Question

a)

Describe the VEGFR signal transduction pathway.
What is a RTK? How does it transmit an external signal to the interior of the cell? What is the response of the cell?

b)

How can signal transduction pathways can be used to explore and develop possible new drugs for breast cancer treatment?

Answer 1

a. The vascular endothelial growth factor signaling pathway (VEGF) is involved in vasculogenesis, angiogenesis and lymphangiogensis. There are 5 VEGF ligands known- VEGF A-D and placental growth factor PIGF. These ligands can bind to any of the three receptors - VEGFR1-3. PIGF binds only to VEGFR1. There are two co-receptors neuropilin (NP)-1 and NP-2.

Binding of VEGF ligand to its receptor, which is a receptor tyrosine kinase, causes dimerization of the receptor. This is followed by autophosphorylation of its tyrosine residues. This phosphorylation of tyrosine residues causes recruitment of adapter proteins such as SHB that have SH2 (Src homology domain 2). This causes the activation of downstream signaling pathways such as PI3K/AKT. In PI3K/AKT pathway activation seen by binding of VEGFA to VEGFR1, PI3K will phosphorylate phosphatidylinositol (4,5)-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PIP3 will then activate Akt. Akt can directly phosphorylate endothelial nitric oxide synthase or eNOS. The eNOS can also be activated by binding of calmodulin, causing increased production of nitric oxide NO. Vascular development can occur in response to NO. Signaling via VEGFR2 activates phospholipase C gamma signaling. Phospholipase C gamma breaks down PIP2 to diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 production results in increased levels of intracellular calcium within the cells and downstream signaling. DAG activates protein kinase C, which then activates phospholipase A, via MAPK pathway. Phospholipase A is involved in increased synthesis of prostacyclin (PGI2). Both PGI2 and NO caused by VEGF signaling increase vascular permeability in cells.

Receptor tyrosine kinases are single pass membrane bound type I receptors. They have an extracellular domain that binds the ligand, a hydrophobic transmembrane domain involved in dimerization and a cytoplasmic protein kinase domain. Binding of ligand to the receptor, causes the dimerization of two RTKs. Dimerization results in auto-phosphorylation of a tyrosine residues in the cytoplasmic domain. Initially catalytic site tyrosines are phosphorylated. A conformational change occurs due to phosphorylation, causing recruitment of either ATP or binding proteins. This activates the kinase activity within the receptor. The kinase domain in the cytoplasmic side of receptor will then phosphorylate other tyrosine residues in the receptor. Autophosphorylation is then followed by binding of adapter proteins that have SH2 domain or PHB domain. This leads to downstream signaling cascades. In case of EGFR signaling, Ras protein is activated, which then activates PI3K-Akt-mTOR or ERK-MAPK pathways. In case of VEGF signaling, PI3K-AKT or phospholipase C gamma pathways are activated. In case of insulin receptor,PI3K-AKT pathway activation leads to increased transcription of glucose transporter genes.

b. Receptor tyrosine kinases and other membrane bound receptors such as GPCRs will activate other signaling cascades. In case of breast cancer, epidermal growth factor, vascular endothelial growth factor receptors (VEGFRs), platelet-derived growth factor receptors (PDGFRs), insulin-like growth factor receptors (IGFRs), and fibroblast growth factor receptors (FGFRs) has been implicated. All these are RTKs signaling cascades that result in increased cell division and proliferation. Gain of function mutation in these RTKs can lead to increased cell proliferation duce to activation of kinase activity. Other ways to activate RTKs for breast cancer are small in frame deletion, point mutations in different domains of the receptor, gene amplification, transcriptional or translational enhancement, chromosomal rearrangements have all lead to activation of these receptors, causing breast cancer.

One way to prevent or treat breast cancer is to develop drugs that target these specific signaling pathways. mTOR inhibitors have been developed that will inhibit the activity of mTOR, which activates genes involved in cell proliferation. PI3K and AKT inhibitors will inhibit all downstream signaling of the PI3K pathways. Phosphatase and tensin homology PTEN is a phosphatase that breaks down PIP3. Increased use of PTEN homologs will downregulate PI3K pathway, thereby reducing proliferation. If drugs can target and inhibit these pathways, then breast cancer proliferation genes are not activated, thereby treating breast cancer.

The ERK-MAPK pathways can also be inhibited using MEK inhibitors. Ras oncogene activates the Raf protein kinase that activates MEK kinase by phosphorylation. MEKK1 or MEKK2 phosphorylates MAP3K which then phosphorylates MAP2K, which phosphorylates ERK. ERK ½ enters the nucleus and activates transcription of cell cycle genes such as c-myc etc. The MEK inhibitor selumetinib has been found to inhibit the motility and invasiveness of the cell lines invitro. Ras inhibitors have also been developed that will inhibit the entire ERK-MAPK pathway.

Other pathway involved is the Wnt-beta catenin family. Binding of Wnt to its receptors causes the destabilization of beta catenin, which then enters the nucleus and induces transcription of downstream genes. Triple negative breast cancer patients show aberrant Wnt beta catenin signaling. The anti-leprosy drug, clofazimine has been able to decrease TNBC by affecting Wnt beta catenin signaling. Thus, by inhibiting these signaling pathways, the drug can prevent increased transcription of cell cycle genes, thereby inhibiting breast cancer development.