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.     

S100A4 regulates cell motility and invasion in an in vitro model for breast cancer metastasis

Elevated levels of the calcium-binding protein S100A4 are associated with poor patient survival in breast cancer patients and induce metastasis in rodent models. To investigate the effects of S100A4 on different components of the metastatic process, epithelial cells lines have been isolated from nonmalignant tumours in neu transgenic mice and from malignant tumours in neu/S100A4 double transgenic mice. Additional cell lines expressing both Neu and S100A4 have also been derived by transfection of rat S100A4 cDNA into tumour cell lines cloned from neu single transgenic mice. Using these cells in transfilter migration assays, it has been shown that increases in either motility or invasive properties correlate with each other and with the level of S100A4 protein. Injection of three of the cell lines separately into the mammary fat pads of nude mice showed that elevated levels of S100A4 correlated with the degree of metastasis to the lungs. In contrast, changes in cell proliferation and cell-substrate adhesion did not correlate with S100A4 levels. Neither motility nor invasiveness correlated with proteolytic degradation of gelatin as measured by zymography. Thus, the results suggest that the main effect of increases in S100A4 levels in metastasis is to generate increased cell motility and invasion and that this latter change is not dependent upon an increased ability to degrade the intercellular matrix.     

Leptin promotes motility and invasiveness in human colon cancer cells by activating multiple signal-transduction pathways

Leptin serum levels are about 5 times higher in obese people than in normal individuals. We aimed at investigating the signaling pathways induced by leptin in the human colonic cell lines LS174T and HM7. Both cells expressed the leptin transmembrane Ob-receptor. Leptin activated the mitogen-activated protein kinase pathway, induced invasion of colonic cells and concomitantly increased the formation of lamellipodial structures. A direct and novel dose- and time-dependent activation of RhoA, Cdc42 and Rac1 by leptin is demonstrated in these aggressive colon cancer cells. The activation of the Rho family of GTPases was amenable to specific inhibition: Wortmannin inhibited leptin-induced Rac1 and Cdc42 activation but did not affect RhoA activation, and inhibited the formation of leptin-induced lamellipodia and cell invasion. The Rac1 inhibitor NSC23766 inhibited only leptin-induced Rac1 activation and concomitantly, lamellipodium formation and cell invasion. The Src kinase inhibitor II (SrcKI-II) exerted a positive effect on RhoA activation, inhibited tyrosine phosphorylation of p190RhoGAP and inhibited leptin-induced Cdc42 activation and leptin-induced lamellopodium formation and cell invasion. The specific JAK2 inhibitor AG490 exerted a positive effect on Rac1 and Cdc42 activation by leptin and concomitantly inhibited RhoA activation. AG490 did not inhibit leptin-induced lamellopodium formation or cell invasion. Our findings clearly indicate that leptin activates PI3K and Src kinase pathways in the metastatic colon cancer cells LS174T and HM7. These signaling pathways induce the activation of Rac1 and Cdc42, lamellopodium formation and concomitantly enhanced cell invasion, but leptin activation of RhoA is not associated with enhanced cell locomotion and invasion. Understanding in-depth the pathways involved in leptin-associated enhanced cell locomotion and invasion may contribute with the design of novel therapeutics to treat obesity-associated advanced colorectal cancer.     

Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion

Inflammatory breast cancer (IBC) is an aggressive form of primary breast cancer characterized by rapid onset and high risk of metastasis and poor clinical outcomes. The biological basis for the aggressiveness of IBC is still not well understood and no IBC‐specific targeted therapies exist. In this study, we report that lipocalin 2 (LCN2), a small secreted glycoprotein belonging to the lipocalin superfamily, is expressed at significantly higher levels in IBC vs non‐IBC tumors, independently of molecular subtype. LCN2 levels were also significantly higher in IBC cell lines and in their culture media than in non‐IBC cell lines. High expression was associated with poor‐prognosis features and shorter overall survival in IBC patients. Depletion of LCN2 in IBC cell lines reduced colony formation, migration, and cancer stem cell populations in vitro and inhibited tumor growth, skin invasion, and brain metastasis in mouse models of IBC. Analysis of our proteomics data showed reduced expression of proteins involved in cell cycle and DNA repair in LCN2‐silenced IBC cells. Our findings support that LCN2 promotes IBC tumor aggressiveness and offer a new potential therapeutic target for IBC.     

Src激酶抑制剂ZD6474在乳腺癌MCF-7细胞转移中的作用和机制

目的:探讨Src激酶抑制剂ZD6474在乳腺癌MCF-7细胞转移中的作用和机制。方法:乳腺癌MCF-7细胞经ZD6474处理后,检测肿瘤细胞体外黏附、侵袭能力的改变,应用Western blot及Real-time PCR观察细胞Src激酶磷酸化水平及E-钙黏素和β-连环蛋白表达的变化,报告基因技术检测Snail在转录水平表达的变化。结果:ZD6474可抑制MCF-7细胞中Src激酶活性,在ZD6474浓度为1×10-5mol/L和1×10-4mol/L时,细胞的抑制率分别为12.2%和27.5%。Src酪氨酸激酶抑制剂可上调E-cadherin在mRNA和蛋白表达水平,下调β-catenin在mRNA和蛋白表达水平及Snail启动子活性。结论:Src激酶与乳腺癌MCF-7细胞肿瘤转移能力密切相关,阻断Src激酶活性可抑制肿瘤细胞转移能力。     

Molecular and cellular analysis of basement membrane invasion by human breast cancer cells in Matrigel-basedin vitro assays

Summary In vitro analyses of basement membrane invasiveness employing Matrigel (a murine tumor extract rich in basement membrane components) have been performed on human breast cancer model systems. Constitutive invasiveness of different human breast cancer (HBC) cell lines has been examined as well as regulation by steroid hormones, growth factors, and oncogenes. Carcinoma cells exhibiting a mesenchymal-like phenotype (vimentin expression, lack of cell border associated uvomorulin) show dramatically increased motility, invasiveness, and metastatic potential in nude mice. These findings support the hypothesis that epithelial to mesenchymal transition (EMT)-like events may be instrumental in the metastatic progression of human breast cancer. The MCF-7 subline MCF-7_ADR appears to have undergone such a transition. The importance of such a transition may be reflected in the emergence of vimentin expression as an indicator of poor prognosis in HBC. Matrix degradation and laminin recognition are highlighted as potential targets for antimetastatic therapy, and analyses of laminin attachment and the matrix metalloproteinase (MMP) family in HBC cell lines are summarized. Matrigel-based assays have proved useful in the study of the molecular mechanisms of basement membrane invasiveness, their regulation in HBC cells, and their potential as targets for antimetastatic therapy.     

环加氧酶-2 通过调控EMT促进乳腺癌MDA-MB-231 细胞的迁移和侵袭

[摘要] 目的：探讨环加氧酶-2（COX-2）在乳腺癌转移中的作用及其可能的机制。方法：收集从2015 年10 月至2018 年4 月在云南省肿瘤医院接受乳腺切除术的患者中获得的原发乳腺癌组织和脑转移乳腺癌组织临床病理样本共45 例,其中原发30 例、脑转移15 例。采用qPCR检测COX-2 在原位乳腺癌和脑转移乳腺癌组织中的表达。将COX-2 过表达重组病毒（LV6-COX2）或敲减COX-2 重组病毒（LV3-COX2 shRNA1、LV3-COX2 shRNA2）感染人乳腺癌MDA-MB-231 细胞并获得稳转细胞株后,CCK-8法检测COX-2 表达对MDA-MB-231 细胞增殖的影响,划痕实验和Transwell 法检测对MDA-MB-231 细胞迁移和侵袭的影响。qPCR和WB实验分析各组细胞中COX-2 mRNA和蛋白的表达水平,qPCR检测COX-2 表达对MDA-MB-231 细胞内EMT相关基因表达的影响。结果：COX-2 表达水平在脑转移乳腺癌患者组织中显著高于原位乳腺癌组织（P<0.01）;并且与乳腺癌患者肿瘤TMN分期有关。成功构建稳定过表达/敲减COX-2 的MDA-MB-231 细胞株。过表达COX-2 促进MDA-MB-231 细胞的迁移和侵袭（均P<0.01）,同时显著提高MMP2、MMP1、N-cadherin 和vimentin 的表达（均P<0.01）,但对细胞增殖无明显影响;而沉默COX-2 则有相反的作用,且可促进细胞增殖（P<0.05）。结论：COX-2 在脑转移乳腺癌组织中高表达,其可能通过调控EMT过程促进乳腺癌MDA-MB-231 细胞的迁移和侵袭。     

Recombinant anthrax lethal toxin inhibits cell motility and invasion in breast cancer cells through the dysregulation of Rho GTPases

Breast cancer is the leading cause of cancer-associated death among women worldwide. Targeting breast cancer cell metastasis is an important therapeutic approach. The MAPK pathway is a key cell signaling pathway that plays a pivotal role in cellular invasion and migration. Numerous studies have identified the MAPK pathway as a way to target cell survival and motility. The present study treated MBA-MD-231 breast cancer cells with anthrax lethal toxin (LeTx), a potent MAPK inhibitor that selectively cleaves and inactivates all MEKs, as a potential therapeutic method to inhibit breast cancer cell migration. LeTx has been demonstrated to affect breast cancer cell migration. Cells treated with LeTx showed a significant decrease in motility, as observed using wound healing and random 2D motility assays. Additionally, cells treated with LeTx showed an increase in adhesion, which would explain the decrease in migration. Pull-down assays examining the activation status of the members of the Rho family of GTPases revealed an increase in RhoA activation accompanied by a decrease in Cdc42 activation following LeTx treatment. Finally, LeTx mediated a decrease in invasion using a Boyden chamber assay, which could be a result of the decrease in Cdc42 activation. The present study reported the effect of LeTx treatment on the migration, adhesion and invasion of breast cancer cells, demonstrating that this effect was associated with the dysregulation of the Rho GTPases, RhoA and Cdc42.     

Cyr61 as mediator of Src signaling in triple negative breast cancer cells

SFKs are involved in tumorigenesis and metastasis. Here we analyzed c-Src contribution to initial steps of metastasis by tetracycline-dependent expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in metastatic MDA-MB-231 cells. c-Src suppression did not alter cell proliferation or survival, but it significantly reduced anchorage-independent growth. Concomitantly with diminished tyrosine-phosphorylation/activation of Fak, caveolin-1, paxillin and p130CAS, c-Src depletion also inhibited cellular migration, invasion and transendothelial migration. Quantitative proteomic analyses of the secretome showed that Cyr61 levels, which were detected in the exosomal fraction, were diminished upon shRNA-c-Src expression. In contrast, Cyr61 expression was unaltered inside cells. Cyr61 partially colocalized with cis-Golgi gp74 marker and with exosomal marker CD63, but c-Src depletion did not alter their cellular distribution. In SUM159PT cells, transient c-Src suppression also reduced secreted exosomal Cyr61 levels. Furthermore, conditional expression of a c-Src dominant negative mutant (SrcDN, c-Src-K295M/Y527F) in MDA-MB-231 and in SUM159PT diminished secreted Cyr61 as well. Cyr61 transient suppression in MDA-MB-231 inhibited invasion and transendothelial migration. Finally, in both MDA-MB-231 and SUM159PT, a neutralizing Cyr61 antibody restrained migration. Collectively, these results suggest that c-Src regulates secreted proteins, including the exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells.     

PAK5通过上皮-间质转化促进人乳腺癌细胞侵袭

目的:探讨p21活化激酶-5（p21-activated kinase 5,PAK5）蛋白对乳腺肿瘤细胞侵袭转移的作用机制。方法:观察PAK5过表达对乳腺癌细胞形态的影响,人乳腺肿瘤细胞MCF-7通过慢病毒感染稳定表达Flag-PAK5。然后提取感染细胞的总蛋白进行蛋白免疫印迹检测上皮-间质转化（EMT）标志物上皮-钙黏蛋白（E-cadherin）,纤维连接蛋白（Fibronectin）和波形蛋白（vimentin）的表达。最后通过Transwell实验检测过表达PAK5对乳腺癌细胞迁移和侵袭的影响。结果:过表达PAK5蛋白能够使细胞形态由鹅卵石样像梭形变化,下调E-cadherin蛋白,促进乳腺癌细胞迁移和侵袭。结论:PAK5可能参与调节乳腺肿瘤细胞发生上皮－间质转化,促进乳腺肿瘤侵袭转移的发生。     

Prostaglandin F(2alpha) stimulates motility and invasion in colorectal tumor cells

Increased expression of cyclooxygenase-2 (COX-2) and subsequent prostaglandin production is an important event in several human malignancies, including colorectal cancer. COX-2 mediated prostanoid synthesis has been shown to play a key role in tumor progression with prostaglandin E(2) (PGE(2)) being shown to promote tumor growth, invasion and angiogenesis. The role of the other prostaglandins produced by COX-2 in tumors remains poorly understood. We have shown that colorectal tumor cells produce prostaglandin F(2alpha) (PGF(2alpha)) and provide evidence that PGF(2alpha) may play an important role in colorectal tumorigenesis. Our data show that PGF(2alpha) is secreted by both colorectal adenoma and carcinoma-derived cell lines at levels in excess of those detected for PGE(2). These cell lines were also found to express the PGF(2alpha) receptor (FP) indicating potential autocrine effects of PGF(2alpha). This finding is further supported by an in vivo immunohistochemical study of FP expression in resected colon tissue. These data show epithelial expression of FP in normal colorectal mucosa and also in colorectal adenomas and carcinomas. We compared the relative abilities of PGF(2alpha) and PGE(2) to induce cell motility in vitro in colorectal tumor cell lines and show the first evidence of prostaglandin-induced cell motility in colorectal adenoma cell lines. PGF(2alpha) induced cell motility with equivalent potency to PGE(2) in all the cell lines tested and was also shown to increase the invasion of carcinoma-derived cells into reconstituted basement membrane. These data show that PGF(2alpha) may play an important role in the malignant progression of colorectal tumors.     

Dual targeting of Src and ER prevents acquired antihormone resistance in breast cancer cells

Acquired resistance to endocrine therapies presents a major obstacle to the successful treatment of breast cancer patients. Previously, we have shown that acquisition of resistance to tamoxifen in breast cancer cells is accompanied by an elevation in Src kinase activity which promotes an aggressive, invasive phenotype in vitro. Here, we have explored the potential therapeutic effects of combining Src inhibition with anti-oestrogen treatment on the development of endocrine insensitivity in breast cancer cells. Treatment of MCF7 and T47D cells with tamoxifen alone resulted in an initial growth inhibitory phase followed by the eventual development of tamoxifen resistance together with an elevation of Src kinase activity, which was central to their increased invasive capacity. Chronic exposure of both cell types to the Src inhibitor, AZD0530, as a monotherapy resulted in outgrowth of AZD0530-resistant cells, in which Src kinase activity remained suppressed as did their in vitro invasive nature. Treatment of both MCF7 and T47D cells with AZD0530 in combination with tamoxifen resulted in a reduction of Src activity together with inhibition of focal adhesion kinase phosphorylation and a complete abrogation of their in vitro invasive behaviour. Furthermore, combination therapy significantly suppressed expression of cyclinD1 and c-myc and prevented cell proliferation and the subsequent emergence of a resistant phenotype, with total cell loss occurring by 12 weeks. These data demonstrate that pharmacological targeting of Src kinase, in conjunction with antihormone therapies, effectively prevents antihormone resistance in breast cancer cells in vitro and suggests a potential novel therapeutic benefit of Src kinase inhibitors clinically.     

High ID2 protein expression correlates with a favourable prognosis in patients with primary breast cancer and reduces cellular invasiveness of breast cancer cells

ID proteins have been implicated in the regulation of cell proliferation and differentiation in various cell types during normal development as well as in the formation of cancer. Our aim was to delineate the expression of ID2 by immunohistochemistry in primary breast cancer in order to detect potential associations with cell cycle regulatory proteins and/or clinicopathologic parameters. We further overexpressed ID2 in a breast cancer cell line to elaborate potential effects on proliferation and invasiveness. We observed large variations in ID2 expression in primary breast cancer, and the protein was localised to both the nucleus and cytoplasm. Interestingly, a high cytoplasmic ID2 protein level correlated with a favourable prognosis. Overexpressing ID2 in the MDA-MB-468 breast cancer cell line generated a marked cytoplasmic localisation of the protein and reduced the invasive capacity of cells. Modest enhancement of cell proliferation was further detected in ID2-overexpressing cells. In conclusion, ID2 protein expression varies substantially within primary breast tumours and high cytoplasmic levels of ID2 might reflect a less aggressive breast tumour phenotype.     

CXCL12 promotes CCR7 ligand–mediated breast cancer cell invasion and migration toward lymphatic vessels

Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. Although there is emerging evidence that multiple chemokines are expressed in tumor tissues and that each chemokine induces receptor‐mediated signaling, their collaboration to regulate tumor invasion and lymph node metastasis has not been fully elucidated. In this study, we examined the effect of CXCL12 on the CCR7‐dependent signaling in MDA‐MB‐231 human breast cancer cells to determine the role of CXCL12 and CCR7 ligand chemokines in breast cancer metastasis to lymph nodes. CXCL12 enhanced the CCR7‐dependent in vitro chemotaxis and cell invasion into collagen gels at suboptimal concentrations of CCL21. CXCL12 promoted CCR7 homodimer formation, ligand binding, CCR7 accumulation into membrane ruffles, and cell response at lower concentrations of CCL19. Immunohistochemistry of MDA‐MB‐231–derived xenograft tumors revealed that CXCL12 is primarily located in the pericellular matrix surrounding tumor cells, whereas the CCR7 ligand, CCL21, mainly associates with LYVE‐1⁺ intratumoral and peritumoral lymphatic vessels. In the three‐dimensional tumor invasion model with lymph networks, CXCL12 stimulation facilitates breast cancer cell migration to CCL21‐reconstituted lymphatic networks. These results indicate that CXCL12/CXCR4 signaling promotes breast cancer cell migration and invasion toward CCR7 ligand–expressing intratumoral lymphatic vessels and supports CCR7 signaling associated with lymph node metastasis.     

人脂联素重组体体外对乳腺癌细胞侵袭和转移的抑制

目的：研究人脂联素重组体对人乳腺癌MDA—MB-231细胞体外侵袭能力的影响。方法：将MDA—MB-231细胞分设对照组和脂联素重组体加药（2．5、5、0、10、20、30μg／ml）组，药物作用一定时间后，分别以MTT法、Transwell小室实验，Chamber小室实验，黏附实验检测脂联素重组体对乳腺癌细胞增殖、侵袭、迁移和黏附能力的影响，以明胶酶谱法检测其对肿瘤细胞分泌MMP-2和MMP-9的影响。结果：脂联素重组体质量浓度高于5．0μg／ml时，对MDA—MB-231细胞生长具有明显的抑制作用（P〈0．01）；能明显地降低肿瘤细胞体外侵袭能力（P〈0．01），侵袭抑制率随脂联素重组体质量浓度的升高而增加，介于22．64％～77．84％之间；脂联素重组体质量浓度高于2．5μg／ml时，明显地抑制肿瘤细胞的迁移能力（P〈0．01）；脂联素重组体对ECM和FN黏附能力影响的程度不同，对FN的敏感性大于ECM；脂联素重组体质量浓度大于2．5μg／ml时，明显抑制肿瘤细胞MMP-2和MMP-9的分泌（P〈0、05或P〈0．01），后者的分泌量随质量浓度的升高而下降。结论：脂联素重组体在大于2．5μg／ml时，可能通过保护基底膜不受破坏而抑制人乳腺癌细胞的侵袭能力。