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Cancer is a phenomenon broadly related to ageing in various ways such as cell cycle deregulation, metabolic defects or telomerases dysfunction as principal processes. Although the tumor cell is the main actor in cancer progression, it is not the only element of the disease. Cells and the matrix surrounding the tumor, called the tumor microenvironment (TME), play key roles in cancer progression. Phenotypic changes of the TME are indispensable for disease progression and a few of these transformations are produced by epigenetic changes including miRNA dysregulation. In this study, we found that a specific group of miRNAs in the liver TME produced by colon cancer called geromiRs, which are miRNAs related to the ageing process, are significantly downregulated. The three principal cell types involved in the liver TME, namely, liver sinusoidal endothelial cells, hepatic stellate (Ito) cells and Kupffer cells, were isolated from a murine hepatic metastasis model, and the miRNA and gene expression profiles were studied. From the 115 geromiRs and their associated hallmarks of aging, which we compiled from the literature, 75 were represented in the used microarrays, 26 out of them were downregulated in the TME cells during colon cancer colonization of the liver, and none of them were upregulated. The histone modification hallmark of the downregulated geromiRs is significantly enriched with the geromiRs miR-15a, miR-16, miR-26a, miR-29a, miR-29b and miR-29c. We built a network of all of the geromiRs downregulated in the TME cells and their gene targets from the MirTarBase database, and we analyzed the expression of these geromiR gene targets in the TME. We found that Cercam and Spsb4, identified as prognostic markers in a few cancer types, are associated with downregulated geromiRs and are upregulated in the TME cells. This work was supported by grants from Instituto de Salud Carlos III (AC17/00012), cofounded by the European Union projects (European Regional Development Fund/European Science Foundation, Investing in your future), (ERA-Net program EracoSysMed, JTC-2 2017) and (H2020-FETOPEN, Circular Vision, Project 899417); Diputación Foral de Gipuzkoa and the Department of Economic Development and Infrastructures of the Basque Government (DFG109/20) and the Department of Economic Development and Infrastructures of the Basque Government (DFG109/Grants Health Department of the Basque Government (Spain), RIS3 call, Exp. No. 2020333039 and 2020333001. 20).

This article belongs to the Special Issue Calcium Signaling Remodeling and Functional Role in Cancer Cells, [Simple Summary] Furin, a proprotein convertase that belongs to a family of Ca2+-dependent serine peptidases, is involved in the maturation of a variety of proproteins, including growth factors, receptors and differentiation factors, adhesion molecules and proteases. Furin have been associated with tumorigenesis and tumor progression and metastasis; therefore, it has been hypothesized that Furin may constitute a new potential target for cancer therapy. In triple negative breast cancer cells, inhibition of Furin by the prodomain ppFurin results in enhancement of Ca2+ influx, which involves both the increase of store-operated calcium entry (SOCE) and the activation of constitutive Ca2+ entry. The latter involves the activation of Orai and TRPC6 channels, while the increase of SOCE observed in ppFurin-expressing cells is entirely dependent on Orai channels. As a result, ppFurin expression reduces triple negative breast cancer cell viability and ability to migrate and enhances their sensitization to hydrogen peroxide-induced apoptosis., [Abstract] The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we showed that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced store-operated calcium entry (SOCE) and reduced the cell malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments revealed that the ppFurin-expressing cells are unable to adequately process the proprotein convertase (PC) substrates vascular endothelial growth factor C (proVEGF-C) and insulin-like growth factor-1 receptor (proIGF-1R). Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 and BT20 cells significantly enhanced SOCE and induced constitutive Ca2+ entry. The enhanced SOCE is impaired by inhibition of Orai channels while the constitutive Ca2+ entry is attenuated by silencing or inhibition of TRPC6 or inhibition of Orai channels. Analysis of TRPC6 activation revealed its upregulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. In addition, while ppFurin had no effect on MDA-MB-435 cell viability, in MDA-MB-231 cells ppFurin expression reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide and similar results were observed in BT20 cells. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca2+ mobilization, leading to breast cancer cells’ malignant phenotype repression and reduction of their resistance to treatments., This research was funded by SIRIC-Brio, La Ligue Contre le Cancer and Region Nouvelle Aquitaine to A.M.K. and by PID2019-104084GB-C21 and PID2019-104084GB-C22/AEI/10.13039/501100011033, MICINN (Grant BFU2016-74932-C2) and Junta de Extremadura-Fondo Europeo de Desarrollo Regional (FEDER; Grants IB16046 and GR18061) to J.A.R. and T.S. J.J.L. is supported by a contract from Junta de Extremadura (TA18011). C.C. is supported by a contract from Junta de Extremadura—FEDER.

Adult stem cells are a partially quiescent cell population responsible for natural cell renewal and are found in many different regions of the body, including the brain, teeth, bones, muscles, skin, and diverse epithelia, such as the epidermal or intestinal epithelium, among others [...]

Despite continuous exertion made, colon cancer still represents a major health problem and its incidence continues being high worldwide. There is growing evidence in support of the cancer stem cells (CSCs) being central in the initiation of this cancer, and CSCs have been the focus of various studies for the identification of new ways of treatment. Lately, the proprotein convertases (PCs) were reported to regulate the maturation and expression of various molecules involved in the malignant phenotype of colon cancer cells, however, the identity of the molecules regulated by these serine proteases in CSCs is unknown. In this study, we used the general PCs inhibitor, the Decanoyl-RVKR-chloromethylketone (Decanoyl-RVKR-CMK) that inhibits all the PCs found in the secretory pathway, and analyzed its effect on CSCs using RNA-seq analysis. Remarkably, from the only 9 up-regulated genes in the human SW620-derived sphere-forming cells, we identified 7 of the 11 human metallothioneins, all of them localized on chromosome 16, and zinc related proteins as downstream effectors of the PCs. The importance of these molecules in the regulation of cell proliferation, differentiation and chemoresistance, and their reported potential tumor suppressor role and loss in colon cancer patients associated with worse prognosis, suggests that targeting PCs in the control of the malignant phenotype of CSCs is a new potential therapeutic strategy in colon cancer.

The intracellular calcium concentration ([Ca2+]i) modulation plays a key role in the regulation of cellular growth and survival in normal cells and failure of [Ca2+]i homeostasis is involved in tumor initiation and progression. Here we show that inhibition of Furin by its naturally occurring inhibitor the prodomain ppFurin in the MDA-MB-231 breast cancer cells resulted in enhanced SOCE through TRPC6 activation that associated reduced cells malignant phenotype. Expression of ppFurin in a stable manner in MDA-MB-231 and the melanoma MDA-MB-435 cell lines inhibits Furin activity as assessed by in vitro digestion assays. Accordingly, cell transfection experiments, revealed that the ppFurin-expressing cells are unable to process adequately the PC substrates proVEGF-C and proIGF-1R. Compared to MDA-MB-435 cells, expression of ppFurin in MDA-MB-231 significantly induces Ca2+ entry which is impaired by silencing of TRPC6 expression. Analysis of TRPC6 activation revealed its up-regulated tyrosine phosphorylation in ppFurin-expressing MDA-MB-231 cells. The expression of ppFurin in MDA-MB-231 cells reduced their viability and ability to migrate and enhanced their sensitization to the apoptosis inducer hydrogen peroxide. These findings suggest that Furin inhibition by ppFurin may be a useful strategy to interfere with Ca2+ mobilization leading to breast cancer cells malignant phenotype repression and reduction of their resistance to treatments.

Colorectal cancer (CRC) is a major health problem in elderly people because of its high incidence and high mortality rate. Despite early screening programs, more than half of CRC patients are diagnosed at advanced stages. Fibroblast activation protein-alpha (FAP) expression in cancer-associated fibroblasts (CAFs) has been associated with a higher risk of metastases and poor survival. Here, we have analyzed the immunohistochemical expression of FAP in 41 adenoma-carcinoma sequences. In addition, FAP expression was analyzed individually and in combination with beta-catenin (BCAT), CD44 and Cyclin-D1 expression in primary tumors and in their corresponding lymph node and liver metastases (n=294). Finally, soluble FAP (sFAP) levels in plasma from CRC patients (n=127) were also analyzed by ELISA. FAP was expressed only in CRC tissue and its expression level was found to be higher in tumors exhibiting deeper local invasion and poorer cancer cell differentiation. FAP and concomitant nuclear BCAT expression in cancer cells at the infiltrating front of primary tumors and in lymph node metastases was independently associated with 5- and 10-year cancer specific and disease-free survival. Moreover, lower sFAP levels correlated with poorer survival. These findings support the potential importance of FAP as a biomarker of CRC development and progression. This work was partially funded by the ELKARTEK 18/10 grant from the Basque Government.

Colon cancer is one of the most frequently occurring types of cancers in the world. Primary tumours are treated very efficiently, but the metastatic cases are known to have severe outcomes. Therefore, the aim of the present study was to obtain a greater understanding of the transformation of primary colon cancer cells into metastatic phenotypes. Small changes in protein expression provoke the metastatic phenotype transformation. More sensitive methods to detect small variations are required. A murine colon cancer cell line with metastatic characteristics in a very early phase was created in order to investigate the first steps of transformation using a murine liver metastasis model. The protein expression patterns of metastatic and non-metastatic cells were compared using the stable isotope labelling by amino acids in cell culture method in combination with mass spectrometry. Quantitative proteomics data indicated that nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase complex I was overexpressed in metastatic cells with respect to non-metastatic cells. Since the NADH dehydrogenase complex catalyses the oxidation of NADH to NAD+, the functionality of the complex was studied by measuring the amount of NADH. The results revealed that metastatic cells accumulate more NADH and reactive oxygen species. In addition, the mitochondrial membrane potential of metastatic cells was lower than that of non-metastatic cells, indicating that the activity of NADH dehydrogenase and the mitochondrial oxidative chain were decreased in metastatic cells. During the incipient transformation of primary cancer cells, NADH dehydrogenase complex I was overexpressed but then became inactive due to the Warburg effect, which inhibits mitochondrial activity. In the first step of transformation, the high energy demand required in an adverse environment is fulfilled by overexpressing components of the respiratory chain, a fact that should be considered for future anti-metastatic therapies.

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.

(1) Background &amp, Aims: The roles of different cells in the tumor microenvironment (TME) are critical to the metastatic process. The phenotypic transformation of the liver cells is one of the most important stages of the hepatic metastasis progression of colorectal cancer (CRC). Our aim was to identify the major molecules (i.e., genes, miRNAs and proteins) involved in this process. (2) Methods: We isolated and performed whole-genome analysis of gene, miRNA, and protein expression in three types of liver cells (Ito cells, Kupffer cells, and liver sinusoidal endothelial cells) from the TME of a murine model of CRC liver metastasis. We selected the statistically significant differentially expressed molecules using the Student's t-test with Benjamini-Hochberg correction and performed functional statistically-significant enrichment analysis of differentially expressed molecules with hypergeometric distribution using the curated collection of molecular signatures, MSigDB. To build a gene-miRNA-protein network centered in Brca1, we developed a software package (miRDiana) that collects miRNA targets from the union of the TargetScan, MicroCosm, mirTarBase, and miRWalk databases. This was used to search for miRNAs targeting Brca1. We validated the most relevant miRNAs with real-time quantitative PCR. To investigate BRCA1 protein expression, we built tissue microarrays (TMAs) from hepatic metastases of 34 CRC patients. (3) Results: Using integrated omics analyses, we observed that the Brca1 gene is among the twenty transcripts simultaneously up-regulated in all three types of TME liver cells during metastasis. Further analysis revealed that Brca1 is the last BRCA1-associated genome surveillance complex (BASC) gene activated in the TME. We confirmed this finding in human reanalyzing transcriptomics datasets from 184 patients from non-tumor colorectal tissue, primary colorectal tumor and colorectal liver metastasis of the GEO database. We found that the most probable sequence of cell activation during metastasis is Endothelial&rarr, Ito&rarr, Kupffer. Immunohistochemical analysis of human liver metastases showed the BRCA1 protein was co-localized in Ito, Kupffer, and endothelial cells in 81.8% of early or synchronous metastases. However, in the greater part of the metachronous liver metastases, this protein was not expressed in any of these TME cells. (4) Conclusions: These results suggest a possible role of the co-expression of BRCA1 in Ito, Kupffer, and sinusoidal endothelial cells in the early occurrence of CRC liver metastases, and point to BRCA1 as a potential TME biomarker.

Liver metastasis depends on the collagenous microenvironment generated by hepatic sinusoidal cells (SCs). DDR1 is an atypical collagen receptor linked to tumor progression, but whether SCs express DDR1 and its implication in liver metastasis remain unknown. Freshly isolated hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs), that conform the SCs, expressed functional DDR1. HSCs expressed the largest amounts. C26 colon carcinoma secretomes increased DDR1 phosphorylation in HSCs and KCs by collagen I. Inhibition of kinase activity by DDR1-IN-1 or mRNA silencing of DDR1 reduced HSCs secretion of MMP2/9 and chemoattractant and proliferative factors for LSECs and C26 cells. DDR1-IN-1 did not modify MMP2/9 in KCs or LSECs secretomes, but decreased the enhancement of C26 migration and proliferation induced by their secretomes. Gene array showed that DDR1 silencing downregulated HSCs genes for collagens, MMPs, interleukins and chemokines. Silencing of DDR1 before tumor inoculation reduced hepatic C26 metastasis in mice. Silenced livers bore less tumor foci than controls. Metastatic foci in DDR1 silenced mice were smaller and contained an altered stroma with fewer SCs, proliferating cells, collagen and MMPs than foci in control mice. In conclusion, hepatic DDR1 promotes C26 liver metastasis and favors the pro-metastatic response of SCs to the tumor. We would like to acknowledge the following core facilities and individuals for their support: CIC bioGUNE Center for Cooperative Research in Biosciences, University of the Basque Country Animal Core Facility and SGIker Advanced Light Microscopy Core Facility. We thank Iratxe Basaldua for the in situ MMPs assays

Simple Summary Did you ever wonder why some tissues can produce very aggressive types of cancer whereas others are apparently immune to this devastating disease? One of the most accepted theories in the scientific community states that tumors are fueled by small numbers of key master cells called cancer stem cells, which mediate tumor relapse and metastasis. Much effort has been made to identify these cells by the characterization of their defining markers, in an attempt to eliminate these cells selectively. However, many of these markers are also present in other healthy stem cells in the body, including those found in some tissues like the dental pulp, which is known to be highly resistant to carcinogenesis. This brings up the question of whether there is indeed a genuine marker that can be used to unequivocally identify cancer stem cells. We set out to address this question by a systematic comparison of healthy stem cells and cancer stem cells of different body locations, and we discuss some key factors that play a role in the resistance of certain types of stem cells to malignant transformation. Abstract The conversion of healthy stem cells into cancer stem cells (CSCs) is believed to underlie tumor relapse after surgical removal and fuel tumor growth and invasiveness. CSCs often arise from the malignant transformation of resident multipotent stem cells, which are present in most human tissues. Some organs, such as the gut and the brain, can give rise to very aggressive types of cancers, contrary to the dental pulp, which is a tissue with a very remarkable resistance to oncogenesis. In this review, we focus on the similarities and differences between gut, brain and dental pulp stem cells and their related CSCs, placing a particular emphasis on both their shared and distinctive cell markers, including the expression of pluripotency core factors. We discuss some of their similarities and differences with regard to oncogenic signaling, telomerase activity and their intrinsic propensity to degenerate to CSCs. We also explore the characteristics of the events and mutations leading to malignant transformation in each case. Importantly, healthy dental pulp stem cells (DPSCs) share a great deal of features with many of the so far reported CSC phenotypes found in malignant neoplasms. However, there exist literally no reports about the contribution of DPSCs to malignant tumors. This raises the question about the particularities of the dental pulp and what specific barriers to malignancy might be present in the case of this tissue. These notable differences warrant further research to decipher the singular properties of DPSCs that make them resistant to transformation, and to unravel new therapeutic targets to treat deadly tumors.

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.

Discoidin Domain Receptor 1 and Discodin Domain Receptor 2 are the two only members of the DDR family. The DDR family is a Tyrosine Kinase Receptor (TKR) family with some peculiarities compared with other Tyrosine Kinase Receptors such as their natural ligand; which in this case is the fibrillar collagen; or the slow phosphorylation pattern. These peculiarities confer a special role to the receptors present in many diseases development processes as cancer, cirrhosis or lung fibrosis. In this review it is described the overview of the DDRs structure and their role in the different disease development and the possibility to consider them as therapeutic targets.

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.

Hepatic fibrosis results from an altered balance of extracellular matrix (ECM) synthesis and degradation that is skewed toward an accumulation of a collagenous ECM following chronic injury. Fibrosis from persistent injury may progress to cirrhosis, an end-stage lesion, which may lead to liver failure and death. Research is focused on receptor tyrosine kinases (RTKs) as new targets for antifibrotic therapies. Among them, discoidin domain receptors (DDRs) attract much attention because they represent a new paradigm of RTK signaling in response to ECM instead of soluble factors.

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.

Epiprofin/Specificity Protein 6 (Epfn) is a Kruppel-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.

Discoidin domain receptors (DD r 1 and DD r 2) 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 respec- tively, compared to mock-transfected cells (A375R2-100). Development of experimental liver metastasis by intra- splenic 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 phosphory- lation, 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.

This study examined the ability of (i) pure nisin, (ii) nisin-producing Lactococcus lactis strain CHCC5826, and (iii) the non-nisin-producing L. lactis strain CHCH2862 to affect the composition of the intestinal microbiota of human flora-associated rats. The presence of both the nisin-producing and the non-nisin-producing L. lactis strains significantly increased the number of Bifidobacterium cells in fecal samples during the first 8 days but decreased the number of enterococci/streptococci in duodenum, ileum, cecum, and colon samples as detected by selective cultivation. No significant changes in the rat fecal microbiota were observed after dosage with nisin. Pearson cluster analysis of denaturing gradient gel electrophoresis profiles of the 16S rRNA genes present in the fecal microbial population revealed that the microbiota of animals dosed with either of the two L. lactis strains were different from that of control animals dosed with saline. However, profiles of the microbiota from animals dosed with nisin did not differ from the controls. The concentrations of nisin estimated by competitive enzyme-linked immunosorbent assay (ELISA) were approximately 10-fold higher in the small intestine and 200-fold higher in feces than the corresponding concentrations estimated by a biological assay. This indicates that nisin was degraded or inactivated in the gastrointestinal tract, since fragments of this bacteriocin are detected by ELISA while an intact molecule is needed to retain biological activity.

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 animal's 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