and IL-8 production is increased. In such conditions, we found p63 to bind to and regulate the expression of the ACE2 gene. We previously showed that ΔNp63α is degraded upon thalidomide treatment and now found that treatment with this drug—or with its analogue lenalidomide—downregulates ACE2 in a p63-dependent manner. Finally, we found that thalidomide treatment reduces in vitro infection by pseudo-SARS-CoV-2, a baculovirus pseudotyped with the SARS-CoV-2 spike protein. Overall, we propose the dual effect of thalidomide in reducing SARS-CoV-2 viral re-entry and inflammation through p63 degradation to weaken SARS-CoV-2 entry into host cells and mitigate lung inflammation, making it a valuable option in clinical management of COVID-19. Key messages: Thalidomide treatment results in p63-dependent ACE2 downregulation. ACE2 is a p63 transcriptional target. Thalidomide reduces the "cytokine storm" associated to COVID-19. Thalidomide prevents viral re-entry of SARS-CoV-2 by p63-dependent ACE2 downregulation. Thalidomide is a modulator of SARS-CoV-2 or other ACE2-dependent infections. ACE2 is modulated by a pharmacological substance. [ABSTRACT FROM AUTHOR]

Abstract Cigarette smoke (CS) has been generally recognized as a chief carcinogenic factor in renal cell carcinoma (RCC). The stimulative effect of CS on renal cancer stem cells (RCSCs) has been described previously. The Sonic Hedgehog (SHH) pathway plays an essential role in self-renewal, cell growth, drug resistance, metastasis, and recurrence of cancer stem cells (CSCs). Renal cancer-related gene ΔNp63α is highly expressed in renal epithelial tissues and contributes to the RCSCs characteristics of tumors. The aim of this study was to elucidate the role of ΔNp63α and the SHH pathway on the activity of RCSCs induced by CS through a series of in vivo and in vitro studies. It was shown that in renal cancer tissues, ΔNp63α and RCSCs markers in smokers are expressed higher than that in non-smokers. RCSCs were effectively enriched by tumor sphere formation assay. Besides, CS increased the expression of RCSCs markers and the capability of sphere-forming in vitro and in vivo. Moreover, the SHH pathway was activated, and the specialized inhibitor alleviated the promotion of CS on RCSCs. ΔNp63α activated the SHH pathway and promoted CS-induced enhancement of RCSCs activity. These findings indicate that ΔNp63α positively regulates the activity of CS-induced RCSCs via the SHH pathway.

Keratinocyte senescence contributes to skin ageing and epidermal dysfunction. According to the existing knowledge, the transcription factor ΔNp63α plays pivotal roles in differentiation and proliferation of keratinocytes. It is traditionally accepted that ΔNp63α exerts its functions via binding to promoter regions to activate or repress gene transcription. However, accumulating evidence demonstrates that ΔNp63α can bind to elements away from promoter regions of its target genes, mediating epigenetic regulation. On the other hand, several epigenetic alterations, including DNA methylation, histone modification and variation, chromatin remodelling, as well as enhancer-promoter looping, are found to be related to cell senescence. To systematically elucidate how ΔNp63α affects keratinocyte senescence via epigenetic regulation, we comprehensively compiled the literatures on the roles of ΔNp63α in keratinocyte senescence, epigenetics in cellular senescence, and the relation between ΔNp63α-mediated epigenetic regulation and keratinocyte senescence. Based on the published data, we conclude that ΔNp63α mediates epigenetic regulation via multiple mechanisms: recruiting epigenetic enzymes to modify DNA or histones, coordinating chromatin remodelling complexes (CRCs) or regulating their expression, and mediating enhancer-promoter looping. Consequently, the expression of genes related to cell cycle is modulated, and proliferation of keratinocytes and renewal of stem cells are maintained, by ΔNp63α. During skin inflammaging, the decline of ΔNp63α may lead to epigenetic dysregulation, resultantly deteriorating keratinocyte senescence.

Abstract Background The DNA damage response (DDR) is a mechanism that protects cells against radiation-induced oxidative DNA damage by causing cell cycle arrest and apoptosis. TP63 is a member of the tumour suppressor TP53 gene family, and ΔNp63α, a TP63 splicing variant, is constitutively expressed in the stem cell-containing basal layer of stratified epithelial tissues, including the mammary gland, where it plays a critical role in stemness and tissue development. ΔNp63α has been reported to transcriptionally inhibit the tumour suppression protein p53. This p53-repressive activity may cause genomic instability in epithelial stem cells exposed to radiation. In this study, we analysed the inhibitory effect of ΔNp63α on radiation-induced DDR. Methods To elucidate the role of the p53-repressive effect of ΔNp63α in radiation response, we performed a p63-siRNA knockdown experiment using human mammary epithelial cells (HMECs) expressing ΔNp63α and then performed ectopic and entopic expression experiments using human induced pluripotent stem cells (hiPSCs). After irradiation, the expression of DDR-related genes and proteins in ΔNp63α-expressing and control cells was analysed by RT–qPCR, Western blotting, and flow cytometry. Results The mRNA/protein expression levels of BAX and p21 were significantly increased in p63-siRNA-treated HMECs (sip63) after X-ray irradiation (4 Gy, 0.7 Gy/min) but not in scramble-siRNA treated HMECs (scr). Transcriptomic analysis showed decreased RNA expression of cell cycle-related genes and increased expression of programmed cell death-related genes in sip63 cells compared to scr cells. Furthermore, flow cytometric analysis revealed an increase in apoptotic cells and a decrease in 5-ethynyl-2´-deoxyuridine uptake in sip63 cells compared to scr cells. On the other hand, both the ectopic and entopic expression of ΔNp63α in apoptosis-sensitive hiPSCs reduced the expression levels of BAX after irradiation and significantly decreased the number of apoptotic cells induced by radiation. Conclusion Taken together, these results indicate that ΔNp63α represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells.

Head and neck squamous cell carcinoma (HNSCC) is one of the most prevalent cancers worldwide. Heat shock factor 1 (HSF1) is a conserved transcriptional factor that plays a critical role in maintaining cellular proteostasis. However, the role of HSF1 in HNSCC development remains largely unclear. Here, we report that HSF1 promotes forkhead box protein O3a (FOXO3a)‐dependent transcription of ΔNp63α (p63 isoform in the p53 family; inhibits cell migration, invasion, and metastasis), which leads to upregulation of cyclin‐dependent kinase 4 expression and HNSCC tumour growth. Ablation of HSF1 or treatment with KRIBB11, a specific pharmacological inhibitor of HSF1, significantly suppresses ΔNp63α expression and HNSCC tumour growth. Clinically, the expression of HSF1 is positively correlated with the expression of ΔNp63α in HNSCC tumours. Together, this study demonstrates that the HSF1–ΔNp63α pathway is critically important for HNSCC tumour growth. [ABSTRACT FROM AUTHOR]

Background: The omnipresence of human phthalate (PAE) exposure is linked to various adverse health issues, including breast cancer. However, the effects of low-dose PAE exposure on breast cancer stem cells (BCSCs) and the underlying mechanism remain unexplored. Methods: BCSCs from breast cancer cell lines (MDA-MB-231 and MCF-7) were enriched using a tumorsphere formation assay. Gene and protein expression was detected by measurement of quantitative real-time reverse transcription PCR, western blot, and immunofluorescence assays. Transient transfection assays were used to evaluate the involvement of Gli1, a signaling pathway molecule and ΔNp63α, an oncogene in influencing the PAE-induced characteristics of BCSCs. Results: PAE (butylbenzyl phthalate, BBP; di-butyl phthalate, DBP; di-2-ethylhexyl phthalate, DEHP) exposure of 10−9 M significantly promoted the tumorsphere formation ability in BCSCs. Breast cancer spheroids with a 10−9 M PAE exposure had higher levels of BCSC marker mRNA and protein expression, activated sonic hedgehog (SHH) pathway, and increased mRNA and protein levels of an oncogene, ΔNp63α. Furthermore, suppression of the SHH pathway attenuated the effects of PAEs on BCSCs. And the overexpression of ΔNp63α enhanced PAE-induced characteristics of BCSCs, while low expression of ΔNp63α inhibited the promotion effects of PAEs on BCSCs and the SHH pathway. Conclusion: Low-dose PAE exposure promoted the stem cell properties of BCSCs in a ΔNp63α- and SHH-dependent manner. The influence of low-dose exposure of PAEs and its relevance for the lowest observed effect concentrations requires further investigation, and the precise underlying mechanism needs to be further explored.

Background: The DNA damage response (DDR) is a mechanism that protects cells against radiation-induced oxidative DNA damage by causing cell cycle arrest and apoptosis. TP63 is a member of the tumour suppressor TP53 gene family, and ΔNp63α, a TP63 splicing variant, is constitutively expressed in the stem cell-containing basal layer of stratified epithelial tissues, including the mammary gland, where it plays a critical role in stemness and tissue development. ΔNp63α has been reported to transcriptionally inhibit the tumour suppression protein p53. This p53-repressive activity may cause genomic instability in epithelial stem cells exposed to radiation. In this study, we analysed the inhibitory effect of ΔNp63α on radiation-induced DDR.Methods: To elucidate the role of the p53-repressive effect of ΔNp63α in radiation response, we performed a p63-siRNA knockdown experiment using human mammary epithelial cells (HMECs) expressing ΔNp63α and then performed ectopic and entopic expression experiments using human induced pluripotent stem cells (hiPSCs). After irradiation, the expression of DDR-related genes and proteins in ΔNp63α-expressing and control cells was analysed by RT-qPCR, Western blotting, and flow cytometry.Results: The mRNA/protein expression levels of BAX and p21 were significantly increased in p63-siRNA-treated HMECs (sip63) after X-ray irradiation (4 Gy, 0.7 Gy/min) but not in scramble-siRNA treated HMECs (scr). Transcriptomic analysis showed decreased RNA expression of cell cycle-related genes and increased expression of programmed cell death-related genes in sip63 cells compared to scr cells. Furthermore, flow cytometric analysis revealed an increase in apoptotic cells and a decrease in 5-ethynyl-2´-deoxyuridine uptake in sip63 cells compared to scr cells. On the other hand, both the ectopic and entopic expression of ΔNp63α in apoptosis-sensitive hiPSCs reduced the expression levels of BAX after irradiation and significantly decreased the number of apoptotic cells induced by radiation.Conclusion: Taken together, these results indicate that ΔNp63α represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells. [ABSTRACT FROM AUTHOR]

Abstract Background p63, a member of the p53 gene family, is an important regulator for epithelial tissue growth and development. ∆Np63α is the main isoform of p63 and highly expressed in Non-melanoma skin cancer (NMSC). Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose biochemical features and cellular regulation are distinct from those of conventional MAPKs such as ERK1/2. While ERK3 has been shown to be upregulated in lung cancers and head and neck cancers, in which it promotes cancer cell migration and invasion, little is known about the implication of ERK3 in NMSCs. Methods Fluorescent immunohistochemistry was performed to evaluate the expression levels of ΔNp63α and ERK3 in normal and NMSC specimens. Dunnett’s test was performed to compare mean fluorescence intensity (MFI, indicator of expression levels) of p63 or ERK3 between normal cutaneous samples and NMSC samples. A mixed effects (ANOVA) test was used to determine the correlation between ΔNp63α and ERK3 expression levels (MFI). The regulation of ERK3 by ΔNp63α was studied by qRT-PCR, Western blot and luciferase assay. The effect of ERK3 regulation by ΔNp63α on cell migration was measured by performing trans-well migration assay. Results The expression level of ∆Np63α is upregulated in NMSCs compared to normal tissue. ERK3 level is significantly upregulated in AK and SCC in comparison to normal tissue and there is a strong positive correlation between ∆Np63α and ERK3 expression in normal skin and skin specimens of patients with AK, SCC or BCC. Further, we found that ∆Np63α positively regulates ERK3 transcript and protein levels in A431 and HaCaT skin cells, underlying the upregulation of ERK3 expression and its positive correlation with ∆Np63α in NMSCs. Moreover, similar to the effect of ∆Np63α depletion, silencing ERK3 greatly enhanced A431 cell migration. Restoration of ERK3 expression under the condition of silencing ∆Np63α counteracted the increase in cell migration induced by the depletion of ∆Np63α. Mechanistically, ERK3 inhibits the phosphorylation of Rac1 G-protein and the formation of filopodia of A431 skin SCC cells. Conclusions ERK3 is positively regulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in NMSC.

The TP63 gene, which encodes the p63 protein, is involved in multiple biological processes, including embryonic development and tumorigenesis. ΔNp63α, the predominant isoform of p63 in epithelial cells, acts as an oncogene in early‐stage tumors, but paradoxically acts as a potent antimetastatic factor in advanced cancers. Here, we report that ΔNp63α is a direct target of hsa‐miR‐522 (miR‐522). Induced expression of miR‐522 reduced the levels of ΔNp63α, predisposing breast epithelial cells to a loss of epithelial and acquisition of mesenchymal morphology, resulting in accelerated collective and single‐cell migration. Restoration of ΔNp63α repressed miR‐522‐induced migration. Interestingly, overexpression of miR‐522 did not affect breast epithelial cell proliferation, suggesting that miR‐522 acts specifically through ΔNp63α in this context. Furthermore, expression of miR‐522‐3p and p63 was negatively correlated in human cancer samples. Thus, miR‐522 might be a causative factor for breast tumorigenesis and cancer metastasis. In summary, our results reveal a novel miR‐522/p63 axis in cell migration and thus suggest a potential strategy for therapeutic treatment of cancer metastasis.

Background: p63, a member of the p53 gene family, is an important regulator for epithelial tissue growth and development. ∆Np63α is the main isoform of p63 and highly expressed in Non-melanoma skin cancer (NMSC). Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose biochemical features and cellular regulation are distinct from those of conventional MAPKs such as ERK1/2. While ERK3 has been shown to be upregulated in lung cancers and head and neck cancers, in which it promotes cancer cell migration and invasion, little is known about the implication of ERK3 in NMSCs.Methods: Fluorescent immunohistochemistry was performed to evaluate the expression levels of ΔNp63α and ERK3 in normal and NMSC specimens. Dunnett's test was performed to compare mean fluorescence intensity (MFI, indicator of expression levels) of p63 or ERK3 between normal cutaneous samples and NMSC samples. A mixed effects (ANOVA) test was used to determine the correlation between ΔNp63α and ERK3 expression levels (MFI). The regulation of ERK3 by ΔNp63α was studied by qRT-PCR, Western blot and luciferase assay. The effect of ERK3 regulation by ΔNp63α on cell migration was measured by performing trans-well migration assay.Results: The expression level of ∆Np63α is upregulated in NMSCs compared to normal tissue. ERK3 level is significantly upregulated in AK and SCC in comparison to normal tissue and there is a strong positive correlation between ∆Np63α and ERK3 expression in normal skin and skin specimens of patients with AK, SCC or BCC. Further, we found that ∆Np63α positively regulates ERK3 transcript and protein levels in A431 and HaCaT skin cells, underlying the upregulation of ERK3 expression and its positive correlation with ∆Np63α in NMSCs. Moreover, similar to the effect of ∆Np63α depletion, silencing ERK3 greatly enhanced A431 cell migration. Restoration of ERK3 expression under the condition of silencing ∆Np63α counteracted the increase in cell migration induced by the depletion of ∆Np63α. Mechanistically, ERK3 inhibits the phosphorylation of Rac1 G-protein and the formation of filopodia of A431 skin SCC cells.Conclusions: ERK3 is positively regulated by ∆Np63α and mediates the role of ∆Np63α in suppressing cell migration in NMSC. [ABSTRACT FROM AUTHOR]

The TP63 gene, which encodes the p63 protein, is involved in multiple biological processes, including embryonic development and tumorigenesis. ΔNp63α, the predominant isoform of p63 in epithelial cells, acts as an oncogene in early‐stage tumors, but paradoxically acts as a potent antimetastatic factor in advanced cancers. Here, we report that ΔNp63α is a direct target of hsa‐miR‐522 (miR‐522). Induced expression of miR‐522 reduced the levels of ΔNp63α, predisposing breast epithelial cells to a loss of epithelial and acquisition of mesenchymal morphology, resulting in accelerated collective and single‐cell migration. Restoration of ΔNp63α repressed miR‐522‐induced migration. Interestingly, overexpression of miR‐522 did not affect breast epithelial cell proliferation, suggesting that miR‐522 acts specifically through ΔNp63α in this context. Furthermore, expression of miR‐522‐3p and p63 was negatively correlated in human cancer samples. Thus, miR‐522 might be a causative factor for breast tumorigenesis and cancer metastasis. In summary, our results reveal a novel miR‐522/p63 axis in cell migration and thus suggest a potential strategy for therapeutic treatment of cancer metastasis. [ABSTRACT FROM AUTHOR]

Objective To screen out the miRNAs regulated by ΔNp63α in squamous cell carcinoma of the cervix and investigate their effect on the function of cervical cancer cells. Methods miRNA microarray was used to screen the miRNAs with significant difference in SiHa cells with overexpressed ΔNp63α and verified by qRT-PCR. The miRNAs with differential expression were validated by qRT-PCR in ME-180 cells with silenced ΔNp63α and the hsa-let-7b-3p was selected as the study object. The effect of hsa-let-7b-3p on the proliferation and apoptosis of SiHa cells was detected by growth curve, Western blot and flow cytometry after the transfection of hsa-let-7b-3p mimic into SiHa cells. Results (1) In SiHa cells with ΔNp63α overexpression, ten miRNAs consistent with the trend of qRT-PCR were screened out from the miRNAs with significant differences in the results of the microarray. (2) Five miRNAs that were consistent with the above change trends were screened out in ME-180 cells with silenced ΔNp63α. (3) Overexpression of hsa-let-7b-3p inhibited the proliferation of SiHa cells. (4) Overexpression of hsa-let-7b-3p promoted the apoptosis of SiHa cells. Conclusion In the cervical cancer cell line, the expression of miRNA hsa-let-7b-3p is positively correlated with the expression of ΔNp63α, and it can inhibit cell proliferation effectively. The mechanism may be related to the induction of cell cycle arrest and the promotion of apoptosis.

Cervical cancer (CC) remains one of the leading causes of mortality of female cancers worldwide, with more than 90% being cervical squamous cell carcinoma (CSCC). ΔNp63α is the predominant isoform expressed in cervical epithelial tissues and exerts its antitumor function in CSCC. In this study, we have identified 39 long noncoding RNAs as ΔNp63α targets in CSCC through RNA sequencing and chromatin immunoprecipitation sequencing, in which we further confirmed and focused on the two tumor‐related long noncoding RNAs, PART1 (lncPART1) and MIR17HG (lncMIR17HG). Experiments from stable overexpression/knockdown cell lines revealed that lncPART1 and lncMIR17HG regulated cell proliferation, migration, and invasion. In vivo experiments further showed that lncPART1 suppresses tumor growth in CSCC‐derived tumors. Examinations of clinical tissues indicated that the expression of lncPART1 was positively correlated with ΔNp63α expression, while lncMIR17HG was negatively correlated with ΔNp63α expression, suggesting that ΔNp63α plays a central role via regulating its direct targets in the progression of CSCC. These findings provide novel insights in targeted therapy of cervical cancers. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase (AMPK) functions as an energy sensor and is pivotal in maintaining cellular metabolic homeostasis. Numerous studies have shown that down-regulation of AMPK kinase activity or protein stability not only lead to abnormality of metabolism but also contribute to tumor development. However, whether transcription regulation of AMPK plays a critical role in cancer metastasis remains unknown. In this study, we demonstrate that AMPKα1 expression is down-regulated in advanced human breast cancer and is associated with poor clinical outcomes. Transcription of AMPKα1 is inhibited on activation of PI3K and HER2 through ΔNp63α. Ablation of AMPKα1 expression or inhibition of AMPK kinase activity leads to disruption of E-cadherin-mediated cell–cell adhesion in vitro and increased tumor metastasis in vivo. Furthermore, restoration of AMPKα1 expression significantly rescues PI3K/HER2-induced disruption of cell–cell adhesion, cell invasion, and cancer metastasis. Together, these results demonstrate that the transcription control is another layer of AMPK regulation and suggest a critical role for AMPK in regulating cell–cell adhesion and cancer metastasis. [ABSTRACT FROM AUTHOR]

The p53 gene family network plays a pivotal role in the control of many biological processes and therefore the right balance between the pro-apoptotic and pro-survival isoforms is key to maintain cellular homeostasis. The stability of the p53 tumor suppressor protein and that of oncogenic ΔNp63α, is crucial to control cell proliferation. The aberrant expression of p53 tumor suppressor protein and oncogenic ΔNp63α contributes to tumorigenesis and significantly affects anticancer drug response. Recently, we demonstrated that TRIM8 increases p53 stability, potentiating its tumor suppressor activity. In this paper, we show that TRIM8 simultaneously reduces the level of the pro-proliferative ΔNp63α protein, in both a proteasomal and caspase-1 dependent way, thereby playing a critical role in the cellular response to DNA damaging agents. Moreover, we provided evidence that ΔNp63α in turn, suppresses TRIM8 gene expression by preventing p53-mediated transactivation of TRIM8, therefore suggesting the existence of a negative feedback loop. These findings indicate that TRIM8 exerts its anticancer power through a joint action that provides on one hand, the activation of the p53 tumor suppressor role, and on the other the quenching of the oncogenic ΔNp63α protein activity. The enhancement of TRIM8 activity may offer therapeutic benefits and improve the management of chemoresistant tumors.

The p53 gene family network plays a pivotal role in the control of many biological processes and therefore the right balance between the pro-apoptotic and pro-survival isoforms is key to maintain cellular homeostasis. The stability of the p53 tumor suppressor protein and that of oncogenic ΔNp63α, is crucial to control cell proliferation. The aberrant expression of p53 tumor suppressor protein and oncogenic ΔNp63α contributes to tumorigenesis and significantly affects anticancer drug response. Recently, we demonstrated that TRIM8 increases p53 stability, potentiating its tumor suppressor activity. In this paper, we show that TRIM8 simultaneously reduces the level of the pro-proliferative ΔNp63α protein, in both a proteasomal and caspase-1 dependent way, thereby playing a critical role in the cellular response to DNA damaging agents. Moreover, we provided evidence that ΔNp63α in turn, suppresses TRIM8 gene expression by preventing p53-mediated transactivation of TRIM8, therefore suggesting the existence of a negative feedback loop. These findings indicate that TRIM8 exerts its anticancer power through a joint action that provides on one hand, the activation of the p53 tumor suppressor role, and on the other the quenching of the oncogenic ΔNp63α protein activity. The enhancement of TRIM8 activity may offer therapeutic benefits and improve the management of chemoresistant tumors. [ABSTRACT FROM AUTHOR]

Tumor protein 63 (TP63) comprises multiple isoforms and plays an important role during embryonic development. It has been shown that TP63 knockdown inhibits myogenic differentiation, but which isoform is involved in the underlying myogenic regulation remains uncertain. Here, we found that two transcripts of TP63, namely, TAp63α and ΔNp63α, are expressed in chicken skeletal muscle. These two transcripts have distinct expression patterns and opposite functions in skeletal muscle development. TAp63 has higher expression in skeletal muscle than in other tissues, and its expression is gradually upregulated during chicken primary myoblast differentiation. ΔNp63 can be expressed in multiple tissues and exhibits stable expression during myoblast differentiation. TAp63α overexpression inhibits myoblast proliferation, induces cell cycle arrest, and enhances myoblast differentiation. However, although ΔNp63α has no significant effect on cell proliferation, the overexpression of ΔNp63α inhibits myoblast differentiation. Using isoform-specific overexpression assays following RNA-sequencing, we identified potential downstream genes of TAp63α and ΔNp63α in myoblast. Bioinformatics analyses and experimental verification results showed that the differentially expressed genes (DEGs) between the TAp63α and control groups were enriched in the cell cycle pathway, whereas the DEGs between the ΔNp63α and control groups were enriched in muscle system process, muscle contraction, and myopathy. These findings provide new insights into the function and expression of TP63 during skeletal muscle development, and indicate that one gene may play two opposite roles during a single cellular process.

Tumor protein 63 (TP63) comprises multiple isoforms and plays an important role during embryonic development. It has been shown that TP63 knockdown inhibits myogenic differentiation, but which isoform is involved in the underlying myogenic regulation remains uncertain. Here, we found that two transcripts of TP63 , namely, TAp63 α and Δ Np63 α, are expressed in chicken skeletal muscle. These two transcripts have distinct expression patterns and opposite functions in skeletal muscle development. TAp63 has higher expression in skeletal muscle than in other tissues, and its expression is gradually upregulated during chicken primary myoblast differentiation. Δ Np63 can be expressed in multiple tissues and exhibits stable expression during myoblast differentiation. TAp63 α overexpression inhibits myoblast proliferation, induces cell cycle arrest, and enhances myoblast differentiation. However, although Δ Np63 α has no significant effect on cell proliferation, the overexpression of Δ Np63 α inhibits myoblast differentiation. Using isoform-specific overexpression assays following RNA-sequencing, we identified potential downstream genes of TAp63 α and Δ Np63 α in myoblast. Bioinformatics analyses and experimental verification results showed that the differentially expressed genes (DEGs) between the TAp63 α and control groups were enriched in the cell cycle pathway, whereas the DEGs between the Δ Np63 α and control groups were enriched in muscle system process, muscle contraction, and myopathy. These findings provide new insights into the function and expression of TP63 during skeletal muscle development, and indicate that one gene may play two opposite roles during a single cellular process. [ABSTRACT FROM AUTHOR]

Potassium channel tetramerization domain containing 5 (KCTD5) was previously documented as a component of the Cullin3‐RING ligase (CRL3). It has been reported that KCTD5 can induce enrichment of polyubiquitinated proteins, and KCTD5‐based CRL3 destabilizes several proteins. In our present study, we report that KCTD5 may physically interact with ΔNp63α, which is a member of the p53 family. Our further investigation revealed that Cullin3/KCTD5 can induce monoubiquitination of ΔNp63α. Cullin3/KCTD5 downregulates the DNA‐binding affinity of ΔNp63α, impairing either its transactivity or its transinhibitory activity. Functionally, Cullin3/KCTD5 abates the proproliferation activity of ΔNp63α. These findings suggest that KCTD5‐based CRL3 may mediate monoubiquitination and is a novel regulator of ΔNp63α. [ABSTRACT FROM AUTHOR]

We aim to determine the lncRNA targets of ΔNp63α in cervical cancer and molecular programs in cancerous differentiation. Different profiles of the lncRNAs were assayed and validated in overexpressing p63 SiHa cells (SiHa/ΔNp63α) and the control cell lines (SiHa/pCon). ENST00000422259, ENST00000447565 (Lnc-LIF-AS) and ENST00000469965, together with their related antisense mRNA DPYD (dihydropyrimidine dehydrogenase, a pyrimidine catabolic pathway gene), LIF (leukemia inhibitor factor) and FLNC (filamin C) were all notably differentially expressed in both ΔNp63α overexpression cells and knockdown cells. Here, we illustrated that ΔNp63α can inhibit the levels of LIF mRNA by direct transcription regulation and decrease LIF mRNA stability by suppressing the expression of Lnc-LIF-AS. An inverse interaction of LIF and ΔNp63α expression was as well validated in clinical samples of cervical cancer, and high level of LIF in cervical cancers was related with poor patient survival. The decrease of ΔNp63α also attenuated the differentiation of cervical cancerous cells. Suggesting that ΔNp63α may be form a complex network in regulation cervical cancerous differentiation. [ABSTRACT FROM AUTHOR]

Activation of phosphatidylinositol 3 kinase (PI3K), Ras, and Her2 signaling plays a critical role in cancer development. Hotspot constitutive activating mutations in oncogenes, such as PIK3CA encoding the p110α catalytic subunit or RAS, as well as over-expression of Her2, are frequently found in human tumors and cancers. It has been well established that activation of these oncogenes profoundly promotes tumor metastasis, whereas decreased expression of ΔNp63α, the major protein isoform of the p53-related p63 expressed in epithelial cells, has been associated with cancer metastasis. In this study, we demonstrate that hotspot oncogenic mutations on PIK3CA and RAS, including p110αH1047R, K-RasG12V, and H-RasG12V, as well as activation of Her2, all led to suppression of ΔNp63α expression via Akt-fork-head transcription factor 3a (Akt-FOXO3a) signaling, resulting in increased cell motility and tumor metastasis. Expression of ΔNp63α effectively reversed p110αH1047R-, K-RasG12V-, H-RasG12V-, or Her2-induced cell motility in vitro and tumor metastasis in mouse models. We show that ΔNp63α was a direct FOXO3a transcriptional target and that expression of FOXO3a and ΔNp63α was correlated in human cancer biopsy samples. Together, these results demonstrate that ΔNp63α is a common inhibitory target of oncogenic PI3K, Ras, and Her2, and that ΔNp63α may function as a critical integrator of oncogenic signaling in cancer metastasis. [ABSTRACT FROM AUTHOR]

The omnipresence of human phthalate (PAE) exposure is linked to various adverse health issues, including breast cancer. However, the effects of low-dose PAE exposure on breast cancer stem cells (BCSCs) and the underlying mechanism remain unexplored. BCSCs from breast cancer cell lines (MDA-MB-231 and MCF-7) were enriched using a tumorsphere formation assay. Gene and protein expression was detected by measurement of quantitative real-time reverse transcription PCR, western blot, and immunofluorescence assays. Transient transfection assays were used to evaluate the involvement of Gli1, a signaling pathway molecule and ΔNp63α, an oncogene in influencing the PAE-induced characteristics of BCSCs. PAE (butylbenzyl phthalate, BBP; di-butyl phthalate, DBP; di-2-ethylhexyl phthalate, DEHP) exposure of 10−9 M significantly promoted the tumorsphere formation ability in BCSCs. Breast cancer spheroids with a 10−9 M PAE exposure had higher levels of BCSC marker mRNA and protein expression, activated sonic hedgehog (SHH) pathway, and increased mRNA and protein levels of an oncogene, ΔNp63α. Furthermore, suppression of the SHH pathway attenuated the effects of PAEs on BCSCs. And the overexpression of ΔNp63α enhanced PAE-induced characteristics of BCSCs, while low expression of ΔNp63α inhibited the promotion effects of PAEs on BCSCs and the SHH pathway. Low-dose PAE exposure promoted the stem cell properties of BCSCs in a ΔNp63α- and SHH-dependent manner. The influence of low-dose exposure of PAEs and its relevance for the lowest observed effect concentrations requires further investigation, and the precise underlying mechanism needs to be further explored. • PAEs promote the stemness of un-invasive and invasive breast cancer cells at environmentally relevant lower doses. • PAEs upregulate ΔNp63α and activate the SHH pathway to enhance the stemness traits of BCSCs. • The ΔNp63α/SHH pathway may be the potential molecular mechanism by which PAEs promote breast cancer, especially BCSCs. [ABSTRACT FROM AUTHOR]

Background: Hotspot mutations occurring in the p110α domain of the PIK3CA gene, specifically p110αH1047R/L increase tumor metastasis and cell motility in triple-negative breast cancer (TNBC). These mutations also affect the transcriptional regulation of ΔNp63α, a significant isoform of the p53 protein involved in cancer progression. This study attempts to investigate the transcriptional impact of p110αH1047R/L mutations on the PIK3CA/ΔNp63α complex in TNBC carcinogenesis., Methods: We performed site-directed mutagenesis to introduce p110αH1047R/L mutations and evaluated their oncogenic effects on the growth, invasion, migration, and apoptosis of three different TNBC cell lines in vitro. We investigated the impact of these mutations on the p110α/ΔNp63α complex and downstream transcriptional signaling pathways at the gene and protein levels. Additionally, we used bioinformatics techniques such as molecular dynamics simulations and protein-protein docking to gain insight into the stability and structural changes induced by the p110αH1047R/L mutations in the p110α/ΔNp63α complex and downstream signaling pathway., Results: The presence of PIK3CA oncogenic hotspot mutations in the p110α/ΔNp63α complex led to increased scattering of TNBC cells during growth, migration, and invasion. Our in vitro mutagenesis assay showed that the p110αH1047R/L mutations activated the PI3K-Akt-mTOR and tyrosine kinase receptor pathways, resulting in increased cell proliferation, invasion, and apoptosis in TNBC cells. These mutations decreased the repressing effect of ΔNp63α on the p110α kinase domain, leading to the enhancement of downstream signaling pathways of PI3K and tyrosine kinase receptors and oncogenic transformation in TNBC. Additionally, our findings suggest that the physical interaction between the DNA binding domain of ΔNp63α and the kinase domain of p110α may be partially impaired, potentially leading to alterations in the conformation of the p110α/ΔNp63α complex., Conclusion: Our findings suggest that targeting the p110αH1047R/L mutations in TNBC could be a promising strategy for developing transcriptional-based therapies. Restoring the interaction between ΔNp63α and the p110α kinase domain, which is disrupted by these mutations, may provide a new approach to treating TNBC.

Background: ΔNp63, a splice variant of p63, is overexpressed and exhibits oncogenic activity in many cancers including pancreatic and breast cancer and promotes cell survival by inhibiting apoptosis. Despite its role in tumorigenesis, mechanistic activity of ΔNp63 mediated oncogenic function in osteosarcoma is poorly understood. Methods: The expression levels of p63 isoforms in osteosarcoma cell lines were identified using quantitative techniques. Expression profiling using microarray, siRNA mediated loss-of-function, and chromatin immunoprecipitation assays were employed to identify novel ΔNp63α targets in p63-null osteosarcoma SaOS-2 cells that were engineered to express ΔNp63α. The phenotype of SaOS-2-ΔNp63α cells was assessed using wound-healing, colony formation, and proliferation assays. Results: The comparative expression analyses identified ΔNp63α as the predominant p63 isoform expressed by invasive OS cell lines. Phenotypic analyses of SaOS-2-ΔNp63α cells in vitro indicate that ΔNp63α imparted tumorigenic attributes upon tumor cells. Further, we show that in osteosarcoma cells ΔNp63α directly regulated the transcription factor GLI2, which is a component of the hedgehog signaling pathway, and that functional interactions between ΔNp63α and GLI2 confer oncogenic properties upon OS cells. Conclusions: Here, we report that GLI2 is the novel target gene of ΔNp63α and that ΔNp63α-GLI2 crosstalk in osteosarcoma cells is a necessary event in osteosarcoma progression. Defining the exact mechanisms involved in this interaction that mediate the pathogenesis of osteosarcoma promises to identify targets for drug therapy. [ABSTRACT FROM AUTHOR]

Abstract: ΔNp63α is a p63 isoform that is predominantly expressed in the epidermal stem cells and in cancer. To find the regulatory pathways of ΔNp63α, we assessed whether ΔNp63α is acetylated and determined the functional implications of acetylation. First, the hinge region of p63 was shown to be acetylated by PCAF, similarly to other p53 family members. Second, acetylation synergistically induced cytoplasmic localization of ΔNp63α. Finally, acetyl-ΔNp63α was induced during high-density culture, suggesting that acetylation of ΔNp63α may reinforce cell cycle arrest upon cell contact. Altogether, these findings suggest that acetylation of ΔNp63α contributes to the epidermal homeostasis. Structured summary of protein interactions: PCAF acetylates ΔNp63α by acetylation assay (View Interaction: 1 , 2 , 3 ) ΔNp63α physically interacts with ΔNp63α by anti tag coimmunoprecipitation ( View interaction ) ΔNp63α physically interacts with p53 by anti bait coimmunoprecipitation ( View interaction ) [Copyright &y& Elsevier]

A dominant negative form of p63, ΔNp63α, is critical for maintaining the proliferative potential of epidermal stem cells and progenitor cells. The expression of ΔNp63α also confers a selective advantage for cancer cell survival, underscoring the importance of ΔNp63α in both normal and neoplastic stratified epithelia. Regulation of ΔNp63α can be achieved at the transcriptional and post-translational levels, the latter being greatly influenced by external stimuli such as UV irradiation. In this study, we have found that tumor necrosis factor-α (TNF-α), a multifunctional cytokine that has been implicated in epidermal homeostasis during normal and pathophysiologic conditions, also triggers the degradation of ΔNp63α in immortalized keratinocytes and cervical cancer cells. Conversely, downregulation of ΔNp63α sensitized cancer cells to TNF-α-induced apoptosis, suggesting a counteractive interaction between TNF-α and ΔNp63α in the regulation of epithelial cell death. The degradation of ΔNp63α by TNF-α was delayed when cells were treated with nuclear factor-κB inhibitors, whereas the induction of apoptosis by TNF-α was accompanied by the dramatic upregulation of the proapoptotic gene Puma. These observations further elucidate the relationship between TNF-α and ΔNp63α, two well-known mediators of epidermal homeostasis, and further suggest crosstalk between the two molecules in normal and pathophysiologic epidermis. [ABSTRACT FROM AUTHOR]

Abstract: ΔNp63α is exclusively expressed in stem cells and progenitor cells of the stratified epithelia. It promotes cell proliferation by antagonizing p53 and related TAp63/TAp73. Here, we report that specific desumoylation by SUMO protease SuPr-1 provides a fine-tuning mechanism for ΔNp63α repressor activity. We found that disrupting the sumoylation site compromised ΔNp63α repressor activity profoundly against TAp63γ and TAp73β-mediated transcription activation, but not to p53-mediated transcription. We further found that SuPr-1 specifically bound to sumoylated ΔNp63α and hydrolyzed SUMO. Consequently, SuPr-1 expression reduced ΔNp63α repressor activity to TAp63γ and TAp73β, whereas p53-mediated transactivation was unaffected. Collectively, these data suggest that SuPr-1-mediated ΔNp63α desumoylation elaborately regulates epithelial growth. [Copyright &y& Elsevier]

Purpose: To compare the histology, phenotype, ultrastructure and barrier function of cultured limbal epithelial cells using two explant culture protocols. Methods: Epithelial cells were cultured for 16 days from limbal explants, positioned with either the stromal side (stromal group) or the epithelial side (epithelial group) on intact amniotic membranes. The cultured epithelium ( n = 56) was examined using light microscopy, immunohistochemistry for K3, Cx43, ABCG2 and p63 expression, Western blot analysis of ΔNp63α, transmission electron microscopy, a horseradish peroxidase (HRP) permeability assay and scanning electron microscopy. Results: The epithelial group demonstrated a significantly higher expression of p63-positive cells (85.7 ± 4.2%) than the stromal group (75.3 ± 8.9%), and Western blots showed a stronger band of ΔNp63α. K3 and ABCG2 were not detected in either group, whereas Cx43 displayed moderate immunostaining in the suprabasal layer. The number of cell layers, the desmosome number and the undulation length in the epithelial group were not significantly different from those in the stromal group. In both groups, HRP accumulated on the apical surface of the superficial cells, and scanning electron microscopy demonstrated tightly apposed superficial cells. Conclusions: Our findings indicate that limbal explants positioned epithelial side down may give rise to cultured epithelia with higher expression of p63 and ΔNp63α. [ABSTRACT FROM AUTHOR]

The p53-regulated growth arrest and DNA damage-inducible gene product Gadd45a has been recently identified as a key factor protecting the epidermis against ultraviolet radiation (UVR)-induced skin tumors by activating p53 via the stress mitogen-activated protein kinase (MAPK) signaling pathway. Herein we identify Gadd45a as an important negative regulator of two oncogenes commonly over-expressed in epithelial tumors: the p53 homologue ?Np63a and ß-catenin. ?Np63a is one of the several p63 isoforms and is the predominant species expressed in basal epidermal keratinocytes. ?Np63a lacks the N-terminal transactivation domain and behaves as a dominant-negative factor blocking expression of several p53-effector genes. ?Np63a also associates with and blocks activation of the adenomatous polyposis coli (APC) destruction complex that targets free cytoplasmic ß-catenin for degradation. While most ß-catenin protein is localized to the cell membrane and is involved in cell-cell adhesion, accumulation of free cytoplasmic ß-catenin will translocate into the nucleus where it functions in a bipartite transcription factor complex, whose targets include invasion and metastasis promoting endopeptidases, matrix metalloproteinases (MMP). We show that Gadd45a not only directly associates with two components of the APC complex, namely protein phosphatase 2A (PP2A) and glycogen synthase kinase 3ß (GSK3ß) but also promotes GSK3ß dephosphorylation at Ser9, which is essential for GSK3ß activation, and resultant activation of the APC destruction complex. We demonstrate that lack of Gadd45a not only prevents ?Np63a suppression and GSK3ß dephosphorylation but also prevents free cytoplasmic ß-catenin degradation after UV irradiation. The inability of Gadd45a-null keratinocytes to suppress ß-catenin may contribute to the resulting observation of increased MMP expression and activity along with significantly faster keratinocyte migration in Matrigel in vitro and accelerated wound closure in vivo. Furthermore, epidermal keratinocytes treated with p38 MAPK inhibitors, both in vivo and in vitro, behave very similarly to Gadd45a-null keratinocytes after UVR. Similarly, Trp53-null mice are unable to attenuate ?Np63a expression in epidermal keratinocytes after such stress. These findings demonstrate a dependence on Gadd45a-mediated p38 MAPK and p53 activation for proper modulation of ?Np63a, GSK3ß, and ß-catenin after irradiation. Taken together, our results indicate that Gadd45a is able to repress ?Np63a, ß-catenin, and consequently MMP expression by two means: by maintaining UVR-induced p38 MAPK and p53 activation and also by associating with the APC complex. This implicates Gadd45a in the negative regulation of cell migration, and invasion.Oncogene (2004) 23, 1829-1837. doi:10.1038/sj.onc.1207301 Published online 1 December 2003 [ABSTRACT FROM AUTHOR]

Pan-histone deacetylase (HDAC) inhibitors can induce the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) protein. However, the underlying mechanism by which this occurs remains unclear. In this study, we show that pan-HDAC inhibitors, including trichostatin A, suberoylanilide hydroxamic acid, and sodium butyrate, were able to induce PTEN mRNA and protein expression via the acetylation of the transcription factor ΔNp63α by inhibiting HDAC1 and HDAC3. ΔNp63α enhanced PTEN promoter activity by binding two newly identified recognition sites on it. Unfortunately, the inhibition of HDAC1 or HDAC3 failed to activate PTEN, as knockdown of HDAC1 inhibited both membrane-bound and nuclear PTEN, and knockdown of HDAC3 only induced cytoplasmic PTEN. Furthermore, the overexpression of ΔNp63α downregulated membrane-bound PTEN but enhanced the nuclear translocation of PTEN, leading to the cisplatin resistance of oral cancer cells. PTEN accumulated in the nuclei of cancerous cells and normal cells when ΔNp63α was highly expressed in specimens from patients with squamous cell carcinoma of the tongue. However, inhibiting either HDAC1 or HDAC6 prevented the nuclear translocation of PTEN and attenuated cisplatin resistance. These results suggest that chemotherapeutic inhibitors of HDAC1 or HDAC6, together with cisplatin, might improve outcomes for patients with squamous cell carcinoma of the tongue., Competing Interests: None., (AJTR Copyright © 2020.)

Targeting metabolic reprogramming is an emerging strategy in cancer therapy. However, clinical attempts to target metabolic reprogramming have been proved to be challenging, with metabolic heterogeneity of cancer being one of many reasons that causes treatment failure. Here, we stratified non-small cell lung cancer (NSCLC) cells, mainly lung adenocarcinoma, based on their metabolic phenotypes and demonstrated that the aerobic glycolysis-preference NSCLC cell subtype was resistant to the OXPHOS-targeting inhibitors. We identified that monocarboxylate transporter 4 (MCT4), a lactate transporter, was highly expressed in the aerobic glycolysis-preference subtype with function supporting the proliferation of these cells. Glucose could induce the expression of MCT4 in these cells through a ΔNp63α and Sp1-dependent pathway. Next, we showed that knockdown of MCT4 increased intracellular lactate concentration and induced a reactive oxygen species (ROS)-dependent cellular apoptosis in the aerobic glycolysis-preference NSCLC cell subtype. By scanning a panel of monoclonal antibodies with MCT4 neutralizing activity, we further identified a MCT4 immunoglobulin M (IgM) monoclonal antibody showing capable anti-proliferation efficacy on the aerobic glycolysis-preference NSCLC cell subtype. Our findings indicate that the metabolic heterogeneity is a critical factor for NSCLC therapy and manipulating the expression or function of MCT4 can be an effective strategy in targeting the aerobic glycolysis-preference NSCLC cell subtype., (© 2020 The Author(s).)

Cell–cell adhesion plays an important role in regulation of cell proliferation, migration, survival, and drug sensitivity. Metformin, a first line drug for type 2 diabetes, has been shown to possess anti-cancer activities. However, whether cell–cell adhesion affects metformin anti-cancer activity is unknown. In this study, Microscopic and FACS analyses showed that metformin induced cancer cell–cell adhesion exemplified by cell aggregation and anoikis under glucose restriction. Furthermore, western blot and QPCR analyses revealed that metformin dramatically upregulated integrin β1 expression. Silencing of integrin β1 significantly disrupted cell aggregation and reduced anoikis induced by metformin. Moreover, we showed that p53 family member ΔNp63α transcriptionally suppressed integrin β1 expression and is responsible for metformin-mediated upregulation of integrin β1. In summary, this study reveals a novel mechanism for metformin anticancer activity and demonstrates that cell–cell adhesion mediated by integrin β1 plays a critical role in metformin-induced anoikis. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS) are key regulators in cell proliferation, survival, tumor initiation and development. However, the role of ROS in tumor metastasis is less clear. Here, we show that oxidative stress inhibited tumor metastasis via activation of Hippo kinase MST1/2, which led to the phosphorylation and nuclear accumulation of FoxO3a, resulting in upregulation of ΔNp63α expression and suppression of cell migration independent of YAP. Strikingly, while loss of MST1 led to and disruption of cell-cell junction exemplified by reduced E-cadherin expression, resulting in scattered cell growth, loss of MST2 led to disruption of cell-matrix adhesion as evidenced by reduced integrin β4, resulting in increased cell migration and tumor metastasis. Furthermore, expression of MST1 and MST2 was down-regulated in human breast carcinoma. Furthermore, oxidative stress inhibited HER2-or PI3K-mediated tumor metastasis via the MST2-FoxO3a-ΔNp63α pathway. Together, these results that this noncanonical Hippo MST2-FoxO3a-ΔNp63α pathway may play a critical role in ROS-mediated regulation of cell migration and tumor metastasis., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Tobacco smoke (TS) critically contributes to the development of lung cancer; however, the underlying molecular mechanisms remain unclear. The induction of cancer stem cells (CSCs) by TS represents an early event in tumor initiation. The lung cancer-related gene ΔNp63α is highly expressed in epithelial tissues and drives tumor formation and cancer stem cell properties. This study investigated the role of ΔNp63α in the long-term acquisition of TS-induced lung CSC-like properties. Methods: The expression levels of ΔNp63α, lung CSC markers, and interleukin (IL)-6 in lung carcinoma specimens were determined by western blotting and enzyme linked immunosorbent assays. Human bronchial epithelial (HBE) cells were chronically exposed to 2 % cigarette smoke extract for 55 passages, following which colony formation capacity, expression of proteins associated with malignant transformation, lung CSC markers, and tumor incidence were investigated. The effects of ΔNp63α on long-term TS exposure-induced lung CSC-like properties and Notch activation were analyzed using tumorsphere formation ability, immunofluorescence assays, luciferase reporter assays, and western blotting. The roles of IL-6 on chronic TS exposure-induced lung CSC-like properties and ΔNp63α expression were also examined. Moreover, the effects of sulforaphane (SFN) on TS-transformed lung CSC-like properties, IL-6 and ΔNp63α expression, and Notch signaling were investigated in vitro and in vivo . Results: Higher levels of ΔNp63α were observed in the lung cancer tissues of smokers than in those of non-smokers, whereas ΔNp63α was positively correlated with CD133 and Oct4 expression in lung cancer tissues. Data from the in vivo and in vitro experiments demonstrated that long-term TS exposure-transformed HBE (THBE) cells acquired lung CSC-like properties. Furthermore, ΔNp63α transcriptionally activated the Notch signaling pathway to promote the acquisition of CSC-like properties by the THBE cells. TS upregulated IL-6, which increased ΔNp63α expression in THBE sphere-forming cells. Finally, SFN inhibited the TS-induced CSC-like properties of THBE cells via the IL-6/ΔNp63α/Notch axis. Conclusion : Our data suggest that the IL-6/ΔNp63α/Notch axis plays an important role in the long-term TS exposure-induced acquisition of lung CSC-like properties and SFN intervention., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.