Schiapparelli, Paula

OBJECTIVE The vertebral column is the most common site for skeletal metastasis, often leading to debilitating pain and weakness. Metastatic cancer has unique genetic drivers that potentiate tumorigenicity. There is an unmet need for novel targeted therapy in patients with spinal metastatic disease. METHODS The authors assessed the effect of verteporfin-induced yes-associated protein (YAP) inhibition on spine metastatic cell tumorigenicity and radiation sensitivity in vitro. Animal studies used a subcutaneous xenograft mouse model to assess the use of systemic intraperitoneal verteporfin (IP-VP) and intratumoral verteporfin microparticles (IT-VP) to inhibit the tumorigenicity of lung and breast spinal metastatic tumors from primary patient-derived tissue. RESULTS Verteporfin led to a dose-dependent decrease in migration, clonogenicity, and cell viability via inhibition of YAP and downstream effectors cyclin D1, CTGF, TOP2A, ANDRD1, MCL-1, FOSL2, KIF14, and KIF23. This was confirmed with knockdown of YAP. Verteporfin has an additive response when combined with radiation, and knockdown of YAP rendered cells more sensitive to radiation. The addition of verteporfin to YAP knockdown cells did not significantly alter migration, clonogenicity, or cell viability. IP-VP and IT-VP led to diminished tumor growth (p < 0.0001), especially when combined with radiation (p < 0.0001). Tissue analysis revealed diminished expression of YAP (p < 0.0001), MCL-1 (p < 0.0001), and Ki-67 (p < 0.0001) in tissue from verteporfin-treated tumors compared with vehicle-treated tumors. CONCLUSIONS This is the first study to demonstrate that verteporfin-mediated inhibition of YAP leads to diminished tumorigenicity in lung and breast spinal metastatic cancer cells. Targeting of YAP with verteporfin offers promising results that could be translated to human clinical trials.

Abstract Glioblastoma (GBM) is the most common and aggressive primary neoplasm of the central nervous system. The location of GBM contributes significantly to patient outcomes, where tumors contacting the lateral ventricles (LVs) have increased expression of stem cell genes, increased incidence of distal recurrence, and decreased overall survival. While the reasons for these findings are not fully understood, we hypothesize they arise due to interactions with the subventricular zone (SVZ), the largest neurogenic niche in mammals. We examined bidirectional signals between GBM cells and neural progenitor cells (NPCs) in vitro and in vivo using a combination of patient-derived intraoperative samples, preclinical animal models, and the methionine tRNA synthetase L274G (MetRS*) nascent proteomic labeling system, which allows for cell-specific proteomics analysis from complex in vivo systems. In co-culture with NPCs, GBM cells increase their viability, proliferation, migration, and expression of malignancy-promoting proteins, including migratory proteins such as TAGLN, PALLD, and STAT1. In vivo, tumor proximity to the SVZ results in increased proliferation, increased expression of stemness markers, decreased survival, and altered tumor-specific proteome. We then examined the reciprocal effect of GBM cells on NPC biology. In vitro and in vivo, GBM cells decreased NPC proliferation and induced decreased neuronal maturation of NPCs. To determine NPC proteomic changes in vivo, we created a transgenic mouse line Nestin-CreERT2; STOPflox R26-MetRS L274G, where upon tamoxifen administration Nestin+ NPCs express GFP and MetRS*. Our results indicate that LV-proximal GBM induces decreased expression of neuronal maturation proteins such as Eml1, VGLUT1, and EAAT2, and an increase in pro-migratory factors such as Lamc1 in NPCs. In conclusion, there are bidirectional proteomic interactions between GBM cells and NPCs that contributes to altered neurogenesis and increased tumor malignancy of LV-proximal GBM. The signaling factors identified in our studies will result in identification of novel therapeutic targets for GBM.

Metabolic rewiring in glioblastoma (GBM) is linked to intra- and extracellular pH regulation. In this study, we sought to characterize the role of melatonin on intracellular pH modulation and metabolic consequences to identify the mechanisms of action underlying melatonin oncostatic effects on GBM tumor initiating cells. GBM tumor initiating cells were treated at different times with melatonin (1.5 and 3.0 mM). We analyzed melatonin’s functional effects on GBM proliferation, cell cycle, viability, stemness, and chemo-radiosensitivity. We then assessed the effects of melatonin on GBM metabolism by analyzing the mitochondrial and glycolytic parameters. We also measured the intracellular and extracellular pH. Finally, we tested the effects of melatonin on a mouse subcutaneous xenograft model. We found that melatonin downregulated LDHA and MCT4, decreasing lactate production and inducing a decrease in intracellular pH that was associated with an increase in ROS and ATP depletion. These changes blocked cell cycle progression and induced cellular death and we observed similar results in vivo. Melatonin’s cytotoxic effects on GBM were due, at least in part, to intracellular pH modulation, which has emerged as a newly identified mechanism, providing new insights into the oncostatic effect of melatonin on GBM.

Abstract A small population of self-renewing stem cells initiate tumors and maintain therapeutic resistance in glioblastoma (GBM). Given the limited treatment options and dismal prognosis for this disease, there is urgent need to identify drivers of stem cells that could be druggable targets. Previous work showed that the endosomal pH regulator NHE9 is upregulated in GBM and correlates with worse survival prognosis. Here, we probed for aberrant signaling pathways in patient-derived GBM cells and found that NHE9 increases cell surface expression and phosphorylation of multiple receptor tyrosine kinases (RTKs) by promoting their escape from lysosomal degradation. Downstream of NHE9-mediated receptor activation, oncogenic signaling pathways converged on the JAK2-STAT3 transduction axis to induce pluripotency genes Oct4 and Nanog and suppress markers of glial differentiation. We used both genetic and chemical approaches to query the role of endosomal pH in GBM phenotypes. Loss-of-function mutations in NHE9 that failed to alkalinize endosomal lumen did not increase self-renewal capacity of gliomaspheres in vitro. However, monensin, a chemical mimetic of Na+/H+ exchanger activity, and the H+ pump inhibitor bafilomycin bypassed NHE9 to directly alkalinize the endosomal lumen resulting in stabilization of RTKs and induction of Oct4 and Nanog. Using orthotopic models of primary GBM cells we found that NHE9 increased tumor initiation in vivo. We propose that NHE9 initiates inside-out signaling from the endosomal lumen, distinct from the established effects of cytosolic and extracellular pH on tumorigenesis. Endosomal pH may be an attractive therapeutic target that diminishes stemness in GBM, agnostic of specific receptor subtype.

Abstract Glioblastoma (GBM) is the most common primary brain cancer in adults where tumor cell heterogeneity and sex differences influence clinical outcomes. Here, we functionally characterize three male and three female patient-derived GBM cell lines, identify protumorigenic BTICs, and create novel male and female preclinical models of GBM. Cell lines were evaluated on the following features: proliferation, stemness, migration, tumorigenesis, clinical characteristics, and sensitivity to radiation, TMZ, rhTNFSF10 (rhTRAIL), and rhBMP4. All cell lines were classified as GBM according to epigenetic subtyping, were heterogenous and functionally distinct from one another, and re-capitulated features of the original patient tumor. In establishing male and female preclinical models, it was found that two male-derived GBM cell lines (QNS108 and QNS120) and one female-derived GBM cell line (QNS315) grew at a faster rate in female mice brains. One male-derived GBM cell line (QNS108) decreased survival in female mice in comparison with male mice. However, no survival differences were observed for mice injected with a female-derived cell line (QNS315). In summary, a panel of six GBM patient-derived cell lines were functionally characterized, and it was shown that BTIC lines can be used to construct sex-specific models with differential phenotypes for additional studies.

Abstract Glioblastoma (GBM) is the most common form of malignant primary brain cancer in adults with a median survival of only 15 months. Therefore, new therapies to suppress malignant brain cancer are needed. Brain Tumor Initiating Cells (BTICs) are a GBM subpopulation of cells with a highly glycolytic profile that are thought to be responsible of the resistance of GBM to treatments. Metabolic reprogramming allows tumor cells to survive in unsupportive microenvironments. Manipulating tumor metabolism to counteract GBM resistance arises as a powerful approach with minimum effects in normal counterparts. At pharmacological concentrations, melatonin displays oncostatic properties. This is thought to be due to an increase in mitochondrial oxidative phosphorylation through the effects of melatonin in mitochondria, key organelle in metabolic homeostasis. We hypothesize that melatonin could alter BTIC metabolism, by inducing an anti-Warburg effect and as consequence, melatonin will decrease the viability of GBM cells and tumor growth. We found that treatment of GBM cell lines with 3mM melatonin significantly altered tumor cell metabolism. We observed that melatonin downregulated the lactate symporter MCT4 (p&lt;0.002), inducing a significant intracellular accumulation of lactate (p&lt;0.002) while decreasing it in the extracellular media (p&lt;0.001). This was followed by a decrease in the internal pH (p&lt;0.002). These effects were compensated by an increase in the oxygen consumption rate (OCR) followed by decay that leaded to an increase in ROS production (p&lt;0.001). All these changes result in a depletion of cellular ATP (p&lt;0.001) and eventually drove to a decrease in the proliferation (p&lt;0.001) and cell death (p&lt;0.001). When applied in vivo we observed a significant reduction in the tumor growth (p&lt;0.001), volume (p&lt;0.002) and weight (p&lt;0.002), as well as a drop in the proliferation marker ki67 (p&lt;0.001) and a fibrosis increase in treated tumors. These results position melatonin as a strong therapeutic candidate for GBM therapy.

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults. Currently, treatment is focused on achieving maximal safe resection, followed by chemo- and radiotherapy. Although surgery remains the most important therapeutic component, complete surgical resection is virtually impossible, as GBM cells readily infiltrate the adjacent brain parenchyma. One fundamental mechanism for GBM cell migration is the ability of these cells to use cell volume regulation as a driving force of cell infiltration and potentially interfering with apoptosis and cell cycle progression. In this work, we targeted the master regulators of cell volume, the kinases STE20/SPS1-Related Proline-Alanine-Rich Protein Kinase (SPAK) and Oxidative stress-responsive kinase-1) (OSR1) by using a novel proprietary small molecule inhibitor. In order to study the effects of inhibiting SPAK and OSR1, we analyzed cell proliferation and migration. First, we determined the IC50 using six different patient-derived GBM cell lines; we found IC50 values ranging from 0.2–2μM. Next, cell proliferation was determined by cell cycle analysis through Edu labeling. We found a significant decrease in S phase and cell cycle arrest in G2M (p= 0.001, n=6). In addition, cell migration was determined using boyden chamber assay. We found a dose-dependent reduction in cell migration (p=0.001, n=3). These results correlated with the phenomenon observed in an orthotopic murine model of GBM, in which the inhibitor showed a decrease in tumor growth (p=0.0026, n=4) and greater survival rates in vivo (p=0.0046, n=8). When used in combination with radiation therapy, this small molecule inhibitor was capable of radio-sensitizing GBM cells decreasing clonogenicity (p=0.01, n=3) were observed in vitro. In summary, by targeting the SPAK/OSR1 kinases with a small molecule inhibitor, GBM cells become less aggressive, mainly by interfering with cell migration and proliferation and becoming more sensitive to radiation.

Abstract Background Glioblastomas (GBMs) are the main primary brain tumors in adults with almost 100% recurrence rate. Patients with lateral ventricle proximal GBMs (LV-GBMs) exhibit worse survival compared to distal locations for unknown reasons. One hypothesis is the proximity of these tumors to the cerebrospinal fluid (CSF) and its chemical cues that can regulate cellular phenotype. We therefore investigated the role of CSF on GBM gene expression and the role of a CSF-induced gene, SERPINA3, in GBM malignancy in vitro and in vivo. Methods We utilized human CSF and GBM brain tumor-initiating cells (BTICs). We determined the impact of SERPINA3 expression in glioma patients using The Cancer Genome Atlas (TCGA) database. SERPINA3 expression changes were evaluated at mRNA and protein levels. The effects of knockdown (KD) and overexpression (OE) of SERPINA3 on cell migration, viability and cell proliferation were evaluated. Stem cell characteristics on KD cells were evaluated by differentiation and colony formation experiments. Tumor growth was studied by intracranial and flank injections. Results GBM-CSF increased BTIC migration accompanied by upregulation of the SERPINA3 gene. In patient samples and TCGA data, we observed SERPINA3 to correlate directly with brain tumor grade and indirectly with GBM patient survival. SERPINA3 KD induced a decrease in cell proliferation, migration, invasion, and stem cell characteristics, while SERPINA3 OE increased cell migration. In vivo, SERPINA3 KD BTICs showed increased survival in a murine model. Conclusions SERPINA3 plays a key role in GBM malignancy and its inhibition results in a better outcome using GBM preclinical models.

Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with a median survival of 14 months despite multimodal treatment. Patients with GBM tumors exhibit a post-resection recurrence rate of almost 100%. The tumor recurrence and treatment resistance properties of GBM reside in a stem-like cell population called Brain Tumor Initiating Cells (BTIC). Among primary GBMs, periventricular tumors exhibit worse survival than tumors distal to the lateral ventricles (LV), the cause for this worse outcome is not known. A potential explanation is the proximity of these tumors to the cerebrospinal fluid (CSF) and the neurogenic niche in the sub-ventricular zone (SVZ). Chemotactic signals present in the cerebrospinal fluid (CSF) regulate neural progenitor cells migratory response and differentiation profile. Our preliminary data show that cancer-derived CSF induces an increase in the migration and proliferation of GBM cells when compared to non-cancer CSF. We evaluated the transcriptome of human GBM cells in response to cancer and non-cancer CSF and identified alpha-1-antichymotrypsin (SERPINA3) as one of the genes that show higher over expression upon cancer CSF-stimulation. SERPINA3 is a circulating anti-protease that is produced and released in different tissues such as liver, prostate, lungs, endometrium and brain that is over-expressed in multiple cancers. We hypothesize that the overexpression of SERPINA3 in response to CSF is a contributing factor to the increase malignancy of periventricular GBM tumors. We tested this hypothesis in silico, in vitro and in vivo. Using TCGA and Rembrandt databases we observed a higher expression of SERPINA3 in brain tumor tissue compared to controls with no differences among GBM subtypes. In GBM patients, expression of SERPINA3 negatively affects survival expectancy. In vitro, Using intraoperative samples, we confirmed that SERPINA3 is overexpressed in brain cancer tissue by western-blot and qRT-PCR. Interestingly, we observed a higher nuclear localization of SERPINA3 in cancer tissue and cells. To determine if SERPINA3 has an effect on the malignancy of GBM cells, we silenced its expression using shRNA. SERPINA3 silencing induced a decrease in cell proliferation (p&lt;0.05), as well as migration (p&lt;0.05), and invasion (p&lt;0.05). Remarkably CD133 expression and stem cell markers were decreased when SERPINA3 is silenced. In contrast, the addition of recombinant SERPINA3 induced an increase in cell migration (p&lt;0.05) of GBM cells. Moreover, cancer CSF is no longer able to induce an increase in GBM cell migration in SERPINA3-KD cells (p&lt;0.05). In vivo, mice orthotopically-injected with SERPINA3-KD cells showed a prolonged overall survival (p&lt;0.05). In summary, our results support the hypothesis that SERPINA3 plays a key role in GBM malignancy in response to CSF and its inhibition results in a better outcome using GBM preclinical models. Targeting the CSF-GBM interaction is a potential therapeutic approach that remains to be explored. Citation Format: Montserrat A. Lara Velazquez, Natanael Zarco, Anna Carrano, Jordan Phillips, Paula Schiapparelli, Emily S. Norton, Stephanie Jeanneret, Teresita Corona, Jose Segovia, Kaisorn Chaichana, Yan Asmann, Alfredo Quinones Hinojosa, Hugo Guerrero-Cazares. Cerebrospinal fluid-responsive factor SERPINA3 induces an increase in the malignancy of glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3780.