Possible role of Pax-6 in promoting breast cancer cell proliferation and tumorigenesis

Pax 6, a member of the paired box (Pax) family, has been implicated in oncogenesis. However, its therapeutic potential has been never examined in breast cancer. To explore the role of Pax6 in breast cancer development, a lentivirus based short hairpin RNA (shRNA) delivery system was used to knockdown Pax6 expression in estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells. Effect of Pax6 silencing on breast cancer cell proliferation and tumorigenesis was analyzed. Pax6-RNAi-lentivirus infection remarkably downregulated the expression levels of Pax6 mRNA and protein in MCF-7 and MDA-MB-231 cells. Accordingly, the cell viability, DNA synthesis, and colony formation were strongly suppressed, and the tumorigenesis in xenograft nude mice was significantly inhibited. Moreover, tumor cells were arrested at G0/G1 phase after Pax6 was knocked down. Pax6 facilitates important regulatory roles in breast cancer cell proliferation and tumor progression, and could serve as a diagnostic marker for clinical investigation.     

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.     

Biological and Clinical Significance of MAD2L1 and BUB1, Genes Frequently Appearing in Expression Signatures for Breast Cancer Prognosis

To investigate the biologic relevance and clinical implication of genes involved in multiple gene expression signatures for breast cancer prognosis, we identified 16 published gene expression signatures, and selected two genes, MAD2L1 and BUB1. These genes appeared in 5 signatures and were involved in cell-cycle regulation. We analyzed the expression of these genes in relation to tumor features and disease outcomes. In vitro experiments were also performed in two breast cancer cell lines, MDA-MB-231 and MDA-MB-468, to assess cell proliferation, migration and invasion after knocking down the expression of these genes. High expression of these genes was found to be associated with aggressive tumors and poor disease-free survival of 203 breast cancer patients in our study, and the association with survival was confirmed in an online database consisting of 914 patients. In vitro experiments demonstrated that lowering the expression of these genes by siRNAs reduced tumor cell growth and inhibited cell migration and invasion. Our investigation suggests that MAD2L1 and BUB1 may play important roles in breast cancer progression, and measuring the expression of these genes may assist the prediction of breast cancer prognosis.     

LncRNA AC009686.2促进乳腺癌细胞增殖和侵袭

长非编码RNAs（long non-coding RNAs,LncRNAs）作为一类基因表达的调控因子,在多种肿瘤的发生发展中发挥关键作用,然而LncRNAs在乳腺癌中的作用及相关机制尚未完全阐明。为了寻找在乳腺癌发生发展中起关键作用的LncRNAs,本研究通过分析TCGA数据库发现,LncRNA AC009686.2在乳腺癌组织中表达明显高于正常组织,并且与乳腺癌病人的预后不良正相关。qRT-PCR分析发现LncRNA AC009686.2在乳腺癌细胞中的表达明显上调,其中在乳腺癌细胞MCF7、T47D、ZR7530、BT549、HCC1937、MDA-MB-231和SKBR3的表达量分别是MCF10A细胞的6.58倍、5.66倍、7.29倍、9.06倍、6.89倍、11.17倍和5.38倍。在相对高表达的MDA-MB-231和BT549细胞中干扰LncRNA AC009686.2能明显抑制细胞的增殖、克隆形成和侵袭能力,并且诱导细胞发生G1 /S 期阻滞,其中干扰LncRNA AC009686.2表达的乳腺癌细胞MDA-MB-321和BT549的克隆抑制率分别为对照组的0.496%,0.438%和0.495%,0.353%。同时干扰LncRNA AC009686.2能下调细胞周期蛋白D2（cyclinD2,CCND2）和锌指E盒结合同源框1（zinc finger E-box binding homeobox1,ZEB1）的蛋白质水平。而在乳腺癌细胞中过表达ZEB1能明显逆转干扰LncRNA AC009686.2引起的细胞侵袭能力下降。进一步通过在线软件JASPAR数据库分析发现LncRNA AC009686.2的启动子存在ZEB1结合位点,过表达ZEB1能上调细胞中LncRNA AC009686.2的表达水平。总之,LncRNA AC009686.2在乳腺癌中高表达,通过上调细胞周期蛋白D2和上皮细胞-间充质转化（epithelial-mesenchymal transition,EMT）相关分子ZEB1促进乳腺癌细胞增殖和侵袭,而ZEB1能正向调控LncRNA AC009686.2的表达。本研究将为阐明AC009686.2在乳腺癌中的作用和相关分子机制提供理论依据。     

Clinicopathological and biological significance of human voltage-gated proton channel Hv1 protein overexpression in breast cancer

In our previous work, we showed for the first time that the voltage-gated proton channel Hv1 is specifically expressed in highly metastatic human breast tumor tissues and cell lines. However, the contribution of Hv1 to breast carcinogenesis is not well known. In this study, we showed that Hv1 expression was significantly correlated with the tumor size (p = 0.001), tumor classification (p = 0.000), lymph node status (p = 0.000), clinical stage (p = 0.000), and Her-2 status (p = 0.045). High Hv1 expression was associated significantly with shorter overall (p = 0.000) and recurrence-free survival (p = 0.000). In vitro, knockdown of Hv1 expression in the highly metastatic MDA-MB-231 cells decreased the cell proliferation and invasiveness, inhibited the cell proton secretion and intracellular pH recovery, and blocked the cell capacity of acidifying extracellular milieu. Furthermore, the gelatinase activity in MDA-MB-231 cells that suppressed Hv1 was reduced. In vivo, the breast tumor size of the implantation of the MDA-MB-231 xenografts in nude mice that were knocked down by Hv1 was dramatically smaller than that in the control groups. The results demonstrated that the inhibition of Hv1 function via knockdown of Hv1 expression can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Based on these results, we came to the conclusion that Hv1 is a potential biomarker for prognosis of breast cancer and a potential target for anticancer drugs in breast cancer therapy.     

CHL1 is involved in human breast tumorigenesis and progression

Neural cell adhesion molecules (CAM) play important roles in the development and regeneration of the nervous system. The L1 family of CAMs is comprised of L1, Close Homolog of L1 (CHL1, L1CAM2), NrCAM, and Neurofascin, which are structurally related trans-membrane proteins in vertebrates. Although the L1CAM has been demonstrated play important role in carcinogenesis and progression, the function of CHL1 in human breast cancer is limited. Here, we found that CHL1 is down-regulated in human breast cancer and related to lower grade. Furthermore, overexpression of CHL1 suppresses proliferation and invasion in MDA-MB-231 cells and knockdown of CHL1 expression results in increased proliferation and invasion in MCF7 cells in vitro. Finally, CHL1 deficiency promotes tumor formation in vivo. Our results may provide a strategy for blocking breast carcinogenesis and progression.     

15-Deoxy-Δ12,14-Prostaglandin J2 Inhibits Osteolytic Breast Cancer Bone Metastasis and Estrogen Deficiency-Induced Bone Loss

Breast cancer is the major cause of cancer death in women worldwide. The most common site of metastasis is bone. Bone metastases obstruct the normal bone remodeling process and aberrantly enhance osteoclast-mediated bone resorption, which results in osteolytic lesions. 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is an endogenous ligand of peroxisome proliferator-activated receptor gamma (PPARγ) that has anti-inflammatory and antitumor activity at micromolar concentrations through PPARγ-dependent and/or PPARγ-independent pathways. We investigated the inhibitory activity of 15d-PGJ₂ on the bone loss that is associated with breast cancer bone metastasis and estrogen deficiency caused by cancer treatment. 15d-PGJ₂ dose-dependently inhibited viability, migration, invasion, and parathyroid hormone-related protein (PTHrP) production in MDA-MB-231 breast cancer cells. 15d-PGJ₂ suppressed receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA levels and normalized osteoprotegerin (OPG) mRNA levels in hFOB1.19 osteoblastic cells treated with culture medium from MDA-MB-231 cells or PTHrP, which decreased the RANKL/OPG ratio. 15d-PGJ₂ blocked RANKL-induced osteoclastogenesis and inhibited the formation of resorption pits by decreasing the activities of cathepsin K and matrix metalloproteinases, which are secreted by mature osteoclasts. 15d-PGJ₂ exerted its effects on breast cancer and bone cells via PPARγ-independent pathways. In Balb/c nu/nu mice that received an intracardiac injection of MDA-MB-231 cells, subcutaneously injected 15d-PGJ₂ substantially decreased metastatic progression, cancer cell-mediated bone destruction in femora, tibiae, and mandibles, and serum PTHrP levels. 15d-PGJ₂ prevented the destruction of femoral trabecular structures in estrogen-deprived ICR mice as measured by bone morphometric parameters and serum biochemical data. Therefore, 15d-PGJ₂ may be beneficial for the prevention and treatment of breast cancer-associated bone diseases.     

STIM1/ORAI1-mediated Ca2+ Influx Regulates Enolase-1 Exteriorization

Tumor cells use broad spectrum proteolytic activity of plasmin to invade tissue and form metastatic foci. Cell surface-associated enolase-1 (ENO-1) enhances plasmin formation and thus participates in the regulation of pericellular proteolysis. Although increased levels of cell surface bound ENO-1 have been described in different types of cancer, the molecular mechanism responsible for ENO-1 exteriorization remains elusive. In the present study, increased ENO-1 protein levels were found in ductal breast carcinoma and on the cell surface of highly metastatic breast cancer cell line MDA-MB-231. Elevated cell surface-associated ENO-1 expression correlated with augmented MDA-MB-231 cell migratory and invasive properties. Exposure of MDA-MB-231 cells to LPS potentiated translocation of ENO-1 to the cell surface and its release into the extracellular space in the form of exosomes. These effects were independent of de novo protein synthesis and did not require the classical endoplasmic reticulum/Golgi pathway. LPS-triggered ENO-1 exteriorization was suppressed by pretreatment of MDA-MB-231 cells with the Ca²⁺ chelator BAPTA or an inhibitor of endoplasmic reticulum Ca²⁺-ATPase pump, cyclopiazonic acid. In line with these observations, the stromal interaction molecule (STIM) 1 and the calcium release-activated calcium modulator (ORAI) 1-mediated store-operated Ca²⁺ entry were found to regulate LPS-induced ENO-1 exteriorization. Pharmacological blockage or knockdown of STIM1 or ORAI1 reduced ENO-1-dependent migration of MDA-MB-231 cells. Collectively, our results demonstrate the pivotal role of store-operated Ca²⁺ channel-mediated Ca²⁺ influx in the regulation of ENO-1 exteriorization and thus in the modulation of cancer cell migratory and invasive properties.     

ADAM9 silencing inhibits breast tumor cell invasion in vitro

ADAM9 (A Disintegrin And Metalloproteinase 9) is a member of the ADAM protein family which contains a disintegrin domain. This protein family plays key roles in many physiological processes, including fertilization, migration, and cell survival. The ADAM proteins have also been implicated in various diseases, including cancer. Specifically, ADAM9 has been suggested to be involved in metastasis. To address this question, we generated ADAM9 knockdown clones of MDA-MB-231 breast tumor cells using silencing RNAs that were tested for cell adhesion, proliferation, migration and invasion assays. In RNAi-mediated ADAM9 silenced MDA-MB-231 cells, the expression of ADAM9 was lower from the third to the sixth day after silencing and inhibited tumor cell invasion in matrigel by approximately 72% when compared to control cells, without affecting cell adhesion, proliferation or migration. In conclusion, the generation of MDA-MB-231 knockdown clones lacking ADAM9 expression inhibited tumor cell invasion in vitro, suggesting that ADAM9 is an important molecule in the processes of invasion and metastasis.     

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.     

Snail1 induced in breast cancer cells in 3D collagen I gel environment suppresses cortactin and impairs effective invadopodia formation

Although an in vitro 3D environment cannot completely mimic the in vivo tumor site, embedding tumor cells in a 3D extracellular matrix (ECM) allows for the study of cancer cell behaviors and the screening of anti-metastatic reagents with a more in vivo-like context. Here we explored the behaviors of MDA-MB-231 breast cancer cells embedded in 3D collagen I. Diverse tumor environmental conditions (including cell density, extracellular acidity, or hypoxia as mimics for a continuous tumor growth) reduced JNKs, enhanced TGFβ1/Smad signaling activity, induced Snail1, and reduced cortactin expression. The reduced JNKs activity blocked efficient formation of invadopodia labeled with actin, cortactin, or MT1-MMP. JNKs inactivation activated Smad2 and Smad4, which were required for Snail1 expression. Snail1 then repressed cortactin expression, causing reduced invadopodia formation and prominent localization of MT1-MMP at perinuclear regions. MDA-MB-231 cells thus exhibited less efficient collagen I degradation and invasion in 3D collagen I upon JNKs inhibition. These observations support a signaling network among JNKs, Smads, Snail1, and cortactin to regulate the invasion of MDA-MB-231 cells embedded in 3D collagen I, which may be targeted during screening of anti-invasion reagents.     

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.     

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaP_M cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis.     

CoREST1 Promotes Tumor Formation and Tumor Stroma Interactions in a Mouse Model of Breast Cancer

Regulators of chromatin structure and gene expression contribute to tumor formation and progression. The co-repressor CoREST1 regulates the localization and activity of associated histone modifying enzymes including lysine specific demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1). Although several CoREST1 associated proteins have been reported to enhance breast cancer progression, the role of CoREST1 in breast cancer is currently unclear. Here we report that knockdown of CoREST1 in the basal-type breast cancer cell line, MDA-MB-231, led to significantly reduced incidence and diminished size of tumors compared to controls in mouse xenograft studies. Notably, CoREST1-depleted cells gave rise to tumors with a marked decrease in angiogenesis. CoREST1 knockdown led to a decrease in secreted angiogenic and inflammatory factors, and mRNA analysis suggests that CoREST1 promotes expression of genes related to angiogenesis and inflammation including VEGF-A and CCL2. CoREST1 knockdown decreased the ability of MDA-MB-231 conditioned media to promote endothelial cell tube formation and migration. Further, tumors derived from CoREST1-depleted cells had reduced macrophage infiltration and the secretome of CoREST1 knockdown cells was deficient in promoting macrophage migration and macrophage-mediated angiogenesis. Taken together, these findings reveal that the epigenetic regulator CoREST1 promotes tumorigenesis in a breast cancer model at least in part through regulation of gene expression patterns in tumor cells that have profound non-cell autonomous effects on endothelial and inflammatory cells in the tumor microenvironment.     

Specific expression of the human voltage-gated proton channel Hv1 in highly metastatic breast cancer cells, promotes tumor progression and metastasis

The newly discovered human voltage-gated proton channel Hv1 is essential for proton transfer, which contains a voltage sensor domain (VSD) without a pore domain. We report here for the first time that Hv1 is specifically expressed in the highly metastatic human breast tumor tissues, but not in poorly metastatic breast cancer tissues, detected by immunohistochemistry. Meanwhile, real-time RT-PCR and immunocytochemistry showed that the expression levels of Hv1 have significant differences among breast cancer cell lines, MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, T-47D and SK-BR-3, in which Hv1 is expressed at a high level in highly metastatic human breast cancer cell line MDA-MB-231, but at a very low level in poorly metastatic human breast cancer cell line MCF-7. Inhibition of Hv1 expression in the highly metastatic MDA-MB-231 cells by small interfering RNA (siRNA) significantly decreases the invasion and migration of the cells. The intracellular pH of MDA-MB-231 cells down-regulated Hv1 expression by siRNA is obviously decreased compared with MDA-MB-231 with the scrambled siRNA. The expression of matrix metalloproteinase-2 and gelatinase activity in MDA-MB-231 cells suppressed Hv1 by siRNA were reduced. Our results strongly suggest that Hv1 regulates breast cancer intracellular pH and exacerbates the migratory ability of metastatic cells.     

Gain-of-function mutant p53-R280K mediates survival of breast cancer cells

Missense mutations in TP53 resulting in the expression of p53-R175H, p53-R273H, or p53-R280K are frequently detected in human breast cancer. Currently, the role of mutant p53-R280K in breast cancer is relatively unknown, and therefore, the present study analyzed the function of mutant p53-R280K in breast cancer cell growth. To this end, we used small interfering RNA to study the role of mutant p53-R280K in MDA-MB-231 cells, which endogenously express the mutant protein. We found that curcumin induced apoptosis in MDA-MB-231 cells and downregulated mutant p53-R280K. We also observed that knockdown of mutant p53 by small interfering RNA induced apoptosis in MDA-MB-231 cells. Curcumin-induced apoptosis was further enhanced by the overexpression of wild-type p53, but was decreased by mutant p53-R280K overexpression. Our findings indicate that mutant p53-R280K has an important role in mediating the survival of triple-negative breast cancer MDA-MB-231 cells. Furthermore, this study suggests mutant p53-R280K could be used as a therapeutic target for breast cancer cells harboring this TP53 missense mutation.     

Cross-talk between endothelial and breast cancer cells regulates reciprocal expression of angiogenic factors in vitro

Reciprocal growth factor exchange between endothelial and malignant cells within the tumor microenvironment may directly stimulate neovascularization; however, the role of host vasculature in regulating tumor cell activity is not well understood. While previous studies have examined the angiogenic response of endothelial cells to tumor-secreted factors, few have explored tumor response to endothelial cells. Using an in vitro co-culture system, we investigated the influence of endothelial cells on the angiogenic phenotype of breast cancer cells. Specifically, VEGF, ANG1, and ANG2 gene and protein expression were assessed. When co-cultured with microvascular endothelial cells (HMEC-1), breast cancer cells (MDA-MB-231) significantly increased expression of ANG2 mRNA (20-fold relative to MDA-MB-231 monoculture). Moreover, MDA-MB-231/HMEC-1 co-cultures produced significantly increased levels of ANG2 (up to 580 pg/ml) and VEGF protein (up to 38,400 pg/ml) while ANG1 protein expression was decreased relative to MDA-MB-231 monocultures. Thus, the ratio of ANG1:ANG2 protein, a critical indicator of neovascularization, shifted in favor of ANG2, a phenomenon known to correlate with vessel destabilization and sprouting in vivo. This angiogenic response was not observed in nonmalignant breast epithelial cells (MCF-10A), where absolute protein levels of MCF-10A/HMEC-1 co-cultures were an order of magnitude less than that of the MDA-MB-231/HMEC-1 co-cultures. Results were further verified with a functional angiogenesis assay demonstrating well-defined microvascular endothelial cell (TIME) tube formation when cultured in media collected from MDA-MB-231/HMEC-1 co-cultures. This study demonstrates that the angiogenic activity of malignant mammary epithelial cells is significantly enhanced by the presence of endothelial cells.     

cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-β. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.