Identification of a miRNA multi-targeting therapeutic strategy in glioblastoma.

Glioblastoma (GBM) is a deadly and the most common primary brain tumor in adults. Due to their regulation of a high number of mRNA transcripts, microRNAs (miRNAs) are key molecules in the control of biological processes and are thereby promising therapeutic targets for GBM patients. In this regard, we recently reported miRNAs as strong modulators of GBM aggressiveness. Here, using an integrative and comprehensive analysis of the TCGA database and the transcriptome of GBM biopsies, we identified three critical and clinically relevant miRNAs for GBM, miR-17-3p, miR-222, and miR-340. In addition, we showed that the combinatorial modulation of three of these miRNAs efficiently inhibited several biological processes in patient-derived GBM cells of all these three GBM subtypes (Mesenchymal, Proneural, Classical), induced cell death, and delayed tumor growth in a mouse tumor model. Finally, in a doxycycline-inducible model, we observed a significant inhibition of GBM stem cell viability and a significant delay of orthotopic tumor growth. Collectively, our results reveal, for the first time, the potential of miR-17-3p, miR-222 and miR-340 multi-targeting as a promising therapeutic strategy for GBM patients.

Correction: Ponzo et al. Nucleolin Therapeutic Targeting Decreases Pancreatic Cancer Immunosuppression. Cancers 2022, 14, 4265.

In the original publication [...].

Nucleolin Therapeutic Targeting Decreases Pancreatic Cancer Immunosuppression.

Background: The pancreatic ductal adenocarcinoma (PDAC) microenvironment is highly fibrotic and hypoxic, with poor immune cell infiltration. Recently, we showed that nucleolin (NCL) inhibition normalizes tumour vessels and impairs PDAC growth. Methods: Immunocompetent mouse models of PDAC were treated by the pseudopeptide N6L, which selectively inhibits NCL. Tumour-infiltrating immune cells and changes in the tumour microenvironment were analysed. Results: N6L reduced the proportion of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) and increased tumour-infiltrated T lymphocytes (TILs) with an activated phenotype. Low-dose anti-VEGFR2 treatment normalized PDAC vessels but did not modulate the immune suppressive microenvironment. RNAseq analysis of N6L-treated PDAC tumours revealed a reduction of cancer-associated fibroblast (CAF) expansion in vivo and in vitro. Notably, N6L treatment decreased IL-6 levels both in tumour tissues and in serum. Treating mPDAC by an antibody blocking IL-6 reduced the proportion of Tregs and MDSCs and increased the amount of TILs, thus mimicking the effects of N6L. Conclusions: These results demonstrate that NCL inhibition blocks the amplification of lymphoid and myeloid immunosuppressive cells and promotes T cell activation in PDAC through a new mechanism of action dependent on the direct inhibition of the tumoral stroma.

Alteration of ribosome function upon 5-fluorouracil treatment favors cancer cell drug-tolerance.

Mechanisms of drug-tolerance remain poorly understood and have been linked to genomic but also to non-genomic processes. 5-fluorouracil (5-FU), the most widely used chemotherapy in oncology is associated with resistance. While prescribed as an inhibitor of DNA replication, 5-FU alters all RNA pathways. Here, we show that 5-FU treatment leads to the production of fluorinated ribosomes exhibiting altered translational activities. 5-FU is incorporated into ribosomal RNAs of mature ribosomes in cancer cell lines, colorectal xenografts, and human tumors. Fluorinated ribosomes appear to be functional, yet, they display a selective translational activity towards mRNAs depending on the nature of their 5'-untranslated region. As a result, we find that sustained translation of IGF-1R mRNA, which encodes one of the most potent cell survival effectors, promotes the survival of 5-FU-treated colorectal cancer cells. Altogether, our results demonstrate that "man-made" fluorinated ribosomes favor the drug-tolerant cellular phenotype by promoting translation of survival genes.

Nucleolin Aptamer N6L Reprograms the Translational Machinery and Acts Synergistically with mTORi to Inhibit Pancreatic Cancer Proliferation.

We previously showed that N6L, a pseudopeptide that targets nucleolin, impairs pancreatic ductal adenocarcinoma (PDAC) growth and normalizes tumor vessels in animal models. In this study, we analyzed the translatome of PDAC cells treated with N6L to identify the pathways that were either repressed or activated. We observed a strong decrease in global protein synthesis. However, about 6% of the mRNAs were enriched in the polysomes. We identified a 5'TOP motif in many of these mRNAs and demonstrated that a chimeric RNA bearing a 5'TOP motif was up-regulated by N6L. We demonstrated that N6L activates the mTOR pathway, which is required for the translation of these mRNAs. An inhibitory synergistic effect in PDAC cell lines, including patient-derived xenografts and tumor-derived organoids, was observed when N6L was combined with mTOR inhibitors. In conclusion, N6L reduces pancreatic cells proliferation, which then undergoes translational reprogramming through activation of the mTOR pathway. N6L and mTOR inhibitors act synergistically to inhibit the proliferation of PDAC and human PDX cell lines. This combotherapy of N6L and mTOR inhibitors could constitute a promising alternative to treat pancreatic cancer.

A novel view on an old drug, 5-fluorouracil: an unexpected RNA modifier with intriguing impact on cancer cell fate.

5-Fluorouracil (5-FU) is a chemotherapeutic drug widely used to treat patients with solid tumours, such as colorectal and pancreatic cancers. Colorectal cancer (CRC) is the second leading cause of cancer-related death and half of patients experience tumour recurrence. Used for over 60 years, 5-FU was long thought to exert its cytotoxic effects by altering DNA metabolism. However, 5-FU mode of action is more complex than previously anticipated since 5-FU is an extrinsic source of RNA modifications through its ability to be incorporated into most classes of RNA. In particular, a recent report highlighted that, by its integration into the most abundant RNA, namely ribosomal RNA (rRNA), 5-FU creates fluorinated active ribosomes and induces translational reprogramming. Here, we review the historical knowledge of 5-FU mode of action and discuss progress in the field of 5-FU-induced RNA modifications. The case of rRNA, the essential component of ribosome and translational activity, and the plasticity of which was recently associated with cancer, is highlighted. We propose that translational reprogramming, induced by 5-FU integration in ribosomes, contributes to 5-FU-driven cell plasticity and ultimately to relapse.

Nucleolin Targeting by N6L Inhibits Wnt/ß-Catenin Pathway Activation in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and resistant cancer with no available effective therapy. We have previously demonstrated that nucleolin targeting by N6L impairs tumor growth and normalizes tumor vessels in PDAC mouse models. Here, we investigated new pathways that are regulated by nucleolin in PDAC. We found that N6L and nucleolin interact with ß-catenin. We found that the Wnt/ß-catenin pathway is activated in PDAC and is necessary for tumor-derived 3D growth. N6L and nucleolin loss of function induced by siRNA inhibited Wnt pathway activation by preventing ß-catenin stabilization in PDAC cells. N6L also inhibited the growth and the activation of the Wnt/ß-catenin pathway in vivo in mice and in 3D cultures derived from MIA PaCa2 tumors. On the other hand, nucleolin overexpression increased ß-catenin stabilization. In conclusion, in this study, we identified ß-catenin as a new nucleolin interactor and suggest that the Wnt/ß-catenin pathway could be a new target of the nucleolin antagonist N6L in PDAC.

AS1411-conjugated gold nanoparticles affect cell proliferation through a mechanism that seems independent of nucleolin.

G-rich oligonucleotide, AS1411, has been shown to interact with nucleolin and to inhibit cancer cell proliferation and tumor growth. This antiproliferative action is increased when AS1411 is conjugated to different types of nanoparticles. However, the molecular mechanisms are not known. In this work, we show in several cell lines that optimized AS1411-conjugated gold nanoparticles (GNS-AS1411) inhibit nucleolin expression at the RNA and protein levels. We observed an alteration of the nucleolar structure with a decrease of ribosomal RNA accumulation comparable to what is observed upon nucleolin knock down. However, the expression of genes involved in cell cycle and the cell cycle blockage by GNS-AS1411 are not regulated in the same way as that in cells where nucleolin has been knocked down. These data suggest that the anti-proliferative activity of GNS-AS1411 is not the only consequence of nucleolin targeting and down-regulation.

Multifaceted Nucleolin Protein and Its Molecular Partners in Oncogenesis.

Discovered in 1973, nucleolin is one of the most abundant phosphoproteins of the nucleolus. The ability of nucleolin to be involved in many cellular processes is probably related to its structural organization and its capability to form many different interactions with other proteins. Many functions of nucleolin affect cellular processes involved in oncogenesis-for instance: in ribosome biogenesis; in DNA repair, remodeling, and genome stability; in cell division and cell survival; in chemokine and growth factor signaling pathways; in angiogenesis and lymphangiogenesis; in epithelial-mesenchymal transition; and in stemness. In this review, we will describe the different functions of nucleolin in oncogenesis through its interaction with other proteins.

Integrated analysis of mRNA and miRNA expression in HeLa cells expressing low levels of Nucleolin.

Nucleolin is an essential protein that plays important roles in the regulation of cell cycle and cell proliferation. Its expression is up regulated in many cancer cells but its molecular functions are not well characterized. Nucleolin is present in the nucleus where it regulates gene expression at the transcriptional and post-transcriptional levels. Using HeLa cells depleted in nucleolin we performed an mRNA and miRNA transcriptomics analysis to identify biological pathways involving nucleolin. Bioinformatic analysis strongly points to a role of nucleolin in lipid metabolism, and in many signaling pathways. Down regulation of nucleolin is associated with lower level of cholesterol while the amount of fatty acids is increased. This could be explained by the decreased and mis-localized expression of the transcription factor SREBP1 and the down-regulation of enzymes involved in the beta-oxidation and degradation of fatty acids. Functional classification of the miRNA-mRNA target genes revealed that deregulated miRNAs target genes involved in apoptosis, proliferation and signaling pathways. Several of these deregulated miRNAs have been shown to control lipid metabolism. This integrated transcriptomic analysis uncovers new unexpected roles for nucleolin in metabolic regulation and signaling pathways paving the way to better understand the global function of nucleolin within the cell.

Pharmacological targeting of the protein synthesis mTOR/4E-BP1 pathway in cancer-associated fibroblasts abrogates pancreatic tumour chemoresistance.

Pancreatic ductal adenocarcinoma (PDAC) is extremely stroma-rich. Cancer-associated fibroblasts (CAFs) secrete proteins that activate survival and promote chemoresistance of cancer cells. Our results demonstrate that CAF secretome-triggered chemoresistance is abolished upon inhibition of the protein synthesis mTOR/4E-BP1 regulatory pathway which we found highly activated in primary cultures of α-SMA-positive CAFs, isolated from human PDAC resections. CAFs selectively express the sst1 somatostatin receptor. The SOM230 analogue (Pasireotide) activates the sst1 receptor and inhibits the mTOR/4E-BP1 pathway and the resultant synthesis of secreted proteins including IL-6. Consequently, tumour growth and chemoresistance in nude mice xenografted with pancreatic cancer cells and CAFs, or with pieces of resected human PDACs, are reduced when chemotherapy (gemcitabine) is combined with SOM230 treatment. While gemcitabine alone has marginal effects, SOM230 is permissive to gemcitabine-induced cancer cell apoptosis and acts as an antifibrotic agent. We propose that selective inhibition of CAF protein synthesis with sst1-directed pharmacological compounds represents an anti-stromal-targeted therapy with promising chemosensitization potential.

Loss of Somatostatin Receptor Subtype 2 Promotes Growth of KRAS-Induced Pancreatic Tumors in Mice by Activating PI3K Signaling and Overexpression of CXCL16.

BACKGROUND & AIMS: The KRAS gene is mutated in most pancreatic ductal adenocarcinomas (PDAC). Expression of this KRAS oncoprotein in mice is sufficient to initiate carcinogenesis but not progression to cancer. Activation of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is required for KRAS for induction and maintenance of PDAC in mice. The somatostatin receptor subtype 2 (sst2) inhibits PI3K, but sst2 expression is lost during the development of human PDAC. We investigated the effects of sst2 loss during KRAS-induced PDAC development in mice. METHODS: We analyzed tumor growth in mice that expressed the oncogenic form of KRAS (KRAS(G12D)) in pancreatic precursor cells, as well as sst2+/- and sst2-/-, and in crossed KRAS(G12D);sst2+/- and KRAS(G12D);sst2-/- mice. Pancreatic tissues and acini were collected and assessed by histologic, immunoblot, immunohistochemical, and reverse-transcription polymerase chain reaction analyses. We also compared protein levels in paraffin-embedded PDAC samples from patients vs heathy pancreatic tissues from individuals without pancreatic cancer. RESULTS: In sst2+/- mice, PI3K was activated and signaled via AKT (PKB; protein kinase B); when these mice were crossed with KRAS(G12D) mice, premalignant lesions, tumors, and lymph node metastases developed more rapidly than in KRAS(G12D) mice. In crossed KRAS(G12D);sst2+/- mice, activation of PI3K signaling via AKT resulted in activation of nuclear factor-κB (NF-κB), which increased KRAS activity and its downstream pathways, promoting initiation and progression of neoplastic lesions. We found this activation loop to be mediated by PI3K-induced production of the chemokine CXCL16. Administration of a CXCL16-neutralizing antibody to KRAS(G12D) mice reduced activation of PI3K signaling to AKT and NF-κB, blocking carcinogenesis. Levels of CXCL16 and its receptor CXCR6 were significantly higher in PDAC tissues and surrounding acini than in healthy pancreatic tissues from mice or human beings. In addition, expression of sst2 was progressively lost, involving increased PI3K activity, in mouse lesions that expressed KRAS(G12D) and progressed to PDAC. CONCLUSIONS: Based on analyses of mice, loss of sst2 from pancreatic tissues activates PI3K signaling via AKT, leading to activation of NF-κB, amplification of oncogenic KRAS signaling, increased expression of CXCL16, and pancreatic tumor formation. CXCL16 might be a therapeutic target for PDAC.