Disruption of the p16INK4A‐CDK4‐pRB Tumor Suppressor Pathway and Telomerase‐Mediated Immortalization Is Sufficient to Initiate the Malignant Conversion of Primary Oral Keratinocytes

Head and neck squamous carcinoma (HNSCC) is the 6 th most common cancer in the developed world. Its 5 year survival rate, less than 50%, has seen only marginal changes within the past 3 decades. To develop a suitable in vitro system to study the process of malignant conversion of oral epithelium, individual keratinocyte cultures were established from normal oral mucosa samples from NIH patients (NIDCR, NIH Protocol #06‐D‐0144) and immortalized with lentiviruses expressing human telomerase (hTERT) and wild type cyclin dependent kinase 4 (wt‐CDK4) (n=25); or infected with lentiviruses expressing hTERT and a mutated CDK4 (Arg residue number 21 mutated to Cys [R21C]) insensitive to p16 INK4A blockade (mut‐CDK4) (n=10). Those infected with mut‐CDK4 underwent progressive phenotypic changes. Two million cells from 4 different stables wt‐CDK4 + hTERT and mut‐CDK4 + hTERT lines were injected into FBV nu−/nu− mice. 65–90 days after injection, anaplastic carcinomas developed in mice implanted with mut‐CDK4 + hTERT. Since decreased expression or activity of the tumor suppressor protein p16 INK4A by mutations, allelic loss, and epigenetic events is one of the earliest events in HNSCC, our results provide evidence that the continuous growth of cells harboring an inactive p16 INK4A ‐CDK4‐pRB tumor suppressor pathway may enable the subsequent accumulation of genetic alterations leading to cancer.