Determination of molecular markers associated with risk for subsequent tumor events in women diagnosed with DCIS
A well-recognized barrier to carcinogenesis is the induction of a senescent cellular response. To determine if this tumor suppressor pathway in DCIS lesions provides mechanistic insight about subsequent tumor events, we examined p16 expression in samples of women without subsequent disease (38 controls) matched to a sample of women with subsequent disease (32 cases), in a DCIS case-control study sufficiently powered to explore pathways that may provide risk stratification. A subsequent tumor event (recurrence) was defined as a subsequent DCIS lesion or invasive cancer lesion diagnosed in the ipsilateral breast or at a distant site at least 6 months following the initial diagnosis of DCIS. Representative p16 staining is illustrated in . We find 26% (18/70) of DCIS lesions show high p16 staining (). This p16 immunopositivity is not associated with any clinicopathological variables such as nuclear grade or hormone receptor status (Supplemental Table 1
). We would predict that high p16 expression induces a cellular growth arrest, and thus DCIS lesions overexpressing p16 would be less likely to precede subsequent disease. This prediction was not substantiated. We find that high p16 expression, as a univariate marker, does not significantly stratify a woman’s risk for developing a subsequent tumor event (DCIS and invasive cancer combined; HR=1.1, 95% CI, 0.5 to 2.5; , ).
p16 overexpression coupled with proliferation increases the risk of subsequent tumor events among women with DCIS
p16 and COX-2 expression coupled with proliferation increases the risk of developing subsequent disease among women with DCIS.
Paradoxically, overexpression of p16 can represent two different biological processes; a response to cellular stress or abrogation of functional Rb signaling (Bates et al., 1994
; Parry et al., 1995
; Serrano et al., 1993
). A cell with functional p16/Rb signaling will initiate stress-induced overexpression of p16 resulting in a proliferative arrest characteristic of cellular senescence. On the other hand, a cell with a compromised Rb pathway will initiate a regulatory-induced overexpression of p16, due to unobstructed negative feedback regulation and disregard the many stress signals that induce senescence and cellular arrest. These later cells proliferate unimpeded and bypass senescence.
To distinguish between these two opposing phenotypes, we also stained serial sections for a proliferation marker, Ki67. Thirty-seven percent (26/70) and 63% (44/70) of the lesions within this case-control study express high and low Ki67, respectively (). High Ki67 alone provides a modest stratification for women that develop a subsequent tumor (DCIS and invasive combined; HR 2.7, 95% CI 1.2 to 5.9; and ). Interestingly, almost half (8/18) of DCIS lesions exhibiting high Ki67 index labelling also show high p16 levels (). We determined if this phenotype, representing deregulated p16/Rb signaling, identifies DCIS associated with subsequent tumor events. We find that all women with DCIS expressing high p16 and high Ki67 develop a subsequent tumor () and thus Ki67 stratifies high p16 expression into two groups: those who develop a subsequent breast cancer (case) versus those that do not (control; HR = 15.1, 95% CI, 1.4 to 161.4; ). The tumors that develop following DCIS with high p16/high Ki67 are often invasive breast cancer (5 of 8; Supplemental Figure 1
). The remaining DCIS lesions showing high p16 immunopositivity (9/18) exhibited low Ki67 index labelling, suggesting these cells have maintained p16/Rb checkpoint regulation. Indeed, most (9 of 10) lesions expressing a high p16/low Ki67 phenotype were not associated with a subsequent tumor event and thus may represent a protective signature (Supplemental Figure 1
). Women with DCIS that exhibit low p16 irrespective of Ki67 are not likely to develop recurrent disease (; HR = 1.1, 95% CI, 0.4 to 3.4; ).
High p16 mRNA levels defines the basal-like subtype of invasive tumors
Previous reports have demonstrated that ipsilateral tumors that develop subsequent to DCIS share many histological and genetic alterations with the primary lesion suggesting a clonal relationship (Bijker et al., 2001
; Lininger et al., 1998
; Millis et al., 2004
; Waldman et al., 2000
). Therefore, one might anticipate that high p16/high Ki67 in the initial DCIS lesion would characterize the subsequent invasive carcinoma. To test this prediction, we explored the prevalence and subtype distribution of p16 and proliferation by evaluating a previously published dataset of gene expression profiles of 130 primary invasive breast tumors (Chin et al., 2006
). Unsupervised clustering with a set of intrinsically variable genes identifies previously reported subtypes (Perou et al., 2000
; Solie et al., 2003
; , Supplemental Table 3
). Molecular subtypes were additionally classified based upon a nearest centroid approach using molecular subtype training data as previously defined (see Supplemental File 1
; (Hu et al., 2006
High p16 mRNA defines the basal-like subtype of invasive tumors
We find increased p16 mRNA expression preferentially characterizes the highly proliferative basal-like tumor subtype (). p16 expression falls within a gene cluster comprised of many well-established basal-like genes, such as keratin 5, 17, SFRP5, and MMP-7 (Supplemental Table 3
). To determine if the increased expression of p16 in actively proliferating cells is a consequence of p16/Rb pathway deregulation, we examined the expression levels of Rb, E2F3, cyclin E and cyclin D1. We find the basal-like sample cluster tends to display relatively low levels of Rb and high levels of E2F3 (). Since E2F3 transcriptional upregulation is a consequence of Rb inactivation (Leone et al., 2000
), we propose that deregulation of Rb signaling is a characteristic feature of basal-like tumors. Loss of Rb signaling can occur as a consequence of genetic alterations that has been observed to account for approximately 35% of breast tumors (Nielsen et al., 1997
; Reis-Filho et al., 2006
). Alternatively, Rb can be negatively regulated through phosphorylation by cyclin-dependent kinases. In examining the transcript levels of cyclin E and cyclin D1 among all tumor subtypes, we observe that cyclin E levels are among the highest and cyclin D1 among the lowest in basal-like tumors (). In concordance with previous observations (Loden et al., 2002
; Reis-Filho et al., 2006
), we find increased cyclin D1 levels to be most consistently elevated in the luminal B subtype. Although deregulation of p16/Rb signaling regulates genes involved in cell cycle progression, altered transcript levels of members of this pathway does not simply reflect proliferating tumor cells since highly proliferating Luminal B tumors do not exhibit the same transcriptional pattern.
To confirm the reproducibility of the observed differential subtype specificity of p16/Rb/Cyclin D1, we analyzed gene expression levels in four publicly available datasets from three different platforms (). In each case, tumors with overexpression of p16 and low transcript levels of both Rb and cyclinD1 were consistently found to be classified as basal-like tumors. Taken together, the observation that Rb transcript levels are among the lowest in basal-like tumors, and that E2F3 and cyclin E are among the highest suggests that loss of functional p16/Rb signaling may play a defining role in the biology of this tumor subtype.
High p16 and COX-2 mRNA levels are enriched in basal-like tumors across multiple microarray platforms
High COX-2 mRNA levels are enriched in basal-like tumors
To further analyze p16 and explore gene expression interactions, hierarchical clustering was performed on the top 6000 variable genes in 130 tumors (for details and data see Supplemental File 1
, Supplemental Figure 2
, and Supplemental Table 4
). As expected, members of the p16/Rb/cyclinD1 pathway showed variable expression, along with many members of the E2F family. We find that COX-2 is a member of the basal-like gene cluster. It is well recognized that high levels of expression of many basal-like genes are also found in normal-like invasive tumors (Hu et al., 2006
; Perou et al., 2000
; Sorlie et al., 2003
). Similarly, we find the 18% of tumors with elevated COX-2 mRNA are restricted to basal-like and normal-like subtypes. While 50% (16/32) of basal-like and 33% (4/12) of normal-like tumors overexpress COX-2, virtually all luminal and HER2 positive tumors express COX-2 mRNA levels below the median. The prevalence of COX-2 overexpression in basal and normal-like tumors is confirmed in independent data sets ().
Concordance between mRNA and protein expression of p16 and COX-2 in tumors
The low levels of COX-2 mRNA expression in HER2 positive tumors is perplexing because previous studies had demonstrated that COX-2 protein levels are enriched in HER2 amplified tumors (Boland et al., 2004
; Cho et al., 2006
; Ristimaki et al., 2002
). Notably, microarray analyses are typically average measurements of numerous cell types that often represent arbitrary units relative to a median value and fail to address post-transcriptional and post-translational regulation. It is therefore, critical to relate thresholds of detection of mRNA by microarray analysis to levels of protein expression as measured by immunohistochemistry (IHC). Further, it is important to determine the contributions of distinct cell types to overall levels of gene expression. We performed p16 and COX-2 IHC on paraffin-embedded tumor blocks representing 54 of the 130 tumors analyzed by microarray. These samples were chosen to represent all 5 molecular subtypes and span a continuum from the lowest to the highest levels of p16 and COX-2 microarray gene expression.
In samples of invasive tumors that showed elevated p16 via microarray analysis, immunopositivity is predominantly found in carcinoma cells. To a lesser extent, heterogeneous foci exhibiting p16 staining are detectable in the morphologically normal epithelial cells. Occasional p16 positivity is also observed in fibroblasts, predominantly those within desmoplastic appearing stroma. Cases with elevated COX-2 show abundant staining within the carcinoma cells as well as in the morphologically normal epithelia. In rare cases, we found intense COX-2 staining in macrophages infiltrating invasive tumors.
For p16, we observe a significant correlation (Wilcoxon rank of sums, p < 0.0001) between mRNA and protein expression (). Basal tumors that express the highest levels of p16 mRNA showed intense p16 protein staining by IHC (score 3+, ). We did observe a fraction of HER2 positive tumors (2/8) that showed intense p16 staining despite low mRNA expression. This concordance between mRNA and protein suggests that p16 protein levels are primarily regulated at the transcriptional level and that p16 protein levels determined by IHC reflect the subtype specificity.
Concordance between p16 or COX-2 mRNA and protein expression in tumors
In contrast, COX-2 demonstrated poor correlation between mRNA and protein levels (Wilcoxon rank of sums, p = 0.161; ). Those cases with the highest levels of COX-2 mRNA, as defined by a greater than 2-fold increase over the median value, exhibited complete concordance and displayed high COX-2 immunoreactivity (). Ten of the 54 invasive tumors show COX-2 mRNA levels greater than 2-fold above the median, eight of these cases are basal and the remaining two are classified as normal-like. In remaining cases (44/54), 10 exhibit low mRNA and were discordant with elevated protein expression as measured by IHC (). We found that 80% of the discordant samples were in the HER2 subtype.
COX-2 overexpression coupled with proliferation is associated with subsequent tumor events among women with DCIS
To determine if elevated COX-2 protein expression in DCIS is also associated with subsequent tumor events, COX-2 IHC was performed in serial sections of 70 cases previously analyzed for p16 and Ki67 (representative staining in ). We find that 56% (39/70) of DCIS lesions show high COX-2 protein levels () which by itself does not stratify risk for subsequent tumor formation and, similar to p16 overexpression by itself, is equally distributed among women that develop subsequent DCIS or invasive cancer (case) and those that do not (control; HR=0.79, 95% CI, 0.4 to 1.7; ; ).
COX-2 overexpression coupled with proliferation increases the risk of subsequent tumor events among women with DCIS
As with p16, stratifying high and low COX-2 DCIS lesions by proliferation identifies those more and less likely to have a subsequent tumor event. A significantly higher fraction of women (13 of 17) with high COX-2/high Ki67 develop a subsequent tumor (; Wilcoxon rank test; p = 0.002) as compared to lesions that show high COX-2/low Ki67 (HR = 4.8, 95% CI, 0.8 to 27.5, ). Correspondingly, we did not observe an increase in subsequent tumor events in women that exhibit low COX-2 expressing DCIS irrespective of Ki67 (; Wilcoxon rank test p = 0.925; HR = 0.86, 95% CI, 0.2 to 3.3; ). In examining the lesions that develop subsequent to high COX-2/high Ki67 DCIS, 7 of 13 cases are invasive breast cancer (Supplemental Figure 1
Similar to our observation that high p16 in the absence of proliferation identifies a protective signature (), 81% (17/21) of women with high COX-2/low Ki67 DCIS do not develop a subsequent tumor event (). Most DCIS lesions (6/7) expressing both high p16 and high COX-2 in the absence of proliferation are not associated with subsequent disease (Supplemental Figure 1
). These observations suggest high COX-2 and/or high p16 mark two clinically different populations of cells in DCIS that can be stratified by proliferation.
COX-2 overexpression causes cell cycle arrest in cells that maintain functional p16/Rb signaling
To determine the cellular context that governs if COX-2 is associated with quiescence or proliferation, we investigated the S-phase fraction in normal mammary epithelial cells and in a series of mammary cell lines (both premalignant and malignant), engineered to constitutively overexpress COX-2. Normal human mammary epithelial cells (HMEC) were propagated from disease-free reduction mammoplasty tissue from three different individuals. Premalignant cells studied include a subpopulation of HMEC (variant HMEC or vHMEC) with an extended but finite lifespan (Crawford et al., 2004
; Gauthier et al., 2005
; McDermott et al., 2006
; Romanov et al., 2001
), vHMEC-hTert, vHMEC immortalized by stably expressing human telomerase, and 184A1, non-tumorigenic immortalized mammary epithelial cells. In addition, we examined 4 malignant cell lines (T47D, MDA-MB-231, BT549, SkBr3) that have compromised p16/Rb signaling through diverse mechanisms including p16 hypermethylation, p16 deletion, and/or Rb deletion.
In HMEC, sustained COX-2 overexpression significantly reduces the number of cycling cells () and produces enlarged flattened morphology (). The proliferative arrest phenocopies that observed with overexpression of p16 (data not shown). In contrast, COX-2 overexpression neither induces nor diminishes proliferation in any of premalignant and malignant cells examined (). Functional p16/Rb signaling is one of the distinguishing features of normal cells compared to all other cells we examined. Therefore, we hypothesized that COX-2-induced growth arrest is dependent on functional p16/Rb signaling. Indeed, we find HMEC overexpressing COX-2 exhibited elevated protein levels of p16, p53 and p21 (). This is in contrast to vHMEC, which did not express p16, where overexpression of COX-2 did not alter the level of p53 or p21. Additionally, targeted degradation of Rb, p107 and p130 (all three Rb family members) by HPV16-E7 (human papilloma virus16-E7) in HMEC resulted in ongoing proliferation in the presence of COX-2 overexpression (). Thus, COX-2 overexpression in cells with functional p16/Rb signaling induces a p16-dependent growth arrest, while cells with disrupted p16/Rb signaling continue to proliferate in the presence of COX-2 overexpression.
p16/Rb pathway regulates COX-2 expression and COX-2-dependent cell cycle arrest
Deregulation of Rb signaling causes COX-2 overexpression
Our finding that the majority of high p16/high Ki67 DCIS lesions overexpress COX-2 suggests that deregulation of p16/Rb may drive COX-2 expression. To test this hypothesis, we modulated the p16/Rb pathway in HMEC generated from three different reduction mammoplasties using different approaches then determined the effect on basal and induced COX-2 protein levels. Downregulation of p16 protein level in HMEC by infecting them with a virus expressing p16-targeted short hairpin RNA (shp16) lead to upregulation of both Rb and E2F1 as expected. ( and (Zhang et al., 2006
)). Reducing p16 protein level did not change the basal level of COX-2 expression appreciable but increased TGF-β treatment-induced COX-2 expression (). Overexpression of cyclin D1 alone did not cause hyperphosphorylation of Rb or alter E2F1 protein levels (), consistent with previous findings (Lundberg and Weinberg, 1998
). Overexpression of cyclin D1 did not increase either basal or TGF-β treatment-induced COX-2 expression (). Silencing of Rb expression by infecting cells with a virus expressing an established short hairpin RNA against Rb (shRb) (Boehm et al., 2005
) resulted in the upregulation of not only E2F1 () but also both basal and induced levels of COX-2 protein (). Similarly, expression of HPV16-E7 elevated both basal and induced levels of COX-2 protein (). These data demonstrate that abrogation of p16/Rb signaling through genetic silencing of p16, Rb and Rb family members sensitizes cells to COX-2 upregulation. Thus, the propensity for COX-2 overexpression in DCIS lesions that exhibit high p16/high Ki67 is most likely a consequence of deregulation of the p16/Rb pathway.