In this study, we have investigated the differences of hydrophobic sub-proteomes between two subtypes of breast cancers (TNBC vs. ER+PR+Her2−). Mass spectrometry (LC/MS/MS) techniques were used to compare the protein profiles identified from the 20 tumors, with 10 in each of the comparison groups. We also report several candidate proteins that may be involved in the aggressive manifestation of TNBC.
An interesting finding of this study was to discover that all nine keratins found in breast cancers were down-regulated in all 10 TNBC cases when compared with the 10 ER+PR+Her2− breast cancer cases. Keratins (previously called cytokeratins) are filament-forming proteins and are essential for normal tissue structure and function [36
]. A major function of epithelial keratins is to provide the mechanical stability and integrity of epithelial cells and tissues [32
], as well as the mechanical support to epithelial cell sheets [37
]. The biological meaning and mechanism of reduced keratins in TNBC has not been previously investigated. Hypothetically, however, less keratin may indicate that TNBC cells are less “stiff” and more “flexible” in changing shapes to facilitate cellular movement, transportation, and migration for its invasive nature and early metastasis. It has been reported that metastatic cancer cells are 70% softer than non-metastatic cells using nanomechanical analysis [38
]. Down-regulated expression of keratins in TNBC was also observed by another study in our laboratory (Whelan et al., submitted for publication), which compares TNBC with Her2+ breast cancers. In this study, keratin 19 was found expressed eightfold lower in TNBC. Thus, change in cell stiffness may be a newly described characteristic of cancer cells that affects the way they spread and the molecules regulate this function.
Annexin family proteins were found over-expressed in TNBC (Table ), which is consistent with our previous finding [14
] in TNBC tumors and cell lines. The protein family of annexins has continued to grow since their association with intracellular membranes was first reported in 1977 [39
]. Annexins are important in various cellular and physiological functions such as providing a membrane scaffold, which is relevant to changes in the cell's shape [40
]. Also, annexins have been shown to be involved in trafficking and organization of vesicles, exocytosis, endocytosis, and calcium ion channel formation [42
]. A literature search indicates that Annexin over-expression correlates with the aggressiveness of cancer. Annexin A3 was significantly up-regulated in invasive lung adenocarcinomas with lymph node metastasis compared to those without lymph node metastasis [43
]. Similarly, annexin A5 and A7 are significantly elevated in lymphatic metastasis of mouse hepatocarcinoma [44
]. Together, these findings provide strong evidence that Annexin family proteins are likely to contribute to the aggressive phenotype and metastatic potential of TNBC.
In this study, we also found an overall up-regulation of heat shock protein expression in TNBC. Heat shock proteins are a class of functionally related proteins whose expression is transcriptionally regulated and over-expressed when cells are exposed to elevated temperatures, hypoxia, or other stress [45
]. Heat shock proteins are named according to their molecular weight. Among them, Hsp60, Hsp70, and Hsp90 are the most widely studied Hsps. Hsps have a dual function depending on their intracellular or extracellular location. The different properties of Hsps allow them to be exploited in therapy. Intracellular Hsps are protective to the cell and are highly expressed in cancerous cells. The elevated Hsp expression has been shown to promote cancer growth through inhibition of programmed cell death (Hsp27, Hsp70), allowance of autonomous tumor growth (Hsp90) and tumor resistance to chemotherapy and hyperthermia [46
]. Since they participate in oncogenesis and in resistance to treatment, the inhibition of Hsps has been tested in clinical trial for cancer treatment. Small molecule inhibitors of Hsps, especially Hsp90, show promise as anticancer agents [35
]. The potent Hsp90 inhibitor 17-AAG is currently in clinical trials for the treatment of several types of cancer [47
]. In contrast to the intracellular Hsps, extracellularly located or membrane-bound Hsps mediate immunological functions. They can elicit an immune response modulated either by the adaptive or innate immune system [35
]. Their immunogenicity can be used to form the basis of anticancer vaccines [46
Our mass spectrometric results showed a significant over-expression of Hsp70 and Hsp90 in the TNBC tumors. Another parallel study comparing TNBC with Her2+ tumors in our laboratory indicates that Hsp70 and Hsp90 are over-expressed in TNBC (He et al., in preparation). This is consistent with other studies reporting Hsp70 and Hsp90 expression associated with tumor of poor differentiation, rapid proliferation, and suppressed apoptosis. Clinically, they are associated with lymph node metastasis and poor clinical outcome [48
Among the nine unique proteins detected exclusively in 10 TNBC cases, two of them were HLA class proteins (Table ): HLA class II histocompatibility antigen DR alpha and HLA class I histocompatibility antigen A-23 alpha were both over-expressed in TNBC but below detection level in ER+PR+Her2− breast cancer specimens. These two proteins belong to the major histocompatibility complex (MHC) class II and I family, respectively. They are membrane proteins, which explain the reason they exist in hydrophobic fractions.
The MHC is a large gene family, which is the most gene-dense region of the mammalian genome and plays an important role in the immune system and autoimmunity [51
]. The MHC proteins display both self antigens (peptide fragments from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms or cancer) to T cells and NK cells. Under normal conditions, expression of MHC molecules inhibits killing by NK cells, while absence of MHC molecules in infected or stressed cells allows cell-mediated cytotoxicity to occur. MHC class I molecules express on all nucleated cells, while MHC class II family proteins express on most immune system cells, specifically on antigen-presenting cells. Thus, over-expression of MHC class II and I proteins in tumor tissue suggests two possibilities. First, antigens may exist on tumor cell surfaces carrying different biological functions to tumor cells. Second, TNBC may have more lymphocytic infiltration, which may be the source of MHC over-expression found in these tumors. Recent studies reported that several HLA protein over-expression are associated with breast cancer [52
] and may favor their escape from anti-tumor immune responses [54
], as well as predict chemotherapy resistance [55
The rapid development of analytical instruments and data mining software in the last two decades has significantly extended the possibilities of studying proteins in life sciences. Proteomic analysis provides deeper insights into qualitative and quantitative changes in protein composition in association with the disease process. The combination of hydrophobic fractionation and nano-LC mass spectrometry analysis of cancer proteome offers a promising tool in the discovery of potential cancer bio-signatures. When sufficiently refined, this approach may prove useful for early detection and better treatment of breast cancer.