Human Invasive Ductal Breast Carcinomas Research Articles

Abstract PR01: Disruption of DDR2-collagen interactions in tumor and stroma with a novel small molecule inhibitor blunts cancer metastasis

Abstract Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase that utilizes the extracellular matrix protein collagen as its ligand. Recently, DDR2 was shown to be critical in facilitating breast cancer metastasis. In tumor cells DDR2 expression, which is absent in normal breast epithelium, is induced during Epithelial Mesenchymal transition (EMT), and serves to stabilize protein levels of the EMT inducing transcription factor, Snail1. DDR2 expression is present in a majority of human invasive ductal breast carcinomas, and expression is predominately localized to the tumor-stroma boundary. In these tumors DDR2 is acting in a positive feedback manner to maintain Snail1 levels and activity at the leading, invasive edge of the tumors, where cells have undergone EMT and come into contact with extracellular matrix (ECM) collagen I. This allows continued invasion through the ECM and contributes to breast cancer metastasis. Interestingly, in genetic models of breast cancer in mice, selectively eliminating DDR2 in either the tumor (MMTV-PyMT; DDR2 fl/fl; K14-Cre) or the fibroblasts within the stroma (MMTV-PyMT; DDR2 fl/fl; FSP-Cre) leads to a dramatic inhibition of tumor metastasis. This indicates that in addition to maintaining EMT in the tumor cells, DDR2 functions within the stromal compartment to facilitate metastasis as well. In culture, the matrix synthesized and remodeled by cancer associated fibroblasts (CAFs) is organized very differently than that of normal fibroblasts. When DDR2 is depleted from CAFS, the matrix more closely resembles that produced by normal fibroblasts, with less organization of matrix proteins. Therefore, it appears that DDR2 engagement by collagen provides a means for both tumor and stromal cells to enhance metastatic potential. As such, DDR2 is a novel RTK target for the treatment of breast cancer metastasis, and we have developed potent and selective small molecule inhibitors of DDR2. These compounds provide a means of blunting breast cancer metastasis by targeting the tumor and stromal cells, thereby disrupting DDR2 signaling in both compartments. The developed inhibitors have nanomolar cellular potency at blocking DDR2 activation. Unlike traditional tyrosine kinase inhibitors (TKIs) that target the intracellular kinase domain, the inhibitors that we have developed act on the extracellular domain of DDR2 and disrupt DDR2 signaling by accelerating the disassembly of the DDR2-collagen complex. Therefore these inhibitors have a unique mode of action that is distinct from the canonical classes of TKIs. These compounds have the ability to attenuate DDR2 signaling both in cell culture, as well as in vivo as demonstrated by using a Snail1-clic beetle green reporter in a syngeneic transplant model. Treating CAFs with the developed inhibitors in culture reverts the organizational phenotype of the resulting matrix to that resembling the matrix produced by normal fibroblasts. Furthermore, using mouse models of late stage breast tumor metastasis, we have shown that these inhibitors reduce metastatic burden in the lungs of mice, to a level comparable to that of genetic knockdown of DDR2. Together these data support further investigation of this novel class of DDR2 inhibitors as anti-metastasis agents, potentially for use in combination with standard of care therapy to halt cancer progression and prevent relapse. Further, it will be important to study the role of these inhibitors in other cancers where DDR2 expression has been shown to promote metastasis, including, but not limited to, ovarian, head and neck, and nonsmall cell lung carcinoma. This abstract is also presented as Poster B13. Citation Format: Whitney R. Grither, Gregory D. Longmore. Disruption of DDR2-collagen interactions in tumor and stroma with a novel small molecule inhibitor blunts cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR01.

Abstract 342: Breast cancer adhesion cascade is influenced by Galectin-1 and E-selectin receptor-ligand interactions

Abstract Invasion of other tissues during bloodborne metastasis requires the adhesion of cancer cells to the endothelium by specific fluid shear-dependent receptor-ligand interactions. It is hypothesized that ligands are shared between the endothelial adhesion molecule known as E-selectin, a transmembrane glycoprotein, and Galectin-1 (Gal-1), a secretory dimer protein, both of which are upregulated during inflammation and cancer. Our previous data strongly indicate that this shear-dependent interaction involves the same or closely related cancer cell-expressed ligands, such as the glycoprotein Mac-2BP. Initial investigation using immuno-staining methods to locate E-selectin and Gal-1 ligands and determine relative proximity to one another revealed that E-selectin and Gal-1 ligands on ZR-75-1 breast cancer cells and breast cancer tissue microarrays had a high percentage of colocalization, as high as 75% in some cases. These results allude to the possibility that these reactive molecules are in close proximity or are in fact the same molecule. Subsequently, E-selectin microspheres were perfused over Gal-1-treated ZR-75-1 cell monolayers and breast cancer tissue microarrays to determine the functional relationship, if any, between Gal-1 and E-selectin ligands during adhesion from flow. Flow assay data show that Gal-1 treatment enhances E-selectin microsphere interactions to a breast cancer monolayer in a shear-dependent manner, with a shear stress of 0.5 dynes/cm2 having an almost 300% increase in binding compared to the untreated monolayer. This amount of binding at a physiological shear stress gives credence to the hypothesis that Gal-1 influences E-selectin receptor-ligand interactions via the same or similar ligands during breast cancer cell adhesion. Dynamic biochemical tissue analysis (DBTA), a novel flow-based tissue interrogation technique developed in our lab, was then implemented to test human invasive ductal breast carcinoma tissue sections. Similar to ZR-75-1 cell line flow assay data, a 142% increase in shear-dependent binding interactions between the E-selectin microspheres and the Gal-1 treated breast cancer tissue sections at 0.5 dynes/cm2 shear stress was observed. Colocalization of reactive signals and increased shear-dependent flow interactions indicate that metastatic binding in breast cancer is aided through E-selectin to Gal-1 interaction via a shared ligand intermediate. Ascertaining the mechanism behind this interaction would lead to a better understanding of the circulating breast cancer cells’ adhesion cascade during metastasis. Citation Format: Nathan M. Reynolds, Claire R. Hall, Sean E. Thomas, Monica M. Burdick. Breast cancer adhesion cascade is influenced by Galectin-1 and E-selectin receptor-ligand interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2015-342

The collagen receptor discoidin domain receptor 2 stabilizes SNAIL1 to facilitate breast cancer metastasis

Increased stromal collagen deposition in human breast tumours correlates with metastases. We show that activation of the collagen I receptor DDR2 (discoidin domain receptor 2) regulates SNAIL1 stability by stimulating ERK2 activity, in a Src-dependent manner. Activated ERK2 directly phosphorylates SNAIL1, leading to SNAIL1 nuclear accumulation, reduced ubiquitylation and increased protein half-life. DDR2-mediated stabilization of SNAIL1 promotes breast cancer cell invasion and migration in vitro, and metastasis in vivo. DDR2 expression was observed in most human invasive ductal breast carcinomas studied, and was associated with nuclear SNAIL1 and absence of E-cadherin expression. We propose that DDR2 maintains SNAIL1 level and activity in tumour cells that have undergone epithelial-mesenchymal transition (EMT), thereby facilitating continued tumour cell invasion through collagen-I-rich extracellular matrices by sustaining the EMT phenotype. As such, DDR2 could be an RTK (receptor tyrosine kinase) target for the treatment of breast cancer metastasis.

Abstract 3939: The collagen receptor discoidin domain receptor 2 facilitates breast cancer metastasis by stabilizing Snail1 protein.

Abstract Mammographic density is a leading risk factor for the development of breast cancer and these cancers are more invasive and metastatic. The increase in breast density is, in part, due to increased fibrillar collagen deposition. In an RNAi human kinome screen to identify post-transcriptional regulators of Snail1 protein stability we found the collagen receptor DDR2, but not related DDR1, to stabilize Snail1 protein as a result of collagen I activated DDR2 stimulation of ERK2 activity. This can occur independent of β1-integrin and TGFβ signals. DDR2 is not required for TGFβ-induced Epithelial Mesenchymal transition (EMT) or EMT-induced Snail1 transcription. Rather DDR2 expression, which is absent in normal breast epithelia, is induced during EMT as breast tumor cells assume invasive, mesenchymal features. DDR2-induced ERK2 activity stabilizes Snail1 protein level in two ways. First, ERK2 interacts with and phosphorylates Snail1 and second ERK2 inhibits GSK3b. DDR2 was found to be critical for breast cancer cell invasion and migration in vitro, in 3D collagen gels, and breast cancer metastasis in vivo, in syngeneic xenograft models. A majority of human invasive ductal breast carcinomas express DDR2 protein and there is a strong association between nuclear Snail1 expression, DDR2 expression, and the absence of E-cadherin expression. The small group of breast cancer patients with tumors harboring increased DDR2 gene copy number has diminished survival. We propose that DDR2 acts in a positive feedback loop to maintain Snail1 protein level and activity in breast tumor cells at the leading, invasive edge where tumor cells that have undergone EMT and invaded through the basement membrane to encounter extracellular matrix collagen I. This facilitates the continued tumor cell invasion and migration through the collagen I-rich ECM, and thereby, contributes to breast cancer metastasis. That the collagen I receptor DDR2 maintains the EMT-inducing Snail1 protein level and activity and contributes to breast cancer metastasis could explain the increase in metastasis of breast cancers in women with dense breasts. As such, DDR2 could be considered a novel RTK target for the treatment of breast cancer metastasis. Citation Format: Kun Zhang, Callie Corsa, Julie Prior, David Piwnica-Worms, Gregory D. Longmore. The collagen receptor discoidin domain receptor 2 facilitates breast cancer metastasis by stabilizing Snail1 protein. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3939. doi:10.1158/1538-7445.AM2013-3939

Abstract 872: Novel potent and orally active STAT3 inhibitors for cancer therapies.

Abstract Signal transducers and activators of transcription (STATs) are proteins which have dual roles as cytoplasmic signaling proteins and transcription factors that activate sets of genes including those contribute to tumorigenesis. Among the STATs family members, STAT3 has been shown to play an important role in breast carcinogenesis and is often overexpressed in primary breast tumors. Increased STAT3 transcriptional activity is reported to correlate with ER-negative phenotype in breast cancer cell lines and in primary human invasive ductal breast carcinomas. Therefore, STAT3 is a promising target for breast cancer therapies. Current strategies of inhibiting STAT3 activity did not sufficiently block STAT3 activation in cancer cells due to their limited potency, poor solubility or bioavailability. Despite several STAT3 inhibitors have been advanced into early clinical trials, there is an urgent need to develop orally active, potent STAT3 inhibitors as cancer therapeutics. We have designed, synthesized and screened a series of novel STAT3 inhibitors utilizing fragment-based drug design strategy. A number of small molecules with novel scaffolds have been discovered with low-micromolar to nanomolar potency in inhibiting the proliferation of various human breast cancer and pancreatic cancer cells. The identified compounds HJC0152, HJC0123, HJC0125, and HJC0416 significantly suppress STAT3 promoter activity and STAT3 phosphorylation (pY705), reduce total STAT3 protein expression, induce cell growth arrest, and early apoptosis in ER-negative breast cancer cell line MDA-MB-231. These novel compounds have demonstrated a better efficacy in suppressing the growth of MDA-MB-231 breast xenograft tumors via intraperitoneal and oral administrative routes, with remarkably improved aqueous solubility and less toxicity than the lead compounds. In conclusion, we have developed a new class of orally active STAT3 inhibitors with superior drug-like properties, for further evaluation in cancer therapeutic and preventive settings. This work was supported by The University of Texas MD Anderson Cancer Center DFI Funding Program. Citation Format: Zhengduo Yang, Haijun Chen, Lili Chu, Yusong Zhang, Chunyong Ding, Huiling Liu, Jia Zhou, Qiang Shen. Novel potent and orally active STAT3 inhibitors for cancer therapies. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 872. doi:10.1158/1538-7445.AM2013-872

Expression of melatonin receptor MT1 in cells of human invasive ductal breast carcinoma

In humans, two main types of membrane melatonin receptors have been identified, MT1 and MT2. Expression of MT1 in neoplastic cells seems to increase the efficacy of melatonin's oncostatic activity. The purpose of this study was to determine the distribution and the intensity of MT1 expression in breast cancer cells and to correlate it with clinicopathological factors. Immunohistochemical studies (IHC) were conducted on 190 cases of invasive ductal breast carcinomas (IDC) and molecular studies were performed on 29 cases of frozen tumor fragments and selected breast cancer cell lines. Most of the studied tumors manifested a membranous/cytoplasmic IHC expression of MT1. In IDC, the MT1 expression was higher than in fibrocystic breast disease. MT1 expression was higher in estrogen receptor positive (ER+) and HER2 positive (HER2+) tumors. Triple negative tumors (TN) manifested the lowest MT1 expression level. The lowest MT1 protein expression level was noted in the TN breast cancer cell line MDA-MB-231 compared with ER+ cell lines MCF-7 and SK-BR-3. MT1 mRNA expression was negatively correlated with the malignancy grade of the studied IDC cases. Moreover, higher MT1 expression was associated with patients' longer overall survival (OS) in the group of ER+ breast cancers and treated with tamoxifen. Multivariate analysis indicated that MT1 was an independent prognostic factor in the ER+ tumors for OS and event-free survival in the ER+ tumors. The results of this study may point to a potential prognostic and therapeutic significance of MT1 in IDC.

Abstract B63: Developing Novel STAT3 Inhibitors for the Treatment and Prevention of Cancer

Abstract Signal transducers and activators of transcription (STATs) are a family of transcription factors involved in the regulation of early embryonic development, immune response, cell proliferation, differentiation, and apoptosis. Particularly, STAT3 regulates lobuloalveolar apoptosis during involution in mammary gland development, and its activation is required for the growth of CD44+CD24- stem cell-like breast cancer cells and the development of multiple cancers including breast cancer. Increased STAT3 transcriptional activity was found to correlate with ER-negative phenotype in breast cancer cell lines and in primary human invasive ductal breast carcinomas. Thus, STAT3 represents a promising target for therapeutic and preventive intervention of breast cancer. Yet current strategies of inhibiting STAT3 activity did not sufficiently block STAT3 activation in cancer cells due to their limited potency, poor solubility or bioavailability. Despite several STAT3 inhibitors have been advanced into early clinical trials, there is an urgent need to develop a more successful strategy targeting STAT3 for cancer therapies, and preventive use in particular. We attempted to identify potent and orally active STAT3 inhibitors for such purposes and have designed, synthesized and screened a series of novel STAT3 inhibitors utilizing fragment-based drug design strategy. A number of small molecules with new scaffolds have been discovered with low-micromolar to nanomolar potency in inhibiting the proliferation of various human breast cancer and pancreatic cancer cells. The identified compound HJC0152 significantly suppresses basal and IL-6-stimulated STAT3 promoter activity and STAT3 phosphorylation, reduces total STAT3 protein expression, induces G2/M cell cycle arrest, and induces early apoptosis. HJC0152 has demonstrated a better potency in suppressing the growth of MDA-MB-231 breast xenograft tumors via intraperitoneal and oral administrative routes, with remarkably improved aqueous solubility (3,300 fold improvement) and less toxicity than the lead compounds. In conclusion, we have developed a new class of orally active STAT3 inhibitors with superior drug-like properties for further preclinical therapeutic and preventive evaluation. Citation Format: Zhengduo Yang, Haijun Chen, Lili Chu, Yusong Zhang, Chunyong Ding, Kristin Terry, Jia Zhou, Qiang Shen. Developing novel STAT3 inhibitors for the treatment and prevention of cancer. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B63.

Abstract 4271: Fibroblast transcriptome sequencing reveals different gene expression profiles among distinct subtypes of breast cancer and normal mammary tissue

Abstract Cancer associated fibroblasts, CAFs, is believed to actively participate in the growth and invasion of tumor cells by providing a unique tumor microenvironment, rendering increasingly clear that the development of cancer cannot be dissociated from its local microenvironment. However, neither the origin of CAFs nor the criteria to distinguish CAFs from normal fibroblasts has been well established. Moreover, the complex nature of the heterogeneity that exists both within and between breast cancers, particularly as related to gene expression profiling, needs more recognition. It must be appreciated that CAFs from different subtypes of breast cancers have distinct gene expression profiles as well as do CAFs in comparison to normal breast fibroblasts (NAFs). The identification of these differentially expressed genes (DEG) may shed light on the molecular interactions of fibroblasts with carcinoma cells and to further understand their signaling pathways that are actively involved. The advent of next generation sequence technologies and the realization that breast cancer is heterogeneous has shifted the focus to the identification of molecular markers of prognostic relevance. In the present study, we isolated total RNA from fibroblasts of frozen tissue samples of human invasive ductal breast carcinoma luminal A and triple negative subtypes and also from normal surrounding tissue from fibroadenoma lesions using laser capture microdissection. Preparing 3′ fragments cDNA libraries from fibroblast samples and sequencing on the Genome Sequencer FLX Titanium 454 Plataform, we obtained a transcriptional snapshot of CAFs and NAF. Sequencing and filtering (removal of adapter, polyA tail etc) of these sequences resulted in a total of 376,528 high quality reads with an average length of 159.43 bp. A total of 14,950 transcripts were sequenced and corresponded to 10,586 genes, representing 53.72% of the human genome repertoire. The analysis of DEG between the distinct samples was investigated by test on proportions (p value ≤ 0.05 and 2.0 fold change difference in expression level). The comparison of expression in NAFs versus triple negative CAF yielded 192 DEG while NAF versus luminal A CAF revealed 88 DEG and triple negative CAF versus luminal A CAF presented 41 DEG. Gene ontology (GO) assignments and KEGG pathways classifications were performed to gain an overview of the biological processes in which these DEG are involved. The results suggest that highly specific genes are expressed in each breast cancer fibroblast subtype. Taken all together, genes will be selected for validation by RT-qPCR and tissue microarray using independent samples. Further understanding of detailed functional mechanisms of the differentially expressed genes, may thus yield additional candidates for prognostic and therapeutic intervention. Supported by FAPESP:05/60333-7, 09/10088-7. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4271. doi:1538-7445.AM2012-4271

Abstract 532: A transcriptional sketch of breast cancer fibroblasts: The validation of cDNA libraries for expression profile study using a high-throughput sequencing technology

Abstract There is now a wide recognition that the cross-talk between cancer and stromal cells may play a crucial role in cancer progression, although the complex underlying molecular mechanisms that occur within the tumor microenvironment are poorly understood. Fibroblasts represent the major cellular component of cancer-associated stroma. Unfortunately at present, a definitive view is lacking on a comparative expression profile between carcinoma associated fibroblasts (CAFs) and fibroblasts derived from normal breast (NAFs). The scarce body of work can not be compared due to differences in methodologies highlighting the importance to evaluate fibroblasts in tumor context and not in culture. Genomic changes in fibroblasts might be the cause for its ability to influence breast cancer cells, hence the identification of differential gene expression through methodologies with high potential of quantitative evaluation not only will complement previous quantitative investigations but could also provide insights on the identification of new prognostic molecular markers for breast cancer. The purpose of this study was the preparation of 3' fragments cDNA libraries from fibroblast samples, its validation on ABI 3130 xl Plataform and the obtainment of a transcriptional snapshot of CAFs and NAFs using the Genome Sequencer FLX Titanium 454 Plataform. Since biological heterogeneity of breast cancers has serious implications on disease treatment and prognostic, total RNAs were isolated from fibroblasts obtained from tissue frozen samples of human invasive ductal breast carcinoma (luminal A subtype), triple negative, fibroadenoma tissues using laser capture microdissection (LCM). Samples were reverse transcribed and amplified, double cDNAs were digested with DpnII restriction enzyme. After recovery of fragments with Streptavidin beads, they were labeled with 454 adaptors containing a specific 6nt TAG. Libraries were pooled before sequencing validation by Sanger method which was held in attempt to confirm the absence of library preparation artefacts (concatamers or linkers with inespecific TAGs). Considering the total high quality sequences presenting a complete adaptor site, 93% also presented complete TAGs and DpnII sites. Moreover, all the studied samples were represented: 19% of total sequences represented luminal A subtype samples while triple negative samples and normal samples presented 40 and 41% of sequences respectively. This work indicates at molecular level, differences among the 3 distinct groups of fibroblasts. The 454 transcriptome sequencing is on going and we expect that this quantitative approach will permit the accurate identification of differential transcriptional portraits associated with changes on breast cancer fibroblasts. Supported by FAPESP: 05/60333-7, 09/10088-7. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 532. doi:10.1158/1538-7445.AM2011-532

Mena invasive (MenaINV) and Mena11a isoforms play distinct roles in breast cancer cell cohesion and association with TMEM

Mena, an actin regulatory protein, functions at the convergence of motility pathways that drive breast cancer cell invasion and migration in vivo. The tumor microenvironment spontaneously induces both increased expression of the Mena invasive (Mena(INV)) and decreased expression of Mena11a isoforms in invasive and migratory tumor cells. Tumor cells with this Mena expression pattern participate with macrophages in migration and intravasation in mouse mammary tumors in vivo. Consistent with these findings, anatomical sites containing tumor cells with high levels of Mena expression associated with perivascular macrophages were identified in human invasive ductal breast carcinomas and called TMEM. The number of TMEM sites positively correlated with the development of distant metastasis in humans. Here we demonstrate that mouse mammary tumors generated from EGFP-Mena(INV) expressing tumor cells are significantly less cohesive and have discontinuous cell-cell contacts compared to Mena11a xenografts. Using the mouse PyMT model we show that metastatic mammary tumors express 8.7 fold more total Mena and 7.5 fold more Mena(INV) mRNA than early non-metastatic ones. Furthermore, Mena(INV) expression in fine needle aspiration biopsy (FNA) samples of human invasive ductal carcinomas correlate with TMEM score while Mena11a does not. These results suggest that Mena(INV) is the isoform associated with breast cancer cell discohesion, invasion and intravasation in mice and in humans. They also imply that Mena(INV) expression and TMEM score measure related aspects of a common tumor cell dissemination mechanism and provide new insight into metastatic risk.

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

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.

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.

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.

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.

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.

396 Chromatin binding sites for the occupied and unoccupied estrogen receptor

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.