Functional characterization of ENPP1 reveals a link between cell cycle progression and stem-like phenotype in glioblastoma

Abstract

A high-throughput phenotypic screen in glioblastoma stem-like cells (GSCs) identified a novel molecular mechanism in which ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) plays an important role in balancing the pool of nucleotides, thus maintaining GSCs in an undifferentiated proliferative state. This finding highlights the connection between cell cycle length and the stem-like tumor state.

Abbreviations

GSCs

glioblastoma stem-like cells

ENPP1

ectonucleotide pyrophosphatase/phosphodiesterase 1

RNAi

interfering RNA

E2F1

E2F transcription factor 1

Glioblastoma is the most common primary brain tumor and among the deadliest human cancers. Because of its location, aggressiveness, and diffuse growth pattern, therapy for glioblastoma is tremendously challenging and only a small improvement in survival has been achieved over the past 30 years. Many patients do not efficiently respond to currently available drugs. This is thought to be mainly due to a sub-population of highly resistant tumor cells, the so-called glioblastoma stem-like cells (GSCs), that can continuously self-renew and regenerate the tumor.^1,2

Since current treatment strategies aim to target the bulk tumor mass they potentially fail to account for the different molecular and clinical properties of this sub-population of GSCs, ultimately causing the therapy to be unsuccessful. This failure might be caused by the different features of bulk tumor cells and GSCs such as variable proliferation rates, differential gene expression including genes that control differentiation, and alterations in programmed cell death. Indeed, major drawbacks of current cancer chemotherapy are the lack of tumor-specific targets and its incapacity to target GSCs. It is therefore critical to discover novel cancer drugs targeting that sub-population of tumor cells in particular.

In the last decade, the advent of high-throughput next-generation sequencing technologies has led to a new era in the study of de novo mutations, helping to uncover disease preconditions and mechanisms. However, although very informative, these analyses do not account for post-translational modifications that lead to oncogene activation or tumor suppressor gene inhibition. The use of a high-throughput phenotypic screen is a means to study the activity of tumor-relevant pathways.^3,4

Using a lentiviral-based RNAi screen, we aimed to identify phosphatases that are implicated in the maintenance of GSCs.³ This screening approach identified many genes that alter the level of the GSC marker CD133 and are as such potential positive regulators of the GSC phenotype. Among these, we highlighted ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), an ectonucleotidase located at the cell surface whose role is to cleave pyrophosphate and phosphodiester bonds from various substrates. Interestingly, ENPP1 is highly expressed in Grade IV glioblastoma and GSCs compared to normal brain and fetal neural stem cells, suggesting an important role in this aggressive brain tumor. In addition to its effect on CD133, knockdown of ENPP1 leads to downregulation of other progenitor/stem cell markers such as CD15, LHX2, and MSI1, as well as increased expression of astrocytic differentiation markers. Moreover, comparison of existing stem cell-associated gene sets with the expression profiling of ENPP1-deficient GSCs revealed a global downregulation of stem cell-associated genes. These data support the important role of ENPP1 for the maintenance of the GSC phenotype. As differentiation of GSCs has previously been shown to increase their response to therapeutic agents, knockdown of ENPP1 increased the apoptotic response of the cells to 1,3-Bis(2-chloroethyl)-1-nitrosourea (BCNU), a cytotoxic agent used in the treatment of malignant gliomas. Additionally, we have shown that knockdown of ENPP1 affects the level of the intracellular nucleotide pool. This deregulation might be attributable to the enzymatic activity of ENPP1 in the extracellular space, resulting in decreased transcriptional function of the cell cycle regulator E2F transcription factor 1 (E2F1) (Fig. 1A). Interestingly, ENPP1 knockdown impaired proliferation and led to the accumulation of cells in G1 phase of the cell cycle. This appears to be a direct consequence of the decreased transcriptional function of E2F1, which is known to positively control S phase entry. Numerous studies have highlighted the correlation between cell cycle progression and cell fate decision in neural stem cells;^5-7 however, our study is the first to suggest the interconnection of a lengthened G1 phase and the induction of differentiation in GSCs. These data are in line with previous findings showing onset of differentiation accompanied by an accumulation of stem-like cells in G1 phase.^8,9 This might have important implications for therapy as the length of the G1 phase of the tumor cells seems to impact tumorigenicity and resistance of these cells to chemotherapeutic agents (Fig. 1B).

Figure 1.

Factors influencing the phenotype of glioblastoma stem-like cells: (A) Model of ENPP1 function in glioblastoma stem-like cells. Acting upstream of the E2F transcription factor 1 (E2F1), ENPP1 is important for the maintenance of a proliferative stem-like phenotype in glioblastoma. A possible implication of a balanced nucleotide pool is indicated by the dashed line. (B) Proposed model for the interconnection of cell cycle state and stem-like phenotype in glioblastoma and its impact on tumorigenicity and response to chemotherapeutics.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.