ONECUT2 regulates proliferation and apoptosis in glioblastoma cell lines

1 Introduction

Glioblastoma multiforme (GBM), or grade IV astrocytoma, is the most aggressive form of nervous system tumor. GBM is characterized by cytological atypia, anaplasia, mitotic division, microvascular proliferation and necrosis, with a generally poor prognosis (Kabel et al., 2018, Wu et al., 2021, Alswailem et al., 2022). GBM accounts for more than 50 % of all diagnosed malignant brain cancers and approximately 20 % of all primary intracranial tumors (Van Meir et al., 2010, Alrfaei et al., 2020). In the United States, the prevalence of GBM increases with age, with the highest rates occurring in individuals aged 75 to 84 years (Wu et al., 2021). Annually, approximately 15,000 new cases of GBM and central nervous system (CNS) malignancies are diagnosed (Alrfaei et al., 2020). Despite the highest level of care, the average patient survival time is less than two years (Kruchko et al., 2018, Alrfaei et al., 2020). In Saudi Arabia, there is an increased incidence of cancer, according to Saudi Cancer Registry (SCR) estimates, which reported 3 % of total cancer patients are brain cancer cases (Al-Rawaji et al., 2018, Moin et al., 2021).

Transcription factors belonging to the ONECUT (OCs) family play crucial roles in CNS development and have been extensively studied across various species, including zebrafish, sea urchins, ascidians, Drosophila species, frogs, and mice (Francius and Clotman, 2010, Espana and Clotman, 2012). These factors are involved in the regulation of neuronal differentiation in some of these organisms (Espana and Clotman, 2012). ONECUT2 (OC-2) is the second member of the OCs families (Lu et al., 2018, Yu et al., 2020), and it was initially identified by Jacquemin et al. in 1999 (Jacquemin et al., 1999, Choi et al., 2022). The OC-2 coding gene is located on chromosome 18 in humans and consists of two exons and one intron. The OC-2 protein binds to specific DNA sequences to stimulate the expression of target genes. It plays a regulatory role in cell proliferation, migration, and differentiation (Choi et al., 2022). In humans, OC-2 is predominantly expressed in the liver, gallbladder, duodenum, and small intestines, with lower expression levels in the brain, testes, stomach, and pancreas (Jacquemin et al., 1999, Yu et al., 2020). OC-2 has also been implicated in angiogenesis and epithelial-mesenchymal transition (EMT) (Sun et al., 2014, Lu et al., 2018, Zhang et al., 2018). Furthermore, OC-2 is involved in the regulation of pancreatic cell identity, retinal horizontal cells, and liver cell differentiation (Vanhorenbeeck et al., 2007, Francius and Clotman, 2010, Wu et al., 2012, Ma et al., 2019). (Sun et al., 2014, Zhang et al., 2018, Yu et al., 2020). Onecut2 is crucial for the proper neuronal formation of neurons. It has a key role in the formation of particular motor neurons and interneurons (Hodge et al., 2007, Thiebes et al., 2015, Kabayiza et al., 2017). Overexpression of OC-2 activates oncogenic pathway genes in neurons, influencing development, angiogenesis, extracellular matrix organization, as well as cell locomotion, migration, and proliferation. Conversely, downregulation of OC-2 primarily impacts genes associated with the inflammatory response and chemotaxis (Ma et al., 2019).

OC-2 is a significant player in cancer, as it is involved in various processes such as tumor cell proliferation, cancer cell manifestation, metastasis, tumorigenesis, angiogenesis, EMT, and potentially tumor stemness. There have been specific studies on OC-2′s involvement in bladder cancer, where it showed high specificity and sensitivity in an epigenetic analysis (Lu et al., 2018). Additionally, OC-2 plays critical roles in neuroendocrine prostate cancer (NEPC), metastatic castration-resistant prostate cancer, and ovarian cell adenocarcinoma (Vanhorenbeeck et al., 2007, Wu et al., 2012, Lu et al., 2018, Ma et al., 2019, Yu et al., 2020). In NEPC, OC-2 interacts with hypoxic conditions to inhibit androgen signaling and induce neuroendocrine plasticity. It also activates the SMAD3 gene, resulting in NEPCs exhibiting higher levels of hypoxia compared to prostate adenocarcinomas (Guo et al., 2019).

However, the specific role of OC-2 in the progression of glioblastoma (GBM) remains unclear. The OC-2 has not been studied in brain cancer even though it involves in many neural activities including malignancies. Therefore, the objective of this study was to explore the pathological relationship between OC-2 and the development of brain tumors, specifically GBM. To accomplish this objective, we conducted experiments to investigate whether OC-2 is implicated in the apoptosis and/or proliferation of human glioblastoma U251 and U87 cells.

2 Results

2.1

2.1 Efficiency of OC-2 transduction

U251 and U87 cells, which are derived from aggressive glioblastoma tumors, were modified by introducing OC-2 overexpression through the use of lentiviruses, as shown in Fig. 1. To estimate how effectively the cells were modified, the level of green fluorescent protein (GFP) expression in infected cells was assessed, and it was found to be approximately 99 %. The cells were viewed using an EVOS XL Core Imaging system. The selection was based on GFP positive cells.

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Fig. 1

Estimation of Transduction efficiency by using GFP expression as indicator of successful transduction. A- Shows U251 transfected cells (Green). B- Shows U87 transfected cells (Green). The bottom images represent light microscope showing the same areas of the Fluorescent micrograph (A) on top (×10 magnification).

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2.2

2.2 Validation of OC-2 down-regulation in GBM cells

The results, depicted in both Fig. 2A and Fig. 2B, revealed a significant decrease in OC-2 expression in both the U251 cells (with a remarkable 98.93 % decrease; P < 0.0001) and the U87 cells (with a 17.17 % decrease; P = 0.0207) when compared to the control group consisting of wild-type cells. These findings provide strong evidence for the successful manipulation of OC-2 expression levels in the U251 and U87 cells, validating the researchers' efforts in constructing cells with reduced OC-2 expression.

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Fig. 2

Confirmed inhibition of OC-2. A and B- show qRT-PCR validation of the OC-2 inhibition in comparison to the expression in the mock transfected (WT) cells. Knockdown of OC-2 was shown as 98.93% inhibition for U251 and 17.17% for U87.

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2.4

2.4 Proliferation assay

The downregulation of OC-2 caused a significant decrease in cell proliferation, with a 65.5 % reduction in U251 cells (P ≤ 0.0001) and a 71.7 % reduction in U87 cells (P ≤ 0.0001) when compared to wild-type (WT) cells, as illustrated in Fig. 4 (A and A1). Conversely, upregulating OC-2 resulted in a substantial increase in cell proliferation, with a 183.2 % increase in U251 cells (P ≤ 0.0001) and a 123.5 % increase in U87 cells (P ≤ 0.0001) when compared to WT cells (Fig 4B and 4B1).

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Fig. 3

Metabolic activities of OC-2 modified cells. A- and B- show OC-2 inhibitor reduction of MTT (activities) within U251 (50%) and U87 (40%) cells compared to the controls. C- and D- show OC-2 overexpression increases MTT (activity) within U251 (21%) and U87 (40%) compared to the controls. The overexpression of OC-2 in U251 and U87 was done at background of 200ug of neomycin to maintain the plasmid with stable OC-2 expression in the cell lines.

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Fig. 4

Proliferation analysis by a real-time cell analyzer (RTCA; xCELLigence, E-Plate 16). A- show U251 cells (Green) with OC-2 down-regulation compared with the WT (Red). A1- show U87 cells (Green) with OC-2 down-regulation compared with the WT (Red). B- show U251 cells (Green) with OC-2 up-regulation compared with the WT (Red). B1- show U87 cells (Green) with OC-2 up-regulation compared with the WT (Red).

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2.5

2.5 Flow cytometry analysis

2.5.1

2.5.1 Effect of OC-2 on cell cycle of GBM cell

To examine the impact of decreasing OC-2 expression on apoptosis in GBM cells, a cell cycle analysis was conducted with flow cytometry. The Flowing software program generated DNA histograms, which enabled determining the distribution of cells across the various stages of the cell cycle. The histograms presented in Fig. 5 illustrates the impact of knocking down OC-2 in U251 and U87 cells. The cells distributed across different phases of cell cycle. In the U251 cells, reducing OC-2 resulted in a decrease in the proportion of cells in the G1 phase (13.6 % compared to control cells), as well as a reduction in the number of cells in the S phase (43.99 % compared to control cells) and the G2/M phase (52.52 % compared to control cells). Similarly, knocking down OC-2 in U87 cells led to a decrease in the proportion of cells in the G1 phase (90.5 % compared to control cells). while the fraction of cells in the G2/M phase increased (76.61 % compared to control cells), and the proportion of cells in the S phase remained unchanged compared to control cells, as shown in Fig. 5. These results indicate that reducing OC-2 expression can obstruct the progression of cells at the S/G2 phase of the cell cycle.

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Fig. 5

Cell cycle distribution analysis. A- Control (U251 mock treated cells) shows cell cycle analysis. A1- Cell cycle analysis of cells possess OC-2 down-regulation and showing less G1, S phase and arrest in G2, P value: 0.0006. B- Control (U87 mock treated cells) shows cell cycle analysis. B1- Cells have OC-2 down-regulated was analyzed for cell cycle and showed decrease in G1, S phase and arrest in G2, P value: 0.0005.

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2.5.2

2.5.2 Effect of OC-2 on apoptosis of GBM cell

To evaluate the effect of OC-2 downregulation on apoptosis in GBM cells, we analyzed the levels of annexin V-FITC/PI dual staining using the Flowing software program v.2.5.1. Annexin V-FITC staining was performed to detect early-stage apoptosis, and its relationship to OC-2 downregulation was examined. The proportion of pre-apoptotic cells in the population was determined, and the resulting histograms are presented in Fig. 6. Our findings revealed that OC-2 downregulation led to a significant increase in apoptotic cells (with degraded DNA), with 95.46 % more apoptotic cells in OC-2-downregulated U251 cells (P = 0.0005) and 19.65 % more apoptotic cells in OC-2-downregulated U87 cells (P = 0.0004) compared to WT cells (Fig. 6A–B). These results strongly indicate that OC-2 has an apoptotic effect on GBM cells.

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Fig. 6

Pre-apoptosis analysis of OC-2 modified cells. A- Represent U251 WT showing minimal pre-apoptotic effect. A1- Represent U251 cells down regulated with OC-2 inhibitor showing high (95.46%) pre-apoptotic effect. B- Represent U87 WT showing minimal pre-apoptotic effect. B1- Represent U87 cells down regulated with OC-2 inhibitor showing high (19.65%) pre-apoptotic effect.

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3 Discussion

Hepatic nuclear factor 6 (Hnf6) is a member of the OCs family of transcription factors. It has been shown to be involved in organogenesis, cell fate, and tumorigenesis (Kropp and Gannon, 2016, Choi et al., 2022). Another OCs family member, OC-2, has been shown to be elevated in several different cancers, such as hepatocellular carcinoma, NETs, lung adenocarcinoma, and gastric, colorectal, prostate, and ovarian cancer (Zhang et al., 2015, Lu et al., 2018, Guo et al., 2019, Ma et al., 2019, Chen et al., 2020, Sun et al., 2021, Choi et al., 2022).

In this research study, our primary focus was to examine the impact of OC-2 function, whether gained or lost, on GBM cells. To achieve this, we employed a range of assays to thoroughly investigate this phenomenon. Noteworthy, we firstly explore the influence of OC-2 on the progression of GBM. To begin our investigation, we utilized qPCR techniques to validate the upregulation and downregulation of OC-2 in both transfected and transduced U251 and U87 cells. The results of the validation process were highly significant, providing compelling evidence that OC-2 expression was downregulated in U251 cells by an impressive 99 % (P < 0.0001), as well as in U87 cells by 18 % (P = 0.0207). Interestingly, despite multiple attempts, we encountered challenges in achieving a 50 % downregulation of OC-2 in U87 cells, the average reduction amounting was just 19 %. This resistance to OC-2 inhibition can likely be attributed to the presence of PTEN deletion in the U87 cells, resulting in diminished effectiveness of OC-2 downregulation. Similarly, in the literature PTEN deletion has been shown to derive resistance against MEK inhibitors (Smith et al., 2019).

In our study, we also investigated the impact of knocking down OC-2 on the metabolic activity of U251 and U87 cells. The results were striking, with a significant decrease of 47 % (P = 0.0056) in U251 cells and 36.4 % (P = 0.0003) in U87 cells (3A and 3B). These findings suggest that OC-2 plays a crucial role in GBM cells, potentially influencing cell survival beyond its effect on metabolic activity. Conversely, when OC-2 was overexpressed in U251 and U87 cells, a noteworthy increase in metabolic activity was observed compared to wild-type cells. Specifically, there was a 94 % (P = 0.0067) increase in U251 cells and a 58 % (P = 0.0028) increase in U87 cells (Fig. 3C and 3D). This indicates that OC-2 plays a significant role in promoting cell proliferation. Interestingly, previous studies have indicated that OC-2 is overexpressed in various cancers such as liver, colon, prostate, and lung cancers, which are known to exhibit increased cell proliferation (Sun et al., 2014, Zhang et al., 2015, Lu et al., 2018, Ma et al., 2019). The fact that similar findings have been reported in different types of cancer suggests that OC-2 likely performs similar functions across different organs.

To further understand the impact of downregulating OC-2 on cell proliferation, we conducted real-time assessments using U251 and U87 cells, with wild-type (WT) cells serving as controls. The results revealed a significant reduction of 65.5 % (P < 0.0001) in U251 cells and 71.7 % (P < 0.0001) in U87 cells (Fig. 4A and 4A1). These findings suggest that OC-2 may play a role in limiting the propagation of GBM cells in vitro. In line with our results, similar studies have reported reduced cell proliferation in gastric cancer and ovarian cancer upon the downregulation of OC-2 (Lu et al., 2018, Chen et al., 2020). In ovarian cancer, the Akt/Erk signaling pathway, which is associated with cell proliferation, migration, and angiogenesis, has been investigated for its relationship with ONECUT2 expression (Lu et al., 2018). Additionally, in neuroendocrine prostate cancer, ONECUT2 was found to regulate cell proliferation and tumor growth (Choi et al., 2022). Extra, ONECUT2 has been shown to control the expression of specific genes involved in cell proliferation. In gastric cancer, ONECUT2 was found to control the expression of ROCK1, a gene associated with tumor proliferation (Chen et al., 2020). This data supports the notion that OC-2 may have a broader influence on cell proliferation across different cancer types. Conversely, when OC-2 was overexpressed in U251 and U87 cells, a significant increase in proliferation was observed compared to WT cells. Specifically, there was a 183.2 % (P < 0.0001) increase in U251 cells and a 123.5 % (P < 0.0001) increase in U87 cells (Fig. 4B and 4B1). Similarly, ONECUT2 was shown by other groups to promote tumor proliferation in gastric and ovarian cancer (Lu et al., 2018, Chen et al., 2020). These findings suggest that OC-2 may stimulate cell proliferation pathways. Interestingly, our proliferation data indicated an initial spike in proliferation during the first 10 h, where the population of OC-2 inhibited cells temporarily outgrew the control. This could be attributed to cellular stress, as the modified cells attempted to compensate by increasing their rate of proliferation. Consequently, we conducted cell cycle assays to further investigate this phenomenon.

In order to gain further insights into the effects of downregulating OC-2 on cell cycle progression, we performed cell cycle analysis on U251 and U87 cells. The results revealed that U251 cells with downregulated OC-2 showed that the cells arrested at G2 phase 52.5 %. Also, downregulation of OC-2 in U87 cells was found to arrest cells at the G2 76.61 % and failed to initiate G1 phase 90.5 %. Furthermore, S phase was significantly low 43.99 % in U251 modified cells only. We concluded that the two type of cell lines probably use two different mechanisms when responding to OC-2 inhibition. Cellular arrest at the S/G2 phase is often associated with the initiation of apoptosis, a programmed cell death process. In light of this, we decided to perform an apoptosis assay to investigate the potential involvement of apoptosis in the observed effects of OC-2 downregulation. By analyzing the cell cycle and apoptosis responses, we aimed to uncover the underlying mechanisms through which OC-2 influences cell cycle progression and cell fate decisions in U251 and U87 cells.

Apoptosis analysis of U251 cells with downregulated OC-2 revealed a significant activation of the pre-apoptotic pathway by 95.46 % (P = 0.0005). This indicates that OC-2 plays a crucial role in promoting cell survival. On the other hand, downregulation of OC-2 in U87 cells resulted in a less pronounced activation of the pre-apoptotic pathway by 19.65 % (P = 0.0004). This suggests that OC-2 is important for the survival of U87 cells. It's worth noting that U87 cells have a PTEN mutation, which might contribute to the reduction of apoptotic initiation by inhibiting OC-2-associated apoptosis. Likewise, Yi et al. has shown OC2 down regulation stimulates apoptosis (Yi et al., 2022). Our apoptosis findings are consistent with the results of our proliferation assay. In the case of U251 cells, proliferation plateaued after 20 h when OC-2 was inhibited. Conversely, U87 cells did not plateau at 20 h but rather after 60 h of proliferation (Fig. 4A and 4B). This further confirms that both cell lines employ different mechanisms or routes to ensure their survival. Taken together, the apoptosis analysis and proliferation assay highlight the importance of OC-2 in promoting cell survival and provide evidence for distinct mechanisms employed by U251 and U87 cells in response to OC-2 downregulation.

These findings highlight the role of OC-2 in GBM and suggest its potential as a therapeutic target in GBM treatment. It is worth noting that this study is the first to demonstrate the impact of OC-2 on GBM cell lines. The involvement of OC-2 in tumor progression suggests its potential as a therapeutic target for GBM. To further understand the role of OC-2 in GBM, scientists should investigate its contribution to angiogenesis and metastasis. In addition, investigating a synergism by combining OC-2 and a new steroidal drug such as Stigmasterol (Ali et al., 2015, Aljarba et al., 2021) may open a new door for therapies. Moreover, preclinical studies including toxicity for assessing OC-2 inhibition are important for understanding GBM therapeutics.

4 Materials and methods

Ethical approval for this study is waived as per institutional regulations’ stating that commercial cell lines are exempt from mandatory IRB application.

Funding

This research was funded by Ministry of Education (MOE), Saudi Arabia, RC20/046/R, and supported by the King Abdullah International Medical Research Center (KAIMRC), Saudi Arabia, RC20/046/R.

6 Institutional review board statement

King Abdullah International Medical Research Center (KAIMRC) Institution Review Board (IRB) Ministry of National Guard – Health Affairs in Riyadh, Saudi Arabia under protocol number RC20/046/R.