3.4.- B. Intracellular signaling pathways

B. 1. Ras and mitogen activated protein kinase (MAPK) pathways.
Ras proteins are small cytoplasmic GTP-binding proteins that serve as critical signal mediators to couple upstream receptor tyrosine kinases to downstream serine/threonine kinases that include the mitogen-activated kinases (MAPKs). These pathways are involved in regulation of proliferation and migration. Mutations in RAS genes are only seen in ~5% of human breast cancers, though the Ha-ras protein is overexpressed in ~70% of breast tumors, and in one study there was a strong correlation with overexpression of ErbB2 (reviewed in Clark and Der, 1995 and Malaney and Daly, 2001). There are conflicting data on the prognostic significance of increased ras expression (reviewed in Malaney and Daly, 2001). To date, no mutational activation of down-stream pathway components Raf, MEKs or MAPKs have been identified in breast cancer, but it is likely that the entire pathway is functionally hyper-activated in a significant fraction of breast cancers, due to activation or overexpression of the upstream receptor tyrosine kinases such as ErbB2.

B. 2. Phosphoinositide-3-kinase (PI3K) pathway and PTEN.
The PI3Ks are cytoplasmic lipid kinases that are involved in transmitting signals from protein tyrosine kinases, such as the receptor tyrosine kinases of the ErbB family, and the cytoplasmic tyrosine kinase c-src (see below). In breast cancer, the PI3K signaling pathway has also been linked to BRCA1 and there is evidence suggesting it may activate the estrogen eceptor independently of estradiol (Fry, 2001). The serine-threonine kinase Akt is an important downstream target of the PI3Ks, and the lipid phosphatase PTEN (phosphatase and tensin homolog on chromosome 10) serves as a negative regulator of the pathway. The PI3Ks appear to be particularly important in proliferation, survival and migration pathways, and are considered to be an important therapeutic target in many tumor types. One study has shown increased levels of the p85 regulatory subunit of PI3K in 80% of breast tumors when compared with adjacent normal tissue, but there was no link with other prognostic indicators (reviewed in Fry, 2001). However, since src and several receptor tyrosine kinases ( eg. erbB2) upstream of PI3K are activated in many breast cancers, it is highly likely that activity of the PI3K pathway will also be increased, even if expression levels of the pathway components are unchanged. This pathway can also be activated by loss of the inhibitor PTEN. Germline mutations in PTEN cause Cowden syndrome, of which breast cancer is a major feature, suggesting that PTEN may be a true tumor suppressor for breast cancer. LOH at the PTEN locus occurs in 30-40% of human sporadic breast cancers, though total inactivation of PTEN by mutation of the second allele is only seen in ~5% of tumors. Overall, loss or decreased expression of PTEN protein was seen in ~35% of sporadic breast cancers (Perren et al., 1999).

B. 3. c-Src pathway.
The proto-oncogene c-Src is an intracellular non-receptor tyrosine kinase that functionally interacts with signals generated by extracellular growth factors. Src is thought to promote tumorigenesis by augmenting signals from extracellular growth factors and by inducing morphogenetic remodeling of the cells (Biscardi et al., 2000). c-Src is overexpressed in up to 70% of breast cancer specimens, and in a subset of breast cancers, c-Src and HER-1 are co-overexpressed and may act synergistically (Biscardi et al., 2000). Src may also be required for the mitogenic effect of estrogen(Castoria et al., 1999). EMS1, which is a Src substrate, is amplified in 15% of breast cancers and was associated with poor prognosis in ER negative patients, but not ER positive patients, probably through enhancement of invasive or metastatic potential (Kairouz and Daly, 2000).