Proteolysis of extracellular matrix by invadopodiafacilitates human breast cancer cell invasion and ismediated by matrix metalloproteinases

Breast cancer cell lines vary in invasive behavior and one highly invasive cell line (MDA-MB-231)proteolytically degrades extracellular matrix with invadopodia (Thompson et al. 1992, J Cell Physiol, 150,534-44; Chen et al. 1994, Breast Cancer Res Treat, 31, 217-26). Invadopodial proteolysis of extracellularmatrix is thought to be necessary for invasion; however, this has not been demonstrated directly. To obtainsuch evidence, normal (HBL-100) and malignant (MCF-7, MDA-MB-231) breast cells were evaluated forinvadopodial proteolysis of extracellular matrix and invasive behavior. We report that invadopodial prote-olysisof immobilized fibronectin is positively correlated with invasion of cells into type I collagen gels.Moreover, reducing the proteolytic activity of invadopodia with the metalloproteinase inhibitor, batimastat(BB-94), also decreases invasion indicating that breast cancer cell invasion is dependent upon proteolyti-callyactive invadopodia. ©Kluwer Academic Publishers     

Proteolysis in colorectal cancer.

BACKGROUND: The process of metastasis is complex, involving many interrelated stages, including proteolysis. Proteolysis occurs in both normal and pathological processes and involves the breakdown of the extracellular matrix and/or basement membrane by proteolytic enzymes. Normally, proteolysis is tightly controlled by specific endogenous proteinase inhibitors. However, in certain disease processes, including cancer, controlled but abnormal proteolysis seems to occur. Proteinases involved in tumour invasion and metastasis include the matrix metalloproteinases (MMPs) and the serine proteinases. AIMS: To gain a greater understanding of the proteolytic process occurring in colorectal cancer and to determine which, if any, proteinases are upregulated. METHODS: The synthesis of proteinases and their inhibitors was compared in paired tumour and normal tissue samples from patients with colorectal cancer (n = 24). Substrate zymography was used to determine the synthesis of MMPs (MMP-2, MMP-9, and MMP-3) and the plasminogen activators (urokinase and tissue-type plasminogen activators); enzyme linked immunosorbent assays (ELISAs) were used to determine the concentrations of MMP-1 and tissue inhibitor of metalloproteinase 1 (TIMP-1); and the technique of quenched fluorescence substrate hydrolysis was performed to determine the total MMP activity of each sample. RESULTS: In general, both proteinase and inhibitor expression was greater in the tumour tissue when compared with the corresponding normal colorectal tissue. The amount of active MMPs was greater in the tumour tissue. CONCLUSIONS: The increased extracellular proteinase concentrations and activity may encourage tumour invasion and metastasis. This study points to MMP-9 as being of potential major importance in the development of this form of cancer.     

Proteolysis in colorectal cancer.

BACKGROUND: The process of metastasis is complex, involving many interrelated stages, including proteolysis. Proteolysis occurs in both normal and pathological processes and involves the breakdown of the extracellular matrix and/or basement membrane by proteolytic enzymes. Normally, proteolysis is tightly controlled by specific endogenous proteinase inhibitors. However, in certain disease processes, including cancer, controlled but abnormal proteolysis seems to occur. Proteinases involved in tumour invasion and metastasis include the matrix metalloproteinases (MMPs) and the serine proteinases. AIMS: To gain a greater understanding of the proteolytic process occurring in colorectal cancer and to determine which, if any, proteinases are upregulated. METHODS: The synthesis of proteinases and their inhibitors was compared in paired tumour and normal tissue samples from patients with colorectal cancer (n = 24). Substrate zymography was used to determine the synthesis of MMPs (MMP-2, MMP-9, and MMP-3) and the plasminogen activators (urokinase and tissue-type plasminogen activators); enzyme linked immunosorbent assays (ELISAs) were used to determine the concentrations of MMP-1 and tissue inhibitor of metalloproteinase 1 (TIMP-1); and the technique of quenched fluorescence substrate hydrolysis was performed to determine the total MMP activity of each sample. RESULTS: In general, both proteinase and inhibitor expression was greater in the tumour tissue when compared with the corresponding normal colorectal tissue. The amount of active MMPs was greater in the tumour tissue. CONCLUSIONS: The increased extracellular proteinase concentrations and activity may encourage tumour invasion and metastasis. This study points to MMP-9 as being of potential major importance in the development of this form of cancer.     

Oestrogen receptors in human breast cancer

Summary Histopathological factors which might explain inconsistency in published data attempting to correlate oestrogen receptor content (ER) and pathological features in primary breast tumours have been investigated in 194 cases. It was found, that unequal assessment of tumour type and of histological grading between observers is one important factor. In terms of grading, however, heterogeneity of growth pattern within the same tumour seems to be of greater significance. No significant correlation was found between histological type of tumour and ER content. However, a trend towards a correlation between the extent of tubule formation (as an indication of differentiation) and ER content was observed.Sponsored by The Danish Cancer Society     

Clonal origins of human breast cancer

Tumours are usually considered as the clonal progeny of single transformed cells. An X-chromosome inactivation assay has been applied to exploring clonal relationships in human breast cancer. Analysis of X-inactivation in DNA extracted from microdissected in situ and invasive breast carcinoma by Hpa II restriction and polymerase chain reaction (PCR) of the androgen receptor exon I CAG polymorphism confirmed monoclonality in 105/133 samples of carcinoma cells from 31/32 informative breast cancers. Clonality was identical in seven cases between in situ and invasive carcinoma. Unexpectedly, 4 of 12 cancers (33%) with two or more monoclonal samples available were mosaic (polyclonal) in respect of X-chromosome inactivation between separate morphologically homogeneous tumour cell samples. Concordant clonality supports a common clonal origin of in situ and invasive breast cancers, but frequent apparently mosaic X-inactivation in breast cancer cannot be explained by non-tumour cell contamination. It is concluded that these carcinomas may be genuinely multiclonal. Possible mechanisms of multiclonality include simultaneous transformation of cell groups straddling X-chromosome inactivation patch boundaries, tumour-initiating mutations prior to X-inactivation, or recruitment of bystander stem cells by DNA transfer from necrotic or apoptotic tumour cells. Collision of independent cancers appears implausible at this frequency. Further studies using independent analytical techniques are required to test the important possibility that a significant proportion of mammary carcinomas are not monoclonal.     

MMTV-like env gene sequences in human breast cancer

Summary.  We have previously detected an MMTV env gene-like 660 bp sequence in 38% of human breast cancers, but not in normal tissues or other tumors. In this communication we report the sequences from eleven tumors and three breast cancer cell lines, and compare them to four strains of MMTV and to the known endogenous retroviral sequences. The breast cancer sequences were highly homogenous to the MMTV’s, but not to the endogenous sequences suggesting an exogenous origin. Received January 11, 2000 Accepted July 3, 2000     

Immunoreactive opioid peptides in human breast cancer.

Opioid peptides have a variety of actions on inter alia pituitary hormone secretion and the immune system. Release of endogenous opioids has been found to stimulate growth of experimental breast cancers and opiate receptor blockers have reduced the growth of chemically induced rat breast tumors. Opioid peptides may therefore play a role in human breast cancer. Invasive ductal carcinomas from 61 premenopausal women were immunocytochemically analyzed for the presence of opioid peptide immunoreactivity. Positive staining was unambiguously identified in 34 of the tumors (56%). In addition, a medullary carcinoma was positive. In a smaller series of tumors, opioid peptide immunoreactive cells were detected in both primary tumors and metastases. Positive tumor cells were usually few and scattered. Therefore, underestimates of their true frequency of occurrence are likely to have occurred, making accurate correlations with clinical behavior and estrogen receptor status difficult. No correlations with estrogen receptors were established for the unambiguously opioid peptide-positive tumors. Many of the positive tumors also stained with antibodies to gamma-endorphin and alpha-melanocyte-stimulating hormone, suggesting the presence of proopiomelanocortin-derived peptides in them. However, peptides derived from other opioid precursors also may be present in breast cancer.     

神经鞘磷脂酶在人乳腺癌组织中的表达

目的通过观察人乳腺癌组织中神经鞘磷脂合成酶SMS1和SMS2的表达,探讨SMS1和SMS2与乳腺癌发生、发展的关系。方法收集手术切除的人乳腺癌组织和癌旁正常乳腺组织,用免疫组织化学方法和免疫印迹法检测20例乳腺癌组织和癌旁正常乳腺组织中SMS1和SMS2的表达水平,并用Image-proplus6.0图像分析系统对实验结果进行分析。结果免疫组化结果显示,乳腺癌组织中SMS1阳性表达强度(MOD:0.34±0.012)明显高于对应癌旁乳腺组织(MOD:0.150±0.021),乳腺癌组织中SMS1阳性表达的细胞数量(SD:19.63±2.41)明显多于对应癌旁乳腺组织(SD:6.32±4.13),P值均0.05;乳腺癌组织中SMS2阳性表达强度(MOD:0.401±0.013)明显高于对应癌旁乳腺组织(MOD:0.175±0.011),乳腺癌组织中SMS2阳性表达的细胞数量(SD:21.45±6.11)明显多于对应癌旁乳腺组织(SD:5.39±1.30),P值均0.05;免疫印迹法检测结果显示,乳腺癌组织中SMS1和SMS2蛋白表达水平均明显高于对应癌旁乳腺组织(P0.05)。结论 SMS1和SMS2在乳腺癌组织中高表达,可能与乳腺癌的发生、发展有关。     

The identification of ''casein'' in human breast cancer

A retrospective study of ninety-six cases of breast cancer was carried out to determine the prognostic values of a casein immunolocalization technique. This was performed using an indirect immunofluorescence method with antisera raised in rabbits to pooled human casein. Fluorescence positivity was graded according to its intensity and distribution. The pathology of each tumour was studied and the tumour type, histological grade and tissue response assessed. The relationship between these observations and the casein content of the tumour was studied. In addition the correlation between casein content and age, menopausal state, clinical staging and survival was investigated. The incidence of casein positivity in our series was 50% with approximately half of the positive cases showing strong fluorescence. There was a relationship between casein content and the age of the patients, with casein being more frequently found in tumours from younger patients. Tumours with a high casein content, in general, show a poorer survival than the group as a whole. This difference in survival was most marked in the first eight years after operation and restricted to those tumours in clinical stage I and histological grade I. The presence of casein in the tumours did not appear to be related to the other factors examined.     

Immunohistochemical localization of transferrin in human breast cancer tissue

The present study was planned to detect the iron binding protein, transferrin (TR) in paraffin sections of the human breast tumors. The distribution of transferrin has been studied in 153 cases (63 benign lesions and 90 malignant tumors). The extent of staining reaction was determined by semiquantitative grading (weak, moderate and consistent). Positivity rate for transferrin was higher (92.2%) in malignant tumors as compared to benign breast lesions (28.5%) with significant p value (P = .0001) for both the groups. The intensity was variable in both the groups, being more intense in the malignant tumors. Tumors with higher grade of malignancy presented consistent positive staining along with the lymph nodes involved. The extent of immunoreactivity revealed a significant positive correlation with axillary lymph node status. However, no significant correlation was found with the age of the patients. Thus the study of transferrin in breast tumors besides being of prognostic significance helps in the further management of malignant lesions of the breast.     

Expression of XB130 in human ductal breast cancer

Objectives: XB130 is involved in gene regulation, cell proliferation, cell survival, cell migration, and tumorigenesis. In the present study, we first evaluated the expression of the XB130 and its prognostic significance in breast cancer. Then we evaluated whether XB130 could be a target for therapy in breast cancer. Materials and methods: Immunohistochemistry was used to assess the level of XB130 protein in surgically resected, formalin-fixed, paraffin-embedded breast cancer specimens. Associations between XB130 and the postoperative prognosis of patients with breast cancer were evaluated. We evaluated the effect of XB130 inhibited by RNA interference on proliferation, invasion and apoptosis in vitro in a metastatic subclone of MCF-7 breast cancer cell line (LM-MCF-7). The effect of XB130 silencing alone or in combination with gemcitabine on LM-MCF-7 cells apoptosis was also investigated. Results: XB130 protein was present in the cytoplasm of malignant cells, and not in the normal breast tissues. There was correlation between the presence of XB130 in tumour cells and lymph node status, tumor classification and clinical stage. XB130 expression level was significantly associated with recurrence-free and overall survival. Furthermore, multivariate Cox regression analyses revealed that positive XB130 was an independent risk factor for overall survival and recurrence free survival. XB130 silencing alone inhibits tumor growth and induces apoptosis in the LM-MCF-7 cells. Depletion of the XB130 in combination with gemcitabine resulted in marked apoptotic and necrotic cell death in LM-MCF-7 cells. Conclusions: XB130 could be useful as a prognostic marker of recurrence-free and overall survival in invasive breast cancer, as well as for the response to chemotherapy.     

Immunohistochemical localization of transferrin in human breast cancer tissue

The present study was planned to detect the iron binding protein, transferrin (TR) in paraffin sections of the human breast tumors. The distribution of transferrin has been studied in 153 cases (63 benign lesions and 90 malignant tumors). The extent of staining reaction was determined by semiquantitative grading (weak, moderate and consistent). Positivity rate for transferrin was higher (92.2%) in malignant tumors as compared to benign breast lesions (28.5%) with significant p value (p = 0.0001) for both the groups. The intensity was variable in both the groups, being more intense in the malignant tumors. Tumors with higher grade of malignancy presented consistent positive staining along with the lymph nodes involved. The extent of immunoreactivity revealed a significant positive correlation with axillary lymph node status. However, no significant correlation was found with the age of the patients. Thus the study of transferrin in breast tumors besides being of prognostic significance helps in the further management of malignant lesions of the breast.     

LKB1 protein expression in human breast cancer.

Peutz-Jeghers syndrome is caused by germline mutations in the LKB1/STK11 gene. Peutz-Jeghers syndrome is associated with an increased risk of developing intestinal and extraintestinal cancers, including pancreatic, lung, and breast carcinomas. LKB1 gene inactivation has recently been demonstrated in a subset of sporadic pancreatic and lung carcinomas. The role of the LKB1 gene in sporadic breast carcinomas remains unclear, though recent studies suggest inactivation only within papillary carcinomas. Using a commercially available polyclonal antibody that has been shown to mirror LKB1 genetic status in gastrointestinal and pulmonary carcinomas, the authors performed IHC on a large series of breast cancers using tissue microarrays (TMAs). All abnormal TMA results were confirmed using whole sections; specifically, whole sections from the donor blocks of lesions demonstrating diminished or absent LKB1 protein expression on TMA were evaluated to compare labeling of the lesion with that of the surrounding normal breast. In all cases, normal breast epithelium demonstrated strong cytoplasmic labeling (providing an internal positive control), whereas the stroma was nonreactive. Luminal cells typically labeled more strongly than myoepithelial cells. Among 70 invasive ductal carcinomas, 3 (4.3%) showed complete loss of LKB1 labeling, whereas 6 others (8.6%) showed diminished labeling. Of the eight intraductal carcinoma lesions adjacent to these invasive carcinomas, one (12.5%) showed complete loss of LKB1 labeling and one other (12.5%) showed diminished labeling; these results were identical to those of the adjacent invasive carcinomas. One of 10 (10%) hematogenous metastases of mammary carcinoma showed loss of LKB1 labeling. Nine of the 10 invasive carcinomas and both of the ductal carcinoma in situ (DCIS) cases showing loss of or diminished LKB1 expression were of high grade. In contrast, all 13 pure nonpapillary DCIS lesions, all 5 invasive lobular carcinomas and 3 accompanying lobular carcinoma in situ lesions, all 7 papillary DCIS lesions, and all 3 papillomas evaluated showed intact LKB1 labeling. Therefore, although frequent methylation of the LKB1 gene has been reported in papillary carcinomas of the breast, the authors did not find loss of protein expression in these lesions. Instead, it was found that loss of LKB1 protein expression occurs in a subset of high-grade in situ and invasive mammary carcinomas. The authors found LKB1 gene methylation in several of these invasive carcinomas. Given recent Western blot results indicating that diminished LKB1 expression in breast carcinomas correlates with shorter relapse-free survival, LKB1 IHC merits evaluation as a potential prognostic marker for breast carcinoma.     

Proteolysis of human hemoglobin by schistosome cathepsin D

Schistosomes feed on human blood. They employ proteases to degrade hemoglobin from ingested erythrocytes, using the residues released for amino acid metabolism. However, the identity and the role of the participating protease(s) are unclear and controversial. Confocal microscopy localized schistosomal cathepsin D to the parasite gastrodermis, and revealed elevated protease expression in females. At sub-cellular level, cathepsin D was localized to superficial digestive vacuoles of the gut and to cisternae of the gastrodermal rough endoplasmic reticulum. Schistosome cathepsin D, expressed in insect cells, autoactivated at pH 3.6 to a approximately 40 kDa form that cleaved the substrates o-aminobenzoyl-Ile-Glu-Phe-nitroPhe-Arg-leu-NH(2) and hemoglobin. The NH(2)-terminal residues of mature cathepsin D of Schistosoma japonicum and Schistosoma mansoni were Asn1 and Gly1, respectively, revealing that the proregion peptide was comprised of 35 residues. The proteases cleaved hemoglobin at pH 2.5--4.6, releasing numerous fragments. S. Japonicum cathepsin D cleaved at 13 sites, S. mansoni cathepsin D at 15 sites. Early cleavage sites were alpha Phe33-Leu34 and beta Phe41-Phe42, while others included alpha Leu109-Ala-110 and beta Leu14-Trp15, demonstrating a preference for bulky hydrophobic residues at P1 and P1'. Most of the schistosomal cathepsin D cleavage sites were discrete from those of human cathepsin D. The gastrodermal location, elevated expression in females, acidic pH optima, similar substrate preferences in two species, and the discrete substrate preferences compared with human cathepsin D together provide compelling support for the hypothesis that schistosomal cathepsin D plays an integral role in hemoglobin proteolysis, and might be selectively targeted by drugs based on protease inhibition.     

Optically imageable metastatic model of human breast cancer

We report an optically imageable orthotopic metastatic nude mouse model of the human breast cancer MDA-MB-435 expressing green fluorescent protein (GFP). We demonstrate fluorescent imaging of primary and metastatic growth in live tissue and in intact animals. Fragments of tumor tissue expressing GFP were sutured into the pocket in the right second mammary gland. Tumor tissue was strongly fluorescent, enabling whole-body imaging of tumor growth by week 5. Neovascularization of the primary tumor was also visualized by whole-body imaging by contrast of the vessels to the fluorescent tumor. At autopsy, the MDA-MB-435-GFP was found to have metastasized to various organs, including the lung in 55% of the animals, the lymph nodes in 15% of the animals including axillary nodes, and the liver in 10% of the animals. These metastases could be visualized in fresh tissue by fluorescent imaging. Detailed fluorescence analysis visualized extensive metastasis in the thoracic cavity and the lymphatic system. Large metastatic nodules in the lung involved most of the pulmonary parenchyma in all lobes. Lymph node metastasis was found mainly in the axillary area. In the liver, fluorescent macroscopic metastatic nodules were found under the capsule. The metastatic pattern in the model thus reflected clinical metastatic breast cancer and provides a powerful model for drug discovery for this disease. This revised version was published online in July 2006 with corrections to the Cover Date.