As an important tumor suppressor, p16 negatively regulates cell proliferation through inhibiting the activity of cyclin-dependent kinase 4/6 (CDK4/6), which promotes phosphorylation and, therefore, inactivate Rb. The p16–cyclinDs–CDK4/6–Rb–E2Fs pathway is abrogated frequently in multiple types of human tumors, either through inactivation of Rb or p16, or through over-expression of cyclin D1 or CDK4. Loss of p16 activity has been widely found in many human cancers due to gene mutation or promoter methylation [10, 11]. However, its aberrant expression and changes in subcellular localization have also been found in some types of cancers, especially in precancerous lesions of cervix and cervical cancers [9, 12, 13]. Evangelou et al. verified that cytoplasmic p16 in non-small cell lung cancer was specific using Electron Microscope Technology [3]. Nilsson et al. showed that p16 indeed expressed in cytoplasm as well as in nucleus by subcellular fractionation of Rb-functional and Rb-inactivated cell lines [4]. Moreover, they also indicated that p16 might have functions in cytoplasm. Studies have demonstrated that cytoplasmic p16 was correlated with the development and prognosis of some tumors. To be exact, in normal breast tissue and fibroadenoma, p16 was present only in nuclei of individual cells, whereas aberrant staining demonstrated in cancer cells, generally in nucleoplasm or only in cytoplasm [9, 14]. In colorectal cancer, p16 presented a similar expression pattern [15]. At present, little was known about the reason for aberrant accumulation of cytoplasmic p16 and its significance in the development of breast cancer.
CDK4, locating at 12q13, plays vital roles in proliferation of mammalian cells. In particular, several tumorigenic events ultimately drive proliferation through CDK4 complexes, underscoring CDK4 as an important therapeutic target in cancer treatment [16]. The importance of interaction between CDK4 and p16 has become apparent with identification of a mutation in CDK4 in patients with familial melanoma [17, 18]. This germline mutation results in the replacement of an arginine residue at position 24 by a cysteine (R24C), making CDK4 lose its affinity for p16 without affecting its ability to bind cyclin D and then form a functional kinase. However, this mutation does not affect the interaction of CDK4 with P21CIP or P27KIP inhibitors.
In this study, we first confirmed co-localization of p16 with CDK4 either in breast cancer tissue or cell lines by immunohistochemistry and immunofluorescence. Further, we observed that subcellular localization of p16 changed significantly with different CDK4 status. p16 protein was mainly in cytoplasm when CDK4 was over-expressed, whereas it mainly located in nucleus when CDK4 was mutated at position 24 (R24C) or knocked down. The above preliminarily confirmed that cytoplasmic p16 could be associated with CDK4, which caused retention of p16 in cytoplasm when CDK4 was up-regulated in some cases. However, more detailed mechanisms need to be further studied.
p16 gene mutations have complicated effects on its subcellular localization. Some studies have shown that various mutants of p16 expressed predominantly in cytoplasm [19–21]. On the other hand, at least one study has shown that cytoplasmic p16 observed in tumor-derived cell lines was not linked to its gene mutations [5]. Therefore, in this study we selected BT549 cell line harboring no p16 mutations to eliminate the influence of mutation on its localization. Besides, as a triple-negative breast cancer cell, BT549 can realistically simulate the expression and subcellular localization of p16 in triple-negative breast cancer tissues. In this way, we can better understand the biological behavior of breast cancer, particularly TNBC, and provide some guidance for the treatment and prognosis of breast cancer patients.
p16, as a tumor biomarker, has been widely used in the pathological diagnosis of many tumors. As is known, abnormal expression of p16 is helpful in differential diagnosis of cervical squamous intraepithelial lesions [12]. Besides, cytoplasmic p16 staining of tumor cells further demonstrated the prognostic value. In primary breast carcinoma, a strong cytoplasmic expression of p16 was associated with a highly malignant phenotype [8], whereas in malignant melanoma, no association between cytoplasmic p16 and patient outcome was indicated [22]. In our study, cytoplasmic p16 was found to promote proliferation, invasion and metastasis of BT549 cells, indicating a poorer prognosis.
However, there are some limitations in this study. Given the heterogeneity of breast cancer, the clinical cohort in this study is relatively modest. Only Chinese ethnicity and one center were involved with no randomization or controls. Besides more detailed mechanisms were needed. Therefore, further investigation is necessary to overcome all these limitations.