NOVEL ROLE OF LYMPHATIC AND BLOOD VASCULATURES IN BREAST CANCER GROWTH AND METASTASIS AND PEPTIDE AGENTS WITH ANTI-LYMPHANGIOGENIC AND ANTI-ANGIOGENIC ACTIVITY

Breast Cancer;Metastasis;Lymphangiogenesis;Angiogenesis;Peptide Agents;Biomedical Engineering

Lee, EsakSukumar, Saraswati ;

Johns Hopkins University

关键词: Breast Cancer;    Metastasis;    Lymphangiogenesis;    Angiogenesis;    Peptide Agents;    Biomedical Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/39501/LEE-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Up to 90% of deaths from breast cancer are a result of metastasis. Metastasis is a very complex process that has been difficult to fully understand as it involves diverse parenchymal and stromal cells and their secreted factors in the tumor and organ microenvironment. Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer for which current therapeutic options are very limited. This dissertation investigates molecular mechanisms of TNBC growth and metastasis by focusing on lymphatic and blood vasculatures in the tumor and organ microenvironment. Crosstalk between TNBC cells and lymphatic or blood endothelial cells reveals under-investigated roles of the microenvironment in pre-metastatic niche formation in distant organs as well as tumor progression in primary sites. The dissertation also focuses on development of novel anti-lymphangiogenic and anti-angiogenic peptides to treat TNBC. For investigating cancer metastasis and testing anti-metastatic therapeutic agents, efficient and reproducible spontaneous metastasis models are needed. Conventional spontaneous breast cancer metastasis models require a long period of observation after establishment of primary tumors to see significant metastatic progression. The dissertation demonstrates that pre-treatment of animals with tumor-conditioned media (TCM) prepared from TNBC cells accelerates spontaneous metastasis in the corresponding TNBC animal models. The TCM contains all the factors secreted by TNBC cells; thus the injection of TCM conditions pre-metastatic niche. An inguinal breast tumor model facilitated by TCM showed robust thoracic metastasis in the lymph nodes (LN) and the lungs, compared to the serum-free media (SFM) treated control group. The TCM-induced metastasis model was further investigated by focusing on molecular crosstalk between lymphatic endothelial cells (LEC) and TNBC cells, as the pre-metastatic organs in TCM-treated animals showed highly enhanced lymphangiogenesis. The dissertation shows that LEC within pre-metastatic niches are educated by TNBC cells to accelerate metastasis in the lungs and the LN. LEC within these organs, educated by tumor secretion secretes a chemokine, CCL5 that is not secreted by either physiological LEC or TNBC cells, directing CCR5-positive TNBC cell dissemination into the tissues. Moreover, tumor-educated LEC promote angiogenesis in these organs by secreting VEGFA, allowing tumor extravasation in the lungs and colonization in the LN. Mechanistically, interleukin-6 (IL6) secreted by the TNBC cells activates Stat3 phosphorylation, causing the formation of a pStat3-pc-Jun-pATF-2 ternary complex, inducing HIF-1α expression in LEC, and ultimately resulting in expression of CCL5 and VEGF. Additional crosstalk between TNBC cells and LEC shows that LEC promote TNBC cell proliferation and induce pericyte recruitment in primary tumor microenvironment by expressing EGF and PDGF-BB, thus promoting tumor growth. Surprisingly, microvascular endothelial cells (MEC) showed an opposite effect by suppressing tumor growth.Motivated by the knowledge that angiogenesis supports tumor growth and metastasis and lymphangiogenesis actively conditions pre-metastatic niches and promotes breast tumor metastasis, novel anti-lymphangiogenic and anti-angiogenic peptides were developed to target tumor growth and metastasis in TNBC. Endogenous peptides derived from proteins containing a conserved somatotropin domain were screened for inhibition of angiogenesis and lymphangiogenesis using in vitro proliferation, migration, adhesion and tube formation assays with blood and lymphatic endothelial cells. A short 14-mer peptide derived from transmembrane protein 45A human shows the most potent multimodal inhibition of angiogenesis and lymphangiogenesis in breast tumor xenografts and tumor-conditioned lymph nodes. Mechanistically, the peptide blocks vascular endothelial growth factor receptors 2 and 3 (VEGFR2/3) and downstream proteins by binding to neuropilin 1/2 (NRP1/2) and inhibiting VEGFR2/3 and NRP1/2 complex formation in the presence of VEGFA/C. A mimetic 20-mer peptide derived from Collagen IV shows synergy with the somatotropin-derived peptide as inhibitors of lymphangiogenesis in vitro and in vivo. The collagen-derived peptide was further optimized, and was evaluated in vitro in lymphangiogenesis and angiogenesis cell assays, and in animal experiments including TNBC breast tumor xenograft and TCM-induced distant metastasis models. In summary, this dissertation investigates molecular mechanisms of breast cancer metastasis, proposing novel roles of lymphatic endothelial cells in pre-metastatic organs and primary tumor microenvironment; identifies key molecules regulating metastatic dissemination and colonization as well as tumor growth. The dissertation also explores several novel anti-angiogenic and anti-lymphangiogenic peptides to effectively treat TNBC tumor growth and metastasis.