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Subject of the thesis

Mechanism of anti-tumor drug therapy on metastatic renal cancer: A study using human organotypic tissue slice cultures

Subject eligible for an allocation from the Foundation for Medical Research (FRM).
Published on 8 February 2019


Background:
The most frequent type of Kidney cancer is Clear Cell Renal Cell Carcinoma (CCRCC). Over the past years, protein kinase-targeted therapies (Sunitinib, Sorafenib, Temsirolimus) have become the main treatment for mRCC, however, most patients acquire resistance. Thus, there is an urgent need to identify new potential therapeutic targets. We performed a dual genetic/drug high-throughput screening choosing 36 potential genes targets and 80 kinase inhibitor drugs. Each of the target gene was silenced by shRNA Lentivirus-mediated transduction into the aggressive 786-O cell line derived from ccRCC that lacks the tumour suppressor VHL. Among the hit combinations that affect cell viability, one of them was chosen because it targeted two important kinases involved in cell survival and DNA repair, namely CK2 and ATM. (European Patent # 15306469.6.)

Aims/Perspectives:
1) Investigation of the molecular pathways affected by the drug combination.
2) Validation of this drug combination in the context of a pre-clinical study promoted by CHUGA Grenoble entitled « COMBOREIN ». The hit combination will be tested on: 1) tissue slices culture from tumors obtained after surgery, 2) models of patient derived xenografts generated on highly immunodeficient mouse strain.
3) Optimization of a microfluidic device to grow, treat with drugs and quantify cell viability of tumor tissue slices.

COMBOREIN: 50 patients/ year will be enrolled. This protocol has been validated by the CPP (Lille Patient Protection Committee, CPP 17/40, N ° 2017 A00702 51) and the collection of tumors began in November 2017. The tumor is brought to the pathology laboratory where three samples are collected. One is kept and frozen at CRB, while the others are carried fresh at 4°C, in a preservation medium to the laboratory where sections are made using a Vibratome® and are then cultured.
Treatments: A portion of the sections will be treated by the combination of ATM and CK2 inhibitors, while the other part will be treated by the current reference first line treatments in metastatic patients (Sunitinib, Pazopanib and Temsirolimus).
Methods: Cell culture, ccRCC cell lines 786-O, RCC4 and ACHN and normal renal proximal tubular epithelial cell line RPTEC; CRISPR Cas9; RT-qPCR; Spheroids culture and 3D assays (Viability, migration; invasion, videomicroscopy; light-sheet microscopy); Fresh Tissue Sectioning using a Vibratome VT1200 (Leica Microsystems), Organotypic Tissue Cultures, Patient-Derived Xenografts.
Analysis:
-Viability test: At the end of the treatment, live/dead cells are quantified in tumor cells grown as spheroids or in tumor slices using a fluorescent viability test. Light sheet microscopy will be used to go deeper inside the 3D organoids.
-Histology: Tissue sections are also included in paraffin to evaluate by immunohistochemistry the effectiveness of the treatment (Ki67 for proliferation, Caspase 3 activated for apoptosis).
-Protein Analysis: In parallel, the proteins extracted from the frozen sections are analyzed by Western Blot (anti-survivin, anti-PARP, anti-VE-cadherin and various signaling pathways affected). The mRNAs isolated from the same sections are also analyzed by RT-qPCR (VEGF, VHL, HIF). In addition, the VHL status of each tumor will be evaluated by PCR. The degree of vascularization of each tumor and the effect of the drug combination on the corresponding vascular network will be analyzed by the detection of cleaved and phosphorylated VE-cadherin.

Keywords:
Kidney cancer, synthetic lethality, 3D culture, protein kinase, targeted combinational therapy

Expected candidate background:
Dynamic and motivated student competent both in biochemistry and cell biology techniques

Three recent publications of the Thesis Director :
• Roelants C, Giacosa S, Pillet C, Bussat R, Champelovier P, Bastien O, Guyon L, Arnoux V, Cochet C and Filhol O
Combined inhibition of PI3K and Src kinases demonstrates synergistic therapeutic efficacy in clear-cell renal carcinoma.
Oncotarget, 2018, 9(53): 30066-30078
• Polena H, Creuzet J, Dufies M, Sidibé A, Khalil-Mgharbel A, Salomon A, Deroux A, Quesada JL, Roelants C, Filhol O, Cochet C, Blanc E, Ferlay-Segura C, Borchiellini D, Ferrero JM, Escudier B, Négrier S, Pages G and Vilgrain I
The tyrosine-kinase inhibitor sunitinib targets vascular endothelial (VE)-cadherin: A marker of response to antitumoural treatment in metastatic renal cell carcinoma.
British Journal of Cancer, 2018, 118(9): 1179-1188
• Burute M, Prioux M, Blin G, Truchet S, Letort G, Tseng Q, Bessy T, Lowell S, Young J, Filhol O and Théry M
Polarity reversal by centrosome repositioning primes cell scattering during epithelial-to-mesenchymal transition.
Developmental Cell, 2017, 40(2): 168-184
• Patent PCT/EP2016/072458 : A synthetic lethal drug combination for treating renal cell carcinoma. Filhol O, Cochet C, Giacosa S, Pillet C, Barette C, Soleilhac E.

Doctors supervised by the Thesis Director who have defended their thesis in the last 5 years.
Sofia Giacosa October 14, 2016 (42 months) Irtelis and FRM fellowship
• Filhol O, Giacosa S, Wallez Y and Cochet C
Protein kinase CK2 in breast cancer: The CK2β regulatory subunit takes center stage in epithelial plasticity.
Cellular and Molecular Life Sciences, 2015, 72(17): 3305-3322
• Giacosa S et al, A synthetic lethal drug combination for treating renal cell carcinoma. Patent PCT/EP2016/07245
• Roelants C, Giacosa S, Pillet C, Bussat R, Champelovier P, Bastien O, Guyon L, Arnoux V, Cochet C and Filhol O
Combined inhibition of PI3K and Src kinases demonstrates synergistic therapeutic efficacy in clear-cell renal carcinoma.
Oncotarget, 2018, 9(53): 30066-30078

Number of researchers and teacher-researchers with HDR in the team: 8

Total number of theses in the team: 2

Thesis supervisor:
Filhol Odile CR1 Inserm, HDR (2000)
BCI/IMAC
Tel: 33 (0)4 38 78 56 45
Fax: 33 (0)4 38 78 50 58

Laboratory:
Feige Jean-Jacques (Director)
U1036, Biologie du Cancer et de l’Infection (BCI)
CEA-Grenoble 17 rue des Martyrs
38054 Grenoble cedex 9
France

Research team within the lab:
Cell invasion in angiogenesis and cancer (IMAC) lead by Isabelle Vilgrain