Hassan El Btaouri

Discoidin Domain Receptors: Potential Actors and Targets in Cancer

[This corrects the article on p. 55 in vol. 7, PMID: 27014069.].

Extracellular Matrix Proteins Modulate Antimigratory and Apoptotic Effects of Doxorubicin

Anticancer drug resistance is a multifactorial process that includes acquired and de novo drug resistances. Acquired resistance develops during treatment, while de novo resistance is the primary way for tumor cells to escape chemotherapy. Tumor microenvironment has been recently shown to be one of the important factors contributing to de novo resistance and called environment-mediated drug resistance (EMDR). Two forms of EMDR have been described: soluble factor-mediated drug resistance (SFM-DR) and cell adhesion-mediated drug resistance (CAM-DR). Anthracyclines, among the most potent chemotherapeutic agents, are widely used in clinics against hematopoietic and solid tumors. Their main mechanism of action relies on the inhibition of topoisomerase I and/or II and the induction of apoptosis. Beyond this well-known antitumor activity, it has been recently demonstrated that anthracyclines may display potent anti-invasive effects when used at subtoxic concentrations. In this paper, we will describe two particular modes of EMDR by which microenvironment may influence tumor-cell response to one of these anthracyclines, doxorubicin. The first one considers the influence of type I collagen on the antimigratory effect of doxorubicin (CAM-DR). The second considers the protection of tumor cells by thrombospondin-I against doxorubicin-induced apoptosis (SFM-DR).

Role of JNK/ATF-2 pathway in inhibition of thrombospondin-1 (TSP-1) expression and apoptosis mediated by doxorubicin and camptothecin in FTC-133 cells.

Our previous studies have shown that camptothecin and doxorubicin triggered ceramide accumulation via de novo synthesis pathway. De novo ceramide generation was responsible for the drug-induced apoptosis through a caspase-3-dependent pathway and a decrease of thrombospondin-1 expression in human thyroid carcinoma FTC-133 cells. Here, we demonstrate that Jun N-terminal kinases play a critical role in camptothecin- and doxorubicin-induced down-regulation of thrombospondin-1 expression: i) de novo ceramide synthesis pathway activates Jun N-terminal kinase 1/2 resulting in activating transcription factor 2 phosphorylation; ii) cell treatment by SP600125, a Jun N-terminal kinase specific inhibitor, strongly reduced activating transcription factor 2 phosphorylation and completely abolished camptothecin and doxorubicin effects; and iii) activating transcription factor 2 expression silencing greatly attenuated camptothecin- and doxorubicin-induced down-regulation of thrombospondin-1 expression and apoptosis. The set of our data established that camptothecin- and doxorubicin-induced activation of Jun N-terminal kinase/activating transcription factor 2 pathway via de novo ceramide synthesis down-regulates thrombospondin-1 expression and apoptosis in human thyroid carcinoma FTC-133 cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Type I collagen aging impairs discoidin domain receptor 2-mediated tumor cell growth suppression.

Tumor cells are confronted to a type I collagen rich environment which regulates cell proliferation and invasion. Biological aging has been associated with structural changes of type I collagen. Here, we address the effect of collagen aging on cell proliferation in a three-dimensional context (3D). We provide evidence for an inhibitory effect of adult collagen, but not of the old one, on proliferation of human fibrosarcoma HT-1080 cells. This effect involves both the activation of the tyrosine kinase Discoidin Domain Receptor 2 (DDR2) and the tyrosine phosphatase SHP-2. DDR2 and SHP-2 were less activated in old collagen. DDR2 inhibition decreased SHP-2 phosphorylation in adult collagen and increased cell proliferation to a level similar to that observed in old collagen. In the presence of old collagen, a high level of JAK2 and ERK1/2 phosphorylation was observed while expression of the cell cycle negative regulator p21CIP1 was decreased. Inhibition of DDR2 kinase function also led to an increase in ERK1/2 phosphorylation and a decrease in p21CIP1 expression. Similar signaling profile was observed when DDR2 was inhibited in adult collagen. Altogether, these data suggest that biological collagen aging could increase tumor cell proliferation by reducingthe activation of the key matrix sensor DDR2.

Accumulation of lactosylceramide and overexpression of a PSC833-resistant P-glycoprotein in multidrug-resistant human sarcoma cells

The selection pressure for resistance to chemotherapy is accompanied by the enhanced expression of ABC proteins and increased cellular glycosphingolipid content. Thus, a possible connection between glycosphingolipid metabolism and ABC proteins in drug resistance has been suggested. In the present study, we established two human multidrug-resistant (MDR) cell lines derived from MESSA sarcoma cells by culturing with increasing concentrations of doxorubicin (DX5 cells) or doxorubicin together with cyclosporin A (GARF cells). Both resistant cell lines overexpressed the MDR1 gene and the wild-type P-glycoprotein at the same level. The cyclosporin derivative PSC833, a potent inhibitor of P-glycoprotein, sensitized DX5 but not GARF cells to the cytotoxic effects of daunorubicin. Moreover, PSC833 increased the nuclear accumulation of daunorubicin and the cellular accumulation of [3H]vinblastine in the DX5 but not in the GARF cells. The cellular incorporation of [3H]-cyclosporin A was lower in DX5 cells compared to MESSA and GARF cells, which incorporated the same level of [3H]-cyclosporin A. Sphingolipid analysis showed that the lactosylceramide level was 2.5- and 5-fold higher in DX5 and GARF cells, respectively, than in MESSA cells. Whereas the pharmacological inhibition of lactosylceramide synthesis was able to reverse only partially the resistance of GARF cells to daunorubicin without significant increase in nuclear accumulation of the drug, the same treatment before the co-treatment with PSC833 and daunorubicin increased the cytotoxic effect of daunorubicin and its nuclear accumulation. These data suggest a possible relationship between lactosylceramide levels and the resistance of P-glycoprotein to modulation by MDR modulators.

Inula Viscosa Extracts Induces Telomere Shortening and Apoptosis in Cancer Cells and Overcome Drug Resistance

ABSTRACT Telomerase is activated in human papillomavirus (HPV) positive cervical cancer and targeting telomeres offers a novel anticancer therapeutic strategy. In this study, the telomere targeting properties, the cytotoxic as well as the pro-apoptotic effects of hexane (IV-HE) and dichloromethane (IV-DF) fractions from Inula viscosa L. extracts were investigated on human cervical HeLa and SiHa cancer cells. Our data demonstrate that IV-HE and IV-DF extracts were able to inhibit cell growth in HeLa and SiHa cells in a dose-dependent manner and studied resistant cell lines exhibited a resistance factor less than 2 when treated with the extracts. IV-HE and IV-DF extracts were able to inhibit telomerase activity and to induce telomere shortening as shown by telomeric repeat amplification protocol and TTAGGG telomere length assay, respectively. The sensitivity of fibroblasts to the extracts was increased when telomerase was expressed. Finally, IV-HE and IV-DF were able to induce apoptosis as evidenced by an increase in annexin-V labeling and caspase-3 activity. This study provides the first evidence that the IV-HE and IV-DF extracts from Inula viscosa L. target telomeres induce apoptosis and overcome drug resistance in tumor cells. Future studies will focus on the identification of the molecules involved in the anticancer activity.

Tomentosin Induces Telomere Shortening and Caspase‐Dependant Apoptosis in Cervical Cancer Cells

Tomentosin, a natural sesquiterpene lactone purified from of Inula viscosa L., was investigated for its anti‐proliferative, telomere shortening, and apoptotic effects on human cervical cancer HeLa and SiHa cell lines. Tomentosin was found to inhibit the growth of SiHa and HeLa cell lines in dose and time‐dependent manner (IC50 values of 7.10 ± 0.78 μM and 5.87 ± 0.36 μM, respectively after 96 h of treatment). As evidenced by TTAGGG telomere length assay, tomentosin target specifically the telomeric overhang lengthening. This was confirmed by the evaluation of the cytotoxic effects of tomentosin in the foetal fibroblast Wi38 and JW10 cells which were derived from Wi38 and express hTERT, the telomerase catalytic subunit. We found that JW10 cells are 4.7‐fold more sensitive to tomentosin which argues for telomere as its specific target. Furthermore, we found that tomentosin mediate this cytotoxic effect by inducing apoptosis and cell cycle arrest at G2/M phase. Morphological features of treated cells, as evidenced by Hoechst 33324 staining, revealed that the cytotoxic effect was due to induction of apoptosis. This was accompanied by pro‐caspase‐3 cleavage, an increase in caspase‐3 activity and a cleavage of poly (ADP‐ribose) polymerase (PARP). Moreover, tomentosin induced a decrease in mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species (ROS), accompanied by a decrease in Bcl‐2 expression. This indicates that tomentosin‐induced apoptosis may involve a mitochondria‐mediated signaling pathway. This study provides the first evidence that tomentosin targets telomere machinery and induces apoptosis in cervical cancer cells. The molecular mechanism underlying tomentosin‐induced apoptosis may involve a mitochondria‐mediated signaling pathway. J. Cell. Biochem. 118: 1689–1698, 2017.

Abstract 3378: Discoidin domain receptor 2 differentially controls HT-1080 cell proliferation in young-adult and old type I collagen 3D matrices

Type I collagen plays a key role in tumor proliferation, adhesion and migration. We have recently demonstrated that this fibrillar and life-long protein undergoes detrimental changes in biochemical and biomechanical properties during aging (1). In addition to a high Advanced Glycation End-products (AGE) and cross-links content, old collagen exhibits a significant loss in fibrillar properties. In this study, we analyzed the effect of collagen aging on human HT-1080 fibrosarcoma cell proliferation in 3D matrix model. For this, type I collagen was extracted from tail tendons of young-adult (2 months) and old (2 years) rats. In 3D matrix, the rate of HT-1080 cell growth was significantly lower in young-adult collagen. This was accompanied by a down-regulation of ERK1/2 phosphorylation and an increase in the cyclin-dependent kinase inhibitor p21 expression. Blocking antibodies and siRNA strategy against β1 integrin and AGEs receptor (RAGE) did not increase cell growth in young-adult collagen. Accumulating evidence suggest that Discoidin Domain Receptor 2 (DDR2) is a receptor tyrosine kinase with the unique ability to be activated by fibrillar collagen. To determine whether differential DDR2 responses are elicited by the two collagens, DDR2 was immunoprecipitated from cell lysates, and its phosphorylated form was detected by immunoblotting. DDR2 phosphorylation level was increased in young-adult collagen. This was accompanied by an increase in the tyrosine phosphatase SHP2 phosphorylation, the primary target of DDR2. Interestingly, the DDR2 inhibitor nilotinib was able to restore cell proliferation in young-adult collagen. In addition to an increase in ERK1/2 phosphorylation, nilotinib decreased SHP2 phosphorylation and p21 expression. Taken together, these data show that age-associated changes of type I collagen organization, especially fibrillar properties, are critical for tumor cell proliferation control by DDR2. References: (1) Wilson SL et al. FASEB J. 2014 28(11):14-25 Citation Format: Charles Saby, Roselyne Garnotel, Hassan El Btaouri, Laurence Van Gulick, Pierre Jeannesson, Hamid Morjani. Discoidin domain receptor 2 differentially controls HT-1080 cell proliferation in young-adult and old type I collagen 3D matrices. [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 3378. doi:10.1158/1538-7445.AM2015-3378

Abstract 1154: Age of type I collagen is critical for regulation of HT-1080 cell proliferation in 3D matrices

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA The tumor microenvironnement consists of cells, soluble factors, signaling molecules, extracellular matrix (EM), and mechanical cues that can promote neoplastic transformation, support tumor growth and invasion, and foster therapeutic resistance. In addition to provide a physical barrier against invasion, type I collagen, the most abundant protein in EM, is also known to play a role in tumor cell proliferation (1). DeClerck et al. (2) has reported a direct inhibitory effect of fibrillar type I collagen on tumor cell proliferation when compared to the monomeric one. Type I collagen is a life-long protein (half-life 15 yrs) susceptible to undergo non enzymatic post-translational modifications such as production of Advanced Glycation End Products (AGEs). These modifications are able to influence the behavior of tumor cells. Here, we investigated whether regulation of HT-1080 cell proliferation is a consequence of type I collagen aging in terms of fibrillar state of the protein (2,3), or of the level of AGEs, also known to stimulate cell proliferation (4). Rat tail type I collagen were prepared from adult (2 months) and old (2 years) animals. A significant increase in AGEs has been observed in collagen extracted from old rats, compared to the adult. Under 3D growth condition, HT-1080 cells proliferate rapidly in old type I collagen relative to the adult one. This effect was not observed in 2D coating culture. The low rate of proliferation in adult collagen is accompanied by a downregulation of ERK1/2 activation, and an upregulation of p21, the inhibitor of cell cycle progression. This age-dependent cell proliferation regulatory effect does not involve α2β1 integrins. Neither Pi3K/Akt nor JNK pathways were involved in this process. Accumulating evidence suggest that Discoidin Domain Receptor 2 (DDR2) is a Receptor Tyrosine Kinase (RTK) with the unique ability among RTKs to respond to fibrillar collagen (5). To determine whether a similar response is elicited by DDR2 in both collagens, DDR2 was immunoprecipitated from cell lysates, and phosphorylated DDR2 was detected by immunoblotting. High level of DDR2 phosphorylation was observed after 3 days culture in adult collagen compared to the old one, whereas DDR2 expression remains unchanged in the presence of both collagens. Moreover, the DDR2 kinase function inhibitor, nilotinib, restored cell proliferation in adult collagen to a level similar to that observed in the old one. Taken together, these data establish a role for DDR2 in critical events during restriction of tumor cell proliferation induced by adult type I collagen. References: (1) Lukashev ME et al. Trends Cell Biol. 1998;8(11):437, (2) Wall SJ et al. J Biol Chem. 2005;280(48):40187, (3) Wilson SL et al. FASEB J. 2013; (accepted), (4) Ishihara K et al. FEBS Lett. 2003;28;550(1-3):107, (5) Rajeshwari R et al. Cancer Metastasis Rev. 2012;31(1-2):295 Citation Format: Charles Saby, Hassan El Btaouri, Julie Routhier, Celine Charpentier, Laurence Van-Gulick, Marie Pierre Courageot, Pierre Jeannesson, Hamid Morjani. Age of type I collagen is critical for regulation of HT-1080 cell proliferation in 3D matrices. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1154. doi:10.1158/1538-7445.AM2014-1154

Abstract 2105: Advanced glycation end products of type I collagen promote cell proliferation and resistance against doxorubicin in HT-1080 cancer cells.

Resistance to apoptosis and chemotherapy is a critical factor in cancer recurrence and patient relapse. Extracellular matrix via its receptors, the integrins, has emerged as a major pathway contributing to cancer cell survival and resistance to chemotherapy (Aoudjit and Vuori, Chemother Res Pract. 2012;2012:283181). However, the effects of extracellular matrix advanced glycation end products (AGE) on cell proliferation and sensitivity to chemotherapy is not well established. In this study, we investigated whether type I collagen and particularly its AGE are involved in cell proliferation and resistance to doxorubicin in human fibrosarcoma HT-1080 cancer cells. Type I collagen was extracted from rats tail tendons (10 days, 8 and 96 weeks-old) and prepared as already described (Millerot-Serrurot et al., 2010, Cancer Cell Int, 13: 10-26). First, we characterized the glycation pattern of the different collagens using SDS-PAGE electrophoresis and fluorescence spectroscopy. A significant increase in AGE level has been observed in collagen extracted from 8 and 96 weeks-old rats. AGE level was significantly higher in collagen extracted from 96 weeks-old rats when compared to 8 weeks-old ones. Only collagen extracted from 96 weeks-old rats was able to induce 3-fold increase in proliferation of HT-1080 cells. Western-blot analysis showed that activation of MAPK/ERK, but not PI3K/AKT, was required for collagen AGE mediated cell proliferation. The MAPK/ERK inhibitor U0126 abolished this effect. Both collagens were able to inhibit doxorubicin-induced apoptosis in HT-1080 cells. Moreover, protection against apoptosis was significantly higher in the presence of collagen extracted from 96 weeks-old rats. Our data suggest extracellular matrix AGE signalling as an important promoter of cancer cell proliferation and that this pathway may contribute to a new mechanism of drug resistance. Citation Format: Charles Saby, Hassan EL Btaouri, Laurence Van Gulick, Marie Guilbert, Pierre Jeannesson, Hamid Morjani. Advanced glycation end products of type I collagen promote cell proliferation and resistance against doxorubicin in HT-1080 cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2105. doi:10.1158/1538-7445.AM2013-2105