Molecules In Breast Cancer Research Articles

Expression of the Classical and Nonclassical HLA Molecules in Breast Cancer

Considering that downregulation of HLA expression could represent a potential mechanism for breast carcinogenesis and metastasis, the aim of the present study was to use immunohistochemical methods to analyze the expression of HLA-Ia, HLA-DR, HLA-DQ, HLA-E, and HLA-G in invasive ductal carcinoma (IDC) of the breast and to relate this HLA profile to anatomopathological parameters. Fifty-two IDC from breast biopsies were stratified according to histological differentiation (well, moderately, and poorly differentiated) and to the presence of metastases in axillary lymph nodes. The expression of HLA molecules was assessed by immunohistochemistry, using a computer-assisted system. Overall, 31 (59.6%) out of the 52 IDC breast biopsies exhibited high expression of HLA-G, but only 14 (26.9%) showed high expression of HLA-E. A large number (41, 78.8%) of the biopsies showed low expression of HLA-Ia, while 45 (86.5%) showed high expression of HLA-DQ and 36 (69.2%) underexpressed HLA-DR. Moreover, 24 (41.2%) of 52 biopsies had both low HLA-Ia expression and high HLA-G expression, while 11 (21.2%) had low HLA-Ia expression and high HLA-E expression. These results suggest that, by different mechanisms, the downregulation of HLA-Ia, HLA-E, and HLA-DR and the upregulation of HLA-G and HLA-DQ are associated with immune response evasion and breast cancer aggressiveness.

Grade Dependent Expression of Growth Factor Receptors and Signaling Molecules in Breast Cancer

Growth factor signaling plays a key role in the growth and development of breast. Aberrant expression and activation of growth factors like insulin like growth factor-I (IGF-I) and epidermal growth factor (EGF) and their downstream signaling has been implicated in breast cancer. The deregulation of growth factor signaling is associated with increased proliferation and cell survival, decreased apoptosis, invasion, angiogenesis and metastasis. The aim of the present study is to survey the different signaling molecules involved in the IGF and EGF signaling pathways, and to find if there are any relationship between breast cancer and their levels and activation. Thirty-nine samples of breast cancer tissues (24 Grade II and 15 Grade III tumours) and sixteen normal breast tissue samples were collected. The expression of the receptors and signaling molecules were investigated using Western blot. IGF-IRβ, AR, pAkt, IKK-α and p38 are upregulated in cancer tissues in a grade depended manner. Further, Akt and β-catenin were also upregulated in cancer samples. Correlation analysis of signaling molecules revealed a disruption in their expression in cancer tissues. The present study shows that various signaling molecules are upregulated or activated in cancer tissues involving IGF-IR and Akt pathway. The expression of signaling molecules in the cancer tissues were deregulated when compared to the control samples. Thus, flawed expression and over activation of Akt pathway is seen in the breast cancer tissues.

P3-18-01: cMET Inhibitor and the Inhibition of Growth of Breast Cancer Cells in Bone Marrow Matrix Environment.

Abstract Background: Hepatocyte growth factor (HGF) is a cytokine that has a diverse but potential role in cancer including breast cancer. HGF, which action is mediated by its specific receptor, cMET, stimulate the aggressiveness of cancer cells by increasing the invasiveness and cellular migration. HGF is also a potent angiogenic factor. Small inhibitors to the HGF receptor are currently investigated in clinical trials of various cancers. In solid tumours which have potency of bone metastasis, HGF and particularly the HGF receptor, cMET, have been found to be particularly over-expressed in tumour cells metastasised to bones. Materials and methods. A panel of breast cancer cell lines were used. Cell growth was determined using a colorimetic method. Cell adhesion and migration were investigated using a ECIS model. Bone matrix proteins were prepared from fresh bones. A small inhibitor to human cMET, PF02341066 was used in the present study in all the cell models. Results. Depending on cell types, cancer cells tended to grow at a faster rate in bone marrow microenvironment than under normal conditions (12% higher for MDA MB 231 cells, for example). While PF02341066 have a concentration dependent effect on the growth of breast cancer, this effect became more marked when cells were growing in the bone marrow matrix. Breast cancer cells migrated rapidly in the presence of bone marrow environment, in comparison with controls. This was also inhibited by the cMET inhibitor. The study further showed that cells over-expressing a molecule linked to bone metastasis, namely ALCAM (1,2), responded more vigorously to cMET inhibitor in the matrix adhesion and cellular migration. Conclusions. The present study has shown that the HGF receptor inhibitor has an inhibitory effect on breast cancer cells. This is particularly so when cells are grown in bone matrix microenvironment. It is concluded therefore that inhibitors to the HGF receptor may have a particular role in the management of bone metastasis in breast cancer.

Aberrant expression and clinical correlation of Notch signaling molecules in breast cancer of Chinese population

Notch signaling molecules play crucial roles in cell proliferation and apoptosis, yet their function in breast cancer remains unclear. Samples and clinical data from 62 breast cancer patients were collected. After total RNA isolation, reverse transcription polymerase chain reaction was applied to analyze the expression of Notch receptors (Notch1, Notch3 and Notch4) and ligands (DLL4 and JAG1), and their clinical association. Immunohistochemical analysis was used to detect the intracellular domain of Notch1 (Notch1-IC) expression. Notch1 was the dominant receptor while DLL4 was the dominant ligand. The Notch molecules expression pattern for infiltrating ductal carcinoma (IDC) was similar to that for infiltrating lobular carcinoma (ILC) except for JAG1, while Notch1 standard coefficients in ILC were statistically higher than that in IDC. Immunohistochemical results showed that Notch1-IC protein expression paralleled the mRNA level. Breast cancer patients' clinical parameters suggested that Notch1 expression was higher in stage II disease and lower in more advanced stages. The Notch3 positive rate was higher in patients with lower levels of Notch1, and the Notch3 positive rate was lower in patients with higher levels of Notch1. No apparent correlation of Notch molecules with estrogen receptor (ER), progesterone receptor (PR) was found. Though high Notch1 and Notch3 RNA levels tended to correlate with c-erbB2 expression, no statistical significance was found. Notch molecules are useful biomarkers in breast cancer especially for Notch1 and DLL4, and Notch1 is expressed differently in different stages of human breast cancer.

Notch, IL-1 and Leptin Crosstalk Outcome (NILCO) Is Critical for Leptin-Induced Proliferation, Migration and VEGF/VEGFR-2 Expression in Breast Cancer

High levels of pro-angiogenic factors, leptin, IL-1, Notch and VEGF (ligands and receptors), are found in breast cancer, which is commonly correlated with metastasis and lower survival of patients. We have previously reported that leptin induces the growth of breast cancer and the expression of VEGF/VEGFR-2 and IL-1 system. We hypothesized that Notch, IL-1 and leptin crosstalk outcome (NILCO) plays an essential role in the regulation of leptin-mediated induction of proliferation/migration and expression of pro-angiogenic molecules in breast cancer. To test this hypothesis, leptin's effects on the expression and activation of Notch signaling pathway and VEGF/VEGFR-2/IL-1 were determined in mouse (4T1, EMT6 and MMT) breast cancer cells. Remarkably, leptin up-regulated Notch1-4/JAG1/Dll-4, Notch target genes: Hey2 and survivin, together with IL-1 and VEGF/VEGFR-2. RNA knockdown and pharmacological inhibitors of leptin signaling significantly abrogated activity of reporter gene-luciferase CSL (RBP-Jk) promoter, showing that it was linked to leptin-activated JAK2/STAT3, MAPK, PI-3K/mTOR, p38 and JNK signaling pathways. Interestingly, leptin upregulatory effects on cell proliferation/migration and pro-angiogenic factors Notch, IL-1 and VEGF/VEGFR-2 were abrogated by a γ-secretase inhibitor, DAPT, as well as siRNA against CSL. In addition, blockade of IL-1R tI inhibited leptin-induced Notch, Hey2 and survivin as well as VEGF/VEGFR-2 expression. These data suggest leptin is an inducer of Notch (expression/activation) and IL-1 signaling modulates leptin effects on Notch and VEGF/VEGFR-2. We show for the first time that a novel unveiled crosstalk between Notch, IL-1 and leptin (NILCO) occurs in breast cancer. Leptin induction of proliferation/migration and upregulation of VEGF/VEGFR-2 in breast cancer cells were related to an intact Notch signaling axis. NILCO could represent the integration of developmental, pro-inflammatory and pro-angiogenic signals critical for leptin-induced cell proliferation/migration and regulation of VEGF/VEGFR-2 in breast cancer. Targeting NILCO might help to design new pharmacological strategies aimed at controlling breast cancer growth and angiogenesis.

Abstract 4086: Notch, IL-1 and leptin crosstalk outcome (NILCO) is critical for leptin regulation of VEGF/VEGFR-2 in breast cancer

Abstract Elevated levels of leptin and its deregulated signals have been strongly associated to obesity-related cancers, including breast cancer. Higher levels of leptin and leptin receptor, OB-R, are expressed by breast cancer cells, which commonly correlated with metastasis and lower survival of breast cancer patients. We have previously reported that leptin signaling induced the growth of breast cancer mainly through the up-regulation of pro-angiogenic molecules, i.e., VEGF/VEGFR-2, IL-1. Disruption of leptin signaling markedly reduced the growth of tumors and the expression of VEGF/VEGFR-2 in syngeneic and human breast cancer xenograft mouse models (ER+ and ER-). We speculate leptin interacts with Notch (essential for tumor progression and angiogenesis) in breast cancer cells through complex molecular mechanisms. We hypothesized that a crosstalk between leptin and Notch signaling could impact the expression of pro-angiogenic molecules in breast cancer. Methods: To test this hypothesis leptin-mediated activation of Notch signaling pathway (Notch1,3,4 NICD and CSL) and VEGF/VEGFR2 expression were determined in mouse (4T1, EMT6 and MMT) and human breast cancer cell lines (MCF-7 and MDA-MB-231) under normoxic conditions. Results: Leptin upregulates Notch receptors/ligands and also Notch-target genes, Hey2 and survivin in breast cancer cells. Notably, these leptin effects were more evident in triple negative breast cancer cells (MDA-MB231). Leptin-induction of CSL promoter activity was linked to multiple signaling pathways (MAPK, PI-3K/mTOR, PKC, p38 and JNK). Interestingly, leptin upregulatory effects on pro-angiogenic factors IL-1, VEGF/VEGFR-2 and Notch were significantly abrogated by a γ-secretase inhibitor, DAPT, as well as siRNA against CSL. In addition, IL-1 tI antibody inhibits leptin induced Notch, Notch target genes, Hey2 and survivin as well as VEGF/VEGFR-2 expression. Conclusions: These data suggest leptin-induced IL-1 signaling is related to leptin effects on Notch signaling. Our results further indicate that a novel unveiled crosstalk between Notch, IL-1 and leptin occurs in breast cancer. Indeed, leptin up-regulation of pro-angiogenic molecules: Notch, VEGF/VEGFR-2, IL-1 requires the activation of Notch signaling in breast cancer cells. Therefore, the integration of developmental, pro-inflammatory and pro-angiogenic signals (Notch, IL-1 and leptin crosstalk outcome (NILCO)] seems to be critical for leptin regulation of VEGF/VEGFR-2 in breast cancer. This novel finding might help to design new pharmacological strategies aimed at controlling breast cancer growth and angiogenesis, especially in triple negative breast cancer patients. [This work was supported in part by NIH/NCI1SC1CA138658-02; NIH/ARRA/3SC1CA138658-02S1and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP)]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4086. doi:10.1158/1538-7445.AM2011-4086

The Endothelin Axis Stimulates the Expression of Pro-Inflammatory Cytokines and Pro-Migratory Molecules in Breast Cancer

ABSTRACTWe investigated the effects of the endothelin-1 (ET-1) receptor dual antagonist (Bosentan®) on the inflammatory cytokines and the chemoattractant molecules associated with breast cancer growth and the development of tumor infiltration in bone explants. Immunocompetent mice implanted with the murine mammary carcinoma 4T1 cells in a skin-fold chamber and treated with Bosentan® had reduced tumor growth (p < .05). ET-1 promoted the secretion of the anti-inflammatory soluble tumor necrosis factor (TNF) receptor and IL12 p40 in vitro. The Bosentan® treatment in vivo was associated with a local increase of the anti-inflammatory IL-1α cytokine concentration and decrease of the pro-inflammatory TNF-α and IL-17 cytokine concentrations (p < .05).

EGFRvIII‐induced estrogen‐independence, tamoxifen‐resistance phenotype correlates with PgR expression and modulation of apoptotic molecules in breast cancer

The tumor-specific, ligand-independent, constitutively active epidermal growth factor receptor (EGFR) variant, EGFRvIII, remains understudied in breast cancer. Here, we report that expression of EGFRvIII in the ErbB-2-overexpressing, estrogen-dependent MDA-MB-361 breast cancer cell line resulted in significant estrogen-independent tumor growth in ovariectomized, athymic nude mice in comparison to MDA-MB-361/wt cells. MDA-MB-361/vIII breast cancer cells maintained estrogen-induced tumor growth, but were tamoxifen-resistant in the presence of estrogen, while MDA-MB-361/wt cells had a significant reduction in tumor growth in the presence of estrogen and tamoxifen. Tamoxifen alone did not have a significant effect on EGFRvIII-mediated estrogen-independent tumor growth. Constitutive signaling from the EGFRvIII receptor resulted in an increased activation of both the Akt and MAPK pathways. Compared to estrogen-dependent, tamoxifen-sensitive MCF-7/vIII breast cancer cells, which had unchanged levels of ERalpha, but an increase in progesterone receptor (PgR) in comparison to MCF-7/wt cells, MDA-MB-361/vIII cells had a reduction in ERalpha expression as well as a more pronounced reduction in PgR compared with MDA-MB-361/wt cells. EGFRvIII expression was also significantly associated with an absence of PgR protein in invasive human breast cancer specimens. Alterations of proapoptotic proteins and antiapoptotic proteins were observed in EGFRvIII transfectants. In conclusion, constitutive signaling through EGFRvIII and its downstream effector proteins crosstalks with the ERalpha pathway, resulting in loss of PgR expression and alterations in the apoptotic pathway, which may result in the estrogen-independent, tamoxifen-resistant phenotype conferred to EGFRvIII-expressing breast cancer cells.

Expression of Indian Hedgehog signaling molecules in breast cancer

To investigate the clinicopathological significance and expression pattern of Hedgehog (Hh) signaling molecules in breast normal glands and invasive ductal carcinoma. A total of 142 cases, including 21 of normal breast and 121 of invasive ductal carcinoma of the breast, were immunohistochemically analyzed for Ihh, Ptch, Smo, Gli-1, Gli-2, and Gli-3 protein expression. All of Hh signaling molecules were greatly enhanced in invasive ductal carcinoma compared with the normal breast epithelia. The expressions of Ihh, Smo, and Gli-2 were increased in PR negative cases, and the expressions of Ihh, Ptch, and Gli-1/2/3 were statistically correlated with increased proliferating index of Ki-67 in invasive ductal carcinoma. Ihh and Gli-1/2/3 expressions were correlated with node metastasis. Additionally, the protein expressions of Ihh, Ptch, and Gli-2 were correlated with the clinical stage of breast cancer. Hedgehog signaling molecules play an important role in the progression of invasive ductal carcinoma of breast.

Small is beautiful: microRNAs and breast cancer—where are we now?

MicroRNAs are a recently discovered class of small regulatory RNAs that influence the stability and translational efficiency of target mRNAs. They have been implicated in an increasing number of biological processes, including neoplasia. Recent studies have shown an involvement for these regulatory molecules in breast cancer. For example, miRNA profiling studies have identified microRNAs that are deregulated in breast cancer. Furthermore, functional studies have uncovered their roles in breast cancer as both tumour suppressor genes (eg miR-335) and oncogenes (eg miR-21). miRNAs deregulated in breast cancer influence the translational regulation of well-established regulatory molecules, such as oestrogen receptor-alpha, which is regulated by miR-206, and novel cancer-related molecules whose functions are not yet fully understood.. Here we present an overview of our current understanding of miRNA in breast cancer.

The Loss of Intercellular Adhesion Molecules in Breast Cancer: Does it Predict a Poor Prognosis?

Background: Intercellular adhesive molecules are essentialfor the integrity of body tissue. A tumor’s potentialfor invasion and metastasis depends on the structuralchanges of those molecules through the mutations oftumor cells. The aim of this study was to evaluate the expressionof E-cadherin and ß-catenin in breast carcinoma,and its effect on the outcome and survival of patients.Material and Methods: Immunohistochemical(IHC) staining of histological sections of different types ofbreast cancer for E-cadherin and ß-catenin was performedin 88 breast tumors. Follow-up of the patients fora period of more than 5 years or until death was possiblein all cases. Results: IHC staining for both E-cadherin andß-catenin was strongly positive in ductal carcinoma insitu (DCIS). Complete loss of expression was shown in71% and 61% respectively, in infiltrating lobular carcinoma.Significant difference in loss of E-cadherin and/orß-catenin expression (p < 0.002) was found betweengrade 2 and grade 3 invasive ductal carcinoma (IDC), regardlessof tumor size, status of axillary nodes, age, orethnicity. Conclusions: The loss of expression of adhesivemolecules is correlated with the grade of IDC tumorsand the type of breast tumor, and it is not an indicator ofprognosis.

Bone morphogenic proteins 1 to 7 in human breast carcinoma

21154 Background: Bone morphological proteins (BMPs) have a diverse role and they act in a time, concentration and cell type specific manner. They have been shown to regulate cellular motility and the cells ability to invade and recently BMP molecules have been shown to have an impact on the biological behaviour of breast cancer cells. In this study we looked, for the first time, at the expression of a panel of BMPs in breast carcinomas. Methods: Fresh frozen primary breast cancer tissues (n =120) and non-neoplastic mammary tissue (n = 32) were used. The distribution and location of BMPs was assessed using immunohistochemical methods and the levels of BMPs (BMP-1, -2, -3, -4, -5, -6, and -7) were determined using quantitative RT-PCR. The results were analysed against the clinical, pathological and follow-up (10 years) data. Results: BMP-2 and BMP-7 had a contrast pattern of expression in normal and tumour tissues, in that BMP-2 transcript level was lower and BMP-7 was higher in breast tumours than normal tissues. BMP-2 transcript was also significantly lower in tumours from patients with a moderate and poor prognosis than from those with a good prognosis (p=0.04). Also both BMP-2 and BMP-7 showed a significant difference between node positive and node negative tumours (p=0.033 and p=0.031 respectively). BMPs 1, 3, 4, 5 and 6 showed an inconsistent variation in transcript levels within the cancer subgroups with no statistically significant results. Conclusions: This study has demonstrated the differential expression pattern of BMP molecules in breast cancer and reveals a potential prognostic value of BMP-2 and BMP-7 for the patients. The findings may also suggest that these BMPs may be potential therapeutic targets. No significant financial relationships to disclose.

Transcriptional deregulation of VEGF, FGF2, TGF-β1, 2, 3 and cognate receptors in breast tumorigenesis

Angiogenesis is an important event during the neoplastic process and is induced by the secretion of numerous growth factors from endothelial cells. Vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (FGF2), and transforming growth factor-β1, β2, β3 (TGF-β1, 2, 3) and cognate receptors (TGF-βRI, II, III) mRNA expression pattern was evaluated by RT-PCR in 25 breast cancer tissue samples and adjacent normal tissues, and correlated to clinicopathological features. Western blot analysis was performed to evaluate VEGF and TGF-β1 protein levels. TGF-β1 and TGF-β3 mRNA levels were significantly different in breast cancer specimens of differing histology (ductal, lobular, other) ( P=0.020 and P=0.043). No statistically significant difference was observed at the mRNA level of VEGF between normal and tumor tissues while elevated VEGF protein levels in tumors were associated with patients' menopausal status. A strong hormonal influence of ER and PR on TGF-β mRNA expression was established. FGF2 transcript levels were substantially decreased in cancer compared to adjacent normal specimens ( P=0.031). A disruption of mRNA co-expression patterns was observed in malignant breast tissues compared to controls. Western blot analysis revealed differences between VEGF and TGFβ1 mRNA and their corresponding protein levels. A substantial negative correlation of TGF-β1 protein and TGF-β1 mRNA levels ( P=0.016) was demonstrated by breast tissue-pair analysis. Summarizing, our findings suggest that transcript levels of the examined markers in breast cancer are associated with menopausal and hormonal status, while their co-expression pattern is altered in malignant tissues compared to controls. In addition the difference between VEGF and TGF-β1 mRNA and protein levels observed, indicates that post-transcriptional mechanisms may regulate expression of these molecules in breast cancer.

Assessing microvessels and angiogenesis in human breast cancer, using VE‐cadherin

Vascular endothelial (VE)-cadherin, also known as cadherin-5 and CD144, is an adhesion molecule uniquely expressed in endothelial cells. We hypothesized that VE-cadherin may be a useful marker for assessing microvessels and angiogenesis in human breast cancer and sought to determine whether a correlation exists between levels of VE-cadherin, angiogenic markers factor VIII and platelet endothelial cell adhesion molecule (PECAM)-1 and patient outcome in breast cancer. Frozen sections from breast cancer primary tumours (tumour n = 114, background n = 30) were immunostained with VE-cadherin, factor VIII and PECAM-1 antibodies and microvessel number was assessed. RNA was reverse transcribed and analysed by quantitative polymerase chain reaction (Q-PCR). VE-cadherin immunostaining showed a significant difference in microvessel number in tumour compared with background. There was no significant difference in the number of microvessels stained with PECAM-1 or factor VIII; there was increased staining of other structures within the sample and higher general background staining. Q-PCR revealed elevated levels of VE-cadherin and PECAM-1 in tumour samples compared with background tissue and in patients with a poor prognosis, as determined by the Nottingham Prognostic Index. There was no difference in levels with factor VIII. Both VE-cadherin and PECAM-1 had significantly reduced expression in lobular compared with ductal carcinomas: there was no difference with factor VIII. Higher levels of angiogenic marker molecules in breast cancer may have an association with poor prognosis in patients. Moreover, VE-cadherin appears to be a preferable marker for such analysis.

Standardized On-Slide Control for Quality Assurance in the Immunohistochemical Assessment of Therapeutic Target Molecules in Breast Cancer

Individualization and optimization of risk-adapted therapy in breast cancer requires molecular profiling of individual cancer specimens. In tissues of heterogeneous cellular composition, such as mammary tissue, cancer prognostic markers and potential therapeutic target molecules are preferentially detected by in situ techniques such as immunohistochemistry. The more therapy decisions are based on immunohistochemical findings, the more histopathologists are confronted with the demand to establish standardized procedures that enable reproducible evaluation independent from the investigating laboratory. Disappointing results of recent national and international immunohistochemistry trials for steroid receptor and HER-2 analysis in breast cancer underline the need to act now beyond recommendations and well-formulated guidelines for standardization. In order to ensure staining quality in every individual immunohistochemical analysis, we have developed an on-slide control with standardized samples which are placed on every slide of a therapy-decisive stain together with the tissue of interest. Novel mini tissue microarrays were constructed comprising cell lines as standardized controls, indicating the sensitivity and reliability of the staining procedure. Standardized control material on the same slide as the patient's specimen require neither additional reagents nor time and are automatically archived on the diagnostic slide to prove the quality of staining even after years.

Activated leukocyte cell adhesion molecule in breast cancer: prognostic indicator.

IntroductionActivated leukocyte cell adhesion molecule (ALCAM) (CD166) is an immunoglobulin molecule that has been implicated in cell migration. The present study examined the expression of ALCAM in human breast cancer and assessed its prognostic value.MethodsThe immunohistochemical distribution and location of ALCAM was assessed in normal breast tissue and carcinoma. The levels of ALCAM transcripts in frozen tissue (normal breast, n = 32; breast cancer, n = 120) were determined using real-time quantitative PCR. The results were then analyzed in relation to clinical data including the tumor type, the grade, the nodal involvement, distant metastases, the tumor, node, metastasis (TNM) stage, the Nottingham Prognostic Index (NPI), and survival over a 6-year follow-up period.ResultsImmunohistochemical staining on tissue sections in ducts/acini in normal breast and in breast carcinoma was ALCAM-positive. Differences in the number of ALCAM transcripts were found in different types of breast cancer. The level of ALCAM transcripts was lower (P = 0.05) in tumors from patients who had metastases to regional lymph nodes compared with those patients without, in higher grade tumors compared with Grade 1 tumors (P < 0.01), and in TNM Stage 3 tumors compared with TNM Stage 1 tumors (P < 0.01). Tumors from patients with poor prognosis (with NPI > 5.4) had significantly lower levels (P = 0.014) of ALCAM transcripts compared with patients with good prognosis (with NPI < 3.4), and tumors from patients with local recurrence had significantly lower levels than those patients without local recurrence or metastases (P = 0.04). Notably, tumors from patients who died of breast cancer had significantly lower levels of ALCAM transcripts (P = 0.0041) than those with primary tumors but no metastatic disease or local recurrence. Patients with low levels of ALCAM transcripts had significantly (P = 0.009) more incidents (metastasis, recurrence, death) compared with patients with primary breast tumors with high levels of ALCAM transcripts.ConclusionsIn the present panel of breast cancer specimens, decreased levels of ALCAM correlated with the nodal involvement, the grade, the TNM stage, the NPI, and the clinical outcome (local recurrence and death). The data suggest that decreased ALCAM expression is of clinical significance in breast cancer, and that reduced expression indicates a more aggressive phenotype and poor prognosis.

Defective antigen presentation resulting from impaired expression of costimulatory molecules in breast cancer

Defective antigen presentation resulting from impaired expression of costimulatory molecules in breast cancer

Bcl-2 over-expression enhances NF-kappaB activity and induces mmp-9 transcription in human MCF7(ADR) breast-cancer cells.

bcl-2 expression is often associated with poor prognosis in several types of tumors; however, the role of this molecule in breast cancer is still controversial. We found earlier that over-expression of bcl-2 in a human breast-cancer cell line (MCF7(ADR)) enhances its tumorigenicity and metastatic potential by inducing metastasis-associated properties such as increased secretion of the matrix metalloproteinase-9 (mmp-9). In the present study, we investigated the effect of bcl-2 over-expression on the activity of the transcription factor NF-kappaB, an important regulator of genes involved in tumor progression and invasion. Transient transfection experiments indicate that over-expression of bcl-2 in the MCF7(ADR) cell line, enhances NF-kappaB-dependent transcriptional activity. Mobility-shift analysis revealed an increase of NF-kappaB DNA-binding in bcl-2-over-expressing clones that correlated with lower levels of the NF-kappaB cytoplasmic inhibitor IkappaBalpha. Moreover, point mutations of 2 highly conserved residues within the BH1 and BH2 domains that abrogate the interaction of bcl-2 with bax, or deletion of the N-terminal BH4 domain, completely eliminate the ability of this molecule to up-regulate NF-kappaB-dependent transactivation. Since mmp-9 is a NF-kappaB-regulated gene, we also investigated whether bcl-2 over-expression up-regulated mmp-9 transcription. We found that induction of mmp-9 mRNA correlates with the activation of an mmp-9-promoter-reporter-gene construct in transient transfection assay, and a mutation of the (-600)mmp-9-NF-kappaB binding element abolishes this effect. The overall data indicate that bcl-2-mediated regulation of NF-kappaB-transcription-factor activity may represent an important mechanism for the promotion of malignant behavior in MCF-7(ADR) cells.

Defective antigen presentation resulting from impaired expression of costimulatory molecules in breast cancer

Previous experiments from our laboratory have shown that immune mechanisms aiming at the destruction of tumour cells including the recognition of target cells and their elimination via the expression of intercellular adhesion molecule-1 (ICAM-1; CD54), the production of tumour necrosis factor-alpha (TNF-alpha) by monocytes and appropriate function of lymphocyte subpopulations were defective in breast cancer. Previous observations were extended to assess expression levels and regulatory mechanisms of costimulatory molecules CD54, CD80 and CD86 on monocytes derived from patients with early breast cancer (EBC). In addition, antigen presentation by antigen-presenting cells (APC) was analyzed within this context. We report that monocytes derived from patients with EBC exhibited significantly decreased expression levels of CD54 (p = 0.0002), CD80 (p = 0.009) and CD 86 (p = 0.002) compared with monocytes derived from healthy females. Simultaneously, lipopolysaccharide (LPS)-induced TNF-alpha production of monocytes was found to be defective in patients with EBC. Finally, T-cell proliferation in response to tetanus toxoid (TT) was significantly decreased in patients with EBC compared with healthy control females (p < 0.0001). Furthermore, T-cell proliferation in response to TT-pulsed APC derived from healthy controls was significantly inhibited in the presence of anti-CD54 and/or anti-CD80 antibodies in a dose-dependent manner, thus corroborating the necessity of the presence of CD54 and CD80 as costimulatory molecules in the present setting. We conclude that monocytes derived from patients with EBC showed a simultaneous defect of expression of CD54 and its regulation via TNF-alpha, CD80 and CD86 as well as T-cell proliferation following exposure to TT-pulsed APC. Based upon these findings, it is speculated that defects in costimulatory molecule expression might contribute to tolerance of the immune system towards the presence of malignant cells in patients with EBC.

Increased level of circulating adhesion molecules in the sera of breast cancer patients with distant metastases.

The adhesion of circulating cancer cells to the vascular endothelium is an important at step in the hematogenous metastasis of cancer. E-selectin expressed on endothelial cells and carbohydrate ligands expressed on cancer cells mediate this adhesion. We investigated the clinical significance of such cell adhesion molecules in breast cancer. The cytosol concentration of sialyl Lewis(x) was found more elevated in cancerous tissue than that in adjacent non-cancerous tissue. In the serum, sialyl Lewis(x) and soluble E-selectin were seen elevated in patients with advanced and recurrent breast cancer, especially in those with distant metastases. From the above, we have concluded that sialyl Lewis(x) and soluble E-selectin could be used as tumor markers with a close relationship to the metastasis of breast cancer.