S100a6 knockdown promotes the differentiation of dental epithelial cells toward the epidermal lineage instead of the odontogenic lineage.

Tooth development is a complex process involving various signaling pathways and genes. Recent findings suggest that ion channels and transporters, including the S100 family of calcium-binding proteins, may be involved in tooth formation. However, our knowledge in this regard is limited. Therefore, this study aimed to investigate the expression of S100 family members and their functions during tooth formation. Tooth germs were extracted from the embryonic and post-natal mice and the expression of S100a6 was examined. Additionally, the effects of S100a6 knockdown and calcium treatment on S100a6 expression and the proliferation of SF2 cells were examined. Microarrays and single-cell RNA-sequencing indicated that S100a6 was highly expressed in ameloblasts. Immunostaining of mouse tooth germs showed that S100a6 was expressed in ameloblasts but not in the undifferentiated dental epithelium. Additionally, S100a6 was localized to the calcification-forming side in enamel-forming ameloblasts. Moreover, siRNA-mediated S100a6 knockdown in ameloblasts reduced intracellular calcium concentration and the expression of ameloblast marker genes, indicating that S100a6 is associated with ameloblast differentiation. Furthermore, S100a6 knockdown inhibited the ERK/PI3K signaling pathway, suppressed ameloblast proliferation, and promoted the differentiation of the dental epithelium toward epidermal lineage. Conclusively, S100a6 knockdown in the dental epithelium suppresses cell proliferation via calcium and intracellular signaling and promotes differentiation of the dental epithelium toward the epidermal lineage.

The Role of Genetically Modified Human Feeder Cells in Maintaining the Integrity of Primary Cultured Human Deciduous Dental Pulp Cells.

Tissue-specific stem cells exist in tissues and organs, such as skin and bone marrow. However, their pluripotency is limited compared to embryonic stem cells. Culturing primary cells on plastic tissue culture dishes can result in the loss of multipotency, because of the inability of tissue-specific stem cells to survive in feeder-less dishes. Recent findings suggest that culturing primary cells in medium containing feeder cells, particularly genetically modified feeder cells expressing growth factors, may be beneficial for their survival and proliferation. Therefore, the aim of this study was to elucidate the role of genetically modified human feeder cells expressing growth factors in maintaining the integrity of primary cultured human deciduous dental pulp cells. Feeder cells expressing leukemia inhibitory factor, bone morphogenetic protein 4, and basic fibroblast growth factor were successfully engineered, as evidenced by PCR. Co-culturing with mitomycin-C-treated feeder cells enhanced the proliferation of newly isolated human deciduous dental pulp cells, promoted their differentiation into adipocytes and neurons, and maintained their stemness properties. Our findings suggest that genetically modified human feeder cells may be used to maintain the integrity of primary cultured human deciduous dental pulp cells.

FLCN alteration drives metabolic reprogramming towards nucleotide synthesis and cyst formation in salivary gland.

FLCN is a tumor suppressor gene which controls energy homeostasis through regulation of a variety of metabolic pathways including mitochondrial oxidative metabolism and autophagy. Birt-Hogg-Dubé (BHD) syndrome which is driven by germline alteration of the FLCN gene, predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas, pulmonary cysts and less frequently, salivary gland tumors. Here, we report metabolic roles for FLCN in the salivary gland as well as their clinical relevance. Screening of salivary glands of BHD patients using ultrasonography demonstrated increased cyst formation in the salivary gland. Salivary gland tumors that developed in BHD patients exhibited an upregulated mTOR-S6R pathway as well as increased GPNMB expression, which are characteristics of FLCN-deficient cells. Salivary gland-targeted Flcn knockout mice developed cytoplasmic clear cell formation in ductal cells with increased mitochondrial biogenesis, upregulated mTOR-S6K pathway, upregulated TFE3-GPNMB axis and upregulated lipid metabolism. Proteomic and metabolite analysis using LC/MS and GC/MS revealed that Flcn inactivation in salivary gland triggers metabolic reprogramming towards the pentose phosphate pathway which consequently upregulates nucleotide synthesis and redox regulation, further supporting that Flcn controls metabolic homeostasis in salivary gland. These data uncover important roles for FLCN in salivary gland; metabolic reprogramming under FLCN deficiency might increase nucleotide production which may feed FLCN-deficient salivary gland cells to trigger tumor initiation and progression, providing mechanistic insight into salivary gland tumorigenesis as well as a foundation for development of novel therapeutics for salivary gland tumors.

BHD-associated kidney cancer exhibits unique molecular characteristics and a wide variety of variants in chromatin remodeling genes.

Birt-Hogg-Dubé (BHD) syndrome is a hereditary kidney cancer syndrome, which predisposes patients to develop kidney cancer, cutaneous fibrofolliculomas and pulmonary cysts. The responsible gene FLCN is a tumor suppressor for kidney cancer, which plays an important role in energy homeostasis through the regulation of mitochondrial oxidative metabolism. However, the process by which FLCN-deficiency leads to renal tumorigenesis is unclear. In order to clarify molecular pathogenesis of BHD-associated kidney cancer, we conducted whole-exome sequencing analysis using next-generation sequencing technology as well as metabolite analysis using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Whole-exome sequencing analysis of BHD-associated kidney cancer revealed that copy number variations of BHD-associated kidney cancer are considerably different from those already reported in sporadic cases. In somatic variant analysis, very few variants were commonly observed in BHD-associated kidney cancer; however, variants in chromatin remodeling genes were frequently observed in BHD-associated kidney cancer (17/29 tumors, 59%). Metabolite analysis of BHD-associated kidney cancer revealed metabolic reprogramming toward upregulated redox regulation which may neutralize reactive oxygen species potentially produced from mitochondria with increased respiratory capacity under FLCN-deficiency. BHD-associated kidney cancer displays unique molecular characteristics that are completely different from sporadic kidney cancer, providing mechanistic insight into tumorigenesis under FLCN-deficiency as well as a foundation for development of novel therapeutics for kidney cancer.

[PATHOLOGICAL COMPLETE RESPONSE WITH EVEROLIMUS FOR MULTIPLE LUNG METASTASES OF RENAL CELL CARCINOMA RESISTANT TO SUNITINIB].

A 55-year-old man was referred to our hospital because of a tumor in his right kidney. A Fluorodeoxyglucose-positron emission tomography (PET)/computed tomography (CT) scan revealed strong abnormal uptake by the tumor in the right kidney and a nodule in the right lung. The patient was diagnosed with stage IV renal cell cancer, for which he underwent transperitoneal nephrectomy. Pathological diagnosis revealed the tumor to be a renal cell carcinoma (clear cell carcinoma, G2, pT3a, v (+), INF-ß). Sunitinib was administered because of the occurrence of multiple lung metastases; however, the therapeutic effect was insufficient, and progressive disease was observed on a CT scan. Therefore, everolimus was immediately administered as a second-line therapeutic agent. After treatment, the lung metastases reduced in size, as observed on a CT scan, and partial response continued for 1 year after therapy. One metastatic lesion persisted in the right lung; therefore, he underwent right upper lobe resection after discontinuing everolimus administration. No viable tumor cells were observed on pathological diagnosis, and the patient achieved pathological complete response. 3 month after discontinuing everolimus administration, no metastatic lesions have been observed.

Early ultrasonographic evaluation of tumor thrombus level during sunitinib therapy for renal cell carcinoma.

A 72-year-old man presenting with a 14-cm left renal mass, an inferior vena cava (IVC) tumor thrombus, and pulmonary metastases underwent renal mass biopsy that revealed clear cell renal cell carcinoma. Because of metastases and the extent of the tumor thrombus, sunitinib was administered, which resulted in a marked reduction in the tumor thrombus (from level III to level II after 11 weeks of treatment). Ultrasonography, preceding computed tomography, showed a slight shrinkage of the tumor thrombus level in the first 2 weeks. Therefore, ultrasound may be advantageous to monitor the IVC tumor thrombus level during the early phase of targeted therapy.