Alvaro G. Alvarado

Cancer Stem Cells: Targeting the Roots of Cancer, Seeds of Metastasis, and Sources of Therapy Resistance

With the goal to remove the roots of cancer, eliminate metastatic seeds, and overcome therapy resistance, the 2014 inaugural International Cancer Stem Cell (CSC) Conference at Cleveland, OH, convened together over 320 investigators, including 55 invited world-class speakers, 25 short oral presenters, and 100 poster presenters, to gain an in-depth understanding of CSCs and explore therapeutic opportunities targeting CSCs. The meeting enabled intriguing discussions on several topics including: genetics and epigenetics; cancer origin and evolution; microenvironment and exosomes; metabolism and inflammation; metastasis and therapy resistance; single cell and heterogeneity; plasticity and reprogramming; as well as other new concepts. Reports of clinical trials targeting CSCs emphasized the urgent need for strategically designing combinational CSC-targeting therapies against cancer.

Cancer Stem Cell-Specific Scavenger Receptor CD36 Drives Glioblastoma Progression

Glioblastoma (GBM) contains a self‐renewing, tumorigenic cancer stem cell (CSC) population which contributes to tumor propagation and therapeutic resistance. While the tumor microenvironment is essential to CSC self‐renewal, the mechanisms by which CSCs sense and respond to microenvironmental conditions are poorly understood. Scavenger receptors are a broad class of membrane receptors well characterized on immune cells and instrumental in sensing apoptotic cellular debris and modified lipids. Here, we provide evidence that CSCs selectively use the scavenger receptor CD36 to promote their maintenance using patient‐derived CSCs and in vivo xenograft models. CD36 expression was observed in GBM cells in addition to previously described cell types including endothelial cells, macrophages, and microglia. CD36 was enriched in CSCs and was able to functionally distinguish self‐renewing cells. CD36 was coexpressed with integrin alpha 6 and CD133, previously described CSC markers, and CD36 reduction resulted in concomitant loss of integrin alpha 6 expression, self‐renewal, and tumor initiation capacity. We confirmed oxidized phospholipids, ligands of CD36, were present in GBM and found that the proliferation of CSCs, but not non‐CSCs, increased with exposure to oxidized low‐density lipoprotein. CD36 was an informative biomarker of malignancy and negatively correlated to patient prognosis. These results provide a paradigm for CSCs to thrive by the selective enhanced expression of scavenger receptors, providing survival, and metabolic advantages. Stem Cells 2014;32:1746–1758

Differential Connexin Function Enhances Self-Renewal in Glioblastoma

SUMMARY The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. Previous reports have suggested that gap junctions are tumor suppressive based on connexin43 (Cx43), but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs) possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression.

Development of a Fluorescent Reporter System to Delineate Cancer Stem Cells in Triple-Negative Breast Cancer.

Advanced cancers display cellular heterogeneity driven by self‐renewing, tumorigenic cancer stem cells (CSCs). The use of cell lines to model CSCs is challenging due to the difficulty of identifying and isolating cell populations that possess differences in self‐renewal and tumor initiation. To overcome these barriers in triple‐negative breast cancer (TNBC), we developed a CSC system using a green fluorescent protein (GFP) reporter for the promoter of the well‐established pluripotency gene NANOG. NANOG‐GFP+ cells gave rise to both GFP+ and GFP− cells, and GFP+ cells possessed increased levels of the embryonic stem cell transcription factors NANOG, SOX2, and OCT4 and elevated self‐renewal and tumor initiation capacities. GFP+ cells also expressed mesenchymal markers and demonstrated increased invasion. Compared with the well‐established CSC markers CD24−/CD44+, CD49f, and aldehyde dehydrogenase (ALDH) activity, our NANOG‐GFP reporter system demonstrated increased enrichment for CSCs. To explore the utility of this system as a screening platform, we performed a flow cytometry screen that confirmed increased CSC marker expression in the GFP+ population and identified new cell surface markers elevated in TNBC CSCs, including junctional adhesion molecule‐A (JAM‐A). JAM‐A was highly expressed in GFP+ cells and patient‐derived xenograft ALDH+ CSCs compared with the GFP− and ALDH− cells, respectively. Depletion of JAM‐A compromised self‐renewal, whereas JAM‐A overexpression induced self‐renewal in GFP− cells. Our data indicate that we have defined and developed a robust system to monitor differences between CSCs and non‐CSCs in TNBC that can be used to identify CSC‐specific targets for the development of future therapeutic strategies. Stem Cells. Stem Cells 2015;33:2114–2125

A Tumor Suppressor Function for Notch Signaling in Forebrain Tumor Subtypes

In the brain, Notch signaling maintains normal neural stem cells, but also brain cancer stem cells, indicating an oncogenic role. Here, we identify an unexpected tumor suppressor function for Notch in forebrain tumor subtypes. Genetic inactivation of RBP-Jκ, a key Notch mediator, or Notch1 and Notch2 receptors accelerates PDGF-driven glioma growth in mice. Conversely, genetic activation of the Notch pathway reduces glioma growth and increases survival. In humans, high Notch activity strongly correlates with distinct glioma subtypes, increased patient survival, and lower tumor grade. Additionally, simultaneous inactivation of RBP-Jκ and p53 induces primitive neuroectodermal-like tumors in mice. Hence, Notch signaling cooperates with p53 to restrict cell proliferation and tumor growth in mouse models of human brain tumors.

Cancer Stem Cell-Secreted Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Suppressor Cell Function and Facilitates Glioblastoma Immune Evasion.

Shifting the balance away from tumor‐mediated immune suppression toward tumor immune rejection is the conceptual foundation for a variety of immunotherapy efforts currently being tested. These efforts largely focus on activating antitumor immune responses but are confounded by multiple immune cell populations, including myeloid‐derived suppressor cells (MDSCs), which serve to suppress immune system function. We have identified immune‐suppressive MDSCs in the brains of GBM patients and found that they were in close proximity to self‐renewing cancer stem cells (CSCs). MDSCs were selectively depleted using 5‐flurouracil (5‐FU) in a low‐dose administration paradigm, which resulted in prolonged survival in a syngeneic mouse model of glioma. In coculture studies, patient‐derived CSCs but not nonstem tumor cells selectively drove MDSC‐mediated immune suppression. A cytokine screen revealed that CSCs secreted multiple factors that promoted this activity, including macrophage migration inhibitory factor (MIF), which was produced at high levels by CSCs. Addition of MIF increased production of the immune‐suppressive enzyme arginase‐1 in MDSCs in a CXCR2‐dependent manner, whereas blocking MIF reduced arginase‐1 production. Similarly to 5‐FU, targeting tumor‐derived MIF conferred a survival advantage to tumor‐bearing animals and increased the cytotoxic T cell response within the tumor. Importantly, tumor cell proliferation, survival, and self‐renewal were not impacted by MIF reduction, demonstrating that MIF is primarily an indirect promoter of GBM progression, working to suppress immune rejection by activating and protecting immune suppressive MDSCs within the GBM tumor microenvironment. Stem Cells 2016;34:2026–2039

Glioblastoma Cancer Stem Cells Evade Innate Immune Suppression of Self-Renewal through Reduced TLR4 Expression

Tumors contain hostile inflammatory signals generated by aberrant proliferation, necrosis, and hypoxia. These signals are sensed and acted upon acutely by the Toll-like receptors (TLRs) to halt proliferation and activate an immune response. Despite the presence of TLR ligands within the microenvironment, tumors progress, and the mechanisms that permit this growth remain largely unknown. We report that self-renewing cancer stem cells (CSCs) in glioblastoma have low TLR4 expression that allows them to survive by disregarding inflammatory signals. Non-CSCs express high levels of TLR4 and respond to ligands. TLR4 signaling suppresses CSC properties by reducing retinoblastoma binding protein 5 (RBBP5), which is elevated in CSCs. RBBP5 activates core stem cell transcription factors, is necessary and sufficient for self-renewal, and is suppressed by TLR4 overexpression in CSCs. Our findings provide a mechanism through which CSCs persist in hostile environments because of an inability to respond to inflammatory signals.

Coordination of self-renewal in glioblastoma by integration of adhesion and microRNA signaling

BACKGROUND Cancer stem cells (CSCs) provide an additional layer of complexity for tumor models and targets for therapeutic development. The balance between CSC self-renewal and differentiation is driven by niche components including adhesion, which is a hallmark of stemness. While studies have demonstrated that the reduction of adhesion molecules, such as integrins and junctional adhesion molecule-A (JAM-A), decreases CSC maintenance. The molecular circuitry underlying these interactions has yet to be resolved. METHODS MicroRNA screening predicted that microRNA-145 (miR-145) would bind to JAM-A. JAM-A overexpression in CSCs was evaluated both in vitro (proliferation and self-renewal) and in vivo (intracranial tumor initiation). miR-145 introduction into CSCs was similarly assessed in vitro. Additionally, The Cancer Genome Atlas dataset was evaluated for expression levels of miR-145 and overall survival of the different molecular groups. RESULTS Using patient-derived glioblastoma CSCs, we confirmed that JAM-A is suppressed by miR-145. CSCs expressed low levels of miR-145, and its introduction decreased self-renewal through reductions in AKT signaling and stem cell marker (SOX2, OCT4, and NANOG) expression; JAM-A overexpression rescued these effects. These findings were predictive of patient survival, with a JAM-A/miR-145 signature robustly predicting poor patient prognosis. CONCLUSIONS Our results link CSC-specific niche signaling to a microRNA regulatory network that is altered in glioblastoma and can be targeted to attenuate CSC self-renewal.

Taking a Toll on Self-Renewal: TLR-Mediated Innate Immune Signaling in Stem Cells

Innate immunity has evolved as the front-line cellular defense mechanism to acutely sense and decisively respond to microenvironmental alterations. The Toll-like receptor (TLR) family activates signaling pathways in response to stimuli and is well-characterized in both resident and infiltrating immune cells during neural inflammation, injury, and degeneration. Innate immune signaling has also been observed in neural cells during development and disease, including in the stem and progenitor cells that build the brain and are responsible for its homeostasis. Recently, the activation of developmental programs in malignant brain tumors has emerged as a driver for growth via cancer stem cells. In this review we discuss how innate immune signaling interfaces with stem cell maintenance in the normal and neoplastic brain.

A 4-miRNA signature to predict survival in glioblastomas

Glioblastomas are among the most lethal cancers; however, recent advances in survival have increased the need for better prognostic markers. microRNAs (miRNAs) hold great prognostic potential being deregulated in glioblastomas and highly stable in stored tissue specimens. Moreover, miRNAs control multiple genes representing an additional level of gene regulation possibly more prognostically powerful than a single gene. The aim of the study was to identify a novel miRNA signature with the ability to separate patients into prognostic subgroups. Samples from 40 glioblastoma patients were included retrospectively; patients were comparable on all clinical aspects except overall survival enabling patients to be categorized as short-term or long-term survivors based on median survival. A miRNome screening was employed, and a prognostic profile was developed using leave-one-out cross-validation. We found that expression patterns of miRNAs; particularly the four miRNAs: hsa-miR-107_st, hsa-miR-548x_st, hsa-miR-3125_st and hsa-miR-331-3p_st could determine short- and long-term survival with a predicted accuracy of 78%. Heatmap dendrograms dichotomized glioblastomas into prognostic subgroups with a significant association to survival in univariate (HR 8.50; 95% CI 3.06–23.62; p<0.001) and multivariate analysis (HR 9.84; 95% CI 2.93–33.06; p<0.001). Similar tendency was seen in The Cancer Genome Atlas (TCGA) using a 2-miRNA signature of miR-107 and miR-331 (miR sum score), which were the only miRNAs available in TCGA. In TCGA, patients with O6-methylguanine-DNA-methyltransferase (MGMT) unmethylated tumors and low miR sum score had the shortest survival. Adjusting for age and MGMT status, low miR sum score was associated with a poorer prognosis (HR 0.66; 95% CI 0.45–0.97; p = 0.033). A Kyoto Encyclopedia of Genes and Genomes analysis predicted the identified miRNAs to regulate genes involved in cell cycle regulation and survival. In conclusion, the biology of miRNAs is complex, but the identified 4-miRNA expression pattern could comprise promising biomarkers in glioblastoma stratifying patients into short- and long-term survivors.