The meaning of p16^ink4a expression in tumors

Abstract

The CDKN2A gene is a tumor suppressor that encodes the CDK4/6 inhibitor p16^ink4a. Loss of this tumor suppressor contributes to the bypass of critical senescent signals and is associated with progression to malignant disease. However, the high-level expression of p16^ink4a in tumors is associated with aggressive subtypes of disease, and in certain clinical settings elevated p16^ink4a expression is an important determinant for disease prognosis and therapeutic response. These seemingly contradictory facets of p16^ink4a expression have lead to confusion related to the meaning of this tumor suppression in tumor pathobiology. As reviewed here, the alternative expression of p16^ink4a represents an ideal marker for considering RB-pathway function, tumor heterogeneity and novel means for directing therapy.

p16^ink4a and Cell Cycle Control

The p16^ink4a protein was identified as a low molecular weight protein bound to cyclin dependent kinase 4 and 6 (CDK4 and CDK6).^1,2 This biochemical characterization was largely facilitated by the finding that in tumor cells transformed by SV40 the sole form of CDK4 that was detectable was in complex with p16^ink4a.¹ Biochemically, the binding of p16^ink4a to CDK4 or CDK6 disrupts the association with cyclins D1, D2 or D3, which are critical for catalytic activity (Fig. 1).^1-3

Figure 1. Schematic of p16^ink4a function.

Cell cycle progression is stimulated through mitogenic signals that coalesce in the activation of CDK4/6 activity.^4,5 Typically, such signals lead to the accumulation of D-type cyclins,⁶ facilitate nuclear localization of the cyclins,⁷ and complex formation with CDK4/6⁸ (Fig. 2). This active catalytic complex initiates the phosphorylation of the RB tumor suppressor in early/mid G₁ phase of the cell cycle.^9,10 The RB protein negatively regulates E2F-transcription factor activities that control the expression of genes that are required for nucleotide biosynthesis (e.g., dihydrofolate reductase, Thymidylate synthase, ribunocleotide reductase), DNA-synthesis (e.g., Cdc6, MCM7, Cyclin A), mitotic progression (e.g., Plk1, Cyclin B1, Cdk1) and DNA damage repair (Rad51, FancA, RPA).^11-14 The induction of this transcriptional program is crucial for cellular proliferation. Therefore, CDK-mediated phosphorylation of RB is critical for subsequent progression through the cell cycle.^15,16 In keeping with this concept, elevated expression of p16^ink4a is a potent mechanism for inhibiting proliferation, and is dominant to a variety of mitogenic and oncogenic signals.^17-19 Thus, p16^ink4a is a particularly potent effector of cell cycle progression that functions in concert with CDK4/Cyclin D and RB in coordinating proliferation.

Figure 2. p16^ink4a functions to activate RB-dependent cell cycle arrest.

Disruption of the p16^ink4a and the RB Pathway in Human Cancer

In parallel with the identification of mechanisms through which p16^ink4a contributed to cell cycle control, a role in tumor suppression was uncovered. Initially, it was observed that the gene encoding CDKN2A was mutated or silenced in a wide spectrum of tumor types and cell lines.^20,21 Particularly, loss or epigenetic silencing of CDKN2A is very common in cell lines, suggesting that the process of cell culture selects against the expression of p16^ink4a. Subsequent analyses revealed that methylation of the CDKN2A locus is a common event in tumors and is believed to be a key target of epigenetic inactivation.²² Lastly, individuals harboring compromised alleles of CDKN2A are predisposed to melanoma.²³ In general, the loss of p16^ink4a has been suggested to facilitate aberrant cell cycle progression by relieving an important aspect of control over G₁/S progression through the RB-pathway. In keeping with this idea, the loss of p16^ink4a is mutually exclusive with the loss of RB or amplification of cyclin D1 in a given tumor.²⁴ Additionally, mutations of CDK4 have been discovered in cancer that specifically preclude binding of p16^ink4a.²⁵ These combined findings indicate the proscribed biochemical function of p16^ink4a through CDK4/6 inhibition is critical for tumor suppression.

Regulation of p16^ink4a Expression

Given the important role of p16^ink4a in limiting proliferation, it is highly regulated. In proliferating normal tissue, the expression of p16^ink4a is generally low. This finding is consistent with the function in CDK4/6 inhibition, which must be relieved for proliferation. However, a number of distinct stresses, including DNA-damage as well as oncogenic stress and physiological aging, can lead to the activation of p16^ink4a expression.^26-30 Particularly, it appears that many events that trigger the process of cellular or induced senescence invoke the expression of p16^ink4a as a critical effector for the stable arrest of cells. For example, during aging there is a general trend to higher p16^ink4a levels in tissue. It is presumed that this mechanism is crucial for limiting tumor development initiated by oncogenes.²⁶ High levels of p16^ink4a are observed in certain pre-malignant lesions, wherein it is believed to contribute to arrest in the progression of the lesion.²⁹ For example, in nevi the expression of p16^ink4a is believed to be induced by oncogenic activation of B-Raf.³¹ These results are all consistent with the role of p16^ink4a as a tumor suppressor and the expected loss during disease progression.

The Meaning of Elevated p16^ink4a in Tumor

While p16^ink4a is clearly a tumor suppressor, aberrant elevation of p16^ink4a is observed in a number of cancers. As was recognized with the identification of p16^ink4a, the expression of viral oncoproteins in particular enables the development of transformed cell populations that express copious levels of p16^ink4a. Correspondingly, human tumors that are driven by specific oncogenic viruses harbor high levels of p16^ink4a. The best example of this phenomenon relates to human papilloma virus (HPV), wherein high levels of p16^ink4a are a hallmark of HPV-positive cervical cancer and head and neck cancers.³² The reason such tumors form with high levels of p16^ink4a is that the viral oncoproteins target RB. In the context of HPV, the E7 protein potently disrupts the function of RB and leads to protein degradation.^33,34 In keeping with this relationship, tumors that lack RB through other means (e.g., genetic loss of RB in small cell lung cancers) harbor high levels of p16^ink4a.³⁵ This relationship appears to be largely absolute and is observed in veritably all tumor cell lines and human patient specimens that have been studied. Functionally, it had been demonstrated that loss of RB function enables the bypass of p16^ink4a mediated cell cycle inhibition.¹⁸ However, it has also been shown that loss of RB generates an oncogenic stress that could lead to the activation of p16^ink4a expression.³⁶ Therefore, at present there are two complementary models for the appearance of tumors with high levels of p16^ink4a. First, oncogenic stresses induce p16^ink4a which limits tumorigenic progression (Fig. 3A). However, this event can be bypassed via the loss of RB. Thus, the inactivation of RB is a secondary event downstream from a given oncogenic stress that facilitates disease progression. Second, loss of RB yields an oncogenic stress which induces p16^ink4a (Fig. 3B). Since RB is already compromised the p16^ink4a induction cannot arrest tumor progression, and thus the tumors develop with high levels of p16^ink4a. It is likely that both mechanisms are at play in different tumor types. For example, in retinoblastoma or cervical cancer loss of RB is part of the etiology of the disease, and thus the induction of p16^ink4a is a secondary event. In contrast, in other tumors where RB loss occurs later in disease it could represent the secondary event facilitating disease progression. Irrespective of mechanism presumably any tumor harboring high levels of p16^ink4a has inactivated RB to facilitate tumorigenic proliferation.

Figure 3. Distinct oncogenic pathways leading to p16^ink4a induction.

Interpretting p16^ink4a Levels

Clearly, there are two discrete tumor-associated states for p16^ink4a, loss/silencing of the tumor suppressor and elevated expression related to RB loss of function. Therefore, it is exceedingly critical to denote what is “meant” by positive staining. In review of the literature many studies only report the presence or absence of p16^ink4a. Of course, this could be interpreted as an RB-deficient tumor vs. an RB-proficient tumor (high vs. low), or as tumors that in fact had lost p16^ink4a. This conundrum, currently clouds interpretation of published work related to p16^ink4a and disease outcome and is likely responsible for a number of disparate conclusions in the literature. As shown in Figure 4, distinct levels of p16^ink4a staining are observed in cancer specimens. In those lesions which are RB-deficient, p16^ink4a staining is highly elevated relative to reference normal tissue. In contrast, other tumors exhibit p16^ink4a loss. In between these two extremes, there are tumors that maintain p16^ink4a levels that are largely consistent with the tissue of origin. As is discussed below, while this provides an important reference point for considering p16^ink4a status in tumors, the use of additional markers is important for ultimately interrogating the functional status of the p16^ink4a/RB pathway in relation to tumor biology.

Figure 4. Representative staining for p16^ink4a and Ki67. (A) High p16 expression in TNBC, 200x. (B) High ki67 proliferation index in TNBC, 200x. (C) Absence of p16 expression in pancreatic carcinoma malignant glands with focal, weak staining present in stroma (200x). (D) High ki67 proliferation index in pancreatic ductal adenocarcinoma, 200x.

Prognostic Features of p16^ink4a in Tumors

Given the central role of the p16^ink4a/RB axis in coordinating cell cycle, it is not surprising that alterations in this pathway are observed in a number premalignant lesions. In this context, such alterations can be associated with distinct prognosis that is modified by the tissue of origin and nature of the originating oncogenic event. In a number of tumor types that are initiated by K-Ras or B-Raf activation loss of p16^ink4a is critical for progression to cancer. Thus, in diseases such as pancreatic cancer or melanoma, loss of p16^ink4a is a relatively common event.³⁷ For example, in pancreatic intraepithelial neoplasia (PanIN), a well-defined precursor to invasive pancreatic carcinoma, genetic loss or epigenetic silencing of p16^ink4a follows KRAS mutations and is directly associated with progression to invasive disease.³⁸ It has been hypothesized that the loss of p16^ink4a is associated with a potent selection to bypass senescence that is ostensibly initiated by the oncogenic insult.^29,39,40 In contrast, breast ductal carcinoma in situ and prostatic interepithelial neoplasia can exhibit overexpression of p16^ink4a.^41,42 This event is likely associated with the disruption of RB function in such lesions and rapidly proliferating p16^ink4a positive lesions are at increased risk for progression to invasive disease.⁴¹ Interestingly, in such tumors there is a critical distinction between those wherein p16^ink4a is suppressing the proliferation of precursor lesions, and those wherein this impact on tumor suppression has been bypassed presumably due to disruption of RB function.⁴¹ Thus, appropriate interpretation of the “meaning” of elevated p16^ink4a expression requires ancillary markers (e.g., the proliferative marker Ki67) to determine the functional state of the pathway.

Not only is p16^ink4a expression heterogeneous between cancers, but within a given cancer it is possible to utilize the level of p16^ink4a as a means to categorize particular sub-types of cancer. This possibility is most obvious in the context of small cell lung cancer that is characterized by high levels of p16^ink4a vs. lung adenocarcinoma with low levels of p16^ink4a.^43,44 While these are histologically distinct forms of disease, the discriminatory capacity of p16^ink4a levels is also significant in defining subtypes of histologically indistinguishable disease. For example, in head and neck cancers those with a specific viral etiology can be defined by p16^ink4a levels and represent a particular clinical manifestation of disease.³² Similarly, basal breast cancer is frequently characterized by elevated p16^ink4a levels, and even as a single marker can be useful in defining these tumors from other breast cancer subtypes.⁴⁵ While this approach has not been extensively analyzed across distinct tumor types, there is data that those tumors harboring elevated p16^ink4a are highly aggressive and are representative of tumors that have inactivated the RB tumor suppressor. Surprisingly, in spite of this evidence, the mechanism through which RB function is actually lost in such tumors remains unclear. These combined findings suggest that p16^ink4a levels can be particularly informative for deciphering subtypes of disease that may harbor intrinsically different disease course and tumor etiology.

p16^ink4a and the Response to Radiation/Chemotherapy

That distinct levels of p16^ink4a occur in cancers is irrefutable; however, the significance thereof to the clinical management of disease and the most appropriate means to treat such cancers remains a challenge. In particular, p16^ink4a expression has been extensively investigated in the context of cervical cancer and head and neck cancer. In cervical cancer, the majority of tumors are HPV positive (~90%) and exhibit elevated levels of p16^ink4a. Because there are so few tumors that exhibit low p16^ink4a expression, no meangingful information related to preferred treatment has emerged. However, in head and neck cancers, ~50% exhibit elevated levels of p16^ink4a. Strikingly, those tumors that exhibit high-levels of p16^ink4a exhibit an improved response to radiation therapy.^46-48 Interestingly, even tumors that are HPV negative but exhibit high p16^ink4a expression are associated with improved therapeutic response.⁴⁹ The reason for this is not fully known, although multiple preclinical models have shown that disruption of RB function renders cells and tumors sensitive to cytotoxic therapies,^50,51 and perhaps both HPV and somatic loss of RB are occurring in head and neck cancers yielding increased sensitivity to radiotherapy. Importantly, these findings have been recapitulated in multiple distinct cohorts and suggest that p16^ink4a status can be deployed prospectively to define head and neck cancers that will exhibit an improved response to radiation. In other cancers the involvement of p16^ink4a levels in the response to radiation therapy or chemotherapy is less well established, but suggestive data are present in the literature. For example, in prostate cancer elevated expression of p16^ink4a was associated with improved response to radiation.⁵² Combined these findings suggest that elevated levels of p16^ink4a could be a relatively general determinant of improved response to radiation or chemotherapy. Of course, there are some highly significant caveats to consider. For example, tumors such as small cell lung cancer that harbor high p16^ink4a expression do respond quite robustly to first-line chemotherapy; however, aggressive recurrence and evolution to therapy resistance is a key feature of this disease.⁵³ Thus, while elevated p16^ink4a can perhaps denote tumors that will respond to radiation or chemotherapy, it does not uniformly presage long therapeutic response or survival (Fig. 5).

Figure 5. Model for differential impact of elevated p16^ink4a in therapeutic response.

Emerging Significance of p16^ink4a in the Response to Targeted Therapies

The role of p16^ink4a levels in the response to targeted therapies has only been marginally explored, although there is a strong rationale for considering it as a significant marker for a host of agents in clinical use and development. Existing analyses of breast cancer cases showed that high p16^ink4a levels in ER-positive breast cancer is associated with the luminal B phenotype that shows more failure with endocrine therapy and benefits from cytotoxic chemotherapy.⁵⁴ Similarly high levels of p16^ink4a are associated with the failure of androgen-deprivation therapy that is deployed in the treatment of prostate cancer.⁵⁵ These combined findings, are in strong agreement with functional assays that demonstrated that the loss of RB-pathway function is associated with resistance to these commonly utilized hormonal therapies.^56,57

While little is known regarding the impact of p16^ink4a levels on other molecular targeted therapies in a clinical setting, high levels of p16^ink4a would be expected to be indicative of poor response to any agent that acts through cytostatic mechanisms that impinge G₁/S cell cycle control (Fig. 5). The primary reason for this is that tumor cells with high p16^ink4a and corresponding loss of RB function would be incapable of effectively arresting in G₁. This has been shown by multiple laboratories using a host of agents in preclinical models.^50,51 In fact, while studies published many years ago seemed far removed from clinical experience, they now have high significance in considering patient treatment. For example, work published initially from Chris Marshall’s laboratory in fibroblast models suggested that a functional RB pathway is required for the response to inhibition of the Raf/Mek/Erk pathway.⁵⁸ Now with highly targeted compounds antagonizing this pathway in clinical use, it would be reasonable to consider elevated p16^ink4a in patient stratification. The most obvious clinical space in which to use elevated p16^ink4a levels for patient inclusion/exclusion is with CDK4/6 inhibitors. Unlike flavopiridol or other first generation CDK-inhibitory compounds, newly developed CDK4/6 inhibitors (e.g., PD-0332991) are highly specific for an intact RB-pathway.⁵⁹ This concept has been demonstrated in multiple preclinical studies, wherein p16^ink4a high and RB loss is specifically associated with therapeutic bypass.^60-62 Presumably, in any tumor with elevated p16^ink4a, CDK4/6 is already inhibited; thus, there is essentially no likelihood of having a positive response with such inhibitors for that tumor. Similar stratification could be critical for a wide range of targeted therapies that engage the cell cycle machinery for a cytostatic effect on disease progression (Fig. 5). Together these findings illustrate the broad association of p16^ink4a expression levels with therapeutic response and underscore the potential utility of employing analyses of p16^ink4a for therapeutic stratification.

Future Considerations

How to employ the collective information regarding p16^ink4a to improve cancer treatment remains a work in progress. That levels of p16^ink4a should be considered in directing use of cytostatic agents and CDK4/6 inhibitors in particular is obvious. However, rigorous scoring approaches and multi-marker combinations will be critical for using such information in the context of tailoring therapy. Certainly, the observed improved therapeutic response with chemotherapy and radiation therapies suggests that such cytotoxic strategies may represent a prudent course of action for tumors deemed to have elevated expression of p16^ink4a. However, because of the ability of such tumors to evolve to therapy resistance and the side effects of such therapy, it would be ideal to define new therapies that specifically target tumors marked by elevated p16^ink4a. In particular synthetic lethal approaches in model systems have recently defined new pathways that could specifically target those tumors harboring deregulation of the p16/RB pathway. Thus, while p16^ink4a is an attractive marker for prognosis and therapeutic response in the clinic, substantial investigation will be required to effectively utilize this information in the management of disease.