Glioblastoma, the most frequent primary malignancy of the central nervous system, is almost universally fatal despite aggressive therapies, such as surgical resection, adjuvant radiation and ...chemotherapy, which remain largely palliative. With increasing evidence showing that glioblastoma cancer stem cells play an important role in tumor escape from conventional therapies and disease recurrence, the targeting of cancer stem cells with different therapeutic strategies provides new avenues of research and confidence for better outcomes. We have previously shown that isolating slow-dividing cells from glioblastoma enriches for a population with cancer stem cell properties. Here, we demonstrate that these slow-dividing cancer stem cells are more invasive and more tolerant to chemotherapy than the rest of the tumor population. Surprisingly, slow-proliferating cells are initially more sensitive to radiation damage. We find a significant overlap between the slow-proliferating compartment and expression of the transcription factor ZEB1, which we have recently identified as a master regulator of stemness and chemoresistance in glioblastoma. Consequently, ZEB1-positive cells also exhibit greater radiosensitivity. Slow proliferating, ZEB1-positive cells accumulate genomic aberrations correlated with retention in the G2/M phase of the cell cycle, rendering these cells more sensitive to radiation damage. However, from this specific subpopulation, a fraction of cells that survive irradiation rebound with a proliferative burst that may contribute to recurrence of more aggressive tumors. This distinct effect of radiation on cancer stem cells points to a previously underappreciated heterogeneity within the cancer stem cell compartment and may open up new avenues of studying and targeting specific cancer stem cell sub-populations.
The coordination of complex tumor processes requires cells to rapidly modify their phenotypes using direct cell-cell communication through gap junction channels composed of connexins. Previous ...reports suggest that gap junctions are tumor suppressors based on connexin 43 (Cx43), but this hypothesis fails to consider the differences in connexin-mediated intercellular communication rate and ion selectivity that drive gap junction diversity. Using patient-derived specimens, we screened connexin proteins and found that glioblastoma cancer stem cells (CSCs) expressed Cx46, while Cx43 was predominantly expressed in non-CSCs. Targeting Cx46 compromised CSC proliferation, self-renewal, and tumor initiation. Consistent with the divergent physiological nature of intercellular communication and ion selectivity between Cx46 and Cx43, CSCs with higher Cx46 had an elevated intercellular communication rate and were more depolarized than non-CSCs. The difference in connexin subtype was responsible for these phenotypic differences; Cx46 knockdown reduced the communication rate of CSCs, and Cx43 knockdown increased the depolarization of non-CSCs. The differences between the two connexins were reflected in GBM patient survival: Cx46 expression correlated with poor prognosis, while Cx43 expression was not informative. Ongoing studies are identifying differentially transported signaling molecules that are responsible for CSC maintenance based on connexin subunits. As clinically relevant gap junction inhibitors including 1-Octanol are being tested for other neurological disorders (essential tremor), we evaluated if these inhibitors were effective in glioblastoma. We confirmed that CSCs possessed functional gap junctions and that inhibitors reduced communication. These inhibitors potently inhibited proliferation and self-renewal of CSCs compared with non-CSCs and neural progenitor cells. In established xenograft tumors, gap junction inhibitors suppressed tumor growth and had an additive effect when combined with Temozolomide. Taken together, our data demonstrate a pro-tumorigenic role of gap junctions that is dependent on connexin subunit expression and provide a rationale for gap junction targeting in glioblastoma.
The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. ...Previous reports have suggested that gap junctions are tumor suppressive based on connexin 43 (Cx43), but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs) possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression.
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•Gap junction targeting potently inhibits GBM growth•Gap junctions have a pro-tumorigenic role that depends on connexin expression•CSCs express Cx46, which is required for self-renewal•Connexin expression dictates intercellular communication and membrane potential
Hitomi et al. demonstrate that glioblastoma cancer stem cells possess functional gap junctions that can be targeted to attenuate self-renewal and tumor growth. The authors also identify connexin 46 as a novel glioblastoma cancer stem cell regulator that is required for stem cell maintenance.
Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies(1). To overcome the issue of tumor ...heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia(2-8). The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma(2,7,8). The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds(9). The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division(10). CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity(2). This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry(2). The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.
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