Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages.

The extracellular serine protease inhibitor serpinE2 is overexpressed in breast cancer and has been shown to foster metastatic spread. Here, we investigated the hypothesis that serpinE2 creates tumor-promoting conditions in the tumor microenvironment (TME) by affecting extracellular matrix remodeling. Using two different breast cancer models, we show that blocking serpinE2, either by knock-down (KD) in tumor cells or in response to a serpinE2 binding antibody, decreases metastatic dissemination from primary tumors to the lungs. We demonstrate that in response to serpinE2 KD or antibody treatment there are dramatic changes in the TME. Multiphoton intravital imaging revealed deposition of a dense extracellular collagen I matrix encapsulating serpinE2 KD or antibody-treated tumors. This is accompanied by a reduction in the population of tumor-promoting macrophages, as well as a decrease in chemokine ligand 2, which is known to affect macrophage abundance and polarization. In addition, TIMP-1 secretion is increased, which may directly inhibit matrix metalloproteases critical for collagen degradation in the tumor. In summary, our findings suggest that serpinE2 is required in the extracellular milieu of tumors where it acts in multiple ways to regulate tumor matrix deposition, thereby controlling tumor cell dissemination.

The serine protease inhibitor protease nexin-1 controls mammary cancer metastasis through LRP-1-mediated MMP-9 expression.

Through their ability to degrade the extracellular matrix, proteases mediate cancer cell invasion and metastasis. Paradoxically, some serine protease inhibitors (serpins) are often overexpressed in human tumors. Using computational analysis, we found that the RNA level of protease nexin-1 (PN-1), a serpin that blocks numerous proteases activity, is significantly elevated in estrogen receptor-alpha-negative and in high-grade breast cancer. The in silico approach was complemented by mechanistic studies on two mammary cancer cell lines, the PN-1-negative 168FARN cells and the PN-1-positive 4T1 cells, both of which form primary mammary tumors, but only 4T1 tumors are able to metastasize to the lungs. We show that treatment of 168FARN cells with PN-1 stimulates extracellular signal-regulated kinase activation via low-density lipoprotein receptor-related protein-1 (LRP-1) binding, resulting in increased matrix metalloproteinase (MMP)-9 RNA, protein, and secreted activity. PN-1-silenced 4T1 cells express low MMP-9 levels. Moreover, injection of PN-1-silenced cells into mice did not affect 4T1 primary mammary tumor outgrowth; however, the tumors had impaired metastatic potential, which could be restored by reexpressing soluble MMP-9 in the PN-1-silenced 4T1 cells. Thus, using mammary tumor models, we describe a novel pathway whereby the serpin PN-1 by binding LRP-1 stimulates extracellular signal-regulated kinase signaling, MMP-9 expression, and metastatic spread of mammary tumors. Importantly, an analysis of 126 breast cancer patients revealed that those whose breast tumors had elevated PN-1 levels had a significantly higher probability to develop lung metastasis, but not metastasis to other sites, on relapse. These results suggest that PN-1 might become a prognostic marker in breast cancer.

Determinants for chromogranin A sorting into the regulated secretory pathway are also sufficient to generate granule-like structures in non-endocrine cells.

In endocrine cells, prohormones and granins are segregated in the TGN (trans-Golgi network) from constitutively secreted proteins, stored in concentrated form in dense-core secretory granules, and released in a regulated manner on specific stimulation. The mechanism of granule formation is only partially understood. Expression of regulated secretory proteins, both peptide hormone precursors and granins, had been found to be sufficient to generate structures that resemble secretory granules in the background of constitutively secreting, non-endocrine cells. To identify which segment of CgA (chromogranin A) is important to induce the formation of such granule-like structures, a series of deletion constructs fused to either GFP (green fluorescent protein) or a short epitope tag was expressed in COS-1 fibroblast cells and analysed by fluorescence and electron microscopy and pulse-chase labelling. Full-length CgA as well as deletion constructs containing the N-terminal 77 residues generated granule-like structures in the cell periphery that co-localized with co-expressed SgII (secretogranin II). These are essentially the same segments of the protein that were previously shown to be required for granule sorting in wild-type PC12 (pheochromocytoma cells) cells and for rescuing a regulated secretory pathway in A35C cells, a variant PC12 line deficient in granule formation. The results support the notion that self-aggregation is at the core of granule formation and sorting into the regulated pathway.

Mice lacking protease nexin-1 show delayed structural and functional recovery after sciatic nerve crush.

Multiple molecular mechanisms influence nerve regeneration. Because serine proteases were shown to affect peripheral nerve regeneration, we performed nerve crush experiments to study synapse reinnervation in adult mice lacking the serpin protease nexin-1 (PN-1). PN-1 is a potent endogenous inhibitor of thrombin, trypsin, tissue plasminogen activators (tPAs), and urokinase plasminogen activators. Compared with the wild type, a significant delay in synapse reinnervation was detected in PN-1 knock-out (KO) animals, which was associated with both reduced proliferation and increased apoptosis of Schwann cells. Various factors known to affect Schwann cells were also altered. Fibrin deposits, tPA activity, mature BDNF, and the low-affinity p75 neurotrophin receptor were increased in injured sciatic nerves of mutant mice. To test whether the absence of PN-1 in Schwann cells or in the axon caused delay in reinnervation, PN-1 was overexpressed exclusively in the nerves of PN-1 KO mice. Neuronal PN-1 expression did not rescue the delayed reinnervation. The results suggest that Schwann cell-derived PN-1 is crucial for proper reinnervation through its contribution to the autocrine control of proliferation and survival. Thus, the precise balance between distinct proteases and serpins such as PN-1 can modulate the overall impact on the kinetics of recovery.

Interleukin-6 induces transcriptional activation of vascular endothelial growth factor (VEGF) in astrocytes in vivo and regulates VEGF promoter activity in glioblastoma cells via direct interaction between STAT3 and Sp1.

Interleukin-6 (IL-6) expression is strongly correlated with the degree of human glioma malignancy and necessary for tumor formation in a mouse model of spontaneous astrocytomas. Yet, exactly how IL-6 contributes to malignant progression of these brain tumors is still unclear. We have scrutinized the mechanism of transcriptional activation of vascular endothelial growth factor (VEGF) expression by IL-6 in the mouse brain and in glioblastoma cells. We demonstrate here that IL-6 drives transcriptional upregulation of VEGF in astrocytes in vivo using glial fibrillary acidic protein (GFAP)-IL-6/VEGF-green fluorescent protein (GFP) double transgenic mice. We further show that IL-6-induced VEGF transcription and VEGF secretion by human glioblastoma cells is dependent on signal transducer and activator of transcription 3 (STAT3). By progressive 5'-deletion analysis we defined the minimal VEGF promoter region for IL-6-responsiveness to nucleotides -88/-50. Surprisingly, this promoter region is rich in GC-boxes and does not contain STAT3 binding elements. Electrophoretic mobility shift and supershift assays revealed binding of Sp1 and Sp3 to the -88/-50 element upon IL-6 stimulation. Interestingly, preincubation with STAT3 antibody prevented the binding of Sp1 and Sp3 to the -88/-50 element, indicating that STAT3 is involved in IL-6-driven Sp1/Sp3 protein-DNA complex formation. Physical interaction of STAT3 and Sp1 was demonstrated by coimmunoprecipitation. The functional relevance of the STAT3/Sp1 association was corroborated by transient transfection experiments, which showed that overexpression of constitutively active STAT3 increased the minimal VEGF promoter activity. Taken together, our study suggests that IL-6 promotes tumor angiogenesis in gliomas and describes a novel transcriptional activation mechanism for STAT3 in the context of a STAT3 binding element (SBE)-free promoter.