Impact of the thyroid hormone T3 and its nuclear receptor TRα1 on colon cancer stem cell phenotypes and response to chemotherapies.

Colorectal cancers (CRCs) are highly heterogeneous and show a hierarchical organization, with cancer stem cells (CSCs) responsible for tumor development, maintenance, and drug resistance. Our previous studies showed the importance of thyroid hormone-dependent signaling on intestinal tumor development and progression through action on stem cells. These results have a translational value, given that the thyroid hormone nuclear receptor TRα1 is upregulated in human CRCs, including in the molecular subtypes associated with CSC features. We used an established spheroid model generated from the human colon adenocarcinoma cell line Caco2 to study the effects of T3 and TRα1 on spheroid formation, growth, and response to conventional chemotherapies. Our results show that T3 treatment and/or increased TRα1 expression in spheroids impaired the response to FOLFIRI and conferred a survival advantage. This was achieved by stimulating drug detoxification pathways and increasing ALDH1A1-expressing cells, including CSCs, within spheroids. These results suggest that clinical evaluation of the thyroid axis and assessing TRα1 levels in CRCs could help to select optimal therapeutic regimens for patients with CRC. Proposed mechanism of action of T3/TRα1 in colon cancer spheroids. In the control condition, TRα1 participates in maintaining homeostatic cell conditions. The presence of T3 in the culture medium activates TRα1 action on target genes, including the drug efflux pumps ABCG2 and ABCB1. In the case of chemotherapy FOLFIRI, the increased expression of ABC transcripts and proteins induced by T3 treatment is responsible for the augmented efflux of 5-FU and Irinotecan from the cancer cells. Taken together, these mechanisms contribute to the decreased efficacy of the chemotherapy and allow cells to escape the treatment. Created with BioRender.com .

SERBP1 interacts with PARP1 and is present in PARylation-dependent protein complexes regulating splicing, cell division, and ribosome biogenesis.

RNA binding proteins (RBPs) containing intrinsically disordered regions (IDRs) are present in diverse molecular complexes where they function as dynamic regulators. Their characteristics promote liquid-liquid phase separation (LLPS) and the formation of membraneless organelles such as stress granules and nucleoli. IDR-RBPs are particularly relevant in the nervous system and their dysfunction is associated with neurodegenerative diseases and brain tumor development. SERBP1 is a unique member of this group, being mostly disordered and lacking canonical RNA-binding domains. Using a proteomics approach followed by functional analysis, we defined SERBP1's interactome. We uncovered novel SERBP1 roles in splicing, cell division, and ribosomal biogenesis and showed its participation in pathological stress granules and Tau aggregates in Alzheimer's disease brains. SERBP1 preferentially interacts with other G-quadruplex (G4) binders, implicated in different stages of gene expression, suggesting that G4 binding is a critical component of SERBP1 function in different settings. Similarly, we identified important associations between SERBP1 and PARP1/polyADP-ribosylation (PARylation). SERBP1 interacts with PARP1 and its associated factors and influences PARylation. Moreover, protein complexes in which SERBP1 participates contain mostly PARylated proteins and PAR binders. Based on these results, we propose a feedback regulatory model in which SERBP1 influences PARP1 function and PARylation, while PARylation modulates SERBP1 functions and participation in regulatory complexes.

The paralogues MAGOH and MAGOHB are oncogenic factors in high-grade gliomas and safeguard the splicing of cell division and cell cycle genes.

The exon junction complex (EJC) plays key roles throughout the lifespan of RNA and is particularly relevant in the nervous system. We investigated the roles of two EJC members, the paralogs MAGOH and MAGOHB, with respect to brain tumour development. High MAGOH/MAGOHB expression was observed in 14 tumour types; glioblastoma (GBM) showed the greatest difference compared to normal tissue. Increased MAGOH/MAGOHB expression was associated with poor prognosis in glioma patients, while knockdown of MAGOH/MAGOHB affected different cancer phenotypes. Reduced MAGOH/MAGOHB expression in GBM cells caused alterations in the splicing profile, including re-splicing and skipping of multiple exons. The binding profiles of EJC proteins indicated that exons affected by MAGOH/MAGOHB knockdown accumulated fewer complexes on average, providing a possible explanation for their sensitivity to MAGOH/MAGOHB knockdown. Transcripts (genes) showing alterations in the splicing profile are mainly implicated in cell division, cell cycle, splicing, and translation. We propose that high MAGOH/MAGOHB levels are required to safeguard the splicing of genes in high demand in scenarios requiring increased cell proliferation (brain development and GBM growth), ensuring efficient cell division, cell cycle regulation, and gene expression (splicing and translation). Since differentiated neuronal cells do not require increased MAGOH/MAGOHB expression, targeting these paralogs is a potential option for treating GBM.

ELF4 is a critical component of a miRNA-transcription factor network and is a bridge regulator of glioblastoma receptor signaling and lipid dynamics.

BACKGROUND: The loss of neurogenic tumor suppressor microRNAs miR-124, miR-128, and miR-137 is associated with glioblastoma's undifferentiated state. Most of their impact comes via the repression of a network of oncogenic transcription factors. We conducted a high-throughput functional siRNA screen in glioblastoma cells and identify E74 like ETS transcription factor 4 (ELF4) as the leading contributor to oncogenic phenotypes. METHODS: In vitro and in vivo assays were used to assess ELF4 impact on cancer phenotypes. We characterized ELF4's mechanism of action via genomic and lipidomic analyses. A MAPK reporter assay verified ELF4's impact on MAPK signaling, and qRT-PCR and western blotting were used to corroborate ELF4 regulatory role on most relevant target genes. RESULTS: ELF4 knockdown resulted in significant proliferation delay and apoptosis in GBM cells and long-term growth delay and morphological changes in glioma stem cells (GSCs). Transcriptomic analyses revealed that ELF4 controls two interlinked pathways: 1) Receptor tyrosine kinase signaling and 2) Lipid dynamics. ELF4 modulation directly affected receptor tyrosine kinase (RTK) signaling, as mitogen-activated protein kinase (MAPK) activity was dependent upon ELF4 levels. Furthermore, shotgun lipidomics revealed that ELF4 depletion disrupted several phospholipid classes, highlighting ELF4's importance in lipid homeostasis. CONCLUSIONS: We found that ELF4 is critical for the GBM cell identity by controlling genes of two dependent pathways: RTK signaling (SRC, PTK2B, and TNK2) and lipid dynamics (LRP1, APOE, ABCA7, PLA2G6, and PITPNM2). Our data suggest that targeting these two pathways simultaneously may be therapeutically beneficial to GBM patients.

Structural Characterization of the RNA-Binding Protein SERBP1 Reveals Intrinsic Disorder and Atypical RNA Binding Modes.

RNA binding proteins (RBPs) are essential for critical biological processes such as translation regulation and mRNA processing, and misfunctions of these proteins are associated with diseases such as cancer and neurodegeneration. SERBP1 (SERPINE1 mRNA Binding Protein 1) is an RBP that comprises two RG/RGG repeat regions yet lacks other recognizable RNA-binding motifs. It is involved in mRNA maturation, and translational regulation. It was initially identified as a hyaluronic acid binding protein, but recent studies have identified central roles for SERBP1 in brain function and development, especially neurogenesis and synaptogenesis. SERBP1 regulates One-carbon metabolism and epigenetic modification of histones, and increased SERBP1 expression in cancers such as leukemia, ovarian, prostate, liver and glioblastoma is correlated with poor patient outcomes. Despite these important regulatory roles for SERBP1, little is known about its structural and dynamic properties, nor about the molecular mechanisms governing its interaction with mRNA. Here, we define SERBP1 as an intrinsically disordered protein, containing highly conserved elements that were shown to be functionally important. The RNA binding activity of SERBP1 was explored using solution NMR and other biophysical techniques. The outcome of these experiments revealed that SERBP1 preferentially samples compact conformations including a central, stable α-helix and show that SERBP1 recognizes G-rich RNA sequences at the C-terminus involving the RGG box and neighboring residues. Despite the role in RNA recognition, the RGG boxes do not seem to stabilize the central helix and the central helix does not participate in RNA binding. Further, SERBP1 undergoes liquid-liquid phase separation, mediated by salt and RNA, and both RGG boxes are necessary for the efficient formation of condensed phases. Together, these results provide a foundation for understanding the molecular mechanisms of SERBP1 functions in physiological and pathological processes.

Altered lipid metabolism marks glioblastoma stem and non-stem cells in separate tumor niches.

Glioblastoma (GBM) displays marked cellular and metabolic heterogeneity that varies among cellular microenvironments within a tumor. Metabolic targeting has long been advocated as a therapy against many tumors including GBM, but how lipid metabolism is altered to suit different microenvironmental conditions and whether cancer stem cells (CSCs) have altered lipid metabolism are outstanding questions in the field. We interrogated gene expression in separate microenvironments of GBM organoid models that mimic the transition between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones using spatial-capture RNA-sequencing. We revealed a striking difference in lipid processing gene expression and total lipid content between diverse cell populations from the same patient, with lipid enrichment in hypoxic organoid cores and also in perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and pseudopalisading regions of clinical GBM specimens, but not lower-grade brain tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis of multiple patient-derived models revealed a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs, but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression which was essential to maintain CSC viability and self-renewal. Our data demonstrate spatially and hierarchically distinct lipid metabolism phenotypes occur clinically in the majority of patients, can be recapitulated in laboratory models, and may represent therapeutic targets for GBM.

Musashi1 Contribution to Glioblastoma Development via Regulation of a Network of DNA Replication, Cell Cycle and Division Genes.

RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in their levels are often observed in tumors with numerous oncogenic RBPs identified in recent years. Musashi1 (Msi1) is an RBP and stem cell gene that controls the balance between self-renewal and differentiation. High Msi1 levels have been observed in multiple tumors including glioblastoma and are often associated with poor patient outcomes and tumor growth. A comprehensive genomic analysis identified a network of cell cycle/division and DNA replication genes and established these processes as Msi1's core regulatory functions in glioblastoma. Msi1 controls this gene network via two mechanisms: direct interaction and indirect regulation mediated by the transcription factors E2F2 and E2F8. Moreover, glioblastoma lines with Msi1 knockout (KO) displayed increased sensitivity to cell cycle and DNA replication inhibitors. Our results suggest that a drug combination strategy (Msi1 + cell cycle/DNA replication inhibitors) could be a viable route to treat glioblastoma.

Synergism of Proneurogenic miRNAs Provides a More Effective Strategy to Target Glioma Stem Cells.

Tumor suppressor microRNAs (miRNAs) have been explored as agents to target cancer stem cells. Most strategies use a single miRNA mimic and present many disadvantages, such as the amount of reagent required and the diluted effect on target genes. miRNAs work in a cooperative fashion to regulate distinct biological processes and pathways. Therefore, we propose that miRNA combinations could provide more efficient ways to target cancer stem cells. We have previously shown that miR-124, miR-128, and miR-137 function synergistically to regulate neurogenesis. We used a combination of these three miRNAs to treat glioma stem cells and showed that this treatment was much more effective than single miRNAs in disrupting cell proliferation and survival and promoting differentiation and response to radiation. Transcriptomic analyses indicated that transcription regulation, angiogenesis, metabolism, and neuronal differentiation are among the main biological processes affected by transfection of this miRNA combination. In conclusion, we demonstrated the value of using combinations of neurogenic miRNAs to disrupt cancer phenotypes and glioma stem cell growth. The synergistic effect of these three miRNA amplified the repression of oncogenic factors and the effect on cancer relevant pathways. Future therapeutic approaches would benefit from utilizing miRNA combinations, especially when targeting cancer-initiating cell populations.

The RNA-Binding Protein Musashi1 Regulates a Network of Cell Cycle Genes in Group 4 Medulloblastoma.

Medulloblastoma is the most common malignant brain tumor in children. Treatment with surgery, irradiation, and chemotherapy has improved survival in recent years, but patients are frequently left with devastating neurocognitive and other sequelae. Patients in molecular subgroups 3 and 4 still experience a high mortality rate. To identify new pathways contributing to medulloblastoma development and create new routes for therapy, we have been studying oncogenic RNA-binding proteins. We defined Musashi1 (Msi1) as one of the main drivers of medulloblastoma development. The high expression of Msi1 is prevalent in Group 4 and correlates with poor prognosis while its knockdown disrupted cancer-relevant phenotypes. Genomic analyses (RNA-seq and RIP-seq) indicated that cell cycle and division are the main biological categories regulated by Msi1 in Group 4 medulloblastoma. The most prominent Msi1 targets include CDK2, CDK6, CCND1, CDKN2A, and CCNA1. The inhibition of Msi1 with luteolin affected the growth of CHLA-01 and CHLA-01R Group 4 medulloblastoma cells and a synergistic effect was observed when luteolin and the mitosis inhibitor, vincristine, were combined. These findings indicate that a combined therapeutic strategy (Msi1 + cell cycle/division inhibitors) could work as an alternative to treat Group 4 medulloblastoma.

MSI1 Promotes the Expression of the GBM Stem Cell Marker CD44 by Impairing miRNA-Dependent Degradation.

The stem cell marker Musashi1 (MSI1) is highly expressed during neurogenesis and in glioblastoma (GBM). MSI1 promotes self-renewal and impairs differentiation in cancer and non-malignant progenitor cells. However, a comprehensive understanding of its role in promoting GBM-driving networks remains to be deciphered. We demonstrate that MSI1 is highly expressed in GBM recurrences, an oncologist's major defiance. For the first time, we provide evidence that MSI1 promotes the expression of stem cell markers like CD44, co-expressed with MSI1 within recurrence-promoting cells at the migrating front of primary GBM samples. With GBM cell models of pediatric and adult origin, including isolated primary tumorspheres, we show that MSI1 promotes stem cell-like characteristics. Importantly, it impairs CD44 downregulation in a 3'UTR- and miRNA-dependent manner by controlling mRNA turnover. This regulation is disturbed by the previously reported MSI1 inhibitor luteolin, providing further evidence for a therapeutic target potential of MSI1 in GBM treatment.

The RNA-binding protein SERBP1 functions as a novel oncogenic factor in glioblastoma by bridging cancer metabolism and epigenetic regulation.

BACKGROUND: RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in RBP expression and function are often observed in cancer and influence critical pathways implicated in tumor initiation and growth. Identification and characterization of oncogenic RBPs and their regulatory networks provide new opportunities for targeted therapy. RESULTS: We identify the RNA-binding protein SERBP1 as a novel regulator of glioblastoma (GBM) development. High SERBP1 expression is prevalent in GBMs and correlates with poor patient survival and poor response to chemo- and radiotherapy. SERBP1 knockdown causes delay in tumor growth and impacts cancer-relevant phenotypes in GBM and glioma stem cell lines. RNAcompete identifies a GC-rich region as SERBP1-binding motif; subsequent genomic and functional analyses establish SERBP1 regulation role in metabolic routes preferentially used by cancer cells. An important consequence of these functions is SERBP1 impact on methionine production. SERBP1 knockdown decreases methionine levels causing a subsequent reduction in histone methylation as shown for H3K27me3 and upregulation of genes associated with neurogenesis, neuronal differentiation, and function. Further analysis demonstrates that several of these genes are downregulated in GBM, potentially through epigenetic silencing as indicated by the presence of H3K27me3 sites. CONCLUSIONS: SERBP1 is the first example of an RNA-binding protein functioning as a central regulator of cancer metabolism and indirect modulator of epigenetic regulation in GBM. By bridging these two processes, SERBP1 enhances glioma stem cell phenotypes and contributes to GBM poorly differentiated state.

Proneural and mesenchymal glioma stem cells display major differences in splicing and lncRNA profiles.

Therapy resistance and recurrence in high-grade gliomas are driven by their populations of glioma stem cells (GSCs). Thus, detailed molecular characterization of GSCs is needed to develop more effective therapies. We conducted a study to identify differences in the splicing profile and expression of long non-coding RNAs in proneural and mesenchymal GSC cell lines. Genes related to cell cycle, DNA repair, cilium assembly, and splicing showed the most differences between GSC subgroups. We also identified genes distinctly associated with survival among patients of mesenchymal or proneural subgroups. We determined that multiple long non-coding RNAs with increased expression in mesenchymal GSCs are associated with poor survival of glioblastoma patients. In summary, our study established critical differences between proneural and mesenchymal GSCs in splicing profiles and expression of long non-coding RNA. These splicing isoforms and lncRNA signatures may contribute to the uniqueness of GSC subgroups, thus contributing to cancer phenotypes and explaining differences in therapeutic responses.

ELF4 Is a Target of miR-124 and Promotes Neuroblastoma Proliferation and Undifferentiated State.

13-Cis-retinoic acid (RA) is typically used in postremission maintenance therapy in patients with neuroblastoma. However, side effects and recurrence are often observed. We investigated the use of miRNAs as a strategy to replace RA as promoters of differentiation. miR-124 was identified as the top candidate in a functional screen. Genomic target analysis indicated that repression of a network of transcription factors (TF) could be mediating most of miR-124's effect in driving differentiation. To advance miR-124 mimic use in therapy and better define its mechanism of action, a high-throughput siRNA morphologic screen focusing on its TF targets was conducted and ELF4 was identified as a leading candidate for miR-124 repression. By altering its expression levels, we showed that ELF4 maintains neuroblastoma in an undifferentiated state and promotes proliferation. Moreover, ELF4 transgenic expression was able to counteract the neurogenic effect of miR-124 in neuroblastoma cells. With RNA sequencing, we established the main role of ELF4 to be regulation of cell-cycle progression, specifically through the DREAM complex. Interestingly, several cell-cycle genes activated by ELF4 are repressed by miR-124, suggesting that they might form a TF-miRNA regulatory loop. Finally, we showed that high ELF4 expression is often observed in neuroblastomas and is associated with poor survival. IMPLICATIONS: miR-124 induces neuroblastoma differentiation partially through the downregulation of TF ELF4, which drives neuroblastoma proliferation and its undifferentiated phenotype.

The Diverse Roles of RNA-Binding Proteins in Glioma Development.

Post-transcriptional regulation of gene expression is fundamental for all forms of life, as it critically contributes to the composition and quantity of a cell's proteome. These processes encompass splicing, polyadenylation, mRNA decay, mRNA editing and modification and translation and are modulated by a variety of RNA-binding proteins (RBPs). Alterations affecting RBP expression and activity contribute to the development of different types of cancer. In this chapter, we discuss current research shedding light on the role of different RBPs in gliomas. These studies place RBPs as modulators of critical signaling pathways, establish their relevance as prognostic markers and open doors for new therapeutic strategies.

Antagonism between the RNA-binding protein Musashi1 and miR-137 and its potential impact on neurogenesis and glioblastoma development.

RNA-binding proteins (RBPs) and miRNAs are critical gene expression regulators that interact with one another in cooperative and antagonistic fashions. We identified Musashi1 (Msi1) and miR-137 as regulators of a molecular switch between self-renewal and differentiation. Msi1 and miR-137 have opposite expression patterns and functions, and Msi1 is repressed by miR-137. Msi1 is a stem-cell protein implicated in self-renewal while miR-137 functions as a proneuronal differentiation miRNA. In gliomas, miR-137 functions as a tumor suppressor while Msi1 is a prooncogenic factor. We suggest that the balance between Msi1 and miR-137 is a key determinant in cell fate decisions and disruption of this balance could contribute to neurodegenerative diseases and glioma development. Genomic analyses revealed that Msi1 and miR-137 share 141 target genes associated with differentiation, development, and morphogenesis. Initial results pointed out that these two regulators have an opposite impact on the expression of their target genes. Therefore, we propose an antagonistic model in which this network of shared targets could be either repressed by miR-137 or activated by Msi1, leading to different outcomes (self-renewal, proliferation, tumorigenesis).

Luteolin inhibits Musashi1 binding to RNA and disrupts cancer phenotypes in glioblastoma cells.

RNA binding proteins have emerged as critical oncogenic factors and potential targets in cancer therapy. In this study, we evaluated Musashi1 (Msi1) targeting as a strategy to treat glioblastoma (GBM); the most aggressive brain tumor type. Msi1 expression levels are often high in GBMs and other tumor types and correlate with poor clinical outcome. Moreover, Msi1 has been implicated in chemo- and radio-resistance. Msi1 modulates a range of cancer relevant processes and pathways and regulates the expression of stem cell markers and oncogenic factors via mRNA translation/stability. To identify Msi1 inhibitors capable of blocking its RNA binding function, we performed a ~ 25,000 compound fluorescence polarization screen. NMR and LSPR were used to confirm direct interaction between Msi1 and luteolin, the leading compound. Luteolin displayed strong interaction with Msi1 RNA binding domain 1 (RBD1). As a likely consequence of this interaction, we observed via western and luciferase assays that luteolin treatment diminished Msi1 positive impact on the expression of pro-oncogenic target genes. We tested the effect of luteolin treatment on GBM cells and showed that it reduced proliferation, cell viability, colony formation, migration and invasion of U251 and U343 GBM cells. Luteolin also decreased the proliferation of patient-derived glioma initiating cells (GICs) and tumor-organoids but did not affect normal astrocytes. Finally, we demonstrated the value of combined treatments with luteolin and olaparib (PARP inhibitor) or ionizing radiation (IR). Our results show that luteolin functions as an inhibitor of Msi1 and demonstrates its potential use in GBM therapy.

Increased expression of the thyroid hormone nuclear receptor TRα1 characterizes intestinal tumors with high Wnt activity.

Our previous work demonstrated a key function of the thyroid hormone nuclear receptor TRα1, a T3-modulated transcription factor, in controlling intestinal development and homeostasis via the Wnt and Notch pathways. Importantly, increased expression of TRα1 in the intestinal epithelium in a mutated Apc genetic background (vil-TRα1/Apc+/1638N mice) accelerated tumorigenesis and contributed to a more aggressive tumor phenotype compared to that of the Apc mutants alone. Therefore, the aim of this study was to determine the relevance of this synergistic effect in human colorectal cancers and to gain insights into the mechanisms involved. We analyzed cohorts of patients by in silico and experimental approaches and observed increased TRα1 expression and a significant correlation between TRα1 levels and Wnt activity. TRα1 loss-of-function and gain-of-function in Caco2 cell lines not only confirmed that TRα1 levels control Wnt activity but also demonstrated the role of TRα1 in regulating cell proliferation and migration. Finally, upon investigation of the molecular mechanisms responsible for the Wnt-TRα1 association, we described the repression by TRα1 of several Wnt inhibitors, including Frzb, Sox17 and Wif1. In conclusion, our results underline an important functional interplay between the thyroid hormone nuclear receptor TRα1 and the canonical Wnt pathway in intestinal cancer initiation and progression. More importantly, we show for the first time that the expression of TRα1 is induced in human colorectal cancers.

Patient-derived conditionally reprogrammed cells maintain intra-tumor genetic heterogeneity.

Preclinical in vitro models provide an essential tool to study cancer cell biology as well as aid in translational research, including drug target identification and drug discovery efforts. For any model to be clinically relevant, it needs to recapitulate the biology and cell heterogeneity of the primary tumor. We recently developed and described a conditional reprogramming (CR) cell technology that addresses many of these needs and avoids the deficiencies of most current cancer cell lines, which are usually clonal in origin. Here, we used the CR cell method to generate a collection of patient-derived cell cultures from non-small cell lung cancers (NSCLC). Whole exome sequencing and copy number variations are used for the first time to address the capability of CR cells to keep their tumor-derived heterogeneity. Our results indicated that these primary cultures largely maintained the molecular characteristics of the original tumors. Using a mutant-allele tumor heterogeneity (MATH) score, we showed that CR cells are able to keep and maintain most of the intra-tumoral heterogeneity, suggesting oligoclonality of these cultures. CR cultures therefore represent a pre-clinical lung cancer model for future basic and translational studies.

The 3' end of the story: deciphering combinatorial interactions that control mRNA fate.

A new study investigates how microRNAs affect the binding of proteins to RNA.