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

Abstract

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.

p53 immunoexpression in stepwise progression of cutaneous squamous cell carcinoma and correlation with angiogenesis and cellular proliferation

“…As recently proposed keratinocytes may have defensive pathways to counterbalance TP53 mutation, to wit mitosis slippage, cell differentiation and shedding. In that sense an additional step would be necessary to allow p53 mutated cells to escape the proliferative block, divide and originate malignant clones [26]. Thus, mutation of a single gene such as TP53 may contribute to stepwise carcinogenesis in multifaceted ways.…”

“…Applying the theory for CSCC, authors believe that a new event would be responsible for bypassing such mitosis block. Thus, cells would accumulate DNA damage leading to high genomic instability and emergence of malignant clones [26]. p53 imunoexpression is frequently more prominent in CSCC periphery with gradual loss of staining as cells differentiate [14], even keeping p53 mutation [11].…”

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

“…9, 10, 11, 12 This DNA damage response (DDR) suppresses cell division but allows extra rounds of DNA replication resulting in polyploidy. This involves mitotic bypass (without intervening mitosis), mitotic slippage (defined as failure to arrest in G2/M) 13 or acytokinetic mitosis.…”

Clasp2 ensures mitotic fidelity and prevents differentiation of epidermal keratinocytes

“…A role for p53 in limiting the proliferation of polyploid cells has long been recognized (Ganem et al . , 2014), and in human keratinocytes its inactivation further potentiates squamous differentiation (Freije et al, 2014). To test if p53 has a similar role in the context of Clasp2 deficiency, we knocked down Clasp2 expression in p53-null mouse keratinocytes (Fig.…”