Dynamics of Internalization and Recycling of the Pro-Metastatic Membrane Type 4-Matrix Metalloproteinase (MT4-MMP) in Breast Cancer Cells
Truong, Alice ; Yip, Cassandre ; PAYE, Alexandra et al
in FEBS Journal (2016), 283(4), 704-22
MT4-MMP (MMP17) is a glycosylphosphatidyl inositol (GPI)-anchored membrane-type MMP expressed on the cell surface of human breast cancer cells. In triple negative breast cancer cells, MT4-MMP promotes ... [more ▼]
MT4-MMP (MMP17) is a glycosylphosphatidyl inositol (GPI)-anchored membrane-type MMP expressed on the cell surface of human breast cancer cells. In triple negative breast cancer cells, MT4-MMP promotes primary tumor growth and lung metastases. Although trafficking and internalization of the transmembrane MT1-MMP have been extensively investigated, little is known about the regulatory mechanisms of the GPI-anchored MT4-MMP. Here, we investigated the fate and cellular trafficking of MT4-MMP by analyzing its homophilic complex interactions, internalization and recycling dynamics compared to an inert form, MT4-MMP-E249A. Oligomeric and dimeric complexes were analyzed by co-transfection of cells with FLAG- or Myc-tagged MT4-MMP by reducing and non-reducing immunoblots and co-immunoprecipitation experiments. The trafficking of MT4-MMP was studied using an antibody feeding assay and confocal microscopy analysis or cell surface protein biotinylation and Western blot analysis. We demonstrate that MT4-MMP forms homophilic complexes at the cell surface, internalizes in early endosomes, and some of the enzyme is either auto-degraded or recycled to the cell surface. Our data indicate that MT4-MMP is internalized by the CLIC/GEEC pathway, a mechanism that differs from other MT-MMP members. Although MT4-MMP localizes with caveolin-1, MT4-MMP internalization was not affected by inhibitors of caveolin-1 or clathrin endocytosis pathways but was reduced by cdc42 or RhoA silencing with siRNA. We provide a new mechanistic insight into the regulatory mechanisms of MT4-MMP, which may have implications in the design of novel therapeutic strategies for metastatic breast cancer. This article is protected by copyright. All rights reserved. [less ▲]Detailed reference viewed: 63 (13 ULg)
Epithelial-mesenchymal transition induces tissue factor and pro-coagulant properties supporting early metastasis of circulating tumor cells.
Bourcy, Morgane ; Suarez-Carmona, Meggy ; Lambert, Justine et al
Poster (2016, January)Detailed reference viewed: 14 (1 ULg)
Quantitative assessment of mouse mammary gland morphology using automated digital image processing and TEB detection.
Blacher, Silvia ; Gérard, Céline ; Gallez, Anne et al
in Endocrinology (2016)
The assessment of rodent mammary gland morphology is largely used to study the molecular mechanisms driving breast development and to analyze the impact of various endocrine disruptors with putative ... [more ▼]
The assessment of rodent mammary gland morphology is largely used to study the molecular mechanisms driving breast development and to analyze the impact of various endocrine disruptors with putative pathological implications. In this work, we propose a methodology relying on fully automated digital image analysis methods including image processing and quantification of the whole ductal tree and of the terminal end buds (TEB) as well. It allows to accurately and objectively measure both growth parameters and fine morphological glandular structures. Mammary gland elongation was characterized by two parameters: the length and the epithelial area of the ductal tree. Ductal tree fine structures were characterized by: 1) branch end-point density, 2) branching density and 3) branch length distribution. The proposed methodology was compared to quantification methods classically used in the literature. This procedure can be transposed to several software and thus largely used by scientists studying rodent mammary gland morphology. [less ▲]Detailed reference viewed: 31 (6 ULg)
Elastin density: Link between histological and biomechanical properties of vaginal tissue in women with pelvic organ prolapse?
DE LANDSHEERE, Laurent ; ; Blacher, Silvia et al
in International Urogynecology Journal & Pelvic Floor Dysfunction (2016)
INTRODUCTION AND HYPOTHESIS: The aim of the study was to correlate histological and biomechanical characteristics of the vaginal wall in women with pelvic organ prolapse (POP). METHODS: Tissue samples ... [more ▼]
INTRODUCTION AND HYPOTHESIS: The aim of the study was to correlate histological and biomechanical characteristics of the vaginal wall in women with pelvic organ prolapse (POP). METHODS: Tissue samples were collected from the anterior [point Ba; POP Questionnaire (POP-Q)] and/or posterior (point Bp; POP-Q) vaginal wall of 15 women who underwent vaginal surgery for POP. Both histological and biomechanical assessments were performed from the same tissue samples in 14 of 15 patients. For histological assessment, the density of collagen and elastin fibers was determined by combining high-resolution virtual imaging and computer-assisted digital image analysis. For biomechanical testing, uniaxial tension tests were performed to evaluate vaginal tissue stiffness at low (C0) and high (C1) deformation rates. RESULTS: Biomechanical testing highlights the hyperelastic behavior of the vaginal wall. At low strains (C0), vaginal tissue appeared stiffer when elastin density was low. We found a statistically significant inverse relationship between C0 and the elastin/collagen ratio (p = 0.048) in the lamina propria. However, at large strain levels (C1), no clear relationship was observed between elastin density or elastin/collagen ratio and stiffness, likely reflecting the large dispersion of the mechanical behavior of the tissue samples. CONCLUSION: Histological and biomechanical properties of the vaginal wall vary from patient to patient. This study suggests that elastin density deserves consideration as a relevant factor of vaginal stiffness in women with POP. [less ▲]Detailed reference viewed: 16 (2 ULg)
Supplementation of transport and freezing media with anti-apoptotic drugs improves ovarian cortex survival
HENRY, Laurie ; Fransolet, Maïté ; LABIED, Soraya et al
in Journal of Ovarian Research (2016)
Background: Ovarian tissue preservation is proposed to patients at risk of premature ovarian failure, but this procedure still needs to be optimized. To limit injury during ovarian tissue cryopreservation ... [more ▼]
Background: Ovarian tissue preservation is proposed to patients at risk of premature ovarian failure, but this procedure still needs to be optimized. To limit injury during ovarian tissue cryopreservation, anti-apoptotic drugs were added to the transport and freezing media of ovarian cortex tissue. Methods: Sheep ovaries were transported, prepared and frozen in solutions containing vehicle or anti-apoptotic drugs (Z-VAD-FMK, a pan-caspase inhibitor, or sphingosine-1-phosphate (S1P), a bioactive lipid). After the tissue was thawed, the ovarian cortex was cultured for 2 or 6 days. Follicular quantification and morphological and proliferation analyses were performed on histological sections. Results: After 2 days of culture, S1P improved the quality of primordial follicles; higher densities of morphologically normal and proliferative primordial follicles were found. Z-VAD-FMK displayed similar effects by preserving global primordial follicular density, but this effect was evident after 6 days of culture. This drug also improved cell proliferation after 2 and 6 days of culture. Conclusions: Our results showed that the addition of S1P or Z-VAD-FMK to the transport and freezing media prior to ovarian tissue cryopreservation improves primordial follicular quality and therefore improves global tissue survival. This should ultimately lead to improved fertility restoration after auto-transplantation. [less ▲]Detailed reference viewed: 18 (1 ULg)
Role of Metabolic Reprogramming in Tumor Adaptation to Anti-angiogenic Therapy
Cimino, Jonathan ; Sounni, Nor Eddine ; Quoirin, Grégory et al
Poster (2015, December 03)Detailed reference viewed: 14 (1 ULg)
Dynamics of Internalization and Recycling of the pro-Metastatic Membrane Type 4-Matrix Metalloproteinase (MT4-MMP) in Breast Cancer cells
Truong, Alice ; Yip, Cassandre ; PAYE, Alexandra et al
Poster (2015, October 26)
MT4-MMP (MMP17) is a glycosyl-phosphatidyl inositol-anchored membrane-type matrix metalloproteinase expressed at the cell surface of human breast cancer cells. In triple negative breast cancer, MT4-MMP ... [more ▼]
MT4-MMP (MMP17) is a glycosyl-phosphatidyl inositol-anchored membrane-type matrix metalloproteinase expressed at the cell surface of human breast cancer cells. In triple negative breast cancer, MT4-MMP promotes primary tumor growth and lung metastases. Recently, we demonstrated that EGFR activation and signaling are enhanced by MT4-MMP in a non-proteolytic dependent manner. While trafficking and internalization of EGFR was extensively investigated, little is known about MT4-MMP. Here, we investigated the dimerization, internalization and recycling dynamics of MT4-MMP and its mutated inactive form MT4-MMP-E249A. We demonstrate that MT4-MMP forms dimers and oligomers at the cell surface, a process that was not inhibited neither by broad-spectrum MMP inhibitors (GM6001 and BB94) nor TIMP-2. MT4-MMP is internalized in early endosomes from 10 minutes to 60 minutes. Once internalized, some amount of MT4-MMP is auto-degraded, whereas its inert form E249A was found intact. Large part of the internalized enzyme was recycled intact at the cell surface. By exploring its endocytosis, we found that MT4-MMP is internalized by the CLIC/GEEC pathway, a mechanism that differs from other MT-MMP members. Overall, we provided a new mechanistic insight on the regulatory mechanisms of MT4-MMP in human breast cancer cells. We also, highlighted unique features of MT4-MMP among membrane-associated MMPs, which may be useful for the design of novel therapeutic strategies for metastatic breast cancer. [less ▲]Detailed reference viewed: 33 (20 ULg)
Exploring Tumor lipid Heterogeneity by High-Resolution Mass Spectrometry Imaging.
Cimino, Jonathan ; Far, Johann ; Debois, Delphine et al
Conference (2015, May 13)Detailed reference viewed: 12 (2 ULg)
Limitation of damage to ovarian cortex during cryopreservation by addition of anti-apoptotic drugs in transport and freezing media
HENRY, Laurie ; Fransolet, Maïté ; LABIED, Soraya et al
Poster (2015, January 27)Detailed reference viewed: 11 (0 ULg)
Mesenchymal stem cells shed amphiregulin at the surface of lung carcinoma cells in a juxtacrine manner .
Carnet, Oriane ; ; et al
in Neoplasia : An International Journal for Oncology Research (2015), 17(7), 552-63
Solid tumors comprise cancer cells and different supportive stromal cells, including mesenchymal stem cells (MSCs), which have recently been shown to enhance tumor growth and metastasis. We provide new ... [more ▼]
Solid tumors comprise cancer cells and different supportive stromal cells, including mesenchymal stem cells (MSCs), which have recently been shown to enhance tumor growth and metastasis. We provide new mechanistic insights into how bone marrow (BM)-derived MSCs co-injected with Lewis lung carcinoma cells promote tumor growth and metastasis in mice. The proinvasive effect of BM-MSCs exerted on tumor cells relies on an unprecedented juxtacrine action of BM-MSC, leading to the trans-shedding of amphiregulin (AREG) from the tumor cell membrane by tumor necrosis factor-α-converting enzyme carried by the BM-MSC plasma membrane. The released soluble AREG activates cancer cells and promotes their invasiveness. This novel concept is supported by the exploitation of different 2D and 3D culture systems and by pharmacological approaches using a tumor necrosis factor-α-converting enzyme inhibitor and AREG-blocking antibodies. Altogether, we here assign a new function to BM-MSC in tumor progression and establish an uncovered link between AREG and BM-MSC. [less ▲]Detailed reference viewed: 77 (18 ULg)
The timing of surgery after neoadjuvant radiotherapy influences tumor dissemination in a preclinical model
Leroi, Natacha ; Sounni, Nor Eddine ; et al
in Oncotarget (2015)
Neoadjuvant radiotherapy (neoRT) used in cancer treatments aims at improving local tumor control and patient overall survival. The neoRT schedule and the timing of the surgical treatment (ST) are ... [more ▼]
Neoadjuvant radiotherapy (neoRT) used in cancer treatments aims at improving local tumor control and patient overall survival. The neoRT schedule and the timing of the surgical treatment (ST) are empirically based and influenced by the clinician’s experience. The current study examines how the sequencing of neoRT and ST affects metastatic dissemination. In a breast carcinoma model, tumors were exposed to different neoRT schedules (2x5Gy or 5x2Gy) followed by surgery at day 4 or 11 post- RT. The impact on the tumor microenvironment and lung metastases was evaluated through immunohistochemical and flow cytometry analyses. After 2x5Gy, early ST (at day 4 post-RT) led to increased size and number of lung metastases as compared to ST performed at day 11. Inversely, after 5x2Gy neoRT, early ST protected the mice against lung metastases. This intriguing relationship between tumor aggressiveness and ST timing could not be explained by differences in classical parameters studied such as hypoxia, vessel density and matrix remodeling. The study of tumor-related inflammation and immunity reveals an increased circulating NK cell percentage following neoRT as compared to non irradiated mice. Then, radiation treatment and surgery were applied to tumor-bearing NOD/SCID mice. In the absence of NK cells, neoRT appears to increase lung metastatic dissemination as compared to non irradiated tumor-bearing mice. Altogether our data demonstrate that the neoRT schedule and the ST timing affect metastasis formation in a pre-clinical model and points out the potential role of NK cells. These findings highlight the importance to cautiously tailor the optimal window for ST following RT. [less ▲]Detailed reference viewed: 50 (30 ULg)
Soluble factors regulated by epithelial-mesenchymal transition mediate tumour angiogenesis and myeloid cell recruitment.
Suarez-Carmona, Meggy ; Bourcy, Morgane ; et al
in Journal of Pathology (The) (2015)
Epithelial-to-mesenchymal transition (EMT) programs provide cancer cells with invasive and survival capacities that might favor metastatic dissemination. Whilst signaling cascades triggering EMT have been ... [more ▼]
Epithelial-to-mesenchymal transition (EMT) programs provide cancer cells with invasive and survival capacities that might favor metastatic dissemination. Whilst signaling cascades triggering EMT have been extensively studied, the impact of EMT on the crosstalk between tumor cells and the tumor microenvironment remains elusive. We aimed to identify EMT-regulated soluble factors that facilitate the recruitment of host cells in the tumor. Our findings indicate that EMT phenotypes relate to the induction of a panel of secreted mediators, namely IL-8, IL-6, sICAM-1, PAI-1 and GM-CSF, and implicate the EMT-transcription factor Snail as a regulator of this process. We further show that EMT-derived soluble factors are pro-angiogenic in vivo (in the mouse ear sponge assay), ex vivo (in the rat aortic ring assay) and in vitro (in a chemotaxis assay). Additionally, conditioned medium from EMT-positive cells stimulates the recruitment of myeloid cells. In a bank of 40 triple-negative breast cancers, tumors presenting features of EMT were significantly more angiogenic and infiltrated by a higher quantity of myeloid cells compared to tumors with little or no EMT. Taken together, our results show that EMT programs trigger the expression of soluble mediators in cancer cells that stimulate angiogenesis and recruit myeloid cells in vivo, which might in turn favor cancer spread. [less ▲]Detailed reference viewed: 65 (23 ULg)
Estetrol is a weak estrogen antagonizing estradiol-dependent mammary gland proliferation.
Gérard, Céline ; Blacher, Silvia ; et al
in Journal of Endocrinology (2015), 224(1), 86-95
Estetrol (E4) is a natural estrogen produced exclusively by the human fetal liver during pregnancy. Its physiological activity remains unknown. In contrast to ethinyl estradiol (EE) and estradiol (E2), E4 ... [more ▼]
Estetrol (E4) is a natural estrogen produced exclusively by the human fetal liver during pregnancy. Its physiological activity remains unknown. In contrast to ethinyl estradiol (EE) and estradiol (E2), E4 has a minimal impact on liver cells activity and could provide a better safety profile in contraception or hormone therapy. The aim of this study was to delineate if E4 exhibits an activity profile distinct from that of E2 on mammary gland. Compared to E2, E4 acted as a low affinity estrogen in both, human in vitro and murine in vivo, models. E4 was 100 times less potent than E2 to stimulate the proliferation of human breast epithelial (HBE) cells and murine mammary gland in vitro and in vivo, respectively. This effect was prevented by fulvestrant and by tamoxifen supporting the notion that ERalpha is the main mediator of the estrogenic effect of E4 on the breast. Interestingly, when E4 was administered along with E2, it significantly antagonized the strong stimulatory effect of E2 on HBE cells proliferation and on the growth of mammary ducts. This study characterizes for the first time the impact of E4 on mammary gland. Our results highlight that E4 is less potent than E2 and exhibits antagonistic properties towards the proliferative effect of E2 on breast epithelial cells. These data support E4 as a potential new estrogen for clinical use with a reduced impact on breast proliferation. [less ▲]Detailed reference viewed: 46 (18 ULg)
Influence of mouse strain on ovarian tissue recovery after engraftment with angiogenic factor.
Fransolet, Maïté ; Henry, Laurie ; et al
in Journal of Ovarian Research (2015), 8(1), 14
BACKGROUND: For women facing gonadotoxic treatment, cryopreservation of ovarian tissue with subsequent retransplantation during remission is a promising technique for fertility preservation. However ... [more ▼]
BACKGROUND: For women facing gonadotoxic treatment, cryopreservation of ovarian tissue with subsequent retransplantation during remission is a promising technique for fertility preservation. However, follicle loss within grafted ovarian tissue can be caused by ischemia and progressive revascularization. Several xenograft models using different immunodeficient rodent lines are suitable for studying ovarian tissue survival and follicular viability after frozen-thawed ovarian cortex transplantation. SCID mice, which are deficient for functional B and T cells, are the most commonly used mice for ovarian xenograft studies. However, due to incomplete immunosuppression, NOD-SCID mice displaying low NK cell function and an absence of circulating complement might be more appropriate. The present study aims to define the most appropriate immunodeficient mouse strain for ovarian tissue xenotransplantation by comparing ovarian graft recovery in SCID and NOD-SCID mice following engraftment in the presence of isoform 111 of vascular endothelial growth factor. METHODS: Sheep ovarian cortex fragments were embedded in a collagen matrix, with or without VEGF111, before being stitched onto the ovaries of SCID and NOD-SCID mice. Transplants were recovered after 3 days to study early revascularization or after 3 weeks to evaluate follicle preservation and tissue fibrosis through histological analyses. RESULTS: At day 3, vessels were largely reorganized in the ovarian grafts of both mouse strains. After 3 weeks, the cortical tissue was clearly identifiable in SCID mice but not in NOD-SCID mice. Upon VEGF111 treatment, vascularization was significantly improved 3 days after transplantation in SCID mice. This increase in vessel density was correlated with better follicular preservation in SCID mice 3 weeks after transplantation. Fibrosis was not decreased by VEGF treatment in either mouse strain. CONCLUSIONS: Tissue architecture and follicular morphology were better preserved in ovarian tissues grafted in SCID mice in comparison with NOD-SCID mice. Moreover, tissue revascularization was improved in SCID mice by VEGF111 graft treatment. Thus, we consider SCID mice to be the best murine model for studying ovarian tissue xenografts. [less ▲]Detailed reference viewed: 22 (3 ULg)
Isoform 165 of vascular endothelial growth factor in collagen matrix improves ovine cryopreserved ovarian tissue revascularisation after xenotransplantation in mice.
Henry, Laurie ; LABIED, Soraya ; Fransolet, Maïté et al
in Reproductive biology and endocrinology (2015), 13(1), 15
BACKGROUND: Aggressive anti-cancer treatments can result in ovarian failure. Ovarian cryopreservation has been developed to preserve the fertility of young women, but early graft revascularisation still ... [more ▼]
BACKGROUND: Aggressive anti-cancer treatments can result in ovarian failure. Ovarian cryopreservation has been developed to preserve the fertility of young women, but early graft revascularisation still requires improvement. METHODS: Frozen/thawed sheep ovarian cortical biopsies were embedded in collagen matrix with or without isoform 165 of vascular endothelial growth factor (VEGF165) and transplanted into ovaries of immunodeficient mice. Ovaries were chosen as transplantation sites to more closely resemble clinical conditions in which orthotopic transplantation has previously allowed several spontaneous pregnancies. RESULTS: We found that VEGF165 significantly increased the number of Dextran-FITC positive functional vessels 3 days after grafting. Dextran- fluorescein isothiocyanate (FITC) positive vessels were detectable in 53% and 29% of the mice in the VEGF-treated and control groups, respectively. Among these positive fragments, 50% in the treated group displayed mature smooth-muscle-actin-alpha (alpha-SMA) positive functional vessels compared with 0% in the control group. CD31 positive murine blood vessels were observed in 40% of the VEGF165 transplants compared with 21% of the controls. After 3 weeks, the density of murine vessels was significantly higher in the VEGF165 group. CONCLUSION: The encapsulation of ovarian tissue in collagen matrix in the presence of VEGF165 before grafting has a positive effect on functional blood vessel recruitment. It can be considered as a useful technique to be improved and further developed before human clinical applications in female cancer patients in the context of fertility preservation. [less ▲]Detailed reference viewed: 21 (1 ULg)
Use of anti-apoptotic drugs in transport and freezing media of cryopreserved sheep ovarian cortex to improve tissue survival
HENRY, Laurie ; Fransolet, Maïté ; LABIED, Soraya et al
Poster (2014, November 14)Detailed reference viewed: 11 (0 ULg)
EGFR activation and signaling in cancer cells are enhanced by the membrane-bound metalloprotease MT4-MMP.
Paye, Alexandra ; Truong, Alice ; Yip, Cassandre et al
in Cancer Research (2014), 74(23), 6758-70
MT4-MMP (MMP-17) is a GPI-anchored matrix metalloprotease expressed on the surface of cancer cells which promotes tumor growth and metastasis. In this report, we identify MT4-MMP as an important driver of ... [more ▼]
MT4-MMP (MMP-17) is a GPI-anchored matrix metalloprotease expressed on the surface of cancer cells which promotes tumor growth and metastasis. In this report, we identify MT4-MMP as an important driver of cancer cell proliferation through CDK4 activation and retinoblastoma protein (Rb) inactivation. We also determine a functional link between MT4-MMP and the growth factor receptor EGFR. Mechanistic experiments revealed direct association of MT4-MMP and its positive effects on EGFR phosphorylation in response to TGF- and EGF in cancer cells. Notably, the effects of MT4-MMP on proliferation and EGFR activation did not rely on metalloprotease activity. Clinically, MT4-MMP and EGFR expression were correlated in human triple negative breast cancer specimens. Altogether our results identify MT4-MMP as a positive modifier of EGFR outside-in signaling that acts to cooperatively drive cancer cell proliferation. [less ▲]Detailed reference viewed: 76 (29 ULg)
Blocking lipid synthesis overcomes tumor re-growth and metastasis after anti-angiogenic therapy withdrawal.
Cimino, Jonathan ; Sounni, Nor Eddine ; BLACHER, Silvia et al
Conference (2014, September 30)Detailed reference viewed: 9 (3 ULg)
Does neoadjuvant radiotherapy and the timing of surgery modify metastatic dissemination?
Leroi, Natacha ; BLACHER, Silvia ; Marée, Raphaël et al
Conference (2014, June 29)Detailed reference viewed: 23 (5 ULg)
Ovarian tissue recovery in SCID and NOD-SCID mice after VEGF111 or VEGF165 graft treatment
Fransolet, Maïté ; HENRY, Laurie ; Blacher, Silvia et al
Poster (2014, May 19)Detailed reference viewed: 9 (0 ULg)