A novel role for 3, 4-dichloropropionanilide (DCPA) in the inhibition of prostate cancer cell migration,proliferation, and hypoxia-inducible factor 1alpha expression

Background

Thrombospondin-1 (TSP-1) is an extracellular matrix component glycoprotein, which is known to be a potent inhibitor of angiogenesis and may be important in cancer invasiveness. We examined the TSP-1 expression in correlation with conventional clinicopathological parameters to clarify its prognostic significance in bladder cancer. In addition, the possible correlation of TSP-1 expression with microvessel count, VEGF expression, p53 expression as well as with the expression of the extracellular matrix components was studied to explore its implication in vascularization and tumour stroma remodeling.

Methods

The immunohistochemical expression of TSP-1 in tumour cells and in the tumour stroma was studied in 148 formalin-fixed paraffin-embedded urothelial cell carcinoma tissue samples.

Results

TSP-1 was detected in perivascular tissue, at the epithelial-stromal junction, in the stroma and in tumour cells in the majority of the cases. In tumour cells, low TSP-1 expression was observed in 43% of the cases, moderate and high in 7%, while 50% showed absence of TSP expression. A higher TSP-1 immunoreactivity in well and moderately differentiated tumours compared to poorly differentiated was noted. PT₁ tumours showed decreased TSP-1 expression in comparison to pTa and pT_2–4 tumours. Increased tumour cell TSP-1 expression was related to increased microvessel density. In the tumour stroma, 37% of the cases showed small amount of TSP-1 expression, 7.5% moderate and high, while 55% of the cases showed absence of TSP-1 stromal immunoreactivity. Stromal TSP-1 expression was inversely correlated with tumour stage and tumour size. This expression was also positively correlated with microvessel density, VEGF expression and extracellular matrix components tenascin and fibronectin. Using univariate and multivariate analysis we didn't find any significant correlation of TSP-1 expression in superficial tumours in both tumour cells and tumour stroma in terns of the risk of recurrence and disease progression

Conclusion

Our data suggest that both tumour and stromal TSP-1 expression may play a role in tumour aggressiveness and angiogenesis. In addition, the correlation of stromal TSP-1 expression with extracellular matrix components fibronectin and tenascin indicate its possible implication in tumour stroma remodeling.     

Expression of tumour necrosis factor (TNFα) and its receptors in benign and malignant breast tissue

As well as having anti-tumour activity in animal models, TNFα has been implicated in the promotion of tumour invasion and metastasis. Expression and localization of TNFα mRNA and protein has been investigated in a series of benign and malignant breast tissues. TNFα mRNA was expressed in a minority of cells (≤0.5%) in 4/11 cases of benign breast tissue and in a higher proportion of invasive carcinomas (43/49 cases). Expression of TNFα mRNA was focal and confined to the tumour stroma. The presence of TNFα protein was confirmed by immunohistochemistry and again was predominantly stromal. Expression of mRNA and protein localized to infiltrating macrophages defined by the antibody EBM/11 in adjacent sections and using 2-colour immunofluoresence. Although the average number of macrophages per high-power field did not vary among invasive carcinomas of different histological type or grade, the number of cells expressing TNFα increased with increasing tumour grade. Immunodetectabfe p75 TNF receptor and to a lesser extent p55 receptor was present on cells of the mononuclear infiltrate and on the endothelium of invasive carcinomas, but not on the tumour cells themselves.     

Presence of endothelial calcium-dependent nitric oxide synthase in breast apocrine metaplasia.

Endothelial calcium-dependent nitric oxide (NO) synthase has been shown to be expressed in human malignant breast tumours, and its presence correlates with tumour grade. Moreover, NO, being synthesised in breast tumour cells, may increase tumour blood flow and promote angiogenesis. In view of these aspects, we have assessed the distribution of NO synthase within a series of benign breast tumours using a monoclonal antibody against human endothelial calcium-dependent NO synthase. Activity was predominantly localised in apocrine metaplastic cells of fibrocystic disease, as well as in endothelia throughout all tissue sections. Consistent with previous reports, no endothelial calcium-dependent NO synthase immunoreactivity was observed in poorly differentiated infiltrating duct carcinoma cells. In conclusion, expression of endothelial calcium-dependent NO synthase in human breast apocrine metaplasia may be of significance in view of the NO''s vascular effects in benign breast disease.     

Prognostic value of syndecan-1 expression in breast cancer

OBJECTIVE: Syndecan-1 is a cell surface heparan sulphate proteoglycan which participates in cell proliferation, cell migration and cell-matrix interactions. Epithelial syndecan-1 expression is reduced in several malignant tumours, but in breast and pancreatic cancer, increased expression has also been described. Loss of epithelial syndecan-1 has been associated with poor prognosis in some forms of cancer, but previous findings in breast cancer have been contradictory. The objective of this study was to evaluate the prognostic value of the immunohistochemical expression of syndecan-1 in a series of 200 patients with invasive breast cancer with a median follow-up of 17 years. METHODS: Formalin-fixed paraffin-embedded specimens were stained using a monoclonal antibody against syndecan-1. RESULTS: Syndecan-1 was expressed in the epithelium in 61% and in the stroma in 67% of the tumours. Epithelial syndecan-1 expression was associated with negative oestrogen receptor (ER) status (p < 0.01), and stromal syndecan-1 expression with positive ER status (p = 0.02). The breast cancer-specific 10-year overall survival for patients with epithelial syndecan-1 expression was 65%, compared with 82% for those with loss of epithelial expression (p = 0.02). Ten-year survival was 66% for those expressing stromal syndecan-1 and 83% for those lacking stromal expression (p = 0.15). Patients with both epithelial and stromal expression had a 10-year survival of only 56%, compared to 78% in patients with other expression pattern combinations (p < 0.002). In Cox multivariate analysis, only axillary involvement and tumour size were significant predictors of breast cancer-specific survival. CONCLUSION: Concomitant expression of syndecan-1 in both epithelium and stroma may be a predictor of unfavourable prognosis in breast cancer, and in contrast with previous studies, loss of epithelial syndecan-1 was associated with a more favourable prognosis.     

Expression of hyaluronan in benign and malignant breast lesions

Hyaluronan (HA) is one of the extracellular-matrix components involved in wound healing, tumour growth and metastasis. Due to the limited data on HA expression in benign and malignant breast lesions, we analyzed its presence in these lesions by using the biotinylated-hyaluronan-binding region and the link-protein complex (bHABC) of cartilage proteoglycan as a specific probe. In all benign breast lesions, the expression of HA was restricted to the stromal connective tissue, the ductal epithelial cells being completely devoid of HA. In malignant breast tumours, the intensity of stromal HA staining was significantly stronger than in benign lesions. In addition, HA was detected on cell membranes or in cytoplasms of adenocarcinoma cells, in some cases of ductal carcinoma in situ and in 31% of malignant tumours. The staining pattern was mostly similar in all breast-cancer types studied, i.e., ductal, lobular, tubular, mucinous and medullary. In ductal breast cancer, intense HA expression in stroma and carcinoma cells correlated statistically significantly to poor differentation of carcinoma, suggesting that altered HA expression may affect the mechanisms of breast-cancer progression. Int. J. Cancer 74:477–481, 1997. © 1997 Wiley-Liss, Inc.     

Stromal influences on breast cancer cell growth.

Paracrine influences from fibroblasts derived from different sources of breast tissue on epithelial breast cancer cell growth in vitro were investigated. Medium conditioned (CM) by fibroblasts derived from tumours, adjacent normal breast tissue, and normal breast tissue obtained from reduction mammoplasty or from skin tissue significantly stimulated the growth of the steroid-receptor positive cell lines MCF-7 and ZR 75.1. The proliferation index (PI) on MCF-7 cells with CM from fibroblasts derived from breast tumour tissue was significantly higher than that obtained with fibroblasts derived from adjacent normal breast tissue (2p less than 0.05, n = 8). The PI obtained with CM from normal fibroblast cultures from reduction mammoplasty tissue, like normal tissue adjacent to the tumour, fell in the lower range of values. Skin fibroblast, like tumour tissue derived fibroblast, CM caused a high range PI. MDA-MB-231 and Evsa-T, two steroid-receptor negative cell lines, showed only a minor growth stimulatory responses with some of the fibroblast CM's. Evsa-T was occasionally inhibited by CM's. In conclusion, stromal factors play a role in the growth regulation of human breast cancer cells. The effects on cancer cell growth are, however, varying depending on the source of the stroma and the characteristics of the epithelial tumour cells.     

Correlation between basic fibroblast growth factor immunostaining of stromal cells and stromelysin-3 mRNA expression in human breast carcinoma.

We examined the localization of basic fibroblast growth factor (bFGF) in a series of human breast carcinomas using immunohistochemistry. Staining was observed in tumour cells in 15 out of 54 (28%) tumours and in the adjacent stroma in 34 out of 54 (63%) tumours examined. No correlation was observed between positive staining of these two compartments. The relationship between bFGF staining and expression of the metalloprotease stromelysin-3, and between bFGF and microvessel density, was examined. A statistically significant correlation (P < 0.003) was observed between bFGF staining of the stromal compartment and high expression of stromelysin-3 (ST-3; MMP-11) metalloprotease mRNA by stromal cells. In contrast, no correlation was observed between bFGF and intratumour microvessel density (IMD). These results raise the possibility that bFGF may be involved in the induction of stromelysin-3 mRNA expression in breast cancer stroma.     

Tissue factor expression in breast cancer tissues: its correlation with prognosis and plasma concentration

Tissue factor (TF), an initiator of the extrinsic coagulation cascade, is expressed in a wide range of cancer cells and plays important roles in cancer progression and metastasis. Recently, the intracellular function of TF has been revealed to be involved in cancer invasion, independent of the blood coagulation pathway. To evaluate the clinical significance of TF expression, we performed an enzyme-linked immunosorbent assay (ELISA) in the plasma of 67 breast cancer patients and immunohistochemistry in 213 breast cancer tissues. In the ELISA study, we showed an up-regulation of plasma TF concentration in breast cancer patients compared with normal controls. Immunohistochemistry demonstrated that TF was expressed in tumour cells and stromal cells and tumour TF expression closely correlated with stromal TF expression (P = 0.0005). The concentration of plasma TF was associated with tissue TF expression in both tumour and stroma. The multivariate analysis demonstrated that tumour TF expression was an independent prognostic indicator for overall survival (P = 0.0452). Our data show that plasma TF concentration reflects tissue TF expression and tumour TF expression can provide some predictive value for prognosis and distant metastasis, which indicates the importance of TF function in tumour progression.     

Tumour-stromal interactions in breast cancer: the role of stroma in tumourigenesis.

Mammary stromal tissue has a major role in the control and regulation of physiological processes in the breast. Recently, the function of stroma in supporting the tumourigenic process as well as responding to the oncogenic lesion has become clearer. This review differs from the conventional view in that it focuses on and discusses the newly available evidence that points to the fact that mammary stroma has a significant contribution in actively generating transformed lesions and tumours. As such, the oncogenic signals can be dependent or independent of genetic mutations in mammary stromal cells. As a supportive and responsive agent in tumourigenesis, the stroma is induced by tumour cells to express critical signals that drive proliferation, angiogenesis, and motility while suppressing cell death. As an oncogenic agent in tumourigenesis, the stroma can provoke tumourigenicity in adjacent cells in the absence of pre-existing tumour cells leading to the acquisition of genomic changes. Investigating the mechanism by which the tumourigenic cues of the stroma facilitate the generation of malignant epithelial cells will provide invaluable insights into the oncogenic process.     

Expression of cyclooxygenase-2 (COX-2) in tumour and stroma compartments in cervical cancer: clinical implications

This study aims at investigating the relationship between cyclooxygenase-2 expression in tumour vs stroma inflammatory compartment and its possible clinical role. The study included 99 stage IB-IV cervical cancer patients: immunostaining of tumour tissue sections was performed with rabbit antiserum against cyclooxygenase-2. CD3, CD4, CD8, CD25, Mast Cell Tryptase monoclonal antibodies were used to characterise stroma inflammatory cells in nine cervical tumours. An inverse relation was found between cyclooxygenase-2 levels (cyclooxygenase-2 IDV) of tumour vs stroma compartment (r=-0.44, P<0.0001). The percentage of cases showing high tumour/stromal cyclooxygenase-2 IDV ratio was significantly higher in patients who did not respond to treatment (93.3%) with respect to patients with partial (60.5%), and complete (43.7%) response (P= 0.009). Cases with a high tumour/stroma cyclooxygenase-2 IDV ratio had a shorter overall survival rate than cases with a low tumour/stroma cyclooxygenase-2 IDV (P<0.0001). In the multivariate analysis advanced stage and the status of tumour/stroma cyclooxygenase-2 IDV ratio retained an independent negative prognostic role. The proportion of CD3(+), CD4(+), and CD25(+) cells was significantly lower in tumours with high tumour/stroma cyclooxygenase-2 IDV ratio, while a higher percentage of mast cells was detected in tumours showing high tumour/stroma cyclooxygenase-2 IDV ratio. Our study showed the usefulness of assessing cyclooxygenase-2 status both in tumour and stroma compartment in order to identify cervical cancer patients endowed with a very poor chance of response to neoadjuvant therapy and unfavourable prognosis.     

A comparative study of cytokine gene transcripts in normal and malignant breast tissue and primary cell cultures derived from the same tissue samples

Using a differential centrifugation method followed by culture in selective medium, we have successfully isolated and maintained individual epithelial and stromal cells from normal (n = 10) and malignant (n = 6) human breast tissue and characterised their phenotype by immunocytochemistry. Further, we have studied expression of the cytokine genes IL-1β, IL-6, IL-8 and TNF-β in each cell fraction by RT-PCR and have compared these results with cytokine gene expression in tissue extracts from which primary cultures were derived. In breast tumours, there was near complete absence of IL-1β in both whole tissue and cell fractions, and in normals it was present in only 3/10 tissue preparations, with increased expression in stromal (6/10) and epithelial (5/10) cell samples. IL-6 was constitutively expressed in all tumour-derived breast tissue samples but down-regulated in tumour cell cultures, with the opposite result in normal breast. Near identical levels of IL-8 expression were found throughout each preparation, irrespective of tissue origin. TNF-β was expressed in all normal tissue samples, in 9/10 epithelial preparations but in only 6/10 stromal preparations. In tumours, TNF-β was associated predominantly with whole tissue or stromal samples, with reduced expression in epithelial preparations. Our data confirm that primary cultures of normal and malignant human breast tissue can be successfully separated into epithelial and mesenchymal cell populations and their phenotype can be maintained in culture for up to 30 days. However, this cellular separation does alter the cytokine profiles; therefore, experimental findings with isolated cells should be treated with a caveat. © 1996 Wiley-Liss, Inc.     

Increased expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product P16 INK4A in ovarian cancer is associated with progression and unfavourable prognosis

Paraffin sections from 190 epithelial ovarian tumours, including 159 malignant and 31 benign epithelial tumours, were analysed immunohistochemically for expression of cyclin-dependent kinase inhibitor 2 (CDKN2A) gene product p16^INK4A (p16). Most benign tumours showed no p16 expression in the tumour cells, whereas only 11% of malignant cancers were p16 negative. A high proportion of p16-positive tumour cells was associated with advanced stage and grade, and with poor prognosis in cancer patients. For FIGO stage 1 tumours, a high proportion of p16-positive tumour cells was associated with poorer survival, suggesting that accumulation of p16 is an early event of ovarian tumorigenesis. In contrast to tumour cells, high expression of p16 in the surrounding stromal cells was not associated with the stage and grade, but was associated with longer survival. When all parameters were combined in multivariate analysis, high p16 expression in stromal cells was not an independent predictor for survival, indicating that low p16 expression in stromal cells is associated with other markers of tumour progression. High expression of p16 survival in the stromal cells of tumours from long-term survivors suggests that tumour growth is limited to some extent by factors associated with p16 expression in the matrix. Int. J. Cancer 74:57–63. © 1997 Wiley-Liss, Inc.     

Expression of CYP3A4 in human breast tumour and non-tumour tissues

Cytochrome P450 3A4 (CYP3A4), a member of multigene superfamily of enzymes, plays a major role in the activation of procarcinogens such as polycyclic hydrocarbon dihydrodiols, aflatoxins and heterocyclic amines as well as of several drugs including tamoxifen which is used in breast cancer therapy. Using immunohistochemistry, the cellular distribution and the level of expression of CYP3A4 was assessed in breast tumour and surrounding tumour free (control) breast tissue in 25 pairs of samples obtained from females with infiltrating ductal carcinoma. Cells staining with the CYP3A4 antibody showed only cytoplasmic positivity with varying intensities in 100% (25/25) of tumours and 68% (17/25) of normal breast tissues. Cytoplasmic staining was present in invasive ductal carcinoma cells but not in tumour stroma. CYP3A4 was detected in normal epithelium and myoepithelium. Only focal staining was noted in some of the non-epithelial cells (e.g. endothelial cells) in normal breast tissues. When CYP3A4 staining was assessed semiquantitatively the mean staining score values of CYP3A4 were found to be significantly higher in tumours compared to that of normal breast tissues. These results show that CYP3A4 protein is expressed in both tumour and normal breast tissue with an increased expression in tumours.     

Prognostic significance of the c-erbB-2 oncogene product in childhood medulloblastoma.

The expression and prognostic significance of the c-erbB-2 oncogene product was studied in 55 cases of childhood medulloblastoma. Forty-six of the 55 tumours (83.6%) expressed the c-erbB-2 product. The percentage of tumour cells expressing the c-erbB-2 product proved to be a significant indicator of patient outcome when analysed as both a categorical and a continuous variable. As a categorical variable, patients with more than 50% positive tumour cells had a significantly worse survival, with only 10% alive at 10 years vs 48% for those with less than 50% positive tumour cells (log rank P = 0.0049). To demonstrate that this observed prognostic significance was both independent and not a result of ''data-driven'' categorisation, it was also entered into the Cox model as a continuous variable. Prognostic significance was retained in P = 0.038.     

Investigation of mammary epithelial cell-bone marrow stroma interactions using primary human cell culture as a model of metastasis

A model has been established using primary human cell culture to study the cell biology of breast cancer metastasis to bone marrow. Mammary epithelia were obtained in single cell suspension from tumour (macroscopically involved), benign (macroscopically uninvolved) and normal (reduction mammoplasty) breast tissue as well as from locally involved lymph nodes. Stromal layers were generated from long-term cultures of human bone marrow or from mammary fibroblasts derived from normal or malignant tissue. The interaction between epithelia and stroma has been studied in terms of adhesion of the epithelia to the stroma and their subsequent growth in co-culture. Our results show that when assayed up to 9 hr after plating, epithelial cells from malignant tissue (14 primary tumours and 9 metastases in lymph nodes) displayed a significant preference for adhesion to bone marrow stroma compared with mammary fibroblasts. In contrast, epithelial cells from 4 normal and 2 of 4 benign samples showed no significant preferential adherence. Subsequent co-culture of mammary epithelia with each of the 3 stromal layers revealed that under serum-free, in vitro conditions, bone marrow stromal layers did not provide an advantageous environment for colony growth, in contrast to their ability to provide a preferential substratum for adhesion. Int. J. Cancer 73:690–696, 1997. © 1997 Wiley-Liss, Inc.     

Association of tumour necrosis factor alpha and its receptors with thymidine phosphorylase expression in invasive breast carcinoma.

Angiogenesis is an essential requirement for tumour growth and metastasis and is regulated by a complex network of factors produced by both stromal cells and neoplastic cells within solid tumours. The cytokine tumour necrosis factor alpha (TNF-alpha) and the enzyme thymidine phosphorylase (TP) are two factors known to promote tumour angiogenesis. We have demonstrated recently that high numbers of tumour-associated macrophages (TAMs) are significantly associated with increased tumour angiogenesis and poor prognosis in invasive carcinoma of the breast. We have also shown that TAMs are a major source of TNF-alpha in invasive breast carcinomas, and that macrophage-like stromal cells as well as tumour cells synthesize TP in such tumours. However, little is known of the factors that regulate the production or activity of these factors in the tumour microenvironment. As TNF-alpha has been shown to up-regulate TP expression in tumour cells in vitro we performed an immunohistochemical study to investigate the possibility that TNF-alpha may be involved in the regulation of TP expression by malignant breast epithelial cells in vivo. To do this, we used a cocktail of non-neutralizing monoclonal anti-TNF-alpha antibodies to visualize both TNF-alpha-expressing macrophages and TNF-alpha bound to its receptors on tumour cells and endothelial cells in a series of 93 invasive carcinomas of the breast. A semiquantitative grading system was then used to compare these staining patterns with that for TP in the same biopsies. TNF-alpha immunoreactivity was also compared with various important tumour variables known to relate to outcome in this disease (microvessel density, node status, grade, stage, receptor status and macrophage infiltration), as well as relapse-free and overall survival data for these patients. Our data show significant positive correlations between TNF-alpha bound to its receptors on tumour cells and: (1) TP protein production by tumour cells, and (2) axillary lymph node status (i.e. metastasis). These results suggest that tumour cell responsiveness to TNF-alpha produced by neighbouring TAMs may play a part in the regulation of TP expression by tumour cells as well as their metastatic behaviour. This may explain, in part, the relationship between increased macrophage infiltration and angiogenesis in breast cancer, and further supports the contention that TAMs may represent an important target for future anti-angiogenic therapies.     

Possible alternative carcinogenesis pathway featuring microsatellite instability in colorectal cancer stroma

Differential microsatellite instability (MSI) in tumour epithelial and stromal compartments has not been well examined for colorectal cancers. Using laser-captured microdissection, separate specimens of these compartments of 40 sporadic colorectal cancers were sampled and MSI was tested with four markers. To examine the relation between the MSI phenotype in the stroma and other genetic events and histopathological features, p53 and K-ras gene mutations were analysed, and the expression of p53, hMLH1, and hMSH2 protein was determined by immunohistochemistry. Microsatellite instability positive results were obtained for both epithelium (34%) and stromal tissue (41%). While MSI in epithelium correlated with differentiation and Dukes' stage, that in stroma demonstrated an inverse relation, being particularly frequent in well-differentiated adenocarcinomas (54%) and Dukes' A lesions (55%). Further, a significant inverse correlation between p53 protein overexpression in the epithelium and MSI in the stroma was found (P=0.02475). The results suggest an alternative pathway of carcinogenesis involving stromal genetic instability in the development of colorectal cancers.     

Infiltration of mononuclear inflammatory cells into primary colorectal carcinomas: an immunohistological analysis.

Local immunoregulation mediated by mononuclear tumour-infiltrating cells is considered of importance for tumour progression of colorectal cancer, although the balance between immunosuppressor and cytotoxic activities is unclear. Colorectal cancers from 26 patients were investigated using a panel of monoclonal antibodies in order to identify subsets of mononuclear inflammatory cells and to study their pattern of distribution in relation to tumour stage and cytotoxic immune reactivity against the tumour. In all but five tumours, mononuclear cells, lymphocytes or monocytes were present in fairly large numbers, particularly in the stroma. The infiltration of CD4+ mononuclear cells predominated over the CD8+ subset. Infiltration near the tumour cells was found in four cancers only. Stromal infiltration of CD11c+ macrophages was found in all but eight tumours. Small regressive areas, in which the histological architecture of the tumours was broken down, were found in 17 tumours with intense or moderate infiltration by CD4+ lymphocytes or CD11c+ macrophages. Probably this destruction of tumour tissue was caused by cytotoxic activity of the tumour-infiltrating mononuclear cells. In Dukes'' class A and B tumours, CD4+ lymphocytes predominated over CD4+ cells with macrophage morphology, but the latter were increasingly found in Dukes'' class C and D disease. The occurrence of MHC II-positive macrophages and lymphocytes in different Dukes'' classes was similar to that of CD4+ cells. In contrast to this, CD11c+ and CD11a+ cells were more frequent in Dukes'' A and B class tumours compared with Dukes'' C and D. Four out of nine tumours of the latter stages showed a poor inflammatory reaction. The interpretation of our results is that the subsets of tumour-infiltrating mononuclear cells change with advancing Dukes'' class and that the local immune control is gradually broken down in progressive tumour growth, even if some cytotoxic activity is still present.