Pyruvate Carboxylase Is Up-Regulated in Breast Cancer and Essential to Support Growth and Invasion of MDA-MB-231 Cells

Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.     

The MicroRNA-23b/27b/24 Cluster Promotes Breast Cancer Lung Metastasis by Targeting Metastasis-suppressive Gene Prosaposin

MicroRNAs (miRNAs) have been shown to function as key regulators of tumor progression and metastasis. Recent studies have indicated that the miRNAs comprising the miR-23b/27b/24 cluster might influence tumor metastasis, although the precise nature of this regulation remains unclear. Here, expression of the miR-23b/27b/24 cluster is found to correlate with metastatic potential in mouse and human breast cancer cell lines and is elevated in metastatic lung lesions in human breast cancer patients. Ectopic expression of the miRNAs in the weakly metastatic mouse 4TO7 mammary tumor cell line had no effect on proliferation or morphology of tumor cells in vitro but was found to increase lung metastasis in a mouse model of breast cancer metastasis. Furthermore, gene expression profiling analysis of miRNA overexpressing 4TO7 cells revealed the direct targeting of prosaposin (PSAP), which encodes a secreted protein found to be inversely correlated with metastatic progression in human breast cancer patients. Importantly, ectopic expression of PSAP was able to suppress the metastatic phenotype in highly metastatic 4T1 and MDA-MB-231 SCP28 cells, as well as in cells ectopically expressing miR-23b/27b/24. These findings support a metastasis-promoting function of the miR-23b/27b/24 cluster of miRNAs, which functions in part through the direct inhibition of PSAP.     

Identification of breast cancer metastasis-associated proteins in an isogenic tumor metastasis model using two-dimensional gel electrophoresis and liquid chromatography-ion trap-mass spectrometry

To better understand the molecular mechanisms underlying breast cancer metastasis and search for potential markers for metastatic progression, we have developed a highly metastatic variant of human MDA-MB-435 breast cancer cell line through in vivo stepwise selection of pulmonary metastatic cells caused by parental MDA-MB-435 cells in the athymic mice. Comparative proteomic analysis using 2-DE and LC-IT-MS revealed that 102 protein spots were reproducibly altered more than three-fold between the selected variant and its parental counterpart. Eleven differentially expressed protein spots were identified with high confidence using SEQUEST with uninterpreted tandem mass raw data. Cathepsin D precursor, peroxiredoxin 6 (PDX6), heat shock protein 27 (HSP27), HSP60, tropomyosin 1 (TPM1), TPM2, TPM3, TPM4, 14-3-3 protein epsilon, and tumor protein D54 were up-regulated in the highly metastatic variant, whereas alpha B-crystalline (CRAB) was only detected in its parental counterpart. Differential expression was confirmed for four proteins including PDX6, CRAB, TPM4, and HSP60 by real-time quantitative PCR and Western blotting analysis in our model. Immunohistochemical analysis in 80 breast cancer donors demonstrated a significant association of TPM4 (p = 0.002), HSP60 (p = 0.001), PDX6 (p = 0.002) but not CRAB (p = 0.113) staining with the presence of lymph node metastasis. In addition, TPM4 staining was also associated with clinical stage (p = 0.000), but no significant association was found between TPM4, PDX6, CRAB, and HSP60 expression and tumor size, hormone receptor, and HER-2 status (p > 0.05). The functional implication of these identified proteins was also discussed. These proteomic data are valuable and informative for understanding breast cancer metastasis and searching for potential markers for metastatic progression.     

Association of increased basement membrane invasiveness with absence of estrogen receptor and expression of vimentin in human breast cancer cell lines.

Lack of estrogen receptor (ER) and presence of vimentin (VIM) associate with poor prognosis in human breast cancer. We have explored the relationships between ER, VIM, and invasiveness in human breast cancer cell lines. In the matrigel outgrowth assay, ER+/VIM- (MCF-7, T47D, ZR-75-1), and ER-/VIM- (MDA-MB-468, SK-Br-3) cell lines were uninvasive, while ER-/VIM+ (BT549, MDA-MB-231, MDA-MB-435, MDA-MB-436, Hs578T) lines formed invasive, penetrating colonies. Similarly, ER-/VIM+ cell lines were significantly more invasive than either the ER+/VIM- or ER-/VIM- cell lines in the Boyden chamber chemoinvasion assay. Invasive activity in nude mice was only seen with ER-/VIM+ cell lines MDA-MB-231, MDA-MB-435 and MDA-MB-436. Hs578T cells (ER-/VIM+) showed hematogenous dissemination to the lungs in one of five mice, but lacked local invasion. The ER-/VIM+ MCF-7ADR subline was significantly more active than the MCF-7 cells in vitro, but resembled the wild-type MCF-7 parent in in vivo activity. Data from these cell lines suggest that human breast cancer progression results first in the loss of ER, and subsequently in VIM acquisition, the latter being associated with increased metastatic potential through enhanced invasiveness. The MCF-7ADR data provide evidence that this transition can occur in human breast cancer cells. Vimentin expression may provide useful insights into mechanisms of invasion and/or breast cancer cell progression.     

Extracellular matrix protein 1 (ECM1) has angiogenic properties and is expressed by breast tumor cells.

Tumor growth and metastasis are critically dependent on the formation of new blood vessels. The present study found that extracellular matrix protein 1 (ECM1), a newly described secretory glycoprotein, promotes angiogenesis. This was initially suggested by in situ hybridization studies of mouse embryos indicating that the ECM1 message was associated with blood vessels and its expression pattern was similar to that of flk-1, a recognized marker for endothelium. More direct evidence for the role of ECM1 in angiogenesis was provided by the fact that highly purified recombinant ECM1 stimulated the proliferation of cultured endothelial cells and promoted blood vessel formation in the chorioallantoic membrane of chicken embryos. Immunohistochemical staining with specific antibodies indicated that ECM1 was expressed by the human breast cancer cell lines MDA-435 and LCC15, both of which are highly tumorigenic. In addition, staining of tissue sections from patients with breast cancer revealed that ECM1 was present in a significant proportion of primary and secondary tumors. Collectively, the results of this study suggest that ECM1 possesses angiogenic properties that may promote tumor progression.     

A humanized anti-osteopontin antibody inhibits breast cancer growth and metastasis in vivo

Osteopontin (OPN) has been implicated as an important mediator of breast cancer progression and metastasis and has been investigated for use as a potential therapeutic target in the treatment of breast cancer. However, the in vivo antitumor effect of anti-OPN antibodies on breast cancer has not been reported. In this study, a mouse anti-human OPN antibody (1A12) was humanized by complementarity-determining region grafting method based on computer-assisted molecular modeling. A humanized version of 1A12, denoted as hu1A12, was shown to possess affinity comparable to that of its parental antibody. The ability of hu1A12 to inhibit cell migration, adhesion, invasion and colony formation was assessed in a highly metastatic human breast cancer cell line MDA-MB-435S. The results indicated that hu1A12 was effective in inhibiting the cell adhesion, migration, invasion and colony formation of MDA-MB-435S cells in vitro. hu1A12 also showed significant efficacy in suppressing primary tumor growth and spontaneous metastasis in a mouse lung metastasis model of human breast cancer. The specific epitope recognized by hu1A12 was identified to be ²¹²NAPSD²¹⁶, adjacent to the calcium binding domain of OPN. Our data strongly support that OPN is a potential target for the antibody-based therapies of breast cancer. The humanized anti-OPN antibody hu1A12 may be a promising therapeutic agent for the treatment of human breast cancer.     

Osteopontin-induced, integrin-dependent migration of human mammary epithelial cells involves activation of the hepatocyte growth factor receptor (Met)

Osteopontin (OPN) is a secreted glycophosphoprotein which induces migration of mammary carcinoma cells, and has been implicated in the malignancy of breast carcinoma. Hepatocyte growth factor (HGF) induces cell migration of several mammary epithelial cell (MEC) lines, via activation of its cognate receptor (Met). This study examines the mechanism of OPN-induced MEC migration, in terms of the cell surface integrins involved and induction of the HGF/Met pathway. Three different MEC cell lines were used, representing different stages of tumor progression: 21PT, non-tumorigenic; 21NT, tumorigenic; non-metastatic; and MDA-MB-435, tumorigenic, highly metastatic. Human recombinant OPN was found to induce the migration of all three lines. OPN-induced migration of 21PT and 21NT cells was alphavbeta5 and beta1-integrin dependent, and alphavbeta3-independent, while that of MDA-MB-435 cells was alphavbeta3-dependent. HGF also induced migration of all three cell lines, and a synergistic response was seen to HGF and OPN together. The increased migration response to OPN was found to be associated with an initial increase in Met kinase activity (within 30 min), followed by an increase in Met mRNA and protein expression. OPN-induced cell migration is thus mediated by different cell surface integrins in MEC lines representing different stages of progression, and involves activation of the HGF receptor, Met.     

Genomic analysis of a spontaneous model of breast cancer metastasis to bone reveals a role for the extracellular matrix

A clinically relevant model of spontaneous breast cancer metastasis to multiple sites, including bone, was characterized and used to identify genes involved in metastatic progression. The metastatic potential of several genetically related tumor lines was assayed using a novel real-time quantitative RT-PCR assay of tumor burden. Based on this assay, the tumor lines were categorized as nonmetastatic (67NR), weakly metastatic to lymph node (168FARN) or lung (66cl4), or highly metastatic to lymph node, lung, and bone (4T1.2 and 4T1.13). In vitro assays that mimic stages of metastasis showed that highly metastatic tumors lines were more adhesive, invasive, and migratory than the less metastatic lines. To identify metastasis-related genes in this model, each metastatic tumor was array profiled against the nonmetastatic 67NR using 15,000 mouse cDNA arrays. A significant proportion of genes relating to the extracellular matrix had elevated expression in highly metastatic tumors. The role of one of these genes, POEM, was further investigated in the model. In situ hybridization showed that POEM expression was specific to the tumor epithelium of highly metastatic tumors. Decreased POEM expression in 4T1.2 tumors significantly inhibited spontaneous metastasis to the lung, bone, and kidney. Taken together, our data support a role for the extracellular matrix in metastatic progression and describe, for the first time, a role for POEM in this process.     

Tumor-host interactions contribute to the elevated expression level of alpha1-antichymotrypsin in metastatic breast tumor xenografts

We investigated alpha1-antichymotrypsin (ACT) gene expression in xenograft tumors generated by two isogenic human breast cancer cell lines derived from the same parent, MDA-MB-435, which display opposite metastatic behaviors. Microarray and real-time PCR experiments showed an overexpression of this serine protease inhibitor in the metastatic tumors (M-4A4T) and its derived metastases (M4-Mets) compared with the weakly metastatic tumors (NM-2C5T), and its release into the blood was confirmed by western-blotting. However, functional assays in vivo using genetically engineered tumor cells demonstrated that ACT up-regulation was not, by itself, responsible for the metastatic phenotype. We also made observations that ACT gene regulation was sensitive to tumor-host interactions: inoculation of these lines into the mouse mammary gland greatly increased ACT production and accentuated the intrinsic difference observed when they are cultured in vitro. Sensitivity of tumor cells to their environment was further analyzed by in vitro experiments, which demonstrated that a purified ECM environment and soluble components from normal host mammary cells were both able to significantly promote ACT expression. In addition, we took advantage of the xenogeneic nature of the model to measure ACT expression by the host cells (mouse) and the tumor cells (human) within the neoplasm using species-specific primers in real-time PCR experiments. It was found that the presence of tumor cells, irrespective of their metastatic capabilities, induced local ACT production by host cells at the primary and secondary tumor sites. Thus, this work indicates that there is a specific association of ACT overexpression with the metastatic phenotype in our breast cancer metastasis model. Moreover, because of the xenogeneic nature of our system, we were able to provide evidence of tumor-host reciprocal regulation of ACT production.     

UCH-L1在乳腺癌中的表达及其与转移关系的研究

目的研究泛素羧基末端水解酶L1(UCH-L1)与磷酸化p38(p-p38)在乳腺癌组织、细胞系中的表达情况、两种蛋白的表达与临床病理特征的关系以及UCH-L1与乳腺癌侵袭转移的关系。方法用免疫组织化学方法检测乳腺癌组织中UCH-L1与p-p38蛋白的表达情况,用Western Blot方法检测乳腺癌组织以及细胞系中UCH-L1与p-p38蛋白的表达情况。应用UCH-L1特异性抑制剂作用于乳腺癌高侵袭高转移细胞系MDA-MB-435s后,用Western Blot观察UCH-L1与p-p38蛋白表达改变的情况,用Transwell实验检测MDA-MB-435s细胞侵袭潜能的改变。结果 UCH-L1和p-p38蛋白在乳腺浸润性导管癌中的表达高于其在癌旁正常乳腺组织中的表达(P=0.012,P=0.001),二者呈正相关(r=0.397,P=0.000),并与乳腺癌的TNM分期(P=0.017,P=0.010)、淋巴结转移情况(P=0.033,P=0.021)相关。乳腺上皮细胞系MCF-10A、乳腺癌低侵袭低转移细胞系MCF-7和乳腺癌高侵袭高转移细胞系MDA-MB-435s中两种蛋白表达水平呈递增趋势(P均<0.05)。UCH-L1特异性抑制剂可以浓度依赖性地下调MDA-MB-435s细胞系中p-p38蛋白的表达水平(P均<0.05),并能抑制乳腺癌细胞的侵袭转移潜能。结论 UCH-L1、p-p38过表达与乳腺癌的TMN分期、淋巴结转移有关。UCH-L1可能通过上调p-p38介导乳腺癌转移。     

Enhanced intrinsic migration of aggressive breast cancer cells by inhibition of Rac1 GTPase

Rac GTPases are known to play a crucial role in regulating cytoskeletal changes necessary for cell migration. Migration has been shown to be positively regulated by Rac in most cell types. However, there is also a large body of conflicting evidence in some other cell types with respect to the role of Rac in migration, suggesting that Rac GTPases regulate cell migration in a cell type-dependent manner. In the present study, we have characterized the effects of Rac1 GTPase inhibition on the migratory abilities of a number of breast cancer cell lines with differential degrees of tumorigenic and metastatic potentials. We show that Rac1 inhibition in non-metastatic (MCF-7, T-47D) or moderately metastatic (Hs578T) cell lines results in inhibition of migration, whereas in highly metastatic cell lines (MDA-MB-435, MDA-MB-231, and C3L5) Rac1 inhibition results in stimulation of migration. This stimulation of migration following Rac1 inhibition is also accompanied by the enhanced RhoA activity, suggesting a possible existence of a dominating role of RhoA over Rac1 in regulating intrinsic migration of the highly metastatic breast cancer cells.     

The LCC15-MB Human Breast Cancer Cell Line Expresses Osteopontin and Exhibits an Invasive and Metastatic Phenotype

We have characterized the LCC15-MB cell line which was recently derived from a breast carcinoma metastasis resected from the femur of a 29-year-old woman. LCC15-MB cells are vimentin (VIM) positive, exhibit a stellate morphology in routine cell culture, and form penetrating colonies when embedded in three-dimensional gels of Matrigel or fibrillar collagen. They show high levels of activity in the Boyden chamber chemomigration and chemoinvasion assays, and like other invasive human breast cancer (HBC) cell lines, LCC15-MB cells activate matrix-metalloproteinase-2 in response to treatment with concanavalin A. In addition, these cells are tumorigenic when implanted subcutaneously in nude mice and recolonize bone after arterial injection. Interestingly, both the primary lesion and the bone metastasis from which LCC15-MB were derived, as well as the resultant cell line, abundantly express the bone matrix protein osteopontin (OPN). OPN is also expressed by the highly metastatic MDA-MB-435 cells, but not other invasive or noninvasive HBC cell lines. Expression of OPN is retained in the subcutaneous xenograft and intraosseous metastases of LCC15-MB as detected by immunohistochemistry. Both VIM and OPN expression have been associated with breast cancer invasion and metastasis, and their expression by the LCC15-MB cell line is consistent with its derivation from a highly aggressive breast cancer. These cells provide a useful model for studying molecular mechanisms important for breast cancer metastasis to bone and, in particular, the implication(s) of OPN and VIM expression in this process.     

Characterization of Spontaneous and TGF-β-Induced Cell Motility of Primary Human Normal and Neoplastic Mammary Cells In Vitro Using Novel Real-Time Technology

The clinical complications derived from metastatic disease are responsible for the majority of all breast cancer related deaths. Since cell migration and invasion are a prerequisite for metastasis their assessment in patient cancer cells in vitro may have prognostic value for the tumor''s metastatic capacity. We employed real-time cell analysis (RTCA) on the xCELLigence DP system to determine in vitro motility of patient-derived primary human breast cancer epithelial cells (HBCEC). Initially, the RTCA assay was validated using established human breast cancer cell lines with either an invasive (MDA-MB-231, MDA-MB-435s) or a non-invasive phenotype (MCF-7, MDA-MB-468), and primary NSCLC cells (Tu459). Previous standard assays of cell migration/invasion revealed that only MDA-MB-231, −435s, and Tu459 cells exhibited spontaneous and TGF-β1-stimulated migration and invasion through a Matrigel barrier. In the present study, the TGF-β1-stimulated activities could be blocked by SB431542, a potent kinase inhibitor of the TGF-β type I receptor ALK5. Application of the RTCA assay to patient-derived tumor cells showed that 4/4 primary HBCEC and primary NSCLC cells, but not normal human mammary epithelial cells (HMEC), displayed high spontaneous migratory and invasive activity which correlated with higher MMP-2 expression and uPA protein levels in HBCEC compared to HMEC. Upon treatment with TGF-β1, HBCEC exhibited morphologic and gene regulatory alterations indicative of epithelial-to-mesenchymal transition. However, exclusively the invasive but not the migratory activity of HBCEC was further enhanced by TGF-β1. This indicates the requirement for molecular, e.g. integrin interactions with Matrigel components in HBCEC in order to become responsive to pro-invasive TGF-β effects. Together, these results show for the first time that tumorigenic HBCEC but not normal HMEC possess a strong basal migratory as well as a basal and TGF-β1-inducible invasive potential. These findings qualify the RTCA assay as an in vitro migration/invasion testing system for patient-specific primary breast cancer cells.     

Bcl-xL promotes metastasis of breast cancer cells by induction of cytokines resistance

Metastasis is a highly complex process involving the survival of tumor cells, both in the blood stream and within specific organs. Cell-death and survival are determined by a number of gene products from an expanding family of the Bcl-2 gene, either promoting or preventing apoptosis. Furthermore, the survival of tumor cells may favor the accumulation of additional genetic alterations causing further growth and invasive opportunities which may lead to metastasis. To examine whether the prevention of cell-death influences the metastatic behavior, we transfected a human breast cancer cell line MDA-MB-435 with the Bcl-xL cDNA and then studied metastatic ability of the selected clones in vivo. Our results show that Bcl-xL-clones had a decreased tumor growth latency and an increased metastatic ability. Apoptosis-resistance to cytokines was induced in 435 cells by Bcl-xL-expression with minor modifications in their proliferation rates. These cells also showed diminished adhesion to extracellular matrix proteins and a survival advantage in suspension over 435/Neo cells. Moreover, to determine survival in blood stream and in cells lodged in the lungs, we injected 435/Bcl-xL and 435/Neo cells at 1:3 proportion i.v., and animals were killed at intervals of 15' to 16 h after injection. Tumor cells were recovered from the lungs and Southern-blot analysis revealed the presence of exogenous Bcl-xL cDNA. These results showed that 435/Bcl-xL cells had a survival advantage in circulation over 435/Neo cells. This advantage in vivo was attributable to Bcl-xL expression. We conclude that Bcl-xL expression in breast cancer cells can increase metastatic activity. This advantage could be created by inducing resistance to apoptosis against cytokines, increasing cell survival in circulation, and enhancing anchorage-independent growth.     

Toll-Like Receptor 4 Prompts Human Breast Cancer Cells Invasiveness via Lipopolysaccharide Stimulation and Is Overexpressed in Patients with Lymph Node Metastasis

Toll-like receptor (TLR)4-mediated signaling has been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study investigated the expression and biological role of TLR4 in human breast cancer metastasis. MCF-7 and MDA-MB-231 are human breast cancer cell lines with low and high metastatic potential, respectively. Using lipopolysaccharide (LPS) to stimulate MCF-7 and MDA-MB-231 cells, expression of TLR4 mRNA and protein increased compared with that in control cells. TLR4 activation notably up-regulated expression of matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor(VEGF) mRNA and their secretion in the supernatants of both cell lines. LPS enhanced invasion of MDA-MB-231 cells by transwell assay and MCF-7 cells by wound healing assay. LPS triggered increased expression of TLR4 downstream signaling pathway protein myeloid differentiation factor 88(MyD88) and resulted in interleukin (IL)-6 and IL-10 higher production by human breast cancer cells. Stimulation of TLR4 with LPS promoted tumorigenesis and formed metastatic lesions in liver of nude mice. Moreover, expression of TLR4 and MyD88 as well as invasiveness and migration of the cells could be blocked by TLR4 antagonist. Combined with clinicopathological parameters, TLR4 was overexpressed in human breast cancer tissue and correlated with lymph node metastasis. These findings indicated that TLR4 may participate in the progression and metastasis of human breast cancer and provide a new therapeutic target.