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Oncogenes and Signal Transduction
The Ras family of proto-oncogenes encode a family of small GTPase proteins that transduce proliferation and survival signals from RTKs at the cell membrane. Activating mutations in the K-ras proto-oncogene are found in about 20-50% of NSCLCs, especially adenocarcinomas, and are associated with smoking (76). Point mutations at codon 12 are the most frequent, followed by mutations at codons 13 and 61, and result in a decreased intrinsic GTPase activity and inappropriate constitutive signaling for cell proliferation.

The Myc proto-oncogene family encodes three basic-helix-loop-helix transcription factors, C-Myc, N-Myc and L-Myc. The MYC proteins regulate the expression of key cell cycle regulators and genes involved in DNA synthesis and RNA metabolism. Activation of MYC occurs through gene amplification or transcriptional dysregulation, both resulting in overexpression of the MYC protein. Results of numerous studies of Myc gene amplification have shown that one Myc family member is amplified in 18-38% of SCLCs and 8-20% of NSCLCs with the lower end of the range representing findings in primary tumors and the upper end in cell lines (75). Furthermore, Myc mRNA expression has been noted in 33-67% of NSCLCs (41). Myc family DNA amplification has been associated with the highly malignant variant class of SCLC (V-SCLC) (50). It is also seen more often in patients that have been previously treated than in untreated patients, and is associated with reduced survival (41).

In relation to involvement of PI3K signal transduction, work by Lee et. al. in Jon Kurie¡¦s group has suggested that the PI3K and MAPKK4 pathways cooperate to maintain lung cancer survival. In their studies, NSCLC cell lines underwent arrest after treatment with an inhibitor of PI3K catalysis (116).

Autocrine/Paracrine Loops
Many growth factors or neuropeptides and their cognate receptors are expressed by individual cancer cells or the adjacent stroma. This results in several autocrine or paracrine loops that provide a driving force for tumor cell proliferation. Autocrine loops involving co-expression of neuropeptides and their specific G-protein coupled receptor (GPCR) are especially common in SCLC. Activated GPCRs have been shown to produce proliferative signals and to elicit a mitogenic response in a variety of cell types (35). Bombesin/gastrin releasing peptide (GRP), bradykinin, cholecystokinin (CCK), gastrin, neurotensin and vasopressin are all thought to be involved in driving SCLC growth (78).

One well characterized autocrine system involves gastrin-releasing peptide or other bombesin-like peptides (GRP /BN) and their receptors. Expression of GRP was demonstrated in 20-60% of SCLC by immunohistochemical analysis. Neutralizing antibodies against GRP/BN and bombesin antagonists inhibit both "in vitro" and "in vivo" growth of SCLC cell lines, and monoclonal antibodies show anti-tumor activity against SCLC in clinical trials (81). Thus GRP /BN autocrine signaling appears to play an important role in stimulating growth of SCLCs. Interestingly, the aberrant expression of these genes does not seem to be linked to gene amplification or rearrangement. GRP /BN is known to be involved in embryonic lung development suggesting that perhaps the cells of these tumors have de-differentiatiated to a more primitive state or have reactivated developmentally important signaling pathways. In addition the high percentage (57%) of SCLCs expressing both gastrin and its receptor CCK-B (74) further support a prominent role of neuropeptide autocrine signaling as a driving force for SCLC proliferation.

Peptide growth factor autocrine loops are more commonly found in NSCLC than SCLC. These growth factors bind to and activate receptor tyrosine kinases (RTKs) which then initiate intracellular signaling cascades. Expression of the Neuregulin receptor ERBB2 (also known as HER2/neu) has been noted in ~30% of NSCLCs and has been associated drug resistance and metastatic potential. The ERBB1 receptor (also known as the Epidermal Growth Factor Receptor) is also commonly overexpressed in NSCLC along with its ligands TGF-a, amphiregulin and EGF (78),(81). Several additional growth factor/RTK autocrine loops may also play a role in SCLC lung cancer proliferation including KIT and its ligand stem cell factor (SCF) as well as insulin like growth factors (IGFs) and their receptor IGF-R which are expressed in both SCLC and NSCLC.

Anti-apoptotic Genes
Bcl-2 was first identified as a proto-oncogene located at a translocation breakpoint in many B cell lymphomas. Bcl-2 is an anti-apoptotic protein that functions at the mitochondrial membrane. It is thought to promote cell survival by inhibiting linker proteins necessary for the activation of caspases. More than 90% of SCLCs express the bcl-2 protein. Most Bcl-2 positive tumors express the protein in a high percentage of the tumor cells (40, 109). A smaller subset of NSCLCs are bcl-2 positive. The bcl-2 protein is expressed in ~25% of squamous cell carcinomas and ~12% of adenocarcinomas (68). Interestingly, bcl-2 expression is thought to correlate with good prognosis in NSCLCs, but does not seem to correlate with prognosis in SCLC.

Tumor Suppressor Genes and Cell Cycle Regulation
Mutations or deletions of p53 are very common in both NSCLC (50%) and SCLC (80%). P53 normally acts to induce cell cycle arrest or apoptosis in response to cellular stresses such as DNA damage. P53 functions as a sequence specific transcription factor activating genes responsible for G1 arrest such as p21 ^Waf1/Cip1, to allow cells to repair damaged DNA before replication. Alternatively p53 can activate the transcription of genes involved in apoptosis such as BAX and PERP others. Mutations in the p53 gene are found in ~70% of SCLC and ~45% of NSCLC (9,31,88).

Alterations in the Rb pathway are also important in both SCLC and NSCLC. The RB protein acts as a growth suppressor by inactivating proteins that promote transcription of genes required for DNA replication, thus blocking the G1/S transition. RB mutations are found in 90% of SCLCs and 15-30% of NSCLCs most of which result in a truncated RB protein (6, 14, 36, 73). Furthermore, Cyclin D1 is overexpressed in up to 47% of NSCLCs (4, 60). Cyclin D1 acts to inhibit RB function by inducing it phosphorylation by Cdk4. Mutations in p16INK4A, an inhibitor of Cdk4 kinase activity, are also common in NSCLC (~60%)(67).

A second protein p14ARF is encoded by the p16INK4A locus. p14ARF is transcribed from an alternate reading frame that largely overlaps that of p16INK4A, but results in a totally unrelated protein. p14ARF prevents p53 degradation by MDM2 , resulting in p53 activation. p14ARF mutations are found in 19-37% of NCLCs (25, 66). The p14ARF protein is frequently lost in SCLC (65%), although the mRNA transcript is still present suggesting a post-transcriptional mechanism of inactivation. Interestingly, loss of p14ARF often occurs in the presence of p53 mutations in both NSCLC and SCLC, suggesting an alternative tumor suppressor function for p14ARF, distinct from that of p16INK4A (25).

Common regions of chromosomal loss and LOH suggest the existence of other tumor suppressor genes involved in lung tumorigenesis. Deletions of 3p are observed in 50% of NSCLCs and 90% of SCLCs. Such deletions often include FHIT, a candidate TSG , and abnormal FHIT mRNAs have been found in 40-80% of lung cancer (85). However many of these tumors also express the wild type FHIT transcript as well, raising the question of whether FHIT acts as a classical tumor suppressor. More recently, several other genes at the 3p locus have been identified. RARƒÒ, RASSF1A, FUS1, SEMA3B, and ROBO1 have been cloned and their expression found to be frequently lost in lung cancer, resulting in their label as potential lung TSGs. (117) RASSF1A is implicated in microtubule stability and when re-expressed in lung carcinoma cells, reduces soft agar colony formation and tumor formation in nude mice (118,119). Despite these findings, the relevance of these genes in lung cancer in vivo remains to be determined. The 3p region lost in human lung cancers maps to at least 3 separate syntenic regions in the mouse genome. Therefore, studies using engineered deletions in mice would be useful to determine which are the most relevant TSG(s) in this region. Loss of the 9p allele, which includes the p16 locus, may be a secondary event to 3p loss. Further cloning and characterization of these and other regions frequently lost in lung cancers will help to elucidate the pathways and the order of genetic changes required for lung tumorigenesis.

Additional sites of chromosomal loss for which the candidate TSGs remain unknown include 4p, 4q, 5p, 5q, 10p, 10q, 13q34 for SCLC; 1p, 6p, 13q11, 18q, 19p and Xq22.1 for NSCLC; and 8p, 9q, Xp for both SCLC and NSCLC (28).

Tumor Vasculature
Small tumors (1-3 mm) can obtain nutrients and oxygen by passive diffusion from their surrounding tissues. However, neovasculariztion is needed to support tumor growth, progression and metastasis. For tumors to induce angiogenesis, tumor cells and stromal cells must secrete factors that induce endothelial migration and proliferation. Vascular endothelial growth factor (VEGF ) stimulates neovasculariztion in a paracrine fashion. It is expressed by >50% of NSCLCs, and is associated with an increase in intratumoral microvascular density (IMD) and poor prognosis (59, 100). Platelet derived endothelial growth factor (PD-ECGF ) was initially identified as a novel angiogenic factor in platelets (98). PD-ECGF is expressed by ~32% of squamous cell carcinomas, 42% of adenocarcinomas and 33% of adenosquamous carcinomas (26). IL-8 is a member of the CXC chemokine family and has been reported to be a potent angiogenic factor (46). IL-8 is expressed in approximately 45% of NSCLCs and is associated with increased IMD (59). However, neither IL-8 nor PD-ECGF is expressed at significant levels in SCLC (110, 108). Finally, 49-70% of pulmonary adenocarcinomas express bFGF and expression correlates with poor prognosis (89, 90).

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