Tumor‐associated macrophages exhibit pro‐ and anti‐inflammatory properties by which they impact on pancreatic tumorigenesis

Pancreatic ductal adenocarcinoma (PDAC) still ranking 4th in the order of fatal tumor diseases is characterized by a profound tumor stroma with high numbers of tumor‐associated macrophages (TAMs). Driven by environmental factors, monocytes differentiate into M1‐ or M2‐macrophages, the latter commonly regarded as being protumorigenic. Because a detailed analysis of TAMs in human PDAC development is still lacking, freshly isolated PDAC‐derived TAMs were analyzed for their phenotype and impact on epithelial‐mesenchymal‐transition (EMT) of benign (H6c7) and malignant (Colo357) pancreatic ductal epithelial cells. TAMs exhibited characteristics of M1‐macrophages (expression of HLA‐DR, IL‐1β, or TNF‐α) and M2‐macrophages (expression of CD163 and IL‐10). In the presence of TAMs, H6c7, and Colo357 cells showed an elongated cell shape along with an increased expression of mesenchymal markers such as vimentin and reduced expression of epithelial E‐cadherin. Similar to TAMs, in vitro generated M1‐ and M2‐macrophages both mediated EMT in H6c7 and Colo357 cells. M1‐macrophages acquired M2‐characteristics during coculture that could be prevented by GM‐CSF treatment. However, M1‐macrophages still potently induced EMT in H6c7 and Colo357 cells although lacking M2‐characteristics. Overall, these data demonstrate that TAMs exhibit anti‐ as well as proinflammatory properties that equally contribute to EMT induction in PDAC initiation and development.     

Tumor‐associated macrophages secrete CC‐chemokine ligand 2 and induce tamoxifen resistance by activating PI3K/Akt/mTOR in breast cancer

Breast cancer is the most prevalent malignancy among women. Although endocrine therapy is effective, the development of endocrine resistance is a major clinical challenge. The tumor microenvironment (TME) promotes tumor malignancy, and tumor‐associated macrophages (TAM) within the TME play a crucial role in endocrine resistance. Herein, we aimed to elucidate the relationship between TAM and the endocrine‐resistant phenotype of breast cancer. Macrophages were cultured with conditioned medium (CM) from tamoxifen‐sensitive (MCF7‐S) or ‐resistant (MCF7‐R) MCF7 breast cancer cells. M2 polarization was detected by CD163 immunofluorescence. To determine the effect on endocrine resistance, MCF7 cells were cultured in the supernatant of different TAM, and then treated with tamoxifen. CC‐chemokine ligand 2 (CCL2) immunohistochemistry was carried out on pathological sections from 100 patients with invasive estrogen receptor‐positive breast cancer. We found that macrophages cultured in the CM of MCF7‐S and MCF7‐R cells were induced into TAM, with a more obvious M2 polarization in the latter. Tamoxifen resistance was increased by culture in TAM medium. TAM secreted CCL2, which increased endocrine resistance in breast cancer cells through activation of the PI3K/Akt/mTOR signaling pathway. High expression of CCL2 was correlated with infiltration of CD163+macrophages (r = 0.548, P < .001), and patients with high CCL2 expression presented shorter progression‐free survival than those with low CCL2 expression (P < .05). We conclude that CCL2 secreted by TAM activates PI3K/Akt/mTOR signaling and promotes an endocrine resistance feedback loop in the TME, suggesting that CCL2 and TAM may be novel therapeutic targets for patients with endocrine‐resistant breast cancer.     

Sphingosine kinase 2 deficient tumor xenografts show impaired growth and fail to polarize macrophages towards an anti‐inflammatory phenotype

A challenging task of the immune system is to fight cancer cells. However, a variety of human cancers educate immune cells to become tumor supportive. This is exemplified for tumor‐associated macrophages (TAMs), which are polarized towards an anti‐inflammatory and cancer promoting phenotype. Mechanistic explanations, how cancer cells influence the macrophage phenotype are urgently needed to address potential anti‐cancer strategies along this line. One potential immune modulating compound, sphingosine‐1‐phosphate (S1P), was recently highlighted in both tumor growth and immune modulation. Using a xenograft model in nude mice, we demonstrate a supportive role of sphingosine kinase 2 (SphK2), one of the S1P‐producing enzymes for tumor progression. The growth of SphK2‐deficient MCF‐7 breast tumor xenografts was markedly delayed when compared with controls. Infiltration of macrophages in SphK2‐deficient and control tumors was comparable. However, TAMs from SphK2‐deficient tumors displayed a pronounced anti‐tumor phenotype, showing an increased expression of pro‐inflammatory markers/mediators such as NO, TNF‐α, IL‐12 and MHCII and a low expression of anti‐inflammatory IL‐10 and CD206. These data suggest a role for S1P, generated by SphK2, in early tumor development by affecting macrophage polarization. © 2009 UICC     

Interleukin‐34 promotes tumor progression and metastatic process in osteosarcoma through induction of angiogenesis and macrophage recruitment

Interleukin‐34 (IL‐34) was recently characterized as the M‐CSF “twin” cytokine, regulating the proliferation/differentiation/survival of myeloid cells. The implication of M‐CSF in oncology was initially suspected by the reduced metastatic dissemination in knock‐out mice, due to angiogenesis impairment. Based on this observation, our work studied the involvement of IL‐34 in the pathogenesis of osteosarcoma. The in vivo effects of IL‐34 were assessed on tissue vasculature and macrophage infiltration in a murine preclinical model based on a paratibial inoculation of human osteosarcoma cells overexpressing or not IL‐34 or M‐CSF. In vitro investigations using endothelial cell precursors and mature HUVEC cells were performed to analyse the involvement of IL‐34 in angiogenesis and myeloid cell adhesion. The data revealed that IL‐34 overexpression was associated with the progression of osteosarcoma (tumor growth, lung metastases) and an increase of neo‐angiogenesis. In vitro analyses demonstrated that IL‐34 stimulated endothelial cell proliferation and vascular cord formation. Pre‐treatment of endothelial cells by chondroitinases/heparinases reduced the formation of vascular tubes and abolished the associated cell signalling. In addition, IL‐34 increased the in vivo recruitment of M2 tumor‐associated macrophages into the tumor tissue. IL‐34 increased in vitro monocyte/CD34⁺ cell adhesion to activated HUVEC monolayers under physiological shear stress conditions. This work also demonstrates that IL‐34 is expressed by osteosarcoma cells, is regulated by TNF‐α, IL‐1β, and contributes to osteosarcoma growth by increasing the neo‐angiogenesis and the recruitment of M2 macrophages. By promoting new vessel formation and extravasation of immune cells, IL‐34 may play a key role in tumor development and inflammatory diseases.     

Tumor necrosis factor alpha induces Warburg‐like metabolism and is reversed by anti‐inflammatory curcumin in breast epithelial cells

The reprogramming of cellular metabolism in cancer cells is a well‐documented effect. It has previously been shown that common oncogene expression can induce aerobic glycolysis in cancer cells. However, the direct effect of an inflammatory microenvironment on cancer cell metabolism is not known. Here, we illustrate that treatment of nonmalignant (MCF‐10a) and malignant (MCF‐7) breast epithelial cells with low‐level (10 ng/ml) tumor necrosis factor alpha (TNF‐α) significantly increased glycolytic reliance, lactate export and expression of the glucose transporter 1 (GLUT1). TNF‐α decreased total mitochondrial content; however, oxygen consumption rate was not significantly altered, suggesting that overall mitochondrial function was increased. Upon glucose starvation, MCF7 cells treated with TNF‐α demonstrated significantly lower viability than nontreated cells. Interestingly, these properties can be partially reversed by coincubation with the anti‐inflammatory agent curcumin in a dose‐dependent manner. This work demonstrates that aerobic glycolysis can be directly induced by an inflammatory microenvironment independent of additional genetic mutations and signals from adjacent cells. Furthermore, we have identified that a natural dietary compound can reverse this effect.     

Breast carcinoma cells modulate the chemoattractive activity of human bone marrow‐derived mesenchymal stromal cells by interfering with CXCL12

We investigated whether breast tumor cells can modulate the function of mesenchymal stromal cells (MSCs) with a special emphasis on their chemoattractive activity towards hematopoietic stem and progenitor cells (HSPCs). Primary MSCs as well as a MSC line (SCP‐1) were cocultured with primary breast cancer cells, MCF‐7, MDA‐MB231 breast carcinoma or MCF‐10A non‐malignant breast epithelial cells or their conditioned medium. In addition, the frequency of circulating clonogenic hematopoietic progenitors was determined in 78 patients with breast cancer and compared with healthy controls. Gene expression analysis of SCP‐1 cells cultured with MCF‐7 medium revealed CXCL12 (SDF‐1) as one of the most significantly downregulated genes. Supernatant from both MCF‐7 and MDA‐MB231 reduced the CXCL12 promoter activity in SCP‐1 cells to 77% and 47%, respectively. Moreover, the CXCL12 mRNA and protein levels were significantly reduced. As functional consequence of lower CXCL12 levels, we detected a decreased trans‐well migration of HSPCs towards MSC/tumor cell cocultures or conditioned medium. The specificity of this effect was confirmed by blocking studies with the CXCR4 antagonist AMD3100. Downregulation of SP1 and increased miR‐23a levels in MSCs after contact with tumor cell medium as well as enhanced TGFβ1 expression were identified as potential molecular regulators of CXCL12 activity in MSCs. Moreover, we observed a significantly higher frequency of circulating colony‐forming hematopoietic progenitors in patients with breast cancer compared with healthy controls. Our in vitro results propose a potential new mechanism by which disseminated tumor cells in the bone marrow may interfere with hematopoiesis by modulating CXCL12 in protected niches.     

miR‐199a‐5p regulates β1 integrin through Ets‐1 to suppress invasion in breast cancer

Increasing evidence has revealed that miR‐199a‐5p is actively involved in tumor invasion and metastasis as well as in the decline of breast cancer tissues. In this research, overexpression of miR‐199a‐5p weakened motility and invasion of breast cancer cells MCF‐7 and MDA‐MB‐231. Upregulation of Ets‐1 increased breast cancer cell invasion, but the mechanism by which miR‐199a‐5p modulates activation of Ets‐1 in breast cancer was not clarified. We investigated the relationship between miR‐199a‐5p and Ets‐1 on the basis of 158 primary breast cancer case specimens, and the results showed that Ets‐1 expression was inversely correlated with endogenous miR‐199a‐5p. Overexpression of miR‐199a‐5p reduced the mRNA and protein levels of Ets‐1 in MCF‐7 and MDA‐MB‐231 cells, whereas anti‐miR‐199a‐5p elevated Ets‐1. siRNA‐mediated Ets‐1 knockdown phenocopied the inhibition invasion of miR‐199a‐5p in vitro. Moreover, luciferase reporter assay revealed that miR‐199a‐5p directly targeted 3′‐UTR of Ets‐1 mRNA. This research revealed that miR‐199a‐5p could descend the levels of β1 integrin by targeting 3′‐UTR of Ets‐1 to alleviate the invasion of breast cancer via FAK/Src/Akt/mTOR signaling pathway. Our results provide insight into the regulation of β1 integrin through miR‐199a‐5p‐mediated Ets‐1 silence and will help in designing new therapeutic strategies to inhibit signal pathways induced by miR‐199a‐5p in breast cancer invasion.     

p14ARF induces apoptosis via an entirely caspase‐3‐dependent mitochondrial amplification loop

The p14^ARF tumor suppressor triggers cell death or cell cycle arrest upon oncogenic stress. In MCF‐7 breast carcinoma cells, expression of the tumor suppressor gene p14^ARF fails to trigger apoptosis but induces an arrest in the G1 and, to a lesser extent, in the G2 phase in the cell division cycle. Here, inhibition of cell cycle arrest resulted in apoptosis induction in caspase‐3 proficient MCF‐7 cells upon expression of p14^ARF. This occurred in the absence of S‐phase progression or mitotic entry. In contrast, syngeneic, caspase‐3‐deficient MCF‐7 cells remained entirely resistant to p14^ARF‐induced apoptosis. Thus, cell cycle checkpoint abrogation overcomes resistance to p14^ARF‐induced cell death and promotes cell death via a caspase‐3‐dependent pathway. Cell death coincided with dissipation of the mitochondrial membrane potential, release of cytochrome c, and was inhibitable by pan‐caspase inhibitors and the caspase‐3/7 inhibitor zDEVD‐fmk. Of note, mitochondrial events of apoptosis execution depended entirely on caspase‐3 proficiency indicating that caspase‐3 either acts “up‐stream” of the mitochondria in a “non‐canonical” pathway or mediates a mitochondrial feedback loop to amplify the apoptotic caspase signal in p14^ARF‐induced stress signaling.     

Colony stimulating factor 1 receptor blockade improves the efficacy of chemotherapy against human neuroblastoma in the absence of T lymphocytes

Tumor‐associated macrophages can promote growth of cancers. In neuroblastoma, tumor‐associated macrophages have greater frequency in metastatic versus loco‐regional tumors, and higher expression of genes associated with macrophages helps to predict poor prognosis in the 60% of high‐risk patients who have MYCN‐non‐amplified disease. The contribution of cytotoxic T‐lymphocytes to anti‐neuroblastoma immune responses may be limited by low MHC class I expression and low exonic mutation frequency. Therefore, we modelled human neuroblastoma in T‐cell deficient mice to examine whether depletion of monocytes/macrophages from the neuroblastoma microenvironment by blockade of CSF‐1R can improve the response to chemotherapy. In vitro, CSF‐1 was released by neuroblastoma cells, and topotecan increased this release. In vivo, neuroblastomas formed by subcutaneous co‐injection of human neuroblastoma cells and human monocytes into immunodeficient NOD/SCID mice had fewer human CD14⁺ and CD163⁺ cells and mouse F4/80⁺ cells after CSF‐1R blockade. In subcutaneous or intra‐renal models in immunodeficient NSG or NOD/SCID mice, CSF‐1R blockade alone did not affect tumor growth or mouse survival. However, when combined with cyclophosphamide plus topotecan, the CSF‐1R inhibitor BLZ945, either without or with anti‐human and anti‐mouse CSF‐1 mAbs, inhibited neuroblastoma growth and synergistically improved mouse survival. These findings indicate that depletion of tumor‐associated macrophages from neuroblastomas can be associated with increased chemotherapeutic efficacy without requiring a contribution from T‐lymphocytes, suggesting the possibility that combination of CSF‐1R blockade with chemotherapy might be effective in patients who have limited anti‐tumor T‐cell responses.     

Distinct chemotherapy‐associated anti‐cancer immunity by myeloid cells inhibition in murine pancreatic cancer models

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy associated with an extremely poor prognosis. Chemotherapy, such as gemcitabine (GEM), is the only treatment for PDAC patients who are not suitable for radical surgical treatment; however, its anti‐tumor efficacy is limited. In this study, we investigated the host immune system response in murine PDAC models undergoing GEM treatment. We found that PDAC tumor tissues were infiltrated with a substantial number of Gr‐1+ myeloid cells and had relatively small numbers of CD4+ and CD8+ cells. In addition, there were increased numbers of myeloid cells expressing CD11b+ and Gr‐1+ in peripheral blood. When mice with PDAC tumors in the intraperitoneal cavity or liver were treated with GEM, numbers of myeloid cells in tumor tissues and in peripheral blood decreased. In contrast, numbers of CD4+ or CD8+ cells increased. In peripheral blood, the numbers of CD8+ cells expressing interferon‐gamma (IFN‐γ) were higher in GEM‐treated mice than in untreated mice. In addition, GEM treatment in combination with myeloid cell depletion further prolonged the survival of PDAC mice. The gene expression profile of peripheral blood in myeloid cell‐depleted PDAC mice treated with GEM showed biological processes related to anti‐cancer immunity, such as natural killer cell‐mediated cytotoxicity, type I IFN signaling, and co‐stimulatory signaling for T cell activation. Thus, in PDAC murine models, GEM treatment was associated with an immune response consistent with an anti‐cancer effect, and depletion of myeloid‐lineage cells played an important role in enhancing anti‐cancer immunity associated with GEM treatment.     

Tissue inhibitor of metalloproteinase‐1 protects MCF‐7 breast cancer cells from paclitaxel‐induced apoptosis by decreasing the stability of cyclin B1

Paclitaxel (PTX) is a very effective drug in treating tumors. It disturbs microtubule dynamics and impairs the transition of cells from metaphase to anaphase in mitosis, leading to cell death by apoptosis. However, the effectiveness of PTX in cancer chemotherapy is hampered by drug resistance in some patients. Tissue inhibitor of metalloproteinase‐1 (TIMP‐1) is well known to be capable of inhibiting apoptosis. Elevated tumor tissue TIMP‐1 levels have been significantly associated with a poor response to chemotherapy. We hypothesized that TIMP‐1 could reduce the sensitivity of breast cancer cells to PTX by inhibiting apoptosis. To test this hypothesis, we first examined the effects of TIMP‐1 on the apoptosis induced by PTX and investigated the effects of TIMP‐1 on the expression and stability of cyclin B1 that critically regulates the metaphase to anaphase transition during mitosis in MCF‐7 breast cancer cells. Our data demonstrate that TIMP‐1 could significantly decrease the sensitivity of MCF‐7 cells to PTX‐induced apoptosis, attenuate mitotic blockage in G₂/M, and enhance the degradation of cyclin B1. To further investigate whether the inhibitory effect of TIMP‐1 on PTX‐induced apoptosis is mediated by lowering levels of cyclin B1, a cyclin B1‐expression plasmid was transfected into clone overexpressing TIMP‐1. The levels of PTX‐induced apoptosis were then analyzed. The data showed that the TIMP‐1‐based decrease in PTX‐induced apoptosis was reversed by cyclin B1. Our data indicate that TIMP‐1 can protect breast cancer cells from PTX‐induced apoptosis by decreasing the stability of cyclin B1.     

Bisphosphonates suppress insulin‐like growth factor 1‐induced angiogenesis via the HIF‐1α/VEGF signaling pathways in human breast cancer cells

Adjunctive chemotherapy with bisphosphonates has been reported to delay bone metastasis and improve overall survival in breast cancer. Aside from its antiresorptive effect, bisphosphonates exhibit antitumor activities, in vitro and in vivo, via several mechanisms, including antiangiogenesis. In this study, we investigated the potential molecular mechanisms underlying the antiangiogenic effect of non–nitrogen‐containing and nitrogen‐containing bisphosphonates, clodronate and pamidronate, respectively, in insulin‐like growth factor (IGF)‐1 responsive human breast cancer cells. We tested whether bisphosphonates had any effects on hypoxia‐inducible factor (HIF)‐1α/vascular endothelial growth factor (VEGF) axis that plays a pivotal role in tumor angiogenesis, and our results showed that both pamidronate and clodronate significantly suppressed IGF‐1‐induced HIF‐1α protein accumulation and VEGF expression in MCF‐7 cells. Mechanistically, we found that either pamidronate or clodronate did not affect mRNA expression of HIF‐1α, but they apparently promoted the degradation of IGF‐1‐induced HIF‐1α protein. Meanwhile, we found that the presence of pamidronate and clodronate led to a dose‐dependent decease in the newly‐synthesized HIF‐1α protein induced by IGF‐1 in breast cancer cells after proteasomal inhibition, thus, indirectly reflecting the inhibition of protein synthesis. In addition, our results indicated that the inhibitory effects of bisphosphonates on the HIF‐1α/VEGF axis are associated with the inhibition of the phosphoinositide 3‐kinase/AKT/mammalian target of rapamycin signaling pathways. Consistently, we demonstrated that pamidronate and clodronate functionally abrogated both in vitro and in vivo tumor angiogenesis induced by IGF‐1‐stimulated MCF‐7 cells. These findings have highlighted an important mechanism of the pharmacological action of bisphosphonates in the inhibition of tumor angiogenesis in breast cancer cells.     

Resveratrol ameliorates Lewis lung carcinoma‐bearing mice development,decreases granulocytic myeloid‐derived suppressor cell accumulation and impairs its suppressive ability

Myeloid‐derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells which consist of 2 subsets: granulocytic MDSC (G‐MDSC) and monocytic MDSC (M‐MDSC). MDSC expand in tumor‐bearing hosts and contribute to immunotherapeutic resistance by remarkably blocking effector T‐cell activation via different mechanisms. Resveratrol (RSV) is a polyphenol and it has been widely used for its various health benefits. However, the underlying mechanism of its anti‐tumor properties remains unclear. In this study, a transplantable mouse model was used to investigate the effects of RSV on MDSC. The results showed that RSV ameliorated tumor development by decreasing G‐MDSC accumulation, impairing its suppressive ability on CD8⁺T cells and promoting M‐MDSC differentiation into CD11c⁺ and F4/80⁺ cells. Our results indicated that RSV should be considered as a modular of MDSC suppressive function and that RSV is a novel booster for tumor immunotherapy.     

Regulation of CD44 expression and focal adhesion by Golgi phosphatidylinositol 4‐phosphate in breast cancer

CD44, a transmembrane receptor, is expressed in the standard or variant form and plays a critical role in tumor progression and metastasis. This protein regulates cell adhesion and migration in breast cancer cells. We previously reported that phosphatidylinositol‐4‐phosphate (PI(4)P) at the Golgi regulates cell migration and invasion in breast cancer cell lines. In this study, we showed that an increase in PI(4)P levels at the Golgi by knockdown of PI(4)P phosphatase SAC1 increased the expression of standard CD44, variant CD44, and ezrin/radixin phosphorylation and enhanced the formation of focal adhesions mediated by CD44 and ezrin/radixin in MCF7 and SK‐BR‐3 cells. In contrast, knockdown of PI 4‐kinase IIIβ in highly invasive MDA‐MB‐231 cells decreased these factors. These results suggest that SAC1 expression and PI(4)P at the Golgi are important in tumor progression and metastasis and are potential prognostic markers of breast cancers.     

Inhibition of constitutively activated phosphoinositide 3‐kinase/AKT pathway enhances antitumor activity of chemotherapeutic agents in breast cancer susceptibility gene 1‐defective breast cancer cells

Loss or decrease of wild type BRCA1 function, by either mutation or reduced expression, has a role in hereditary and sporadic human breast and ovarian cancers. We report here that the PI3K/AKT pathway is constitutively active in BRCA1‐defective human breast cancer cells. Levels of phospho‐AKT are sustained even after serum starvation in breast cancer cells carrying deleterious BRCA1 mutations. Knockdown of BRCA1 in MCF7 cells increases the amount of phospho‐AKT and sensitizes cells to small molecule protein kinase inhibitors (PKIs) targeting the PI3K/AKT pathway. Restoration of wild type BRCA1 inhibits the activated PI3K/AKT pathway and de‐sensitizes cells to PKIs targeting this pathway in BRCA1 mutant breast cancer cells, regardless of PTEN mutations. In addition, clinical PI3K/mTOR inhibitors, PI‐103, and BEZ235, showed anti‐proliferative effects on BRCA1 mutant breast cancer cell lines and synergism in combination with chemotherapeutic drugs, cisplatin, doxorubicin, topotecan, and gemcitabine. BEZ235 synergizes with the anti‐proliferative effects of gemcitabine by enhancing caspase‐3/7 activity. Our results suggest that the PI3K/AKT pathway can be an important signaling pathway for the survival of BRCA1‐defective breast cancer cells and pharmacological inhibition of this pathway is a plausible treatment for a subset of breast cancers. © 2012 Wiley Periodicals, Inc.     

Targeting claudin‐4 enhances chemosensitivity in breast cancer

Triple negative breast cancer (TNBC) is characterized by highly aggressive phenotype, limited treatment options and a poor prognosis. In the present study, we examined the therapeutic effect of anti–claudin (CLDN)‐4 extracellular domain antibody, 4D3, on TNBC. When the expression of CLDN4 and CLDN1 in invasive ductal carcinoma (IDC) was examined in 114 IDC (78 cases from 2004 to 2009 in a single center and 36 cases of tissues array), CLDN1 had lower expression than CLDN4 and was correlated with histological grade. In contrast, expression of CLDN4 was correlated with histological grade, receptor subtype, and stage. CLDN4 expression in human IDC cell lines MCF‐7 (luminal subtype) and MDA‐468 (TNBC) was at the same level. In both cells, paclitaxel (PTX)‐induced growth suppression was enhanced by 4D3. Furthermore, 4D3 increased both intracellular PTX concentration (in both cells) and apoptosis. In the mouse model, 4D3 promoted the antitumor effect of PTX on subcutaneous tumors and reduced lung metastasis. The combination of PTX and 4D3 reduced M2 macrophages and mesenchymal stem cells in the tumor. 4D3 also reduced stemness of the tumors and increased the intratumoral pH. Moreover, concurrent treatment with 4D3, PTX and tamoxifen, or with PTX and tamoxifen in MDA‐468 also showed the same level of antitumor activity and survival as MCF‐7. Furthermore, in a bone metastasis model, combination of PTX and bisphosphonate with 4D3 promoted tumor growth in both cells. Thus, CLDN4 targeting of the antibody facilitated existing therapeutic effects.     

Receptor activator for nuclear factor‐κB ligand signaling promotes progesterone‐mediated estrogen‐induced mammary carcinogenesis

Breast cancer is a leading cause of cancer‐related death in women. Prolonged exposure to the ovarian hormones estrogen and progesterone increases the risk of breast cancer. Although estrogen is known as a primary factor in mammary carcinogenesis, very few studies have investigated the role of progesterone. Receptor activator for nuclear factor‐κB (NF‐κB) ligand (RANKL) plays an important role in progesterone‐induced mammary carcinogenesis. However, the molecular mechanism underlying RANKL‐induced mammary carcinogenesis remains unknown. In our current study, we show that RANKL induces glioma‐associated oncogene homolog 1 (GLI‐1) in estrogen‐induced progesterone‐mediated mammary carcinogenesis. In vivo experiments were carried out using ACI rats and in vitro experiments were carried out in MCF‐7 cells. In ACI rats, mifepristone significantly reduced the incidence of mammary tumors. Likewise, mifepristone also inhibited the proliferation of MCF‐7 cells. Hormone treatments induced RANKL, receptor activator of NF‐κB (RANK), and NF‐κB in a protein kinase B‐dependent manner and inhibited apoptosis by activation of anti‐apoptotic protein Bcl2 in mammary tumors and MCF‐7 cells. Mechanistic studies in MCF‐7 cells reveal that RANKL induced upstream stimulatory factor‐1 and NF‐κB, resulting in subsequent activation of their downstream target GLI‐1. We have identified that progesterone mediates estrogen‐induced mammary carcinogenesis through activation of GLI‐1 in a RANKL‐dependent manner.     

In vivo imaging of membrane type‐1 matrix metalloproteinase with a novel activatable near‐infrared fluorescence probe

Membrane type‐1 matrix metalloproteinase (MT1‐MMP) is a protease activating MMP‐2 that mediates cleavage of extracellular matrix components and plays pivotal roles in tumor migration, invasion and metastasis. Because in vivo noninvasive imaging of MT1‐MMP would be useful for tumor diagnosis, we developed a novel near‐infrared (NIR) fluorescence probe that can be activated following interaction with MT1‐MMP in vivo. MT1‐hIC7L is an activatable fluorescence probe comprised of anti‐MT1‐MMP monoclonal antibodies conjugated to self‐assembling polymer micelles that encapsulate NIR dyes (IC7‐1, λ_em: 858 nm) at concentrations sufficient to cause fluorescence self‐quenching. In aqueous buffer, MT1‐hIC7L fluorescence was suppressed to background levels and increased approximately 35.5‐fold in the presence of detergent. Cellular uptake experiments revealed that in MT1‐MMP positive C6 glioma cells, MT1‐hIC7L showed significantly higher fluorescence that increased with time as compared to hIC7L, a negative control probe lacking the anti‐MT1‐MMP monoclonal antibody. In MT1‐MMP negative MCF‐7 breast adenocarcinoma cells, both MT1‐hIC7L and hIC7L showed no obvious fluorescence. In addition, the fluorescence intensity of C6 cells treated with MT1‐hIC7L was suppressed by pre‐treatment with an MT1‐MMP endocytosis inhibitor (P < 0.05). In vivo optical imaging using probes intravenously administered to tumor‐bearing mice showed that MT1‐hIC7L specifically visualized C6 tumors (tumor‐to‐background ratios: 3.8 ± 0.3 [MT1‐hIC7L] vs 3.1 ± 0.2 [hIC7L] 48 h after administration, P < 0.05), while the probes showed similarly low fluorescence in MCF‐7 tumors. Together, these results show that MT1‐hIC7L would be a potential activatable NIR probe for specifically detecting MT1‐MMP‐expressing tumors.