LIN28 expression and function in medulloblastoma

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Current treatment modalities are not completely effective and can lead to severe neurological and cognitive adverse effects. In addition to urgently needing better treatment approaches, new diagnostic and prognostic biomarkers are required to improve the therapy outcomes of MB patients. The RNA‐binding proteins, LIN28A and LIN28B, are known to regulate invasive phenotypes in many different cancer types. However, the expression and function of these proteins in MB had not been studied to date. This study identified the expression of LIN28A and LIN28B in MB patient samples and cell lines and assessed the effect of LIN28 inhibition on MB cell growth, metabolism and stemness. LIN28B expression was significantly upregulated in MB tissues compared to normal brain tissues. This upregulation, which was not observed in other brain tumors, was specific for the aggressive MB subgroups and correlated with patient survival and metastasis rates. Functionally, pharmacological inhibition of LIN28 activity concentration‐dependently reduced LIN28B expression, as well as the growth of D283 MB cells. While LIN28 inhibition did not affect the levels of intracellular ATP, it reduced the expression of the stemness marker CD133 in D283 cells and the sphere formation of CHLA‐01R cells. LIN28B, which is highly expressed in the human cerebellum during the first few months after birth, subsequently decreased with age. The results of this study highlight the potential of LIN28B as a diagnostic and prognostic marker for MB and open the possibility to utilize LIN28 as a pharmacological target to suppress MB cell growth and stemness.

molecular aetiology (Ellison, 2010;Taylor et al., 2012). Groups 3 and 4 are characterized by higher rates of metastasis, lower survival and poorer prognosis compared to the other two MB subgroups (Cavalli et al., 2017;Taylor et al., 2012). Current MB treatments include surgery, radiotherapy and chemotherapy, which are frequently combined to maximize treatment efficacy (Hildebrand & Balériaux, 2002). However, due to the harsh nature of the current treatments, surviving children often suffer from long-term neurological adverse effects (Crawford et al., 2007;Maddrey et al., 2005). In addition, in about half of the children treated with chemotherapy alone (generally those less than 4 years of age), MB reoccurs (Hildebrand & Balériaux, 2002). Therefore, new therapeutic options are urgently needed to treat MB.
Although the physiological importance of LIN28A and LIN28B in tissue regeneration and pluripotency in embryonal stem cells is well established, both genes have not been studied in MB. Therefore, this study systematically investigated the expression of LIN28A and LIN28B in the different molecular MB subgroups and other brain tumors and correlated the expression levels with tumor metastasis and patient survival. These results were subsequently extended to correlate LIN28A and LIN28B expression in multiple MB cell lines with cell viability, metabolism and stemness in vitro. For the first time, this study addressed the effects of LIN28A and LIN28B in MB, the results of which may lead to a new approach to target this devastating disease.

| MATERIALS AND METHODS
2.1 | In silico analysis of MB patient data 2.1.1 | Platforms and data sets LIN28A and LIN28B mRNA expression levels in multiple data sets were visualized, using R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl) and GlioVis data portal (only used for patient survival analysis) (Bowman et al., 2017). The data sets used in this study include Harris (Harris et al., 2009), Sun (Sun et al., 2006), Donson (Griesinger et al., 2013), den Boer (de Bont et al., 2008, Gilbertson (Robinson et al., 2012), Pfister (Northcott et al., 2017) and Cavalli et al. (2017). Data sets with either unclassified or not specified tissues were excluded to avoid interpretation bias and to keep data as consistent as possible among the data sets used (Table 1). 2.1.2 | Expression analysis in brain tumors and normal brain tissue The data sets by Harris (Harris et al., 2009), Sun (Sun et al., 2006), Donson (Griesinger et al., 2013), den Boer (de Bont et al., 2008), Gilbertson (Robinson et al., 2012) and Pfister (Northcott et al., 2017) were used to assess the expression of LIN28A and LIN28B genes in MB tissues compared to other brain tumors and normal brain tissues. Data were extracted from the R2 platform and plotted in GraphPad Prism version 9.1 software for Windows (GraphPad). Expression values are presented as log 2 values.

| Expression in MB subgroups and normal brain samples
The data sets by Harris (Harris et al., 2009), Gilbertson (Robinson et al., 2012) and Pfister (Northcott et al., 2017)  Log-rank test compared between high and low expression groups.
The hazard ratios with 95% confidence interval, for LIN28A and LIN28B, are shown in Kaplan-Meier plots. Data were extracted from the GlioVis portal and plotted in GraphPad Prism.     are presented in reads per kilobase of transcript.

| Statistical analysis
All statistical analysis was performed using GraphPad Prism legend. Data were reported as mean ± SD, with p < 0.05 considered to be statistically significant.

| RESULTS
3.1 | Expression of LIN28A and LIN28B in brain tumors and MB molecular subgroups Expression levels of LIN28A and LIN28B in normal brain and different brain tumors, including glioblastoma, astrocytoma, oligodendroglioma, ependymoma and MB, were compared. LIN28A expression was comparable across the tested groups and was only significantly higher (p < 0.0001) in oligodendrogliomas compared to the nontumor tissues ( Figure 1a). In contrast, among tumor tissues, LIN28B expression was significantly higher only in MB tissues compared to tissues from normal brain tissue (Figure 1b). MB is classified into four molecular subgroups such as WNT, SHH, Group 3 and Group 4.
These molecular subgroups influence the prognostic variability between MB patients (Ramaswamy et al., 2016). Therefore, LIN28A and LIN28B expression in the normal brain were compared to the different molecular MB subgroups. LIN28A was expressed homogeneously across the different MB groups with a significantly lower expression in the SHH subgroup compared to normal tissue ( Figure 1c). In contrast, LIN28B was highly overexpressed (p < 0.0001) in MB subgroups 3 and 4 compared to MB subgroups 1 and 2 and normal brain tissue (Figure 1d). with the LIN28 inhibitor did not significantly alter relative intracellular ATP levels (Figure 5a), suggesting that LIN28 does not affect ATP synthesis in these cells.

| LIN28 inhibition suppresses stemness of D283 and CHLA-01R MB cells
LIN28 was reported to regulate the expression of CD133 (King, Cuatrecasas, et al., 2011;Zhong et al., 2020), a stemness marker that is also involved in cell proliferation (Tang et al., 2012). Given the observed suppression of cell proliferation by LIN28 inhibition, the effect of the LIN28 inhibitor on CD133 expression in D283 cells was assessed. Treatment with Lin28 1632, concentrationdependently, reduced the expression of LIN28B and reduced cell growth. LIN28 inhibition significantly reduced the expression of CD133 at 150 µM in D283 cells (Figure 5b,c). Since CD133 is also regulating cellular stemness, we also assessed if LIN28 inhibition affected sphere formation of CHLA-01R cancer cells, which is associated with cellular stemness (L. Cao, Zhou, et al., 2011;Pastrana et al., 2011;Yakisich et al., 2016).
Treatment of CHLA-01R cells with the LIN28 inhibitor, Lin28 1632, significantly suppressed the formation of cell spheres in a concentration-dependent manner (Figure 5d,e).
3.6 | Expression of LIN28A and LIN28B in the developing brain Our previous results pointed toward a potential role of LIN28 in cellular stemness. As cells age, they reduce their stemness features (Ahmed et al., 2017;Bose & Shenoy, 2016). MB is a primary malignant tumor of the cerebellum that is rarely seen in adults and predominantly occurs in children when cells possess enhanced stemness characteristics. Therefore, LIN28A and LIN28B expression F I G U R E 1 LIN28A and LIN28B expression in different brain tumors and normal brain tissue. (a) LIN28A and (b) LIN28B mRNA expression in MB (Donson, den Boer, Gilbertson and Pfister data sets; n = 345) compared to nontumor brain tissues (Harris, Sun, Donson and den Boer data sets; n = 84) and different brain tumors, glioblastoma (Sun and Donson data sets; n = 111), astrocytoma (Sun and Donson data sets; n = 41), oligodendroglioma (Sun data set, n = 50) and ependymoma (Donson and den Boer data set, n = 59) (ns: not significant p > 0.05; ****p < 0.0001, nonparametric test, with Kruskal-Wallis test multiple comparisons, mean with SD, n = 690). (c) LIN28A and (d) LIN28B mRNA expression in different MB subgroups (Gilbertson and Pfister data sets; n = 296) and normal brain tissues (Harris data set, n = 43) (ns: not significant p > 0.05; ****p < 0.0001, nonparametric test, with Kruskal-Wallis test multiple comparisons, mean with SD, n = 339). G3, group 3; G4, group 4; MB, medulloblastoma; mRNA, messenger RNA; SHH, sonic hedgehog; WNT, wingless.
| 539 was measured in the cerebellum of the developing brain at different time points. Using the Atlas of the Developing Human Brain data set that includes cerebellar expression profiles from individuals between 12 weeks to 40 years of age (https://www.brainspan.org), LIN28A expression remained steady (Figure 6a). In contrast, cerebellar LIN28B showed higher levels in the newborn brain that gradually decreased to very low or nondetectable levels at 37 weeks of age, which remained at this level into adulthood (Figure 6b).

| DISCUSSION
This study assessed the expression of LIN28A and LIN28B in MB patient samples and cell lines and observed the effect of LIN28 inhibition on MB cell growth, metabolism and stemness. Our data demonstrate a significant upregulation of LIN28B gene expression in MB tissues compared to normal brain tissues. This upregulation, which was only observed in MB and not in other brain tumors, was specific for aggressive MB subgroups 3 and 4. LIN28B expression levels correlated with MB patient survival and metastasis rates.
LIN28A levels, on the other hand, were not upregulated in MB and did not show any association with MB subgroups, patient survival or metastasis rates. These data suggest the value of LIN28B as a potential diagnostic biomarker for MB and its molecular subgroups, as well as a prognostic indicator of survival time. Several studies have correlated LIN28A and/or LIN28B levels with patient prognosis in other cancers. High LIN28 expressions were shown to be associated with tumor aggressiveness and poor patient prognosis in oesophagus cancer (Hamano et al., 2012). Similarly, LIN28A expression was shown to serve as a prognostic marker for gastric carcinoma (C. Xu et al., 2013) and HCC (Qiu et al., 2012), while LIN28B expression was proposed to be a novel prognostic marker in gastric adenocarcinoma  and oral squamous cell carcinoma (Wu et al., 2013).
To test if targeting LIN28 has the potential to control MB cell growth, we also assessed the effect of pharmacological inhibition of LIN28 in vitro. LIN28 inhibition was most effective in D283 cells, where concentration-dependently reduced LIN28B expression as well as MB cell growth and the expression of stemness marker CD133. Since LIN28 inhibition did not affect the levels of intracellular ATP in D283 cells, it is more likely that reduced CD133 expression, which was reported to be involved in cell proliferation (Tang et al., 2012), accounted for the reduced cell growth observed in this study.
Our data also showed that LIN28    way (Huang et al., 2016). It is therefore critical to target these cancer  (Huang et al., 2016). Future studies can assess whether LIN28 inhibition affects the metastatic characteristics of MB cells, such as cell migration, invasion and epithelial-to-mesenchymal transition (EMT). EMT is a process that plays a key role in the progression, dissemination and therapy resistance of epithelial tumors; however, recent evidence suggests that EMT also promotes malignancy of nonepithelial tumors (Kahlert et al., 2017).
The present study showed that LIN28B is highly expressed in the cerebellum during the first few months after birth and subsequently shows a clear decrease with age. This is an intriguing observation since MB was shown to arise from disruptions in cerebellar F I G U R E 5 Effect of Lin28 1632 on CD133 protein expression in D283 cells and on the sphere formation of CHLA-01R cells. (a) Intracellular ATP levels normalized to protein content in D283 cells (ns: not significant). (b) Representative immunoblot of CD133 and β-actin proteins in D283 cells treated with Lin28 1632 (0-150 µM). (c) Densitometric analysis of relative CD133 protein levels (normalized to β-actin as the loading control) in D283 cells (ns: not significant p > 0.05; **p < 0.01, one-way ANOVA, with Tukey's multiple comparisons). Bars represent mean ± SD from three independent experiments (three biological replicates). (d) Representative images of three-dimensional spheroids. Scale bar, 300 μm. (e) Concentration-dependent response of Lin28 1632 on sphere size and number. Sphere size and numbers were quantified for all acquired images using ImageJ software. The results obtained at each concentration were normalized by comparing them to the untreated control (0.3% DMSO) and expressing the results as a fold change. The results are shown as the mean ± SD for three independent repeats, each of which had three replicates (ns: not significant p > 0.05; *p < 0.05; ***p < 0.001, one-way ANOVA with Tukey's multiple comparisons). ANOVA, analysis of variance; ATP, adenosine triphosphate; conc., concentration; DMSO, dimethyl sulfoxide; µM, micromole; kDa, kilodaltons.
Samples used for this study, however, do not distinguish the specific cell type within the cerebellum. Given that cerebellum is composed of several cell types including granule, Purkinje, Golgi, stellate and basket cells (Consalez et al., 2021), further studies are warranted to know which cell type(s) specifically express LIN28A and LIN28B in the developing cerebellum. Indeed, LIN28B was reported to regulate neuronal differentiation by modulating the expression of Staufen1, a protein that initiates posttranscriptional regulation, including mRNA export, relocation, translation and decay (Oh et al., 2018). In addition, LIN28B promotes neural crest cell migration and leads to the transformation of the trunk region of the developing embryo (Corallo et al., 2020). Future studies are warranted to assess if the observed altered expression of LIN28B in the developing cerebellum contributes to MB initiation.
To understand the role of LIN28 in MB cells, this study used the only commercially available pharmacological inhibitor of this protein to date, Lin28 1632 (Roos et al., 2016). To better understand the isoformspecific roles of LIN28 proteins, siRNA-mediated silencing and/or plasmid overexpression studies, specifically targeting LIN28A and LIN28B genes, are needed in future studies. In addition, it is important to note that Lin28 1632, only inhibits the LIN28/let-7 interaction, while LIN28 proteins are known to regulate key biological functions via let-7-dependent and let-7-independent mechanisms (Balzeau et al., 2017). Therefore, mechanistic studies are needed to delineate whether the observed LIN28 activities in MB cells observed in this study occur via let-7-dependent or let-7-independent pathways. However, at present, there are no pharmacological inhibitors available commercially F I G U R E 6 Age-dependent cerebellar LIN28 expression. Cerebellar microarray data were collected from the BrainSpan Atlas of the Developing Human Brain (https://www.brainspan.org). Thirty-one cerebellar samples ranged in age from 12 weeks after conception to 40 years. Both (a) LIN28A and (b) LIN28B expression levels are indicated in reads per kilobase of transcript (RPKM). Donor IDs have been indicated in brackets for each sample.
to inhibit the let-7-independent activities of LIN28. Therefore, the development of such compounds in the future will be invaluable to selectively modulate these two distinct signalling activities of LIN28.
Overall, the present study demonstrates the potential value of LIN28B as a diagnostic and prognostic marker for MB. It also, for the first time, highlights the potential to use a small-molecule pharmacological antagonist of LIN28 to suppress MB cell growth and stemness, which could lead to a new therapeutic approach for the treatment of MB. Consequently, future studies need to test LIN28 inhibition in preclinical models of MB to explore the therapeutic potential of this approach as a future treatment strategy against MB. Nuri Gueven and Iman Azimi.