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.
Specific reports have linked GPC3 with cancer. Its usefulness as a marker has been proved for hepatocarcinoma, melanoma and ovary carcinoma. However, there are no studies analyzing GPC3 usefulness as a biomarker in mammary tumors. The aim of this work was to analyze GPC3 expression in breast tissues and to determine whether it might be useful as a biomarker in breast cancer patients. Expression level of GPC3 mRNA in Brazilian and Argentine human breast tumor (n=121) and peritumoral "normal" tissue (n=77) samples was analyzed using qRT-PCR. GPC3 protein expression was analyzed from 69 breast cancer and 10 peritumoral samples using IHC. Statistical analyses were done to evaluate the clinical-pathological significance of GPC3 expression. We found that Brazilian and Argentine populations are statistically different regarding GPC3 mRNA expression. In Argentine patients a lower GPC3 mRNA expression was found in tumors as compared to peritumoral tissues. No association was found between GPC3 mRNA and protein expression and the clinical-pathological parameters. The Kaplan-Meier curves suggested that elevated levels of GPC3 mRNA are associated with relapse. Our results indicate differential expression of GPC3 in mammary tumors in comparasion to normal breast tissues. They also suggest the potential role of GPC3 as a biomarker and the importance of deepening the study.
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 Joffé, 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
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<0.0001). So, the p38 inhibitor SB203580 was able to revert the inhibitory action of GPC3 on metastasis development. 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 Joffé, María 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
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/Höescht 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 Joffé, 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
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