The liver kinase B1 supports mammary epithelial morphogenesis by inhibiting critical factors that mediate epithelial-mesenchymal transition.

The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor ß (TGFß) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional (3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFß, including TGFß auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFß signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFß type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFß family members.

The association between clinical symptoms and later subjective quality of life in individuals with ultra-high risk for psychosis and recent-onset psychotic disorder: A longitudinal investigation.

AIM: Subjective quality of life is a clinically relevant outcome that is strongly associated with the severity of clinical symptoms in individuals with ultra-high risk for psychosis and patients with recent-onset psychotic disorder. Our objective was to examine whether longitudinal changes in clinical symptoms are associated with quality of life in ultra-high risk individuals and patients with recent-onset psychotic disorder. METHODS: Individuals with ultra-high risk and patients with recent-onset psychosis disorder were recruited in the same clinical settings at baseline and were followed up with more than 6 months and less than 5 years later. We assessed five factors of clinical symptoms using the positive and negative syndrome scale, and quality of life using the World Health Organization quality of life questionnaire-short form. We used multiple regression to examine the relationships between clinical symptoms and quality of life while controlling for diagnosis, follow-up period, age, and sex. RESULTS: Data were collected from 22 individuals with ultra-high risk and 27 patients with recent-onset psychosis disorder. The multiple regression analysis results indicated that the more severe anxiety/depression was at baseline, the poorer the quality of life at follow-up. Further, improvement of anxiety/depression and disorganized thoughts were associated with improvement in quality of life. The difference in diagnosis did not affect the association between clinical symptoms and quality of life. CONCLUSION: These findings suggest that the improvement of anxiety/depression and disorganized thoughts is important in the early stages of psychosis before it becomes severe, affecting the quality of life.

Left supraclavicular (Virchow's) node metastasis detected before primary infradiaphragmatic tumor: a case series.

BACKGROUND: Metastasis of infradiaphragmatic tumors to the left supraclavicular lymph node is reported to be rare. When metastasis is detected in the left supraclavicular node in patients with head and neck carcinoma, locating the primary cancer remains a difficult and time-consuming challenge despite the dramatic development of screening technologies and treatment methods. CASE PRESENTATION: We report three cases of malignant infradiaphragmatic tumor diagnosed following an initial finding of left supraclavicular node metastasis after surgery for tongue squamous cell carcinoma (follow-up period, range 18-62 months). In these cases, adenocarcinoma was diagnosed based on left supraclavicular node biopsies, and a second primary tumor was found, in a 78-year-old Japanese woman with a diagnosis of cholangiocarcinoma, a 64-year-old Japanese man with a diagnosis of bladder carcinoma, and a 61-year-old Japanese man with a diagnosis of prostate carcinoma. In the cholangiocarcinoma case, carbohydrate antigen 19-9 and alpha-fetoprotein levels helped to diagnose cholangiocarcinoma. Palliative care only was given, with survival for 11 months after diagnosis of lymph node metastasis. In the bladder carcinoma case, pathological analysis of fine-needle aspiration biopsy specimen of the metastatic cervical lymph node showed atypical cells with slight squamous differentiation. Hematoxylin-eosin staining of the bladder lesion did not identify a clear glandular or squamous component, and we could not make a definitive diagnosis of whether the lesion was poorly differentiated squamous cell carcinoma, adenocarcinoma, or high-grade urothelial carcinoma. GATA3 staining aided in the diagnosis of urothelial bladder cancer with left supraclavicular node metastasis. He survived for 2 months after diagnosis of left supraclavicular lymph node metastasis. In the prostate carcinoma case, 18F-fluorodeoxyglucose uptake was weak. Prostate-specific antigen levels and magnetic resonance imaging findings aided the diagnostic process. This patient underwent bilateral orchiectomy and adjuvant hormonal therapy and survived for 47 months after diagnosis of left supraclavicular node metastasis. CONCLUSIONS: Pathological diagnosis on the basis of immunohistochemistry and specific diagnosis methods such as radiological and serological assessments are important for providing rapid diagnosis and appropriate treatment.

IL-1 Generated by Oral Squamous Cell Carcinoma Stimulates Tumor-Induced and RANKL-Induced Osteoclastogenesis: A Possible Mechanism of Bone Resorption Induced by the Infiltration of Oral Squamous Cell Carcinoma.

We previously observed a novel osteoclastogenesis system that is induced by oral squamous cell carcinoma (OSCC) cells, which target osteoclast precursor cells (OPC) without upregulation of the master transcriptional factor of osteoclastogenesis, NFATc1. Here, we analyzed inflammatory cytokines that were preferentially expressed in one of the osteoclastogenic OSCC cell lines, namely NEM, compared with the subclone that had lost its osteoclastogenic properties. Based on a gene expression microarray and a protein array analyses, IL-1, IL-6, IL-8, and CXCL1 were chosen as candidates responsible for tumor-induced osteoclastogenesis. From the results of the in vitro osteoclastogenesis assay using OPCs cultured with OSCC cells or their culture supernatants, IL-1 was selected as a stimulator of both OSCC-induced and RANKL-induced osteoclastogenesis. The IL-1 receptor antagonist significantly attenuated osteoclastogenesis induced by NEM cells. The stimulatory effects of IL-1 for OSCC-induced and RANKL-induced osteoclastogenesis were effectively attenuated with cannabidiol and denosumab, respectively. These results suggest that IL-1 secreted from OSCC cells stimulates not only tumor-induced osteoclastogenesis targeting OPCs but also physiological RANKL-induced osteoclastogenesis, and this may be the biological mechanism of bone resorption induced by the infiltration of OSCC. These results also suggest that IL-1 inhibitors are candidates for therapeutic agents against bone resorption induced by OSCC.

Multicenter retrospective analysis of clinicopathological features and prognosis of oral tongue squamous cell carcinoma in adolescent and young adult patients.

ABSTRACT: The aim of this study is to report the differences in clinicopathological features of oral tongue squamous cell carcinoma (OTSCC) and survival between adolescent and young adult (AYA) patients and elderly patients and to find the prognosticators. The medical records of 101 AYA patients and 175 control patients with OTSCC who underwent surgery were reviewed. Variables related to prognosis and their clinicopathological associations were analyzed. The 5-year overall survival (5y-OS) rates of AYA and control patients with stage I and II OTSCC were 94.4% and 89.6% (P = .353), respectively, and their 5-year disease-free survival (5y-DFS) rates were 82.0% and 76.6%, respectively (P = .476). The 5y-OS rates of patients with stages III and IV OTSCC were 83.3% and 66.7% (P = .333), respectively, and their 5y-DFS rates were 75.0% and 57.1% (P = .335), respectively. Logistic regression analysis revealed that there was no significant clinicopathological difference in AYA and control group. Furthermore, there was no significant difference in 5y-OS rates between patients who underwent elective neck dissection (END) and those who underwent therapeutic neck dissection (TND) in both group (P = 0.717 and 0.688). Overall, the present study revealed the clinicopathological features and prognosis of OTSCC were similar in AYA patients and elderly patients. Moreover, as there was no significant difference in OS between patients who underwent END and those who underwent TND in AYA and control groups, our results suggest that the indication for END in AYA patients with clinical N0 OTSCC is similar to that for elderly patients.

Epithelial-Myoepithelial Carcinoma of the Minor Salivary Glands: Case Series with Comprehensive Review.

Epithelial-myoepithelial carcinoma (EMC) is a rare salivary gland tumor that is histologically characterized by biphasic tubular structures composed of inner ductal and outer clear myoepithelial cells, which is especially uncommon in the minor salivary glands (MSG). Because of its histologic variety, complexity, and heterogeneity, it is sometimes challenging to make the accurate diagnosis. Here, we report a literature review of EMC of the MSGs with our experience of two cases. Incisional biopsy was suggestive of pleomorphic adenoma in Case 1 and pleomorphic adenoma or a low-grade salivary gland carcinoma in Case 2. Both cases were performed intraoral tumor resection, and they have good postoperative courses and are alive with no evidence of local recurrence or metastasis at 31 and 16 months, respectively. Considering that the anatomy, structure, and size of salivary glands are quite different from MSGs, it might be difficult to predict EMCs of the MSG similarly to EMCs of the major salivary glands. This comprehensive review also reports the features of EMC of the MSG cases and the trends of diagnosis and discusses treatment strategy.

Primordial odontogenic tumor occurred in the maxilla with unique calcifications and its crucial points for differential diagnosis.

Primordial odontogenic tumor (POT) is a newly classified, mixed epithelial and mesenchymal odontogenic tumor, with only 17 reported cases to date. Herein, we report a case of POT that occurred in the right maxilla of a 10-year-old boy and reveal unique features in comparison with those previously reported. Radiologically, the lesion presented as a well-defined, unilocular radiolucency with notable radiopaque foci on the periphery. Microscopically, the tumor was mainly composed of dental papilla-like myxoid fibrous connective tissue, largely surrounded by non-keratinized squamous epithelium with numerous calcified particles, and partly enclosed by inner enamel epithelium-like columnar cells and enamel organ-like structures accompanied with cuboidal and/or stellate reticulum-like cells. Immunohistochemically, the epithelium tested positive for cytokeratin 14 and 19. Moreover, amelogenin and ameloblastin, matrix proteins relating to enamel formation, were positive in the covering epithelium. The tumor was enucleated as a whole, and no recurrence was recorded thereafter. Although the presence of numerous calcified particles was unique, we diagnosed this lesion as POT based on the above-described features. Furthermore, we emphasize the importance of the differential diagnosis of POT and other odontogenic tumors that resemble corresponding tooth germ components.

Genetic and histopathological analysis of a case of primary intraosseous carcinoma, NOS with features of both ameloblastic carcinoma and squamous cell carcinoma.

BACKGROUND: Primary intraosseous carcinoma (PIOC), NOS is an odontogenic carcinoma with unknown etiology. Its diagnosis may be used when central jaw carcinoma cannot be categorized as any other type of carcinoma. Further information on this extremely rare tumor is needed to improve our understanding and evaluate the classification of odontogenic carcinomas. CASE PRESENTATION: We herein presented two patients with PIOC, NOS with different clinical and histopathological features and analyzed gene mutations in these patients using next-generation sequencing (NGS). The typical PIOC, NOS case had many histopathological similarities to oral squamous cell carcinoma (OSCC), including the missense point mutations of TP53 Glu285Val, KDR Gln472His, and APC Pro1433Leu, which are similar to those in other cancers; however, no mutations were detected in the other patient with an atypical presentation of PIOC, NOS, which was derived from a precursor cystic lesion with similarities to both ameloblastic carcinoma and OSCC. CONCLUSIONS: Genetic analysis suggested that these two PIOC, NOS cases have different features and can be subcategorized.

Intracellular claudin-1 at the invasive front of tongue squamous cell carcinoma is associated with lymph node metastasis.

Claudins are the major component of tight junctions, which form a primary barrier to paracellular diffusion and maintain cell polarity in normal epithelia and endothelia. In cancer cells, claudins play additional roles besides serving as components of the tight junctions, and participate in anoikis or invasion. Among the claudin family proteins, claudin-1 has the most promising potential, both diagnostically and prognostically, in many types of cancers, including oral, gastric, liver, and colon cancers. However, conflicting results have been reported in relation to the degree of claudin-1 expression and the prognosis, suggesting that the expression level of claudin-1 alone is not sufficient to analyze the relationship between claudin-1 and cancer progression. As endocytic trafficking of claudin-1 has been reported in several epithelial cell types in vitro, we aimed to determine whether intracellular localization of claudin-1 is the missing aspect between claudin-1 and cancer. We investigated the expression of claudin-1 in 83 tongue squamous cell carcinoma (TSCC) pathological specimens. Although the expression level of claudin-1 based on immunohistochemistry was not associated with TSCC progression, within the high claudin-1 expression group, the incidence of intracellular localization of claudin-1 was correlated with cervical lymph node metastasis. In an in vitro experiment, claudin-1 was constitutively internalized in TSCC-derived cells. Motility of TSCC-derived cells was increased by deficiency of claudin-1, suggesting that the decrease in cell-surface claudin-1 promoted the cell migration. Therefore, intracellular localization of claudin-1 at the invasion front may represent a promising diagnostic marker of TSCC.

A Novel, Tumor-Induced Osteoclastogenesis Pathway Insensitive to Denosumab but Interfered by Cannabidiol.

Bone metabolism is strictly regulated, and impaired regulation caused by hormonal imbalances induces systemic bone loss. Local bone loss caused by tumor invasion into bone is suggested to be induced by the generation of cytokines, which affect bone metabolism, by tumor cells. The major cause of systemic and local bone losses is excess bone resorption by osteoclasts, which differentiate from macrophages by receptor activator of nuclear factor kappa-B ligand (RANKL) or tumor necrosis factor-alpha (TNF-α). We previously found a novel pathway for tumor-induced osteoclastogenesis targeting osteoclast precursor cells (OPCs). Tumor-induced osteoclastogenesis was resistant to RANKL and TNF-α inhibitors. In the present study, we confirmed that exosomes derived from oral squamous cell carcinoma (OSCC) cells induced osteoclasts from OPCs. We also showed that the depletion of exosomes from culture supernatants of OSCC cells partially interfered with osteoclastogenesis, and cannabidiol, an innoxious cannabinoid without psychotropic effects, almost completely suppressed tumor-induced osteoclastogenesis. Osteoclastogenesis and its interference by cannabidiol were independent of the expression of nuclear factor of T cell c1 (NFATc1). These results show that osteoclastogenesis induced by OSCC cells targeting OPCs is a novel osteoclastogenic pathway independent of NFATc1 expression that is partially caused by tumor-derived exosomes and suppressed by cannabidiol.

Glucose metabolism changes during the development and progression of oral tongue squamous cell carcinomas.

Previous studies have revealed several genes involved in the carcinogenesis of oral cancer. However, the detailed mechanisms underlying this process are poorly understood. Previously, we established a database cataloging the transcriptional progression profile of oral carcinogenesis and identified several candidate genes with continuously increasing or decreasing expression, which specifically promote the transition of oral premalignant lesions to invasive carcinomas. In this study, using our microarray database, we attempted to determine significant genes that may contribute to metabolic alterations during oral carcinogenesis. After performing a literature survey, we focused on 15 candidate genes associated with glucose metabolism changes, particularly the tri-carboxylic acid cycle, and investigated the mRNA-expression status of these genes with our database. Only the solute carrier family 2 member 1 gene (also known as GLUT1), showed significantly increased mRNA expression during oral tumorigenesis. Immunohistochemical analysis confirmed that GLUT1 protein expression significantly increased during oral carcinogenesis. In addition, tumors with high expression of this protein significantly correlated with nodal status (P=0.002). Kaplan-Meier survival curves clearly demonstrated the adverse impact of high GLUT1 protein expression on disease-free survival (P=0.004). GLUT1 mRNA and protein expression increased in the order of normal mucosal tissues, epithelial dysplastic lesions and invasive carcinomas. Therefore, metabolic alterations, especially in glucose metabolism, occurred at the very early stage of development of oral malignancies. In addition, GLUT1 played a significant role in oral cancer, acquiring a malignant phenotype.

A new osteoclastogenesis pathway induced by cancer cells targeting osteoclast precursor cells.

The precise mechanism of osteolysis induced by tumors infiltrating into the bone remains unclear. The main hypothesis is that tumor cells generate receptor activator of nuclear factor kappa-B ligand (RANKL), tumor necrosis factor-alpha (TNF-α), or other molecules that activate the expression of RANKL in osteoblasts, osteocytes, or bone marrow stromal cells. Administration of bisphosphonates or anti-RANKL antibody drugs, which suppress systemic bone resorption, prevents osteolysis induced by tumors infiltrating into the bone. However, these therapeutic agents may cause medication-related osteonecrosis of the jaw. In this study, we found a novel tumor-associated osteoclastogenesis pathway in osteoclast precursor cells. Co-culture with human oral squamous cell carcinoma cells, 3A or NEM, or culture with each of their conditioned medium induced many osteoclasts from osteoclast precursor cells, which were generated by a 24-h pretreatment of RANKL or TNF-α. Osteoprotegerin, a decoy RANKL receptor, denosumab, an anti-RANKL antibody drug, and infliximab, an anti-TNF-α antibody drug, did not prevent this tumor-associated osteoclastogenesis. Quantitative RT-PCR analysis showed that the expression of NFATc1 was decreased in this tumor-associated osteoclastogenesis, which was suggested to be independent of NFATc1. These results revealed a novel pathway for tumor-associated osteoclastogenesis, which may be a new therapeutic target for osteolysis induced by tumors infiltrating into the bone without affecting systemic bone metabolism.

Electrophysiological evidence for abnormal glutamate-GABA association following psychosis onset.

Previous studies have shown glutamatergic dysfunction and γ-aminobutyric acid (GABA)-ergic dysfunction in schizophrenia. Animal studies suggest that N-methyl-D-aspartate receptor (NMDAR) dysfunction and GABA-ergic dysfunction interact with each other and lead to alterations in excitatory/inhibitory balance. The NMDAR and GABAergic-interneuron functions may be indexed by mismatch negativity (MMN) and auditory steady-state gamma-band response (ASSR), respectively. However, no previous studies have tested the hypothesis of an abnormal association between MMN and gamma-band ASSR in the same patients to identify the in vivo evidence of NMDAR-GABA association during the early stages of psychosis. Participants were individuals with recent-onset schizophrenia (ROSZ; N = 21), ultra-high risk (UHR; N = 27), and healthy controls (HCs; N = 24). The MMN amplitude was significantly impaired in ROSZ (p = 0.001, d = 1.20) and UHR (p = 0.003, d = 1.01) compared with HCs. The intertrial phase coherence (ITC) index of gamma-band ASSR was significantly reduced in ROSZ compared with HCs (p < 0.001, d = -1.27) and UHR (p = 0.032, d = -0.75). The event-related spectral perturbation (ERSP) index of gamma-band ASSR was significantly smaller in ROSZ compared with HCs (p < 0.001, d = -1.21). The MMN amplitude was significantly correlated with the ITC in ROSZ (r = -0.69, p < 0.001). These findings provide the first in vivo evidence that an abnormal association of the electrophysiological indices of NMDAR and GABA dysfunctions may be present in recent-onset schizophrenia.

Auditory gamma oscillations predict global symptomatic outcome in the early stages of psychosis: A longitudinal investigation.

OBJECTIVES: The gamma-band auditory steady-state response (ASSR) is thought to reflect the function of parvalbumin-positive γ-aminobutyric acid (GABA)-ergic interneurons and may be a candidate biomarker in early psychosis. Although previous cross-sectional studies have shown that gamma-band ASSR is reduced in early psychosis, whether reduced gamma-band ASSR could be a predictor of the long-term prognosis remains unknown. METHODS: In this longitudinal study, we investigated the association between gamma-band ASSR reduction and future global symptomatic or functional outcome in early psychosis. We measured 40-Hz ASSR in 34 patients with recent-onset schizophrenia (ROSZ), 28 ultra-high risk (UHR) individuals, and 30 healthy controls (HCs) at baseline. After 1-2 years, we evaluated the global assessment of functioning (GAF) in the ROSZ (N = 20) and UHR (N = 20) groups. RESULTS: The 40-Hz ASSR was significantly reduced in the ROSZ and UHR groups. The attenuated 40-Hz ASSR was correlated with the future global symptomatic outcome in the ROSZ, but not in the UHR groups. CONCLUSIONS: A reduction in the gamma-band ASSR after the onset of psychosis may predict symptomatic outcomes in early psychosis. SIGNIFICANCE: Gamma-band ASSR may be a potentially useful biomarker of the long-term prognosis in patients with recent-onset schizophrenia.

Leukemia inhibitory factor produced by fibroblasts within tumor stroma participates in invasion of oral squamous cell carcinoma.

The interaction between cancer cells and the cancer stroma plays a crucial role in tumor progression and metastasis in diverse malignancies, including oral cancer. However, the mechanism underlying this interaction remains incompletely elucidated. Here, to investigate the interaction between oral cancer cells and fibroblasts, which are major cellular components of the tumor stroma, we conducted an in vitro study by using human oral squamous cell carcinoma (OSCC) cell lines and normal human dermal fibroblasts (NHDFs). The results of transwell assays revealed that the migration and invasion of 2 OSCC cell lines, HO1-N-1 and HSC3, were markedly stimulated upon coculturing with NHDFs. To investigate the factors that promote tumor invasion, we isolated NHDFs from cocultures prepared with HO1-N-1 cells and performed microarray analysis. Among the various genes that were upregulated, we identified the gene encoding leukemia inhibitory factor (LIF), and we focused on LIF in further analyses. We confirmed that all OSCC-derived conditioned media potently upregulated LIF expression in NHDFs, and the results of our transwell analysis demonstrated that NHDF-induced OSCC migration and invasion were inhibited by LIF-neutralizing antibodies. Furthermore, immunohistochemical analysis of patient samples revealed that in 44 out of 112 OSCC cases, LIF was expressed in the tumor stroma, particularly in cancer-associated fibroblasts (CAFs), and, notably, clinicopathological analyses confirmed that LIF expression in CAFs was significantly correlated with increased depth of tumor invasion. Collectively, our results suggest that OSCC stimulates fibroblasts to produce LIF, which, in turn, participates in cancer-cell invasion. Our finding offers a potential therapeutic strategy targeting the cancer stroma for the treatment of OSCC patients.

Extraction of DNA from human embryos after long-term preservation in formalin and Bouin's solutions.

The "Kyoto Collection of Human Embryos" at Kyoto University was begun in 1961. Although morphological analyses of samples in the Kyoto Collection have been performed, these embryos have been considered difficult to genetically analyze because they have been preserved in formalin or Bouin's solution for 20-50 years. Owing to the recent advances in molecular biology, it has become possible to extract DNA from long-term fixed tissues. The purpose of this study was to extract DNA from wet preparations of human embryo samples after long-term preservation in fixing solution. We optimized the DNA extraction protocol to be suitable for tissues that have been damaged by long-term fixation, including DNA-protein crosslinking damage. Diluting Li2 CO3 with 70% ethanol effectively removed picric acid from samples fixed in Bouin's solution. Additionally, 20.0 mg/mL proteinase was valuable to lyse the long-term fixed samples. The extracted DNA was checked with PCR amplification using several sets of primers and sequence analysis. The PCR products included at least 295- and 838-bp amplicons. These results show that the extracted DNA is applicable for genetic analyses, and indicate that old embryos in the Kyoto Collection should be made available for future studies. The protocol described in this study can successfully extract DNA from old specimens and, with improvements, should be applicable in research aiming to understand the molecular mechanisms of human congenital anomalies.