Maheedhara R. Guda

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.

Cancer stem cell molecular reprogramming of the Warburg effect in glioblastomas: a new target gleaned from an old concept

Prior targeted treatment for glioblastoma multiforme (GBM) with anti-angiogenic agents, such as bevacizumab, has been met with limited success potentially owing to GBM tumors ability to develop a hypoxia-induced escape mechanism--a glycolytic switch from oxidative phosphorylation to glycolysis, an old concept known as the Warburg effect. New studies points to a subpopulation of cells as a source for treatment-resistance, cancer stem cells (CSCs). Taken together, the induction of the Warburg effect leads to the promotion of CSC self-renewal and undifferentiation. In response to hypoxia, hypoxia-inducible transcription factor is upregulated and is the central driver in setting off the cascade of events in CSC metabolic reprogramming. Hypoxia-inducible transcription factor upregulates GLUT1 to increase glucose uptake into the cell, upregulates HK2 and PK during glycolysis, upregulates LDHA in the termination of glycolysis, and downregulates PDH to redirect energy production toward glycolysis. This review aims to unite these old and new concepts simultaneously and examine potential enzyme targets driven by hypoxia in the glycolytic phenotype of CSCs to reverse the metabolic shift induced by the Warburg effect.

Metabolic targeting of EGFRvIII/PDK1 axis in temozolomide resistant glioblastoma

Glioblastomas are characterized by amplification of EGFR. Approximately half of tumors with EGFR over-expression also express a constitutively active ligand independent EGFR variant III (EGFRvIII). While current treatments emphasize surgery followed by radiation and chemotherapy with Temozolomide (TMZ), acquired chemoresistance is a universal feature of recurrent GBMs. To mimic the GBM resistant state, we generated an in vitro TMZ resistant model and demonstrated that dichloroacetate (DCA), a metabolic inhibitor of pyruvate dehydrogenase kinase 1 (PDK1), reverses the Warburg effect. Microarray analysis conducted on the TMZ resistant cells with their subsequent treatment with DCA revealed PDK1 as its sole target. DCA treatment also induced mitochondrial membrane potential change and apoptosis as evidenced by JC-1 staining and electron microscopic studies. Computational homology modeling and docking studies confirmed DCA binding to EGFR, EGFRvIII and PDK1 with high affinity. In addition, expression of EGFRvIII was comparable to PDK1 when compared to EGFR in GBM surgical specimens supporting our in silico prediction data. Collectively our current study provides the first in vitro proof of concept that DCA reverses the Warburg effect in the setting of EGFRvIII positivity and TMZ resistance leading to GBM cytotoxicity, implicating cellular tyrosine kinase signaling in cancer cell metabolism.

Modified tunicamycins with reduced eukaryotic toxicity that enhance the antibacterial activity of β-lactams

Tunicamycins (TUN) are inhibitors of the UDP-HexNAc: polyprenol-P HexNAc-1-P transferase family of enzymes, which initiate the biosynthesis of bacterial peptidoglycan and catalyze the first step in eukaryotic protein N-glycosylation. The TUN are therefore general and potent toxins to both eukaryotes and prokaryotes. Screening a library of synthetic TUN against Bacillus and yeast identified TUN that are antibacterial, but have significantly reduced eukaryotic toxicity. One of these (Tun-15:0) differs from the native TUN control only by the lack of the conjugated double bond in the tunicaminyl N-acyl group. Tun-15:0 also showed reduced inhibition for protein N-glycosylation in a Pichia-based bioassay. Natural TUN was subsequently modified by chemically reducing the N-acyl double bond (TunR1) or both the N-acyl and uridyl double bonds (TunR2). TunR1 and TunR2 retain their antibacterial activity, but with considerably reduced eukaryotic toxicity. In protein N-glycosylation bioassays, TunR1 is a less potent inhibitor than native TUN and TunR2 is entirely inactive. Importantly, the less toxic TunR1 and TunR2 both enhance the antibacterial activity of β-lactams: oxacillin by 32- to 64-fold, comparable with native TUN, and with similar enhancements for methicillin and penicillin G. Hence, the modified TUNs, TunR1 and TunR2, are potentially important as less-toxic synergistic enhancers of the β-lactams.

Methylation regulates HEY1 expression in glioblastoma

Glioblastoma (GBM) remains one of the most lethal and difficult-to-treat cancers of the central nervous system. The poor prognosis in GBM patients is due in part to its resistance to available treatments, which calls for identifying novel molecular therapeutic targets. In this study, we identified a mediator of Notch signaling, HEY1, whose methylation status contributes to the pathogenesis of GBM. Datamining studies, immunohistochemistry and immunoblot analysis showed that HEY1 is highly expressed in GBM patient specimens. Since methylation status of HEY1 may control its expression, we conducted bisulphite sequencing on patient samples and found that the HEY1 promoter region was hypermethylated in normal brain when compared to GBM specimens. Treatment on 4910 and 5310 xenograft cell lines with sodium butyrate (NaB) significantly decreased HEY1 expression with a concomitant increase in DNMT1 expression, confirming that promoter methylation may regulate HEY1 expression in GBM. NaB treatment also induced apoptosis of GBM cells as measured by flow cytometric analysis. Further, silencing of HEY1 reduced invasion, migration and proliferation in 4910 and 5310 cells. Furthermore, immunoblot and q-PCR analysis showed the existence of a potential positive regulatory loop between HEY1 and p53. Additionally, transcription factor interaction array with HEY1 recombinant protein predicted a correlation with p53 and provided various bonafide targets of HEY1. Collectively, these studies suggest HEY1 may be an important predictive marker for GBM and potential target for future GBM therapy.

Characterization of brain tumor initiating cells isolated from an animal model of CNS primitive neuroectodermal tumors

CNS Primitive Neuroectodermal tumors (CNS-PNETs) are members of the embryonal family of malignant childhood brain tumors, which remain refractory to current therapeutic treatments. Current paradigm of brain tumorigenesis implicates brain tumor-initiating cells (BTIC) in the onset of tumorigenesis and tumor maintenance. However, despite their significance, there is currently no comprehensive characterization of CNS-PNETs BTICs. Recently, we described an animal model of CNS-PNET generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult neural stem cells (NSC) - into NOD-SCID mice brain and proposed that BTICs may play a role in the maintenance of these tumors. Here we report the characterization of BTIC lines derived from this CNS-PNET animal model. BTIC’s orthotopic transplantation generated highly aggressive tumors also characterized as CNS-PNETs. The BTICs have the hallmarks of NSCs as they demonstrate self-renewing capacity and have the ability to differentiate into astrocytes and early migrating neurons. Moreover, the cells demonstrate aberrant accumulation of wild type tumor-suppressor protein p53, indicating its functional inactivation, highly up-regulated levels of onco-protein cMYC and the BTIC marker OCT3/4, along with metabolic switch to glycolysis - suggesting that these changes occurred in the early stages of tumorigenesis. Furthermore, based on RNA- and DNA-seq data, the BTICs did not acquire any transcriptome-changing genomic alterations indicating that the onset of tumorigenesis may be epigenetically driven. The study of these BTIC self-renewing cells in our model may enable uncovering the molecular alterations that are responsible for the onset and maintenance of the malignant PNET phenotype.

Abstract 1153: Hand-1 overexpression inhibits medulloblastoma cell invasion and metastatic ability via Oct-3/4 / β-catenin interaction

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Medulloblastoma (MB) is the most common malignant brain tumor in children. Treatment of medulloblastoma includes surgery, radiation and chemotherapy. Despite long-term survival for the majority of children, 50% of medulloblastomas recur. Recently, medulloblastomas are classified into four distinct molecular subgroups, viz., WNT, SHH, group 3, and group 4 based on constitutive activation of WNT/β-catenin, SHH, and NOTCH signaling pathways that regulate cancer stem cell pluripotency, tumor growth, progression and metastasis. Previously, we investigated the molecular machinery underlying uPAR and WNT/β-catenin interdependent signaling pathways in medulloblastoma stem cells. Further, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. Complementing to this we demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand-1. Here, we show that Hand-1 expression is observed to be downregulated in all the molecular subgroups of medulloblastoma. We also showed that the pluripotency marker, Oct-3/4 (encoded by the Pou5f1 gene) interacts with β-catenin and Hand-1 that ultimately regulated the uPAR expression. Our preliminary study demonstrated that Oct-3/4 preferentially cooperated with β-catenin in medulloblastoma to maintain tumor progression. Immunohistostaining of clinical specimens also confirmed our aforementioned results. Taken together, our studies show that overexpression of Hand-1 in medulloblastoma cells may effectively inhibit uPAR-induced invasion and metastatic ability, thereby regulating Oct-3/4/β-catenin balance. Citation Format: Swapna Asuthkar, Maheedhara R. Guda, Andrew J. Tsung, Reuben Antony, Dzung H. Dinh, Kiran K. Velpula. Hand-1 overexpression inhibits medulloblastoma cell invasion and metastatic ability via Oct-3/4 / β-catenin interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1153.

Abstract 1920: miR-211 directly targets pyruvate dehydrogenase kinase 4 to inhibit cellular growth and glucose metabolism in triple negative breast cancer

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Deregulated metabolism and aerobic glycolysis are common to many kinds of tumors including, breast cancers driven by a plethora of transcription factors. One such is hypoxia-inducible factor-1α (HIF-1α) known to drive the expression of glycolytic target genes. Here, we identify PDK4 (pyruvate dehydrogenase kinase 4) as a key regulator of HIF-1α signaling. In triple-negative human breast cancer (TNBC) cell lines, we investigated the contribution of PDK4 and HIF-1α to experimental tumor growth, core glucose metabolism and ionizing radiation effects. When these TNBC cells were exposed to 4, 6 and 8Gy treatment, we observed a linear increase in the expression levels of PDK4 and HIF-1α genes. In this study we also report that miR-211, a microRNA predicted to target PDK4 is suppressed in TNBC patient specimens as evidenced by in situ hybridization analysis. The expression levels of PDK4, HIF-1α and RICTOR were reduced with miR-211 over expression while the levels of PDH and FH were observed to be increased. Further, we observed that miR-211 conjugated to cerium oxide nanoparticles (nanoceria) showed increased cytotoxicity. Similar effects were observed when these TNBCs were treated with 5mM DCA, a potent metabolic inhibitor of PDK. Combinatory suppression of PDK4 and HIF-1α exerted synergistic inhibition of lactate released suggesting of reversal of aerobic glycolysis. Alternatively, TCGA datasets were predictive of patient survival, with a PDK4/miR-211 signature robustly predicting poor patient prognosis. Overall, our findings suggest that combined targeting of PDK4 signaling cascade with miR-211 and DCA in combination specifically regulate core glucose metabolism in TNBC. Citation Format: Maheedhara R. Guda, Swapna Asuthkar, Soumen Das, Sudipta Seal, Andrew J. Tsung, Kiran K. Velpula. miR-211 directly targets pyruvate dehydrogenase kinase 4 to inhibit cellular growth and glucose metabolism in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1920.

Abstract 41: DCA bind to EGFR/EGFRvIII/PDK1 and affect the proliferation and growth in TMZ resistant glioblastoma model

Glioblastomas are characterized by amplification of epidermal growth factor receptor (EGFR). Approximately half of the GBM tumors with EGFR over-expression also express a constitutively active ligand independent EGFR variant III (EGFRvIII). This phenotype represents a tumor specific target correlating with a high growth potential. Despite advances in treatment, whether surgery, chemotherapeutics or molecular targeted therapies, very few have shown promise. Temozolomide (TMZ), as standard of care alkylation therapy, demonstrates improved survival when administered with concomitant radiotherapy. Unfortunately, acquired TMZ chemo-resistance is observed in more than 90% of recurrent GBMs. To understand the mechanisms in the context of TMZ resistance, we generated an in vitro TMZ resistant model by continuous exposure of U373 cells constitutively expressing EGFRvIII (U373vIII) to 150uM TMZ for 6 months (U373vIIIR). Dicholoroacetate (DCA) is a known metabolic inhibitor of pyruvate dehydrogenase kinase 1 (PDK1). Its application to cancer has recently been revived in the realm of metabolic oncology, where reversal of the Warburg effect has resulted in decreased tumor growth. Treatment of U373, U373vIII, and U373vIIIR GBM cell lines with DCA was found to induce cytotoxicity and reduced cell survival across all cell lines. Further, micro array studies conducted on U373vIII or U373vIIIR and their subsequent treatment with DCA revealed that PDK1 is the sole target in TMZ resistant tumors expressing EGFRvIII. Additionally, we demonstrated that 1mM DCA induced mitochondrial membrane potential change as evidenced in JC-1 staining and electron microscopic studies confirming mitochondrial apoptosis. Consistent with our previous findings that EGFR interacts with PDK1, our computational analysis revealed that DCA aligns itself to the binding sites of both EGFR and EGFRvIII with strong binding affinity apart from its binding to PDK1. Clinically, expression of EGFRvIII correlated with PDK1 when compared to EGFR in GBM surgical specimens. Collectively, our studies demonstrate that principles of metabolic oncology and DCA are active in GBM treatment despite resistance providing new insight into the development of alternative treatment in TMZ failure. Citation Format: Kiran Kumar Velpula, Kamlesh Sahu, Jack Tuszynski, Maheedhara R. Guda, Swapna Asuthkar, Sarah E. Martin, Justin D. Lathia, Andrew J. Tsung. DCA bind to EGFR/EGFRvIII/PDK1 and affect the proliferation and growth in TMZ resistant glioblastoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 41.

Hand1 overexpression inhibits medulloblastoma metastasis.

Medulloblastoma (MB) is the most frequent malignant pediatric brain tumor. Current treatment includes surgery, radiation and chemotherapy. However, ongoing treatment in patients is further classified according to the presence or absence of metastasis. Since metastatic medulloblastoma are refractory to current treatments, there is need to identify novel biomarkers that could be used to reduce metastatic potential, and more importantly be targeted therapeutically. Previously, we showed that ionizing radiation-induced uPAR overexpression is associated with increased accumulation of β-catenin in the nucleus. We further demonstrated that uPAR protein act as cytoplasmic sequestration factor for a novel basic helix-loop-helix transcription factor, Hand1. Among the histological subtypes classical and desmoplastic subtypes account for the majority while large cell/anaplastic variant is most commonly associated with metastatic disease. In this present study using immunohistochemical approach and patient data mining for the first time, we demonstrated that Hand1 expression is observed to be downregulated in all the subtypes of medulloblastoma. Previously we showed that Hand1 overexpression regulated medulloblastoma angiogenesis and here we investigated the role of Hand1 in the context of Epithelial-Mesenchymal Transition (EMT). Moreover, UW228 and D283 cells overexpressing Hand1 demonstrated decreased-expression of mesenchymal markers (N-cadherin, β-catenin and SOX2); metastatic marker (SMA); and increased expression of epithelial marker (E-cadherin). Strikingly, human pluripotent stem cell antibody array showed that Hand1 overexpression resulted in substantial decrease in pluripotency markers (Nanog, Oct3/4, Otx2, Flk1) suggesting that Hand1 expression may be essential to attenuate the EMT and our findings underscore a novel role for Hand1 in medulloblastoma metastasis.