Syndecan-1 induction in lung microenvironment supports the establishment of breast tumor metastases.

BACKGROUND: Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan normally expressed primarily by epithelia and plasma cells, is aberrantly induced in stromal fibroblasts of breast carcinomas. Stromal fibroblast-derived Sdc1 participates in paracrine growth stimulation of breast carcinoma cells and orchestrates stromal extracellular matrix fiber alignment, thereby creating a migration and invasion-permissive microenvironment. Here, we specifically tested the role of stromal Sdc1 in metastasis. METHODS: The metastatic potential of the aggressive mouse mammary carcinoma cell lines, 4T1 and E0776, was tested in wild-type and genetically Sdc1-deficient host animals. Metastatic lesions were characterized by immunohistochemical analysis. RESULTS: After orthotopic inoculation, the lung metastatic burden was reduced in Sdc1-/- animals by 97% and more than 99%, in BALB/cJ and C57BL/6 animals, respectively. The difference in metastatic efficiency was maintained when the tumor cells were injected into the tail vein, suggesting that host Sdc1 exerts its effect during later stages of the metastatic cascade. Co-localization studies identified Sdc1 expression in stromal fibroblasts within the metastatic microenvironment and in normal airway epithelial cells but not in other cells (endothelial cells, α-smooth muscle actin positive cells, leucocytes, macrophages). The Ki67 proliferation index and the rate of apoptosis of the metastatic tumor cells were diminished in Sdc1-/- vs. Sdc1+/+ animals, and leucocyte density was indistinguishable. Sdc1-mediated metastatic efficiency was abolished when the animals were housed at a thermoneutral ambient temperature of 31 °C, suggesting that the host Sdc1 effect on metastasis requires mild cold stress. CONCLUSIONS: In summary, Sdc1 is induced in the lung microenvironment after mammary carcinoma cell dissemination and promotes outgrowth of metastases in a temperature-dependent manner.

Malignant peripheral nerve sheath tumor (MPNST) in the spine: a retrospective analysis of clinical and molecular prognostic factors.

Spinal malignant peripheral nerve sheath tumors (MPNSTs) are relatively rare. There is little information published in the literature regarding this subject. The aim of this retrospective study was to evaluate factors that may affect the outcomes of patients with spinal MPNSTs by reviewing 43 patients with spinal MPNST who were treated in our hospital between 2001 and 2012. Univariate and multivariate analyses were performed to identify prognostic variables relative to patient and tumor characteristics, treatment modality and molecules. All 43 MPNST patients (25 men and 18 women; median age 49 years) underwent surgical resection, of whom 15 patients also underwent postoperative radiotherapy. Local recurrence was found in 21 (48.8 %) patients. Twenty-two (51.2 %) patients died during the follow-up periods with a median survival time of 49 months. The 5-year recurrence and survival rate was 53 and 44 % respectively. The statistical analyses suggested that high-grade malignancy and osteolytic destruction were closely associated with recurrence and death. A total of 38 cases accepted postoperative immunohistochemisty examine. S-100 was identified as an independent factor related to both recurrence and survival, adjusting for clinical factors. In conclusion, we confirmed that malignant grade and osteolytic destruction were the two independent factors for both recurrence and survival, while patients with S-100 protein negative had a higher recurrence rate and a lower survival rate.

A positive feedback loop between prolactin and STAT5 promotes angiogenesis.

The signal transduction events that orchestrate cellular activities required for angiogenesis remain incompletely understood. We and others recently described that proangiogenic mediators such as fibroblast growth factors can activate members of the signal transducers and activators of transcription (STAT) family. STAT5 activation is necessary and sufficient to induce migration, invasion and tube formation of endothelial cells. STAT5 effects on endothelial cells require the secretion of the prolactin (PRL) family member proliferin-1 (PLF1) in mice and PRL in humans. In human endothelial cells, PRL activates the PRL receptor (PRLR) resulting in MAPK and STAT5 activation, thus closing a positive feedback loop. In vivo, endothelial cell-derived PRL is expected to combine with PRL of tumor cell and pituitary origin to raise the concentration of this polypeptide hormone in the tumor microenvironment. Thus, PRL may stimulate tumor angiogenesis via autocrine, paracrine, and endocrine pathways. The disruption of tumor angiogenesis by interfering with PRL signaling may offer an attractive target for therapeutic intervention.

STAT5 and prolactin participate in a positive autocrine feedback loop that promotes angiogenesis.

We have shown previously that the murine prolactin/growth hormone family member proliferin plays a pivotal role in angiogenesis induced by the FGF2/STAT5 signaling cascade. To delineate the signaling pathway downstream of STAT5 in the human system, where proliferin does not exist, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in hCMEC/D3 human brain endothelial cells. We found that conditioned medium from CA-STAT5A- but not from DN-STAT5A-overexpressing endothelial cells (EC) is sufficient to induce EC migration and tube formation but not proliferation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. We identified prolactin (PRL) as a candidate autocrine factor. CA-STAT5A expression stimulates PRL production at the RNA and protein level, and STAT5A binds to the PRL promoter region, suggesting direct transcriptional regulation. Medium conditioned by CA-STAT5A-overexpressing EC induces phosphorylation of the PRL receptor and activates MAPK. Knockdown of PRL expression by shRNA or blocking of PRL activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. The addition of recombinant PRL restores this activity. STAT5A-induced PRL in the conditioned medium can activate STAT5, STAT1, and to a lesser extent STAT3 in hCMEC/D3 cells, suggesting the existence of a positive feedback loop between STAT5 and PRL that promotes angiogenesis. Furthermore, we find that VEGF, a potent proangiogenic factor, is induced by activation of STAT5A, and VEGF induction depends on PRL expression. These observations demonstrate a STAT5/PRL/VEGF signaling cascade in human brain EC and implicate PRL and VEGF as autocrine regulators of EC migration, invasion, and tube formation.

Angiogenesis induced by signal transducer and activator of transcription 5A (STAT5A) is dependent on autocrine activity of proliferin.

Multiple secreted factors induce the formation of new blood vessels (angiogenesis). The signal transduction events that orchestrate the numerous cellular activities required for angiogenesis remain incompletely understood. We have shown previously that STAT5 plays a pivotal role in angiogenesis induced by FGF2 and FGF8b. To delineate the signaling pathway downstream of STAT5, we expressed constitutively active (CA) or dominant-negative (DN) mutant STAT5A in mouse brain endothelial cells (EC). We found that the conditioned medium from CA-STAT5A but not from dominant-negative STAT5A overexpressing EC is sufficient to induce EC invasion and tube formation, indicating that STAT5A regulates the secretion of autocrine proangiogenic factors. Conversely, CA-STAT5A-induced conditioned medium had no effect on EC proliferation. Using a comparative genome-wide transcription array screen, we identified the prolactin family member proliferin (PLF1 and PLF4) as a candidate autocrine factor. The CA-STAT5A-dependent transcription and secretion of PLF by EC was confirmed by quantitative RT-PCR and Western blotting, respectively. CA-STAT5A binds to the PLF1 promoter region, suggesting a direct transcriptional regulation. Knockdown of PLF expression by shRNA or by blocking of PLF activity with neutralizing antibodies removed the CA-STAT5A-dependent proangiogenic activity from the conditioned medium of EC. Similarly, the ability of concentrated conditioned medium from CA-STAT5A transfected EC to induce angiogenesis in Matrigel plugs in vivo was abolished when PLF was depleted from the medium. These observations demonstrate a FGF/STAT5/PLF signaling cascade in EC and implicate PLF as autocrine regulator of EC invasion and tube formation.

Scaffold stiffness influences breast cancer cell invasion via EGFR-linked Mena upregulation and matrix remodeling.

Clinically, increased breast tumor stiffness is associated with metastasis and poorer outcomes. Yet, in vitro studies of tumor cells in 3D scaffolds have found decreased invasion in stiffer environments. To resolve this apparent contradiction, MDA-MB-231 breast tumor spheroids were embedded in 'low' (2 kPa) and 'high' (12 kPa) stiffness 3D hydrogels comprised of methacrylated gelatin/collagen I, a material that allows for physiologically-relevant changes in stiffness while matrix density is held constant. Cells in high stiffness materials exhibited delayed invasion, but more abundant actin-enriched protrusions, compared to those in low stiffness. We find that cells in high stiffness had increased expression of Mena, an invadopodia protein associated with metastasis in breast cancer, as a result of EGFR and PLCγ1 activation. As invadopodia promote invasion through matrix remodeling, we examined matrix organization and determined that spheroids in high stiffness displayed a large fibronectin halo. Interestingly, this halo did not result from increased fibronectin production, but rather from Mena/α5 integrin dependent organization. In high stiffness environments, FN1 knockout inhibited invasion while addition of exogenous cellular fibronectin lessened the invasion delay. Analysis of fibronectin isoforms demonstrated that EDA-fibronectin promoted invasion and that clinical invasive breast cancer specimens displayed elevated EDA-fibronectin. Combined, our data support a mechanism by which breast cancer cells respond to stiffness and render the environment conducive to invasion. More broadly, these findings provide important insight on the roles of matrix stiffness, composition, and organization in promoting tumor invasion.

Leader cell PLCγ1 activation during keratinocyte collective migration is induced by EGFR localization and clustering.

Re-epithelialization is a critical step in wound healing and results from the collective migration of keratinocytes. Previous work demonstrated that immobilized, but not soluble, epidermal growth factor (EGF) resulted in leader cell-specific activation of phospholipase C gamma 1 (PLCγ1) in HaCaT keratinocytes, and that this PLCγ1 activation was necessary to drive persistent cell migration. To determine the mechanism responsible for wound edge-localized PLCγ1 activation, we examined differences in cell area, cell-cell interactions, and EGF receptor (EGFR) localization between wound edge and bulk cells treated with vehicle, soluble EGF, or immobilized EGF. Our results support a multistep mechanism where EGFR translocation from the lateral membrane to the basolateral/basal membrane allows clustering in response to immobilized EGF. This analysis of factors regulating PLCγ1 activation is a crucial step toward developing therapies or wound dressings capable of modulating this signal and, consequently, cell migration.

Constitutive regulation of CYP1B1 by the aryl hydrocarbon receptor (AhR) in pre-malignant and malignant mammary tissue.

The aryl hydrocarbon receptor (AhR) is a receptor/transcription factor which regulates cytochrome P450 (CYP) gene transcription and which is activated by environmental carcinogens, some of which are associated with increased breast cancer risk. Here, we show that the AhR is over-expressed and constitutively active in human and rodent mammary tumors, suggesting its ongoing contribution to tumorigenesis regardless of tumor etiology. AhR regulation of CYP1A1 and CYP1B1 was studied to determine if constitutively active AhR effects the same transcriptional outcomes as environmental chemical-activated AhR. Elevated AhR and CYP1B1 but not CYP1A1 before tumor formation in a rat model of mammary tumorigenesis suggested differential CYP1B1 regulation by a constitutively active AhR. This hypothesis was tested with human mammary gland cell lines which hyper-express AhR and CYP1B1 but which express little or no CYP1A1. CYP1B1 expression was diminished by repression of AhR activity or by AhR knockdown, demonstrating AhR control of basal CYP1B1 levels. ChIP assays demonstrated constitutive AhR binding to both CYP1A1 and CYP1B1 promoters, demonstrating that differential CYP1A1 and CYP1B1 regulation by constitutively active AhR does not result from different amounts of promoter-bound AhR. While increasing AhR binding to both CYP1A1 and CYP1B1, 2,3,7,8-tetrachlorodibenzo-p-dioxin induced CYP1A1 mRNA in both a malignant and non-malignant line but increased only CYP1B1 mRNA in the malignant line, again demonstrating that the level of promoter binding does not necessarily correlate with gene mRNA levels. These studies suggest that constitutively active AhR mediates different molecular outcomes than environmental chemical-activated AhR, and further implicate the AhR in mammary tumorigenesis.

The aryl hydrocarbon receptor constitutively represses c-myc transcription in human mammary tumor cells.

The aryl hydrocarbon receptor (AhR) is an environmental carcinogen-activated transcription factor associated with tumorigenesis. High levels of apparently active AhR characterize a variety of tumors, even in the absence of environmental ligands. Despite this association between transformation and AhR upregulation, little is known of the transcriptional consequences of constitutive AhR activation. Here, the effects of constitutively active and environmental ligand-induced AhR on c-myc, an oncogene whose promoter contains six AhR-binding sites (AhREs (aryl hydrocarbon response elements)), were investigated. A reporter containing the human c-myc promoter, with its six AhREs and two NF-kappaB-binding sites, was constructed. This vector, and variants with deletions in the NF-kappaB and/or AhR-binding sites, was transfected into a human breast cancer cell line, Hs578T, which expresses high levels of apparently active, nuclear AhR. Results indicate that: (1) the AhR constitutively binds the c-myc promoter; (2) there is a low but significant baseline level of c-myc promoter activity, which is not regulated by NF-kappaB and is not affected by an environmental AhR ligand; (3) deletion of any one of the AhREs has no effect on constitutive reporter activity, while deletion of all six increases reporter activity approximately fivefold; (4) a similar increase in reporter activity occurs when constitutively active AhR is suppressed by transfection with an AhR repressor plasmid (AhRR); (5) AhRR transfection significantly increases background levels of endogenous c-myc mRNA and c-Myc protein. These results suggest that the AhR influences the expression of c-Myc, a protein critical to malignant transformation.

Growth of a human mammary tumor cell line is blocked by galangin, a naturally occurring bioflavonoid, and is accompanied by down-regulation of cyclins D3, E, and A.

INTRODUCTION: This study was designed to determine if and how a non-toxic, naturally occurring bioflavonoid, galangin, affects proliferation of human mammary tumor cells. Our previous studies demonstrated that, in other cell types, galangin is a potent inhibitor of the aryl hydrocarbon receptor (AhR), an environmental carcinogen-responsive transcription factor implicated in mammary tumor initiation and growth control. Because some current breast cancer therapeutics are ineffective in estrogen receptor (ER) negative tumors and since the AhR may be involved in breast cancer proliferation, the effects of galangin on the proliferation of an ER-, AhRhigh line, Hs578T, were studied. METHODS: AhR expression and function in the presence or absence of galangin, a second AhR inhibitor, alpha-naphthoflavone (alpha-NF), an AhR agonist, indole-3-carbinol, and a transfected AhR repressor-encoding plasmid (FhAhRR) were studied in Hs578T cells by western blotting for nuclear (for instance, constitutively activated) AhR and by transfection of an AhR-driven reporter construct, pGudLuc. The effects of these agents on cell proliferation were studied by 3H-thymidine incorporation and by flow cytometry. The effects on cyclins implicated in mammary tumorigenesis were evaluated by western blotting. RESULTS: Hs578T cells were shown to express high levels of constitutively active AhR. Constitutive and environmental chemical-induced AhR activity was profoundly suppressed by galangin as was cell proliferation. However, the failure of alpha-NF or FhAhRR transfection to block proliferation indicated that galangin-mediated AhR inhibition was either insufficient or unrelated to its ability to significantly block cell proliferation at therapeutically relevant doses (IC50 = 11 microM). Galangin inhibited transition of cells from the G0/G1 to the S phases of cell growth, likely through the nearly total elimination of cyclin D3. Expression of cyclins A and E was also suppressed. CONCLUSION: Galangin is a strong inhibitor of Hs578T cell proliferation that likely mediates this effect through a relatively unique mechanism, suppression of cyclin D3, and not through the AhR. The results suggest that this non-toxic bioflavonoid may be useful as a chemotherapeutic, particularly in combination with agents that target other components of the tumor cell cycle and in situations where estrogen receptor-specific therapeutics are ineffective.