- Top of page
- Materials and Methods
- Disclosure Statement
Tumor-initiating stem cells (also referred to as cancer stem cells, CSCs) are a subpopulation of cancer cells that play unique roles in tumor propagation, therapeutic resistance and tumor recurrence. It is increasingly important to understand how molecular signaling regulates the self-renewal and differentiation of CSCs. Basic helix-loop-helix (bHLH) transcription factors are critical for the differentiation of normal stem cells, yet their roles in neoplastic stem cells are not well understood. In glioblastoma neurosphere cultures that contain cancer stem cells (GBM-CSCs), the bHLH family member inhibitors of DNA binding protein 2 and 4 (Id2 and Id4) were found to be upregulated during the differentiation of GBM-CSCs in response to histone deacetylase inhibitors. In this study, we examined the functions of Id2 and Id4 in GBM neurosphere cells and identified Id proteins as efficient differentiation regulators of GBM-CSCs. Overexpression of Id2 and Id4 promoted the lineage-specific differentiation of GBM neurosphere cells as evidenced by the induction of neuronal/astroglial differentiation markers Tuj1 and GFAP and the inhibition of the oligodendroglial marker GalC. Id protein overexpression also reduced both stem cell marker expression and neurosphere formation potential, a biological marker of cancer cell “stemness.” We further showed that Id2 and Id4 regulated GBM neurosphere differentiation through downregulating of another bHLH family member, the oligodendroglial lineage-associated transcription factors (Olig) 1 and 2. Our results provide evidence for distinct functions of Id proteins in neoplastic stem cells, which supports Id proteins and their downstream targets as potential candidates for differentiation therapy in CSCs. (Cancer Sci 2012; 103: 1028–1037)
Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults, with a 2-year survival rate of <30% following surgical resection, chemotherapy and radiotherapy. Recurrent GBM growth is nearly certain after initial treatment and there is no therapy proven to prolong survival after tumor recurrence. The dismal prognosis associated with GBM has motivated intensive investigation into alternative therapeutic paradigms, such as differentiation therapy.
Recent findings support the theory that cancer stem cells (CSCs) play a fundamental role in therapeutic resistance and cancer recurrence. CSCs represent a small subset of neoplastic cells within clinical and experimental tumors that possess stem-like properties, including self-renewal, multipotency and the capacity to efficiently initiate tumors when implanted in the appropriate host.[2-4] Stem-like cancer cells have been isolated from a variety of malignancies, including breast and prostate cancer, leukemia and glioblastoma.[5-8] GBM-CSCs are typically propagated in vitro based on their ability to grow as neurospheres when cultured in serum-free medium supplemented with epidermal growth factor and fibroblast growth factor.[7, 9] GBM-CSCs also express certain stem cell-associated markers, including CD133, aldehyde dehydrogenase (ALDH) and specific ABC transporters, such as ABCG2. Given the increasing evidence that GBM-CSCs are major culprits in GBM therapeutic resistance and recurrence, there is considerable interest in understanding the cellular and molecular determinants of the stem cell phenotype and developing cytotoxic and differentiation strategies that efficiently target the GBM-CSC pool. Differentiation therapies in oncology are broadly defined as those that induce malignant reversion, which is likely to be reevaluated on the basis of the emerging concept of neoplastic stem cell.[13, 14] Various approaches have been tested to differentiate GBM-CSCs to reduce their tumor-initiation potential. These include using bone morphogenic proteins (BMP), histone deacetylase inhibitors, retinoic acid and Krüppel-like factor 9.
Cellular differentiation programs are tightly controlled through the coordinated regulation of gene expression by proteins called basic helix-loop-helix (bHLH) transcription factors, which regulate the differentiation programs of multiple cell lineages. Of particular interest are the inhibitors of DNA binding proteins (Id), which belong to the bHLH superfamily. To date, four members of the Id protein family have been described in mammals.[20-22] Among them, Id1, 2 and 3 are expressed ubiquitously, whereas Id4 is expressed predominantly in testis, brain and kidney. All the Id protein family members lack the domain necessary for DNA binding and, hence, act as dominant negative regulators by forming heterodimers with other DNA-binding proteins, such as oligodendroglial lineage-associated transcription factors (Olig).[20, 23, 24] Olig1 and Olig2 are specifically expressed in regions of the central nervous system enriched for oligodendrocytes and oligodendrocyte progenitors.[25-27] Several lines of evidence link Olig to neural stem cell growth and oligodendroglial lineage-dependent differentiation.[26, 27] Olig1 and Olig2 are expressed by oligodendrogliomas and by subsets of cells, including CD133+ stem-like cells found in malignant astrocytomas.[25, 28] Through these interactions, Id proteins play crucial roles in regulating cell proliferation, survival, lineage-dependent differentiation, and cell–cell interaction.[29-32] In addition, inappropriate regulation of Id proteins in differentiated cells can contribute to tumorigenesis, including invasion and angiogenesis.[20, 22, 29]
Evidence points to a fundamental role of bHLH proteins during GBM-CSC differentiation. In our previous work, we found that Id2 and Id4 proteins were significantly upregulated during the differentiation of GBM-CSCs by histone deacetylase inhibitors. We further identified that Olig1 and Olig2 were significantly downregulated in GBM-CSCs in response to retinoic acid-induced differentiation. In the present study, to better understand the functions of these bHLH proteins, we examine the effects of Id2 and Id4 gain-of-function in GBM neurosphere cell growth and differentiation. We show that overexpression of Id2 and Id4 in GBM neurosphere cells inhibits oligodendroglial differentiation, but promotes neuronal/astroglial differentiation. The differentiation effect of Id proteins decreases stem cell marker expression and depletes the CSC pool. The biological effects of Id protein expression are found to be mediated by Olig1 and Olig2.
- Top of page
- Materials and Methods
- Disclosure Statement
In our previous work, we identified changes in the expression of Id2 and Id4 during transition of GBM stem-like cells (GBM-CSCs) from conditions of growth and self-renewal to conditions that induce their differentiation. These results suggested that Id2 and Id4 play a role in differentiation regulation of GBM-CSCs. In this work, we studied gain-of-function of Id2 and Id4 in various GBM neurosphere cultures. We found that overexpression of Id2 and Id4 inhibit GBM neurosphere cell differentiation along oligodendroglial lineage while promoting neuronal/astroglial differentiation. Differentiation of GBM neurosphere cells leads to the depletion of the CSC pool, evident through the decrease in expression of stem cell markers and neurosphere formation ability. The effect of Id2 and Id4 is likely mediated by downregulation of Olig1 and Olig2, another family member of the bHLH transcription factor.
Glioblastoma multiforme neurospheres, enriched for tumor-initiating cells, possess stem-like characteristics sufficient to warrant comparisons with non-neoplastic neural stem cells (NSCs). Recent work from Hide et al. reveals that GBM-CSCs can arise from tissue-specific stem cells (NSCs) or committed precursor cells (oligodendrocyte precursor cells).[41, 42] Not surprisingly, GBM-CSCs and NSCs share common pathways (i.e. Notch, Hedgehog and Wnt) that control their biological functions.[43, 44] bHLH transcription factor family members play a critical role during differentiation of NSCs.[19-22, 25-27] In addition, cells of the oligodendrocyte lineage express Id proteins and the bHLH factors Olig1 and Olig2.[25-27] In Olig1 and Olig2 double-mutant mice, there is a complete failure of oligodendrocyte development in all areas of the brain, along with an apparent increase in astrocytogenesis in the spinal cord.[45, 46] This indicates that Olig1 and Olig2 expression is essential for oligodendrogliogenesis and suggests that repression of oligodendrocyte development might be sufficient to cause astrogliogenesis. Expression of Id4 in oligodendrocyte precursor cells progressively decreases as the precursor cells differentiate in vivo and in vitro. Overexpression of Id2 inhibits oligodendrocyte differentiation, where its absence induces premature oligodendrocyte differentiation in vitro. In our work, we found that overexpressing Id proteins or downregulating Olig1 and Olig2 in GBM neurosphere cells negatively regulates oligodendrocyte differentiation and induces neuronal/astroglial differentiation. These findings mirror the function of Id proteins, Olig1 and Olig2 transcription factors in NSCs and oligodendrocyte precursor cells.
In our work, we found that overexpressing Id2 and Id4 decrease GalC expression in GBM neurosphere cells. GalC is the first oligodendrocyte-specific marker to be expressed by differentiating oligodendrocyte precursor cells and marks immature oligodendrocytes. GBM neurosphere cultures are heterogeneous, consisting of tumor-initiating stem cells, transit amplifying progenitors and differentiated cells. The expression of GalC in control HSR-GBM1A and HSR-GBM1B cells might indicate an oligodendrocyte lineage-related origin of these CSCs. Consistent with these results are the findings of Liu et al. that identify oligodendrocyte progenitors as the cell origin of gliomas. Our results further support these findings.
Inhibitor of DNA binding proteins are regulated by a plethora of growth factors and mitogens, one being BMP. BMP has been proposed to be a potent differentiation reagent in normal and neoplastic stem cells where Id proteins and Olig transcription factors act as its downstream effectors.[15, 24, 49, 50] It has been reported that BMP, specifically BMP4, elicits the strongest effect to induce neuronal/astroglial differentiation of GBM-CSCs, followed by a significant reduction in the stem cell marker expression and tumor-initiating properties.[15, 50] Samanta and Kessler further demonstrate that in neural progenitor cells treated with BMP4, interactions between Id proteins and Olig transcription factors inhibit oligodendrocyte differentiation while enhancing astrogliogenesis. All these findings point to a role of Id and Olig proteins as differentiators of GBM-CSCs in response to BMP. In our work, we found that Id protein overexpression induced the downregulation of Olig1 and Olig2 at the protein level. We further revealed that Olig downregulation by Id proteins probably occurs at the transcriptional level because Id proteins reduce levels of Olig1 and Olig2 mRNA. Olig downregulation might not result directly from Id protein expression because Id proteins do not function directly as transcriptional repressors. As dominant negative regulators of gene expression, it is likely that Id proteins downregulate Olig expression by interacting with other transcription factors and driving cell differentiation along neuronal and astroglial lineages while away from the oligodendroglial lineage. Further studies of the interactions between the Id–Olig axis in GBM neurosphere cells will shed light on deep mechanisms of CSC differentiation.
Inhibitor of DNA binding proteins proteins generally act as positive regulators of cell proliferation. In GBM-CSCs, we found that Id2 or Id4 overexpression did not change cell cycle regulation and affect cell proliferation. This might be explained by the fact that the neurosphere cultures are heterogeneous, including tumor-initiating CSCs, transiently amplifying progenitors and differentiated cells.[16-18, 43] Id proteins might elicit different mitogenic actions in different cell populations. This is substantiated by the fact that the exact function of Id proteins in cell growth, differentiation and cell death largely depends upon cellular context. For example, both pro-apoptotic and anti-apoptotic actions of Id proteins have been reported. In addition, Id proteins have been viewed as negative regulators of cellular differentiation, yet Id proteins can act as positive regulators of differentiation depending on cell lineage and developmental stage. Id1 has been found to prevent premature differentiation of stem cells and to induce cell proliferation in hematopoietic stem cells. High expression of Id2 protein is found in terminally differentiated, nonproliferating cells, suggesting a role of Id2 in promoting differentiation and maintenance of differentiated cells. In our GBM neurosphere models, Id proteins function as negative regulators of oligodendrocyte differentiation, but positive regulators of neuronal/astroglial differentiation, further supporting the versatility of Id proteins in cellular differentiation. Detailed analysis of the mitogenic function of Id proteins in different populations of GBM neurosphere cells will help us to better understand the role of Id proteins in cancer biology. In summary, our results provide evidence of a distinct function of Id proteins in neoplastic stem cells: to promote neuronal/glial differentiation by suppressing oligodendrocyte differentiation. Our findings expand on the role of bHLH family members in stem cell and cancer biology, supporting Id proteins and their downstream targets as potential determinants of GBM cell/tumor subtypes and as potential candidates for differentiation therapy in CSCs.
In summary, our results provide evidence of a distinct function of Id proteins in neoplastic stem cells: to promote neuronal/glial differentiation by suppressing oligodendrocyte differentiation. Our findings expand the role of bHLH family members in stem cell and cancer biology, supporting Id proteins and their downstream targets as potential candidates for differentiation therapy in CSCs. The aim of differentiation therapy is to force the neoplastic stem cell to resume the process of maturation and move to a stage of differentiation at which it no longer divides, and senesces or undergoes apoptosis.[14, 55] Although differentiation therapy does not directly destroy the neoplastic stem cells, it restrains their growth and prevents them from generating more proliferating or transit amplifying cancer cells, thereby improving the long-term efficacy of conventional therapies such as radiation and chemotherapy. In the future, we will test the function of Id proteins in vivo and investigate the effect of combining Id protein overexpression with anti-proliferative drugs which might synergize to elicit a strong anti-tumor effect.