Antiestrogens inhibit the mitogenic effect of growth factors on breast cancer cells in the total absence of estrogens

The antiproliferative effect of antiestrogens in breast cancer is believed to be entirely due to the inhibition of estrogen induced growth. We show here that non-steroidal antiestrogens inhibit the growth of the human breast cancer MCF7 cells in the complete absence of estrogens (phenol-red-free medium) when cell proliferation is stimulated by insulin or epidermal growth factor. This non-antiestrogenic effect of antiestrogens is, however, mediated by accessible estrogen receptor sites, as it is not observed in receptor negative hormone-independent breast cancers, and is rescued by estradiol but not by insulin. We conclude that antiestrogens inhibit cell proliferation by inhibiting growth factor action as well as estrogen action and that in both cases, accessible estrogen receptors are required.     

Growth regulatory peptide production by human breast carcinoma cells

The mechanisms by which human breast cancers regulate their own growth have been studied by us in an in vitro model system. We showed that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells. A variety of experiments suggest that they are involved in tumor growth and progression. These activities are induced by estradiol in hormone-dependent breast cancer cells and secreted constitutively by estrogen-independent cells. Concentrates of conditioned medium derived from breast cancer cells can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta. TGF beta is growth inhibitory. Growth inhibitors such as antiestrogens or glucocorticoids increase TGF beta secretion. An antiestrogen-resistant mutant of MCF-7 cells does not secrete TGF beta when treated with antiestrogen, but is growth inhibited when treated with exogenous TGF beta. Thus, TGF beta functions as a negative autocrine growth regulator and is probably responsible for some of the growth inhibitory effects of antiestrogens.     

Regulation of transforming growth factor alpha and transforming growth factor beta messenger ribonucleic acid abundance in T-47D, human breast cancer cells

Both transforming growth factor beta (TGF beta) and TGF alpha mRNA are expressed in human breast cancer cell lines. We have investigated the relationship of mRNA abundance for these growth modulators to the proliferation rate of a number of human breast cancer cell lines. Furthermore, we have investigated the relationship of regulation of TGF beta and TGF alpha mRNA to growth inhibition caused by progestins and nonsteroidal antiestrogens in T-47D human breast cancer cells. The abundance of TGF beta and TGF alpha mRNA in human breast cancer cell lines was not related directly to proliferation rate of the cells in culture or estrogen receptor positivity or negativity. The relationship of TGF beta and TGF alpha mRNA to growth inhibition caused by antiestrogens and progestins was investigated in T-47D human breast cancer cells. We observed that in T-47D human breast cancer cells the abundance of TGF beta mRNA is decreased in a time- and dose-dependent fashion by progestins but remains unaltered by nonsteroidal antiestrogens. Treatment of T-47D cells for 24 h with 10 nM medroxyprogesterone acetate (MPA) reduced the level of TGF beta mRNA to one third that present in untreated cells. The same treatment increased TGF alpha mRNA 3-fold above untreated controls in a time- and dose-dependent fashion and nonsteroidal antiestrogens caused a small decrease. The regulation of both TGF alpha and TGF beta mRNA was not directly related to inhibition of growth by progestins and antiestrogens in T-47D cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of estrogen receptor-positive breast cancer tumorspheres to antiestrogen treatments

Estrogen signaling plays a critical role in the pathogenesis of breast cancer. Because the majority of breast carcinomas express the estrogen receptor ERα, endocrine therapy that impedes estrogen-ER signaling reduces breast cancer mortality and has become a mainstay of breast cancer treatment. However, patients remain at continued risk of relapse for many years after endocrine treatment. It has been proposed that cancer recurrence may be attributed to cancer stem cells (CSCs)/tumor-initiating cells (TICs). Previous studies in breast cancer have shown that such cells can be enriched and propagated in vitro by culturing the cells in suspension as mammospheres/tumorspheres. Here we established tumorspheres from ERα-positive human breast cancer cell line MCF7 and investigated their response to antiestrogens Tamoxifen and Fulvestrant. The tumorsphere cells express lower levels of ERα and are more tumorigenic in xenograft assays than the parental cells. Both 4-hydroxytamoxifen (4-OHT) and Fulvestrant attenuate tumorsphere cell proliferation, but only 4-OHT at high concentrations interferes with sphere formation. However, treated tumorsphere cells retain the self-renewal capacity. Upon withdrawal of antiestrogens, the treated cells resume tumorsphere formation and their tumorigenic potential remains undamaged. Depletion of ERα shows that ERα is dispensable for tumorsphere formation and xenograft tumor growth in mice. Surprisingly, ERα-depleted tumorspheres display heightened sensitivity to 4-OHT and their sphere-forming capacity is diminished after the drug is removed. These results imply that 4-OHT may inhibit cellular targets besides ERα that are essential for tumorsphere growth, and provide a potential strategy to sensitize tumorspheres to endocrine treatment.     

Growth regulation of human breast carcinoma occurs through regulated growth factor secretion

We describe studies on human breast cancer in which it is shown that specific growth factors (IGF-I, TGF alpha, PDGF) are secreted by human breast cancer cells and likely to be involved in tumor growth and progression. These activities are regulated by estradiol in hormone-dependent breast cancer and secreted constitutively by hormone-independent cells. These growth factor activities can induce the growth of hormone-dependent cells in vivo in athymic nude mice. Hormone-dependent breast cancer cells also secrete TGF beta, a growth-inhibitory substance, when treated with antiestrogens. TGF beta functions as a negative autocrine growth regulator and is responsible for some of the growth-inhibitory effects of antiestrogens.     

Nonsteroidal antiestrogens are estrogen-receptor-targeted growth inhibitors that can act in the absence of estrogens

The mechanism of the antiproliferative effect of nonsteroidal antiestrogens (tamoxifen, hydroxytamoxifen) is discussed from studies performed in human breast cancer cell lines. At least two types of mechanism have been evidenced. In the presence of estrogens, antiestrogens behave as classical antihormones and their inhibition of cell proliferation is likely due to inhibition of the synthesis and release of several estrogen-induced mitogens (growth factors and proteases). In the absence of estrogens (cells cultured in phenol-red-free medium), antiestrogens can still inhibit the effect of growth factors (EGF, insulin). At concentrations less than or equal to 4 microM, antiestrogens are also cytotoxic and they require accessible estrogen receptors for their action. 'Estrogen-receptor-targeted drugs' is therefore a better general term than 'antiestrogens' to describe the mechanism of action of these drugs, which can also function without inhibiting estrogen action.     

Livin promotes progression of breast cancer through induction of epithelial–mesenchymal transition and activation of AKT signaling

The inhibitor of apoptosis proteins (IAP) are closely correlated with proliferation, apoptosis, motility, and metastasis. Livin is the most recently identified IAP, and its role in breast progression remains unknown. In our study, analyses of 50 patients with breast cancer revealed that the positive expression rate of Livin was higher in breast cancer tissues (62%) relative to that in adjacent (35%) and normal tissues (25%). Livin expression in breast cancer correlated with the clinical stage and axillary lymph node metastasis and could be used as a prognostic marker. Our in vitro experiment revealed that Livin was highly expressed in high-invasive MDA-MB-231 cells as compared to low-invasive cells (MCF-7). Suppression of Livin by short-hairpin RNA reduced the Livin expression of MDA-MB-231 cells and subsequently inhibited tumor cell growth, proliferation, and colony formation and induced tumor cell apoptosis, motility, migration, and invasion. Overexpression of Livin in MCF7 cells resulted in increased migration and invasion capabilities of the cells without affecting proliferation and apoptosis. In addition, epithelial–mesenchymal transition (EMT) was induced by Livin expression in breast cancer cell lines. The high level of phosphorylated AKT in MDA-MB-231 cells was suppressed by Livin knockdown. Further, Livin-induced migration and invasion could be abolished by either the application of the phosphoinositide-3-kinase inhibitor LY294002 or knockdown of AKT expression using small-interfering RNA. In conclusion, Livin serves as an independent prognostic indicator for breast cancer. Livin expression promotes breast cancer metastasis through the activation of AKT signaling and induction of EMT in breast cancer cells both in vitro and in vivo.     

Multihormonal regulation of insulin-like growth factor-I-related protein in MCF-7 human breast cancer cells

MCF-7 human breast cancer cells have been studied for hormonal regulation of secretion of an insulin growth factor-I (IGF-I)-related growth factor. 17 beta-Estradiol, which is required for tumorigenesis of the cell line in the nude mouse and which stimulates proliferation in vitro, was able to significantly induce IGF-I secretion at 10(-13) M, with maximal induction at 10(-11) M. Under optimal conditions IGF-I could be induced 4-fold after 4 days. Demonstration of estrogenic stimulations required removal of phenol red, a weak estrogen, from the cell culture medium. In addition to estrogen, insulin, epidermal growth factor, and transforming growth factor alpha induce both cellular proliferation and IGF-I secretion, while growth inhibitory antiestrogens, transforming growth factor beta, and glucocorticoids have the opposite effect. In each case, modulations in IGF-I secretion preceeded effects on cellular proliferation. IGF-I was not regulated by human GH, basic fibroblast growth factor, platelet-derived growth factor, or PRL, none of which affected proliferation rate. Thus, regulation of IGF-I secretion in human breast cancer is controlled by different hormones from those previously reported in human fibroblasts. Regulation of IGF-I by neither estrogen nor antiestrogen was associated with changes in steady-state mRNA levels; thus regulation may occur at a step beyond mRNA. We conclude that IGF-I production is tightly coupled to growth regulation by estrogens, antiestrogens, and other hormones and may contribute to autocrine and/or paracrine growth regulation by these agents in breast cancer.     

Tumor Suppressor Function of Syk in Human MCF10A In Vitro and Normal Mouse Mammary Epithelium In Vivo

The normal function of Syk in epithelium of the developing or adult breast is not known, however, Syk suppresses tumor growth, invasion, and metastasis in breast cancer cells. Here, we demonstrate that in the mouse mammary gland, loss of one Syk allele profoundly increases proliferation and ductal branching and invasion of epithelial cells through the mammary fat pad during puberty. Mammary carcinomas develop by one year. Syk also suppresses proliferation and invasion in vitro. siRNA or shRNA knockdown of Syk in MCF10A breast epithelial cells dramatically increased proliferation, anchorage independent growth, cellular motility, and invasion, with formation of functional, extracellular matrix-degrading invadopodia. Morphological and gene microarray analysis following Syk knockdown revealed a loss of luminal and differentiated epithelial features with epithelial to mesenchymal transition and a gain in invadopodial cell surface markers CD44, CD49F, and MMP14. These results support the role of Syk in limiting proliferation and invasion of epithelial cells during normal morphogenesis, and emphasize the critical role of Syk as a tumor suppressor for breast cancer. The question of breast cancer risk following systemic anti-Syk therapy is raised since only partial loss of Syk was sufficient to induce mammary carcinomas.     

Oncogenic microRNA-765 promotes the growth and metastasis of breast carcinoma by directly targeting ING4

Previous investigations have proved that microRNA (miR)-765 is significantly overexpressed in multiple tumor types. Nevertheless, the underlying molecular mechanism of miR-765 in mediating breast carcinoma cell growth and metastasis remains unclear. Quantitative real-time polymerase chain reaction was used to determine the levels of miR-765 and inhibitor of growth 4 (ING4) in breast carcinoma tissues and breast carcinoma cells. Cell proliferation, colony formation, wound healing, and Transwell invasion assays were used to analysis the role of miR-765 on breast carcinoma cell growth and aggressiveness. The expressions of ING4 were determined using Western blot analysis and immunohistochemical staining. The direct target of ING4 and miR-765 was confirmed using the luciferase reporter assay. Nude mice were subcutaneously implanted with miR-765 inhibitor transfected MDA-MB-231 cells to determine the potential role of miR-765 in tumor growth in vivo. We observed that miR-765 is overexpressed in breast carcinoma tissue and breast cancer cells. By using luciferase reporter gene bioassay, we find that ING4 is the direct target of miR-765 in breast carcinoma. The level of ING4 is inversely associated with the level of miR-765. The gain-of-function and loss-of-function experiments in vitro indicate that the downregulation of miR-765 suppresses the growth, mobility, and invasion abilities of breast cancer cells by inhibiting ING4. In addition, overexpression of ING4 suppresses the aggressiveness of the MDA-BA-231 cell that is induced by miR-761 in vitro. In this study, we prove that miR-765 regulates the growth and metastasis of breast cancer via modulating miR-765-ING4-negative feedback loop.     

Upregulation of HYAL1 expression in breast cancer promoted tumor cell proliferation, migration, invasion and angiogenesis

Hyaluronic acid (HA) is a component of the Extra-cellular matrix (ECM), it is closely correlated with tumor cell growth, proliferation, metastasis and angiogenesis, etc. Hyaluronidase (HAase) is a HA-degrading endoglycosidase, levels of HAase are elevated in many cancers. Hyaluronidase-1 (HYAL1) is the major tumor-derived HAase. We previously demonstrated that HYAL1 were overexpression in human breast cancer. Breast cancer cells with higher HAase expression, exhibited significantly higher invasion ability through matrigel than those cells with lower HAase expression, and knockdown of HYAL1 expression in breast cancer cells resulted in decreased cell growth, adhesion, invasion and angiogenesis. Here, to further elucidate the function of HYAL1 in breast cancer, we investigated the consequences of forcing HYAL1 expression in breast cancer cells by transfection of expression plasmid. Compared with control, HYAL1 up-regulated cells showed increased the HAase activity, and reduced the expression of HA in vitro. Meantime, upregulation of HYAL1 promoted the cell growth, migration, invasion and angiogenesis in vitro. Moreover, in nude mice model, forcing HYAL1 expression induced breast cancer cell xenograft tumor growth and angiogenesis. Interestingly, the HA expression was upregulated by forcing HYAL1 expression in vivo. These findings suggested that HYAL1-HA system is correlated with the malignant behavior of breast cancer.     

Additive growth-inhibitory effects of DL-alpha-difluoromethylornithine and antiestrogens on MCF-7 breast cancer cell line

We studied the growth inhibitory effects of DL-alpha-difluoromethylornithine, and antiestrogens (tamoxifen, 4-hydroxytamoxifen, trioxifene, keoxifene, and LY117018) as single agents and in combinations on the proliferation of a breast cancer cell line, MCF-7. At 0.1 mM difluoromethylornithine, the proliferation of MCF-7 cells was inhibited to 75 +/- 6% of the controls. Treatment of the cells with 0.1 microM 4-hydroxytamoxifen reduced cell growth to 72 +/- 4%. Combination of 0.1 mM difluoromethylornithine and 0.1 microM 4-hydroxytamoxifen reduced cell growth to 38 +/- 5%, indicating additive growth inhibitory effects. Similar additive effects were observed with all 5 antiestrogens in combination with difluoromethylornithine.     

Regulation of proliferation, invasion and growth factor synthesis in breast cancer by steroids

Endogenous ovarian estrogens and progestins appear to play a critical role in the development and progression of breast cancer. Local productions of growth factors probably also contribute to malignant proliferation, while production and activation of collagenolytic enzymes may be equally critical for local invasive processes. The current review focuses on characterization of growth factor-receptor systems operant in normal and malignant breast epithelium. In addition, the determinants of local invasion are reviewed: attachment, modality, and proteose secretion. Finally, data are discussed concerning the regulation of both proliferation and invasion by hormones and antihormonal agents in hormone-dependent breast cancer. The results suggest new potential pharmacologic targets to explore to suppress onset and progression of breast cancer.     

Ezrin蛋白在乳腺癌细胞迁移侵袭中的作用的研究进展

Ezrin蛋白是ERM(Ezrin;Radixin;Moesin)蛋白家族成员之一。作为酪氨酸激酶的底物,Ezrin蛋白能在细胞膜蛋白与肌动蛋白骨架之间起到桥梁的作用。近年,大量研究表明Ezrin蛋白广泛参与乳腺癌细胞增殖、凋亡、黏附、侵袭转移以及新生血管发生的调节过程。上述过程不仅与Ezrin蛋白自身表达水平、亚细胞定位等改变密切相关,而且受到原发乳腺癌细胞微环境改变的影响,同时涉及胞膜粘附分子(CD44、ICAM、E-cadherin)、EGF、HGF、PDGF、E2及其相应受体所介导的多条信号通路的交叉互动。明确Ezrin蛋白在乳腺癌细胞迁移侵袭过程中的作用及机制,可为乳腺癌的防治提供潜在的药物干预靶点,并为肿瘤转移机制的阐明提供进一步的理论依据。本文就Ezrin蛋白在乳腺癌细胞迁移侵袭过程中的作用作一综述。     

Role of the IGF-I receptor in the regulation of cell-cell adhesion: implications in cancer development and progression

The insulin-like growth factor-I receptor (IGF-IR) is a ubiquitous multifunctional tyrosine kinase that has an important role in normal cell growth and development. However, abnormal stimulation of IGF-IR signaling has been implicated in the development of different types of tumors. The strong antiapoptotic activity of IGF-IR has been recognized as critical in IGF-I-dependent tumorigenesis, however, the impact of other IGF-IR functions, such as regulation of cell-cell and cell-matrix adhesion are also increasingly acknowledged. Here, on the model of breast cancer cells, we discuss how IGF-IR-dependent regulation of intercellular adhesion may affect cell survival, resistance to antiestrogens, and invasion.     

The emerging role of the LIV-1 subfamily of zinc transporters in breast cancer

Zinc transporter LIV-1 (SLC39A6) is estrogen regulated and present in increased amounts in estrogen receptor-positive breast cancer as well as in tumors that spread to the lymph nodes. The LIV-1 subfamily of ZIP zinc transporters consists of nine human sequences that share considerable homology across transmembrane domains. Many of these sequences have been shown to transport zinc and/or other ions across cell membranes. Increasingly, studies have implicated members of the LIV-1 transporter subfamily in a variety of diseases. We review these studies and report our own investigations of the role in breast cancer of the nine LIV-1 zinc transporters. We have documented the response of these transporters to estrogen and antiestrogens, and also their presence in our models of resistance to antiestrogens. Resistance to antiestrogen drugs such as tamoxifen and fulvestrant often occurs in advanced breast cancer. In these models we observed differential expression of individual LIV-1 family members, which may be related to their observed variable tissue expression. We were unable detect ZIP4, which is known to be expressed in the intestine. HKE4/SLC39A7 had elevated expression in both antiestrogen-resistant cell lines, and ZIP8 had elevated expression in fulvestrant-resistant cells. In addition, we investigated the expression of the nine LIV-1 family members in a clinical breast cancer series. Although a number of different LIV-1 family members showed some association with growth factor receptors, LIV-1 was solely associated with estrogen receptor and a variety of growth factors commonly associated with clinical breast cancer. HKE4, however, did show an association with the marker of cell proliferation Ki67 the spread of breast cancer to lymph nodes.     

乳腺癌细胞QSOX1的CRISPR/Cas9基因编辑及其对增殖侵袭的影响研究*

目的:乳腺癌细胞的恶性增殖和易于侵袭转移特性与其对患者的危害直接相关,因此,探究其产生的分子机制,对其有效防治具有重要意义。静息巯基氧化酶-1(QSOX1)是巯基氧化酶家族成员之一,有研究证明其对细胞内蛋白质折叠过程中二硫键形成及细胞外基质的形成发挥重要作用。由于QSOX1在乳腺癌和胰腺癌等多种癌细胞中过表达,将探索QSOX1对乳腺癌细胞过度增殖和侵袭转移方面的可能作用。方法:通过利用CRISPR/Cas9技术构建QSOX1基因敲除和敲入的乳腺癌细胞模型,检测分析QSOX1对乳腺癌细胞MCF-7的分裂增殖、侵袭迁移能力等方面的影响。结果:利用CRISPR/Cas9基因编辑技术成功构建了QSOX1基因敲除和敲入的乳腺癌MCF-7细胞株,其与对照野生型组细胞相比,QSOX1基因敲除株的增殖能力显著下降,癌细胞在体外的迁移和侵袭能力受到明显抑制;而QSOX1基因敲入株的增殖能力和体外迁移侵袭能力却明显有提高。结论:初步揭示了QSOX1在癌症发生与发展中的作用,为进一步阐明其作用的分子机制和设计靶向药物奠定了重要基础。     

Role of TGF-β signaling in the mechanisms of tamoxifen resistance

The transforming growth factor beta (TGF-β) signaling pathway plays complex role in the regulation of cell proliferation, apoptosis and differentiation in breast cancer. TGF-β activation can lead to multiple cellular responses mediating the drug resistance evolution, including the resistance to antiestrogens. Tamoxifen is the most commonly prescribed antiestrogen that functionally involved in regulation of TGF-β activity. In this review, we focus on the role of TGF-β signaling in the mechanisms of tamoxifen resistance, including its interaction with estrogen receptors alfa (ERα) pathway and breast cancer stem cells (BCSCs). We summarize the current reported data regarding TGF-β signaling components as markers of tamoxifen resistance and review current approaches to overcoming tamoxifen resistance based on studies of TGF-β signaling.