Physiomimetic Fluidic Culture Platform on Microwell-Patterned Porous Collagen Scaffold for Human Pancreatic Islets.

Pancreatic islet transplantation is one of the clinical options for certain types of diabetes. However, difficulty in maintaining islets prior to transplantation limits the clinical expansion of islet transplantations. Our study introduces a dynamic culture platform developed specifically for primary human islets by mimicking the physiological microenvironment, including tissue fluidics and extracellular matrix support. We engineered the dynamic culture system by incorporating our distinctive microwell-patterned porous collagen scaffolds for loading isolated human islets, enabling vertical medium flow through the scaffolds. The dynamic culture system featured four 12 mm diameter islet culture chambers, each capable of accommodating 500 islet equivalents (IEQ) per chamber. This configuration calculates > five-fold higher seeding density than the conventional islet culture in flasks prior to the clinical transplantations (442 vs 86 IEQ/cm2). We tested our culture platform with three separate batches of human islets isolated from deceased donors for an extended period of 2 weeks, exceeding the limits of conventional culture methods for preserving islet quality. Static cultures served as controls. The computational simulation revealed that the dynamic culture reduced the islet volume exposed to the lethal hypoxia (< 10 mmHg) to ~1/3 of the static culture. Dynamic culture ameliorated the morphological islet degradation in long-term culture and maintained islet viability, with reduced expressions of hypoxia markers. Furthermore, dynamic culture maintained the islet metabolism and insulin-secreting function over static culture in a long-term culture. Collectively, the physiological microenvironment-mimetic culture platform supported the viability and quality of isolated human islets at high-seeding density. Such a platform has a high potential for broad applications in cell therapies and tissue engineering, including extended islet culture prior to clinical islet transplantations and extended culture of stem cell-derived islets for maturation.

Downregulation of SPARC Expression Enhances the Fusion of BeWo Choriocarcinoma Cells.

Syncytiotrophoblasts, which are formed by the fusion of villous cytotrophoblasts, play an essential role in maintaining a successful pregnancy. Secreted protein acidic and rich in cysteine (SPARC) is a non-structural Ca2+-binding extracellular matrix glycoprotein involved in tissue remodeling and cell proliferation, differentiation, and migration. Previous studies have revealed that SPARC is expressed in villous and extravillous cytotrophoblasts in the first trimester and that RNA interference targeted at SPARC significantly inhibited invasion of human extravillous trophoblast HTR8/SVneo cells. However, the involvement of SPARC in cytotrophoblast fusion remains unknown. This study aimed to investigate the role of SPARC in cytotrophoblast fusion, using the BeWo choriocarcinoma cell line as a model of villous cytotrophoblasts. Immunohistochemical analysis was conducted to assess SPARC expression in normal human placentas using placental tissues obtained during the first and third trimesters of pregnancy. We investigated the effects of SPARC knockdown on trophoblast differentiation markers and cell fusion in BeWo cells using small interfering RNA. Immunohistochemical analysis revealed that SPARC expression was high in the early gestational chorionic villi and low in the late gestational chorionic villi. SPARC knockdown increased the expressions of human chorionic gonadotropin and Ovo-like transcriptional repressor 1; however, glial cells missing transcription factor 1, syncytin-1, and syncytin-2 showed no significant changes. The assessment revealed that SPARC knockdown significantly enhanced cell fusion compared to the non-silencing control. Our data suggest that SPARC plays a vital role in regulating trophoblast fusion and differentiation during placental development.

Recent trends and perspectives in reconstruction and regeneration of intra/extra-oral wounds using tissue-engineered oral mucosa equivalents.

Many conditions, including cancer, trauma, and congenital anomalies, can damage the oral mucosa. Multiple cultures of oral mucosal cells have been used for biocompatibility tests and oral biology studies. In recent decades, the clinical translation of tissue-engineered products has progressed significantly in developing tangible therapies and inspiring advancements in medical science. However, the reconstruction of an intraoral mucosa defect remains a significant challenge. Despite the drawbacks of donor-site morbidity and limited tissue supply, the use of autologous oral mucosa remains the gold standard for oral mucosa reconstruction and repair. Tissue engineering offers a promising solution for repairing and reconstructing oral mucosa tissues. Cell- and scaffold-based tissue engineering approaches have been employed to treat various soft tissue defects, suggesting the potential clinical use of tissue-engineered oral mucosa (TEOMs). In this review, we first cover the recent trends in the reconstruction and regeneration of extra-/intra-oral wounds using TEOMs. Next, we describe the current status and challenges of TEOMs. Finally, future strategic approaches and potential technologies to support the advancement of TEOMs for clinical use are discussed.

Predictive Ability of the Combination of White Blood Cell and C Reactive Protein Levels for Infections Following Laparoscopic Hysterectomy.

OBJECTIVE: In the present retrospective study, we evaluated potentially predictive factors and determined receiver operating characteristic (ROC) curve cut-off values for effective detection of patients at higher risk of re-hospitalization due to postoperative infection after total laparoscopic hysterectomy. MATERIALS AND METHODS: We included 168 patients who had undergone laparoscopic hysterectomy. Data were collected from medical records. Patients were classified into non-infection (n = 161) and infection (n = 7) groups based on whether they developed fever after hospital discharge. We evaluated factors conventionally known to affect postoperative infection in general, and values of white blood cell (WBC) and C-reactive protein (CRP) determined on postoperative days 1 and 3-5. RESULTS: There were significant differences in WBC 3-5 days postoperatively (WBC POD3-5) (p = 0.049), CRP 3-5 days postoperatively (CRP POD3-5) (p = 0.018) and CRP POD3-5 × WBC 1 day postoperatively (WBC POD1) (p = 0.002). Area under the ROC curves for CRP POD3-5 and CRP POD3-5 × WBC POD1 were 0.81 and 0.84, and cutoffs were 4.46 mg/dL and 46885.5, respectively. CONCLUSION: If CRP POD3-5 or CRP POD3-5 × WBC POD1 is high, the physician should be alert to postoperative infection, and the patient should be under careful management and supervision.

Development of a lip vermilion epithelium reconstruction model using keratinocytes from skin and oral mucosa.

Lip vermilion is unique and can be distinguished from the adjacent skin and oral mucosa. However, because of the lack of appropriate evaluation tools, skin and/or oral mucosa substitutes such as in vitro vermilion epithelial models have been used for lip product testing. We aimed to develop and characterize a lip vermilion epithelium reconstruction model (LVERM) using skin and oral keratinocytes. LVERM was manufactured by co-culturing primary skin and oral keratinocytes, using a device that allowed the separation of cell seeding, and created an intercalated cell-free zone, referred to as the vermilion part. After removing the device, LVERM construction was completed in 8 days, in a submerged condition. Subsequently, they were placed in an air-liquid interface for 7 days. To determine the epithelial characteristics of LVERM, keratin 2e (KRT2) and small proline-rich protein 3 (SPRR3) expression patterns were examined. The in vivo expression profiles of KRT2 and SPRR3 genes in vermilion were also examined. We found that a continuous multi-layered epithelium was generated in the LVERM that exhibited ortho- and para-keratinization in the skin and oral mucosa parts, respectively. Although an intermediate keratinization pattern was observed in the vermilion part, KRT2 and SPRR3 were co-expressed in the suprabasal layer, consistent with the expression pattern of a single vermilion epithelial model. Clustering analysis revealed that KRT2 and SPRR3 gene expression in vermilion was location-dependent within the sample. Therefore, LVERM can be used as an evaluation tool for lip products and has great importance in innovative approaches for cosmetic testing.

The EGF/EGFR axis and its downstream signaling pathways regulate the motility and proliferation of cultured oral keratinocytes.

We previously reported that the cell and colony motion of oral keratinocytes are correlated with proliferative capacity, and speculated that this may be a specific index for monitoring cell quality. However, how cell motility and proliferation are regulated by signaling pathways remains unelucidated. Here, we found that the regulation of cell motility and proliferative capacity of oral keratinocytes can be attributed to the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) axis. The EGFR downstream cascade involving the Src/PI3K/Akt/mTOR signaling pathway showed a major effect on cell motility and proliferative capacity in oral keratinocytes. Furthermore, both EGFR and Src attenuated E-cadherin expression. Taken together, these findings provide a potential basis for future quality control of cells for therapeutic use.

Development of the Follow-Up Human 3D Oral Cancer Model in Cancer Treatment.

As function preservation cancer therapy, targeted radiation therapies have been developed for the quality of life of cancer patients. However, preclinical animal studies evaluating the safety and efficacy of targeted radiation therapy is challenging from the viewpoints of animal welfare and animal protection, as well as the management of animal in radiation-controlled areas under the regulations. We fabricated the human 3D oral cancer model that considers the time axis of the follow up in cancer treatment. Therefore, in this study, the 3D model with human oral cancer cells and normal oral fibroblasts was treated based on clinical protocol. After cancer treatment, the histological findings of the 3D oral cancer model indicated the clinical correlation between tumor response and surrounding normal tissue. This 3D model has potential as a tool for preclinical studies alternative to animal studies.

Cholesterol Is a Regulator of CAV1 Localization and Cell Migration in Oral Squamous Cell Carcinoma.

Cholesterol plays an important role in cancer progression, as it is utilized in membrane biogenesis and cell signaling. Cholesterol-lowering drugs have exhibited tumor-suppressive effects in oral squamous cell carcinoma (OSCC), suggesting that cholesterol is also essential in OSCC pathogenesis. However, the direct effects of cholesterol on OSCC cells remain unclear. Here, we investigated the role of cholesterol in OSCC with respect to caveolin-1 (CAV1), a cholesterol-binding protein involved in intracellular cholesterol transport. Cholesterol levels in OSCC cell lines were depleted using methyl-ß-cyclodextrin and increased using the methyl-ß-cyclodextrin-cholesterol complex. Functional analysis was performed using timelapse imaging, and CAV1 expression in cholesterol-manipulated cells was investigated using immunofluorescence and immunoblotting assays. CAV1 immunohistochemistry was performed on surgical OSCC samples. We observed that cholesterol addition induced polarized cell morphology, along with CAV1 localization at the trailing edge, and promoted cell migration. Moreover, CAV1 was upregulated in the lipid rafts and formed aggregates in the plasma membrane in cholesterol-added cells. High membranous CAV1 expression in tissue specimens was associated with OSCC recurrence. Therefore, cholesterol promotes the migration of OSCC cells by regulating cell polarity and CAV1 localization to the lipid raft. Furthermore, membranous CAV1 expression is a potential prognostic marker for OSCC patients.

A novel approach for the endothelialization of xenogeneic decellularized vascular tissues by human cells utilizing surface modification and dynamic culture.

Decellularized xenogeneic vascular grafts can be used in revascularization surgeries. We have developed decellularization methods using high hydrostatic pressure (HHP), which preserves the extracellular structure. Here, we attempted ex vivo endothelialization of HHP-decellularized xenogeneic tissues using human endothelial cells (ECs) to prevent clot formation against human blood. Slices of porcine aortic endothelium were decellularized using HHP and coated with gelatin. Human umbilical vein ECs were directly seeded and cultured under dynamic flow or static conditions for 14 days. Dynamic flow cultures tend to demonstrate higher cell coverage. We then coated the tissues with the E8 fragment of human laminin-411 (hL411), which has high affinity for ECs, and found that Dynamic/hL411showed high area coverage, almost reaching 100% (Dynamic/Gelatin vs Dynamic/hL411; 58.7 ± 11.4 vs 97.5 ± 1.9%, P = 0.0017). Immunostaining revealed sufficient endothelial cell coverage as a single cell layer in Dynamic/hL411. A clot formation assay using human whole blood showed low clot formation in Dynamic/hL411, almost similar to that in the negative control, polytetrafluoroethylene. Surface modification of HHP-decellularized xenogeneic endothelial tissues combined with dynamic culture achieved sufficient ex vivo endothelialization along with prevention of clot formation, indicating their potential for clinical use as vascular grafts in the future.

Micropyramid-patterned, oxygen-permeable bottomed dish for high density culture of pancreatic islets.

The need for maintaining cell-spheroid viability and function within high-density cultures is unmet for various clinical and experimental applications, including cell therapies. One immediate application is for transplantation of pancreatic islets, a clinically recognized treatment option to cure type 1 diabetes; islets are isolated from a donor for subsequent culture prior to transplantation. However, high seeding conditions cause unsolicited fusion of multiple spheroids, thereby limiting oxygen diffusion to induce hypoxic cell death. Here we introduce a culture dish incorporating a micropyramid-patterned surface to prevent the unsolicited fusion and oxygen-permeable bottom for optimal oxygen environment. A 400µm-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400µm-base and 200µm-height was fabricated to apply to the 24-well plate format. The micropyramid pattern separated the individual pancreatic islets to prevent the fusion of multiple islets. This platform supported the high oxygen demand of islets at high seeding density at 260 islet equivalents cm-2, a 2-3-fold higher seeding density compared to the conventional islet culture used in a preparation for the clinical islet transplantations, demonstrating improved islet morphology, metabolism and function in a 4 d-culture. Transplantation of these islets into immunodeficient diabetic mice exhibited significantly improved engraftment to achieve euglycemia compared to islets cultured in the conventional culture wells. Collectively, this simple design modification allows for high-density cultures of three-dimensional cell spheroids to improve the viability and function for an array of investigational and clinical replacement tissues.

Adhesion Characteristics of Human Pancreatic Islets, Duct Epithelial Cells, and Acinar Cells to a Polymer Scaffold.

We reported in 2018 that among several extracellular matrices, fibronectin, type I collagen, type IV collagen, laminin I, fibrinogen, and bovine serum albumin, fibronectin is particularly useful for adhesion of porcine pancreatic tissue. Subsequently, we developed a technology that enables the chemical coating of the constituent motifs of fibronectin onto cell culture dishes. In this experiment, we used islets (purity ≥ 90%), duct epithelial cells (purity ≥ 60%), and acinar cells (purity ≥ 99%) isolated from human pancreas according to the Edmonton protocol published in 2000 and achieved adhesion to the constituent motifs of fibronectin. A solution including cGMP Prodo Islet Media was used as the assay solution. In islets, adhesion was enhanced with the constitutive motifs of fibronectin compared with uncoated islets. In the functional evaluation of islets, insulin mRNA expression and insulin secretion were enhanced by the constitutive motif of fibronectin compared with non-coated islets. The stimulation index was comparable between non-coated islets and fibronectin motifs. In duct epithelial cells, adhesion was mildly promoted by the fibronectin component compared with non-coated component, while in acinar cells, adhesion was inhibited by the fibronectin component compared with the non-coated component. These data suggest that the constitutive motifs of fibronectin are useful for the adhesion of islets and duct epithelial cells.

Laminin Isoforms in Human Dental Pulp: Lymphatic Vessels Express Laminin-332, and Schwann Cell-Associated Laminin-211 Modulates CD163 Expression of M2-like Macrophages.

Laminin, a basement membrane heterotrimeric glycoprotein composed of α/ß/γ subunits, has important tissue-specific functions in the control of cellular behavior. Our recent study showed the colocalization of CD163+ M2-like macrophages with Schwann cells in human dental pulp, leading us to hypothesize that the laminin isoform of Schwann cells is associated with CD163 expression. The present study investigated the distribution of laminin isoforms in human dental pulp and the underlying mechanisms that affect macrophage phenotypes. Immunofluorescence analysis indicated that blood vessels were exclusively positive for laminin α4 and α5, whereas laminin α2 was associated with Schwann cells. Unexpectedly, laminin α3/laminin-332 (α3ß3γ2) was detected on lymphatic vessels. In intact and carious teeth, CD163+ cells were associated with laminin α2, whereas CD206 single-positive cells were present inside, outside, and along blood vessels. In vitro incubation of THP-1 macrophages in plates coated with laminin-211/511 or its functionally analogous E8 fragments of α-chain (E8-α) indicated that cell shapes differed between macrophages grown on laminin-211/E8-α2 and macrophages grown on laminin-511/E8-α5. Laminin-211/E8-α2-coated plates upregulated CD163 expression, compared with laminin-511/E8-α5-coated plates. Integrin α3- and integrin α6-neutralizing Abs altered the shape of THP-1 macrophages and upregulated mRNA levels of CD206 and CD163 in macrophages grown on laminin-511; the neutralizing Abs did not affect macrophages grown on laminin-211. These findings suggest that laminin isoforms differentially regulate macrophage behavior via distinct integrin-laminin affinities. Of note, laminin-332 is expressed by pulpal lymphatic vessels, the existence of which has been debated; laminin-211 might have a role in maintaining CD163 expression on macrophages.

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate.

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and α6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFRα. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

Crosstalk between oral squamous cell carcinoma cells and cancer-associated fibroblasts via the TGF-ß/SOX9 axis in cancer progression.

Cancer-associated fibroblasts (CAFs) have important roles in promoting cancer development and progression. We previously reported that high expression of sex-determining region Y (SRY)-box9 (SOX9) in oral squamous cell carcinoma (OSCC) cells was positively correlated with poor prognosis. This study developed three-dimensional (3D) in vitro models co-cultured with OSCC cells and CAFs to examine CAF-mediated cancer migration and invasion in vitro and in vivo. Moreover, we performed an immunohistochemical analysis of alpha-smooth muscle actin and SOX9 expression in surgical specimens from 65 OSCC patients. The results indicated that CAFs promote cancer migration and invasion in migration assays and 3D in vitro models. The invading OSCC cells exhibited significant SOX9 expression and changes in the expression of epithelial-mesenchymal transition (EMT) markers, suggesting that SOX9 promotes EMT. TGF-ß1 signalling inhibition reduced SOX9 expression and cancer invasion in vitro and in vivo, indicating that TGF-ß1-mediated invasion is dependent on SOX9. In surgical specimens, the presence of CAFs was correlated with SOX9 expression in the invasive cancer nests and had a significant impact on regional recurrence. These findings demonstrate that CAFs promote cancer migration and invasion via the TGF-ß/SOX9 axis.

The human vermilion surface contains a rich amount of cholesterol sulfate than the skin.

BACKGROUND: The vermilion of the human lip presents characteristic features and undergoes aging faster than the skin. Therefore, knowledge of the vermilion surface-specific functional molecules is important to understand lip aging and formulate lip care products. Previously, we analyzed the free fatty acids distributions and showed that docosahexaenoic acid highly accumulated in the vermilion's epithelium than in the skin. OBJECTIVE: We aimed to explore the functional molecules other than the free fatty acids on the vermilion's surface. METHODS: Human lip tissues from children and tape-stripped samples from smooth and rough lips of adults were measured by desorption electrospray ionization-mass spectrometry imaging (DESI-MSI). RESULTS: DESI-MSI of children's lip sections revealed a major distribution of five phospholipid species in the viable layer, but not in the superficial area, of both the vermilion and the skin than that in the underlying tissue. Interestingly, a remarkably higher distribution of cholesterol sulfate was observed in the vermilion's superficial area compared to that in the skin in all subjects under this study. Furthermore, DESI-MSI of tape-stripped lip samples showed an overall higher accumulation of cholesterol sulfate in the stratum corneum of the rough lips than that in the smooth lips. CONCLUSION: Our study concluded that cholesterol sulfate has a characteristic distribution to the vermilion's surface and showed an association with the roughness of the lip.

Perivascular Hedgehog responsive cells play a critical role in peripheral nerve regeneration via controlling angiogenesis.

Hh signaling has been shown to be activated in intact and injured peripheral nerve. However, the role of Hh signaling in peripheral nerve is not fully understood. In the present study, we observed that Hh signaling responsive cells [Gli1(+) cells] in both the perineurium and endoneurium. In the endoneurium, Gli1(+) cells were classified as blood vessel associated or non-associated. After injury, Gli1(+) cells around blood vessels mainly proliferated to then accumulate into the injury site along with endothelial cells. Hh signaling activity was retained in Gli1(+) cells during nerve regeneration. To understand the role of Hedgehog signaling in Gli1(+) cells during nerve regeneration, we examined mice with Gli1(+) cells-specific inactivation of Hh signaling (Smo cKO). After injury, Smo cKO mice showed significantly reduced numbers of accumulated Gli1(+) cells along with disorganized vascularization at an early stage of nerve regeneration, which subsequently led to an abnormal extension of the axon. Thus, Hh signaling in Gli1(+) cells appears to be involved in nerve regeneration through controlling new blood vessel formation at an early stage.

Cells/colony motion of oral keratinocytes determined by non-invasive and quantitative measurement using optical flow predicts epithelial regenerative capacity.

Cells/colony motion determined by non-invasive, quantitative measurements using the optical flow (OF) algorithm can indicate the oral keratinocyte proliferative capacity in early-phase primary cultures. This study aimed to determine a threshold for the cells/colony motion index to detect substandard cell populations in a subsequent subculture before manufacturing a tissue-engineered oral mucosa graft and to investigate the correlation with the epithelial regenerative capacity. The distinctive proliferating pattern of first-passage [passage 1 (p1)] cells reveals the motion of p1 cells/colonies, which can be measured in a non-invasive, quantitative manner using OF with fewer full-screen imaging analyses and cell segmentations. Our results demonstrate that the motion index lower than 40 µm/h reflects cellular damages by experimental metabolic challenges although this value shall only apply in case of our culture system. Nonetheless, the motion index can be used as the threshold to determine the quality of cultured cells while it may be affected by any different culture conditions. Because the p1 cells/colony motion index is correlated with epithelial regenerative capacity, it is a reliable index for quality control of oral keratinocytes.

Metabolomic Alteration of Oral Keratinocytes and Fibroblasts in Hypoxia.

The oxygen concentration in normal human tissue under physiologic conditions is lower than the atmospheric oxygen concentration. The more hypoxic condition has been observed in the cells with wound healing and cancer. Somatic stem cells reside in a hypoxic microenvironment in vivo and prefer hypoxic culture conditions in vitro. Oral mucosa contains tissue-specific stem cells, which is an excellent tissue source for regenerative medicine. For clinical usage, maintaining the stem cell in cultured cells is important. We previously reported that hypoxic culture conditions maintained primary oral keratinocytes in an undifferentiated and quiescent state and enhanced their clonogenicity. However, the metabolic mechanism of these cells is unclear. Stem cell biological and pathological findings have shown that metabolic reprogramming is important in hypoxic culture conditions, but there has been no report on oral mucosal keratinocytes and fibroblasts. Herein, we conducted metabolomic analyses of oral mucosal keratinocytes and fibroblasts under hypoxic conditions. Hypoxic oral keratinocytes and fibroblasts showed a drastic change of metabolite concentrations in urea cycle metabolites and polyamine pathways. The changes of metabolic profiles in glycolysis and the pentose phosphate pathway under hypoxic conditions in the oral keratinocytes were consistent with those of other somatic stem cells. The metabolic profiles in oral fibroblasts showed only little changes in any pathway under hypoxia except for a significant increase in the antioxidant 2-oxoglutaric acid. This report firstly provides the holistic changes of various metabolic pathways of hypoxic cultured oral keratinocytes and fibroblasts.

HEATR1, a novel interactor of Pontin/Reptin, stabilizes Pontin/Reptin and promotes cell proliferation of oral squamous cell carcinoma.

Pontin and Reptin are closely related proteins belonging to the AAA+ (ATPases Associated with various cellular Activities) family. They form a hetero-oligomeric complex, Pontin/Reptin, which is involved in protein stability and assembly of the protein complexes as a molecular chaperone. Overexpression of Pontin and Reptin in tumor cells has been reported and is implicated in the development of various cancers. However, the molecular mechanism of Pontin/Reptin function in oral squamous cell carcinoma (OSCC) development remains unclear. Here, we identify HEAT repeat-containing protein 1 (HEATR1) as a novel binding factor of Pontin/Reptin. Functionally, HEATR1 stabilizes Pontin/Reptin and positively regulates OSCC cell proliferation by activating mTOR and pre-rRNA synthesis. We also find that HEATR1 expression is markedly upregulated in tumor region of OSCC tissue. Hence, we propose that HEATR1 is involved in the regulation of mTOR and ribosome biogenesis as a potential protein stabilizer of Pontin/Reptin in OSCC.

Identification and characterization of R2TP in the development of oral squamous cell carcinoma.

R2TP is a well-conserved molecular chaperone complex, composed of Pontin, Reptin, RPAP3, and PIH1D, in eukaryotes. Recent studies have suggested an involvement of R2TP in cancer development. However, it remains unclear if it is related to the development of oral squamous cell carcinoma (OSCC), which is the most common type of oral cancer. Here, we identify and investigate the function of R2TP in OSCC development. Immunohistochemical analysis reveals that all of the R2TP components are strongly expressed in normal oral epithelia and OSCC tissues, where actively proliferating cells are abundant. Co-immunoprecipitation assay identifies that R2TP components form a protein complex in OSCC-derived HSC4-cells. Knockdown experiments show that all R2TP components, except for RPAP3, are required for the cell proliferation and migration of HSC-4 cells. Furthermore, we reveal that Pontin contributes to a gain-of-function (GOF) activity of mutp53-R248Q in HSC-4 cells by regulating phosphorylation levels of mutp53 at Ser15 and Ser46. To our knowledge, this study is the first to report the functional involvement of R2TP and its components in the malignant characteristics of OSCC cells.