Tumor‐secreted products repress B‐cell lymphopoiesis in a murine model of breast cancer

Growing cancers are known to modify immune responses through suppressive mechanisms manifested within the local tumor microenvironment. Accumulating evidence indicates that secreted tumor products can also influence on distant immunological compartments, including myelopoiesis in the bone marrow. However, it is unknown if a similar effect can occur to regulate B‐cell lymphopoiesis in breast cancer. Examining the MMTV‐PyMT murine model of breast cancer, we show a complete block in bone marrow B‐cell lymphopoiesis, which is dependent on tumor burden. We also observed an increase in the total number of splenic B cells and an elevated frequency of marginal zone B cells. By using in vitro assays of B‐cell lymphopoiesis, we show that tumor‐secreted molecules directly inhibit B‐cell progenitor proliferation and favor maturation. These data demonstrate a profound sensitivity of B‐cell lymphopoiesis to the accumulation of ectopically produced molecules during tumor growth in PyMT.     

Collaboration of cancer‐associated fibroblasts and tumour‐associated macrophages for neuroblastoma development

Neuroblastoma is the most common extracranial solid tumour in children and is histologically classified by its Schwannian stromal cells. Although having fewer Schwannian stromal cells is generally associated with more aggressive phenotypes, the exact roles of other stromal cells (mainly macrophages and fibroblasts) are unclear. Here, we examined 41 cases of neuroblastoma using immunohistochemistry for the tumour‐associated macrophage (TAM) markers CD68, CD163, and CD204, and a cancer‐associated fibroblast (CAF) marker, alpha smooth muscle actin (αSMA). Each case was assigned to low/high groups on the basis of the number of TAMs or three groups on the basis of the αSMA‐staining area for CAFs. Both the number of TAMs and the area of CAFs were significantly correlated with clinical stage, MYCN amplification, bone marrow metastasis, histological classification, histological type, and risk classification. Furthermore, TAM settled in the vicinity of the CAF area, suggesting their close interaction within the tumour microenvironment. We next determined the effects of conditioned medium of a neuroblastoma cell line (NBCM) on bone marrow‐derived mesenchymal stem cells (BM‐MSCs) and peripheral blood mononuclear cell (PBMC)‐derived macrophages in vitro. The TAM markers CD163 and CD204 were significantly up‐regulated in PBMC‐derived macrophages treated with NBCM. The expression of αSMA by BM‐MSCs was increased in NBCM‐treated cells. Co‐culturing with CAF‐like BM‐MSCs did not enhance the invasive ability but supported the proliferation of tumour cells, whereas tumour cells co‐cultured with TAM‐like macrophages had the opposite effect. Intriguingly, TAM‐like macrophages enhanced not only the invasive abilities of tumour cells and BM‐MSCs but also the proliferation of BM‐MSCs. CXCL2 secreted from TAM‐like macrophages plays an important role in tumour invasiveness. Taken together, these results indicate that PBMC‐derived macrophages and BM‐MSCs are recruited to a tumour site and activated into TAMs and CAFs, respectively, followed by the formation of favourable environments for neuroblastoma progression. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Protease‐activated receptor 2 suppresses lymphangiogenesis and subsequent lymph node metastasis in a murine pancreatic cancer model

Protease‐activated receptor‐2 (PAR‐2) is a G protein‐coupled receptor that functions as a cell‐surface sensor for coagulation factors and other proteases associated with the tumour microenvironment. Pancreatic cancer cells express high levels of PAR‐2 and activation of PAR‐2 may induce their proliferation and migration. Interestingly, however, PAR‐2 expression is increased in stroma‐rich pancreatic cancer regions, suggesting a potential role of PAR‐2 in the tumour microenvironment. Here, we assessed the importance of PAR‐2 in the stromal compartment by utilizing an orthotopic pancreatic cancer model, in which tumour cells are PAR‐2‐positive, whereas stromal cells are PAR‐2‐negative. We assessed tumour weight and volume and analysed proliferation and (lymph)angiogenesis both in vivo and in vitro. We show that genetic ablation of PAR‐2 from the stromal compartment inhibits primary tumour growth, which is accompanied by reduced vascularization in primary tumours and reduced in tube formation of vascular endothelial cells in vitro. In contrast to smaller primary tumours, the number of lymph node metastases was increased in PAR‐2‐deficient animals, which was accompanied by an increased number of lymphatic vessels. In vitro tube‐formation assays show that PAR‐2 does not inhibit the intrinsic tube‐forming capacity of lymphatic endothelial cells, but that PAR‐2 actually inhibits cancer cell‐induced tube formation. Overall, stromal PAR‐2 thus plays a dual role in pancreatic cancer development by potentiating primary tumour growth but limiting lymphangiogenesis and subsequent lymph node metastasis. Our data identify a novel role of PAR‐2 in the tumour microenvironment and pinpoint PAR‐2 as a negative regulator of lymphangiogenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

A tissue reconstitution model to study cancer cell‐intrinsic and ‐extrinsic factors in mammary tumourigenesis

The contribution of cancer cell‐intrinsic and ‐extrinsic factors to metastatic breast cancer is still poorly understood, hampering development of novel therapeutic strategies that decrease breast cancer mortality. Cre/loxP‐based conditional mouse models of breast cancer present unique opportunities to study sporadic tumour formation and progression in a controlled setting. Unfortunately, the generation of mouse strains carrying multiple mutant alleles needed for such studies is very time‐consuming. Moreover, conditional mouse tumour models do not permit independent manipulation of tumour cell‐intrinsic and ‐extrinsic factors. Although the latter can be achieved by cleared fat‐pad transplantation of mouse mammary epithelial cells (MMECs) from tumour suppressor gene (TSG) knockouts into wild‐type or mutant recipients, this procedure is not possible for mutations that cause embryonic lethality or preclude mammary gland development. Here we show that cleared fat‐pad transplantations with MMECs isolated from K14cre;Cdh1^F/F; Trp53^F/F mice expressing Cre recombinase under control of the cytokeratin‐14 promoter and carrying conditional null alleles for p53 and E‐cadherin (Cdh1) first resulted in the formation of phenotypically normal mammary glands, followed by the development of invasive metastatic mammary tumours. Tumour formation in the recipients mimicked tumour latency, spectrum, morphology, immunophenotype, and metastatic characteristics of the original mammary tumour model. This transplantation system, which can be expanded to other conditional TSG knockouts, permits independent genetic analysis of stromal factors and testing of additional cancer cell‐intrinsic mutations that would otherwise be embryonic lethal or require intensive breeding. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Lipocalin‐2 ensures host defense against Salmonella Typhimurium by controlling macrophage iron homeostasis and immune response

Lipocalin‐2 (Lcn2) is an innate immune peptide with pleiotropic effects. Lcn2 binds iron‐laden bacterial siderophores, chemo‐attracts neutrophils and has immunomodulatory and apoptosis‐regulating effects. In this study, we show that upon infection with Salmonella enterica serovar Typhimurium, Lcn2 promotes iron export from Salmonella‐infected macrophages, which reduces cellular iron content and enhances the generation of pro‐inflammatory cytokines. Lcn2 represses IL‐10 production while augmenting Nos2, TNF‐α, and IL‐6 expression. Lcn2^?/? macrophages have elevated IL‐10 levels as a consequence of increased iron content. The crucial role of Lcn‐2/IL‐10 interactions was further demonstrated by the greater ability of Lcn2^?/? IL‐10^?/? macrophages and mice to control intracellular Salmonella proliferation in comparison to Lcn2^?/? counterparts. Overexpression of the iron exporter ferroportin‐1 in Lcn2^?/? macrophages represses IL‐10 and restores TNF‐α and IL‐6 production to the levels found in wild‐type macrophages, so that killing and clearance of intracellular Salmonella is promoted. Our observations suggest that Lcn2 promotes host resistance to Salmonella Typhimurium infection by binding bacterial siderophores and suppressing IL‐10 production, and that both functions are linked to its ability to shuttle iron from macrophages.     

CD169 identifies an anti‐tumour macrophage subpopulation in human hepatocellular carcinoma

Macrophages are a major component of most solid tumours and can exert both anti‐ and pro‐tumourigenic functions. Although the immunosuppressive/pro‐tumour roles of macrophages have been widely examined, significantly less is known about macrophage subpopulations that have potential anti‐tumour properties in humans. In the present study, a population of CD169⁺ macrophages with relatively high expression levels of HLA‐DR and CD86 was identified in human hepatocellular carcinoma tissues. The frequency of CD169‐expressing macrophages within cancer nests was significantly lower than that found in paired non‐tumour areas. In vitro experiments revealed that in the presence of anti‐CD3 stimulation, CD169⁺ macrophages could significantly enhance the proliferation, cytotoxicity, and cytokine production capacity of CD8⁺ T cells in a CD169 molecule‐dependent manner. Autocrine TGF‐β produced by tumour‐stimulated macrophages was involved in the down‐regulation of CD169 expression on these cells. Moreover, the accumulation of CD169⁺ macrophages in tumour tissues was negatively associated with disease progression and predicted favourable survival in hepatocellular carcinoma patients, which was in contrast to the trend observed for total CD68⁺ macrophages. Therefore, CD169 might act as a co‐stimulatory molecule for cytotoxic T‐cell activation, and could define a population of tumour‐infiltrating macrophages with potential anti‐tumour properties in human hepatocellular carcinoma tissues. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Sensitization of human breast cancer cells to natural killer cell‐mediated cytotoxicity by proteasome inhibition

The proteasome inhibitor, bortezomib, has direct anti‐tumour effects and has been demonstrated to sensitize tumour cells to tumour necrosis factor‐related apoptosis‐inducing ligand‐mediated apoptosis. Natural killer (NK) cells are effective mediators of anti‐tumour responses, both through cytotoxic granule killing and apoptosis‐inducing pathways. We therefore investigated if bortezomib sensitized human breast cancer cells to killing by the human NK cell line, NK‐92. Bortezomib was unable to sensitize MDA‐231 breast cancer cells to NK cell‐mediated killing in short‐term in vitro assays. However, bortezomib did cause these cells to up‐regulate apoptosis‐related mRNA as well as death receptors on the cell surface. In a long‐term in vitro tumour outgrowth assay that allows NK cells to use their full repertoire of killing pathways, bortezomib sensitized three breast cancer cell lines to NK cell‐mediated killing, which led to greater anti‐tumour effects than either treatment alone. We then used a xenogeneic mouse model in which CB‐17 SCID mice were injected with human breast cancer cells. This model displayed the effectiveness of NK‐92 cells, but the addition of bortezomib did not increase the survival further or reduce the number of lung metastases in tumour‐bearing mice. However, while bortezomib was highly cytotoxic to NK‐92 cells in vitro, bortezomib treatment in vivo did not decrease NK‐92 function, suggesting that through alternative dosing or timing of bortezomib, greater efficacy may occur from combined therapy. These data demonstrate that combined treatment of human breast cancer with bortezomib and NK cells has the potential to generate superior anti‐tumour responses than either therapy alone.     

The ADAMTS1 protease gene is required for mammary tumor growth and metastasis

A disintegrin and metalloprotease with thrombospondin motifs protein 1 (ADAMTS1) is a protease commonly up-regulated in metastatic carcinoma. Its overexpression in cancer cells promotes experimental metastasis, but whether ADAMTS1 is essential for metastatic progression is unknown. To address this question, we investigated mammary cancer progression and spontaneous metastasis in the MMTV-PyMT mouse mammary tumor model in Adamts1 knockout mice. Adamts1(-/-)/PyMT mice displayed significantly reduced mammary tumor and lung metastatic tumor burden and increased survival, compared with their wild-type and heterozygous littermates. Histological examination revealed an increased proportion of tumors with ductal carcinoma in situ and a lower proportion of high-grade invasive tumors in Adamts1(-/-)/PyMT mice, compared with Adamts1(+/+)/PyMT mice. Increased apoptosis with unaltered proliferation and vascular density in the Adamts1(-/-)/PyMT tumors suggested that reduced cell survival accounts for the lower tumor burden in ADAMTS1-deficient mice. Furthermore, Adamts1(-/-) tumor stroma had significantly lesser amounts of proteolytically cleaved versican and increased numbers of CD45(+) leukocytes. Characterization of immune cell gene expression indicated that cytotoxic cell activation was increased in Adamts1(-/-) tumors, compared with Adamts1(+/+) tumors. This finding is supported by significantly elevated IL-12(+) cell numbers in Adamts1(-/-) tumors. Thus, in vivo ADAMTS1 may promote mammary tumor growth and progression to metastasis in the PyMT model and is a potential therapeutic target to prevent metastatic breast cancer.     

MiR‐26b is down‐regulated in carcinoma‐associated fibroblasts from ER‐positive breast cancers leading to enhanced cell migration and invasion

Carcinoma‐associated fibroblasts (CAFs) influence the behaviour of cancer cells but the roles of microRNAs in this interaction are unknown. We report microRNAs that are differentially expressed between breast normal fibroblasts and CAFs of oestrogen receptor‐positive cancers, and explore the influences of one of these, miR‐26b, on breast cancer biology. We identified differentially expressed microRNAs by expression profiling of clinical samples and a tissue culture model: miR‐26b was the most highly deregulated microRNA. Using qPCR, miR‐26b was confirmed as down‐regulated in fibroblasts from 15 of 18 further breast cancers. Next, we examined whether manipulation of miR‐26b expression changed breast fibroblast behaviour. Reduced miR‐26b expression caused fibroblast migration and invasion to increase by up to three‐fold in scratch‐closure and trans‐well assays. Furthermore, in co‐culture with MCF7 breast cancer epithelial cells, fibroblasts with reduced miR‐26b expression enhanced both MCF7 migration in trans‐well assays and MCF7 invasion from three‐dimensional spheroids by up to five‐fold. Mass spectrometry was used to identify expression changes associated with the reduction of miR‐26b expression in fibroblasts. Pathway analyses of differentially expressed proteins revealed that glycolysis/TCA cycle and cytoskeletal regulation by Rho GTPases are downstream of miR‐26b. In addition, three novel miR‐26b targets were identified (TNKS1BP1, CPSF7, COL12A1) and the expression of each in cancer stroma was shown to be significantly associated with breast cancer recurrence. MiR‐26b in breast CAFs is a potent regulator of cancer behaviour in oestrogen receptor‐positive cancers, and we have identified key genes and molecular pathways that act downstream of miR‐26b in CAFs. © 2013 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Comprehensive genetic and functional characterization of IPH‐926: a novel CDH1‐null tumour cell line from human lobular breast cancer

Infiltrating lobular breast cancer (ILBC) is a clinically and biologically distinct tumour entity defined by a characteristic linear cord invasion pattern and inactivation of the CDH1 tumour suppressor gene encoding for E‐cadherin. ILBCs also lack β‐catenin expression and show aberrant cytoplasmic localization of the E‐cadherin binding protein p120‐catenin. The lack of a well‐characterized ILBC cell line has hampered the functional characterization of ILBC cells in vitro. We report the establishment of a permanent ILBC cell line, named IPH‐926, which was derived from a patient with metastatic ILBC. The DNA fingerprint of IPH‐926 verified genetic identity with the patient and had no match among the human cell line collections of several international biological resource banks. IPH‐926 expressed various epithelial cell markers but lacked expression of E‐cadherin due to a previously unreported, homozygous CDH1 241ins4 frameshift mutation. Detection of the same CDH1 241ins4 mutation in archival tumour tissue of the corresponding primary ILBC proved the clonal origin of IPH‐926 from this particular tumour. IPH‐926 also lacked β‐catenin expression and showed aberrant cytoplasmic localization of p120‐catenin. Array‐CGH analysis of IPH‐926 revealed a profile of genomic imbalances that included many distinct alterations previously observed in primary ILBCs. Spectral karyotyping of IPH‐926 showed a hyperdiploid chromosome complement and numerous clonal, structural aberrations. IPH‐926 cells were anti‐cancer drug‐resistant, clonogenic in soft agar, and tumourigenic in SCID mice. In xenograft tumours, IPH‐926 cells recapitulated the linear cord invasion pattern that defines ILBCs. In summary, IPH‐926 significantly extends the biological spectrum of the established breast cancer cell lines and will facilitate functional analyses of genuine human ILBC cells in vitro and in vivo. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Mammosphere‐derived gene set predicts outcome in patients with ER‐positive breast cancer

Tumourigenic subpopulations with stem cell‐like features have been identified in breast tumours and breast cancer cell lines. The hormone receptor status, molecular characteristics and clinical significance of these cells are still matters of debate. Enrichment for tumourigenic cells without the requirement of surface markers can be achieved by the in vitro mammosphere culture assay. Here we compared the hormone receptor status and genome‐wide gene expression profiles of mammospheres derived from four oestrogen‐receptor (ER) positive breast cancer cell lines with those of the respective parental cells. Immunohistochemistry and gene expression profiling revealed a significant reduction in the expression of progesterone receptor, proliferation and cell cycle regulated genes in mammospheres when compared to parental cell lines. The 200 most differentially expressed genes between mammospheres and parental cell lines were used to generate a ‘mammosphere‐derived’ gene set. Hierarchical clustering of gene expression profiles of two independent cohorts of primary ER‐positive cancers based on the ‘mammosphere‐derived’ gene set revealed that the subgroup of breast cancers with profiles similar to those of mammospheres has a significantly longer overall survival. In conclusion, tumour‐initiating breast cancer cells grown in mammospheres seem to reside in a quiescent state. ER‐positive breast cancers with expression profiles similar to those of mammospheres have a better outcome, providing evidence in support of the concept that outcome of patients with ER‐positive disease is for a large part determined by cell cycle and proliferation activity. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The invasive and metastatic properties of hormone-independent but hormone-responsive variants of MCF-7 human breast cancer cells

We have previously isolated a series of MCF-7 human breast cancer cell variants which no longer require estrogen-supplementation for tumor growth in nude mice (Clarkeet al. Proc Natl Acad Sci USA 86: 3649–3653, 1989). We now report that these hormone-independent and hormone-responsive variants (MIII, MCF7/LCC1) can invade locally from solid mammary fat pad tumors, and produce primary extensions on the surface of intraperitoneal structures including liver, pancreas, and diaphragm. Both lymphatic and hematogenous dissemination are observed, resulting in the establishing of pulmonary, bone, and renal metastases. The pattern of metastasis by MIII and MCF7/LCC1 cells closely resembles that frequently observed in breast cancer patients, and provides the first evidence of metastasis from MCF-7 cells growingin vivo without supplementary estrogen. The interexperimental incidence of metastases, and the time from cell inoculation to the appearance of metastatic disease are variable. The increased metastatic potential is not associated with an increase in either the level of laminin attachment, laminin receptor mRNA expression, or secreted type IV collagenolytic activity. We also did not detect a significant decrease in the steady-state mRNA levels of the metastasis inhibitor nm23 gene. However, when growing without estrogenin vitro, MCF7/LCC1 cells produce elevated levels of the estrogen-inducible cathepsin D enzyme.     

SKA1 over‐expression promotes centriole over‐duplication,centrosome amplification and prostate tumourigenesis

Although spindle‐ and kinetochore‐associated protein 1 (Ska1) has previously been identified as essential for proper chromosome segregation, it is unknown whether it plays a role in tumour development. Here, we report that Ska1 over‐expression promotes prostate tumourigenesis. Immunohistochemistry and quantitative RT–PCR analysis revealed that Ska1 was over‐expressed in human prostatic intra‐epithelial neoplasia (PIN), the most likely prostate cancer precursor, and adenocarcinomas. Up‐regulation of Ska1 protein was also found to be tumour‐specific in breast, lung and other common human cancers. Importantly, prostate‐specific up‐regulation of Ska1 in a transgenic mouse model resulted in spontaneous tumourigenesis. Furthermore, in addition to its abundance in spindle microtubules and the outer kinetochore interface during mitosis, Ska1 was enriched at centrosomes in cultured cells. Depletion of Ska1 caused a failure of centrosome duplication, whilst Ska1 over‐expression led to centrosome amplification in human prostate epithelial cells via the induction of centriole over‐duplication. These epithelial cells harbouring extra centrosomes switched from a non‐tumourigenic to a tumourigenic state in nude mice. Taken together, these data indicate that Ska1 over‐expression promotes tumourigenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Lentivirus‐Mediated Small Interfering RNA Targeting VEGF‐C Inhibited Tumor Lymphangiogenesis and Growth in Breast Carcinoma

Lymph node metastasis is a major prognostic factor for patients with breast cancer. The activation of vascular endothelial growth factor (VEGF)‐C plays a key role in lymph node metastasis through promoting lymphangiogenesis. Thus, we attempted to elucidate whether small interfering RNAs (siRNA) targeting VEGF‐C could suppress lymphangiogenesis and lymph node metastasis in vivo. A lentivirus‐based VEGF‐C siRNA vector was infected into breast cancer cells and a xenograft model. The expression of VEGF‐C mRNA and protein were quantified by quantitative real‐time polymerase chain reaction (QRT‐PCR), immunohistochemistry, and western blot analysis. The effect of VEGF‐C siRNA on breast cancer cells was investigated by an invasion assay. Lymphangiogenesis was analyzed with anti‐LYVE‐1 and anti‐D2‐40 by immunohistochemical analysis. Lentivirus‐mediated VEGF‐C siRNA stably reduced VEGF‐C mRNA and protein expression. VEGF‐C siRNA inhibited the invasive ability of breast cancer cells in vitro. Five weeks after intratumoral injection, the tumor volume was significantly smaller in the VEGF‐C siRNA group than in the control scramble siRNA group in the MDA‐MB‐231 cell xenograft model. The numbers of LYVE‐1 and D2‐40 positive vessels per microscopic field were significantly decreased in the VEGF‐C siRNA group, which indicates that VEGF‐C siRNA inhibited lymphangiogenesis. Moreover, lymph node metastasis was significantly suppressed by VEGF‐C siRNA in vivo. In conclusion, these results indicate that lentivirus‐mediated VEGF‐C siRNA offers a new approach for therapeutic intervention to prevent tumor growth and lymphatic metastasis of breast cancer. Anat Rec, 292:633–639, 2009. © 2009 Wiley‐Liss, Inc.     

Issue Information ‐ Journal Information

Periostin (POSTN) is a limiting factor in the metastatic colonization of disseminated tumour cells. However, the role of POSTN in regulating the immunosuppressive function of immature myeloid cells in tumour metastasis has not been documented. Here, we demonstrate that POSTN promotes the pulmonary accumulation of myeloid‐derived suppressor cells (MDSCs) during the early stage of breast tumour metastasis. Postn deletion decreases neutrophil and monocytic cell populations in the bone marrow of mice and suppresses the accumulation of MDSCs to premetastatic sites. We also found that POSTN‐deficient MDSCs display reduced activation of ERK, AKT and STAT3 and that POSTN deficiency decreases the immunosuppressive functions of MDSCs during tumour progression. Moreover, the pro‐metastatic role of POSTN is largely limited to ER‐negative breast cancer patients. Lysyl oxidase contributes to POSTN‐promoted premetastatic niche formation and tumour metastasis. Our findings indicate that POSTN is essential for immunosuppressive premetastatic niche formation in the lungs during breast tumour metastasis and is a potential target for the prevention and treatment of breast tumour metastasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Cancer‐associated fibroblasts predict poor outcome and promote periostin‐dependent invasion in oesophageal adenocarcinoma

Interactions between cancer cells and cancer‐associated fibroblasts (CAFs) play an important role in tumour development and progression. In this study we investigated the functional role of CAFs in oesophageal adenocarcinoma (EAC). We used immunochemistry to analyse a cohort of 183 EAC patients for CAF markers related to disease mortality. We characterized CAFs and normal oesophageal fibroblasts (NOFs) using western blotting, immunofluorescence and gel contraction. Transwell assays, 3D organotypic culture and xenograft models were used to examine the effects on EAC cell function and to dissect molecular mechanisms regulating invasion. Most EACs (93%) contained CAFs with a myofibroblastic (α‐SMA‐positive) phenotype, which correlated significantly with poor survival [p = 0.016; HR 7. 1 (1.7–29.4)]. Primary CAFs isolated from EACs have a contractile, myofibroblastic phenotype and promote EAC cell invasion in vitro (Transwell assays, p ≤ 0.05; organotypic culture, p < 0.001) and in vivo (p ≤ 0.05). In vitro, this pro‐invasive effect is modulated through the matricellular protein periostin. Periostin is secreted by CAFs and acts as a ligand for EAC cell integrins αvβ3 and αvβ5, promoting activation of the PI3kinase–Akt pathway. In patient samples, periostin expression at the tumour cell–stromal interface correlates with poor overall and disease‐free survival. Our study highlights the importance of the tumour stroma in EAC progression. Paracrine interaction between CAF‐secreted periostin and EAC‐expressed integrins results in PI3 kinase–Akt activation and increased tumour cell invasion. Most EACs contain a myofibroblastic CAF‐rich stroma; this may explain the aggressive, highly infiltrative nature of the disease, and suggests that stromal targeting may produce therapeutic benefit in EAC patients. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

EMILIN2 down‐modulates the Wnt signalling pathway and suppresses breast cancer cell growth and migration

EMILIN2 is an extracellular matrix (ECM) protein that exerts contradictory effects within the tumour microenvironment: it induces apoptosis in a number of tumour cells, but it also enhances tumour neo‐angiogenesis. In this study, we describe a new mechanism by which EMILIN2 attenuates tumour cell viability. Based on sequence homology with the cysteine‐rich domain (CRD) of the Frizzled receptors, we hypothesized that EMILIN2 could affect Wnt signalling activation and demonstrate direct interaction with the Wnt1 ligand. This physical binding leads to decreased LRP6 phosphorylation and to the down‐modulation of β‐catenin, TAZ and their target genes. As a consequence, EMILIN2 negatively affects the viability, migration and tumourigenic potential of MDA‐MB‐231 breast cancer cells in a number of two‐ and three‐dimensional in vitro assays. EMILIN2 does not modulate Wnt signalling downstream of the Wnt–Frizzled interaction, since it does not affect the activation of the pathway following treatment with the GSK3 inhibitors LiCl and CHIR99021. The interaction with Wnt1 and the subsequent biological effects require the presence of the EMI domain, as there is no effect with a deletion mutant lacking this domain. Moreover, in vivo experiments show that the ectopic expression of EMILIN2, as well as treatment with the recombinant protein, significantly reduce tumour growth and dissemination of cancer cells in nude mice. Accordingly, the tumour samples are characterized by a significant down‐regulation of the Wnt signalling pathway. Altogether, these findings provide further evidence of the complex regulations governed by EMILIN2 in the tumour microenvironment, and they identify a key extracellular regulator of the Wnt signalling pathway. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Whole‐exome DNA sequence analysis of Brca2‐ and Trp53‐deficient mouse mammary gland tumours

Germline mutations in the tumour suppressor BRCA2 predispose to breast, ovarian and a number of other human cancers. Brca2‐deficient mouse models are used for preclinical studies but the pattern of genomic alterations in these tumours has not yet been described in detail. We have performed whole‐exome DNA sequencing analysis of mouse mammary tumours from Blg–Cre Brca2^f/f Trp53^f/f animals, a model of BRCA2‐deficient human cancer. We also used the sequencing data to estimate DNA copy number alterations in these tumours and identified a recurrent copy number gain in Met, which has been found amplified in other mouse mammary cancer models. Through a comparative genomic analysis, we identified several mouse Blg–Cre Brca2^f/f Trp53^f/f mammary tumour somatic mutations in genes that are also mutated in human cancer, but few of these genes have been found frequently mutated in human breast cancer. A more detailed analysis of these somatic mutations revealed a set of genes that are mutated in human BRCA2 mutant breast and ovarian tumours and that are also mutated in mouse Brca2‐null, Trp53‐null mammary tumours. Finally, a DNA deletion surrounded by microhomology signature found in human BRCA1/2‐deficient cancers was not common in the genome of these mouse tumours. Although a useful model, there are some differences in the genomic landscape of tumours arising in Blg–Cre Brca2^f/f Trp53^f/f mice compared to human BRCA‐mutated breast cancers. Therefore, this needs to be taken into account in the use of this model. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Tumor‐associated macrophages induce lymphangiogenesis in cervical cancer via interaction with tumor cells

Our studies were conducted to investigate the clinical and functional significance of tumor‐associated macrophages (TAMs) in cervical tumor lymphatic metastasis. We found that the increase in macrophages in tumor stroma is significantly associated with lymphatic metastasis (p = 0.017), through performing immunohistochemical staining in 111 cervical samples (55 invasive squamous carcinomas of uterine cervix, 27 cervical intraepithelial neoplasms III, and 29 normal cervix). The human lymphatic endothelial cells (HLEC), which were cultured in conditioned medium of cervical cancer cell‐macrophage coculture, formed more tube‐like structures in vitro, when compared with those in conditioned mediums of LEC, normal cervical epithelium, single macrophage, and single cervical cancer cell (all p < 0.001). The mRNA expressions of IL‐1β and IL‐8 in cervical cancer cells cocultured with macrophages were increased, compared with those in cervical cancer cell cultured alone (p_IL‐1β < 0.05 and p_IL‐8 < 0.01). Meanwhile, the mRNA expression of VEGF‐C and VEGF‐A was increased in macrophages cocultured with cervical cancer cells, compared with the expression in those macrophages cultured alone (both p < 0.05). Taken together, the results suggest that TAMs promote lymphangiogenesis mainly through interaction with surrounding cervical cancer cells.