Aberrant expression of p16 INK4a in human cancers – a new biomarker?

The ARF and INK4a genes are located in the same CDKN2a locus, both showing its tumor suppressive activity. ARF has been shown to detect potentially harmful oncogenic signals, making incipient cancer cells undergo senescence or apoptosis. INK4a, on the other hand, responds to signals from aging in a variety of tissues including islets of Langerhans, neuronal cells, and cancer stem cells in general. It also detects oncogenic signals from incipient cancer cells to induce them senescent to prevent neoplastic transformation. Both of these genes are inactivated by gene deletion, promoter methylation, frame shift, and aberrant splicing although mutations changing the amino acid sequences affect only the latter. Recent studies indicated that polycomb gene products EZH2 and BMI1 repressed p16 INK4a expression in primary cells, but not in cells deficient for pRB protein function. It was also reported that that p14 ARF inhibits the stability of the p16 INK4a protein in human cancer cell lines and mouse embryonic fibroblasts through its interaction with regenerating islet-derived protein 3 γ . Overexpression of INK4a is associated with better prognosis of cancer when it is associated with human papilloma virus infection. However, it has a worse prognostic value in other tumors since it is an indicator of pRB loss. The p16 INK4a tumor suppressive protein can thus be used as a biomarker to detect early stage cancer cells as well as advanced tumor cells with pRB inactivation since it is not expressed in normal cells. and HER2 in the murine system.


Introduction
Since the discovery as a product of the alternate reading frame of the mouse Arf/Ink4a locus signals, the Arf tumor suppressor has been identified as a key sensor of hyperproliferative stimuli such as those originating from mutant Ras and c-Myc oncoproteins to prevent early stage cancer cells to undergo neoplastic transformation by inducing senescence or apoptosis (reviewed in 1, 2). p19 Arf and p16 Ink4a are transcribed from separate and unique first exons 1β and 1α which splice into two shared exons 2 and 3 (Fig. 1). These two genes are different tumor suppressors since p19 Arf uses only exons 1 and 2 while p16 Ink4a uses all of the exons 1-3 for production of the protein (3,4). This locus has a very unique genomic structure not found in other mammalian genes due to the unprecedented splicing utilized by Arf which causes an alternate reading frame in the coding region of exon two. Of note this ARF-INK4a (CDKN2a) locus is located 11.5 kbp apart from the genomic locus for CDKN2b that encodes for p15 INK4b in humans (Fig. 1). The transcriptional regulation for the ARF-INK4a locus has been described (5,6). The aberrant transcripts from CDKN2a locus have also been reviewed (7). Both p14 ARF and p16 INK4a function as tumor suppressors (8)(9)(10)(11) despite the lack of amino acid sequence similarity. Consistent with the findings in mice, frequent mutation, promoter methylation, or deletion of the ARF/INK4a locus in human cancers has been reported (5,12,13) second only to p53 in frequency.
ARF is a highly basic (the predicted pI is 11), insoluble protein which exhibits little structure apart from a pair of α helices at its amino terminus (14). Ectopic Arf is capable of arresting immortal mouse cell lines such as NIH 3T3 as well as transformed human cells (3,4,15), a classic and requisite property of tumor suppressors. Arf sequesters MDM2 in the nucleolus, preventing p53 degradation (1,2). Additionally, it inhibits transcription factor E2F activity. These actions lead to cell cycle arrest at G1 and G2 (4). Importantly, Arf has both p53-dependent and independent functions (16; reviewed in 1, 2, 17).
Expression of p16 INK4a functions to limit cell-cycle progression and to promote cellular senescence in response to multiple stressors, including oncogene activation, telomere erosion, reactive oxygen species, and stalled replication forks (reviewed in 18-22; Fig. 1). Expression of p16 INK4a in healthy cells is low, but once induced, p16 INK4a binds and inhibits cyclin-dependent kinase 4/6 (CDK4/6) activity, thereby promoting a retinoblastoma (RB)dependent cell-cycle arrest. This tumor suppressive mechanism is believed to limit the growth of early stage neoplasms, and accordingly, the p16 INK4a -CDK4/6-RB signaling axis is disrupted in most, if not all, human cancers, with inactivation of p16 INK4a being the most common lesion of this pathway (12; Table 1). Although induction of p16 INK4a in response to oncogenic stimuli results in a beneficial, anti-cancer mechanism, expression of this tumor suppressor also accelerates mammalian cell aging (19)(20)(21)(22). Both senescent cells and levels of p16 INK4a progressively accumulate with age (23,24) and are associated with a decline in the replicative capacity of many tissue types (25,26). p16 Ink4a overexpression has been reported in human cancer and senescent fibroblasts in response to oxidative stress, DNA damage, and changes in chromatin structure (27). Although gene knock-out mice for p15 Ink4b does not show striking phenotypes, it acts as a tumor suppressor when the Arf-Ink4a locus is simultaneously inactivated (28; Fig. 1). Hybrid proteins that encode for p15 Ink4b and p16 Ink4a have been reported, indicating the synergy of co-deletion for tumor suppressor genes (29).
This review is focused on the mechanism of regulation of INK4a, and its expression in human cancers. Special interests have been put on its value for early stage tumor detection for a potential biomarker since it is not expressed in normal tissues.

Transcriptional regulation of p16 INK4a
Transcriptional regulation plays a major role in p16 INK4a regulation as the half-life is long (8 hrs; 30). A progressive increase of p16 INK4a expression has been described in passaged MEFs (8,31), transformation from normal tissue to pre-neoplastic lesions, and from preneoplastic lesions to carcinomas (32)(33)(34). Human cells lacking functional pRB contain high levels of p16 INK4a mRNA and protein, suggesting a negative feedback loop by which pRB regulates p16 INK4a expression in late G1. Hara et al. conducted nuclear run-on assays and promoter characterization of p16 INK4a in human fibroblasts (35). They showed that p16 INK4a transcription was affected by the status of RB and defined the p16 INK4a promoter that was required for this response. p16 INK4a RNA was extremely stable, and the levels did not change during the cell cycle consistent with that fact that it lacked E2F or p53 consensus sequences (35). Primary human fibroblasts expressed very low levels of p16 INK4a , but the mRNA and protein accumulated in late passage, senescent cells (35). The overexpression of p16 INK4a in RB-negative cells is caused by 1) loss of repression by RB, and 2) an increase in the number of population doublings. The mouse p16 Ink4a promoter has the Dmp1-binding site in its proximal region, which can be transactivated by Dmp1 in response to cyclin D1 overexpression since cyclin D1 does not have the transactivation domain (36). The mouse p16 Ink4a promoter is repressed by E2F1-3, indicating that p16 Ink4a is not overexpressed in RB-deficient cells due to E2F activation. This is consistent with that fact that the Dmp1 upregulates both p19 Arf and p16 Ink4a , and the p16 Ink4a promoter is repressed by E2Fs  (42). The induction of p16 INK4a by Ets2, which was abundant in young human diploid fibroblasts, was potentiated by signaling through the Ras-Raf-MEK kinase cascade, and inhibited by a direct interaction with the helix-loop-helix protein Id1 (42). In senescent cells, where the ETS2 levels and MEK signaling decline, they saw a marked increase in p16 INK4a expression consistent with the reciprocal reduction of ID1 and accumulation of ETS1 (42). These results indicated the opposing effects of Ets and Id proteins on p16 INK4a expression during cellular senescence. Type 1 diabetes is associated with loss of functional pancreatic β-cells, and restoration of βcells is a major goal for regenerative therapies (43). The regenerative capacity of β-cells declines rapidly with age due to accumulation of p16 INK4a , resulting in limited capacity for adult endocrine pancreas regeneration (24). Dhawan et al. showed that TGFβ signaling via Smad3 integrated with the trithorax complex to activate and maintain Ink4a expression to prevent β-cell replication (43). Importantly, inhibition of TGFβ signaling resulted in repression of the ARF/INK4a locus, resulting in increased β-cell replication in adult mice. These data revealed a novel role for TGF-β signaling in the regulation of the ARF/INK4a locus. In mice homozygous for a hypomorphic allele of the α-klotho ageing-suppressor gene, accelerated ageing phenotypes were rescued by p16 Ink4a deletion, suggesting its dependency (44). Indeed, p16 Ink4a repressed α-klotho promoter activity by blocking the functions of E2Fs, indicating that p16 Ink4a plays a role in downregulating α-klotho expression during ageing (44). Inoue

PRC (polycomb repressor complex) mediated inhibition of p16 INK4a expression
Both the expression of p16 INK4a and p14 ARF are regulated by promoter hypermethylation through proteins of the polycomb repressor complex (PRC1) and PRC2 complexes (reviewed in 45, 46; Fig. 2). The polycomb group (PcG) of transcriptional repressor proteins was originally characterized in drosophila for maintenance proteins of pluripotency (47). PRC2 functions as an initiator of transcription repression while PRC1 functions as a repressor maintenance complex. Thus the methylation mediated by PRC2 is a prerequisite for the binding of PRC1 to the chromatin (45,46,48). The PRC1 complex is comprised of Polycomb (CXB2, 4, 6-8), Bmi1, HPH (HPH1-3), and RING (RING1 and 2) proteins. The primary mechanism of transcriptional repression by the PRC1 involves mono-ubiquitination of histone H2A K119 by histone H2A ubiquitin ligase (48; Fig. 2). The PRC2 complex is composed of three core proteins, EZH2 which mediates tri-methylation of histone H3K27, EED, SUZ12, and RbAp46 (49; Fig. 2). Histone methyltransferase EZH2 plays a critical role in epigenetic regulation as a bridge between histone methylation/deacetylation and DNA methylation. EZH2 is frequently overexpressed and considered to be an oncogene in cancers (49). The SET domain of EZH2 possesses methyltransferase activity specific for histone H3K27 (50,51). The role of deregulated PRC2 in tumor suppressor gene expression, DNA damage response, and the fidelity of DNA replication has been suggested (52), resulting in long-term reversible suppression p14 ARF and p16 INK4a .
The ARF/INK4a tumor suppressor locus, which is a key executor of cellular senescence, is regulated by members of the PcG of transcriptional repressors (Fig. 2). Barradas et al. (53) showed that signaling from oncogenic RAS overrides PcG-mediated repression of INK4a by activating the H3K27 demethylase JMJD3, and down-regulating the methyltransferase EZH2. In human fibroblasts, JMJD3 activated p16 INK4a , but not p14 ARF , and caused p16 INK4a -dependent cell cycle arrest. In MEFs, Jmjd3 activated both Ink4a and Arf and causes a p53-dependent arrest, echoing the effects of Ras in this system. Their findings directly implicate JMJD3 in the regulation of ARF/INK4a during oncogene-induced senescence (OIS), suggesting that JMJD3 has the capacity to act as a tumor suppressor (53). Similar findings were also reported from two other groups, indicating the importance of EZH2 -mediated repression of the ARF/INK4a locus in cellular senescence (54,55).

Mechanisms of upregulation of p16 Ink4a in Rb-deficient cells
The p16 Ink4a protein is always upregulated in Rb-deficient cells (35,56). So what are the molecular mechanisms? It was believed that dysregulated activities of E2F proteins were responsible for overexpression of p16 Ink4a in Rb-deficient cells, but it is the p19 Arf promoter that is transactivated with E2F1-3 overexpression, not p16 Ink4a . Indeed E2F1-3 repressed the murine p16 Ink4a promoter and E2F4, even combined with nuclear localization signal, had no

Regulation of p16 INK4a at the protein level
Kobayashi et al. (30) found that that p14 ARF regulates the stability of the p16 INK4a protein in human cancer cell lines, as well as in MEFs. In particular, ARF promoted rapid degradation of p16 INK4a protein (the half-life became 3.5 hrs from 8 hrs), which was mediated by the proteasome and, more specifically, by interaction of ARF with one of its subunits, regenerating islet-derived protein 3γ (REGγ). Furthermore, this ARF-dependent destabilization of p16 INK4a was abrogated by knock-down of REGγ or by pharmacological blockade of its nuclear export. Thus their findings uncovered a novel crosstalk of two key tumor suppressors mediated by a REGγ-dependent mechanism (30).

p16 INK4a overexpression in pre-malignant lesions
Several pieces of evidence suggested that the ability to bypass senescence is the main molecular mechanism in the progression of pre-malignant to fully malignant cells (58,59). This hypothesis is based on the concept of OIS, which was established after demonstration of p53-and p16 Ink4a -mediated senescent-like growth arrest in response to expression of oncogenic Ras in normal primary cells (18,(58)(59)(60), which has been considered as highly possible mechanism to prevent proliferation of incipient cancer cells. Consistently, senescent cells have been reported in a number of different benign lesions, including nevi and neurofibromas (61, 62), but not in cancer. Since p16 Ink4a is involved in OIS, the overexpression has been found in benign and pre-malignant lesions with senescent cells where the ARF/INK4a locus plays its role (61)(62)(63). Indeed, human nevus lesions remain in a growth-arrested state, and rarely progress to malignant melanomas (64,65). A mutation in the downstream effector of Ras, BRAF (BRAF V600E ) is often found in malignant melanomas, the expression of which in human melanocytes induces cell cycle arrest, followed by p16 INK4a induction (61)(62)(63)(64)(65). A similar finding has been reported in schwannomas/neurofibromas (64). Schwannomas express high levels of p16 INK4a , and show senescence-associated β-galactosidase activity, BRAF mutations, and very low proliferative activity (66). Conversely, the malignant counterparts for these tumors are negative for p16 INK4a , and loss of p16 INK4a has thus been implicated in the development of both types of Inoue  malignant neoplasms (64)(65)(66)(67)(68). In other words, benign tumors overexpress p16 INK4a , which seems to inhibit cell proliferation in response to oncogenic stimuli, protecting cells from malignant transformation. p16 INK4a methylation could thus have a diagnostic value; it can be used in the differential diagnosis from pre-malignant and malignant lesions (69). In fact, p16 INK4a methylation is found of 53.2 % of non-small cell lung cancer while p14 ARF methylation is found 6.5 % (38), the frequency 8 times higher than that in ARF.
OIS is emerging as a potent cancer-protective response to oncogenic events, serving to eliminate early neoplastic cells from the neoplastic tissue. Kuilman et al. (70) reported a unique role of interleukin-6 (IL-6) in OIS of cancer cells. They found that OIS was linked specifically to the activation of an inflammatory transcriptome. Induced genes included IL-6, which upon secretion by senescent cells acted mitogenically in a paracrine fashion (70). IL-6 was also required for the execution of OIS, but in a cell-autonomous fashion. Its depletion caused the inflammatory network to collapse and abolished senescence entry and maintenance. They also demonstrated that C/EBPβ cooperated with IL-6 to amplify the activation of the inflammatory network, including IL-8. In human colon adenomas, IL-8 specifically colocalized with p16 INK4a -positive epithelium (70). They proposed a model in which interleukins connect senescence with an inflammatory phenotype and cancer. The role of IL-6 in OIS has been confirmed by later studies (71,72), indicating the unique role of this cytokine in cancer prevention.  (73). Activation of p16 Ink4a was noted in the emerging neoplasm and surrounding stromal cells. They concluded that p16 Ink4a activation is a characteristic of all emerging cancers, making the p16 LUC allele a sensitive, unbiased reporter of neoplastic transformation. It seems p16 Ink4a is a gateway to detect oncogenic signals to force incipient cancer cells into senescence and/or apoptosis from this study; however Zindy et al. had reported that p19 Arf was the major player that played its role in vivo and in vitro in response to c-Myc overexpression since p16 Ink4a was not induced in response to c-Myc (74,75). Our data indicate that the p16 Ink4a promoter is repressed in response to E2F1-3 although the p19 Arf promoter was strongly activated by these E2Fs (6,76). Moreover, our HER2 study showed that p16 Ink4a was induced 2-16 folds (median 3 folds) while p19 Arf was induced 7-39 folds (median 12 folds) in wild type Dmp1 ND tumors (40) indicating that p19 Arf induction was the predominant player for quenching the oncogenic stress in tumors driven by c-Myc, E2Fs, and HER2 in the murine system. It should be noted that the ARF protein is always overexpressed whenever the mRNA is high. However, that is not the case in p16 INK4a . Kobayashi et al. reported that the p16 INK4a protein is unstable due to the increased turnover in ARF overexpressing cells, suggesting the crosstalk between ARF and p16 INK4a , and suggested possible discrepancy between p16 INK4a mRNA and protein (30). Hence, analysis of p16 INK4a protein levels is necessary to establish its role in tumor suppression in those overexpressing ARF. The impact of aging should also be considered in the assessment of p16 Ink4a in aged mice (19)(20)(21)(22). As a matter of fact, p16 Ink4a remains high in MEFs that are passaged for more than 12 days (passage 4) regardless of the protocol (8,31); it remains consistently elevated in later passages regardless of the mechanism of immortalization indicating the Cdk4 inhibition is not enough to inhibit cell proliferation in immortalized MEFs. Whether ARF or INK4a is major player in silencing oncogenic signals remains a big question that should be resolved in human systems.

p16 INK4a overexpression in HPV-infected human cancers and their prognostic values
The RB protein is inactivated by interaction with the high-risk HPV oncoprotein E7 (77,78), and oncoprotein E6 induces degradation of the tumor suppressor p53. RB inactivation releases p16 INK4a from its negative feedback control, causing a paradoxical increase in the levels of this protein, which attempts to inhibit uncontrolled cellular replication. As a consequence, p16 INK4a is overexpressed in HPV-expressing tumors such as cervical cancer and head and neck tumors (79-81; Table 1).   (84). In breast cancer, p16 INK4a was negative or low in normal ducal epithelium, but a progressive increase was reported in benign lesions and carcinoma (33,34). Increased nuclear p16 INK4a protein expression compared with normal epithelium has also been reported in pre-neoplastic and tumor tissues of the gallbladder (85

Research in progress and future directions
We have reviewed p16 INK4 alterations in cancer. Both ARF and INK4a genes are inactivated in human cancer by gene deletion, promoter methylation, gene frame shift, and splicing errors although mutations mainly affect only the latter (12,13). It has also been reported that many human cancers overexpress p16 INK4a (84)(85)(86)(92)(93)(94). The mechanism of p16 INK4a overexpression in RB-null cells is related to aberrant PRC activity (57)  the prognosis of such patients is better than the others since they respond to therapies. In summary, we have to be cautious in using p16 INK4a as a biomarker since it can be associated with either favorable (early stage or HPV -positive tumors) or worse prognosis (HPV -negative tumors). The structure for the human p15 INK4b -p14 ARF -p16 INK4a locus.
The genomic structure is well-conserved between human and mice, and thus gene knockout studies have been extensively conducted in mice. The distance between exon 1β and exon 1α is 19.4 kbp in humans and 12.4 kbp in mice. The exon 1α is 3.8 kbp upstream of exon 2 in humans; 5.2 kbp in mice. The ARF-INK4a (CDKN2a) locus is located 11.5 kbp apart from the genomic locus for CDKN2b that encodes for p15 INK4b in humans. All of p15 Ink4b , p19 Arf , and p16 Ink4a genes act as tumor suppressors as reported by Krimpenfort   PRC1 and PRC2 repress transcription from the ARF-INK4a locus. Both the expression of p14 ARF and p16 INK4a are regulated by promoter hypermethylation through proteins of the polycomb repressor complex (PRC1) and PRC2 complexes (45)(46)(47). The polycomb group of transcriptional repressor proteins was originally characterized in drosophila for maintenance proteins of pluripotency (47). PRC1 functions as a repressor of the maintenance complex while PRC2 functions as an initiator of transcriptional repression. Thus the methylation mediated by PRC2 is a prerequisite for the binding of PRC1 to the chromatin (48). The PRC1 complex is comprised of Polycomb (CXB2, 4, 6, 8), Bmi1, HPH (HPH1-3), and RING (RING1 and 2) proteins. The most important mechanism for transcriptional repression by the PRC1 complex involves mono-ubiquitination of histone H2A K119 by histone H2A ubiquitin ligase (48). The PRC2 complex is composed of three core proteins, EZH2 which mediates tri-methylation of histone H3K27, EED, SUZ12, and RbAp46 (49). Inoue Cancer Rep Rev. Author manuscript; available in PMC 2018 June 25.