International Journal of Neurooncology

: 2021  |  Volume : 4  |  Issue : 2  |  Page : 80--83

Award Paper Basic Science - Translational Research


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. Award Paper Basic Science - Translational Research.Int J Neurooncol 2021;4:80-83

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. Award Paper Basic Science - Translational Research. Int J Neurooncol [serial online] 2021 [cited 2022 Jun 25 ];4:80-83
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The tumor suppressor MTUS1/ATIP1 modulates tumor promotion in glioma: Association with epigenetics and DNA repair

Nikhil Ranjan, Vimal Pandey, Manas Kumar Panigrahi, Lukas Klumpp, Ulrike Naumann, Phanithi Prakash Babu; University of Hyderabad

Glioblastoma (GBM) is a highly aggressive brain tumor. Resistance mechanisms in GBM present an array of challenges to understand its biology and to develop novel therapeutic strategies. We investigated the role of a TSG, MTUS1/ATIP1 in glioma. Glioma specimen, cells, and low-passage GBM sphere cultures (GSCs) were analyzed for MTUS1/ATIP1 expression at the RNA and protein level. Methylation analyses were done by bisulfite sequencing (BSS). The consequence of chemotherapy and irradiation on ATIP1 expression and the influence of different cellular ATIP1 levels on survival were examined in vitro and in vivo. MTUS1/ATIP1 was downregulated in high-grade glioma (HGG), GSC, and GBM cells, and hypermethylation at the ATIP1 promoter region seems to be at least partially responsible for this downregulation. ATIP1 overexpression significantly reduced glioma progression by mitigating cell motility, proliferation and facilitating cell death. In glioma-bearing mice, elevated MTUS1/ATIP1 expression prolonged their survival. Chemotherapy, as well as irradiation, recovered ATIP1 expression both in vitro and in vivo. Surprisingly, ATIP1 overexpression increased irradiation-induced DNA-damage repair, resulting in radio resistance. Our findings indicate that MTUS1/ATIP1 serves as TSG-regulating gliomagenesis, progression, and therapy resistance. In HGG, higher MTUS1/ATIP1 expression might interfere with tumor irradiation therapy.

MiR-193a, a WNT subgroup-specific microRNA, destabilizes MYC and acts as a tumor suppressor in MYC amplified Group 3 medulloblastoma

Harish Shrikrishna Bharambe, Sadaf Kazi, Neelam Vishwanath Shirsat; Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai, Maharashtra, India

Objective: The WNT subgroup medulloblastomas are characterized by the constitutive activation of the canonical WNT signaling pathway. MYC, a potent oncogene, is a downstream target of WNT signaling and is expressed in both WNT and Group 3 tumors. Although the WNT subgroup has an excellent long-term survival of > 90%, Group 3 has a poor 5-year survival of 50%. WNT subgroup medulloblastomas have a distinctive microRNA expression profile with several microRNAs specifically upregulated in this subgroup. The molecular mechanism underlying WNT subgroup-specific expression of miR-193a and its functional role in medulloblastoma biology was studied in detail.

Materials and Methods: The regulation of miR-193a promoter was studied by the luciferase reporter assay. Genome-wide DNA methylation data were analyzed for the methylation status of miR-193a promoter in medulloblastoma subgroups. The expression of miR-193a was restored in MYC amplified Group 3 medulloblastoma cell lines in a doxycycline-inducible manner and its effect on growth and malignant behavior was studied. The genes differentially expressed upon miR-193a expression were identified by transcriptome profiling and the direct target genes of miR-193a were identified and validated by the luciferase reporter assay. Half-life of MYC protein levels was studied by the pulse-chase experiments.

Results: MiR-193a is silenced in non-WNT medulloblastoms and Group 3 medulloblastoma cell lines as a result of promoter hypermethylation. Restoration of miR-193a expression in MYC overexpressing Group 3 medulloblastoma cell lines inhibited proliferation, anchorage-independence, tumorigenic potential, and increased radiation sensitivity of medulloblastoma cells indicating its tumor-suppressive role in medulloblastoma. Restoration of miR-193a expression resulted in widespread repression of genes involved in multiple cellular pathways. MAX, DCAF7, and STMN1 were identified as novel target genes of miR-193a. Furthermore, miR-193a-mediated downregulation of MAX was found to destabilize the MYC oncoprotein. On the contrary, MYC was found to upregulate miR-193a expression. Thus, miR-193a appears to act as an inhibitory feedback of WNT/MYC signaling.

Conclusion: MYC, a potent oncogene, is deregulated in at least 50% of all cancers and is associated with aggressive cancers with poor survival. In cancer cells having high MYC expression, MYC brings about transcriptional amplification of all active genes. On the contrary, MiR-193a brought about global repression of gene expression resulting in substantial reduction in malignant potential of MYC amplified medulloblastoma cells. Therefore, miR-193a is likely to contribute to excellent survival of WNT subgroup and has therapeutic potential in the treatment of not only Group 3 medulloblastomas but possibly other MYC overexpressing aggressive cancers as well.

MiR-592 activates the mTOR kinase, ERK1/ERK2 kinase signaling, and imparts neuronal differentiation signature characteristic of Group 4 medulloblastoma

Raikamal Paul, Purna Bapat, Akash Deogharkar, Sadaf Kazi, Tejpal Gupta, Epari Sridhar, Aliasgar Moiyadi, Prakash Shetty, Neelam Shirsat; Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai, Maharashtra, India

Objective: Medulloblastoma consists of four core molecular subgroups: WNT, SHH, Group 3, and Group 4. Group 3 and Group 4 tumors have considerable overlap in their gene expression profiles but differ significantly in their survival rates. The molecular mechanism underlying pathogenesis of these two subgroups is not understood well. MiR-592 is primarily expressed in Group 4 tumors. The functional role of miR-592 in the biology of medulloblastoma cells was studied in detail.

Materials and Methods: The expression level of miR-592 was studied in 200 medulloblastomas of the Indian cohort. MiR-592 was exogenously expressed in multiple established Group 3 cell lines to levels similar to that in Group 4 medulloblastomas using an inducible, lentiviral vector. The effect of expression of miR-592 on growth and malignant potential of medulloblastoma cells was studied. Transcriptome sequencing was performed to identify the genes differentially expressed on miR-592 expression and the novel target genes were identified and validated by the luciferase reporter assay. Western blot analysis and the protein–protein interaction network analysis were performed to delineate the molecular mechanism underlying miR-592-mediated pathogenesis of Group 4 medulloblastomas.

Results: MiR-592 expression reduced the malignant potential of Group 3 medulloblastoma cells. DEPTOR, an endogenous inhibitor of the mTOR kinase, was identified as a novel target of miR-592. MiR-592 expression resulted in the upregulation of both mTORC1 and mTORC2 activity but a decrease in the AKT kinase activity. The reduction in the AKT kinase activity due to the inhibitory feedback of mTORC1 signaling is consistent with the decrease in the malignant potential upon miR-592 expression using several assays. MiR-592 expression resulted in the upregulation of several neuronal-differentiation-related genes in Group 3 cells. The protein–protein interaction analysis of the genes upregulated upon miR-592 expression identified ERK1/ERK2 kinases as hub proteins connected to neuronal differentiation-related pathways. The ERK1/ERK2 kinase activity was found to be elevated in multiple medulloblastoma cell lines upon miR-592 expression. ERK1/ERK2 inhibitor but not mTOR inhibitor treatment abrogated miR-592 mediated upregulation of neuronal differentiation-related genes.

Conclusion: The expression of neuronal differentiation-related genes is the characteristic that distinguishes Group 4 medulloblastomas from Group 3 tumors. MiR-592 expression imparting Group 4 characteristic to Group 3 medulloblastoma cells is the first experimental demonstration of Group 3, Group 4 continuum. The study also identified the role of MAPK and mTOR signaling in the pathogenesis of Group 4 medulloblastoma. This study gives an important insight into the pathogenesis of Group 4 medulloblastoma.

Deep learning-based automated classification of brain metastases

Gurukrishna B, Abhishek Mahajan, Sanjay Talbar, Ujjwal Baid, Shwetha Wadhwa, Amit Kumar Janu, Naveen Mummudi, Anil Tibdewal, JP Agarwal, Vanita Noronha, Vijay Patil, Amit Joshi, Subash Yadav, Rajiv Kaushal, Trupti Pai, Ameya Puranik, Kumar Prabhash; Tata Memorial Hospital

Objective: The aim of this study was to investigate the accuracy of deep learning-based convolutional neural network (CNN) architecture to classify brain metastases from non-small-cell lung cancer based on EGFR and ALK mutation groups and to compare classification based on semantic features.

Materials and Methods: Dataset was classified into three groups: namely, EGFR positive, ALK positive, and both negative. CNN EfficientNet architecture was used to study the accuracy of classification using T1, T2, T1post, and FLAIR MRI sequences. Dataset was divided into 80% training and 20% test. Further, statistical analysis was performed on the classified dataset using MR and clinical semantic features assuming 95% confidence interval.

Results: In this study of 117 patients, accuracy of CNN EfficientNet: pre-manual segmentation: 76%; post-segmentation: 89%. Smokers are 79.2% less likely to be ALK-positive comparing both negative groups, p-value 0.03. T2W hypointense lesions 10 times more odds of being ALK positive comparing EGFR positive, p-value 0.025. Hemorrhagic lesions have 6.5 times more odds of being EGFR positive comparing both negative groups, P = 0.025. Peripherally restricting lesions are 95% more odds of being ALK-positive comparing EGFR positive, P = 0.005. Ring enhancing lesions have 10 times more odds of being ALK positive comparing EGFR positive, P =0.015. Meningeal involvement is 6.4 times in being ALK positive comparing both negative groups, P =0.004.

Conclusion: Both semantic features and the deep learning model showed comparable accuracy in classifying EGFR and ALK mutations. Both methods can be clinically used useful to predict mutations while biopsy or genetic testing is awaited.

Expression of immune checkpoint regulators in brain metastases

Nufina TA, Shilpa Rao, Nandeesh BN, Anita Mahadevan, TC Yasha, Vani Santosh; National Institute of Mental Health and Neuro-Sciences (NIMHANS)

Objectives: The aim of this work was to study the expression of immune checkpoint regulators––programmed cell death ligand-1 (PDL-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4)––in brain metastases with known primaries.

Materials and Methods: A retrospective cohort study was conducted on all cases of brain metastases with known primaries, diagnosed at the Department of Neuropathology, NIMHANS between January 2017 and December 2019. Histomorphology and immunohistochemistry for PDL-1 and CTLA-4 were carried out, and their expression was assessed in tumor cells as well as the tumor infiltrating lymphocytes on all the cases, with clinicopathological correlation.

Results: Overall, there were 227 histopathologically diagnosed cases of brain metastases, of which 44 had a known primary. The mean age at diagnosis was 49.1 years (range: 27–64 years) with a female preponderance (3.4:1). The most common site of the primary was breast (43.2%, n = 19) followed by lung and ovary (11.4% each, n = 5). Adenocarcinoma was the predominant histological type (65.9%, n = 29), with the cerebellum being the most common metastatic site (36.4%, n = 16). Adjuvant therapy in the form of chemotherapy and/or radiotherapy was given before the histological diagnosis in 29 (65.9%) patients. Tumor infiltrating lymphocytes were noted in 14 (31.8%) cases. Immunohistochemistry for PDL-1 was positive in 20 (45.5%) cases (tumor cells only [n = 3], tumor-infiltrating lymphocytes only [n = 7], both [n = 10]), while CTLA-4 was positive in 30 (68.2%) cases (tumor cells only [n = 1], tumor-infiltrating lymphocytes only [n = 10], both [n = 19]). Nineteen cases (43.2%) were found to have a combined positivity of PDL-1 and CTLA-4 in the tumor cells and/or tumor infiltrating lymphocytes in the stroma.

Conclusion: Immune checkpoint inhibitors (anti-CLTA-4 antibodies and anti-PD-1/PD-L1 antibodies) potentiate the host’s own anti-tumor immune response and have shown impressive clinical efficacy in brain metastases. Our study shows that a significant proportion of brain metastases express PDL-1 and CTLA-4. Expression of these immune checkpoint regulators would probably identify a subset of patients who could benefit from targeted immuno-oncological therapy and have the potential to be useful prognostic and predictive biomarkers.

Transcription factor expression profile in immunonegative pituitary adenomas

Akhilesh Ravichandran, Honavar AG, Chacko AG, Rajshekhar V, Joseph BV, Rajaratnam S, Thomas N, Asha HS, Mannam PR, Joseph J, Ranjani J, Gowri M, Chacko G; Christian Medical College, Vellore, Tamil Nadu, India

Objectives: The aim of this study was to identify true null cell adenomas among the immunonegative pituitary adenomas using the transcription factors steroidogenic factor-1 (SF-1), T-Pit, and Pit-1, and to ascertain if their clinical behavior is truly aggressive.

Materials and Methods: This study included a prospective and retrospective cohort and was carried out in the Department of General Pathology between 2009 and 2020. Slides of each biopsy including their hematoxylin and eosin sections and hormonal markers were reviewed. The hormonally immunonegative cases were subjected to immunohistochemical staining for SF-1, T-Pit, and Pit-1. The tumor was considered positive for a transcription factor if >10% of tumor cells showed nuclear positivity. Statistical analysis was done with continuous variables and presented with mean (SD)/ median (IQR). Categorical data were expressed as a frequency (%). The risk for invasion, recurrence, and MIB-1 were analyzed with logistic regression analysis and presented as an odds ratio (95% CI).

Results: There were 994 cases of pituitary adenomas of which 92 (9.25%) were hormonally immunonegative. Among the latter, 48 were positive for SF-1, 9 were positive for T-pit, and 3 were positive for Pit-1. Following the application of transcription factors, the proportion of null cell adenomas dropped to 3.21% from the earlier prevalence of 9.25%, prior to the application of transcription factors. Null cell adenomas had a significantly higher prevalence of invasion (84.38%) as well as recurrence (54.84%) when compared with most other subtypes. Among the “high-risk” aggressive adenomas, the sparsely granulated somatotroph adenomas and silent corticotroph adenomas had a significantly higher prevalence of invasion as compared to the other subtypes. Silent corticotroph adenomas had significantly higher recurrence rates as compared to the functional corticotroph adenomas. The prevalence of invasion in lactotroph adenomas was higher in males (90%) than in females (37.50%).

Conclusion: The use of transcription factors better defines subtypes of pituitary adenomas. This is of extreme clinical significance as one is then able to identify patients with “high-risk” adenomas which can be followed up more closely and provided with adjuvant therapy in addition to surgical resection.

Aurora kinase B inhibition abrogate MNGC division by neosis and delay GBM recurrence

Tejashree Mahaddalkar, Anuradha Kumari, Dulal Panda, Shilpee Dutt; Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai, Maharashtra, India

Objectives: We have previously shown that residual cells of GBM are the cause of recurrence in glioblastoma. To understand the mechanism of their survival, we generated in vitro radio resistant cellular model that mimics clinical scenario of GBM resistance. We found residual population to be enriched in multinucleated and giant cells (MNGCs) that were capable of cell division forming aggressive relapse. Therefore, here we aim to understand the molecular mechanism of MNGCs division with the hypothesis that abrogation of MNGC division will prevent recurrence.

Materials and Methods:In vitro cellular model from cell lines and patient samples was used to capture MNGCs. MNGCs were characterized by β-tubulin and beta-galactosidase staining. Stable cell lines expressing H2B-GFP and mcherry α-tubulin were generated to study MNGC division by immunofluorescence and live-cell imaging. RNA sequencing and WGCNA analysis of parent, MNGCs, and relapse cells were performed followed by functional assays and pharmacological inhibition of candidate genes to gain mechanistic insights of MNGC division.

Results: MNGCs are shown to die of mitotic-catastrophe while attempting multipolar mitosis. However, we show by single-cell dilution of MNGCs that they divide and form viable mononucleated daughter cells, at a rate similar to mononucleated cells. Interestingly, beta-tubulin staining showed absence of spindles during MNGC division. This was confirmed by monitoring live-cell imaging of dividing MNGCs labeled with H2B-GFP and mcherry α-tubulin. Furthermore, immunofluorescence showed absence of F-actin-rich contractile ring required for normal cytokinesis. Additionally, live-cell imaging revealed MNGCs undergoing asymmetric cytokinesis, and this division cycle is repeated on exposing the daughter cells to radiation. MNGCs also showed γ-H2AX and β-galactosidase staining. These data confirm unconventional division of MNGCs by a phenomenon called “neosis.” For mechanistic understanding of neosis, RNA sequencing of parent, non-dividing and dividing MNGCs and recurrent cells was performed and analyzed by WGCNA. Aurora kinase B was identified as a candidate gene significantly upregulated from nondividing to dividing MNGCs. Immunofluorescence and western blots of nuclear and cytoplasmic fraction of MNGCs confirmed increased nuclear localization of Aurora kinase B during MNGC division. Indeed, pharmacological inhibition of Aurora kinase B lead to abrogation of MNGC neosis and significantly delayed GBM recurrence.

Conclusions: In this study, we identified that MNGCs do not divide by conventional mitosis but by “Neosis.” Further, we found Aurora kinase B as a novel regulator of “Neotic” cell division, inhibition of which abrogated residual multinucleated giant cell division and prevented recurrence in GBM.

ER stress response and GCN5 are novel molecular targets for GBM senotherapy

Madhura Ketkar, Shilpee Dutt; Shilpee Dutt Laboratory, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Navi Mumbai, India, Homi Bhabha National Institute, Training School Complex, Mumbai, Maharashtra, India

Objectives: Therapy resistance and relapse is a clinical challenge in glioblastoma (GBM). We previously reported that residual GBM cells survive radiotherapy. We also showed that radiation therapy induces senescence in the residual cells. Senescence is an irreversible cell cycle arrest, and thus is a favorable cancer therapeutic outcome. Unfortunately, we showed that GBM residual cells escape senescence and cause relapse. Therefore, here we aim to understand the mechanisms of senescence escape in residual cells and induction of permanent senescence in GBM that can be exploited for senotherapy.

Materials and Methods: Radiation-resistant residual (RR) cells were captured from primary patient samples and cell lines of GBM. RNA sequencing of 40 samples post-irradiation at different time points was done and the transcriptome data were analyzed to identify molecular pathways of senescence escape. Real-time qPCR, western blot analysis, beta-galactosidase assays, and genetic perturbation of gene expression were performed for mechanistic studies.

Results: Lethal dose of radiation to GBM cell lines and primary patient samples induced cell death in more than 90% of cells. But unfortunately, 10% residual cells survived and showed hallmarks of cellular senescence with high beta-galactosidase activity, high p21, and p16 transcripts and SASP (senescence-associated secretory phenotype) production. Molecular features of senescence were reversed during the escape from senescence. Transcriptome sequencing and DGE analysis of dynamic senescent stages after irradiation obtained 742, 907, 338, and 3868 genes differential in U87MG, SF268, and patient samples, respectively. GO Biological function and Pathway enrichment analysis of the significantly downregulated genes during reversal of senescence (n=1082) using Reactome and Panther database indicated enrichment of cytokine signaling and endoplasmic reticulum stress-related pathways. ER stress response pathway components ATF4, CHOP, Grp78, GADD34, sXBP1alpha, PDI, and ERO1alpha showed reduced expression accompanying reversal of senescence on mRNA and protein levels. Similarly, expression and secretion of SASP factors, CXCL8, IL6, STC1, TIMP1, and MMP2 exhibited a direct correlation with senescence induction and reversal post-irradiation at transcript and protein levels. Importantly, GCN5 a histone acetyltransferase knockdown induced an irreversible senescence in GBM with p16high, p21high, SASPlow, and Beta galhigh phenotype. Conditioned media from radiation-induced senescent cells but not from GCN5 knockdown induced senescent cells could stimulate paracrine senescence as well as ER stress response in parent GBM.

Conclusion: Taken together, we have identified GCN5 as a novel target for senotherapy in GBM. Furthermore, we also identified ER stress pathway as a potential molecular switch for reversal of radiation-induced senescence in GBM.