Action anti-tumorale de la mélatonine par une augmentation du stress oxydatif des cellules de choriocarcinome humaines
Fatma KHARRAT
Adaptive metabolic reprogramming of cancer cells in three dimensional cultures
Maia Al-Masri
Algorithmic Quantification of Radiation Induced Pulmonary Fibrosis
Li Ming Wang
Are ovarian cancer human avatar models useful to probe therapy-induced senescence?
Véronique Tu
Cell surface changes associated with chemotherapy-induced senescence in ovarian clear cell carcinoma
Alizée Gouronnec
Compréhension des mécanismes cellulaires, induit par le virus de la mosaïque de la papaye (PapMV), conduisant au développement d’une réponse immunitaire anti-tumorale
Léa Bourguignon
Developing RNA Therapeutics Against Hypoxic Cancer Cells
Jordan Quenneville
Disease-specific costs of non- metastatic and metastatic castration-resistant prostate cancer in Quebec
Ivan Yanev
Dopamine Receptor 2 (DRD2) antagonism and gemcitabine promising therapeutic approach for Pancreatic cancer
Rowa Bakadlag1
EIF4F Links Translation to Energy Stress Response in Cancer
Laura Hulea
Epithelial polarity remodeling and luminal collapse in early mammary tumourigenesis
Ruba Halaoui
From senescence to surfaceome to death - Immune clearance of ovarian cancer cells
Yu Zhan
Gleason score evaluation of the tissue microarray series from the Canadian Prostate Cancer Biomarker Network
Mame-Kany Diop
GRHL2 as a master regulator of the epithelial cell fate in breast cancer cell lines
Salwa Haidar
Identification of endogenous retroelement-derived MHC I-associated peptides in ovarian cancer
Jean-David Larouche
Molecular imaging Raman microscopy for the diagnosis and the prognosis of prostate cancer patients
Andrée-Anne Grosset
Multi-Institutional Expansion of the Opal Patient Portal Smartphone App
Stacey Beard
Nouvelle cible thérapeutique pour la prise en charge des patients atteints de leucémie aiguë lymphoblastique : Implication du DFF40
Merve Kulbay
Oncolytic viruses as adjuvants for personalized anti-cancer vaccination in mice
Victor Mullins-Dansereau
Telomere-initiated senescence requires homologous recombination-mediated sister chromatid fusions and genomic instability
Marc-Alexandre Olivier
The impact of rectal filling on rectal dose during hypofractionated radiotherapy of the prostate
Haley Patrick
The Mnk1/2-eIF4E axis facilitates invasion and metastasis of pregnancy associated breast cancer (PABC)
Qianyu Guo

Action anti-tumorale de la mélatonine par une augmentation du stress oxydatif des cellules de choriocarcinome humaines


Fatma KHARRAT1,2, Cathy Vaillancourt1,2,3,4
1INRS-Institut Armand-Frappier 2CINBIOSE 3BioMed 4RQR

Contexte: La mélatonine est un puissant antioxydant produit par les cellules trophoblastiques placentaires et les protégeant du stress oxydatif. Dans les cellules tumorales placentaires, elle induit plutôt la mort cellulaire par apoptose, mais le mécanisme impliqué dans cette action de la mélatonine n’a jamais été investigué. Hypothèse: La mélatonine augmente le stress oxydatif dans les cellules de choriocarcinome placentaire entraînant l’apoptose. Objectifs: Déterminer l’effet de la mélatonine sur 1) le taux d’espèces réactives de l’oxygène (EROs); 2) l’expression des enzymes oxydantes (xanthine oxydase (XO)) et antioxydantes (superoxydes dismutases (SODs), glutathionne peroxydase (GPx) et la catalase (CAT)); 3) la peroxydation des lipides et la carbonylation des protéines dans une lignée de cellules de choriocarcinome placentaire humaine, BeWo. Méthodes: Les cellules BeWo ont été exposées en normoxie (8% O2) à la mélatonine (0–1mM) et exposées ou non à un contrôle de stress par une hypoxie (0,5% O2) suivie d’une réoxygénation (8% O2). Les niveaux des EROs ont été mesurés par Carboxy-DCFDA, l’expression des enzymes a été analysée par immunobuvardage de type western-Blot. La peroxydation lipidique a été analysée par le test TBARS et la carbonylation des protéines par fluorimétrie. Résultats: La mélatonine à 1mM augmente le taux des EROs (P < 0,01), la peroxydation des lipides ainsi que la carbonylation des protéines par rapport au véhicule (DMSO), mimant l’effet de l’hypoxie/réoxygénation (H/R). L’expression de la XO, des SODs, de la GPx et de la CAT est aussi augmentée en condition d’H/R et cet effet ne semble pas être renversé par la mélatonine. Conclusion: Nos résultats montrent que la mélatonine augmente le stress oxydatif dans les cellules de choriocarcinome placentaire, mais le mécanisme exact par lequel elle exerce son activité anti-tumorale est à confirmer.

 

Adaptive metabolic reprogramming of cancer cells in three dimensional cultures


Maia Al-Masri1,2, Li Ting1,2, Ruba Halaoui1,2, Luke McCaffrey1,2,3
1Goodman Cancer Research Center 2McGill University 3Department of Oncology

Background An exciting development in cancer therapy is the exploitation of abnormal tumor metabolism. Cancer cells propagated in three-dimensional (3D) culture systems exhibit physiologically relevant cell-cell and cell-matrix interactions that reflect structural complexity of in vivo tumors, more so than two-dimensional (2D) monolayers. However, whether metabolism differs between 2D and 3D systems has not been systematically characterized. The purpose of this project is to understand metabolic differences between 2D- and 3D-cultures and to explore their underlying mechanisms. Results Cancer cells from multiple cancer types, including intestinal, breast, lung and ovarian cancer were grown as 3D organoids in hydrogel or as 2D monolayers. The dependency of each cell line on glucose, a major energy source, was examined by growing cells in glucose deficient medium.  In 2D cultures, all cell lines were completely dependent on glucose for proliferation. Surprisingly, the same cell lines showed no dependency on glucose when cultured in a 3D environment as organoids, and growth was indistinguishable from those grown in complete medium. The intracellular uptake of glucose was tracked using live cell imagining and the fluorescent tracer 2-NBDG, and it showed that the uptake of glucose is far more significant in 2D cell cultures than 3D. Consistent with their differences in glucose consumption, cells in 2D had significantly higher extracellular acidification rates, an indicator of metabolism through glucose-dependent glycolytic pathway. Surprisingly, functional metabolic measurements revealed that when energy production by oxidative phosphorylation is blocked, cells in 3D have the capacity to utilize the glycolytic pathway. This indicates that cancer cells in a 3D environment appear to prefer non-glycolytic energy pathways, but are highly adaptable. Furthermore, 3D Caco-2 organoids grown in culture medium lacking essential amino acids, showed a significant growth decrease, suggesting that amino acid metabolism is altered between 2D and 3D cultures. Conclusion The above data clearly highlights major metabolic differences between 2D and 3D culture conditions in substrates uptake and requirements that are reflected in the energy pathways upregulated in each culture condition.

Algorithmic Quantification of Radiation Induced Pulmonary Fibrosis


Li Ming Wang1,2,5, Dr. Alexandre Semionov1,3, Dr. James Tsui1,4, Dr. Avishek Chatterjee1,2, Dr. Sangkyu Lee6, Dr. Jan Seuntjens1,2, Dr. Issac Yang1,4, Dr. Yarab Al Bulushi1,4, Dr. Norma Ybarra1,2,5
1McGill University 2Medical Physics Unit - Cedar's Cancer Centre 3Department of Diagnostic Radiology - Royal Victoria Hospital 4Department of Radiation Oncology - Cedar's Cancer Centre 5Research Institute - Montreal University Healthcare Centre Glen Site 6Memorial Sloan Kettering Cancer Centre

Radiation-induced pulmonary fibrosis (RIPF) is a frequent complication of radiotherapy (RT) when applied to the thorax for intrathoracic malignancies. After RT, the onset of RIPF is late and permanent. Currently, the assessment of RIPF by computer tomography (CT) imaging is dependent on the qualitative appraisals of radiologists, while symptoms of RIPF are evaluated only after patient self-reports. As a result, RIPF is poorly identified, assessed with variability, and poorly quantified. This leads to a lack of available accurate techniques to study RIPF prevalence and severity post-RT of all modalities. Taking advantage of radiodensity measures, we analyzed CT imaging by extracting voxel densities in Hounsfield Units for pre-treatment planning and follow-up post-treatment patient image sets (n=68). Changes in pulmonary density distribution, in regions of high-density fibrosis tissue, were correlated with scoring generated by a cohort of radiologists and radiation oncologists for the same image set and showed a significant (p<<0.05) correlation of r2= 0.705. With the most extreme grades correlating best and moderate grades being more difficult to differentiate via density values. We also noted that some patients with grades 0 and 1 exhibit decreased average densities as opposed to no density or minimum density increases. As a result, we were able to verify the feasibility of using radiodensity as a characteristic for quantifying fibrosis while establishing a thorough, non-traditional, highly replicable and quantitative method for the measurement of fibrosis that is semi-automated and can take into consideration the entirety of the treated and untreated lung volumes for analysis.

Are ovarian cancer human avatar models useful to probe therapy-induced senescence?


Véronique Tu1, Michael Skulimowski1, Pavel Chrobak1, Loise Gilbert1, Euridice Carmona1, Anne-Marie Mes-Masson1, Diane Provencher1, John Stagg1, Francis Rodier1
1Centre de recherche du CHUM (CRCHUM) and Institut du Cancer de Montréal

Epithelial ovarian cancer (EOC) is the deadliest gynecological malignancy in Canada. Despite new therapy modalities, low 5-year survival rates and minimal improvements in the past decades demonstrate the necessity of better understanding the cellular mechanisms brought on by treatment. Damaged cells undergo different cell fates including death or senescence. Evaluating the role of therapy-induced senescence (TIS) could guide new therapeutic opportunities. Among other effects, TIS induces a proinflammatory secretome associated with senescence (SASP) that can influence the tumoral microenvironment as well as the identity and activity of the immune cells recruited to the tumor site leading to context-dependent beneficial or detrimental responses. Immune response being important in the hopes of cancer recovery, it is imperative to further characterize the immune cells’ interactions with TIS EOC cells. Here we propose to examine these interactions at a molecular level and in real-time using live biopsies from patients adapted to different avatars: microdissected tumors (MDTs) in lab-on-a-chip systems as well as patient-derived xenografts (PDX). PDX have been successfully generated from several EOC patients and MDTs were recovered pre/post-treatment (carboplatin/paclitaxel combination). Cell fate decision markers have been characterized, using different techniques including qPCR and multiplexed secretome profiling. Not surprisingly, TIS responses differ between patients. Nevertheless, treated EOC tissues present SASP profiles and proliferation arrest responses. Moreover, PDX and patient plasma display similar circulating blood SASP profiles seven days post-treatment. These observations in patient-derived models support our previous in vitro results as to EOC cells having the ability to undergo senescence following chemotherapy treatment.

Cell surface changes associated with chemotherapy-induced senescence in ovarian clear cell carcinoma


Alizée Gouronnec1, Léo Aubert1, Yu Zhan2, Shuofei Cheng2, Francis Rodier1,2, Philippe Roux1
1Université de Montréal 2CRCHUM

Introduction: Ovarian cancer is a major health problem, as less than half of patients survive 5 years past initial diagnosis. One of the best therapeutic options for ovarian cancer is chemotherapy, which consists of a combination of carboplatin/paclitaxel (C/P). While these drugs result in mitotic catastrophe and cell death, recent evidence suggests that ovarian cancer cells also respond by becoming growth arrested (i.e., senescent). Therapy‐induced senescence (TIS) may thus provide an effective mean to induce persistent growth inhibitory responses, but little is known about the properties of senescent cells and few markers exist to study them.

Methods and Results: To better understand TIS in ovarian cancer and identify potential markers for senescent cells, we characterized cell‐surface changes induced by chemotherapeutic drugs. For this, we used an ovarian clear cell carcinoma cell line (TOV21G) that undergoes prolonged growth arrest with many characteristics of senescence upon C/P treatment. When compared to untreated cells, C/P‐treated cells express transcriptional signatures that are highly characteristic of senescence. Using a cutting‐edge proteomics approach, we found that 174 and 157 cell surface proteins were significantly upregulated and downregulated, respectively, in senescent cells. Many of these proteins have no known roles in senescence, suggesting many new research directions.

Conclusion and Relevance: Overall, our results demonstrate that TIS alters the cell surface distribution of many proteins involved in diverse biological functions. Careful analysis of these changes will improve our understanding of TIS, but also help in the design of improved chemotherapy for ovarian cancer.

Compréhension des mécanismes cellulaires, induit par le virus de la mosaïque de la papaye (PapMV), conduisant au développement d’une réponse immunitaire anti-tumorale


Léa Bourguignon1, Denis Leclerc2, Alain Lamarre1
1INRS-IAF 2CHU de Québec/U Laval

Les thérapies actuelles utilisées contre le cancer ne sont pas toujours efficaces et ne permettent pas une protection contre les métastases ou les récidives. De plus elles sont néfastes pour l'organisme entier.

Une alternative est de faire intervenir son propre organisme, en stimulant et éduquant son système immunitaire afin de cibler spécifiquement les cellules cancéreuses.

Les nanoparticules de virus de plantes (VLP), ayant la même structure virale mais ne contenant pas le matériel génétique infectieux, provoquent une réponse immunitaire dirigée contre les cellules tumorales. Elles sont reconnues par le système immunitaire grâce à leurs épitopes de surface, et entrainent une réponse immunitaire cellulaire et humorale.

Au laboratoire nous étudions le PapMV. Nous avons déjà montré que des injections intratumorales de PapMV, dans un mélanome murin B16, diminue la vitesse de croissance de la tumeur, en augmentant le nombre de cellules immunitaires recrutées, notamment des lymphocytes T CD8+ antigène tumoral-spécifique. De plus une injection systémique de PapMV 6h avant ou 2 jours après l’injection intraveineuse de B16 réduit les métastases pulmonaires.

Il a aussi été montré que PapMV, en tant que virus à ARN simple brin, est reconnu par TLR7 et entraine la production d’interféron alpha via MyD-88 et IRF7. Cependant, lors d’une injection intratumorale ceux sont principalement les macrophages qui captent PapMV et qui produisent l’interféron alpha.

Récemment nous nous sommes penchés sur le rôle des macrophages, connus comme marqueurs de mauvais pronostiques, dans l’effet de PapMV.  Nous observons une diminution de la proportion de ces derniers lors d’un traitement avec PapMV.

De plus, nous observons que PapMV permet un ralentissement de la croissance tumorale dans un cancer mammaire murin 4T1 et un carcinome du colon murin CT26, implantés en sous cutanée. Et que PapMV protège des métastases pulmonaires pour ces cancers également. Ce qui montre un effet universel de PapMV.

Ces données nous encouragent à poursuivre les recherches afin de décortiquer le mécanisme de PapMV et de le considérer comme un candidat potentiel d'immunothérapie des cancers.

Developing RNA Therapeutics Against Hypoxic Cancer Cells


Jordan Quenneville1,2, Zohra Saci2, Albert Feghaly2, Francois Major1,2, Etienne Gagnon1,2
1Université de Montréal 2IRIC

Introduction:

            Anti-cancer therapy relapses are primarily due intratumoral heterogeneity, particularly local oxygen levels. In response to hypoxia, tumour cells stabilize the transcription factor HIF1 leading to: metabolic changes, angiogenesis, increased metastatic potential, and resistance to chemotherapy/radiotherapy. Our project aims to transcriptionally describe and counter the HIF1 program, sensitizing hypoxic tumour cells to other therapies through a novel type of RNA-based therapy: small artificial RNAs.

Methods & Results:

Our therapeutic strategy requires acquiring transcriptomic data of tumour cells using the HIF1 program.

To this end, a mouse melanoma cell line expressing a HIF1a-eGFP fusion protein was validated in vitro using chemical HIF1 stabilization and hypoxic cell culture. We have analyzed RNAseq data from these cells after exposure to hypoxic conditions, hypoxic conditions in conjunction with starvation, and a 14-day hypoxia timecourse.

We have identified unique adaptation signatures in each case, highlighting the variety of changes to subtypes of hypoxia. We have also integrated microRNA expression and targeting across biological pathways through the miRBooking software developed by Dr. Major.

Impact:

These integrative transcriptomic analyses allows for a more complete understanding of the interplay between microRNAs and their targets within biological pathways in a given cell-state. It identifies genes vulnerable to RNAi and allows us to design new multi-targeting microRNAs, called small artificial RNAs. The invention of single-molecule, multi-target based RNA therapy creates new possibilities for RNA therapy in all therapeutic contexts. This project also deepens our understanding of the tumour microenvironment, and will help inform research in cancer progression and therapy resistance.

Disease-specific costs of non- metastatic and metastatic castration-resistant prostate cancer in Quebec


Ivan Yanev1, Armen Aprikian1, Emma Nablsi1, Jonathan Primiani1, Zachary Vaillancourt1, Karen Bremner2, Steven Carcone2, Nicholas Mitsakakis2, Welson Ryan2, Murray Krahn2, Alice Dragomir1
1McGill University 2Toronto Health Economics and Technology Assessment (THETA) Collaborative

OBJECTIVESEstimate the disease specific costs of prostate cancer patients during the health states of nonmetastatic castration-resistant prostate cancer (NM-CRPC) and metastatic castration-resistant prostate cancer (M-CRPC).

 

METHODS: This cohort analysis contains 211 prostate cancer patients from the MUHC. The usage of imagery tests, hospital visits and treatments were extracted from the patients files and the mean usage per resource was calculated for 30 days in the given health state. Resources price were obtained from the RAMQ List of Medications when available, when unavailable prices were obtained from the MUHC internal prices lists. This cost analysis was performed by health care system perspective.

 

RESULTSMean duration of NM-CRPC was 26.07 months while duration of M-CRPC was 20.79 months, with 62 and 68 patients per health state respectfully. The average disease specific resource utilisation per patient for 30 days was $786 for NM-CRPC and $2,210 for M-CRPC health states with the cost driver being chemotherapy or prescription drugs different than ADT. The total average cost for NM-CRPC health state was $20,457 compared to $45,956 for M-CRPC health state.

 

CONCLUSIONSThe disease specific resource utilisation costs for a patient in M-CRPC are significantly higher than the costs of NM-CRPC. This being said it would be interesting to study the total healthcare costs from a societal perspective in order the improve the cost management of PCa.

Dopamine Receptor 2 (DRD2) antagonism and gemcitabine promising therapeutic approach for Pancreatic cancer


Rowa Bakadlag11,2, Pouria Jandaghi11,2, Yasser Riazalhosseini11,2
1Department of Human Genetics, McGill University 2McGill University and Genome Quebec Innovation Center

Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is characterized by frequent mutations of the KRAS proto-oncogene in ~90% of sporadic cases. Gemcitabine is a widely used chemotherapy treatment for PDAC and is often used in combination therapy, but limitations have emerged from the development of drug resistance. To improve survival and prognosis, identification of new targetable candidates is necessary, which can be achieved by better understanding the molecular basis of PDAC. One such novel potential targetable protein for therapy is the dopamine receptor 2 (DRD2). Pharmacological blockade of DRD2 activity with haloperidol has been shown to suppress proliferation of pancreatic cancer in vitro and in vivo. To increase the efficacy of DRD2 antagonists in PDAC treatment, we aim to test the outcome of haloperidol and gemcitabine combinational treatment. Multiple approaches were used to understand the interaction between these drugs and determine their combined effects against viability of cancer cells in vitro. Our studies demonstrated different responses to the combination treatment in multiple PDAC cell lines, which differentially express DRD2 – Panc-1, MiaPaCa-2, and BxPC-3. Further, synergism, antagonism, or additive combination effect, determined by calculating the combination index (CI), were studied. Our data suggest that some combination doses act synergistically in BxPC-3 contrasting with Panc-1 and MiaPaCa-2, which both showed either additive or antagonism combination effect.

Keywords: combination therapy – gemcitabine & haloperidol, dopamine receptor 2 (DRD2), Pancreatic ductal adenocarcinoma (PDAC)

EIF4F Links Translation to Energy Stress Response in Cancer


Laura Hulea1,2, Simon-Pierre Gravel3, Masahiro Morita4, Oro Uchenunu1,2, Marie Cargnello5, Young Kyuen Im1,2, Shannon McLaughlan2, Ola Larsson6, Josie Ursini-Siegel1,2, Julie St-Pierre7, Michael Pollak1,2, Ivan Topisirovic1,2
11McGill University, Montreal, Canada 2Lady Davis Institute, Montreal, Canada 3Universite de Montreal, Montreal, Canada 4University of Texas Health Science Center, San Antonio, Texas, USA 5INSERM, Toulouse, France 6Karolinska Institutet, Solna, Sweden 7University of Ottawa, Ottawa, Canada

Protein synthesis is one of the most energy consuming process in the cell. Oncogenic kinases (e.g. EGFR/HER2, BCR/ABL and BRAF) play a central role in reprogramming translation and energy metabolism in neoplasia. The downstream mechanisms that link translational machinery and energy homeostasis in cancer, however, remain largely unknown.

 

We found that widely used anti-diabetics (biguanides) abrogate adaptations to kinase inhibitor-induced energetic stress, which results in synergistic anti-neoplastic effects both in vitro and in vivo and the inhibition of mTORC1/4E-BP pathway by the drug combination. In turn, cancer cells in which 4E-BP1/2 expression was abrogated by CRISPR were partially resistant to the combination of kinase inhibitors and biguanides. This was paralleled by the inability of the drugs to inhibit the translation of mRNAs encoding important metabolic regulators including those involved in serine (PHGDH, PSAT1) and aspartate and asparagine synthesis (PC, ASNS), and by an increase in these key metabolites, as well as citric acid cycle intermediates, in 4E-BP KO cells.

 

We also identified HIF-1α as a mediator of the lapatinib-biguanide effect, as high HIF-1α expression caused by VHL loss led to decreased sensitivity to the combination and was paralleled by an upregulation of glutamine reductive metabolism at the metabolic level.

 

Together our findings demonstrate that HIF-1α and the mTORC1/4E-BP/eIF4E axis are important mediator of metabolic adaptation in response to the combination of biguanides and clinically-used kinase inhibitors and suggest that the metabolic plasticity of cancer cells is an important factor that can influence the efficiency of such anti-cancer strategies.

Epithelial polarity remodeling and luminal collapse in early mammary tumourigenesis


Ruba Halaoui1,2, Joseph Szymborski1,2, Carlis Rejon1, Sudipa Chatterjee1,2, Sarkis Meterissian4,5,6, William J. Muller1,3, Atilla Omeroglu6,7, Luke McCaffrey1,2,4
1Goodman Cancer Research Centre 2Division of Experimental Medicine, McGill University 3Department of Biochemistry, McGill University 4Department of Oncology, McGill University 5Department of Surgery, McGill University 6McGill University Health Centre, Montreal 7Department of Pathology, McGill University

Breast cancer is a leading cancer type in women and although breast cancer mortality has reduced, incidence is increasing. It progresses through benign and malignant stages, presenting with loss of ductal lumen and cell polarity. How apical-basal polarity is disrupted, however, is incompletely understood. Here, our aim is to better characterize early stages of progression, focusing on describing the mechanisms by which luminal filling with non-polarized cells occurs.

Looking at biopsy samples from human breast that contained pre-invasive breast lesions , there were different polarity phenotypes present at DCIS, each having different modes of progression towards polarity loss. In fact, apical polarity was lost progressively in the absence of intra-luminal filling on these early stages of breast cancer progression. Cords of polarized cells move into the lumen, splitting it into smaller secondary lumen. Further investigations using a mouse model that presents with early disease show that instead of luminal filling, asymmetric cell divisions produce multilayered ducts with a population of basally positioned cells lacking cell polarity. The remaining polarized cells are subsequently eliminated following luminal collapse, with defects in apical tension marked by reduced activity of myosin II and RhoA-GTP. At later stages, the luminal space disappears, and cells exhibit an apical patch at cell-cell contacts. Re-establishment of tension at the apical membrane through restoration of RhoA activity, enables ducts to maintain lumen shape and cell polarity. Finally, de-induction of the tumour in mice leads to not only a regression of the tumour, but also to reversion of polarity in the resulting ducts.

This reveals that there exist different modes of polarity loss in early breast cancer progression. The most prevalent mode is a progressive loss through different pre-malignant to malignant stages, through the cooperation of multiple mechanisms such as mis-oriented divisions, increased proliferation, and loosened apical tension, leading to luminal collapse, rather than filling. Notably, this series of morphological steps leading to the formation of solid ducts is in fact reversible, and ducts are able to re-establish proper organization and polarity upon cessation of oncogenic activity.

 

From senescence to surfaceome to death - Immune clearance of ovarian cancer cells


Yu Zhan1, Leo Aubert2, Alizée Gouronnec2, Shuofei Cheng1, David P1, Philippe Roux2, Rejean Lapointe1, Francis Rodier1
1CRCHUM 2IRIC

      Senescence is an important tumor suppression mechanism and senescent cells develop many senescence-associated phenotypes which are responsible for their biological functions. Among those, the stable senescence-associated proliferation arrest (SAPA) removes damaged cells from the proliferative pool, thus prevents their expansion. In addition to apoptosis or mitotic catastrophe, therapy induced senescence (TIS) is one of the major response to anti-cancer treatments, including chemotherapy and ionizing radiation (IR).

     Senescent cells secrete various cytokines and chemokines which constitute the senescence-associated secretory phenotype (SASP). SASP is important for immune cell attraction and then clearance of senescent cells. To better understand cellular senescence and to elucidate the targets for senescent cell clearance by immune cells or senolytic drugs, we are processing proteomic analysis of surfaceome of chemotherapy induced senescent cancer cells.

       Our hypothesis is that the surfacesome of senescent cells will reveal a batch of proteins on plasma membrane or in extracellular matrix which play a key function of secretory and signaling pathway in senescent cells. Our preliminary results revealed that the expression of amino acid transporters in these senescent cells are significantly down-regulated and this intriguing finding indicates a novel amino acid transport pathway in senescent cells for the production and secretion of massive SASP. Another interesting finding is the upregulation of anti-inflammatory factor MFGE8 which could imply the mechanisms of the shielding of senescent cells from immuno-surveillance and -clearance. Deep analysis of the proteomic data will offer a set of candidates for immune-clearance and senolytic drug clearance of senescent cells. 

Gleason score evaluation of the tissue microarray series from the Canadian Prostate Cancer Biomarker Network


Sami-Ramzi Leyh-Bannurah1,2, Dominique Trudel3,4,5, Mathieu Latour3,4, Emanuele Zaffuto1,6, Andree-Anne Grosset5, Veronique Ouellet5, Mame-Kany Diop5, Christine Tam5, Markus Graefen2, Lars Budäus2, Armen G. Aprikian7, Louis Lacombe8, Neil E. Fleshner9, Martin E. Gleave10, Anne-Marie Mes-Masson4,5, Fred Saad5,11, Pierre I. Karakiewicz1,11
1Cancer Prognostics and Health Outcomes Unit, University of Montreal Health Center, Montreal, Quebec, Canada 2Martini-clinic, Prostate Cancer Center Hamburg-Eppendorf, Hamburg, Germany 3Department of Pathology and Cellular Biology, Université de Montréal, Montreal, Quebec, Canada 4Department of Medecine, Université de Montréal, Montreal, Quebec, Canada 5Institut du Cancer de Montreal and Centre de recherche du centre hospitalier de l’Université de Montréal, Montreal, Quebec, Canada 6Department of Urology and Division of Experimental Oncology, URI, Urological Research Institute, IRCCS San Raffaele Scientific Institute, Milan, Italy 7Research Institute of McGill University Health Center and Department of Surgery (Urology), McGill University, Montreal, Quebec, Canada 8CHU de Québec, Quebec City, Quebec, Canada 9Division of Urology, Department of Surgery, University Health Network, University of Toronto, Toronto, Ontario, Canada 10Vancouver Prostate Centre & Department of Urologic Science, University of British Columbia, Vancouver, British Columbia, Canada 11Department of Surgery, Université de Montréal, Montreal, Quebec, Canada

Background: Validation of good concordance between tissue microarray (TMA) and radical prostatectomy (RP) high-grade prostate cancer (PCa) is crucial because the latter determines the treated natural history of PCa. We hypothesized that TMA derived Gleason score is in agreement with RP pathology and is capable of accurately predicting BCR.

Objective: To test the agreement rate between high grade PCa at RP and TMA, and the ability of TMA Gleason score to predict BCR.

Design, Setting and Participants: Data were provided from a multi-institutional Canadian sample of 1,262 RP specimens arrayed on TMAs with complete clinical and pathological data, including biopsy Gleason score. The rate of agreement between TMA and high grade Gleason at RP or biopsy and RP was tested. Then the ability of Gleason score, from TMA cores and biopsy to predict BCR were evaluated using univariate and multivariate Cox regression models.

Results and limitations: Agreement between RP and TMA as well as between RP and biopsy were 72.6% (95% CI:69.7-75.5) and 60.4% (95% CI:57.2-63.6), respectively. In multivariate analysis predicting BCR, the accuracy rates for RP, TMA and biopsy were 0.73 (95% CI 0.70-0.76), 0.72 (95% CI:0.69-0.75) and 0.68 (95% CI:0.69-0.75), respectively.

Conclusions: TMA Gleason grade accurately reflects presence of high grade Gleason in RP specimen and accurately predicts BCR rates after RP. 

GRHL2 as a master regulator of the epithelial cell fate in breast cancer cell lines


Salwa Haidar1, Houssam Ismail1, Eric Audemard1, Sylvie Mader1,2
1Institute for Research in Immunology and Cancer (IRIC), University of Montreal, Montreal, Canada 2Biochemistry Department, University of Montreal, Montreal, Canada

Breast cancer is a heterogeneous disease with different molecular subtypes. "Luminal" and "HER2 +" tumors benefit from targeted therapies, while "basal-like" and "claudin-low" tumors do not currently benefit from targeted therapies and are treated by chemotherapy. It is therefore important to better understand the molecular causes underlying the emergence of different subtypes of breast cancer in order to improve existing treatments.

The transcription factor (TF) GRHL2 controls normal mammary epithelial differentiation and inhibits the epithelial-mesenchymal transition (EMT) observed in the claudin-low subtype. In order to elucidate the role of GRHL2 in EMT and in breast epithelial differentiation, we stably expressed it in the claudin-low MDA-MB-231 cells. Our RNA-seq results demonstrate the induction of epithelial genes (CDH1, CLDN4, RAB25), including the EMT suppressors OVOL1/OVOL2 but also induction of the basal transcriptional cofactor VGLL1. Conversely, GRHL2 knockdown by shRNAs in the HCC70 basal-like cells leads to induction of an EMT phenotype including increased expression of vimentin and downregulation of VGLL1. RNA-seq profiling identified 100 common genes positively regulated in both cell lines. Our ChIP-Seq results show that GRHL2 binds to regulatory regions of genes involved in the maintenance of an epithelial phenotype, and directly regulates OVOL1/OVOL2 and VGLL1. These results suggest a role of GRHL2 in the regulation of basal and luminal mammary epithelial differentiation, possibly modulated by the expression of TFs specific to each phenotype. This project elucidates the role of GRHL2 in the control of the EMT and emphasizes its role as a master regulator of the epithelial cell fate.

Identification of endogenous retroelement-derived MHC I-associated peptides in ovarian cancer


Jean-David Larouche1,2, Qingchuan Zhao1,2, Leslie Hesnard1,2, Assya Trofimov1,3, Joël Lanoix1, Chantal Durette1, Caroline Côté1, Patrick Gendron1, Jean-Philippe Laverdure1, Mathieu Courcelles1, Sébastien Lemieux1,3, Pierre Thibault1,4, Claude Perreault1,2,5
1Institute of Research in Immunology and Cancer, Université de Montréal, Montréal, Qc, Canada. 2Department of Medicine, Université de Montréal, Montréal, Qc, Canada 3Department of Informatics and Operational Research, Université de Montréal, Montréal, Qc, Canada 4Department of Chemistry, Université de Montréal, Montréal, Qc, Canada 5Division of Hematology-Oncology, Hôpital Maisonneuve-Rosemont, Montréal, Qc, Canada

MHC-I molecules present peptides at the cell surface for CD8 T cell recognition, which is crucial for cancer immunosurveillance. Consequently, tumor-infiltrating CD8 T lymphocytes (TILs) are associated with superior prognosis and mutational burden. However, TILs can also recognize non-mutated, aberrantly expressed MHC I-associated peptides (MAPs), which derive from the re-expression of sequences epigenetically silenced in normal cells, such as endogenous retroelements (EREs). Despite their expected immunogenicity, ERE-derived MAPs (ereMAPs) are difficult to identify as EREs sequences are typically not included in conventional approaches.

 

Since ovarian cancers are characterized by a high level of TILs, low mutational burden and re-expression of ERE transcripts, they are expected to produce ereMAPs. To identify them, we developed an approach combining mass spectrometry and RNA-sequencing. RNA-sequencing reads were filtered against reference ERE sequences, followed by in silico translation to generate a sample-specific proteome. Mass spectrometry data were analyzed using this database, which enabled us to identify 31 ereMAPs from OVCAR3 cells and 4 tumor samples. We compared ERE expression in a cohort of ovarian cancer patients from TCGA to generalize our results to a larger population. Finally, the RNA-seq data of medullary thymic epithelial cells, which are responsible for the establishment of self tolerance of CD8 T cells, has been analyzed to estimate the immunogenicity of ereMAPs. 

 

This study is the first to show the contribution of EREs to the immunopeptidome of ovarian cancer. From a clinical perspective, ereMAPs might represent ideal targets for cancer immunotherapy as they are non-mutated and might be shared between patients.

Molecular imaging Raman microscopy for the diagnosis and the prognosis of prostate cancer patients


Andrée-Anne Grosset1,2, Catherine St-Pierre1,3, Karl St-Arnaud1,3, Kelly Aubertin1, Michael Jermyn3,4, Frédéric Leblond1,3, Dominique Trudel1,2,5
1Centre de recherche du Centre hospitalier de l'Université de Montréal (CRCHUM), Institut du cancer de Montréal, Montreal, Qc, Canada 2Dept of Pathology and Cellular Biology, Université de Montréal, Montreal, Qc, Canada 3Dept of Engineering Physics, Polytechnique Montreal, Montreal, Qc, Canada 4Thayer School of Engineering, Dartmouth College, Hanover, NH, United States 5Dept of Pathology, Centre hospitalier de l'Université de Montréal (CHUM), Montreal, Qc, Canada

Background: To better determine the diagnosis and prognosis of prostate cancer patients, especially to identify high-risk prostate cancer, integration of complementary analyses are needed. Molecular imaging Raman microscopy, a non-destructive and label-free technique, is a promising histopathological diagnostic complement. To adapt this imaging technique to the clinical workflow, we previously developed a rapid standardized protocol for the preparation and the analysis of diagnostic tissues by Raman microscopy. In this study, we evaluated the potential of molecular imaging Raman microscopy for the identification of prostate cancer diagnosis and prognosis.

 

Methods: Tissue microarrays (TMAs) of prostate cancer tissues from Centre hospitalier de l'Université de Montréal (CHUM) were used in this study. TMA sections of 4 µm on low-cost Raman-neutral slides were dewaxed following the standardized protocol of the CHUM in 8 minutes and dried in a vacuum desiccator for 20 minutes. For Raman imaging, the Renishaw inVia Reflex confocal Raman microscope was used. The capacity to predict biochemical recurrence at 18 months using Raman microscopy was tested using artificial intelligence. The analysis was performed with Wire 4.4 software and MATLAB.

 

Results: From prostate cancer TMAs, 80 patients were analyzed by molecular Raman microscopy. We developed two methods of molecular imaging analysis: 1) we extracted chemical information of the Raman image to identify the stroma (collagen in red), cytoplasm (phenylalanine in green) and nuclei (DNA in blue) to create a virtual staining, and 2) we calculated Raman peak ratios to specifically recognize benign (blue) and cancer (green) prostate tissues. Using the Support Vector Machine (SVM) classifier on 320 Raman spectra, recurrence within 18 months after surgery was identified with an accuracy of 84%.

 

Conclusion: Molecular imaging Raman microscopy is a promising complementary analysis for physicians. Integration of this technique in the hospital could help for patient follow-up and therapeutic approaches.

Multi-Institutional Expansion of the Opal Patient Portal Smartphone App


Stacey Beard2, Jamil Asselah1,2, Laurie Hendren2, Tarek Hijal1,2, John Kildea1,2
1McGill University Health Centre 2Université McGill

Introduction:

Opal is a smartphone app that allows cancer patients to view their medical data and enables clinicians to send them personalized educational material and patient-reported outcome questionnaires (opalmedapps.com). Currently, Opal is in use at the McGill University Health Centre. Our objective is to expand Opal to multiple hospitals simultaneously, allowing patients to access all of their medical data via one login. Six cancer centres in the Montreal area are participating in the project, with funding from the Canadian Partnership Against Cancer.

Methods:

The existing Opal platform was studied to determine necessary changes for a multi-institutional implementation. The data storage, the app's data request process, and the user registration system were revised after comparing the pros and cons of various design options.

Results:

A model for a multi-institutional Opal was designed, and a prototype partially implemented. The new data storage system involves replicating the existing Opal database and backend software identically in each connected hospital, with an additional shared database and software for central data storage. A mockup was developed to demonstrate how patients' data can be displayed together with identifying information indicating the source hospital for each data element.

Discussion:

Opal's new design reuses much of the existing system, but involves modifications at each level (database(s), backend software(s), registration system, frontend application). The development of a prototype implementing the new design is partially completed. This proof-of-concept prototype will be used to guide the development of a production version which will be deployed at the six participating institutions.

Nouvelle cible thérapeutique pour la prise en charge des patients atteints de leucémie aiguë lymphoblastique : Implication du DFF40


Merve Kulbay1, Bruno Johnson1, Jacques Bernier1,2
1INRS-Institut Armand-Frappier 2INRS

La résistance aux médicaments de chimiothérapie est un obstacle majeur dans la prise en charge des patients atteint de cancer. Il a été démontré dans la littérature que l’altération au sein des cellules tumorales de l’expression du DFF40 (le facteur de fragmentation de l’ADN), responsable de la fragmentation internucleosomale de l’ADN lors de l’apoptose, expliquait les mauvais pronostics. Son expression est régulée de manière négative dans plusieurs types de tumeurs malins. Notre laboratoire a récemment démontré qu’une délétion génomique du DFF40 retardait l’activation de l’apoptose induit par des perturbateurs endocriniens. L’hypothèse de recherche suggère qu’une altération de l’expression du DFF40 dans les cellules cancéreuses pourrait altérer la réponse pharmacologique engendrée aux agents de chimiothérapie.L’objectif vise à déterminer le rôle du DFF dans la toxicité cellulaire engendrée par les médicaments de chimiothérapie. Méthodes Les cellules Jurkat ont été délités du gène DFF40 par la méthode CRISPR-cas9, puis traité avec des agents inhibiteurs de la topoïsomérase II (inhibiteur-Top2), ou antimétabolites. Divers essais d’apoptose ont ensuite été réalisés. Résultats Les cellules DFF40KO ont une résistance à l’apoptose induit par les agents antimétabolites, exposé pendant 24h(p<0.001). En revanche, le traitement avec des inhibiteur-Top2 pendant 24h induit une mortalité cellulaire supérieure chez les cellules DFF40KO(p<0.05). L’incorporation de nucléotides dans les cellules DFF40KO permet d’augmenter la prolifération cellulaire(p<0.05). Ces effets sont dus à une modulation des ROS, de la caspase-3 et de H2AX. Conclusion La caractérisation de l’expression du DFF40 au sein des cancers malins permettrait d’individualiser la thérapie pharmacologique et proposer une thérapie combinatoire. 

Oncolytic viruses as adjuvants for personalized anti-cancer vaccination in mice


Victor Mullins-Dansereau1,2, Dominic Guy Roy3,4, Nikolas Tim Martin3,4, Monique Marguerie3,4, John Cameron Bell3,4, Marie-Claude Bourgeois-Daigneault1,2
1CRCHUM: Centre Hospitalier de l’Université de Montréal Research Centre, Montreal, H2X 0A9 Canada 2Département de Microbiologie, d’Infectiologie et d’Immunologie, Faculté de Médecine, Université de Montréal, Montreal, H3C3J7, Canada 3Ottawa Hospital Research Institute, Centre for Innovative Cancer Research, Ottawa, K1H 8L6, Canada 4University of Ottawa, Department of Biochemistry, Microbiology and Immunology, Ottawa, K1H 8M5, Canada

Immunotherapeutic approaches like anti-cancer vaccines are emerging as long-term solutions for cancer treatment as they confer systemic protection and durable benefits. One such vaccination strategy that is currently under clinical investigation is a heterologous virus prime-boost approach using an Adenovirus (Ad) and the oncolytic virus (OV) Maraba (MRB) to vaccinate against tumor antigens. The concept of this platform is to engineer two different viruses to encode the same tumor antigen to prime and boost anti-tumor immunity. Given that the antigen is expressed by the viruses, peptide production is triggered upon robust infection. The vaccination efficiency is therefore partially dependent on the intrinsic sensitivity of tumors to virus infection, which varies from patient to patient and dictates the magnitude of antigen expression. In this study, we demonstrate that the co-administration of OVs with antigenic peptides is as efficient at inducing antigen-specific immunity as the use of antigen-engineered viruses. Our strategy is particularly attractive for personalized anti-cancer vaccines targeting patient-specific mutations as it has the advantage of avoiding the need to engineer patient-tailored viruses, a process that would be costly and time consuming. Our results demonstrate that off-the-shelf viruses encoding common tumor antigens can be efficiently co-administered with peptides corresponding to cancer-specific mutations as personalized cancer vaccines. We suggest that the use of OVs as vaccine adjuvant platforms warrants testing for anti-cancer vaccination.

Telomere-initiated senescence requires homologous recombination-mediated sister chromatid fusions and genomic instability


Marc-Alexandre Olivier1, Sabrina Ghadaouia1, Aurélie Martinez1, Guillaume B Cardin1, Nicolas Malaquin1, Francis Rodier1,2
1Centre de recherche du CHUM (CRCHUM) and Institut du cancer de Montréal (ICM), Montreal, QC, Canada 2Université de Montréal, Département de radiologie, radio-oncologie et médecine nucléaire, Montreal, QC, Canada

Introduction: Replicative senescence is the permanent proliferative arrest caused by gradual telomere attrition at each round of genome replication. Telomeric DNA loss leads to telomere uncapping (TU), which occurs when critically shortened telomeres lose their protective shelterin complex, revealing free chromosome ends recognized as DNA double-strand breaks. TU is proposed to directly trigger a p53-associated DNA damage response (DDR) that actively sustains a stable senescence-associated (SA) proliferation arrest (SAPA). Telomeres are heterogeneous in length within single cells, and the number of TU necessary for senescence onset remains poorly defined. Indeed, accumulating evidence suggests that normal cells tolerate a certain level of TU before entering SAPA, which lead us to hypothesize that TU cannot, in and of itself, trigger stable SAPA.

Method: We use controlled shelterin inactivation to trigger TU and subsequent SAPA in normal human fibroblasts, and validate our observations during natural replicative senescence.

Results: We show that TU alone cannot trigger senescence. While TU generates persistent DDR activation at telomeric ends, cells rapidly bypass primary cell cycle checkpoints, and the subsequent S-phase allows replicated sister chromatid fusions at telomeres mediated by homologous recombination. During the ensuing mitosis, fused telomeres lead to additional DNA breaks and to genomic instability (GI) including chromosome bridges or micronuclei, which then sustains a definitive p53-mediated secondary SAPA. During naturally occurring replicative senescence, interphase cells that have already undergone SAPA display GI, while near senescent cells captured in mitosis with TU have not yet undergone telomere fusions explaining their continued, albeit slowed, proliferation.

Progress for the field: We refine a novel multistep model defining entry into telomere-mediated replicative senescence, which is not directly induced by TU, but rather by an amplification of DNA lesions caused by critically short telomere fusions leading to permanent irreparable genome damage. Paradoxically, if replicative senescence serves as a tumor suppression mechanism, a stage of genomic instability susceptible to cancer initiation is now inherently associated with it.

The impact of rectal filling on rectal dose during hypofractionated radiotherapy of the prostate


Haley Patrick1, Kelly Agnew1, Luis Souhami1,2, John Kildea1,2
1McGill University 2McGill University Health Centre

Understanding the relationship between radiation doses delivered to patients during radiotherapy and the toxicities they experience is crucial in the endeavour to improve radiotherapy outcomes. In prostate cancer radiotherapy, motion of the prostate, bladder, and rectum between treatment days can impact radiation dose delivered to the organs of the pelvis, but the degree at which this occurs is not measured in regular clinical practice. Using a custom platform, we calculated the average dose delivered the rectums of 15 patients undergoing hypofractionated prostate radiotherapy. The volume of the rectum, along with the density composition of the volume was assessed using patient CT images taken over the course of treatment. The rectum’s composition and dose distribution at the time of treatment planning were compared to the volume and dose distributions over the course of treatment and the differences quantified using Z-statistics and bivariate correlation coefficients. Eight patients were found to have received significantly different dose distributions in comparison to what was planned. Of those eight, four possessed significantly different rectal volumes at the time of planning compared to at the time of delivery, which was found to be correlated to the volume of gas in the rectum (Pearson R^2 0.641). The remaining patient differences were attributed to variations in rectal curvature at the level of the prostate. This highlights the relationship between rectal filling and dose delivery consistency, and supports the hypothesis  that regulation of bowel filling may improve treatment accuracy.

The Mnk1/2-eIF4E axis facilitates invasion and metastasis of pregnancy associated breast cancer (PABC)


Qianyu Guo1,2, Fang Huang1,2, Vivian Zihui Li2, Samuel Preston1,2, Hanne Lefrere2,3, Audrey Emond2, Claudia Duerr4, Yirui Gui2, Christophe Goncalves2, Dany Plourde2, Jie Su2, William Yang1,2, Elie Khoury1,2, Valeria Narykina2, Jessica Nichol2, Mark Basik1,2, Frederic Amant3,5, Jorg Fritz4, Sonia del Rincon1,2, Wilson Miller1,2,6
1Department of Experimental Medicine, Faculty of Medicine, McGill University 2Segal Cancer Center, Lady Davis Institute & Jewish General Hospital 3Department of Oncology, Faculty of Medicine, University of Leuven 4Microbiology & Immunology, Faculty of Medicine, McGill University 5Netherlands Cancer Institute 6Montreal Rossy Cancer Network

Purpose: Breast cancer diagnosed within 5 years of a last pregnancy is defined as PABC, and is highly metastatic. Mammary gland (MG) involution induces tumor cells to adopt invasive characteristics. The MNK/eIF4E axis is a master regulator of translation of mRNAs that encode for proteins that promote cell invasion. We hypothesize that the MNK/eIF4E axis is required for the establishment of pre-metastatic niche, and could serve as a novel druggable target in PABC.

Methods: The mouse models used include BALB/c and C57BL/6 female wild-type (WT) and phospho-eIF4E deficient (eIF4ES209A/S209A knock-in) KI mice. Mice lactated for 10-14 days, and pups weaned to initiate involution. Breast cancer cells were implanted into the MGs 1 day post weaning. Lungs were harvested at 2 and 5 weeks post tumor cell implantation for histology and immune cell profiling.

Results: WT mice have higher lung metastatic burden than KI mice, suggesting that stromal phospho-eIF4E facilitates PABC metastasis. Immune cell profiling of WT and KI lungs 2 weeks post tumor cell injection revealed less type 2 innate lymphoid cells (ILC2) in the KI background. The KI tumor bearing lungs 5 weeks post tumor injection have more cytotoxic T cells and reduced myeloid-derived suppressor cells, compared to WT lungs. Expression of IL33, a hallmark ILC2-activating cytokine, is regulated by phospho-eIF4E. IL33 facilitates chronic inflammation and immunosuppression by inducing CXCL1, GM-CSF, CCL17, IL6 and PD-L1 on tumor cells. 

Conclusion: We propose that activation of the MNK/eIF4E axis supports a microenvironment that impairs tumor immune surveillance to facilitate PABC metastasis.