Endogenous osteonectin/SPARC/BM-40 expression inhibits MDA-MB-231 breast cancer cell metastasis

Skeletal metastases occur with high incidence in patients with breast cancer and cause long-term skeletal morbidity. Osteonectin (SPARC, BM-40) is a bone matrix factor that is an in vitro chemoattractant for breast and prostate cancer cells. Increased expression of osteonectin is found in malignant breast tumors. We infected MDA-231 breast cancer cells with an adenovirus expressing osteonectin to examine the role of osteonectin expression in breast cancer cells and its effect on metastasis, in particular to bone. Expression of osteonectin did not affect MDA-231 cell proliferation, apoptosis, migration, cell aggregation, or protease cleavage of collagen IV. However, in vitro invasion of these osteonectin-infected cells through Matrigel and colony formation on Matrigel was decreased. Interestingly, high osteonectin expression in MDA-231 cells inhibited metastasis in a dose-dependent manner to many different organs including bone. The reduction in metastasis may be due to decreased platelet-tumor cell aggregation, because exogenous osteonectin inhibited platelet aggregation in vitro and the high osteonectin expression in MDA-231 cells reduced tumor cell-induced thrombocytopenia in vivo compared with control-infected cells. These studies suggest that high endogenous expression of osteonectin in breast cancer cells may reduce metastasis via reduced invasive activity and reduced tumor cell-platelet aggregation.     

Knockdown of contactin-1 expression suppresses invasion and metastasis of lung adenocarcinoma

Numerous genetic changes are associated with cancer cell metastasis and invasion. In search for key regulators of invasion and metastasis, a panel of lung cancer cell lines with different invasive ability was screened. The gene for contactin-1 was found to play an essential role in tumor invasion and metastasis. Suppression of contactin-1 expression abolished the ability of lung adenocarcinoma cells to invade Matrigel in vitro as well as the polymerization of filamentous-actin and the formation of focal adhesion structures. Furthermore, knockdown of contactin-1 resulted in extensive inhibition of tumor metastasis and in increased survival in an animal model. RhoA but not Cdc42 or Rac1 was found to serve a critical role in contactin-1-mediated invasion and metastasis. Contactin-1-specific RNA interference resulted in loss of metastatic and invasive capacity in both in vitro and in vivo models. This loss was overturned by constitutive expression of the active form of RhoA. Contactin-1 was differentially expressed in tumor tissues, and its expression correlated with tumor stage, lymph node metastasis, and patient survival. Contactin-1 is proposed to function importantly in the invasion and metastasis of lung adenocarcinoma cells via RhoA-mediated mechanisms.     

Gab2通过GSK-3β/Snail信号通路促进乳腺癌的上皮-间质转化

背景与目的：越来越多的证据显示,Grb2协同结合蛋白2(Grb2 binding protein-2,Gab2)与肿瘤的侵袭转移相关,但Gab2与乳腺癌上皮-间质转化(epithelial-mesenchymal transition,EMT)的关系尚不清楚。本研究旨在探讨Gab2对乳腺癌EMT标志物的影响,明确Gab2在乳腺癌侵袭和转移中的作用机制。方法：采用免疫组织化学染色法检测80例乳腺癌组织中Gab2及EMT标记物上皮性钙黏着蛋白(E-cadherin)、波形蛋白(vimentin)的表达情况并分析其相关性,用蛋白[质]印迹法(Western blot)检测乳腺组织Gab2的表达情况,采用小干扰RNA(siRNA)技术降低乳腺癌细胞系MDA-MB-231中Gab2的表达,采用划痕实验检测表皮生长因子(epithelial growth factor,EGF)刺激后转染细胞的侵袭能力变化,用Western blot检测敲低Gab2后MDA-MB-231细胞中E-cadherin及vimentin的表达情况,同时检测p-GSK-3β的表达情况、转录因子Snail转核情况。结果：Gab2在乳腺癌组织中的表达与E-cadherin的表达呈负相关,而与vimentin的表达呈正相关(P<0.05);乳腺癌组织中Gab2的表达量明显高于正常乳腺组织;siRNA质粒转染后,SiGab2/MDA-MB-231细胞组中Gab2蛋白的表达量明显降低,结果显示转染成功,划痕实验显示细胞的侵袭能力减弱,表明Gab2影响乳腺癌细胞系的侵袭能力;敲低Gab2后,MDA-MB-231细胞中的E-cadherin的表达明显升高,而vimentin的表达明显降低;GSK-3β的磷酸化受到抑制,而Snail在敲低Gab2的细胞核中的表达明显下调。结论：Gab2可以通过GSK-3β/Snail信号通路促进乳腺癌的EMT,从而促进乳腺癌的侵袭和转移。     

Heparanase promotes bone destruction and invasiveness in prostate cancer

Heparanase is an endoglycosidase that plays an important role in angiogenesis and metastasis of cancer. Herein we evaluate the effect of heparanase overexpression on invasiveness and bone destruction in prostate cancer bone metastases. The human prostate cancer cell line PC-3 was stably transfected with a plasmid containing the cDNA for human heparanase or with the vector alone as a control. Overexpression of heparanase did not affect the growth of PC-3 cells, but did promote invasiveness of the cells in an in vitro assay. Both cell types were injected into the tibias of nude mice. Four weeks later, the mice were examined radiologically prior to sacrifice and samples of leg tissue were taken to investigate bone destruction and metastasis. Mice injected with PC-3 cells overexpressing heparanase had more severe bone destruction and larger, more invasive, tumors. These results demonstrate that heparanase overexpression can facilitate tumor invasion and accelerate bone destruction caused by prostate cancer bone metastasis.     

A secreted isoform of ErbB3 promotes osteonectin expression in bone and enhances the invasiveness of prostate cancer cells

The propensity for prostate cancer to metastasize to bone led us and others to propose that bidirectional interactions between prostate cancer cells and bone are critical for the preferential metastasis of prostate cancer to bone. We identified previously a secreted isoform of ErbB3 (p45-sErbB3) in bone marrow supernatant samples from men with prostate cancer and bone metastasis and showed by immunohistochemical analysis of human tissue specimens that p45-sErbB3 was highly expressed in metastatic prostate cancer cells in bone. Here, we show that p45-sErbB3 stimulated mouse calvaria to secrete factors that increased the invasiveness of prostate cancer cells in a Boyden chamber invasion assay. Using gene array analysis to identify p45-sErbB3-responsive genes, we found that p45-sErbB3 up-regulated the expression of osteonectin/SPARC, biglycan, and type I collagen in calvaria. We further show that recombinant osteonectin increased the invasiveness of PC-3 cells, whereas osteonectin-neutralizing antibodies blocked this p45-sErbB3-induced invasiveness. These results indicate that p45-sErbB3 enhances the invasiveness of PC-3 cells in part by stimulating the secretion of osteonectin by bone. Thus, p45-sErbB3 may mediate the bidirectional interactions between prostate cancer cells and bone via osteonectin.     

Bone morphogenetic protein-6 promotes osteoblastic prostate cancer bone metastases through a dual mechanism

Prostate cancer frequently metastasizes to bone where it forms osteoblastic lesions through unknown mechanisms. Bone morphogenetic proteins (BMP) are mediators of skeletal formation. Prostate cancer produces a variety of BMPs, including BMP-6. We tested the hypothesis that BMP-6 contributes to prostate cancer-induced osteosclerosis at bone metastatic sites. Prostate cancer cells and clinical tissues produced BMP-6 that increased with aggressiveness of the tumor. Prostate cancer-conditioned medium induced SMAD phosphorylation in the preosteoblast MC3T3 cells, and phosphorylation was diminished by anti-BMP-6 antibody. Prostate cancer-conditioned medium induced mineralization of MC3T3 cells, which was blocked by both the BMP inhibitor noggin and anti-BMP-6. Human fetal bones were implanted in severe combined immunodeficient mice and after 4 weeks, LuCaP 23.1 prostate cancer cells were injected both s.c. and into the bone implants. Anti-BMP-6 or isotype antibody administration was then initiated. Anti-BMP-6 reduced LuCaP 23.1-induced osteoblastic activity, but had no effect on its osteolytic activity. This was associated with increased osteoblast numbers and osteoblast activity based on bone histomorphometric evaluation. As endothelin-1 has been implicated in bone metastases, we measured serum endothelin-1 levels but found they were not different among the treatment groups. In addition to decreased bone production, anti-BMP-6 reduced intraosseous, but not s.c., tumor size. We found that BMP-2, BMP-4, BMP-6, and BMP-7 had no direct effect on prostate cancer cell growth, but BMP-2 and BMP-6 increased the in vitro invasive ability of prostate cancer cell. These data show that prostate cancer promotes osteoblastic activity through BMP-6 and that, in addition to its bone effects, suggest that BMPs promote the ability of the prostate cancer cells to invade the bone microenvironment.     

Prostate-derived ETS factor is a mediator of metastatic potential through the inhibition of migration and invasion in breast cancer

Cell migration and invasion are critical events during the progression to metastasis. Without motile function, cancer cells are unable to leave the primary tumor site, invade through the basement membrane, and form secondary tumors. Expression of the epithelial-specific ETS factor prostate-derived ETS factor (PDEF) is reduced in human invasive breast tissue and lost in invasive breast cancer cell lines. Gain-of-function studies that examine different aspects of cell migration show that constitutive or inducible PDEF reexpression inhibits migration and invasion in multiple breast cancer cell lines, and loss-of-function studies show a stimulation of migration in noninvasive breast cancer cells. Furthermore, the introduction of PDEF into invasive breast cancer cells led to a remodeling of the actin cytoskeleton and altered focal adhesion localization and adherence levels. Cells expressing PDEF no longer form the defined morphologic polarity required for efficient, directional migration. Collectively, these data indicate that PDEF down-regulation in invasive breast cancer may promote actin-mediated cell migration through the extracellular matrix.     

Matrix metalloproteinase-1 promotes breast cancer angiogenesis and osteolysis in a novel in vivo model

Matrix metalloproteinase-1 (MMP-1) is critical for mediating breast cancer metastasis to bone. We investigated the role of MMP-1 in breast cancer invasion of soft tissues and bone using human MDA MB-231 breast cancer cells stably transfected with shRNAs against MMP-1 and a novel murine model of bone invasion. MMP-1 produced by breast cancer cells with control shRNA facilitated invasion of tumors into soft tissue in vivo, which correlated with enhanced blood vessel formation at the invasive edge, compared to tumors with silenced MMP-1 expression. Tumors expressing MMP-1 were also associated with osteolysis in vivo, whereas tumors with inhibited MMP-1 levels were not. Additionally, tumor-secreted MMP-1 activated bone-resorbing osteoclasts in vitro. Together, these data suggest a mechanism for MMP-1 in the activation of osteoclasts in vivo. We conclude that breast cancer-derived MMP-1 mediates invasion through soft tissues and bone via mechanisms involving matrix degradation, angiogenesis, and osteoclast activation.     

Invadopodia formation by bladder tumor cells

A major cause of death in patients with bladder tumors is recurrence with metastasis. Bladder tumor metastasis is largely dependent upon the invasive capacity of tumor cells. Tumor cell invasion is mainly mediated by actin-rich protrusive membrane structures called invadopodia. The formation of invadopodia was observed in various types of invasive tumors such as breast cancer and melanomas. However, invadopodia formation so far has not been described in bladder tumor cells. We here report that human bladder tumor cells form functionally active invadopodia. By using a confocal laser scanning microscope, we demonstrated that invasive bladder tumor cell lines, YTS-1 and T24, with high Matrigel degradation activity form invadopodia but that noninvasive bladder tumor cell lines, RT4 and KK-47, form no detectable invadopodia. Invadopodia formed by YTS-1 cells had the ability to secrete matrix metalloproteases and degrade extracellular matrix to invade surrounding areas. Moreover, we observed that primary tumor cells obtained from patients with invasive bladder tumors also form invadopodia, validating the results from bladder tumor cell lines. Our results provide evidence that invasive human bladder tumor cells form invadopodia for tumor invasion.     

乙酰肝素酶在乳腺癌侵袭转移中的作用及调控机制*

乙酰肝素酶（Heparanase ,HPSE）是目前发现的哺乳动物细胞中唯一能降解细胞外基质和基底膜中硫酸肝素蛋白多糖侧链—硫酸乙酰肝素的内源性糖苷酶,是目前抗肿瘤转移的理想靶点。HPSE在乳腺癌中常有高表达,并与肿瘤大小和淋巴结转移等密切相关。体外研究表明,乳腺癌细胞中HPSE启动子活性增高,转染HPSE的乳腺癌细胞在体内成瘤后,其肿瘤体积、重量、微血管密度以及癌细胞存活时间均明显高于阴性对照组。应用反义核酸技术或RNA干扰技术封闭或沉默HPSE基因表达后,乳腺癌细胞黏附和侵袭能力显著降低,表明HPSE在乳腺癌侵袭转移中发挥极其重要的作用。HPSE在乳腺癌侵袭转移中受多种机制调控,主要有:雌激素与其受体结合后作用于HPSE基因特定区域,从而提高HPSE转录活性,增强其基因和蛋白的表达;HPSE可使破骨细胞刺激因子产生增加,从而导致骨质溶解破坏,为乳腺癌骨转移奠定基础;HPSE诱导循环淋巴细胞产生刺激因子,从而促进乳腺癌的侵袭转移。此外,HPSE基因还受p53基因、ETS 基因、EGR 1 基因、PI-88因子、COX-2 等的调控。本文就HPSE在乳腺癌中的表达状况、在侵袭转移中的作用及调控机制进行综述。      

Bone sialoprotein‐αvβ3 integrin axis promotes breast cancer metastasis to the bone

The underlying mechanisms of breast cancer cells metastasizing to distant sites are complex and multifactorial. Bone sialoprotein (BSP) and αvβ3 integrin were reported to promote the metastatic progress of breast cancer cells, particularly metastasis to bone. Most theories presume that BSP promotes breast cancer metastasis by binding to αvβ3 integrin. Interestingly, we found the αvβ3 integrin decreased in BSP silenced cells (BSPi), which have weak ability to form bone metastases. However, the relevance of their expression in primary tumor and the way they participate in metastasis are not clear. In this study, we evaluated the relationship between BSP, αvβ3 integrin levels, and the bone metastatic ability of breast cancer cells in patient tissues, and the data indicated that the αvβ3 integrin level is closely correlated to BSP level and metastatic potential. Overexpression of αvβ3 integrin in cancer cells could reverse the effect of BSPi in vitro and promote bone metastasis in a mouse model, whereas knockdown of αvβ3 integrin have effects just like BSPi. Moreover, The Cancer Genome Atlas data and RT‐PCR analysis have also shown that SPP1, KCNK2, and PTK2B might be involved in this process. Thus, we propose that αvβ3 integrin is one of the downstream factors regulated by BSP in the breast cancer‐bone metastatic cascade.     

Matrix metalloproteinase-1 promotes prostate tumor growth and metastasis

Cell migration and invasion are critical events during the progression to metastasis. Matrix metalloproteinase-1 (MMP-1) is involved in the progression of human malignancies, but the precise role of MMP-1 in tumor invasion and metastasis remains unclear. In the present study, we investigated the role of MMP-1 in tumor cell invasion and metastasis by overexpressing MMP-1 in prostate cancer cells. Overexpression of MMP-1 in prostate cancer cells increases cell invasion and migration as measured by modified transwell assays. Furthermore, the results from a bioluminescence tumor/metastasis model showed that the overexpression of MMP-1 significantly induces prostate tumor growth and the incidence of lung metastasis. We observed that this increase in tumor growth correlates with an increase in tumor angiogenesis. In addition, we assessed the importance of MMP-1 expression in cell invasion and migration by inhibiting MMP-1 activity with specific inhibitor and antibodies. Blockade of MMP-1 activity inhibited prostate cancer cell migration and invasion in vitro. Treatment of mice with an MMP-1 specific inhibitor significantly decreased prostate tumor growth and incidence of lung metastasis in vivo. Collectively, our findings suggest that MMP-1 plays an important role in prostate cancer progression during the invasive and metastatic stages of the disease.     

ATP‐P2Y2‐β‐catenin axis promotes cell invasion in breast cancer cells

Extracellular adenosine 5′‐triphosphate (ATP), secreted by living cancer cells or released by necrotic tumor cells, plays an important role in tumor invasion and metastasis. Our previous study demonstrated that ATP treatment in vitro could promote invasion in human prostate cancer cells via P2Y2, a preferred receptor for ATP, by enhancing EMT process. However, the pro‐invasion mechanisms of ATP and P2Y2 are still poorly studied in breast cancer. In this study, we found that P2Y2 was highly expressed in breast cancer cells and associated with human breast cancer metastasis. ATP could promote the in vitro invasion of breast cancer cells and enhance the expression of β‐catenin as well as its downstream target genes CD44, c‐Myc and cyclin D1, while P2Y2 knockdown attenuated above ATP‐driven events in vitro and in vivo. Furthermore, iCRT14, a β‐catenin/TCF complex inhibitor, could also suppress ATP‐driven migration and invasion in vitro. These results suggest that ATP promoted breast cancer cell invasion via P2Y2‐β‐catenin axis. Thus blockade of the ATP‐P2Y2‐β‐catenin axis could suppress the invasive and metastatic potential of breast cancer cells and may serve as potential targets for therapeutic interventions of breast cancer.     

RRR-alpha-tocopheryl succinate inhibits human prostate cancer cell invasiveness

RRR-alpha-tocopheryl succinate (alpha-vitamin E succinate, VES), one of the vitamin E derivatives, can effectively inhibit the proliferation of human prostate cancer cells. However, little is known about its effect on prostate cancer cell invasive ability. Tumor metastasis is a complex process and the extracellular matrix (ECM) is the first barrier that tumor cells encounter. Therefore, we tested the effect of VES on the invasion of different prostate tumor cells, PC-3, DU-145, and LNCaP, through Matrigel, a reconstituted ECM, using an in vitro cell invasion assay. The invasion of PC-3 and DU-145 cells through Matrigel was inhibited by 20 microM VES after treating for 24 h. The condition did not alter cell survival, cell cycle, cell adhesion or cell motility. We further investigated whether the ability of VES to inhibit prostate cancer cell invasiveness was associated with its ability to inhibit the activity of matrix metalloproteinases (MMPs), the key enzymes in the proteolysis of basement membrane during invasion. PC-3 and DU-145 cells that were treated with VES showed a significant reduction in the levels of MMP-9 in the culture medium. In contrast, LNCaP cells, which did not secrete MMP-9, were poorly invasive in Matrigel and were hardly affected by treatment with VES. This is the first report suggesting that VES inhibits human prostate cancer cell invasiveness and the reduction of secreted MMP-9 activity could be one of the contributory factors, which points to the potential use of VES in the prevention and therapy of prostate cancer invasion.     

Suppression of invasion and metastasis of prostate cancer cells by overexpression of NDRG2 gene

N-myc downstream regulated gene 2 (NDRG2) is involved in invasion and metastasis of cancer, furthermore it is frequently down-regulated in prostate cancer. Herein we evaluated the effect of NDRG2 overexpression on invasiveness and bone destruction in prostate cancer. The human prostate cancer cell line PC-3 and DU145 were infected with Ad-NDRG2 or Ad-LacZ. Overexpression of NDRG2 not only inhibited the growth of the cells, but also suppressed invasiveness of the cells in an in vitro assay. PC-3 cells infected with Ad-NDRG2 or Ad-LacZ were injected into the tibias of nude mice. Four weeks later, we found the mice injected with PC-3 cells overexpressing NDRG2 had smaller tumors and less bone destruction. These results demonstrate that NDRG2 overexpression can inhibit tumor growth and invasion, furthermore, it can decrease bone destruction caused by prostate cancer bone metastasis.     

Cellular interactions in the tropism of prostate cancer to bone

At autopsy >or=80% of prostate cancers have established macrometastases in marrow containing bone. The mechanism(s) to explain this remarkable level of bone involvement remain to be elucidated. We examined the adhesive and invasive behavior of prostate cancer cells to osteoblastic and human bone marrow endothelial cells (HBME-1) in an attempt to explain the tropism of prostate cells for bone. We found an inverse relationship between adhesion and prostate cell tumorigenicity and metastatic potential. Relative cell adhesion of P69 between cell lines was 1.74-fold (95% confidence interval [CI] = 1.15-2.64) and 1.58-fold (95% CI = 0.94-2.68) greater at 1 hr and 2 hr, respectively, than LNCaP that was essentially equivalent to C4-2 cells when using an osteoblastic cell line, D1 as the substrate. Similar results were acquired when HBME-1 were used as substratum. There was a marked increase in adhesion of the poorly tumorigenic cell line P69 as compared to the cancer cells to HBME-1. P69 adhesion was 2.78-fold (95% CI = 1.87-4.84) and 2.0-fold (95% CI = 1.43-2.80) greater at 1 hr and 2 hr, respectively when compared to LNCaP or C4-2 cells. D1 cells, a bone homing osteoblastic precursor, behaved contrary to the metastatic, bone-colonizing C4-2 cell line and bound best to other bone cells but not as well as a non-homing fetal bone marrow-derived cell line, D2. Invasion of prostate cancer cells through HBME-1 lawns was examined at 8 hr and 16 hr. In contrast to the adhesion studies, the invasion of the more aggressive C4-2 cells was 3.46-fold (95% CI = 1.18-10.17) and 2.65-fold (95% CI = 1.26-5.56) greater at 8 hr and 16 hr, respectively than LNCaP cells. Similarly, LNCaP cell invasion was 1.73-fold (95% CI = 0.69-4.37) and 2.35-fold (95% CI = 1.41-3.93) greater at 8 hr and 16 hr, respectively than P69 cells at the invasion of HBME-1 monolayers. At 8 hr, C4-2 cells had 6.0-fold (95% CI = 2.63,13.65) higher invasive potential than P69 cells. Phage display biopanning of LNCaP cells versus C4-2 cells in vitro using 4 separate techniques repeatedly identified the same peptide in support of minimal cell surface changes associated with the ability of C4-2 cells to metastasize to bone. As integrins are vital to cell adhesion and migration, we examined the integrin subunit expression in the prostate cell lines. The expression of integrin subunits is much higher in the nontumorigenic cell line, P69, whereas the differences in integrin expression between LNCaP and C4-2 are negligible. Only alpha(2) and beta(5) integrin subunits increase from LNCaP to C4-2. Given that C4-2 cells spontaneously metastasize to bone in vivo and LNCaP cells do not, these studies imply that the ability of a metastatic prostate cancer cell to colonize the bone is not completely dependent upon the ability of the cancer cell to adhere to either osteoblastic cells or to the bone marrow endothelial cell lining. Therefore, the initial interaction between the bone endothelium or stroma and prostate cells is not accurately referred to as a tropic or homing response. The invasion assay results indicate that the invasive potential of the cell more accurately reflects the bone colonizing potential of a prostate cancer cell. It is likely that bidirectional paracrine interactions, subsequent to marrow adhesion, between prostate cancer cells and the bone microenvironment are what determine the successful colonization of the bone by prostate cancer cells. Further, functional changes in surface proteins that are involved in invasion are likely to occur without major changes in levels of cell surface protein expression. Functional integrin association, substratum usage and outside in signaling are more likely to predict metastatic behavior.     

Bone marrow stromal cells enhance prostate cancer cell invasion through type I collagen in an MMP-12 dependent manner

At the cellular level, the process of bone metastasis involves many steps. Circulating cancer cells enter the marrow, proliferate, induce neovascularization, and ultimately expand into a clinically detectable, often symptomatic, metastatic deposit. Although the initial establishment and later expansion of the metastatic deposit in bone require tumor cells to possess invasive capability, the exact proteases responsible for this phenotype are not well known. The objective of our study was to take an unbiased approach to determine which proteases were expressed and functional during the initial interactions between prostate cancer cells and bone marrow stromal (BMS) cells. We found that the combination of human prostate cancer PC3 and BMS cells stimulates the invasive ability of cancer cells through type I collagen. The use of inhibitors for each of the major protease families indicated that 1 or more MMPs was/were responsible for the BMS-induced invasion. Gene profiling and semiquantitative RT-PCR analysis revealed an increased expression of several MMP genes because of PC3/BMS cell interaction. However, only MMP-12 showed an increase in protein expression. Downregulation of MMP-12 expression in PC3 cells by siRNA inhibited the enhanced invasion induced by PC3/BMS cell interaction. In vivo, MMP-12 was found to be primarily expressed by prostate cancer cells growing in bone. Our data suggest that BMS cells induce MMP-12 expression in prostate cancer cells, which results in invasive cells capable of degradation of type I collagen.