Lilian F. Castillo

α2-Adrenoceptor action on cell proliferation and mammary tumour growth in mice

Breast cancer, the most common cancer in women in most countries, is a highly stressful disease. Catecholamines released during stress bind to adrenoceptors and we have recently described α2‐adrenoceptors in human breast cell lines, linked to enhanced cell proliferation. The purpose was to assess the in vivo effects of compounds acting on α2‐adrenoceptors in a reliable model of breast cancer.

Involvement of α2- and β2-adrenoceptors on breast cancer cell proliferation and tumour growth regulation.

BACKGROUND AND PURPOSE β‐Adrenoceptors are expressed in human and experimental animal breast cancer cells. However, the effect of the agonists and antagonists reported on cell proliferation and tumour growth was paradoxical, precluding their utilization as possible adjuvant therapy, mainly in the cases of refractory tumours.

Adrenoceptors: Non Conventional Target for Breast Cancer?

Epinephrine and Norepinephrine, typically released during stress bind to nine different adrenoceptors (AR) which classically control the cardiovascular and respiratory systems. New targets were described for the many agonists and antagonists developed for these AR, as the central nervous system. During the last three decades, AR expression and action on the mammary gland/breast were extensively investigated. In the cow mammary gland, good milkability was associated with low density of beta(2)-AR and high density of alpha(2)-AR. In the rat normal mammary gland, beta-AR are expressed in the epithelial cells, alveoli, ducts, and adipocytes showing an exquisite regulation by steroid hormones and prolactin. In rat dimethylbenz(a)anthracene (DMBA) tumors, a close correlation was observed between tumor growth and beta-AR concentration. beta(2)-AR were described in numerous human cell lines and breast tumors. The action of beta-adrenergic compounds on cell proliferation is contradictory. While some authors found that beta-agonists significantly inhibit cancer cell proliferation and tumor growth in mice, others described a significant reduction in DNA synthesis by beta-blockers. Also, positive effects of beta-AR on human carcinoma cell migration have been described. alpha(2)-AR are expressed in human breast cancer and non-cancer cell lines, their stimulation being associated with increased cell proliferation. In vivo clonidine increased tumor growth and alpha (2)-adrenergic antagonists completely reversed this effect. When administered alone, rauwolscine inhibited tumor growth behaving as an inverse agonist. Therefore, the numerous adrenergic beta- and alpha-AR agonists or antagonists could prove to be unexpected therapeutic options for mammary gland/ breast and mainly breast cancer.

Expression analysis of epithelial cadherin and related proteins in IBH-6 and IBH-4 human breast cancer cell lines.

Epithelial cadherin (E‐cadherin) is a 120 kDa cell–cell adhesion molecule involved in the establishment of epithelial adherens junctions. It is connected to the actin cytoskeleton by adaptor proteins such as β‐catenin. Loss of E‐cadherin expression/function has been related to tumor progression and metastasis. Several molecules associated with down‐regulation of E‐cadherin have been described, within them neural cadherin, Twist and dysadherin. Human breast cancer cell lines IBH‐6 and IBH‐4 were developed from ductal primary tumors and show characteristic features of malignant epithelial cells. In this study expression of E‐cadherin and related proteins in IBH‐6 and IBH‐4 cell lines was evaluated. In IBH‐6 and IBH‐4 cell extracts, only an 89 kDa E‐cadherin form (Ecad89) was detected, which is truncated at the C‐terminus and is present at low levels. Moreover, no accumulation of the 86 kDa E‐cadherin ectodomain and of the 38 kDa CTF1 fragment was observed. IBH‐6 and IBH‐4 cells showed an intracellular scattered E‐cadherin localization. β‐catenin accompanied E‐cadherin localization, and actin stress fibers were identified in both cell types. E‐cadherin mRNA levels were remarkably low in IBH‐6 and IBH‐4 cells. The E‐cadherin mRNA and genomic sequence encoding exons 14–16 could not be amplified in either cell line. Neither the mRNA nor the protein of neural cadherin and dysadherin were detected. Up‐regulation of Twist mRNA was found in both cell lines. In conclusion, IBH‐6 and IBH‐4 breast cancer cells show down‐regulation of E‐cadherin expression with aberrant protein localization, and up‐regulation of Twist; these features can be related to their invasive/metastatic characteristics. J. Cell. Physiol. 222: 596–605, 2010.

Contribution of α2-adrenoceptors to the mitogenic effect of catecholestrogen in human breast cancer MCF-7 cells

Catecholestrogens are estrogen metabolites formed by hydroxylation of 17beta-estradiol and estrone at either the C-2 or C-4 position, rivaling the parent estrogens in concentration. The objective of the present work was to assess if their catechol group could make them induce proliferation of human breast cancer cells via alpha(2)-adrenoceptors. In competition studies in human breast cancer MCF-7 cells, high concentrations of 2-hydroxy-estradiol (2-OH-E(2)), 2-hydroxy-estrone (2-OH-E(1)) and 4-hydroxy-estrone (4-OH-E(1)) competed for [(3)H]-rauwolscine binding, whereas 4-hydroxy-estradiol (4-OH-E(2)) did not. The contribution of alpha(2)-adrenoceptors and estrogen receptors (ERs) in proliferation enhancement was analyzed with specific antagonists. The specific alpha(2)-adrenergic antagonist yohimbine partially reversed the effect of catecholestrogens except 4-OH-E(2). The selective ER downregulator ICI-182780 or fulvestrant partially or totally reversed the effect of all hydroxylated catecholestrogens. When analyzing the effect of the combination of both antagonists in MCF-7, the contribution of the alpha(2)-adrenoceptors and ERs for 2-OH-E(2), 2-OH-E(1) and 4-OH-E(1) was mixed, whereas for 4-OH-E(2), the only receptor implied was an ER. In MDA-MB-231 cells (ER-alpha negative) the proliferation stimulation by these three catecholestrogens and reversal by the adrenergic antagonist was also observed. It can be concluded that alpha(2)-adrenoceptors contribute at least in part to the mitogenic effect of 2-OH-E(2), 2-OH-E(1) and 4-OH-E(1).

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.

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

Alpha2-adrenoceptor agonists trigger prolactin signaling in breast cancer cells

Breast cancer is the most frequent malignancy among women worldwide. We have described the expression of α2-adrenoceptors in breast cancer cell lines, associated with increased cell proliferation and tumor growth. A mitogenic autocrine/paracrine loop of prolactin (Prl) has been described in breast cancer cells. We hypothesized that the α2-adrenergic enhancement of proliferation could be mediated, at least in part, by this Prl loop. In both T47D and MCF-7 cell lines, the incubation with the α2-adrenergic agonist dexmedetomidine significantly increased Prl release into the culture medium (measured by the Nb2 bioassay), this effect being reversed by the α2-adrenergic antagonist rauwolscine. No change in Prl receptors (PrlR) was observed by RT-qPCR in these cell lines. In IBH-6 cells a decrease in Prl secretion was observed at the lower dexmedetomidine concentration. The signaling pathways involved in ovine Prl (oPrl) and dexmedetomidine action were also assessed. Both compounds significantly activated STAT5 and ERK in all three cell lines. In T47D and MCF-7 cell lines also AKT was activated by both Prl and dexmedetomidine. We therefore describe the STAT5 phosphorylation by an α2-adrenergic agonist, dexmedetomidine. In T47D cells, the α2-adrenergic stimulation of cell proliferation is probably mediated, at least in part, by the Prl autocrine/paracrine loop, because this effect is abrogated by the specific PrlR antagonist Δ1-9-G129R-hPrl. The implication of Prl loop describes a novel mechanism of action of this GPCR.

Abstract 1686: Glypican-3 (GPC3) induces a mesenchymal-epithelial transition in human breast cancer cells lines

Glypicans constitute a family of heparan sulphate proteoglycans which are linked to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol anchor. Since GPC3 has been linked to cancer, herein we generated and characterized a pre-clinical human breast cancer cell model to evaluate the role of GPC3 in human mammary tumor progression. GPC3 expression was blocked in MCF-7 cells (poorly-metastatic, GPC3 +) by siRNA (generating MCF-7-shGPC3 sublines), while it was overexpressed in MDA-MB231cells (metastatic, GPC3 -) by viral infection (producing MDA-MB231-GPC3 sublines). We performed in vitro and in vivo characterization of this model. GPC3 silenced MCF-7 cells acquired F-actin stress fibers, enhanced their clonogenic ability (Colony number 130 -shGPC3 vs. 37 -sh Negative Control (NC)) as well as their migration (Wound coverage (%): 15 -shGPC3 vs. 2 -shNC), were less susceptible to cell death induction (Cell death (%): 21.6 -shGPC3 vs. 34 -shNC), diminished the expression of the epithelial marker E-Cadherin while acquired the mesenchymal marker N-Cadherin, and were more invasive and metastatic in vivo. These cells exhibited an upregulation of the EMT-transcription factor ZEB1, and the canonical Wnt/beta-Catenin signaling pathway was activated. GPC3 overexpressing MDA-MB231 cells misplaced their fibroblast-like appearance as well as their F-actin stress fibers, repressed their clonogenicity (Colony number: 8 -GPC3 vs. 40 -vector) and migratory capacity (Wound coverage (%): 10 -GPC3 vs. 90 -vector), were more sensitive to death in starving condition (Cell death (%) 30 -GPC3 vs. 6.5 -vector), got the ability to form E-Cadherin dependent-spheroids, reexpressed E-Cadherin in cell-cell junctions and downregulated the mesenchymal markers N-Cadherin and vimentin, while were less invasive and metastatic in vivo. The expression of the E-Cadherin repressor ZEB1 was diminished, as well as the activity of the canonical Wnt/beta-Catenin signaling pathway. However, when canonical Wnt signaling was activated by LiCl, the expression of E-Cadherin did not change. In summary, we present in vitro and in vivo experimental evidence supporting the hypothesis that GPC3 has a protective role of human breast cancer progression by inducing mesenchymal-epithelial transition (MET). The canonical Wnt/beta-Catenin signaling inhibition would not be required for MET process induced by GPC3. Citation Format: Lilian F. Castillo, Rocio Tascon, Elisa Bal de Kier Joffe, Giselle Peters. Glypican-3 (GPC3) induces a mesenchymal-epithelial transition in human breast cancer cells lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1686.