Cases Of Normal Breast Tissue Research Articles

Investigation of IDO1 and TDO2 expression in breast tumors by immunohistochemistry and real-time polymerase chain reaction methods

Although diagnostic and therapeutic advances have been made in the treatment of breast cancer, the challenge of effectively controlling tumor progression persists. The objective of this study was to determine the relationship between IDO1 and TDO2 expression in breast cancer by immunohistochemistry and real-time polymerase chain reaction, hormone receptor status, Ki67 proliferation index, molecular classification, metastasis, and to investigate whether IDO1 and TDO2 expression can be used in combination with targeted therapy or as a marker to increase treatment efficacy in selected cases. The study included 74 cases of breast cancer and 14 cases of normal breast tissue as controls. All cases were analyzed for IDO1 and TDO2 by both immunohistochemistry and real-time polymerase chain reaction. The immunohistochemical analysis revealed that IDO1 immunoreactivity was significantly higher in tumor tissue compared to normal breast tissue. A statistically significant correlation was observed between IDO1 immunoreactivity and histologic subtypes. Furthermore, IDO1 gene expression was correlated with IDO1 immunoreactivity. TDO2 immune reactivity did not differ between tumor and non-tumor tissues and no correlation was found between histological subtypes. There was no correlation between TDO2 immunoreactivity and gene expression. The significant increase in IDO1 levels in tumor tissue and high positivity in age, HER2 positive and triple negative cases compared to other cases suggest that IDO1 inhibitors may be suitable for the target patient group in treatment selection.

Identification of prognostic genes for early basal-like breast cancer with weighted gene co-expression network analysis.

Background:Breast cancer (BC) has become the leading cause of death for women’s malignancies and increasingly threatens the health of women worldwide. However, there is a lack of effective targeted drugs for basal-like BC. Therefore, biomarkers related to the prognosis of early BC need to be identified.Methods:The RNA-seq data of 87 cases of early basal-like BC and 111 cases of normal breast tissue from The Cancer Genome Atlas were explored by the weighted gene co-expression network analysis method and Limma package. Then, intersected genes were identified, and hub genes were selected by the maximal clique centrality method. The prognostic effect of the hub genes was also evaluated in early basal-like BC.Results:In total, 601 IGs were identified in this study. An APPI network was constructed, and the top 10 hub genes were selected, namely, cyclin B1, cyclin A2, cyclin-dependent kinase 1, cell division cycle 20, DNA topoisomerase II alpha, BUB1 mitotic checkpoint serine/threonine kinase, aurora kinase B (AURKB), cyclin B2, kinesin family member 11, and assembly factor for spindle microtubules. Only AURKB was found to be significantly associated with the overall prognosis of early basal-like BC. The immune cell infiltration analysis showed that the infiltration numbers of CD4 + T cells and naïve CD8 + T cells were positively correlated with the AURKB expression level, while those of naïve B cells and macrophage M2 cells were negatively correlated with the AURKB expression level in basal-like BC.Conclusion:AURKB might be a potential prognostic indicator in early basal-like BC.

Screening potential immune signatures for early-stage basal-like/triple-negative breast cancer

BackgroundBreast cancer (BC) is a highly heterogeneous disease. Among the BC molecular subtypes, basal-like/triple-negative BC (TNBC) is characterized by a high propensity for relatively early metastases and a lack of available endocrine and targeted therapies. Therefore, this study aimed to discover potential signatures for predicting the immune response in early-stage basal-like/triple-negative BC.MethodA total of 86 cases of early-stage TNBC from the TCGA and 459 cases of normal breast tissue from GTEx were enrolled and analyzed to screen out differentially expressed genes (DEGs). Then, the prognostic effect and tumor immune cell infiltration relationship with the basal-like-specific DEGs were also evaluated.ResultsA total of 1556 DEGs, including 929 upregulated genes and 627 downregulated genes, were screened in early-stage basal-like BC. Two prognosis-associated DEGs, GAL and TTC36, were finally found to be basal-like BC specific. However, only GAL was significantly correlated with tumor immune-infiltrating cells, especially CD8+ T cells. The expressions of GAL and TTC36 were revalidated by using the GEO dataset.ConclusionGAL might be an immune signature for the response to immune checkpoint therapy in early basal-like/triple-negative BC.

Characterization of 5-inflammatory-gene signature to affect the immune status and predict prognosis in breast cancer

Breast cancer (BC) is associated with an inflammatory microenvironment. In BC, epidemiological evidence suggests that inflammation is associated with a poor prognosis. However, approaches to determine the extent of inflammation in the tumor microenvironment remain unclear. We downloaded the expression profiles and corresponding clinicopathological information of 1050 BC tissues and 59 cases of normal breast tissue from The Cancer Genome Atlas (TCGA) dataset. Similarly, data of 1050 BC tissues were downloaded from Gene Expression Omnibus (GEO) and 200 inflammation-related genes were downloaded from the MSigDB database. We developed an inflammatory risk model to reflect the immune microenvironment in BC. Multivariate Cox analysis showed that the risk score was an independent predictor of overall survival (OS). Inflammatory signature was significantly associated with clinical and molecular features and could serve as an independent prognostic factor for BC patients. Furthermore, most immune cells were significantly less infiltrated in the high-risk group than in the low-risk group. There was a significant difference in survival time between the group with a high and low tumor mutational burden (TMB) score, and the survival time of the patients with a low TMB was significantly higher than that of the high-risk group. The risk scores were significantly lower in patients who responded to immunotherapy (complete response/partial response - CR/PR) than in patients who did not respond to immunotherapy (stable disease/progressive disease - SD/PD). We developed and validated an inflammatory risk model, which served as an independent prognostic indicator and reflected immune response intensity in the BC microenvironment.

14-3-3ζ promoted invasion and lymph node metastasis of breast invasive ductal carcinoma with HER2 overexpression

BackgroundHER2 was a recognized oncogene that promoted the development and metastasis of breast cancer, but its positive expression rate in invasive ductal carcinoma (IDC) was much lower than that in ductal carcinoma in situ (DCIS). The correlation between the occurrence and development of breast cancer and the amplification and overexpression of HER2 gene alone was still controversial. 14–3–3ζ had a strong protein binding ability and a variety of functions, mainly through the interaction with other proteins to exert its unique biological activities. However, influence and interaction relationship of the two proteins on the development of IDC was not clear. Furthermore, the mutual effect mechanism of synergy effect on lymph node metastasis of IDC was not known well too. MethodsImmunohistochemistry experiment was performed to detect expression status of 14–3–3ζ, HER2, TGF-β, p53 and Gli2 in paraffin-embedded samples respectively, including 30 cases of normal breast tissue, 30 cases of usual ductal hyperplasia (UDH), 30 cases of atypical ductal hyperplasia (ADH), 30 cases of DCIS and 120 cases of IDC. ResultsThe positive expression rates of 14–3–3ζ/HER2 in Normal group, UDH group, ADH group, DCIS group and IDC group were 30%/0.00%, 26.7%/0.00%, 53.3%/33.3%, 46.7%/53.3% and 50%/24.2%, respectively. Compared with Normal group or UDH group, the expression of 14–3–3ζ was significantly increased in ADH, DCIS and IDC groups. 14–3–3ζ was overexpressed in only 4 of the 16 DCIS cases with HER2 overexpression (25.0%, 4/16), but it was overexpressed in 7 of the 9 IDC cases with DCIS (77.8%, 7/9). Among HER2 overexpression cases, 14–3–3ζ overexpression was significantly different between DCIS group and IDC with DCIS group (P = 0.017). In 18 IDC cases with lymph node metastasis and HER2 overexpression, 14–3–3ζ was overexpressed in 15 cases (83.3%, 15/18), while in the 11 IDC cases without lymph node metastasis, 14–3–3ζ and HER2 were overexpressed in only 5 cases (45.5%, 5/11). Co-overexpression of 14–3–3ζ and HER2 was positively correlated with occurrence of lymph node metastasis (P = 0.048). TGF-β was overexpressed in both precancerous lesion group and IDC group compared with normal group. Compared with the IDC group without lymph node metastasis, TGF-β expression was significantly increased in the IDC group with lymph node metastasis (P = 0.015). In IDC cases with 14–3–3ζ and HER2 co-overexpression, the expression of p53 in IDC with lymph node metastasis was significantly decreased (P = 0.010), while the expression of Gli2 was significantly increased compared with IDC cases without lymph node metastasis (P = 0.038). The co-overexpression of 14–3–3ζ and HER2 was positively correlated with ER negative expression (P < 0.001) and PR negative expression (P = 0.038), respectively. Conclusion14–3–3ζ synergistic with HER2 could promote the occurrence and development of breast IDC and induce the lymph node metastasis of IDC, suggesting that combined overexpression of 14–3–3ζ and HER2 would lead to higher invasion and metastasis risk of breast cancer. It was speculated that the combined detection of 14–3–3ζ and HER2 would be one of the key factors affecting the clinical treatment decision and prognosis.

The effects of apoptosis and apelin on lymph node metastasis in invasive breast carcinomas.

This study aimed to evaluate the biological and clinical significance of apelin-36 in breast cancer and to compare apelin-36 expression and apoptotic index in both breast tissue and metastatic lymph nodes in patients with invasive breast carcinoma. In this study, both tumor tissue and metastatic lymph nodes of the same patient were collected from 60 cases of invasive breast carcinoma patients (IDC, ILC) and 20 cases of normal breast tissue with no tumor from mammoplasty were used as the control group. The expression of apelin was examined with immunohistochemically, and the apoptotic index was examined with TUNEL methods. According to Kruskal-Wallis analysis, there was a significant difference between IDC and the control group when the apelin expression was compared between the breast tissues (p = 0.001). There were significant differences between the three groups when comparing relationships with apoptotic index (p < 0.001). According to the Mann-Whitney U test, both tumor size and expression of apelin in lymph nodes in ILCs were significantly higher than IDCs. (p = 0.026, p = 0.024, respectively). According to correlation analysis, there was a good correlation between the expression of apelin in breast tissue and apelin expression in lymph nodes (p = 0.000). It is also found a similar relationship in terms of the apoptotic index (p = 0.000). In addition, the negative correlation was found between apelin expression and the apoptotic index in breast tissues (p = 0.003). Based on these results, apelin-36 can be used as a marker for determining the metastasis potential in invasive breast cancer.

Abstract P6-06-18: Promyelocytic leukemia (PML) is induced by tumor-associated macrophages (TAMs) in breast cancer

Abstract Introduction: Tumor-associated macrophages (TAMs) are pivotal orchestrators of the tumor microenvironment. Clinical evidences show that density of TAMs increases with lymph node metastasis, which is associated with poor prognosis in breast cancer patients. The underlying mechanism has not been clearly elucidated. Methods: Transcriptome of MCF-7 cells cocultured with TAMs or monocyte-derived macrophages (MDMs) was analyzed and compared by RNA-seq analysis, which showed that the promyelocytic leukemia (PML) gene was significantly upregulated by TAMs (P&amp;lt;0.001). The upregulation of PML was further verified by qRT-PCR and immunohistochemistry (IHC) in tissue microarrays (TMAs) containing 294 cases of breast cancer tissue with duplicate cores and 28 cases of normal breast tissue with single core. The age, sex, pathological type, histologic grade and stage of the patients were well recorded. Results: RNA-seq analysis revealed that TAMs significantly upregulated PML expression, which was confirmed by qRT-PCR. Further, the protein level of PML was detected by IHC in TMAs. As expected, 57 cores of benign breast tissue, including normal breast tissue, adjacent normal breast tissue, adenosis and fibrous tissue in invasive ductal carcinoma (IDC) and lobular carcinoma in situ (LCIS), showed absent (80.70%) or low (19.30%) staining scores. In 508 (99.03%) cores of invasive breast cancer tissue, PML expression was positively correlated with lymph node metastasis (P= 0.003). However, no significant associations were observed between PML expression and tumor stage, primary tumor size, histologic tumor grade in IDC, or PML cellular localization. Conclusion:Recently, PML, upregulated in a subset of breast cancer, has been identified as a breast cancer oncogene. Here, we demonstrated PML expression was induced by TAMs and positively correlated with lymph node metastasis. Thus, we conclude PML mediates TAMs function in breast tumorigenesis and TAMs stimulates the expression of PML in breast cancer. Citation Format: Meijun Long, Mei Yang, Jun An, Renbin Liu. Promyelocytic leukemia (PML) is induced by tumor-associated macrophages (TAMs) in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-18.

Expression of VEGF in breast lesions: an immunohistochemical study

Background: Breast cancer is the most frequent cancer in India. During the last few years, several investigators have focused on tumor angiogenesis as a critical step in cancer development and progression. Among these, vascular endothelial growth factor (VEGF) is emerging as a prognostic marker in patients with several type of cancer including breast cancer. The aim of the study was to analyse the expression of VEGF in human breast cancer as compared to normal breast tissue and benign breast lesions by immunohistochemistry. Also, to assess the usefulness of VEGF as a predictor of aggressiveness of breast lesions.Methods: Formalin fixed paraffin embedded sections of 10 cases of normal breast tissue, 20 cases of benign breast lesions and 20 cases of malignant breast lesions were taken up for the study and subjected to immunohistochemistry using VEGF.Results: The intensity of VEGF immunostaining in normal breast, benign and malignant breast lesions was evaluated and scoring was graded as 0, 1+, 2+, 3+ and 4+. Statistical analysis was performed with Chi-Square test and significant differences were noted between these 3 groups (p value &lt;0.05).Conclusions:VEGF expression correlated well with the grade and stage of tumor indicating that VEGF positive tumors are biologically aggressive and are associated with poor prognosis but little is known about the implication of genetic alterations of VEGF in benign breast lesions.

Expressions of FHIT, BCRP and Bcl-2 Proteins in Breast Invasive Ductal Carcinoma

Objective: To explore the expressions of FHIT (Human fragile histidine triad), BCRP (breast cancer resistance protein) and Bcl-2 proteins in breast invasive ductal carcinoma (IDC) and its relationship with clinical pathological factors. Methods: The expressions of FHIT, BCRP and Bcl-2 proteins in 84 cases of intraoperative resection of primary breast IDC and 36 cases of normal breast tissue about 3 cm away from the neoplastci foci were detected with the method of immunohistochemical streptavidin-peroxidase (SP) to analyze the relationship between the three proteins and the pathological features of breast IDC. Results: The positive expression rates of FHIT, of BCRP and Bcl-2 were 42.86%, 72.62% and 61.90% respectively in the breast IDC group and 72.22%, 36.11% and 38.89 % respectively in the normal breast tissue control group, between which the differences were statistically significant (P <0.05). In the IDC group, the expression of the three proteins was related to the lymph nodes metastasis and tumor recurrence of breast IDC, but was not significantly correlated with the expression of estrogen receptor (ER), progesterone receptor (PR) and proto-oncogene human epidermal growth factor receptor 2 (Her-2). The expression of FHIT was negatively correlated with the expression of and Bcl-2 (r = -0.322, -0.389), but BCRP and Bcl-2 expression were positively correlated (r = 0.276). Conclusion: The abnormal expressions of FHIT, BCRP and Bcl-2 proteins in breast IDC may play an important role in its onset and development.

Expression and clinical significance of OTUB1 in breast cancer

Objective To explore the expression level of OTUB1 and its clinical significance in breast cancer. Methods Immunohistochemistry was used to detect the expression level of OTUB1 in 78 cases of breast cancer tissues and 30 cases of normal breast tissue adjacent to carcinoma, and the relationships between OTUB1 and the clinical pathological features of breast cancer were analyzed. Results The positive expression rate of OTUB1 in breast cancer tissues [66.7% (52/78)] was significantly higher than that in adjacent normal breast tissues [30.0% (9/30)], with a statistically significant difference (χ2=11.851, P=0.001). OTUB1 expression level was related to the lymph node metastasis (χ2=5.029, P=0.025), postoperative TNM staging (χ2=4.478, P=0.034), expression of human epidermal growth factor receptor-2 (HER-2) (χ2=8.775, P=0.003), expression of P53 (χ2=4.708, P=0.030), expression of estrogen receptor (ER) (χ2=10.364, P=0.001) and molecular subtypes (χ2=10.934, P=0.012). However, OTUB1 expression level in breast cancer was not related to the age (χ2=2.194, P=0.139), menopausal status (χ2=1.843, P=0.175), tumor size (χ2=0.643, P=0.423), histological grade (χ2=3.580, P=0.167), expression of progestin receptor (PR) (χ2=3.371, P=0.066) and expression of Ki-67 (χ2=1.345, P=0.246). Conclusion OTUB1 expression level increases in breast cancer, which is associated with the lymph node metastasis, TNM staging, expressions of HER-2, P53, ER and molecular subtypes of breast cancer. It suggests that the expression of OTUB1 is related to the progression and metastasis of breast cancer. Key words: Breast neoplasms; Immunohistochemistry; OTUB1

Correlations between the expressions of Smad4, estrogen receptor and the clinicopathological features of breast cancer

Objective To explore the expressions of Smad4 and estrogen receptor (ER) and their interrelation, and the relationship with the clinicopathological features of breast cancer. Methods The immunohistochemical SP method was used to detect the expressions of Smad4 and ER in 50 case of invasive cancer, 12 cases of carcinoma in situ and 15 cases of normal breast tissues. The differences in different clinical stages, diffe-rentiation degrees and nodal metastases were analyzed. The correlation between Smad4 and ER was explored. Results The positive expression rate of Smad4 in invasive cancer was 52.00%, which lower than that in normal breast tissue (93.33%), with a significant difference (χ2=8.329, P=0.004), positive expression rates of ER were 60.00% and 40.00% respectively, with no significant difference (χ2=1.868, P=0.172). The positive expression rates of Smad4 in carcinoma in situ and invasive cancer were 75.00% and 52.00% respectively, with no significant difference (χ2=2.082, P=0.149). The positive expression rates of ER were 58.33% and 60.00% respectively, with no significant difference (χ2=0.011, P=0.916). The positive expression of Smad4 was related to the TNM stage (χ2=6.392, P=0.011) and the lymph node metastasis(χ2=6.738, P=0.009), but it was not associated with the histologic grade (χ2=0.542, P=0.462). The positive expression of ER was related to the lymph node metastasis (χ2=4.133, P=0.042) and histologic grade (χ2=5.357, P=0.021), but it was not associated with the TNM stage (χ2=1.159, P=0.282). There was positive correlation between Smad4 and ER in breast cancer tissue (r=0.263, P=0.032). Conclusion Smad4 is expressed at lower level in breast cancer than in normal breast tissue. The expressions of Smad4 and ER are related to the different clinicopathological features of breast cancer with positive correlation. Key words: Breast neoplasms; Receptors, estrogen; Smad4 protein

Expression of CCL5 and S100A4 protein in breast cancer tissues and their relationship with clinicopathological features and prognosis

Objective To investigate the relationship between the expression of CC chemokine ligand 5(CCL5) and S100 calcium binding protein A4 (S100A4) protein in breast cancer tissues with clinicopathological features and prognosis. Methods The expression of CCL5 and S100A4 in 40 cases of normal breast tissues and 120 cases of breast cancer were detected by immunohistochemistry, and the relationship between the degree of expression and the clinicopathological features and prognosis of breast cancer was analyzed. Results The expression positive rates of CCL5 and S100A4 in breast cancer tissues were 56.67% and 62.50% respectively, which were not expressed in normal breast tissues, and the differences were statistically significant(χCCL52=39.403, P 0.05). And the expression of S100A4 was statistically correlated with clinical staging(χ2=44.311, P 0.05). The expression of CCL5 and S100A4 in breast cancer was positively correlated(r=0.301, P<0.01). CCL5 was positively correlated with the recurrence of breast cancer(OR=6.270, P<0.01), and S100A4 was not correlated with the recurrence of breast cancer(OR=1.103, P=0.768). Survival analysis showed that the disease-free survival time of patients with positive CCL5 expression was significantly shorter than the patients with negative CCL5 expression(χ2=11.851, P<0.01), and the disease-free survival time of patients with positive S100A4 expression was significantly shorter than the patients with negative S100A4 expression(χ2=5.433, P=0.021). The joint detection showed that the disease-free survival time in CCL5(+ )+ S100A4(+ ) group was significantly lower than that of CCL5(+ ) or S100A4(+ ) group(χ2=15.341, P<0.01) and CCL5(-)+ S100A4(-)group(χ2=15.341, P<0.01). Conclusion The expression of CCL5 and S100A4 in breast cancer can reflect the metastasis and staging of breast cancer, which can be used to judge the clinical pathological characteristics and prognosis of breast cancer. Key words: Breast neoplasms; Chemokine CCL5; Calcium-binding proteins; Immunohistochemistry

The expression of PI3K/AKT signal pathway in breast tissue and it’s clinical significance

This work aimed to investigate the expression of p-AKT in breast tissue and its clinical significance. Methods: Western blot and RT-PCR were used to detect the expression of p-AKT in 100 cases of breast cancer and 30 cases of normal breast tissue. The expression levels of p-AKT in breast cancer were significant higher than that in normal breast tissue. The expressions of p-AKT had correlation with Clinical grade(P&lt;0.05), but it was irrelevant to the age, tumor size, lymph node metastasis and histological differentiating degree. PI3K/AKT Signal Pathway may take a part in the development and progression of breast cancer.

Expression and clinical significance of mTOR/p70S6K signaling pathway in breast carcinoma

Objective To investigate the expression of TOR(mammalian target of rapamycinm)/p70S6K(70 kDa ribosomal protein S6 kinase) signaling pathway in human breast carcinoma. Methods From January 2013 to January 2016, 40 cases of breast carcinom tissues,40 cases of paracancerous tissues,32 cases of normal breast tissues were selected in Binzhou Central Hospital Affiliated to Binzhou Medical College.RT-PCR and Immunohistochemistry were employed to determine the expression of mTOR/p70S6K signaling pathway in cancerous and paracancerous tissuess of 40 cases and normal healthy tissues of 32 cases. The relationship between expression of the genes mRNA in breast carcinom tissues and related parameters was analyzed. Results The expression level of mTOR was markedly higher in the breast carcinoma tissues (0.611±0.098) than in the paracancerous tissues (0.419±0.101) or normal breast tissues (0.381±0.043) .The expression of p70S6K mRNA was also higher in the breast carcinoma tissues(0.625±0.109) than in the paracancerous tissues(0.471±0.129) or normal breast tissues(0.441±0.130), with a significant difference among groups (P<0.05). There was a positive correlation between mTOR mRNA and p70S6K mRNA expressions in breast carcinom tissues (γ=0.491, P=0.019). The expression of mTOR/p70S6K signaling pathway in the breast carcinom tissues was significantly correlated with the pathological grading, lymph node metastasis, but not with the diameter, and age. Conclusions The mTOR/P70S6K signaling pathway is specifically activated in breast carcinom tissues. The mTOR/p70S6K signaling pathway might play an important role in the pathogenesis of breast carcinom. Key words: Breast carcinoma; Mammalian target of rapamycinm(mTOR); 70 kDa ribosomal protein S6 kinase(P70S6K); RT-PCR; Immunohistochemistry

Expressions and significances of apoptosis related protein Bcl-2 and Bax in basal-like breast carcinoma

Objective To investigate the expressions and significances of Bcl-2 and Bax in basal-like breast carcinoma (BLBC). Methods The expressions of Bcl-2 and Bax were detected in 43 cases of BLBC, 57 cases of non-BLBC and 60 cases of normal breast tissues by immunohistochemistry, and their relationships with physiological and pathological characteristics of patients were analysized. Results The positive rate of Bcl-2 in BLBC was 69.77%, higher than 43.86% in non-BLBC (χ2=6.647, P=0.010) and 21.67% in normal breast tissues (χ2=23.831, P=0.001). The positive rate of Bax in BLBC was 20.93%, lower than 45.61% in non-BLBC (χ2=6.564, P=0.010) and 76.67% in normal breast tissues (χ2=31.270, P=0.001). The expressions of Bcl-2 and Bax were correlated with lymphnode metastasis (χ2=6.927, P=0.008; χ2=6.203, P=0.013) and pTNM staging of BLBC (χ2=6.331, P=0.012; χ2=5.972, P=0.015). There was negative correlation between the expression of Bcl-2 and Bax in BLBC (r=-0.408, P<0.010). Conclusion High expression of Bcl-2 and low expression of Bax interact with each other leading to unbalance of cell deferation and apoptosis, resulting in promoting genesis and progress of BLBC. Key words: Breast neoplasms; Proto-oncogene proteins c-bcl-2; Bax

Clinicopathological significance of dual-specificity protein phosphatase 4 expression in invasive ductal carcinoma of the breast.

PurposeDual-specificity protein phosphatase 4 (DUSP4), also known as mitogen-activated protein kinase phosphatase (MKP) 2 is a member of the inducible nuclear MKP group. The role of DUSP4 in cancer development and progression appears to vary with the type of malignancy. The purpose of this study was to investigate DUSP4 expression in a case series of invasive ductal carcinoma of the breast.MethodsWe constructed tissue microarrays consisting of 16, 14, 47, and 266 cases of normal breast tissue, usual ductal hyperplasia, ductal carcinoma in situ, and invasive ductal carcinoma, respectively. DUSP4 expression was investigated by immunohistochemistry.ResultsCytoplasmic DUSP4 expression was observed. DUSP4 was more frequently expressed in malignant than in benign cases (p=0.024). The mean DUSP4 expression score was significantly higher in malignant tumors than in benign lesions (p=0.019). DUSP4 expression was significantly correlated with a larger tumor size (>2 cm, p=0.015). There was no significant correlation between overall survival or disease-free survival and DUSP4 expression in all 266 patients. We evaluated the impact of DUSP4 expression on the survival of 120 patients with T1-stage tumors. Interestingly, Kaplan-Meier survival curves revealed that DUSP4 expression had a significant effect on both overall patient survival (p=0.034, log-rank test) and disease-free survival (p=0.045, log-rank test). In early T-stage breast cancer, DUSP4 expression was associated with a worse prognosis.ConclusionDUSP4 is frequently upregulated in breast malignancy, and may play an important role in cancer development and progression. In addition, it may be a marker of adverse prognosis, especially in patients with early T1-stage cancer.

Expression of Fascin-1 protein in breast cancer and its clinicopathologic correlation

To study the expression of fascin-1 protein in breast cancer and to evaluate its correlation with clinicopathologic features of the tumor. Immunohistochemical EnVision method was performed to evaluate the expression of fascin-1 in 23 cases of normal breast tissues, 69 cases of benign breast lesions, 58 cases of usual ductal hyperplasia (UDH), 61 cases of ductal carcinoma in situ (DCIS) and 221 cases of breast cancer from March 2007 to December 2011. Fascin-1 protein expression rates in normal breast tissues, benign breast lesions, UDH, DCIS and breast cancer were 100.0% (23/23), 89.9% (62/69), 13.8% (8/58), 19.7% (12/61), and 42.1% (93/221), respectively. Fascin-1 expression in normal breast tissues and benign breast lesions was significantly higher than those in UDH, DCIS and breast cancer (P < 0.01); Fascin-1 expression in breast cancer was significantly higher than those in UDH and DCIS (P < 0.01). There was a tendency of increased fascin-1 expression in DCIS compared to UDH, but the difference was not statistically significant (P > 0.05). Fascin-1 positive rates in patients with DCIS grade III (26.8%, 11/41) was significantly higher than that in patients with DCIS grade I-II (1/20, P < 0.05). Fascin-1 protein expression in breast cancer increased with increasing histologic grade and clinical stage (P < 0.01). Fascin-1 protein expression was also significantly higher in tumors with negative estrogen receptor (ER) and progestone receptor (PR) status and > 3 axillary lymph node metastases compared to tumors that were ER and PR positive and ≤ 3 axillary lymph node metastases (P < 0.01 and P < 0.05, respectively). Logistic regression analysis showed that fascin-1 expression correlated positively with high clinical stage (OR = 1.568, 95% CI = 1.029-2.387, P < 0.05) , but negatively with ER expression (OR = 0.149, 95% CI = 0.079-0.281, P < 0.01) . Fascin-1 is highly expressed in normal breast tissues and benign breast lesions, suggesting that it may be a biological marker of mature mammary ductal epithelium. Fascin-1 protein expression shows a significantly increasing trend from UDH, DCIS to invasive breast cancer, suggesting that fascin-1 plays an important role in breast carcinogenesis and may be a potential target for therapy.

Inhibition of ErbB-2 induces TFF3 downregulation in breast cancer cell lines

ErbB-2 gene plays an important role in carcinoma formation whose overexpression was observed in many types of tumors, including breast cancer. Dysregulation of Trefoil factor 3 (TFF3), which is thought to function in the development and progression of breast cancer, was found to be upregulated in ErbB2-overexpressing breast cancers and cells. However, a putative interaction between ErbB-2 and TFF3 in breast cancer remains unknown. To determine whether TFF3 has an important role in breast tumor, its levels were measured by immunohistochemistry in 130 cases of breast infiltrating duct carcinoma and 30 cases of normal breast tissue with a specific monoclonal antibody raised against human TFF3. Patients who were positive for ErbB-2 also had high expression levels of TFF3 (p < 0.05). Also, after infecting the SK-BR-3 cells with lentivirus-mediated ErbB2-specific shRNA (Lenti-ShERBB2), we detected the expressions of ErbB-2 and TFF3 by real-time polymerase chain reaction and Western blotting, respectively. Compared with the control groups, ErbB-2 mRNA expression was decreased in the Lenti-ShERBB2 infection group, and Western blotting indicated a concordant ErbB-2 protein reduction. On the other hand, TFF3 expression at both mRNA and protein levels was significantly downregulated by ErbB-2 silencing in SK-BR-3. These findings are a proof of the foundation for a certain relationships of ErbB-2 and TFF3, which may serve as novel therapeutic markers of ErbB2-overexpressing breast cancers in the future.

PTEN mutation, methylation and expression in breast cancer patients

The tumor suppressor gene, PTEN, has previously been demonstrated to be involved in breast tumorigenesis and tumor progression. The aim of the present study was to investigate the expression and significance of PTEN in breast carcinomas, to detect the mutation frequency of PTEN in sporadic breast carcinoma tissues and to determine the association between PTEN promoter methylation and gene expression. Immunohistochemical methods were used to analyze the expression of the PTEN gene in 146 cases of breast carcinoma and 10 cases of normal breast tissue closely adjacent to the carcinoma. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis was used to analyze conformation polymorphisms in 45 breast carcinoma and 10 normal breast tissues. Point mutations of abnormal single stranded conformation were detected by DNA sequencing. The methylation of the PTEN promoter was analyzed by methylation-specific PCR. Expression of PTEN was detected in 57.5% (84/146) of patients with breast carcinoma. By contrast, PTEN expression was detected in 100% of normal samples. Expression of PTEN was found to negatively correlate with the tumor size, the pathological stage and the expression of the estrogen receptor (ER) and the progesterone receptor (PR) in breast cancer. The 2-year disease-free survival of patients with a high expression of PTEN was higher compared with those with low PTEN expression (P<0.05). Missense mutations in exon 2 of PTEN were identified in 1/45 breast cancer cases. PTEN promoter methylation was detected in 31.1% (14/45) of breast carcinomas, of which 64.3% (9/14) were associated with a loss of PTEN expression. The tumor suppressor gene, PTEN, was abnormally expressed in the breast carcinomas. The number of PTEN mutations were low (1/45) in the sporadic breast cancer cases analyzed in the present study and PTEN promoter methylation may have been the main mechanism leading to the decreased expression of PTEN. These results indicate that PTEN is important for the tumorigenesis, development and prognosis of breast cancer.

Lactate Dehydrogenase-B Is Silenced by Promoter Methylation in a High Frequency of Human Breast Cancers

ObjectiveUnder normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer.Experimental designLactate Dehydrogenase (LDH) isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA.ResultsAbsent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/ 25 cases of breast cancer tissues, but not in 5/ 5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained), was seen in 23/ 26 (88%) breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O2), for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002), and T-47D cells (2.9 fold, p = 0.009), but not in MDA-MB-436 (-0.9 fold, p = 0.229), or MCF10AT (1.2 fold, p = 0.09) cells.ConclusionsLoss of LDH-B expression is an early and frequent event in human breast cancer occurring due to promoter methylation, and is likely to contribute to an enhanced glycolysis of cancer cells under hypoxia.