In Cellulo Evaluation of the Therapeutic Potential of NHC Platinum Compounds in Metastatic Cutaneous Melanoma.

We describe here the evaluation of the cytotoxic efficacy of two platinum (II) complexes bearing an N-heterocyclic carbene (NHC) ligand, a pyridine ligand and bromide or iodide ligands on a panel of human metastatic cutaneous melanoma cell lines representing different genetic subsets including BRAF-inhibitor-resistant cell lines, namely A375, SK-MEL-28, MeWo, HMCB, A375-R, SK-MEL-5-R and 501MEL-R. Cisplatin and dacarbazine were also studied for comparison purposes. Remarkably, the iodine-labelled Pt-NHC complex strongly inhibited proliferation of all tested melanoma cells after 1-h exposure, likely due to its rapid uptake by melanoma cells. The mechanism of this inhibitory activity involves the formation of DNA double-strand breaks and apoptosis. Considering the intrinsic chemoresistance of metastatic melanoma cells of current systemic treatments, these findings are promising and could give research opportunities in the future to improve the prognosis of patients suffering from unresectable metastatic melanoma that are not eligible or that do not respond to the most effective drugs available to date, namely BRAF inhibitors and the anti-PD-1 monoclonal antibody (mAb).

High-Frequency Mechanical Properties of Tumors Measured by Brillouin Light Scattering.

The structure of tumors can be recapitulated as an elastic frame formed by the connected cytoskeletons of the cells invaded by interstitial and intracellular fluids. The low-frequency mechanics of this poroelastic system, dictated by the elastic skeleton only, control tumor growth, penetration of therapeutic agents, and invasiveness. The high-frequency mechanical properties containing the additional contribution of the internal fluids have also been posited to participate in tumor progression and drug resistance, but they remain largely unexplored. Here we use Brillouin light scattering to produce label-free images of tumor microtissues based on the high-frequency viscoelastic modulus as a contrast mechanism. In this regime, we demonstrate that the modulus discriminates between tissues with altered tumorigenic properties. Our micrometric maps also reveal that the modulus is heterogeneously altered across the tissue by drug therapy, revealing a lag of efficacy in the core of the tumor. Exploiting high-frequency poromechanics should advance present theories based on viscoelasticity and lead to integrated descriptions of tumor response to drugs.

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.

Spatial sequestration of activated-caspase 3 in aggresomes mediates resistance of neuroblastoma cell to bortezomib treatment.

Neuroblastoma (NB) is the most common pediatric tumor and is currently treated by several types of therapies including chemotherapies, such as bortezomib treatment. However, resistance to bortezomib is frequently observed by mechanisms that remain to be deciphered. Bortezomib treatment leads to caspase activation and aggresome formation. Using models of patients-derived NB cell lines with different levels of sensitivity to bortezomib, we show that the activated form of caspase 3 accumulates within aggresomes of NB resistant cells leading to an impairment of bortezomib-induced apoptosis and increased cell survival. Our findings unveil a new mechanism of resistance to chemotherapy based on an altered subcellular distribution of the executioner caspase 3. This mechanism could explain the resistance developed in NB patients treated with bortezomib, emphasizing the potential of drugs targeting aggresomes.

HSV-1 cellular model reveals links between aggresome formation and early step of Alzheimer's disease.

Many studies highlight the potential link between the chronic degenerative Alzheimer's disease and the infection by the herpes simplex virus type-1 (HSV-1). However, the molecular mechanisms making possible this HSV-1-dependent process remain to be understood. Using neuronal cells expressing the wild type form of amyloid precursor protein (APP) infected by HSV-1, we characterized a representative cellular model of the early stage of the sporadic form of the disease and unraveled a molecular mechanism sustaining this HSV-1- Alzheimer's disease interplay. Here, we show that HSV-1 induces caspase-dependent production of the 42 amino-acid long amyloid peptide (Aß42) oligomers followed by their accumulation in neuronal cells. Aß42 oligomers and activated caspase 3 (casp3A) concentrate into intracytoplasmic structures observed in Alzheimer's disease neuronal cells called aggresomes. This casp3A accumulation in aggresomes during HSV-1 infection limits the execution of apoptosis until its term, similarly to an abortosis-like event occurring in Alzheimer's disease neuronal cells patients. Indeed, this particular HSV-1 driven cellular context, representative of early stages of the disease, sustains a failed apoptosis mechanism that could explain the chronic amplification of Aß42 production characteristic of Alzheimer's disease patients. Finally, we show that combination of flurbiprofen, a non-steroidal anti-inflammatory drug (NSAID), with caspase inhibitor reduced drastically HSV-1-induced Aß42 oligomers production. This provided mechanistic insights supporting the conclusion of clinical trials showing that NSAIDs reduced Alzheimer's disease incidence in early stage of the disease. Therefore, from our study we propose that caspase-dependent production of Aß42 oligomers together with the abortosis-like event represents a vicious circle in early Alzheimer's disease stages leading to a chronic amplification of Aß42 oligomers that contributes to the establishment of degenerative disorder like Alzheimer's disease in patients infected by HSV-1. Interestingly this process could be targeted by an association of NSAID with caspase inhibitors.

Heterogeneity and dynamic of EMT through the plasticity of ribosome and mRNA translation.

Growing evidence exposes translation and its translational machinery as key players in establishing and maintaining physiological and pathological biological processes. Examining translation may not only provide new biological insight but also identify novel innovative therapeutic targets in several fields of biology, including that of epithelial-to-mesenchymal transition (EMT). EMT is currently considered as a dynamic and reversible transdifferentiation process sustaining the transition from an epithelial to mesenchymal phenotype, known to be mainly driven by transcriptional reprogramming. However, it seems that the characterization of EMT plasticity is challenging, relying exclusively on transcriptomic and epigenetic approaches. Indeed, heterogeneity in EMT programs was reported to depend on the biological context. Here, by reviewing the involvement of translational control, translational machinery and ribosome biogenesis characterizing the different types of EMT, from embryonic and adult physiological to pathological contexts, we discuss the added value of integrating translational control and its machinery to depict the heterogeneity and dynamics of EMT programs.

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.

Ribosome Biogenesis Alterations in Colorectal Cancer.

Many studies have focused on understanding the regulation and functions of aberrant protein synthesis in colorectal cancer (CRC), leaving the ribosome, its main effector, relatively underappreciated in CRC. The production of functional ribosomes is initiated in the nucleolus, requires coordinated ribosomal RNA (rRNA) processing and ribosomal protein (RP) assembly, and is frequently hyperactivated to support the needs in protein synthesis essential to withstand unremitting cancer cell growth. This elevated ribosome production in cancer cells includes a strong alteration of ribosome biogenesis homeostasis that represents one of the hallmarks of cancer cells. None of the ribosome production steps escape this cancer-specific dysregulation. This review summarizes the early and late steps of ribosome biogenesis dysregulations described in CRC cell lines, intestinal organoids, CRC stem cells and mouse models, and their possible clinical implications. We highlight how this cancer-related ribosome biogenesis, both at quantitative and qualitative levels, can lead to the synthesis of ribosomes favoring the translation of mRNAs encoding hyperproliferative and survival factors. We also discuss whether cancer-related ribosome biogenesis is a mere consequence of cancer progression or is a causal factor in CRC, and how altered ribosome biogenesis pathways can represent effective targets to kill CRC cells. The association between exacerbated CRC cell growth and alteration of specific steps of ribosome biogenesis is highlighted as a key driver of tumorigenesis, providing promising perspectives for the implementation of predictive biomarkers and the development of new therapeutic drugs.

Iron and hepcidin mediate human colorectal cancer cell growth.

High dietary iron intake is a risk factor for the development of colorectal cancer. However, how iron subsequently impacts the proliferation of colorectal cancer cells remains unclear. This study determined the expression of six iron regulatory genes in twenty-one human colorectal cancer (CRC) biopsies and matched normal colonic tissue. The results show that only hepcidin and ferritin heavy chain expression were increased in CRC biopsies as compared to matched normal tissues. Four established human CRC cell lines, HT-29, HCT-116, SW-620 and SW-480 were subsequently examined for their growth in response to increasing concentrations of iron, and iron depletion. Real time cell growth assay showed a significant inhibitory effect of acute iron loading in HCT-116 cells (IC50 = 258.25 µM at 72 h), and no significant effects in other cell types. However, ten week treatment with iron significantly reduced HT-29 and SW-620 cell growth, whereas no effect was seen in HCT-116 and SW-480 cells. Intracellular labile iron depletion induced the complete growth arrest and detachment of all of the CRC cell types except for the SW-620 cell line which was not affected in its growth. Treatment of starved CRC cells with hepcidin, the major regulator of iron metabolism, induced a significant stimulation of HT-29 cell growth but did not affect the growth of the other cell types. Collectively these results show that iron is central to CRC cell growth in a manner that is not identical between acute and chronic loading, and that is specific to the CRC cell type.

The contribution of growth hormone to mammary neoplasia.

While the effects of growth hormone (GH) on longitudinal growth are well established, the observation that GH contributes to neoplastic progression is more recent. Accumulating literature implicates GH-mediated signal transduction in the development and progression of a wide range malignancies including breast cancer. Recently autocrine human GH been demonstrated to be an orthotopically expressed oncogene for the human mammary gland. This review will highlight recent evidence linking GH and mammary carcinoma and discuss GH-antagonism as a potential therapeutic approach for treatment of breast cancer.

Oncogenic transformation of human mammary epithelial cells by autocrine human growth hormone.

The human growth hormone (hGH) gene is expressed in the normal human mammary epithelial cell and its expression increases concomitant with the acquisition of proliferative lesions. Herein we demonstrate that autocrine production of hGH in human mammary carcinoma cells dramatically enhances anchorage-independent growth in a Janus kinase 2-dependent manner. Forced expression of the hGH gene in immortalized human mammary epithelial cells increased proliferation, decreased apoptosis, altered the cellular morphology and resulted in oncogenic transformation. Autocrine hGH was therefore sufficient to support anchorage-independent growth of immortalized human mammary epithelial cells and tumor formation in vivo. Moreover, autocrine hGH disrupted normal mammary acinar architecture with luminal filling and deregulated proliferation in three-dimensional epithelial cell culture. Autocrine hGH utilized homeobox A1 to govern the transcriptional program required for autocrine hGH-stimulated oncogenic transformation of human mammary epithelial cells, including transcriptional up-regulation of c-Myc, cyclin D1, and Bcl-2. Forced expression of a single orthotopically expressed wild-type gene is therefore sufficient for oncogenic transformation of the immortalized human mammary epithelial cell.

Involvement of the specific nucleolar protein SURF6 in regulation of proliferation and ribosome biogenesis in mouse NIH/3T3 fibroblasts.

The nucleolar proteins which link cell proliferation to ribosome biogenesis are regarded to be potentially oncogenic. Here, in order to examine the involvement of an evolutionary conserved nucleolar protein SURF6/Rrp14 in proliferation and ribosome biogenesis in mammalian cells, we established stably transfected mouse NIH/3T3 fibroblasts capable of conditional overexpression of the protein. Cell proliferation was monitored in real-time, and various cell cycle parameters were quantified based on flow cytometry, Br-dU-labeling and conventional microscopy data. We show that overexpression of SURF6 accelerates cell proliferation and promotes transition through all cell cycle phases. The most prominent SURF6 pro-proliferative effects include a significant reduction of the population doubling time, from 19.8 ± 0.7 to 16.2 ± 0.5 hours (t-test, p < 0.001), and of the length of cell division cycle, from 17.6 ± 0.6 to 14.0 ± 0.4 hours (t-test, p < 0.001). The later was due to the shortening of all cell cycle phases but the length of G1 period was reduced most, from 5.7 ± 0.4 to 3.8 ± 0.3 hours, or by ∼30%, (t-test, p < 0.05). By Northern blots and qRT-PCR, we further showed that the acceleration of cell proliferation was concomitant with an accumulation of rRNA species along both ribosomal subunit maturation pathways. It is evident, therefore, that like the yeast homologue Rrp14, mammalian SURF6 is involved in various steps of rRNA processing during ribosome biogenesis. We concluded that SURF6 is a novel positive regulator of proliferation and G1/S transition in mammals, implicating that SURF6 is a potential oncogenic protein, which can be further studied as a putative target in anti-cancer therapy.

Tumour-Derived Human Growth Hormone As a Therapeutic Target in Oncology.

The growth hormone (GH) and insulin-like growth factor-1 (IGF1) axis is the key regulator of longitudinal growth, promoting postnatal bone and muscle growth. The available data suggest that GH expression by tumour cells is associated with the aetiology and progression of various cancers such as endometrial, breast, liver, prostate, and colon cancer. Accordingly there has been increased interest in targeting GH-mediated signal transduction in a therapeutic setting. Because GH has endocrine, autocrine, and paracrine actions, therapeutic strategies will need to take into account systemic and local functions. Activation of related hormone receptors and crosstalk with other signalling pathways are also key considerations.

In-depth phenotypic characterization of multicellular tumor spheroids: Effects of 5-Fluorouracil.

MultiCellular Tumor Spheroids (MCTS), which mimic the 3-Dimensional (3D) organization of a tumor, are considered as better models than conventional cultures in 2-Dimensions (2D) to study cancer cell biology and to evaluate the response to chemotherapeutic drugs. A real time and quantitative follow-up of MCTS with simple and robust readouts to evaluate drug efficacy is still missing. Here, we evaluate the chemotherapeutic drug 5-Fluorouracil (5-FU) response on the growth and integrity of MCTS two days after treatment of MCTS and for three colorectal carcinoma cell lines with different cohesive properties (HT29, HCT116 and SW480). We found different sensitivity to 5-FU for the three CRC cell lines, ranging from high (SW480), intermediate (HCT116) and low (HT29) and the same hierarchy of CRC cell lines sensitivity is conserved in 2D. We also evidence that 5-FU has a strong impact on spheroid cohesion, with the apparition of a number of single detaching cells from the spheroid in a 5-FU dose- and cell line-dependent manner. We propose an innovative methodology for the chemosensitivity evaluation in 3D MCTS that recapitulates and regionalizes the 5-FU-induced changes within MCTS over time. These robust phenotypic read-outs could be easily scalable for high-throughput drug screening that may include different types of cancer cells to take into account tumor heterogeneity and resistance to treatment.

Hypermethylation of 28S ribosomal RNA in ß-thalassemia trait carriers.

Ribosome biogenesis is the process of synthesis of the cellular ribosomes which mediate protein translation. Integral with the ribosomes are four cytoplasmic ribosomal RNAs (rRNAs) which show extensive post-transcriptional modifications including 2'-O-methylation and pseudouridylation. Several hereditary hematologic diseases including Diamond-Blackfan anemia have been shown to be associated with defects in ribosome biogenesis. Thalassemia is the most important hematologic inherited genetic disease worldwide, and this study examined the post-transcriptional ribose methylation status of three specific active sites of the 28S rRNA molecule at positions 1858, 4197 and 4506 of ß-thalassemia trait carriers and normal controls. Samples from whole blood and cultured erythroid cells were examined. Results showed that site 4506 was hypermethylated in ß-thalassemia trait carriers in both cohorts. Expression of fibrillarin, the ribosomal RNA methyltransferase as well as snoRNAs were additionally quantified by RT-qPCR and evidence of dysregulation was seen. Hemoglobin E trait carriers also showed evidence of dysregulation. These results provide the first evidence that ribosome biogenesis is dysregulated in ß-thalassemia trait carriers.

Translational reprogramming of colorectal cancer cells induced by 5-fluorouracil through a miRNA-dependent mechanism.

5-Fluorouracil (5-FU) is a widely used chemotherapeutic drug in colorectal cancer. Previous studies showed that 5-FU modulates RNA metabolism and mRNA expression. In addition, it has been reported that 5-FU incorporates into the RNAs constituting the translational machinery and that 5-FU affects the amount of some mRNAs associated with ribosomes. However, the impact of 5-FU on translational regulation remains unclear. Using translatome profiling, we report that a clinically relevant dose of 5-FU induces a translational reprogramming in colorectal cancer cell lines. Comparison of mRNA distribution between polysomal and non-polysomal fractions in response to 5-FU treatment using microarray quantification identified 313 genes whose translation was selectively regulated. These regulations were mostly stimulatory (91%). Among these genes, we showed that 5-FU increases the mRNA translation of HIVEP2, which encodes a transcription factor whose translation in normal condition is known to be inhibited by mir-155. In response to 5-FU, the expression of mir-155 decreases thus stimulating the translation of HIVEP2 mRNA. Interestingly, the 5-FU-induced increase in specific mRNA translation was associated with reduction of global protein synthesis. Altogether, these findings indicate that 5-FU promotes a translational reprogramming leading to the increased translation of a subset of mRNAs that involves at least for some of them, miRNA-dependent mechanisms. This study supports a still poorly evaluated role of translational control in drug response.

p44/42 MAP kinase-dependent regulation of catalase by autocrine human growth hormone protects human mammary carcinoma cells from oxidative stress-induced apoptosis.

Previous microarray expression analyses have indicated autocrine human growth hormone (hGH) regulation of genes involved in the oxidative stress response. Expression analysis of antioxidant enzymes revealed that autocrine hGH increased both the mRNA and protein levels of catalase, superoxide dismutase 1 (SOD1), glutathione peroxidase and glutamylcysteine synthetase but not that of SOD2. As a consequence, autocrine hGH increased the antioxidant capacity of mammary carcinoma cells and protected against oxidative stress-induced apoptosis. Catalase activity was increased by autocrine production of hGH in mammary carcinoma cells and a catalase inhibitor abrogated protection from oxidative stress afforded by autocrine hGH. Autocrine hGH transcriptionally regulated catalase gene expression in a p44/42 MAP kinase-dependent manner and inhibition of MEK concordantly abrogated the protective effect of autocrine hGH against oxidative stress-induced apoptosis. Given that increased cellular oxidative stress is a key effector mechanism of specific chemotherapeutic agents, we propose that antagonism of autocrine hGH will improve the efficacy of chemotherapeutic regimes utilized for human mammary carcinoma.