Subhas Mukherjee

Drosophila Brat and Human Ortholog TRIM3 Maintain Stem Cell Equilibrium and Suppress Brain Tumorigenesis by Attenuating Notch Nuclear Transport

Cancer stem cells exert enormous influence on neoplastic behavior, in part by governing asymmetric cell division and the balance between self-renewal and multipotent differentiation. Growth is favored by deregulated stem cell division, which enhances the self-renewing population and diminishes the differentiation program. Mutation of a single gene in Drosophila, Brain Tumor (Brat), leads to disrupted asymmetric cell division resulting in dramatic neoplastic proliferation of neuroblasts and massive larval brain overgrowth. To uncover the mechanisms relevant to deregulated cell division in human glioma stem cells, we first developed a novel adult Drosophila brain tumor model using brat-RNAi driven by the neuroblast-specific promoter inscuteable Suppressing Brat in this population led to the accumulation of actively proliferating neuroblasts and a lethal brain tumor phenotype. brat-RNAi caused upregulation of Notch signaling, a node critical for self-renewal, by increasing protein expression and enhancing nuclear transport of Notch intracellular domain (NICD). In human glioblastoma, we demonstrated that the human ortholog of Drosophila Brat, tripartite motif-containing protein 3 (TRIM3), similarly suppressed NOTCH1 signaling and markedly attenuated the stem cell component. We also found that TRIM3 suppressed nuclear transport of active NOTCH1 (NICD) in glioblastoma and demonstrated that these effects are mediated by direct binding of TRIM3 to the Importin complex. Together, our results support a novel role for Brat/TRIM3 in maintaining stem cell equilibrium and suppressing tumor growth by regulating NICD nuclear transport. Cancer Res; 76(8); 2443-52. ©2016 AACR.

Cancer stem cell division: when the rules of asymmetry are broken.

Asymmetric division of stem cells is a highly conserved and tightly regulated process by which a single stem cell produces two daughter cells and simultaneously directs the differential fate of both: one retains its stem cell identity while the other becomes specialized and loses stem cell properties. Coordinating these events requires control over numerous intra- and extracellular biological processes and signaling networks. In the initial stages, critical events include the compartmentalization of fate determining proteins within the mother cell and their subsequent passage to the appropriate daughter cell. Disturbance of these events results in an altered dynamic of self-renewing and differentiation within the cell population, which is highly relevant to the growth and progression of cancer. Other critical events include proper asymmetric spindle assembly, extrinsic regulation through micro-environmental cues, and noncanonical signaling networks that impact cell division and fate determination. In this review, we discuss mechanisms that maintain the delicate balance of asymmetric cell division in normal tissues and describe the current understanding how some of these mechanisms are deregulated in cancer. The universe is asymmetric and I am persuaded that life, as it is known to us, is a direct result of the asymmetry of the universe or of its indirect consequences. The universe is asymmetric. -Louis Pasteur.

TRIM8 regulates stemness in glioblastoma through PIAS3-STAT3

Glioblastoma (GBM) is the most malignant form of primary brain tumor, and GBM stem‐like cells (GSCs) contribute to the rapid growth, therapeutic resistance, and clinical recurrence of these fatal tumors. STAT3 signaling supports the maintenance and proliferation of GSCs, yet regulatory mechanisms are not completely understood. Here, we report that tri‐partite motif‐containing protein 8 (TRIM8) activates STAT3 signaling to maintain stemness and self‐renewing capabilities of GSCs. TRIM8 (also known as ‘glioblastoma‐expressed ring finger protein’) is expressed equally in GBM and normal brain tissues, despite its hemizygous deletion in the large majority of GBMs, and its expression is highly correlated with stem cell markers. Experimental knockdown of TRIM8 reduced GSC self‐renewal and expression of SOX2, NESTIN, and p‐STAT3, and promoted glial differentiation. Overexpression of TRIM8 led to higher expression of p‐STAT3, c‐MYC, SOX2, NESTIN, and CD133, and enhanced GSC self‐renewal. We found that TRIM8 activates STAT3 by suppressing the expression of PIAS3, an inhibitor of STAT3, most likely through E3‐mediated ubiquitination and proteasomal degradation. Interestingly, we also found that STAT3 activation upregulates TRIM8, providing a mechanism for normalized TRIM8 expression in the setting of hemizygous gene deletion. These data demonstrate that bidirectional TRIM8‐STAT3 signaling regulates stemness in GSC.

Molecular Programs Underlying Asymmetric Stem Cell Division and Their Disruption in Malignancy

Asymmetric division of stem cells is a highly conserved and tightly regulated process by which a single stem cell produces two unequal daughter cells. One retains its stem cell identity while the other becomes specialized through a differentiation program and loses stem cell properties. Coordinating these events requires control over numerous intra- and extracellular biological processes and signaling networks. In the initial stages, critical events include the compartmentalization of fate determining proteins within the mother cell and their subsequent passage to the appropriate daughter cell in order to direct their destiny. Disturbance of these events results in an altered dynamic of self-renewing and differentiation within the cell population, which is highly relevant to the growth and progression of cancer. Other critical events include proper asymmetric spindle assembly, extrinsic regulation through micro-environmental cues, and non-canonical signaling networks that impact cell division and fate determination. In this review, we discuss mechanisms that maintain the delicate balance of asymmetric cell division in normal tissues and describe the current understanding how some of these mechanisms are deregulated in cancer.

CDK5 Inhibition Resolves PKA/cAMP-Independent Activation of CREB1 Signaling in Glioma Stem Cells

SUMMARY Cancer stem cells promote neoplastic growth, in part by deregulating asymmetric cell division and enhancing self-renewal. To uncover mechanisms and potential therapeutic targets in glioma stem cell (GSC) self-renewal, we performed a genetic suppressor screen for kinases to reverse the tumor phenotype of our Drosophila brain tumor model and identified dCdk5 as a critical regulator. CDK5, the human ortholog of dCdk5 (79% identity), is aberrantly activated in GBMs and tightly aligned with both chromosome 7 gains and stem cell markers affecting tumor-propagation. Our investigation revealed that pharmaceutical inhibition of CDK5 prevents GSC self-renewal in vitro and in xenografted tumors, at least partially by suppressing CREB1 activation independently of PKA/cAMP. Finally, our TCGA GBM data analysis revealed that CDK5, stem cell, and asymmetric cell division markers segregate within non-mesenchymal patient clusters, which may indicate preferential dependence on CDK5 signaling and sensitivity to its inhibition in this group.

Abstract 5217: Microenvironmental influences on glioma stem cell migration

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Glioblastoma (GBM) is the most aggressive of the primary brain tumors and carries a poor prognosis due to its highly infiltrative nature and tendency to progress. Glioma stem cells are a small tumor-initiating population in GBM whose properties are incompletely understood. Autocrine and paracrine factors associated with specific microenvironmental niches, such as the perivascular space and hypoxic regions, are thought to attract and support this stem cell component. Precise mechanisms guiding the homing to these niches have not been defined. Glioma stem cells express the receptor CXCR4, which initiates cell migration in the presence of the chemokine, stromal cell-derived factor-1α (SDF-1α). Both hypoxic and vascular regions in the brain express SDF-1α. Therefore, we hypothesize that glioma stem cells transplanted into the brain are driven by SDF-1α chemoattraction towards vascular and hypoxic niches. Cells were sorted for the stem cell marker CD133 using MACS for two explanted human GBM cell lines grown as neurospheres (N08-74 and N08-30). Sorted cells were compared to unsorted cells by Western blot for expression of CD133, hypoxia inducible factor-1α (HIF-1α), CXCR4 and stem cell markers under normoxia and hypoxia. Two million CD133-sorted cells suspended in PBS were delivered into the nasal mucosa of NOD/SCID mice to test cellular migration into the brain via intranasal transplantation. Coronal sections of brain tissue were analyzed at 12 hours after intranasal cell delivery. In both GBM cell lines (N08-30 and N08-74), we confirmed that sorted cells expressed more CD133 than unsorted cells and that both cell lines expressed CXCR4. Further, CXCR4 expression was significantly greater in the CD133-sorted N08-30 cells with a trend in an increase of c-Myc expression compared to unsorted cells. Hypoxic exposure of N08-74 CD133-sorted cells showed a trend of increased HIF-1α and c-Myc expression. In mice, we demonstrated that CD133-sorted cells can migrate into the brain when delivered intranasally. To show the potential for migration towards the vasculature, we show that SDF-1α is expressed in the brain vasculature in vivo. These data demonstrate that GBM cells can be sorted for CD133-positive glioma stem cells, respond to hypoxia, and express the SDF-1α receptor, CXCR4. In one cell line, CXCR4 expression increased after CD133 cell sorting. With intranasal delivery, CD133-sorted cells can migrate into the brain. Understanding the migration behavior of glioma stem cells relative to the diverse microenvironments has implications in treatment for targeting certain migratory populations or microenvironments. These data prepare the conditions to test the migratory potential of glioma stem cells in hypoxic and vascular environments in the brain. Citation Format: Monica J. Chau, Myles R. McCrary, Subhas Mukherjee, Daniel J. Brat. Microenvironmental influences on glioma stem cell migration. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5217. doi:10.1158/1538-7445.AM2015-5217

Abstract LB-50: TRIM3, the human homolog of Drosophila Brat, is a tumor suppressor that regulates asymmetric cell division (ACD) in glioblastoma stem cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Cancer stem cells, capable of self-renewal and multipotent differentiation, influence tumor behavior through a complex balance of symmetric and asymmetric cell divisions. Mechanisms regulating the dynamics of stem cell and their progeny in human cancer are poorly understood. In Drosophila, mutation of brain tumor (brat) leads to loss of normal asymmetric cell division by developing neural cells and results in a massively enlarged brain composed of neuroblasts with neoplastic properties. Brat promotes asymmetric cell division and directs neural differentiation at least partially through its suppression on Myc. We identified TRIM3 (11p15.5) as a human ortholog of Drosophila brat and demonstrate its regulation of asymmetric cell division and stem cell properties of glioblastoma (GBM), a highly malignant human brain tumor. TRIM3 gene expression is markedly reduced in human GBM samples, neurosphere cultures and cell lines and its reconstitution impairs growth properties in vitro and in vivo. TRIM3 expression attenuates stem-like qualities of primary GBM cultures, including neurosphere formation and the expression of stem cell markers CD133, Nestin and Nanog. In GBM stem cells, TRIM3 expression leads to a greater percentage dividing asymmetrically rather than symmetrically. As with Brat in Drosophila, TRIM3 suppresses c-Myc expression and activity in human glioma cell lines. We also demonstrate a strong regulation of Musashi-Notch signaling by TRIM3 in GBM neurospheres and neural stem cells that may better explain its effect on stem cell dynamics. We conclude that TRIM3 acts as a tumor suppressor in GBM by restoring asymmetric cell division. Citation Format: Subhas Mukherjee, Gang Chen, Monika Anand, Jun Kong, Carol Tucker-Burden, Yuan Rong, Fahmia Rahman, Carlos Moreno, Erwin Van Meir, Constantinos Hadjipanayis, Daniel Brat. TRIM3, the human homolog of Drosophila Brat, is a tumor suppressor that regulates asymmetric cell division (ACD) in glioblastoma stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-50. doi:10.1158/1538-7445.AM2014-LB-50