Catherine Gaudin

Prion protein prevents human breast carcinoma cell line from tumor necrosis factor α-induced cell death

To define genetic determinants of tumor cell resistance to the cytotoxic action of tumor necrosis factor α (TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrPc), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrPc is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrPc, our data demonstrate that PrPc overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation.

Analysis of T-cell immune response in renal cell carcinoma: Polarization to type 1-like differentiation pattern, clonal T-cell expansion and tumor-specific cytotoxicity

We assessed the naturally occurring T‐cell immune response in primary renal cell carcinoma (RCC) tumors from 12 unselected patients. A predominance of CD3+ T‐cell receptor (TCR)α/β+ T cells was observed in tumor‐infiltrating lymphocytes (TILs), in contrast with peripheral blood lymphopenia found in some patients. Activation antigen expression on TILs revealed an imbalance in the activation status, with a significant percentage of CD69+ and HLA‐DR+ and a low percentage of CD25+ and CD71+ TILs. The lymphocyte activation gene‐3 (LAG‐3) was detected in some TIL subpopulations and especially in one patient in whom TILs were predominantly TCRα/β+CD8+DR+LAG‐3+. In addition, we found that RCC TILs are polarized to a global type 1‐like (Th1/Tc1) differentiation pattern (strong secretion of interferon‐γ and interleukin‐2 (IL‐2) following CD3/TCR cross‐linking) but are under the influence of the down‐modulatory cytokines IL‐6 (secreted by tumor cells) and IL‐10, within the tumor microenvironment. In 3 of 5 patients, clonal T‐cell expansion at the tumor site was found for several Vβ specificities, suggesting that in situ stimulation of specific clonotypes in response to potential tumor antigens is a frequent event in RCC. Furthermore, in one patient, selective intratumor amplification of a Vβ1 subpopulation (5% of TCR α/β+ cells) corresponding to 2 distinct Vβ1‐Jβ1.6 and Vβ1‐Jβ2.3 tumor‐specific MHC class I‐restricted cytotoxic T lymphocytes supports the view that discrete T‐cell subsets contribute readily to in situ immunosurveillance. Int. J. Cancer 72:431–440, 1997.

Identification of target actin content and polymerization status as a mechanism of tumor resistance after cytolytic T lymphocyte pressure

To investigate tumor resistance to T cell lysis, a resistant variant was selected after specific cytolytic T lymphocytes (CTL) selection pressure. Although the resistant variant triggered perforin and granzyme B transcription in specific CTLs, as well as their degranulation, it exhibited a dramatic resistance to cytotoxic T cell killing. It also displayed strong morphological changes with alterations of the actin cytoskeleton. Electron microscopy analysis revealed a loosen interaction between CTLs and the resistant variant despite the formation of apparently normal conjugates. Transcriptional profiling identified a gene expression signature that distinguished sensitive from resistant tumor targets. More notably, we found that actin-related genes ephrin-A1 and scinderin were overexpressed in resistant target. Silencing of these genes using RNA interference resulted in a restoration of normal cell morphology and a significant attenuation of variant resistance to CTL killing. Our present study shows that a shift in cytoskeletal organization can be used, by tumor cells, as a strategy to promote their resistance after CTL selection pressure.

Human NK Cells Lyse Organ-Specific Endothelial Cells: Analysis of Adhesion and Cytotoxic Mechanisms

Human organ-specific microvascular endothelial cells (ECs) were established and used in the present study to investigate their susceptibility to natural killer cell line (NKL)-induced lysis. Our data indicate that although IL-2-stimulated NKL (NKL2) cells adhered to the human peripheral (HPLNEC.B3), mesenteric lymph node (HMLNEC), brain (HBrMEC), and lung (HLMEC) and skin (HSkMEC.2) ECs, they significantly killed these cells quite differently. A more pronounced lysis of OSECs was also observed when IL-2-stimulated, purified peripheral blood NK cells were used as effector cells. In line with the correlation observed between adhesion pattern and the susceptibility to NKL2-mediated killing, we demonstrated using different chelators that the necessary adhesion step was governed by an Mg2+-dependent, but Ca2+-independent, mechanism as opposed to the subsequent Ca2+-dependent killing. To identify the cytotoxic pathway used by NKL2 cells, the involvement of the classical and alternate pathways was examined. Blocking of the Ca2+-dependent cytotoxicity pathway by EGTA/MgCl2 significantly inhibited endothelial target cell killing, suggesting a predominant role for the perforin/granzyme pathway. Furthermore, using confocal microscopy, we demonstrated that the interaction between NKL2 effectors and ECs induced cytochrome c release and Bid translocation in target cells, indicating an involvement of the mitochondrial pathway in NKL2-induced EC death. In addition, although all tested cells were sensitive to the cytotoxic action of TNF, no susceptibility to TRAIL or anti-Fas mAb was observed. The present studies emphasize that human NK cell cytotoxicity toward ECs may be a potential target to block vascular injury.

Clonal T cell expansion induced by interleukin 2 therapy in blood and tumors.

In a phase I clinical trial on the effects of preoperative adjuvant IL-2 therapy given to patients undergoing hepatic resection of colorectal adenocarcinoma metastases, we monitored the putative induction of T cell clonal expansion in both tissues and blood. The presence of T cell clonotypes was analyzed with a PCR-based method that determines V-D-J junction size patterns in T cell receptor (TCR) V beta subfamilies in samples before and after a 5-d IL-2 infusion. This high resolution method analyzing CDR3 sizes of TCR transcripts was used in conjunction with FACS analysis of the corresponding T cell subpopulations with TCR V beta-specific mAb. At time of surgery (day 8 after starting IL-2), we found in the three patients analyzed with V beta-C beta primers multiple dominant T cell clonotypes in the tumor and peritumoral tissues which had probably expanded as a result of therapy. In three control patients not treated with IL-2, multiple oligoclonal patterns were not observed with this set of primers. In the fourth control patient a unique V beta 21-C beta CDR3 pattern which corresponds to two dominant clonotypes was found in the tumor. The same dominant clonotypes identified in the tumor after IL-2 were also detectable in the blood and comparison of the profiles obtained before and after IL-2 therapy indicates that they were induced by IL-2. The relative expansion of the corresponding T cell subpopulations was maintained for varying periods of time after surgery (4-7 d and almost 2 yr in one case). Together, these results indicate that IL-2 induces marked expansion of several T cell clones. Systemic IL-2 administration may represent, either alone or as a vaccine adjuvant, an appropriate way of boosting antigen-specific immune responses.

Abstract 2995: Loss of SHISA3 is an early event of the epithelial-to-mesenchymal transition associated with chemoresistance in prostate cancer

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Despite scientific advances over the past decade, prostate cancer still remains the fifth leading cause of death from cancer in men worldwide. Docetaxel has an established role in the treatment of metastatic castrate-resistant prostate cancer (CRPC). However, malignant cells frequently acquire Docetaxel resistance. Therefore, further research is required to understand the molecular mechanisms underlying Docetaxel-resistance, which could be helpful in formulating alternative and superior therapeutic strategies. Methods: Two Docetaxel-resistant prostate cancer cell lines (IGR-CaP1 and PC3) were obtained by continuous exposure to Docetaxel. By comparing genes and miRs expression profiles established in these two Docetaxel-resistant models, we observed an enrichment of genes involved in the epithelial-to-mesenchymal transition (EMT) in the chemoresistant models and identified new genes potentially implicated in the resistance mechanism. Results: We identified the SHISA3 gene as a highly down-regulated gene in resistant cells. This gene was originally identified as an inhibitor of Wnt and FGF signaling during development in Xenopus. We showed that SHISA3 was lost during the acquisition of resistance to Docetaxel possibly via an EMT mechanism. Knockdown of SHISA3 in the parental IGR-CaP1 and PC3 cells triggered loss of the tight junction protein Occludin, engaged the Cadherin switch and increased the migratory properties of cells in vitro. Loss of SHISA3, along with loss of E-cadherin expression and expression of the mesenchymal marker N-cadherin were also observed in Docetaxel-resistant tumors obtained from IGR-CaP1-R xenografted mice. Phylogenetic comparison of SHISA3 gene sequences suggested that it may correspond to transmembrane adapters capable of regulating the activity of membrane receptors such as growth factor receptors. We are currently studying the binding partners of SHISA3 in our models by a proteomic approach to identify the implicated signaling pathway in prostate cancer. Conclusion: Our results show that loss of SHISA3 is an early event linked to the EMT process associated with chemoresistance and suggest that SHISA3 could be a useful biomarker to identify chemoresistant cells. Moreover, identification of its mechanism of action may lead to the identification of new therapeutic targets to overcome Docetaxel resistance. Citation Format: Nicolas J-p Martin, Sophie Cotteret, Catherine Gaudin, Marine Garrido, Safae Aarab-Terrisse, Nader al Nakouzi, Lucas Gentilini, Daniel Compagno, Vasily Ogryzko, Guillaume Meurice, Karim Fizazi, Anne Chauchereau. Loss of SHISA3 is an early event of the epithelial-to-mesenchymal transition associated with chemoresistance in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2995. doi:10.1158/1538-7445.AM2015-2995

Regulation of eIF4F complex by the peptidyl prolyl isomerase FKBP7 in taxane-resistant prostate cancer

Targeted therapies that exploit the signaling pathways involved in prostate cancer are required to overcome chemoresistance and improve treatment outcomes for men. Molecular chaperones play a key role in the regulation of protein homeostasis and are potential targets to alleviate chemoresistance. Using image-based high content siRNA functional screening based on a gene expression signature, we identified FKBP7, a molecular chaperone overexpressed in docetaxel-resistant and in cabazitaxel-resistant prostate cancer cells. FKBP7 was upregulated in human prostate cancers and correlated with the recurrence in patients receiving Docetaxel. FKBP7 silencing showed that FKBP7 is required to maintain the growth of chemoresistant cell lines and of chemoresistant tumors in mice. Mass spectrometry analysis revealed that FKBP7 interacts with the eIF4G component of the eIF4F translation initiation complex to mediate survival of chemoresistant cells. Using small molecule inhibitors of eIF4A, the RNA helicase component of eIF4F, we were able to overcome docetaxel and cabazitaxel resistance.

Abstract 3948: Identification of a new therapeutic target in prostate cancer from siRNA screening in Docetaxel-resistant cells

Background: Prostate cancer is the most common male cancer and the second leading cause of death. Docetaxel is a chemotherapy used to manage the Metastatic Castrate-Resistant Prostate Cancer (mCRPC). Inexorably, patients will develop resistance to this treatment. Therefore, new efforts are needed to understand the molecular mechanisms of resistance, enabling the discovery of specific therapeutic targets to avoid this resistance in patients. Methods: We developed a series of Docetaxel-resistant cell lines (IGR-CaP1-R, PC3-R, 22RV1-R, LNCaP-R) and we generated a signature of 1,006 highly differentially expressed genes potentially implicated in chemoresistance. Moreover, we performed a high throughput siRNA screening in IGR-CaP1, allowing us to select 60 genes with a functional role in Docetaxel resistance. Results: We focused our attention on the role of a chaperone protein, which is localized in the endoplasmic reticulum and belongs to the FKBP-type peptidyl-prolyl cis/trans isomerase (PPIase) family. This protein is highly overexpressed in Docetaxel-resistant prostate cancer cell lines and its overexpression is specifically induced by agents targeting microtubules such as taxanes (Docetaxel, Cabazitaxel, and Paclitaxel) or Nocodazole. Induction of its expression was also observed in tumors obtained from sub-cutaneous injection of IGR-CaP1 after Docetaxel treatment in vivo. Specific inhibition with siRNA leads to cell death of the Docetaxel-resistant IGR-CaP1-R and PC3-R cell lines as well as the Cabazitaxel-resistant IGR-CaP1 cells. In LNCaP-R and 22RV1-R resistant cell lines, it decreases the proliferation rate. Moreover, knockdown of this protein restores Docetaxel response of all the resistant cell lines. In addition, although this chaperone is highly expressed in several non cancerous cells, its extinction does not affect cell growth suggesting a different function of the protein between chemoresistant cells and non cancerous cells. Preliminary observations suggest that this protein could be mislocalized in Docetaxel-resistant cells in some cytoplasmic vesicles, as a consequence of taxane treatment. Conclusion: We identified a new chaperone harbouring an enzymatic activity which could be a relevant therapeutic target in chemoresistant CRPC. We are now focusing on the identification of a specific inhibitor in order to validate the role of this target in two Docetaxel-resistant prostate cancer mice models established within the laboratory. Citation Format: Marine Garrido, Nicolas J-p Martin, Catherine Gaudin, Elaine Del Nery, Jacques Camonis, Franck Perez, Stephanie Lerondel, Alain Le Pape, Karim Fizazi, Anne Chauchereau. Identification of a new therapeutic target in prostate cancer from siRNA screening in Docetaxel-resistant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3948. doi:10.1158/1538-7445.AM2015-3948