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Intitulé du sujet: Functional consequences and therapeutic potential arising from the identification of CD160-TM as a tumor marker of breast cancers

Sujet

Codirection:

Nombre de mois: 48 mois

Ecole Doctorale: ED 561 - Hématologie, Oncogénèse et Biothérapies

Unité de recherche et équipe:

INSERM U976 / HIPI - Team 1 (Onco-dermatology and Therapies)

Coordonnées de l’équipe:

Saint Louis Hospital

Bazin Pavillion

1 avenue Claude Vellefaux

75010 Paris

France

Secteur: Sciences de la vie / Life Sciences

Langue attendue: Anglais

Niveau de langue attendu: C2

Description

Description du sujet:

Background

Breast cancer (BC) is the most commonly diagnosed and the first leading cause of cancer death for women world- wide [1]. The implementation of prevention programs and the improvement of the effectiveness of treatments considerably reduced the mortality associated with this pathology. Nevertheless, the 10-year survival rate for metastatic breast cancer is estimated at 13% and many patients develop resistance to conventional treatments.

Although major progresses have been achieved regarding the quality and the quantity of current treatments, the therapeutic scheme remains complex and dependent on the disease stage (early vs metastatic) and the BC molecular subtype. Indeed, BC is a heterogeneous disease that can be classified according to histopathological criterions, staging, genetic alterations or molecular status [2, 3]. Molecular classification based on immuno-histological staining is routinely used and relies on the detection of estrogen, progesterone and human epidermal growth factor receptor-2 (ER, PR and HER2, respectively), or Ki-67 amplification. Accordingly, BC are classified in four main subtypes, namely luminal A (ER+, PR+, HER2−, Ki-67−), luminal B (ER+, PR±, HER2±, Ki-67 ±), HER2+ (ER−, PR−, HER2+), and triple negative (TNBC; ER−, PR−, HER2−) [4]. Given their distinct molecular and cellular characteristics, each subtype presents its own therapeutic scheme and highly variable responses to treatments, the main observed limits being:

- a high risk of overall relapse. It is estimated that approximately 10% of the treated patients will relapse (without metastases) within 5 years of diagnosis, all subtypes combined [5];

- a persistent risk of relapse in the long term (>20 years) for patients with ER+ BC [6];

- the lack of therapeutic options for the treatment of TNBC tumors [7];

- the low diversification or non-specificity of tumor targets which reinforce the appearance of resistance and the impossibility of counteracting them [3];

- the strong molecular heterogeneity and cellular plasticity associated with breast cancers. The use of treatments combination can lead to the appearance of double resistance and the absence of other treatments in the event of relapse

These clinical outcomes illustrate a critical need for new innovative therapeutic approaches in the treatment of breast cancers.

CD160 has been originally identified as a receptor expressed by cytotoxic NK and CD8+ T lymphocytes, anchored at the plasma membrane through a glycosyl-phosphatidyl-inositol motif (CD160-GPI) [8, 9]. Its engagement is accompanied by an induction of the cytotoxicity of NK lymphocytes and the production of a Th1 type cytokine response (production of IFNγ, TNFα, IL-6) [10]. Its preponderant role in the development of an anti -tumor response has also been demonstrated [11]. A transmembrane isoform (CD160-TM) has also been characterized, resulting from an alternative splicing of CD160 gene, whose expression by NK lymphocytes is induced by activation [12]. Its recruitment then allows an increase in the NK cell cytotoxic response. Remarkably, the GPI and TM isoforms of CD160 present 98% of homology in their extracellular domains but display distinct activating signaling pathways in NK cells.

Recently, we reported the abnormal expression of CD160-TM, but not CD160-GPI, by TNBC [13]. We further established in in vitro functional assays that a specific anti-CD160-TM antibody (Ab) is able to mediate antibody-dependent cell cytotoxicity (ADCC) and phagocytosis (ADCP) and consequently TNBC cell line depletion. We also demonstrated that this antibody exerts an efficient anti-tumor activity in vivo, in a TNBC mouse model. Altogether, these data identify CD160‐TM as a tumor marker for TNBC and provide a rationale for the use of anti‐CD160‐TM antibodies as therapeutic tools in this tumor context.

Preliminary experiments, performed on patients' biopsies and cell lines from other BC subtypes (luminal A/B and HER2+), led to the detection of CD160-TM tumor expression, as observed for TNBC. This atypical expression of CD160-TM might therefore be a hallmark for breast tumors and raises the question of its functional implications at the tumor level (tumor proliferative or invasive potential and/or establishment of an immunosuppressive microenvironment) but also of its use in the context of the development of anti-CD160 antibodies capable of promoting tumor depletion or growth arrest.

Project

Based on our previous results, the main objectives of the project will be:

1- to characterize the function of CD160-TM in BC

2- to evaluate CD160-TM potential as a therapeutic target

Part 1: Functional characterization of CD160-TM in BC.

BC cell lines (luminal A/B or HER2+), unable to express or over-expressing CD160-TM, will be generated. The impact of the gain or loss of CD160-TM expression will then be evaluated by:

- Comparing the tumorigenesis processes exhibited by WT cell lines and their genetically modified counterparts. The proliferation, migration and invasion of the different cell lines will be assessed in bi- and tri-dimensional settings (single cells vs spheroids) using a live cell imaging system (Incucyte). The potential role of CD160-TM in the processes leading to BC resistance to clinically used chemotherapies (e.g. doxorubicin; paclitaxel) will also be explored by evaluating the induction of apoptosis upon treatment.

- Analyzing the genetic and molecular modifications resulting from CD160-TM expression. The RNA content of the different cell lines will be analyzed by RNA sequencing to further identified the mostly impacted proteins and corresponding pathways. A deeper analysis of the signaling pathways involving the identified proteins will be performed to confirm their implication in CD160-TM-mediated pathways (e.g. proteome, chemokines/cytokines or kinases activation status analyses by Western blot).

Part 2 : Use of CD160-TM as a tumor target in BC.

Both in vitro and in vivo experiments will be conducted to establish the proof-of-concept for the use of anti-CD160-TM antibodies as novel therapeutic tools for the treatment of BC:

2.1- in vitro testing of anti-CD160-TM antibody to promote BC tumor cell depletion by ADCC or ADCP.

BC cell lines will be mixed with NK cells or phagocytes in the presence of an anti-CD160-TM Ab or its corresponding control isotype. Target cells' apoptosis or phagocytosis will be assessed by flow cytometry. Eventually, combinations of anti-CD160-TM Ab with chemotherapies or antibodies agonist of immune checkpoints (PD1/PDL1, CTLA4) and currently used to treat patients, will also be tested. Such experiments will also be performed by live cell imaging (Incucyte) by using tumor spheroids as cellular targets and lymphocytes, isolated from the blood of healthy donors, as effector cells, as a tri-dimensional model better mimics the physiological reality, especially in terms of lymphocytes infiltration. The phenotype and activation status of the infiltrating lymphocytes will then be monitored by flow cytometry.

2.2- in vivo testing of anti-CD160-TM antibody to promote BC tumor cell depletion.

In order to evaluate the anti-tumor effectiveness of the anti-CD160-TM antibody in vivo, intra-mammary graft of breast cancer cell lines (luminal A/B or HER2+) will be performed on mice with severe combined immunodeficiency (SCID). These mice do not have an adaptive immune response but retain normal NK lymphocyte activity, making ADCC generation possible. After establishment of the tumor, the anti-CD160-TM antibody or a control isotype will be injected. Mice survival and tumor volume will be monitored. A regression of breast tumors, or even their depletion, would be expected, as observed for TNBC [13].

Expected outcomes

The corresponding results should provide new information on the pathophysiology of breast cancer, with the possible involvement of CD160-TM in the mechanisms governing the progression and the invasion of the tumor. They should also bring the first proof-of-concept for the use of an anti-CD160-TM antibody as a possible therapeutic tool in this pathology.

Compétences requises:

Technical skills:

  • cell biology (cell culture, transfection, proliferation, apoptosis),
  • flow cytometry,
  • biochemistry (protein extraction, Western blot),
  • previous experience with mouse models (animals handling, tumor cells engraftment).

Scientific knowledge:

  • strong background in the field of breast cancers.
  • knowledge in anti-tumor immune response and tumor microenvironment will be appreciated.

Professional skills:

  • Motivation and perseverance
  • Strong ethic and intellectual honesty
  • Ability to recognize his/her mistakes and to inform his/her supervisor
  • Ability to perform adequate and regular bibliographic survey and to present results with clarity and objectivity
  • Ability to integrate a research team and to communicate with its members
  • Use of basic softwares (Word, Excel, Power Point, Prism)

Références bibliographiques:

  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2021;71:209–49.
  2. Eliyatkin N, Yalcin E, Zengel B, Aktas S, Vardar E. Molecular classification of breast carcinoma: from traditional, old‐fashioned way to a new age, and a new way. J Breast Health. 2015;11:59–66.
  3. Lukasiewicz S, Czeczelewski M, Forma A, Baj J, Sitarz R, Stanislawek A. Breast Cancer‐epidemiology, risk factors, classification, prognostic mark‐ ers, and current treatment strategies‐an updated review. Cancers (Basel). 2021;13
  4. Cardoso F, Kyriakides S, Ohno S, Penault‐Llorca F, Poortmans P, Rubio IT, et al. Early breast cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow‐updagger. Ann Oncol. 2019;30:1194–220.
  5. Maughan KL, Lutterbie MA, Ham P. Treatment of Breast Cancer. Am Fam Physician. 2010;81(11):1339-1346.
  6. Pan H, Gray R, Braybrooke J, et al. 20-Year Risks of Breast-Cancer Recurrence after Stopping Endocrine Therapy at 5 Years. N Engl J Med. 2017;377(19):1836-1846. doi:10.1056/NEJMoa1701830
  7. Lee J. Current treatment landscape for early triple‐negative breast cancer (TNBC). J Clin Med. 2023;12.
  8. Anumanthan A, Bensussan A, Boumsell L, Christ AD, Blumberg RS, Voss SD, et al. Cloning of BY55, a novel Ig superfamily member expressed on NK cells, CTL, and intestinal intraepithelial lymphocytes. J Immunol. 1998;161:2780–90.
  9. Bensussan A, Gluckman E, el Marsafy S, Schiavon V, Mansur IG, Dausset J,
    et al. BY55 monoclonal antibody delineates within human cord blood and bone marrow lymphocytes distinct cell subsets mediating cytotoxic activity. Proc Natl Acad Sci USA. 1994;91:9136–40
  10. Barakonyi A, Rabot M, Marie‐Cardine A, Aguerre‐Girr M, Polgar B, Schiavon V, et al. Cutting edge: engagement of CD160 by its HLA‐C physiological ligand triggers a unique cytokine profile secretion in the cytotoxic peripheral blood NK cell subset. J Immunol. 2004;173:5349–54.
  11. Tu TC, Brown NK, Kim TJ, Wroblewska J, Yang X, Guo X, et al. CD160 is essential for NK‐mediated IFN‐gamma production. J Exp Med. 2015;212:415–29.
  12. Giustiniani J, Bensussan A, Marie‐Cardine A. Identification and characterization of a transmembrane isoform of CD160 (CD160‐TM), a unique activating receptor selectively expressed upon human NK cell activation. J Immunol. 2009;182:63–71.
  13. Scheffges C, Devy J, Giustiniani J, Francois S, Cartier L, Merrouche Y, Foussat A, Potteaux S, Bensussan A, Marie-Cardine A. Identification of CD160-TM as a tumor target on triple negative breast cancers: possible therapeutic applications. Breast Cancer Res. 2024; 26, 28. https://doi.org/10.1186/s13058-024-01785-x