Paracrine Interaction of Cancer Stem Cell Populations Is Regulated by the Senescence-Associated Secretory Phenotype (SASP).

Dyskeratosis congenita is a telomere DNA damage syndrome characterized by defective telomere maintenance, bone marrow failure, and increased head and neck cancer risk. The Pot1b-/-;Terc+/- mouse exhibits some features of dyskeratosis congenita, but head and neck cancer was not reported in this model. To model the head and neck cancer phenotype, we created unique Pot1b- and p53-null-mutant models which allow genetic lineage tracing of two distinct stem cell populations. Loss of Pot1b expression depleted stem cells via ATR/Chk1/p53 signaling. Tumorigenesis was inhibited in Pot1b-/-;p53+/+ mice due to cellular senescence. Pot1b-/-;p53-/- tumors also exhibited senescence, but proliferated and metastasized with expansion of Lgr6+ stem cells indicative of senescence-associated secretory phenotype. Selective depletion of the small K15+ stem cell fraction resulted in reduction of Lgr6+ cells and inhibition of tumorigenesis via senescence. Gene expression studies revealed that K15+ cancer stem cells regulate Lgr6+ cancer stem cell expansion via chemokine signaling. Genetic ablation of the chemokine receptor Cxcr2 inhibited cancer stem cell expansion and tumorigenesis via senescence. The effects of chemokines were primarily mediated by PI3K signaling, which is a therapeutic target in head and neck cancer. IMPLICATIONS: Paracrine interactions of cancer stem cell populations impact therapeutic options and patient outcomes.

Histone Methylation Mechanisms Modulate the Inflammatory Response of Periodontal Ligament Progenitors.

Inflammatory conditions affect periodontal ligament (PDL) homeostasis and diminish its regenerative capacity. The complexity of biological activities during an inflammatory response depends on genetic and epigenetic mechanisms. To characterize the epigenetic changes in response to periodontal pathogens we have focused on histone lysine methylation as a relatively stable chromatin modification involved in the epigenetic activation and repression of transcription and a prime candidate mechanism responsible for the exacerbated and prolonged response of periodontal cells and tissues to dental plaque biofilm. To determine the effect of inflammatory conditions on histone methylation profiles, related gene expression and cellular functions of human periodontal ligament (hPDL) progenitor cells, a hPDL cell culture system was subjected to bacterial cell wall toxin exposure [lipopolysaccharide (LPS)]. Chromatin immunoprecipitation-on-chip analysis revealed that healthy PDL cells featured high enrichment levels for the active H3K4me3 mark at COL1A1, COL3, and RUNX2 gene promoters, whereas there were high occupancy levels for the repressive H3K27me3 marks at DEFA4, CCL5, and IL-1ß gene promoters. In response to LPS, H3K27me3 enrichment increased on extracellular matrix and osteogenesis lineage gene promoters, whereas H3K4me3 enrichment increased on the promoters of inflammatory response genes, suggestive of an involvement of epigenetic mechanisms in periodontal lineage differentiation and in the coordination of the periodontal inflammatory response. On a gene expression level, LPS treatment downregulated COL1A1, COL3A1, and RUNX2 expression and upregulated CCL5, DEFA4, and IL-1ß gene expression. LPS also greatly affected PDL progenitor function, including a reduction in proliferation and differentiation potential and an increase in cell migration capacity. Confirming the role of epigenetic mechanisms in periodontal inflammatory conditions, our studies highlight the significant role of histone methylation mechanisms and modification enzymes in the inflammatory response to LPS bacterial cell wall toxins and periodontal stem cell function.

MicroRNAs and immunity in periodontal health and disease.

MicroRNAs (miRNAs) are critical regulators of the host immune and inflammatory response against bacterial pathogens. In the present review, we discuss target genes, target gene functions, the potential regulatory role of miRNAs in periodontal tissues, and the potential role of miRNAs as biomarkers and therapeutics. In periodontal disease, miRNAs exert control over all aspects of innate and adaptive immunity, including the functions of neutrophils, macrophages, dendritic cells and T and B cells. Previous human studies have highlighted some key miRNAs that are dysregulated in periodontitis patients. In the present study, we mapped the major miRNAs that were altered in our reproducible periodontitis mouse model relative to control animals. The miRNAs that were upregulated as a result of periodontal disease in both human and mouse studies included miR-15a, miR-29b, miR-125a, miR-146a, miR-148/148a and miR-223, whereas miR-92 was downregulated. The association of individual miRNAs with unique aspects of periodontal disease and their stability in gingival crevicular fluid underscores their potential as markers for periodontal disease progression or healthy restitution. Moreover, miRNA therapeutics hold great promise for the future of periodontal therapy because of their ability to modulate the immune response to infection when applied in conjunction with synthetic antagomirs and/or relatively straightforward delivery strategies.

Differential Apoptosis in Mucosal and Dermal Wound Healing.

Objectives: Dermal and mucosal healing are mechanistically similar. However, scarring and closure rates are dramatically improved in mucosal healing, possibly due to differences in apoptosis. Apoptosis, nature's preprogrammed form of cell death, occurs via two major pathways, extrinsic and intrinsic, which intersect at caspase3 (Casp3) cleavage and activation. The purpose of this experiment was to identify the predominant pathways of apoptosis in mucosal and dermal wound healing. Approach: Wounds (1 mm biopsy punch) were made in the dorsal skin (n=3) or tongue (n=3) of female Balb/C mice aged 6 weeks. Wounds were harvested at 6 h, 24 h, day 3 (D3), D5, D7, and D10. RNA was isolated and analyzed using real time reverse transcriptase-polymerase chain reaction. Expression levels for genes in the intrinsic and extrinsic apoptotic pathways were compared in dermal and mucosal wounds. Results: Compared to mucosal healing, dermal wounds exhibited significantly higher expression of Casp3 (at D5; p<0.05), Casp7 (at D5; p<0.05), Trp53 (at 24 h and D5; p<0.05), Tnfrsf1b (at 24 h; p<0.05), FasR (at 24 h, D5, and D7; p<0.05), and Casp8 (at 24 h; p<0.05) and significantly lower gene expression of Tradd (at 24 h; p<0.05). Innovation: Our observations indicate differential execution of apoptosis in oral wound healing compared to skin. Conclusion: Expression patterns of key regulators of apoptosis in wound healing indicate that apoptosis occurs predominantly through the intrinsic pathway in the healing mucosa, but predominantly through the extrinsic pathway in the healing skin. The identification of differences in the apoptotic pathways in skin and mucosal wounds may allow the development of therapeutics to improve skin healing.