Dorsomorphin reverses the mesenchymal phenotype of breast cancer initiating cells by inhibition of bone morphogenetic protein signaling

Increasing evidence supports the theory that tumor growth, homeostasis, and recurrence are dependent on a small subset of cells with stem cell properties, redefined cancer initiating cells (CICs) or cancer stem cells. Bone morphogenetic proteins (BMPs) are involved in cell-fate specification during embryogenesis, in the maintenance of developmental potency in adult stem cells and may contribute to sustain CIC populations in breast carcinoma. Using the mouse A17 cell model previously related to mesenchymal cancer stem cells and displaying properties of CICs, we investigated the role of BMPs in the control of breast cancer cell plasticity. We showed that an autocrine activation of BMP signaling is crucial for the maintenance of mesenchymal stem cell phenotype and tumorigenic potential of A17 cells. Pharmacological inhibition of BMP signaling cascade by Dorsomorphin resulted in the acquisition of epithelial-like traits by A17 cells, including expression of Citokeratin-18 and E-cadherin, through downregulation of Snail and Slug transcriptional factors and Cyclooxygenase-2 (COX2) expression, and in the loss of their stem-features and self-renewal ability. This phenotypic switch compromised A17 cell motility, invasiveness and in vitro tumor growth. These results reveal that BMPs are key molecules at the crossroad between stemness and cancer

HER2-Driven Carcinogenesis: New Mouse Models for Novel Immunotherapies

HER2 overexpression is a hallmark of aggressive breast cancer subtypes, and HER2-targeted therapies, such as passive immunotherapy with the humanized monoclonal antibody Trastuzumab, have become standard treatments for these tumor subtypes. However, increasing evidence points to a major role for the Δ16HER2 splice variant, which is commonly coexpressed with the wild-type protein, in cancer progression, metastatic potential and resistance to Trastuzumab treatment. Using our recently derived mouse strain transgenically expressing human Δ16HER2 under the transcriptional control of the MMTV promoter, we showed that this HER2 isoform per se can transform mammary epithelium in vivo. Thus, Δ16HER2 mice provide a new preclinical model in which to study mammary carcinogenesis and the metastatic process, as well as new therapies, including immune-based DNA vaccines. Such vaccines, by virtue of the polyclonal response they induce, might synergize with standard treatments and might ensure targeting of HER2 variants no longer recognized by monoclonal antibodies. In addition, immunological memory might provide long-term anticancer immune protection without side effects associated with many conventional therapies. The efficacy of DNA vaccination against the HER2 oncoantigen has been widely demonstrated in BALB-neuT mice transgenically expressing the activated rat neu oncogene and recapitulating several features of human breast cancers; however, HER2 is a self-tolerated molecule and an effective response to it must circumvent tolerance mechanisms. Here, we retrace the findings that have led to our most promising DNA vaccines encoding human/rat chimeric forms of the HER2 molecule bearing both xenogeneic and syngeneic portions of the protein and able to overcome peripheral tolerance. Preclinical data obtained with our DNA vaccines have provided the rationale for their use in an ongoing phase I clinical trial

Inhibition of Asaia in adult mosquitoes causes male-specific mortality and diverse transcriptome changes

Mosquitoes can transmit many infectious diseases, such as malaria, dengue, Zika, yellow fever, and lymphatic filariasis. Current mosquito control strategies are failing to reduce the severity of outbreaks that still cause high human morbidity and mortality worldwide. Great expectations have been placed on genetic control methods. Among other methods, genetic modification of the bacteria colonizing different mosquito species and expressing anti-pathogen molecules may represent an innovative tool to combat mosquito-borne diseases. Nevertheless, this emerging approach, known as paratransgenesis, requires a detailed understanding of the mosquito microbiota and an accurate characterization of selected bacteria candidates. The acetic acid bacteria Asaia is a promising candidate for paratransgenic approaches. We have previously reported that Asaia symbionts play a beneficial role in the normal development of Anopheles mosquito larvae, but no study has yet investigated the role(s) of Asaia in adult mosquito biology. Here we report evidence on how treatment with a highly specific anti-Asaia monoclonal antibody impacts the survival and physiology of adult Anopheles stephensi mosquitoes. Our findings offer useful insight on the role of Asaia in several physiological systems of adult mosquitoes, where the influence differs between males and females

Identification of Relevant Conformational Epitopes on the HER2 Oncoprotein by Using Large Fragment Phage Display (LFPD)

We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines

The Human Splice Variant Δ16HER2 Induces Rapid Tumor Onset in a Reporter Transgenic Mouse

Several transgenic mice models solidly support the hypothesis that HER2 (ERBB2) overexpression or mutation promotes tumorigenesis. Recently, a HER2 splice variant lacking exon-16 (Δ16HER2) has been detected in human breast carcinomas. This alternative protein, a normal byproduct of HER2, has an increased transforming potency compared to wild-type (wt) HER2 receptors. To examine the ability of Δ16HER2 to transform mammary epithelium in vivo and to monitor Δ16HER2-driven tumorigenesis in live mice, we generated and characterized a mouse line that transgenically expresses both human Δ16HER2 and firefly luciferase under the transcriptional control of the MMTV promoter. All the transgenic females developed multifocal mammary tumors with a rapid onset and an average latency of 15.11 weeks. Immunohistochemical analysis revealed the concurrent expression of luciferase and the human Δ16HER2 oncogene only in the mammary gland and in strict correlation with tumor development. Transgenic Δ16HER2 expressed on the tumor cell plasma membrane from spontaneous mammary adenocarcinomas formed constitutively active homodimers able to activate the oncogenic signal transduction pathway mediated through Src kinase. These new transgenic animals demonstrate the ability of the human Δ16HER2 isoform to transform “per se” mammary epithelium in vivo. The high tumor incidence as well as the short latency strongly suggests that the Δ16HER2 splice variant represents the transforming form of the HER2 oncoprotein

Calcium channels and pumps in cancer: changes and consequences

Increases in intracellular free Ca2+ play a major role in many cellular processes. The deregulation of Ca2+ signaling is a feature of a variety of diseases, and modulators of Ca2+ signaling are used to treat conditions as diverse as hypertension to pain. The Ca2+ signal also plays a role in processes important in cancer, such as proliferation and migration. Many studies in cancer have identified alterations in the expression of proteins involved in the movement of Ca2+ across the plasma membrane and subcellular organelles. In some cases, these Ca2+ channels or pumps are potential therapeutic targets for specific cancer subtypes or correlate with prognosis

Extra-virgin olive oil contains a metabolo-epigenetic inhibitor of cancer stem cells

We are grateful to Custodio Borrego for giving us free use of the photograph he took of EVOO and olive trees in Granada (Spain), which have been included in Figure 7. This work has been awarded with the IV Premio Internacional Castillo de Canena de Investigación Oleícola ‘LUIS VAÑÓ’(IV Edition of Castillo de Canena LUIS VAÑÓ Award for Research on Olive Cultivation and Olive Oil; UC Davis Olive Center, Castillo de Canena, and Universidad de Jaén).The authors would like to thank Dr Kenneth McCreath for editorial support. We are greatly indebted to Prof Robert A. Weinberg (Whitehead Institute for Biomedical Research, Cambridge, MA) for providing the HMLERshCntrol/HMLERshEcad cells used in this work.Targeting tumor-initiating, drug-resistant populations of cancer stem cells (CSC) with phytochemicals is a novel paradigm for cancer prevention and treatment. We herein employed a phenotypic drug discovery approach coupled to mechanism-of-action profiling and target deconvolution to identify phenolic components of extra virgin olive oil (EVOO) capable of suppressing the functional traits of CSC in breast cancer (BC). In vitro screening revealed that the secoiridoid decarboxymethyl oleuropein aglycone (DOA) could selectively target subpopulations of epithelial-like, aldehyde dehydrogenase (ALDH)-positive and mesenchymal-like, CD44+CD24−/low CSC. DOA could potently block the formation of multicellular tumorspheres generated from single-founder stem-like cells in a panel of genetically diverse BC models. Pretreatment of BC populations with noncytotoxic doses of DOA dramatically reduced subsequent tumor-forming capacity in vivo. Mice orthotopically injected with CSC-enriched BC-cell populations pretreated with DOA remained tumor-free for several months. Phenotype microarray-based screening pointed to a synergistic interaction of DOA with the mTOR inhibitor rapamycin and the DNA methyltransferase (DNMT) inhibitor 5-azacytidine. In silico computational studies indicated that DOA binds and inhibits the ATP-binding kinase domain site of mTOR and the S-adenosyl-l-methionine (SAM) cofactorbinding pocket of DNMTs. FRET-based Z-LYTE™ and AlphaScreen-based in vitro assays confirmed the ability of DOA to function as an ATP-competitive mTOR inhibitor and to block the SAM-dependent methylation activity of DNMTs. Our systematic in vitro, in vivo and in silico approaches establish the phenol-conjugated oleoside DOA as a dual mTOR/DNMT inhibitor naturally occurring in EVOO that functionally suppresses CSC-like states responsible for maintaining tumorinitiating cell properties within BC populations.This work was supported by grants from the Ministerio de Ciencia e Innovación (Grant SAF2016-80639-P to J.A.M.), Plan Nacional de I+D+I, Spain, the Agència de Gestió d’Ajuts Universitaris i de Recerca (AGAUR; Grant 2014 SGR229 to J.A.M.), Departament d’Economia i Coneixement, Catalonia, Spain, the Andalusian Regional Government Council of Innovation and Science (Grant P11-CTS-7625 to A.S.-C.), the Ministerio de Economía, Industria y Competitividad, Spain (Grants AGL2015- 67995-C2-3-R and AGL2015-67995-C3-1-R to A.S.-C. and V.M.) and Conselleria d’Educació, Investigació, Cultura I Esport, Generalitat Valenciana, Spain (Grant PROMETEO/2016/006 to V.M). E.C. is supported by the Sara Borrell post doctoral contract (CD15/00033) from the Ministerio de Sanidad y Consumo, Fondo de Investigación Sanitaria (FIS), Spain

Sanguinarine anti-tumor activity on a model of basal-like breast cancer

The aim of this work is to study the potential anti-proliferative effects of sanguinarine on mesenchymal cancer basal-like A17 cells isolated from FVB/neuT mammary carcinomas. Studies that have reviewed the histological presentation of basal-like breast cancer demonstrate that >90% of these tumors arise from the breast ducts and are often associated with higher nuclear and histological grade, high mitotic index and more aggressive phenotypic features. This clinical subset represents one of the most important treatment challenges today because these tumors are not likely to respond to hormonal maneuvers (tamoxifen or aromatase inhibitors) nor to drugs targeting HER2 over expression (trastuzumab) [6-17]. Marchini et al compared crucial molecular pathways of A17 derived-carcinoma with that of both carcinomas and other mesenchymal phenotypes, such as mesenchymal stem cells (MSCs), breast stroma, and various types of sarcomas. They identified three mesenchymal/stromal-signatures which A17 cells share with MSCs and breast stroma, showing that these signatures significantly relates to basal-like breast cancer subtypes and significantly relates to bone metastasis [19, 20]. Sanguinarine, a natural benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis and other poppy species, could truly be an excellent candidate as a possible anticancer drug, since many different animals and human cancer cell lines have demonstrated to be highly sensitive to this alkaloid. In this work, sanguinarine shows its efficacy on A17 cell line model, both in in vitro and in in vivo experiments.\ud Mtt, Wound-healing, motility assay and Annexin-V staining revealed that sanguinarine is able to reduce A17 viability, motility, and causes apoptotic and necrotic cell death.\ud Most important, this alkaloid reduces Dihydrofolate Reductase (DHFR) activity, an enzyme implicated in the synthesis of Dna bases.\ud Encouraged by these results, we managed an in vivo experiment by injecting A17 cells in FVB syngenic mice and treating them with sanguinarine: obtained results encouraged us to considered sanguinarine as a promising anticancer drug candidate in the treatment of basal-like breast cancer