Human Invasive Ductal Carcinoma Research Articles

Pleiotrophin Secreted from Human Breast Cancer MCF-7-Ptn Cells Activates Stromal Fibroblasts, Induces Epithelial Island Formation, and Remodels the Microenvironment.

Pleiotrophin (PTN, Ptn) is an 18 kD cytokine that is expressed in many human breast cancers and its gene is inappropriately expressed in cell lines derived from these breast cancers. To study the siginificance of inappropriate expression of Ptn in human breast cancer cells on surrounding stromal cells, we first compared nude mouse xenografts of MCF-7 and MCF-7-Ptn cells. MCF-7-Ptn cells lack the Receptor Protein Tyrosine Phosphatase (RPTP)b/z, the PTN receptor, and thus are not responsive to PTN through autocrine or paracrine stimulation. The MCF-7-Ptn cell xenografts grew rapidly whereas MCF-7 cells xenografts were barely detectable 6 weeks after injection. MCF-7-Ptn cells that were co-injected with equal numbers of NIH3T3 cells grew even more rapidly in the flanks of the nude mice. Surprisingly, the MCF-7-Ptn cell explants developed a morphological phenotype remarkably similar to that of the human invasive ductal carcinoma. We then co-cultured MCF-7 cells that express Ptn (MCF-7-Ptn cells) with NIH 3T3 cells. Secretion of PTN from MCF-7-Ptn cells induced formation of sharply defined clusters of MCF-7-Ptn cells, termed “epithelial islands”, that were surrounded by dense fibrous bands interspersed with NIH 3T3 cells that morphologically closely resemble carcinoma associated fibroblasts (CAFs). A striking increase in tropoelastin and expression of type IV procollagen mRNA was identified in NIH3T3 cells co-cultured with MCF-7-Ptn cells. Furthermore, different markers often resulting from stromal cell-carcinoma cell interactions in breast cancer, including protein kinase C (PKC)-d, and both human and murine matrix metalloproteinase (MMP) 9 were identified either in cells or in the culture media taken from MCF-7-Ptn/NIH3T3 cell co-cultures. The induction of these biochemical and morphological features in the co-cultures of MCF-7-Ptn and NIH3T3 cells was demonstrated to be Ptn expression dependent, PTN-secretion dependent, and NIH3T3 cell dependent. The data suggest that PTN secretion alone from human breast cancer cells with inappropriate expression of Ptn is sufficient to markedly remodel the microenvironment of the breast cancer cell and induce a morphological transition of the MCF-7-Ptn cells and NIH3T3 cells to patterns resembling breast carcinomas through activation of the PTN/RPTPb/z signaling pathway in NIH3T3 cells and reciprocal signaling between the carcinoma stromal cells and the PTN secreting breast cancer cells.

Essential Role of Angiogenic Factors and Bone Marrow-Derived Endothelial/Hematopoietic Cells in the Growth of Solid Tumors

The current thesis examines the role of vascular endothelial growth factor receptor-1 (VEGFR-1) autocrine and paracrine loops in different subsets of cells, namely, breast tumor, endothelial and bone marrow (BM)-derived cells, and their role on tumor growth regulation. These studies highlight the functional relevance of the biological pathways mediated by this receptor and the importance of therapies against VEGFRs as antitumor and antiangiogenesis targets. These findings open new perspectives for the study and development of novel and more effective strategies to fight tumor growth and invasion, and lay the foundation for further development of clinical trials. It is well established that angiogenesis is essential for tumor development. Although the process is controlled by several molecules, the vascular endothelial growth factor (VEGF), by signaling through its receptors, VEGFR-1 and VEGFR-2, seems to be the main mediator. Whereas VEGFR-2 biological functions on tumor endothelium are clearly defined, VEGFR-1 roles are mostly unknown. Moreover, besides being initially described as endothelial specific, VEGFR-1 has also been reported in other types of cells of nonendothelial origin, such as subsets of breast tumor cells and hematopoietic stem and progenitor cells. These observations led us to hypothesize the following: (1) If breast tumor cells express VEGFR-1 and produce VEGF, VEGF/VEGFR-1 autocrine loops could occur and promote breast tumor growth in an endothelial cell-independent fashion. (2) Tumorproduced VEGF could also activate, in a paracrine fashion, VEGFR-1 on tumorassociated endothelium promoting angiogenesis, a feature still unclarified. (3) Tumor cells, by releasing VEGF, may be able to induce in a paracrine way the mobilization of populations of VEGFR-1, expressing BM-derived hematopoietic cells that, in addition to VEGFR-2+ endothelial progenitor cells (EPCs) recruitment to tumor sites, might influence malignant cell growth and neovascularization. The in vitro and in vivo functional expression of VEGFR-1 in human breast cancer cells, and the relevance of the specific impairment of this receptor as a therapeutic target, using monoclonal antibodies (mAbs) as strategy, was first evaluated. VEGFR-1 was functionally expressed on subsets of primary human invasive ductal breast carcinoma tissues and breast cancer cell lines, directly promoting their in vitro proliferation by the

Study of COX-2, Ki67, and p53 expression to predict effectiveness of 5-flurouracil, epirubicin and cyclophosphamide with celecoxib treatment in breast cancer patients

Background. - Cyclooxygenase-2 (COX-2) affects cell proliferation, apoptosis, and metastasis of breast cancer, and may also be involved in tumor angiogenesis through vascular endothelial growth factor. Ki67 and p53 are common markers of proliferation and apoptosis in tumor cells. This study investigated the change in expression of COX-2, Ki67, and p53 in solid tumors after the administration of chemotherapeutic drugs. Materials and Methods. - Fifty patients were eligible to be treated with preoperative 5-fluorouracil, epirubicin, and cyclophosphamide, with celecoxib (FECC). Tumor tissue samples from 10 patients who, diagnosed with invasive ductal carcinoma, completed chemotherapy were examined immunohistochemically for COX-2, Ki67, and p53. Results. - From the 60% of patients who expressed COX-2 and 90% who expressed Ki67 and p53 before treatment, 90% of patients revealed a lower intensity staining for each marker after FECC treatment. However, changes in expression of the three markers did not significantly correlate with tumor size, grade, axillary lymph node status. Immunostained slides clearly showed that the diaminobenzidine intensity was markedly reduced after the three-cycle FECC treatment, which implied the combined regimens be effective to the cancer patients. Conclusions. - This study demonstrates a novel relationship between COX-2, Ki67, and p53 expression of human breast invasive ductal carcinomas. This functional relationship provides support for a potential therapeutic role of COX-2 inhibitors in human breast cancer.

PC-cell derived growth factor (PCDGF/GP88): A novel circulating biomarker in breast cancer (BC) patients

551 Background: The identification of a measurable biomarker that is also a therapeutic target of malignant transformation or malignant progression of breast carcinoma (BC) is needed. PCDGF/GP88, an 88 kDa glycoprotein autocrine growth factor is expressed in human BC tumors in a positive correlation with their tumorigenicity. In estrogen receptor positive (ER+) cells, PCDGF/GP88 expression is low and is stimulated by estradiol whereas in ER negative cells, it is constitutively overexpressed. In ER+ cells, PCDGF/GP88 mediates estrogen mitogenic activity and its overexpression renders cells estrogen-independent and tamoxifen-resistant. Inhibition of PCDGF/GP88 expression by antisense transfection leads to inhibition of breast tumor incidence and growth in nude mice. PCDGF/GP88 is expressed in 80% of human invasive ductal carcinomas and correlates with expression of markers of poor prognosis whereas normal tissues and benign breast lesions are negative. Methods: A blood sampling study was conducted to determine the serum level of PCDGF/GP88 in healthy volunteers (HV) and BC pts. Approximately 10 ml of blood was drawn every three months and processed to obtain serum. PCDGF/GP88 serum concentration was determined in triplicate by quantitative enzyme immunoassay using human PCDGF/GP88 as standard. One hundred twenty three subjects were accrued: 16 HV and 107 BC pts. BC Patient characteristics: race, Caucasian- 51, African American-51, Asian-5; median age, 52.5 (range 29–84); stage I - 24, II - 29, III - 13, IV - 41. Results: Circulating PCDGF/GP88 was measurable in the serum. Mean level of PCDGF was 33.6 ng/ml (range 23.8–42.8) in HV and 47.4 ng/ml (range 19.9–158.4) in BC pts, (p- value= 0.004). Conclusions: PCDGF/GP88 is measurable in the sera of HV and BC pts. Comparison between the two groups of subjects indicates that PCDGF/GP88 level is significantly higher in the sera of BC pts. Future studies will examine the correlation of PCDGF/GP88 level with BC prognostic factors such as stage, tumor size, lymph node involvement, tumor grade and presence of ER and HER-2neu. We will also attempt to correlate the serum level of PCDGF/GP88 with median survival of BC pts. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration A&G A&G

Clinicopathological significance of Bcl-2 and Bax protein expression in human pancreatic cancer

To assess the clinicopathological significance of the expression of the apoptosis-inhibitory Bcl-2 protein (pBcl-2) and the apoptosis-promoting Bax protein (pBax) in human invasive ductal carcinomas (IDCs) of the pancreas. Fifty-nine surgical specimens of IDCs of the pancreas were stained immunohistochemically to detect pBcl-2 and pBax expressions whose correlation to tumor classification, staging, and prognosis was analyzed by univariate and multivariate analyses. The expression of pBcl-2 and pBax was detected in 21 of 59 (35.6%) and in 29 of 59 (49.2%) patients with IDCs of the pancreas, respectively. Neither pBcl-2 nor pBax alone was correlated to TNM staging and differentiation degree of IDCs of the pancreas according to univariate analysis. By Mantel-Cox test, the median survival time after surgery for pBcl-2(+) and pBcl-2(-) groups were 14.3 and 7.3 mo, respectively (chi(2) = 9.357, P = 0.002) and that for pBax(+) and pBax(-) groups were 12.9 and 10.2 mo, respectively (chi(2) = 0.285, P>0.05). Contingency coefficient between pBcl-2 and pBax expression was 0.298, indicating that there was correlation between them (chi(2) = 5.74, P<0.05). The median survival time after surgery for pBcl-2(+)pBax(+) and pBcl-2(+)pBax(-) groups were 14.3 and 14.1 mo, respectively, and that for pBcl-2(-)pBax(+) and pBcl-2(-)pBax(-) groups were 5.9 and 9.9 mo, respectively. There was a significant difference between pBcl-2(+)pBax(+) and pBcl-2(-)pBax(+) (chi(2) = 5.06, P<0.05), such was the case for pBcl-2(+)pBax(+) and pBcl-2(-)pBax(-) (chi(2) = 7.18, P<0.01). Cox proportional hazards model for multivariate analysis was applied, indicating that pBcl-2, TNM staging, age and pBax were high risk factors of post-surgical survival time. Both pBcl-2 and pBax have high expression in IDCs of the pancreas, indicating that co-expression of pBcl-2 and pBax is a good indicator of favorable prognosis in IDCs of the pancreas.

Over-expression of lysophosphatidic acid receptor-2 in human invasive ductal carcinoma

IntroductionLysophosphatidic acid (LPA) is a bioactive phospholipid with diverse effects on various cells. It interacts with at least three G-protein-coupled transmembrane receptors, namely LPA1, LPA2 and LPA3, whose expression in various tumours has not been fully characterized. In the present study we characterized the expression profile of LPA receptors in human breast cancer tissue and assessed the possible roles of each receptor.MethodsThe relative expression levels of each receptor's mRNA against β-actin mRNA was examined in surgically resected invasive ductal carcinomas and normal gland tissue using real-time RT-PCR. LPA2 expression was also examined immunohistochemically using a rat anti-LPA2 monoclonal antibody.ResultsIn 25 cases normal and cancer tissue contained LPA1 mRNA at similar levels, whereas the expression level of LPA2 mRNA was significantly increased in cancer tissue as compared with its normal counterpart (3479.0 ± 426.6 versus 1287.3 ± 466.8; P < 0.05). LPA3 was weakly expressed in both cancer and normal gland tissue. In 48 (57%) out of 84 cases, enhanced expression of LPA2 protein was confirmed in carcinoma cells as compared with normal mammary epithelium by immunohistochemistry. Over-expression of LPA2 was detected in 17 (45%) out of 38 premenopausal women, as compared with 31 (67%) out of 46 postmenopausal women, and the difference was statistically significant (P < 0.05).ConclusionThese findings suggest that upregulation of LPA2 may play a role in carcinogenesis, particularly in postmenopausal breast cancer.

Expression pattern of CXC chemokine receptor-4 is correlated with lymph node metastasis in human invasive ductal carcinoma.

The stromal cell-derived factor-1/CXC chemokine receptor-4 (SDF-1/CXCR4) signal has been shown to be important in various immunological reactions. Recent studies have suggested that CXCR4 is expressed in certain cancer cells and that they use this chemokine receptor efficiently for metastasis formation. The expression of CXCR4 was evaluated by immunohistochemical study in 79 surgically resected invasive ductal carcinomas, and the relation between the staining pattern and clinicopathological features was examined. CXCR4 was diffusely and homogeneously expressed in 59 cancers, which were further divided into 28 high-expression and 31 low-expression cancers by their staining intensity. The other 20 cancers showed heterogeneous immunoreactivity in tumor tissue, which was defined as focal type. In comparison with the diffuse type, focal type tumors showed significantly more extensive lymph node metastasis, because the number and extent of metastatic nodes were larger in the focal than the diffuse type. In the diffuse type, the rate of node-positive cases did not show a difference in staining intensity. However, high-CXCR4 tumors showed more extensive nodal metastasis in comparison with low-expression tumors. In contrast, the expression pattern of CXCR4 did not have a significant correlation with hematogeneous metastasis. The overall survival of these patients tended to be better in the diffuse type than in the focal type, although the difference was not statistically significant. The expression pattern of CXCR4 was significantly correlated with the degree of lymph node metastasis in breast cancers. Our data suggest that CXCR4 might be particularly important in facilitating metastasis through the lymphatic system.

The molecular biology of mammary intraepithelial neoplasia outgrowths

Genetically engineered mice have been used extensively to model human breast cancer. Yet few have been developed and characterized to model the progression from hyperplasia to cancer. We have developed a transplantable model for mammary hyperplasia that progresses to invasive carcinoma, effectively mimicking human ductal carcinoma in situ (DCIS). As such, this provides a unique model for chemoprevention trials for high-risk premalignant breast lesions. These transplantable lines, also known as mammary intraepihelial neoplasia outgrowths (MIN-Os), were developed by our group and are derived from the mammary hyperplasia of mouse mammary tumor virus-polyomavirus middle T (mT) transgenic mice. The polyomavirus transgene provides an attractive model for human mammary carcinoma, as it is capable of transforming cells by triggering signal transduction pathways that have been implicated to be activated by erbB2, through interactions between its mT gene product and key cellular signaling proteins–such as c-Src, Shc, and phosphatidylinositol 3-kinase, which have all been implicated as important in human breast cancer. These transplanted lines are heterogeneous; however, within a line, through multiple generations, the MIN-Os show consistent growth rate, histopathology, and latency to tumor formation. The lines and the tumors that arise from them maintain their defining characteristics in 'tests by transplantation'. Histopathologically, the MIN-Os resemble human DCIS, and the resulting mammary invasive carcinoma resembles human invasive ductal carcinoma. With gene expression studies, we have found dysregulated genes and pathways that have been shown to be similarly altered in human DCIS, suggesting that our model is related to DCIS not only at a histopathological level, but also at a molecular level. These gene expression studies suggest the importance of stromal–epithelial interactions, the extracellular matrix proteoglycan-mediated regulation of cell proliferation signaling, actin cytoskeleton organization, and the insulin-like growth factors and their effectors in the transition from hyperplasia to transformed invasive carcinoma. We have begun using this human DCIS model for developing chemoprevention strategies for high-risk breast lesions. With in vitro assays, we have ranked inhibitors for effectiveness and inclusion in in vivo studies. We have demonstrated that inhibitors to phosphatidylinositol 3-kinase and related downstream mediators are effective in inhibiting growth. This mouse model provides an attractive platform that is amenable to interventional studies and chemoprevention preclinical trials, with easily measurable end-points for testing effectiveness of agents while providing tissue for correlative molecular studies.

Immunohistochemical expression of receptor-tyrosine kinase c-kit protein in invasive ductal carcinoma of the pancreas.

The expression of receptor tyrosine kinase c-kit and its biologic significance in pancreatic cancer are unclear. We studied the expression of c-kit protein (c-KIT) in resectable invasive ductal carcinomas (IDCs) of the pancreas, in order to assess whether a selective c-kit inhibitor, STI571 (Glivec), may be applied for the treatment of pancreatic IDCs. This study included 72 pancreatic IDC patients who received a pancreatectomy between 1982 and 2002. The expression of c-KIT was analyzed retrospectively by immunohistochemistry. c-KIT was expressed in 78% (56/72) of the pancreatic IDCs. c-KIT expression did not correlate with any clinicopathological factor of pancreatic IDC and c-KIT expression had no significant influence on the survival of the patients. The survival rate of the adjuvant chemotherapy (ACT) (+) group was significantly higher than that of the ACT (-) group, but c-KIT expression had no significant effects on the efficacy of the ACT. Multivariate analysis indicated that the pTNM stage, grade and ACT were all significant variables for survival in IDCs overall. As c-KIT was expressed in 78% of the pancreatic IDCs, it suggests that STI571 may be a beneficial agent for chemotherapy against human pancreatic IDCs.

Metallothionein 2A expression is associated with cell proliferation in breast cancer.

Metallothioneins (MTs) belong to a family of cysteine-rich, metal-binding intracellular proteins, which have been linked with cell proliferation. In this study, expression levels of the 8 known MT-1 and MT-2 functional isoforms in human invasive ductal breast cancer specimens were determined by RT-PCR. The expression profiles of the MT protein and MT-2A mRNA were further evaluated in 79 cases of human invasive ductal breast carcinoma by immunohistochemistry and in situ hybridization, and correlated with cancer cell proliferation (determined by Ki-67 nuclear antigen immunolabeling). MT-1A, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X and MT-2A but not MT-1B, were detected in breast cancer tissue samples. The MT-2A mRNA transcript was the highest among all the isoforms detected. A positive correlation was observed between MT-2A mRNA and MT protein expression with Ki-67 labeling (P = 0.0003 and P < 0.0001, respectively) but not with apoptosis (P = 0.1244 and P = 0.8189, respectively). Co-localization of the MT protein and Ki-67 nuclear antigen in breast cancer cells was demonstrated by double immunofluorescence staining. There was also significantly higher MT protein and MT-2A mRNA expression in histological grade 3 tumors than in histological grade 1 and 2 tumors. The finding that MT 2A appears to be the main isoform associated with cell proliferation in invasive ductal breast cancer tissues, may have therapeutic implications.

Correlation between vascular endothelial growth factor, angiogenesis, and tumor-associated macrophages in invasive ductal breast carcinoma.

Angiogenic and anti-angiogenic factors, secreted by tumor, inflammatory, and stromal cells play an important role in regulation of neovascularization. Among the most important of these is vascular endothelial growth factor (VEGF), a specific mitogen for endothelium, which increases vascular permeability and induces proteolytic enzymes necessary for vascular remodeling. Tumor-associated macrophages (TAMs) can express complex functions related to tumor biology, including growth, proliferative rate, stroma formation and dissolution, and neovascularization. The aim of this study was to define, using immunohistochemical analysis, the microvessel density (MVD), VEGF expression, and TAMs level in 97 human invasive ductal breast carcinomas not otherwise specified (NOS), investigate a possible relationship between them and then correlate their values with tumor grade, mitotic activity index (MAI), tumor size and lymph-node status. Statistical analysis showed a strong positive relationship between MVD and VEGF expression ( P<0.001). Furthermore, both MVD and VEGF expression were significantly correlated with tumor grade and lymph-node status, and TAMs infiltration with MAI. TAM level showed a significant positive connection with VEGF expression and MVD. These in situ observations suggest that VEGF stimulates angiogenesis in human invasive ductal breast carcinoma NOS and attracts macrophages to the tumor locus, which then may be involved in angiogenesis promotion. The expression of this angiogenic molecule, and MVD and TAM level, can provide additional prognostic significance and help in the identification of patients who need postoperative adjuvant therapy.

Heterogeneic distribution of thymidine phosphorylase between primary tumors and metastatic lesions of human pancreatic ductal carcinoma: implications for the efficacy of chemotherapy with 5-FU or its derivatives.

It has been suggested that the expression of thymidine phosphorylase (TdRPase) correlates with the malignant potential of various cancers, but its involvement in human invasive ductal carcinoma (IDC) of the pancreas has not been reported. In the present study, the distribution and clinical significance of TdRPase in IDCs and benign diseases of the pancreas were assessed, especially in relation to the efficacy of chemotherapy with 5-FU or its derivatives. The expression of TdRPase in 148 specimens of pancreatic IDCs (66 primary lesions, 46 nodal lesions and 36 distant metastases from 126 patients) and in 24 specimens of benign diseases (4 cystadenomas, 3 hyperplasias, and 17 chronic pancreatitises) was examined by immunohistochemical staining with anti-TdRPase monoclonal antibody and evaluated in terms of three grades of immunoreactivity: negative 0, low 1, or high 2. Positive TdRPase staining (low and high immunoreactivity) was detected in 71% (47/66) of the primary lesions, in 46% (21/46) of the involved nodes, in 53% (19/36) of various lesions of distant metastasis, and in 37% (9/24) of the benign diseases. The staining intensity was significantly higher in the IDC tissues than in the benign disease tissues, and significantly lower in the metastatic lesions than in the primary lesions. TdRPase reactivity did not correlate with the survival rate in both resectable and unresectable IDCs. In patients with both primary tumor and nodal involvement, however, high TdRPase activity in involved nodes was significantly associated with a poor prognosis. On the other hand, although adjuvant chemotherapy was found to improve the survival of patients, TdRPase activity in the tumor did not show any significant relationship with the efficacy of chemotherapy with 5-FU or its derivatives. The present study suggested that in pancreatic IDC the activity of TdRPase in primary lesions is different from that in metastatic lesions, and that DNA is synthesized mainly through the salvage pathway in primary lesions and through a de novo pathway in metastatic lesions. This may be one of the reasons for the heterogeneity in chemosensitivity of human pancreatic IDC.

Apoptosis and expression of Bcl-2 and Bax proteins in invasive ductal carcinoma of the pancreas.

The Bcl-2 family of genes plays important roles in the regulation of apoptosis. The present study was designed to assess the clinicopathologic significance of apoptosis and the expression of the apoptosis-inhibitory Bcl-2 protein (pBcl-2) and the apoptosis-promoting Bax protein (pBax) in human invasive ductal carcinomas (IDCs) of the pancreas. The present study included 66 IDCs that were resected between 1982 and 1998. Apoptosis was assessed by the in situ nick end labeling method and pBcl-2 and pBax were stained immunohistochemically. Apoptosis was quantified as the apoptotic index (AI, the percentage of apoptotic cells of the total tumor cells), and a high AI (>10%) was observed in 26 of the 66 (39%) IDCs. The AI correlated significantly with the extent of nodal involvement. pBax immunoreactivity was detected in 42 of 66 IDCs (64%), and pBax expression was significantly correlated with female gender and showed a significant negative correlation with the extent of nodal involvement. pBcl-2 was expressed in 16 IDCs (24%) but did not show any correlation with the clinicopathologic factors. The AI did not correlate with the expression of pBcl-2 or pBax, but there was a significant correlation between the expression of pBcl-2 and that of pBax; 15 of the 16 pBcl-2(+)IDCs were also pBax(+), and only one pBcl-2(+)IDC was pBax(-). Univariate analysis demonstrated that the degree of apoptosis had no significant influence on the patients' prognosis, pBax or pBcl-2 expression was significantly associated with a better prognosis, and in particular, the pBax(+)pBcl-2(+) group had a significantly higher survival than the other groups. On the other hand, the survival curve of the adjuvant chemotherapy (ACT) group was also higher than that of the surgery alone (SA) group, with borderline statistical signfiicance. The ACT group showed a significantly better survival rate than the SA group for the pBax(+)IDC patients, but the AI and pBcl-2 expression were not correlated with an improved survival rate in the ACT group. Multivariate analysis showed that the AI. pBcl-2 expression, and pBax expression by themselves did not represent significant variables for death owing to IDC, but pBax expression was significantly associated with the efficacy of ACT. In conclusion, pBax expression may be essential for pBcl-2 expression. pBcl-2 and pBax expressions are not significant prognostic factors for patients with IDC, but pBax expression may be beneficial in predicting the effects of ACT on patients with IDC.

Retinoid receptors in human breast carcinoma: possible modulators of in situ estrogen metabolism.

Retinoid receptors (retinoic acid (RARs) and retinoid X (RXRs) receptors) were immunolocalized in 32 human invasive ductal breast carcinomas. These findings were correlated with clinicopathological parameters to study their biological significance in breast carcinoma. Retinoid receptor immunoreactivity, except for RXRgamma, was detected in the nuclei of carcinoma cells. Percentage of positive cases were RARalpha; 81%, RARbeta; 6%, RARgamma; 28%, RXRalpha; 81%, and RXRbeta; 59%. A significant correlation was detected between RARalpha labeling index (LI), and RXRalpha LI (r = 0.667, p < 0.001). Results from immunoblotting performed in three cases were consistent with those of immunohistochemistry. There was a significant correlation between RARalpha LI and 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 1 immunoreactivity (p < 0.05). A significant correlation was also detected between RARalpha (r= 0.413, p = 0.019) or RXRalpha (r = 0.429, p = 0.014) LI, and estrogen receptor (ER) LI. In T-47D breast cancer cells, which express RARalpha, RXRalpha and ER, 17beta-HSD reductive activity increased 1.76-fold (p < 0.001), five days following treatment with 10 nM retinoic acid. These data suggest that retinoid receptors modulate various effects of retinoids, including estrogen metabolism in human breast carcinomas.

Induction of inflammatory angiogenesis by monocyte chemoattractant protein-1.

Almost any growth of tumors is to some extent associated with an inflammatory reaction which may be anti-tumorigenic by acting directly on tumor cells or protumorigenic cells presumably by inducing tumor-associated angiogenesis. In this study, we have analyzed the angiogenesis-inducing capacity of monocyte chemoattractant protein-1 (MCP-1), a key regulatory molecule of monocyte trafficking to sites of inflammation. MCP-1 was found to be potently angiogenic when implanted into rabbit cornea, exerting potency similar to the specific angiogenic vascular endothelial growth factor (VEGF)-A(121). MCP-1-induced angiogenesis in the cornea is associated with prominent recruitment of macrophages, whereas VEGF-A(121)-induced corneal angiogenesis is devoid of inflammatory cell recruitment. Based on these findings, we studied MCP-1 expression and macrophage recruitment in human invasive ductal mammary carcinomas in comparison with the physiological angiogenic processes in bovine ovarian corpus luteum. Macrophage recruitment was always associated with MCP-1 expression. High macrophage counts in mammary tumors corresponded with poor prognosis. In contrast, physiological ovarian angiogenesis was associated with only minimal inflammatory recruitment of macrophages. Our data show that MCP-1 is an indirect inflammation-associated inducer of angiogenesis and demonstrate distinct qualitative differences between tumor angiogenesis in human mammary tumors and physiological angiogenesis in the ovary.

Abnormal levels and minimal activity of the dsRNA-activated protein kinase, PKR, in breast carcinoma cells

The interferon induced, dsRNA-activated, protein kinase, PKR, is a key regulator of translational initiation, playing an important role in the regulation of cell proliferation, apoptosis and transformation. PKR levels correlate inversely with proliferative activity in several human tumor systems. This inverse relationship breaks down in human invasive ductal breast carcinomas which exhibit high levels of PKR (Haines et al., Tumor Biol. 17 (1996) 5-12). Consistent with the data from human tumors, the levels of PKR in several breast carcinoma cell lines, MCF7, T47D, BT20, MDAMB231 and MDAMB468, are paradoxically high compared to those found in the normal breast cell lines MCF10A and Hs578Bst. The activity of affinity- or immuno-purified PKR from MCF7, T47D, and BT20 cells appears to be severely attenuated, as judged by its ability to autophosphorylate, or phosphorylate eIF2α. Furthermore, the activity of the kinase from breast carcinoma cells is refractory to stimulation by dsRNA or heparin. However, PKR from breast carcinoma cells remains functional with respect to its ability to bind dsRNA. The activity of PKR from MCF10A cells is reduced by prior incubation with extracts from MCF7 cells, suggesting that MCF7 extracts contain a transdominant inhibitor of PKR. Deregulation of PKR may therefore provide a mechanism for the development or maintenance of a transformed phenotype of human breast carcinomas, mimicking the effects of manipulation of PKR or eIF2 activity observed in experimental systems. Thus, breast carcinomas may provide the first indication of a role for PKR in the pathogenesis of a naturally occurring human cancer.

Expression of monocyte chemotactic protein-1 in human invasive ductal breast cancer

Monocyte chemotactic protein-1 (MCP-1) is a chemokine involved in the macrophage infiltration of tumor tissue. Tumor associated macrophages (TAMs) are a population of mononuclear-phagocytic cells, which can express complex functions related to tumor biology. The present study was designed to analyse the expression of MCP-1 in parenchymal and stromal elements on frozen sections of 27 breast invasive ductal carcinomas not otherwise specified (NOS) by immunohistochemistry. The expression of MCP-1 in tumor parenchyma and the degree of tumor differentiation were assessed. MCP-1 was detected in the parenchyma in 15 of 27 ductal carcinomas. Positive immunoreactivity manifested as diffuse, homogeneous, moderate or strong, cytoplasmic staining, confined to tumor epithelium. Generally, MCP-1-negative tumors tended to be well differentiated, while chemokine-positive tumors exhibited a low level of differentiation. MCP-1 immunoreactivity was also present in TAMs (CD68 positive cells) in 23 of 27 tumors, and in endothelial cells in 11 of 27 tumors. These results indicate that parenchymal and, more variably, stromal elements of human invasive ductal carcinomas NOS can express MCP-1 in vivo. Additionally, these findings suggest that MCP-1 expression in tumor parenchyma is correlated with the histological grade of ductal invasive breast carcinoma.

Correlation between apoptosis and tumor-associated macrophages in human invasive ductal breast carcinoma

The relationship between apoptosis and tumor-associated macrophages (TAM) was studied in situ in 60 breast carcinomas (18 G1, 28 G2 and 14 G3 carcinomas) using simultaneous apoptosis (TUNEL method) and macrophage staining (anti-CD68 antibody). Apoptotic tumor cell rate (ATCR), total TAM content (TAM(TOT)) and the proportion of TAM that had either cell-to-cell contact with apoptotic tumor cells or that were phagocytosing them (TAM/APO cells) were quantified. ATCR correlated significantly with TAM(TOT). Within all apoptotic tumor cells, the proportion of TAM/APO cells was lower than 20%. Considering the fact that cell-to-cell contact is essential for macrophage-mediated tumor cell killing, our data suggest that the majority of apoptoses occurring in breast cancer may not be caused by macrophage tumoricidal activity. TAM/APO cells accounted for only 1.7% of all TAM. Thus, tumor cell killing and apoptotic tumor cell phagocytosis seem to be quantitatively less important functions of TAM in human breast cancer in vivo.

Expression of connective tissue growth factor mRNA in the fibrous stroma of mammary tumors

Desmoplasia, the formation of highly cellular, excessive connective tissue stroma associated with some cancers, shares many features with the wound healing response. Since connective tissue growth factor (CTGF) has previously been demonstrated to play a role in wound repair, we wanted to determine if it might be involved in the pathogenesis of stromal desmoplasia in mammary cancer. We assayed 11 human invasive mammary ductal carcinomas by Northern blot and 7 out of 11 were positive for both CTGF expression and transforming growth factor-beta 1 (TGF-β1, a principal CTGF inducer). One specimen was positive only for TGF-β1. The remaining 3 tumors lacked significant stromal involvement and were negative for either factor. In every case we assayed, in which there was marked connective tissue involvement, both CTGF and TGF-β1 messages were found. We also assayed 3 murine mammary tumor models. The GI-101 xenograft model had marked stroma and was positive for both factors in-vivo, but positive for only TGF-β1 mRNA expression in culture where fibroblasts were absent. The DMBA murine tumor lacked significant stroma and was negative for CTGF and TGF-β1 expression by Northern blot, while the stromal rich DMBA-MMTV tumor contained multifocal desmoplasia and was positive for both factors. We performed in-situ hybridization for CTGF and TGF-β1 on the GI-101 and DMBA-MMTV tumors. CTGF message was observed only in the fibroblasts of the stroma, while TGF-β1 mRNA hybridization was present in tumor epithelial cells and leukocytes. These results suggest that cancer stroma formation involves induction of similar fibroproliferative growth factors (TGF-β1 and CTGF) as wound repair.

397 Progestin-regulated proteins released by the T47D human breast cancer cell line

The interferon induced, dsRNA-activated, protein kinase, PKR, is a key regulator of translational initiation, playing an important role in the regulation of cell proliferation, apoptosis and transformation. PKR levels correlate inversely with proliferative activity in several human tumor systems. This inverse relationship breaks down in human invasive ductal breast carcinomas which exhibit high levels of PKR (Haines et al., Tumor Biol. 17 (1996) 5-12). Consistent with the data from human tumors, the levels of PKR in several breast carcinoma cell lines, MCF7, T47D, BT20, MDAMB231 and MDAMB468, are paradoxically high compared to those found in the normal breast cell lines MCF10A and Hs578Bst. The activity of affinity- or immuno-purified PKR from MCF7, T47D, and BT20 cells appears to be severely attenuated, as judged by its ability to autophosphorylate, or phosphorylate eIF2α. Furthermore, the activity of the kinase from breast carcinoma cells is refractory to stimulation by dsRNA or heparin. However, PKR from breast carcinoma cells remains functional with respect to its ability to bind dsRNA. The activity of PKR from MCF10A cells is reduced by prior incubation with extracts from MCF7 cells, suggesting that MCF7 extracts contain a transdominant inhibitor of PKR. Deregulation of PKR may therefore provide a mechanism for the development or maintenance of a transformed phenotype of human breast carcinomas, mimicking the effects of manipulation of PKR or eIF2 activity observed in experimental systems. Thus, breast carcinomas may provide the first indication of a role for PKR in the pathogenesis of a naturally occurring human cancer.