Various effects of hepatoma-derived growth factor on cell growth, migration and invasion of breast cancer and prostate cancer cells

Hepatoma-derived growth factor (HDGF) has been implicated in the growth and metastasis of various types of human cancer, but the role of HDGF expression in prostate cancer or breast cancer has not been documented. To assess the role of HDGF in the proliferation, migration and invasion by prostate and breast cancer cells, HDGF expression in DU145 and MCF7 cells was knocked down using siRNA, and the effect of such knockdown was assessed by MTS and [3H]-thymidine incorporation Transwell assays. Moreover, we identified differentially expressed genes that might mediate the HDGF-induced cellular effects. Our results demonstrate that down-regulation of HDGF expression significantly reduces the proliferation of both DU145 and MCF7 cells. However, down-regulation of HDGF expression in DU145 inhibited cell migration and invasion, but in MCF7 cells it stimulated cell migration and invasion. This differential effect might result from the differential induction of PIK3R1 or SERPINE1 in the two cell lines upon HDGF-siRNA treatment. In conclusion, HDGF may participate in the pathogenesis of prostate and breast cancer by promoting cell growth and it may be a therapeutic target for these cancers.     

Expression of bone morphogenetic protein 6 in normal mammary tissue and breast cancer cell lines and its regulation by epidermal growth factor

Bone morphogenetic proteins (BMPs) are multifunctional regulators of proliferation, differentiation and apoptosis. BMP‐6 is involved in numerous developmental processes. We have demonstrated expression of BMP‐6 in breast cancer cell lines by RT‐PCR and immuno‐histochemistry. The level of BMP‐6 mRNA decreased upon serum starvation, whereas epidermal growth factor (EGF) treatment led to elevation of BMP‐6 mRNA levels in a dose‐dependent manner, with a maximum at 50 ng/ml EGF under serum‐free conditions in hormone‐sensitive (MCF‐7) and in hormone‐insensitive (SK‐BR‐3) breast cancer cell lines. The EGF‐like growth factors transforming growth factor‐α, amphiregulin and betacellulin were also able to elevate the BMP‐6 mRNA level after 24 hr. Inhibition of EGF receptor tyrosine kinase with tyrphostine AG1517 repressed the inductive effect of these growth factors, indicating an EGF receptor‐mediated regulation of BMP‐6 mRNA. In addition, BMP‐6 mRNA was detected in tumor samples from breast carcinoma patients. However, levels were reduced in 18/44 samples compared with tumor‐free resection margins. In 12 of these 18 patients, at least a 10‐fold reduction of EGF receptor mRNA levels in tumor samples vs. tumor‐free samples was observed. This suggests a putative relationship between EGF receptor and BMP‐6 mRNA levels in breast cancer. Int. J. Cancer 80:250–256, 1999. © 1999 Wiley‐Liss, Inc.     

miR-340 inhibition of breast cancer cell migration and invasion through targeting of oncoprotein c-Met

BACKGROUND:

Different microRNAs have been shown to have oncogenic and tumor‐suppressive functions in human cancers. Detection of their expression may lead to identifying novel markers for breast cancer.

METHODS:

The authors detected miR‐340 expression in 4 human breast cell lines and then focused on its role in regulation of tumor cell growth, migration, and invasion and target gene expression. They then analyzed miR‐340 expression in benign and cancerous breast tissue specimens.

RESULTS:

Endogenous miR‐340 expression was down‐regulated in the more aggressive breast cancer cell lines, which was confirmed in breast cancer tissue specimens by using quantitative real‐time polymerase chain reaction. Further studies showed that induction of miR‐340 expression was able to suppress tumor cell migration and invasion, whereas knockdown of miR‐340 expression induced breast cancer cell migration and invasion. At the gene level, the authors identified c‐Met as a direct miR‐340 target to mediate cell migration and invasion through regulation of MMP‐2 and MMP‐9 expression. Ex vivo, loss of miR‐340 expression was associated with lymph node metastasis, high tumor histological grade, clinical stage, and shorter overall survival of breast cancer as well as increased c‐Met expression in breast cancer tissue specimens.

CONCLUSIONS:

miR‐340 may play an important role in breast cancer progression, suggesting that miR‐340 should be further evaluated as a novel biomarker for breast cancer metastasis and prognosis, and potentially a therapeutic target. Cancer 2011. © 2011 American Cancer Society.     

乳腺癌组织中血管生成素样蛋白4与骨形成蛋白7的表达及临床意义

目的:研究乳腺癌组织中血管生成素样蛋白4（angiopoietin-like protein 4,ANGPTL4）与骨形成蛋白7（bone morphogenetic protein 7,BMP7）的表达,探讨其临床意义。方法:选取2014年01月至2015年07月我院120例乳腺癌及癌旁正常组织石蜡标本,采用免疫组化检测ANGPTL4和BMP7蛋白表达水平,分析与临床病理参数间的关系。收集32例新鲜乳腺癌及癌旁正常组织,采用Western blot检测ANGPTL4和BMP7蛋白表达水平。结果:石蜡组织中,乳腺癌ANGPTL4蛋白和BMP7蛋白阳性率均高于癌旁正常组织。ANGPTL4蛋白表达水平与肿瘤大小、TNM分期有关（P＜0.05）。BMP7蛋白表达水平与淋巴结转移、TNM分期有关（P＜0.05）。ANGPTL4和BMP7蛋白阳性表达患者5年生存率均低于阴性表达患者（P＜0.05）。ANGPTL4和BMP7蛋白是影响乳腺癌预后的独立危险因素。新鲜组织中,乳腺癌组织ANGPTL4和BMP7蛋白表达量分别为2.55±0.17和3.03±0.16,高于癌旁正常组织的2.42±0.28和2.89±0.12（P＜0.05）。乳腺癌组织中,ANGPTL4和BMP7蛋白表达量呈正相关（P＜0.05）。结论:ANGPTL4与BMP7蛋白可能协同促进乳腺癌的发生发展,是判断乳腺癌预后的重要指标,可能为乳腺癌的治疗提供新的分子靶点。     

Human Sprouty1 suppresses growth,migration, and invasion in human breast cancer cells

Breast cancer is the most common cancer and the leading cause of cancer death in women worldwide. Expression of human Sprouty1 (hSpry1) gene is downregulated in most breast cancer patients, implicating it as an important tumor suppressor gene. So, we hypothesized that overexpression of hSpry1 gene may suppress breast cancer cell growth, migration, and invasion. Here, we demonstrate that in breast cancer cell lines, MDA-MB-231 and T47D, transfection-induced overexpression of hSpry1 reduced cell population, proliferation, and colony formation in vitro without affecting cell apoptosis. Adhesion molecules act as both positive and negative modulators of cellular migration and invasion. Here, we found that overexpression of hSpry1 enhances the initial establishment events in breast cancer cell adhesion to type IV collagen and vitronectin. Moreover, the overexpression of hSpry1 in the highly invasive MDA-MB-231 breast cancer cells causes a significant reduction in cellular migration and invasion through Matrigel membranes. In addition, we showed that hSpry1 overexpression prevents VEGF secretion. VEGF is essential for primary tumor growth, migration, and invasion. Thus, our study provides a novel mechanism of tumor suppression activity of hSpry1.     

Expression patterns of bone morphogenetic protein antagonists in colorectal cancer desmoplastic invasion fronts

Bone morphogenetic proteins (BMPs) are a group of growth factors with dual functions in cancer development and progression. Recently, certain tumor‐promoting roles have been identified for selected antagonists/inhibitors (BMPIs) of this developmental pathway. A recent focus on the implication of BMP in colorectal cancer progression has emerged, mainly due to the presence of inactivating mutations in several members of the canonical signaling cascade. However, the detailed expression profiles of BMPIs remain largely unknown. Based on our previous work, whereby three specific BMPIs, gremlin‐1 (GREM1), high‐temperature requirement A3 (HTRA3) and follistatin (FST) were collectively overexpressed in desmoplastic cocultures of colorectal cancer (CRC), here, we undertook an immunohistochemical approach to describe the patterns of their expression in CRC patients. Two major characteristics described the BMPI expression signature: First, the synchronous and coordinated stromal and epithelial overexpression of individual BMPIs in desmoplastic lesions, which demonstrated that all three of them contribute to increasing levels of BMP antagonism in such areas. Second, the presence of microenvironmental polarity in the BMPI pattern of expression, which was indicated through the preferential expression of HTRA3 in the stromal, and the parallel FST/GREM1 expression in the epithelial component of the investigated sections. In addition, expression of HTRA3 in the epithelial compartment of the tumors demonstrated a significant predictive power to discriminate between tumor‐budding‐bearing and tumor‐budding‐free desmoplastic microenvironments. Together, these findings contribute to the understanding of signaling dynamics of BMP antagonism in the colorectal cancer desmoplastic invasion front.     

Harmine suppresses breast cancer cell migration and invasion by regulating TAZ-mediated epithelial-mesenchymal transition

Breast cancer is a highly lethal disease due to cancer metastasis. Harmine (HM), a β-carboline alkaloid, is present in various medicinal plants. Our previous study demonstrated that HM suppresses cell proliferation and migration by regulating TAZ in breast cancer cells and accelerates apoptosis. Epithelial-mesenchymal transition (EMT) plays an important role in the development of breast cancer by inducing the characteristics of cancer stem cells, cancer metastasis and recurrence. Overexpression of TAZ was shown to mediate EMT in breast cancer cells. We aimed to investigate whether HM inhibits EMT and metastasis of breast cancer cells by targeting TAZ. In this study, the cells treated with HM or with downregulated expression of TAZ showed an increase in epithelial markers and decrease in mesenchymal markers in breast cancer cells. Consistently, the breast cancer cells treated with HM or with downregulated expression of TAZ showed suppressed migration and proliferation. Moreover, TAZ overexpression reversed EMT and metastasis induced by HM in breast cancer cells. Thus, HM suppresses EMT and metastasis and invasion by targeting TAZ in breast cancer cells. HM can be used as an anticancer drug for breast cancer treatment and chemoprevention.     

Oncogenic beta-catenin is required for bone morphogenetic protein 4 expression in human cancer cells

Somatic cell gene targeting was used to create an isogenic set of human colon cancer cells that differs only in the presence or absence of their endogenous activated beta-catenin oncogene. Affymetrix Genechip expression profiling of parental cells and gene-targeted derivatives identified numerous novel genes whose expression was dependent on the presence of oncogenic beta-catenin. The transforming growth factor-beta family member bone morphogenetic protein 4 (BMP4), whose receptor is mutated in a rare inherited gastrointestinal cancer predisposition syndrome, was the most highly differentially expressed gene. Additional experiments revealed that the oncogenic allele of beta-catenin specifically is absolutely required for BMP4 expression and secretion by human cancer cells and that BMP4 is overexpressed and secreted by human colon cancer cells with mutant adenomatous polyposis coli genes. These data identify the presence of regulatory interactions between the Wnt and BMP signaling pathways in cancer pathogenesis, providing an intriguing connection between the sporadic and inherited forms of a common human malignancy.     

Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion

Kruppel-like factor 4 (KLF4) is highly expressed in more than 70% of breast cancers and functions as an oncogene. However, an exact mechanism by which KLF4 enhances tumorigenesis of breast cancer remains unknown. In this study, we show that KLF4 was highly expressed in cancer stem cell (CSC)-enriched populations in mouse primary mammary tumor and breast cancer cell lines. Knockdown of KLF4 in breast cancer cells (MCF-7 and MDA-MB-231) decreased the proportion of stem/progenitor cells as demonstrated by expression of stem cell surface markers such as aldehyde dehydrogenase 1, side population and by in vitro mammosphere assay. Consistently KLF4 overexpression led to an increase of the cancer stem cell population. KLF4 knockdown also suppressed cell migration and invasion in MCF-7 and MDA-MB-231 cells. Furthermore, knockdown of KLF4 reduced colony formation in vitro and inhibited tumorigenesis in immunocompromised non-obese diabetic/severe combined immunodeficiency mice, supporting an oncogenic role for KLF4 in breast cancer development. Further mechanistic studies revealed that the Notch signaling pathway was required for KLF4-mediated cell migration and invasion, but not for CSC maintenance. Taken together, our study provides evidence that KLF4 has a potent oncogenic role in mammary tumorigenesis likely by maintaining stem cell-like features and by promoting cell migration and invasion. Thus, targeting KLF4 may provide an effective therapeutic approach to suppress tumorigenicity in breast cancer.     

三阴性乳腺癌细胞源性外泌体活化的巨噬细胞促进乳腺癌细胞的迁移和侵袭

目的:研究三阴性乳腺癌（triple-negative breast cancer,TNBC）细胞来源的外泌体对巨噬细胞极化作用以及经外泌体活化的巨噬细胞对TNBC细胞迁移和侵袭能力的影响及作用机制。方法:提取TNBC细胞BT-549和MDA-MB-231外泌体,透射电镜、纳米颗粒跟踪分析（nanoparticle tracking analysis,NTA）、蛋白质印记(Western blot)鉴定外泌体;流式细胞术检测经外泌体处理后巨噬细胞分子标志物CD206、CD80的表达;实时荧光定量PCR检测单核细胞THP-1、巨噬细胞M0及外泌体处理后巨噬细胞中CD163、转化生长因子β(transforming growth factor β,TGF-β)、一氧化氮合酶(nitric oxide synthase,iNOS)、肿瘤坏死因子α(tumor necrosis factor α,TNF-α)的mRNA水平;Transwell实验分析外泌体活化的巨噬细胞对TNBC细胞迁移和侵袭的影响,Western blot方法检测MMP-2、MMP-9、N-cadherin、E-cadherin的蛋白水平。结果:透射电镜下BT-549和MDA-MB-231细胞的外泌体具有膜结构囊状小泡并表达外泌体的分子标记物CD81、CD63;BT-549细胞来源的外泌体直径分布为80~150 nm,MDA-MB-231细胞来源的外泌体直径分布为100~200 nm;经BT-549和MDA-MB-231细胞源性外泌体处理的巨噬细胞与M0相比,CD206均有稳定表达（P＜0.05),而CD80表达未见明显差异;外泌体处理后巨噬细胞的CD163、TGF-β mRNA表达水平均增加（P＜0.01),iNOS mRNA表达水平降低（P＜0.05),TNF-α mRNA表达水平未见明显变化;外泌体处理后的巨噬细胞使得TNBC细胞的迁移、侵袭穿膜数目增加(P＜0.01),N-cadherin、MMP-2和MMP-9蛋白表达增加（P＜0.05),E-cadherin 蛋白表达降低（P＜0.05)。结论:TNBC细胞分泌的外泌体可以诱导巨噬细胞向M2极化,进而促进TNBC细胞间质转化,增强TNBC细胞的迁移和侵袭能力。     

Role of Src in breast cancer cell migration and invasion in a breast cell/bone-derived cell microenvironment

The preferential metastasis of breast cancer cells to bone comprises a complex set of events including homing and preferential growth, which may require unique factors produced by bone or other cells in the immediate microenvironment. In this study, an in vitro co-culture system composed of bone mesenchymal stem cells and breast cancer cell lines is used to examine the role of Src kinase on breast cancer cell migration and invasion in the presence of bone-derived cells. This research shows that Src kinase activity in breast cancer cell lines with either high or low levels of endogenous Src activity is increased by bone-derived cell-conditioned medium but not HS68 fibroblast-conditioned medium. Breast cancer cells exhibit enhanced migration in co-culture with bone-derived cells but not HS68 fibroblasts or no co-cultured cells. Inhibition of Src kinase activity using the inhibitors PP2 or saracatinib or using siRNA abrogates the preferential migration of the breast cancer cell lines in response to bone-derived cells. Inhibition of Src activity with saracatinib does not have any significant effect on breast cancer cell invasion in the presence of bone-derived cells. Factors are identified that are produced preferentially by bone-derived cells over HS68 cells that may impact breast cancer cell behavior. This research implicates Src kinase as an important effector of bone-derived cell signals on breast cancer cell migration.     

Dasatinib synergizes with doxorubicin to block growth, migration, and invasion of breast cancer cells

Background:

Src family kinases control multiple cancer cell properties including cell cycle progression, survival, and metastasis. Recent studies suggest that the Src inhibitor dasatinib blocks these critical cancer cell functions.

Methods:

Because the molecular mechanism of action of dasatinib in breast cancers has not been investigated, we evaluated the effects of dasatinib as a single agent and in combination with the commonly used chemotherapeutic doxorubicin, on the proliferation, viability, and invasive capacity of breast cancer cells lines earlier categorised as dasatinib-sensitive (MDA-MB-231) and moderately resistant (MCF7 and T47D). We also tested the effects of these drugs on the actin cytoskeleton and associated signalling pathways.

Results:

The cell lines tested varied widely in sensitivity to growth inhibition (IC₅₀=0.16–12.3 μM), despite comparable Src kinase inhibition by dasatinib (IC₅₀=17–37 nM). In the most sensitive cell line, MDA-MB-231, dasatinib treatment induced significant G₁ accumulation with little apoptosis, disrupted cellular morphology, blocked migration, inhibited invasion through Matrigel (P<0.01), and blocked the formation of invadopodia (P<0.001). Importantly, combination treatment with doxorubicin resulted in synergistic growth inhibition in all cell lines and blocked the migration and invasion of the highly metastatic, triple-negative MDA-MB-231 cell line.

Conclusion:

The observed synergy between dasatinib and doxorubicin warrants the re-evaluation of dasatinib as an effective agent in multi-drug regimens for the treatment of invasive breast cancers.     

miR-21通过靶向PDCD4调控三阴性乳腺癌细胞的迁移和侵袭

目的： 研究miR-21在三阴性乳腺癌细胞MDA-MB-231中的表达,以及其是否通过调控PDCD4影响MDA-MB-231细胞的迁移和侵袭。方法： 采用实时定量PCR（qPCR）法检测MDA-MB-231细胞和正常乳腺细胞MCF-10A中miR-21和PDCD4 mRNA的表达。将MDA-MB-231细胞随机分为5组：空白对照组,转染miR-21模拟物组,模拟物对照组,转染miR-21抑制物组和抑制物对照组。采用Western blot法检测MDA-MB-231细胞PDCD4蛋白的表达;采用荧光素酶报告基因试剂盒检测转染不同载体后荧光强度的变化来判断miR-21的靶标;采用Transwell实验检测各组细胞的迁移和侵袭数目。结果： miR-21和PDCD4 mRNA在MDAMB-231细胞中的表达水平分别明显高于和低于MCF-10A细胞（P均 < 0.01）。过表达或抑制miR-21可调节PDCD4的表达水平。荧光素酶报告基因试剂盒检测结果显示miR-21可直接靶向调控PDCD4的表达。Transwell实验结果表明过表达miR-21表达能增强MDA-MB-231细胞的迁移和侵袭能力。结论： 在MDA-MB-231细胞中,miR-21通过靶向调控PDCD4表达影响细胞的迁移和侵袭。miR-21可能成为抑制三阴性乳腺癌迁移和侵袭的靶点。