Introduction
Breast cancer continues to be the most frequent cancer type and the second leading cause of cancer death in females in Western Europe and North America. Although we have significantly improved diagnostic tools and therapeutic interventions in breast cancer over the last decade, breast oncologists still face paramount challenges in hormone-negative breast cancer, especially triple-negative breast cancer, the most aggressive breast cancer subtype, for which we do not yet have any molecular-targeted treatments apart from cytotoxic chemotherapy. Several large studies have identified RNA expression signatures that are prognostic for disease recurrence and metastasis early-stage hormone-positive breast cancer (HPBC) [
1-
3]. No such biomarkers exist currently for the more aggressive hormone-negative breast cancer (HNBC). In addition, mounting evidence indicates that levels of proteins that are immediately relevant to cell growth and metabolism are often not particularly well correlated to mRNA levels [
4], suggesting that it may be more informative to directly measure protein levels as a indicator of biological behavior. These biomarkers likely reflect genetic signatures of early cancer formation and evolution of cancer aggressiveness.
Ataxia telangiectasia mutated (ATM) belongs to a family of so-called phosphatidyl inositol-3 kinase (PI3K)-like serine/threonine protein kinases (PIKKs), most of which are involved in the cellular response to various stresses [
5]. In response to DNA double-strand breaks, ATM phosphorylates multiple key downstream proteins including p53 and BRCA1/2, the most common sporadic and inheritable mutated genes in breast cancer [
6], thereby orchestrating complex signaling pathways involved in cell cycle arrest, DNA repair, senescence and apoptosis [
7]. Germline mutation of ATM genes causes ataxia telangiectasia, a rare pleiotropic autosomal recessive disorder characterized by heightened radiation sensitivity and a predisposition to cancers including breast cancer [
6,
8]. The incidence of sporadic ATM mutations in breast cancer is low (2 to 3%), as compared to p53 and PIK3A mutations whose incidence rates are 30 to 40% [
9]. Although we have some evidence supporting ATM loss in malignant breast tumors [
10-
17], and its prognostic significance in breast cancer was recently uncovered [
16,
17], the protein level of ATM in breast cancer-associated stromal tissues remains unknown.
In this study, using fluorescent immunohistochemistry (IHC) and automated quantitative analysis (AQUA), we measured ATM protein expression in both malignant tumor and stromal compartments of HPBC and HNBC. We hypothesized that reduced ATM expression correlates with increased tumor aggressiveness and poorer clinical outcome. Remarkably, we also identified a strong association between stromal expression of ATM and clinical outcome, suggesting that the tumor microenvironment contributes significantly to the overall course of disease.
Discussion
In this study, we show that ATM expression was reduced in HNBC, as compared to either HPBC or normal breast epithelium. In both HPBC and HNBC cohorts, reduced ATM expression was associated with poorer disease-specific outcomes. Interestingly, stromal ATM expression was higher in both cohorts as compared to normal epithelium, though lower expression in this compartment was also associated with poorer clinical outcome. These expression studies were enhanced by fluorescent IHC and AQUAnalysis, which allowed us to describe ATM expression as a continuous variable across a broader range than is possible with brightfield IHC. The dynamic range of protein quantification that is possible in the fluorescent realm allows the analysis, in a research context, of samples with widely varying degrees of protein expression. It should be noted that fluorescent IHC and the associated automated image analysis workflows are not currently used in clinical practice; therefore some data presented in this study may not be directly applicable. It is however possible that they could be translated into clinically relevant assays upon identification of the levels of ATM expression that offer prognostic value. To the best of our knowledge, this is the first study to examine ATM protein expression in malignant tumor and cancer-associated stromal tissues simultaneously.
About 60 to 80% of the EBC patients in both cohorts had ATM scores (tATM or nATM) lower than the mean values for the same compartment in normal breast epithelium. This suggests that loss of ATM expression is an important early event signaling malignant transformation of breast epithelial cells as the majority of EBC has some degree of ATM deficiency. In addition, ATM loss continues when breast cancer evolves and becomes more aggressive, to a certain threshold that significantly impacts on cancer-specific survival. Reduced ATM expression within malignant breast epithelial tissues was predominant in HNBC, especially TNBC, the most aggressive breast cancer type. These results, along with similar outcomes reported by others [
10-
17], suggest that ATM loss not only occurs early during breast tumorigenesis, but also may promote cancer progression by permitting the accumulation of genetic mutations due to genome instability as a result of ATM loss, thereby selecting cancer cells bearing aggressive phenotypes.
In HPBC, low ATM within malignant breast epithelial cells and stroma is associated with aggressive characteristics of breast cancer including large size, high grade and LN involvement. It is an interesting observation that this association is not seen in HNBC, which is a more aggressive tumor type. It is likely that ATM loss predominates the aggressive nature regardless of other worse features in this aggressive tumor type, which is consistent with our later finding of independent prognostic ability of ATM in only the HNBC cohort. Previous studies [
16,
17] demonstrated the significant association between low ATM expression and worse clinicopathological features in an undifferentiated breast cancer cohort containing both HPBC and HNBC cases. We believe that our data, consistent with others [
10-
17], support the notion that ATM loss contributes to the aggressive nature of breast tumors as they evolve.
It is interesting that ATM is higher in cancer-associated stromal tissues compared with stromal tissues around normal breast epithelium. Although this is consistent with another study [
24], validation is needed using a larger distinct cohort. To the best of our knowledge, the mechanisms of regulation and functional roles of ATM in malignant stroma are presently unknown. Although it may seem contradictory that the ATM level is increased in cancer-associated stroma compared to normal stroma, albeit the ATM level is decreased in malignant epithelium compared to normal epithelium, we believe that this could be a reflection of the complexity of ATM regulation in diverse cell types. We speculate that the heightened level of DNA damage and oxidative stress [
25] that are predominant in malignancy may initially lead to significant induction of ATM in the malignant epithelial tumor as well as in the stroma, Subsequently, a range of other factors may lead to differentially active downregulation of ATM, more so within the epithelial tumor and less so within the stroma. For example, one of such factors may include caspases that are activated particularly in the necrotic cores of aggressive tumors, which were recently shown to proteolytically cleave ATM [
26]. Other regulatory factors are discussed later. Furthermore, we speculate that ATM may have context-dependent roles in stroma that are distinct from its well-known conventional function in maintaining genomic stability in response to DNA damage within the epithelial component. For example, it may regulate inflammatory response such as chemokine release (for example interleukin (IL)-6, IL-8) to promote/inhibit tumor growth in a paracrine-mediated fashion [
27]. In addition, it may serve as a contributor to extracellular matrix stiffness (in addition to many other cytokines) and regulates tumor invasion and metastasis through bidirectional signaling between tumor cells and stroma microenvironment [
28]. Therefore, it is not surprising that low level of ATM in cancer-associated stroma is also associated with a poor survival (Figure
4E and F).
It should be noted that cut-points used to define positive and negative ATM tumors varied widely in previous studies despite all having used conventional IHC as there is no well-defined method for choosing cut-points for biomarker analysis. For example, Bueno’s group [
16] defined ATM IHC scores as negative or positive according to the absence or presence of nuclear staining in epithelial cells evaluated by two independent pathologists who had been blinded to the outcome. Abdel-Fatah’s group [
17] used a cutoff of <25% cells being classed as low and ≥25% as high for nuclear ATM protein level. Because ATM expression in our study is a continuous variable that is generated automatically by the analysis software, we chose to use the X-tile program [
21], which is a validated methodology to define our cut-point in two independent cohorts. The other advantage of using such digital image analysis as compared to conventional IHC is that it can potentially avoid human bias (inter- and intra- observer variability) as the intensity scores are objective representations of protein expression. In addition, this platform allows us to define the specific localization of ATM expression within the tumor. Therefore, we can evaluate tumor nuclear and cytoplasmic expression of ATM concurrently, in a similar fashion as the above tumor and stromal ATM analysis. We examined nuclear versus cytoplasmic ratio of ATM expression (ncATM) in both EBC cohorts as we speculated that aberrant localization of ATM may also contribute to loss of ATM function thereby resulting in tumor aggressiveness and poor prognosis. Although we did observe a significantly decreased ncATM in more aggressive breast tumor, HNBC compared with HPBC (Figure S1 in Additional file
4), the prognostic value of ncATM for clinical outcome (DFS and DSS) was not as significant as tATM, nATM and csATM in both cohorts (data not shown). We speculate that this may be due to our small sample size.
Lastly, our study illustrates the independent prognostic ability of ATM protein expression levels for DSS in both malignant tumor and stromal tissues in HNBC. Bueno
et al. found that ATM protein expression associated significantly with both DFS and DSS [
16]. We also observed a strong trend that low ATM group (tATM, nATM and csATM) had worse DFS than high ATM (tATM, nATM and csATM) group with a
P value of 0.05 to 0.09 based on log-rank tests of KM analysis (data not shown). The lack of statistical significance may be an outcome of relative small sample size as well as the different methodologies of evaluating ATM protein expression and defining cut-points to divide ATM low and high groups. We, and others [
16,
17], clearly demonstrated that ATM protein has potential use as a novel biomarker in EBC, in addition to the known clinicopathological prognostic factors such as tumor size, grade, LVI and LN status. This could be a much simpler tool than several biomarker tools deployed in HPBC such as Oncotype DX, PAM50 and MammaPrint, which are all RNA-based assays [
1-
3].
Our study did not seek to investigate the mechanisms underlying the regulation of ATM expression in EBC. ATM gene mutations have been described in the literature, but since the incidence of ATM mutations in breast cancer is low [
9], we would not expect this to explain the full range of our results. Other mechanisms such as gene copy number loss [
16,
29], posttranscriptional microRNA regulation [
16,
30,
31], epigenetic modifications [
32,
33] and direct caspase cleavage [
26] for altering ATM levels have been described. It should also be noted that a simple measure of ATM protein expression by IHC cannot establish the functional capability of ATM within the tumor. Measuring of phosphorylated ATM protein and its targets within the ATM signaling pathways may provide more insight with regard to ATM functions in breast cancer. Our results taken in the context of what is known about the biological role of ATM in protecting the genome suggest that it is very likely that a substantial proportion of breast cancers, which have lost ATM expression at the protein level, are in fact deficient also in ATM function [
34].
We attempted to investigate the predictive value of ATM protein for the efficacy of adjuvant RT and chemotherapy in our cohorts. Due to small sample size, we cannot draw any conclusions. We noted that other studies have showed that ATM loss seems to associate with resistance to anthracycline chemotherapy in breast cancer [
16,
17,
29], which seems counterintuitive to many preclinical studies revealing that ATM-deficient cells are sensitive to cytotoxic chemotherapy [
7]. More interestingly, we and others have recently shown that ATM loss can predict sensitivity to poly (ADP-ribose) polymerase (PARP) inhibitors, a novel drug that may exploit this deficiency, in breast cancer [
35] as well as other cancers [
19,
36,
37]. Prospective clinical trials are warranted to validate its predicative value.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
XF designed the study, interpreted the results, and drafted the manuscript. HL performed the statistical analysis, helped prepare most of the figures and critically revised the manuscript. MD and HW carried out the TMA staining and data analysis in the two cohorts and helped prepare some figures. EK carried out ER/PR/HER2 staining and data analysis in the HPBC cohort. EE participated in optimizing antibodies and techniques for TMA staining, and helped to draft the manuscript. AM generated two TMA cohorts, participated in the study design and critically revised the manuscript. PT participated in generating the HNBC cohort TMA and critically revising the manuscript. AP, SPL-M and GB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. In addition, all authors agreed to be accountable for the accuracy and integrity of the results from this study.
Xiaolan Feng MD PhD FRCPC, Locum Medical Oncologist and Research Scientist.
Haocheng Li PhD, Biostatistician.
Michelle Dean BSc, Technician.
Holly Wilson BSc, Technician.
Elizabeth Kornaga MSc, Senior Technician.
Emeka Enwere PhD, Research Scientist.
Patricia Tang MD FRCPC, Medical Oncologist.
Alexander Paterson MD FRCPC, Medical Oncologist.
Susan P Lees-Miller PhD, Senior Research Scientist.
Anthony M Magliocco MD, Pathologist and Senior Research Scientist.
Gwyn Bebb MD PhD FRCPC, Medical Oncologist and Senior Research Scientist.