Involvement of CXCR4 Chemokine Receptor in Metastastic HER2-Positive Esophageal Cancer

A functional linkage of the structurally unrelated receptors HER2 and CXCR4 has been suggested for breast cancer but has not been evaluated for esophageal carcinoma. The inhibition of HER2 leads to a reduction of primary tumor growth and metastases in an orthotopic model of esophageal carcinoma. The chemokine receptor CXCR4 has been implicated in metastatic dissemination of various tumors and correlates with poor survival in esophageal carcinoma. The aim of this study was to investigate a correlation between the expression levels of HER2 and CXCR4 and to evaluate the involvemnent of CXCR4-expression in HER2-positive esophageal carcinoma. The effects of HER2-inhibition with trastuzumab and of CXCR4-inhibition with AMD3100 on primary tumor growth, metastatic homing, and receptor expression were evaluated in vitro and in an orthotopic model of metastatic esophageal carcinoma using MRI for imaging. The clinical relevance of HER2- and CXCR4-expression was examined in esophageal carcinoma patients. A significant correlation of HER2- and CXCR4-expression in primary tumor and metastases exists in the orthotopic model. Trastuzumab and AMD3100 treatment led to a significant reduction of primary tumor growth, metastases and micrometastases. HER2-expression was significantly elevated under AMD3100 treatment in the primary tumor and particularly in the metastases. The positive correlation between HER2- and CXCR4-expression was validated in esophageal cancer patients. The correlation of CXCR4- and HER2-expression and the elevation of HER2-expression and reduction of metastases through CXCR4-inhibition suggest a possible functional linkage and a role in tumor dissemination in HER2-positive esophageal carcinoma.     

Colonization of experimental murine breast tumours by Escherichia coli K-12 significantly alters the tumour microenvironment

The successful application of live bacteria in cancer therapy requires a more detailed understanding of bacterial interaction with the tumour microenvironment. Here, we analysed the effect of Escherichia coli K-12 colonization on the tumour microenvironment by immunohistochemistry and fluorescence microscopy in the murine 4T1 breast carcinoma model. We described the colonization of tumour-bearing mice, as well as the spatiotemporal distribution of E. coli K-12 in the 4T1 tumour tissue over a period of 14 days. The colonization resulted within 3 days in large avascular necrotic tissue, redistribution of hypoxic areas and an enhanced collagen IV deposition within the colonized tumour tissue, which changed the tumoral perfusion of systemically injected immunoglobulins. In addition, E. coli K-12 colonization led to the redistribution of tumour-associated macrophages, forming a granulation tissue around bacterial colonies, and also to an increase in TNFalpha and matrix metalloproteinase 9 expression. Colonization of 4T1 tumours by E. coli K-12 resulted in strong reduction of pulmonary metastatic events. These new insights will contribute to the general understanding of the tumour-microbe cross-talk and to the design of bacterial strains with enhanced anticancer efficiency.     

Targeting ion transport in cancer

The metabolism of cancer cells differs substantially from normal cells, including ion transport. Although this phenomenon has been long recognized, ion transporters have not been viewed as suitable therapeutic targets. However, the acidic pH values present in tumours which are well outside of normal limits are now becoming recognized as an important therapeutic target. Carbonic anhydrase IX (CAIX) is fundamental to tumour pH regulation. CAIX is commonly expressed in cancer, but lowly expressed in normal tissues and that presents an attractive target. Here, we discuss the possibilities of exploiting the acidic, hypoxic tumour environment as possible target for therapy. Additionally, clinical experience with CAIX targeting in cancer patients is discussed.     

Hypoxia increases cytoplasmic expression of NDRG1, but is insufficient for its membrane localization in human hepatocellular carcinoma

NDRG1 is a hypoxia-inducible protein, whose modulated expression is associated with the progression of human cancers. Here, we reveal that NDRG1 is markedly upregulated in the cytoplasm and on the membrane in human hepatocellular carcinoma (HCC). We demonstrate further that hypoxic stress increases the cytoplasmic expression of NDRG1 in vitro, but does not result in its localization on the plasma membrane. However, grown within an HCC-xenograft in vivo, cells express NDRG1 in the cytoplasm and on the plasma membrane. In conclusion, hypoxia is a potent inducer of NDRG1 in HCCs, albeit requiring additional stimuli within the tumour microenvironment for its recruitment to the membrane.     

TFPI‐2 silencing increases tumour progression and promotes metalloproteinase 1 and 3 induction through tumour‐stromal cell interactions

Tissue factor pathway inhibitor‐2 (TFPI‐2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumour microenvironment makes TFPI‐2 a potential inhibitor of tumour invasion and metastasis. As demonstrated in aggressive cancers, TFPI‐2 is frequently down‐regulated in cancer cells, but the mechanisms involved in the inhibition of tumour progression remained unclear. We showed in this study that stable TFPI‐2 down‐regulation in the National Cancer Institute (NCI)‐H460 non‐small cell lung cancer cell line using specific micro interfering micro‐interfering RNA promoted tumour progression in a nude mice orthotopic model that resulted in an increase in cell invasion. Moreover, TFPI‐2 down‐regulation enhanced cell adhesion to collagen IV and laminin via an increase in α₁ integrin on cell surface, and increased MMP expression (mainly MMP‐1 and ‐3) contributing to cancer cell invasion through basement membrane components. This study also reveals for the first time that pulmonary fibroblasts incubated with conditioned media from TFPI‐2 silencing cancer cells exhibited increased expression of MMPs, particularly MMP‐1, ‐3 and ‐7, that are likely involved in lung cancer cell invasion through the surrounding stromal tissue, thus enhancing formation of metastases.     

Extracellular AGR2 triggers lung tumour cell proliferation through repression of p21CIP1

The human Anterior GRadient 2 (AGR2) protein is an Endoplasmic Reticulum (ER)-resident protein which belongs to the Protein-Disulfide Isomerase (PDI) superfamily and is involved to productive protein folding in the ER. As such AGR2, often found overexpressed in adenocarcinomas, contributes to tumour development by enhancing ER proteostasis. We previously demonstrated that AGR2 is secreted (extracellular AGR2 (eAGR2)) in the tumour microenvironment and plays extracellular roles independent of its ER functions. Herein, we show that eAGR2 triggers cell proliferation and characterize the underlying molecular mechanisms. We demonstrate that eAGR2 enhances tumour cell growth by repressing the tumour suppressor p21^CIP1. Our findings shed light on a novel mechanism through which eAGR2 behaves as a growth factor in the tumour microenvironment, independently of its ER function, thus promoting tumour cell growth through repression of p21^CIP1. Our results provide a rationale for targeting eAGR2/p21^CIP1-based signalling as a potential therapeutic target to impede tumour growth.     

Activation of blood coagulation in cancer: implications for tumour progression

Several studies have suggested a role for blood coagulation proteins in tumour progression. Herein, we discuss (1) the activation of the blood clotting cascade in the tumour microenvironment and its impact on primary tumour growth; (2) the intravascular activation of blood coagulation and its impact on tumour metastasis and cancer-associated thrombosis; and (3) antitumour therapies that target blood-coagulation-associated proteins. Expression levels of the clotting initiator protein TF (tissue factor) have been correlated with tumour cell aggressiveness. Simultaneous TF expression and PS (phosphatidylserine) exposure by tumour cells promote the extravascular activation of blood coagulation. The generation of blood coagulation enzymes in the tumour microenvironment may trigger the activation of PARs (protease-activated receptors). In particular, PAR1 and PAR2 have been associated with many aspects of tumour biology. The procoagulant activity of circulating tumour cells favours metastasis, whereas the release of TF-bearing MVs (microvesicles) into the circulation has been correlated with cancer-associated thrombosis. Given the role of coagulation proteins in tumour progression, it has been proposed that they could be targets for the development of new antitumour therapies.     

Phase-shifted pentafluorobutane nanoparticles for ultrasound imaging and ultrasound-mediated hypoxia modulation

The integration of ultrasound (US) contrast enhancement with oxygen-loading nanoagents provide the synergistic strategy for simultaneously US imaging and hypoxic microenvironment modulation. Herein, we synthesize pentafluorobutane (PFB)-loading methoxy poly(ethylene glycol)-b-poly(l -lactide) (PLLA) nanoparticle as the novel US-contrast-enhanced agent and demonstrate that PFB@PLLA effectively loads oxygen. We characterize the nanosize, phase-transformation property and oxygen-loading amount of PFB@PLLA and investigate the effectiveness of these nanoagents in US-contrast-enhanced imaging. The PFB@PLLA displays a perfect temperature-responsive phase-transition property and its liquid-to-gas phase transition temperature is 45°C, which produces microbubbles in the targeted regions. Moreover, PFB@PLLA loads high amount of oxygen and US-triggering PFB@PLLA reoxygenation effectively inhibits the expression of hypoxia-related proteins (HIF-1α and CAIX), reduces lactate secretion and glycolysis, which modulates hypoxic microenvironment and inhibits cancer cell migration and invasion in vitro. This study demonstrates that the US contrast-enhanced activity of PFB@PLLAs and the promising utility of oxygen-loading nanoagents to improve hypoxic microenvironment.     

Effective suppression of tumour cells by oligoclonal HER2-targeted delivery of liposomal doxorubicin

Synergistic effect of combined antibodies targeting distinct epitopes of a particular tumour antigen has encouraged some clinical trial studies and is now considered as an effective platform for cancer therapy. Providing several advantages over conventional antibodies, variable domain of heavy chain of heavy chain antibodies (V_HH) is now major tools in diagnostic and therapeutic applications. Active targeting of liposomal drugs is a promising strategy, resulting in enhanced binding and improved cytotoxicity of tumour cells. In the present study, we produced four anti-HER2 recombinant VHHs and purified them via native and refolding method. ELISA and flow cytometry analysis confirmed almost identical function of VHHs in refolded and native states. Using a mixture of four purified VHHs, PEGylated liposomal doxorubicin was targeted against HER2-overexpressing cells. The drug release was analyzed at pH 7.4, 6.4 and 5.5 and dynamic light-scattering detector and TEM micrograph was applied to characterize the produced nanoparticles. The binding efficiency of these nanoparticles to BT474 and SKBR3 as HER2-positive and MCF10A as HER2-negative cell line was examined by flow cytometry. Our results indicated effective encapsulation of about 94% of the total drug in immunoliposomes. Flow cytometry results verified receptor-specific binding of targeted liposomes to SKBR3 and BT474 cell lines and more efficient binding was observed for liposomes conjugated with oligoclonal VHHs mixture compared with monoclonal VHH-targeted liposomes. Oligoclonal nanoparticles also showed more cytotoxicity compared with non-targeted liposomes against HER2-positive tumour cells. Oligoclonal targeting of liposomes was represented as a promising strategy for the treatment of HER2-overexpressing breast cancers.     

ShcA signalling is essential for tumour progression in mouse models of human breast cancer

To explore the in vivo significance of ShcA during mammary tumorigenesis, we used mice expressing several phosphotyrosine-deficient ShcA alleles under the control of their endogenous promoter. We show that all three ShcA tyrosine phosphorylation sites are involved in the early stages of mammary tumour progression, including loss of the myoepithelial cell layer surrounding hyperplasias and during progression to carcinoma. We have determined that signals emanating from Y313 are important for tumour cell survival, whereas Y239/240 transduce signals promoting tumour vascularization. We further demonstrate that loss of ShcA expression in mammary epithelial cells abrogates tumour development. This study is the first to directly demonstrate that signalling downstream from the ShcA adaptor protein is critical for breast cancer development.     

Radiosensitization of a mouse tumour by Ro 03-8799: acute and protracted administration

The nitroimidazole Ro 03-8799 has been tested as a sensitizer of hypoxic tumour cells, using regrowth delay of a mouse mammary carcinoma. This drug has a short biological half-life and has previously proved to be less promising in tumour experiments than was predicted from in vitro studies and from artificially hypoxic skin. The postulate that this might result from the delay in penetrating to poorly vascularized tumour regions has been tested by maintaining constant blood and tumour levels for 2 hours before irradiation, using an infusion pump. For equivalent gross tumour concentrations at the time of irradiation there was no significant difference in the radiosensitization achieved with a single dose or with prolonged administration. This indicates that slow penetration of the drug is not a problem.     

Targeting tumour hypoxia to prevent cancer metastasis. From biology,biosensing and technology to drug development: the METOXIA consortium

Abstract

The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation–deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009–2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [¹⁸F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O₂), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.     

Role of zinc in catalytic activity of carbonic anhydrase IX

The carbonic anhydrases (CAs) in the α class are zinc-dependent metalloenzymes. Previous studies have reported that recombinant forms of carbonic anhydrase IX (CAIX), a membrane-bound form of CA expressed in solid tumors, appear to be activated by low levels of zinc independent of its well-studied role at the catalytic site. In this study, we sought to determine if CAIX is stimulated by zinc in its native environment. MDA-MB-231 breast cancer cells express CAIX in response to hypoxia. We compared CAIX activity associated with membrane ghosts isolated from hypoxic cells with that in intact hypoxic cells. We measured CA activity directly using (18)O exchange from (13)CO(2) into water determined by membrane inlet mass spectrometry. In membrane ghosts, there was little effect of zinc at low concentrations on CAIX activity, although at high concentration zinc was inhibitory. In intact cells, zinc had no significant effect on CAIX activity. This suggests that there is an appreciable decrease in sensitivity to zinc when CAIX is in its natural membrane milieu compared to the purified forms.     

Oncometabolites in cancer aggressiveness and tumour repopulation

Tumour repopulation is recognized as a crucial event in tumour relapse where therapy‐sensitive dying cancer cells influence the tumour microenvironment to sustain therapy‐resistant cancer cell growth. Recent studies highlight the role of the oncometabolites succinate, fumarate, and 2‐hydroxyglutarate in the aggressiveness of cancer cells and in the worsening of the patient's clinical outcome. These oncometabolites can be produced and secreted by cancer and/or surrounding cells, modifying the tumour microenvironment and sustaining an invasive neoplastic phenotype. In this review, we report recent findings concerning the role in cancer development of succinate, fumarate, and 2‐hydroxyglutarate and the regulation of their related enzymes succinate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase. We propose that oncometabolites are crucially involved in tumour repopulation. The study of the mechanisms underlying the relationship between oncometabolites and tumour repopulation is fundamental for identifying efficient anti‐cancer therapeutic strategies and novel serum biomarkers in order to overcome cancer relapse.     

Impact of tumour associated macrophages in pancreatic cancer

During cancer progression, bone marrow derived myeloid cells, including immature myeloid cells and macrophages, progressively accumulate at the primary tumour site where they contribute to the establishment of a tumour promoting microenvironment. A marked infiltration of macrophages into the stromal compartment and the generation of a desmoplastic stromal reaction is a particular characteristic of pancreatic ductal adenocarcinoma (PDA) and is thought to play a key role in disease progression and its response to therapy. Tumour associated macrophages (TAMs) foster PDA tumour progression by promoting angiogenesis, metastasis, and by suppressing an anti-tumourigenic immune response. Recent work also suggests that TAMs contribute to resistance to chemotherapy and to the emergence of cancer stem-like cells. Here we will review the current understanding of the biology and the pro-tumourigenic functions of TAMs in cancer and specifically in PDA, and highlight potential therapeutic strategies to target TAMs and to improve current therapies for pancreatic cancer. [BMB Reports 2013; 46(3): 131-138]     

Coupled modelling of tumour angiogenesis, tumour growth and blood perfusion

We propose a mathematical modelling system to investigate the dynamic process of tumour cell proliferation, death and tumour angiogenesis by fully coupling the vessel growth, tumour growth and blood perfusion. Tumour growth and angiogenesis are coupled by the chemical microenvironment and the cell-matrix interaction. The haemodynamic calculation is carried out on the updated vasculature. The domains of intravascular, transcapillary and interstitial fluid flow were coupled in the model to provide a comprehensive solution of blood perfusion variables. An estimation of vessel collapse is made according to the wall shear stress criterion to provide feedback on vasculature remodelling. The simulation can show the process of tumour angiogenesis and the spatial distribution of tumour cells for periods of up to 24 days. It can show the major features of tumour and tumour microvasculature during the period such as the formation of a large necrotic core in the tumour centre with few functional vessels passing through, and a well circulated tumour periphery regions in which the microvascular density is high and associated with more aggressive proliferating cells of the growing tumour which are all consistent with physiological observations. The study also demonstrated that the simulation results are not dependent on the initial tumour and networks, which further confirms the application of the coupled model feedback mechanisms. The model enables us to examine the interactions between angiogenesis and tumour growth, and to study the dynamic response of a solid tumour to the changes in the microenvironment. This simulation framework can be a foundation for further applications such as drug delivery and anti-angiogenic therapies.     

Skin squamous cell carcinoma propagating cells increase with tumour progression and invasiveness

Cancer stem cells have been described in various cancers including squamous tumours of the skin by their ability to reform secondary tumours upon transplantation into immunodeficient mice. Here, we used transplantation of limiting dilution of different populations of FACS‐isolated tumour cells from four distinct mouse models of squamous skin tumours to investigate the frequency of tumour propagating cells (TPCs) at different stages of tumour progression. We found that benign papillomas, despite growing rapidly in vivo and being clonogenic in vitro, reformed secondary tumours upon transplantation at very low frequency and only when tumour cells were co‐transplanted together with tumour‐associated fibroblasts or endothelial cells. In two models of skin squamous cell carcinoma (SCC), TPCs increased with tumour invasiveness. Interestingly, the frequency of TPCs increased in CD34^HI but not in CD34^LO SCC cells with serial transplantations, while the two populations initially gave rise to secondary tumours with the same frequency. Our results illustrate the progressive increase of squamous skin TPCs with tumour progression and invasiveness and reveal that serial transplantation may be required to define the long‐term renewal potential of TPCs.