Cigarette smoking causes cycles of injury and repair of the airway and is a known cause of lung cancer (24
). We, and others, have shown that K14+ progenitor cells are a reparative cell population and contribute to repair of the epithelium of the cartilaginous airways and in the more distant bronchioles after injury, such as hypoxic-ischemic injury, naphthalene injection and sulfur dioxide inhalation (12, 16
). Here we propose that in the context of injury, K5+K14+ cells originate from the submucosal gland K5+K14+ cells and/or from the K5+K14- basal cells that then acquire K14 expression on the repairing surface airway epithelium. However, once normal repair is completed, K14 expression is no longer seen in the mature basal cells of the pseudostratified columnar epithelium. This implies that K14 expression is tightly regulated at steady state and the persistence of K5+K14+ cells on the surface airway epithelium after injury represents self-renewing cells that do not differentiate to mature airway epithelial cell types and represent dysregulated repair. Our data are, therefore, consistent with the development of dysregulated repair after injury leading to a self-renewing K14+ progenitor cell population in premalignant lesions. These cells could therefore potentially survive long enough to accumulate the genetic and epigenetic mutations that are thought to be necessary to develop a tumor (3
). We found that the presence of dysregulated K14+ progenitor cells in NSCLC after chronic smoking injury was associated with increased mortality from lung cancer. This implies that there could be a novel putative tumor-initiating cell population in a subset of smoking-related NSCLCs with a poor prognosis.
A self-renewing tumor-initiating cell population associated with poor prognosis in human NSCLC has not yet been described. Kim et al
isolated a putative lung stem cell termed the bronchoalveolar stem cell or BASC, which expressed markers of both Clara cells (CCSP) and type II pneumocytes (SP-C), proliferated for repair and were seen in the earliest cancerous lesions and increased as the tumors advanced (25
). However, these studies were performed in mice and it is not clear what the equivalent human cell surface markers are that would enable the purification and propagation of these cells in xenograft models to determine whether these are truly CSCs in lung cancer patients. In addition, the heterogeneity of lung cancers suggests that there are likely to be multiple tumor-initiating cell populations for different lung cancer histologic subtypes and locations. K14-expressing cells have been found for repair in the distal bronchioles16
and we found K14 mRNA and protein expression in adenocarcinomas as well as squamous cell cancers. In addition, K14 expression correlated with poor prognosis in all NSCLC histologic subtypes, although it only correlated with metastases in non-adenocarcinoma histologies.
Classical validation of a CSC tumor-initiating cell population involves reconstituting the human tumor in an immunodeficient mouse, followed by the indefinite serial xenotransplantation of these CSCs. Eramo et al
found CD133 expression in both small cell and non-small cell lung tumors. High numbers of CD133+epCAm+ cells isolated from fresh lung tumor specimens were capable of generating tumor xenografts upon subcutaneous injection. However, the self-renewal capacity of CD133+ cells was not evident and CD133 expression was found not to be prognostic in NSCLC although it did correlate with expression of chemotherapy resistance genes (26
). In order to demonstrate the tumor-initiating potential of K14-expressing cells in NSCLC, by current definitions, the development and serial transplantation of NSCLC in immunodeficient mice is required (27
). However, no surface markers have as yet been identified to allow the isolation of live K14-expressing cells from tumors. It is therefore not currently possible to evaluate the tumor-initiating ability of K14-expressing cells in NSCLC in a serial xenotransplantation model. We are therefore not functionally able to test the tumor-initiating potential of K14-expressing cells.
Precursor lesions of squamous lung cancer are known to have high levels of K14 expression, from basal/reserve cell hyperplasia to squamous metaplasia and dysplasia to carcinoma in situ
as well as invasive carcinoma itself (28
). Our data suggest that K14-expressing cells in the airway epithelium in premalignant lesions may represent self-renewing, reparative progenitor cells, that may have the potential to be tumor-initiating cells. We also believe that K14 expression alone is not sufficient to generate a malignancy and that subsequent genetic and epigenetic changes are needed to develop NSCLC. This is illustrated by work from Dakir et al
who used a mouse Clara cell specific 10kDa protein promoter (CC10) to constitutively express human K14 in bronchial epithelium. The CC10-hK14 overexpressing transgenic mouse developed a squamous differentiation program in the mouse lung, but failed to promote squamous maturation with rare squamous metaplastic lesions and squamous carcinomas in old age mice (28
). This supports the idea that K14 expression in airway epithelial cells is a marker of a self-renewing progenitor cell, and is a putative tumor-initiating cell, which requires genetic and/or epigenetic changes in order to be sufficient for carcinogenesis. While we found no difference in the proliferative capacity of K14-expressing cells compared to non-K14-expressing cells in premalignant lesions and in NSCLC, it is possible that the K14+ cells are an important subset of tumor cells as the keratin14 cytoskeletal protein may allow for changes in cell shape and motility with an increased potential for cell migration.
In summary, the presence of K14+ cells in NSCLC is a biomarker of tumors with a worse prognosis. This was especially predictive in smokers, and furthermore these patients had an increased likelihood of metastases. K14 expression in NSCLC in smokers may therefore be useful as a biomarker of poor prognosis and of metastases in squamous lung cancer. Furthermore, identifying the genetic and epigenetic changes that occur in the K14+ cell population that lead to dysregulated repair may result in the discovery of novel biomarkers and therapeutic targets for chemoprevention in smokers(29