Purpose: Lymphatic vessels are mainly regarded as passive conduits for the dissemination of cancer cells. In this study, we investigate whether and how the tumor-associated lymphatic vessels may play an active role in tumor metastasis.Experimental Design: In situ laser capture microdissection of lymphatic vessels followed by cDNA microarray analysis was used to determine the expression profiling of lymphatic endothelial cells (LEC). Gene expression levels and activity of signaling pathways were measured by real-time RT-PCR, ELISA, or immunoblotting. Lymphangiogenesis was assessed by IHC. Lymph node metastasis was measured using fluorescence imaging. The effects of SEMA4C on lymphangiogenesis in vitro were evaluated using migration assay and tube-formation assay of LECs.Results: Tumor-associated LECs are molecularly and functionally different from their normal counterparts. In addition to expressing high levels of membrane-bound SEMA4C, tumorassociated LECs also produced soluble SEMA4C (sSEMA4C). Increased serum sSEMA4C was detected in patients with breast cancer and cervical cancer. Patients with metastasis had much higher levels of serum sSEMA4C. sSEMA4C promoted lymphangiogenesis by activating PlexinB2-ERBB2 signaling in LECs, and promoted the proliferation and migration of tumor cells by activating PlexinB2-MET signaling, thus promoting lymphatic metastasis. Although the SEMA4C signaling pathways differ between LECs and tumor cells, RHOA activation was necessary for the effects of SEMA4C in both types of cells.Conclusions: Tumor-associated LECs produce sSEMA4C to promote lymphatic metastasis of tumors. Our results suggest that SEMA4C and RHOA might be potential therapeutic targets, and that higher serum sSEMA4C could be a marker for breast cancer and cervical cancer.

Background: No effective approaches to target mutant Kras have yet been developed. Immunoprevention using KRAS-specific antigenic peptides to trigger T cells capable of targeting tumor cells relies heavily on lipid metabolism. To facilitate better TCR/peptide/MHC interactions that result in better cancer preventive efficacy, we combined KVax with avasimibe, a specific ACAT1 inhibitor, tested their anti-cancer efficacy in mouse lung cancer models, where Kras mutation was induced before vaccination. Methods: Control of tumor growth utilizing a multi-peptide Kras vaccine was tested in combination with avasimibe in a syngeneic lung cancer mouse model and a genetically engineered mouse model (GEMM). Activation of immune responses after administration of Kras vaccine and avasimibe was also assessed by flow cytometry, ELISpot and IHC. Findings: We found that Kras vaccine combined with avasimibe significantly decreased the presence of regulatory T cells in the tumor microenvironment and facilitated CD8 + T cell infiltration in tumor sites. Avasimibe also enhanced the efficacy of Kras vaccines target mutant Kras. Whereas the Kras vaccine significantly increased antigen-specific intracellular IFN-γ and granzyme B levels in CD8 + T cells, avasimibe significantly increased the number of tumor-infiltrating CD8 + T cells. Additionally, modulation of cholesterol metabolism was found to specifically impact in T cells, and not in cancer cells. Interpretation: Avasimibe complements the efficacy of a multi-peptide Kras vaccine in controlling lung cancer development and growth. This treatment regimen represents a novel immunoprevention approach to prevent lung cancer.

Early growth response 1 (EGR-1) contributes to the development of chronic obstructive pulmonary disease in the lungs of smokers by mediating pulmonary inflammatory responses, but the direct downstream genes of EGR-1 that regulate this process remain unknown. We show that a new EGR-1 target gene, geranylgeranyl diphosphate synthase (GGPPS), which controls protein prenylation, can regulate the proinflammatory function of EGR-1 by activating MAPK signaling. When C57BL/6 mice were exposed to cigarette smoke, EGR-1 and GGPPS levels increased in their lungs, and the inflammatory responses were augmented, whereas these effects could be reversed by the down-regulation of EGR-1 transcription activity. The accumulation of EGR-1 and GGPPS was induced by MAPK/ERK pathway activation when Beas-2B human bronchial epithelial cells were exposed to cigarette smoke extract (CSE). Further examination showed that EGR-1 in turn regulated Erk1/2 activity because inhibition of EGR-1 transcription activity decreased CSE-induced Erk1/2 phosphorylation. Furthermore, EGR-1-promoted Erk1/2 activation was dependent on GGPPS transcription. Knockdown of GGPPS expression with small-interfering RNA abolished the EGR-1-activated Erk1/2 activity. Both EGR-1 transcription inhibition and GGPPS expression knockdown decreased the inflammatory response induced by CSE in Beas-2B cells. Our results reveal a new EGR-1/GGPPS/MAPK signaling pathway that controls cigarette smoke-induced pulmonary inflammation, and this may shed light on our understanding of the mechanism of cigarette smoke-related pulmonary diseases such as chronic obstructive pulmonary disease.

Intestinal ischemia-reperfusion (I/R) is a critical event in the pathogenesis of multiple organ dysfunction syndromes (MODS). The lungs are some of the most vulnerable organs that are impacted by intestinal I/R. The aim of this study is to investigate whether ginsenoside Rb1 can ameliorate remote lung injury induced by intestinal I/R. Adult male Wistar rats were randomly divided into four groups: (1) a control, sham-operated group (sham group); (2) an intestinal I/R group subjected to 1 h intestinal ischemia and 2 h reperfusion (I/R group); (3) a group treated with 20 mg/kg ginsenoside Rb1 before reperfusion (Rb1-20 group); and (4) a group treated with 40 mg/kg ginsenoside Rb1 before reperfusion (Rb1-40 group). Intestinal and lung histology was observed. The malondialdehyde (MDA) levels in intestinal tissues were measured. Myeloperoxidase (MPO), TNF-α, MDA levels, wet/dry weight ratio and immunohistochemical expression of intracellular adhesion molecule-1 (ICAM-1) in lung tissues were assayed. In addition, a western blot of lung NF-kB was performed. Results indicated that intestinal I/R induced intestinal and lung injury, which was characterized by increase of MDA levels and pathological scores in intestinal tissues and MPO, TNF-α , MDA levels, wet/dry weight ratio and ICAM-1, NF-kB expression in the lung tissues. Ginsenoside Rb1 (20, 40 mg/kg) ameliorated intestinal and lung injury, decreased MPO, TNF-α, MDA levels, wet/dry weight ratio, ICAM-1 and NF-kB expression in lung tissues. In conclusion, ginsenoside Rb1 ameliorated the lung injuries by decreasing the NF-kB activation-induced inflammatory response.