Iker Badiola

Discoidin domain receptor 2 deficiency predisposes hepatic tissue to colon carcinoma metastasis

Background The transdifferentiation of hepatic stellate cells (HSCs) into myofibroblasts is a major mechanism for stroma development in hepatic metastasis, but their regulatory pathways remain unclear. Transdifferentiated HSCs from fibrotic liver express high levels of the fibrillar collagen receptor discoidin domain receptor 2 (DDR2), but it is unclear if DDR2 plays a direct profibrogenic role in the tumour microenvironment. Aim To assess the impact of DDR2 on the prometastatic role of HSC-derived myofibroblasts. Methods Hepatic metastases were induced in DDR2−/− and DDR2+/+ mice by intrasplenic injection of MCA38 colon carcinoma cells, and their growth and features were characterised. Stromagenic, angiogenic and cancer cell proliferation responses were quantified in metastases by immunohistochemistry. The adhesion-, migration- and proliferation-stimulating activities of supernatants from primary cultured DDR2−/− and DDR2+/+ HSCs, incubated in MCA38 cell-conditioned medium, were evaluated in primary cultured liver sinusoidal endothelium cells (LSECs) and MCA38 cells. Gene expression signatures from freshly isolated DDR2−/− and DDR2+/+ HSCs were compared and DDR2-regulated genes were studied by RT-PCR under basal conditions and after stimulation with MCA38 tumour-conditioned media. Results Metastases were increased three fold in DDR2−/− livers, and contained a higher density of α−smooth muscle actin-expressing myofibroblasts, CD31-expressing microvessels and Ki67-expressing MCA38 cells than metastases in DDR2+/+ livers. Media conditioned by MCA38-activated DDR2−/− HSCs significantly increased adhesion, migration and proliferation of LSECs and MCA38 cells, compared with DDR2+/+ HSCs. DDR2 deficiency in HSCs led to decreased gene expression of interferon γ-inducing factor interleukin (IL)-18 and insulin-like growth factor-I; and increased gene expression of prometastatic factors IL-10, transforming growth factor (TGF)β and vascular endothelial growth factor (VEGF), bone morphogenetic protein-7 and syndecan-1. MC38 tumour-conditioned media further exacerbated expression changes in DDR2-dependent IL-10, TGFβ and VEGF genes. Conclusion DDR2 deficiency fosters the myofibroblast transdifferentiation of tumour-activated HSCs, generating a prometastatic microenvironment in the liver via HSC-derived factors. These findings underscore the role of stromal cells in conditioning the hepatic microenvironment for metastases through altered receptor–stroma interactions.

Downregulation of discoidin domain receptor 2 in A375 human melanoma cells reduces its experimental liver metastasis ability

Discoidin domain receptors (DDR1 and DDR2) are tyrosine kinase receptors for fibrillar collagen implicated in postnatal development, tissue repair, and primary and metastatic cancer progression. While DDR1 has been described in tumor cells, DDR2 has been localized in the tumor stroma, but its presence in the tumor cells remains unknown. The aim of this study was to elucidate the role of DDR2 signaling in tumor cells during hepatic metastasis progression. DDR2 expression and phosphorylation in cultured human A375 melanoma cells was documented by Western blot analysis. A375 cells were stably transfected with a small interfering RNA (siRNA) against DDR2 and two clones were selected: A375R2-70 and A375R2-40, with 70 and 40% of the DDR2 protein expression respectively, compared to mock-transfected cells (A375R2-100). Development of experimental liver metastasis by intrasplenic inoculation of A375R2-70 and A37R2-40 clones was reduced by 60 and 75%, respectively, measured as tumor volume, compared to livers injected with A375R2-100 cells. Accordingly, A375R2-70 and A37R2-40 clones showed reduced in vitro gelatinase activity and JNK phosphorylation, compared to mock transfected cells, with maximal inhibition in A375R2-40. Additionally, A375 melanoma, SK-HEP hepatoma and HT-29 colon carcinoma human cell lines transiently transfected with siRNA against DDR2 also showed reduced proliferation and migration rates compared to mock-transfected ones. In conclusion, DDR2 promotes A375 melanoma metastasis to the liver and the underlying mechanism implicates regulation of metalloproteinase release, cell growth and chemotactic invasion of the host tissue.

Nanocarriers for microRNA delivery in cancer medicine

The number of deaths caused by cancer is expected to increase partly due to the lack of selectivity and undesirable systemic effects of current treatments. Advances in the understanding of microRNA (miRNA) functions and the ideal properties of nanosystems have brought increasing attention to the application of nanomedicine to cancer therapy. This review covers the different miRNA therapeutic strategies and delivery challenges for its application in cancer medicine. Current trends in inorganic, polymeric and lipid nanocarrier development for miRNA replacement or inhibition are summarized. To achieve clinical success, in-depth knowledge of the effects of the promotion or inhibition of specific miRNAs is required. To establish the dose and the length of treatment, it will be necessary to study the duration of gene silencing. Additionally, efforts should be made to develop specifically targeted delivery systems to cancer cells to reduce doses and unwanted effects. In the near future, the combination of miRNAs with other therapeutic approaches is likely to play an important role in addressing the heterogeneity of cancer.

Epiprofin/Sp6 regulates Wnt-BMP signaling and the establishment of cellular junctions during the bell stage of tooth development

Epiprofin/Specificity Protein 6 (Epfn) is a Krüppel-like family (KLF) transcription factor that is critically involved in tooth morphogenesis and dental cell differentiation. However, its mechanism of action is still not fully understood. We have employed both loss-of-function and gain-of-function approaches to address the role of Epfn in the formation of cell junctions in dental cells and in the regulation of junction-associated signal transduction pathways. We have evaluated the expression of junction proteins in bell-stage incisor and molar tooth sections from Epfn(−/−) mice and in dental pulp MDPC-23 cells overexpressing Epfn. In Epfn(−/−) mice, a dramatic reduction occurs in the expression of tight junction and adherens junction proteins and of the adherens-junction-associated β-catenin protein, a major effector of canonical Wnt signaling. Loss of cell junctions and β-catenin in Epfn(−/−) mice is correlated with a clear decrease in bone morphogenetic protein 4 (BMP-4) expression, a decrease in nestin in the tooth mesenchyme, altered cell proliferation, and failure of ameloblast cell differentiation. Overexpression of Epfn in MDPC-23 cells results in an increased cellular accumulation of β-catenin protein, indicative of upregulation of canonical Wnt signaling. Together, these results suggest that Epfn enhances canonical Wnt/β-catenin signaling in the developing dental pulp mesenchyme, a condition that promotes the activity of other downstream signaling pathways, such as BMP, which are fundamental for cellular induction and ameloblast differentiation. These altered signaling events might underlie some of the most prominent dental defects observed in Epfn(−/−) mice, such as the absence of ameloblasts and enamel, and might throw light on developmental malformations of the tooth, including hyperdontia.

Biomolecular bases of the senescence process and cancer. A new approach to oncological treatment linked to ageing

Human ageing is associated with a gradual decline in the physiological functions of the body at multiple levels and it is a key risk factor for many diseases, including cancer. Ageing process is intimately related to widespread cellular senescence, characterised by an irreversible loss of proliferative capacity and altered functioning associated with telomere attrition, accumulation of DNA damage and compromised mitochondrial and metabolic function. Tumour and senescent cells may be generated in response to the same stimuli, where either cellular senescence or transformation would constitute two opposite outcomes of the same degenerative process. This paper aims to review the state of knowledge on the biomolecular relationship between cellular senescence, ageing and cancer. Importantly, many of the cell signalling pathways that are found to be altered during both cellular senescence and tumourigenesis are regulated through shared epigenetic mechanisms and, therefore, they are potentially reversible. MicroRNAs are emerging as pivotal players linking ageing and cancer. These small RNA molecules have generated great interest from the point of view of future clinical therapy for cancer because successful experimental results have been obtained in animal models. Micro-RNA therapies for cancer are already being tested in clinical phase trials. These findings have potential importance in cancer treatment in aged people although further research-based knowledge is needed to convert them into an effective molecular therapies for cancer linked to ageing.

Inhibition of the metastatic progression of breast and colorectal cancer in vitro and in vivo in murine model by the oxidovanadium(IV) complex with luteolin

The anticancer and antimetastatic behavior of the flavonoid luteolin and its oxidovanadium(IV) complex [VO(lut)(H2O)2]Na·3H2O (VOlut) has been investigated. Considering that the complex displayed strong anticancer activity on MDAMB231 human breast cancer cell line we herein determined through in vitro assays that the complex would probably reduce breast cancer cell metastasis in a higher extent than the natural antioxidant. In the CT26 colon cancer cell line a stronger anticancer effect has also been determined for the complex (IC50 0.9μM) and in addition it did not exert toxic effects on normal colon epithelial cells at concentrations up to 10μM. Working with a murine model of highly aggressive, orthotopic colon cancer model (CT26 cancer cell lines) it has been determined that the complex might prevent metastatic dissemination of the colon cancer cells to the liver. The flavonoid luteolin also exerted anticancer effects (at a low degree, IC50 5.9μM) on CT26 cell line and produced a 24% reduction of colon cancer liver metastasis.

Abstract 5347: MiRNA expression profile of tumor microenvironment in murine liver metastatic model.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Colorectal cancer (CRC) is the second most recurrent cancer type and one of the most lethal diseases around the worldwide. The lethality of the CRC is due to the metastasis and the principal target is the liver. Although the main responsible of the metastasis is the cancer cell we can not obviate the crucial role of the host organ and how the cells of the host organ support the progression of the disease. The crosstalk of the tumor microenvironment with the tumor has been deeply studied from the cellular point of view but the regulatory pathways of these processes still remain unknown. The epigenetic regulation has been postulated as one important process implicated in the regulation of cellular changes. The microRNA (miRNA) expression is one of the variants of the epigenetic regulation. In this work we study the deregulation of the miRNA expression in the metastasized liver respect to healthy condition using a murine in vivo liver metastasis model. We inoculated CT26 murine colon carcinoma cell intrasplenically in BALB/c mice and after 15 days we sacrificed the animals to obtain four liver cell types (hepatocytes, liver sinusoidal endothelial cells, Kupffer cells and hepatic stellate cells). Then we isolated the total RNA and performed gene expression and miRNA expression microarrays (Agilent) comparing the tumor received cells with control cells (PBS injected animals cells). The gene expression microarray helps us finding the biological and cellular sense of the deregulated miRNAs and could be an important data source to elucidate the therapeutic option of each deregulated miRNAs. An in vivo approach is a more realistic model that can show more elements implicated in the crosstalk of the different liver cell types. This fact show us several results in the different cell types with expected and unexpected new miRNA deregulations. For instance downregulation of miR-16 and miR-15b and upregulation of miR-138 has been found in hepatic stellate cells with important role in activation proliferation and apoptosis of this cell type and we also observed deregulation of miR-135 in liver sinusoidal cells. These are some of the examples of a large amount of miRNA identified. Nowadays we are checking the role of each deregulated miRNA in the cellular processes to verify their implication in the metastatic progression and postulate them as therapeutic targets. Citation Format: Joana Marquez, Arianne Schaub, Maite Unzurrunzaga, Patricia Garcia, Iker Badiola. MiRNA expression profile of tumor microenvironment in murine liver metastatic model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5347. doi:10.1158/1538-7445.AM2013-5347

Targeting liver sinusoidal endothelial cells with miR-20a-loaded nanoparticles reduces murine colon cancer metastasis to the liver: Nanoparticles loaded with miR-20a reduces liver metastasis

Phenotypic transformation of liver sinusoidal endothelial cells is one of the most important stages of liver metastasis progression. The miRNA effects on liver sinusoidal endothelial cells during liver metastasis have not yet been studied. Herein, whole genome analysis of miRNA expression in these cells during colorectal liver metastasis revealed repressed expression of microRNA‐20a. Importantly, downregulation of miR‐20a occurs in parallel with upregulation of its known protein targets. To restore normal miR‐20a levels in liver sinusoidal endothelial cells, we developed chondroitin sulfate‐sorbitan ester nanoparticles conjugated with miR‐20a in a delivery system that specifically targets liver sinusoidal endothelial cells. The restoration of normal mir‐20a levels in these cells induced downregulation of the expression of its protein targets, and this also resulted in a reduction of in vitro LSEC migration and a reduction of in vivo activation and tumor‐infiltrating capacity and ability of the tumor decreased by ∼80% in a murine liver metastasis model.

Regulation of the proprotein convertases expression and activity during regenerative angiogenesis: Role of hypoxia-inducible factor (HIF)

Proprotein convertases (PCs) are involved in various physiological and pathological processes ranging from embryogenesis to carcinogenesis. Here, using the zebrafish fin regeneration model, we report induced expression of furin and PC5 but not PACE4 and PC7 during fin regeneration that is associated with increased PC activity. Stabilization of HIF by cobalt chloride (CoCl2) further increases these processes. The use of the general PC-inhibitor decanoyl-RVKR-cholromethyl ketone (CMK) inhibited control and CoCl2-induced PC activity. CoCl2 inhibits embryonic zebrafish ZF4 cell proliferation and caudal fin regeneration that is associated with the expression of the anti-proliferative genes P21, P16, PC3 and P53 in ZF4 cells and in non-regenerating stump tissues. In contrast, during fin regeneration, HIF stabilization failed to promote the expression of these anti-proliferative genes and maintained high expression of cyclin D. Further analysis revealed that CoCl2 maintained the formation of immature regenerating vasculature that was associated with amplified expression of OCT4 and various angiogenic factors reported to be PC substrates and/or downstream effectors. These findings revealed that while furin and PC5 expression/activity and their substrates/effectors are regulated during fin regeneration, HIF stabilization by CoCl2 has the potential to modulate these processes and impact on the regenerative process and vessels organization.

Development and characterisation of chondroitin sulfate- and hyaluronic acid-incorporated sorbitan ester nanoparticles as gene delivery systems

Graphical abstract Figure. No caption available. ABSTRACT Glycosaminoglycans (GAGs) are natural polymers that are broadly used in gene delivery systems to increase stability as well as decrease toxicity and nonspecific interactions, thereby increasing transfection efficiency. In this work, we propose sorbitan ester‐based lipid nanoparticles (SENS) functionalised with the GAGs chondroitin sulfate (CS) and hyaluronic acid (HA) as gene delivery systems. For this purpose, we describe the design and evaluation of these nanosystems loaded with plasmid DNA, including an evaluation of their physicochemical characteristics, stability properties, ability to protect and efficiently transfect cells with Enhanced Green Fluorescent Protein plasmid (pEGFP) in vitro, and biocompatibility both in vitro and in vivo. We confirm that molecules with high biological value and targeting potential, such as HA and CS, can be successfully incorporated into our recently developed sorbitan ester‐based nanoparticles (SENS) and that this incorporation leads to effective stabilisation of both nanosystems as well as protects plasmid DNA. We demonstrated that the aforementioned incorporation of HA and CS enables long‐term stability of the nanosystems in both liquid and lyophilised states, which is a remarkable property that can aid in their transfer to industry. The ability of these functionalised nanosystems to transfect the A549 cell line without compromising cell viability was also shown, as well as their innocuous safety profile in vivo. Thus, we provide valuable evidence of the suitable properties and potential of these hybrid nanoparticles as gene delivery systems.