Oral cancer cell to endothelial cell communication via exosomal miR-21/RMND5A pathway.

Required for meiotic nuclear division 5 homolog A (RMND5A), a novel ubiquitin E3 Ligase, has been reported to correlate with poor prognosis of several cancers. However, its role in endothelial cells has not been reported. In this study, overexpression of RMND5A in human umbilical vein endothelial cells (HUVECs) was performed via lentiviral infection, followed by MTT, would healing and tube formation assay as well as signaling analysis. Moreover, crosstalk between HUVECs and oral squamous cell carcinoma (OSCC) cells was investigated by indirect co-culture with condition medium or tumor cell derived exosomes. Our results showed that overexpression of RMND5A reduced the proliferation, migration and tube formation ability of HUVECs by inhibiting the activation of ERK and NF-κB pathway. Interestingly, OSCC cells can inhibit RMND5A expression of endothelial cells via exosomal miR-21. In summary, our present study unveils that OSCC cells can activate endothelial cells via exosomal miR-21/RMND5A pathway to promote angiogenesis, which may provide novel therapeutic targets for the treatment of OSCC.

Overexpression of RAB27A in Oral Squamous Cell Carcinoma Promotes Tumor Migration and Invasion via Modulation of EGFR Membrane Stability.

Oral squamous cell carcinoma (OSCC) is the most prevalent subtype of head and neck tumors, highly prone to lymph node metastasis. This study aims to examine the expression pattern of Ras-related protein Rab-27A (RAB27A) and explore its potential implications in OSCC. The expression of RAB27A was assessed through immunohistochemical analysis utilizing tissue microarrays. In vitro experiments were conducted using RAB27A-knockdown cells to investigate its impact on OSCC tumor cells. Additionally, transcriptome sequencing was performed to elucidate potential underlying mechanisms. RAB27A was significantly overexpressed in OSCC, and particularly in metastatic lymph nodes. It was positively correlated with the clinical progression and poor survival prognosis. Silencing RAB27A notably decreased the proliferation, migration, and invasion abilities of OSCC cells in vitro. A Gene Ontology (GO) enrichment analysis indicated a strong association between RAB27A and the epidermal growth factor receptor (EGFR) signaling pathway. Further investigations revealed that RAB27A regulated the palmitoylation of EGFR via zinc finger DHHC-type containing 13 (ZDHHC13). These findings provide insights into OSCC progression and highlight RAB27A as a potential therapeutic target for combating this aggressive cancer.

Is it safe and effective to extract impacted maxillary tooth adjacent to maxillary sinus via submaxillary sinus membrane space approach?-a randomized controlled trial.

OBJECTIVE: To evaluate the clinical outcomes following extraction of impacted maxillary tooth adjacent to maxillary via submaxillary sinus membrane space approach. MATERIALS AND METHODS: Seventy-two patients were enrolled in our study. The positions of the maxillary impacted tooth were confirmed by cone-beam computed tomography (CBCT). Cases were randomly divided into two groups: the "submaxillary sinus membrane space approach" was applied in the new method (NM) group, and the conventional "avoid maxillary sinus membrane exposure" strategy was executed in the traditional method (TM) group. The clinical and follow-up data were recorded. RESULTS: The duration of the procedure in the TM group was significantly longer than those in the NM group (P < 0.05). Four teeth were accidentally displaced into the maxillary sinus with MSM perforation. The MSM perforation rate was slightly higher in the TM group than in the NM group, however, without significant difference between the two groups (8/36 vs. 3/36, P = 0.19). The maxillary sinus membrane perforation was associated with the displacement of tooth into the maxillary sinus (OR = 16.2, P = 0.026). The root tip exposure of the adjacent tooth was significantly higher in the TM group than in the NM group (10/36 vs. 1/36, P = 0.006). The incidence of reduced pulp vitality of the adjacent tooth was significantly higher in the TM group (10/36 vs. 1/36, P = 0.006), and it was associated with the exposure of the root tip intraoperatively (OR = 456.5, P < 0.001). The incidence of external root resorption was significantly lower in the NM group, and there was no significant association with the root exposure intraoperatively (OR = 3.7, P = 0.47). CONCLUSIONS: Submaxillary sinus membrane space approach is a safe and efficient approach in extraction of impacted maxillary tooth. It is an alternative way for cases which are in close proximity to the maxillary sinus. CLINICAL RELEVANCE: A novel method to extract impacted maxillary tooth adjacent to maxillary sinus.

RUNX1 is required in granulocyte-monocyte progenitors to attenuate inflammatory cytokine production by neutrophils.

The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancy (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 was shown to regulate inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to epigenetically repress two inflammatory signaling pathways in neutrophils: Toll-like receptor 4 (TLR4) and type I interferon (IFN) signaling. RUNX1 loss in GMPs augments neutrophils' inflammatory response to the TLR4 ligand lipopolysaccharide through increased expression of the TLR4 coreceptor CD14. RUNX1 binds Cd14 and other genes encoding proteins in the TLR4 and type I IFN signaling pathways whose chromatin accessibility increases when RUNX1 is deleted. Transcription factor footprints for the effectors of type I IFN signaling-the signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRFs)-were enriched in chromatin that gained accessibility in both GMPs and neutrophils when RUNX1 was lost. STAT1::STAT2 and IRF motifs were also enriched in the chromatin of retrotransposons that were derepressed in RUNX1-deficient GMPs and neutrophils. We conclude that a major direct effect of RUNX1 loss in GMPs is the derepression of type I IFN and TLR4 signaling, resulting in a state of fixed maladaptive innate immunity.

RAB27B controls palmitoylation-dependent NRAS trafficking and signaling in myeloid leukemia.

RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation. Our proteomic studies revealed RAB27B upregulation in CBL- or JAK2-mutated myeloid malignancies, and its expression correlated with poor prognosis in acute myeloid leukemias (AMLs). RAB27B depletion inhibited the growth of CBL-deficient or NRAS-mutant cell lines. Strikingly, Rab27b deficiency in mice abrogated mutant but not WT NRAS-mediated progenitor cell growth, ERK signaling, and NRAS palmitoylation. Further, Rab27b deficiency significantly reduced myelomonocytic leukemia development in vivo. Mechanistically, RAB27B interacted with ZDHHC9, a palmitoyl acyltransferase that modifies NRAS. By regulating palmitoylation, RAB27B controlled c-RAF/MEK/ERK signaling and affected leukemia development. Importantly, RAB27B depletion in primary human AMLs inhibited oncogenic NRAS signaling and leukemic growth. We further revealed a significant correlation between RAB27B expression and sensitivity to MEK inhibitors in AMLs. Thus, our studies presented a link between RAB proteins and fundamental aspects of RAS posttranslational modification and trafficking, highlighting future therapeutic strategies for RAS-driven cancers.

RUNX1 is required in granulocyte-monocyte progenitors to attenuate inflammatory cytokine production by neutrophils.

The transcription factor RUNX1 is mutated in familial platelet disorder with associated myeloid malignancies (FPDMM) and in sporadic myelodysplastic syndrome and leukemia. RUNX1 regulates inflammation in multiple cell types. Here we show that RUNX1 is required in granulocyte-monocyte progenitors (GMPs) to restrict the inflammatory response of neutrophils to toll-like receptor 4 (TLR4) signaling. Loss of RUNX1 in GMPs increased the TLR4 coreceptor CD14 on neutrophils, which contributed to neutrophils’ increased inflammatory cytokine production in response to the TLR4 ligand lipopolysaccharide. RUNX1 loss increased the chromatin accessibility of retrotransposons in GMPs and neutrophils and induced a type I interferon signature characterized by enriched footprints for signal transducer and activator of transcription (STAT1::STAT2) and interferon regulatory factors (IRF) in opened chromatin, and increased expression of interferon-stimulated genes. The overproduction of inflammatory cytokines by neutrophils was reversed by inhibitors of type I IFN signaling. We conclude that RUNX1 restrains the chromatin accessibility of retrotransposons in GMPs and neutrophils, and that loss of RUNX1 increases proinflammatory cytokine production by elevating tonic type I interferon signaling.

Depalmitoylation rewires FLT3-ITD signaling and exacerbates leukemia progression.

Internal tandem duplication within FLT3 (FLT3-ITD) is one of the most frequent mutations in acute myeloid leukemia (AML) and correlates with a poor prognosis. Whereas the FLT3 receptor tyrosine kinase is activated at the plasma membrane to transduce PI3K/AKT and RAS/MAPK signaling, FLT3-ITD resides in the endoplasmic reticulum and triggers constitutive STAT5 phosphorylation. Mechanisms underlying this aberrant FLT3-ITD subcellular localization or its impact on leukemogenesis remain poorly established. In this study, we discovered that FLT3-ITD is S-palmitoylated by the palmitoyl acyltransferase ZDHHC6. Disruption of palmitoylation redirected FLT3-ITD to the plasma membrane and rewired its downstream signaling by activating AKT and extracellular signal-regulated kinase pathways in addition to STAT5. Consequently, abrogation of palmitoylation increased FLT3-ITD-mediated progression of leukemia in xenotransplant-recipient mouse models. We further demonstrate that FLT3 proteins were palmitoylated in primary human AML cells. ZDHHC6-mediated palmitoylation restrained FLT3-ITD surface expression, signaling, and colonogenic growth of primary FLT3-ITD+ AML. More important, pharmacological inhibition of FLT3-ITD depalmitoylation synergized with the US Food and Drug Administration-approved FLT3 kinase inhibitor gilteritinib in abrogating the growth of primary FLT3-ITD+ AML cells. These findings provide novel insights into lipid-dependent compartmentalization of FLT3-ITD signaling in AML and suggest targeting depalmitoylation as a new therapeutic strategy to treat FLT3-ITD+ leukemias.

Surviving Inhibition Induces Cell Cycle Arrest and Disrupts Multipotency in Haemangioma Stem Cells.

OBJECTIVE: To explore the potential therapies for infantile haemangiomas by targeting survivin, a member of the inhibitor of apoptosis protein family, using its specific small molecule inhibitor YM155. METHODS: The expression of survivin in human haemangioma tissue was explored using immunohistochemistry and immunohistofluorescence. Cell cycle analysis and EdU assays were used to measure cell proliferation. Heochst33342 and Annexin V/PI double staining were performed to measure cell apoptosis. The capacity for self-renewal and multilineage differentiation potential of haemangioma stem cells (HemSCs) were measured by clone formation assays and multiple differentiation assays. Murine haemangioma models were established to explore the therapeutic efficacy of YM155 in vivo. RESULTS: Strong staining of survivin in stromal cells was observed in the proliferative haemangioma tissue. In vitro studies demonstrated that YM155 induced cell cycle arrest and proliferation suppression of HemSCs, and also caused cell apoptosis at a higher concentration. YM155 impaired the self-renewal capacities and damaged multiple differentiation potentials of HemSCs. Importantly, YM155 suppressed blood vessel formation and cell proliferation, and induced cell apoptosis in murine haemangioma models. CONCLUSION: The present study demonstrated that targeting survivin using its specific suppressant, YM155, prevented the progression of infantile haemangioma by suppressing cell proliferation, inducing cell apoptosis and disrupting the differentiation potential of HemSCs. These results indicate a novel and promising therapeutic approach for the treatment of infantile haemangioma.

HectD1 controls hematopoietic stem cell regeneration by coordinating ribosome assembly and protein synthesis.

Impaired ribosome function is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. However, how ribosomes are regulated remains poorly understood, as are approaches to restore hematopoietic stem cell (HSC) function loss because of defective ribosome biogenesis. Here we reveal a role of the E3 ubiquitin ligase HectD1 in regulating HSC function via ribosome assembly and protein translation. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and ex vivo maintenance concomitant with reduced protein synthesis and growth rate under stress conditions. Mechanistically, HectD1 ubiquitinates and degrades ZNF622, an assembly factor for the ribosomal 60S subunit. Hectd1 loss leads to accumulation of ZNF622 and the anti-association factor eIF6 on 60S, resulting in 60S/40S joining defects. Importantly, Znf622 depletion in Hectd1-deficient HSCs restored ribosomal subunit joining, protein synthesis, and HSC reconstitution capacity. These findings highlight the importance of ubiquitin-coordinated ribosome assembly in HSC regeneration.

Bleomycin: A novel osteogenesis inhibitor of dental follicle cells via a TGF-ß1/SMAD7/RUNX2 pathway.

BACKGROUND AND PURPOSE: Tooth eruption is a complicated process regulated by the dental follicles (DF). Our recent study discovered that tooth eruption was inhibited upon injection of bleomycin into DF. However, the mechanisms were unknown. EXPERIMENTAL APPROACH: Human dental follicle cells (hDFCs) were treated by bleomycin or exogenous TGF-ß1 or transfected by plasmids loading SMAD7 or shRNA targeting SMAD7, followed by osteogenesis induction assay and signalling analysis. Human fresh DF tissues and Wistar rats were used to further confirm bleomycin function. KEY RESULTS: Bleomycin decreased expression of RUNX2 and osteogenic genes in hDFCs, reducing osteogenic capacity. TGF-ß1 expression was up-regulated in bleomycin-treated hDFCs. The effects of exogenous TGF-ß1 were similar to those of bleomycin in hDFCs. Additionally, compared to SMAD2/3, SMAD7 expression increased more in bleomycin- or TGF-ß1-treated hDFCs. Overexpression of SMAD7 likewise significantly decreased RUNX2 expression and osteogenic capacity of hDFCs. Knockdown of SMAD7 markedly attenuated the inhibitory effects of bleomycin and TGF-ß1 on osteogenic capacity and RUNX2 expression of hDFCs. Most importantly, changes in TGF-ß1, SMAD7, and RUNX2 expressions were similar in the DF of rats and humans treated with bleomycin. CONCLUSION AND IMPLICATIONS: SMAD7 was a negative regulator of osteogenic differentiation in DFCs through suppressing RUNX2 expression. Bleomycin or TGF-ß1 inhibited osteogenic differentiation of DFCs via a TGF-ß1/SMAD7/RUNX2 pathway. Our findings might be beneficial for enhancing the osteogenic activity of DFCs or inhibiting the eruption of undesirable teeth.

Effects of bleomycin on tooth eruption: a novel potential application.

There are many kinds of potentially undesirable teeth. At present, surgical extraction is the most efficient way to eliminate these teeth, but it's very complex and invasive. In this study, we investigated the effects of bleomycin (BLM) on dental follicle and tooth eruption as a potential conservative therapy for undesirable teeth. Our data showed that local injection of 0.2 U/kg BLM had no significant effects on tooth eruption compared to the control group in Wistar rats. With higher dose of BLM (0.5 or 2 U/kg), the eruption of treated teeth was interrupted and their root formation failed until 4 weeks postnatal without significant systemic toxicity. Additionally, those effects were not depending on the toxicity of overdose evidenced by TUNEL assay. In summary, injecting BLM into dental follicle at an early stage could interrupt tooth development and eruption, and may prevent the potentially clinical problems resulting from undesirable teeth instead of surgical removal.

Magnetic Chip Based Extracorporeal Circulation: A New Tool for Circulating Tumor Cell in Vivo Detection.

In vivo detection of circulating tumor cells (CTCs) which inspect all of the circulating blood in body seems to have more advantages on cell capture, especially in earlier cancer diagnosis. Herein, based on in vivo microfluidic chip detection system (IV-chip-system), an extracorporeal circulation was constructed to effectively detect and monitor CTCs in vivo. Combined with microfluidic chip and immunomagnetic nanosphere (IMN), this system not only acts as a window for CTC monitoring but also serves as a collector for further cancer diagnosis and research on CTCs. Compared with the current in vivo detection method, this system can capture and detect CTCs in the bloodstream without any pretreatments, and it also has a higher CTC capture efficiency. It is worth mentioning that this system is stable and biocompatible without any irreversible damage to living animals. Taking use of this system, the mimicked CTC cleanup process in the blood vessel is monitored, which may open new insights in cancer research and early cancer diagnosis.

Increased salivary microvesicles are associated with the prognosis of patients with oral squamous cell carcinoma.

Microvesicles (MVs), which are cell-derived membrane vesicles present in body fluids, are closely associated with the development of malignant tumours. Saliva, one of the most versatile body fluids, is an important source of MVs. However, the association between salivary MVs (SMVs) and oral squamous cell carcinoma (OSCC), which is directly immersed in the salivary milieu, remains unclear. SMVs from 65 patients with OSCC, 21 patients with oral ulcer (OU), and 42 healthy donors were purified, quantified and analysed for their correlations with the clinicopathologic features and prognosis of OSCC patients. The results showed that the level of SMVs was significantly elevated in patients with OSCC compared to healthy donors and OU patients. Meanwhile, the level of SMVs showed close correlations with the lymph node status, and the clinical stage of OSCC patients. Additionally, the ratio of apoptotic to non-apoptotic SMVs was significantly decreased in OSCC patients with higher pathological grade. Consistently, poorer overall survival was observed in patients with lower ratio of apoptotic to non-apoptotic SMVs. In conclusion, the elevated level of SMVs is associated with clinicopathologic features and decreased survival in patients with OSCC, suggesting that SMVs are a potential biomarker and/or regulator of the malignant progression of OSCC.

Lymphotoxin-α promotes tumor angiogenesis in HNSCC by modulating glycolysis in a PFKFB3-dependent manner.

Tumor angiogenesis is critical for tumor progression as the new blood vessels supply nutrients and facilitate metastasis. Previous studies indicate tumor associated lymphocytes, including B cells and T cells, contribute to tumor angiogenesis and tumor progression. The present study aims to identify the function of Lymphotoxin-α (LT-α), which is secreted by the activated lymphocytes, in the tumor angiogenesis of head and neck squamous cell carcinoma (HNSCC). The coculture system between HNSCC cell line Cal27 and primary lymphocytes revealed that tumor cells promoted the LT-α secretion in the cocultured lymphocytes. In vitro data further demonstrated that LT-α promoted the proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) by enhancing the PFKFB3-mediated glycolytic flux. Genetic and pharmacological inhibition of PFKFB3 suppressed the enhanced proliferation and migration of HUVECs. We further identified that LT-α induced PFKFB3 expression was dependent on the TNFR/NF-κB signaling pathway. In addition, we proved that PFKFB3 blockade decreased the density of CD31 positive blood vessels in HNSCC xenografts. Finally, the results from the human HNSCC tissue array revealed that the expression of LT-α in HNSCC samples positively correlated with microvessel density, lymphocytes infiltration and endothelial PFKFB3 expression. In conclusion, infiltrated lymphocyte secreted LT-α enhances the glycolysis of ECs in a PFKFB3-dependent manner through the classical NF-κB pathway and promotes the proliferation and migration of ECs, which may contribute to the aberrant angiogenesis in HNSCCs. Our study suggests that PFKFB3 blockade is a promising therapeutic approach for HNSCCs by targeting tumor angiogenesis.

Lnk/Sh2b3 deficiency restores hematopoietic stem cell function and genome integrity in Fancd2 deficient Fanconi anemia.

Fanconi anemia (FA) is a bone marrow failure (BMF) syndrome that arises from mutations in a network of FA genes essential for DNA interstrand crosslink (ICL) repair and replication stress tolerance. While allogeneic stem cell transplantation can replace defective HSCs, interventions to mitigate HSC defects in FA do not exist. Remarkably, we reveal here that Lnk (Sh2b3) deficiency restores HSC function in Fancd2-/- mice. Lnk deficiency does not impact ICL repair, but instead stabilizes stalled replication forks in a manner, in part, dependent upon alleviating blocks to cytokine-mediated JAK2 signaling. Lnk deficiency restores proliferation and survival of Fancd2-/- HSCs, while reducing replication stress and genomic instability. Furthermore, deletion of LNK in human FA-like HSCs promotes clonogenic growth. These findings highlight a new role for cytokine/JAK signaling in promoting replication fork stability, illuminate replication stress as a major underlying origin of BMF in FA, and have strong therapeutic implications.

Increased level of cell-derived microparticles in the cyst fluids of odontogenic keratocysts.

The aim of this study was to examine the level and basic characteristics of cell­derived microparticles (MPs) in the cyst fluids of odontogenic keratocysts (OKCs). For this purpose, MPs from the cyst fluids (CFMPs) of OKCs were purified by a classic differential centrifugation method and characterized by a transmission electron microscope and fluorescence microscope. Flow cytometric analysis was used to determine the size, concentration and cellular origins of the CFMPs. Moreover, the expression level of receptor activator for nuclear factor­κB ligand in the OKCs was evaluated by immunohistochemical staining and then analyzed for its correlation with the concentration of CFMPs by Spearman's rank correlation test. In addition, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and tartaric­resistant acid phosphatase (TRAP) staining were performed to examine the osteoclastogenesis of mouse bone marrow­derived macrophages (BMMs) in response to CFMPs. The results revealed that the levels of total CFMPs were significantly elevated in OKCs compared with dentigerous cysts (DCs) and radicular cysts (RCs). In addition, in vitro experiments further revealed that CFMPs derived from the OKCs of patients could be taken up by BMMs, leading to a significant increase in the mRNA expression levels of nuclear factor of activated T­cells 1 (NFATc1) and TRAP. Moreover, TRAP­positive multinucleated osteoclasts were successfully cultured in the presence of macrophage colony­stimulating factor (M­CSF) and CFMPs with BMMs. On the whole, our findings indicate that patients with OKCs have higher levels of CFMPs compared with patients with DCs and RCs, which may be associated with the bone resorption of OKCs.

Lymphotoxins Promote the Progression of Human Lymphatic Malformation by Enhancing Lymphatic Endothelial Cell Proliferation.

Formation of inflammation-related tertiary lymphoid organs promotes human lymphatic malformation (LM) development. However, the role of lymphotoxins (LTs) and LT-related inducible ligand, the crucial mediators for tertiary lymphoid organ formation, is undetermined in LMs. Herein, we show that LTs and LT-related inducible ligand promote LM development by enhancing lymphatic endothelial cell (LEC) proliferation via activating NF-κB pathways. The expression of LTs and their receptors was increased in LMs, especially the infected ones, when compared with normal skins. Nuclear translocation of p65, p52, and RelB in the LECs of LMs indicated the activation of classic and alternative NF-κB pathways. Pearson's correlation and cluster analysis suggested the close relationship between LEC proliferation and NF-κB activation. Moreover, in vitro data demonstrated LTs accelerated the proliferation of human dermal LECs (HdLECs) through activation of NF-κB. In addition, lipopolysaccharide (LPS) up-regulated LT receptor expression in HdLECs, leading to increased sensitivity to LTs. Suppression of LT receptors hampered LPS-enhanced HdLEC proliferation, indicating the crucial role of LT pathways in inflammatory lymphangiogenesis. Besides, evidence from the LM rat models demonstrated LTα and LPS enhanced LEC proliferation, therefore promoting LM development. Blocking LT pathways by neutralizing antibodies against LTα and lymphotoxin ß receptor may decelerate the growth of the disease. In summary, our present study demonstrated activation of LT signaling pathways in LECs contributed to the progression of LMs.

Down-regulation of polycystin in lymphatic malformations: possible role in the proliferation of lymphatic endothelial cells.

Lymphatic malformations (LMs) are composed of aberrant lymphatic vessels and regarded as benign growths of the lymphatic system. Recent studies have demonstrated that the mutant embryos of PKD1 and PKD2, encoding polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively, result in aberrant lymphatic vessels similar to those observed in LMs. In this study, for the first time, we investigated PC-1 and PC-2 expression and assessed their roles in the development of LMs. Our results demonstrated that PC-1 and PC-2 gene and protein expressions were obviously decreased in LMs compared with normal skin tissues. In addition, the expression of phosphorylated ERK but not total ERK was up-regulated in LMs and negatively correlated with the expression of PC-1 and PC-2. Moreover, up-regulation of Ki67 was detected in LMs and positively correlated with ERK phosphorylation levels. Furthermore, cluster analysis better reflected close correlation between these signals. All of the above results provided strong evidence suggesting that the hyperactivation of the ERK pathway may be caused by down-regulation of PC-1 and PC-2 in LMs, contributing to increased proliferation of lymphatic endothelial cells in LMs. Our present study sheds light on novel potential mechanisms involved in LMs and may help to explore novel treatments for LMs.

Association of ATF4 Expression With Tissue Hypoxia and M2 Macrophage Infiltration in Infantile Hemangioma.

Accumulating studies have revealed the hypoxic condition and its crucial role in the distinctive progression of infantile hemangioma (IH), the most common benign tumor in infancy. Activating transcription factor 4 (ATF4), an important gene mediating cellular adaptation to various stress signals, could confer a survival advantage for tumor cells under hypoxia and regulate tumor progression. However, the potential role of ATF4 in IH was still unknown. In this study, the expression of hypoxia inducible factor (HIF)-1α, ATF4, and macrophage colony-stimulating factor (M-CSF) in 27 specimens of IH was measured by immunochemistry and double-labeling immunofluorescence, followed by the Spearman rank correlation test. Our results showed that the expression of HIF-1α, ATF4, and M-CSF was significantly upregulated in proliferating IH compared with involuting IH. Meanwhile, HIF-1α and ATF4, in parallel with ATF4 and M-CSF, exhibited positive correlation and synchronous expression. In addition, our in vitro studies demonstrated that hypoxia obviously upregulated the expression of HIF-1α, ATF4, and M-CSF in hemangioma stem cells. Most importantly, their expression was uniformly correlated with the percentage of M2-polarized macrophages in IH. All those results and established evidence indicated that hypoxia-induced ATF4 expression may promote progression of proliferating IH through M-CSF-induced M2-polarized macrophages infiltration.