Esak Lee

Polyproline‐type helical‐structured low‐molecular weight heparin (LMWH)‐taurocholate conjugate as a new angiogenesis inhibitor

Although heparin can regulate angiogenesis, tumor growth and metastasis, its clinical application, as well as that of low‐molecular heparin (LMWH), for treating cancer are limited because of heparins anticoagulant activity and risk of hemorrhages. LMWH‐taurocholate conjugates (LHT7), which have low anticoagulant activity, were synthesized. The structural property of LHT was evaluated by circular dichroism and the binding affinity of LHT7 to vascular endothelial growth factor 165 (VEGF165) was measured by isothermal titration calorimetry. The inhibitory effect of LHT7 on VEGF‐mediated KDR (VEGF‐receptor 2) phosphorylation in Human umbilical vein endothelial cells was evaluated. The VEGF165 dependent Matrigel plug assay was performed to verify the antiangiogenic potential of LHT7 on a VEGF165 inhibitor. Finally, tumor growth inhibition effects of LHT7 on SCC7 and the survival rate of animal models were investigated. Moreover, MDA‐MB231 xenograft mouse model was additionally used to confirm the therapeutic effect of LHT7 on human breast cancer cell line. As a result, LHT7 which has 12.7% of anticoagulant activity of the original LMWH showed a peculiar polyproline‐type helical structure. LHT7 binds to VEGF strongly and inhibits VEGF dependent KDR phosphorylation. The results of Matrigel plug assay proved LHT7 as a strong antiangiogenic agent inhibiting VEGF165. Remarkably, LHT7 showed a significant tumor growth inhibition potential on SCC7 with an increased survival rate. LHT7 also delayed tumor growth in MDA‐MB231 human breast cancer cell lines.

Breast cancer cells condition lymphatic endothelial cells within pre-metastatic niches to promote metastasis.

Breast cancer metastasis involves lymphatic dissemination in addition to hematogenous spreading. Although stromal lymphatic vessels (LVs) serve as initial metastatic routes, roles of organ-residing LVs are under-investigated. Here we show that lymphatic endothelial cells (LECs), a component of LVs within pre-metastatic niches, are conditioned by triple-negative breast cancer (TNBC) cells to accelerate metastasis. LECs within the lungs and lymph nodes, conditioned by tumor-secreted factors express CCL5 that is not expressed either in normal LECs or cancer cells, and direct tumor dissemination into these tissues. Moreover, tumor-conditioned LECs promote angiogenesis in these organs, allowing tumor extravasation and colonization. Mechanistically, tumor cell-secreted IL6 causes Stat3 phosphorylation in LECs. This pStat3 induces HIF-1α and VEGF, and a pStat3-pc-Jun-pATF-2 ternary complex induces CCL5 expression in LECs. This study demonstrates anti-metastatic activities of multiple repurposed drugs, blocking a self-reinforcing paracrine loop between breast cancer cells and LECs.

Crosstalk between cancer cells and blood endothelial and lymphatic endothelial cells in tumour and organ microenvironment.

Tumour and organ microenvironments are crucial for cancer progression and metastasis. Crosstalk between multiple non-malignant cell types in the microenvironments and cancer cells promotes tumour growth and metastasis. Blood and lymphatic endothelial cells (BEC and LEC) are two of the components in the microenvironments. Tumour blood vessels (BV), comprising BEC, serve as conduits for blood supply into the tumour, and are important for tumour growth as well as haematogenous tumour dissemination. Lymphatic vessels (LV), comprising LEC, which are relatively leaky compared with BV, are essential for lymphogenous tumour dissemination. In addition to describing the conventional roles of the BV and LV, we also discuss newly emerging roles of these endothelial cells: their crosstalk with cancer cells via molecules secreted by the BEC and LEC (also called angiocrine and lymphangiocrine factors). This review suggests that BEC and LEC in various microenvironments can be orchestrators of tumour progression and proposes new mechanism-based strategies to discover new therapies to supplement conventional anti-angiogenic and anti-lymphangiogenic therapies.

Small peptides derived from somatotropin domain-containing proteins inhibit blood and lymphatic endothelial cell proliferation, migration, adhesion and tube formation

Angiogenesis is thoroughly balanced and regulated in health; however, it is dysregulated in many diseases including cancer, age-related macular degeneration, cardiovascular diseases such as coronary and peripheral artery diseases and stroke, abnormal embryonic development, and abnormal wound healing. In addition to angiogenesis, lymphangiogenesis is pivotal for maintaining the immune system, homeostasis of body fluids and lymphoid organs; dysregulated lymphangiogenesis may cause inflammatory diseases and lymph node mediated tumor metastasis. Anti-angiogenic or anti-lymphangiogenic small peptides may play an important role as therapeutic agents normalizing angiogenesis or lymphangiogenesis in disease conditions. Several novel endogenous peptides derived from proteins containing a conserved somatotropin domain have been previously identified with the help of our bioinformatics-based methodology. These somatotropin peptides were screened for inhibition of angiogenesis and lymphangiogenesis using in vitro proliferation, migration, adhesion and tube formation assays with blood and lymphatic endothelial cells. We found that the peptides have the potential for inhibiting both angiogenesis and lymphangiogenesis. Focusing the study on the inhibition of lymphangiogenesis, we found that a peptide derived from the somatotropin conserved domain of transmembrane protein 45A human was the most potent lymphangiogenesis inhibitor, blocking lymphatic endothelial cell migration, adhesion, and tube formation.

Laminar flow downregulates Notch activity to promote lymphatic sprouting

The major function of the lymphatic system is to drain interstitial fluid from tissue. Functional drainage causes increased fluid flow that triggers lymphatic expansion, which is conceptually similar to hypoxia-triggered angiogenesis. Here, we have identified a mechanotransduction pathway that translates laminar flow–induced shear stress to activation of lymphatic sprouting. While low-rate laminar flow commonly induces the classic shear stress responses in blood endothelial cells and lymphatic endothelial cells (LECs), only LECs display reduced Notch activity and increased sprouting capacity. In response to flow, the plasma membrane calcium channel ORAI1 mediates calcium influx in LECs and activates calmodulin to facilitate a physical interaction between Krüppel-like factor 2 (KLF2), the major regulator of shear responses, and PROX1, the master regulator of lymphatic development. The PROX1/KLF2 complex upregulates the expression of DTX1 and DTX3L. DTX1 and DTX3L, functioning as a heterodimeric Notch E3 ligase, concertedly downregulate NOTCH1 activity and enhance lymphatic sprouting. Notably, overexpression of the calcium reporter GCaMP3 unexpectedly inhibited lymphatic sprouting, presumably by disturbing calcium signaling. Endothelial-specific knockouts of Orai1 and Klf2 also markedly impaired lymphatic sprouting. Moreover, Dtx3l loss of function led to defective lymphatic sprouting, while Dtx3l gain of function rescued impaired sprouting in Orai1 KO embryos. Together, the data reveal a molecular mechanism underlying laminar flow–induced lymphatic sprouting.

Analysis of gene expression of secreted factors associated with breast cancer metastases in breast cancer subtypes.

Breast cancer is a heterogeneous disease, having multiple subtypes with different malignant phenotypes. The triple-negative breast cancer, or basal breast cancer, is highly aggressive, metastatic, and difficult to treat. Previously, we identified that key molecules (IL6, CSF2, CCL5, VEGFA, and VEGFC) secreted by tumor cells and stromal cells in basal breast cancer can promote metastasis. It remains to assess whether these molecules function similarly in other subtypes of breast cancer. Here, we characterize the relative gene expression of the five secreted molecules and their associated receptors (GP130, GMRA, GMRB, CCR5, VEGFR2, NRP1, VEGFR3, NRP2) in the basal, HER2 (human epidermal growth factor receptor 2) positive, luminal A, and luminal B subtypes using high throughput data from tumor samples in The Cancer Genome Atlas (TCGA) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC). IL6 and CCL5 gene expression are basal breast cancer specific, whereas high gene expression of GP130 was observed in luminal A/B. VEGFA/C and CSF2 mRNA are overexpressed in HER2 positive breast cancer, with VEGFA and CSF2 also overexpressed in basal breast cancer. Further study of the specific protein function of these factors within their associated cancer subtypes may yield personalized biomarkers and treatment modalities.

Inhibition of breast cancer growth and metastasis by a biomimetic peptide

Metastasis is the main cause of mortality in cancer patients. Though there are many anti-cancer drugs targeting primary tumor growth, anti-metastatic agents are rarely developed. Angiogenesis and lymphangiogenesis are crucial for cancer progression, particularly, lymphangiogenesis is pivotal for metastasis in breast cancer. Here we report that a novel collagen IV derived biomimetic peptide inhibits breast cancer growth and metastasis by blocking angiogenesis and lymphangiogenesis. The peptide inhibits blood and lymphatic endothelial cell viability, migration, adhesion, and tube formation by targeting IGF1R and Met signals. The peptide blocks MDA-MB-231 tumor growth by inhibiting tumor angiogenesis in vivo. Moreover, the peptide inhibits lymphangiogenesis in primary tumors. MDA-MB-231 tumor conditioned media (TCM) was employed to accelerate spontaneous metastasis in tumor xenografts, and the anti-metastatic activity of the peptide was tested in this model. The peptide prevents metastasis to the lungs and lymph nodes by inhibiting TCM-induced lymphangiogenesis and angiogenesis in the pre-metastatic organs. In summary, a novel biomimetic peptide inhibits breast cancer growth and metastasis by blocking angiogenesis and lymphangiogenesis in the pre-metastatic organs as well as primary tumors.

Pre-treatment of mice with tumor-conditioned media accelerates metastasis to lymph nodes and lungs: a new spontaneous breast cancer metastasis model

Current spontaneous metastasis models require a long period of observation after establishment of primary tumors to see significant metastatic progression. The degree of metastasis is not consistent among animals: this is problematic since it requires the use of a large number of animals to obtain reliable statistics. Here we report that pre-treatment of animals with tumor-conditioned media (TCM) consistently accelerates spontaneous metastasis in breast cancer. An inguinal breast tumor model facilitated by TCM showed robust anterior metastasis to the axillary and brachial lymph nodes (LN), and the lungs compared to the serum-free media treated group. The LN in TCM-treated animals showed enhanced angiogenesis and lymphangiogenesis. Primary tumors and lungs in TCM-treated animals showed enhanced lymphangiogenesis with no significant change in angiogenesis. TCM-treated animals also showed metastatic dissemination to abdomen from the primary injection site: this would generally enhance metastasis to other organs. In sum, the addition of TCM pre-treatment to current metastasis models results in accelerated and robust metastasis which would enable more efficient evaluation of anti-metastatic agents.

Synergy between a collagen IV mimetic peptide and a somatotropin-domain derived peptide as angiogenesis and lymphangiogenesis inhibitors

Angiogenesis is central to many physiological and pathological processes. Here we show two potent bioinformatically-identified peptides, one derived from collagen IV and translationally optimized, and one from a somatotropin domain-containing protein, synergize in angiogenesis and lymphangiogenesis assays including cell adhesion, migration and in vivo Matrigel plugs. Peptide-peptide combination therapies have recently been applied to diseases such as human immunodeficiency virus (HIV), but remain uncommon thus far in cancer, age-related macular degeneration and other angiogenesis-dependent diseases. Previous work from our group has shown that the collagen IV-derived peptide primarily binds β1 integrins, while the receptor for the somatotropin-derived peptide remains unknown. We investigate these peptides’ mechanisms of action and find both peptides affect the vascular endothelial growth factor (VEGF) pathway as well as focal adhesion kinase (FAK) by changes in phosphorylation level and total protein content. Blocking of FAK both through binding of β1 integrins and through inhibition of VEGFR2 accounts for the synergy we observe. Since resistance through activation of multiple signaling pathways is a central problem of anti-angiogenic therapies in diseases such as cancer, we suggest that peptide combinations such as these are an approach that should be considered as a means to sustain anti-angiogenic and anti-lymphangiogenic therapy and improve efficacy of treatment.

Angiogenesis interactome and time course microarray data reveal the distinct activation patterns in endothelial cells.

Angiogenesis involves stimulation of endothelial cells (EC) by various cytokines and growth factors, but the signaling mechanisms are not completely understood. Combining dynamic gene expression time-course data for stimulated EC with protein-protein interactions associated with angiogenesis (the “angiome”) could reveal how different stimuli result in different patterns of network activation and could implicate signaling intermediates as points for control or intervention. We constructed the protein-protein interaction networks of positive and negative regulation of angiogenesis comprising 367 and 245 proteins, respectively. We used five published gene expression datasets derived from in vitro assays using different types of blood endothelial cells stimulated by VEGFA (vascular endothelial growth factor A). We used the Short Time-series Expression Miner (STEM) to identify significant temporal gene expression profiles. The statistically significant patterns between 2D fibronectin and 3D type I collagen substrates for telomerase-immortalized EC (TIME) show that different substrates could influence the temporal gene activation patterns in the same cell line. We investigated the different activation patterns among 18 transmembrane tyrosine kinase receptors, and experimentally measured the protein level of the tyrosine-kinase receptors VEGFR1, VEGFR2 and VEGFR3 in human umbilical vein EC (HUVEC) and human microvascular EC (MEC). The results show that VEGFR1–VEGFR2 levels are more closely coupled than VEGFR1–VEGFR3 or VEGFR2–VEGFR3 in HUVEC and MEC. This computational methodology can be extended to investigate other molecules or biological processes such as cell cycle.