A novel role of kynureninase in the growth control of breast cancer cells and its relationships with breast cancer.

Breast cancer is the most common malignancy among women worldwide. Kynureninase (KYNU) located in 2q22.2, which was associated with tryptophan utilization and metabolic diseases including cardiac, renal and limb defects syndrome 2. However, the role of KYNU in breast cancer (BC) development remains unclear. The expression of KYNU was examined by immunohistochemistry (IHC) in 137 primary BC tissues, and the correlation of KYNU expression with clinical pathological characteristics and the biomarkers (ER, PR, HER2, E-cad and Ki-67) was analysed. The role of KYNU in cancer cell proliferation, tumour growth and development was evaluated by MTT assay, soft agar colony formation assay and xenograft mouse models. Among 137 primary BC tissues, 46.7% (64/137) had high KYNU expression (IHC scores >4) while 53.3% (73/137) had low KYNU expression (IHC scores ≤4). The expression of KYNU was positively correlated with the expressions of ER (P = .002), PR (P = .007) and E-cad (P = .03), while negatively associated with tumour grade (P = .008), tumour stage (P < .001) and the expressions of HER2 (P = .04) and Ki-67 (P = .019). Overexpression of KYNU significantly inhibited cell proliferation in cell culture, colony formation in soft agar and xenograft BC development in NOD/SCID mice. Kynureninase suppresses BC cell proliferation, tumour growth and development. Kynureninase may function as a tumour suppressor in BC.

Evaluation of the Expression of Amine Oxidase Proteins in Breast Cancer.

We aimed to evaluate the expression of amine oxidase proteins in breast cancer and their clinical implications. We performed immunohistochemical staining of amine oxidase proteins (LOX, lysyl oxidase, AOC3, amine oxidase, MAOA, monoamine oxidase A, MAOB, monoamine oxidase B). Based on their hormone receptors, such as estrogen receptor (ER) and progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2), and Ki-67 immunohistochemical staining, breast cancer was divided into four molecular subtypes: luminal A, luminal B, HER-2 type, and triple-negative breast cancer (TNBC). Luminal A was observed in 380 cases (49.4%), luminal B in 224 (29.1%), HER-2 type in 68 (8.8%), and TNBC in 98 (12.7%). Stromal AOC3, MAO-A, and MAO-B expression varied according to molecular subtypes. Stromal AOC3 expression was high in luminal B and HER-2 type and MAO-A expression was high in luminal A and luminal B (p < 0.001). MAO-B expression was higher in TNBC than in other subtypes (p = 0.020). LOX positivity was associated with high histological grade (p < 0.001) and high Ki-67 labeling index (LI) (p = 0.009), and stromal AOC3 positivity was associated with high histological grade (p = 0.001), high Ki-67 LI (p < 0.001), and HER-2 positivity (p = 0.002). MAO-A positivity was related to low histological grade (p < 0.001), ER positivity, PR positivity (p < 0.001), and low Ki-67 LI (p < 0.001). In univariate analysis, MAO-A positivity was related to short disease-free survival in HER-2 type (p = 0.013), AOC3 negativity was related to short disease-free survival and overall survival in ER-positive breast cancer, PR-positive breast cancer, HER-2-negative breast cancer, and lymph node metastasis. In conclusion, the expression of amine oxidase proteins varies depending on the molecular subtype of breast cancer. Stromal AOC3 expression was high in luminal B and HER-2 type, and MAO-A expression was high in luminal A and luminal B.

Aromatase expression increases the survival and malignancy of estrogen receptor positive breast cancer cells.

In postmenopausal women, local estrogen produced by adipose stromal cells in the breast is believed to support estrogen receptor alpha (ERα) positive breast cancer cell survival and growth. This raises the question of how the ERα positive metastatic breast cancer cells survive after they enter blood and lymph circulation, where estrogen level is very low in postmenopausal women. In this study, we show that the aromatase expression increased when ERα positive breast cancer cells were cultured in suspension. Furthermore, treatment with the aromatase substrate, testosterone, inhibited suspension culture-induced apoptosis whereas an aromatase inhibitor attenuated the effect of testosterone suggesting that suspended circulating ERα positive breast cancer cells may up-regulate intracrine estrogen activity for survival. Consistent with this notion, a moderate level of ectopic aromatase expression rendered a non-tumorigenic ERα positive breast cancer cell line not only tumorigenic but also metastatic in female nude mice without exogenous estrogen supplementation. The increased malignant phenotype was confirmed to be due to aromatase expression as the growth of orthotopic tumors regressed with systemic administration of an aromatase inhibitor. Thus, our study provides experimental evidence that aromatase plays an important role in the survival of metastatic ERα breast cancer cells by suppressing anoikis.

Identification of upregulated phosphoinositide 3-kinase γ as a target to suppress breast cancer cell migration and invasion.

Metastasis is the major cause of breast cancer mortality. We recently reported that aberrant G-protein coupled receptor (GPCR) signaling promotes breast cancer metastasis by enhancing cancer cell migration and invasion. Phosphatidylinositol 3-kinase γ (PI3Kγ) is specifically activated by GPCRs. The goal of the present study was to determine the role of PI3Kγ in breast cancer cell migration and invasion. Immunohistochemical staining showed that the expression of PI3Kγ protein was significantly increased in invasive human breast carcinoma when compared to adjacent benign breast tissue or ductal carcinoma in situ. PI3Kγ was also detected in metastatic breast cancer cells, but not in normal breast epithelial cell line or in non-metastatic breast cancer cells. In contrast, PI3K isoforms α, ß and δ were ubiquitously expressed in these cell lines. Overexpression of recombinant PI3Kγ enhanced the metastatic ability of non-metastatic breast cancer cells. Conversely, migration and invasion of metastatic breast cancer cells were inhibited by a PI3Kγ inhibitor or by siRNA knockdown of PI3Kγ but not by inhibitors or siRNAs of PI3Kα or PI3Kß. Lamellipodia formation is a key step in cancer metastasis, and PI3Kγ blockade disrupted lamellipodia formation induced by the activation of GPCRs such as CXC chemokine receptor 4 and protease-activated receptor 1, but not by the epidermal growth factor tyrosine kinase receptor. Taken together, these results indicate that upregulated PI3Kγ conveys the metastatic signal initiated by GPCRs in breast cancer cells, and suggest that PI3Kγ may be a novel therapeutic target for development of chemotherapeutic agents to prevent breast cancer metastasis.

Lactate dehydrogenase-B is silenced by promoter methylation in a high frequency of human breast cancers.

OBJECTIVE: Under 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 DESIGN: Lactate 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. RESULTS: Absent 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% O(2)), 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. CONCLUSIONS: Loss 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.

Trastuzumab for the treatment of salivary duct carcinoma.

OBJECTIVE: Salivary duct carcinoma (SDC) is a rare and aggressive malignancy with high mortality and poor response to treatment. A significant fraction of SDCs are HER2 positive. This retrospective review examines HER2 testing in SDC and the outcome of trastuzumab-based therapy in adjuvant and palliative settings. METHODS: A total of 13 patients with SDC and HER2/neu expression by immunohistochemistry of 1-3+ were treated with trastuzumab in adjuvant (n = 8) or palliative (n = 5) setting. Adjuvant therapy consisted of concurrent radiation and chemotherapy with weekly paclitaxel, carboplatin, and trastuzumab (TCH) for 6 weeks followed by TCH for 12 weeks and trastuzumab alone for 1 year. Palliative treatment for metastatic disease consisted of TCH every 3 weeks for 6 cycles followed by trastuzumab for variable time periods with or without second-line chemotherapy for progression. All patients had fluorescence in situ hybridization testing for HER2/neu gene amplification. RESULTS: The median duration of follow-up was 27 months (range: 8-48 months). In all, 62% of adjuvant patients (5/8) had no evidence of disease more than 2 years from completion of therapy. All patients with metastatic disease (5/5 patients) responded to treatment with TCH. One patient achieved a complete response and remains with no evidence of disease 52 months after initiation of TCH. The median duration of response was 18 months (range: 8-52 months). CONCLUSION: HER2/neu positivity and treatment with trastuzumab correlated well with long-term survival and response in our patients. Based on this data, we propose that HER2/neu status be examined routinely in all patients with SDCs and the treatment be directed accordingly.

Phosphorylation of caspase-7 by p21-activated protein kinase (PAK) 2 inhibits chemotherapeutic drug-induced apoptosis of breast cancer cell lines.

p21-activated kinase (PAK) 2, a member of the PAK family of serine/threonine protein kinases, plays an important role in physiological processes such as motility, survival, mitosis, and apoptosis. However, the role of PAK2 in resistance to chemotherapy is unclear. Here we report that PAK2 is highly expressed in human breast cancer cell lines and human breast invasive carcinoma tissue compared with a human non-tumorigenic mammary epithelial cell line and adjacent normal breast tissue, respectively. Interestingly, we found that PAK2 can bind with caspase-7 and phosphorylate caspase-7 at the Ser-30, Thr-173, and Ser-239 sites. Functionally, the phosphorylation of caspase-7 decreases its activity, thereby inhibiting cellular apoptosis. Our data indicate that highly expressed PAK2 mediates chemotherapeutic resistance in human breast invasive ductal carcinoma by negatively regulating caspase-7 activity.

Expression of cyclooxygenase-2 and its relation to histological grade, inducible nitric oxide synthase, matrix metalloproteinase-2, CD-34, Caspase-3, and CD8 in invasive ductal carcinoma of the breast.

OBJECTIVE: To assess by immunohistochemistry the cyclooxygenase-2 (COX-2) expression in invasive ductal carcinoma and its possible correlation with the histological grade, inducible nitric oxide synthase (iNOS), matrix metalloproteinase-2 (MMP2), and other common immunohistochemical parameters (CD-34, Caspase-3, and CD8). METHODS: This was a retrospective pathology archive study including 50 female patients and was performed in Mustafa Kemal University, Hatay, and Sutcu Imam University, Kahraman Maras, Turkey. The routine hematoxylin-eosin staining and COX-2, iNOS, MMP-2, CD-34, CASP-3, and CD8 immunoperoxidase techniques were performed on paraffin-embedded tissues. RESULTS: The mean value of COX-2 was 274.02+/-54.49 and MMP-2 was 263.42+/-54.30, whereas the mean iNOS values were 258.10+/-56.05. CD-34 staining also yielded positive results as 26.18+/-8.00. The mean value of CASP-3 was 284.06+/-41.2 and CD8 was 164.17+/-69.5. This reveals an inverse correlation between CASP-3 reactivity and CD8 (Spearman correlation [r]= -0.33, p=0.01). There was also an inverse correlation between iNOS reactivity and patients age (r = -0.29, p=0.03). There was a positive correlation with COX-2 and MMP-2 (p=0.00), but there was no relation with COX-2 and other parameters. CONCLUSION: COX-2 expression is an important parameter for invasive ductal carcinoma of the breast. We found a positive correlation between COX-2 and MMP-2, whereas, we could not show direct correlation between COX-2 and iNOS, CD-34, CASP-3, and CD8.

Increased ceramide synthase 2 and 6 mRNA levels in breast cancer tissues and correlation with sphingosine kinase expression.

Intervention in the ceramide metabolic pathway is emerging as a novel means to regulate cancer and to modify the activity of chemotherapeutic drugs. We now study mRNA expression levels of the six ceramide synthase (CerS) genes in breast cancer tissue. CerS2 and CerS6 mRNA was significantly elevated in breast cancer tissue compared to paired normal tissue, with approximately half of the individuals showing elevated CerS2 and CerS6 mRNA. A significant correlation was found between CerS2 and CerS6 expression, and between CerS4 and CerS2/CerS6 expression. Moreover, patients that expressed higher CerS2 or 4 mRNA levels tended to show no changes in sphingosine kinase 1 levels, and likewise patients that expressed no change in CerS2 or CerS4 mRNA levels tended to express higher levels of sphingosine kinase 1. Together these results suggest an important role for the CerS genes in breast cancer etiology or diagnosis.

Aromatase and in situ estrogen production in DCIS (ductal carcinoma in situ) of human breast.

Ductal carcinoma in situ or DCIS belongs to intraductal proliferative lesions, which are a group of cytologically and architecturally diverse ductal proliferations, typically originating from the terminal duct-lobular units. In these intraductal proliferative diseases, estrogens are considered to be involved in the progression of the disease especially from ductal non-neoplastic hyperplasia to DCIS and possibly development of invasive carcinoma from DCIS. Estrogen receptor (ER) alpha is abundantly expressed in atypical ductal hyperplasia and low grade DCIS. Suppression of estrogenic actions using tamoxifen resulted in inhibition of recurrence of DCIS and/or of progression into invasive carcinoma. Intratumoral estrogen concentration in DCIS determined by liquid chromatography/electrospray tandem mass spectrometry is significantly higher than that in non-neoplastic breast tissues with statistically not lower than that in invasive carcinoma. Aromatase mRNA expression in both stromal and parenchymal cells of DCIS determined by quantitative RT-PCR following laser capture microdissection was also much higher than that in non-neoplastic breast, although lower than that in invasive carcinoma. Immunohistochemistry of aromatase also revealed the similar patterns of immunolocalization as in invasive carcinoma. Aromatase is overexpressed in noninvasive breast malignancies including DCIS and results in elevated concentrations of intratumoral estradiol. These findings could provide the scientific rationale as to employing aromatase inhibitors in the management of ER positive DCIS patients.

Histamine and its metabolizing enzymes in tissues of primary ductal breast cancer.

OBJECTIVES: The aim of the study was to evaluate histamine concentrations in plasma and tissues of breast cancers depending on the activity of histamine metabolic enzymes in neoplasmatic tissues of the breast gland. MATERIAL AND METHODS: In 95 women aged 38-70 years the concentration of histamine in the plasma by the immunoenzymatic method, the concentration of histamine in breast cancer tissues and metabolism enzymes of histamine: histidine decarboxylase, decarboxylase of aromatic L-amino acid, N-histamine methyltransferase, monoamine oxydase B, diamine oxydase determined using an isotope technique were assessed. The 24-hour excretion of N-methylimidazolacetate acid was evaluated by the chromatography method. RESULTS: Significant increases were found of histamine concentrations in plasma tissues of ductal breast cancers, activity of histidine decarboxylase, aromatic L-amino acid and histamine methyltransferase. CONCLUSIONS: 1. Concentrations of histamine in plasma is dependent on the concentration of histamine in the tissues of ductal breast cancers. 2. Significant increases of histamine in cancerous tissues of ductal breast cancer could suggest the participation of this monoamine in the development of breast cancer. 3. The increase of histamine concentrations in ductal breast cancer tissues can be connected with disturbances in the balance between synthesis and enzymatic activation of this monoamine. 4. The concentration of histamine in plasma of women with ductal breast cancers is dependent on the number of lymph nodes and grade of histological malignancy.

Role of COX-2 in epithelial-stromal cell interactions and progression of ductal carcinoma in situ of the breast.

Epithelial-stromal cell interactions have an important role in breast tumor progression, but the molecular mechanisms underlying these effects are just beginning to be understood. We previously described that fibroblasts promote, whereas normal myoepithelial cells inhibit, the progression of ductal carcinoma in situ (DCIS) to invasive breast carcinomas by using a xenograft model of human DCIS. Here, we report that the tumor growth and progression-promoting effects of fibroblasts are at least in part due to increased COX-2 expression in tumor epithelial cells provoked by their interaction with fibroblasts. Up-regulation of COX-2 in DCIS xenografts resulted in increased VEGF and MMP14 expression, which may contribute to the larger weight and invasive histology of COX-2-expressing tumors. Administration of celecoxib, a selective COX-2 inhibitor, to tumor-bearing mice decreased xenograft tumor weight and inhibited progression to invasion. Coculture of fibroblasts with DCIS epithelial cells enhanced their motility and invasion, and this change was associated with increased MMP14 expression and MMP9 protease activity. We identified the NF-kappaB pathway as one of the mediators of stromal fibroblast-derived signals regulating COX-2 expression in tumor epithelial cells. Inhibition of NF-kappaB and COX-2 activity and down-regulation of MMP9 expression attenuated the invasion-promoting effects of fibroblasts. These findings support a role for COX-2 in promoting the progression of DCIS to invasive breast carcinomas, and suggest that therapeutic targeting of the NF-kappaB and prostaglandin signaling pathways might be used for the treatment and prevention of breast cancer.

17Beta-hydroxysteroid dehydrogenase type 12 in human breast carcinoma: a prognostic factor via potential regulation of fatty acid synthesis.

17beta-Hydroxysteroid dehydrogenase type 12 (17beta-HSD12) has been shown to be involved in elongation of very long chain fatty acid (VLCFA) as well as in biosynthesis of estradiol (E2). 17beta-HSD12 expression was also reported in breast carcinomas but its functions have remained unknown. In this study, we examined the correlation between mRNA expression profiles determined by microarray analysis and tissue E2 concentrations obtained from 16 postmenopausal breast carcinoma cases. No significant correlations were detected between 17beta-HSD12 expression and E2 concentration. We then immunolocalized this enzyme in 110 cases of invasive ductal carcinoma. 17beta-HSD12 immunoreactivity in breast carcinoma cells was significantly associated with poor prognosis of the patients. We further examined the biological significance of 17beta-HSD12 using cell-based studies. Small interfering RNA-mediated knockdown of 17beta-HSD12 in SK-BR-3 (estrogen receptor-negative breast carcinoma cell line) resulted in significant growth inhibition, which was recovered by the addition of VLCFAs such as arachidonic acid. The status of 17beta-HSD12 immunoreactivity was also correlated with adverse clinical outcome in cyclooxygenase 2 (COX2)-positive breast cancer patients but not in COX2-negative patients. Therefore, these findings indicated that 17beta-HSD12 was not necessarily related to intratumoral E2 biosynthesis, at least in human breast carcinoma, but was rather correlated with production of VLCFAs such as arachidonic acid, which may subsequently be metabolized to prostaglandins by COX2 and result in tumor progression of the patients.

Mel-18 negatively regulates INK4a/ARF-independent cell cycle progression via Akt inactivation in breast cancer.

Mel-18, a polycomb group (PcG) protein, has been suggested as a tumor suppressor in human breast cancer. Previously, we reported that Mel-18 has antiproliferative activity in breast cancer cells. However, its functional mechanism has not been fully elucidated. Here, we investigated the role of Mel-18 in human breast cancer. We saw an inverse correlation between Mel-18 and phospho-Akt, which were expressed at low and high levels, respectively, in primary breast tumor tissues from 40 breast cancer patients. The effect of Mel-18 on cell growth was examined in two breast cancer cell lines, SK-BR-3 and T-47D, which express relatively low and high levels of endogenous Mel-18, respectively. On Mel-18 overexpression in SK-BR-3 cells, cell growth was attenuated and G(1) arrest was observed. Likewise, suppression of Mel-18 by antisense expression in T-47D cells led to enhanced cell growth and accelerated G(1)-S phase transition. In these cells, cyclin-dependent kinase (Cdk)-4 and Cdk2 activities were affected by Mel-18, which were mediated by changes in cyclin D1 expression and p27(Kip1) phosphorylation at Thr(157), but not by INK4a/ARF genes. The changes were both dependent on the phosphatidylinositol 3-kinase/Akt signaling pathway. Akt phosphorylation at Ser(473) was reduced by Mel-18 overexpression in SK-BR-3 cells and enhanced by Mel-18 suppression in T-47D cells. Akt-mediated cytoplasmic localization of p27(Kip1) was inhibited by Mel-18 in SK-BR-3 cells. Moreover, Mel-18 overexpression showed reduced glycogen synthase kinase-3beta phosphorylation, beta-catenin nuclear localization, T-cell factor/lymphoid enhancer factor promoter activity, and cyclin D1 mRNA level. Taken together, we established a linear relationship between Mel-18-->Akt-->G(1) phase regulators.

Enhanced activation of epidermal growth factor receptor caused by tumor-derived E-cadherin mutations.

Mutations of the tumor suppressor E-cadherin and overexpression of the receptor tyrosine kinase epidermal growth factor receptor (EGFR) are among the most frequent genetic alterations associated with diffuse-type gastric carcinoma. Accumulating evidence suggests a functional relationship between E-cadherin and EGFR that regulates both proteins. We report that somatic mutation of E-cadherin is associated with increased activation of EGFR followed by enhanced recruitment of the downstream acting signaling components growth factor receptor binding protein 2 and Shc, and activation of Ras. Reduced complex formation of mutant E-cadherin - with an in frame deletion of exon 8 in the extracellular domain resulting in reduced adhesion and increased motility - with EGFR was observed compared with wild-type E-cadherin. We conclude that reduced binding of mutant E-cadherin to EGFR in a multicomponent complex or reduced stability of the complex may enhance EGFR surface motility, thereby facilitating EGFR dimerization and activation. Furthermore, reduced surface localization due to enhanced internalization of mutant E-cadherin compared with the wild-type protein was observed. The internalization of EGFR was decreased in response to epidermal growth factor stimulation in cells expressing mutant E-cadherin, suggesting that mutation of E-cadherin also influences the endocytosis of EGFR. Moreover, we show increased activation of EGFR in gastric carcinoma samples with mutant E-cadherin lacking exons 8 or 9. In summary, we describe activation of EGFR by mutant E-cadherin as a novel mechanism in tumor cells that explains the enhanced motility of tumor cells in the presence of an extracellular mutation of E-cadherin.

Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse.

An immunohistochemical study was performed using tissue microarrays and specific antibodies against matrix metalloproteinase (MMP)-1, -2, -7, -9, -11, -13 and -14, tissular inhibitors of metalloproteinase (TIMP)-1, -2 and -3. More than 2600 determinations on cancer specimens from 131 patients with primary ductal invasive tumours of the breast were performed. To identify specific groups of tumours with distinct expression profiles the data were analysed by unsupervised hierarchical cluster analysis by each cellular type. We did not find well-defined cluster of cases for tumour cells or fibroblastic cells. However, for mononuclear inflammatory cells the dendogram shows a first-order division of the tumours into two distinct MMP/TIMP molecular profiles, designated group 1 (n=89) and group 2 (n=42). Matrix metalloproteinase-7, -9, -11, -13 and -14, and TIMP-1 and -2, were identified as showing significant high expression in group 2 compared with group 1. Multivariate analysis demonstrated that clustering for mononuclear inflammatory cells was the most potent independent factor associated with distant relapse-free survival (group 2: 5.6 (3.5-9.6), P<0.001). We identify a phenotype of mononuclear inflammatory cells infiltrating tumours, which is associated with the development of distant metastasis. Therefore, this finding suggests that these host inflammatory cells could be a possible target for inhibition of metastasis.

Expression of nm23, MMP-2, TIMP-2 in breast neoplasm in Zhengzhou Center Hospital, China.

BACKGROUND: In recent years, the carcinoma of the breast threatens to women's health heavily. Invasion and metastasis is the main reason that results in the patients death. OBJECTIVE: A prospective study is made in the center hospital of zhengzhou, in order to approach the expression of nm23, MMP-2 (Matrix metallo-proteinase-2), TIMP-2 (it's tissue-inhibitor of the metalloproteinase-2) in the breast neoplasm and the relationship with invasion and metastasis. METHODOLOGY: This study applied the immunohistochemistry technique SP method RESULTS: In fibroadenoma, ductal carcinoma in situ and invasive ductal carcinoma of the breast 4 groups, the positive immunostaining rate of nm23, MMP-2 and TIMP-2 have significant difference among 4 groups (p < 0.05). In the breast invasive ductal carcinoma, the expression of nm23 and TIMP-2 decreased or the expression of MMP-2 increased CONCLUSION: This suggested that invasion and metastasis is ability of the neoplasm. MMP-2 in the breast ductal carcinoma in situ appears of high expression and this suggested that the positive expression of this onco-proteins was the early incident in the genetic course of the breast cancer The unite detection of nm23, MMP-2 and TIMP-2 expression would contribute to the early diagnosis and prognostic assessment of the carcinoma of the breast.

Study of matrix metalloproteinases and their inhibitors in breast cancer.

An immunohistochemical study was performed using tissue microarrays and specific antibodies against matrix metalloproteinases (MMPs) 1, 2, 7, 9, 11, 13, 14, and their tisullar inhibitors (TIMPs) 1, 2, and 3. More than 2600 determinations on cancer specimens from 131 patients with primary ductal invasive tumours of the breast (65 with and 66 without distant metastasis) and controls were performed. Staining results were categorised using a score based on the intensity of the staining and a specific software program calculated the percentage of immunostained cells automatically. We observed a broad variation of the total immunostaining scores and the cell type expressing each protein. There were multiple and significant associations between the expression of the different MMPs and TIMPs evaluated and some parameters indicative of tumour aggressiveness, such as large tumour size, advanced tumour grade, high Nottinham prognostic index, negative oestrogen receptor status, peritumoural inflammation, desmoplastic reaction, and infiltrating tumoural edge. Likewise, the detection of elevated immunohistochemical scores for MMP-9, 11, TIMP-1, and TIMP-2, was significantly associated with a higher rate of distant metastases. The expression of MMP-9 or TIMP-2 by tumour cells, MMP-1, 7, 9, 11, 13, or TIMP-3 by fibroblastic cells, and MMP-7, 9, 11, 13, 14, TIMP-1, or TIMP-2 by mononuclear inflammatory cells, was also significantly associated with a higher rate of distant metastases.

Cyclooxygenase-2 expression in brain metastases.

BACKGROUND: Elevated Cyclooxygenase-2 (COX-2) expression is thought to increase metastatic potential of many tumors. Furthermore, elevated COX-2 expression correlates with radiation resistance in many tumor types. We evaluated whether: (i) the degree of COX-2 expression correlated with either metastatic tumor type or with the presence of necrosis and whether (ii) radiation-resistant tumors (renal cell and melanoma) had higher expression of COX-2 than did relatively radiation-sensitive tumors (breast and lung). MATERIALS AND METHODS: Specimens from sixteen patients who underwent resection of brain metastases were analyzed for COX-2 expression using a COX-2 antibody-based immunoassay. Specimens consisted of brain metastases from lung tumors, breast adenocarcinomas, melanomas and renal cell carcinomas. All specimens were analyzed for the presence or absence of necrosis. RESULTS: Ten of sixteen brain metastasis specimens had ten percent or less Cox-2 immunostaining. Statistical analyses showed no correlation between Cox-2 immunostaining and metastatic tumor type or between Cox-2 immunostaining and necrosis in this study. Furthermore, renal cell carcinoma and melanoma showed variable Cox-2 immunostaining. CONCLUSION: Cox-2 is not consistently expressed in metastases to the brain. The degree of Cox-2 expression does not correlate with metastatic tumor type or with the presence of necrosis. Radioresistant tumors did not have statistically different expression of Cox-2 than radiosensitive specimens studied in this analysis.

Altered expression of the hormone- and xenobiotic-metabolizing sulfotransferase enzymes 1A2 and 1C1 in malignant breast tissue.

Cytosolic sulfotransferases (SULTs) catalyze the biotransformation of steroid hormones as well as drugs and environmental toxins. Mostly, sulfonation leads to an inactivation of parent compounds, although formation of more toxic and cancerogenic metabolites also occurs. To assess possible alterations in the SULT enzyme expression pattern between malignant and non-malignant tissue, we studied the presence of 9 SULT enzymes of family 1 and 2 by semi-quantitative RT-PCR. Forty-two specimens from ductal and lobular breast carcinomas, lymph node metastasis, mastopathy and normal breast tissue were derived from 29 patients. Substantial expression of SULT 1A1, 1A2, 1A3, 1B1, 1C1, 1E1, 2A1, 2B1a and 2B1b mRNAs was observed in malignant and non-malignant tissue, although the pattern of the individual SULTs varied between the patients, and SULT1C1 mRNA was present in a greater number of malignant than non-malignant tissues (p<0.05). A major finding was that unspliced SULT1A2 mRNA, containing the complete intron between exons 7 and 8, was found in 4 of 16 non-malignant specimens, but was undetectable in the 26 malignant samples investigated. Taken together, the presence of various SULT enzymes in normal, premalignant and malignant breast tissue suggests an important role of SULT-mediated biotransformation in the breast. While the increased expression of SULT1C1 in malignant tissue seems to reflect tumor dedifferentiation, our finding of unspliced SULT1A2 mRNA in non-malignant tissue offers additional aspects regarding the search for breast cancer risk factors.