Loss of annexin A1 expression in human breast cancer detected by multiple high-throughput analyses

To test the efficacy of combined high-throughput analyses (HTA) in target gene identification, screening criteria were set using >fivefold difference by microarray and statistically significant changes (p<0.01) in SAGE and EST. Microarray analysis of two normal and seven breast cancer samples found 129 genes with >fivefold changes. Further SAGE and EST analyses of these genes identified four qualified genes, ERBB2, GATA3, AGR2, and ANXA1. Their expression pattern was validated by RT-PCR in both breast cell lines and tissue samples. Loss of ANXA1 in breast cancer was further confirmed at mRNA level by Human Breast Cancer Tissue Profiling Array and at protein level by immunohistochemical staining. This study demonstrated that combined HTA effectively narrowed the number of genes for further study, while retaining the sensitivity in identifying biologically important genes such as ERBB2 and ANXA1. A distinctive loss of ANXA1 in breast cancer suggests its involvement in maintaining normal breast biology.     

Tissue Aluminum Concentration Does Not Affect the Genomic Stability of ERBB2, C-MYC, and CCND1 Genes in Breast Cancer

It has long been hypothesized that body tissue uptake of aluminum may have biological implications in breast cancer. In vitro and in vivo studies have shown that aluminum may trigger genomic instability by interfering with DNA strands. The objective of this study was to examine the relationship between aluminum concentrations in the peripheral and central areas of breast tumors with the instability of three key genes in breast cancer, ERBB2, C-MYC, and CCND1 and aneuploidy of the chromosomes harboring these genes. Tissue samples of 118 women treated for breast cancer were obtained. Evaluation of aluminum content was carried out using graphite furnace atomic absorption spectrometry. A tissue microarray slide containing the tumor samples was used in FISH assays to assess ERBB2, C-MYC, and CCND1 expressions as well as the statuses of their respective chromosomes 17, 8, and 11. Clinicopathological data were obtained from patient’s records. Aluminum levels of >2.0 mg/kg were found in 20.3 and 22.1 % of the central and peripheral breast tumor areas, respectively. Amplification and/or aneuploid-positive statuses for ERBB2/CEP17, C-MYC/CEP8, and CCND1/CEP11 were detected in 24, 36.7, and 29.3 % of the tumors, respectively. We found that aluminum concentration was not related to these altered gene statuses. Our findings suggest that aluminum concentration does not affect genomic stability in breast tissues. Tissue microenvironment modifications, due to the presence of aluminum compounds, seem more appealing as a possible target for future studies to determine the implications of aluminum in breast carcinogenesis.     

DNA structural features on borders of ERBB2 amplicons in breast cancer

In 25–30% of cases of breast cancer tumors, the amplification of the chromosome fragment around ERBB2 underlies the increased expression of genes adjacent to ERBB2. The increased expression of genes within ERBB2-containing amplicons may impact not only the growth and development of the tumor, but also the sensitivity of the tumor to different types of anti-cancer therapies. The initial cause of the amplification and the exact borders of ERBB2-amplified chromosome fragment are still not completely characterized. No specific DNA sequences were found on the junction regions during intrachromosomal DNA amplification. We hypothesized that amplification borders can be specified by the structural peculiarities of DNA, rather than the particular DNA sequence. This study focused on the mapping of ERBB2 amplification borders in breast cancer and the search for unusual structural features of DNA at the borders of the identified amplicons. The copy number of ten genes adjacent to ERBB2 was evaluated by real time PCR in 162 breast cancer samples. Several ERBB2-containing amplicons of various lengths were revealed. In the majority of the analyzed samples, the borders of these amplicons were located within ZNFN1A3 and RARA genes. A bioinformatics analysis of the nucleotide sequence peculiarities around ERBB2 gene revealed the presence of AT-rich DNA regions with a high degree of flexibility. These regions were able to form stable secondary structures. Positions of these sites strongly coincide with the positions of the ERBB2-containing amplicon borders found in real time PCR experiments. Based on the obtained results, one can suppose that the structural features of DNA are involved in the formation of ERBB2-containing amplicon borders in breast cancer cells and the data are of importance for understanding the mechanisms of oncogene amplification.     

Association of functional FEN1 genetic variants and haplotypes and breast cancer risk

Aim

As a tumor suppressor, FEN1 plays an essential role in preventing tumorigenesis. Two functional germline variants (-69G > A and 4150G > T) in the FEN1 gene have been associated with DNA damage levels in coke-oven workers and multiple cancer risk in general populations. However, it is still unknown how these genetic variants are involved in breast cancer susceptibility.

Methods

We investigated the association between these polymorphisms and breast cancer risk in two independent case–control sets consisted of a total of 1100 breast cancer cases and 1400 controls. The influence of these variations on FEN1 expression was also examined using breast normal tissues.

Results

It was found that the FEN1-69GG genotypes were significantly correlated to increased risk for developing breast cancer compared with the -69AA genotype in both sets [Jinan set: odds ratios (OR) = 1.41, 95% confidence interval (CI) = 1.20–1.65, P = 1.9×10^− 5; Huaian set: OR = 1.51, 95% CI = 1.22–1.86, P = 1.7×10^− 4]. Similar results were observed for 4150G > T polymorphism. The genotype–phenotype correlation analyses demonstrated that the -69G or 4150G allele carriers had more than 2-fold decreased FEN1 expression in breast tissues compared with -69A or 4150T carriers, suggesting that lower FEN1 expression may lead to higher risk for malignant transformation of breast cells.

Conclusion

Our findings highlight FEN1 as an important gene in human breast carcinogenesis and genetic variants in FEN1 confer susceptibility to breast cancer.     

ERBB2 overexpression suppresses stress-induced autophagy and renders ERBB2-induced mammary tumorigenesis independent of monoallelic Becn1 loss

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1^+/− mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1^+/+ and Becn1^+/− immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.     

Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer

Immunotherapy is emerging as a supplement to conventional cancer treatment, and identifying antigen targets for specific types of cancer is critical to optimizing therapeutic efficacy. Cancer/testis antigens are highly promising targets for immunotherapy due to their cancer-specific expression and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many other CT antigens, such as MAGE-A proteins. Surprisingly, we detected no ADAM2, CALR3 and SAGE1 in the 67 lung cancers (mainly non-small lung cancer) and 189 breast cancers, while MAGE-A proteins were present in 15% and 7–16% of these tumor types, respectively. Treatment with DNA methyltransferase inhibitors has been proposed as an attractive strategy to increase the expression of cancer/testis antigens in tumors before immunotargeting; however, neither ADAM2, CALR3 nor SAGE1 could be significantly induced in lung and breast cancer cell lines using this strategy. Our results suggest that ADAM2, CALR3 and SAGE1 cancer/testis antigens are not promising targets for immunotherapy of breast and lung cancer.     

Inflammation and Cancer: Role of Annexin A1 and FPR2/ALX in Proliferation and Metastasis in Human Laryngeal Squamous Cell Carcinoma

The anti-inflammatory protein annexin A1 (ANXA1) has been associated with cancer progression and metastasis, suggesting its role in regulating tumor cell proliferation. We investigated the mechanism of ANXA1 interaction with formylated peptide receptor 2 (FPR2/ALX) in control, peritumoral and tumor larynx tissue samples from 20 patients, to quantitate the neutrophils and mast cells, and to evaluate the protein expression and co-localization of ANXA1/FPR2 in these inflammatory cells and laryngeal squamous cells by immunocytochemistry. In addition, we performed in vitro experiments to further investigate the functional role of ANXA1/FPR2 in the proliferation and metastasis of Hep-2 cells, a cell line from larynx epidermoid carcinoma, after treatment with ANXA1_2–26 (annexin A1 N-terminal-derived peptide), Boc2 (antagonist of FPR) and/or dexamethasone. Under these treatments, the level of Hep-2 cell proliferation, pro-inflammatory cytokines, ANXA1/FPR2 co-localization, and the prostaglandin signalling were analyzed using ELISA, immunocytochemistry and real-time PCR. An influx of neutrophils and degranulated mast cells was detected in tumor samples. In these inflammatory cells of peritumoral and tumor samples, ANXA1/FPR2 expression was markedly exacerbated, however, in laryngeal carcinoma cells, this expression was down-regulated. ANXA1_2–26 treatment reduced the proliferation of the Hep-2 cells, an effect that was blocked by Boc2, and up-regulated ANXA1/FPR2 expression. ANXA1_2–26 treatment also reduced the levels of pro-inflammatory cytokines and affected the expression of metalloproteinases and EP receptors, which are involved in the prostaglandin signalling. Overall, this study identified potential roles for the molecular mechanism of the ANXA1/FPR2 interaction in laryngeal cancer, including its relationship with the prostaglandin pathway, providing promising starting points for future research. ANXA1 may contribute to the regulation of tumor growth and metastasis through paracrine mechanisms that are mediated by FPR2/ALX. These data may lead to new biological targets for therapeutic intervention in human laryngeal cancer.     

ERBB4 Promoter Polymorphism Is Associated with Poor Distant Disease-Free Survival in High-Risk Early Breast Cancer

A number of genetic variants have been linked to increased risk of breast cancer. Little is, however, known about the prognostic significance of hereditary factors. Here, we investigated the frequency and prognostic significance of two ERBB4 promoter region variants, −782G>T (rs62626348) and −815A>T (rs62626347), in a cohort of 1010 breast cancer patients. The frequency of nine previously described somatic ERBB4 kinase domain mutations was also analyzed. Clinical material used in the study consisted of samples from the phase III, adjuvant, FinHer breast cancer trial involving 1010 women. Tumor DNA samples were genotyped for ERBB4 variants and somatic mutations using matrix-assisted laser desorption ionization/time of flight mass spectrometry. Paraffin-embedded tumor sections from all patients were immunohistochemically stained for ErbB4 expression. Association of ERBB4 genotype to distant disease-free survival (DDFS) was assessed using Kaplan-Meier and Cox regression analyses. Genotyping was successful for 91–93% of the 1010 samples. Frequencies observed for the ERBB4 variants were 2.5% and 1.3% for −782G>T and −815A>T, respectively. Variant −815A>T was significantly associated with poor survival (HR  = 2.86 [95% CI 1.15–6.67], P = 0.017). In contrast, variant −782G>T was associated with well-differentiated cancer (P = 0.019). Two (0.2%) ERBB4 kinase domain mutations were found, both of which have previously been shown to be functional and promote cancer cell growth in vitro. These data present the germ-line ERBB4 variant −815A>T as a novel prognostic marker in high-risk early breast cancer and indicate the presence of rare but potentially oncogenic somatic ERBB4 mutations in breast cancer.