Stem cells are of great interest to the scientific community due to their potential role in regenerative and rejuvenative medicine. However, their role in the aging process and carcinogenesis remains unclear. Because DNA replication in stem cells may contribute to the background mutation rate and thereby to cancer, reducing proliferation and establishing a relatively quiescent stem cell compartment has been hypothesized to limit DNA replication-associated mutagenesis. On the other hand, as the main function of stem cells is to provide daughter cells to build and maintain tissues, the idea of a quiescent stem cell compartment appears counterintuitive. Intriguing observations in mice have led to the idea of separated stem cell compartments that consist of cells with different proliferative activity. Some epithelia of short-lived rodents appear to lack quiescent stem cells. Comparing stem cells of different species and different organs (comparative stem cell biology) may allow us to elucidate the evolutionary pressures such as the balance between cancer and longevity that govern stem cell biology (evolutionary stem cell biology). The oral mucosa and its stem cells are an exciting model system to explore the characteristics of quiescent stem cells that have eluded biologists for decades.

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While proliferation is decreased in aged tissues, no change in SC number was detected by our group or Bickenbachs. Lechler et al. examined types of SC divisions. Divisions perpendicular to the basement membrane were 8% in single-layered embryonic epidermis, 66% in multilayer epidermis and 85% in adult mice. In humans there is a decrease in cells positive for the proliferation marker Ki67. Our aim was to quantify mitoses of basal keratinocytes in vivo, and examine the types of division, over the human lifespan. We quantified divisions in the basal layer in neonatal (0-7 days), adult (30-45 years) and aged (70-80 years) skin. Asymmetric segregation of polarity proteins is a defining characteristic of SCs. We visualized division orientation in the basal layer using gtubulin (spindle) and NUMA (polarity protein) in biopsies. Because of the limited divisions seen in vivo, we also quantified division type in keratinocytes from freshly obtained biopsies, in vitro, using Numb segregation to identify asymmetric SC divisions. The number of divisions/10cm of basal layer was significantly decreased with age [neonatal 7.6AE2 (n¼5) vs. adult 3.2AE0.5 (n¼4) vs. aged 1.5AE 0.4 (n¼8), p¼0.001]. Both perpendicular (asymmetric) and parallel (symmetric) divisions were decreased with age. No significant change in the proportion of perpendicular (asymmetric) SC divisions was detected (neonatal 41.7AE14.4% vs. adult 27.5AE15.9% vs. aged 36.8AE8%). Similar results were found in vitro [neonatal 29.1AE7.4% (n¼11) vs. adult 32.2% (n¼ 2) vs. aged 31.2AE11.6% (n¼ 7)]. We conclude that epidermal basal cell divisions decrease over the human lifespan such that the number of divisions in 70+ year olds is 20% of that in neonates. However, the ratio of asymmetric/symmetric divisions remains unchanged. These findings could reflect a decrease in SC number, an increase in SC quiescence, and/or longer cell cycle duration in human epidermis.

In humans, tissue homeostasis has to be maintained for decades without significant loss of functionality. Epithelia such as the oral mucosa achieve this through the self-renewing capabilities of stem cells. These life-long residents of the oral mucosa maintain homeostasis of the epithelium and at the same time have to be able to protect themselves from the main threats to longevity, i.e., stem cell exhaustion and malignant conversion and carcinogenesis. A key pathway in mediating cell cycle arrest in oral keratinocytes is mediated by TGFB superfamily signaling. There is good evidence implicating TGFB signaling as a tumor suppressor pathway in oral cancer. We have previously shown that disruption of TGFB signaling and cell adhesion can lead to oral squamous cell carcinoma. In vitro using oral keratinocytes, we found that TGFB regulates the stem cell compartment, inducing gene expression of quiescent stem cell markers MECP2, XPC, ITGB1 and PDPN. Using multiplex immunofluorescence, we have stained a tissue microarray containing 28 cases of squamous cell carcinoma, 4 adenocarcinoma, 4 metastatic carcinoma, 6 hyperplasia, and 5 adjacent inflammatory as well as normal adjacent and normal tissues. The antibodies selected were MECP2, XPC, ITGB1 and K15 in combination with the proliferation marker ki67 to determine if TGFB signaling regulates markers of stem cell quiescence. This quiescent gene signature highlighted a basal niche for slow-cycling cells, reserve stem cells. During oral disease progression, marker expression was abandoned in favor of increased proliferation. In vivo, a mouse model in which KrasG12D expression was induced and the Smad4 gene deleted specifically in the adult epithelium showed invasive cancers in the oral cavity. These Smad4-negative tumors express Sox9, a marker of human OSCCs and a TGFB target, which we have also observed to be upregulated in human premalignant lesions. Sox9-positive areas in the mouse tumors are associated with a downregulation of quiescent oral stem cell markers Krt15 and Mecp2, same as we observed in immunostainings of human tissues. In conclusion, TGFB and SMAD4 regulate quiescent stem cells and loss of this pathways allows invasive cancers to develop in the setting of oncogene expression. Furthermore, we could show that MECP2 in combination with other markers identifies quiescent basal cells in the human oral mucosa and is lost as TGFB signaling is disrupted in tumorigenesis. Citation Format: Claudia D. Andl, Anna L. Means, James S. Lewis, Alexandra Rothaus, Thomas Andl. TGFB signaling disruption in the formation of oral pre-neoplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4682.