The identification of breast cancer markers has been the focus of extensive research in past decades, there still an urgent need for good cancer biomarkers remains. Moreover, characterization of these proteins and identification of the alterations that distinguish cancer cells from normal ones will help to understand the molecular mechanisms of cancerogenesis. In previous study we identified by SEREX-approach 41 novel medullary breast carcinoma autoantigens, 18 of which had cancer-related serological profile
]. During this study expression pattern of 6 from 18 potential MBC-derived antigens were studied by immunohistochemistry in tissue samples obtained from patients with different histological types of breast cancer and fibrocystic disease using specifically generated polyclonal antibodies. Medullary breast carcinoma, lobular and ductal invasive breast carcinomas’ tissue samples were taken for immunohistochemical investigation of expression patterns of above mentioned proteins. It’s known that lobular and ductal carcinomas derive both from the breast terminal duct lobular units
] and the terminology “ductal” and “lobular” is still being used for historical reasons and to date there is no evidence to suggest that these tumors arise from ductal or lobular epithelial cells. Thus, the differences in their morphology are likely to reflect the different mechanisms of carcinogenesis rather than the anatomical origin of the lesions
]. For example, it was demonstrated that 5.8% of the transcriptionally regulated genes were differentially expressed in ILCs compared to grade- and molecular subtype-matched IDCs
]. Identification and characterization of molecular targets specific for each type of breast cancer will help to design more precise therapeutic and diagnostic approaches for curing of this type of malignancy.
During this preliminary study, we investigated expression pattern of 6 MBC autoantigens, including secretory protein LGALS3BP, 2 nuclear proteins (RAD50, FAM50A), one cytoplasmic protein (PABPC4) and 2 tratscription factors (RBPJ, LRRFIP1), in different histological types of breast cancer and non-cancerous tissue samples by immunohistochemical analysis. Despite the fact that automated measurement using digital slide scanners and computer-aid methods are increasingly used for immunohystochemical analysis, during this study we used visual semi-quantitative evaluation of MBC antigens expression pattern by expert pathologist because high correlation between these two approaches was recently shown
It was shown that all analyzed proteins expressed in the most cells of non-cancerous and cancer tissue samples. However distinct expression pattern was revealed for some antigens in breast carcinomas of different histological types.
LGALS3BP (also known as MAC-2BP and 90K protein) was originally identified as a tumor-associated antigen in the culture supernatant of human breast cancer cells
]. Early works focused on study of LGALS3BP showed its expression in more than 80% of breast cancer tissues, but not in non-cancerous normal mammary gland surrounding the cancer cells
]. Immunohistochemical analysis of LGALS3BP expression in several tumors, including breast carcinomas, revealed LGALS3BP positive staining predominantly in cytoplasm
]. During our study, we also, observed moderate to strong LGALS3BP positive staining in cytoplasm and weak nuclear staining of IDC and MBC cells. Interestingly, that in ILC tumors we detected opposite pattern of LGALS3BP expression with moderate nuclear and weak cytoplasmic staining. Moreover, during this study we observed an explicit apical membrane staining of LGALS3BP in some of epithelial cells in breast tissues of patients with fibrocystic disease and epithelial cells lining the normal ducts in ILCs, contrary to mentioned above literature data
]. This strong apical membrane staining of LGALS3BP may partly be explained by its ability to be secreted by different types of cells including epithelial cells
RAD50 is a highly conserved DNA double-strand break repair factor
]. Its aberrantly reduced protein expression was reported in 3% of breast tumors (predominantly ER/PR/ERBB2 triple-negative and higher-grade familial breast tumors)
]. In our investigation we also showed strong RAD50 nuclear staining of non-cancerous and cancer cells. However, in 4 from 5 ILC cases we observed rather moderate than strong RAD50 positive staining in the nucleus of cancer cells. Since, down-regulation of some genes including RAD50 was shown in ILCs
]; our results partially confirmed the data about reduced RAD50 expression in ILC tumors. However, this fact should to be further investigated on a large number of samples.
FAM50A (also known as XAP5) is a poorly studied protein with unknown function having signal for nuclear localization
]. Here, we describe for the first time its subcellular localization and expression pattern in cancer and non-cancerous breast tissues. According to our findings FAM50A protein is expressed both in nucleus and cytoplasm in the most cells of cancer and non-cancerous breast tissues without any significant differences. However we found stronger FAM50A nuclear staining in immunocytes compared with cancer and non-cancerous breast cells.
PABPC4 (or inducible PABP (iPABP)) a homolog of PABP, have critical roles in RNA processing beyond simply binding to poly(A) sequences
]. According to the literature data PABPC4 is a diffusely cytoplasmic protein that can be localized to stress granules
]; its expression is induced upon stimulation of peripheral T cells
]. The fact that induction of PABPC4 coincides with the induction of lymphokine mRNA in activated T cells suggests that perhaps iPABP is necessary for regulation of stability of labile mRNA species
]. In our study, we observed moderate to strong PABPC4 positive staining in cytoplasm in the most of cancer and normal cells of all tissue samples and in part of immunocytes. One may be speculated that PABPC4 expression can be restricted to activated T cells at least in MBC tumors, and possibly contributes to a more effective immune response that can be associated with good prognosis for patients with this tumor type. However, to confirm this assumption additional study has to be performed.
RBPJ is a primary transcription mediator of canonical Notch signaling
], which participates in cell fate determination, and is involved in the regulation of tumor behavior in multiple decisions
]. It has been shown previously, that RBPJ protein can be detected in the nucleus as well as in the cytoplasm and it subcellular distribution changes in defined physiological contexts
In all cancer and non-cancerous cases predominantly moderate nuclear RBPJ and weak cytoplasmic staining was detected, however in some MBC and IDC cases strong nuclear staining was revealed. These differences may be associated with histological and molecular features of these tumor types.
LRRFIP1 is ubiquitously expressed and found in the nuclear and cytoplasmic compartments
]. This protein acts as transcriptional repressor of some genes
] and indirectly throught interaction with Flightless 1 protein involved in actin severing, capping and cytoskeletal rearrangement
]. Notably, LRRFIP1 can be mutated in breast tumors and soft tissue sarcomas
], and may be involved in the regulation of cell growth
]. We observed strong nuclear and weak cytoplasmic distribution of LRRFIP1 in cancer and normal cells except for MBC tumors, which showed moderate cytoplasmic staining in addition to strong nuclear staining.
Thus, during this study we described for the first time expression patterns of 6 potential MBC-associated antigens, including LGALS3BP, RAD50, FAM50A, RBPJ, PABPC4, LRRFIP1 in different histological types of breast carcinomas and non-cancerous breast tissues by immunohistochemical analysis. Our data indicate that all 6 antigens predominantly expressed in the most cells of all histological types of breast tumors, non-cancerous tissues, and immunocytes with slight differences in intensity of staining and subcellular localization. The most significant differences were observed for RAD50 and LGALS3BP antigens in different histological types of breast carcinomas. During this study we described for the first time expression pattern of poorly studied antigen FAM50A and observed its more intensive nuclear staining in the immune cells compared with cancer and non-cancerous cells. Additionally increased expression of other antigen PABPC4 was observed in restricted number of immune cells infiltrating breast tumors. These observations possibly reflect molecular changes that occur during breast carcinogenesis in different histological types of breast cancer and indicate that RAD50 and LGALS3BP antigens are promising candidates for more comprehensive research as potential molecular markers for breast cancer diagnostics and therapy. Moreover, antigens FAM50A and PABPC4 are intriguing targets for investigation of the features of the immune response in patients with highly infiltrating breast tumors including MBC, which despite anaplastic features has favorable prognosis.