Slit protein-mediated inhibition of CXCR4-induced chemotactic and chemoinvasive signaling pathways in breast cancer cells

Slit, which mediates its function by binding to the Roundabout (Robo) receptor, has been shown to regulate neuronal and CXCR4-mediated leukocyte migration. Slit-2 was shown to be frequently inactivated in lung and breast cancers because of hypermethylation of its promoter region. Furthermore, the CXCR4/CXCL12 axis has been reported recently to be actively involved in breast cancer metastasis to target organs such as lymph nodes, lung, and bone. In this study, we sought to characterize the effect of Slit (=Slit-2) on the CXCL12/CXCR4-mediated metastatic properties of breast cancer cells. We demonstrate here that breast cancer cells and tissues derived from breast cancer patients express Robo 1 and 2 receptors. We also show that Slit treatment inhibits CXCL12/CXCR4-induced breast cancer cell chemotaxis, chemoinvasion, and adhesion, the fundamental components that promote metastasis. Slit had no significant effect on the CXCL12-induced internalization process of CXCR4. In addition, characterization of signaling events revealed that Slit inhibits CXCL12-induced tyrosine phosphorylation of focal adhesion components such as RAFTK/Pyk2 at residues 580 and 881, focal adhesion kinase at residue 576, and paxillin. We found that Slit also inhibits CXCL12-induced phosphatidylinositol 3-kinase, p44/42 MAP kinase, and metalloproteinase 2 and 9 activities. However, it showed no effect on JNK and p38 MAP kinase activities. To our knowledge, this is the first report to analyze in detail the effect of Slit on breast cancer cell motility as well as its effect on the critical components of the cancer cell chemotactic machinery. Studies of the Slit-Robo complex may foster new anti-chemotactic approaches to block cancer cell metastasis.     

Epigenetic changes of CXCR4 and its ligand CXCL12 as prognostic factors for sporadic breast cancer

Chemokines and their receptors are involved in the development and cancer progression. The chemokine CXCL12 interacts with its receptor, CXCR4, to promote cellular adhesion, survival, proliferation and migration. The CXCR4 gene is upregulated in several types of cancers, including skin, lung, pancreas, brain and breast tumors. In pancreatic cancer and melanoma, CXCR4 expression is regulated by DNA methylation within its promoter region. In this study we examined the role of cytosine methylation in the regulation of CXCR4 expression in breast cancer cell lines and also correlated the methylation pattern with the clinicopathological aspects of sixty-nine primary breast tumors from a cohort of Brazilian women. RT-PCR showed that the PMC-42, MCF7 and MDA-MB-436 breast tumor cell lines expressed high levels of CXCR4. Conversely, the MDA-MB-435 cell line only expressed CXCR4 after treatment with 5-Aza-CdR, which suggests that CXCR4 expression is regulated by DNA methylation. To confirm this hypothesis, a 184 bp fragment of the CXCR4 gene promoter region was cloned after sodium bisulfite DNA treatment. Sequencing data showed that cell lines that expressed CXCR4 had only 15% of methylated CpG dinucleotides, while the cell line that not have CXCR4 expression, had a high density of methylation (91%). Loss of DNA methylation in the CXCR4 promoter was detected in 67% of the breast cancer analyzed. The absence of CXCR4 methylation was associated with the tumor stage, size, histological grade, lymph node status, ESR1 methylation and CXCL12 methylation, metastasis and patient death. Kaplan-Meier curves demonstrated that patients with an unmethylated CXCR4 promoter had a poorer overall survival and disease-free survival. Furthermore, patients with both CXCL12 methylation and unmethylated CXCR4 had a shorter overall survival and disease-free survival. These findings suggest that the DNA methylation status of both CXCR4 and CXCL12 genes could be used as a biomarker for prognosis in breast cancer.     

Aquaporin-3 Controls Breast Cancer Cell Migration by Regulating Hydrogen Peroxide Transport and Its Downstream Cell Signaling

Most breast cancer mortality is due to clinical relapse associated with metastasis. CXCL12/CXCR4-dependent cell migration is a critical process in breast cancer progression; however, its underlying mechanism remains to be elucidated. Here, we show that the water/glycerol channel protein aquaporin-3 (AQP3) is required for CXCL12/CXCR4-dependent breast cancer cell migration through a mechanism involving its hydrogen peroxide (H₂O₂) transport function. Extracellular H₂O₂, produced by CXCL12-activated membrane NADPH oxidase 2 (Nox2), was transported into breast cancer cells via AQP3. Transient H₂O₂ accumulation was observed around the membrane during CXCL12-induced migration, which may be facilitated by the association of AQP3 with Nox2. Intracellular H₂O₂ then oxidized PTEN and protein tyrosine phosphatase 1B (PTP1B) followed by activation of the Akt pathway. This contributed to directional cell migration. The expression level of AQP3 in breast cancer cells was related to their migration ability both in vitro and in vivo through CXCL12/CXCR4- or H₂O₂-dependent pathways. Coincidentally, spontaneous metastasis of orthotopic xenografts to the lung was reduced upon AQP3 knockdown. These findings underscore the importance of AQP3-transported H₂O₂ in CXCL12/CXCR4-dependent signaling and migration in breast cancer cells and suggest that AQP3 has potential as a therapeutic target for breast cancer.     

A Comprehensive Analysis of CXCL12 Isoforms in Breast Cancer1,2

CXCL12-CXCR4-CXCR7 signaling promotes tumor growth and metastasis in breast cancer. Alternative splicing of CXCL12 produces isoforms with distinct structural and biochemical properties, but little is known about isoform-specific differences in breast cancer subtypes and patient outcomes. We investigated global expression profiles of the six CXCL12 isoforms, CXCR4, and CXCR7 in The Cancer Genome Atlas breast cancer cohort using next-generation RNA sequencing in 948 breast cancer and benign samples and seven breast cancer cell lines. We compared expression levels with several clinical parameters, as well as metastasis, recurrence, and overall survival (OS). CXCL12-α, -β, and -γ are highly co-expressed, with low expression correlating with more aggressive subtypes, higher stage disease, and worse clinical outcomes. CXCL12-δ did not correlate with other isoforms but was prognostic for OS and showed the same trend for metastasis and recurrence-free survival. Effects of CXCL12-δ remained independently prognostic when taking into account expression of CXCL12, CXCR4, and CXCR7. These results were also reflected when comparing CXCL12-α, -β, and -γ in breast cancer cell lines. We summarized expression of all CXCL12 isoforms in an important chemokine signaling pathway in breast cancer in a large clinical cohort and common breast cancer cell lines, establishing differences among isoforms in multiple clinical, pathologic, and molecular subgroups. We identified for the first time the clinical importance of a previously unstudied isoform, CXCL12-δ.     

Crosstalk between chemokine receptor CXCR4 and cannabinoid receptor CB2 in modulating breast cancer growth and invasion

Background

Cannabinoids bind to cannabinoid receptors CB₁ and CB₂ and have been reported to possess anti-tumorigenic activity in various cancers. However, the mechanisms through which cannabinoids modulate tumor growth are not well known. In this study, we report that a synthetic non-psychoactive cannabinoid that specifically binds to cannabinoid receptor CB₂ may modulate breast tumor growth and metastasis by inhibiting signaling of the chemokine receptor CXCR4 and its ligand CXCL12. This signaling pathway has been shown to play an important role in regulating breast cancer progression and metastasis.

Methodology/Principal Findings

We observed high expression of both CB₂ and CXCR4 receptors in breast cancer patient tissues by immunohistochemical analysis. We further found that CB₂-specific agonist JWH-015 inhibits the CXCL12-induced chemotaxis and wound healing of MCF7 overexpressing CXCR4 (MCF7/CXCR4), highly metastatic clone of MDA-MB-231 (SCP2) and NT 2.5 cells (derived from MMTV-neu) by using chemotactic and wound healing assays. Elucidation of the molecular mechanisms using various biochemical techniques and confocal microscopy revealed that JWH-015 treatment inhibited CXCL12-induced P44/P42 ERK activation, cytoskeletal focal adhesion and stress fiber formation, which play a critical role in breast cancer invasion and metastasis. In addition, we have shown that JWH-015 significantly inhibits orthotopic tumor growth in syngenic mice in vivo using NT 2.5 cells. Furthermore, our studies have revealed that JWH-015 significantly inhibits phosphorylation of CXCR4 and its downstream signaling in vivo in orthotopic and spontaneous breast cancer MMTV-PyMT mouse model systems.

Conclusions/Significance

This study provides novel insights into the crosstalk between CB₂ and CXCR4/CXCL12-signaling pathways in the modulation of breast tumor growth and metastasis. Furthermore, these studies indicate that CB₂ receptors could be used for developing innovative therapeutic strategies against breast cancer.     

CXCR7/CXCR4 heterodimer constitutively recruits beta-arrestin to enhance cell migration

G protein-coupled receptor hetero-oligomerization is emerging as an important regulator of ligand-dependent transmembrane signaling, but precisely how receptor heteromers affect receptor pharmacology remains largely unknown. In this study, we have attempted to identify the functional significance of the heteromeric complex between CXCR4 and CXCR7 chemokine receptors. We demonstrate that co-expression of CXCR7 with CXCR4 results in constitutive recruitment of β-arrestin to the CXCR4·CXCR7 complex and simultaneous impairment of G(i)-mediated signaling. CXCR7/CXCR4 co-expression also results in potentiation of CXCL12 (SDF-1)-mediated downstream β-arrestin-dependent cell signaling pathways, including ERK1/2, p38 MAPK, and SAPK as judged from the results of experiments using siRNA knockdown to deplete β-arrestin. Interestingly, CXCR7/CXCR4 co-expression enhances cell migration in response to CXCL12 stimulation. Again, inhibition of β-arrestin using either siRNA knockdown or a dominant negative mutant abrogates the enhanced CXCL12-dependent migration of CXCR4/CXCR7-expressing cells. These results show how CXCR7, which cannot signal directly through G protein-linked pathways, can nevertheless affect cellular signaling networks by forming a heteromeric complex with CXCR4. The CXCR4·CXCR7 heterodimer complex recruits β-arrestin, resulting in preferential activation of β-arrestin-linked signaling pathways over canonical G protein pathways. CXCL12-dependent signaling of CXCR4 and its role in cellular physiology, including cancer metastasis, should be evaluated in the context of potential functional hetero-oligomerization with CXCR7.     

TNF receptor-associated factor 2-dependent canonical pathway is crucial for the development of Peyer's patches

Activation of the noncanonical pathway through the interaction of lymphotoxin (LT)-alpha(1)beta(2) and LT-betaR is essential for the development of secondary lymphoid organs including lymph nodes (LN) and Peyer's patches (PP). Although TNFR-associated factor (TRAF) 2 and TRAF5 were identified as signal transducers for the LT-betaR, roles for TRAF2 and TRAF5 in the development of secondary lymphoid organs remain obscure. In this study, we show that PP but not mesenteric LN development is severely impaired in traf2(-/-) and traf2(-/-)traf5(-/-) mice. Development of VCAM-1(+) and ICAM-1(+) mesenchymal cells and expression of CXCL13, a crucial chemokine for the development of PP, are severely impaired in PP anlagen in the intestines of traf2(-/-) mice. Surprisingly, TNF-alpha stimulation potently up-regulates cxcl13 mRNA expression in wild-type murine embryonic fibroblasts, which is impaired in traf2(-/-) and relA(-/-) murine embryonic fibroblasts. Moreover, RelA is recruited to the promoter of cxcl13 gene upon TNF-alpha stimulation and PP development is impaired in TNFR type 1 (tnfr1)(-/-) mice. These results underscore a crucial role for the TNFR1-TRAF2-RelA-dependent canonical pathway in the development of PP through up-regulation of cxcl13 mRNA.     

Microfluidic Endothelium for Studying the Intravascular Adhesion of Metastatic Breast Cancer Cells

Background

The ability to properly model intravascular steps in metastasis is essential in identifying key physical, cellular, and molecular determinants that can be targeted therapeutically to prevent metastatic disease. Research on the vascular microenvironment has been hindered by challenges in studying this compartment in metastasis under conditions that reproduce in vivo physiology while allowing facile experimental manipulation.

Methodology/Principal Findings

We present a microfluidic vasculature system to model interactions between circulating breast cancer cells with microvascular endothelium at potential sites of metastasis. The microfluidic vasculature produces spatially-restricted stimulation from the basal side of the endothelium that models both organ-specific localization and polarization of chemokines and many other signaling molecules under variable flow conditions. We used this microfluidic system to produce site-specific stimulation of microvascular endothelium with CXCL12, a chemokine strongly implicated in metastasis.

Conclusions/Significance

When added from the basal side, CXCL12 acts through receptor CXCR4 on endothelium to promote adhesion of circulating breast cancer cells, independent of CXCL12 receptors CXCR4 or CXCR7 on tumor cells. These studies suggest that targeting CXCL12-CXCR4 signaling in endothelium may limit metastases in breast and other cancers and highlight the unique capabilities of our microfluidic device to advance studies of the intravascular microenvironment in metastasis.     

The role of CXC chemokine ligand 4/CXC chemokine receptor 3-B in breast cancer progression

Chemokines and their receptors participate in the development of cancers by enhancing tumor cell proliferation, angiogenesis, invasion, metastasis and penetration of tumor immune cells. It remains unclear whether CXC chemokine ligand 4 (CXCL4)/CXC chemokine receptor 3-B (CXCR3-B) can be used as an independent molecular marker for establishing prognosis for breast cancer patients. We evaluated CXCL4 and CXCR3-B expression in 114 breast cancer tissues and 30 matched noncancerous tissues using immunohistochemistry and western blot, and determined the correlation between their expression and clinicopathologic findings. We observed that breast cancer tissues express CXCL4 strongly and CXCR3-B weakly compared to noncancerous tissues. Strong CXCL4 expression was detected in 94.7% and weak CXCR3-B expression was detected in 78.9% of the tissues. Therefore, CXCL4/CXCR3-B might play a crucial role in breast cancer progression. We found no significant correlation between CXCL4 and age, tumor stage, tumor grade or TNM stage. CXCR3-B was associated significantly with tumor grade. Moreover, the Chi-square test of association showed that the expression of CXCL4/CXCR3-B might be an independent prognostic marker for breast cancer. Therefore, we suggest that CXCR3-B is an indicator of poor prognosis and may also be a chemotherapeutic target.     

Therapeutic efficacy and molecular mechanisms of snake (Walterinnesia aegyptia) venom-loaded silica nanoparticles in the treatment of breast cancer- and prostate cancer-bearing experimental mouse models

The treatment of drug-resistant cancer is a clinical challenge, and thus screening for novel anticancer drugs is critically important. We recently demonstrated a strong enhancement of the antitumor activity of snake (Walterinnesia aegyptia) venom (WEV) in vitro in breast carcinoma, prostate cancer, and multiple myeloma cell lines but not in normal cells when the venom was combined with silica nanoparticles (WEV+NP). In the present study, we investigated the in vivo therapeutic efficacy of WEV+NP in breast cancer- and prostate cancer-bearing experimental mouse models. Xenograft breast and prostate tumor mice models were randomized into 4 groups for each cancer model (10 mice per group) and were treated with vehicle (control), NP, WEV, or WEV+NP daily for 28 days post tumor inoculation. The tumor volumes were monitored throughout the experiment. On Day 28 post tumor inoculation, breast and prostate tumor cells were collected and either directly cultured for flow cytometry analysis or lysed for Western blot and ELISA analysis. Treatment with WEV+NP or WEV alone significantly reduced both breast and prostate tumor volumes compared to treatment with NP or vehicle alone. Compared to treatment with WEV alone, treatment of breast and prostate cancer cells with WEV+NP induced marked elevations in the levels of reactive oxygen species (ROS), hydroperoxides, and nitric oxide; robust reductions in the levels of the chemokines CXCL9, CXCL10, CXCL12, CXCL13, and CXCL16 and decreased surface expression of their cognate chemokine receptors CXCR3, CXCR4, CXCR5, and CXCR6; and subsequent reductions in the chemokine-dependent migration of both breast and prostate cancer cells. Furthermore, we found that WEV+NP strongly inhibited insulin-like growth factor 1 (IGF-1)- and epidermal growth factor (EGF)-mediated proliferation of breast and prostate cancer cells, respectively, and enhanced the induction of apoptosis by increasing the activity of caspase-3,–8, and -9 in both breast and prostate cancer cells. In addition, treatment of breast and prostate cancer cells with WEV+NP or WEV alone revealed that the combination of WEV with NP robustly decreased the phosphorylation of AKT, ERK, and IκBα; decreased the expression of cyclin D1, surviving, and the antiapoptotic Bcl-2 family members Bcl-2, Bcl-_XL, and Mcl-1; markedly increased the expression of cyclin B1 and the proapoptotic Bcl-2 family members Bak, Bax, and Bim; altered the mitochondrial membrane potential; and subsequently sensitized tumor cells to growth arrest. Our data reveal the therapeutic potential of the nanoparticle-sustained delivery of snake venom against different cancer cell types.     

Cell,Isoform, and Environment Factors Shape Gradients and Modulate Chemotaxis

Chemokine gradient formation requires multiple processes that include ligand secretion and diffusion, receptor binding and internalization, and immobilization of ligand to surfaces. To understand how these events dynamically shape gradients and influence ensuing cell chemotaxis, we built a multi-scale hybrid agent-based model linking gradient formation, cell responses, and receptor-level information. The CXCL12/CXCR4/CXCR7 signaling axis is highly implicated in metastasis of many cancers. We model CXCL12 gradient formation as it is impacted by CXCR4 and CXCR7, with particular focus on the three most highly expressed isoforms of CXCL12. We trained and validated our model using data from an in vitro microfluidic source-sink device. Our simulations demonstrate how isoform differences on the molecular level affect gradient formation and cell responses. We determine that ligand properties specific to CXCL12 isoforms (binding to the migration surface and to CXCR4) significantly impact migration and explain differences in in vitro chemotaxis data. We extend our model to analyze CXCL12 gradient formation in a tumor environment and find that short distance, steep gradients characteristic of the CXCL12-γ isoform are effective at driving chemotaxis. We highlight the importance of CXCL12-γ in cancer cell migration: its high effective affinity for both extracellular surface sites and CXCR4 strongly promote CXCR4+ cell migration. CXCL12-γ is also more difficult to inhibit, and we predict that co-inhibition of CXCR4 and CXCR7 is necessary to effectively hinder CXCL12-γ-induced migration. These findings support the growing importance of understanding differences in protein isoforms, and in particular their implications for cancer treatment.     

Baohuoside i suppresses breast cancer metastasis by downregulating the tumor-associated macrophages/C-X-C motif chemokine ligand 1 pathway

BackgroundBreast cancer is the most common malignancy in women and metastasis is the leading cause of breast cancer-related deaths. Our previous studies have shown that XIAOPI formula, a newly approved drug by the State Food and Drug Administration of China (SFDA), can dramatically inhibit breast cancer metastasis by modulating the tumor-associated macrophages/C-X-C motif chemokine ligand 1 (TAMs/CXCL1) pathway. However, the bioactive compound accounting for the anti-metastatic effect of XIAOPI formula remains unclear.PurposeThis study was designed to separate the anti-metastatic bioactive compound from XIAOPI formula and to elucidate its action mechanisms.Study Design/MethodsTAMs/CXCL1 promoter activity-guided fractionation and multiple chemical structure identification approaches were conducted to screen the bioactive compound from XIAOPI formula. Breast cancer cells and TAMs were co-cultured in vitro or co-injected in vivo to simulate their coexistence. Multiple molecular biology experiments, zebrafish breast cancer xenotransplantation model and mouse breast cancer xenografts were applied to validate the anti-metastatic activity of the screened compound.ResultsBioactivity-guided fractionation identified baohuoside I (BHS) as the key bioactive compound of XIAOPI formula in inhibiting TAMs/CXCL1 promoter activity. Functional studies revealed that BHS could significantly inhibit the migration and invasion as well as the expression of metastasis-related proteins in both human and mouse breast cancer cells, along with decreasing the proportion of breast cancer stem cells (CSCs). Furthermore, BHS could suppress the M2 phenotype polarization of TAMs and therefore attenuate their CXCL1 expression and secretion. Notably, mechanistic investigations validated TAMs/CXCL1 as the crucial target of BHS in suppressing breast cancer metastasis as exogenous addition of CXCL1 significantly abrogated the anti-metastatic effect of BHS on breast cancer cells. Moreover, BHS was highly safe in vivo as it exhibited no observable embryotoxicity or teratogenic effect on zebrafish embryos. More importantly, BHS remarkably suppressed breast cancer metastasis and TAMs/CXCL1 activity in both zebrafish breast cancer xenotransplantation model and mouse breast cancer xenografts.ConclusionThis study not only provides novel insights into TAMs/CXCL1 as a reliable screening target for anti-metastatic drug discovery, but also suggests BHS as a promising candidate drug for metastatic breast cancer treatment.