Astrocytes Directly Influence Tumor Cell Invasion and Metastasis In Vivo

Brain metastasis is a defining component of tumor pathophysiology, and the underlying mechanisms responsible for this phenomenon are not well understood. Current dogma is that tumor cells stimulate and activate astrocytes, and this mutual relationship is critical for tumor cell sustenance in the brain. Here, we provide evidence that primary rat neonatal and adult astrocytes secrete factors that proactively induced human lung and breast tumor cell invasion and metastasis capabilities. Among which, tumor invasion factors namely matrix metalloprotease-2 (MMP-2) and MMP-9 were partly responsible for the astrocyte media-induced tumor cell invasion. Inhibiting MMPs reduced the ability of tumor cell to migrate and invade in vitro. Further, injection of astrocyte media-conditioned breast cancer cells in mice showed increased invasive activity to the brain and other distant sites. More importantly, blocking the preconditioned tumor cells with broad spectrum MMP inhibitor decreased the invasion and metastasis of the tumor cells, in particular to the brain in vivo. Collectively, our data implicate astrocyte-derived MMP-2 and MMP-9 as critical players that facilitate tumor cell migration and invasion leading to brain metastasis.     

Chromodomain Helicase/ATPase DNA-Binding Protein 1-Like Gene (CHD1L) Expression and Implications for Invasion and Metastasis of Breast Cancer

Background

Chromodomain helicase/ATPase DNA-binding protein 1-like gene (CHD1L), also known as ALC1 (amplified in liver cancer 1 gene), is a new oncogene amplified in many solid tumors. Whether this gene plays a role in invasion and metastasis of breast cancer is unknown.

Methods

Immunohistochemistry was performed to detect the expression of CHD1L in patients with invasive ductal carcinoma and normal mammary glands. Chemotaxis, wound healing, and Transwell invasion assays were also performed to examine cell migration and invasion. Western blot analysis was conducted to detect the expression of CHD1L, MMP-2, MMP-9, pAkt/Akt, pARK5/ARK5, and pmTOR/mTOR. Moreover, ELISA was carried out to detect the expression levels of MMP-2 and MMP-9. Nude mice xenograft model was used to detect the invasion and metastasis of breast cancer cell lines.

Results

CHD1L overexpression was observed in 112 of 268 patients (41.8%). This overexpression was associated with lymph node metastasis (P = 0.008), tumor differentiation (P = 0.020), distant metastasis (P = 0.026), MMP-2 (P = 0.035), and MMP-9 expression (P = 0.022). In the cell experiment, reduction of CHD1L inhibited the invasion and metastasis of breast cancer cells by mediating MMP-2 and MMP-9 expression. CHD1L knockdown via siRNA suppressed EGF-induced pAkt, pARK5, and pmTOR. This knockdown inhibited the metastasis of breast cancer cells into the lungs of SCID mice.

Conclusions

CHD1L promoted the invasion and metastasis of breast cancer cells via the PI3K/Akt/ARK5/mTOR/MMP signaling pathway. This study identified CHD1L as a potential anti-metastasis target for therapeutic intervention in breast cancer.     

N-Cadherin Promotes Adhesion Between Invasive Breast Cancer Cells and the Stroma

Calcium-dependent cell adhesion molecules (cadherins) are involved in maintaining the epithelial structure of a number of tissues including the mammary gland. In breast and other tumor types, loss of E-cadherin expression has been seen in high grade tumors and correlates with increased invasiveness. Here we show high levels of expression of N-cadherin in the most invasive breast cancer cell lines which was inversely correlated with their expression of E-cadherin. A stromal cell line also expressed N-cadherin in accordance with its fibroblastic morphology. N-cadherin localized to areas of cell-cell contact in all cells that expressed it. Calcium-dependent intercellular adhesion of N-cadherin-expressing breast cancer and stromal cells was specifically inhibited by an anti N-cadherin monoclonal antibody. In addition, N-cadherin promoted the interaction of invasive breast cancer cells with mammary stromal cells: in contrast, E-cadherin expressing cell lines did not co-aggregate with stromal cells. The combined results suggest a functional role for N-cadherin in cohesion of breast tumor cells which, in addition promotes their interaction with the surrounding stromal cells, thereby facilitating invasion and metastasis.     

N-Cadherin Promotes Adhesion Between Invasive Breast Cancer Cells and the Stroma

Calcium-dependent cell adhesion molecules (cadherins) are involved in maintaining the epithelial structure of a number of tissues including the mammary gland. In breast and other tumor types, loss of E-cadherin expression has been seen in high grade tumors and correlates with increased invasiveness. Here we show high levels of expression of N-cadherin in the most invasive breast cancer cell lines which was inversely correlated with their expression of E-cadherin. A stromal cell line also expressed N-cadherin in accordance with its fibroblastic morphology. N-cadherin localized to areas of cell-cell contact in all cells that expressed it. Calcium-dependent intercellular adhesion of N-cadherin-expressing breast cancer and stromal cells was specifically inhibited by an anti N-cadherin monoclonal antibody. In addition, N-cadherin promoted the interaction of invasive breast cancer cells with mammary stromal cells: in contrast, E-cadherin expressing cell lines did not co-aggregate with stromal cells. The combined results suggest a functional role for N-cadherin in cohesion of breast tumor cells which, in addition promotes their interaction with the surrounding stromal cells, thereby facilitating invasion and metastasis.     

ERBB2 overexpression suppresses stress-induced autophagy and renders ERBB2-induced mammary tumorigenesis independent of monoallelic Becn1 loss

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1^+/− mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1^+/+ and Becn1^+/− immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.     

A Cell Motility Screen Reveals Role for MARCKS-Related Protein in Adherens Junction Formation and Tumorigenesis

Invasion through the extracellular matrix (ECM) is important for wound healing, immunological responses and metastasis. We established an invasion-based cell motility screen using Boyden chambers overlaid with Matrigel to select for pro-invasive genes. By this method we identified antisense to MARCKS related protein (MRP), whose family member MARCKS is a target of miR-21, a microRNA involved in tumor growth, invasion and metastasis in multiple human cancers. We confirmed that targeted knockdown of MRP, in both EpRas mammary epithelial cells and PC3 prostate cancer cells, promoted in vitro cell migration that was blocked by trifluoperazine. Additionally, we observed increased immunofluoresence of E-cadherin, β-catenin and APC at sites of cell-cell contact in EpRas cells with MRP knockdown suggesting formation of adherens junctions. By wound healing assay we observed that reduced MRP supported collective cell migration, a type of cell movement where adherens junctions are maintained. However, destabilized adherens junctions, like those seen in EpRas cells, are frequently important for oncogenic signaling. Consequently, knockdown of MRP in EpRas caused loss of tumorigenesis in vivo, and reduced Wnt3a induced TCF reporter signaling in vitro. Together our data suggest that reducing MRP expression promotes formation of adherens junctions in EpRas cells, allowing collective cell migration, but interferes with oncogenic β-catenin signaling and tumorigenesis.     

Activation of EGFR promotes squamous carcinoma SCC10A cell migration and invasion via inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin

EGFR is a potent stimulator of invasion and metastasis in head and neck squamous cell carcinomas (HNSCC). However, the mechanism by which EGFR may stimulate tumor cell invasion and metastasis still need to be elucidated. In this study, we showed that activation of EGFR by EGF in HNSCC cell line SCC10A enhanced cell migration and invasion, and induced loss of epitheloid phenotype in parallel with downregulation of E-cadherin and upregulation of N-cadherin and vimentin, indicating that EGFR promoted SCC10A cell migration and invasion possibly by an epithelial to mesenchymal transition (EMT)-like phenotype change. Interestingly, activation of EGFR by EGF induced production of matrix metalloproteinase-9 (MMP-9) and soluble E-cadherin (sE-cad), and knockdown of MMP-9 by siRNA inhibited sE-cad production induced by EGF in SCC10A. Moreover, both MMP-9 knockdown and E-cadherin overexpression inhibited cell migration and invasion induced by EGF in SCC10A. The results indicate that EGFR activation promoted cell migration and invasion through inducing MMP-9-mediated degradation of E-cadherin into sE-cad. Pharmacologic inhibition of EGFR, MEK, and PI3K kinase activity in SCC10A reduced phosphorylated levels of ERK-1/2 and AKT, production of MMP-9 and sE-cad, cell migration and invasion, and expressional changes of EMT markers (E-cadherin and N-cadherin) induced by EGF, indicating that EGFR activation promotes cell migration and invasion via ERK-1/2 and PI3K-regulated MMP-9/E-cadherin signaling pathways. Taken together, the data suggest that EGFR activation promotes HNSCC SCC10A cell migration and invasion by inducing EMT-like phenotype change and MMP-9-mediated degradation of E-cadherin into sE-cad related to activation of ERK-1/2 and PI3K signaling pathways.     

Protein tyrosine phosphatase controls breast cancer invasion through the expression of matrix metalloproteinase-9

The expression of matrix metalloproteinases (MMPs) produced by cancer cells has been associated with the high potential of metastasis in several human carcinomas, including breast cancer. Several pieces of evidence demonstrate that protein tyrosine phosphatases (PTP) have functions that promote cell migration and metastasis in breast cancer. We analyzed whether PTP inhibitor might control breast cancer invasion through MMP expression. Herein, we investigate the effect of 4-hydroxy-3,3-dimethyl-2H benzo[g]indole-2,5(3H)-dione (BVT948), a novel PTP inhibitor, on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. The expression of MMP-9 and cell invasion increased after TPA treatment, whereas TPA-induced MMP-9 expression and cell invasion were decreased by BVT948 pretreatment. Also, BVT948 suppressed NF-κB activation in TPA-treated MCF-7 cells. However, BVT948 didn’t block TPA-induced AP-1 activation in MCF-7 cells. Our results suggest that the PTP inhibitor blocks breast cancer invasion via suppression of the expression of MMP-9. [BMB Reports 2013; 46(11): 533-538]     

Linoleic acid induces an EMT-like process in mammary epithelial cells MCF10A

Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of developing breast cancer. Epithelial-mesenchymal-transition (EMT) is a process, by which epithelial cells are transdifferentiated to a mesenchymal state, and it has been implicated in cancer progression, including invasion and metastasis. Linoleic acid (LA) induces proliferation and invasion in breast cancer cells. However, the role of LA on the EMT process in human mammary epithelial cells remains to be studied. In the present study, we demonstrate that LA induces a transient down-regulation of E-cadherin expression, accompanied with an increase of Snail1, Snail2, Twist1, Twist2 and Sip1 expressions. Furthermore, LA induces FAK and NFκB activation, MMP-2 and -9 secretions, migration and invasion. In summary, our findings demonstrate, for the first time, that LA promotes an EMT-like process in MCF10A human mammary epithelial cells.     

Exogenous expression of N-cadherin in breast cancer cells induces cell migration, invasion, and metastasis

E- and N-cadherin are calcium-dependent cell adhesion molecules that mediate cell-cell adhesion and also modulate cell migration and tumor invasiveness. The loss of E-cadherin-mediated adhesion has been shown to play an important role in the transition of epithelial tumors from a benign to an invasive state. However, recent evidence indicates that another member of the cadherin family, N-cadherin, is expressed in highly invasive tumor cell lines that lacked E-cadherin expression. These findings have raised the possibility that N-cadherin contributes to the invasive phenotype. To determine whether N-cadherin promotes invasion and metastasis, we transfected a weakly metastatic and E-cadherin-expressing breast cancer cell line, MCF-7, with N-cadherin and analyzed the effects on cell migration, invasion, and metastasis. Transfected cells expressed both E- and N-cadherin and exhibited homotypic cell adhesion from both molecules. In vitro, N-cadherin-expressing cells migrated more efficiently, showed an increased invasion of Matrigel, and adhered more efficiently to monolayers of endothelial cells. All cells produced low levels of the matrix metalloproteinase MMP-9, which was dramatically upregulated by treatment with FGF-2 only in N-cadherin-expressing cells. Migration and invasion of Matrigel were also greatly enhanced by this treatment. When injected into the mammary fat pad of nude mice, N-cadherin-expressing cells, but not control MCF-7 cells, metastasized widely to the liver, pancreas, salivary gland, omentum, lung, lymph nodes, and lumbar spinal muscle. The expression of both E- and N-cadherin was maintained both in the primary tumors and metastatic lesions. These results demonstrate that N-cadherin promotes motility, invasion, and metastasis even in the presence of the normally suppressive E-cadherin. The increase in MMP-9 production by N-cadherin-expressing cells in response to a growth factor may endow them with a greater ability to penetrate matrix protein barriers, while the increase in their adherence to endothelium may improve their ability to enter and exit the vasculature, two properties that may be responsible for metastasis of N-cadherin-expressing cells.     

Metadherin mediates lipopolysaccharide-induced migration and invasion of breast cancer cells

Background

Breast cancer is the most prevalent cancer in women worldwide and metastatic breast cancer has very poor prognosis. Inflammation has been implicated in migration and metastasis of breast cancer, although the exact molecular mechanism remains elusive.

Principal Findings

We show that the pro-inflammatory endotoxin Lipopolysaccharide (LPS) upregulates the expression of Metadherin (MTDH), a recently identified oncogene, in a number of breast cancer lines. Stable knockdown of MTDH by shRNA in human breast MDA-MB-231 cells abolishes LPS-induced cell migration and invasion as determined by several in vitro assays. In addition, knockdown of MTDH diminishes Nuclear Factor-kappa B (NF-κB) activation by LPS and inhibited LPS-induced IL-8 and MMP-9 production.

Conclusions

These results strongly suggest that MTDH is a pivotal molecule in inflammation-mediated tumor metastasis. Since NF-κB, IL-8 and MMP-9 play roles in LPS-induced invasion or metastasis, the mechanism of MTDH-promoted invasion and metastasis may be through the activation of NF-κB, IL-8 and MMP-9, also suggesting a role of MTDH in regulating both inflammatory responses and inflammation-associated tumor invasion. These findings indicate that MTDH is involved in inflammation-induced tumor progression, and support that MTDH targeting therapy may hold promising prospects in treating breast cancer.     

CREB3L1 Is a Metastasis Suppressor That Represses Expression of Genes Regulating Metastasis,Invasion, and Angiogenesis

The unfolded protein response (UPR) is activated in response to hypoxia-induced stress such as in the tumor microenvironment. This study examined the role of CREB3L1 (cyclic AMP [cAMP]-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis. Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low-metastasis or nonmetastatic cell lines. When metastatic cells were transfected with CREB3L1, they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under nonadherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, not only did CREB3L1-expressing cells fail to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and chromatin immunoprecipitation with microarray technology (ChIP on Chip) analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis. These data suggest that CREB3L1 plays an important role in suppressing tumorigenesis and that loss of expression is required for the development of a metastatic phenotype.     

Compensatory function of Pyk2 protein in the promotion of focal adhesion kinase (FAK)-null mammary cancer stem cell tumorigenicity and metastatic activity

Mammary cancer stem cells (MaCSCs) have been identified as a rare population of cells capable of self-renewal to drive mammary tumorigenesis and metastasis. Nevertheless, relatively little is known about the intracellular signaling pathways regulating self-renewal and metastatic activities of MaCSCs in vivo. Using a recently developed breast cancer mouse model with focal adhesion kinase (FAK) deletion in mammary tumor cells (MFCKO-MT mice), here we present evidence suggesting a compensatory function of Pyk2, a FAK-related kinase, in the regulation of MaCSCs and metastasis in these mice. Increased expression of Pyk2 was found selectively in pulmonary metastatic nodules of MFCKO-MT mice, and its inhibition significantly reduced mammary tumor development and metastasis in these mice. Consistent with the idea of metastasis driven by MaCSCs, we detected selective up-regulation of Pyk2 in MaCSCs, but not bulk mammary tumor cells, of primary tumors developed in MFCKO-MT mice. We further showed that inhibition of Pyk2 in FAK-null MaCSCs significantly decreased their tumorsphere formation and migration in vitro as well as self-renewal, tumorigenicity, and metastatic activity in vivo. Last, we identified PI3K/Akt signaling as a major mediator of FAK regulation of MaCSCs as well as a target for the compensatory function of Pyk2 in FAK-null MaCSCs. Together, these results further advance our understanding of FAK and its related tyrosine kinase Pyk2 in regulation of MaCSCs in breast cancer and suggest that pharmaceutically targeting these kinases may hold promise as a novel treatment for the disease by targeting and eradicating MaCSCs.