The molecular contribution of TNF-α in the link between obesity and breast cancer

Obesity is a growing worldwide medical problem, as it pre-disposes the affected hosts to a number of severe diseases, including postmenopausal breast cancer. Obesity development is characterised, amongst others, by aberrant secretion of adipokines. White fat tissue infiltrating macrophages secrete tumour necrosis factor-α (TNF-α) so that its circulating levels correlate positively with body mass index (BMI). In the study presented here, the effect of TNF-α on cell proliferation, cell signalling pathway activation and cell cycle in two breast cancer cell lines and one breast epithelial cell lines was assessed to determine the contribution of TNF-α on breast cancer progression and aetiology, respectively. TNF-α acted differently on all three cell lines. In MDA-MB-231 breast cancer cells, cell proliferation and PI3-kinase activation were not affected, while MAP-kinase activation and cell cycle progression were decreased, with indications of increased apoptosis. This suggests a growth inhibitory function of TNF-α in these cells. In SK-BR-3 breast cancer cells, cell proliferation and cell signalling pathway activation increased, while cell cycle progression decreased, which contradictorily suggests both growth promoting and growth inhibiting properties of TNF-α on these cells. This makes TNF-α an unlikely candidate for a general contribution to the link between obesity and breast cancer progression, however, individual tumours may be responsive to a proliferative signal of TNF-α. In MCF-10A breast epithelial cells, cell proliferation and MAP-kinase activation increased, while cell cycle progression was unaffected. This suggests a strong proliferative response in these cells, suggesting the possibility that TNF-α may contribute to breast cancer aetiology as a novel link between obesity and increased risk of breast cancer development.     

TNF-α promotes breast cancer cell migration and enhances the concentration of membrane-associated proteases in lipid rafts

Purpose

Tumor progression is associated with cell migration, invasion and metastasis. These processes are accompanied by the activation of specific proteases that are either linked to cellular membranes or are secreted into extracellular spaces. TNF-α is known to play an important role in various aspects of tumor progression. The aim of this work was to assess the effect of TNF-α on the migration of breast cancer cells and, in addition, to assess its association with the location of membrane-associated proteases in lipid rafts.

Methods

Wound scratch healing and Transwell migration assays were used to study the effect of TNF-α on the migration of both hormone-dependent and hormone-independent breast cancer-derived cells, i.e., MCF7 and MDA-MB-231, respectively. The expression and secretion of three matrix metalloproteases, MMP9, MMP2 and MT1-MMP, and two dipeptidyl peptidases, CD26 and FAP-α, was investigated using RT-PCR, Western blotting and gelatin zymography. In addition, activation of the MAPK/ERK signaling pathway was investigated by Western blotting.

Results

We found that a TNF-α-induced enhancement of breast cancer cell migration was accompanied by an increased secretion of MMP9, but not MMP2, into the culture media. We also found that TNF-α upregulated the expression of the dipeptidyl peptidases CD26 and FAP-α in a dose-dependent manner and, in addition, enhanced the concentration of all five proteases in lipid rafts in the breast cancer-derived cells tested, regardless of cell type. Furthermore, we found that TNF-α activated the MAPK/ERK signaling pathway by increasing the ERK1/2 phosphorylation level. Application of the MEK/ERK1/2 inhibitor U-0126 resulted in down-regulation of TNF-α-induced MMP9 secretion and abrogation of the enhanced concentration of proteases in the lipid rafts.

Conclusions

From our results we conclude that TNF-α-induced activation of the MAPK/ERK signaling pathway may promote breast cancer cell migration via both upregulation of MMP9, CD26 and FAP-α and concentration of these proteases, as also MT1-MMP and MMP2, in the lipid rafts. TNF-α may serve as a potential therapeutic target in breast cancers susceptible to TNF-α stimulation.

     

Is there a role of breast pathologist in diagnostic challenges of discordances in ER,PR, and HER2 between primary breast cancer and brain metastasis?

Journal of Neuro-Oncology - In Table 2 of the original publication, there were errors in the baseline scores for the PedsQL TM 3.0 Cancer Module questionnaire, so a corrected version of...     

Expression of HIF-1α in breast cancer and precancerous lesions and the relationship to clinicopathological features简

Objective： The aim of this study was to observe the expressions and clinical significance of HIF-1a in breast cancer and precancerous lesions, and analyze the relationship between the expressions and clinicopathological features in breast cancer. Methods： We analyzed the HIF-1a expression in 128 cases of invasive ductal carcinomas, 146 precancerous lesions patients including 89 cases of ductal carcinoma in situ and 57 cases of atypical ductal hyperplasia. 53 cases of usual ductal hyperplasia breast tissues were selected as a control group. The specimens were evaluated for HIF-1a, estrogen receptor （ER） ＆ progesterone receptor （PR）, epidermal growth factor receptor type 2 （HER2/neu） and Ki-67. Immunoreactivity was semi-quantitatively evaluated in at least 1000 cells examined under the microscope at 40 x magnification and recorded as the percentage of positive tumor cells over the total number of cells examined in the same area. The percentage scores were subsequently categorized. The express of HIF-1a and their relationship with multiple biological parameters including ER ＆ PR, HER2/neu and Ki-67, the biomarkers levels of CA153, CA125 TSGF, and CEA in blood serum and nipple discharge, histological grade, region lymph node metastasis, distant metastasis and recurrence on files were also assessed. Results： Compared with usual ductal hyperplasia, the positive expression rate of HIF-1a in atypical ductal hyperplasia, ductal carcinoma in situ and invasive ductal carcinomas group was significantly increased （P 〈 0.01）. The positive rates of HIF-1a in invasive ductal carcinomas were 68.75%, which were significantly higher than that in ductal carcinoma in situ （43.8%）, atypical ductal hyperplasia （31.6%）, usual ductal hyperplasia （9.4%; X2 = 13.44, 22.27, 52.79, respectively, P 〈 0.01）. Statistical analysis showed that difference of abnormal expression rate of HIF-1a between ductal carcinoma in situ and usual ductal hyperplasia （X2 = 18.37, P = 0.00）, atypical ductal hyperplasia and usual ductal hyperplasia （x2 = 8.14, P = 0.00） was significant （P = 0.00）. However, no significant difference in the positive expression rate of HIF-1a was found between atypical ductal hyperplasia and ductal carcinoma in situ tissue （X2 = 2.19, P = 0.14）. There was a significantly difference in the mean HIF-1a frequency between ER ＆ PR positive invasive ductal carcinomas group and negative group, epidermal growth factor receptor type 2 （HER2/neu） positive and negative groups, Ki-67 proliferation index 〈 14% and 〉 14% groups, histological grade （I ＋ II） and grade III invasive ductal carcinomas groups, with lymph node metastasis, distant metastasis and recurrence groups （P 〈 0.05） and without groups （P 〈 0.05）. However, there was not difference in the mean HIF-1a between age （〈 50 years vs 〉 50 years）, tumor diameter （〈 2 cm vs 〉 2 cm; P 〉 0.05）. The nipple discharge and serum levels of CA153, TSGF, CA125 and CEA in invasive ductal carcinomas HIF-1a positive patients were significantly higher than those in the negative patients （P 〈 0.05）. Conclusion： In breast cancer, HIF-1a expressibn was abnormally increased. The aberration of HIF-1a may play a key role during oncogenesis （atypical ductal hyperplasia or ductal carcinoma in situ） and promote breast cellular transformation into malignancy, a finding useful for further understanding of tumorigenesis. The abnormal expression of HIF-1a may be as an early event in the development of breast tumor. The over-expression of HIF-1a might be important biological markers for invasion, metastasis and recurrence of breast cancer.     

Antisense depletion of RIα subunit of protein kinase A induces apoptosis and growth arrest in human breast cancer cells

In recent years, several laboratories have explored the possibility of using antisense oligodeoxynucleotides for specific manipulation of gene expression leading to cancer treatment. The enhanced expression of the RI subunit of cyclic AMP-dependent protein kinase type I (PKA-I) has been correlated with cancer cell growth. In the present study, the effects of an antisense oligodeoxynucleotide targeted against RI subunit of PKA-I on growth inhibition and apoptosis in MDA-MB-231 human breast cancer cells were investigated. The growth inhibitory effects of RI antisense oligodeoxynucleotide correlated with a decrease in the RI mRNA and protein levels. The growth inhibition was accompanied by changes in the cell cycle phase distribution, cell morpbology, cleavage of poly (ADP-ribose) polymerase (PARP), and appearance of apoptotic nuclei. By comparison, mismatched control oligodeoxynucleotide had no effect. On the basis of these results, it can be suggested that the RI antisense oligodeoxynucleotide, which efficiently depletes the growth stimulatory RI and induces apoptosis/differentiation, could be used as a therapeutic agent for breast cancer treatment.     

Polyamine involvement in the secretion and action of TGF-α in hormone sensitive human breast cancer cells in culture

These experiments were designed to test polyamine (PA)* involvement in the secretion and action of transforming growth factor (TGF-) in hormone responsive MCF-7 breast cancer cells in liquid culture. At the same time, we evaluated the influence of culture conditions (with serum vs. serum depleted) and subclonality of MCF-7 cells on PA involvement in estrogen (E₂) and TGF- stimulated cell proliferation. Despite inducing a profound suppression of cellular PA levels and inhibiting basal and E₂-stimulated growth, administration of the PA synthesis inhibitor -difluoromethylornithine (DFMO) did not influence either basal or E₂-induced TGF- secretion. In the same experiments, on the other hand, addition of DFMO completely blocked the growth stimulatory effect of exogenous TGF-. However, when the culture conditions were changed to serum-free medium, TGF- and E₂-induced cell proliferation was affected modestly or not at all by DFMO administration, despite similar suppression of cellular ornithine decarboxylase (ODC) activity and PA levels. In addition, different clones of MCF-7 cells differed in their sensitivity to the antiproliferative effect of DFMO as well as in basal levels of ODC activity and PA. We conclude that PAs are not involved in basal or E₂-stimulated TGF- secretion in MCF-7 breast cancer cells. On the other hand, PAs do seem to be important mediators of TGF- and E₂-induced breast cancer cell proliferation, though the degree of such involvement appears to be influenced by serum factors and clonal variability of MCF-7 cells.     

Role of ethnic variations in TNF-α and TNF-β polymorphisms and risk of breast cancer in India

TNF-α and -β, the multi-functional pro-inflammatory cytokines, are known to play important roles in both tumor progression and destruction based on their concentrations. Growth factors and various stimuli such as cytokines regulate proliferation of the breast epithelial cells. Therefore, the polymorphisms in the genes encoding these signaling molecules could affect the risk of breast cancer. We have investigated selected genetic polymorphisms in TNF-α promoter (rs1800629, −308 G>A and rs361525, −238 G>A) and TNF-β intron 1 (rs909253, +252 A>G) in ethnically two different case–control groups from India. The study included 200 cases and 200 controls from an Indo-European (North Indian) group, and 265 cases and 237 controls from a Dravidian (South Indian) group. Genotyping of a total of 902 individuals was done by direct DNA sequencing. None of the polymorphisms showed significant association with breast cancer in the Indo-European group; however, all the three polymorphisms showed strong association with breast cancer in the Dravidian group. Further, sub-group analysis in the Indo-European group showed no significant difference between pre-menopausal cases and controls or between post-menopausal cases and controls at any of the loci analyzed. However, all the polymorphisms in the Dravidian group were significantly associated with pre-menopausal but not with post-menopausal breast cancer. In conclusion, TNF-α and -β polymorphisms are strongly associated with breast cancer in the Dravidian but not in the Indo-European group.     

Association of ErbB1–4 expression in invasive breast cancer with clinicopathological characteristics and prognosis

Background

Human epidermal growth factor receptor type 2 (Her2)/ErbB2 plays a key role in the initiation and progression of invasive breast cancer. However, the prognostic relevance to breast cancer patients of the other ErbB family members has long been a matter of debate.

Methods

In a series of 250 primary invasive breast cancer patients, we performed a comprehensive analysis of ErbB1–4 at the levels of mRNA expression and gene copy number using real-time quantitative PCR. The relationship between the status of ErbB1–4 and the clinicopathological characteristics or prognosis was evaluated.

Results

The mRNA expression of ErbB2, but not the other ErbB genes, was significantly correlated to copy number (P = 0.0005). ErbB3 and ErbB4 mRNA expression were positively correlated to each other (P < 0.0001). The mRNA expression of ErbB1/2 was inversely correlated to estrogen receptor (ER) and progesterone receptor (PgR) positivity, although mRNA expression of ErbB3/4 was positively correlated to ER and PgR positivity. Kaplan-Meier survival analysis showed that ErbB1 mRNA expression was associated with reduced survival. Neither ErbB2 nor ErbB3 mRNA expression had any association with survival, because half of the patients with Her2-positive tumors were treated with trastuzumab. High ErbB4 mRNA expression showed good prognosis with respect to breast cancer-specific survival

Conclusions

ErbB3 and ErbB4 mRNA expression, as well as well as that of ErbB1 and ErbB2, could be histopathological factors. ErbB3 mRNA was highly expressed in ER-positive tumors and has controversial prognostic value. ErbB4 mRNA expression was well correlated with ER positivity and good prognosis, indicating that ErbB4 may contribute to ER-dependent growth.     

ADSCs and adipocytes are the main producers in the autotaxin–lysophosphatidic acid axis of breast cancer and healthy mammary tissue in vitro

Background

Breast cancer is the most common malignancy in women affecting one out of eight females throughout their lives. Autotaxin (ATX) is upregulated in breast cancer which results in increased lysophosphatidic acid (LPA) formation within the tumor. This study’s aim was to identify the role of different mammary cell populations within the ATX–LPA axis.

Methods

Epithelial-cell-adhesion-molecule-positive (EpCAM) and -negative cells from breast tumors, adipose-derived stem cells (ADSCs) of tumor-adjacent and tumor-distant mammary fat were isolated and compared to healthy ADSCs, mammary epithelial cells (HMECs), and mesenchymal cells (MES) of healthy mammary tissue (n =?4 each) and further to well-established breast (cancer) cell lines.

Results

mRNA expression analyses revealed that ADSCs and MES largely expressed LPA receptor 1 (LPAR1) while epithelial cells mainly expressed LPAR6. LPA 18:1 activated all the cell populations and cell lines by rise in cytosolic free calcium concentrations. MES and ADSCs expressed ATX whereas epithelial cells did not. ADSCs revealed the highest expression in ATX with a significant decline after adipogenic differentiation in healthy ADSCs, whereas ATX expression increased in ADSCs from tumor patients. Breast (cancer) cell lines did not express ATX. Transmigration of MES was stimulated by LPA whereas an inhibitory effect was observed in epithelial cells with no differences between tumors and healthy cells. Triple-negative breast cancer (TNBC) cell lines were also stimulated and the transmigration partly inhibited using the LPA receptor antagonist Ki16425.

Conclusions

We here show that each mammary cell population plays a different role in the ATX–LPA axis with ADSCs and adipocytes being the main source of ATX in tumor patients in our experimental setting. Inhibitors of this axis may therefore present a valuable target for pharmacological therapies.

     

Detection of human epidermal growth factor receptor 2 protein and gene in fine needle aspiration cytology specimens and tissue sections from invasive breast cancer: can cytology specimens take the place of tissue sections?

Overexpression of HER2 protein and HER2 gene amplification in breast cancer are prognostic factors for the response to specific medical treatments such as trastuzumab, endocrine therapy, and chemotherapy. Whereas HER2 expression and gene amplification are generally examined in tissue sections, we investigated whether specimens from fine needle aspiration cytology (FNAC) are adequate for these analyses. HER2 protein overexpression and HER2 gene amplification were assessed in both FNAC specimens and tissue sections from 58 cases of invasive breast cancer. Immunohistochemistry assay for HER2 protein expression was performed according to the HercepTest protocol, and HER2 gene amplification was examined with the Spot-light CISH (chromogenic in situ hybridization) Detection kit. There was a significant positive correlation between assessments of HER2 protein status in the cytology specimens and tissue sections. The sensitivity, specificity, and accuracy of HER2 gene amplification detection in cytology specimens in relation to those in tissue sections were 84.0% (21/25 cases), 87.9% (29/33 cases), and 86.2% (50/58 cases), respectively. FNAC specimens are suitable for detection of HER2 overexpression and HER2 gene amplification in invasive breast cancer.     

Predictive value of apoptosis,proliferation, HER-2, and topoisomerase IIα for anthracycline chemotherapy in locally advanced breast cancer

Purpose.Laboratory evidence indicates that tumor growth depends on the balance between cell proliferation and cell death, and many anticancer agents may exert their therapeutic effect by decreasing proliferation and increasing apoptosis. Additionally, clinical observations indicate that overexpression of HER-2 or topoisomerase IIα (topo IIα) may be predictors of better response to anthracyclines in breast cancer. The objective of this study was to determine if proliferation (Ki-67), apoptosis (TUNEL), and expression of HER-2 and topo IIα are affected by anthracycline treatment, and if these molecular markers predict anthracycline responsiveness.Experimental design.Thirty-three women with primary breast tumors ≥3 cm received either doxorubicin (75 mg/m²) or epirubicin (120 mg/m²) for 4 cycles before surgery. Clinical response was evaluated after 4 cycles of treatment. Changes in molecular markers were assessed from core needle taken before treatment (D0), at 24–48 h (Dl) and on day 7 (D7) while on treatment, and from the surgical specimen excised on day 84 (D84) after the fourth cycle of chemotherapy.Results.The overall response rate was 51% (17 of 33 patients), with a 12% complete clinical response rate (4 of 33 patients). There were trends for tumors with higher apoptosis and topo IIα at baseline (D0) to be more responsive to anthracyclines, p = 0.1 and p = 0.08, respectively. Median apoptosis increased from D0 to Dl (p = 0.06) while median Ki-67 decreased (p = 0.07). Overall, expression of HER-2 remained stable throughout the chemotherapy administration. By Day 84, topo IIα had significantly decreased from baseline in responders, while it increased in non-responders, p = 0.03.Conclusions.In human primary breast cancer, anthracycline treatment causes an early increase in apoptosis and a decrease in proliferation. In this pilot study, higher apoptosis and topo IIαa levels in primary tumors were associated with greater responsiveness to anthracyclines, and topo IIα levels declined in responsive tumors.     

TNF-α in promotion and progression of cancer

Tumour necrosis factor alpha is a member of the TNF/TNFR cytokine superfamily. In common with other family members, TNF-α is involved in maintenance and homeostasis of the immune system, inflammation and host defence. However, there is a ‘dark side’ to this powerful cytokine; it is now clear that, especially in middle and old age, TNF-α is involved in pathological processes such as chronic inflammation, autoimmunity and, in apparent contradiction to its name, malignant disease. This article will discuss the involvement of TNF-α in the inflammatory network that contributes to all stages of the malignant process, and consider the possibility that TNF-α may be a target for cancer therapy.     

Three-dimensional in vitro tissue culture models of breast cancer — a review

Three-dimensional (3D) in vitro breast tumour models have an invaluable role in tumour biology today providing some very important insights into breast cancer. As well as increasing our understanding of homeostasis, cellular differentiation and tissue organization they provide a well defined environment for cancer research in contrast to the complex host environment of an in vivo model. With the recent availability of relevant stromal elements together with the vast array of extracellular matrix constituents available, in vivo like microenvironments can be recreated. These tissue like structures more realistically model the structural architecture and differentiated function of breast cancer than a cellular monolayer providing in vivo like responses to therapeutic agents. Three dimensional in vitro models allow the study of cell-cell and cell-extracellular matrix interactions, in addition to the influence of the microenvironment on cellular differentiation, proliferation, apoptosis and gene expression. Due to their enormous potential 3D cultures are currently being exploited by many other branches of biomedical science with therapeutically orientated studies becoming the major focus of research. In return great progress in 3D culture techniques have been made, largely due to this greater interaction. At present they are being used in studies ranging from investigating the role of adhesion molecules (e.g., E-cadherin) in invasion/metastasis; VEGF and angiogenesis, to tissue modelling and remodelling. Progress in the development of complex 3D culture systems is more productive than ever, however further research is vital.     

Crosstalk between TGF-β signaling and miRNAs in breast cancer metastasis

Transforming growth factor-β (TGF-β) signaling pathway is a key regulator of various cancer biologies, including cancer cell migration, invasion, angiogenesis, proliferation, as well as apoptosis, and it is one of indispensable signaling pathways during cancer metastasis. TGF-β signaling pathway can regulate and be regulated by a series of molecular and signaling pathways where microRNAs (miRNAs) seem to play important roles. miRNAs are small non-coding RNAs that can regulate expressions of their target genes. Emerging evidence suggest that miRNAs participate in various biological and pathologic processes such as cancer cells apoptosis, proliferation, invasion, migration, and metastasis by influencing multiple signaling pathways. In this article, we focus on the interaction between miRNAs and TGF-β in breast cancer (BC) metastasis through modulating invasion-metastasis-related factors, including epithelial-to-mesenchymal transition (EMT), cancer stem cells (CSCs), matrix metalloproteinase (MMP), tissue inhibitors of MMPs (TIMPs), cell adhesion molecules (CAMs), and tumor microenvironment (TME). Through a clear understanding of the complicated links between TGF-β pathway and miRNAs, it may provide a novel and safer therapeutic target to prevent BC metastasis.