Browsing by Author "Michelhaugh, Sharon K."

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    3D models of glioblastoma interaction with cortical cells
    Abedin, Md Joynal; Michelhaugh, Sharon K.; Mittal, Sandeep; Berdichevsky, Yevgeny (Frontiers, 2023-03-09)
    Introduction: Glioblastoma (GBM) invasiveness and ability to infiltrate deep into the brain tissue is a major reason for the poor patient prognosis for this type of brain cancer. Behavior of glioblastoma cells, including their motility, and expression of invasion-promoting genes such as matrix metalloprotease-2 (MMP2), are strongly influenced by normal cells found in the brain parenchyma. Cells such as neurons may also be influenced by the tumor, as many glioblastoma patients develop epilepsy. In vitro models of glioblastoma invasiveness are used to supplement animal models in a search for better treatments, and need to combine capability for high-throughput experiments with capturing bidirectional interactions between GBM and brain cells.Methods: In this work, two 3D in vitro models of GBM-cortical interactions were investigated. A matrix-free model was created by co-culturing GBM and cortical spheroids, and a matrix-based model was created by embedding cortical cells and a GBM spheroid in Matrigel.Results: Rapid GBM invasion occurred in the matrix-based model, and was enhanced by the presence of cortical cells. Little invasion occurred in the matrix-free model. In both types of models, presence of GBM cells resulted in a significant increase in paroxysmal neuronal activity.Discussion: Matrix-based model may be better suited for studying GBM invasion in an environment that includes cortical cells, while matrix-free model may be useful in investigation of tumor-associated epilepsy.
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    Alternative Splicing of RAD6B and Not RAD6A is Selectively Increased in Melanoma: Identification and Functional Characterization
    Gajan, Ambikai; Martin, Carly E.; Kim, Seongho; Joshi, Milap; Michelhaugh, Sharon K.; Sloma, Ido; Mittal, Sandeep; Firestine , Steven; Shekhar, Malathy P. V. (MDPI, 2019-11-01)
    Rad6B, a principal component of the translesion synthesis pathway, and activator of canonical Wnt signaling, plays an essential role in cutaneous melanoma development and progression. As Rad6 is encoded by two genes, namely, UBE2A (RAD6A) and UBE2B (RAD6B), in humans, we compared their expressions in melanomas and normal melanocytes. While both genes are weakly expressed in normal melanocytes, Rad6B is more robustly expressed in melanoma lines and patient-derived metastatic melanomas than RAD6A. The characterization of RAD6B transcripts revealed coexpression of various splice variants representing truncated or modified functional versions of wild-type RAD6B in melanomas, but not in normal melanocytes. Notably, two RAD6B isoforms with intact catalytic domains, RAD6BΔexon4 and RAD6Bintron5ins, were identified. We confirmed that RAD6BΔexon4 and RAD6Bintron5ins variants are expressed as 14 and 15 kDa proteins, respectively, with functional in vivo ubiquitin conjugating activity. Whole exome sequence analysis of 30 patient-derived melanomas showed RAD6B variants coexpressed with wild-type RAD6B in all samples analyzed, and RAD6Bintron5ins variants were found in half the cases. These variants constitute the majority of the RAD6B transcriptome in contrast to RAD6A, which was predominantly wild-type. The expression of functional RAD6B variants only in melanomas reveals RAD6B’s molecular heterogeneity and its association with melanoma pathogenesis.
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    Gliosarcoma vs. glioblastoma: a retrospective case series using molecular profiling
    Dardis, Christopher; Donner, David; Sanai, Nader; Xiu, Joanne; Mittal, Sandeep; Michelhaugh, Sharon K.; Pandey, Manjari; Kesari, Santosh; Heimberger, Amy B.; Gatalica, Zoran; Korn, Michael W.; Sumrall, Ashley L.; Phuphanich, Surasak (2021-06-23)
    Abstract Background Gliosarcoma (GS) refers to the presence of mesenchymal differentiation (as seen using light microscopy) in the setting of glioblastoma (GB, an astrocytoma, WHO Grade 4). Although the same approach to treatment is typically adopted for GS and GB, there remains some debate as to whether GS should be considered a discrete pathological entity. Differences between these tumors have not been clearly established at the molecular level. Methods Patients with GS (n=48) or GB (n=1229) underwent molecular profiling (MP) with a pan-cancer panel of tests as part of their clinical care. The methods employed included next-generation sequencing (NGS) of DNA and RNA, copy number variation (CNV) of DNA and immunohistochemistry (IHC). The MP comprised 1153 tests in total, although results for each test were not available for every tumor profiled. We analyzed this data retrospectively in order to determine if our results were in keeping with what is known about the pathogenesis of GS by contrast with GB. We also sought novel associations between the MP and GS vs. GB which might improve our understanding of pathogenesis of GS. Results Potentially meaningful associations (p<0.1, Fisher’s exact test (FET)) were found for 14 of these tests in GS vs. GB. A novel finding was higher levels of proteins mediating immuno-evasion (PD-1, PD-L1) in GS. All of the differences we observed have been associated with epithelial-to-mesenchymal transition (EMT) in other tumor types. Many of the changes we saw in GS are novel in the setting of glial tumors, including copy number amplification in LYL1 and mutations in PTPN11. Conclusions GS shows certain characteristics of EMT, by contrast with GB. Treatments targeting immuno-evasion may be of greater therapeutic value in GS relative to GB.
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    Omeprazole Inhibits Glioblastoma Cell Invasion and Tumor Growth
    Jin, Un-Ho; Michelhaugh, Sharon K.; Polin, Lisa A.; Shrestha, Rupesh; Mittal, Sandeep; Safe, Stephen (MDPI, 2020-07-28)
    Background: The aryl hydrocarbon receptor (AhR) is expressed in gliomas and the highest staining is observed in glioblastomas. A recent study showed that the AhR exhibited tumor suppressor-like activity in established and patient-derived glioblastoma cells and genomic analysis showed that this was due, in part, to suppression of CXCL12, CXCR4 and MMP9. Methods: Selective AhR modulators (SAhRMs) including AhR-active pharmaceuticals were screened for their inhibition of invasion using a spheroid invasion assay in patient-derived AhR-expressing 15-037 glioblastoma cells and in AhR-silenced 15-037 cells. Invasion, migration and cell proliferation were determined using spheroid invasion, Boyden chambers and scratch assay, and XTT metabolic assays for cell growth. Changes in gene and gene product expression were determined by real-time PCR and Western blot assays, respectively. In vivo antitumorigenic activity of omeprazole was determined in SCID mice bearing subcutaneous patient-derived 15-037 cells. Results: Results of a screening assay using patient-derived 15-037 cells (wild-type and AhR knockout) identified the AhR-active proton pump inhibitor omeprazole as an inhibitor of glioblastoma cell invasion and migration only AhR-expressing cells but not in cells where the AhR was downregulated. Omeprazole also enhanced AhR-dependent repression of the pro-invasion CXCL12, CXCR4 and MMP9 genes, and interactions and effectiveness of omeprazole plus temozolomide were response-dependent. Omeprazole (100 mg/kg/injection) inhibited and delayed tumors in SCID mice bearing patient-derived 15-037 cells injected subcutaneously. Conclusion: Our results demonstrate that omeprazole enhances AhR-dependent inhibition of glioblastoma invasion and highlights a potential new avenue for development of a novel therapeutic mechanism-based approach for treating glioblastoma.