• Introduction
• EGF Chemotaxis in Breast Cancer Cells
• Epidermal Growth Factor Receptor (EGFR)
• EGFR: Ras Mediated Signaling
• MAPK Phosphorylation Underlies Cell Spreading and Motility
• EGFR Signaling: PLCγ Pathway
• Final Comments

No two cancers are alike. More to the point, as we will see a malignant tumor is a collection of a diversity of cells of different clonal origins. One goal is to find a common functional aspect of cancer cells that can serve as a target for developing drugs and other anti-cancer therapies. Recently it was believed that angiogenesis-the formation of new blood vessels-was that target but inhibiting blood vessel growth into tumours did not stop tumour development. In fact it sometimes helped it. That's because some of the maligant cells were more than happy in at low oxygen levels. This should have been anticipated because it has been known for well over half a century that most cancer cells express high levels of glycolytic (anaerobic metabolism) enzymes through which they generate ATP for cellular processes. Here we will look at another a target of anti-cancer therapies, the epidermal growth factor receptor (EGFR) and its role in events underlying invasiveness, motility and metastasis. We will look at EGFR signaling pathways and then detail some events in a breast cancer cell line. In doing this we will be completing our analysis of signaling mechanism (i.e., receptor tyrosine kinases) and showing how signaling mechanisms are tied to a specific cellular process, cell motility. Breast Cancer Cells: Cell Spreading and Motility Let's now look at the role of some of these events in some breast cancer cell lines. The first event a cell undergoes prior to movement is cell spreading. This occurs in normal as well as cancer cells. The following immunofluorescence picture shows the effects of epidermal growth factor (EGF) on the actin cytoskeleton and its nucleation sites that underlie cell spreading.

The function of EGF as a chemoattractant is shown in the next figure. EGF contained in a micropipette diffuses out forming a gradient to which the cancer cell responds by spreading and forming large lamellipodia oriented towards the EGF source.

In addition to regulating cell spreading, motility and chemotaxis, EGF also regulates cell division and growth in various breast cancer cell lines. The functions of EGF are mediated through the epidermal growth factor receptor (EGFR). In keeping with its function as a receptor tyrosine kinase, EGFR forms dimers upon ligand binding and undergoes autophosphorylation to become fully active.

As mentioned in the previous lecture, Ras is the most common oncogene product found in human tumors. It mediates cancer cell growth as well as motility. The next figure takes us step-wise through the binding of EGF to its receptor leading to its autophosphorylation followed by the sequential addition of the upstream ras-signaling components. Ras-linked RTKs use a highly conserved signaling pathway. The EGFR serves to demonstrate these signaling events. EGF binding to the EGFR leads to dimerization and autophosphorylation of specific tyrosine residues. This results in changes that allow Grb2 and Sos to bind providing the binding site for inactive Ras-GDP. Grb2 is an adaptor protein while Sos is a target for Ras. Ras and other monomeric GTP-binding proteins were discussed in the earlier lecture "Vesicle Traffic: COPs, SNARES & Other Things". The binding of Ras-GDP directly to Sos leads to a conformational change resulting in the exchange of GTP for GDP and the activation of the Ras. The active Ras mediates downstream kinase cascade that can lead to different results depending on the cell type. These are summarized in the next figure. The cascade initiated by Ras involves a sequential series of a kinases that phosphorylate their targets which are typically the next kinase in the cascade. The first component of the cascade is the kinase Raf, a serine/threonine kinase (phosphorylates serine and kinase residues on target proteins). Ras binds to and activates Raf. Raf then binds to and phosphorylates and activates MEK. MEK is a dual kinase that can phosphorylate tyrosine and serine residues. MEK phosphorylates and activates MAPK, a ser/thr kinase. MAPK breaks the routine by phosphorylating a diversity of proteins including transcription factors as well as components that mediate cytoskeletal function and cell adhesion mechanisms. Ras signaling leading to the phosphorylation of specific transcription factors (TCF, SRF) and the subsequent activation of transcription is shown in the next picture.

Scientists use different cell lines isolated from human cancers to study cancer cell behaviour. For breast cancer, MCF-7 and some MDA cell lines are actively studied. MCF-7 have estrogen receptors, grow slowly and are slow to metastasize. MDA-MB-231 is a highly metastatic, actively motile cell line derived from a human breast tumour. It has unique attributes since it lacks the estrogen receptor allowing investigators to study signaling events without interference from estrogen signaling. The following figure shows the time course of MAPK phosphorylation after EGF treatment in several different breast cancer cell lines. In MCF-7 cells, the total levels of MAPK don't change but the level of phosphorylation of MAPK changes markedly. Little MAPK phosphorylation is evident in control cultures but after 15min treatment with EGF significant phosphorylation occurs which peaks at 2h and then levels off. This increase in phosphorylation correlates with the increase in cell spreading and motility observed in the culture. In MDA cell lines, this sequence does not occur because the MAPK is highly phosphorylated even in untreated cells. The might be one reason that these cells are so highly invasive.

For the most part, the next figure is fundamentally a review of previous information with a few minor modifications. The EGFR also mediates cell function via phosphoinositide signaling involving phospholipase Cg. Dimerization, phosphorlation and activation of the EGFR occurs upon ligand binding leading to a conformational change in the receptor. This allows binding to PLCg. Hydrolysis of phosphoinositol 4,5 bisphosphate (PIP2) leads to the production of diacylglycerol (DAG) and inositol 1,4,5 trisphosphate (IP3) as detailed in our lecture on the Dual Signaling Pathway. IP3 binds to the IP3 Receptor on the endoplasmic reticulum releasing Ca2+ into the cytoplasm. DAG at the membrane plus Ca2+ can activate various PKC isoforms. Ca2+ also binds to various calcium-binding proteins such as calmodulin (CaM). The calcium-mediated results include changes in the cytoskeleton and in cell adhesion. In support of the role of CaM in EGFR-mediated signal transduction, inhibitors of CaM inhibit cell spreading. Similarly, tamoxifen also inhibits cell spreading via its inhibition of CaM in the estrogen receptor-negative breast cancer cell line MDA-MB-231. Before we get the wrong idea about EGF, it should be noted that the EGF mediated chemotaxis is important for normal brain development.

• Development of cancer involves many mutations
• Metastasis is a highly orchestrated process
• Signaling pathways differ between cancers and cell-lines
• Elucidation of these signaling pathways may assist in clinical treatment