DNA damage triggers squamous metaplasia in human lung and mammary cells via mitotic checkpoints.

Epithelial transdifferentiation is frequent in tissue hyperplasia and contributes to disease in various degrees. Squamous metaplasia (SQM) precedes epidermoid lung cancer, an aggressive and frequent malignancy, but it is rare in the epithelium of the mammary gland. The mechanisms leading to SQM in the lung have been very poorly investigated. We have studied this issue on human freshly isolated cells and organoids. Here we show that human lung or mammary cells strikingly undergo SQM with polyploidisation when they are exposed to genotoxic or mitotic drugs, such as Doxorubicin or the cigarette carcinogen DMBA, Nocodazole, Taxol or inhibitors of Aurora-B kinase or Polo-like kinase. To note, the epidermoid response was attenuated when DNA repair was enhanced by Enoxacin or when mitotic checkpoints where abrogated by inhibition of Chk1 and Chk2. The results show that DNA damage has the potential to drive SQM via mitotic checkpoints, thus providing novel molecular candidate targets to tackle lung SCC. Our findings might also explain why SCC is frequent in the lung, but not in the mammary gland and why chemotherapy often causes complicating skin toxicity.

p21CIP1 controls the squamous differentiation response to replication stress.

The control of cell fate is critical to homeostasis and cancer. Cell cycle cdk inhibitor p21CIP1 has a central and paradoxical role in the regulatory crossroads leading to senescence, apoptosis, or differentiation. p21 is an essential target of tumor suppressor p53, but it also is regulated independently. In squamous self-renewal epithelia continuously exposed to mutagenesis, p21 controls cell fate by mechanisms still intriguing. We previously identified a novel epidermoid DNA damage-differentiation response. We here show that p21 intervenes in the mitosis block that is required for the squamous differentiation response to cell cycle deregulation and replication stress. The inactivation of endogenous p21 in human primary keratinocytes alleviated the differentiation response to oncogenic loss of p53 or overexpression of the DNA replication major regulator Cyclin E. The bypass of p21-induced mitotic block involving upregulation of Cyclin B allowed DNA damaged cells to escape differentiation and continue to proliferate. In addition, loss of p21 drove keratinocytes from differentiation to apoptosis upon moderate UV irradiation. The results show that p21 is required to drive keratinocytes towards differentiation in response to genomic stress and shed light into its dual and paradoxical role in carcinogenesis.

The DNA damage response links human squamous proliferation with differentiation.

How rapid cell multiplication leads to cell differentiation in developing tissues is still enigmatic. This question is central to morphogenesis, cell number control, and homeostasis. Self-renewal epidermoid epithelia are continuously exposed to mutagens and are the most common target of cancer. Unknown mechanisms commit rapidly proliferating cells to post-mitotic terminal differentiation. We have over-activated or inhibited the endogenous DNA damage response (DDR) pathways by combinations of activating TopBP1 protein, specific shRNAs, or chemical inhibitors for ATR, ATM, and/or DNA-PK. The results dissect and demonstrate that these signals control keratinocyte differentiation in proliferating cells independently of actual DNA damage. The DDR limits keratinocyte multiplication upon hyperproliferative stimuli. Moreover, knocking down H2AX, a common target of the DDR pathways, inhibits the epidermoid phenotype. The results altogether show that the DDR is required to maintain the balance proliferation differentiation and suggest that is part of the squamous program. We propose a homeostatic model where genetic damage is automatically and continuously cleansed by cell-autonomous mechanisms.

Keratinocyte Differentiation by Flow Cytometry.

The epidermis is continuously exposed to environmental hazard and undergoes continuous cell renewal. The maintenance of the epidermal balance between proliferation and differentiation is essential for the homeostasis of the skin. Proliferation and terminal differentiation are compartmentalized in basal and suprabasal layers, respectively. These compartments can be identified by different patterns of protein expression that can be used as differentiation markers. For instance, components of the intermediate filament cytoskeleton keratins K5 and K14 are confined to the proliferative basal layer, while keratins K1 and K10, keratins K6 and K16, or precursors of the cornified envelope such as involucrin are expressed by suprabasal terminally differentiating cells. The analysis of the expression of these markers allows studying the imbalance typical of disease. Although these markers have been traditionally analyzed on skin microsections, on attached cells by immunostaining or by western blotting, it is possible and advantageous to quantify them by flow cytometry. We have extensively applied this technology onto human and mouse keratinocytes. Here we describe detailed flow cytometry methods to determine the differentiation status of keratinocyte populations.

Genetic Modification of Human Primary Keratinocytes by Lentiviral Vectors.

Keratinocytes are hard to transfect. Viral vectors are a good alternative to genetically modify primary keratinocytes. A classical method is the use of retroviral vectors by co-culture of keratinocytes with virus-producer cells. This method is efficient in high-calcium conditions with feeder cells. However, sometimes co-culture is not possible and is more laborious as producer cells need to be replaced by feeder cells. Our solution is the use of lentiviral vectors, far more efficient as supernatant on keratinocytes. In this chapter we describe improved detailed protocols for stable genetic modification of human primary keratinocytes of the skin or head and neck, in both low- and high-calcium conditions by lentiviral vectors.

Response of head and neck epithelial cells to a DNA damage-differentiation checkpoint involving polyploidization.

BACKGROUND: Squamous epithelia of the head and neck undergo continuous cell renewal and are continuously exposed to mutagenic hazard, the main cause of cancer. How they maintain homeostasis upon cell cycle deregulation is unclear. METHODS: To elucidate how head and neck epithelia respond to cell cycle stress, we studied human keratinocytes from various locations (oral mucosa, tonsil, pharynx, larynx, and trachea). We made use of genotoxic or mitotic drugs (doxorubicin [DOXO], paclitaxel, and nocodazole), or chemical inhibitors of the mitotic checkpoint kinases, Aurora B and polo-like-1. We further tested the response to inactivation of p53, ectopic cyclin E, or to the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). RESULTS: All treatments provoked DNA damage or mitosis impairment and strikingly triggered squamous differentiation and polyploidization, resulting in irreversible loss of clonogenic capacity. CONCLUSION: Keratinocytes from head and neck epithelia share a cell-autonomous squamous DNA damage-differentiation response that is common to the epidermis and might continuously protect them from cancer.

Sublethal UV irradiation induces squamous differentiation via a p53-independent, DNA damage-mitosis checkpoint.

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.

Inefficient differentiation response to cell cycle stress leads to genomic instability and malignant progression of squamous carcinoma cells.

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.

The mitosis-differentiation checkpoint, another guardian of the epidermal genome.

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.

Inactivation of p53 in Human Keratinocytes Leads to Squamous Differentiation and Shedding via Replication Stress and Mitotic Slippage.

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.

Cycling up the epidermis: reconciling 100 years of debate.

There is likely general consensus within the skin research community that cell cycle control is critical to epidermal homeostasis and disease. The current predominant model proposes that keratinocytes switch off DNA replication and undergo cell cycle and cell growth arrest as they initiate terminal differentiation. However, this model cannot explain key physiological features of the skin, mainly why squamous differentiation prevails over proliferation in benign hyperproliferative disorders. In recent years, we have proposed an alternative model that involves mitotic slippage and endoreplication. This new model is controversial and has encountered resistance within the field. However, looking back at history, the epidermal cell cycle has been a matter of controversy and debate for around 100 years now. The accumulated data are confusing and contradictory. Our present model can explain and reconcile both old and new paradoxical observations. Here, we explain and discuss the endoreplicative cell cycle, the evidence for and against its existence in human epidermis and the important implications for skin homeostasis and disease. We show that regardless of the strengths or weaknesses of the Endoreplication Model, the existing evidence in support of the Cell Cycle Arrest Model is very weak.

A mitosis block links active cell cycle with human epidermal differentiation and results in endoreplication.

How human self-renewal tissues co-ordinate proliferation with differentiation is unclear. Human epidermis undergoes continuous cell growth and differentiation and is permanently exposed to mutagenic hazard. Keratinocytes are thought to arrest cell growth and cell cycle prior to terminal differentiation. However, a growing body of evidence does not satisfy this model. For instance, it does not explain how skin maintains tissue structure in hyperproliferative benign lesions. We have developed and applied novel cell cycle techniques to human skin in situ and determined the dynamics of key cell cycle regulators of DNA replication or mitosis, such as cyclins E, A and B, or members of the anaphase promoting complex pathway: cdc14A, Ndc80/Hec1 and Aurora kinase B. The results show that actively cycling keratinocytes initiate terminal differentiation, arrest in mitosis, continue DNA replication in a special G2/M state, and become polyploid by mitotic slippage. They unambiguously demonstrate that cell cycle progression coexists with terminal differentiation, thus explaining how differentiating cells increase in size. Epidermal differentiating cells arrest in mitosis and a genotoxic-induced mitosis block rapidly pushes epidermal basal cells into differentiation and polyploidy. These observations unravel a novel mitosis-differentiation link that provides new insight into skin homeostasis and cancer. It might constitute a self-defence mechanism against oncogenic alterations such as Myc deregulation.