Rocio Sampayo

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.

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.

Reversal of the Migratory and Invasive Phenotype of Ras-Transfected Mammary Cells by Photodynamic Therapy Treatment

Photodynamic therapy (PDT) is a non‐thermal technique for inducing tumor damage following administration of a light‐activated photosensitizing drug (PS). In a previous work we found that PDT induces cytoskeleton changes in HB4a‐Ras cells (human mammary breast carcinoma HB4a cells transfected with the RAS oncogene). In the present work we have studied the migratory and invasive features and the expression of proteins related to these processes on HB4a‐Ras cells after three successive cycles of PDT using different PSs: 5‐aminolevulinic acid (ALA), Verteporfin (Verte), m‐tetrahydroxyphenylchlorin (m‐THPC), and Merocyanine 540 (MC). A slight (1.25‐ to 2‐fold) degree of resistance was acquired in cell populations subjected to the three successive PDT treatments. However, complete cell killing was achieved after a light dose increase. Regardless of the PS employed, all the PDT‐treated populations had shorter stress fibres than the untreated control HB4a‐Ras cells, and the number of dorsal stress fibres was decreased in the PDT‐treated populations. E‐Cadherin distribution, which was already aberrant in HB4a‐Ras cells, became even more diffuse in the PDT‐treated populations, though its expression was increased in some of them. The strong migratory and invasive ability of HB4a‐Ras cells in vitro was impaired in all the PDT‐treated populations, with a behavior that was similar to the parental non‐tumoral HB4a cells. MMP‐2 and ‐9 metalloproteinase activities were also impaired in the PDT‐treated populations. The evidence presented herein suggests that the cells surviving PDT would be less metastatic than the initial population. These findings encourage the use of PDT in combination with other treatments such as intraoperative or post‐surgery therapeutic procedures. J. Cell. Biochem. 118: 464–477, 2017.

Abstract 2240: Progesterone receptor isoform ratio regulates the stem cell population in the mouse mammary gland

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA The ovarian steroid hormones progesterone and estrogen play vital roles in the development of normal mammary gland and are likewise linked to mammary carcinogenesis via their receptors. Progesterone receptor (PR) is expressed as two isoforms, PR-A and PR-B, both in human and mouse. It has been reported that in human breast tumors the PR-A/PR-B expression ratio is altered. To further study the role of PR isoforms on mammary gland biology, transgenic mice carrying either an additional A form of PR (PR-A) or the B form of PR (PR-B) were generated. Both mice strains present abnormal development in their mammary glands. Overexpression of the A isoform of PR leads to increased side branching and multilayered ducts, while overexpressing the B isoform leads to limited ductal growth. The characteristics of mammary glands from these mice provide direct evidence that an aberration in the mechanisms regulating the differential expression of the two isoforms of PR can have major implications to mammary carcinogenesis and might affect the stem cell population of the gland. Recent studies have shown that a hierarchy of mammary stem/progenitor cells exists among the mammary epithelium in both human and mouse and PR signaling has been shown to play a major role in the maintenance of the stem cell population. So far, however, the differential role of the PR isoforms in the regulation of the stem cell population in the breast has not been established. We propose that the altered ratio of PR A/B might affect the stem cell population expansion and self renewal in the mammary gland. We found that mammary glands of mice overexpressing the B isoform presented a higher percentage of cells positive for stem cell markers (CD24+/CD29+) than those derived from PR-A and WT mice. Furthermore cells derived from PR-B mammary glands had increased mammosphere forming capacity compared to both PR-A and WT. Moreover, when mammosphere cultures were carried out in the presence of 4-OH-tamoxifen 10-8M, the number of mammospheres formed from mammary glands of WT and PR-A mice was reduced compared to control cultures treated with vehicle, whereas there were no differences for cultures derived from mammary glands of PR-B mice. Additionally, upon ovariectomy (OVX), mammary glands from PR-A and WT mice showed a higher percentage of cells positive for stem cell markers by flow cytometry, and increase mammosphere forming capacity than control sham mice, while in mammary glands derived from PR-B OVX mice neither parameters changed. Our results suggest that the B isoform of PR, may play a predominant role in the maintenance of stem cell population of the mammary gland and in the development of hormone resistance given that ovarian regulation affected mammary glands of WT and PR-A mice, but not those derived from PR-B mice. Citation Format: Maria S. Recouvreux, Rocio Sampayo, Marina Simian. Progesterone receptor isoform ratio regulates the stem cell population in the mouse mammary gland. [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 2240. doi:10.1158/1538-7445.AM2015-2240

Mammary Gland Organoids

The study of the mechanisms that regulate development and tumorigenesis is a complex undertaking that requires a variety of model systems to test hypothesis that embrace all levels of organization: from single cells to organs. In the mammary gland field, the use of three-dimensional culture systems has provided a platform to study, in a physiologically relevant setting, cell biology in context. In the late 50’s methods to isolate primary mammary organoids were established and since then they have been increasingly used to understand cell behavior. In this chapter we embrace, in a historical perspective, the key findings carried out using primary mammary organoids considering that the broadening of our knowledge will, in the future, rely increasingly on this kind of tridimensional culture setting.

Fibronectin rescues estrogen receptor α from lysosomal degradation in breast cancer cells

Estrogen receptor &agr; (ER&agr;) is expressed in tissues as diverse as brains and mammary glands. In breast cancer, ER&agr; is a key regulator of tumor progression. Therefore, understanding what activates ER&agr; is critical for cancer treatment in particular and cell biology in general. Using biochemical approaches and superresolution microscopy, we show that estrogen drives membrane ER&agr; into endosomes in breast cancer cells and that its fate is determined by the presence of fibronectin (FN) in the extracellular matrix; it is trafficked to lysosomes in the absence of FN and avoids the lysosomal compartment in its presence. In this context, FN prolongs ER&agr; half-life and strengthens its transcriptional activity. We show that ER&agr; is associated with &bgr;1-integrin at the membrane, and this integrin follows the same endocytosis and subcellular trafficking pathway triggered by estrogen. Moreover, ER&agr;+ vesicles are present within human breast tissues, and colocalization with &bgr;1-integrin is detected primarily in tumors. Our work unravels a key, clinically relevant mechanism of microenvironmental regulation of ER&agr; signaling.

Distinct ErbB2 receptor populations differentially interact with beta1 integrin in breast cancer cell models

ErbB2 is a member of the ErbB family of tyrosine kinase receptors that plays a major role in breast cancer progression. Located at the plasma membrane, ErbB2 forms large clusters in spite of the presence of growth factors. Beta1 integrin, membrane receptor of extracellular matrix proteins, regulates adhesion, migration and invasiveness of breast cancer cells. Physical interaction between beta1 integrin and ErbB2 has been suggested although published data are contradictory. The aim of the present work was to study the interaction between ErbB2 and beta1 integrin in different scenarios of expression and activation. We determined that beta1 integrin and ErbB2 colocalization is dependent on the expression level of both receptors exclusively in adherent cells. In suspension cells, lack of focal adhesions leave integrins free to diffuse on the plasma membrane and interact with ErbB2 even at low expression levels of both receptors. In adherent cells, high expression of beta1 integrin leaves unbound receptors outside focal complexes that diffuse within the plasma membrane and interact with ErbB2 membrane domains. Superresolution imaging showed the existence of two distinct populations of ErbB2: a major population located in large clusters and a minor population outside these structures. Upon ErbB2 overexpression, receptors outside large clusters can freely diffuse at the membrane and interact with integrins. These results reveal how expression levels of beta1 integrin and ErbB2 determine their frequency of colocalization and show that extracellular matrix proteins shape membrane clusters distribution, regulating ErbB2 and beta1 integrin activity in breast cancer cells.

Abstract 3477: Tamoxifen leads to an increase in tumor cells with self-renewing capacity in a mouse model of estrogen dependent breast cancer

About 75% of breast cancers are positive for estrogen receptor (ER). Tamoxifen (TAM) has been the main treatment for the last 30 years, however, 1/3 of patients that initially respond develop resistance. Recent discoveries have led to hypothesis that considers a direct involvement of stem cell populations (SCP) in cancer progression and resistance to therapy. The aim of this work was to analyze the impact of endocrine therapy on SCP in breast cancer. To do so we used the M05 mouse mammary tumor (Simian et al., BCRT, 2009), a syngeneic model of estrogen dependent, TAM sensitive breast cancer, together with the LM05-E cell line derived from it and MCF-7 cells. When treated with TAM for 30 days tumor growth was inhibited and immunofluorescence (IF) staining showed a decrease in ER+/E-cadherin+ tumor cells together with an increase in the stromal compartment and deposition of extracellular matrix (ECM) components, compared to vehicle treated controls. Tumors were digested until a single cell suspension was obtained. Mammosphere assays (MA) were carried out and quantified after 7 days in culture. A statistically significant increase in mammosphere formation was achieved in the cultures of tumors derived from TAM treated mice, compared to controls. Next we tested whether this was true for tumors derived from TAM treated and untreated animals that were subsequently passaged to untreated mice and allowed to regrow. MA revealed that this was the case. IF studies showed that those tumors that derived from the TAM treated mice contained higher levels of β1 and β6 integrins as well as increased phopho-ser118-ER. Next, to further establish a link between TAM and self-renewal capacity in vitro, we treated LM05-E and MCF-7 cells with 4-OH-TAM 10-6M or vehicle for 5 days and carried out MA. TAM led to an increase in mammosphere formation. However, when TAM was added directly to the MA no differences were detected between the treated and control groups suggesting that only those cells that resist the treatment are able to grow in suspension. Finally we had previously shown that fibronectin and laminin conferred TAM resistance through β1 and β6 integrins respectively. To test whether these factors lead to an increase in cells with self-renewing capacity we treated LM05-E and MCF-7 cells with these ECM components for 48 hours and then carried out MA and measured ALDH activity. Compared to the control group we found a decrease in the % of mammosphere forming cells as well as a decrease in ALDH activity in the treated groups. Our results show that TAM leads to an increase in cells with self-renewal capacity both in vitro and in vivo, and that microenvironmental factors seem to have the opposite effect. Understanding the effects of tamoxifen on the breast cancer SCP has important implications for the development of strategies that could lead to a decrease in recurrence in patients with ER+ tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3477. doi:1538-7445.AM2012-3477

Abstract 545: Laminin induces tamoxifen resistance through alfa 6 integrin in breast cancer cells

Even though it is now well established that the tumor microenvironment plays a central role in modulating tumor progression and response to therapy, its function as a regulator of endocrine resistance in breast cancer has not yet been thoroughly explored. We previously showed that fibronectin through beta 1 integrin induces tamoxifen resistance in breast cancer cells. The aim of this study was to determine whether other extracellular matrix components induce tamoxifen resistance and what mechanisms are involved. We found that laminin, but not collagen IV, induced tamoxifen resistance in both estrogen receptor positive (ER+) human MCF-7 and mouse LM05-E breast cancer cells (P −8 M) and 4-OH-tamoxifen (10 −6 M) with the addition of either the MAPK/ERK1/2 inhibitor PD98059 (10µM) or the PI3K/AKT inhibitor LY294002 (10µM). Only LY294002 reversed the protective effect of laminin (P −8 M induced a significant stimulation of ER activity (P −6 M 4-OH-tamoxifen (P Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 545. doi:10.1158/1538-7445.AM2011-545