The increased expression of cytokeratin 13 leads to an increase in radiosensitivity of nasopharyngeal carcinoma HNE-3 cells by upregulating ERRFI1.

The main factors contributing to the unfavorable outcome in the clinical treatment of patients with nasopharyngeal carcinoma (NPC) patients are radiation resistance and recurrence. This study aimed to investigate the sensitivity and molecular foundation of cytokeratin 13 (CK13) in the radiotherapy of NPC. To achieve this, a human NPC cell line overexpressing CK13, HNE-3-CK13, was constructed. The effects of CK13 overexpression on cell viability and apoptosis under radiotherapy conditions were evaluated using the CCK-8 assay, immunofluorescence, and western blotting (WB). Next-generation sequencing was performed to identify the downstream genes and signaling pathways of CK13 that mediate radiotherapy response. The potential role of the candidate gene ERRFI1 in CK13-induced enhancement of radiosensitivity was investigated through rescue experiments using clone formation and WB. The effects of ERRFI1 on cell viability, cell apoptosis, cell cycle, and the related key genes were further evaluated using CCK-8, immunofluorescence, flow cytometry, quantitative polymerase chain reaction and WB. The results showed that CK13 overexpression in HNE-3 significantly inhibited cell survival under radiotherapy and promoted apoptosis marker γH2AX expression, leading to a significant increase of ERRFI1. Knockdown of ERRFI1 rescued the decreased cell viability and proliferation and the increased cell apoptosis that were caused by CK13 overexpression-mediated radiotherapy sensitization of NPC cells. In this process, EGFR, AKT, and GSK-3ß were found involved. In the end, ERRFI1 was proven to inhibit expression levels of CDK1, CDK2, cyclin B1, and cyclin D1, resulting an increased G2/M cell ratio. Overexpression of CK13 enhances the radiosensitivity of NPC cells, which is characterized by decreased cell viability and proliferation and increased apoptosis. This regulation may affect the survival of HNE-3 cells by increasing the expression of ERRFI1 and activating the EGFR/Akt/GSK-3ß signaling pathway, providing new potential therapeutic targets for the treatment of NPC.

KRT13 promotes stemness and drives metastasis in breast cancer through a plakoglobin/c-Myc signaling pathway.

BACKGROUND: Keratins (KRTs) are intermediate filament proteins that interact with multiple regulatory proteins to initiate signaling cascades. Keratin 13 (KRT13) plays an important role in breast cancer progression and metastasis. The objective of this study is to elucidate the mechanism by which KRT13 promotes breast cancer growth and metastasis. METHODS: The function and mechanisms of KRT13 in breast cancer progression and metastasis were assessed by overexpression and knockdown followed by examination of altered behaviors in breast cancer cells and in xenograft tumor formation in mouse mammary fat pad. Human breast cancer specimens were examined by immunohistochemistry and multiplexed quantum dot labeling analysis to correlate KRT13 expression to breast cancer progression and metastasis. RESULTS: KRT13-overexpressing MCF7 cells displayed increased proliferation, invasion, migration and in vivo tumor growth and metastasis to bone and lung. Conversely, KRT13 knockdown inhibited the aggressive behaviors of HCC1954 cells. At the molecular level, KRT13 directly interacted with plakoglobin (PG, γ-catenin) to form complexes with desmoplakin (DSP). This complex interfered with PG expression and nuclear translocation and abrogated PG-mediated suppression of c-Myc expression, while the KRT13/PG/c-Myc signaling pathway increased epithelial to mesenchymal transition and stem cell-like phenotype. KRT13 expression in 58 human breast cancer tissues was up-regulated especially at the invasive front and in metastatic specimens (12/18) (p < 0.05). KRT13 up-regulation in primary breast cancer was associated with decreased overall patient survival. CONCLUSIONS: This study reveals that KRT13 promotes breast cancer cell growth and metastasis via a plakoglobin/c-Myc pathway. Our findings reveal a potential novel pathway for therapeutic targeting of breast cancer progression and metastasis.

Cytokeratin 13 promotes radiotherapy sensitivity of nasopharyngeal carcinoma by downregulating the MEK/ERK pathway.

BACKGROUND: Radiation therapy is the first treatment choice for nasopharyngeal carcinoma (NPC), while radiation resistance and recurrence have become the primary factors and are associated with poor prognosis in the clinical treatment of NPC patients. The purpose of the present study was to explore the sensitivity and molecular basis of cytokeratin 13 (CK13) that regulates NPC radiotherapy. METHODS: HNE-3 or C666-1 cell line was used for overexpression and knockdown tests. Under radiotherapy conditions, CCK-8 assay, clone formation assay, and flow cytometry analyzed the effects of CK13 overexpression on cell proliferation, apoptosis, and cell cycle, respectively. In addition, Western blotting detected CK13-mediated downregulation of cell cycle-related genes. The mouse subcutaneous tumor-bearing experiment identified the effects of CK13 overexpression on the treatment of NPC in vivo. Further, Western blotting, CCK-8 assay, and flow cytometry investigated whether the CK13-mediated cell apoptosis involves the MEK/ERK signaling pathway. RESULTS: Overexpression of CK13 significantly inhibited the survival of HNE-3 cells under radiotherapy in vitro and in vivo, and there was a substantial decrease in cyclin-dependent kinase 4 and 6 (CDK4/6) levels promoting the cell percentage number in the G2/M phase and, subsequently, the ratio of the apoptotic cells. In contrast, the knockdown of CK13 showed the opposite partial regulatory effect. Interestingly, CK13 overexpression also showed a reduction in the survival of C666-1 cells and an increased ratio of the apoptotic cells under radiotherapy treatment. Furthermore, higher levels of CK13 downregulated the MEK/ERK signaling pathway, resulting in decreased HNE-3 cell proliferation and increased apoptosis. However, ERK activators were able to rescue the process partially. CONCLUSIONS: Together, these results showed that CK13 promoted the radiosensitivity of NPC cells by downregulating the MEK/ERK signaling pathway. Thus, targeting CK13 provided insights into the treatment of NPC radiotherapy.

Keratin 13 deficiency causes white sponge nevus in mice.

White sponge nevus (WSN) is a benign autosomal dominant disorder characterized by the formation of white spongy plaques in the oral mucosa. Keratin (KRT) 13 is highly expressed in the mucosa, and mutations in this gene have been commonly associated with WSN patients. However, it remains unknown whether there is a causal relationship between KRT13 mutations and WSN and what the underlying mechanisms might be. Here, we use mouse genetic models to demonstrate that Krt13 is crucial for the maintenance of epithelial integrity. Krt13 knockout mice show a WSN-like phenotype in several tissues, including the tongue, buccal mucosa, and esophagus. Transcriptome analyses uncover that Krt13 regulates a cohort of gene networks in tongue epithelial cells, including epithelial differentiation, immune responses, stress-activated kinase signaling, and metabolic processes. We also provide evidence that epithelial cells without Krt13 are susceptible to mechanical stresses experienced during postnatal life, resulting in unbalanced cell proliferation and differentiation. These data demonstrate that Krt13 is essential for maintaining epithelial homeostasis and loss of Krt13 causes the WSN-like phenotype in mice.

Usefulness of fluorescence visualization-guided surgery for early-stage tongue squamous cell carcinoma compared to iodine vital staining.

BACKGROUND: In the most cases of oral squamous cell carcinoma (OSCC), oral epithelial dysplasia (OED) is found adjacent to the primary tumor. The delineation of surgical margins for OSCC is critical to minimize the risk for local recurrence. The aim of this study is to demonstrate that the fluorescence visualization (FV)- device can delineated the lesion visualizes OED of adjacent primary tumors by histopathologically comparison to conventional iodine vital staining. MATERIAL AND METHODS: The study involved 40 patients with superficial tongue squamous cell carcinoma treated from July 2016 to July 2018 at the Oral Cancer Center, Tokyo Dental College. RESULTS: Cytokeratin 13 (CK13) expression rate in the area of fluorescence visualization loss (FVL) was significantly lower than that in the area of fluorescence visualization retention (FVR). In addition, CK17, Ki-67, and p53 expression rates were significantly higher in FVL than FVR. There was no significant difference in the delineation rate or area between FVL and iodine-unstained area. High-grade dysplasia was observed most frequently at the FV and iodine-unstained boundary, but no significant pathological differences were found. CONCLUSION: We strongly suggest the FV-guided surgery is a useful method for accurate resection in early-stage tongue squamous cell carcinoma.

Keratin 13 gene is epigenetically suppressed during transforming growth factor-ß1-induced epithelial-mesenchymal transition in a human keratinocyte cell line.

Epithelial-mesenchymal transition (EMT) is a biological event in which epithelial cells lose their polarity and cell-cell adhesions and concomitantly acquire mesenchymal traits, and is thought to play an important role in pathological processes such as wound healing and cancer progression. In this study, we evaluated transforming growth factor (TGF)-ß1-treated human keratinocyte HaCaT cells as an in vitro model of EMT. HaCaT cells were changed into an elongated fibroblast-like morphology, which is indicative of EMT in response to TGF-ß1. Phalloidin staining demonstrated the formation of actin stress fibers in TGF-ß1-treated cells. Quantitative RT-PCR analysis revealed that TGF-ß1 increased the mRNA levels of EMT transcription factors (SNAI2, TWIST1, and ZEB1) and mesenchymal markers (CDH2, VIM, and FN1), while it decreased the transcripts of epithelial phenotypic genes (CLDN1, OCLN, KRT5, KRT15, KRT13, and TGM1). Furthermore, we found that KRT13 was drastically suppressed through the reduction of RNA polymerase II occupancy of its promoter, which was accompanied by a decrease in active histone marks (H3K4me3 and H3K27ac) and an increase in a repressive mark (H3K27me3) during EMT. These findings indicate that the TGF-ß1-induced EMT program regulates a subset of epithelial and mesenchymal marker genes, and that KRT13 is transcriptionally suppressed through the modulation of the chromatin state at the KRT13 promoter in HaCaT cells.

Establishment of dermal sheath cell line from Cashmere goat and characterizing cytokeratin 13 as its novel biomarker.

OBJECTIVE: To establish a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from Cashmere goat and clarify the similarities and differences among them. RESULTS: We established a dermal sheath cell line, a dermal papilla cell line and a outer root sheath cell line from the pelage skin hair follicles of Cashmere goat. The growth rate of dermal sheath cells was intermediate between that of dermal papilla cells and outer root sheath cells. Immunofluorescence experiments and reverse transcription-polymerase chain reaction analysis showed that at both the transcriptional and translational levels, the dermal sheath cells were alpha-smooth muscle actin (α-SMA)+/cytokeratin 13+, while the dermal papilla cells were α-SMA+/cytokeratin 13- and the outer root sheath cells were α-SMA-/cytokeratin 13+. Patterns of cytokeratin 13 expression could distinguish the dermal sheath cells from the dermal papilla cells. CONCLUSIONS: These results suggest that cytokeratin 13 could serve as a novel biomarker for dermal sheath cells of Cashmere goat, and should prove useful for researchers investigating dermal stem cells or interaction of different types of cells during hair cycle.

A loss of profilin-1 in late-stage oral squamous cell carcinoma.

BACKGROUND: The genes for PFN1 and TMSB4 are both highly expressed in oral tissue and both encode actin monomer binding proteins thought to play a role in cell motility and possibly other crucial parts of tumor progression. METHODS: Oral brush cytology of epithelium from oral squamous cell carcinoma (OSCC) was used to measure PFN1 and TMSB4 mRNA in OSCC, while immunohistochemical analysis of tissue was used to check protein levels. RESULTS: High but variable expression of mRNAs encoding these two proteins was observed suggesting they may contribute to tumor characteristics in a subset of OSCCs. Both proteins were highly expressed in normal appearing basal epithelium, in the cytoplasm, and perinuclear area, while expression was minimal in upper epithelial layers. In OSCCs, expression of these proteins varied. In tumors classified as later stage, based on size and/or lymph node involvement, PFN1 levels were lower in tumor epithelium. A control gene, KRT13, showed expression in normal differentiated basal and suprabasal oral mucosa epithelial cells and as reported was lost in OSCC cells. CONCLUSION: Loss of PFN1 in tumor cells has been associated with lymph node invasion and metastasis in other tumor types, strengthening the argument that the protein has the potential to be a tumor suppressor in late-stage OSCC.

Krüppel-like Factor 4 Promotes Esophageal Squamous Cell Carcinoma Differentiation by Up-regulating Keratin 13 Expression.

Squamous cell differentiation requires the coordinated activation and repression of genes specific to the differentiation process; disruption of this program accompanies malignant transformation of epithelium. The exploration of genes that control epidermal proliferation and terminal differentiation is vital to better understand esophageal carcinogenesis. KLF4 is a member of the KLF family of transcription factors and is involved in both cellular proliferation and differentiation. This study using immunohistochemistry analysis of KLF4 in clinical specimens of esophageal squamous cell carcinoma (ESCC) demonstrated that decreased KLF4 was substantially associated with poor differentiation. Moreover, we determined that both KLF4 and KRT13 levels were undoubtedly augmented upon sodium butyrate-induced ESCC differentiation and G1 phase arrest. Conversely, silencing of KLF4 and KRT13 abrogated the inhibition of G1-S transition induced by sodium butyrate. Molecular investigation demonstrated that KLF4 transcriptionally regulated KRT13 and the expression of the two molecules appreciably correlated in ESCC tissues and cell lines. Collectively, these results suggest that KLF4 transcriptionally regulates KRT13 and is invovled in ESCC cell differentiation.

Measuring the regulation of keratin filament network dynamics.

The organization of the keratin intermediate filament cytoskeleton is closely linked to epithelial function. To study keratin network plasticity and its regulation at different levels, tools are needed to localize and measure local network dynamics. In this paper, we present image analysis methods designed to determine the speed and direction of keratin filament motion and to identify locations of keratin filament polymerization and depolymerization at subcellular resolution. Using these methods, we have analyzed time-lapse fluorescence recordings of fluorescent keratin 13 in human vulva carcinoma-derived A431 cells. The fluorescent keratins integrated into the endogenous keratin cytoskeleton, and thereby served as reliable markers of keratin dynamics. We found that increased times after seeding correlated with down-regulation of inward-directed keratin filament movement. Bulk flow analyses further revealed that keratin filament polymerization in the cell periphery and keratin depolymerization in the more central cytoplasm were both reduced. Treating these cells and other human keratinocyte-derived cells with EGF reversed all these processes within a few minutes, coinciding with increased keratin phosphorylation. These results highlight the value of the newly developed tools for identifying modulators of keratin filament network dynamics and characterizing their mode of action, which, in turn, contributes to understanding the close link between keratin filament network plasticity and epithelial physiology.

Remodeling of epithelial cells and basement membranes in a corneal deficiency model with long-term follow-up.

The ocular surface consists of the cornea, conjunctiva, and the limbus that is located in the transitional zone between the cornea and conjunctiva. The corneal epithelial cells are generated through the mitosis of corneal epithelial stem cells in the limbus. This study investigated a rabbit corneal deficiency model prepared by the surgical removal of the corneal and limbal epithelia, which express cytokeratin 12 (K12). After the surgery, K13-expressing conjunctival epithelium migrated onto the corneal surface and completely covered the surface, leading to neovascularization and corneal opacification. However, at 24 and 48 weeks after the surgery, K12-expressing cornea-like cells reappeared on the model ocular surface. These cells formed an island surrounded by invaded conjunctiva and were isolated from the limbus. Interestingly, in the 24-week model surface, α1(IV) and α2(IV) collagen chains, which are normally found in the basement membrane of the native limbus and conjunctiva, and not in the cornea, were continuously deposited throughout the entire basement membrane, including the basement membrane under cornea-like cells. By contrast, in the 48-week model surface, α1(IV) and α2(IV) collagen chains were absent from the basement membrane beneath the central part of cornea-like cells and were localized below the invaded conjunctiva and the transitional zone between cornea-like cells and the invaded conjunctiva, which had similar distribution to the native ocular basement membrane. Moreover, K12, K14, p63, vimentin, and α1(IV) and α2(IV) collagen chains, which are colocalized in the native limbus, were all present at the transitional zone of the 48-week model surface. Therefore, a limbus-like structure appeared to be reconstructed on the surface of the 48-week model as a stem cell niche. This study should aid in the understanding of human corneal deficiency, the correlation between the epithelial cell phenotype and the composition of the basement membrane, and the epithelial stem cell niche.

Cytokeratin 13, Cytokeratin 17, Ki-67 and p53 Expression in Upper Layers of Epithelial Dysplasia Surrounding Tongue Squamous Cell Carcinoma.

Early detection of oral squamous cell carcinoma (OSCC) improves its prognosis and aids in selecting the appropriate treatment, which may also have a positive effect on quality of life. Early detection, therefore, is an important issue in the treatment of this disease. The purpose of this study was to investigate expression of cytokeratin 13 (CK13), CK17, Ki-67 and p53 as potential markers of tongue SCC. Five areas in 12 specimens were examined: the upper and lower layers of normal epithelium; those of dysplastic epithelial tissue surrounding the cancerous lesion; and the lesion itself. Strong expression of each of the following mRNAs and proteins was observed; CK13 in upper layers of normal epithelium; Ki-67 and p53 in lower layers of normal epithelium; CK13 and CK17 in upper layer of epithelial dysplasia; and CK17, Ki-67, and p53 in lower layer of epithelial dysplasia and cancerous lesions. These results indicate that the characteristic pattern of expression of CK13 and CK17 differs between normal and dysplastic oral epithelium. Oral epithelial dysplasia adjacent to OSCC has high malignant potential, and is similar to early-stage OSCC. This suggests that evaluation of these markers could be a useful secondary procedure for improving detection of early-stage OSCC.

Epigenetic alterations of the keratin 13 gene in oral squamous cell carcinoma.

BACKGROUND: Epigenetic modifications play important roles in the regulation of gene expression determining cellular phenotype as well as various pathologies such as cancer. Although the loss of keratin 13 (KRT13) is reportedly linked to malignant transformation of oral epithelial cells, the molecular mechanisms through which KRT13 is repressed in oral squamous cell carcinoma (OSCC) remain unclear. The aim of this study is to identify the epigenetic alterations of the KRT13 gene in OSCCs. METHODS: We investigated KRT13 expression levels and chromatin modifications of the KRT13 promoter in the three OSCC cell lines (HSC4, HSC3, and SAS). The expression levels of KRT13 protein and mRNA were analyzed by western blotting and quantitative reverse-transcription polymerase chain reaction, respectively, and the localization of KRT13 protein was detected by immunofluorescence. DNA methylation and histone modifications in the KRT13 promoter were determined by bisulfite sequencing and chromatin immunoprecipitation (ChIP), respectively. For the pharmacological depletion of Polycomb repressive complex 2 (PRC2), cells were treated with 3-deazaneplanocin A (DZNep). RESULTS: KRT13 expression was transcriptionally silenced in the HSC3 and SAS cells and post-transcriptionally repressed in the HSC4 cells, while the KRT13 promoter was hypermethylated in all of the three OSCC cell lines. ChIP analysis revealed that PRC2-mediated trimethylation of Lys 27 on histone H3 (H3K27me3) was increased in the KRT13 promoter in the HSC3 and SAS cells. Finally, we demonstrated that the treatment of SAS cells with DZNep reactivated the transcription of KRT13 gene. CONCLUSIONS: Our data provide mechanistic insights into the epigenetic silencing of KRT13 genes in OSCC cells and might be useful for the development of diagnostic markers and novel therapeutic approaches against OSCCs.

Immunohistochemical staining patterns of cytokeratins 13, 14, and 17 in oral epithelial dysplasia including orthokeratotic dysplasia.

Diagnosis of the exact grade of oral epithelial dysplasia is difficult, and interobserver variations in grading are common. The aim of this study was to investigate the expression patterns of cytokeratins (CKs) in dysplastic oral epithelia, to identify useful double immunostaining diagnostic markers. Immunoexpression of CK13, CK14, CK17, and Ki-67 were investigated in 21 normal epithelial specimens and 146 epithelial dysplasia specimens. In epithelial dysplasia specimens, orthokeratotic dysplasia (OKD) was identified using CK10 immunostaining. Most mild dysplasia specimens were CK13+ and CK17-. In moderate dysplasia, CK13 expression tended to be lower and CK17 expression tended to be higher than in mild dysplasia. All carcinoma in situ (CIS) specimens were CK17+. In differentiated type CIS specimens, CK13 expression was weakly positive. Most epithelial dysplasia specimens were CK14+. There were no significant differences in the expression patterns of CKs between OKD and non-OKD specimens in any of the grades of dysplasia. These results indicate that CK14 expression can be used to detect early epithelial dysplasia, and that CK13 and CK17 expression are useful for detecting neoplastic changes.

Effect of porcine chondrocyte-derived extracellular matrix on the pterygium in mouse model.

PURPOSE: To investigate the effect of porcine chondrocyte-derived extracellular matrix (PCDECM) on an experimental mouse model of human pterygial epithelial cells. METHODS: Cultured human pterygial epithelial cells (hPECs) were stained with pan-cytokeratin (CK), CK3/2p, vimentin, and CK13 antibodies to characterize the cells. A pterygium mouse model was developed by injecting 1X104 hPECs into the nasal subconjunctival space in athymic nude mice. PCDECM (25 mg/mL, 10 µL) was injected into the nasal subconjunctival space in the right eye 7, 10 and 14 days after the epithelial cell injection (PCDECM group). Image analysis was performed using ImageJ® to compare the lesion size. A histopathological analysis of the cornea was conducted to evaluate the state of the epithelium and the expression of pterygial epithelial cell markers. RESULTS: The isolated pterygial cells were positive for pan-CK, CK3/2p and vimentin, and they were negative for CK13 under immunofluorescence microscopy. On day 17 after epithelial cell injection, the size of the lesion compared to the entire cornea was increased to 37.1 % in the control group. However, in the PCDECM group, the lesion covered only 26.3 % of the entire cornea. The corneas of the pterygium mice showed an epithelium of irregular thickness, proliferation of the stroma, extracellular matrix breakdown and overexpression of pterygium-positive markers. However, these changes were significantly suppressed by the application of PCDEDM. CONCLUSIONS: Our findings suggest that PCDECM seems to suppress pterygial epithelial cell growth and it could be used as a promising biomaterial for the noninvasive treatment of pterygium.

Establishment and characterization of a telomerase immortalized human gingival epithelial cell line.

BACKGROUND AND OBJECTIVE: Gingival keratinocytes are used in model systems to investigate the interaction between periodontal bacteria and the epithelium in the initial stages of the periodontal disease process. Primary gingival epithelial cells (GECs) have a finite lifespan in culture before they enter senescence and cease to replicate, while epithelial cells immortalized with viral proteins can exhibit chromosomal rearrangements. The aim of this study was to generate a telomerase immortalized human gingival epithelial cell line and compare its in vitro behaviour to that of human GECs. MATERIAL AND METHODS: Human primary GECs were immortalized with a bmi1/hTERT combination to prevent cell cycle triggers of senescence and telomere shortening. The resultant cell-line, telomerase immortalized gingival keratinocytes (TIGKs), were compared to GECs for cell morphology, karyotype, growth and cytokeratin expression, and further characterized for replicative lifespan, expression of toll-like receptors and invasion by P. gingivalis. RESULTS: TIGKs showed morphologies, karyotype, proliferation rates and expression of characteristic cytokeratin proteins comparable to GECs. TIGKs underwent 36 passages without signs of senescence and expressed transcripts for toll-like receptors 1-6, 8 and 9. A subpopulation of cells underwent stratification after extended time in culture. The cytokeratin profiles of TIGK monolayers were consistent with basal cells. When allowed to stratify, cytokeratin profiles of TIGKs were consistent with suprabasal cells of the junctional epithelium. Further, TIGKs were comparable to GECs in previously reported levels and kinetics of invasion by wild-type P. gingivalis and an invasion defective ΔserB mutant. CONCLUSION: Results confirm bmi1/hTERT immortalization of primary GECs generated a robust cell line with similar characteristics to the parental cell type. TIGKs represent a valuable model system for the study of oral bacteria interactions with host gingival cells.

Organotypic culture of normal, dysplastic and squamous cell carcinoma-derived oral cell lines reveals loss of spatial regulation of CD44 and p75 NTR in malignancy.

Oral squamous cell carcinomas (OSCC) often arise from dysplastic lesions. The role of cancer stem cells in tumour initiation is widely accepted, yet the potential existence of pre-cancerous stem cells in dysplastic tissue has received little attention. Cell lines from oral diseases ranging in severity from dysplasia to malignancy provide opportunity to investigate the involvement of stem cells in malignant progression from dysplasia. Stem cells are functionally defined by their ability to generate hierarchical tissue structures in consortium with spatial regulation. Organotypic cultures readily display tissue hierarchy in vitro; hence, in this study, we compared hierarchical expression of stem cell-associated markers in dermis-based organotypic cultures of oral epithelial cells from normal tissue (OKF6-TERT2), mild dysplasia (DOK), severe dysplasia (POE-9n) and OSCC (PE/CA P J15). Expression of CD44, p75(NTR), CD24 and ALDH was studied in monolayers by flow cytometry and in organotypic cultures by immunohistochemistry. Spatial regulation of CD44 and p75(NTR) was evident for organotypic cultures of normal (OKF6-TERT2) and dysplasia (DOK and POE-9n) but was lacking for OSCC (PE/CA PJ15)-derived cells. Spatial regulation of CD24 was not evident. All monolayer cultures exhibited CD44, p75(NTR), CD24 antigens and ALDH activity (ALDEFLUOR(®) assay), with a trend towards loss of population heterogeneity that mirrored disease severity. In monolayer, increased FOXA1 and decreased FOXA2 expression correlated with disease severity, but OCT3/4, Sox2 and NANOG did not. We conclude that dermis-based organotypic cultures give opportunity to investigate the mechanisms that underlie loss of spatial regulation of stem cell markers seen with OSCC-derived cells.

KRT13 is upregulated in pancreatic cancer stem-like cells and associated with radioresistance.

Pancreatic cancer is one of the most aggressive cancers and the seventh leading cause of cancer-associated death in the world. Radiation is performed as an adjuvant therapy as well as anti-cancer drugs. Because cancer stem-like cells (CSCs) are considered to be radioresistant and cause recurrence and metastasis, understanding their properties is required for the development of novel therapeutic strategies. To investigate the CSC properties of pancreatic cancer cells, we used a pancreatic CSC model, degron (++) cells, which have low proteasome activity. Degron (++) cells displayed radioresistance in comparison with control cells. Using Ribonucleic acid (RNA) sequencing, we successfully identified KRT13 as a candidate gene responsible for radioresistance. Knockdown of KRT13 sensitized the degron (++) cells to radiation. Furthermore, a database search revealed that KRT13 is upregulated in pancreatic cancer cell lines and that high expression of KRT13 is associated with poorer prognosis. These results indicate that a combination therapy of KRT13 knockdown and radiation could hold therapeutic promise in pancreatic cancer.

KRT13 and UPK1B for differential diagnosis between metastatic lung carcinoma from oral squamous cell carcinoma and lung squamous cell carcinoma.

Oral squamous cell carcinomas unusually show distant metastasis to the lung after primary treatment, which can be difficult to differentiate from primary squamous cell carcinoma of the lung. While the location and number of tumor nodules is helpful in diagnosing cases, differential diagnosis may be difficult even with histopathological examination. Therefore, we attempted to identify molecules that can facilitate accurate differential diagnosis. First, we performed a comprehensive gene expression analysis using microarray data for OSCC-LM and LSCC, and searched for genes showing significantly different expression levels. We then identified KRT13, UPK1B, and nuclear receptor subfamily 0, group B, member 1 (NR0B1) as genes that were significantly upregulated in LSCC and quantified the expression levels of these genes by real-time quantitative RT-PCR. The expression of KRT13 and UPK1B proteins were then examined by immunohistochemical staining. While OSCC-LM showed no KRT13 and UPK1B expression, some tumor cells of LSCC showed KRT13 and UPK1B expression in 10 of 12 cases (83.3%). All LSCC cases were positive for at least one of these markers. Thus, KRT13 and UPK1B might contribute in differentiating OSCC-LM from LSCC.

Reduction of NOTCH1 expression pertains to maturation abnormalities of keratinocytes in squamous neoplasms.

Notch is a transmembrane receptor functioning in the determination of cell fate. Abnormal Notch signaling promotes tumor development, showing either oncogenic or tumor suppressive activity. The uncertainty about the exact role of Notch signaling, partially, stems from inconsistencies in descriptions of Notch expression in human cancers. Here, we clarified basal-cell dominant expression of NOTCH1 in squamous epithelium. NOTCH1 was downregulated in squamous neoplasms of oral mucosa, esophagus and uterine cervix, compared with the normal basal cells, although the expression tended to be retained in cervical lesions. NOTCH1 downregulation was observed even in precancers, and there was little difference between cancers and high-grade precancerous lesions, suggesting its minor contribution to cancer-specific events such as invasion. In culture experiments, reduction of NOTCH1 expression resulted in downregulation of keratin 13 and keratin 15, and upregulation of keratin 17, and NOTCH1 knockdown cells formed a dysplastic stratified epithelium mimicking a precancerous lesion. The NOTCH1 downregulation and the concomitant alterations of those keratin expressions were confirmed in the squamous neoplasms both by immunohistochemical and cDNA microarray analyses. Our data indicate that reduction of NOTCH1 expression directs the basal cells to cease terminal differentiation and to form an immature epithelium, thereby playing a major role in the histopathogenesis of epithelial dysplasia. Furthermore, downregulation of NOTCH1 expression seems to be an inherent mechanism for switching the epithelium from a normal and mature state to an activated and immature state, suggesting its essential role in maintaining the epithelial integrity.