Identification of a miRNA multi-targeting therapeutic strategy in glioblastoma.

Glioblastoma (GBM) is a deadly and the most common primary brain tumor in adults. Due to their regulation of a high number of mRNA transcripts, microRNAs (miRNAs) are key molecules in the control of biological processes and are thereby promising therapeutic targets for GBM patients. In this regard, we recently reported miRNAs as strong modulators of GBM aggressiveness. Here, using an integrative and comprehensive analysis of the TCGA database and the transcriptome of GBM biopsies, we identified three critical and clinically relevant miRNAs for GBM, miR-17-3p, miR-222, and miR-340. In addition, we showed that the combinatorial modulation of three of these miRNAs efficiently inhibited several biological processes in patient-derived GBM cells of all these three GBM subtypes (Mesenchymal, Proneural, Classical), induced cell death, and delayed tumor growth in a mouse tumor model. Finally, in a doxycycline-inducible model, we observed a significant inhibition of GBM stem cell viability and a significant delay of orthotopic tumor growth. Collectively, our results reveal, for the first time, the potential of miR-17-3p, miR-222 and miR-340 multi-targeting as a promising therapeutic strategy for GBM patients.

Macropinocytosis requires Gal-3 in a subset of patient-derived glioblastoma stem cells.

Recently, we involved the carbohydrate-binding protein Galectin-3 (Gal-3) as a druggable target for KRAS-mutant-addicted lung and pancreatic cancers. Here, using glioblastoma patient-derived stem cells (GSCs), we identify and characterize a subset of Gal-3high glioblastoma (GBM) tumors mainly within the mesenchymal subtype that are addicted to Gal-3-mediated macropinocytosis. Using both genetic and pharmacologic inhibition of Gal-3, we showed a significant decrease of GSC macropinocytosis activity, cell survival and invasion, in vitro and in vivo. Mechanistically, we demonstrate that Gal-3 binds to RAB10, a member of the RAS superfamily of small GTPases, and ß1 integrin, which are both required for macropinocytosis activity and cell survival. Finally, by defining a Gal-3/macropinocytosis molecular signature, we could predict sensitivity to this dependency pathway and provide proof-of-principle for innovative therapeutic strategies to exploit this Achilles' heel for a significant and unique subset of GBM patients.

Phase I/II trial testing safety and immunogenicity of the multipeptide IMA950/poly-ICLC vaccine in newly diagnosed adult malignant astrocytoma patients.

BACKGROUND: Peptide vaccines offer the opportunity to elicit glioma-specific T cells with tumor killing ability. Using antigens eluted from the surface of glioblastoma samples, we designed a phase I/II study to test safety and immunogenicity of the IMA950 multipeptide vaccine adjuvanted with poly-ICLC (polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose) in human leukocyte antigen A2+ glioma patients. METHODS: Adult patients with newly diagnosed glioblastoma (n = 16) and grade III astrocytoma (n = 3) were treated with radiochemotherapy followed by IMA950/poly-ICLC vaccination. The first 6 patients received IMA950 (9 major histocompatibility complex [MHC] class I and 2 MHC class II peptides) intradermally and poly-ICLC intramuscularly (i.m.). After protocol amendment, IMA950 and poly-ICLC were mixed and injected subcutaneously (n = 7) or i.m. (n = 6). Primary endpoints were safety and immunogenicity. Secondary endpoints were overall survival, progression-free survival at 6 and 9 months, and vaccine-specific peripheral cluster of differentiation (CD)4 and CD8 T-cell responses. RESULTS: The IMA950/poly-ICLC vaccine was safe and well tolerated. Four patients presented cerebral edema with rapid recovery. For the first 6 patients, vaccine-induced CD8 T-cell responses were restricted to a single peptide and CD4 responses were absent. After optimization of vaccine formulation, we observed multipeptide CD8 and sustained T helper 1 CD4 T-cell responses. For the entire cohort, CD8 T-cell responses to a single or multiple peptides were observed in 63.2% and 36.8% of patients, respectively. Median overall survival was 19 months for glioblastoma patients. CONCLUSION: We provide, in a clinical trial, using cell surface-presented antigens, insights into optimization of vaccines generating effector T cells for glioma patients. TRIAL REGISTRATION: Clinicaltrials.gov NCT01920191.

Harnessing 3D models of mammary epithelial morphogenesis: An off the beaten path approach to identify candidate biomarkers of early stage breast cancer.

Regardless of the etiological factor, an aberrant morphology is the common hallmark of ductal carcinoma in situ (DCIS), which is a highly heterogeneous disease. To test if critical core morphogenetic mechanisms are compromised by different mutations, we performed proteomics analysis of five mammary epithelial HME1 mutant lines that develop a DCIS-like morphology in three dimensional (3D) culture. Here we show first, that all HME1 mutant lines share a common protein signature highlighting an inverse deregulation of two annexins, ANXA2 and ANXA8. Either ANXA2 downregulation or ANXA8 upregulation in the HME1 cell context are per se sufficient to confer a 3D DCIS-like morphology. Seemingly, different mutations impinged on a common mechanism that differentially regulates the two annexins. Second, we show that ANXA8 expression is significantly higher in DCIS tissue samples versus normal breast tissue and atypical ductal hyperplasia (ADH). Apparently, ANXA8 expression is significantly more upregulated in ER-negative versus ER-positive cases, and significantly correlates with tumor stage, grade and positive lymph node. Based on our study, 3D mammary morphogenesis models can be an alternate/complementary strategy for unraveling new DCIS mechanisms and biomarkers.

Tracing anti-cancer and cancer-promoting actions of all-trans retinoic acid in breast cancer to a RARα epigenetic mechanism of mammary epithelial cell fate.

A hallmark of cancer cells is the ability to evade the growth inhibitory/pro-apoptotic action of physiological all-trans retinoic acid (RA) signal, the bioactive derivative of Vitamin A. However, as we and others reported, RA can also promote cancer cell growth and invasion. Here we show that anticancer and cancer-promoting RA actions in breast cancer have roots in a mechanism of mammary epithelial cell morphogenesis that involves both transcriptional (epigenetic) and non-transcriptional RARα (RARA) functions. We found that the mammary epithelial cell-context specific degree of functionality of the RARA transcriptional (epigenetic) component of this mechanism, by tuning the effects of the non-transcriptional RARA component, determines different cell fate decisions during mammary morphogenesis. Indeed, factors that hamper the RARA epigenetic function make physiological RA drive aberrant morphogenesis via non-transcriptional RARA, thus leading to cell transformation. Remarkably, also the cell context-specific degree of functionality of the RARA epigenetic component retained by breast cancer cells is critical to determine cell fate decisions in response to physiological as well as supraphysiological RA variation. Overall this study supports the proof of principle that the epigenetic functional plasticity of the mammary epithelial cell RARA mechanism, which is essential for normal morphogenetic processes, is necessary to deter breast cancer onset/progression consequent to the insidious action of physiological RA.

Entrainment of breast (cancer) epithelial cells detects distinct circadian oscillation patterns for clock and hormone receptor genes.

Most physiological and biological processes are regulated by endogenous circadian rhythms under the control of both a master clock, which acts systemically and individual cellular clocks, which act at the single cell level. The cellular clock is based on a network of core clock genes, which drive the circadian expression of non-clock genes involved in many cellular processes. Circadian deregulation of gene expression has emerged to be as important as deregulation of estrogen signaling in breast tumorigenesis. Whether there is a mutual deregulation of circadian and hormone signaling is the question that we address in this study. Here we show that, upon entrainment by serum shock, cultured human mammary epithelial cells maintain an inner circadian oscillator, with key clock genes oscillating in a circadian fashion. In the same cells, the expression of the estrogen receptor α (ER A) gene also oscillates in a circadian fashion. In contrast, ER A-positive and -negative breast cancer epithelial cells show disruption of the inner clock. Further, ER A-positive breast cancer cells do not display circadian oscillation of ER A expression. Our findings suggest that estrogen signaling could be affected not only in ER A-negative breast cancer, but also in ER A-positive breast cancer due to lack of circadian availability of ER A. Entrainment of the inner clock of breast epithelial cells, by taking into consideration the biological time component, provides a novel tool to test mechanistically whether defective circadian mechanisms can affect hormone signaling relevant to breast cancer.

Identification of an estrogen-regulated circadian mechanism necessary for breast acinar morphogenesis.

Altered estrogen receptor α (ERA) signaling and altered circadian rhythms are both features of breast cancer. By using a method to entrain circadian oscillations in human cultured cells, we recently reported that the expression of key clock genes oscillates in a circadian fashion in ERA-positive breast epithelial cells but not in breast cancer cells, regardless of their ERA status. Moreover, we reported that ERA mRNA oscillates in a circadian fashion in ERA-positive breast epithelial cells, but not in ERA-positive breast cancer cells. By using ERA-positive HME1 breast epithelial cells, which can be both entrained in vitro and can form mammary gland-like acinar structures in three-dimensional (3D) culture, first we identified a circuit encompassing ERA and an estrogen-regulated loop consisting of two circadian clock genes, PER2 and BMAL1. Further, we demonstrated that this estrogen-regulated circuit is necessary for breast epithelial acinar morphogenesis. Disruption of this circuit due to ERA-knockdown, negatively affects the estrogen-sustained circadian PER2-BMAL1 mechanism as well as the formation of 3D HME1 acini. Conversely, knockdown of either PER2 or BMAL1, by hampering the PER2-BMAL1 loop of the circadian clock, negatively affects ERA circadian oscillations and 3D breast acinar morphogenesis. To our knowledge, this study provides the first evidence of the implication of an ERA-circadian clock mechanism in the breast acinar morphogenetic process.

Regeneration-associated WNT signaling is activated in long-term reconstituting AC133bright acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a genetically heterogeneous clonal disorder characterized by two molecularly distinct self-renewing leukemic stem cell (LSC) populations most closely related to normal progenitors and organized as a hierarchy. A requirement for WNT/ß-catenin signaling in the pathogenesis of AML has recently been suggested by a mouse model. However, its relationship to a specific molecular function promoting retention of self-renewing leukemia-initiating cells (LICs) in human remains elusive. To identify transcriptional programs involved in the maintenance of a self-renewing state in LICs, we performed the expression profiling in normal (n = 10) and leukemic (n = 33) human long-term reconstituting AC133(+) cells, which represent an expanded cell population in most AML patients. This study reveals the ligand-dependent WNT pathway activation in AC133(bright) AML cells and shows a diffuse expression and release of WNT10B, a hematopoietic stem cell regenerative-associated molecule. The establishment of a primary AC133(+) AML cell culture (A46) demonstrated that leukemia cells synthesize and secrete WNT ligands, increasing the levels of dephosphorylated ß-catenin in vivo. We tested the LSC functional activity in AC133(+) cells and found significant levels of engraftment upon transplantation of A46 cells into irradiated Rag2(-/-)γc(-/-) mice. Owing to the link between hematopoietic regeneration and developmental signaling, we transplanted A46 cells into developing zebrafish. This system revealed the formation of ectopic structures by activating dorsal organizer markers that act downstream of the WNT pathway. In conclusion, our findings suggest that AC133(bright) LSCs are promoted by misappropriating homeostatic WNT programs that control hematopoietic regeneration.

Down-regulation of microRNAs 222/221 in acute myelogenous leukemia with deranged core-binding factor subunits.

Core-binding factor leukemia (CBFL) is a subgroup of acute myeloid leukemia (AML) characterized by genetic mutations involving the subunits of the core-binding factor (CBF). The leukemogenesis model for CBFL posits that one, or more, gene mutations inducing increased cell proliferation and/or inhibition of apoptosis cooperate with CBF mutations for leukemia development. One of the most common mutations associated with CBF mutations involves the KIT receptor. A high expression of KIT is a hallmark of a high proportion of CBFL. Previous studies indicate that microRNA (MIR) 222/221 targets the 3' untranslated region of the KIT messenger RNA and our observation that AML1 can bind the MIR-222/221 promoter, we hypothesized that MIR-222/221 represents the link between CBF and KIT. Here, we show that MIR-222/221 expression is upregulated after myeloid differentiation of normal bone marrow AC133(+) stem progenitor cells. CBFL blasts with either t(8;21) or inv(16) CBF rearrangements with high expression levels of KIT (CD117) display a significantly lower level of MIR-222/221 expression than non-CBFL blasts. Consistently, we found that the t(8;21) AML1-MTG8 fusion protein binds the MIR-222/221 promoter and induces transcriptional repression of a MIR-222/221-LUC reporter. Because of the highly conserved sequence homology, we demonstrated concomitant MIR-222/221 down-regulation and KIT up-regulation in the 32D/WT1 mouse cell model carrying the AML1-MTG16 fusion protein. This study provides the first hint that CBFL-associated fusion proteins may lead to up-regulation of the KIT receptor by down-regulating MIR-222/221, thus explaining the concomitant occurrence of CBF genetic rearrangements and overexpression of wild type or mutant KIT in AML.

Derangement of a factor upstream of RARalpha triggers the repression of a pleiotropic epigenetic network.

BACKGROUND: Chromatin adapts and responds to extrinsic and intrinsic cues. We hypothesize that inheritable aberrant chromatin states in cancer and aging are caused by genetic/environmental factors. In previous studies we demonstrated that either genetic mutations, or loss, of retinoic acid receptor alpha (RARalpha), can impair the integration of the retinoic acid (RA) signal at the chromatin of RA-responsive genes downstream of RARalpha, and can lead to aberrant repressive chromatin states marked by epigenetic modifications. In this study we tested whether the mere interference with the availability of RA signal at RARalpha, in cells with an otherwise functional RARalpha, can also induce epigenetic repression at RA-responsive genes downstream of RARalpha. METHODOLOGY/PRINCIPAL FINDINGS: To hamper the availability of RA at RARalpha in untransformed human mammary epithelial cells, we targeted the cellular RA-binding protein 2 (CRABP2), which transports RA from the cytoplasm onto the nuclear RARs. Stable ectopic expression of a CRABP2 mutant unable to enter the nucleus, as well as stable knock down of endogenous CRABP2, led to the coordinated transcriptional repression of a few RA-responsive genes downstream of RARalpha. The chromatin at these genes acquired an exacerbated repressed state, or state "of no return". This aberrant state is unresponsive to RA, and therefore differs from the physiologically repressed, yet "poised" state, which is responsive to RA. Consistent with development of homozygosis for epigenetically repressed loci, a significant proportion of cells with a defective CRABP2-mediated RA transport developed heritable phenotypes indicative of loss of function. CONCLUSION/SIGNIFICANCE: Derangement/lack of a critical factor necessary for RARalpha function induces epigenetic repression of a RA-regulated gene network downstream of RARalpha, with major pleiotropic biological outcomes.