Characterization of the subcellular localization of the TGF-β receptors in the wing imaginal disc

The TGF-β pathway has been extensively studied in vivo in the wing imaginal disc of D. melanogaster. In particular, many investigations focus on the TGF-β ligand Dpp (BMP2/4 orthologue) that acts as a morphogen, regulating patterning and growth of wing disc cells. It was reported that Dpp forms an extracellular gradient consisting of an apical and a basolateral fraction (Entchev, Schwabedissen, & González-Gaitán, 2000; Gibson, Lehman, & Schubiger, 2002; Teleman & Cohen, 2000). However the function of these distinct fractions and the subcellular localization of the molecules responsible for ligand perception have not yet been characterized in this developmental context. Therefore, in this thesis I investigated the localization of BMP receptors and co-receptors expressed in the wing disc. I found that the D. melanogaster TGF-β superfamily type-I receptors have different subcellular localizations: while the BMP receptors Tkv and Sax distribute along the apical and the basolateral side of the wing disc epithelium, the TGF-β/Activin receptor Babo is only basolaterally localized. This subcellular bias in localization is also found in the TGF-β type-II receptors, Punt and Wit. Punt localizes only to the basolateral compartment, whereas Wit is also present at the apical compartment and shows a clear enrichment in this domain. The glypicans Dally and Dlp localize to both apical and basolateral compartments, as previously reported (Ayers, Gallet, Staccini-Lavenant, & Thérond, 2010; Gallet, Staccini-Lavenant, & Thérond, 2008). In order to understand the functional importance of the polarized subcellular localization of TGF-β receptors, I tried to mislocalize the TGF-β receptors using different approaches. In a first approach I have been using membrane-bound nanobodies. Despite the fact that these nanobody-tools were designed to trap secreted GFP-tagged proteins, I will show that they are also potential tools to mislocalize polarized membrane proteins. In another approach, I tried to mislocalize the TGF-β receptors through the mutation of “targeting domains”. Finally, to address whether signalling polarization has important consequences on physiological development, I attempted to modify the subcellular localization of the endogenous type-I receptor Tkv. To this end, I used a landing site in the tkv locus, generated by replacing the last two exons of tkv with an attP landing site and resulting in a tkv null allele. This tool allows manipulation of the receptor at the endogenous locus, important to understand the developmental impact of its proper subcellular localization. I achieved to generate different tagged versions of Tkv, with either an extracellular or a C-terminal mCherry tag. Moreover, I deleted a conserved targeting domain in the Tkv protein and obtained apical enrichment of Tkv localization. In the following, I will discuss these different methods and their implications for studying TGF-β signalling polarization in the wing disc

Brown Tumors Belong to the Spectrum of KRAS-driven Neoplasms

Brown tumors are rare and generally self-limiting mass lesions of bone occurring in the context of hyperparathyroidism. Although commonly regarded as endocrine-driven tumor-like lesions, we detected pathogenic hotspot KRAS mutations in 10/16 brown tumors (62%) with similar frequencies found in cases affecting the peripheral and axial skeleton. Pathogenic mutations in other driver genes of the RAS-MAPK pathway were not identified. Our findings suggest brown tumors to represent true neoplasms driven by the activation of the RAS-MAPK signaling pathway. The frequent regression of brown tumors after normalization of hyperparathyroidism points to a second hit mediated by endocrine stimulation to be required for tumor development. Our findings underline the pathogenic relation of brown tumors to nonossifying fibroma and giant cell granuloma of the jaws which both appear histologically similar to brown tumors and are also driven by RAS-MAPK signaling pathway activation

Long non-coding RNAs are key players in Prostate cancer tumorigenesis and drug resistance

"Long non-coding RNAs (lncRNAs) have been characterized as key players in several cancer-associated processes such as tumorigenesis and drug resistance. Emerging evidence indicates that lncRNAs affect initiation and progression of several cancers, including prostate cancer (PCa) and its advanced forms, such as metastatic castration resistant prostate cancer (mCRPC). Among others, H19 is one of the most studied oncogenic lncRNAs in cancer and has been associated with cancer tumorigenesis and progression, via mediating several pathways an acting in different mechanisms of action, including epigenetic and miRNA regulation. We have investigated lncRNA roles in PCa tumorigenesis by analysing the E006AA cell model. Emerging evidence has shown that parental E006AA are not tumorigenic in nude mice, while its derived E006AA-ht, is highly tumorigenic. Via high-throughput RNA sequencing we have shown that E006AA-ht overexpress different lncRNAs compared to E006AA. Interestingly, we found a high upregulation of H19 in E006AA-ht vs the parental cell line. Our in vitro validation has confirmed the sequencing data and further research could unravel a crucial role of H19 in PCa tumorigenesis, opening a new challenging chapter of lncRNAs research. We hypothesize that H19 could be involved in in vivo immunity suppression and our further studies aim at investigating H19 in this molecular and clinical context. Furthermore, we have studied lncRNAs as key players in cancer aggressiveness by using cellular models of mCRPC. From our studies, HORAS5 (i.e. linc00161) emerged as the most consistently upregulated lncRNA in CRPC patient-derived xenografts. This lncRNAs was previously associated with cancer drug-response in osteosarcoma, ovarian cancer and hepatocellular carcinoma. Our study has shown for the first time that HORAS5 promotes drug resistance in CRPC. After a preliminary drug screen, we have selected the chemotherapeutics cabazitaxel for further investigation. Via lentiviral-mediated overexpression and siRNA-based silencing we have regulated HORAS5 expression and analysed cell count and apoptosis of CRPC cells exposed to clinically achievable concentrations of cabazitaxel. The overexpression of HORAS5 increases cabazitaxel resistance, while HORAS5 silencing has an opposite effect, via inhibition of apoptosis. RNA sequencing and RT-qPCR revealed that BCL2A1 is the most upregulated transcript in HORAS5 overexpressing cells exposed to cabazitaxel, and that BCL2A1 silencing decreases cell count and increases caspase activity. Our data suggest that BCL2A1 expression is induced by HORAS5, thereby enhancing CRPC cells resistance to cabazitaxel. Transfection of CRPC cells with HORAS5-targeting ASOs can effectively silence this lncRNA and determine a decrease of cabazitaxel resistance. We have also shown that both HORAS5 and BCL2A1 upregulation results in decreased survival in PCa patients and samples from mCRPC patients treated with taxanes have upregulation of HORAS5. Overall, our studies bring novel insights into crucial roles of lncRNAs in PCa progression and aggressiveness making them emerging targets for cancer treatment at different stages.

Cancer Diagnosis Using a Liquid Biopsy: Challenges and Expectations

The field of cancer diagnostics has recently been impacted by new and exciting developments in the area of liquid biopsy. A liquid biopsy is a minimally invasive alternative to surgical biopsies of solid tissues, typically achieved through the withdrawal of a blood sample or other body fluids, allowing the interrogation of tumor-derived material including circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) fragments that are present at a given time point. In this short review, we discuss a few studies that summarize the state-of-the-art in the liquid biopsy field from a diagnostic perspective, and speculate on current challenges and expectations of implementing liquid biopsy testing for cancer diagnosis and monitoring in the clinical setting

LncRNA HORAS5 promotes taxane resistance in castration-resistant prostate cancer via a BCL2A1-dependent mechanism.

Background: Castration-resistant prostate cancer (CRPC) is an incurable malignancy. Long noncoding RNAs (lncRNAs) play key roles in drug resistance. Materials & methods: LncRNA HORAS5 role in cabazitaxel resistance (i.e., cell-count, IC50 and caspase activity) was studied via lentiviral-mediated overexpression and siRNA-based knockdown. Genes expression was analyzed with RNA-sequencing, reverse transcription quantitative PCR (RT-qPCR) and western blot. HORAS5 expression was queried in clinical database. Results: Cabazitaxel increased HORAS5 expression that upregulated BCL2A1, thereby protecting CRPC cells from cabazitaxel-induced apoptosis. BCL2A1 knockdown decreased cell-count and increased apoptosis in CRPC cells. HORAS5-targeting antisense oligonucleotide decreased cabazitaxel IC50. In CRPC clinical samples, HORAS5 expression increased upon taxane treatment. Conclusion:HORAS5 stimulates the expression of BCL2A1 thereby decreasing apoptosis and enhancing cabazitaxel resistance in CRPC cells

Fibroblast growth factor receptor (FGFR) inhibitor rogaratinib in patients with advanced pretreated squamous-cell non-small cell lung cancer over-expressing FGFR mRNA: The SAKK 19/18 phase II study.

BACKGROUND Patients with advanced squamous-cell lung cancer (SQCLC) frequently (46%) exhibit tumor overexpression of fibroblast growth factor receptor (FGFR) messenger ribonucleic acid (mRNA). Rogaratinib is a novel oral pan-FGFR inhibitor with a good safety profile and anti-tumor activity in early clinical trials as a single agent in FGFR pathway-addicted tumors. SAKK 19/18 determined clinical activity of rogaratinib in patients with advanced SQCLC overexpressing FGFR1-3 mRNA. METHODS Patients with advanced SQCLC failing standard systemic treatment and with FGFR1-3 mRNA tumor overexpression as defined in the protocol received rogaratinib 600 mg BID until disease progression or intolerable toxicity. A 6-months progression-free survival rate (6mPFS) ≤15 % was considered uninteresting (H0), whereas a 6mPFS ≥38 % was considered promising (H1). According to a Simon 2-stage design, 2 out of 10 patients of the first stage were required to be progression-free at 6 months. Comprehensive Genomic Profiling was performedusing the Oncomine Comprehensive Assay Plus (Thermo Fisher Scientific). RESULTS Between July 2019 and November 2020, 49 patients were screened and 20 were classified FGFR-positive. Among a total of 15 patients, 6mPFS was reached in 1 patient (6.7 %), resulting in trial closure for futility after the first stage. There were 7 (46.7 %) patients with stable disease and 5 (33.3 %) patients with progressive disease. Median PFS was 1.6 (95 % CI 0.9-3.5) months and median overall survival (OS) 3.5 (95 % CI 1.0-5.9) months. Most frequent treatment-related adverse events (TRAEs) included hyperphosphatemia in 8 (53 %), diarrhea in 5 (33 %), stomatitis in 3 (20 %) and nail changes in 3 (20 %) patients. Grade ≥3 TRAEs occurred in 6 (40 %) patients. No associations between mutational profile and treatment outcome were observed. CONCLUSION Despite preliminary signals of activity, rogaratinib failed to improve PFS in patients with advanced SQCLC overexpressing FGFR mRNA. FGFR inhibitors in SQCLC remain a challenging field, and more in-depth understanding of pathway crosstalks may lead to the development of drug combinations with FGFR inhibitors resulting in improved outcomes

The Long Non-Coding RNA H19 Drives the Proliferation of Diffuse Intrinsic Pontine Glioma with H3K27 Mutation

Diffuse intrinsic pontine glioma (DIPG) is an incurable paediatric malignancy. Identifying the molecular drivers of DIPG progression is of the utmost importance. Long non-coding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, whose functions have not yet been elucidated in DIPG. Herein, we studied the oncogenic role of the development-associated H19 lncRNA in DIPG. Bioinformatic analyses of clinical datasets were used to measure the expression of H19 lncRNA in paediatric high-grade gliomas (pedHGGs). The expression and sub-cellular location of H19 lncRNA were validated in DIPG cell lines. Locked nucleic acid antisense oligonucleotides were designed to test the function of H19 in DIPG cells. We found that H19 expression was higher in DIPG vs. normal brain tissue and other pedHGGs. H19 knockdown resulted in decreased cell proliferation and survival in DIPG cells. Mechanistically, H19 buffers let-7 microRNAs, resulting in the up-regulation of oncogenic let-7 target (e.g., SULF2 and OSMR). H19 is the first functionally characterized lncRNA in DIPG and a promising therapeutic candidate for treating this incurable cancer

A highly conserved SOX6 double binding site mediates SOX6 gene downregulation in erythroid cells

The Sox6 transcription factor plays critical roles in various cell types, including erythroid cells. Sox6-deficient mice are anemic due to impaired red cell maturation and show inappropriate globin gene expression in definitive erythrocytes. To identify new Sox6 target genes in erythroid cells, we used the known repressive double Sox6 consensus within the εy-globin promoter to perform a bioinformatic genome-wide search for similar, evolutionarily conserved motifs located within genes whose expression changes during erythropoiesis. We found a highly conserved Sox6 consensus within the Sox6 human gene promoter itself. This sequence is bound by Sox6 in vitro and in vivo, and mediates transcriptional repression in transient transfections in human erythroleukemic K562 cells and in primary erythroblasts. The binding of a lentiviral transduced Sox6FLAG protein to the endogenous Sox6 promoter is accompanied, in erythroid cells, by strong downregulation of the endogenous Sox6 transcript and by decreased in vivo chromatin accessibility of this region to the PstI restriction enzyme. These observations suggest that the negative Sox6 autoregulation, mediated by the double Sox6 binding site within its own promoter, may be relevant to control the Sox6 transcriptional downregulation that we observe in human erythroid cultures and in mouse bone marrow cells in late erythroid maturation

The evolutionarily conserved long non‐coding RNA <i>LINC00261</i> drives neuroendocrine prostate cancer proliferation and metastasis <i>via</i> distinct nuclear and cytoplasmic mechanisms

Metastatic neuroendocrine prostate cancer (NEPC) is a highly aggressive disease, whose incidence is rising. Long noncoding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, most of which are still functionally uncharacterized. Thus, we set out to identify the highly conserved lncRNAs that play a central role in NEPC pathogenesis. To this end, we performed transcriptomic analyses of donor-matched patient-derived xenograft models (PDXs) with immunohistologic features of prostate adenocarcinoma (AR+/PSA+) or NEPC (AR-/SYN+/CHGA+ ) and through differential expression analyses identified lncRNAs that were upregulated upon neuroendocrine transdifferentiation. These genes were prioritized for functional assessment based on the level of conservation in vertebrates. Here, LINC00261 emerged as the top gene with over 3229-fold upregulation in NEPC. Consistently, LINC00261 expression was significantly upregulated in NEPC specimens in multiple patient cohorts. Knockdown of LINC00261 in PC-3 cells dramatically attenuated its proliferative and metastatic abilities, which are explained by parallel downregulation of CBX2 and FOXA2 through distinct molecular mechanisms. In the cell cytoplasm, LINC00261 binds to and sequesters miR-8485 from targeting the CBX2 mRNA, while inside the nucleus, LINC00261 functions as a transcriptional scaffold to induce SMAD-driven expression of the FOXA2 gene. For the first time, these results demonstrate hyperactivation of the LINC00261-CBX2-FOXA2 axes in NEPC to drive proliferation and metastasis, and that LINC00261 may be utilized as a therapeutic target and a biomarker for this incurable disease

The Origin Recognition Complex Interacts with a Subset of Metabolic Genes Tightly Linked to Origins of Replication

The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell (such as in the orc2-1 mutant) only sites that bind ORC tightly (such as HMR-E) remain fully occupied by ORC, while lower affinity sites, including many origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC “affinity” genome-wide by performing an ORC ChIP–on–chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1–resistant ORC–interacting chromosomal sites (ORF–ORC sites) that did not function as replication origins or silencers. Instead, ORF–ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC–silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF–ORC genes were enriched in proximity to origins of replication and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection among ORC, replication origins, and cellular metabolism