Background The oral microbiome serves as an effector of bidirectional promotion between periodontitis and type 2 diabetes mellitus (T2DM). However, the association between diabetes status and oral microbiota alterations and whether these patterns explained increased periodontitis severity remains unclearly illuminated.Objective In this study, an investigation was conducted into the association of T2DM with periodontitis severity from the perspective of the oral microbiome.Methods This cross-sectional study enrolled cohorts of patients with and without T2DM presenting periodontitis. Combined with bioinformatics and statistical analyses, 16S rRNA gene sequencing was utilized for characterizing the oral microbiome across four oral niches in patients with different T2DM statuses and periodontitis severity.Results Oral microbiome composition was dysregulated in the context of periodontitis with or without T2DM. The variation pattern of the oral microbiome showed obvious differences. Capnocytophaga sputigena, Fusobacterium hwasookii, and Capnocytophaga gingivalis demonstrated a significant down-regulation exclusively in T2DM subjects. Compared with non-diabetic (ND) subjects, T2DM subjects exhibited markedly altered correlation patterns between Filifactor alocis, Fusobacterium nucleatum, and other periodontitis-associated differential microbes and clinical parameters. Solobacterium moorei, Catonella morbi, and several additional taxa were potential biomarkers of periodontitis severity in T2DM subjects. In addition, T2DM altered microbial interaction between plaque (Pla) and gingival crevicular fluid (GCF) communities, which may form an oral microbial environment facilitating periodontitis severity.Conclusion T2DM greatly reshapes periodontitis-associated oral microbial dysbiosis patterns, which additionally display T2DM-specific microbial traits. This highlights the unique regulatory role and significant impact of T2DM on oral microbiome alterations in periodontitis.

Abstract Periodontitis development is strongly associated with the succession of the oral microbiome. However, the knowledge about the succession of the oral microbiome in the development of periodontitis remains insufficient. In the present study, an analysis was conducted on the succession of tongue back, the saliva (Sal) microbiome, and gingival crevicular fluid (GCF) from healthy individuals and patients with mild (CPL), moderate (CPM), severe chronic (CPH), and generalized aggressive periodontitis (GAgP). The composition and structure of the oral microbiome gradually changed with the increasing severity of periodontitis, among which GCF showed the highest correlation with periodontitis. With an ecological preference, pathogens in the mouth varied with the development of periodontitis. In healthy and CPL patients, Sal‐derived microorganisms accounted for a large proportion of GCF but exhibited a decrease in patients with CPM, CPH, and GAgP. Permutation and time course sequencing analysis revealed that a variety of microorganisms changed with the severity of periodontitis. A majority of these microorganisms are closely related to clinical periodontal indices. Ecological analysis suggested that the composition of oral microbial communities at different stages of periodontitis is controlled by random processes. The comparison of microbial interaction networks demonstrated that a series of key microorganisms drive oral health to severe periodontitis. In this study, the relationship between the succession process of the oral microbiota and the development of periodontitis was revealed.

Cancer-associated fibroblasts (CAFs) are a heterogeneous cell population that plays a crucial role in remodeling the tumor microenvironment (TME). Here, through the integrated analysis of spatial and single-cell transcriptomics data across six common cancer types, we identified four distinct functional subgroups of CAFs and described their spatial distribution characteristics. Additionally, the analysis of single-cell RNA sequencing (scRNA-seq) data from three additional common cancer types and two newly generated scRNA-seq datasets of rare cancer types, namely epithelial-myoepithelial carcinoma (EMC) and mucoepidermoid carcinoma (MEC), expanded our understanding of CAF heterogeneity. Cell-cell interaction analysis conducted within the spatial context highlighted the pivotal roles of matrix CAFs (mCAFs) in tumor angiogenesis and inflammatory CAFs (iCAFs) in shaping the immunosuppressive microenvironment. In patients with breast cancer (BRCA) undergoing anti-PD-1 immunotherapy, iCAFs demonstrated heightened capacity in facilitating cancer cell proliferation, promoting epithelial-mesenchymal transition (EMT), and contributing to the establishment of an immunosuppressive microenvironment. Furthermore, a scoring system based on iCAFs showed a significant correlation with immune therapy response in melanoma patients. Lastly, we provided a web interface ( https://chenxisd.shinyapps.io/pancaf/ ) for the research community to investigate CAFs in the context of pan-cancer.

Background Anti-angiogenic therapy has been shown to be a promising strategy for anti-tumor treatment. Increasing evidence indicates that tumor angiogenesis is affected by exosomes that are secreted by mesenchymal stem cells (MSCs), but whether exosomes derived from MSCs suppress or promote angiogenesis remain paradoxical. The purpose of this study focused on understanding the potential role of exosomes derived from stem cells of human deciduous exfoliated teeth (SHED-Exos) in regulating angiogenesis and the underlying molecular mechanism. Methods Exosomes were isolated from supernatants of SHED cells using an exosome purification kit and were characterized by transmission electron microscopy, nanoparticle tracking analysis and western blot analysis. Cell Counting Kit-8, flow cytometric assays, western blots, wound healing and transwell migration assays were performed to characterize the roles of SHED-Exos on cell proliferation, apoptosis and migration of human umbilical vein endothelial cells (HUVECs). The anti-angiogenic activity of SHED-Exos was assessed via a tube formation assay of endothelial cells and angiogenesis-related factors were analyzed by western blotting. In vivo, we used the chick chorioallantoic membrane (CAM) assay and an oral squamous cell carcinoma (OSCC) xenograft transplantation model with nude mice that received multi-point injections at three-day intervals to evaluate the effects on angiogenesis. Furthermore, the sequencing of microRNAs (miRNAs) in SHED-Exos was performed to investigate the underlying anti-angiogenic mechanism. Results The results showed that SHED-Exos inhibit cell proliferation and migration and induce apoptosis in HUVECs. SHED-Exos suppress the tube-like structure formation of HUVECs in vitro. SHED-Exos downregulate several angiogenesis-related factors, including VEGFA, MMP-9 and ANGPT1. In vivo, the chick CAM assay verified that treatment with SHED-Exos inhibits micro-vascular formation, and importantly, significantly reduces the micro-vascular formation of tumors generated from xenografted OSCC cells, which was associated with the inhibition of tumor growth in vivo. Mechanistically, our data suggested that SHED-Exos are enriched with miR-100-5p and miR-1246 and are transferred to endothelial cells, which results in decreased tube formation via the down-regulation of VEGFA expression. Conclusions These results demonstrate that SHED-Exos inhibit angiogenesis in vitro and in vivo, which suggests that SHED-Exos could potentially serve as a novel and effective therapeutic approach for anti-angiogenic treatment.

Physiological root resorption of deciduous teeth is a normal phenomenon. How the angiogenesis process is regulated to provide adequate levels of oxygen and nutrients in hypoxic conditions when the dental pulp tissue is reduced at the stage of root resorption is not fully understood. In this study, we designed hypoxic preconditioning (2%) to mimic the physiological conditions. We isolated exosomes from hypoxic-preconditioned SHED (Hypo-exos) cells and from normally cultured SHED cells (Norm-exos). We found that treatment with Hypo-exos significantly enhanced the growth, migration and tube formation of endothelial cells in vitro compared with Norm-exos. We also performed matrigel plug assays in vivo and higher expression of VEGF and higher number of lumenal structures that stained positive for CD31 were found in the Hypo-exos treated group. To understand the potential molecular mechanism responsible for the positive effects of Hypo-exos, we performed exosomal miRNA sequencing and validated that Hypo-exos transferred both let-7f-5p and miR-210-3p to promote the tube formation of endothelial cells. Further study revealed that those two miRNAs regulate angiogenesis via the let-7f-5p/AGO1/VEGF and/or miR-210-3p/ephrinA3 signal pathways. Finally, we found that the increased release of exosomes regulated by hypoxia treatment may be related to Rab27a. Taking these data together, the present study demonstrates that exosomes derived from hypoxic-preconditioned SHED cells promote angiogenesis by transferring let-7f-5p and miR-210-3p, which suggests that they can potentially be developed as a novel therapeutic approach for pro-angiogenic therapy in tissue regeneration engineering.

Mesenchymal stem cells (MSCs) within the periodontal ligament (PDL), termed periodontal ligament stem cells (PDLSCs), have a self-renewing capability and a multidirectional differentiation potential. The molecular mechanisms that regulate multidirectional differentiation, such as the osteogenic differentiation of PDLSCs, remain to be elucidated. Cullin 4B (CUL4B), which assembles the CUL4B-RING ubiquitin ligase (CRL4B) complex, is involved in regulating a variety of developmental and physiological processes including the skeletal development and stemness of cancer stem cells. However, nothing is known about the possible role of CUL4B in the osteogenic differentiation of PDLSCs. Here, we found that knockdown of CUL4B decreased the proliferation, migration, stemness and osteogenic differentiation ability of PDLSCs. Mechanistically, we demonstrate that CUL4B cooperates with the PRC2 complex to repress the expression of miR-320c and miR-372/373-3p, which results in the upregulation of RUNX2, a master transcription factor (TF) that regulates osteogenic differentiation. In brief, the present study reveals the role of CUL4B as a new regulator of osteogenic differentiation in PDLSCs.

Exosomes secreted by cancer cells are important components in the tumor microenvironment, enabling cancer cells to communicate with each other and with noncancerous cells to play important roles in tumor progression and metastasis. Phenformin, a biguanide antidiabetic drug, has been reported to have a strong antitumor function in multiple types of cancer cells, however little research has been reported about whether phenformin can regulate the secretion of exosomes by cancer cells to regulate the tumor microenvironment and contribute to its antitumor function. Here we found that exosomes (Phen-Exo) derived from phenformin-treated oral squamous cell carcinoma (OSCC) cells significantly suppress the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. The inhibition of angiogenesis by Phen-Exo was verified in vivo by matrigel plug angiogenesis assays and by chick chorioallantoic membrane assays. Mechanistically, we discovered that the expression of microRNA-1246 (miR-1246) and microRNA-205 (miR-205) was significantly increased in exosomes secreted by OSCC cells treated with phenformin, while high expression levels of miR-1246 or miR-205 in vascular endothelial cells inhibited their angiogenic effects and decreased expression of the angiogenic factor VEGFA. In conclusion, these results reveal that phenformin can inhibit angiogenesis by regulating the levels of miR-1246 and miR-205 in exosomes secreted by OSCC cells, suggesting that phenformin has the potential to alter the tumor microenvironment to antagonize the growth of OSCCs, which provides a theoretical basis for developing new strategies to treat OSCCs in the future.

Fusobacterium nucleatum (F. nucleatum) is an oral anaerobe that plays a role in several oral diseases. However, F. nucleatum is also found in other tissues of the digestive tract, and several studies have recently reported that the level of F. nucleatum is significantly elevated in malignant tumors of the digestive tract. F. nucleatum is proposed as one of the risk factors in the initiation and progression of digestive tract malignant tumors. In this review, we summarize recent reports on F. nucleatum and its role in digestive tract cancers and evaluate the mechanisms underlying the action of F. nucleatum in digestive tract cancers.

Activating transcription factor 3 (ATF3) is a key transcription factor involved in regulating cellular stress responses, with different expression levels and functions in different tissues. ATF3 has also been shown to play crucial roles in regulating tumor development and progression, however its potential role in oral squamous cell carcinomas has not been fully explored. In this study, we examined biopsies of tongue squamous cell carcinomas (TSCCs) and found that the nuclear expression level of ATF3 correlated negatively with the differentiation status of TSCCs, which was validated by analysis of the ATGC database. By using gain- or loss- of function analyses of ATF3 in four different TSCC cell lines, we demonstrated that ATF3 negatively regulates the growth and migration of human TSCC cells in vitro. RNA-seq analysis identified two new downstream targets of ATF3, interferon alpha inducible proteins 6 (IFI6) and 27 (IFI27), which were upregulated in ATF3-deleted cells and were downregulated in ATF3-overexpressing cells. Chromatin immunoprecipitation assays showed that ATF3 binds the promoter regions of the IFI6 and IFI27 genes. Both IFI6 and IFI27 were highly expressed in TSCC biopsies and knockdown of either IFI6 or IFI27 in TSCC cells blocked the cell growth and migration induced by the deletion of ATF3. Conversely, overexpression of either IFI6 or IFI27 counteracted the inhibition of TSCC cell growth and migration induced by the overexpression of ATF3. Finally, an in vivo study in mice confirmed those in vitro findings. Our study suggests that ATF3 plays an anti-tumor function in TSCCs through the negative regulation of its downstream targets, IFI6 and IFI27.

Abstract Objective: SOX4, a transcription factor, has been found to contribute to tumorigenesis in several cancers. This study was performed to determine whether SOX4 mediates BRAF inhibitor resistance in melanoma. Methods: Melanoma cell lines with acquired resistance to BRAF inhibitor (SK-MEL-5R, SK-MEL-28R, and A375R) were generated by adding escalating concentrations of PLX4032 into parental SK-MEL-5, SK-MEL-28, and A375 cells for >6 months. The expression of SOX4 and insulin-like growth factor 1 receptor (IGF-1R) was measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The downstream signaling of IGF-1R was detected by Western blotting. SOX4 and IGF-1R overexpression or knockdown was conducted by lentivirus transfection. Cell viability and apoptosis were demonstrated by MTT and flow cytometry, respectively. The binding ability of SOX4 to IGF-1R promoter was determined by chromatin immunoprecipitation quantitative PCR assay. Results: SOX4 was upregulated in BRAF inhibitor-resistant melanoma cells as compared with parental cells (SK-MEL-5 group, 1.02 vs. 6.33; SK-MEL-28 group, 1.03 vs. 3.22; A375 group, 1.00 vs. 1.86; t =°7.069, 29.26, and 5.291, respectively; all P < 0.01), and PLX4032 treatment could not alter the expression of SOX4 in resistant cells. SOX4 overexpression attenuated the response of parental cells to PLX4032 (for cell viability, SK-MEL-5 group: 77.76% vs. 104.28%, F = 91.50; SK-MEL-28 group: 60.59% vs. 93.13%, F = 171.8; A375 group: 62.50% vs. 80.87%, F = 47.15. For apoptosis rates, SK-MEL-5 group: 34.90% vs. 14.31%, F = 4.781; SK-MEL-28 group, 40.8% vs. 29.4%, F = 13.32, P = 0.063; A375 group: 40.20% vs. 17.09%, F = 11.39; all P < 0.05, otherwise indicated). While SOX4 knockdown enhanced the response of resistant cells to PLX4032 (for cell viability, SK-MEL-5R group: 93.75% vs. 69.53%, F = 94.45, SK-MEL-28R group: 95.60% vs. 66.79%, F = 30.41, A375R group: 95.51% vs. 59.98%, F = 111.6; for apoptosis rates, SK-MEL-5R group: 16.2% vs. 44.4%, F = 25.67, SK-MEL-28R group: 26.59% vs. 44.20%, F = 158.0, A375R group: 5.98% vs. 31.51%, F = 14.35, and all P < 0.01). Chromatin immunoprecipitation quantitative PCR assay demonstrated that SOX4 binded to the promoter of IGF-1R (1.04 vs. 1.94 [−1044 to −920 bp] and 0.110 vs. 0.139 [GAPDH], F = 534.5, P < 0.01). In addition, SOX4 overexpression increased IGF-1R and its downstream phosphorylated ERK, phosphorylated AKT, and phosphorylated STAT3 expression, while SOX4 knockdown exerted the opposite effects. Moreover, IGF-1R knockdown overcame SOX4 overexpression-induced PLX4032 resistance (cell viability: 35.85% vs. 52.79% vs. 37.84% [A375 group, negative control group vs. SOX4 overexpressing group vs. SOX4 overexpressing + sh-IGF-1R group]; apoptosis rates: 25.30% vs. 9.56% vs. 22.26 [A375 group, negative control group vs. SOX4 overexpressing group vs. SOX4 overexpressing + sh-IGF-1R group]; F = 13.01 and 41.87, respectively; all P < 0.01), while IGF-1R overexpression abrogated SOX4 knockdown-induced response enhancement to PLX4032 for comparison of negative control group, sh-SOX4 group and sh-SOX4 + IGF-1R overexpressing group (cell viability: 96.62% vs. 86.86% vs. 97.26% (A375R), 98.15% vs. 81.63% vs. 98.49% [SK-MEL-5R]; apoptosis rates: 13.81% vs. 32.00% vs. 12.16 [A375R], 29.70% vs. 41.40% vs. 26.10% [SK-MEL-5R]; F = 13.56, 12.86, 38.81, and 39.85, respectively; all P < 0.01). Conclusion: SOX4 mediates BRAF inhibitor resistance in melanoma through regulation of IGF-1R signaling. SOX4 might serve as a potential target for the treatment of BRAF inhibitor-resistant melanoma.

The isolation and culture of primary melanocytes from skin tissues is very important for biological research and has been widely used for clinical applications. Isolating primary melanocytes from skin tissues by the conventional method usually takes about 3 to 4 weeks to passage sufficiently. More importantly, the tissues used are usually newborn foreskins and it is still a challenge to efficiently isolate primary melanocytes from adult tissues. We recently developed a new isolation method for melanocytes that adds Y-27632, a Rho kinase inhibitor, to the initial culture medium for 48 h. Compared with the conventional protocol, this new method dramatically increases the yield of melanocytes and shortens the time required to isolate melanocytes from foreskin tissues. We now describe this new method in more detail using adult epidermis to efficiently culture primary melanocytes. Importantly, we show that melanocytes obtained from adult tissues prepared by this new method can function normally. This new protocol will significantly benefit studies of pigmentation defects and melanomas using primary melanocytes prepared from easily accessed adult skin tissues.

The treatment of melanoma has remained a difficult challenge. Targeting the tumor stroma has recently attracted attention for developing novel strategies for melanoma therapy. Activating transcription factor 3 (ATF3) plays a crucial role in regulating tumorigenesis and development, but whether the expression of ATF3 in human dermal fibroblasts (HDFs) can affect melanoma development hasn't been studied. Our results show that ATF3 expression is downregulated in stromal cells of human melanoma. HDFs expressing high levels of ATF3 suppressed the growth and migration of melanoma cells in association with downregulation of different cytokines including IL-6 in vitro. In vivo, HDFs with high ATF3 expression reduced tumor formation. Adding recombinant IL-6 to melanoma cells reversed those in vitro and in vivo effects, suggesting that ATF3 expression by HDFs regulates melanoma progression through the IL-6/STAT3 pathway. More importantly, HDFs pretreated with cyclosporine A or phenformin to induce ATF3 expression inhibited melanoma cell growth in vitro and in vivo. In summary, our study reveals that ATF3 suppresses human melanoma growth and that inducing the expression of ATF3 in HDFs can inhibit melanoma growth, a new potential melanoma therapeutic approach.

The last two years have seen new tissue-engineered skin substitutes come onto the market and begin to resolve the various roles to which each is best suited. It is becoming evident that some of the very expensive cell-based products have cost-benefit advantage despite their high price and are valuable within the restricted applications for which they are intended. The use of skin substitutes for testing purposes has extended from epidermal keratinocytes to other integumentary epithelia and into preparations containing multiple cell types in which reactions resulting from paracrine interactions can be examined. Challenges remain in the application of gene therapy techniques to skin substitutes, both the control of transgene expression and in the selection of suitable genes to transfect. A coming challenge is the production of tissue-engineered products without the use of animal products other than human cells. A challenge that may be diminishing is the importance of acute rejection of allogeneic tissue-engineered skin substitutes.

Background: BRAF inhibitors have successfully treated melanoma patients harboring BRAF mutation. However, nearly all these patients developed resistance to BRAF inhibitors. The molecular mechanisms of BRAF inhibitor resistance in melanoma are not fully understood.Methods: BRAF inhibitor acquired resistant melanoma cell lines were generated by continuous exposure to increasing concentration of PLX4032. E2F1, IGF-1R and AKT overexpression/knockdown were performed by lentivirus transfection. The binding activity of E2F1 on the promoter of IGF-1R were determined by CHIP and CHIP-qPCR.Findings: E2F1 was highly expressed in BRAF inhibitor resistant melanoma. The increases of E2F1 in resistant melanoma was partially caused by PI3K/AKT activation. Overexpressed E2F1 mediated resistance to BRAF inhibitor in melanoma, while knockdown of E2F1 reversed BRAF inhibitor resistance. E2F1 can bind to the gene promoter of IGF-1R. Then, IGF-1R knockdown completely overcome E2F1 overexpression-induced BRAF inhibitor resistance, while IGF-1R overexpression completely reversed E2F1 knockdown-induced response to BRAF inhibitor. Next, overexpression of IGF-1R markedly upregulated E2F1 in parental cells, while knockdown of IGF-1R notably downregulated E2F1 in resistant cells. In addition, HLM006474, an E2F inhibitor, was proved to potentiate the effects of BRAF inhibitor in resistant melanoma cells. AG1024, an IGF-1R inhibitor, further improved HLM006474-induced response of resistant melanoma cells to BRAF inhibitor.Interpretation: E2F1 and IGF-1R forms a positive feedback loop in melanoma, and this loop mediates BRAF inhibitor resistance. Furthermore, combined E2F1 inhibitor and IGF-1R inhibitor might be a novel strategy to enhance the therapeutic benefits of BRAF inhibitor in resistant melanoma.Funding Statement: This work is supported by grants from the National Natural Science Foundation of China (81673917).Declaration of Interests: The authors stated: "No conflict of interests is needed to declare."Ethics Approval Statement: Not required.

E2F1, a transcription factor, is involved in the regulation of apoptosis, cell cycle, and senescence (Denechaud et al., 2017; Ginsberg, 2002; Stevens and La Thangue, 2004). It plays diverse roles in the tumorigenesis and progression of multiple cancers (Meng and Ghosh, 2014). In addition, E2F1 contributes to chemotherapy and radiotherapy resistance in several types of cancers (Fang et al., 2018; Stoleriu et al., 2014; Yan et al., 2014; Zheng et al., 2009). Cyclin D1/E2F pathway is a key regulator of the critical G1 to S phase transition of the cell cycle (Jia et al., 2006).

Currently, no ideal in vivo skin model, to exactly mimic the native human skin, has been utilized for laboratory and clinical application. Here, we describe a method to in vivo reconstitute a human skin model, so-called hRSK, by using culture-expanded skin cells. We grafted a mixture of dissociated human epidermal and dermal cells onto an excision wound on the back of immunodeficient mouse to generate the hRSK, and the hRSK, containing epidermis, dermis, and subcutis and also appendages such as hair follicles, histologically mirrors in situ human skin.

Primary melanocytes isolated from skin and expanded in culture have been widely used for laboratory research and clinical applications. The conventional method to isolate primary melanocytes from skin usually requires about 3-4 weeks of culture for melanocytes to grow sufficiently to passage. Considering that melanocytes comprise only 3%-7% of epidermal cells in normal human skin, it would be extremely helpful to increase the isolation efficiency and shorten the initial culture time to quickly meet various application needs. Here, we report that adding Y-27632, a Rho kinase inhibitor, into the initial culture medium for 2 days can dramatically increase the yield of melanocytes. We found that Y-27632 can promote keratinocyte attachment and survival in the melanocyte culture system, resulting in not only better recovery, but also increased proliferation of melanocytes by a paracrine signaling pathway. More specifically, Y-27632 significantly induced keratinocyte expression of stem cell factor, which played an important role in enhancing the growth of melanocytes. In summary, Y-27632 could profoundly enhance the yield of primary melanocytes in the initial culture through paracrine effects on keratinocytes.

An increased incidence of skin inflammatory diseases is frequently observed in organtransplanted patients being treated with calcineurin inhibitor-based immunosuppressive agents. The mechanism of increased skin inflammation in this context has however not yet been clarified. Here we report an increased inflammation following inhibition of calcineurin signaling seen in both chemically induced mouse skin tumors and in tumors grafted from H-rasV12 expressing primary human keratinocytes (HKCs). Following UVB or TPA treatment, we specifically found that deletion of the calcineurin gene in mouse keratinocytes (MKCs) resulted in increased inflammation, and this was accompanied by the enhanced production of pro-inflammatory cytokines, such as TNFα, IL-8 and CXCL1. Furthermore, expression of the RNA-binding protein, tristetraprolin (TTP) was down-regulated in response to calcineurin inhibition, wherein TTP was shown to negatively regulate the production of pro-inflammatory cytokines in keratinocytes. The induction of TTP following TPA or UVB treatment was attenuated by calcineurin inhibition in keratinocytes, and correspondingly, disruption of calcineurin signaling down-regulated the amounts of TTP in both clinical and H-rasV12-transformed keratinocyte tumor models. Our results further demonstrated that calcineurin positively controls the stabilization of TTP in keratinocytes through a proteasome-dependent mechanism. Reducing the expression of TTP functionally promoted tumor growth of H-rasV12 expressing HKCs, while stabilizing TTP expression counteracted the tumor-promoting effects of calcineurin inhibition. Collectively these results suggest that calcineurin signaling, acting through TTP protein level stabilization, suppresses keratinocyte tumors by downregulating skin inflammation.

Rho-associated protein kinase (ROCK) plays crucial roles in the proliferation and migration of different types of cells. ROCK inhibitor Y-27632 was previously reported to inhibit melanoma cell growth, and ROCK signaling was suggested to be a therapeutic target for treating melanoma. However, the negative effect of Y-27632 on melanoma cells was mainly seen in studies on murine B16 melanoma cells. Here, we reported that ROCK inhibitor actually promoted human melanoma cell growth and migration in vitro. Y-27632 increased the growth and migration of BRAF-mutated melanoma cells but had a negative effect on wild-type melanoma cells or primary melanocytes. We discovered that Y-27632 enhanced the growth of BRAF-mutated melanoma cells through increased ATK and ERK activity. The in vivo study further confirmed the in vitro finding. These data suggested that the effect of ROCK inhibitor on melanoma cells is cell-context dependent, and the application of ROCK inhibitor in the treatment of melanoma requires further study.