Marina Simian

The tumor microenvironment modulates tamoxifen resistance in breast cancer: a role for soluble stromal factors and fibronectin through β1 integrin

Tamoxifen resistance has been largely attributed to genetic alterations in the epithelial tumor cells themselves, such as overexpression of HER-2/Neu. However, in the clinic, only about 15–20% of cases of HER-2/Neu amplification has actually been correlated to the acquisition of endocrine resistance, suggesting that other mechanisms must be involved as well. Using the epithelial LM05-E and the fibroblastic LM05-F cell lines, derived from the estrogen dependent spontaneous M05 mouse mammary tumor, as well as MCF-7 cells, we analyzed whether soluble stromal factors or extracellular matrix components protected against tamoxifen induced cell death. Involvement of signaling pathways was determined by using specific inhibitors and western blot, and phosphorylation of the estrogen receptor alpha by western blot and immunofluorescence. Soluble factors produced by the fibroblastic cells protect the epithelial tumor cells from tamoxifen-induced cell death through a mechanism that involves EGFR and matrix metalloproteinases upstream of PI3K/AKT. Exogenous fibronectin by itself confers endocrine resistance through interaction with β1 integrin and activation of PI3K/AKT and MAPK/ERK 1/2 pathways. The conferred resistance is reversed by blocking β1 integrin. We show also that treatment with both conditioned medium and fibronectin leads to the phosphorylation of the estrogen receptor at serine-118, suggesting stromal factors as modulators of ER activity. Our results show that the tumor microenvironment can modulate tamoxifen resistance, providing an alternative explanation for why patients become refractory to hormone-therapy.

Organoids: A historical perspective of thinking in three dimensions

In this perspective, Simian and Bissell discuss the evolution of the 3D culture and organoid research field up to now as well as its future directions.

Regulation of cell growth of a progestin-dependent murine mammary carcinoma in vitro: progesterone receptor involvement in serum or growth factor-induced cell proliferation

Primary cultures of the medroxyprogesterone acetate-induced mouse mammary tumor line C4-HD are stimulated by medroxyprogesterone acetate (MPA) or progesterone. Serum obtained from ovariectomized, MPA-treated animals (OVX-MPA) exerts a stimulatory effect that is significantly higher than that induced by serum obtained from OVX mice with the exogenous addition of MPA, suggesting the involvement of MPA-induced serum factors potentiating the proliferative effect of MPA. The object of this paper is to further explore the stimulatory effect of mouse serum and to investigate the role of aFGF and bFGF on cell proliferation. The role of PR as possible mediators was tested using two different antiprogestins and antisense oligodeoxynucleotides of PR A isoform. Serum was obtained from OVX untreated or MPA-treated mice and was charcoalized and/or heat-inactivated. The effect of MPA or mifepristone at 10 nM concentrations was tested. Charcoalization and heat inactivation exerted a stimulatory effect (P<0.01) when OVX-serum was used. This effect was potentiated by MPA. Charcoalized OVX-MPA serum induced a significant inhibition of cell proliferation that was restored by the exogenous addition of MPA or by heat inactivation. Mifepristone induced an inhibition of 3H-thymidine uptake when OVX-MPA serum was used. These results suggest that serum factors activated by different manipulations may replace the stimulatory effect of MPA. When charcoalized fetal calf serum (chFCS) was used, a higher proliferative activity was obtained using higher serum concentrations. Mifepristone and onapristone 10 nM also inhibited this effect. aFGF and bFGF 100 ng/ml were both able to stimulate 3H-thymidine uptake. MPA exerted an additive effect. Mifepristone 10 nM inhibited bFGF and MPA+bFGF induced cell proliferation. Antisense oligodeoxynucleotides of PR (ASPR) were used to further confirm the participation of PR in the proliferative pathway of these cells. They inhibited serum and bFGF-induced cell proliferation in a specific dose-dependent manner. Our results suggest that PR play a central role in proliferation and suggest the existence of a cross-talk between steroid and growth factor signaling pathways.

Decreased metastatic phenotype in cells resistant to aminolevulinic acid-photodynamic therapy

Photodynamic therapy (PDT) is a novel cancer treatment utilising a photosensitiser, visible light and oxygen. PDT often leaves a significant number of surviving tumour cells. In a previous work, we isolated and studied two PDT resistant clones derived from the mammary adenocarcinoma LM3 line (Int. J. Oncol. 29 (2006) 397-405). The isolated Clon 4 and Clon 8 exhibited a more fibroblastic, dendritic pattern and were larger than the parentals. In the present work we studied the metastatic potential of the two clones in comparison with LM3. We found that 100% of LM3 invaded Matrigel, whereas only 19+/-6% and 24+/-7% of Clon 4 and Clon 8 cells invaded. In addition, 100% of LM3 cells migrated towards a chemotactic stimulus whereas 38+/-8% and 73+/-10% of Clones 4 and 8, respectively, were able to migrate. In vivo, 100% of the LM3 injected mice developed spontaneous lung metastasis, whereas none of the Clon 8 did, and only one of the mice injected with Clon 4 did. No differences were found in the proteolytic enzyme profiles among the cells. Anchorage-dependent adhesion was also impaired in vivo in the resistant clones, evidenced by the lower tumour take, latency time and growth rates, although both clones showed in vitro higher binding to collagen I without overexpression of beta1 integrin. This is the first work where the metastatic potential of cells surviving to PDT has been studied. PDT strongly affects the invasive phenotype of these cells, probably related to a higher binding to collagen. These findings may be crucial for the outcome of ALA-PDT of metastatic tumours, although further studies are needed to extrapolate the results to the clinic employing another photosensitisers and cell types.

Establishment of an in vitro estrogen-dependent mouse mammary tumor model: a new tool to understand estrogen responsiveness and development of tamoxifen resistance in the context of stromal–epithelial interactions

Currently, to our knowledge, there are no continuous cell lines derived from estrogen dependent, tamoxifen sensitive spontaneous mouse mammary carcinomas. We describe here the establishment and characterization of a cell line derived from the M05 mouse mammary tumor, LM05-Mix, composed of both an epithelial and a fibroblastic component. From it the respective epithelial LM05-E and fibroblastic LM05-F cell lines were generated by limiting dilution. Immunofluorescence studies confirmed that the epithelial cells were positive for E-cadherin, cytokeratins and vimentin whereas the fibroblastic cells were negative for the epithelial markers and positive for α-smooth muscle actin and vimentin. Both cell types expressed estrogen and progesterone receptors, although only the epithelial LM05-E cells were stimulated by estradiol and inhibited by tamoxifen. In the bicellular LM05-Mix cell line estradiol proved to stimulate cell proliferation whereas the response to tamoxifen was dependent on confluency and the degree of epithelial-fibroblastic interactions. The presence of membrane estrogen receptors in both cell types was suggested by the achievement of non-genomic responses to short treatments with estradiol, leading to the phosphorylation of ERK1/2. Finally, cytogenetic studies suggest that these two cell types represent independent cell populations within the tumor and would not be the result of an epithelial-mesenchymal transition. This model presents itself as a valuable alternative for the study of estrogen responsiveness and tamoxifen resistance in the context of epithelial-stromal interactions.

Involvement of EGF in medroxyprogesterone acetate (MPA)-induced mammary gland hyperplasia and its role in MPA-induced mammary tumors in BALB/c mice

In previous papers we have demonstrated that sialoadenectomy inhibited MPA-induced mammary tumorigenesis in BALB/c mice. To further explore the role of EGF in this experimental model, we evaluated its effects on mammary glands of sialoadenectomized (sialox) MPA-treated female mice and on tumor growth. MPA-treated sialox mice were injected s.c. (n = 3) or not (n = 6) with 5 microg EGF every 36 h for 45 days; MPA-treated sham-operated mice were used as controls (n = 6). Mammary glands from sialox MPA-treated mice are considerably less developed as compared with sham-operated animals. The exogenous administration of EGF restores the usual MPA-induced growth pattern of the glands, thus confirming a role for EGF either in mediating or cooperating with MPA in inducing the mammary architectural changes observed in MPA-treated mice. On the other hand, primary cultures of progestin-dependent (PD) ductal mammary adenocarcinoma in vivo tumor lines and of lobular progestin-independent (PI) tumor lines were used to evaluate the effect of EGF on tumor growth. In vitro EGF was found to stimulate cell proliferation of lobular PI tumor cells and of fibroblastic cells from both types of tumors at concentrations higher than 0.1-0.5 ng/ml and in the presence of 1-5% of charcoal-stripped fetal calf serum. Conversely, no proliferative effects were observed in ductal PD cells under the same experimental conditions, regardless of the presence of 10 nM MPA. It can be concluded that although EGF plays an important role in MPA-induced mammary carcinogenesis, it is not necessary in PD tumor growth.

Isolation of a stromal cell line from an early passage of a mouse mammary tumor line: a model for stromal parenchymal interactions.

We have developed a murine mammary tumor cell line, MC4‐L4, and after 15 passages, a spindle‐shaped population became evident. The cuboidal cells, MC4‐L4E, cloned by limit dilution, proved to be epithelial tumor cells. When inoculated in syngeneic mice, they gave rise to invasive metastatic carcinomas expressing estrogen and progesterone receptors. These tumors regressed after anti‐progestin treatment and stopped growing after 17‐β‐estradiol administration. In vitro, they were insensitive to medroxyprogesterone acetate (MPA), 17‐β‐estradiol, and EGF and were inhibited by TGFβ1. They expressed mutated p53 and estrogen receptors α; progesterone receptors were undetectable. Cells were polyploid and shared the same four common marker chromosomes present in the parental tumor in addition to an exclusive marker. Spindle‐shaped cells, MC4‐L4F, were selected by differential attachment and detachment and proved to be non‐epithelial non‐tumorigenic cells. They were cytokeratin negative, showed mesenchymal features by electron microscopy, differentiated to adipocytes when treated with an adipogenic cocktail, were stimulated by TGFβ1 and EGF, showed a wild‐type p53, and did not exhibit the marker chromosomes of the parental tumor. Although they expressed estrogen receptors α, they were insensitive to 17‐β‐estradiol in proliferation assays. Co‐cultures of both cell types had a synergic effect on progesterone receptors expression and on cell proliferation, being the epithelial cells, the most responsive ones, and 17‐β‐estradiol increased cell proliferation only in co‐cultures. Cytogenetic studies and data on p53 mutations rule out the possibility of an epithelial mesenchymal transition. Their unique characteristics make them an excellent model to be used in studies of epithelial–stromal interactions in the context of hormone responsiveness in hormone related tumors.

Microenvironment and endocrine resistance in breast cancer: Friend or foe?

Breast cancer affects one in eight women around the world. Seventy five percent of these patients have tumors that are estrogen receptor positive and as a consequence receive endocrine therapy. However, about one third eventually develop resistance and cancer reappears. In the last decade our vision of cancer has evolved to consider it more of a tissue-related disease than a cell-centered one. This editorial argues that we are only starting to understand the role the tumor microenvironment plays in therapy resistance in breast cancer. The development of new therapeutic strategies that target the microenvironment will come when we clearly understand this extremely complicated scenario. As such, and as a scientific community, we have extremely challenging work ahead. We share our views regarding these matters.

Angiotensin-(1-7) counteracts the transforming effects triggered by angiotensin II in breast cancer cells

Angiotensin (Ang) II, the main effector peptide of the renin-angiotensin system, has been implicated in multiple aspects of cancer progression such as proliferation, migration, invasion, angiogenesis and metastasis. Ang-(1-7), is a biologically active heptapeptide, generated predominantly from AngII by the enzymatic activity of angiotensin converting enzyme 2. Previous studies have shown that Ang-(1-7) counterbalances AngII actions in different pathophysiological settings. In this study, we have analysed the impact of Ang-(1-7) on AngII-induced pro-tumorigenic features on normal murine mammary epithelial cells NMuMG and breast cancer cells MDA-MB-231. AngII stimulated the activation of the survival factor AKT in NMuMG cells mainly through the AT1 receptor. This PI3K/AKT pathway activation also promoted epithelial–mesenchymal transition (EMT). Concomitant treatment of NMuMG cells with AngII and Ang-(1-7) completely abolished EMT features induced by AngII. Furthermore, Ang-(1-7) abrogated AngII induced migration and invasion of the MDA-MB-231 cells as well as pro-angiogenic events such as the stimulation of MMP-9 activity and VEGF expression. Together, these results demonstrate for the first time that Ang-(1-7) counteracts tumor aggressive signals stimulated by AngII in breast cancer cells emerging the peptide as a potential therapy to prevent breast cancer progression.

AT1 receptor blockade delays postlactational mammary gland involution: a novel role for the renin angiotensin system

Angiotensin II (AngII), the main effector peptide of the renin‐angiotensin system (RAS), participates in multiple biological processes, including cell growth, apoptosis, and tissue remodeling. Since AngII activates, in different cell types, signal transducing pathways that are critical for mammary gland postlactational regression, we investigated the role of the RAS during this process. We found that exogenous administration of AngII in mammary glands of lactating Balb/c mice induced epithelium apoptosis [2.9±0.5% (control) vs. 9.6±1.1% (AngII); P < 0.001] and activation of the proapoptotic factor STAT3, an effect inhibited by irbesartan, an AT1 receptor blocker. Subsequently, we studied the expression kinetics of RAS components during involution. We found that angiotensin‐converting enzyme (ACE) mRNA expression peaked 6 h after weaning (5.7‐fold; P<0.01), while induction of angiotensinogen and AT1 and AT2 receptors expression was detected 96 h after weaning (6.2‐, 10‐, and 6.2‐fold increase, respectively; P<0.01). To assess the role of endogenously generated AngII, mice were treated with losartan, an AT1 receptor blocker, during mammary involution. Mammary glands from losartan‐treated mice showed activation of the survival factors AKT and BCL‐XL, significantly lower LIF and TNF‐α mRNA expression (P<0.05), reduced apoptosis [12.1±2.1% (control) vs. 4.8±0.7% (losartan); P<0.001] and shedding of epithelial cells, inhibition of MMP‐9 activity in a dose‐dependent manner (80%; P<0.05; with losartan IC50 value of 6.9 mg/kg/d] and lower collagen deposition and adipocyte invasion causing a delayed involution compared to vehicle‐treated mice. Furthermore, mammary glands of forced weaned AT1A‐ and/or AT1B‐deficient mice exhibited retarded apoptosis of epithelial cells [6.3±0.95% (WT) vs. 3.3±0.56% (AT1A/AT1B DKO); P<0.05] with remarkable delayed postlactational regression compared to wild‐type animals. Taken together, these results strongly suggest that AngII, via the AT1 receptor, plays a major role in mouse mammary gland involution identifying a novel role for the RAS.—Nahmod, K. A., Walther, T., Cambados, N., Fernandez, N., Meiss, R., Tappenbeck, N., Wang, Y., Raffo, D., Simian, M., Schwiebs, A., Pozner, R. G., Fuxman Bass, J. I., Pozzi, A. G., Geffner, J. R., Kordon, E. C., Schere‐Levy, C. AT1 receptor blockade delays postlactational mammary gland involution: a novel role for the renin angiotensin system. FASEB J. 26, 1982‐1994 (2012). www.fasebj.org