1 Molecular Origins of Cancer Tumor Angiogenesis N Engl J Med 2008;358:

2 Introduction (I) Tumor angiogenesis: therapeutic implications. - Folkman J. in 1971 Bevacizumab, humanized monoclonal antibody against VEFG –FDA Approves First Angiogenesis Inhibitor to Treat Colorectal Cancer in 2004 –Combination therapy with conventional chemotherapy for metastatic colorectal cancer –First-line treatment of advanced NSCLC in combination with standard chemotherapy Sorafenib and sunitinib, RTKIs –Antiangiogenic drugs approved by FDA –VEGF receptors and PDGF receptors –Sorafenib and sunitinib in the treatment of metastatic renal-cell cancer –Sorafenib monotherapy in the treatment of hepatocellular carcinoma

3 Introduction (II) Questions with respect to improving antiangiogenic therapy –How do such drugs work? –How does bevacizumab increase the efficacy of chemotherapy? –How do tumors become resistant to antiangiogenic drugs? –Are there clinically useful markers that can predict the efficacy of this class of drug? –Are there promising surrogate pharmacodynamic biomarkers that will help to determine the best dose of a particular agent? –Will antiangiogenic RTKIs such as sunitinib or sorafenib consistently enhance the efficacy of chemotherapy? –What accounts for the side effects of these agents? Many recent discoveries over the past 5 years –Answer some of these questions –New therapeutic targets and treatment strategies Purpose of this review –To summarize a number of these discoveries –To point out their potential clinical impact

4 VEGF and VEGF-Receptor Family in Tumor Angiogenesis

5 VEGF family of growth factors and the receptor tyrosine kinases –Proangiogenic effects –VEGF-A, VEGF-B, VEGF-C, VEGF-D, PlGF –Elevated level by genetic and epigenetic ways VEGF action mechanism –Paracrine –Autocrine –Intracrine VEGFR –VEGFR-2 Endothelial cells engaged in angiogenesis Circulating bone marrow–derived endothelial progenitor cells –VEGFR-1 is a mystery with respect to VEGF-mediated angiogenesis. Approximately 10 times the affinity of VEGFR-2 binding Extremely weak signal-transducing properties Mice with a mutant VEGF gene –Severe cardiac defects –Gastrointestinal perforation, thrombotic events → bevacizumab adverse effects

6 RTKIs –Ability to penetrate cells → myelosuppression caused by targeting other receptor tyrosine kinases such as c-kit and fms-like tyrosine kinase 3 –More effective than antibodies in treating with functional intracellular autocrine VEGF receptors Neurophilin –Transmembrane receptor for semaphorin 3A –Lack tyrosine kinase activity –Axon guidance –Binding with VEGF to modulate angiogensis –Emerging potentially promising antiangiogenic targets Monoclonal antibodies to PIGF –Few side effects unlike anti-VEGF –Minimally expressed by most normal cell and tissue Tie-2, a receptor tyrosine kinase –Two major ligands Angiopoietin-1 (ang-1) as an agonist Angiopoietin-2 (ang-2) as an anatagoist –Stabilization and maturation of new capillaries in concert with VEGF

7 Pericyte, an accessory cell –Secretion of ang-1 –Expression of PDGF receptor –RTKIs that target PDGF receptor

8 Circulating VEGF and a soluble form of VEGFR-2 Markers of antiangiogenic drug activity Elevated VEGF levels in blood or even tumor tissue do not predict a benefit in patients receiving drugs that target the VEGF–VEGFR-2 pathway! –Tumor cells produce VEGF but cannot respond to it directly since they do not have cellsurface VEGF receptors. –VEGF in amounts sufficient to drive tumor angiogenesis originates from various host cells in the body such as platelets and muscle cells Measurement in blood used as a preclinical means of establishing the optimal biologic dose of drugs that target VEGFR-2, including antibodies and small-molecule RTKIs Assessment of circulating VEGF in a complex with a VEGF antagonist such as the soluble “decoy” receptor drug called the “VEGF trap” for predicting the blockade of angiogenesis

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10 Notch–Deltalike Ligand 4 Signaling Pathway Notch cell surface receptors (i.e., notch 1, 2, 3, and 4) involved in cell fate, differentiation, and proliferation Transmembrane ligands (jagged 1, jagged 2, and Dll1, Dll3, and Dll4) on adjacent cells Vascular endothelial cells express notch 1 and notch 4 receptors and the jagged 1, Dll1, and Dll4 ligands; among these, Dll4 is expressed exclusively by endothelial cells. Notch–Dll4 signaling is essential for vascular development in the embryo shown by experimental mice Notch–Dll4 signaling system –A major stimulator of angiogenesis –Potential drug target Paradoxically, drugs that target Dll4, including neutralizing antibodies, actually increase tumor angiogenesis, but most of the newly formed blood vessels are abnormal and functionally compromised in ways that drastically reduce blood flow. The question of whether they need to be combined with chemotherapy, radiation therapy, or other antiangiogenic drugs is important.

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12 Angiogenesis and Circulating Bone Marrow–Derived Cells

13 Cell types from the bone marrow –Hematopoietic-cell (CD45+) Monocytic or myeloid cells Endothelial-cell markers as VE-cadherin, VEGFR-1, VEGFR-2, and tie-2 Chemokine receptors such as CXC chemokine receptor 4 (CXCR4) → Binding with stromal-cell– derived factor 1 (SDF-1, also called CXCL12) → Lymphocytes, neutrophils and macrophages ( proangiogenic properties) –Nonhematopoietic (CD45−) bone marrow cell Circulating endothelial progenitor cells Paracrine mechanisms such as local secretion of VEGF Differentiation into endothelial cell → wall of a growing blood vessel Study on the general role of circulating endothelial progenitors –Various cell-surface marker –Detection methods Treatment with vascular disrupting agents (VDAs) in preclinical studies → acute, marked mobilization of the progenitors from the bone marrow → ‘Increased circulating endothelial progenitors invade and colonize the viable rim of the tumor’ → rapid re-growth of the tumor

14 Clinical implications in the contribution of circulating bone marrow-derived cells to angiogenesis Chemotherapy drug such as cyclophosphamide –Mobilization of circulating endothelial progenitors –Closely spaced, regular administration of relatively less toxic doses of chemotherapy drugs with no prolonged breaks — ‘metronomic chemotherapy’ Use of hematopoietic growth factors in cancer patients –Recombinant granulocyte colonystimulating factor (G-CSF) can mobilize not only endothelial-cell progenitors, but also CD11b+ granulocyte differentiation antigen (Gr1)+ myeloid suppressor cells that can promote angiogenesis VEGFR-1 and CXCR4 as new target receptors to inhibit angiogenesis

15 Intrinsic resistance –Anti-VEGF Ab. resistant tumor cell line from mice Transplantation into mice by bone marrow-derived cell colonization Resistant cell mixed with normally sensitive tumor cells → transplantation → transplanted tumors resistance –Tumor cells using existing blood vessels in vasculature-rich organs such as the lungs –Absence of VEGF or VEGF receptors Acquired resistance –Redundancy of angiogenesis stimulators Up-regulation of the angiogenesis stimulator basic fibroblast growth factor within the tumor after treatment with anti–VEGFR-2 antibody therapy Bevacizumab → PlGF  Sunitinib → PlGF and VEGF  Two or more such induced growth factors could act in a synergistic manner to promote tumor angiogenesis. –Selection and overgrowth of mouse tumor cells with mutations in genes such as Tp53, which cause relative resistance to hypoxia. –Rapid vascular remodeling of tumor-associated vessels The mature remodeled vessels are resistant to antiangiogenic drugs, which usually target relatively immature vessels. Resistance to Antiangiogenic Drugs

16 “Tumor initiating” cancer stem cells –Tumor growth –Seeds of resistance to treatment Transplantation of selectively enriched populations of cells into immunodeficient mice – ‘strong proangiogenic property’ –Extremely small numbers of such putative human cancer stem cells –Much larger numbers of cancer cells lacking stem cell characteristics Putative cancer stem cells in brain tumors reside in close proximity to blood vessels in a “vascular niche.” –Treatment of orthotopic transplanted gliomas in mice with antibodies to VEGF disrupts the vascular niche and targets the stem-cell population. Anti-VEGF treatment Low-dose metronomic chemotherapy Angiogenesis and Cancer Stem Cells

17 Summary Antiangiogenic drugs for the treatment of cancer –Increasing use, but relatively modest benefits Molecular and cellular mechanisms governing tumor angiogenesis and the response to antiangiogenic therapies Strategies for improving the clinical benefits of antiangiogenic therapy –Better preclinical models to study the biology of tumor angiogenesis and antiangiogenic therapies Long-term antiangiogenic therapy in the adjuvant setting in patients with early-stage disease With respect to the treatment of metastatic disease, antiangiogenic drug combinations