SMARCA4 Loss and Mutated ß-Catenin Induce Proliferative Lesions in the Murine Embryonic Cerebellum.

Almost all medulloblastomas (MB) of the Wingless/Int-1 (WNT) type are characterized by hotspot mutations in CTNNB1, and mouse models have convincingly demonstrated the tumor-initiating role of these mutations. Additional alterations in SMARCA4 are detected in ∼20% of WNT MB, but their functional role is mostly unknown. We, therefore, amended previously described brain lipid binding protein (Blbp)-cre::Ctnnb1(ex3)fl/wt mice by the introduction of floxed Smarca4 alleles. Unexpectedly, mutated and thereby stabilized ß-catenin on its own induced severe developmental phenotypes in male and female Blbp-cre::Ctnnb1(ex3)fl/wt mice in our hands, including a thinned cerebral cortex, hydrocephalus, missing cerebellar layering, and cell accumulations in the brainstem and cerebellum. An additional loss of SMARCA4 even resulted in prenatal death for most mice. Respective Blbp-cre::Ctnnb1(ex3)fl/wt::Smarca4fl/rec mutants (male and female) developed large proliferative lesions in the cerebellum evolving from E13.5 to E16.5. Histological and molecular analysis of these lesions by DNA methylation profiling and single-cell RNA sequencing suggested an origin in early undifferentiated SOX2-positive cerebellar progenitors. Furthermore, upregulated WNT signaling, altered actin/cytoskeleton organization, and reduced neuronal differentiation were evident in mutant cells. In vitro, cells harboring alterations in both Ctnnb1 and Smarca4 were negatively selected and did not show tumorigenic potential after transplantation in adult female recipient mice. However, in cerebellar explant cultures, mutant cells displayed significantly increased proliferation, suggesting an important role of the embryonic microenvironment in the development of lesions. Altogether, these results represent an important first step toward the unraveling of tumorigenic mechanisms induced by aberrant WNT signaling and SMARCA4 deficiency.

Molecular refinement of pilocytic astrocytoma in adult patients.

AIM: Pilocytic astrocytomas (PA) in adults are rare and may be challenging to identify based only on histomorphology. Compared to their paediatric counterparts, they are reportedly molecularly more diverse and associated with a worse prognosis. We aimed to describe the characteristics of adult PAs more precisely by comprehensively profiling a series of 79 histologically diagnosed adult cases (≥18 years). METHODS: We performed global DNA methylation profiling and DNA and RNA panel sequencing, and integrated the results with clinical data. We further compared the molecular characteristics of adult and paediatric PAs that had a significant match to one of the established PA methylation classes in the Heidelberg brain tumour classifier. RESULTS: The mean age in our cohort was 33 years, and 43% of the tumours were located supratentorially. Based on methylation profiling, only 39% of the cases received a significant match to a PA methylation class. Sixteen per cent matched a different tumour type and 45% had a Heidelberg classifier score <0.9 with an affiliation to diverse established methylation classes in t-SNE analyses. Although the KIAA1549::BRAF fusion was found in 98% of paediatric PAs, this was true for only 27% of histologically defined and 55% of adult PAs defined by methylation profiling. CONCLUSIONS: A particularly high fraction of adult tumours with histological features of PA do not match current PA methylation classes, indicating ambiguous histology and an urgent need for molecular profiling. Moreover, even in adult PAs with a match to a PA methylation class, the distribution of genetic drivers differs significantly from their paediatric counterparts (p<0.01).

Mouse models of pediatric high-grade gliomas with MYCN amplification reveal intratumoral heterogeneity and lineage signatures.

Pediatric high-grade gliomas of the subclass MYCN (HGG-MYCN) are highly aggressive tumors frequently carrying MYCN amplifications, TP53 mutations, or both alterations. Due to their rarity, such tumors have only recently been identified as a distinct entity, and biological as well as clinical characteristics have not been addressed specifically. To gain insights into tumorigenesis and molecular profiles of these tumors, and to ultimately suggest alternative treatment options, we generated a genetically engineered mouse model by breeding hGFAP-cre::Trp53Fl/Fl::lsl-MYCN mice. All mice developed aggressive forebrain tumors early in their lifetime that mimic human HGG-MYCN regarding histology, DNA methylation, and gene expression. Single-cell RNA sequencing revealed a high intratumoral heterogeneity with neuronal and oligodendroglial lineage signatures. High-throughput drug screening using both mouse and human tumor cells finally indicated high efficacy of Doxorubicin, Irinotecan, and Etoposide as possible therapy options that children with HGG-MYCN might benefit from.

MYC overexpression and SMARCA4 loss cooperate to drive medulloblastoma formation in mice.

Group 3 medulloblastoma is one of the most aggressive types of childhood brain tumors. Roughly 30% of cases carry genetic alterations in MYC, SMARCA4, or both genes combined. While overexpression of MYC has previously been shown to drive medulloblastoma formation in mice, the functional significance of SMARCA4 mutations and their suitability as a therapeutic target remain largely unclear. To address this issue, we combined overexpression of MYC with a loss of SMARCA4 in granule cell precursors. Both alterations did not increase proliferation of granule cell precursors in vitro. However, combined MYC overexpression and SMARCA4 loss successfully induced tumor formation in vivo after orthotopic transplantation in recipient mice. Resulting tumors displayed anaplastic histology and exclusively consisted of SMARCA4-negative cells although a mixture of recombined and non-recombined cells was injected. These observations provide first evidence for a tumor-promoting role of a SMARCA4 deficiency in the development of medulloblastoma. In comparing the transcriptome of tumors to the cells of origin and an established Sonic Hedgehog medulloblastoma model, we gathered first hints on deregulated gene expression that could be specifically involved in SMARCA4/MYC driven tumorigenesis. Finally, an integration of RNA sequencing and DNA methylation data of murine tumors with human samples revealed a high resemblance to human Group 3 medulloblastoma on the molecular level. Altogether, the development of SMARCA4-deficient medulloblastomas in mice paves the way to deciphering the role of frequently occurring SMARCA4 alterations in Group 3 medulloblastoma with the perspective to explore targeted therapeutic options.

The tumor suppressor CREBBP and the oncogene MYCN cooperate to induce malignant brain tumors in mice.

The tumor suppressor and chromatin modifier cAMP response element-binding protein binding protein (CREBBP) and v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN), a member of the MYC oncogene family, are critically involved in brain development. Both genes are frequently mutated in the same tumor entities, including high-grade glioma and medulloblastoma. Therefore, we hypothesized that alterations in both genes cooperate to induce brain tumor formation. For further investigation, hGFAP-cre::CrebbpFl/Fl::lsl-MYCN mice were generated, which combine Crebbp deletion with overexpression of MYCN in neural stem cells (NSCs). Within eight months, these animals developed aggressive forebrain tumors. The first tumors were detectable in the olfactory bulbs of seven-day-old mice. This location raises the possibility that presumptive founder cells are derived from the ventricular-subventricular zone (V-SVZ). To examine the cellular biology of these tumors, single-cell RNA sequencing was performed, which revealed high intratumoral heterogeneity. Data comparison with reference CNS cell types indicated the highest similarity of tumor cells with transit-amplifying NSCs or activated NSCs of the V-SVZ. Consequently, we analyzed V-SVZ NSCs of our mouse model aiming to confirm that the tumors originate from this stem cell niche. Mutant V-SVZ NSCs showed significantly increased cell viability and proliferation as well as reduced glial and neural differentiation in vitro compared to control cells. In summary, we demonstrate the oncogenic potential of a combined loss of function of CREBBP and overexpression of MYCN in this cell population. hGFAP-cre::CrebbpFl/Fl::lsl-MYCN mice thus provide a valuable tool to study tumor-driving mechanisms in a key neural stem/ progenitor cell niche.

A Carboxy-terminal Smarcb1 Point Mutation Induces Hydrocephalus Formation and Affects AP-1 and Neuronal Signalling Pathways in Mice.

The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb11148del/1148del mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.

OLIG2 Is a Determinant for the Relapse of MYC-Amplified Medulloblastoma.

PURPOSE: Patients with MYC-amplified medulloblastoma (MB) have poor prognosis and frequently develop recurrence, thus new therapeutic approaches to prevent recurrence are needed. EXPERIMENTAL DESIGN: We evaluated OLIG2 expression in a panel of mouse Myc-driven MB tumors, patient MB samples, and patient-derived xenograft (PDX) tumors and analyzed radiation sensitivity in OLIG2-high and OLIG2-low tumors in PDX lines. We assessed the effect of inhibition of OLIG2 by OLIG2-CRISPR or the small molecule inhibitor CT-179 combined with radiotherapy on tumor progression in PDX models. RESULTS: We found that MYC-associated MB can be stratified into OLIG2-high and OLIG2-low tumors based on OLIG2 protein expression. In MYC-amplified MB PDX models, OLIG2-low tumors were sensitive to radiation and rarely relapsed, whereas OLIG2-high tumors were resistant to radiation and consistently developed recurrence. In OLIG2-high tumors, irradiation eliminated the bulk of tumor cells; however, a small number of tumor cells comprising OLIG2- tumor cells and rare OLIG2+ tumor cells remained in the cerebellar tumor bed when examined immediately post-irradiation. All animals harboring residual-resistant tumor cells developed relapse. The relapsed tumors mirrored the cellular composition of the primary tumors with enriched OLIG2 expression. Further studies demonstrated that OLIG2 was essential for recurrence, as OLIG2 disruption with CRISPR-mediated deletion or with the small molecule inhibitor CT-179 prevented recurrence from the residual radioresistant tumor cells. CONCLUSIONS: Our studies reveal that OLIG2 is a biomarker and an effective therapeutic target in a high-risk subset of MYC-amplified MB, and OLIG2 inhibitor combined with radiotherapy represents a novel effective approach for treating this devastating disease.

Single-cell transcriptomics identifies potential cells of origin of MYC rhabdoid tumors.

Rhabdoid tumors (RT) are rare and highly aggressive pediatric neoplasms. Their epigenetically-driven intertumoral heterogeneity is well described; however, the cellular origin of RT remains an enigma. Here, we establish and characterize different genetically engineered mouse models driven under the control of distinct promoters and being active in early progenitor cell types with diverse embryonic onsets. From all models only Sox2-positive progenitor cells give rise to murine RT. Using single-cell analyses, we identify distinct cells of origin for the SHH and MYC subgroups of RT, rooting in early stages of embryogenesis. Intra- and extracranial MYC tumors harbor common genetic programs and potentially originate from fetal primordial germ cells (PGCs). Using PGC specific Smarcb1 knockout mouse models we validate that MYC RT originate from these progenitor cells. We uncover an epigenetic imbalance in MYC tumors compared to PGCs being sustained by epigenetically-driven subpopulations. Importantly, treatments with the DNA demethylating agent decitabine successfully impair tumor growth in vitro and in vivo. In summary, our work sheds light on the origin of RT and supports the clinical relevance of DNA methyltransferase inhibitors against this disease.

Brahma-related gene 1 has time-specific roles during brain and eye development.

During development, gene expression is tightly controlled to facilitate the generation of the diverse cell types that form the central nervous system. Brahma-related gene 1 (Brg1, also known as Smarca4) is the catalytic subunit of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex that regulates transcription. We investigated the role of Brg1 between embryonic day 6.5 (E6.5) and E14.5 in Sox2-positive neural stem cells (NSCs). Being without major consequences at E6.5 and E14.5, loss of Brg1 between E7.5 and E12.5 resulted in the formation of rosette-like structures in the subventricular zone, as well as morphological alterations and enlargement of neural retina (NR). Additionally, Brg1-deficient cells showed decreased survival in vitro and in vivo. Furthermore, we uncovered distinct changes in gene expression upon Brg1 loss, pointing towards impaired neuron functions, especially those involving synaptic communication and altered composition of the extracellular matrix. Comparison with mice deficient for integrase interactor 1 (Ini1, also known as Smarcb1) revealed that the enlarged NR was Brg1 specific and was not caused by a general dysfunction of the SWI/SNF complex. These results suggest a crucial role for Brg1 in NSCs during brain and eye development.

SMARCA4-deficient rhabdoid tumours show intermediate molecular features between SMARCB1-deficient rhabdoid tumours and small cell carcinomas of the ovary, hypercalcaemic type.

Extracranial rhabdoid tumours (ECRTs) are an aggressive malignancy of infancy and early childhood. The vast majority of cases demonstrate inactivation of SMARCB1 (ECRTSMARCB1 ) on a background of a remarkably stable genome, a low mutational burden, and no other recurrent mutations. Rarely, ECRTs can harbour the alternative inactivation of SMARCA4 (ECRTSMARCA4 ) instead of SMARCB1. However, very few ECRTSMARCA4 cases have been published to date, and a systematic characterization of ECRTSMARCA4 is missing from the literature. In this study, we report the clinical, pathological, and genomic features of additional cases of ECRTSMARCA4 and show that they are comparable to those of ECRTSMARCB1. We also assess whether ECRTSMARCB1 , ECRTSMARCA4 , and small cell carcinomas of the ovary, hypercalcaemic type (SCCOHT) represent distinct or overlapping entities at a molecular level. Using DNA methylation and transcriptomics-based tumour classification approaches, we demonstrate that ECRTSMARCA4 display molecular features intermediate between SCCOHT and ECRTSMARCB1 ; however, ECRTSMARCA4 appear to be more closely related to SCCOHT by DNA methylation. Conversely, both transcriptomics and DNA methylation show a larger gap between SCCOHT and ECRTSMARCB1 , potentially supporting their continuous separate classification. Lastly, we show that ECRTSMARCA4 display concomitant lack of SMARCA4 (BRG1) and SMARCA2 (BRM) expression at the protein level, similar to what is seen in SCCOHT. Overall, these results expand our knowledge on this rare tumour type and explore the similarities and differences among entities from the 'rhabdoid tumour' spectrum. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Simultaneous Brg1 Knockout and MYCN Overexpression in Cerebellar Granule Neuron Precursors Is Insufficient to Drive Tumor Formation but Temporarily Enhances their Proliferation and Delays their Migration.

Medulloblastoma (MB) is the most common malignant brain tumor in childhood. According to the World Health Organization (WHO) classification of central nervous system (CNS) tumors, this embryonal tumor is divided into a wingless (WNT)-activated, Sonic hedgehog (SHH)-activated, and non-WNT/non-SHH entity. The latter is poorly defined but frequently carries mutations in Brahma-related gene 1 (BRG1) or amplifications of MYCN. Here, we investigated whether a combination of a Brg1 knockout and an overexpression of MYCN in cerebellar granule neuron precursors or multipotent neural stem cells is sufficient to drive brain tumor formation in mice. To this end, we generated Math1-creERT2::Brg1fl/fl::lslMYCN and hGFAP-cre::Brg1fl/fl::lslMYCN mice, respectively. We did not observe brain tumor formation in any of these models. hGFAP-cre::Brg1fl/fl::lslMYCN mice revealed severe CNS abnormalities with short survival, similar to the situation with a sole loss of Brg1, as we previously described. Investigation of Math1-creERT2::Brg1fl/fl::lslMYCN mice with a tamoxifen induction at postnatal day 3 revealed a regular survival but significant increase in cerebellar granule neuron precursor proliferation, followed by a delayed inward migration of these cells. This is in stark contrast to the hypoplastic cerebellum that we previously observed after embryonic deletion of Brg1 in Math1 positive cerebellar granule neurons. Our results indicate a time-specific function of Brg1 in cerebellar granule neuron precursors. Yet, the exact temporal and spatial origin of non-WNT/non-SHH MB remains unclear.

Atypical teratoid/rhabdoid tumors (ATRTs) with SMARCA4 mutation are molecularly distinct from SMARCB1-deficient cases.

Atypical teratoid/rhabdoid tumors (ATRTs) are very aggressive childhood malignancies of the central nervous system. The underlying genetic cause are inactivating bi-allelic mutations in SMARCB1 or (rarely) in SMARCA4. ATRT-SMARCA4 have been associated with a higher frequency of germline mutations, younger age, and an inferior prognosis in comparison to SMARCB1 mutated cases. Based on their DNA methylation profiles and transcriptomics, SMARCB1 mutated ATRTs have been divided into three distinct molecular subgroups: ATRT-TYR, ATRT-SHH, and ATRT-MYC. These subgroups differ in terms of age at diagnosis, tumor location, type of SMARCB1 alterations, and overall survival. ATRT-SMARCA4 are, however, less well understood, and it remains unknown, whether they belong to one of the described ATRT subgroups. Here, we examined 14 ATRT-SMARCA4 by global DNA methylation analyses. We show that they form a separate group segregating from SMARCB1 mutated ATRTs and from other SMARCA4-deficient tumors like small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) or SMARCA4 mutated extra-cranial malignant rhabdoid tumors. In contrast, medulloblastoma (MB) samples with heterozygous SMARCA4 mutations do not group separately, but with established MB subgroups. RNA sequencing of ATRT-SMARCA4 confirmed the clustering results based on DNA methylation profiling and displayed an absence of typical signature genes upregulated in SMARCB1 deleted ATRT. In summary, our results suggest that, in line with previous clinical observations, ATRT-SMARCA4 should be regarded as a distinct molecular subgroup.

An extracellular vesicle-related gene expression signature identifies high-risk patients in medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is a malignant brain tumor in childhood. It comprises 4 subgroups with different clinical behaviors. The aim of this study was to characterize the transcriptomic landscape of MB, both at the level of individual tumors as well as in large patient cohorts. METHODS: We used a combination of single-cell transcriptomics, cell culture models and biophysical methods such as nanoparticle tracking analysis and electron microscopy to investigate intercellular communication in the MB tumor niche. RESULTS: Tumor cells of the sonic hedgehog (SHH)-MB subgroup show a differentiation blockade. These cells undergo extensive metabolic reprogramming. The gene expression profiles of individual tumor cells show a partial convergence with those of tumor-associated glial and immune cells. One possible cause is the transfer of extracellular vesicles (EVs) between cells in the tumor niche. We were able to detect EVs in co-culture models of MB tumor cells and oligodendrocytes. We also identified a gene expression signature, EVS, which shows overlap with the proteome profile of large oncosomes from prostate cancer cells. This signature is also present in MB patient samples. A high EVS expression is one common characteristic of tumors that occur in high-risk patients from different MB subgroups or subtypes. CONCLUSIONS: With EVS, our study uncovered a novel gene expression signature that has a high prognostic significance across MB subgroups.

The basic helix-loop-helix transcription factor TCF4 impacts brain architecture as well as neuronal morphology and differentiation.

Germline mutations in the basic helix-loop-helix transcription factor 4 (TCF4) cause the Pitt-Hopkins syndrome (PTHS), a developmental disorder with severe intellectual disability. Here, we report findings from a new mouse model with a central nervous system-specific truncation of Tcf4 leading to severe phenotypic abnormalities. Furthermore, it allows the study of a complete TCF4 knockout in adult mice, circumventing early postnatal lethality of previously published mouse models. Our data suggest that a TCF4 truncation results in an impaired hippocampal architecture affecting both the dentate gyrus as well as the cornu ammonis. In the cerebral cortex, loss of TCF4 generates a severe differentiation delay of neural precursors. Furthermore, neuronal morphology was critically affected with shortened apical dendrites and significantly increased branching of dendrites. Our data provide novel information about the role of Tcf4 in brain development and may help to understand the mechanisms leading to intellectual deficits observed in patients suffering from PTHS.

hGFAP-Positive Stem Cells Depend on Brg1 for Proper Formation of Cerebral and Cerebellar Structures.

Brahma-related gene 1 (Brg1) is one of the two mutually exclusive catalytic subunits of the SWItch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. Several roles of Brg1 have been described including acting as a tumor suppressor but also functioning in neural stem cell (NSC) maintenance, neural crest development, or differentiation of oligodendrocytes and Schwann cells. Here, we generated human glial fibrillary acidic protein (hGFAP)-cre::Brg1fl/fl mice to analyze the function of Brg1 in multipotential NSCs during late stages of neural development. hGFAP-cre::Brg1fl/fl mice died approximately 2 weeks after birth. Macroscopic examination revealed a severe hydrocephalus and a decreased brain weight caused by the loss of Brg1. The cerebellum of hGFAP-cre::Brg1fl/fl mice displayed disorganized cortical layers as well as a massive hypoplasia due to a dramatically reduced number of granule neurons. The cerebrum presented with less proliferative and more apoptotic precursor cells in the subventricular zone (SVZ). Furthermore, the cerebral cortex stood out with significantly thinned upper layers and with impressive dendrite pathology. Finally, the hippocampus was severely underdeveloped with only a sparse number of detectable neurons. We conclude that NSCs depend on Brg1 to give rise to major essential brain structures including the cerebellum, the cerebral cortex, and the hippocampus.

The transcriptional coactivator and histone acetyltransferase CBP regulates neural precursor cell development and migration.

CREB (cyclic AMP response element binding protein) binding protein (CBP, CREBBP) is a ubiquitously expressed transcription coactivator with intrinsic histone acetyltransferase (KAT) activity. Germline mutations within the CBP gene are known to cause Rubinstein-Taybi syndrome (RSTS), a developmental disorder characterized by intellectual disability, specific facial features and physical anomalies. Here, we investigate mechanisms of CBP function during brain development in order to elucidate morphological and functional mechanisms underlying the development of RSTS. Due to the embryonic lethality of conventional CBP knockout mice, we employed a tissue specific knockout mouse model (hGFAP-cre::CBPFl/Fl, mutant mouse) to achieve a homozygous deletion of CBP in neural precursor cells of the central nervous system.Our findings suggest that CBP plays a central role in brain size regulation, correct neural cell differentiation and neural precursor cell migration. We provide evidence that CBP is both important for stem cell viability within the ventricular germinal zone during embryonic development and for unhindered establishment of adult neurogenesis. Prominent histological findings in adult animals include a significantly smaller hippocampus with fewer neural stem cells. In the subventricular zone, we observe large cell aggregations at the beginning of the rostral migratory stream due to a migration deficit caused by impaired attraction from the CBP-deficient olfactory bulb. The cerebral cortex of mutant mice is characterized by a shorter dendrite length, a diminished spine number, and a relatively decreased number of mature spines as well as a reduced number of synapses.In conclusion, we provide evidence that CBP is important for neurogenesis, shaping neuronal morphology, neural connectivity and that it is involved in neuronal cell migration. These findings may help to understand the molecular basis of intellectual disability in RSTS patients and may be employed to establish treatment options to improve patients' quality of life.

The Hepatic Microenvironment and TRAIL-R2 Impact Outgrowth of Liver Metastases in Pancreatic Cancer after Surgical Resection.

Most patients with pancreatic ductal adenocarcinoma (PDAC) undergoing curative resection relapse within months, often with liver metastases. The hepatic microenvironment determines induction and reversal of dormancy during metastasis. Both tumor growth and metastasis depend on the Tumor necrosis factor (TNF)-related apoptosis-inducing ligand-receptor 2 (TRAIL-R2). This study investigated the interplay of TRAIL-R2 and the hepatic microenvironment in liver metastases formation and the impact of surgical resection. Although TRAIL-R2-knockdown (PancTu-I shTR2) decreased local relapses and number of macroscopic liver metastases after primary tumor resection in an orthotopic PDAC model, the number of micrometastases was increased. Moreover, abdominal surgery induced liver inflammation involving activation of hepatic stellate cells (HSCs) into hepatic myofibroblasts (HMFs). In coculture with HSCs, proliferation of PancTu-I shTR2 cells was significantly lower compared to PancTu-I shCtrl cells, an effect still observed after switching coculture from HSC to HMF, mimicking surgery-mediated liver inflammation and enhancing cell proliferation. CXCL-8/IL-8 blockade diminished HSC-mediated growth inhibition in PancTu-I shTR2 cells, while Vascular Endothelial Growth Factor (VEGF) neutralization decreased HMF-mediated proliferation. Overall, this study points to an important role of TRAIL-R2 in PDAC cells in the interplay with the hepatic microenvironment during metastasis. Resection of primary PDAC seems to induce liver inflammation, which might contribute to outgrowth of liver metastases.

TCF4 (E2-2) harbors tumor suppressive functions in SHH medulloblastoma.

The TCF4 gene encodes for the basic helix-loop-helix transcription factor 4 (TCF4), which plays an important role in the development of the central nervous system (CNS). Haploinsufficiency of TCF4 was found to cause Pitt-Hopkins syndrome (PTHS), a severe neurodevelopmental disorder. Recently, the screening of a large cohort of medulloblastoma (MB), a highly aggressive embryonal brain tumor, revealed almost 20% of adult patients with MB of the Sonic hedgehog (SHH) subtype carrying somatic TCF4 mutations. Interestingly, many of these mutations have previously been detected as germline mutations in patients with PTHS. We show here that overexpression of wild-type TCF4 in vitro significantly suppresses cell proliferation in MB cells, whereas mutant TCF4 proteins do not to the same extent. Furthermore, RNA sequencing revealed significant upregulation of multiple well-known tumor suppressors upon expression of wild-type TCF4. In vivo, a prenatal knockout of Tcf4 in mice caused a significant increase in apoptosis accompanied by a decreased proliferation and failed migration of cerebellar granule neuron precursor cells (CGNP), which are thought to be the cells of origin for SHH MB. In contrast, postnatal in vitro and in vivo knockouts of Tcf4 with and without an additional constitutive activation of the SHH pathway led to significantly increased proliferation of CGNP or MB cells. Finally, publicly available data from human MB show that relatively low expression levels of TCF4 significantly correlate with a worse clinical outcome. These results not only point to time-specific roles of Tcf4 during cerebellar development but also suggest a functional linkage between TCF4 mutations and the formation of SHH MB, proposing that TCF4 acts as a tumor suppressor during postnatal stages of cerebellar development.