Patricia Fernández-Nogueira

Targeting of substance P induces cancer cell death and decreases the steady state of EGFR and Her2

NK1 is a tachykinin receptor highly relevant to tumorigenesis and metastasis development in breast cancer and other carcinomas. Despite the substantial efforts done to develop potent NK1 receptor antagonists, none of these antagonists had shown good antitumor activity in clinical trials. Now, we have tested the effect of inhibition of the neuropeptide Substance P (SP), a NK1 ligand, as a potential therapeutic approach in cancer. We found that the inhibition of SP with antibodies strongly inhibit cell growth and induce apoptosis in breast, colon, and prostate cancer cell lines. These effects were accompained by a decrease in the mitogen‐activated kinase singaling pathway. Interestingly, in some cell lines SP abrogation decreased the steady state of Her2 and EGFR, suggesting that SP‐mediated signaling is important for the basal activity of these ErbB receptors. In consequence, we observed a blockade of the cell cycle progression and the inhibition of several cell cycle‐related proteins including mTOR. SP inhibition also induced cell death in cell lines resistant to Lapatinib and Trastuzumab that have increased levels of active Her2, suggesting that this therapeutic approach could be also effective for those cancers resistant to current anti‐ErbB therapies. Thus, we propose a new therapeutic strategy for those cancers that express NK1 receptor and/or other tachykinin receptors, based in the immuno‐blockade of the neuropeptide SP. J. Cell. Physiol. 227: 1358–1366, 2012.

Substance P autocrine signaling contributes to persistent HER2 activation that drives malignant progression and drug resistance in breast cancer

ERBB receptor transmodulation by heterologous G-protein-coupled receptors (GPCR) generates functional diversity in signal transduction. Tachykinins are neuropeptides and proinflammatory cytokines that promote cell survival and cancer progression by activating several GPCRs. In this work, we found that the pain-associated tachykinin Substance P (SP) contributes to persistent transmodulation of the ERBB receptors, EGFR and HER2, in breast cancer, acting to enhance malignancy and therapeutic resistance. SP and its high-affinity receptor NK-1R were highly expressed in HER2(+) primary breast tumors (relative to the luminal and triple-negative subtypes) and were overall correlated with poor prognosis factors. In breast cancer cell lines and primary cultures derived from breast cancer samples, we found that SP could activate HER2. Conversely, RNA interference-mediated attenuation of NK-1R, or its chemical inhibition, or suppression of overall GPCR-mediated signaling, all strongly decreased steady-state expression of EGFR and HER2, establishing that their basal activity relied upon transdirectional activation by GPCR. Thus, SP exposure affected cellular responses to anti-ERBB therapies. Our work reveals an important oncogenic cooperation between NK-1R and HER2, thereby adding a novel link between inflammation and cancer progression that may be targetable by SP antagonists that have been clinically explored.

Tumor promoting effects of CD95 signaling in chemoresistant cells.

BackgroundCD95 is a death receptor controlling not only apoptotic pathways but also activating mechanisms promoting tumor growth. During the acquisition of chemoresistance to oxaliplatin there is a progressive loss of CD95 expression in colon cancer cells and a decreased ability of this receptor to induce cell death. The aim of this study was to characterize some key cellular responses controlled by CD95 signaling in oxaliplatin-resistant colon cancer cells.ResultsWe show that CD95 triggering results in an increased metastatic ability in resistant cells. Moreover, oxaliplatin treatment itself stimulates cell migration and decreases cell adhesion through CD95 activation, since CD95 expression inhibition by siRNA blocks the promigratory effects of oxaliplatin. These promigratory effects are related to the epithelia-to-mesenchymal transition (EMT) phenomenon, as evidenced by the up-regulation of some transcription factors and mesenchymal markers both in vitro and in vivo.ConclusionsWe conclude that oxaliplatin treatment in cells that have acquired resistance to oxaliplatin-induced apoptosis results in tumor-promoting effects through the activation of CD95 signaling and by inducing EMT, all these events jointly contributing to a metastatic phenotype.

Differential expression of neurogenes among breast cancer subtypes identifies high risk patients

The nervous system is now recognized to be a relevant component of the tumor microenvironment. Receptors for neuropeptides and neurotransmitters have been identified in breast cancer. However, very little is known about the role of neurogenes in regulating breast cancer progression. Our purpose was to identify neurogenes associated with breast cancer tumorigenesis with a potential to be used as biomarker and/or targets for treatment. We used three databases of human genes: GeneGo, GeneCards and Eugenes to generate a list of 1266 relevant neurogenes. Then we used bioinformatics tools to interrogate two published breast cancer databases SAGE and MicMa (n=96) and generated a list of 7 neurogenes that are differentially express among breast cancer subtypes. The clinical potential was further investigated using the GOBO database (n=1881). We identified 6 neurogenes that are differentially expressed among breast cancer subtypes and whose expression correlates with prognosis. Histamine receptor1 (HRH1), neuropilin2 (NRP2), ephrin-B1 (EFNB1), neural growth factor receptor (NGFR) and amyloid precursor protein (APP) were differentially overexpressed in basal and HER2-enriched tumor samples and syntaxin 1A (STX1A) was overexpressed in HER2-enriched and luminal B tumors. Analysis of HRH1, NRP2, and STX1A expression using the GOBO database showed that their expression significantly correlated with a shorter overall survival (p < 0.0001) and distant metastasis-free survival (p < 0.0001). In contrast, elevated co-expression of NGFR, EFNB1 and APP was associated with longer overall (p < 0.0001) and metastasis-free survival (p < 0.0001). We propose that HRH1, NRP2, and STX1A can be used as prognostic biomarkers and therapeutic targets for basal and HER2-enriched breast cancer subtypes.

Glucocorticoids promote transition of ductal carcinoma in situ to invasive ductal carcinoma by inducing myoepithelial cell apoptosis

BackgroundThe microenvironment and stress factors like glucocorticoids have a strong influence on breast cancer progression but their role in the first stages of breast cancer and, particularly, in myoepithelial cell regulation remains unclear. Consequently, we investigated the role of glucocorticoids in ductal carcinoma in situ (DCIS) in breast cancer, focusing specially on myoepithelial cells.MethodsTo clarify the role of glucocorticoids at breast cancer onset, we evaluated the effects of cortisol and corticosterone on epithelial and myoepithelial cells using 2D and 3D in vitro and in vivo approaches and human samples.ResultsGlucocorticoids induce a reduction in laminin levels and favour the disruption of the basement membrane by promotion of myoepithelial cell apoptosis in vitro. In an in vivo stress murine model, increased corticosterone levels fostered the transition from DCIS to invasive ductal carcinoma (IDC) via myoepithelial cell apoptosis and disappearance of the basement membrane. RU486 is able to partially block the effects of cortisol in vitro and in vivo. We found that myoepithelial cell apoptosis is more frequent in patients with DCIS+IDC than in patients with DCIS.ConclusionsOur findings show that physiological stress, through increased glucocorticoid blood levels, promotes the transition from DCIS to IDC, particularly by inducing myoepithelial cell apoptosis. Since this would be a prerequisite for invasive features in patients with DCIS breast cancer, its clinical management could help to prevent breast cancer progression to IDC.

Abstract 1574: Neuropilin 2 regulates lung disseminated tumor cell escape from dormancy and progression into metastasis

Despite considerable advances in the treatment of cancer, about 50% of patients will still eventually develop metastatic disease which remains the main cause of cancer-related death. Disseminated tumor cells (DTCs) are the precursors of metastasis and understanding their biology is one of the most important challenges for the future of cancer research. Our previous studies have shown that TGFβ2 through binding to TGFβR3 regulates DTCs quiescence through the activation of p38αMAPK and induction of the dormant genes DEC2 and p27. However, these DTCs dormancy may be reversed when micro-environmental conditions shift to support DTCs expansion. Nerve fibers and neural mediators are present in organs that serve as key targets for breast and head and neck cancer metastasis, including lungs and bone. In addition, neuropeptides and neurotransmitters receptors are expressed by both, nerve and cancer cells, which suggests that neurotrophic factors can act as messengers between the nervous system and DTCs and influence metastasis progression. Therefore, we hypothesized that in situations of chronic stress or inflammation, neural mediators might influence DTCs fate at secondary organs. Using bioinformatics tools we have found that the semaphorins receptor Neuropilin 2 (NRP2), a co-receptor for TGFβ ligands, is overexpressed in basal and HER2+ breast cancer patient samples and its expression in primary tumors correlate with worst prognosis. In basal breast cancer cell lines, NRP2 expression correlates with lower levels of DEC2 and p27 dormancy genes. Furthermore, we have found that NRP2 is downregulated in dormant cells and overexpressed in proliferative lung DTCs derived cell lines that express low levels of P-p38, DEC2 and p27 dormancy markers. Our results show that in lung DTCs derived cell lines, NRP2 inhibits TGFβR3 expression favoring TGFβ1 signaling and promoting DTCs growth. In agreement with this, breast cancer lung metastasis express higher levels of NRP2 than quiescence lung DTCs in vivo, suggesting that NRP2 might play a role in lung DTCs proliferation. Therefore, we conclude that NRP2 can regulate breast cancer lung DTCs progression from a dormant state to a proliferative state and promote metastasis formation. Citation Format: Paloma Bragado Domingo, Raul Alonso, Gemma Fuster, Mario Mancino, Patricia Fernandez-Nogueira, Julio Aguirre-Ghiso, Pere Gascon. Neuropilin 2 regulates lung disseminated tumor cell escape from dormancy and progression into metastasis. [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 1574.

Abstract A150: A methodology for the isolation, culture, and immortalization of human breast epithelial and myoepithelial cells.

Introduction: Breast tissue is composed by different cell types including epithelial and myoepithelial cells, macrophages, fibroblasts, adipocytes, and cells from the immune system. Two main lineages, epithelial and myoepithelial cells, are the most essential cell populations in the normal mammary gland and also in breast cancer. Traditionally, cancer research has been performed using commercial cell lines, but primary cell cultures suppose a powerful tool to study with a higher reliability new aspects of mammary gland biology including normal and diseased tissue. However, normal primary human epithelial cells in culture achieve senescence at 10 to 40 population doubling, hampering their long-term culture. In addition, some studies have described lineage-specific marker limitations to be used in order to isolate these two cell types, since some of the markers tend to change during cell culture. Experimental Procedures: In this protocol we compared two different methodologies in an effort to improve isolation, culture and immortalization of different cell populations of human normal mammary epithelium. We based our approaches in previously described methods incorporating additional steps to improve cell survival, to preserve cellular antigens used for isolation and validation of cell subpopulations and to enhance cellular transformation. For these purposes, we determined cell growth and viability, and also cell-lineage specific markers of cytospinned cells in both methodologies used. Results: One of the goals of the present work to yield on cell survival and antigens preservation is in fact overnight digestion at low enzymatic concentration. Other crucial steps are cell immortalization and also addition of critical compounds to the cell culture media. Conclusions: In the present work, we propose some guidelines to establish more efficiently mammary epithelial cell lines allowing long-term cell culture system and providing us a strong instrument to better understand the role of the different epithelial cell types and the origins of breast cancer. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A150. Citation Format: Gemma Fuster, Arantzazu Zubeldia-Plazaola, Mario Mancino, Patricia Fernandez-Nogueira, Elisabet Ametller, Pedro Gascon, Vanessa Almendro. A methodology for the isolation, culture, and immortalization of human breast epithelial and myoepithelial cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A150.

Abstract B100: Subtype specific expression of HRH1 contributes to increased chemoresistance of breast cancer cells

Background: Histamine is a monoamine produced by α-decarboxylation of histidine by the enzyme histidine decarboxylase (HDC). Histamine acts as an autocrine regulator of cell proliferation through its binding to HRH1-4 receptors. In breast cancer (BC), there are controversial results about the therapeutic efficacy of HRH1 or HRH2 inhibitors and the role of histamine receptors in tumor progression. Given the phenotypic heterogeneity of BC and the presence of different molecular subtypes with different clinical outcome, we aimed to investigate the contribution of histamine receptors in the proliferation, migration and cell survival of BC cells of different molecular subtypes. Methods: The expression of histamine receptors HRH1-4 and HDC in human breast tumors of different subtypes, and their correlation with clinical variables were gathered through informatics analysis using the “Gene expression based Outcome for Breast cancer Online” (GOBO) web-based tool. The measurement of the mRNA levels of HRH1-4 and HDC in a panel of breast cancer cell lines was used to confirm the subtype specific expression of each protein. Using specific HRH inhibitors in luminal and basal-like breast cancer cell lines we assessed the functional role of histamine receptors in cell proliferation, migration and survival. Results: HRH1 was highly expressed in ER+ tumors while HDC was highly expressed in the ER- tumors. In any case, the high expression of HRH1 or HDC was significantly correlated with lower overall survival only in ER+ tumors. The same pattern of expression was observed in BC cell lines, where HRH1 was highly expressed in basal-like BC cells compared to the luminal ones. HRH2 and HRH3 were highly expressed in the estrogen-dependent MCF7 cell line, while HRH4 was not detected in any breast cancer cell line. Only the antagonism of HRH1 induced apoptosis, and decreased proliferation and migration, while HRH2 or HRH3 inhibition had no effect on cell survival. Interestingly, the luminal cell lines were more sensitive than the basal-like cell lines to HRH1 antagonism, even though the low expression of HRH1 in these cells. Conclusion: Taken together, these findings suggest a complex role of the autocrine histamine signaling system in the progression of BC tumors of different subtype through the activation of HRH1. Citation Format: Mario Mancino, Patricia Fernandez-Nogueira, Estel Enreig, Elisabet Ametller, Pedro Gascon, Vanessa Almendro. Subtype specific expression of HRH1 contributes to increased chemoresistance of breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B100.