Epithelial-mesenchymal transition in cancer metastasis through the lymphatic syste

It was already in the 18th century when the French surgeon LeDran first noted that breast cancer patients with spread of tumor cells to their axillary lymph nodes had a drastically worse prognosis than patients without spread (LeDran et al., ). Since then, metastatic spread of cancer cells to regional lymph nodes has been established as the most important prognostic factor in many types of cancer (Carter et al., ; Elston and Ellis, ). However, despite its clinical importance, lymph metastasis remains an underexplored area of tumor biology. Fundamental questions, such as when, how, and perhaps most importantly, why tumor cells disseminate through the lymphatic system, remain largely unanswered. Accordingly, no treatment strategies exist that specifically target lymph metastasis. The identification of epithelial-mesenchymal transition (EMT) as a mechanism, which allows cancer cells to dedifferentiate and acquire enhanced migratory and invasive properties, has been a game changer in cancer research. Conceptually, EMT provides an explanation for why epithelial cancers with poor differentiation status are generally more aggressive and prone to metastasize than more differentiated cancers. Inflammatory cytokines, such as TGF-β, which are produced and secreted by tumor-infiltrating immune cells, are potent inducers of EMT. Thus, reactivation of EMT also links cancer-related inflammation to invasive and metastatic disease. Recently, we found that breast cancer cells undergoing TGF-β- induced EMT acquire properties of immune cells allowing them to disseminate in a targeted fashion through the lymphatic system similar to activated dendritic cells during inflammation. Here, we review our current understanding of the mechanisms by which cancer cells spread through the lymphatic system and the links to inflammation and the immune system. We also emphasize how imaging techniques have the potential to further expand our knowledge of the mechanisms of lymph metastasis, and how lymph nodes serve as an interface between cancer and the immune system

Functionally specialized junctions between endothelial cells of lymphatic vessels

Recirculation of fluid and cells through lymphatic vessels plays a key role in normal tissue homeostasis, inflammatory diseases, and cancer. Despite recent advances in understanding lymphatic function (Alitalo, K., T. Tammela, and T.V. Petrova. 2005. Nature. 438:946–953), the cellular features responsible for entry of fluid and cells into lymphatics are incompletely understood. We report the presence of novel junctions between endothelial cells of initial lymphatics at likely sites of fluid entry. Overlapping flaps at borders of oak leaf–shaped endothelial cells of initial lymphatics lacked junctions at the tip but were anchored on the sides by discontinuous button-like junctions (buttons) that differed from conventional, continuous, zipper-like junctions (zippers) in collecting lymphatics and blood vessels. However, both buttons and zippers were composed of vascular endothelial cadherin (VE-cadherin) and tight junction–associated proteins, including occludin, claudin-5, zonula occludens–1, junctional adhesion molecule–A, and endothelial cell–selective adhesion molecule. In C57BL/6 mice, VE-cadherin was required for maintenance of junctional integrity, but platelet/endothelial cell adhesion molecule–1 was not. Growing tips of lymphatic sprouts had zippers, not buttons, suggesting that buttons are specialized junctions rather than immature ones. Our findings suggest that fluid enters throughout initial lymphatics via openings between buttons, which open and close without disrupting junctional integrity, but most leukocytes enter the proximal half of initial lymphatics

Pericytes contribute to airway remodeling in a mouse model of chronic allergic asthma

Myofibroblast accumulation, subepithelial fibrosis, and vascular remodeling are complicating features of chronic asthma, but the mechanisms are not clear. Platelet-derived growth factors (PDGFs) regulate the fate and function of various mesenchymal cells and have been implicated as mediators of lung fibrosis. However, it is not known whether PDGF-BB signaling via PDGFRβ, which is critical for the recruitment of pericytes to blood vessels, plays a role in airway remodeling in chronic asthma. In the present study, we used a selective PDGFRβ inhibitor (CP-673451) to investigate the role of PDGFRβ signaling in the development of airway remodeling and lung dysfunction in an established mouse model of house dust mite-induced chronic allergic asthma. Unexpectedly, we found that pharmacological inhibition of PDGFRβ signaling in the context of chronic aeroallergen exposure led to exacerbated lung dysfunction and airway smooth muscle thickening. Further studies revealed that the inflammatory response to aeroallergen challenge in mice was associated with decreased PDGF-BB expression and the loss of pericytes from the airway microvasculature. In parallel, cells positive for pericyte markers accumulated in the subepithelial region of chronically inflamed airways. This process was exacerbated in animals treated with CP-673451. The results indicate that perturbed PDGF-BB/PDGFRβ signaling and pericyte accumulation in the airway wall may contribute to airway remodeling in chronic allergic asthma

Essential Role of the Coxsackie - and Adenovirus Receptor (CAR) in Development of the Lymphatic System in Mice.

Peer reviewe

Chronic Respiratory Aeroallergen Exposure in Mice Induces Epithelial-Mesenchymal Transition in the Large Airways

Chronic allergic asthma is characterized by Th2-polarized inflammation and leads to airway remodeling and fibrosis but the mechanisms involved are not clear. To determine whether epithelial-mesenchymal transition contributes to airway remodeling in asthma, we induced allergic airway inflammation in mice by intranasal administration of house dust mite (HDM) extract for up to 15 consecutive weeks. We report that respiratory exposure to HDM led to significant airway inflammation and thickening of the smooth muscle layer in the wall of the large airways. Transforming growth factor beta-1 (TGF-β1) levels increased in mouse airways while epithelial cells lost expression of E-cadherin and occludin and gained expression of the mesenchymal proteins vimentin, alpha-smooth muscle actin (α-SMA) and pro-collagen I. We also observed increased expression and nuclear translocation of Snail1, a transcriptional repressor of E-cadherin and a potent inducer of EMT, in the airway epithelial cells of HDM-exposed mice. Furthermore, fate-mapping studies revealed migration of airway epithelial cells into the sub-epithelial regions of the airway wall. These results show the contribution of EMT to airway remodeling in chronic asthma-like inflammation and suggest that Th2-polarized airway inflammation can trigger invasion of epithelial cells into the subepithelial regions of the airway wall where they contribute to fibrosis, demonstrating a previously unknown plasticity of the airway epithelium in allergic airway disease

Downregulation of miR-92a Is Associated with Aggressive Breast Cancer Features and Increased Tumour Macrophage Infiltration

BACKGROUND: MicroRNAs are small non-coding RNAs involved in the regulation of gene expression on a posttranscriptional level. These regulatory RNAs have been implicated in numerous cellular processes and are further deregulated in different cancer types, including breast cancer. MiR-92a is part of the miR-17∼92 cluster, which was first reported to be linked to tumourigenesis. However, little is known about the expression of miR-92a in breast cancer and potential associations to tumour properties. The expression of miR-92a was therefore characterized in 144 invasive breast cancer samples using in situ hybridization and related to clinico-pathological data as well as to selected key properties of the tumour stroma, including the presence of macrophages (CD68) and cancer activated fibroblasts (alpha-SMA). METHODOLOGY/PRINCIPAL FINDINGS: To measure miR-92a levels, an in situ hybridisation protocol was developed and validated using cell lines and miR-92a inhibitors. The expression in the tumour samples was objectively evaluated using digital image analysis program subtracting background activities. We found that the miR-92a expression varied between tumours and was inversely correlated to tumour grade (r = -0.276, p = 0.003) and recurrence-free survival (p = 0.008) and provided independent prognostic information in multivariate Cox analysis (HR: 0.375, CI: 0.145-0.972, p = 0.043). MiR-92a was moreover inversely correlated to the number of infiltrating macrophages in the tumour stroma (r = -0.357, p<0.001), and downregulation of miR-92a promoted cell migration (p<0.01). CONCLUSIONS/SIGNIFICANCE: This study demonstrates that downregulation of miR-92a in breast cancer is linked to key epithelial and stromal properties as well as clinical outcome

Rodent models of focal cerebral ischemia: procedural pitfalls and translational problems

Rodent models of focal cerebral ischemia are essential tools in experimental stroke research. They have added tremendously to our understanding of injury mechanisms in stroke and have helped to identify potential therapeutic targets. A plethora of substances, however, in particular an overwhelming number of putative neuroprotective agents, have been shown to be effective in preclinical stroke research, but have failed in clinical trials. A lot of factors may have contributed to this failure of translation from bench to bedside. Often, deficits in the quality of experimental stroke research seem to be involved. In this article, we review the commonest rodent models of focal cerebral ischemia - middle cerebral artery occlusion, photothrombosis, and embolic stroke models - with their respective advantages and problems, and we address the issue of quality in preclinical stroke modeling as well as potential reasons for translational failure

Deficiency for endoglin in tumor vasculature weakens the endothelial barrier to metastatic dissemination

Therapy-induced resistance remains a significant hurdle to achieve long-lasting responses and cures in cancer patients. We investigated the long-term consequences of genetically impaired angiogenesis by engineering multiple tumor models deprived of endoglin, a co-receptor for TGF-β in endothelial cells actively engaged in angiogenesis. Tumors from endoglin-deficient mice adapted to the weakened angiogenic response, and refractoriness to diminished endoglin signaling was accompanied by increased metastatic capability. Mechanistic studies in multiple mouse models of cancer revealed that deficiency for endoglin resulted in a tumor vasculature that displayed hallmarks of endothelial-to-mesenchymal transition, a process of previously unknown significance in cancer biology, but shown by us to be associated with a reduced capacity of the vasculature to avert tumor cell intra- and extravasation. Nevertheless, tumors deprived of endoglin exhibited a delayed onset of resistance to anti-VEGF (vascular endothelial growth factor) agents, illustrating the therapeutic utility of combinatorial targeting of multiple angiogenic pathways for the treatment of cancer

p16INK4A and p15INK4B in senescence, immortalization and cancer : Gene transfer by adenovirus vectors

The astrocytic gliomas are the most common form of brain tumors in humans. The malignant progression from low grade (astrocytoma, grade II) to high-grade (glioblastoma multiforme, grade, IV) astrocytomas is well documented and is accompanied by an increasing number of genetic aberrations. Many of these genetic alterations center round genes that regulate the G 1/S transition of the cell cycle. This is particularly the case with the genes encoding the cyclin dependent kinase inhibitors (CDKIs), p16 INK4A (p16) and p15 INK4B (p15). They are homozygously deleted in 40-50% of primary gliomas and in an even higher percentage in glioma cell lines. In addition, the p16 gene is frequently inactivated by point mutations and by DNA methylation, and mice that are deficient for the p16 gene spontaneously develop tumors within their first year of life. While the role of p16 in senescence and tumor suppression has been amply documented, the role of p 15 remained less clear. Recombinant adenoviruses (Ad) expressing p15 (Adp15) or p16 (Adp16) were constructed and used to infect human glioma cells with different status of the p15, p16, and pRB genes. The results indicated that p15 is as potent as p16 in inducing cell cycle arrest and senescence-like changes in human glioma cells with an intact pRB. In addition, both p 15 and p 16 strongly inhibited telomerase activity in these cells. This shows the potential of p15 to function as a tumor suppressor and mediator of senescence. Together with the fact that p15 accumulates in T lymphocytes as they approach senescence and that p15 deficient mice display tumors of the hematopoietic cell system, these results suggest that p15 plays a role in regulating homeostasis in these (and perhaps other) cell systems. The mechanism behind the synthesis of an alternative isoform of p 15 was also elucidated. This protein, termed p15.5, was found to be an N-terminally extended variant of p15, initiated from an upstream GUG codon. It was shown to separately, or in combination with p15, interact with both CDK4 and CDK6, and to induce cell cycle arrest and a senescent-like phenotype when transfected into human glioma cells. The involvement of the p16/pRB pathway components in telomerase-mediated immortalization of bovine capillary endothelial (BCE) cells was determined. In these studies, transfection of the catalytic subunit of human telomerase (hTERT) alone was sufficient to immortalize BCE cells. Surprisingly, the telomere lengths in the hTERT-BCE cells were consistently shorter than in the senescent parental cells. pRB was hyperphosphorylated, and the expression of p16 (and p21CIP1) was repressed by promoter methylation. Reactivation of p16 by either Ad-mediated expression or by demethylation reversed the immortalized phenotype and induced senescence-like changes. These results suggest that the immortalization of BCE cells by hTERT is mediated through inactivation of crucial cellular senescence machineries including p161pR13 and p21CIP1, proteins that also by others have been implicated in inducing and maintaining a senescent phenotype in mammalian cells. The ability of type C Ad to infect human cells is dependent on the expression of the coxsackie- and adenovirus receptor, hCAR. Since very little is known about the expression of hCAR in primary human gliomas and since hCAR has been implicated to function as a tumor suppressor, such expression studies were performed. A great variation in hCAR expression was detected, both in glioma cell lines and in the primary tumors, and the expression correlated well with Ad infectability. A significant decrease in the mean CAR expression levels was detected in the grade IV tumors as compared to the values for the grade II and grade III tumors. Moreover, a mean 12-fold higher expression of hCAR was detected in xenografts derived from glioblastomas, compared to the parental tumors. Interestingly, the two xenografts that did not show any upregulation of hCAR expression grew much faster than the hCAR-expressing cells. This points to an inverse correlation between growth rate and hCAR expression, and suggests that hCAR may contribute to suppression of tumor growth