A Haspin-ARHGAP11A axis regulates epithelial morphogenesis through Rho-ROCK dependent modulation of LIMK1-Cofilin.

Throughout mitosis, a plethora of processes must be efficiently concerted to ensure cell proliferation and tissue functionality. The mitotic spindle does not only mediate chromosome segregation, but also defines the axis of cellular division, thus determining tissue morphology. Functional spindle orientation relies on precise actin dynamics, shaped in mitosis by the LIMK1-Cofilin axis. The kinase Haspin acts as a guardian of faithful chromosome segregation that ensures amphitelic chromosome attachment and prevents unscheduled cohesin cleavage. Here, we report an unprecedented role for Haspin in the determination of spindle orientation in mitosis. We show that, during mitosis, Haspin regulates Rho-ROCK activity through ARHGAP11A, a poorly characterized GAP, and that ROCK is in turn responsible for the mitotic activation of LIMK1 and stabilization of the actin cytoskeleton, thus supporting a functional spindle orientation. By exploiting 3D cell cultures, we show that this pathway is pivotal for the establishment of a morphologically functional tissue.

Photobiomodulation and Inorganic Bovine Bone in Guided Bone Regeneration: Histomorphometric Analysis in Rats.

The objective of this study was to evaluate the efficacy of photobiomodulation in the bone regeneration of critical-sized defects (CSD) filled with inorganic bovine bone associated or not with collagen membranes. The study has been conducted on 40 critical defects in the calvaria of male rats, divided into four experimental groups (n = 10): (1) DBBM (deproteinized bovine bone mineral); (2) GBR (DBBM+collagen membrane); (3) DBBM+P (DBBM+photobiomodulation); and (4) GBR+P (GBR+photobiomodulation). At 30 days postoperative, the animals were euthanized, and after the tissue had been processed, histological, histometric, and statistical analyses were performed. The analyses have taken into account newly formed bone area (NBA), linear bone extension (LBE), and residual particle area (RPA) as variables. The Kruskal-Wallis test has been performed, followed by the Dwass-Steel-Critchlow-Fligner test for comparison between groups (p < 0.05). When the DBBM+P group was compared to the DBBM group, it was possible to observe significant statistical differences in all the variables analyzed (p < 0.05). The application of photobiomodulation in guided bone regeneration (GBR+P) has shown a decrease in the median value for the RPA variable (26.8) when compared to the GBR group (32.4), with a significant statistical difference; however, for NBA and LBE, the therapy has not provided significant results.

The Underestimated Role of the p53 Pathway in Renal Cancer.

The TP53 tumor suppressor gene is known as the guardian of the genome, playing a pivotal role in controlling genome integrity, and its functions are lost in more than 50% of human tumors due to somatic mutations. This percentage rises to 90% if mutations and alterations in the genes that code for regulators of p53 stability and activity are taken into account. Renal cell carcinoma (RCC) is a clear example of cancer that despite having a wild-type p53 shows poor prognosis because of the high rate of resistance to radiotherapy or chemotherapy, which leads to recurrence, metastasis and death. Remarkably, the fact that p53 is poorly mutated does not mean that it is functionally active, and increasing experimental evidences have demonstrated this. Therefore, RCC represents an extraordinary example of the importance of p53 pathway alterations in therapy resistance. The search for novel molecular biomarkers involved in the pathways that regulate altered p53 in RCC is mandatory for improving early diagnosis, evaluating the prognosis and developing novel potential therapeutic targets for better RCC treatment.

Evaluation of Different Photobiomodulation Therapy Protocols as Adjuncts in the Healing of Bone Defects Grafted with Inorganic Bovine Bone.

PURPOSE: This study evaluated the effect of two photobiomodulation therapy protocols on bone regeneration in criticalsize bone defects grafted with inorganic bovine bone. MATERIALS AND METHODS: A critical-size defect was created in 30 adult male rat calvaria, which were divided equally and randomly into three experimental groups (n = 10): (1) DBBM (deproteinized bovine bone mineral); (2) DBBM + PBMT 4 J (4 J; photobiomodulation therapy; GaAlAs, 730 nm, 100 mW, 140 J/cm2); and (3) DBBM + PBMT 6 J (6 J; GaAlAs, 730 nm, 100 mW, 210 J/cm2). Animals were euthanized after 30 days. The neoformed bone area (NBA), linear bone extension (LBE), and area of the remaining particles (ARP) were evaluated. The data were subjected to nonparametric Kolmogorov-Smirnov test and ANOVA, followed by Tukey post hoc test to identify differences between the groups (P < .05). RESULTS: The 6 J group showed the highest average NBA (48.57% ± 28.22%) and demonstrated a statistically significant difference in NBA and LBE. A higher mean ARP was found in the DBBM group (38.73 ± 6.95) than in the groups irradiated by photobiomodulation therapy, with statistically significant differences (P < .05). CONCLUSION: The 6 J protocol showed the best results, promoting greater bone formation with greater resorption of residual particles.

Fotobiomodulação sistêmica transdérmica: uma revisão de literatura / Transdermal systemic photobiomodulation: a literature review

A fotobiomodulação sistêmica (FBM-S) consiste em uma técnica que utiliza o laser de baixa intensidade no espectro vermelho da luz para irradiação sistêmica. Seus benefícios incluem efeito analgésico, antioxidante sistêmico e anti-inflamatório, ativação de células imunológicas, melhora na cicatrização, vasodilatação e aumento da microcirculação. A técnica original, que utiliza cateter e fibra óptica para irradiação sistêmica, é uma técnica invasiva, por isso a fotobiomodulação sistêmica transdérmica foi desenvolvida como uma alternativa. Assim, o objetivo dessa revisão de literatura é discutir os efeitos, aplicações, protocolos e efeitos colaterais desta terapia modificada. Para tanto, foi realizada uma busca na literatura nas bases de dados Pubmed, Bireme, Embase, Scopus, Science Direct, Web of Science e CENTRAL, sem restrição de idioma no período entre 2010 e 2021. Encontraram-se seis estudos sendo um na área da Odontologia. Os resultados desses estudos sugerem que a FBS-S pode ser utilizada para o tratamento de condições sistêmicas. Em Odontologia, no entanto, a literatura ainda é escassa e mais estudos clínicos randomizados controlados são necessários para comprovar seus efeitos e estabelecer um protocolo clínico para sua utilização.

Systemic photobiomodulation (PBM-S) is a technique that uses low-level laser in the red spectrum of light for systemic irradiation. Its benefits include analgesic, systemic antioxi-dant, and anti-inflammatory effect, activation of immune cells, improved healing, vasodilation, and increased microcirculation. The original technique, which uses catheter and optical fibers for systemic irradiation is an invasive technique. Thus, the transdermal systemic photobiomodulation was developed as an alternative. The purpose of this literature review is to discuss the effects, applications, protocols, and side effects of this modified therapy. A literature search was carried out on Pubmed, Bireme, Embase, Scopus, Science Direct, Web of Science, and CENTRAL databases, with no language restriction in the period be-tween 2010 and 2021. Six studies were found, one in the area of Dentistry. The results of these studies suggest that PBM-S can be used for the treatment of systemic conditions. In Dentistry, however, the literature is still scarce and more randomized controlled clinical trials are needed to prove its effects and establish a protocol for its use.

Emerging Roles of TRIM8 in Health and Disease.

The superfamily of TRIM (TRIpartite Motif-containing) proteins is one of the largest groups of E3 ubiquitin ligases. Among them, interest in TRIM8 has greatly increased in recent years. In this review, we analyze the regulation of TRIM8 gene expression and how it is involved in many cell reactions in response to different stimuli such as genotoxic stress and attacks by viruses or bacteria, playing a central role in the immune response and orchestrating various fundamental biological processes such as cell survival, carcinogenesis, autophagy, apoptosis, differentiation and inflammation. Moreover, we show how TRIM8 functions are not limited to ubiquitination, and contrasting data highlight its role either as an oncogene or as a tumor suppressor gene, acting as a "double-edged weapon". This is linked to its involvement in the selective regulation of three pivotal cellular signaling pathways: the p53 tumor suppressor, NF-κB and JAK-STAT pathways. Lastly, we describe how TRIM8 dysfunctions are linked to inflammatory processes, autoimmune disorders, rare developmental and cardiovascular diseases, ischemia, intellectual disability and cancer.

TRIM8 Blunts the Pro-proliferative Action of ΔNp63α in a p53 Wild-Type Background.

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.

p63 is a cereblon substrate involved in thalidomide teratogenicity.

Cereblon (CRBN) is a primary target of thalidomide and mediates its multiple pharmacological activities, including teratogenic and antimyeloma activities. CRBN functions as a substrate receptor of the E3 ubiquitin ligase CRL4, whose substrate specificity is modulated by thalidomide and its analogs. Although a number of CRL4CRBN substrates have recently been identified, the substrate involved in thalidomide teratogenicity is unclear. Here we show that p63 isoforms are thalidomide-dependent CRL4CRBN neosubstrates that are responsible, at least in part, for its teratogenic effects. The p53 family member p63 is associated with multiple developmental processes. ∆Np63α is essential for limb development, while TAp63α is important for cochlea development and hearing. Using a zebrafish model, we demonstrate that thalidomide exerts its teratogenic effects on pectoral fins and otic vesicles by inducing the degradation of ∆Np63α and TAp63α, respectively. These results may contribute to the invention of new thalidomide analogs lacking teratogenic activity.

Evaluation of photobiomodulation therapy associated with guided bone regeneration in critical size defects. In vivo study.

The repair of bone defects raises the interest of investigators in several health specialties. Grafting techniques with bone substitutes and laser therapies have been investigated to replace autogenous bone and accelerate the bone healing process. Objective To evaluate the effect of photobiomodulation therapy (PBMT) associated with guided bone regeneration (GBR) in critical size defects. Material and Methods The study was conducted on 80 male rats (Rattus norvegicus albinus, Wistar) submitted to surgical creation of a critical size defect on the calvaria, divided into eight study groups: group C (control - only blood clot); group M (collagen membrane); group PBMT (photobiomodulation therapy); group AB (autogenous bone); group AB+PBMT; group AB+M; group PBMT+M; group AB+PBMT+M. The animals were killed 30 days postoperatively. After tissue processing, bone regeneration was evaluated by histomorphometric analysis and statistical analyses were performed (Tukey test, p<0.05). Results All groups had greater area of newly formed bone compared to group C (9.96±4.49%). The group PBMT+M (achieved the greater quantity of new bone (64.09±7.62%), followed by groups PBMT (47.67±8.66%), M (47.43±15.73%), AB+PBMT (39.15±16.72%) and AB+PBMT+M (35.82±7.68%). After group C, the groups AB (25.10±16.59%) and AB+M (22.72±13.83%) had the smallest quantities of newly formed bone. The area of remaining particles did not have statistically significant difference between groups AB+M (14.93±8.92%) and AB+PBMT+M (14.76±6.58%). Conclusion The PBMT utilization may be effective for bone repair, when associated with bone regeneration techniques.

Sumoylation and ubiquitylation crosstalk in the control of ΔNp63α protein stability.

ΔNp63α is finely and strictly regulated during embryogenesis and differentiation. ΔNp63α is the only p63 isoform degraded by the proteasome after Ubiquitin and SUMO (Small Ubiquitin-like MOdifier) conjugation. Here, we show that p63 ubiquitylation per se is not the signal triggering p63 proteasomal degradation. Taking advantage of natural ΔNp63α mutants isolated by patients with Split Hand and Foot Malformation IV syndrome, we found that SUMO and Ub modifications are not redundant and both are required to guarantee efficient ΔNp63α degradation. Here, we present evidence that sumoylation and ubiquitylation of ΔNp63α are strongly intertwined, and none of the two can efficiently occur if the other is impaired.

Evaluation of photobiomodulation therapy associated with guided bone regeneration in critical size defects. In vivo study

Abstract The repair of bone defects raises the interest of investigators in several health specialties. Grafting techniques with bone substitutes and laser therapies have been investigated to replace autogenous bone and accelerate the bone healing process. Objective To evaluate the effect of photobiomodulation therapy (PBMT) associated with guided bone regeneration (GBR) in critical size defects. Material and Methods The study was conducted on 80 male rats (Rattus norvegicus albinus, Wistar) submitted to surgical creation of a critical size defect on the calvaria, divided into eight study groups: group C (control - only blood clot); group M (collagen membrane); group PBMT (photobiomodulation therapy); group AB (autogenous bone); group AB+PBMT; group AB+M; group PBMT+M; group AB+PBMT+M. The animals were killed 30 days postoperatively. After tissue processing, bone regeneration was evaluated by histomorphometric analysis and statistical analyses were performed (Tukey test, p<0.05). Results All groups had greater area of newly formed bone compared to group C (9.96±4.49%). The group PBMT+M (achieved the greater quantity of new bone (64.09±7.62%), followed by groups PBMT (47.67±8.66%), M (47.43±15.73%), AB+PBMT (39.15±16.72%) and AB+PBMT+M (35.82±7.68%). After group C, the groups AB (25.10±16.59%) and AB+M (22.72±13.83%) had the smallest quantities of newly formed bone. The area of remaining particles did not have statistically significant difference between groups AB+M (14.93±8.92%) and AB+PBMT+M (14.76±6.58%). Conclusion The PBMT utilization may be effective for bone repair, when associated with bone regeneration techniques.

TRIM8 restores p53 tumour suppressor function by blunting N-MYC activity in chemo-resistant tumours.

BACKGROUND: TRIM8 plays a key role in controlling the p53 molecular switch that sustains the transcriptional activation of cell cycle arrest genes and response to chemotherapeutic drugs. The mechanisms that regulate TRIM8, especially in cancers like clear cell Renal Cell Carcinoma (ccRCC) and colorectal cancer (CRC) where it is low expressed, are still unknown. However, recent studies suggest the potential involvement of some microRNAs belonging to miR-17-92 and its paralogous clusters, which could include TRIM8 in a more complex pathway. METHODS: We used RCC and CRC cell models for in-vitro experiments, and ccRCC patients and xenograft transplanted mice for in vivo assessments. To measure microRNAs levels we performed RT-qPCR, while steady-states of TRIM8, p53, p21 and N-MYC were quantified at protein level by Western Blotting as well as at transcript level by RT-qPCR. Luciferase reporter assays were performed to assess the interaction between TRIM8 and specific miRNAs, and the potential effects of this interaction on TRIM8 expression. Moreover, we treated our cell models with conventional chemotherapeutic drugs or tyrosine kinase inhibitors, and measured their response in terms of cell proliferation by MTT and colony suppression assays. RESULTS: We showed that TRIM8 is a target of miR-17-5p and miR-106b-5p, whose expression is promoted by N-MYC, and that alterations of their levels affect cell proliferation, acting on the TRIM8 transcripts stability, as confirmed in ccRCC patients and cell lines. In addition, reducing the levels of miR-17-5p/miR-106b-5p, we increased the chemo-sensitivity of RCC/CRC-derived cells to anti-tumour drugs used in the clinic. Intriguingly, this occurs, on one hand, by recovering the p53 tumour suppressor activity in a TRIM8-dependent fashion and, on the other hand, by promoting the transcription of miR-34a that turns off the oncogenic action of N-MYC. This ultimately leads to cell proliferation reduction or block, observed also in colon cancer xenografts overexpressing TRIM8. CONCLUSIONS: In this paper we provided evidence that TRIM8 and its regulators miR-17-5p and miR-106b-5 participate to a feedback loop controlling cell proliferation through the reciprocal modulation of p53, miR-34a and N-MYC. Our experiments pointed out that this axis is pivotal in defining drug responsiveness of cancers such ccRCC and CRC.

Effect of different salivary exposure times on the rehardening of acid-softened enamel.

This in situ study assessed the effect of different times of salivary exposure on the rehardening of acid-softened enamel. Bovine enamel blocks were subjected in vitro to a short-term acidic exposure by immersion in 0.05 M (pH 2.5) citric acid for 30 s, resulting in surface softening. Then, 40 selected eroded enamel blocks were randomly assigned to 10 volunteers. Intraoral palatal appliances containing 4 enamel blocks were constructed for each volunteer, who wore the appliance for 12 nonconsecutive hours: initial 30 min, followed by an additional 30, and then by an additional 1 hour. For the last additional 10 hours the appliances were used at night, during the volunteers' sleep. Surface hardness was analyzed in the same blocks at baseline, after erosion and after each period of salivary exposure, enabling percentage of surface hardness recovery calculation (%SHR). The data were tested using repeated measures ANOVA and Tukey's test (α = 0.05). Increasing periods of salivary action promoted a progressive increase in the surface hardness (p < 0.001). However a similar degree of enamel rehardening (p = 0.641) was observed between 2 hours (49.9%) and 12 hours (53.3%) of salivary exposure. Two hours of salivary exposure seems to be appropriate for partial rehardening of the softened enamel surface. The use of the intraoral appliance during sleep did not improve the enamel rehardening after erosion.

Effect of different salivary exposure times on the rehardening of acid-softened enamel

Abstract This in situ study assessed the effect of different times of salivary exposure on the rehardening of acid-softened enamel. Bovine enamel blocks were subjected in vitro to a short-term acidic exposure by immersion in 0.05 M (pH 2.5) citric acid for 30 s, resulting in surface softening. Then, 40 selected eroded enamel blocks were randomly assigned to 10 volunteers. Intraoral palatal appliances containing 4 enamel blocks were constructed for each volunteer, who wore the appliance for 12 nonconsecutive hours: initial 30 min, followed by an additional 30, and then by an additional 1 hour. For the last additional 10 hours the appliances were used at night, during the volunteers' sleep. Surface hardness was analyzed in the same blocks at baseline, after erosion and after each period of salivary exposure, enabling percentage of surface hardness recovery calculation (%SHR). The data were tested using repeated measures ANOVA and Tukey's test (α = 0.05). Increasing periods of salivary action promoted a progressive increase in the surface hardness (p < 0.001). However a similar degree of enamel rehardening (p = 0.641) was observed between 2 hours (49.9%) and 12 hours (53.3%) of salivary exposure. Two hours of salivary exposure seems to be appropriate for partial rehardening of the softened enamel surface. The use of the intraoral appliance during sleep did not improve the enamel rehardening after erosion.

FGF8, c-Abl and p300 participate in a pathway that controls stability and function of the ΔNp63α protein.

The p63 transcription factor, homolog to the p53 tumor suppressor gene, plays a crucial role in epidermal and limb development, as its mutations are associated to human congenital syndromes characterized by skin, craniofacial and limb defects. While limb and skin-specific p63 transcriptional targets are being discovered, little is known of the post-translation modifications controlling ΔNp63α functions. Here we show that the p300 acetyl-transferase physically interacts in vivo with ΔNp63α and catalyzes its acetylation on lysine 193 (K193) inducing ΔNp63α stabilization and activating specific transcriptional functions. Furthermore we show that Fibroblast Growth Factor-8 (FGF8), a morphogenetic signaling molecule essential for embryonic limb development, increases the binding of ΔNp63α to the tyrosine kinase c-Abl as well as the levels of ΔNp63α acetylation. Notably, the natural mutant ΔNp63α-K193E, associated to the Split-Hand/Foot Malformation-IV syndrome, cannot be acetylated by this pathway. This mutant ΔNp63α protein displays promoter-specific loss of DNA binding activity and consequent altered expression of development-associated ΔNp63α target genes. Our results link FGF8, c-Abl and p300 in a regulatory pathway that controls ΔNp63α protein stability and transcriptional activity. Hence, limb malformation-causing p63 mutations, such as the K193E mutation, are likely to result in aberrant limb development via the combined action of altered protein stability and altered promoter occupancy.

DLX5, FGF8 and the Pin1 isomerase control ΔNp63α protein stability during limb development: a regulatory loop at the basis of the SHFM and EEC congenital malformations.

Ectrodactyly, or Split-Hand/Foot Malformation (SHFM), is a congenital condition characterized by the loss of central rays of hands and feet. The p63 and the DLX5;DLX6 transcription factors, expressed in the embryonic limb buds and ectoderm, are disease genes for these conditions. Mutations of p63 also cause the ectodermal dysplasia-ectrodactyly-cleft lip/palate (EEC) syndrome, comprising SHFM. Ectrodactyly is linked to defects of the apical ectodermal ridge (AER) of the developing limb buds. FGF8 is the key signaling molecule in this process, able to direct proximo-distal growth and patterning of the skeletal primordial of the limbs. In the limb buds of both p63 and Dlx5;Dlx6 murine models of SHFM, the AER is poorly stratified and FGF8 expression is severely reduced. We show here that the FGF8 locus is a downstream target of DLX5 and that FGF8 counteracts Pin1-ΔNp63α interaction. In vivo, lack of Pin1 leads to accumulation of the p63 protein in the embryonic limbs and ectoderm. We show also that ΔNp63α protein stability is negatively regulated by the interaction with the prolyl-isomerase Pin1, via proteasome-mediated degradation; p63 mutant proteins associated with SHFM or EEC syndromes are resistant to Pin1 action. Thus, DLX5, p63, Pin1 and FGF8 participate to the same time- and location-restricted regulatory loop essential for AER stratification, hence for normal patterning and skeletal morphogenesis of the limb buds. These results shed new light on the molecular mechanisms at the basis of the SHFM and EEC limb malformations.

BMP-mediated functional cooperation between Dlx5;Dlx6 and Msx1;Msx2 during mammalian limb development.

The Dlx and Msx homeodomain transcription factors play important roles in the control of limb development. The combined disruption of Msx1 and Msx2, as well as that of Dlx5 and Dlx6, lead to limb patterning defects with anomalies in digit number and shape. Msx1;Msx2 double mutants are characterized by the loss of derivatives of the anterior limb mesoderm which is not observed in either of the simple mutants. Dlx5;Dlx6 double mutants exhibit hindlimb ectrodactyly. While the morphogenetic action of Msx genes seems to involve the BMP molecules, the mode of action of Dlx genes still remains elusive. Here, examining the limb phenotypes of combined Dlx and Msx mutants we reveal a new Dlx-Msx regulatory loop directly involving BMPs. In Msx1;Dlx5;Dlx6 triple mutant mice (TKO), beside the expected ectrodactyly, we also observe the hallmark morphological anomalies of Msx1;Msx2 double mutants suggesting an epistatic role of Dlx5 and Dlx6 over Msx2. In Msx2;Dlx5;Dlx6 TKO mice we only observe an aggravation of the ectrodactyly defect without changes in the number of the individual components of the limb. Using a combination of qPCR, ChIP and bioinformatic analyses, we identify two Dlx/Msx regulatory pathways: 1) in the anterior limb mesoderm a non-cell autonomous Msx-Dlx regulatory loop involves BMP molecules through the AER and 2) in AER cells and, at later stages, in the limb mesoderm the regulation of Msx2 by Dlx5 and Dlx6 occurs also cell autonomously. These data bring new elements to decipher the complex AER-mesoderm dialogue that takes place during limb development and provide clues to understanding the etiology of congenital limb malformations.

TRIM8 modulates p53 activity to dictate cell cycle arrest.

p53 is a central hub in controlling cell proliferation. To maintain genome integrity in response to cellular stress, p53 directly regulates the transcription of genes involved in cell cycle arrest, DNA repair, apoptosis and/or senescence. An array of post-translational modifications and protein-protein interactions modulates its stability and activities in order to avoid malignant transformation. However, to date it is still not clear how cells decide their own fate in response to different types of stress. We described here that the human TRIM8 protein, a member of the TRIM family, is a new modulator of the p53-mediated tumor suppression mechanism. We showed that under stress conditions, such as UV exposure, p53 induced the expression of TRIM8, which in turn stabilized p53 leading to cell cycle arrest and reduction of cell proliferation through enhancement of CDKN1A (p21) and GADD45 expression. TRIM8 silencing reduced the capacity of p53 to activate genes involved in cell cycle arrest and DNA repair, in response to cellular stress. Concurrently, TRIM8 overexpression induced the degradation of the MDM2 protein, the principal regulator of p53 stability. Co-immunoprecipitation experiments showed that TRIM8 physically interacted with p53, impairing its interaction with MDM2. Altogether, our results reveal a previously unknown regulatory pathway controlling p53 activity and suggest TRIM8 as a novel therapeutic target to enhance p53 tumor suppressor activity.

Developmental factor IRF6 exhibits tumor suppressor activity in squamous cell carcinomas.

The transcription factor interferon regulatory factor 6 (IRF6) regulates craniofacial development and epidermal proliferation. We recently showed that IRF6 is a component of a regulatory feedback loop that controls the proliferative potential of epidermal cells. IRF6 is transcriptionally activated by p63 and induces its proteasome-mediated down-regulation, thereby limiting keratinocyte proliferative potential. We hypothesized that IRF6 may also be involved in skin carcinogenesis. Hence, we analyzed IRF6 expression in a large series of squamous cell carcinomas (SCCs) and found a strong down-regulation of IRF6 that correlated with tumor invasive and differentiation status. IRF6 down-regulation in SCC cell lines and primary tumors correlates with methylation on a CpG dinucleotide island located in its promoter region. To identify the molecular mechanisms regulating IRF6 potential tumor suppressive activity, we performed a genome-wide analysis by combining ChIP sequencing for IRF6 binding sites and gene expression profiling in primary human keratinocytes after siRNA-mediated IRF6 depletion. We observed dysregulation of cell cycle-related genes and genes involved in differentiation, cell adhesion, and cell-cell contact. Many of these genes were direct IRF6 targets. We also performed in vitro invasion assays showing that IRF6 down-regulation promotes invasive behavior and that reintroduction of IRF6 into SCC cells strongly inhibits cell growth. These results indicate a function for IRF6 in suppression of tumorigenesis in stratified epithelia.