Mimetics of extra virgin olive oil phenols with anti-cancer stem cell activity.

The extra virgin olive oil (EVOO) dihydroxy-phenol oleacein is a natural inhibitor of multiple metabolic and epigenetic enzymes capable of suppressing the functional traits of cancer stem cells (CSC). Here, we used a natural product-inspired drug discovery approach to identify new compounds that phenotypically mimic the anti-CSC activity of oleacein. We coupled 3D quantitative structure-activity relationship-based virtual profiling with phenotypic analysis using 3D tumorsphere formation as a gold standard for assessing the presence of CSC. Among the top 20 computationally-predicted oleacein mimetics, four fulfilled the phenotypic endpoint of specifically suppressing the tumorsphere-initiating capacity of CSC, in the absence of significant cytotoxicity against differentiated cancer cells growing in 2D cultures in the same low micromolar concentration range. Of these, 3,4-dihydrophenetyl butyrate -a lipophilic ester conjugate of the hydroxytyrosol moiety of oleacein- and (E)-N-allyl-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide) -an inhibitor of Trypanosoma cruzi triosephosphate isomerase- were also highly effective at significantly reducing the proportion of aldehyde dehydrogenase (ALDH)-positive CSC-like proliferating cells. Preservation of the mTOR/DNMT binding mode of oleacein was dispensable for suppression of the ALDH+-CSC functional phenotype in hydroxytyrosol-unrelated mimetics. The anti-CSC chemistry of complex EVOO phenols such as oleacein can be phenocopied through the use of mimetics capturing its physico-chemical properties.

The LSD1 inhibitor iadademstat (ORY-1001) targets SOX2-driven breast cancer stem cells: a potential epigenetic therapy in luminal-B and HER2-positive breast cancer subtypes.

SOX2 is a core pluripotency-associated transcription factor causally related to cancer initiation, aggressiveness, and drug resistance by driving the self-renewal and seeding capacity of cancer stem cells (CSC). Here, we tested the ability of the clinically proven inhibitor of the lysine-specific demethylase 1 (LSD1/KDM1A) iadademstat (ORY-100) to target SOX2-driven CSC in breast cancer. Iadademstat blocked CSC-driven mammosphere formation in breast cancer cell lines that are dependent on SOX2 expression to maintain their CSC phenotype. Iadademstat prevented the activation of an LSD1-targeted stemness-specific SOX2 enhancer in CSC-enriched 3-dimensional spheroids. Using high-throughput transcriptional data available from the METABRIC dataset, high expression of SOX2 was significantly more common in luminal-B and HER2-enriched subtypes according to PAM50 classifier and in IntClust1 (high proliferating luminal-B) and IntClust 5 (luminal-B and HER2-amplified) according to integrative clustering. Iadademstat significantly reduced mammospheres formation by CSC-like cells from a multidrug-resistant luminal-B breast cancer patient-derived xenograft but not of those from a treatment-naïve luminal-A patient. Iadademstat reduced the expression of SOX2 in luminal-B but not in luminal-A mammospheres, likely indicating a selective targeting of SOX2-driven CSC. The therapeutic relevance of targeting SOX2-driven breast CSC suggests the potential clinical use of iadademstat as an epigenetic therapy in luminal-B and HER2-positive subtypes.

Extra Virgin Olive Oil Contains a Phenolic Inhibitor of the Histone Demethylase LSD1/KDM1A.

The lysine-specific histone demethylase 1A (LSD1) also known as lysine (K)-specific demethylase 1A (KDM1A) is a central epigenetic regulator of metabolic reprogramming in obesity-associated diseases, neurological disorders, and cancer. Here, we evaluated the ability of oleacein, a biophenol secoiridoid naturally present in extra virgin olive oil (EVOO), to target LSD1. Molecular docking and dynamic simulation approaches revealed that oleacein could target the binding site of the LSD1 cofactor flavin adenosine dinucleotide with high affinity and at low concentrations. At higher concentrations, oleacein was predicted to target the interaction of LSD1 with histone H3 and the LSD1 co-repressor (RCOR1/CoREST), likely disturbing the anchorage of LSD1 to chromatin. AlphaScreen-based in vitro assays confirmed the ability of oleacein to act as a direct inhibitor of recombinant LSD1, with an IC50 as low as 2.5 µmol/L. Further, oleacein fully suppressed the expression of the transcription factor SOX2 (SEX determining Region Y-box 2) in cancer stem-like and induced pluripotent stem (iPS) cells, which specifically occurs under the control of an LSD1-targeted distal enhancer. Conversely, oleacein failed to modify ectopic SOX2 overexpression driven by a constitutive promoter. Overall, our findings provide the first evidence that EVOO contains a naturally occurring phenolic inhibitor of LSD1, and support the use of oleacein as a template to design new secoiridoid-based LSD1 inhibitors.

Mitostemness.

Unraveling the key mechanisms governing the retention versus loss of the cancer stem cell (CSC) state would open new therapeutic avenues to eradicate cancer. Mitochondria are increasingly recognized key drivers in the origin and development of CSC functional traits. We here propose the new term "mitostemness" to designate the mitochondria-dependent signaling functions that, evolutionary rooted in the bacterial origin of mitochondria, regulate the maintenance of CSC self-renewal and resistance to differentiation. Mitostemness traits, namely mitonuclear communication, mitoproteome components, and mitochondrial fission/fusion dynamics, can be therapeutically exploited to target the CSC state. We briefly review the pre-clinical evidence of action of investigational compounds on mitostemness traits and discuss ongoing strategies to accelerate the clinical translation of new mitostemness drugs. The recognition that the bacterial origin of present-day mitochondria can drive decision-making signaling phenomena may open up a new therapeutic dimension against life-threatening CSCs. New therapeutics aimed to target mitochondria not only as biochemical but also as biophysical and morpho-physiological hallmarks of CSC might certainly guide improvements to cancer treatment.

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

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) cofactor-binding 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 tumor-initiating cell properties within BC populations.

Information Management.

The collection and storage of human tissue samples has been undertaken in medicine for centuries; however, biobanking has only recently become a dedicated activity. The technological developments that have allowed the procurement and long-term storage of viable human cells ex vivo, and to obtain relevant scientific information, including genetic information, provide tremendous possibilities for advancing biomedical research. At the same time, these possibilities have raised complex information management issues regarding samples, processing, donor information, traceability, and use of the sample. This chapter considers the requirements for managing information within biobanks, critical to their operation. Special consideration is given to Laboratory Information Managing Systems (LIMS) as a tool for comprehensive access and storage of information.

Isolation, Culture, and Expansion of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs), together with hematopoietic stem cells (HSCs), are the most frequently used cell type for cell-based therapeutics. As for other cell types intended for research and translational use, it is important to establish correctly typed cell lines from human tissue donations. Here, we describe methods for isolating, culturing, and identifying MSCs from various tissues obtained through human tissue donation. The methods have been used in the context of a biobank, prepared as standard operating procedures (SOPs), ensuring traceability and reproducibility of cell production.

Cryobanking Mesenchymal Stem Cells.

Cryopreservation and storage of culture-expanded mesenchymal stem cells (MSCs) is essential for a biobank to maintain a collection of cell lines for research and clinical use. Optimization of cryopreservation protocols and methods to minimize damage to cells during freezing and thawing is critical to ensure reliable availability of viable cells. Controlling the freezing rate and the use of appropriate cryoprotectant, as well as stable storage temperature, can minimize the negative effects on cell viability. In this chapter, protocols for cryopreserving MSCs are described.

Linking Pluripotency Reprogramming and Cancer.

Tumor development and the generation of induced pluripotent stem cells are highly comparable processes with striking similarities. Cellular plasticity is inherent to tumor evolution, rendering cells that acquire a stem cell-like phenotype, for which Sox2 activation has proved instrumental for the plastic acquisition of stemness properties in tumor cells. Understanding the molecular mechanisms underlying both events might uncover novel approaches for the development of anticancer therapeutics and constitute model systems for understanding tumor generation and ensuring the biosafety of cell-based therapies. Stem Cells Translational Medicine 2017;6:335-339.

Neural-competent cells of adult human dermis belong to the Schwann lineage.

Resident neural precursor cells (NPCs) have been reported for a number of adult tissues. Understanding their physiological function or, alternatively, their activation after tissue damage or in vitro manipulation remains an unsolved issue. Here, we investigated the source of human dermal NPCs in adult tissue. By following an unbiased, comprehensive approach employing cell-surface marker screening, cell separation, transcriptomic characterization, and in vivo fate analyses, we found that p75NTR(+) precursors of human foreskin can be ascribed to the Schwann (CD56(+)) and perivascular (CD56(-)) cell lineages. Moreover, neural differentiation potential was restricted to the p75NTR(+)CD56(+) Schwann cells and mediated by SOX2 expression levels. Double-positive NPCs were similarly obtained from human cardiospheres, indicating that this phenomenon might be widespread.

Chemical inhibition of acetyl-CoA carboxylase suppresses self-renewal growth of cancer stem cells.

Cancer stem cells (CSC) may take advantage of the Warburg effect-induced siphoning of metabolic intermediates into de novo fatty acid biosynthesis to increase self-renewal growth. We examined the anti-CSC effects of the antifungal polyketide soraphen A, a specific inhibitor of the first committed step of lipid biosynthesis catalyzed by acetyl-CoA carboxylase (ACACA). The mammosphere formation capability of MCF-7 cells was reduced following treatment with soraphen A in a dose-dependent manner. MCF-7 cells engineered to overexpress the oncogene HER2 (MCF-7/HER2 cells) were 5-fold more sensitive than MCF-7 parental cells to soraphen A-induced reductions in mammosphere-forming efficiency. Soraphen A treatment notably decreased aldehyde dehydrogenase (ALDH)-positive CSC-like cells and impeded the HER2's ability to increase the ALDH+-stem cell population. The following results confirmed that soraphen A-induced suppression of CSC populations occurred throughACACA-driven lipogenesis: a.) exogenous supplementation with supraphysiological concentrations of oleic acid fully rescued mammosphere formation in the presence of soraphen A and b.) mammosphere cultures of MCF-7 cells with stably silenced expression of the cytosolic isoform ACACA1, which specifically participates in de novo lipogenesis, were mostly refractory to soraphen A treatment. Our findings reveal for the first time that ACACA may constitute a previously unrecognized target for novel anti-breast CSC therapies.

Xenopatients 2.0: reprogramming the epigenetic landscapes of patient-derived cancer genomes.

In the science-fiction thriller film Minority Report, a specialized police department called "PreCrime" apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called "PreCogs". We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized "stemotoxic" cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge "chromosome therapies" aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors following the activation of phenotypic copies of specific cancer diseases might crucially evaluate a "reprogramming cure" for cancer. A new era of xenopatients 2.0 generated via nuclear reprogramming of the epigenetic landscapes of patient-derived cancer genomes might revolutionize the current personalized translational platforms in cancer research.

Mammosphere formation in breast carcinoma cell lines depends upon expression of E-cadherin.

Tumors are heterogeneous at the cellular level where the ability to maintain tumor growth resides in discrete cell populations. Floating sphere-forming assays are broadly used to test stem cell activity in tissues, tumors and cell lines. Spheroids are originated from a small population of cells with stem cell features able to grow in suspension culture and behaving as tumorigenic in mice. We tested the ability of eleven common breast cancer cell lines representing the major breast cancer subtypes to grow as mammospheres, measuring the ability to maintain cell viability upon serial non-adherent passage. Only MCF7, T47D, BT474, MDA-MB-436 and JIMT1 were successfully propagated as long-term mammosphere cultures, measured as the increase in the number of viable cells upon serial non-adherent passages. Other cell lines tested (SKBR3, MDA-MB-231, MDA-MB-468 and MDA-MB-435) formed cell clumps that can be disaggregated mechanically, but cell viability drops dramatically on their second passage. HCC1937 and HCC1569 cells formed typical mammospheres, although they could not be propagated as long-term mammosphere cultures. All the sphere forming lines but MDA-MB-436 express E-cadherin on their surface. Knock down of E-cadherin expression in MCF-7 cells abrogated its ability to grow as mammospheres, while re-expression of E-cadherin in SKBR3 cells allow them to form mammospheres. Therefore, the mammosphere assay is suitable to reveal stem like features in breast cancer cell lines that express E-cadherin.

Reprogramming of non-genomic estrogen signaling by the stemness factor SOX2 enhances the tumor-initiating capacity of breast cancer cells.

The restoration of pluripotency circuits by the reactivation of endogenous stemness factors, such as SOX2, may provide a new paradigm in cancer development. The tumoral stem cell reprogramming hypothesis, i.e., the ability of stemness factors to redirect normal and differentiated tumor cells toward a less-differentiated and stem-like state, adds new layers of complexity to cancer biology, because the effects of such reprogramming may remain dormant until engaged later in response to (epi)genetic and/or (micro)environmental events. To test this hypothesis, we utilized an in vitro model of a SOX2-overexpressing cancer stem cell (CSC)-like cellular state that was recently developed in our laboratory by employing Yamanaka's nuclear reprogramming technology in the estrogen receptor α (ERα)-positive MCF-7 breast cancer cell line. Despite the acquisition of distinct molecular features that were compatible with a breast CSC-like cellular state, such as strong aldehyde dehydrogenase activity, as detected by ALDEFLUOR, and overexpression of the SSEA-4 and CD44 breast CSC markers, the tumor growth-initiating ability of SOX2-overexpressing CSC-like MCF-7 cells solely occurred in female nude mice supplemented with estradiol when compared with MCF-7 parental cells. Ser118 phosphorylation of estrogen receptor α (ERα), which is a pivotal integrator of the genomic and nongenomic E 2/ERα signaling pathways, drastically accumulated in nuclear speckles in the interphase nuclei of SOX2-driven CSC-like cell populations. Moreover, SOX2-positive CSC-like cells accumulated significantly higher numbers of actively dividing cells, and the highest levels of phospho-Ser118-ERα occurred when chromosomes lined up on a metaphase plate. The previously unrecognized link between E 2/ERα signaling and SOX2-driven stem cell circuitry may significantly impact our current understanding of breast cancer initiation and progression, i.e., SOX2 can promote non-genomic E 2 signaling that leads to nuclear phospho-Ser118-ERα, which ultimately exacerbates genomic ER signaling in response to E 2. Because E 2 stimulation has been recently shown to enhance breast tumor-initiating cell survival by downregulating miR-140, which targets SOX2, the establishment of a bidirectional cross-talk interaction between the stem cell self-renewal regulator, SOX2, and the local and systemic ability of E 2 to increase breast CSC activity may have profound implications for the development of new CSC-directed strategies for breast cancer prevention and therapy.

Nuclear reprogramming of luminal-like breast cancer cells generates Sox2-overexpressing cancer stem-like cellular states harboring transcriptional activation of the mTOR pathway.

Energy metabolism plasticity enables stemness programs during the reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state. This relationship may introduce a new era in the understanding of Warburg's theory on the metabolic origin of cancer at the level of cancer stem cells (CSCs). Here, we used Yamanaka's stem cell technology in an attempt to create stable CSC research lines in which to dissect the transcriptional control of mTOR--the master switch of cellular catabolism and anabolism--in CSC-like states. The rare colonies with iPSC-like morphology, obtained following the viral transduction of the Oct4, Sox2, Klf4, and c-Myc (OSKM) stemness factors into MCF-7 luminal-like breast cancer cells (MCF-7/Rep), demonstrated an intermediate state between cancer cells and bona fide iPSCs. MCF-7/Rep cells notably overexpressed SOX2 and stage-specific embryonic antigen (SSEA)-4 proteins; however, other stemness-related markers (OCT4, NANOG, SSEA-1, TRA-1-60, and TRA-1-81) were found at low to moderate levels. The transcriptional analyses of OSKM factors confirmed the strong but unique reactivation of the endogenous Sox2 stemness gene accompanied by the silencing of the exogenous Sox2 transgene in MCF-7/Rep cells. Some but not all MCF-7/Rep cells acquired strong alkaline phosphatase (AP) activity compared with MCF-7 parental cells. SOX2-overexpressing MCF-7/Rep cells contained drastically higher percentages of CD44(+) and ALDEFLUOR-stained ALDH(bright) cells than MCF-7 parental cells. The overlap between differentially expressed mTOR signaling-related genes in 3 different SOX2-overexpressing CSC-like cell lines revealed a notable downregulation of 3 genes, PRKAA1 (which codes for the catalytic α 1 subunit of AMPK), DDIT4/REDD1 (a stress response gene that operates as a negative regulator of mTOR), and DEPTOR (a naturally occurring endogenous inhibitor of mTOR activity). The insulin-receptor gene (INSR) was differentially upregulated in MCF-7/Rep cells. Consistent with the downregulation of AMPK expression, immunoblotting procedures confirmed upregulation of p70S6K and increased phosphorylation of mTOR in Sox2-overexpressing CSC-like cell populations. Using an in vitro model of the de novo generation of CSC-like states through the nuclear reprogramming of an established breast cancer cell line, we reveal that the transcriptional suppression of mTOR repressors is an intrinsic process occurring during the acquisition of CSC-like properties by differentiated populations of luminal-like breast cancer cells. This approach may provide a new path for obtaining information about preventing the appearance of CSCs through the modulation of the AMPK/mTOR pathway.

Basal/HER2 breast carcinomas: integrating molecular taxonomy with cancer stem cell dynamics to predict primary resistance to trastuzumab (Herceptin).

High rates of inherent primary resistance to the humanized monoclonal antibody trastuzumab (Herceptin) are frequent among HER2 gene-amplified breast carcinomas in both metastatic and adjuvant settings. The clinical efficacy of trastuzumab is highly correlated with its ability to specifically and efficiently target HER2-driven populations of breast cancer stem cells (CSCs). Intriguingly, many of the possible mechanisms by which cancer cells escape trastuzumab involve many of the same biomarkers that have been implicated in the biology of CS-like tumor-initiating cells. In the traditional, one-way hierarchy of CSCs in which all cancer cells descend from special self-renewing CSCs, HER2-positive CSCs can occur solely by self-renewal. Therefore, by targeting CSC self-renewal and resistance, trastuzumab is expected to induce tumor shrinkage and further reduce breast cancer recurrence rates when used alongside traditional therapies. In a new, alternate model, more differentiated non-stem cancer cells can revert to trastuzumab-refractory, CS-like cells via the activation of intrinsic or microenvironmental paths-to-stemness, such as the epithelial-to-mesenchymal transition (EMT). Alternatively, stochastic transitions of trastuzumab-responsive CSCs might also give rise to non-CSC cellular states that lack major attributes of CSCs and, therefore, can remain "hidden" from trastuzumab activity. Here, we hypothesize that a better understanding of the CSC/non-CSC social structure within HER2-overexpressing breast carcinomas is critical for trastuzumab-based treatment decisions in the clinic. First, we decipher the biological significance of CSC features and the EMT on the molecular effects and efficacy of trastuzumab in HER2-positive breast cancer cells. Second, we reinterpret the genetic heterogeneity that differentiates trastuzumab-responders from non-responders in terms of CSC cellular states. Finally, we propose that novel predictive approaches aimed at better forecasting early tumor responses to trastuzumab should identify biological determinants that causally underlie the intrinsic flexibility of HER2-positive CSCs to "enter" into or "exit" from trastuzumab-sensitive states. An accurate integration of CSC cellular states and EMT-related biomarkers with the currently available breast cancer molecular taxonomy may significantly improve our ability to make a priori decisions about whether patients belonging to HER2 subtypes differentially enriched with a "mesenchymal transition signature" (e.g., luminal/HER2 vs. basal/HER2) would distinctly benefit from trastuzumab-based therapy ab initio.

Sp1 transcription factor interaction with accumulated prelamin a impairs adipose lineage differentiation in human mesenchymal stem cells: essential role of sp1 in the integrity of lipid vesicles.

Lamin A (LMNA)-linked lipodystrophies may be either genetic (associated with LMNA mutations) or acquired (associated with the use of human immunodeficiency virus protease inhibitors [PIs]), and in both cases they share clinical features such as anomalous distribution of body fat or generalized loss of adipose tissue, metabolic alterations, and early cardiovascular complications. Both LMNA-linked lipodystrophies are characterized by the accumulation of the lamin A precursor prelamin A. The pathological mechanism by which prelamin A accumulation induces the lipodystrophy associated phenotypes remains unclear. Since the affected tissues in these disorders are of mesenchymal origin, we have generated an LMNA-linked experimental model using human mesenchymal stem cells treated with a PI, which recapitulates the phenotypes observed in patient biopsies. This model has been demonstrated to be a useful tool to unravel the pathological mechanism of the LMNA-linked lipodystrophies, providing an ideal system to identify potential targets to generate new therapies for drug discovery screening. We report for the first time that impaired adipogenesis is a consequence of the interaction between accumulated prelamin A and Sp1 transcription factor, sequestration of which results in altered extracellular matrix gene expression. In fact, our study shows a novel, essential, and finely tuned role for Sp1 in adipose lineage differentiation in human mesenchymal stem cells. These findings define a new physiological experimental model to elucidate the pathological mechanisms LMNA-linked lipodystrophies, creating new opportunities for research and treatment not only of LMNA-linked lipodystrophies but also of other adipogenesis-associated metabolic diseases.

Epithelial-to-mesenchymal transition (EMT) confers primary resistance to trastuzumab (Herceptin).

The rate of inherent resistance to single-agent trastuzumab in HER2-overexpressing metastatic breast carcinomas is impressive at above 70%. Unfortunately, little is known regarding the distinctive genetic signatures that could predict trastuzumab refractoriness ab initio. The epithelial-to-mesenchymal transition (EMT) molecular features, HER2 expression status and primary responses to trastuzumab were explored in the public Lawrence Berkeley Laboratory (LBL) Breast Cancer Collection. Lentivirus-delivered small hairpin RNAs were employed to reduce specifically and stably the expression of EMT transcription factors in trastuzumab-refractory basal/HER2+ cells. Cell proliferation assays and pre-clinical nude mice xenograft-based studies were performed to assess the contribution of specific EMT transcription factors to inherent trastuzumab resistance. Primary sensitivity to trastuzumab was restricted to the SLUG/SNAIL2-negative subset of luminal/HER2+ cell lines, whereas all of the SLUG/SNAIL2-positive basal/HER2+ cell lines exhibited an inherent resistance to trastuzumab. The specific knockdown of SLUG/SNAIL2 suppressed the stem-related CD44+CD24(-/low) mesenchymal immunophenotype by transcriptionally upregulating the luminal epithelial marker CD24 in basal/HER2+ cells. Basal/HER2+ cells gained sensitivity to the growth-inhibitory effects of trastuzumab following SLUG/SNAIL2 gene depletion, which induced the expression of the mesenchymal-to-epithelial transition (MET) genes involved in promoting an epithelial phenotype. The isolation of CD44+CD24(-/low) mesenchymal cells by magnetic-activated cell sorting (MACS) confirmed their intrinsic unresponsiveness to trastuzumab. A reduction in tumor growth and dramatic gain in sensitivity to trastuzumab in vivo were confirmed when the SLUG/SNAIL2 knockdown basal/HER2+ cells were injected into nude mice. HER2 overexpression in a basal, rather than in a luminal molecular background, results in a basal/HER2+ breast cancer subtype that is intrinsically resistant to trastuzumab. EMT transcription factors might induce an enhanced phenotypic plasticity that would allow basal/HER2+ breast cancer cells to "enter" into and "exit" dynamically from trastuzumab-responsive stem cell-like states. The systematic determination of SLUG/SNAIL2 as a stem/CD44+CD24(-/low) cell-associated protein may improve the therapeutic management of HER2+ breast carcinomas.

Activation of AMP-activated protein kinase (AMPK) provides a metabolic barrier to reprogramming somatic cells into stem cells.

The ability of somatic cells to reprogram their ATP-generating machinery into a Warburg-like glycolytic metabotype while overexpressing stemness genes facilitates their conversion into either induced pluripotent stem cells (iPSCs) or tumor-propagating cells. AMP-activated protein kinase (AMPK) is a metabolic master switch that senses and decodes intracellular changes in energy status; thus, we have evaluated the impact of AMPK activation in regulating the generation of iPSCs from nonstem cells of somatic origin. The indirect and direct activation of AMPK with the antidiabetic biguanide metformin and the thienopyridone A-769662, respectively, impeded the reprogramming of mouse embryonic and human diploid fibroblasts into iPSCs. The AMPK activators established a metabolic barrier to reprogramming that could not be bypassed, even through p53 deficiency, a fundamental mechanism to greatly improve the efficiency of stem-cell production. Treatment with metformin or A-769662 before the generation of iPSC colonies was sufficient to drastically decrease iPSC generation, suggesting that AMPK activation impedes early stem cell genetic reprogramming. Monitoring the transcriptional activation status of each individual reprogramming factor (i.e., Oct4, Sox2, Klf4 and c-Myc) revealed that AMPK activation notably prevented the transcriptional activation of Oct4, the master regulator of the pluripotent state. AMPK activation appears to impose a normalized metabolic flow away from the required pro-immortalizing glycolysis that fuels the induction of stemness and pluripotency, endowing somatic cells with an energetic infrastructure that is protected against reprogramming. AMPK-activating anti-reprogramming strategies may provide a roadmap for the generation of novel cancer therapies that metabolically target tumor-propagating cells.

Xenografts in zebrafish embryos as a rapid functional assay for breast cancer stem-like cell identification.

The cancer stem cell is defined by its capacity to self-renew, the potential to differentiate into all cells of the tumor and the ability to proliferate and drive the expansion of the tumor. Thus, targeting these cells may provide novel anti-cancer treatment strategies. Breast cancer stem cells have been isolated according to surface marker expression, ability to efflux fluorescent dyes, increased activity of aldehyde dehydrogenase or the capacity to form spheres in non-adherent culture conditions. In order to test novel drugs directed towards modulating self-renewal of cancer stem cells, rapid, easy and inexpensive assays must be developed. Using 2 days-post-fertilization (dpf) zebrafish embryos as transplant recipients, we show that cells grown in mammospheres from breast carcinoma cell lines migrate to the tail of the embryo and form masses with a significantly higher frequency than parental monolayer populations. When stem-like self-renewal was targeted in the parental population by the use of the dietary supplement curcumin, cell migration and mass formation were reduced, indicating that these effects were associated with stem-like cell content. This is a proof of principle report that proposes a rapid and inexpensive assay to target in vivo cancer stem-like cells, which may be used to unravel basic cancer stem cell biology and for drug screening.