Les chercheurs et cliniciens du SIRIC publient !

Int. J. of Oncol. 59: 43, 2021

Anti-Müllerian hormone concentration regulates activin receptor-like kinase-2/3 expression levels with opposing effects on ovarian cancer cell survival

Maëva Chauvin , Véronique Garambois , Sylvie Choblet , Pierre-Emmanuel Colombo, Myriam Chentouf , Laurent Gros , David-Paul De Brauwere , Martine Duonor-Cerutti , Karen Dumas , Bruno Robert , Marta Jarlier , Pierre Martineau , Isabelle Navarro-Teulon , David Pépin, Thierry Chardès and André Pèlegrin

Anti‑Müllerian hormone (AMH) type II receptor (AMHRII) and the AMH/AMHRII signaling pathway are potential therapeutic targets in ovarian carcinoma. Conversely, the role of the three AMH type I receptors (AMHRIs), namely activin receptor‑like kinase (ALK)2, ALK3 and ALK6, in ovarian cancer remains to be clarified. To determine the respective roles of these three AMHRIs, the present study used four ovarian cancer cell lines (COV434‑AMHRII, SKOV3‑AMHRII, OVCAR8, KGN) and primary cells isolated from tumor ascites from patients with ovarian cancer. The results demonstrated that ALK2 and ALK3 may be the two main AMHRIs involved in AMH signaling at physiological endogenous and supraphysiological exogenous AMH concen‑ trations, respectively. Supraphysiological AMH concentrations (25 nM recombinant AMH) were associated with apoptosis in all four cell lines and decreased clonogenic survival in COV434‑AMHRII and SKOV3‑AMHRII cells. These biolog‑ ical effects were induced via ALK3 recruitment by AMHRII, as ALK3‑AMHRII dimerization was favored at increasing AMH concentrations. By contrast, ALK2 was associated with AMHRII at physiological endogenous concentrations of AMH (10 pM). Based on these results, tetravalent IgG1‑like bispecific antibodies (BsAbs) against AMHRII and ALK2, and against AMHRII and ALK3 were designed and evaluated. In vivo, COV434‑AMHRII tumor cell xenograft growth was signifi‑ cantly reduced in all BsAb‑treated groups compared with that in the vehicle group (P=0.018 for BsAb 12G4‑3D7; P=0.001 for all other BsAbs). However, the growth of COV434‑AMHRII tumor cell xenografts was slower in mice treated with the anti‑AMRII‑ALK2 BsAb 12G4‑2F9 compared with that in animals that received a control BsAb that targeted AMHRII and CD5 (P=0.048). These results provide new insights into type I receptor specificity in AMH signaling pathways and may lead to an innovative therapeutic approach to modulate AMH signaling using anti‑AMHRII/anti‑AMHRI BsAbs.

Nat Commun. 2021 Mar 19;12(1):1716

FTO-mediated cytoplasmic m6Am demethylation adjusts stem-like properties in colorectal cancer cell

Sébastien Relier, Julie Ripoll , Hélène Guillorit , Amandine Amalric, Cyrinne Achou , Florence Boissière , Jérôme Vialaret , Aurore Attina , Françoise Debart, Armelle Choquet, Françoise Macari, Virginie Marchand, Yuri Motorin, Emmanuelle Samalin, Jean-Jacques Vasseur, Julie Pannequin, Francesca Aguilo, Evelyne Lopez-Crapez, Christophe Hirtz, Eric Rivals , Amandine Bastide , Alexandre David 

Cancer stem cells (CSCs) are a small but critical cell population for cancer biology since they display inherent resistance to standard therapies and give rise to metastases. Despite accruing evidence establishing a link between deregulation of epitranscriptome-related players and tumorigenic process, the role of messenger RNA (mRNA) modifications in the regulation of CSC properties remains poorly understood. Here, we show that the cytoplasmic pool of fat mass and obesity-associated protein (FTO) impedes CSC abilities in colorectal cancer through its N6,2′-O-dimethyladenosine (m6Am) demethylase activity. While m6Am is strategically located next to the m7G-mRNA cap, its biological function is not well understood and has not been addressed in cancer. Low FTO expression in patient-derived cell lines elevates m6Am level in mRNA which results in enhanced in vivo tumorigenicity and chemoresistance. Inhibition of the nuclear m6Am methyltransferase, PCIF1/CAPAM, fully reverses this phenotype, stressing the role of m6Am modification in stem-like properties acquisition. FTO-mediated regulation of m6Am marking constitutes a reversible pathway controlling CSC abilities. Altogether, our findings bring to light the first biological function of the m6Am modification and its potential adverse consequences for colorectal cancer management.

https://www.igf.cnrs.fr/index.php/fr/actus-fr/253-actus-29-03-2021-fr

Mol Cell. 2021 Mar 18;81(6):1231-1245

TopBP1 assembles nuclear condensates to switch on ATR signaling

Frattini C, Promonet A, Alghoul E, Urbach S, Basbous J, Constantinou A

ATR checkpoint signaling is crucial for cellular responses to DNA replication impediments. Using an optogenetic platform, we show that TopBP1, the main activator of ATR, self-assembles extensively to yield micrometer-sized condensates. These opto-TopBP1 condensates are functional entities organized in tightly packed clusters of spherical nano-particles. TopBP1 condensates are reversible, occasionally fuse, and co-localize with TopBP1 partner proteins. We provide evidence that TopBP1 condensation is a molecular switch that amplifies ATR activity to phosphorylate checkpoint kinase 1 (Chk1) and slow down replication forks. Single amino acid substitutions of key residues in the intrinsically disordered ATR activation domain disrupt TopBP1 condensation and consequently ATR/Chk1 signaling. In physiologic salt concentration and pH, purified TopBP1 undergoes liquid-liquid phase separation in vitro. We propose that the actuation mechanism of ATR signaling is the assembly of TopBP1 condensates driven by highly regulated multivalent and cooperative interactions.

https://pubmed.ncbi.nlm.nih.gov/33503405/

Lancet Oncol. 2021 Feb;22(2):256-266

Cytoreductive surgery plus hyperthermic intraperitoneal chemotherapy versus cytoreductive surgery alone for colorectal peritoneal metastases (PRODIGE 7) : a multicentre, randomised, open-label, phase 3 trial

Quénet F, Elias D, Roca L, Goéré D, Ghouti L, Pocard M, Facy O, Arvieux  C, Lorimier G, Pezet D, Marchal F, Loi V, Meeus P, Juzyna B, de Forges H, Paineau J, Glehen O, UNICANCER-GI Group and BIG Renape Group

Background: The addition of hyperthermic intraperitoneal chemotherapy (HIPEC) to cytoreductive surgery has been associated with encouraging survival results in some patients with colorectal peritoneal metastases who were eligible for complete macroscopic resection. We aimed to assess the specific benefit of adding HIPEC to cytoreductive surgery compared with receiving cytoreductive surgery alone.

Methods: We did a randomised, open-label, phase 3 trial at 17 cancer centres in France. Eligible patients were aged 18-70 years and had histologically proven colorectal cancer with peritoneal metastases, WHO performance status of 0 or 1, a Peritoneal Cancer Index of 25 or less, and were eligible to receive systemic chemotherapy for 6 months (ie, they had adequate organ function and life expectancy of at least 12 weeks). Patients in whom complete macroscopic resection or surgical resection with less than 1 mm residual tumour tissue was completed were randomly assigned (1:1) to cytoreductive surgery with or without oxaliplatin-based HIPEC. Randomisation was done centrally using minimisation, and stratified by centre, completeness of cytoreduction, number of previous systemic chemotherapy lines, and timing of protocol-mandated systemic chemotherapy. Oxaliplatin HIPEC was administered by the closed (360 mg/m2) or open (460 mg/m2) abdomen techniques, and systemic chemotherapy (400 mg/m2 fluorouracil and 20 mg/m2 folinic acid) was delivered intravenously 20 min before HIPEC. All individuals received systemic chemotherapy (of investigators’ choosing) with or without targeted therapy before or after surgery, or both. The primary endpoint was overall survival, which was analysed in the intention-to-treat population. Safety was assessed in all patients who received surgery. This trial is registed with ClinicalTrials.gov, NCT00769405, and is now completed.

Findings: Between Feb 11, 2008, and Jan 6, 2014, 265 patients were included and randomly assigned, 133 to the cytoreductive surgery plus HIPEC group and 132 to the cytoreductive surgery alone group. After median follow-up of 63·8 months (IQR 53·0-77·1), median overall survival was 41·7 months (95% CI 36·2-53·8) in the cytoreductive surgery plus HIPEC group and 41·2 months (35·1-49·7) in the cytoreductive surgery group (hazard ratio 1·00 [95·37% CI 0·63-1·58]; stratified log-rank p=0·99). At 30 days, two (2%) treatment-related deaths had occurred in each group.. Grade 3 or worse adverse events at 30 days were similar in frequency between groups (56 [42%] of 133 patients in the cytoreductive surgery plus HIPEC group vs 42 [32%] of 132 patients in the cytoreductive surgery group; p=0·083); however, at 60 days, grade 3 or worse adverse events were more common in the cytoreductive surgery plus HIPEC group (34 [26%] of 131 vs 20 [15%] of 130; p=0·035).

Interpretation: Considering the absence of an overall survival benefit after adding HIPEC to cytoreductive surgery and more frequent postoperative late complications with this combination, our data suggest that cytoreductive surgery alone should be the cornerstone of therapeutic strategies with curative intent for colorectal peritoneal metastases.

https://pubmed.ncbi.nlm.nih.gov/33476595/

Cells. 2021 Feb 22;10(2):472

Lipid Droplets Are a Physiological Nucleoporin Reservoir

Kumanski S, Viart B T, Kossida S, and Moriel-Carretero M

Lipid Droplets (LD) are dynamic organelles that originate in the Endoplasmic Reticulum and mostly bud off toward the cytoplasm, where they store neutral lipids for energy and protection purposes. LD also have diverse proteins on their surface, many of which are necessary for the their correct homeostasis. However, these organelles also act as reservoirs of proteins that can be made available elsewhere in the cell. In this sense, they act as sinks that titrate key regulators of many cellular processes. Among the specialized factors that reside on cytoplasmic LD are proteins destined for functions in the nucleus, but little is known about them and their impact on nuclear processes. By screening for nuclear proteins in publicly available LD proteomes, we found that they contain a subset of nucleoporins from the Nuclear Pore Complex (NPC). Exploring this, we demonstrate that LD act as a physiological reservoir, for nucleoporins, that impacts the conformation of NPCs and hence their function in nucleo-cytoplasmic transport, chromatin configuration, and genome stability. Furthermore, our in silico modeling predicts a role for LD-released fatty acids in regulating the transit of nucleoporins from LD through the cytoplasm and to nuclear pores.

https://pubmed.ncbi.nlm.nih.gov/33671805/

Theranostics. 2021 Jan 1;11(4):1626-1640.

Metastatic colorectal cancer cells maintain the TGFβ program and use TGFBI to fuel angiogenesis

Barbara Chiavarina , Brunella ostanza, Roberto Ronca , Arnaud Blomme , Sara Rezzola, Paola Chiodelli , Ambre Giguelay , Guillame Belthier, Gilles Doumont , Gaetan Van Simaeys , Simon Lacroix , Takehiko Yokobor , Bilguun Erkhem-Ochir, Patrick Balaguer, Vincent Cavailles, Eric Fabbrizio, Emmanuel Di Valentin, Stephanie Gofflot, Olivier Detry, Guy Jerusalem , Serge Goldman , Philippe Delvenne , Akeila Bellahcène , Julie Pannequin , Vincent Castronovo, Andrei Turtoi

Colorectal cancer (CRC) cells are traditionally considered unresponsive to TGFβ due to mutations in the receptors and/or downstream signaling molecules. TGFβ influences CRC cells only indirectly via stromal cells, such as cancer-associated fibroblasts. However, CRC cell ability to directly respond to TGFβ currently remains unexplored. This represents a missed opportunity for diagnostic and therapeutic interventions. Methods: We examined whether cancer cells from primary CRC and liver metastases respond to TGFβ by inducing TGFβ-induced protein ig-h3 (TGFBI) expression, and the contribution of canonical and non-canonical TGFβ signaling pathways to this effect. We then investigated in vitro and in vivo TGFBI impact on metastasis formation and angiogenesis. Using patient serum samples and an orthotopic mouse model of CRC liver metastases we assessed the diagnostic/tumor targeting value of novel antibodies against TGFBI. Results: Metastatic CRC cells, such as circulating tumor cells, directly respond to TGFβ. These cells were characterized by the absence of TGFβ receptor mutations and the frequent presence of p53 mutations. The pro-tumorigenic program orchestrated by TGFβ in CRC cells was mediated through TGFBI, the expression of which was positively regulated by non-canonical TGFβ signaling cascades. TGFBI inhibition was sufficient to significantly reduce liver metastasis formation in vivo. Moreover, TGFBI pro-tumorigenic function was linked to its ability to stimulate angiogenesis. TGFBI levels were higher in serum samples from untreated patients with CRC than in patients who were receiving chemotherapy. A radiolabeled anti-TGFBI antibody selectively targeted metastatic lesions in vivo, underscoring its diagnostic and therapeutic potential. Conclusions: TGFβ signaling in CRC cells directly contributes to their metastatic potential and stromal cell-independence. Proteins downstream of activated TGFβ, such as TGFBI, represent novel diagnostic and therapeutic targets for more specific anti-metastatic therapies.

https://pubmed.ncbi.nlm.nih.gov/33408771/

Cancers (Basel) 2020;12(8):E2014

Src Family Tyrosine Kinases in Intestinal Homeostasis, Regeneration and Tumorigenesis

Sirvent A, Mevizou R, Naim D, Lafitte m, Roche S

Src, originally identified as an oncogene, is a membrane-anchored tyrosine kinase and the Src family kinase (SFK) prototype. SFKs regulate the signalling induced by a wide range of cell surface receptors leading to epithelial cell growth and adhesion. In the intestine, the SFK members Src, Fyn and Yes regulate epithelial cell proliferation and migration during tissue regeneration and transformation, thus implicating conserved and specific functions. In patients with colon cancer, SFK activity is a marker of poor clinical prognosis and a potent driver of metastasis formation. These tumorigenic activities are linked to SFK capacity to promote the dissemination and tumour-initiating capacities of epithelial tumour cells. However, it is unclear how SFKs promote colon tumour formation and metastatic progression because SFK-encoding genes are unfrequently mutated in human cancer. Here, we review recent findings on SFK signalling during intestinal homeostasis, regeneration and tumorigenesis. We also describe the key nongenetic mechanisms underlying SFK tumour activities in colorectal cancer, and discuss how these mechanisms could be exploited in therapeutic strategies to target SFK signalling in metastatic colon cancer.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464719/

Nature Communications 2020 Aug 7;11(1):3940. doi: 10.1038/s41467-020-17858-2.

Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites

Promonet A, Padioleau I, Liu Y, Sanz L, Biernacka A, Schmitz AL, Skrzypczak M, Sarrazin A, Mettling C, Rowicka M, Ginalski K, Chedin F, Chen CL, Lin YL, Pasero P.

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.

https://www.nature.com/articles/s41467-020-17858-2

Cells 2020 Jun; 9(6): 1537.

Diversity of tumor-infiltrating γδ T cell abundance in solid cancers.

Chabab G, Boissière-Michot F, Mollevi C, Ramos J, Lopez-Crapez E, Colombo PE, Jacot W, Bonnefoy N, Lafont V

γδ T-cells contribute to the immune response against many tumor types through their direct cytolytic functions and their capacity to recruit and regulate the biological functions of other immune cells. As potent effectors of the anti-tumor immune response, they are considered an attractive therapeutic target for immunotherapies, but their presence and abundance in the tumor microenvironment are not routinely assessed in patients with cancer. Here, we validated an antibody for immunohistochemistry analysis that specifically detects all γδ T-cell subpopulations in healthy tissues and in the microenvironment of different cancer types. Tissue microarray analysis of breast, colon, ovarian, and pancreatic tumors showed that γδ T-cell density varies among cancer types. Moreover, the abundance of γδ tumor-infiltrating lymphocytes was variably associated with the outcome depending on the cancer type, suggesting that γδ T-cell recruitment is influenced by the context. These findings also suggest that γδ T-cell detection and analysis might represent a new and interesting diagnostic or prognostic marker.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348937/

Leukemia. 2020; 34(5): 1315–1328.

The therapeutic effectiveness of 177Lu-lilotomab in B-cell non-Hodgkin lymphoma involves modulation of G2/M cell cycle arrest.

Pichard A, Marcatili S, Karam J, Constanzo J, Ladjohounlou R, Courteau A, Jarlier M, Bonnefoy N, Patzke S, Stenberg V, Coopman P, Cartron G, Navarro-Teulon I, Repetto-Llamazares A, Heyerdahl H, Dahle J, Bardiès M, Pouget JP

Some patients with B-cell non-Hodkin lymphoma Lymphoma (NHL) become refractory to rituximab (anti-CD20 antibody) therapy associated with chemotherapy. Here, the effect of the anti-CD37 antibody-radionuclide conjugate lutetium-177 (¹⁷⁷Lu)-lilotomab (Betalutin^®) was investigated in preclinical models of NHL. In SCID mice bearing DOHH2 (transformed follicular lymphoma, FL) cell xenografts, ¹⁷⁷Lu-lilotomab significantly delayed tumor growth, even at low activity (100 MBq/kg). In athymic mice bearing OCI-Ly8 (diffuse large B-cell lymphoma, DLBCL) or Ramos (Burkitt’s lymphoma) cell xenografts, ¹⁷⁷Lu-lilotomab activity had to be increased to 500 MBq/kg to show a significant tumor growth delay. Clonogenic and proliferation assays showed that DOHH2 cells were highly sensitive to ¹⁷⁷Lu-lilotomab, while Ramos cells were the least sensitive, and U2932 (DLBCL), OCI-Ly8, and Rec-1 (mantle cell lymphoma) cells displayed intermediate sensitivity. The strong ¹⁷⁷Lu-lilotomab cytotoxicity observed in DOHH2 cells correlated with reduced G2/M cell cycle arrest, lower WEE-1- and MYT-1-mediated phosphorylation of cyclin-dependent kinase-1 (CDK1), and higher apoptosis. In agreement, ¹⁷⁷Lu-lilotomab efficacy in vitro, in vivo, and in patient samples was increased when combined with G2/M cell cycle arrest inhibitors (MK-1775 and PD-166285). These results indicate that ¹⁷⁷Lu-lilotomab is particularly efficient in treating tumors with reduced inhibitory CDK1 phosphorylation, such as transformed FL.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192854/

Int J Radiat Oncol Biol Phys 2020

Higher Anti-Tumor Efficacy of the Dual HER3-EGFR Antibody MEHD7945a Combined with Ionizing Irradiation in Cervical Cancer Cells.

Bourillon L, Demontoy S, Lenglet A, Zampieri A, Fraisse J, Jarlier M, Boissière-Michot F, Perrochia H, Rathat G, Garambois V, Bonnefoy N, Michaud H-A, Chardès T, Tosi D, Pèlegrin A, Azria D, Larbouret C, Bourgier C.

Life Sci Alliance. 2020 Apr 17;3(6).

Ubiquitin and SUMO conjugation as biomarkers of acute myeloid leukemias response to chemotherapies.

Gâtel P, Brockly F, Reynes C, Pastore M, Hicheri Y, Cartron G, Piechaczyk M, Bossis G.

Ubiquitin and the ubiquitin-like SUMO are covalently conjugated to thousands of proteins to modulate their function and fate. Many of the enzymes involved in their conjugation are dysregulated in cancers and involved in cancer cell response to therapies. We describe here the identification of biomarkers of the activity of these enzymes and their use to predict acute myeloid leukemias (AML) response to standard chemotherapy (daunorubicin-DNR and cytarabine-Ara-C). We compared the ability of extracts from chemosensitive and chemoresistant AML cells to conjugate ubiquitin or SUMO-1 on 9,000 proteins spotted on protein arrays. We identified 122 proteins whose conjugation by these posttranslational modifiers marks AML resistance to DNR and/or Ara-C. Based on this signature, we defined a statistical score predicting AML patient response to standard chemotherapy. We finally developed a miniaturized assay allowing for easy assessment of modification levels of the selected biomarkers and validated it in patient cell extracts. Thus, our work identifies a new type of ubiquitin-based biomarkers that could be used to predict cancer patient response to treatments.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7167290/

FASEB J. 2020 Mar;34(3):3616-3630

Blood contains circulating cell-free respiratory competent mitochondria

Al Amir Dache Z, Otandault A, Tanos R, Pastor B, Meddeb R, Sanchez C, Arena G, Lasorsa L, Bennett E A, Grange T, El Messaoudi S, Mazard T, Prevostel C, and. Thierry. A R

Mitochondria are considered as the power-generating units of the cell due to their key role in energy metabolism and cell signaling. However, mitochondrial components could be found in the extracellular space, as fragments or encapsulated in vesicles. In addition, this intact organelle has been recently reported to be released by platelets exclusively in specific conditions. Here, we demonstrate for the first time, that blood preparation with resting platelets, contains whole functional mitochondria in normal physiological state. Likewise, we show, that normal and tumor cultured cells are able to secrete their mitochondria. Using serial centrifugation or filtration followed by polymerase chain reaction-based methods, and Whole Genome Sequencing, we detect extracellular full-length mitochondrial DNA in particles over 0.22 µm holding specific mitochondrial membrane proteins. We identify these particles as intact cell-free mitochondria using fluorescence-activated cell sorting analysis, fluorescence microscopy, and transmission electron microscopy. Oxygen consumption analysis revealed that these mitochondria are respiratory competent. In view of previously described mitochondrial potential in intercellular transfer, this discovery could greatly widen the scope of cell-cell communication biology. Further steps should be developed to investigate the potential role of mitochondria as a signaling organelle outside the cell and to determine whether these circulating units could be relevant for early detection and prognosis of various diseases.

https://pubmed.ncbi.nlm.nih.gov/31957088/

British J. of Cancer 2020 Mar / vol 122 (5) 715-725

Hypoxia differently modulates the release of mitochondrial and nuclear DNA

A. Otandault, J. D. Abraham, Z. Al Amir Dache, A. Khalyfa, I. Jariel-Encontre, T. Forné, C. Prévostel, S. Chouaib, D. Gozal and A. R. Thierry

Nat Rev Clin Oncol. 2020 Jan;17(1):9-10.

Use of genomics to balance cure and complications.

Azria D, Rosenstein B

The identification of biomarkers and the development of genomics-based assays predictive of outcomes following radiotherapy, in an effort to help guide the treatment of patients with cancer, is an area of increasing research interest. Here, we discuss the validity of one such classifier, ARTIC, in the context of complementary genomic approaches designed to predict both tumour response and the susceptibility of nonmalignant tissues to toxicities resulting from radiotherapy.

https://www.nature.com/articles/s41571-019-0306-1