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The epidermis is a self-renewal epithelium continuously exposed to the genotoxic effects of ultraviolet (UV) light, the main cause of skin cancer. Therefore, it needs robust self-protective mechanisms facing genomic damage. p53 has been shown to mediate apoptosis in sunburn cells of the epidermis. However, epidermal cells daily receive sublethal mutagenic doses of UV and massive apoptosis would be deleterious. We have recently unravelled an anti-oncogenic keratinocyte DNA damage-differentiation response to cell cycle stress. We now have studied this response to high or moderate single doses of UV irradiation. Whereas, as expected, high levels of UV induced p53-dependent apoptosis, moderate levels triggered squamous differentiation. UV-induced differentiation was not mediated by endogenous p53. Overexpression of the mitosis global regulator FOXM1 alleviated the proliferative loss caused by UV. Conversely, knocking-down the mitotic checkpoint protein Wee1 drove UV-induced differentiation into apoptosis. Therefore, the results indicate that mitosis checkpoints determine the response to UV irradiation. The differentiation response was also found in cells of head and neck epithelia thus uncovering a common regulation in squamous tissues upon chronic exposure to mutagens, with implications into homeostasis and disease.

Squamous cell carcinoma (SCC) or epidermoid cancer is a frequent and aggressive malignancy. However in apparent paradox it retains the squamous differentiation phenotype except for very dysplastic lesions. We have shown that cell cycle stress in normal epidermal keratinocytes triggers a squamous differentiation response involving irreversible mitosis block and polyploidisation. Here we show that cutaneous SCC cells conserve a partial squamous DNA damage-induced differentiation response that allows them to overcome the cell division block. The capacity to divide in spite of drug-induced mitotic stress and DNA damage made well-differentiated SCC cells more genomically instable and more malignant in vivo. Consistently, in a series of human biopsies, non-metastatic SCCs displayed a higher degree of chromosomal alterations and higher expression of the S phase regulator Cyclin E and the DNA damage signal γH2AX than the less aggressive, non-squamous, basal cell carcinomas. However, metastatic SCCs lost the γH2AX signal and Cyclin E, or accumulated cytoplasmic Cyclin E. Conversely, inhibition of endogenous Cyclin E in well-differentiated SCC cells interfered with the squamous phenotype. The results suggest a dual role of cell cycle stress-induced differentiation in squamous cancer: the resulting mitotic blocks would impose, when irreversible, a proliferative barrier, when reversible, a source of genomic instability, thus contributing to malignancy. AG is grateful to Jean-Jeaques Guilhou, Jean-Claude Rossi and the INSERM for professional support and to Renata Polakowska for the generous gift of precious BCCP cell line. We thank Lucía Barbier, Tania Lobato, Evelyn Andrades, Alicia Noriega and María Aramburu for technical assistance and Natalia Sanz for critical reading of the MS. To AG: National grants from Instituto de Salud Carlos III, Fondo de Investigación Sanitaria (ISCIII-FIS/FEDER, Spain): PI08/0890, PI11/02070, PI14/00900; Ligue Nationale Contre la Cancer (La Ligue; France). To AT: ISCIII-FIS PI10/00785. To JP: MINECO grant SAF2015-66015-R; AES grant ISCIII-RETIC RD12/0036/0009. VC was funded by a fellowship from La Ligue (France), PA by IDIVAL (Spain), RM and IdP by AG lab and ISCIII-FIS-FEDER PI11/02070 (Spain).

This is an open-access article distributed under the terms of the Creative Commons Attribution License., CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, down-regulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis., This study was supported by the Instituto de Salud Carlos III-Fondo de Investigaciones Sanitarias (ISCIII-FIS; National Grants, Spain): FIS-PI08/0829, FIS-PI11/00397 and Red Temática de Investigación Cooperativa en Cáncer-RTICC-RD06/0020/017 (MDD); FIS-PI08/0890, FIS-PI11/02070 and Programa Regiones Emergentes-EMER (AG); by the Société Française de Dermatologie (SFD, France; AG); and by a co-operative grant from Universidad de Cantabria-Fundación Marqués de Valdecilla-IFIMAV (Cantabria, Spain; Javier León-AG). MRG was recipient of a fellowship from University of Cantabria. Both MRG and PA were also funded by Fundación Leonardo Torres Quevedo and Fundación Marqués de Valdecilla-IFIMAV. AG is ‘on leave’ at the Institut National de la Santé et de la Recherche Médicale (INSERM, France).

The role of p53, the original “guardian of the genome”, in skin has remained elusive. We have explored p53 function in human epidermal cells and demonstrated the importance of a mitosis-differentiation checkpoint to suppress potentially precancerous cells. This model places epidermal endoreplication as an antioncogenic mechanism in the face of irreparable genetic alterations.

Summary Tumor suppressor p53 is a major cellular guardian of genome integrity, and its inactivation is the most frequent genetic alteration in cancer, rising up to 80% in squamous cell carcinoma (SCC). By adapting the small hairpin RNA (shRNA) technology, we inactivated endogenous p53 in primary epithelial cells from the epidermis of human skin. We show that either loss of endogenous p53 or overexpression of a temperature-sensitive dominant-negative conformation triggers a self-protective differentiation response, resulting in cell stratification and expulsion. These effects follow DNA damage and exit from mitosis without cell division. p53 preserves the proliferative potential of the stem cell compartment and limits the power of proto-oncogene MYC to drive cell cycle stress and differentiation. The results provide insight into the role of p53 in self-renewal homeostasis and help explain why p53 mutations do not initiate skin cancer but increase the likelihood that cancer cells will appear.