Lecture 3 - BS3540 X

The Extracellular Matrix (ECM)

• Components:
  • Fibrous glycoproteins (collagen, fibrin, elastin).
  • Large glycoproteins (fibronectin and laminin).
  • Large proteoglycans (e.g., perlecan).
    • Proteoglycans consist of polypeptides carrying glycosaminoglycans (long, unbranched, highly negatively charged polysaccharide chains).
• Cells interact with the ECM via:
  • Cell surface proteoglycans.
  • Adhesion proteins (e.g., integrins).
• Some cells reside on prominent ECM layers (basement membranes).

Angiogenesis and Metastasis

• The process of metastasis involves several steps:
  • Transformation.
  • Induction of angiogenesis.
  • Motility and invasion.
  • Adherence.
  • Arrest in capillary beds.
  • Embolism and circulation.
  • Growth extravasation into organ parenchyma.
  • Specific response to the organ microenvironment.
  • Tumor cell proliferation and induction of organ-specific angiogenesis.

Fibronectin

• Glycoprotein: ~$250kD$.
• Homodimeric, but with splice variants.
• Multiple domains allow for multiple interactions.
• RGD motif (Arg-Gly-Asp) is a binding site for integrins on the cell surface.
• Contains multiple cell recognition sites and functional domains that bind ligands such as heparin, collagen, and fibrin.
• Specific sites include: C51, RCD (Gly-Arg-Gly-Asp-Ser), H (heparin-binding domain), CS1, and REDV.

Laminin

• Structural model includes various domains designated by Roman numerals on different chains (A, B1, B2).
• Has heparin-binding domains: Hep-1, Hep-2, and Hep-3.
• Molecular weight: M, ~$850kD$.

Structure of Glycosaminoglycans

• Hyaluronate

• Chondroitin sulfates

• Dermatan sulfate

• Heparan sulfate and Heparin

• Keratan sulfate

• GlcUA: glucuronic acid

• IdUA: iduronic acid

• GlcN: N-acetylglucosamine

• GalN: N-acetylgalactosamine

Aggrecan

• Core polypeptide: $300,000 kD$.
• Three globular domains.
• Largely Ser-Gly repeats.
• Typically 80-100 chondroitin sulfate chains attached.
• Forms a large aggregate with hyaluronic acid (Christmas tree-like structure).
• Molecular weight: Mr ~ $2.5 x 10^6$

Heparin

• Most widely used anticoagulant.
• Mechanism involves Antithrombin III (ATIII).
• Heparin binds to ATIII, enhancing its activity.
• ATIII inhibits clotting enzymes (e.g., XIIa, XIa, IXa, Xa, IIa/Thrombin).
• Forms a ternary complex with ATIII and the clotting enzyme.
• Heparin can dissociate from the complex and be reused.

Sequence of the Antithrombin Binding Pentasaccharide

• Unusual 3-O-sulfate is essential for activity.
• Sulphate groups are essential for activity.
• Residue IV is a uronic acid that has been epimerized to iduronic and 2-O-sulfated.
• Compares with the unmodified glucuronic acid (residue II).

Perlecan

• Mouse: $400kD$
• Human: $470kD$
• Contains domains such as signal peptide, LDL-receptor, Laminin short arm, Laminin A globular region, cysteine-rich repeat, Ig-repeat, NCAM, and EGF repeat.

Syndecan-1

• Core protein: $33kD$
• Contains 1-3 chains of heparan sulfate ($40-46kD$) and 2 chains of chondroitin sulfate ($17-30KD$).
• Located on the plasma membrane.

Glypicans

• Family of 6 heparan sulfate proteoglycans.
• $70kD$ core protein, cleaved by furin.
• Attached to the cell membrane via a GPI anchor.

Heparan Sulfate Proteoglycan Expression in Cancer

• Perlecan: Upregulated in invasive melanomas.
• Syndecan-1:
  • Expression lost in epithelial tumors.
  • Overexpressed in breast cancer and invasive multiple myeloma.
• Glypican-3:
  • Overexpressed in 70% of patients with hepatocellular carcinoma (50% show soluble glypican-3 in serum), squamous cell carcinoma of lung, and testicular germ cell tumors.
  • Downregulated in breast cancer, epithelial ovarian cancer, and mesothelioma.

Degradation of Syndecan-1 Promotes Metastasis

• Involves enzymes such as heparanase, 6-O sulfatase, and sheddase.
• Leads to the release of heparan sulfate and the core protein.

Proteoglycans and Cytokine Activity

• Many cytokines bind to heparin and heparan sulfate.
• Heparan sulfate acts as a co-receptor for fibroblast growth factors.
• Involved in cytokine activity within the BMP and chemokine cytokine families.

The Fibroblast Growth Factors (FGFs) and Heparin/Heparan Sulfate

• FGFs are potent mitogens in many cell types.
• Two FGFs identified by purification, both $18kD$ polypeptides:
  • FGF-1 (acidic FGF): pI 5.6
  • FGF-2 (basic FGF): pI 9.0
• Both bind to columns of immobilized heparin.
• FGF-2 elutes at 1.5M NaCl, FGF-1 elutes at 0.5M NaCl (physiological salt conc. 0.18M).
• Heparin stabilizes FGFs against heat, pH, and proteolytic degradation.
• FGFs are ‘stored’ in the ECM of tissues subject to injury and act in a paracrine manner.
• Currently 23 FGFs – all but 3 bind to heparin/HS.
• Heparin/HS act as co-receptors (i.e., FGF tyrosine kinase signaling receptors do not function in their absence).
• Promote cell division of many cancer cell types, thus promoting tumor growth in a paracrine manner.

Crystal Structure of the FGF1-FGFR2-heparin oligosaccharide complex

• FGF-1 in green, Heparin oligosaccharide in CPK.

Chemokines in Cancer

• Chemotactic cytokines recruit white blood cells into infected tissues.
• Approximately 50 known chemokines.
• Bind to heparan sulfate.
• Major families: CCL, CXCL, CX3CL
• 20 Chemokine receptors (CCRs, CXCRs) are G-protein coupled receptors.
• Roles in cancer:
  • Recruit stromal cells, e.g., tumor-associated macrophages.
  • Metastasis.
  • Angiogenesis.
• Examples:
  • CXCL1, CXCL2, CXCL3, and CXCL8 promote tumor cell proliferation, angiogenesis, and resistance to apoptosis in melanoma and other cancers.
  • CXCR4 is commonly highly expressed in various cancers and promotes metastasis.

TGF-β Cytokine Superfamily

• Transforming growth factor-β.
• Initially thought to have a key role in cell transformation.
• Has a characteristic polypeptide fold based on intrachain disulphide bridges (S-S bridged dimers).
• Ancient lineage, but ~30 superfamily members in mammals.
• 12 BMPs (bone morphogenetic proteins).
• 4 neurotrophic factors.
• So far 10 known to bind to heparin/heparan sulfate – paracrine activity.
• Signal via cell surface tyrosine kinase receptors, control expression of multiple genes via the Smad transcription factors.
• Roles in regulating cell proliferation, differentiation, cell survival, embryonic development (morphogenesis and organogenesis).
• These activities may be subverted in cancer, e.g., BMPs inhibit proliferation of breast cancer cells but promote invasiveness, migration, and metastasis to bone.

The BMP Antagonist Coco Promotes Growth of Breast Cancer Metastases in Lung

• Compared to non-metastatic breast cancer cells, metastatic variants over-express Coco.
• Localized pericellular BMPs in the lung suppress metastatic growth in the lung – dormant micrometastases only.
• Secreted Coco relieves inhibition of cancer cell proliferation – metastases expand.