The current study describes, for the first time, expression levels of APM components in primary vs. brain metastasis of breast cancer. Our results suggest that low or defective expression of APM components β2-microglobulin, TAP1 and calnexin as well as paucity of CD8+ T cell infiltration in the primary breast cancers may dictate high risks of developing brain metastasis. Our results from in vitro and in vivo functional studies support the role of TAP1 in CTL mediated lysis and reduction of brain metastasis.
Our results showed that defective or low expression of HLA class I APM components occurred frequently in both primary breast cancer and brain metastasis. There have been only few reports on the APM status and cancer metastasis [28
], and to our knowledge, our report is the first specifically evaluating the APM status in brain metastases. Although no significant differences were found in the expression levels of APM components between primary breast and metastatic brain lesions in 15 paired cases, these primary breast lesions with known history of brain metastasis showed lower levels of β2-microgloblin, TAP1 and calnexin compared with breast lesions without known brain metastases. These results suggest that metastases do not originate from a subclone of tumor cells that undergo down-regulation of APM expression in the primary site (“acquired” phenotype for tumor immune escape), but rather that an entire primary tumor with lower or defective expression of β2-microgloblin, TAP1 and calnexin might be more likely to spread to the brain (“inherent” phenotype for immune escape). Prospecitve studies in patients with breast cancer are needed to prove this hypothesis.
The three molecules (β2-microglobulin, TAP1 and calnexin) which are down-regulated in the cases with known metastases have critical functions in the complex cascade where peptides are processed and loaded on HLA class I molecules [30
]. The processing and presentation of HLA class I antigen-derived peptides is accomplished through a series of intracellular events involving multiple APMs [32
]. Following proteosome-mediated degradation and cytosolic cleavage of antigen proteins, peptides are transported via TAP1 and TAP2 into the endoplasmic reticulum (ER). In the ER, the HLA class I heavy chain and β2-microglobulin assembly occurs, which is coordinated by the chaperones calnexin, calreticulin, and the thiol oxidoreductase ERp57. Upon peptide loading, the trimer consisting of the HLA class I heavy chain, β2-microglobulin and antigen-peptide is released and transported via Golgi to the cell surface and there exposed to the CD8+ CTL. Down-regulation of TAP1 inhibits the transportation of peptide to the ER, whereas down-regulation of calnexin impedes proper folding of HLA class I heavy chain-β2-microglobulin complex. β2-microglobulin and TAP1 were also often found to be down-regulated among APM components in a variety of tumors [33
]. Based on these known mechanisms underlying the assembly of HLA class I complexes, our results demonstrating a positive correlation of HLA class I heavy chain expression level with β2-microglobulin and TAP1 are reasonable, although we recognize that the immunohistochemical analysis in this study does not completely distinguish intracellular vs. surface expression of HLA class I. Although our data with mouse models demonstrate the functional significance of TAP1 in CTL recognition and metastasis, further mechanistic studies are warranted to better understand the molecular mechanisms responsible for APM/HLA down-regulation in cancer cells.
CTLs play an active role in the recognition and destruction of tumor cells. Their activation is initiated by the interaction with HLA class I molecules that present cognate antigen peptides. The lack or decreased expression of single or multiple components of the HLA class I antigen processing pathway can allow tumor cells to escape from recognition by CD8+ CTLs. Indeed, down-regulation of APM components, such as TAP1, TAP2 and tapasin, has been found to be associated with failure of CTL recognition in squamous cell carcinoma of the head and neck [34
]. In accordance with these previous studies, in the current study, the extent of CD8+ T cell infiltration in primary breast cancer was positively associated with expression of TAP1 and calnexin. Significance of our results in the clinical samples was further potentiated by the suppressed CTL-mediated lysis of B16-TAP1KO cells. This finding is not unique to a specific cell line, because similar results have also been described in both mouse and human tumor cells [34
]. These data imply that future immunotherapy studies ought to consider TAP1 expression levels in tumors as a biomarker and/or develop strategies to enhance TAP1 expression, such as gene transfer.
Presence and extent of T cell infiltration were associated with longer survival of cancer patients [37
]. Our results show that primary breast lesions with known brain metastases were infiltrated by lower numbers of CD8+ T cells compared with breast lesions without known metastases. These results suggest that low or absent CD8+ T cell infiltration in the primary breast cancer could also be a biomarker for breast cancers that have a higher risk of brain metastasis. We also found a trend that patients with more advanced stages had fewer CD8+ T cell infiltration compared to lower stages in their primary breast cancer site, although the trend did not meet the criteria for statistical significance. The limited amounts of available tissues did not allow us to evaluate the status of CD8+ T cell infiltration in the brain metastases. However, recent reports have demonstrated that the extent of CD8+ T cell infiltration in glioblastoma multiforme tissues correlates with long-term survival of patients [40
]. Future studies will address whether the degree of CD8+ T cell infiltration in metastatic cancers in the brain provide us with any additional prognostic information.
Dendritic cells (DCs) and other antigen presenting cells are important initiator and modulator of anti-tumor immune responses. Unfortunately, we were unable to evaluate the status of DC infiltration in primary and metastatic breast cancer tissues due to the limited amounts of available tumor materials. Nonetheless, the literature suggests that metastatic tumor cells in patients’ lymph nodes may affect the maturation of DCs, thereby affecting the antigen-presentation process. Although the number of DC infiltration in breast cancer did not correlate with lymph node metastasis, pathologic stage, or relapse-free survival [42
], sentinel lymph nodes with metastatic tumor cells contained fewer mature DCs in than those without metastasis [43
]. In addition to DCs, there is a variety of immune response mediators that can significantly impact the immune surveillance against cancer metastasis, such as chemokines, cytokines and T cell effector molecules [44
]. Additional studies are warranted to gain more comprehensive understanding on the immunological factors that affect the risk and development of cancer metastasis.
In our analyses of clinicopathologic factors and their relation to APM expression levels, lower expression of TAP1 was significantly associated with early onset of the cancers in both primary and metastatic lesions. It is well known that breast cancers in young patients tend to behave more aggressively and possess poorer prognosis compared with cases in old patients [47
]. Furthermore, in our study, lower TAP1 or TAP2 in primary breast cancers were both associated with poorer AJCC stage. Thus, based on our results that TAP1 is down-regulated in primary breast cancers with known brain metastases and that its expression level is positively associated with intratumoral CD8+ T cell infiltration, TAP1 may be considered as a biomarker that dictates higher risks for developing brain metastasis in patients with breast cancer. Further studies, especially prospective studies, are warranted to establish the value of TAP1 as such a biomarker. Indeed, our experiment using 4T1-TAP1KO cells indicates that the knockdown of TAP1 in the primary breast cancer promotes brain metastases. Since the extent of CD8+ T cell infiltration in primary breast cancer was positively associated with TAP1 expression, it is reasonable to postulate that down-regulation or lack of TAP1 allows breast cancer cells to escape from T cell-mediated immuno-surveillance, thereby promoting the brain metastasis.
In addition to TAP1, lower expression of TAP2 in the primary breast cancer was associated with higher AJCC stage and higher nuclear grade which were poor prognostic indicators. On the other hand, TAP2 expression was negatively associated with ER and PR expression in the primary lesions. As negative ER and PR status has been determined to be a strong predictor of poor prognosis [49
], these results suggested that TAP2 and ER/PR status might be two independent indicators of breast cancer prognosis and function in independent mechanisms. Recent studies have shown some degrees of discordance in ER and PR status between primary and metastatic lesions [52
]. In fact, in brain metastases, there were no associations between the TAP2 expression levels and ER/PR status, likely supporting our notion that these are two independent factors. Although previous studies by others have shown that over-expression of HER2 in tumor cells leads to markedly reduced levels of APM components [54
], we didn’t find either positive or inverse correlations between HER2 and APM expression levels in our cases.
Although there is a growing body of evidence demonstrating down-regulation of APM components in a variety of cancer types [12
], the current study now shed lights on the potential roles of these molecules in the metastatic processes of cancer. Extension of these studies will likely allow us to develop novel biomarkers dictating the risks for metastasis and delineate key underlying mechanisms upon which we can develop strategies to prevent and/or reduce the risk of cancer metastasis.