P14^ARF Is Down-regulated During Tumour Progression and Predicts the Clinical Outcome in Human Breast Cancer

Discussion

The INK4A/ARF/INK4B locus at chromosome 9p21 encodes for three proteins with a role in the control of the cell cycle. Two of these, p16 and p15, are products of the INK4A and INK4B regions of this locus, which are separated by an intervening exon termed 1β. Both of these proteins have a role in the control of the retinoblastoma (Rb) family of cycle regulators (pRb, p107 and p130) (5).

Under normal conditions, the Rb proteins inhibit DNA synthesis. In the course of the cell cycle, this action is inhibited when Rb proteins are phosphorylated by cyclin dependent kinase (CDK) -4 and -6, thereby facilitating progression of the cell from the first gap (G₁) phase to the synthetic (S) phase of the cell cycle. p16 and p15 inhibit this action of CDK4/6 on Rb, thus enabling exit from the cell cycle (5, 6).

This Rb pathway is known to be downstream of the rat sarcoma (RAS) cascade, and the phosphatidylinositide 3-kinase (PI3K) signalling pathway. These pathways have well-known roles in a number of neoplastic diseases (7, 8). p16 has been found to have a role in a number of malignancies, including uveal melanoma, head and neck neoplasia, mesothelioma, and acute lymphoid leukaemia (9-13).

In the context of breast cancer, p16 is of interest as a member of the wider Rb pathway, which is known to have a role in invasive breast cancer, as well as in ductal carcinoma in situ (DCIS) (14). In a study of public microarray databases, Ertel et al. determined that Rb-related pathway related disruption was seen more commonly in cancerous tissue. In sub-group analysis, it was further determined that oestrogen receptor (ER)-negative tumours were more likely to have elevated p16 expression in conjunction with pRb loss, possibly reflecting a defect in Rb-mediated transcription despite elevated p16 levels. The role of the Rb pathway in oncogenesis has led to interest in p16 and other members of the pathway as markers for prognosis (15).

Witkiewicz et al. found elevated expression of p16 to be associated with Rb pathway disruptions in the context of DCIS. In addition, they confirmed an association between increased levels of p16 and ER-negative lesions. High p16 expression in conjunction with high Ki76 expression, as determined by histological staining, was found to be significantly predictive for the recurrence of DCIS (6).

However, in a study of breast tissues with atypical hyperplasia, a precursor to carcinoma in situ, Radisky et al. were unable to find an independent association between immunohistochemical staining for p16 and long-term disease prognosis and progression (16). Our findings corroborate this with regards to the mRNA expression of p16, as determined by quantitative PCR.

p16 has been characterized as a marker of cell senescence. Several studies have shown an association between p16 expression and patient age (16). p16 has been studied for its role in cell senescence. Cells can undergo replicative senescence mediated by the erosion of telomeres after a number of cell cycles, or be subject to senescence induced by cellular stress in a telomere-independent manner. The latter is termed as stress-induced premature senescence (SIPS), and is believed to be mediated by p16 (17). Studies in human lung fibroblasts have demonstrated that p16–related senescence is unaffected by the actions of hTERT (17, 18). In addition, suppression of p16 did not prevent replicative senescence in human fibroblasts (17, 19). Our findings are in keeping with these observations, with the weak correlation between p16 and hTERT likely to be due to upstream convergence in the PI3K pathway (20).

Transcription of the INK4A/ARF by a specific promoter invokes an alternative reading frame resulting in an additional protein termed p14, or ARF. p14 has a role in cell- cycle regulation, cell senescence, and apoptosis which is distinct from that of p16. It is characterized as a 15-kDa nucleolar protein, believed to exert the majority of its function through the regulation of p53, which is has a key role in DNA repair, cell-cycle regulation and metabolism (21).

The actions of p53 are suppressed by mouse double minute-2 (MDM2, also known as HDM2), which facilitates the translocation and eventual destruction of p53 in the cytosol. In turn, the transcription of MDM2 is controlled by p53, thereby completing a negative feedback loop. p14 inhibits the effect of MDM2 on p53 by sequestering MDM2 in the nucleolus (22). In addition, p14 inhibits the action of ARF-BP1, a E3 ubiquitin ligase which targets p53 (23).

Disruptions in the p53 pathway are widely observed in human oncogenesis, including breast cancer. The p53 pathway disruptions due deletion or silencing of p14 have been found in a number of neoplasias including those of the lungs, head and neck, colon, liver, and breast (21). Vestey et al. found a weak association between cytosolic p14 protein expressions in human breast cancer and overall survival (24). Aberrant methylation of p14 and p16 were shown to be associated with worse prognosis in colonic, breast and bladder carcinomas (25). Our findings regarding the prognostic implications of p14 expression are broadly in keeping with previous studies. However, our study is the first in recent literature to suggest a surge in p14 expression in early–stage human breast cancer.

In addition, p14 may form a part of a cell senescence pathway via p53, which stimulates pRb through p21. In contrast to the p16-Rb pathway, the p53-p21-pRb pathway mediates replicative senescence rather than SIPS (26). Shamanin et al. found that breast cell lines immortalised by hTERT underexpressed p14, and underwent p53-induced senescence if p14 expression was enforced (27).

Our results suggest a strong-positive correlation of p14 with hTERT. Taken together with the contrasting prognostic implications of hTERT and p14 (28), it may be surmised that p14 may be induced in order to offset the oncogenic effects of immortalisation due to hTERT.