Osteoprotegrin and the bone homing and colonization potential of breast cancer cells

Breast cancer cells preferentially metastasize to bone, leading to the formation of primarily osteolytic lesions. Osteoprotegerin (OPG) plays multifactorial roles in the development of osteolytic bone metastases. An increase in the ratio of receptor activator of nuclear factor kappaB ligand (RANKL) to OPG increases osteoclastogenesis within the bone microenvironment. OPG also acts as a survival factor for cancer cells by protecting them from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apoptosis. This study compares OPG production in vitro in a number of breast cancer cell lines exhibiting both differences in metastatic capacity and in preferential metastasis to bone. Our studies demonstrated that OPG expression by MDA-231, MDA-MET, and MDA-231/K cancer cells was directly correlated with bone specific homing and colonization potential but not with metastasis of cancer cells to other organs; both in IL-1 beta stimulated and control cells. We also demonstrated expression of other bone-related markers including type I collagen, osteocalcin, osteopontin, and Runx2 in these cells. However, the generally lower expression of these markers in the bone selective cell line MDA-MET suggested that increased OPG expression in the bone specific variant was not merely a consequence of enhanced osteomimicry by these cells but that it has a significant role in the metastatic process. Co-culture of breast cancer cells with osteoblastic cells (hFOB 1.19) led to an overall downregulation in OPG production, which was not affected by the bone homing and colonization potential of the cell lines, suggesting that OPG alone is not indicative of osteolytic bone activity by breast cancer cells.     

Failure to detect intra-abdominal metastases from breast cancer: a case for staging laparotomy

Two studies were performed to assess the accuracy of non-invasive methods in detecting intra-abdominal metastases from breast cancer. Firstly, the sites of spread detected at the time of first presentation with metastases were compared with the sites of spread shown at necropsy in the same patients. Although about two-thirds of the patients with bone and lung metastases at necropsy had had metastases detected at these sites when they first presented with metastases, only a third of the patients with liver metastases and none of those with other intra-abdominal metastases had had evidence of disease at first presentation with metastases. The second study confirmed a poor detection rate of liver and other intra-abdominal metastases in patients with breast cancer undergoing laparotomy and oophorectomy who were staged immediately before operation.Pre-mastectomy staging laparotomy should be considered in those patients with primary breast cancer who are most likely to have disseminated disease beyond the regional nodes. In the presence of occult gross metastases detected by staging laparotomy, mastectomy will not provide additional protection against loca recurrence of disease. Patients with occult gross metastases should also be excluded from studies on adjuvant chemotherapy (designed to treat micrometastases). Aggressive methods of staging are justified to protect the patient as far as possible against unnecessary mastectomy and to identify those patients who should be treated by therapeutic chemotherapy rather than adjuvant chemotherapy.     

Claudin-2 promotes breast cancer liver metastasis by facilitating tumor cell interactions with hepatocytes

We previously identified claudin-2 as a functional mediator of breast cancer liver metastasis. We now confirm that claudin-2 levels are elevated in liver metastases, but not in skin metastases, compared to levels in their matched primary tumors in patients with breast cancer. Moreover, claudin-2 is specifically expressed in liver-metastatic breast cancer cells compared to populations derived from bone or lung metastases. The increased liver tropism exhibited by claudin-2-expressing breast cancer cells requires claudin-2-mediated interactions between breast cancer cells and primary hepatocytes. Furthermore, the reduction of the claudin-2 expression level, either in cancer cells or in primary hepatocytes, diminishes these heterotypic cell-cell interactions. Finally, we demonstrate that the first claudin-2 extracellular loop is essential for mediating tumor cell-hepatocyte interactions and the ability of breast cancer cells to form liver metastases in vivo. Thus, during breast cancer liver metastasis, claudin-2 shifts from acting within tight-junctional complexes to functioning as an adhesion molecule between breast cancer cells and hepatocytes.     

The tumor macroenvironment and systemic regulation of breast cancer progression

Breast cancer is the most common malignancy among women worldwide and is the most common cause of death for women between 35 and 50 years of age. Women with breast cancer are at risk of developing metastases for their entire lifetime and, despite local and systemic therapies, approximately 30% of breast cancer patients will relapse (Jemal et al., 2010). Nearly all breast cancer related deaths are due to metastatic disease, even though metastasis is considered to be an inefficient process. In some cases, tumor cells disseminate from primary sites at an early stage, but remain indolent for protracted periods of time before becoming overt, life-threatening tumors. Little is known about the mechanisms that cause these indolent tumors to grow into malignant disease. Because of this gap in our understanding, we are unable to predict which breast cancer patients are likely to experience disease relapse or develop metastases years after treatment of their primary tumor. A better understanding of the mechanisms and signals involved in the exit of tumor cells from dormancy would not only allow for more accurate selection of patients that would benefit from systemic therapy, but could also lead to the development of more targeted therapies to inhibit the signals that promote disease progression. In this review, we address the systemic, or "macroenvironmental", contribution to tumor initiation and progression and what is known about how a pro-tumorigenic systemic environment is established.     

Tumor-supportive and Osteoclastogenic Changes Induced by Breast Cancer-derived Factors Are Reversed by Inhibition of γ-Secretase

During breast cancer metastasis to bone, tumor cells home to bone marrow, likely targeting the stem cell niche, and stimulate osteoclasts, which mediate osteolysis required for tumor expansion. Although osteoblasts contribute to the regulation of the hematopoietic stem cell niche and control osteoclastogenesis through production of proresorptive cytokine RANKL (receptor activator of NF-κB ligand), their role in cancer metastases to bone is not fully understood. C57BL/6J mouse bone marrow cells were treated for 3–12 days with ascorbic acid (50 μg/ml) in the presence or absence of 10% medium conditioned by breast carcinoma cells MDA-MB-231, 4T1, or MCF7. Treatment with cancer-derived factors resulted in a sustained 40–60% decrease in osteoblast differentiation markers, compared with treatment with ascorbic acid alone, and induced an osteoclastogenic change in the RANKL/osteoprotegerin ratio. Importantly, exposure of bone cells to breast cancer-derived factors stimulated the subsequent attachment of cancer cells to immature osteoblasts. Inhibition of γ-secretase using pharmacological inhibitors DAPT and Compound E completely reversed cancer-induced osteoclastogenesis as well as cancer-induced enhancement of cancer cell attachment, identifying γ-secretase activity as a key mediator of these effects. Thus, we have uncovered osteoblasts as critical intermediary of premetastatic signaling by breast cancer cells and pinpointed γ-secretase as a robust target for developing therapeutics potentially capable of reducing both homing and progression of cancer metastases to bone.     

Micrometastases in bone marrow in patients with primary breast cancer: evaluation as an early predictor of bone metastases.

The bone marrow of 307 patients with primary breast cancer was examined for tumour cells by immunocytochemistry using an antiserum to epithelial membrane antigen. Micrometastases were found in 81 cases (26.4%) and their presence was related to various poor prognostic factors: spread to lymph nodes, vascular invasion, T stage, and pathological size. The median duration of follow up was 28 months. Seventy five patients relapsed, 60 at distant sites. Of these 60 patients, 26 had micrometastases detected at presentation and 34 were free of micrometastases initially. The relapse free interval was significantly shorter for patients with micrometastases, and these patients had a shorter survival. Analysis of the sites of relapse showed that the test predicted bone metastases only. Thus 10 out of 19 patients (53%) who developed bone metastases at first relapse had micrometastases at presentation compared with only 41 out of 288 patients (14%) who remained free of bone metastases or relapsed in non-skeletal sites. The presence of micrometastases detected at the time of initial surgery in a patient with primary breast cancer is a useful predictor of early relapse in bone and may help in selecting patients for subsequent systemic treatment.     

Neural Stem Cells as a Novel Platform for Tumor-Specific Delivery of Therapeutic Antibodies

Background

Recombinant monoclonal antibodies have emerged as important tools for cancer therapy. Despite the promise shown by antibody-based therapies, the large molecular size of antibodies limits their ability to efficiently penetrate solid tumors and precludes efficient crossing of the blood-brain-barrier into the central nervous system (CNS). Consequently, poorly vascularized solid tumors and CNS metastases cannot be effectively treated by intravenously-injected antibodies. The inherent tumor-tropic properties of human neural stem cells (NSCs) can potentially be harnessed to overcome these obstacles and significantly improve cancer immunotherapy. Intravenously-delivered NSCs preferentially migrate to primary and metastatic tumor sites within and outside the CNS. Therefore, we hypothesized that NSCs could serve as an ideal cellular delivery platform for targeting antibodies to malignant tumors.

Methods and Findings

As proof-of-concept, we selected Herceptin™ (trastuzumab), a monoclonal antibody widely used to treat HER2-overexpressing breast cancer. HER2 overexpression in breast cancer is highly correlated with CNS metastases, which are inaccessible to trastuzumab therapy. Therefore, NSC-mediated delivery of trastuzumab may improve its therapeutic efficacy. Here we report, for the first time, that human NSCs can be genetically modified to secrete anti-HER2 immunoglobulin molecules. These NSC-secreted antibodies assemble properly, possess tumor cell-binding affinity and specificity, and can effectively inhibit the proliferation of HER2-overexpressing breast cancer cells in vitro. We also demonstrate that immunoglobulin-secreting NSCs exhibit preferential tropism to tumor cells in vivo, and can deliver antibodies to human breast cancer xenografts in mice.

Conclusions

Taken together, these results suggest that NSCs modified to secrete HER2-targeting antibodies constitute a promising novel platform for targeted cancer immunotherapy. Specifically, this NSC-mediated antibody delivery system has the potential to significantly improve clinical outcome for patients with HER2-overexpressing breast cancer.     

Breast cancer cells induce osteoblast apoptosis: a possible contributor to bone degradation

Breast cancer cells exhibit a predilection for metastasis to bone. There, the metastases usually bring about bone loss with accompanying pain and loss of function. One way that breast cancer cells disrupt the normal pattern of bone remodeling is by activating osteoclasts, the bone degrading cells. Nevertheless, targeting the osteoclasts does not cure the disease or result in bone repair. These observations indicate that osteoblast function also may be compromised. The objective of this study was to investigate the interaction of metastatic breast cancer cells with osteoblasts. Human metastatic breast cancer cells, MDA-MB-435 or MDA-MB-231, or their conditioned media were co-cultured with a human osteoblast line hFOB1.19. The breast cancer cells caused an increase in the prevalence of apoptotic osteoblasts. Apoptotic osteoblasts detected by the TUNEL assay or by caspase activity increased approximately two to fivefold. This increase was not seen with non-metastatic MDA-MB-468 cells. In an investigation of the mechanism, it was determined that the hFOB1.19 cells expressed fas and that fas was functional. Likewise the hFOB1.19 cells were susceptible to TNF-alpha, but this cytokine was not detected in the conditioned medium of the breast cancer cells. This study indicates that osteoblasts are the target of breast cancer cell-induced apoptosis, but fas/fas-ligand and TNF-alpha, two common initiators of cell death, are probably not involved in this aspect of the metastases/bone cell axis. There are several mechanisms that remain to be explored in order to determine how breast cancer cells bring about osteoblast apoptosis. Even though the specific initiator of apoptosis remains to be identified, the results of this study suggest that the mechanism is likely to be novel.     

Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells

Claudins and occludin constitute the major transmembrane proteins of tight junctions (TJs). We have previously identified the human homologue of the murine Cldn1, CLDN1 (SEMP1) that is expressed in normal, mammary gland-derived epithelial cells but is absent in most human breast cancer cell lines. To investigate the potential functions of CLDN1 protein in tumor and normal epithelial cells, we developed an I-NGFR retroviral vector and monoclonal anti-CLDN1 antibody. In subconfluent and confluent breast cancer cells, MDA-MB-435 and MDA-MB-361, endogenous CLDN1 expression was not detected by an anti-CLDN1 monoclonal antibody by Western blot analysis or quantitative RT-PCR. When CLDN1-negative breast cancer cell lines were transduced with a CLDN1 retrovirus the cells express CLDN1 mRNA constitutively as shown by quantitative RT-PCR. Immunofluorescence analyses of the CLDN1 retroviral transduced breast tumor cells using monoclonal antibodies against CLDN1 reveals a subcellular distribution at cell-cell contact sites similar to the CLDN1 homing pattern in T47-D cells, which express endogenous CLDN1. This cell-cell contact co-localization of CLDN1 was evident in CLDN1-transduced breast tumor cells which fail to express occludin protein (MDA-MB-361 and MDA-MB-435) and express relatively little ZO-1 protein (MDA-MB-435), suggesting that other proteins may be responsible for targeting of CLDN1 to cell-cell contact sites. The re-expression of CLDN1 decreases the paracellular flux of 3 and 40 kDa dextran despite the absence of occludin in the MDA-MB-361 tumor cells. Our findings indicate that in CLDN1-negative breast tumor cells, the basal protein partner requirements for physiological homing of the CLDN1 protein are intact, and that CLDN1 gene transfer and protein expression itself might be sufficient to exert a TJ-mediate gate function in metastatic tumor cells even in the absence of other TJ-associated proteins, such as occludin.     

Biochemical-markers for the diagnosis of bone metastasis: a clinical review

The preferential metastasis of cancer cells to skeleton not only disrupts the process of bone remodeling and influences the therapeutic decision, but also results in severe complications. Although the current diagnosis of bone metastases (BM) relies on bone imaging techniques, they are not sensitive enough for early detection as well as they are invasive and expensive to use. Since factors derived from bone metabolism are potentially useful to diagnose metastatic bone disease in cancer patients, a number of clinical trials have been carried out on this area. Results suggest that higher levels of bone biomarkers are associated with an increased risk of BM. As a result, biochemical-markers are showing prospects in early diagnosis of BM. This review summarizes the available evidence on the clinical use of biochemical-markers in the diagnosis of various cancers with high incidence of BM including breast, prostate and lung.     

The CXCR4 Antagonist AMD3465 Regulates Oncogenic Signaling and Invasiveness In Vitro and Prevents Breast Cancer Growth and Metastasis In Vivo

CXCR4, the receptor for stromal-derived factor-1, is reportedly involved in breast carcinogenesis. However, the mechanisms through which CXCR4 contributes to breast cancer cell growth and metastases are poorly understood. In this study, we examined the putative in vitro and in vivo anti-cancer effects of the specific CXCR4 inhibitor AMD3465. Here, we report that AMD3465 triggers a reduction in breast cancer cell invasiveness in vitro, and promotes marked changes in oncogenic signaling proteins including a reduction in STAT3, JAK2, AKT, and CXCR4 phosphorylation and the reduced expression of GSK3 and cMYC. Using three breast cancer cell lines as murine syngeneic immunocompetent breast cancer models, we found that AMD3465 inhibited breast tumor formation and reduced tumor cell metastases to the lung and liver. Furthermore, treatment with AMD3465 significantly reduced the infiltration of myeloid CD11b positive cells at the aforementioned metastatic sites as well as the spleen implying this agent could regulate the formation of the tumor microenvironment and conceivably the premetastatic niche. In conclusion, our studies suggest that AMD3465 inhibits breast cancer growth and metastases by acting on tumor cells as well as immune cells that constitute the tumor microenvironment. This process appears to be regulated, at least in part, through the modulation of oncogenic signaling that includes the STAT3 pathway. Thus, CXCR4 could be a novel target for breast cancer therapy.     

Mechanically stimulated osteocytes reduce the bone-metastatic potential of breast cancer cells in vitro by signaling through endothelial cells

Bone metastases occur in 65% to 75% of patients with advanced breast cancer and significantly worsen their survival and quality of life. We previously showed that conditioned medium (CM) from osteocytes stimulated with oscillatory fluid flow, mimicking bone mechanical loading during routine physical activities, reduced the transendothelial migration of breast cancer cells. Endothelial cells are situated at an ideal location to mediate signals between osteocytes in the bone matrix and metastasizing cancer cells in the blood vessels. In this study, we investigated the specific effects of flow-stimulated osteocytes on the interaction between endothelial cells and breast cancer cells in vitro. We observed that CM from flow-stimulated osteocytes reduced endothelial permeability by 15% and breast cancer cell adhesion onto endothelial monolayers by 18%. The difference in adhesion was abolished with anti-intercellular adhesion molecule 1 (ICAM-1) neutralizing antibodies. Furthermore, CM from endothelial cells conditioned in CM from flow-stimulated osteocytes significantly altered the gene expression in bone-metastatic breast cancer cells, as shown by RNA sequencing. Specifically, breast cancer cell expression of matrix metallopeptidase 9 (MMP-9) was downregulated by 62%, and frizzled-4 (FZD4) by 61%, when the osteocytes were stimulated with flow. The invasion of these breast cancer cells across Matrigel was also reduced by 47%, and this difference was abolished by MMP-9 inhibitors. In conclusion, we demonstrated that flow-stimulated osteocytes downregulate the bone-metastatic potential of breast cancer cells by signaling through endothelial cells. This provides insights into the capability of bone mechanical regulation in preventing bone metastases; and may assist in prescribing exercise or bone-loading regimens to patients with breast cancers.     

A New Mouse Model for the Study of Human Breast Cancer Metastasis

Breast cancer is the most common cancer in women, and this prevalence has a major impact on health worldwide. Localized breast cancer has an excellent prognosis, with a 5-year relative survival rate of 85%. However, the survival rate drops to only 23% for women with distant metastases. To date, the study of breast cancer metastasis has been hampered by a lack of reliable metastatic models. Here we describe a novel in vivo model using human breast cancer xenografts in NOD scid gamma (NSG) mice; in this model human breast cancer cells reliably metastasize to distant organs from primary tumors grown within the mammary fat pad. This model enables the study of the entire metastatic process from the proper anatomical site, providing an important new approach to examine the mechanisms underlying breast cancer metastasis. We used this model to identify gene expression changes that occur at metastatic sites relative to the primary mammary fat pad tumor. By comparing multiple metastatic sites and independent cell lines, we have identified several gene expression changes that may be important for tumor growth at distant sites.     

Imaging of specific activation of photodynamic molecular beacons in breast cancer vertebral metastases

Breast cancer is the second leading cause of cancer-related death in women. Approximately 85% of patients with advanced cases will develop spinal metastases. The vertebral column is the most common site of breast cancer metastases, where overexpression of matrix metalloproteinases (MMPs) promotes the spread of cancer. Current therapies have significant limitations due to the high associated risk of damaging the spinal cord. An attractive alternative is photodynamic therapy providing noninvasive and site-selective treatment. However, current photosensitizers are limited by their nonspecific accumulation. Photodynamic molecular beacons (PP(MMP)B), activated by MMPs, offer another level of PDT selectivity and image-guidance preserving criticial tissues, specifically the spinal cord. Metastatic human breast carcinoma cells, MT-1, were used to model the metastatic behavior of spinal lesions. In vitro and in vivo evidence demonstrates MMP specific activation of PP(MMP)B in MT-1 cells. Using a clinically relevant metastatic model, fluorescent imaging establishes the specific activation of PP(MMP)B by vertebral metastases versus normal tissue (i.e., spinal cord) demonstrating the specificity of these beacons. Here, we validate that the metastasis-selective mechanism of PP(MMP)Bs can specifically image breast cancer vertebral metastases, thereby differentiating tumor and healthy tissue.     

Transmigration characteristics of breast cancer and melanoma cells through the brain endothelium: Role of Rac and PI3K

Brain metastases are common and devastating complications of both breast cancer and melanoma. Although mammary carcinoma brain metastases are more frequent than those originating from melanoma, this latter has the highest tropism to the brain. Using static and dynamic in vitro approaches, here we show that melanoma cells have increased adhesion to the brain endothelium in comparison to breast cancer cells. Moreover, melanoma cells can transmigrate more rapidly and in a higher number through brain endothelial monolayers than breast cancer cells. In addition, melanoma cells have increased ability to impair tight junctions of cerebral endothelial cells. We also show that inhibition of Rac or PI3K impedes adhesion of breast cancer cells and melanoma cells to the brain endothelium. In addition, inhibition of Rac or PI3K inhibits the late phase of transmigration of breast cancer cells and the early phase of transmigration of melanoma cells. On the other hand, the Rac inhibitor EHT1864 impairs the junctional integrity of the brain endothelium, while the PI3K inhibitor LY294002 has no damaging effect on interendothelial junctions. We suggest that targeting the PI3K/Akt pathway may represent a novel opportunity in preventing the formation of brain metastases of melanoma and breast cancer.     

Mechanisms of cancer metastasis to the bone

Some of the most common human cancers, including breast cancer, prostate cancer, and lung cancer, metastasize with avidity to bone. What is the basis for their preferential growth within the bone microenvironment? Bidirectional interactions between tumor cells and cells that make up bone result in a selective advantage for tumor growth and can lead to bone destruction or new bone matrix deposition. This review discusses our current understanding of the molecular components and mechanisms that are responsible for those interactions.     

Breast Cancer and Obesity: In Vitro Interferences between Adipokines and Proangiogenic Features and/or Antitumor Therapies?

Obesity is now considered as a risk factor for breast cancer in postmenopausal women. Adipokine levels are modulated in obesity, and may play a role in carcinogenesis. Moreover, obesity increases risk of cancer mortality. Here, we hypothesized that this increase could be due to a modification in angiogenesis, capital event in the development of metastases, and/or in effectiveness of cancer treatments. To test these assumptions, following a same experimental design and simultaneously the effects of leptin and adiponectin on angiogenesis were investigated, and the impact of hyperleptinemia on anticancer drug effectiveness was measured in physiological and obesity situations. Focusing on angiogenesis, the proliferation of endothelial cells (HUVEC), which expressed leptin and adiponectin receptors, was stimulated by leptin and inhibited by adiponectin. Both adipokines globally reduced apoptosis and caspase activity. Leptin increased migration whereas adiponectin decreased migration, and leptin enhanced the area of the tubes formed by HUVEC cells while adiponectin inhibited their formation. MCF7 and MDA-MB-231 cells treated with leptin secreted more VEGF than untreated cells, whereas adiponectin treatment inhibited VEGF secretion. Finally, MCF7 cells pre-treated with leptin were more invasive than untreated cells. This effect was not reproduced in MDA-MB-231 cells. In the MCF7 breast cancer cell line, leptin could induce cell proliferation and reduced the efficacy of all breast cancer therapies (tamoxifen, 5-fluorouracil, taxol and vinblastin). These results suggest that, in obesity situation, leptin– in contrast to adiponectin – may promote tumor invasion and angiogenesis, leading to metastases ‘apparition, and reduce treatment efficacy, which could explain the increased risk of cancer mortality in cases of overweight. The evidence suggests adipokines influence breast cancer issue and could play a significant role, especially in obese patients for which hyperleptinemia, hypoadiponectinemia and increased metastatic potential are described.