María Giselle Peters

Inhibition of Invasion and Metastasis by Glypican-3 in a Syngeneic Breast Cancer Model

Glypican-3 (GPC3), a proteoglycan bound to the cell membrane through a GPI anchor, is widely expressed in the embryo but down regulated in most adult tissues, with some exceptions as mammary cells. GPC3 is involved in the regulation of cell proliferation and survival in specific cell types. LM3, a murine mammary tumor cell line unable to express GPC3, was stably transfected with the rat GPC3 gene to analyze its role in tumor progression. Upon injection into syngeneic BALB/c mice LM3-GPC3 clones showed less local invasiveness and developed fewer spontaneous and experimental lung metastasis than controls. GPC3-expressing cells were more sensitive to apoptosis induced by serum depletion, exhibited a delay in the first steps of spreading and were less motile than controls. On the other hand, LM3-GPC3 cells were significantly more adherent to FN than control ones. We observed that GPC3 transfectants presented a higher expression of E-cadherin and β-catenin, molecules whose down regulation has been associated with tumor progression. Exogenous TGF-β increased MMP-9 activity in both control and GPC3-expressing cells, but did not modulate MMP-2. Contrarily, GPC3 expression prevented the increase of MMP-2 activity induced by IGF-II. Our results suggest that GPC3 has a protective role against mammary cancer progression.

Progestin-induced caveolin-1 expression mediates breast cancer cell proliferation.

Progestin regulation of gene expression was assessed in the progestin-dependent murine tumor line C4HD which requires MPA, a synthetic progestin, for in vivo growth and expresses high levels of progesterone receptor (PR). By using suppressive subtractive hybridization, caveolin-1 was identified as a gene whose expression was increased with in vivo MPA treatment. By Northern and Western blot analysis, we further confirmed that caveolin-1 mRNA and protein expression increased in MPA-treated tumors as compared with untreated tumors. When primary cultures of C4HD cells were treated in vitro with MPA, caveolin-1 levels also increased, effect that was abolished by pre-treatment with progestin antagonist RU486. In addition, MPA promoted strong caveolin-1 promoter transcriptional activation both in mouse and human breast cancer cells. We also showed that MPA regulation of caveolin-1 expression involved in activation of two signaling pathways: MAPK and PI-3K. Short-term MPA treatment of C4HD cells led to tyrosine phosphorylation of caveolin-1 protein, where Src was the kinase involved. Additionally, we showed that MPA-induced association of caveolin-1 and PR, which was detected by coimmunoprecipitation and by confocal microscopy. Finally, we proved that MPA-induced proliferation of C4HD cells was inhibited by suppression of caveolin-1 expression with antisense oligodeoxynucleotides to caveolin-1 mRNA. Furthermore, we observed that inhibition of caveolin-1 expression abrogated PR capacity to induced luciferase activity from a progesterone response element-driven reporter plasmid. Comprehensively, our results demonstrated for the first time that caveolin-1 expression is upregulated by progestin in breast cancer. We also demonstrated that caveolin-1 is a downstream effector of MPA that is partially responsible for the stimulation of growth of breast cancer cells.

Glypican-3 reexpression regulates apoptosis in murine adenocarcinoma mammary cells modulating PI3K/Akt and p38MAPK signaling pathways

Glypican-3 (GPC3) is a proteoglycan involved in proliferation and cell survival. Several reports demonstrated that GPC3 is downregulated in some tumors, such as breast cancer. Previously, we determined that GPC3 reexpression in the murine mammary adenocarcinoma LM3 cells induced an impairment of their invasive and metastatic capacities, associated with a decrease of their motility and an increase of their cell death. We demonstrated that GPC3 inhibits canonical Wnt signaling, as well as it activates non canonical pathway. Now, we identified signaling pathways responsible for the pro-apoptotic role of GPC3 in LM3 cells. We found for the first time that GPC3 inhibits the PI3K/Akt anti-apoptotic pathway while it stimulates the p38MAPK stress-activated one. We report a concomitant modulation of CDK inhibitors as well as of pro- and anti-apoptotic molecules. Our results provide new clues regarding the mechanism involved in the modulation induced by GPC3 of mammary tumor cell growth and survival.

Generation and characterization of human insulin-releasing cell lines

BackgroundThe in vitro culture of insulinomas provides an attractive tool to study cell proliferation and insulin synthesis and secretion. However, only a few human beta cell lines have been described, with long-term passage resulting in loss of insulin secretion. Therefore, we set out to establish and characterize human insulin-releasing cell lines.ResultsWe generated ex-vivo primary cultures from two independent human insulinomas and from a human nesidioblastosis, all of which were cultured up to passage number 20. All cell lines secreted human insulin and C-peptide. These cell lines expressed neuroendocrine and islets markers, confirming the expression profile found in the biopsies. Although all beta cell lineages survived an anchorage independent culture, none of them were able to invade an extracellular matrix substrate.ConclusionWe have established three human insulin-releasing cell lines which maintain antigenic characteristics and insulin secretion profiles of the original tumors. These cell lines represent valuable tools for the study of molecular events underlying beta cell function and dysfunction.

Glypican-3 induces a mesenchymal to epithelial transition in human breast cancer cells

Breast cancer is the disease with the highest impact on global health, being metastasis the main cause of death. To metastasize, carcinoma cells must reactivate a latent program called epithelial-mesenchymal transition (EMT), through which epithelial cancer cells acquire mesenchymal-like traits. Glypican-3 (GPC3), a proteoglycan involved in the regulation of proliferation and survival, has been associated with cancer. In this study we observed that the expression of GPC3 is opposite to the invasive/metastatic ability of Hs578T, MDA-MB231, ZR-75-1 and MCF-7 human breast cancer cell lines. GPC3 silencing activated growth, cell death resistance, migration, and invasive/metastatic capacity of MCF-7 cancer cells, while GPC3 overexpression inhibited these properties in MDA-MB231 tumor cell line. Moreover, silencing of GPC3 deepened the MCF-7 breast cancer cells mesenchymal characteristics, decreasing the expression of the epithelial marker E-Cadherin. On the other side, GPC3 overexpression induced the mesenchymal-epithelial transition (MET) of MDA-MB231 breast cancer cells, which re-expressed E-Cadherin and reduced the expression of vimentin and N-Cadherin. While GPC3 inhibited the canonical Wnt/β-Catenin pathway in the breast cancer cells, this inhibition did not have effect on E-Cadherin expression. We demonstrated that the transcriptional repressor of E-Cadherin - ZEB1 - is upregulated in GPC3 silenced MCF-7 cells, while it is downregulated when GPC3 was overexpressed in MDA-MB231 cells. We presented experimental evidences showing that GPC3 induces the E-Cadherin re-expression in MDA-MB231 cells through the downregulation of ZEB1. Our data indicate that GPC3 is an important regulator of EMT in breast cancer, and a potential target for procedures against breast cancer metastasis.

Metastasis Suppressors: Basic and Translational Advances

Tumor metastasis is a main contributor to death in cancer patients. In the last years, a new class of molecules that reduces the metastatic propensity has been identified: metastasis suppressors. These proteins regulate multiple steps in the metastatic cascade, including cell invasion, survival in the vascular and lymphatic circulation, and colonization of distant organ sites. As a consequence, they are very important therapeutic targets. This review discusses our current understanding of metastasis suppressors and how this knowledge might be useful to improve the treatment of cancer patients.

Abstract 3256: Glypican-3 (GPC3) inhibits the metastasis development in a murine breast cancer model through the activation of p38MAPK signaling pathway

GPC3 reexpression into the murine mammary adenocarcinoma LM3 cells (GPC3 negative) induced an inhibition of the metastatic ability in vivo, while in vitro caused a reversion of the epithelial to mesenchymal transition (EMT), suggesting its role as a metastasis suppressor. Although GPC3 signaling mechanism is unknown, we found that LM3-GPC3 cells exhibit an inhibition of Akt and canonical Wnt pathways, while non-canonical Wnt (PCP) and p38 pathways are activated. In relation to cell parameters, GPC3 reexpression caused a significant increase in the apoptosis induced by nutrient depletion. Moreover, when LM3-GPC3 starved cells were treated with the p38 inhibitor SB203580, we observed a decrease in the apoptosis, suggesting that p38 activation is responsible of the increase in the susceptibility to apoptotic death detected in LM3-GPC3 cells. The aim of this work was to analyze if the effects of GPC3 on in vivo cell behavior are due to p38 activation. Our assays showed that p38 inhibition had no effect on s.c. primary tumor growth but modulated spontaneous metastasis. Thus, about 60% of animals treated with SB203580 developed lung spontaneous metastases while no LM3-GPC3 tumor-bearing mice did so. We decided to confirm whether the activation of p38 is necessary for the inhibitory effect of GPC3 on metastasis development through an experimental metastasis assay. LM3-GPC3 #1 and #2, and LM3-vector #1 and #2 cells were inoculated i.v. into female BALB/c mice. Simultaneously, the p38 inhibitor SB203580 or the vehicle DMSO, were injected i.p. into mice. Our results confirmed that mice inoculated with LM3-GPC3 cells show a lower number of lung nodules than animals inoculated with LM3-vector cells. On the other hand, when mice injected with LM3-GPC3 cells were treated with SB203580, they showed a significantly higher number of lung nodules, similar to those found in animals inoculated with control cells (Lung nodules Md [Rg]: 31 [10-52] LM3-vector #1; 27 [22-30]] LM3-vector #2; 11 [0-17] LM3-GPC3 #1 +DMSO; 13 [2-19] LM3-GPC3 #2 +DMSO; 20 [11-64] LM3-GPC3 #1 +SB203589; 35 [17-57] LM3-GPC3 #2 +SB203589; U-Mann Whitney test p In sum, we have confirmed that GPC3 acts as metastasis suppressor in this murine breast cancer model. We also demonstrated that GPC3 would be able to inhibit metastatic development through p38 signaling pathway activation. Citation Format: Rocio S. Tascon, Lilian Castillo, Elisa Bal de Kier Joffe, Maria G. Peters. Glypican-3 (GPC3) inhibits the metastasis development in a murine breast cancer model through the activation of p38MAPK signaling pathway. [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 3256. doi:10.1158/1538-7445.AM2015-3256

Abstract 133: Role of Glypican-3 (GPC3) on tumor progression of the human mammary gland

We have previously shown that the reexpression of Glypican-3 (GPC3), a proteoglycan downregulated in breast cancer, leads to the impairment of the in vivo metastatic capacity of the murine LM3 mammary adenocarcinoma cells. On the basis of clinical and translational potential of GPC3, the aim of this work was to assess whether GPC3 acts as a metastasis suppressor in human cells. So, we generated a preclinical breast cancer cell model. We chose MCF-7 cell line (poorly-metastatic, GPC3 +) and MDA-MB231 cell line (metastatic, GPC3 -) to be genetically modified. GPC3 expression was blocked in MCF-7 cells by siRNA. We demonstrated that MCF-7-shGPC3 cells proliferate faster than controls (Population doubling time (h): 72 -shGPC3 vs 87 -shNC). In agreement, silencing of GPC3 increased clonogenic capacity (Colony number: 130 -shGPC3 vs 37 -shNC). No differences were found in viability. By wound healing assay we determined that -shGPC3 cells are significantly more motile than controls (Wound coverage (%): 15 -shGPC3 vs 2 -shNC). GPC3 blocking induced an increase in homotypic MCF-7 cell adhesion (Cellular aggregation (%): 56 -shGPC3 vs 21 -shNC). We performed an in vivo tumor growth assay, by sc inoculation of MCF-7 engineered cells into nude mice. MCF-7-shGPC3 tumor-bearing mice showed shorter tumor latency than controls (Md [Rg] (days): 30 [30-44] -shGPC3 vs 49 [30-71] -shNC), and an increase in tumorigenic ability (Tumorigenicity (%): 80 -shGPC3 vs 60 -shNC), as well as in the tumor growth rate (mm3/day: 26±2.2 -shGPC3 vs 0.2±0.02). Spontaneous metastases were not detected in any of the MCF-7 tumor-bearing mice. On the other hand, GPC3 was reexpressed in MDA-MB231 cells by lentiviral infection. Surprisingly, GPC3 reexpressing cells showed a higher proliferation rate (Population doubling time (h): 24 -GPC3 vs 45 -vector), but their clonogenic capacity was less (Colony number: 8 -GPC3 vs 40 -vector). While GPC3 reexpression induced a 30% inhibition on MDA-MB231 viability, controls remained 100% viable. We also determined that -GPC3 cells are less migrant than controls (Wound coverage (%): 10 -GPC3 vs 90 -vector), and also they exhibit a reduction in their homotypic adhesion (Cellular aggregation (%): 33 -GPC3 vs 81 -vector). In relation to in vivo behavior, MDA-MB231-GPC3 tumor-bearing mice showed shorter tumor latency than controls (Md (days): 14 -GPC3 vs 97 -vector). However, GPC3 reexpression induced a reduction in tumorigenicity: 40% -GPC3 vs 60% -vector. The tumor growth rate was higher for GPC3 reexpressing cells (mm3/day: 72.73±9.46 -GPC3 vs 1.9±0.21). Spontaneous metastases were detected in lungs, showing an incidence of 20% for -GPC3 vs 40% -vector cells. In sum, we generated a preclinical human breast cancer cell model with differential GPC3 expression. Our in vitro and in vivo results reveal the key role of GPC3 in the biology of breast cancer cells. Citation Format: Lilian F. Castillo, Rocio S. Tascon, Elisa Bal de Kier Joffe, Maria G. Peters. Role of Glypican-3 (GPC3) on tumor progression of the human mammary gland. [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 133. doi:10.1158/1538-7445.AM2014-133

Signaling network involved in the GPC3-induced inhibition of breast cancer progression: role of canonical Wnt pathway

PurposeWe have shown that GPC3 overexpression in breast cancer cells inhibits in vivo tumor progression, by acting as a metastatic suppressor. GPC3-overexpressing cells are less clonogenic, viable and motile, while their homotypic adhesion is increased. We have presented evidences indicating that GPC3 inhibits canonical Wnt and Akt pathways, while non-canonical Wnt and p38MAPK cascades are activated. In this study, we aimed to investigate whether GPC3-induced Wnt signaling inhibition modulates breast cancer cell properties as well as to describe the interactions among pathways modulated by GPC3.MethodsFluorescence microscopy, qRT-PCR microarray, gene reporter assay and Western blotting were performed to determine gene expression levels, signaling pathway activities and molecule localization. Lithium was employed to activate canonical Wnt pathway and treated LM3-GPC3 cell viability, migration, cytoskeleton organization and homotypic adhesion were assessed using MTS, wound healing, phalloidin staining and suspension growth assays, respectively.ResultsWe provide new data demonstrating that GPC3 blocks—also at a transcriptional level—both autocrine and paracrine canonical Wnt activities, and that this inhibition is required for GPC3 to modulate migration and homotypic adhesion. Our results indicate that GPC3 is secreted into the extracellular media, suggesting that secreted GPC3 competes with Wnt factors or interacts with them and thus prevents Wnt binding to Fz receptors. We also describe the complex network of interactions among GPC3-modulated signaling pathways.ConclusionGPC3 is operating through an intricate molecular signaling network. From the balance of these interactions, the inhibition of breast metastatic spread induced by GPC3 emerges.

Abstract 1419: Glypican-3 (GPC3) inhibits metastatic dissemination in a preclinical human breast cancer model

Although GPC3 has been linked to cancer, its role during mammary tumor progression is barely known. With the aim to develop a pre-clinical breast cancer model, we genetically modified MCF-7 (poorly-metastatic, GPC3 +) and MDA-MB231 (metastatic, GPC3 -) cell lines. GPC3 expression was blocked in MCF-7 by siRNA (generating MCF-7-shGPC3 sublines), while it was overexpressed in MDA-MB231 by viral infection (producing MDA-MB231-GPC3 sublines). First, we analyzed the in vivo tumor growth by s.c. inoculation of MCF-7 and MDA-MB231 engineered cells into nude mice. Silencing GPC3 stimulated tumorigenicity, while its overexpression inhibited it. The histological analysis of MCF-7-shGPC3 tumors revealed extensive invasion of the muscle and the dermis, while control tumors did not invade. In addition, MDA-MB231-GPC3 tumors were less invasive than MDA-MB231-vector ones. We also evaluated spontaneous metastasis capacity by lung histological analysis. While no metastases were found in MCF-7-sh scramble tumor-bearing mice, lung nodules were detected in mice inoculated with GPC3 silenced cells. On the other hand, mice bearing MDA-MB231-GPC3 tumors showed a decrease in the incidence of metastasis. Next, we did a panel of in vitro tests. Although MCF-7-shGPC3 and control cells were morphologically similar, an increase in the number of actin stress fibers was found in GPC3 silenced cells. In association with this mesenchymal characteristic, this subline exhibited a decrease in E-Cadherin expression. While MDA-MB231-vector cells presented a fibroblastic appearance, GPC3 overexpression induced a drastic change in cell morphology turning to an epithelial phenotype. In addition, although control cells showed large stress fibers, GPC3 overexpressing cells localized their actin in cortical position. Moreover, GPC3 induced a reexpression of the epithelial marker E-Cadherin. We tested anchorage-independent growth, finding that GPC3 silencing disorganized MCF-7 spheroids. MDA-MB231-GPC3 cells formed larger spheroids than those generated by controls. Blocking E-cadherin, employing a neutralizing antibody, reversed the 3-D growth ability of GPC3 overexpressing cells. The sensitivity to nutrient depletion was evaluated. We found, through propidium iodide/Hoescht staining, a reduction in starved MCF-7-shGPC3 cell death. While MCF-7-sh scramble cells showed morphological alterations characteristic of apoptotic death as detected by orange acridine/ethidium bromide staining, no apoptosis was found in MCF-7-shGPC3 cells. GPC3 overexpression increased MDA-MB-231 cell susceptibility to death. Although there was no apoptosis in MDA-MB231 control cells, the overexpression of GPC3 promoted an increase in the number of cells with nuclear and cytoplasmic manifestations of apoptosis. We demonstrate a central role of GPC3 in human breast cancer biology. Our results indicate that it acts a metastasis suppressor in human mammary cancer. Citation Format: Lilian F. Castillo, Rocio Tascon, Elisa Bal de Kier Joffe, Maria G. Peters. Glypican-3 (GPC3) inhibits metastatic dissemination in a preclinical human breast cancer model. [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 1419. doi:10.1158/1538-7445.AM2015-1419