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Cancer spread: targeting platelets to counter metastasis?

Scanning electron microscopy. Here we see how platelets (in blue/purple) attach to two tumour cells (in red) in a pre-clinical mouse model. © Maria Jesus Garcia Leon (unit 1109 Inserm/Université de Strasbourg)

What if our blood platelets[1], which play a major role in maintaining the integrity of our circulatory system, were not always on our side? Research teams from Inserm, Université de Strasbourg and the French Blood Establishment have studied their role in the process of metastasis formation. Their findings suggest that platelets, by binding specifically to circulating cancer cells, promote their survival not just in the bloodstream, but also within metastases. This research, published in Nature Communications, also shows that using treatments to target this binding could fight the formation of metastases without the same bleeding risk[2] as with conventional antiplatelets.

A metastasis is a ‘secondary’ tumour which is usually formed from cancer cells that have broken off from a ‘primary’ tumour before migrating through the blood or lymphatic vessels to settle elsewhere in the body. During their migration, these cancer cells encounter the blood platelets – which prove to be unexpected allies. By binding to the cancer cells, the platelets help them to survive the immune cells in the blood environment and exit the bloodstream to reach their metastasis site.

However, not all cancer cells receive the same protection because some bind to the platelets more easily than others. This dictates their capacity to survive in the blood circulation, target certain vascular regions and, as such, their ability to metastasise. Furthermore, in-depth analyses of lung metastases have shown the presence of large numbers of platelets which may play a role that differs from or complements the role they play in blood vessels.

Two teams led by Inserm researchers Jacky Goetz, from the Molecular Immuno-Rheumatology Unit (Inserm/Université de Strasbourg) and Pierre Mangin, from the Biology and Pharmacology of Blood Platelets: Haemostasis, Thrombosis, Transfusion Unit (Inserm/French Blood Establishment/Université de Strasbourg), studied the moments at which the platelets intervened during the migration of cancer cells to promote their survival and spread. They also looked at how to counter this alliance without using conventional antiplatelet drugs which, by altering bleeding cessation, present the risk of haemorrhage.

In a mouse model, the researchers artificially induced controlled falls in the number of platelets at different stages in the formation of lung metastases. They saw that by removing platelets early (while they were still circulating in the blood), they limited the exit from the blood vessel of the cancer cells with a high affinity for the platelets, and thereby inhibited the formation of metastases. Those cancer cells with low levels of platelet binding were also affected, but only when the platelet level was decreased later, when the lung metastases were already formed.

These observations suggest that as well as protecting the cancer cells in the bloodstream, the platelets could also protect them against the immune system later on – i.e. within the metastases themselves, helping them to proliferate there, explains Goetz. So the aim of our future research will be to understand how the platelets colonise growing metastases.’

 

But how can we circumvent the issue of bleeding risk associated with antiplatelet treatments?

One initial avenue could involve a specific protein found on the platelet surface: glycoprotein VI (GPVI). Previous research has suggested that it could modulate the pro-metastatic activity of platelets without causing bleeding. The expression of this protein could be inhibited by glenzocimab, a molecule currently being evaluated in patients for the treatment of stroke. When they used glenzocimab in their animal model, the scientists saw that it effectively reduced the development of already established lung metastases, without affecting the cessation of bleeding.

‘These observations reinforce the idea of the contribution of platelets to the formation of metastases after cancer cells leave the circulation, explains Mangin. In addition, our research highlights the possibilities of developing new therapeutic strategies which, unlike conventional antiplatelet treatments, would not disrupt bleeding cessation and could therefore be considered in oncology, particularly to reduce the progression of lung metastases. Our two teams are currently working to explore this potential,’ adds the researcher.

This study, carried out on experimental animal models, reconciles previous contradictory data on the role of platelets in the metastatic process.

Studies in humans, for example on cohorts of patients with long-term exposure to antiplatelet agents for cardiovascular indications, or to evaluate the efficacy of oncology treatments in patients on antiplatelet therapy, could be excellent indicators for verifying these findings,’ concludes Goetz.

 

[1]Platelets (thrombocytes) present in the blood are not cells per se, but fragments of large bone marrow cells: megakaryocytes. They play a major role in the rapid cessation of bleeding (haemostasis). As such, platelet counts that are too low can lead to clotting disorders and therefore a risk of bleeding (in the event of injury, for example).

[2] See footnote 1

A neural organoid with an immune environment

organoïde neuronal _ CP Gustave RousyNeural organoid with immune environment magnified twice on the left, 20 times on the right: green macrophages, red and blue neural progenitor cells (fluorescence microscopy). © Gustave Roussy

French, Singaporean and British researchers, led by Prof. Florent Ginhoux, head of a research team at Gustave Roussy/Inserm, have succeeded in demonstrating in a neuronal organoid the role of the brain’s immune environment in its formation and development. The development of these three-dimensional structures integrating neuronal cells and the immune environment is, to date, one of the most complete in vitro models of the human brain. This work is published in Nature.

At Gustave Roussy, these organoids are used to model the development of childhood brain cancers, to understand their mechanisms and discover new avenues of treatment.

“Although microglial cells, immune cells derived from the evolution (differentiation) of primitive macrophages present in the embryonic brain, are known to contribute to multiple aspects of brain development and function, their precise role remains poorly understood and little studied”, says Prof. Florent Ginhoux, director of a research team at Gustave Roussy/Inserm and Senior Principal Investigatorat A*STAR’s Singapore Immunology Network (SIgN).

The use of neuronal organoids to study their functions is one of the avenues currently favored by research.

An organoid is a 3D structure grown in the laboratory which reproduces certain morphological and functional characteristics of a human body organ or tissue. In research, these cell-cultured pseudo-organisms are a new biological model in full development in various fields, notably neurology; most studies of neuron formation (neurogenesis) are based on animal models.

With their 3D structure, the function and properties of these organoids are close to those of a real organ, but not quite as advanced. They measure just one millimeter and have no thoughts, consciousness or emotions. By generating neural organoids from human induced pluripotent stem cells (iPS cells), it is possible to model some of the key features of early human brain development. “However, current approaches do not include microglial cells, explains Prof. Florent Ginhoux.

The international team of researchers led by Prof. Florent Ginhoux has succeeded in producing a new type of model: neuronal organoids with microglia, by cultivating together organoids and primitive-type macrophages, all generated from the same culture of iPS induced stem cells.

Organoids and primitive macrophages are prepared separately. It takes around 25 days to obtain them. The macrophages are then placed in contact with the organoids for a further 15 to 20 days.

In the model developed by the researchers, macrophages colonized the organoids. In this 3D environment, in contact with immature neuronal cells, they differentiated into microglial cells expressing the genes and functions specific to this cell type. These microglial cells proved capable of controlling the differentiation of neuronal precursors (so-called neuronal progenitor cells), thus limiting their multiplication (proliferation), while promoting synapse creation (synaptogenesis) and axon growth (axonogenesis), two key elements in the transmission of the nerve message from neuron to neuron.

A discovery within a discovery

Prof. Florent Ginhoux’s team also observed that the organoids’ microglial cells contain high levels of perilipin-2, a molecule belonging to a family of proteins that coat lipids – including cholesterol – in droplets, enabling them to be stored in and exported from the cells. Armed with these perilipin-2-laden droplets, microglial cells facilitate cholesterol transport to the organoids. The neural progenitor cells that absorb this cholesterol undergo metabolic reprogramming as they differentiate into nerve cells.

The approach developed by Prof. Florent Ginhoux and his colleagues has significantly advanced the complexification of organoid models by integrating microglial cells. This progress is illustrated by the discovery of a key lipid-mediated pathway between microglia and neural progenitor cells, essential for the synthesis of new neurons.

With microglia cells incorporated, the neural organoids we have succeeded in generating are a new, more complete 3D model, closer to reality. We know that the immune system plays a fundamental role in the development of cancers, so at Gustave Roussy we’re going to use them to better understand and discover the mechanisms that regulate the development of pediatric brain tumors“, concludes Prof. Florent Ginhoux.

This work has been supported by the Gustave Roussy Foundation’s “Curing childhood cancer in the 21st century” campaign.

Countering the effects of aging and the occurrence of cancers: new and promising results

By studying immune cells in the lung, researchers from Institut Curie and Inserm have provided new knowledge on the topic.© Fotolia

Cancer and aging are closely linked processes, but the mechanisms underlying this relationship are still not well understood. By studying immune cells in the lung, researchers from Institut Curie and Inserm have provided new knowledge on the topic. They show that targeting ruptures of the nuclear envelope of these cells would represent a new opportunity for therapeutic intervention in age-related diseases, in particular cancer, thus improving the quality of life of the elderly in the long term. Funded by the Fondation ARC, this work has just been published in the journal Nature Aging.

Age is one of the main risk factors for the development of a number of diseases, such as viral or bacterial infections and neuro-degenerative diseases, but also cancers. The economic and societal issues related to the overall aging of the population constitute a major challenge. Furthermore, the notion of “healthy aging” increasingly suggests that targeting aging rather than its consequences is a far better strategy for reducing morbidity among the elderly.

More than two thirds of new cancers diagnosed in France occur in people over the age of 65[1]. The appearance of cancers with age can be explained by the accumulation of genetic alterations during a lifetime, less effective DNA repair mechanisms, and also by an aging immune system with diminished protective functions (immunosenescence). What are the mechanisms that govern this phenomenon? How can we develop strategies to counter immunosenescence?

 

The nucleus of immune cells sensitive to deformations

It is these questions that the Inserm and Institut Curie researchers attempted to answer. With time, DNA becomes fragile and one of the characteristic markers of cell aging is genome instability. When they patrol through the various tissues within the body, the immune system cells are sensitive to deformations which weaken their nucleus and promote DNA breakage. To maintain the structure of the nucleus and thus the genome’s integrity, the cell relies on a dense network of proteins, which include lamins. Among them lamin A/C is studied in particular since it undergoes alterations over the course of aging. In addition, mutations in the gene coding for this protein are known to cause early aging syndromes.

Repeated ruptures of the nuclear envelope lead to DNA damage. It is vital to understand the processes at work in this regard since they promote not just aging of the body, but also the development of cancers. For example, ruptures of the nucleus make the DNA “visible” by damaging proteins, thus triggering a response from the cell that will promote the development of metastases“, explains Dr. Nicolas Manel, Inserm research director and team leader at Institut Curie.

 

Observation en microscopie « 2-photons » de la déformation extrême d’un macrophage alvéolaire, lors de son passage entre deux alvéoles.“Two-photon” microscopy of the extreme deformation of an alveolar macrophage, when it passes between two alveoli. During these migrations the nucleus is also deformed, and it is at this point that the DNA can be damaged.

 

A protein identified in the lung: lamin A/C

At Institut Curie, the Innate Immunity team of Dr. Nicolas Manel, Inserm research director, studied a new experimental model in which the immune system’s cells are deficient in lamin A/C. Researchers looked closely at a population of lung macrophages – alveolar macrophages – which are highly dependent on lamin A/C for their survival. The role of these alveolar macrophages is to constantly monitor the lungs, and they are one of the main entry points for a number of pathogens.

The researchers showed that without lamin A/C, the alveolar macrophages show serious signs of fractures in their nucleus and damage to the DNA, leading to a dramatic drop in their number in the lungs. Furthermore, the surviving alveolar macrophages have many characteristics similar to those of aged alveolar macrophages, and accumulate markers characteristic of aging.

The team also revealed that without lamin A/C in the macrophages, the implantation and growth of lung tumors is a lot faster, encouraged by the malfunction of the aged macrophages.

The loss of lamin A/C would therefore constitute a mechanism for alveolar macrophage aging and a prime study model for understanding how lung cancers develop in the elderly.

Our results open up many new opportunities for studying aging of the immune system, caused by rupture of the nuclear envelope and the decrease in its effectiveness against infections and tumors, in the lungs but also in other organs“, concludes Dr. Nicolas Manel.

These studies are funded in the amount of 2.5 million euros as part of the call for projects “Cancer and Aging” of Fondation ARC for cancer research.

[1] Source INCa : https://www.e-cancer.fr/Professionnels-de-sante/L-organisation-de-l-offre-de-soins/Oncogeriatrie/Epidemiologie

Discovery of an original DNA repair system, bringing new hope for the treatment of breast and ovarian cancer

3D cancer cell © Fotalia

Almost half of breast and ovarian cancers are connected to deficiencies in the biological systems that repair DNA breaks. Researchers from Institut Curie, Inserm and CEA reveal a hitherto unknown DNA repair mechanism involving a protein: Pol, which is able to act during cell division. Published in Nature on 6 September, 2023, their results pave the way for the development of new therapeutic targets for treating cancers, particularly breast and ovarian cancer.

Ultraviolet rays, alcohol, tobacco, genetic predisposition, spontaneous mutations… so many factors constantly damage our genome. Among these lesions, breaks that affect both DNA strands simultaneously are the most harmful. Our body is constantly repairing this damage through several repair systems, including homologous recombination. However, when these mechanisms fail (due to a genetic mutation, for example), they may cause cancer to occur. Also, the proven correlation between these homologous recombination deficiencies and the aggressiveness of cancers or their resistance to current chemotherapies underlines the pressing need for targeted cancer-fighting therapies.

 

A new major player in DNA repair: Pol

A few years ago, a new player in DNA repair (polymerase theta or PolꝊ) was identified as a therapeutic hope for treating these cancers[1]. The “Alternative DNA Repair Mechanisms in Human Cancers”[2] team headed by Dr. Raphaël Ceccaldi, Inserm researcher at Institut Curie, has just clarified the mechanism for the action of this polymerase and the reason why this enzyme is vital to the development of breast and ovarian cancers.

For the first time scientists have shown that PolꝊ works where other DNA repair pathways do not. While it was believed that DNA repair was impossible during cell division (when DNA is extremely compacted), the team from Institut Curie showed that PolꝊ is active specifically during mitosis when the other contributors to repair were proven ineffective.

“Along with my team at Institut Curie, we looked closely at the mechanisms that the cell puts in place to repair its DNA, enabling cancer cells to survive. It is by understanding such mechanisms that we can build new options to thwart cancer“, explains Dr. Raphaël Ceccaldi, Inserm researcher and team leader at Institut Curie. “Our discoveries on the role and functioning of polymerase theta in maintaining integrity of the genome gives us hope for new therapeutic strategies against cancer, particularly breast and ovarian cancer.“

 

Genome integrity highly preserved by Pol

Through a collaboration with the team of Dr. Sophie Zinn-Justin, researcher at the CEA (Laboratoire de Biologie Structurale et Radiobiologie), researchers went even further, showing that, in order to repair DNA, PolꝊ had to be activated by an enzyme specifically present during cell division. In addition, the mechanisms that enable this activation of PolꝊ seem to have been extremely well-preserved throughout evolution. This suggests that they play an important role in maintaining the stability of the genome needed for development of eukaryotic organisms.

 

A therapeutic hope for breast and ovarian cancer

The team of Dr. Raphaël Ceccaldi also discovered that inhibiting PolꝊ during cell division by mitosis prevents the proper repair of DNA and as a result leads to the death of cancer cells. With almost half of breast and ovarian cancers showing DNA repair deficiencies by homologous recombination, this step therefore represents a milestone in the fight against these cancers. Clarifying the molecular mechanisms governing the use and regulation of PolꝊ could ultimately lead to the development of new therapeutic targets for treating these cancers.

CancerPolꝊ (green) marks DNA breaks (gH2AX, red) in the mitotic chromosomes (DAPI, blue) – Scale 5μM

 

[1] Ceccaldi R, Liu JC, Amunugama R, Hajdu I, Primack B, Petalcorin M, O’Connor KW, Konstantinopoulos PA, Elledge SJ, Boulton SJ, Yusufzai T, D’Andrea AD. Homologous recombination (HR)-deficient tumors are hyper-dependent on POLQ-mediated repair. Nature. 2015 Feb 12;518(7538):258-62.

[2]Cancer, Heterogeneity, Instability and Plasticity unit – CHIP (U830, Institut Curie/Inserm)

Immunotherapy for blood cancer: remote destruction of tumor cells demonstrated

Attaque d’une tumeur par des cellules CAR TCAR T cells attacking a tumor, visualized using intravital imaging. Live tumor cells are shown in white, apoptotic tumor cells in blue and CAR T cells in green. © Morgane Boulch, Philippe Bousso / Institut Pasteur

The aim of immunotherapy strategies is to use cells in the patient’s own immune system to destroy tumor cells. CAR T cell therapy is an immunotherapy that is effective in treating blood cancer. Around 35,000 people are affected by blood cancer each year in France, with 1.24 million cases worldwide. By closely investigating some of the immune cells generated during this therapy, known as CD4 T cells, scientists from the Institut Pasteur and Inserm, in collaboration with clinicians from the Paris Public Hospital Network (AP-HP), discovered that these cells are capable of remotely neutralizing tumor cells by producing interferon gamma (IFN-γ). This study raises new hopes for blood cancer patients with an incomplete response to CAR T cell therapy and for cancers sensitive to IFN-γ. The results were published on May 29, 2023 in the journal Nature Cancer.

CAR T cell therapy is an immunotherapy that has produced remarkable results in treating certain types of leukemia or lymphoma. But some patients who receive this treatment relapse because their tumor cells evade the therapy. A multidisciplinary team of scientists from the Institut Pasteur and Inserm and clinicians from the Paris Public Hospital Network (AP-HP) sought to shed light on how the therapy works with the aim of obtaining even more effective responses.

The principle of CAR T cell therapy is to isolate the patient’s T cells, genetically modify them so that they specifically target tumor cells, and multiply them before injecting them back into the patient in large numbers. This army of killer CAR T cells is composed of CD4 T cells and CD8 T cells in varying proportions from one patient to the next. While we know that CD8 killer T cells need to come into direct contact with tumor cells to destroy them, the mechanism of action of CD4 T cells had not previously been fully explored.

The research team studied these CD4 CAR T cells more closely and revealed a very interesting property: their ability to kill tumor cells remotely by secreting a molecule involved in the immune response, interferon gamma (IFN-γ).

For some types of cancer that are sensitive to IFN-γ, this destruction mechanism is highly efficient. We also observed that, among patients with a high quantity of CD4 T cells, those that produce a large amount of IFN-γ respond better to treatment,” explains Philippe Bousso, Head of the Institut Pasteur’s Dynamics of Immune Responses Unit (Inserm 1223) and last author of the study.

To reveal the novel mechanism of action of these remote killer cells, the scientists began by exploring preclinical models to analyze the mechanism in detail, in particular using in vivo imaging techniques, then they checked the relevance of the results on samples from patients.

CAR T cells attacking a tumor, visualized using intravital imaging. Live tumor cells are shown in white, apoptotic tumor cells in blue and CAR T cells in green. The white circles indicate the destruction of tumor cells when they come into direct contact with CAR T cells, and the red circles show the remote destruction of tumor cells. © Morgane Boulch, Philippe Bousso / Institut Pasteur

 

This discovery opens new avenues for adjusting treatments to prevent tumor cells evading CAR T therapy. It also raises new therapeutic hopes for patients, offering the possibility of a more personalized treatment approach whereby a larger quantity of CD4 CAR T cells can be used to activate IFN-γ depending on tumor cell sensitivity,” comments Philippe Bousso.

By developing a better understanding of how CD4 T killer cells work, it may also be possible to extend the scope of this therapy to other solid tumor cancers that are sensitive to IFN-γ. The clinical data will be confirmed on other cohorts.

 

This research was funded by the institutes cited above, and also by the French National Cancer Institute (INCa) and the European Research Council.

Inflammation and cancer: identifying the role of copper paves the way for new therapeutic applications

équipe CurieThe research team developed a “drug prototype” capable of mitigating both the mechanisms of inflammation and the processes potentially involved in metastatic spread. © Institut Curie / BELONCLE Frank

For the first time, researchers from Institut Curie, the CNRS and Inserm have uncovered a previously unknown chain of biochemical reactions. This chain involves copper and leads to metabolic and epigenetic alterations[1] that activate inflammation and tumorigenesis. But there is more; the research team developed a “drug prototype” capable of mitigating both the mechanisms of inflammation and the processes potentially involved in metastatic spread. Published in the journal Nature on April 26, 2023, these results provide hope for new therapeutic opportunities to control inflammation and cancer.

Inflammation is a complex biological process that can eradicate pathogens and promotes repair of damaged tissues. However, deregulation of the immune system can lead to uncontrolled inflammation and produce lesions instead. Inflammation is also involved in cancer. The molecular mechanisms underlying inflammation are not fully understood, and so developing new drugs represents a significant challenge.

As far back as 2020, Dr. Raphaël Rodriguez, CNRS research director and head of the Chemical Biology team at Institut Curie (Equipe Labellisé Ligue Contre le Cancer) at the Cellular and Chemical Biology laboratory (Institut Curie/CNRS/Inserm), had shed new light on a membrane receptor called CD44, which marks immune responses, inflammation and cancer progression. Dr. Rodriquez and his team showed that CD44 helped import iron into cell[2], triggering a series of reactions leading to activation of genes involved in the metastatic process.

“This is a cell plasticity phenomenon we continued to study, investigating other metals potentially internalized by CD44, notably copper,” he explains.

 

Copper causing epigenetic alterations

Along with his colleagues[3], Dr. Rodriguez has now reached a new milestone.

The research team managed to identify a signaling pathway involving copper and leading to the expression of pro-inflammatory genes in macrophages, the cells present in all tissues and playing an important role in innate immunity.

Once internalized in macrophages, copper enters into the mitochondria (the organelle responsible for cell respiration and energy production), where it catalyzes the oxidation of NADH into NAD+  (nicotinamide adenine dinucleotide, a molecule needed for the activity of certain enzymes). The increase of NAD+ in cells enables the activity of certain enzymes involved in the production of metabolites essential for epigenetic regulation. These metabolites thus, contribute to the activation of genes involved in inflammation.

 

Inflammation and cancer: shared molecular mechanisms

The scientists did not stop there, they also designed molecules able to bind to copper, inspired from the structure of metformin.[4] By testing these new molecules on models of acute inflammation, they found that a synthetic dimer of metformin, LCC-12 (also termed Supformin), reduced activation of macrophages and attenuated inflammation.

“Our work has enabled us to develop a drug prototype that inactivates copper chemistry in the cell’s metabolic machinery, thus blocking expression of the genes involved in inflammation”, explains Dr. Rodriguez.

To finish, they applied this therapeutic strategy to cancer cell models engaged in an epithelial-mesenchymal transition[5]. Here again, Supformin blocked the cellular mechanism and thus the cell transformation.

“The genes activated in cancer cells are not the same as those expressed in immune cells, but the chain reaction leading to epigenetic alterations is identical”, explains Dr. Rodriguez.

These results thus reveal the role of copper in cancer cells and their ability to adopt a metastatic nature.

Dr. Raphaël Rodriguez concludes: “Our study reveals that the inflammatory and cancer processes depend on similar molecular mechanisms and could therefore in the future benefit from similar innovative therapies, such as those tested with Supformin.”

The explanations of Dr. Raphaël Rodriguez in video :

 

[1]Epigenetics is the study of the mechanisms at play in gene regulation, which is essential to the action of cells and to maintaining their identity. Unlike genetic mutations, which are permanent, epigenetic modifications on DNA or histones are reversible.

[2] Read the press release “Cancer: a new mechanism that regulates cell activity involving iron”: https://curie.fr/sites/default/files/medias/documents/2020-08/CPCNRS-CD44ferCancer-FR-emb.pdf

[3] This study was conducted at Institut Curie, in the Cellular and Chemical Biology unit (Institut Curie, CNRS, Inserm), in collaboration with UVSQ, Raymond Poincaré hospital (AP-HP), Gustave Roussy hospital, the Institut de chimie moléculaire et des matériaux d’Orsay (CNRS/University Paris-Saclay), the Multimodal Imaging Center (CNRS/Institut Curie/Inserm/University Paris-Saclay), the Center for Infection and Immunity of Lille (CNRS/Inserm/Institut Pasteur de Lille/CHU of Lille/University of Lille), Institute of Pharmacology and Structural Biology (CNRS/University of Toulouse III) along with British and Australian researchers.

[4]Metformin is a treatment used for Type-2 diabetes, and is able to form a bimolecular complex with copper.

[5] Epithelial-mesenchymal transition is the first step in enabling cancer cells to metastasize.

Cystic Fibrosis: A New Therapeutic Avenue Thanks to Research Into an Edible Mushroom

Lepista flaccida, champignon comestible

Lepista flaccida, an edible mushroom found in the northern hemisphere, was the focus of research by French teams into ways of correcting certain genetic mutations known as nonsense mutations. © MNHN/CNRS – Christine Bailly

A molecule obtained from an edible mushroom could open up therapeutic avenues for patients with cystic fibrosis, the most frequent rare genetic disease. A team led by Fabrice Lejeune, Inserm researcher at the Cancer Heterogeneity, Plasticity and Resistance to Therapies laboratory[1] (Inserm/ CNRS/ Université de Lille/Institut Pasteur de Lille/University Hospital Lille) tested the effects of 2,6-diaminopurine (DAP), one of the active principles contained in the Lepista flaccida mushroom, in different experimental models of the disease.  The scientists have shown that this molecule could be of therapeutic value in patients with cystic fibrosis linked to a particular type of mutation known as a nonsense mutation. Their findings have been published in Molecular Therapy.

Around 6,000 people in France have cystic fibrosis, a genetic disease that primarily affects digestive and respiratory function, and has a 40 to 50-year life expectancy. Nevertheless, therapeutic innovations have improved patient prognosis in recent years. Treatments are now available for the vast majority of patients – those whose disease is caused by the delta F508 mutation of the CFTR gene. In these patients, the CFTR protein (coded by the CFTR gene) is present in small amounts but is dysfunctional. The molecules currently available are able to correct this dysfunction and significantly improve their clinical symptoms.

However, they are not effective in the 10% of patients for whom the protein is completely absent, as is the case when the disease is linked to a nonsense mutation (see box).

Nonsense Mutations and Genetic Diseases

DNA is made up of nucleotides, organic compounds that code the amino acids implicated in the synthesis of the proteins needed for the body to function correctly. In practice, nonsense mutations introduce a “stop codon” in the mutated gene, i.e. a sequence of nucleotides that brings the synthesis of the corresponding protein to a premature halt. From that point, the protein is no longer produced, leading to the onset of the clinical symptoms of the disease.

Identifying ways to correct nonsense mutations is therefore an important challenge for researchers studying genetic diseases and who hope to develop new therapeutic options against cystic fibrosis.

In this context, Inserm researcher Fabrice Lejeune and his team[2] made an innovative finding in 2017 by showing that extracts of a commonplace edible mushroom known as Lepista flaccida could repair nonsense mutations in three cell lines isolated from cystic fibrosis patients. A few years later in 2020, Lejeune and his team published a study identifying the active principle in the mushroom that is capable of correcting the nonsense mutations associated with the UGA stop codon – the most common of the three stop codons of the human genetic code. The active principle concerned was 2,6 diaminopurine (DAP).

In their latest research, the scientists tested the effects of this molecule in four experimental models of cystic fibrosis: animal models of the disease, developed in the laboratory; cell lines; patient cells and organoids. This diversity of models makes it possible to be as close as possible to what is happening in the patient’s body, in order to assess the potential therapeutic benefits they may obtain.

The results obtained by the team suggest that DAP corrects the nonsense mutation in the different models studied, by re-establishing protein production and effectively restoring the function of the mutated gene.

In clinical terms, this results in an improvement in symptoms in animals. The treatment with DAP makes it possible to restore CFTR expression in the lungs and intestines as well as the function of this protein, significantly reducing the premature mortality observed prior to administration of this molecule.

In addition, the research team has also shown that DAP can be given orally and that it is distributed effectively throughout the body for around two hours. These characteristics are also a positive point when it comes to considering DAP as a serious therapeutic avenue, as this means that we could reach all the tissues in the body while limiting the duration of exposure to the molecule, thereby reducing possible side effects.

“DAP could represent the first molecule capable of providing therapeutic benefit to patients with cystic fibrosis linked to a nonsense mutation and, more broadly, to patients with a genetic disease linked to a nonsense mutation,” explains Lejeune.

These results pave the way for a potential clinical trial in the coming years to test the efficacy of the molecule in patients. Before this, the goal is to develop the best possible formulation for the drug and to carry out toxicity tests to ensure its safety in humans. In the shorter term, the teams also want to test DAP in models of other rare genetic diseases, particularly Duchenne muscular dystrophy and Rett’s syndrome, for which over 60% of patients are affected by nonsense mutations.

 

[1] Cancer Heterogeneity, Plasticity and Resistance to Therapies laboratory at the ONCOLille institute

[2]The following research units also contributed to these findings: Communication Molecules and Adaptation of Micro-organisms (CNRS/MNHN), Biometrics and Evolutional Biology Laboratory (CNRS/Université Claude Bernard Lyon 1/VetAgro Sup), Lille Platforms in Biology and Health (PLBS) (CNRS/University Hospital Lille/Inserm/Institut Pasteur Lille/Université Lille), Strasbourg Platform for Integrative Biological Chemistry (CNRS/Université de Strasbourg).

A potential therapy to reduce the side effects of a chemotherapy

Convergent effect of cisplatin and KW6002 on DNA double-strand breaks in lung tumor cells. Blue corresponds to cell nuclei and red to a protein that marks DNA damage © Dewaeles et al

Cisplatin is a chemotherapy indicated to fight tumors in many types of cancer. However, it does have major side effects – especially kidney toxicity, that can lead to acute kidney failure. In addition, patients treated with cisplatin also often report high levels of neuropathic pain. Scientists from Inserm, Université de Lille, University Hospital Lille, CNRS and Institut Pasteur de Lille within the CANTHER and Lille Neuroscience & Cognition laboratories, in collaboration with researchers from Michigan State University (USA), have identified a drug that could be a game changer for patients. Istradefylline, which is already approved for Parkinson’s disease, could not only reduce the harmful effects of cisplatin but also improve its anti-tumor properties. These findings will now need to be confirmed in a clinical trial. The study is published in The Journal of Clinical Investigation.

Cisplatin is a chemotherapy used to treat several types of cancer, in particular lung, ovarian and testicular cancers. While its anti-tumor efficacy has been proven, cisplatin promotes side effects. These include intense pain (peripheral neuropathy) and kidney damage, leading to acute kidney failure in one third of cases. Currently, there is no effective solutions to limit side effects for patients exposed to cisplatin.

An international work conducted by Christelle Cauffiez, David Blum and Geoffroy Laumet[1] have now identified a molecule that reduces cisplatin-induced side effects, while preserving or even potentiating its anti-tumor properties.

 

A Parkinson’s disease drug

The scientists focused on a drug called istradefylline, which is already approved in the USA and Japan for the treatment of Parkinson’s disease. Biologically, this compound blocks the adenosine receptors receptors at the surface of cells.

Blum’s team, which is working on neurodegenerative diseases, had previously observed an increased density of these receptors in the brains of patients with dementia, a phenomenon involved in the development of these diseases. Interestingly, a comparable increase of adenosine receptors was also observed by Cauffiez’s team in the kidneys, under exposure to cisplatin.

With this in mind, the scientists decided to join forces with Laumet’s lab, a specialist of  cisplatin-induced neuropathic pain, to test the impact of istradefylline to mitigate the harmful effects of cisplatin.

 

Findings to confirm in a clinical trial

Their experiments, conducted on animal and cellular models, indeed pointed towards a beneficial role of istradefylline. In mice exposed to cisplatin, the molecule not only reduced kidney damages but also prevented neuropathic pain.

In addition, cisplatin’s ability to reduce tumor growth was increased in the animals receiving istradefylline – an effect subsequently confirmed in cell models.

Before considering the widespread application of this therapeutic approach to patients with cancer, these findings must however first be consolidated by organizing a rigorous clinical trial. The fact that istradefylline is already used in humans to treat another disease already constitutes an interesting perspective.

“In fact, we already have a lot of clinical data showing that this molecule is safe. While it is necessary to conduct a clinical study to test its efficacy in reducing the side effects of the chemotherapy, the possibility of therapeutic repositioning is a promising perspective for improving patient care in the short term,” the researchers point out.

 

[1] from the CANTHER laboratory (Inserm/Institut Pasteur de Lille/CNRS/Université de Lille/University Hospital Lille), the Lille Neuroscience & Cognition laboratory (Inserm/Université de Lille/University Hospital Lille) and the Department of Physiology of Michigan State University

Infertility: New Avenues to Understand the Harmful Effects of Chemotherapy

Immunostaining of a mouse testicle section

Immunostaining of a mouse testicle section, with (in red) the undifferentiated germ cells and (in green) the GFP protein reflecting TGR5 receptor expression in this study model. ©David Volle/Inserm

Infertility is a public health problem affecting millions of couples in France. Among the possible causes, chemotherapy has been singled out as having particularly harmful effects on the fertility of both women and men. In order to better prevent and restore fertility in cancer survivors, understanding the mechanisms behind these negative effects is a priority. In a new study, researchers from Inserm, CNRS and Université Clermont Auvergne investigated a receptor found on male germ cells that produce gametes, their aim being to find out more about its role in chemotherapy-related infertility. Their findings, published in Advanced Science, pave the way for a better understanding of male infertility and the development of treatments to reduce the risk of sterility from chemotherapy.

Around 3.3 million people in France are directly affected by infertility. Concerning both men and women, it has continued to increase in recent years, making it a major public health problem [1].

While there are many causes of infertility, it is currently well established that cancer treatments, including chemotherapy, can have particularly harmful effects on male and female fertility. Although cancer therapies have improved in recent years, tackling this issue is becoming a matter of urgency, as an increasing number of cancer survivors will be affected by infertility problems.

For almost 15 years, Inserm researcher David Volle and his team at the Genetics, Reproduction and Development Laboratory (Inserm/CNRS/Université Clermont Auvergne) have sought to improve their understanding of the biological mechanisms underlying infertility. Part of their research focuses on the impact of chemotherapy on male fertility, with the longer-term objective of identifying avenues to counter the adverse effects of this treatment.

In their new study, the researchers looked at TGR5 receptors, which are present on cell membranes, in order to understand their role in the harmful effects of chemotherapy.

TGR5 receptors are widely studied in the context of metabolic diseases, such as diabetes and obesity. They are activated by bile acids – molecules produced in the liver that regulate certain physiological functions, including blood glucose and energy expenditure.

 However, previous research by the team had shown that these receptors are also present in germ cells, the cells that produce gametes. In mouse models mimicking liver disease, with elevated bile acid levels, the scientists had found that the TGR5 receptors on germ cells were activated – which was associated with increased sterility in animals.

Germ cell death

To further understand the impact of TGR5 on fertility in the context of chemotherapy, the scientists in their latest study exposed mice to a chemotherapy agent called busulfan. They then showed that the chemotherapy induces the death of some of the germ cells in healthy mice, thereby affecting their fertility. “The fact that it is the germ cells, at that point undifferentiated, which are affected is particularly problematic because we are talking about the reserve of cells that produce gametes. This can reduce their renewal and contribute to post-chemotherapy infertility,” says Volle.

However, in mice that have been genetically modified to have an absence of TGR5 receptors, the effects of chemotherapy on germ cells are attenuated. This results in an accelerated return of fertility in these busulfan-treated mice compared with the control mice.

Our study has therefore improved our understanding of the molecular mechanisms involved in the harmful effects of chemotherapies on germ cells and fertility. These findings show that TGR5 receptors play an important role in the harmful effects of chemotherapy on infertility,” adds Volle.

In the longer term, the objective is to develop methods to modulate TGR5 receptor activation in a targeted manner within germ cells, in order to protect them and restore fertility after chemotherapy.

The idea is also to assess whether these data can be extrapolated to other disease contexts in which TGR5 receptor activity could be modulated, such as obesity and diabetes, conditions known to impair fertility.

In addition, in parallel to this research, the team observed that even when fertility was maintained in mice exposed to chemotherapy, the quality of the gametes was affected. The scientists will therefore now endeavor to understand both the quantitative and qualitative impacts on germ cells in order to limit not just fertility disorders but also the longer-term consequences on the offspring of animals.

 

[1] A report requested by the French Minister of Health and the Secretary of State for Childhood and Family in February 2022 outlines a national strategy to combat infertility: https://solidarites- sante.gouv.fr/IMG/pdf/rapport_sur_les_causes_d_infertilite.pdf

Colon cancer: how mutation of the APC gene disrupts lymphocyte migration

Migrating human T lymphocytes

In patients with familial adenomatous polyposis, a genetic disease predisposing to colon cancer, mutations of the APC gene induce the formation of intestinal polyps, but also reduce immune system activity. In a new study, researchers from the Institut Pasteur, INSERM(1) and Université Paris Cité describe the mechanisms that modify the structure of T lymphocytes and hinder their migration towards the tumors to be destroyed. This discovery, published in the journal Science Advances on April 13, 2022, provides new perspectives on the migration of immune cells, a key process in antitumor immune defense.

As its name suggests, familial adenomatous polyposis is transmitted from generation to generation. The cause: mutations of the tumor suppressor gene APC (adenomatous polyposis coli). People who inherit these mutations develop hundreds, possibly thousands, of polyps in their colon from adolescence, then colorectal cancer(2) in adulthood if the polyps are not surgically removed. “As it’s a hereditary disease, all of the body’s cells carry the mutation and can be affected in different ways”, explains Andrés Alcover, Head of the Lymphocyte Cell Biology Unit at the Institut Pasteur and joint senior author of the study. “Today we know that these mutations disrupt the functioning of colon cells but also cells of the immune system”.

In previous studies, the team of researchers from the Institut Pasteur, CNRS and Inserm – funded by the French Cancer League since 2018(3) – demonstrated the dual impact of APC mutations.

Not only do these mutations prevent intestinal epithelial cells from differentiating correctly and cause them to form tissue growths (polyps), they also adversely affect the functioning of immune cells, thereby preventing them from effectively combating polyps and tumors. Two mechanisms that together promote the growth of tumors.

In order to better understand what prevents immune cells from fulfilling their role, the researchers this time decided to take a closer look at the T lymphocytes whose mission is to detect and destroy tumors by infiltrating them. To this end, biologists and clinical research physicians of the Institut Pasteur’s ICAReB platform, Dr. Hélène Laude and Dr. Marie-Noëlle Ungeheuer, approached the patient association POLYPOSES FAMILIALES France. A new clinical research project involving the association recruited patient volunteers for the collection of blood samples. “Thanks to the association, we met patients and also clinicians specialized in polyposis. We learned a lot about this complex pathological condition, the experience of patients and families, and the different levels of disease severity. We recognize the valuable role of the patients, who were highly motivated to take part in the study, and the input of specialists”, pointed out Andrés Alcover.

The naturally mutated T lymphocytes present in the blood of these patients were cultured then subjected to several in vitro experiments. Using several microdevices – filters, channels, protein substrates and layers of vascular endothelial cells – the researchers could compare the behavior of diseased lymphocytes with that of lymphocytes from healthy volunteers.

They studied how lymphocytes moved along biological surfaces similar to blood vessel walls, but also how easily they could separate cells and cross tightly packed cell layers.

“In order to move along blood vessel walls, cross them and reach the tumor to be infiltrated, healthy lymphocytes change their morphology. Something akin to a large adhesive foot, supported by the lymphocyte’s cytoskeleton, grows longer in the direction of migration. This polarization is essential for movement in the right direction,” explains Marta Mastrogiovanni, researcher in the Institut Pasteur’s Lymphocyte Cell Biology Unit and lead author of the study. In mutated lymphocytes, the microtubules making up the cytoskeleton are disorganized and there are fewer adhesion proteins. The cells lose their polarity and their ‘muscles'”.

Although the mutated T lymphocytes are not necessarily moving more slowly than healthy lymphocytes, they adhere less well to the walls and have more difficulty moving in a given direction and passing through the walls. In short, this research showed their migration to be less effective. “This discovery is important because the motility of immune cells is a key process in antitumor immune defense. “We know that the immune system is very important in combating pathogens but we sometimes forget that it also contributes to combating cancer cells”, concludes Vincenzo Di Bartolo, researcher in the Institut Pasteur’s Lymphocyte Cell Biology Unit and joint senior author of the study.

 

(1) Collaborative project: Institut Pasteur, Department of Immunology and  and Center for Translational Science (CRT, ICAReB), and Institut Pasteur, Institut Cochin, Institut Curie, and Institut Pierre-Gilles de Gennes.

(2) Familial adenomatous polyposis accounts for 1% of all colorectal cancers. 

(3) Funding via the French Cancer League (La Ligue Contre Le Cancer), 2018-2022 “Équipe Labellisée” program, the Institut Pasteur and Inserm. Marta Mastrogiovanni was funded by the Pasteur-Paris University International Doctoral Program and the European Union Horizon 2020 Research and Innovation Programme under Marie Sklodowska-Curie grant agreement 665807 and La Ligue Contre Le Cancer, doctoral grant 4th year of PhD.

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