p14^ARF expression in invasive breast cancers and ductal carcinoma in situ– relationships to p53 and Hdm2

The development and growth of breast cancers result from the inactivation of p53 or retinoblastoma (pRb) tumour suppressor proteins that regulate cell cycle control. Such regulatory pathways trigger cell cycle arrest or apoptosis in response to intracellular challenges such as DNA damage, hypoxia and oncogene activation, with preservation of genomic stability [1]. pRb maintains checkpoint integrity through binding and blocking E2F transcription factors, a process reinforced by the G1 cyclin-dependent kinase inhibitor p16^INK4a [2]. Similarly, p53 accumulates to transcriptionally activate the cyclin-dependent kinase inhibitor p21^WAFI/CIPI, as well as its own negative regulator Hdm2 (human Mdm2; murine double minute 2), which terminates the p53 response. Nuclear retention of p53 underlines its tumour suppressor response and suggests an additional pathway for disabling p53 over and above its mutation in more than 50% of human cancers [3‐5]. In an autoregulatory feedback loop, Hdm2 maintains low levels of p53 in normal non-stressed cells and inhibits nuclear p53 through multiple and diverse mechanisms. Hdm2 binds p53 to inhibit its transactivation function and shuttles p53 from the nucleus to the cytoplasm to facilitate its degradation [6‐8]. Hdm2 is also an E3 ubiquitin ligase that targets p53 for the ubiquitin-dependent 26S proteosome in the cytoplasm [9].

The INK4a/ARF gene locus on chromosome 9p21 encodes p16^INK4a and p14^ARF, both of which act in tumour surveillance and link the pRb and p53 pathways [2, 10‐14]. p14^ARF is encoded from an alternative, but partly overlapping, reading frame together with p16^INK4a [15], such that ARF has a separate first exon (1β) that splices into common exons 2 and 3, shared with p16^INK4a. These proteins independently target two cell cycle control pathways, with p16^INK4a inhibiting cyclin D1/cyclin-dependent kinases within the pRb pathway and p14^ARF inhibiting the oncoprotein Hdm2 within the p53 pathway [10‐14]. ARF expression and activation occur through mitogenic signals such as Myc, E1A, E2F1, Ras and v-Abl, stabilising p53, followed by cell cycle arrest or apoptosis [16, 17]. Consequently, ARF connects the pRb and p53 pathways, with excessive proliferative signalling via pRb activating arrest mechanisms by p53. This suggests that loss of ARF function is a major contributor to carcinogenesis in humans [17]. p14^ARF is a true tumour suppressor protein; tumours develop spontaneously in ARF-null mice [14, 18]. Promoter hypermethylation of the INK4a and ARF genes is a major mechanism of their inactivation, followed by hemizygous deletions [2, 10, 11, 19]. Breast cancers rarely demonstrate homozygous deletions of either gene, with no mutations of ARF [2, 19‐21].

ARF expression in vitro and in vivo is associated with a G1 or G2 cell cycle arrest or apoptosis after inappropriate mitogenic stimuli or DNA damage [10‐13, 22, 23]. Although these effects in large part relate to activation of the p53 pathway, recent reports suggest that p14^ARF inhibits growth independently of p53 [12, 17, 23, 24]. Low or undetectable p14^ARF expression in normal tissues and its function independently of p53 in highly proliferating, homeostatic tissues, in comparison with tumours, suggests that p14^ARF function has yet to be completely established [17, 24]. Subcellular localisation of p14^ARF is preferentially nucleolar or nuclear, where it binds Hdm2 to inhibit the latter's activities towards p53 [15, 25‐27]. ARF-bound Hdm2 blocks Hdm2-dependent nucleocytoplasmic shuttling of p53, to produce nuclear retention and activation of p53 [13, 27]. Further, ARF averts degradation of p53 by inhibiting Hdm2-dependent ubiquitination of p53 in vivo [13, 28, 29]. p14^ARF acts upstream of p53 and is subject to its negative feedback regulation, suggesting that p53 mutations or its inactivation by HDM2 amplification are often accompanied by overexpression of ARF [10‐14, 30]. p53-positive tumours are also likely to have sustained epistatic mutations such as HDM2 amplification or ARF loss [11]. Inactivation of ARF might occur through the overexpression of several ARF repressors, including Twist and Tbx-2 in breast cancers and Bmi-1 in other tumours [31, 32].

Nuclear import and export is a feature of both p53 and Hdm2 with implications for their functional regulation, such that cytoplasmic p53 is associated with tumours with a poor prognosis [3‐6, 8]. Just as Hdm2 regulates the nuclear export of p53, in vivo evidence suggests that ARF influences the subcellular localisation of Hdm2 [21]. Cytoplasmic ARF occurs through binding to Pex19p with evidence of weak to moderate cytoplasmic staining in human tumours, whereas two studies disregarded cytoplasmic p14^ARF expression [33‐37]. In a single in vitro study, mouse p19^ARF binds a cytoplasmic protein Pex19p (cloned from mouse testis) in normal cells (NIH 3T3) [33]. Consequently, the subcellular localisation of these proteins and the relationship between their levels of expression are likely to be important in evaluating breast cancers and their development from pre-invasive ductal carcinoma in situ (DCIS) to invasive disease.

Previous studies have examined p14^ARF mRNA expression in breast cancers, with evidence suggesting altered expression and an association with p53 [38, 39]. The aim of this study was to evaluate the levels of protein expression for p14^ARF in relation to Hdm2 and p53 using immunohistochemistry in DCIS and invasive ductal breast cancers (IDCs), including the study of their subcellular localisation. So far, no studies have examined p14^ARF/Hdm2 and p53 in relation to clinicopathological parameters and prognosis in breast cancer. We have shown nuclear p14^ARF expression in 79% of IDCs and in associated DCIS in 74 patients. Cytoplasmic p14^ARF was detectable in 23 breast cancers. Levels of expression and subcellular localisation for p14^ARF, Hdm2 and p53 were similar in IDCs and DCIS. ARF expression showed no correlation with p53 immunoreactivity and was associated with Hdm2 nuclear and cytoplasmic expression. HER-2-positive breast cancers were associated with nuclear p14^ARF and nuclear Hdm2 immunoreactivity. Our preliminary findings suggest that cytoplasmic p14^ARF and cytoplasmic Hdm2-expressing breast cancers might be associated with a better outcome.

p14^ARF suppresses cancer cell growth, and several in vitro studies have reported its inhibition of Hdm2 to stabilise and activate p53, with a loss of ARF increasing p53 degradation. The relevance of these mechanisms in vivo requires further investigation, in view of ARF's proposed function as a tumour suppressor and the suggestion that p14^ARF overexpression is a surrogate marker of dysregulation of the pRb and p53 pathways [30]. p14^ARF mRNA expression in breast cancers underlines a variability with overexpression in 17–19% and underexpression in 24–26% [38, 39]. There are few studies clarifying p14^ARF protein levels in vivo, with low ARF expression in non-neoplastic epithelium [17, 34, 35]. The present study suggests an increased frequency of nuclear p14^ARF overexpression in 47% of invasive and non-invasive breast cancers. Nuclear p14^ARF overexpression has been shown by immunohistochemistry in 22% of B cell lymphomas to predict tumour aggression and outcome [30]. Possible abnormalities in the p53 pathways might be implicated [10‐14, 30, 34]. p53-independent mechanisms might also contribute through oncogenic stimulation of ARF [16, 17, 30].

The lack of p14^ARF expression in 21% of tumours is not dissimilar to other studies of ARF mRNA levels in breast cancers [38, 39]. B cell lymphomas lacked nuclear p14^ARF in 11% of cases, in association in large part with promoter hypermethylation, as reported for breast cancers [21, 30, 38]. Predominant nucleolar localisation of p14^ARF is determined by amino-terminus and exon 2 carboxy-terminus, with evidence of this after immunofluorescence of a number of cancer cell lines [25, 26] and exclusive nucleolar p14^ARF in a subset of lymphomas and non-tumour tissue [25, 26, 30]. The 4C6 monoclonal antibody and other p14^ARF monoclonal antibodies have verified the nuclear/nucleoplasmic localisation of ARF with intactness of its functional pathway, as well as its implications as a prognostic surrogate marker compared with nucleolar ARF [27, 30].

This study shows nuclear p14^ARF in the majority of breast cancers, with 24% cytoplasmic detection. Few other studies have attempted to analyse cytoplasmic p14^ARF, although detectable on immunohistochemistry, with in vitro evidence of an ARF-binding cytoplasmic protein [33‐37]. Cytoplasmic p14^ARF has been observed in non-small cell lung cancers, in oral squamous carcinomas and in another study of lung and pancreatic tumours with the use of several monoclonal and polyclonal antibodies [34‐37]. This study analysed cytoplasmic ARF in the context of positive and sero-matched negative controls as described, with no evidence of ARF expression in surrounding normal breast epithelial cells. Such observations might invoke an additional pathway of ARF regulation through changes in its subcellular localisation as for other tumour suppressors, such as p53 and p21 ^WAF1/CIP1 [5, 8, 51, 52]. Similar genomic aberrations might occur in DCIS and IDC, highlighting possible similarities in protein expression [53, 54]. This study shows a consistent similarity in levels of protein expression and subcellular localisation for p14^ARF, p53 and Hdm2 in DCIS and IDCs.

It is suggested that the upregulation of nuclear p14^ARF expression is a consequence of cell cycle malfunction involving p53 and Hdm2 [30]. Increasing levels of nuclear ARF on immunohistochemistry are a measure of p53 inactivation by mutation, or by Hdm2 overexpression resulting in disruption of the p53–p14^ARF negative feedback loop [30]. The implication is that p14^ARF expression is associated with P53-deficient cell lines, suggesting a p53-mediated downregulation of ARF [10‐14]. Although p53 immunostaining is not necessarily tightly correlated with TP53 gene function, 57% (17 cases) of p53-positive tumours in this study strongly expressed ARF, reflecting similar in vivo evidence showing concomitant p14^ARF mRNA expression and p53 immunostaining in breast cancers, with a similar relationship between increasing levels of ARF protein and p53 mutations in B cell lymphomas (Table 3) [30, 34, 39]. Others have shown a lack of an inverse relationship between the two genes, suggesting that the ARF–p53 pathways are not strictly linear and that decreased ARF expression and TP53 mutations are not mutually exclusive [39, 55]. ARF stabilises nuclear p53 and is postulated to congregate with Hdm2 and p53 in ternary complexes or 'nuclear bodies' that have yet to be demonstrated in vivo [10, 25, 27, 30].

Double-fluorescent immunolabelling in B cell lymphomas revealed a partial co-localisation of nuclear p14^ARF and p53, with no Hdm2 association, suggesting that the mechanisms underpinning ARF's role in inhibiting the nuclear export of p53 by Hdm2 inhibition remains to be defined [30]. Studies in vivo have not previously examined a possible association between ARF expression and p53 subcellular localisation in breast cancer. In large part, this interpretation depends on the accumulation of p53 that is likely to reflect mutant p53 as inferred from immunohistochemistry [30, 34, 49]. Proportionately more (55%) p53-expressing breast cancers showed exclusive nuclear p53 in association with nuclear ARF expression (data not shown).

Hdm2 expression is a feature of tumorigenesis, with overexpression attributed in part to gene amplifications in the minority of breast cancers [56‐59]. Other contributing mechanisms include enhanced transcription and translation and an extended protein half-life. This study demonstrates a minority (11%) of Hdm2-overexpressing (MQS 6–8) invasive breast cancers and 16% of non-invasive breast cancers. Recent evidence shows that p14^ARF overexpression is associated with increased levels of Hdm2 in cancer cells, with a similar finding in oral cancers and B cell lymphomas that indicate a direct association between p14^ARF and Hdm2 expression determined by immunohistochemistry [27, 30, 35]. Similar findings are reflected in this series of breast cancers (Table 3).

An additional mechanism of Hdm2-mediated regulation by p14^ARF involves intracellular compartmentation [21]. The subcellular localisation of Hdm2 involves its nucleolar localisation signal, as well as both nuclear import and export receptors [59]. In vitro immunofluorescent studies verify the ARF-mediated localisation of Hdm2 in the nucleolus and nucleoplasm [27]. Not uncommonly in colorectal cell lines and primary tumours, in vivo inactivation of ARF by methylation is associated with increasing cytoplasmic Hdm2 expression, emphasising the importance of p14^ARF in nuclear Hdm2 localisation [21]. ARF-negative cancers, implying inactivation in this series, show no specific association with increased cytoplasmic Hdm2 expression (Fig. 2). There is a further suggestion in this series of breast cancers that nuclear ARF expression is associated with both nuclear and cytoplasmic Hdm2-expressing breast cancers, underlining the absence of a preferential nuclear localisation of Hdm2 (data not shown). Similarly, a study of colorectal cancers shows the lack of an exclusive relationship between ARF function and the subcellular localisation of Hdm2, in comparison with cell lines [21]. HER-2/neuneu might also regulate the subcellular localisation of Hdm2, with better prognostic HER-2-negative breast cancers associated with Hdm2 in both the nucleus and cytoplasm, in contrast to HER-2-mediated phosphorylation of Hdm2 to produce its nuclear localisation and the degradation of p53 [60]. Such findings could imply that the relationship between p14^ARF and the subcellular localisation of Hdm2 is not as exclusive as that found in cell lines.

Clinically, the implications of p14^ARF levels of expression in breast cancer are unknown; two previous studies examined p14^ARF mRNA expression in relation to prognostic parameters [38, 39]. Variable p14^ARF mRNA expression including both overexpression as well as decreased mRNA levels are reported to be significantly associated with poor prognostic criteria including p53 mutational status, peritumoural vessel invasion, lymph node metastases and negative progesterone receptors [38, 39]. By comparison, nuclear p14^ARF overexpression in B cell lymphomas with the use of immunohistochemistry predicts tumour aggression and reduced overall survival [30]. We find no clear associations between ARF levels and prognostic parameters or outcome, although the presence of cytoplasmic p14^ARF as opposed to its absence suggests a better outcome (Fig. 3b). At present, a poor understanding of the implications of cytoplasmic ARF expression in tumours requires further study [35‐37]. In vitro, HER-2/neuneu interacts with the p14^ARF/Hdm2 pathway; some in vivo studies show that p14^ARF mRNA overexpression in breast cancers is correlated with HER-2 negativity [38, 39, 61]. In vitro, HER-2 promotes Hdm2-mediated p53 degradation through the inactivation of ARF, and HER-2 further enhances mammary tumorigenesis in ARF heterozygous mice [60, 61]. Our finding of an association between ARF and HER-2 expression is preliminary and suggests the importance of HER-2 in the ARF pathway.

Inactivation of p53 might occur through pathways other than mutation, involving p14^ARF/Hdm2 and its degradation through subcellular localisation. Immunohistochemical p53 expression might detect up to 89% of TP53 point mutations in breast carcinoma specimens, although it does not always correlate with specific mutations in exons 5 to 9 [34, 49]. Despite an established body of literature regarding the implications of p53 nuclear immunoreactivity, few or no in vivo studies have evaluated cytoplasmic p53. The cytoplasmic localisation of p53 is a prerequisite for its proteosomal degradation and has been implicated in patient prognosis [5, 62]. A study of inflammatory breast cancers showed the presence of wild-type cytoplasmic p53 in 37% of cases [5]. We found a significant association of cytoplasmic p53 with increased tumour proliferation in DCIS, suggesting the value of its further investigation in future studies. Similarly, Hdm2 undergoes nucleocytoplasmic shuttling; highly proliferative, invasive breast cancers were associated with increasing levels of nuclear or cytoplasmic Hdm2 expression in this study. Immunohistochemical p53 and Hdm2 expression were also shown to be correlated with ki67 in B cell lymphomas [30]. A possible functional relevance of subcellular Hdm2 expression is suggested, although Ki67 was not an independent prognosticator in this study.

Our findings of an association between HER-2 and nuclear Hdm2 expression might support in vivo findings in which HER-2, through phosphoinositide 3-kinase/Akt-mediated phosphorylation, is preferentially associated with nuclear Hdm2 [60]. In the present study the association between the presence of cytoplasmic Hdm2 and improved outcome would initially seem counterintuitive, yet it is substantiated by recent evidence that HER-2-negative and Akt-negative cell lines are associated predominantly with cytoplasmic Hdm2 [60]. In vivo, the study of a small subset of breast cancers (21 tumours) confirmed these in vitro findings, suggesting an important relationship between HER-2 activation and the p14^ARF/Hdm2 and p53 pathways [60].

Defining relevant pathways regulating p53 function are fundamental to an understanding of its tumour suppressor role and mechanisms of p53 inactivation, which might be independent of a p53 mutation. p14^ARF and Hdm2 are key upstream and downstream regulators of p53, although in part they might be independent of p53. We have demonstrated a spectrum of tumour-specific p14^ARF upregulation with predominant nuclear ARF expression in the absence of exclusive nucleolar staining in invasive and non-invasive breast cancers; we suggest that the presence of cytoplasmic p14^ARF might be favourable in terms of breast cancer outcome. Predominant nuclear ARF levels showed no clear clinical implications about the degree of ARF expression versus its negativity. Clearly our in vivo findings do not completely mirror in vitro relationships between p14^ARF/Hdm2 and p53 to suggest a greater complexity of these pathways in human solid tumours. Increasingly, evidence suggests the importance of evaluating the subcellular localisation of checkpoint proteins to include p53, Hdm2 and p14^ARF [51, 52]. Potentially, changes in the cellular distribution of these proteins might be driven by key tumorigenic processes to which HER-2 and other growth factor pathways might contribute.