Reverse Transfection of Functional RNA Rings into Cancer Cells Followed by in Vitro Irradiation.

RNA nanoparticles are promising therapeutic platforms to improve radiotherapy since they can be functionalized with multiple small interfering RNAs (RNAi) to simultaneously silence critical radioresistance genes. Here we describe the transfer of RNA rings to mammalian cancer cells through reverse transfection, followed by in vitro irradiation and biological assays as surrogates' endpoints for radiotherapy efficacy.

Sulforaphane: An emergent anti-cancer stem cell agent.

Cancer is a major public health concern worldwide responsible for high morbidity and mortality rates. Alternative therapies have been extensively investigated, and plant-derived compounds have caught the attention of the scientific community due to their chemopreventive and anticancer effects. Sulforaphane (SFN) is one of these naturally occurring agents, and studies have shown that it is able to target a specific cancer cell population displaying stem-like properties, known as cancer stem cells (CSCs). These cells can self-renewal and differentiate to form highly heterogeneous tumor masses. Notably, most of the conventional chemotherapeutic agents cannot target CSCs once they usually exist in a quiescent state and overall, the available cytotoxic drugs focus on highly dividing cells. This is, at least in part, one of the reasons why some oncologic patients relapse after standard therapy. In this review we bring together studies supporting not only the chemopreventive and anticancer properties of SFN, but especially the emerging anti-CSCs effects of this natural product and its potential to be used with conventional antineoplastic drugs in the clinical setting.

Simultaneous silencing of lysophosphatidylcholine acyltransferases 1-4 by nucleic acid nanoparticles (NANPs) improves radiation response of melanoma cells.

Radiation induces the generation of platelet-activating factor receptor (PAF-R) ligands, including PAF and oxidized phospholipids. Alternatively, PAF is also synthesized by the biosynthetic enzymes lysophosphatidylcholine acyltransferases (LPCATs) which are expressed by tumor cells including melanoma. The activation of PAF-R by PAF and oxidized lipids triggers a survival response protecting tumor cells from radiation-induced cell death, suggesting the involvement of the PAF/PAF-R axis in radioresistance. Here, we investigated the role of LPCATs in the melanoma cell radiotherapy response. LPCAT is a family of four enzymes, LPCAT1-4, and modular nucleic acid nanoparticles (NANPs) allowed for the simultaneous silencing of all four LPCATs. We found that the in vitro simultaneous silencing of all four LPCAT transcripts by NANPs enhanced the therapeutic effects of radiation in melanoma cells by increasing cell death, reducing long-term cell survival, and activating apoptosis. Thus, we propose that NANPs are an effective strategy for improving radiotherapy efficacy in melanomas.

Emerging Autophagy Functions Shape the Tumor Microenvironment and Play a Role in Cancer Progression - Implications for Cancer Therapy.

The tumor microenvironment (TME) is a complex environment where cancer cells reside and interact with different types of cells, secreted factors, and the extracellular matrix. Additionally, TME is shaped by several processes, such as autophagy. Autophagy has emerged as a conserved intracellular degradation pathway for clearance of damaged organelles or aberrant proteins. With its central role, autophagy maintains the cellular homeostasis and orchestrates stress responses, playing opposite roles in tumorigenesis. During tumor development, autophagy also mediates autophagy-independent functions associated with several hallmarks of cancer, and therefore exerting several effects on tumor suppression and/or tumor promotion mechanisms. Beyond the concept of degradation, new different forms of autophagy have been described as modulators of cancer progression, such as secretory autophagy enabling intercellular communication in the TME by cargo release. In this context, the synthesis of senescence-associated secretory proteins by autophagy lead to a senescent phenotype. Besides disturbing tumor treatment responses, autophagy also participates in innate and adaptive immune signaling. Furthermore, recent studies have indicated intricate crosstalk between autophagy and the epithelial-mesenchymal transition (EMT), by which cancer cells obtain an invasive phenotype and metastatic potential. Thus, autophagy in the cancer context is far broader and complex than just a cell energy sensing mechanism. In this scenario, we will discuss the key roles of autophagy in the TME and surrounding cells, contributing to cancer development and progression/EMT. Finally, the potential intervention in autophagy processes as a strategy for cancer therapy will be addressed.

Identification of two novel adenoviruses in smooth-billed ani and tropical screech owl.

Avian adenoviruses (AdVs) are a very diverse group of pathogens causing diseases in poultry and wild birds. Wild birds, endangered by habitat loss and habitat fragmentation in the tropical forests, are recognised to play a role in the transmission of various AdVs. In this study, two novel, hitherto unknown AdVs were described from faecal samples of smooth-billed ani and tropical screech owl. The former was classified into genus Aviadenovirus, the latter into genus Atadenovirus, and both viruses most probably represent new AdV species as well. These results show that there is very limited information about the biodiversity of AdVs in tropical wild birds, though viruses might have a major effect on the population of their hosts or endanger even domesticated animals. Surveys like this provide new insights into the diversity, evolution, host variety, and distribution of avian AdVs.

Developmental signaling pathways regulating mammary stem cells and contributing to the etiology of triple-negative breast cancer.

Cancer has been considered as temporal and spatial aberrations of normal development in tissues. Similarities between mammary embryonic development and cell transformation suggest that the underlying processes required for mammary gland development are also those perturbed during various stages of mammary tumorigenesis and breast cancer (BC) development. The master regulators of embryonic development Cripto-1, Notch/CSL, and Wnt/ß-catenin play key roles in modulating mammary gland morphogenesis and cell fate specification in the embryo through fetal mammary stem cells (fMaSC) and in the adult organism particularly within the adult mammary stem cells (aMaSC), which determine mammary progenitor cell lineages that generate the basal/myoepithelial and luminal compartments of the adult mammary gland. Together with recognized transcription factors and embryonic stem cell markers, these embryonic regulatory molecules can be inappropriately augmented during tumorigenesis to support the tumor-initiating cell (TIC)/cancer stem cell (CSC) compartment, and the effects of their deregulation may contribute for the etiology of BC, in particular the most aggressive subtype of BC, triple-negative breast cancer (TNBC). This in depth review will present evidence of the involvement of Cripto-1, Notch/CSL, and Wnt/ß-catenin in the normal mammary gland morphogenesis and tumorigenesis, from fMaSC/aMaSC regulation to TIC generation and maintenance in TNBC. Specific therapies for treating TNBC by targeting these embryonic pathways in TICs will be further discussed, providing new opportunities to destroy not only the bulk tumor, but also TICs that initiate and promote the metastatic spread and recurrence of this aggressive subtype of BC.

Cripto-1 ablation disrupts alveolar development in the mouse mammary gland through a progesterone receptor-mediated pathway.

Cripto-1, a member of the epidermal growth factor-Cripto-1/FRL-1/Cryptic family, is critical for early embryonic development. Together with its ligand Nodal, Cripto-1 has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Several studies have clearly shown that Cripto-1 is involved in regulating branching morphogenesis and epithelial-mesenchymal transition of the mammary gland both in vitro and in vivo and together with the cofactor GRP78 is critical for the maintenance of mammary stem cells ex vivo. Our previous studies showed that mammary-specific overexpression of human Cripto-1 exhibited dramatic morphological alterations in nulliparous mice mammary glands. The present study shows a novel mechanism for Cripto-1 regulation of mammary gland development through direct effects on progesterone receptor expression and pathways regulated by progesterone in the mammary gland. We demonstrate a strict temporal regulation of mouse Cripto-1 (mCripto-1) expression that occurs during mammary gland development and a stage-specific function of mCripto-1 signaling during mammary gland development. Our data suggest that Cripto-1, like the progesterone receptor, is not required for the initial ductal growth but is essential for subsequent side branching and alveologenesis during the initial stages of pregnancy. Dissection of the mechanism by which this occurs indicates that mCripto-1 activates receptor activator NF-κB/receptor activator NF-κB ligand, and NF-κB signaling pathways.

Cripto-1: an extracellular protein - connecting the sequestered biological dots.

Cripto-1 (CR-1) is a multifunctional embryonic protein that is re-expressed during inflammation, wound repair, and malignant transformation. CR-1 can function either as a tethered co-receptor or shed as a free ligand underpinning its flexible role in cell physiology. CR-1 has been shown to mediate cell growth, migration, invasion, and induce epithelial to mesenchymal transition (EMT). The main signaling pathways mediating CR-1 effects include Nodal-dependent (Smad2/3) and Nodal-independent (Src/p44/42/Akt) signaling transduction pathways. In addition, there are several naturally occurring binding partner proteins (BPPs) for CR-1 that can either agonize or antagonize its bioactivity. We will review the collective role of CR-1 as an extracellular protein, discuss caveats to consider in developing a quantitation assay, define possible mechanistic avenues applicable for drug discovery, and report on our experimental approaches to overcome these problematic issues.

Cripto-1 as a novel therapeutic target for triple negative breast cancer.

Triple-negative breast cancer (TNBC) presents the poorest prognosis among the breast cancer subtypes and no current standard therapy. Here, we performed an in-depth molecular analysis of a mouse model that establishes spontaneous lung metastasis from JygMC(A) cells. These primary tumors resembled the triple-negative breast cancer (TNBC) both phenotypically and molecularly. Morphologically, primary tumors presented both epithelial and spindle-like cells but displayed only adenocarcinoma-like features in lung parenchyma. The use of laser-capture microdissection combined with Nanostring mRNA and microRNA analysis revealed overexpression of either epithelial and miRNA-200 family or mesenchymal markers in adenocarcinoma and mesenchymal regions, respectively. Cripto-1, an embryonic stem cell marker, was present in spindle-like areas and its promoter showed activity in primary tumors. Cripto-1 knockout by the CRISPR-Cas9 system inhibited tumor growth and pulmonary metastasis. Our findings show characterization of a novel mouse model that mimics the TNBC and reveal Cripto-1 as a TNBC target hence may offer alternative treatment strategies for TNBC.

The multifaceted role of the embryonic gene Cripto-1 in cancer, stem cells and epithelial-mesenchymal transition.

Cripto-1 (CR-1)/Teratocarcinoma-derived growth factor1 (TDGF-1) is a cell surface glycosylphosphatidylinositol (GPI)-linked glycoprotein that can function either in cis (autocrine) or in trans (paracrine). The cell membrane cis form is found in lipid rafts and endosomes while the trans acting form lacking the GPI anchor is soluble. As a member of the epidermal growth factor (EGF)/Cripto-1-FRL-1-Cryptic (CFC) family, CR-1 functions as an obligatory co-receptor for the transforming growth factor-ß (TGF-ß) family members, Nodal and growth and differentiation factors 1 and 3 (GDF1/3) by activating Alk4/Alk7 signaling pathways that involve Smads 2, 3 and 4. In addition, CR-1 can activate non-Smad-dependent signaling elements such as PI3K, Akt and MAPK. Both of these pathways depend upon the 78kDa glucose regulated protein (GRP78). Finally, CR-1 can facilitate signaling through the canonical Wnt/ß-catenin and Notch/Cbf-1 pathways by functioning as a chaperone protein for LRP5/6 and Notch, respectively. CR-1 is essential for early embryonic development and maintains embryonic stem cell pluripotentiality. CR-1 performs an essential role in the etiology and progression of several types of human tumors where it is expressed in a population of cancer stem cells (CSCs) and facilitates epithelial-mesenchymal transition (EMT). In this context, CR-1 can significantly enhance tumor cell migration, invasion and angiogenesis. Collectively, these facts suggest that CR-1 may be an attractive target in the diagnosis, prognosis and therapy of several types of human cancer.

Regulation of human Cripto-1 expression by nuclear receptors and DNA promoter methylation in human embryonal and breast cancer cells.

Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However, mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study, we investigated the effects of two nuclear receptors, liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter, while GCNF strongly suppressed CR-1 expression in these cells. In addition, the CR-1 promoter was unmethylated in NTERA-2 cells, while T47D, ZR75-1, and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells, promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors, suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively, these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer.

Cripto-1 enhances the canonical Wnt/ß-catenin signaling pathway by binding to LRP5 and LRP6 co-receptors.

Cripto-1 is implicated in multiple cellular events, including cell proliferation, motility and angiogenesis, through the activation of an intricate network of signaling pathways. A crosstalk between Cripto-1 and the canonical Wnt/ß-catenin signaling pathway has been previously described. In fact, Cripto-1 is a downstream target gene of the canonical Wnt/ß-catenin signaling pathway in the embryo and in colon cancer cells and T-cell factor (Tcf)/lymphoid enhancer factor binding sites have been identified in the promoter and the first intronic region of the mouse and human Cripto-1 genes. We now demonstrate that Cripto-1 modulates signaling through the canonical Wnt/ß-catenin/Tcf pathway by binding to the Wnt co-receptors low-density lipoprotein receptor-related protein (LRP) 5 and LRP6, which facilitates Wnt3a binding to LRP5 and LRP6. Cripto-1 functionally enhances Wnt3a signaling through cytoplasmic stabilization of ß-catenin and elevated ß-catenin/Tcf transcriptional activation. Conversely, Wnt3a further increases Cripto-1 stimulation of migration, invasion and colony formation in soft agar of HC11 mouse mammary epithelial cells, indicating that Cripto-1 and the canonical Wnt/ß-catenin signaling co-operate in regulating motility and in vitro transformation of mammary epithelial cells.

An evolving web of signaling networks regulated by Cripto-1.

Over the past few decades, our understanding of the embryonic gene Cripto-1 has considerably advanced through biochemical, cell biology, and animal studies. Cripto-1 performs key functions during embryonic development, while it dramatically disappears in adult tissues, except possibly in adult tissue stem cells. Cripto-1 is re-expressed in human tumors promoting cell proliferation, migration, invasion, epithelial to mesenchymal transition, and tumor angiogenesis. This diversity of biological effects is dependent upon interaction of Cripto-1 with an extensive array of signaling molecules. In fact, Cripto-1 modulates signaling of transforming growth factor-ß family members, including Nodal, GDF-1/-3, Activin, and TGF-ß1, activates c-src/MAPK/Protein Kinase B (AKT) pathway in a Glypican-1 and GRP78-dependent manner, and cross-talks with erbB4, Wnt/ß-catenin, Notch, Caveolin-1, and Apelin/putative receptor protein related to Angiotensin-type I receptor (APJ) pathways. This article provides an updated survey of the various signaling pathways modulated by Cripto-1 with a focus on mechanistic insights in our understanding of the biological function of Cripto-1 in eukaryotic cells.

Role of Cripto-1 in stem cell maintenance and malignant progression.

Cripto-1 is critical for early embryonic development and, together with its ligand Nodal, has been found to be associated with the undifferentiated status of mouse and human embryonic stem cells. Like other embryonic genes, Cripto-1 performs important roles in the formation and progression of several types of human tumors, stimulating cell proliferation, migration, epithelial to mesenchymal transition, and tumor angiogenesis. Several studies have demonstrated that cell fate regulation during embryonic development and cell transformation during oncogenesis share common signaling pathways, suggesting that uncontrolled activation of embryonic signaling pathways might drive cell transformation and tumor progression in adult tissues. Here we review our current understanding of how Cripto-1 controls stem cell biology and how it integrates with other major embryonic signaling pathways. Because many cancers are thought to derive from a subpopulation of cancer stem-like cells, which may re-express embryonic genes, Cripto-1 signaling may drive tumor growth through the generation or expansion of tumor initiating cells bearing stem-like characteristics. Therefore, the Cripto-1/Nodal signaling may represent an attractive target for treatment in cancer, leading to the elimination of undifferentiated stem-like tumor initiating cells.

Cripto-1: an embryonic gene that promotes tumorigenesis.

Several studies have shown that cell fate regulation during embryonic development and oncogenic transformation share common regulatory mechanisms and signaling pathways. Indeed, an embryonic gene member of the EGF-Cripto-1/FRL1/Cryptic family, Cripto-1, has been implicated in embryogenesis and in carcinogenesis. Cripto-1 together with the TGF-beta ligand Nodal is a key regulator of embryonic development and is a marker of undifferentiated human and mouse embryonic stem cells. While Cripto-1 expression is very low in normal adult tissues, Cripto-1 is re-expressed at high levels in several different human tumors, modulating cancer cell proliferation, migration, epithelial-to-mesenchymal transition and stimulating tumor angiogenesis. Therefore, inhibition of Cripto-1 expression using blocking antibodies or antisense expression vectors might be a useful modality not only to target fully differentiated cancer cells but also to target a subpopulation of tumor cells with stem-like characteristics.

Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1.

Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor-like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum-Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the posttranslational maturation of Notch receptors.

Identificação de marcadores moleculares em câncer de mama através da técnica de microarray utilizando uma plataforma de exons tumor-associados / Identification of breast cancer molecular markers through microarray technology using a tumor-associated exons platform

Análises recentes têm mostrado a ocorrência de splicing alternativo (AS) do mRNA em pelo menos 60% dos genes humanos, sendo que 80% desses eventos ocorrem dentro da região codificadora, aumentando a diversidade proteômica. ... Para identificar variantes de splicing diferencialmente reguladas em câncer de mama, 270 exons expressos em tecidos ... Esses exons foram imobilizados em membranas de nylon juntamente com controles positivos e negativos, e hibridizados contra amostras tumorais e normais de mama. ... Para validação técnica dos exons selecionados como superexpressos de acordo com os critérios estabelecidos, foi empregada a técnica de RT-PCR quantitativo (qRT-PCR), usando o mesmo grupo de amostras já utilizadas anteriormente. ... Os resultados mostraram que a razão do nível de expressão entre as 3 VCE e a expressão constitutiva do gene (VCE/EC) foi significativamente maior em amostras tumorais de mama quando comparadas às amostras normais (p<0,05), sugerindo que TRIM37-VCE, MK-STYX-VCE e BRRN1-VCE são, de fato, variantes de splicing superexpressas em câncer de mama. Essas variantes foram também avaliadas em um grupo independente de 40 amostras tumorais de mama para validar biologicamente a superexpressão da VCE em amostras tumorais de mama quando comparadas às amostras normais. Todos os dados foram correlacionados com características clínicas e histopatológicas das amostras.

Current analyses have shown that alternative mRNA splicing (AS) appears in at least 60% of human genes and 80% of these events occurs within the coding region, increasing the proteomic diversity. Some AS variants have been preferentially expressed in human tumors and are potential molecular markers, contributing to the development of more accurate diagnostic and prognostic factors as well as therapeutic targets. To identify differentially regulated splicing variants in breast cancer, 270 exons expressed in tumor tissues were selected by a computational analysis, of which 75 were associated with breast, because they are found to be expressed in libraries that originated from breast tumors and they were not found in the corresponding normal libraries. These exons were immobilized on nylon membranes together with positive and negative controls, and hybridized against tumor and normal breast samples. To identify the most highly expressed exons in tumor tissues, 3 comparisons were performed: 4 tumor against 2 normal breast cell lines (LTxLN), 27 tumor against 5 non-neoplasic breast tissues (TxN) and 4 matched tumor-normal samples (PTxPN). A Tstudent test was used to select for differentially expressed exons (p<0.05) in each comparison (LTxLN; TxN; PTxPN). Here, 24 were selected as over expressed exons in the LTxLN comparison, 79 exons in the TxN comparison and 195 exons in the PTxPN comparison. For technical validation, those exons having a fold change of ≥ 3 in tumor samples and being present in at least 2 comparisons were selected. Fourteen exons were identified by microarray experiments and evaluated through quantitative RT-PCR (qRTPCR), using the same sample set utilized previously. In order to test whether the exons selected by microarray experiments belonged to a prone over expressed splicing variant, 2 criteria were adopted: (1) Validation by qRTPCR of the variant that comprises the selected over expressed exon (VCE) (fold change ≥ 3); (2) For those exons confirmed in criterion (1), evaluation of whole gene expression (through the analysis of constitutive gene expression) is adopted as a secondary criterium. Three of the VCE's were confirmed through qRT-PCR as being over expressed in breast tumor, such as TRIM37-VCE, MK-STYX-VCE and BRRN1-VCE. The constitutive expression of the gene (EC) was analyzed by qRT-PCR, through the primer design within constitutive exons, that is, present in all variants of the gene. The results showed that the expression level ratio between the 3 VCE's and the constitutive expression of genes (VCE/EC) was significantly higher in tumor samples when compared to normal samples (p<0.05), suggesting that TRIM37-VCE, MK-STYX-VCE and BRRN1-VCE are indeed breast tumor associated variants. These variants were also evaluated in an independent set of 40 breast tumor samples to biologically validate the VCE over expression in tumor samples as compared to normal samples. All data were correlated with clinical and histopathological samples features, and some significative associations were found, such as the expression of estrogen (ER+) and progesterone (PgR+) receptors with TRIM37-VCE. Although an increase of the experimental data is required for the complete exploration of this study, the results suggest 3 splicing variants that are over expressed in ductal carcinoma of the breast, and are candidates for molecular markers.