The molecular balancing act of p16(INK4a) in cancer and aging

Abstract

p16(INK4a), located on chromosome 9p21.3, is lost among a cluster of neighboring tumor suppressor genes. Although it is classically known for its capacity to inhibit cyclin-dependent kinase (CDK) activity, p16(INK4a) is not just a one-trick pony. Long-term p16(INK4a) expression pushes cells to enter senescence, an irreversible cell-cycle arrest that precludes the growth of would-be cancer cells but also contributes to cellular aging. Importantly, loss of p16(INK4a) is one of the most frequent events in human tumors and allows precancerous lesions to bypass senescence. Therefore, precise regulation of p16(INK4a) is essential to tissue homeostasis, maintaining a coordinated balance between tumor suppression and aging. This review outlines the molecular pathways critical for proper p16(INK4a) regulation and emphasizes the indispensable functions of p16(INK4a) in cancer, aging, and human physiology that make this gene special.

Figure 1

Function, Structure and Polymorphisms of the INK4/ARF Locus. A, p15^INK4b and p16^INK4a both function in the RB tumor suppressor pathway through inhibition of CDK4/6 activity. Expression of p14^ARF inhibits the E3 ubiquitin ligase activity of MDM2, leading to stabilization of p53. The p53 and RB pathways play integral roles in blocking inappropriate cellular proliferation. B, Packed into 35 kilobases of chromosome 9p21.3 are three well-characterized tumor suppressor genes: p14^ARF, p15^INK4b and p16^INK4a. GWAS have implicated 9p21.3 SNPs in cancer, heart disease, glaucoma, type II diabetes, autism and endometriosis. The majority of the SNPs lie outside of transcript and coding regions in a recently discovered long, non-coding RNA, ANRIL. Of the identified SNPs, those that have been shown to correlate with CDKN2A expression in at least one study are filled with grey. Other SNPs that have not been correlated with CDKN2A expression in validation studies, or have yet to be examined are filled with black or white, respectively.

Figure 2

Mechanisms of p16^INK4a Regulation by Chromatin Modification. A, p16^INK4a is negatively regulated by the histone modifying complexes, PRC1 and PRC2, which combine to lay down repressive marks (e.g. H3K27me3, H2AK119ub) throughout the INK4/ARF locus. Several associated proteins (APs), including RNA binding proteins (RBPs), are reported to facilitate PRC1/2 interaction with the p16^INK4a promoter. APs and RBPs discussed in the text are shown. Elevated expression of the PRC2 component, EZH2, is also reported to enhance INK4/ARF gene silencing. Proteins reported to transactivate EZH2, leading to PRC-mediated silencing of p16^INK4a are depicted above. B, Activation of p16^INK4a is associated with decreases in PRC1/2 levels and the removal of repressive histone marks. JMJD3 demethylates H3K27me3 and subsequent chromatin decondensation promotes access by transcription factors and p16^INK4a transcription while JDP2 binds H3K27 and prevents further methylation. CTCF maintains chrosomal boundaries and 3-dimension structure of the chromatin surrounding p16^INK4a.

Figure 3

Transcriptional Regulation of p16^INK4a. Expression of p16^INK4a requires the action of transcription factors that recruit and/or facilitate RNA polymerase association with the promoter (shown in green). Opposing this action are transcriptional repressors (shown in red). Direct interactions with the p16^INK4a promoter are depicted by a solid line, indirect interactions with a dotted line. The numbers below each binding site indicate the position of protein interaction relative to the p16^INK4a transcriptional start site. All locations correspond to the human genome unless designated by an ‘(m)’, which signifies the mouse genome. Proteins predicted to share a common binding site are depicted over top of one another.

Figure 4

Alterations in the p16^INK4a Tumor Suppressor Pathway are Frequent in Human Cancer. A, Data from TCGA was obtained and analyzed using cBioPortal (112, 113). The tumors analyzed are as follows: Bladder Urothelial Carcinoma (BLUCA), Glioblastoma Multiforme (GBM), Head and Neck Squamous Cell Carcinoma (HNSC), Lung Squamous Cell Carcinoma (LUSC), Skin Cutaneous Melanoma (SKCM), Ovarian Serous Cystadenocarcinoma (OV), Lung Adenocarcinoma (LUAD), Stomach Adenocarcinoma (STAD), Breast Invasive Cancer (BRCA), Uterine Corpus Endometrial Carcinoma (UCEC), Brain Lower Grade Glioma (LGG), Colon and Rectum Adenocarcinoma (COAD/READ), Prostate Adenocarcinoma (PRAD), Kidney Renal Papillary Cell Carcinoma (KIRP), Kidney Renal Clear Cell Carcinoma (KIRC), Acute Myeloid Leukemia (AML). The percent of tumors with mutations or copy number changes in the p16^INK4a tumor suppressor pathway are shown. For the purposes of this analysis the p16^INK4a tumor suppressor pathway was defined to contain: CDKN2A (p16^INK4a), RB1 (RB), CCND1 (CYCLIN D1), CCND2 (CYCLIN D2), CCND3 (CYCLIN D3), CCNE1 (CYCLIN E1), and CCNE2 (CYCLIN E2). B, OncoPrints from cBioPortal show aberrations in individual tumors across the X-axis. C, Methylation of CDKN2A (black bars) and RB1 (grey bars) was quantified using HM27 or HM450 TCGA data. Genes were considered methylated if β-values exceeded 0.2.

Figure 5

Extrinsic Versus Intrinsic Activation of p16^INK4a. A, Injection of Lkb1 null endometrial cancer cells into a syngenic mouse heterozygous for the p16^LUC reporter causes stromal luciferase expression upon tumor growth. Injection of Matrigel vehicle on the opposite flank does not alter p16^INK4a expression. See also: (130) B, Intrinsic signals induces p16^INK4a expression in damaged, senescent or transformed cells. Alterations in surrounding the cellular milieu can trigger the induction of p16^INK4a in nearby undamaged cells through an unknown pathway.