Angiogenesis of breast cancer focusing on the angiogenic factors, HIF-1𝛼 and VEGF Ye Young Shin Biochemistry lab Supervised by Beum-soo An 2017.04.18

Angiogenesis Definition of angiogenesis Roles of angiogenesis The process of new blood vessel development from existing blood vessels. The first vessels in the developing embryo form through vasculogenesis, after which angiogenesis is responsible for most. The process of new blood vessel development from existing blood vessels. The first vessels in the developing embryo form through vasculogenesis, after which angiogenesis is responsible for most. Forms new blood vessels during healing at site of injury Develops collateral circulation during ischemia Allow tumor to grow Roles of angiogenesis Inflammation or ischemia Macrophage TNF 𝛼 ORM 1 VEGF Mural cell Angiogenesis Tumor growth Small tumor Sprouting capillary Angiogenesis Growing tumor

Angiogenesis Types of angiogenesis Angiogenic sprouting: Controlled by the balance between pro-angiogenic signals such as vascular endothelial growth factor (VEGF). Intussusception: The splitting of vessels through the insertion of tissue pillars. Little is known about the function or regulation of intussusceptive growth. Angiogenic sprouting Intussusception

Angiogenesis Steps of angiogenesis (sprouting) Stimulation of endothelial cells by angiogenic factors. Vascular Endothelial Growth Factor (VEGF) Highly specific for endothelial cells.  Inducer of extracellular proteinase expression. Binds to endo-specific receptors Flt-1and Flk-1. Expression of VEGF potentiated by hypoxia and  inactivation of p53. Degradation of the capillary basal lamina by activated endothelial cells (via extracellular proteinases). Matrix Metalloproteinases (MMPs)  A family of 22 zinc-dependent endopeptidases that degrade  all extracellular matrix components.  Subgroups of collagenases, stromelysins, gelatinases, and  membrane-type MMPs.  MMP1 (a collagenase), MMP2 and MT1-MMP (basal  lamina degradation) are all expressed during angiogenesis.         Vascular Endothelial Growth Factor (VEGF) Highly specific for endothelial cells.  Inducer of extracellular proteinase expression, increased  expression of specific integrins for migration, and initiation of cell proliferation and migration.  Binds to endo-specific receptors Flt-1 and Flk-1. Expression of VEGF potentiated by hypoxia and  inactivation of p53.

Angiogenesis Steps of angiogenesis (sprouting) Capillary sprout formation and migration of  endothelial cells. Integrin Expression  Integrin proteins on newly forming vessels.  Allow migrating endothelial cells to interact with specific components of the surrounding  matrix. Extracellular proteinases MMPs and urokinase faciliate migration of endothelium cells into surrounding matrix.    New Vessel Maturation. Angioprotin (Ang 1) Produced by the surrounding stromal cells.  Induces endothelial cell survival and stabilization of new capillary tubes.          

Angiogenesis Angiogenesis in cancer Solid tumor growth is dependent upon angiogenesis, the growth of new blood vessels. Critical event during early stages of tumorigenesis. Well established in breast cancer cell line (MCF-7) about its characteristics.

Breast cancer What is breast cancer Symptom The most frequently diagnosed life-threatening cancer in women. Invasive ductal carcinoma (IDC), sometimes called infiltrating ductal carcinoma, is the most common type of breast cancer. Symptom • Lump • Pulled in nippled • Dimpling • Dripping • Redness/ Rash • Skin Changes

Non-proliferative changes Proliferative disease Breast cancer Development of breast cancer Sex steroid hormones such as estrogen and progesterone play a central role in the development and progression of prostate and breast cancers. Breast cancer cells can avoid cellular response by disabling the apoptotic pathways. Angiogenesis is regarded as a central mechanism of breast tumor growth. Normal/ Non-proliferative changes Proliferative disease Atypical hyperplasia Carcinoma in situ Invasive carcinoma Luminal cells Germline mutations Myoepithelial cell Loss of appotosis Genome Instabiltity Loss of grwoth inhibition Self-sufficient growth Angiogenesis Limitless replication Tissue invasion Loss of function Stromal cells

Angiogenesis in breast cancer First evident at the pre-invasive stage of high-grade ductal carcinoma Angiogenesis in breast cancer Help to meet growing metabolic demands of the tumor by supplying additional nutrients. Provide potential routes for tumor dissemination and metastasis. Microvascular density (MVD) is prognostic factor in invasive breast cancer. Growth of cancer Metastasis of cancer

Hypoxia-inducible factor (HIF) Fibroblast growth factor (FGF) Angiogenesis in breast cancer Several factors of angiogenesis in breast cancer FGF signaling has also been shown to be important in a number of malignancies, including prostate, endometrial, and breast cancer. Over-expression of HIF-1𝛼 protein has been identified in various tumor types, with high levels influencing the growth rate and metastatic potential of these cancers. Hypoxia-inducible factor (HIF) Including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, PDGF. VEGF expression has been found to correlate with risk and outcomes in breast cancer. VEGF family Angioproteins with Tie-2 receptor controls vessel maturation ,and breast cancer progrression and invasion. Ang Fibroblast growth factor (FGF)

Angiogenesis in breast cancer Hypoxia-inducible factor (HIF) Hypoxia is a key signal for the induction of angiogenesis. HIF are heterodimeric transcription factors consisting of 𝛼 and 𝛽 subunits. Angioproteins Fibroblast Growth Factor (FGF) Transforming growth factor beta-1 (TGF𝛽-1) Normoxia The 𝛽 subunit is constitutively expressed while the 𝛼 subunit is protected from degradation. The 𝛼 subunit contains two hydroxylated pronyl residues which tumor surprressor protein (pVHL) binds to. Also, the 𝛼 subunit is degraded by ubiquitination. Hypoxia In hypoxia condition, the pVHL can not bind to 𝛼 subunit and ubiqutination does not occur. Binding HIF-1 complex to HRE leads angiogenesis in breast cancer.

Angiogenesis in breast cancer Hypoxia-inducible factor (HIF) The low tissue oxygen tension arises from masses of tightly packed, rapidly growing cancer cells. HIF-1𝛼 is stabilized in hypoxic conditions, and proangiogenic genes are produced. HIF-1𝛼 plays a significant role in experimental tumor growth and tumor-associated angiogenesis. Mice deficient in factor have markedly reduced angiogenic responses. In human, HIF1-𝛼 is overexpressed in ductal carcinomas.

Angiogenesis in breast cancer VEGF Type Function VEGF-A Angiogenesis Migration of endothelial cells Mitosis of endothelial cells Methane monooxygenase activity 𝛼𝛾𝛽3 activity Creation of blood vessel lumen Creates fenestrations Chemotactic for macrophages and granulocytes Vasodilation (indirectly by NO release) VEGF-B Embryonic angiogenesis (myocardial tissue, specifically) VEGF-C Lymphangiogenesis VEGF-D Needed for the development of lymphatic vasculature surrounding lung bronchioles PGF Important for vasculogenesis, also needed for angiogenesis during ishemia, inflammation, wound healing, and cancer

Angiogenesis in breast cancer VEGF HIF-1𝛼 expression is associated with increased expression of the VEGFR. Dimerized VEGF stimulate cellular responses by binding to tyrosine kinase receptors on the cell surface. VEGFR becomes activated through transphosphorylation, inducing signaling pathways. - ERK/MAPK - (PI3K)/(PKB/AKT) - P38mapk.

Angiogenesis in breast cancer VEGF Angiogenesis induced by breast cancer has been linked to an increased production of VEGF. Increased expression of VEGF corresponds with the earliest visible breast-tumor-induced angiogenesis that is evident in pre-invasive highgrade ductal carcinoma. Stabilized HIF-1𝛼 induces expression of VEGF as well as one of its receptors, VEGF receptor 1 (VEGFR1) in breast cancer. Increase in expression of VEGFR2 has also been noted in adjacent beast tumor endothelial cells. Junichi K et al., Jpn.J. Cancer Res. 1999

Anti-Angiogenesis therapies Anti-HIF therapy IPAS Dimerizing with HIF-1𝛼 Inhibiting HIF mediated transactivation FIH Asparagin hydroxylase activitiy Bind with HIF-1𝛼 Inhibit HIF transcription under hypoxia Cited 2 Nuclear regulatory protein Bind to CBP/p300 Competitively inhibiting its interaction with HIF-1𝛼 Several trials of agents that decreases or block HIF-1𝛼 expression. Rapamycin/CCI779, quinocarmycin, toppoisomerase inhibitors, anti-microtubular agents, YC-1, 17-AAG, thioredoxin inhibitors and 2ME2. Obstructing the interaction between HIF-1𝛼 and the co-activator CBP/p300 led to attenuation of HIF-induced gene expression and inhibition of tumor growth. Tirapazamine, which inhibits DNA repair under hypoxic condition.

Anti-Angiogenesis therapies Anti-VEGF therapy Many therapies have directly targeted the VEGF pathway because of its critical role in pathological angiogenesis. Anti-VEGF therapy usually targets VEGF or VEGF receptor Inhibit VEGF Inhibit VEGFR

Anti-Angiogenesis therapies Anti-VEGF therapy Targeting VEGF Recombinant VEGF antibody derived from a humanized mouse monoclonal antibody. Recognizes all isoforms of VEGF-A. Prevents receptor binding, which leads to inhibition of angiogenesis and breast tumor growth. Bevacizumab Targeting VEGF-R VEGFR antibody or tyrosine kinase inhibitors of VEGF (TKIs). A variety of other small molecule TKIs targeting the VEGFRs are being evaluated. Rybozyme (catalytic RNA molecules that specifically cleave VEGFR mRNAs) and antisense strategies Prevents receptor binding, which leads to inhibition of angiogenesis and breast tumor growth. Cabozantinib, Pazopanib, Regorafenib etc.

Anti-angiogenesis therapies Side-effects of anti-angiogenesis therapies Anti-angiogenesis therapies Diarrhea and dizziness Hypertension Off-target effect Skin toxity Mucositis VEGFR PDGFR Only the combination of agents are effective to cancer. Difficult to identify optimal dosing and scheduling of molecular-targeted agent. Unknown negative drug interactions or the presence of off-target. Long-term toxicity of agent. Lack knowledge of angiogenic signaling such as VEGF-E.

Conclusion Angiogenesis plays a central role in the process of tumor growth and metastatic dissemination. Targeting angiogenic factors could inhibit growth of cancer. Angiogenesis The full complexity of the mechanisms of tumor angiogenesis and their regulators need to be defined. Recognizing the correlations between the biology and clinical outcomes are required. Targeting multiple pathways of angiogenesis will hopefully shed light on the true potentials of agents.

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