Therapeutic Role and Immunogenic Characteristics of Cancer Testis Antigens in Breast Cancer: A Review.

Roles of cancer/testis antigens (CTAs) in breast cancer

Backround: Cancer Testis Antigens (CTA) are a family of proteins normally expressed in the human germ cells such as in the testis. They also have been found in various types of malignant tumors including breast cancer. Because of their restricted expression pattern they are frequently able to elicit T-cell immune responses, and therefore they are considered as ideal targets for cancer immunotherapy. Breast differentiation antigen NY-BR-1 has also immunogenic properties. The aim of this study was to explore the CTA expression in breast cancer and detect possible clinical correlations. Materials and Methods: The expression patterns of 6 CTAs (MAGE A1- MA454, MAGE A3-M3H67, MAGE A4-57B, NY-ESO-1-E978, GAGE, MAGE A-6C1) and NY-BR-1 were examined by immunohistochemistry in a series of 210 non-selected patients with primary invasive breast cancer using the tissue microarray technique. The intensity of the stain was scored semi-quantitatively in a scale 0 to+3. The expression of the antigens was correlated to established clinicopathological parameters as well as disease-free (DFS) and overall (OS) survival. Results: CTA expression exhibited a predominantly cytoplasmic and occasionally nuclear localization. At least one CTA was identified in 37.2% of cases with expression of each antigen varying from 4.5 to 15%. NY-BR-1 was positive in 46.6% of tumors, with the well differentiated tumors showing more frequent expression. MAGE A4-57B (p=0.028 and 0.015) and MAGE A3-M3H67 (p=0.001 and 0.004, respectively) positivestaining was significantly correlated to shorter OS and DFS. MAGE A1-MA454 correlated significantly only with OS (p=0.028). GAGE and MAGE A-6C1 displayed a clear trend, but not statistically significant prognostic value concerning shorter DFS and OS. In multivariate analysis only MAGE A3-M3H67 (p=0.007 OS) and MAGE A4-57B (p=0.017 DFS, 0.052 OS) showed and independent prognostic relevance. Additionally, the mortality rate increased substantially if co-expression of any 3 or more CTAs was observed. Discussion: Our findings suggest that CTAs could serve as potential prognostic markers in primary breast cancer patients. The exclusive expression of CTAs in tumor tissues as well as the frequent expression of NY-BR-1 could define potential new targets for specific breast cancer therapies. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-40.

e21126 Background: Cancer testis antigens (CTAs) are expressed in a variety of malignant tumors. In normal adult tissues CTA expression is restricted predominantly to germs cells of the adult testis and placenta. Based on their tumor-restricted expression pattern, CTAs are regarded as valuable targets for cancer immunotherapy. The prognostic significance of CTAs in breast cancer has not been analyzed previously. Methods: To evaluate the potential prognostic significance of two member of this family, MAGE-A10 and NY-ESO-1 antigens, we examined their expression in breast cancer patients who underwent curative surgery at our hospital between 2002 and 2003. Paraffin embedded tumor sections were collected retrospectively from 165 breast cancer patients and immunohistochemical staining for MAGE-A10 and NY-ESO-1 was performed. Impact of MAGE-A10 and NY-ESO-1 expression on disease free survival (DFS) and overall survival (OS) was analyzed by the Kaplan-Meier method using 8-year follow up data. Results: MAGE-A10 expression (score ≥2+) was detected in 105/164 (64%) and NY-ESO-1 expression (score ≥2+) was observed in 14/164 (8.5%) patients. 8-year DFS for MAGE-A10 positive patients was 69% and 73% for MAGE-A10 negative (p=0.452). 8-year OS for MAGE-A10 positive patients was 80% and 90% for MAGE-A10 negative (p=0.134). For NY-ESO-1 positive patients 8-year DFS was 67% and 70% for NY-ESO-1 negative patients (p=0.837). 8-year OS for NY-ESO-1 positive patients was 83% and 84% for NY-ESO-1 negative patients. (p=0.991) Conclusions: To our knowledge this is the first study analyzing prognostic significance CTAs in breast cancer. In this retrospective study we did not show statistically significant correlation between MAGE-A10 and NY-ESO-1 expression and clinical outcome. Additional studies are warranted to determine weather this antigens might have prognostic value in breast cancer patients.

Simple SummaryTriple-negative breast cancer (TNBC) has been associated with worse prognoses due to the limited treatment options. Thus, there is a need to characterise new biomarkers or treatment targets to improve patient outcomes. Cancer testis antigens (CTAs) are a group of antigens that are preferentially expressed in tumours and exhibit strong immunogenicity, as such, CTAs hold great promise as potential treatment targets and biomarkers in cancer. Previous reports have implicated roles for CTAs in different subtypes of breast cancer, including TNBC. Multiple clinical trials are in progress investigating CTAs as treatment targets in various cancers. This review aims to discuss the roles of CTAs in TNBC and discuss the potential applications and benefits of incorporating CTAs in clinical practice. Breast cancer cells commonly express tumour-associated antigens that can induce immune responses to eradicate the tumour. Triple-negative breast cancer (TNBC) is a form of breast cancer lacking the expression of hormone receptors and cerbB2 (HER2) and tends to be more aggressive and associated with poorer prognoses due to the limited treatment options. Characterisation of biomarkers or treatment targets is thus of great significance in revealing additional therapeutic options. Cancer-testis antigens (CTAs) are tumour-associated antigens that have garnered strong attention as potential clinical biomarkers in targeted immunotherapy due to their cancer-restricted expressions and robust immunogenicity. Previous clinical studies reported that CTAs correlated with negative hormonal status, advanced tumour behaviour and a poor prognosis in a variety of cancers. Various studies also demonstrated the oncogenic potential of CTAs in cell proliferation by inhibiting cell death and inducing metastasis. Multiple clinical trials are in progress to evaluate the role of CTAs as treatment targets in various cancers. CTAs hold great promise as potential treatment targets and biomarkers in cancer, and further research could be conducted on elucidating the mechanism of actions of CTAs in breast cancer or combination therapy with other immune modulators. In the current review, we summarise the current understandings of CTAs in TNBC, addressing the role and utility of CTAs in TNBC, as well as discussing the potential applications and advantage of incorporating CTAs in clinical practise.

Ghrelin and obestatin are gastrointestinal peptides, encoded by the same preproghrelin gene. Both are expressed in breast cancer tissue and ghrelin has been implicated in breast cancer tumorigenesis. Despite recent advances in breast cancer management the need for new prognostic markers and potential therapeutic targets in breast cancer remains high. We studied the prognostic impact of ghrelin and obestatin in women with node negative breast cancer.Within a cohort of women with breast cancer with tumor size ≤ 50 mm, no lymph node metastases and no initiation of adjuvant chemotherapy, 190 women were identified who died from breast cancer and randomly selected 190 women alive at the corresponding time as controls. Tumor tissues were immunostained with antibodies versus the peptides.Ghrelin expression was associated with better breast cancer specific survival in univariate analyses (OR 0.55, 95% CI 0.36–0.84) and in multivariate models, adjusted for endocrine treatment and age (OR 0.57, 95% CI 0.36–0.89). Obestatin expression was non-informative (OR 1.2, 95% CI 0.60–2.46). Ghrelin expression is independent prognostic factor for breast cancer death in node negative patients—halving the risk for dying of breast cancer. Our data implies that ghrelin could be a potential therapeutic target in breast cancer treatment.

Post-translational modification by covalent attachment of isoprenoid lipids (prenylation) regulates the functions and biological activities of several proteins implicated in the oncogenic transformation and metastatic progression of cancer. The largest group of prenylated proteins contains a CAAX motif at the C-terminal that serves as a substrate for a series of post-translational modifications that convert these otherwise hydrophilic proteins to lipidated proteins, thus facilitating membrane association. C17orf37 (chromosome 17 open reading frame 37), also known as C35/Rdx12/MGC14832, located in the 17q12 amplicon, is overexpressed in human cancer, and its expression correlates with the migratory and invasive phenotype of cancer cells. Here we show that C17orf37 contains a functional CAAX motif and is post-translationally modified by protein geranylgeranyltransferase-I (GGTase-I). Geranylgeranylation of C17orf37 at the CAAX motif facilitates association of the protein to the inner leaflet of plasma membrane, enhances migratory phenotype of cells by inducing increased filopodia formation, and potentiates directional migration. A prenylation-deficient mutant of C17orf37 is functionally inactive and fails to trigger dissemination of tail vein-injected cells in a mouse model of metastasis. These findings demonstrate that prenylation is required for the function of the C17orf37 protein in cancer cells and imply that the post-translational modification may functionally regulate metastatic progression of disease.

Objective: The six transmembrane epithelial antigen of the prostate (STEAP1) is predominantly overexpressed in human prostate cancer. STEAP1 was first identified as a prostate-specific cell-surface antigen and found to be up-regulated in various cancers including lung, bladder, colon, and ovarian with little expression in normal tissue. An anti-STEAP1 monoclonal antibody linked to an antimitotic agent is currently in Phase I clinical trials for prostate cancer patients. Microarray data from our lab suggested that STEAP1 is also highly expressed in human breast cancers and bone marrow disseminated tumor cells. In this study we evaluate expression STEAP1 in primary tumors, and bone marrow (BM) from breast cancer patients. Experimental procedures: RNA was isolated from primary tumor, non-malignant breast tissue and bone marrow (BM) from stage II and III breast cancer patients, healthy volunteers, breast cancer cell lines and BM from patient derived xenographs (PDX). Disseminated tumor cells (DTCs) from patient BM were enriched by microfiltration and analyzed by RNA-in situ hybridization (ISH). STEAP1 RNA expression was analyzed by Nanostring nCounter and qRT-PCR using human specific probes. STEAP1 immunohistochemical (IHC) staining of human tissue was performed using standard protocols. Knockdown of steap1 expression was accomplished using a lentiviral system. Results: STEAP1 mRNA was up-regulated in 77% of tumors (28/36) compared to the corresponding normal tissue. STEAP1 protein was expressed in 100% of tumors (8/8) and was absent in non-malignant breast tissue (7/7) by IHC staining. STEAP1 mRNA was not expressed normal BM, but was detected in 8% (6/74) of BM from patients with early stage breast cancer. STEAP1 expression in the BM was associated with triple negative disease (3/6) and recurrent disease development (4/6, p=.028). STEAP1 expression was observed in individual DTCs isolated from patients BM, while no expression was observed in normal BM. In a PDX model of breast cancer, STEAP1 expression in BM was only observed in mouse who developed metastatic disease associated (7/10, p=.004). Knockdown of STEAP1 in the breast cancer cell line MDA-MB231 cells inhibited cell growth by 80-90%. Conclusion: STEAP1 is expressed in human breast tumors and disseminated tumor cells found in the bone marrow of breast cancer patients. Expression of STEAP1 in the BM is significantly associated with the development of metastatic disease in patients as well as in a mouse model of breast cancer. Our data indicate that STEAP1 could serve as a therapeutic target for the treatment of minimal residual disease in breast cancer. Citation Format: Aft R, Mudalagiriyappa C, Pillai S, Watson M. Analysis of STEAP1 expression as a therapeutic target in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-04-06.

Recent Advances and Current Challenges in Tumor Immunology and Immunotherapy

e11098 Background: Novel breast cancer risk-reducing strategies for individuals with germline mutations of the BRCA1 and/or BRCA2 genes are urgently needed. Identification of antigenic targets which are expressed in early cancers, in situ or invasive, but absent in normal breast epithelium of these high-risk individuals could provide the basis for the development of effective immunoprohylactic strategies. Cancer Testes (CT) antigens are potential candidates because their expression is restricted to tumors and accumulating data suggest that they play important roles in cellular proliferation, stem cell function and carcinogenesis of at least some tumors. The objective of this study was to examine the expression of CT antigens and their frequency in BRCA-associated breast cancers. Methods: Archived breast cancer tissues (n=26) as well as morphologically normal breast tissues (n=7) from women carrying deleterious BRCA 1 and or 2 mutations were obtained for antigen expression analysis by immunohistochemistry. Expression of the following CT antigens was examined: MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1.CT7, NY-ESO-1, MAGE-C2/CT10 and GAGE. Results: CT antigens were expressed in 61.5% (16/26) of BRCA-associated cancers. 50% (13/26) of tumors expressed two or more CT antigens; three cancers expressed all seven CT antigens. CT antigens were expressed across all tumor types and stages including DCIS. In contrast, none of the CT antigens were expressed in adjacent or contralateral normal breast epithelium (p<0.01). Conclusions: We report a high CT antigen expression rate in BRCA-associated breast cancer as well as the lack of expression of these antigens in benign breast tissue of carriers, identifying CT antigens as potential vaccine targets for breast cancer prevention in these high-risk individuals.

Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing1 (CMTM1) consists of at least 23 alternatively spliced isoforms designated CMTM1_v1-v23. In the present study, we detected CMTM1_v17 expression in multiple human normal and tumor tissues and found that CMTM1_v17 was highly expressed in testis and many tumor tissues including breast tumor. The overexpression of CMTM1_v17 in the breast cancer cell line MDA-MB-231 promoted cell proliferation and resistance to tumor necrosis factor-α (TNF-α)-induced apoptosis. Moreover, siRNA-mediated silencing of CMTM1_v17 sensitized MDA-MB-231 cells to TNF-α-induced apoptosis. We propose that CMTM1_v17 may be a novel potential target for therapy in breast cancer patients. The present study provides insight into a novel mechanism by which CMTM1_v17 enhances cellular proliferation and abrogates TNF-α-induced apoptosis. These findings also have implications for clinical practice as they highlight the potential for therapeutic targeting of CMTM1_v17 for the treatment of breast and other cancers in which CMTM1_v17 impacts cellular proliferation and survival.

Analysis of autophagy-related gene expression data identified RUBCN as a novel biomarker influencing the pathogenesis and progression of breast cancer, underscoring its potential as a therapeutic target. We analyzed multiple breast cancer sample datasets using bioinformatics tools and databases. A consensus prognostic model was constructed and validated across several independent datasets to further examine its association with patient outcomes. A series of bioinformatics analyses focused on RUBCN were conducted, including expression profiling, independent prognostic evaluation, immune correlation analysis, and survival analysis. RUBCN expression was verified in breast cancer cell lines and clinical tissue specimens via Western blotting, quantitative real-time reverse transcription PCR, and immunohistochemistry. Functional assays, such as the Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine incorporation assay, wound healing assay, and Transwell invasion assay, were employed to evaluate the effects of RUBCN knockdown on breast cancer cell proliferation and invasion. Autophagic activity, indicated by LC3 and P62 levels, was measured via Western blot in RUBCN-knockdown breast cancer cells with or without chloroquine treatment. Elevated expression of multiple autophagy-related genes was observed in breast cancer. The consensus prognostic model accurately predicted survival across multiple datasets, with RUBCN emerging as a key gene whose expression levels were significantly correlated with patient prognosis. Enrichment analysis indicated that RUBCN likely promotes breast cancer progression by regulating cell cycle and invasion processes. Further investigation revealed a negative correlation between RUBCN expression levels and immune cell infiltration, suggesting a potential role in mammary tumorigenesis through mediating immune evasion by suppressing immune cell infiltration. Immunohistochemical results confirmed upregulated RUBCN expression in carcinoma tissues. Knockdown of RUBCN was shown to suppress the proliferative and invasive abilities of breast cancer cells. Mechanistically, RUBCN knockdown impaired autophagic flux, as evidenced by altered LC3 and P62 levels upon chloroquine treatment. Together, these findings establish RUBCN as a promising therapeutic target in breast cancer. Future studies should emphasize in vivo functional validation using animal models and screen for targeted agents capable of modulating RUBCN expression or activity, thereby facilitating the development of innovative therapeutic strategies for breast cancer treatment.

BackgroundBreast cancer is a leading cause of cancer‐related morbidity and mortality in women worldwide. Among its subtypes, triple‐negative breast cancer (TNBC) poses the greatest therapeutic challenge due to its aggressive nature and lack of targeted treatments. Holocytochrome c synthase (HCCS), a mitochondrial enzyme essential for cytochrome c maturation, may play a pivotal role in cancer pathogenesis.ObjectiveThis study aimed to investigate the expression profile, epigenetic regulation, immune interactions, prognostic significance, and molecular networks of HCCS across cancers, with a particular focus on breast cancer and its subtypes.MethodsPublicly available datasets and bioinformatics tools were employed to analyze HCCS expression, methylation, survival outcomes, immune infiltration, and interaction networks. Expression and clinical outcomes were examined using TCGA, while methylation and expression patterns were assessed via UALCAN and TNMplot. Survival analyses were performed using Kaplan–Meier Plotter, and immune infiltration was evaluated with TIMER2.0. Protein–protein interaction networks were generated with STRING, and functional enrichment was conducted through g:Profiler. Key findings were validated in independent breast cancer cohorts from GEO and the GOBO platform.ResultsHCCS was significantly overexpressed in multiple cancers, with the highest upregulation observed in breast cancer, particularly TNBC. Hypomethylation of the HCCS promoter was associated with increased expression. High HCCS expression correlated with poorer relapse‐free survival and greater immune infiltration, including CD4+ T cells, CD8+ T cells, macrophages (M1/M2), mast cells, and regulatory T cells. Protein–protein interaction analysis revealed HCCS‐associated genes enriched in mitochondrial and apoptotic pathways. Validation across independent datasets consistently supported the association of elevated HCCS expression with poor prognosis in breast cancer.ConclusionThis integrated bioinformatics analysis highlights HCCS as a potential prognostic biomarker and therapeutic target in breast cancer, particularly in TNBC, although further experimental validation is required before clinical application.

Bromodomain and extra-terminal (BET) proteins are recognized acetylated lysine of histone 4 and act as scaffolds to recruit many other proteins to promoters and enhancers of active genes, especially at the super-enhancers of key genes, driving the transcription process and have been identified as potential therapeutic targets in breast cancer. However, the efficacy of BET inhibitors such as JQ1 in breast cancer therapy is impeded by interleukin-6 (IL-6) through an as-yet-defined mechanism. We investigated the interplay between IL-6 and JQ1 in MCF-7 and MDA-MB-231 human breast cancer cells. The results demonstrate that the efficacy of JQ1 on the inhibition of cell growth and apoptosis was stronger in MDA-MB-231 cells than in MCF-7 cells. Further, MCF-7 cells, but not MDA-MB-231 cells, exhibited increased expression of CXCR4 following IL-6 treatment. JQ1 significantly reduced CXCR4 surface expression in both cell lines and diminished the effects of IL-6 pre-treatment on MCF-7 cells. While IL-6 suppressed the extension of breast cancer stem cells in MCF-7 cells, JQ1 impeded its inhibitory effect. In MCF-7 cells JQ1 increased the number of senescent cells in a time-dependent manner. Analysis of gene expression indicated that JQ1 and IL-6 synergistically increase SNAIL expression and decrease c-MYC expression in MCF-7 cells. So, the BET proteins are promising, novel therapeutic targets in late-stage breast cancers. BET inhibitors similar to JQ1 show promise as therapeutic candidates for breast cancers, especially when triple-negative breast cancer cells are increased and/or tumor-promoting factors like IL-6 exist in the tumor microenvironment.

IntroductionNovel breast cancer risk-reducing strategies for individuals with germline mutations of the BRCA1 and/or BRCA2 genes are urgently needed. Identification of antigenic targets that are expressed in early cancers, but absent in normal breast epithelium of these high-risk individuals, could provide the basis for the development of effective immunoprophylactic strategies. Cancer testis (CT) antigens are potential candidates because their expression is restricted to tumors, and accumulating data suggest that they play important roles in cellular proliferation, stem cell function, and carcinogenesis. The objective of this study was to examine the expression of CT antigens and their frequency in BRCA-associated breast cancers.MethodsArchived breast cancer tissues (n = 26) as well as morphologically normal breast tissues (n = 7) from women carrying deleterious BRCA 1 and/or 2 mutations were obtained for antigen expression analysis by immunohistochemistry. Expression of the following CT antigens was examined: MAGE-A1, MAGE-A3, MAGE-A4, MAGE-C1. CT7, NY-ESO-1, MAGE-C2/CT10, and GAGE.ResultsCT antigens were expressed in 16/26 (61.5%, 95% CI 43–80%) of BRCA-associated cancers, including in situ tumors. Thirteen of twenty-six (50%) breast cancers expressed two or more CT antigens; three cancers expressed all seven CT antigens. MAGE-A was expressed in 13/26 (50%) of cancers, NY-ESO-1 was expressed in 10/26 (38%) of tumors. In contrast, none of the CT antigens were expressed in adjacent or contralateral normal breast epithelium (P = 0.003).ConclusionsWe report a high CT antigen expression rate in BRCA-associated breast cancer as well as the lack of expression of these antigens in benign breast tissue of carriers, identifying CT antigens as potential vaccine targets for breast cancer prevention in these high-risk individuals.

Breast cancer is the second leading cause of cancer‑related deaths in female patients, and the main reasons are late diagnosis, limited therapeutic options and metastasis. Therefore, development of molecular therapeutic targets for breast cancer to suppress tumorigenesis, growth and metastasis may improve the therapeutic options and be of great benefit to patients. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) is a novel molecule for maintaining immune homeostasis and is involved in cancer development. Previous studies have shown that TIPE2 plays a suppressive role in the development of several types of human cancers. However, its role in breast cancer is still not clear. In the present study, we detected TIPE2 expression in human breast cancer and adjacent normal tissues. The expression of TIPE2 was reduced in breast cancer tissues compared to the level in adjacent normal tissues. We then established a breast cancer cell line stably expressing TIPE2 to investigate the role of TIPE2 in breast cancer carcinogenesis. Our results showed that overexpression of TIPE2 significantly inhibited the proliferation of MDA-MB‑231 cells as detected by Cell Counting Kit-8 assay and suppressed the migration and invasion of breast cancer cells as detected by Transwell migration and invasion assays invitro. TIPE2 also promoted cell apoptosis as detected by flow cytometry analysis. Moreover, TIPE2 inhibited the tumorigenesis of breast cancer invivo. Mechanistically, TIPE2 inhibited the phosphorylation of AKT and p38 as detected by western blot analysis. Taken together, TIPE2 suppressed breast cancer tumorigenesis, growth and metastasis possibly via regulation of the AKT and p38 signaling pathways. The results indicate that TIPE2 may be a potential therapeutic target for breast cancer therapy.