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Lung cancer results from the uncontrolled lung cell proliferation mainly from the epithelial cells of the lungs hence termed carcinomas. Lung cancer is mainly divided into non small cell lung cancer (NSCLC) and the small cell lung cancer ( SCLC) whereby NSCLC is the major killer in cancer resposnsible for over 1.2 million deaths a year. The main forms of NSCLC are adenocarcinoma, squamous cell carcinoma and large cell carcinoma. According to Brambilla and Gazdar 2009, 85% of lung cancers are due to tobacco smoke which causes the accumulation of epigenetic and genetic abnormalities resulting in the multistep epithelial carcinogenesis destabilise the normal cell growth whilst 25% of lung cancers result independent of tobacco smoke. The progression of lung cancer follows the 'Hallmarks of cancer' described by (Hanahan and Weinberg, 2000) as growth signal autonomy, evasion of growth inhibitory signals, evasion of apoptosis, unlimited replicative potential, sustained angiogenesis, and tissue invasion and metastasis. Fong et al, (2003) went on to support these findings whereby they found that abnormalities in the tumour suppressor genes and overactivity of the growth proto-oncogenes led to the 'Hallmarks of lungs cancer'. Ignacio et al, 2006 explains that lung cancers are mainly due to a variety of complex epigenetic and genetic factors, which inactivate the tumour suppressor genes and activate the oncogenes.
Normal lung cells require growth factors to enable them to grow and regenerate. Most of the growth factors are provided by other surrounding cells and their release is tightly regulated to maintain the proper regulation of cell cycle control, but the lung cancer tumour cells find ways to abolish the proper signalling of growth factors. In the non small-cell lung cancer (NSCLC) and the small cell lung cancer (SCLC), abnormal growth factor signalling and expression involving insulin growth factor like (IGF-1), epidermal growth factor receptor (EGFR) expression, RAS proto-oncogene and c-Myc proto-oncogene signalling has been observed.
The small cell lung cancers (SCLCs) exhibit abnormal increase in the expression of insulin growth factor like (IGF-1) and its receptors therefore initiating a self autocrine signalling loop. The tumour cells lose their dependency on the growth factor autocrine homeostasis and become self-sufficient leading to the overexpression of IGF-1 and subsequently cell proliferation. The overexpressed IGF-1 ligands (IGF-I and II) bind to the IGF-IR and IGF-IIR respectively which are cell surface receptor tyrosine kinases. These IGFRs function primarily through the MAPK pathway and the PI3-Kinase pathway leading to increased cell growth and proliferation, downregulation of apoptosis and increased cell migration.
In non small cell lung cancer (NSCLC) there is overexpression of the receptor tyrosine kinases (RTKs) such as the epidermal growth factor receptors (EGFRs). Fong et al, 2003 also mentioned that the ERB family RTKs (ERB1, EGFR) and (ERB2, Her2/neu) are expressed in lung cancers mainly in NSCLC. When the ERB family RTKs bind their ligands they can either homodimerise or heterodimerise, become activated and transduce downstream signalling of kinase cascades enhancing tumour survival and proliferation. (Fong et al, 2003). Under normal physiological conditions EGFR functions through different downstream signalling pathways including the MAPK pathway, activation of the PI3-kinase/AKT (PKB) and the phospholipase C-γ pathway as shown in figure 1 to maintain normal cellular growth and proliferation.
In lung cancer cells EGFRs are overexpressed and according to Poulsen et al, 2008, EGFR is found to be overexpressed in about 50 to 90% of all NSCLCs, mainly in the squamous cell carcinoma. Lung tumour cells also express transforming growth factor α (TNFα) and epidermal growth factor (EGF) which are EGFR ligands. The expression of these ligands stimulates an autocrine growth loop making the tumour cells self sufficient in growth signals. Therefore overexpression of growth factor receptors and growth factor self sufficiency of tumour cell is vital in lung cancer pathogenesis.
In NSCLC abnormal growth factor function has also been found to be caused by mutation in the EGFR gene. Poulsen et al (2008) states that a mutant EGFR termed EGFRvIII is found in approximately 16% of the NSCLC and this receptor has the intracellular and the membrane spanning domain only. Therefore EGFRvIII cannot bind its ligand due to the missing extracellular ligand binding domain but is constitutively active and can activate downstream signalling pathways to drive uncontrolled cell proliferation.
The upregulation of the PI3-kinase and AKT (PKB) pathway enhances the survival of cell due to the inhibition of pro-apoptotic proteins such as Bad and Bax, and the activation of anti-apoptotic proteins such as Bcl-2. PKB also inhibits GSK3 which is an inhibitor of cyclin D1 therefore enhancing progressing of the G1/S transition as shown in figure 1.
Mutations have been observed in the RAS proto-oncogene mainly due to cigarette smoke. G-T point mutations are the main type of mutations found in the RAS gene, Kirsten-ras(K-RAS) which is mutated in about 30% of NSCLC. K-RAS is important in promoting cell proliferation due to mutations in codons 12, 13 and 61which are found to be frequently expressed in NSCLC mainly in the adenocarcinoma. c-MYC is another proto-oncogene which is also activated in both the NSCLC and SCLC. c-MYC is found to be overexpressed in cancer cell due to increased gene amplification, transcriptional dysregulation and Myc mRNA stabilisation. Both the RAS and c-MYC proto-oncogenes are very potent growth promoters and their overexpression stimulate uncontrolled cell proliferation.
Lung cancer cells are able to evade growth inhibitory signals (Hanahan and Weinberg, 2000) by causing the inactivation of tumour suppressor genes such as P53, retinoblastoma (Rb), P16INK4, TGFRII and the loss of heterozygosity (LOH) at chromosme 3p. For the suppressor genes to be fully inactivated, both alleles of the gene must be mutated. This occurs through the two-hit hypothesis whereby the first allele undergoes deletion or chromosomal translocation subsequently leading to loss of heterozygosity (LOH). The other allele undergoes inactivation by single point mutation or due to epigenetic hypermethylation of the promoter.
Under normal conditions the P53 is bound to MDM2 which inhibits the function of P53, but when DNA damaged is experienced, p14ARF binds and inhibits MDM2 releasing an active p53 as shown in figure 2. The ATR and ATM kinases also phosphorylate P53 on ser15 to activate it and an activated P53 senses DNA damage and stimulate DNA damage checkpoints to arrest the tumour cells. P53 which is located in chromosome 17p13, is mutated in the vast majority of lung cancers more specifically in the SCLC and squamous cell carcinoma. P53 undergoes various changes in lung cancer from the more severe loss of heterozygosity to localised point mutations. The P53 inactivation has been observed in about 50% of NSCLC and 80% of SCLC according to Poulsen et al, 2008. The mutated P53 acquires some stability which makes it stable and accumulates in lung cancer cells, resulting in the loss of G1/S and G2/M transition DNA damage checkpoints. When P53 is mutated the transcription factor P53 becomes unable to transcribe genes such as cyclin-dependent kinase (Minna, 1993) inhibitors (CKIs) which arrest the cell cycle when it encounters DNA damage.
Retinoblastoma (RB) is another tumour suppressor which is commonly found to be mutated in lung cancer. RB is a transcription factor involved in the regulation of the cell cycle at the G1/S transition phase and it is found on chromosome 13q14. It exerts its tumour suppression function when not phosphorylated, whereby it binds proteins including those of the E2F family and therefore preventing transcription of genes required for G1/S transition. Therefore when the RB becomes mutated or inactivated, they lose the ability to interact with the E2F family of proteins and these are released leading to the transcription of genes required for G1/S transition. Poulsen et al, 2008, states that in 90% of the SCLC tumours RB is found to be inactivated either by the loss of heterozygosity or by single mutations.
p16INK4a is a member of the INK4 family inhibitors of CDK4 located on the chromosome 9p21, (Minna 1993) which acts as a tumour suppressor by indirectly inhibiting the phosphorylation of RB. p16INK4a inhibits the formation of the cyclin D-CDK4/6 complexes which are responsible for phosphorylating Rb and allow progression of the G1/S transition phase. The p16INK4a gene is commonly found to be inactivated in NSCLC mainly due to the loss of heterozygosity at chromosome 9p21. When the p16INK4a gene becomes mutated, the tumour suppressor becomes unable to inhibit the formation of cyclin D-CDK4/6 complex which then phosphorylates Rb. Phosphorylated Rb loses its interaction with E2F which goes on to transcribe genes required for the G1/S transition (Weinberg, 2007) as shown in figure 3 below.
Poulsen et al, 2008 explains that these mutations of Rb and p16INK4 contribute to the inactivation of Rb and that a dysfunctional Rb tumour suppressor is found in almost every lung cancers. In support to the loss of tumour suppressor genes in lung cancer, Estelle et al, 1999 conducted experiments with DNA from the plasma and serum of patients with NSCLC and normal patients to investigate epigenetic factors which induce lung cancer. The main epigenetic factor detected was the aberrant promoter hypermethylation observed in 68% of NSCLC tumours but not in normal patients. This data supported the findings that hypermethyation of the normally unmethylated CpG islands of tumour suppressor genes was involved in lung cancer pathogenesis, (Baylin et al, 1998) and that the hypermethylation of p16 is involved in the early pathogenesis events of lung cancer.
TGFβ (transforming growth factor β) is a growth factor which is involved in the inhibition of cell cycle progression. TGFβ binds to its serine/threonine kinase receptors, TGFβRI and TGFβRII to inhibit Cyclin-CDK complexes responsible for phosphorylating Rb. TGFβRII has been observed to be absent in SCLCs resulting in no response to TGFβ, also known as TGFβ resistance. Poulsen et al, 2008 explained that a functional TGFβRII was introduced into receptor negative lung cancer cells and TGFβ response was restored confirming the lack of TGFβRII in SCLC.
The loss of genes within chromosome 3p has been showed to be arguably the most common form of chromosomal abnormality in all lung cancers. The chromosome 3p region contains genes such as the fragile histidine triad (FHIT) and RASSFIA located at chromosome 3p14.2 and 3p21 respectively. These genes are thought to act as tumour suppressors and therefore inhibit cell proliferation under normal physiological conditions. The loss of function of chromosome 3 is mainly due to the loss of heterozygosity and this has been observed in 70 to 100% of NSCLC and in over 90% of SCLC.
The FHIT mutations have been shown to be very frequent in lung cancer. Their importance was shown by mice studies, whereby by a functional FHIT was inserted into NSCLC cell lines and in mice. The results showed tumour suppression and induction of apoptosis. FHIT is also believed to prevent the ubiquitinylation of p53 by binding to MDM2 and therefore promoting apoptosis. Therefore when FHIT becomes mutated in lung cancers, the tumour cells cannot undergo apoptosis and continue to proliferate indefinitely. RASSFIA is an inhibitor of DNA synthesis and also decreases cyclin D1 expression. Its function can be lost by promoter hypermethylation in both NSCLC and SCLC but most frequently in SCLCs.
Tumour cells acquire the ability to evade apoptosis which is a programmed cell death necessary for controlled cell growth and proliferation. Apoptosis is controlled by pro-apoptotic factors such as (Bax), anti-apoptotic (Bcl-2), death receptors (Fas) and p53 tumour suppressor genes. According to Brambilla and Gazdar 2009, BCL-2, an anti-apoptotic factor has been found to be overexpressed in 95% of SCLC and 25% of NSCLC. Fong et al, 2003 also supported the idea that BCL2 is found frequently in SCLC than in NSCLC. BCL2 is found to be expressed in higher levels compared to Bax in the majority of SCLC cells with a dysfunctional p53. A dysfunctional p53 inhibits apoptosis because the inactive p53 becomes unable to activate pro-apoptotic proteins such as Bax which mediate cell apoptosis or to inactivate the anti-apoptotic Bcl-2 as shown in Figure 4.
Fas receptor (CD95) is a death receptor which stimulates apoptosis by the extrinsic binding upon the binding of its ligand called FasL. Binding of FasL induces a Fas conformational change and the formation of the death-inducing signalling complex (DISC), resulting in the formation of the apoptosome leading to apoptosis as illustrated in figure 5. According to Brambilla and Gazdar 2009, the Fas receptor and FasL have been found to be dowregulated in 70% of NSCLCs enabling the lung tumour cells to evade apoptosis. High levels of FasL expression but with a low or zero expression of the Fas (CD95) receptor have been observed in 50% of SCLC again allowing these cells to evade apoptosis by disrupting the Fas-FasL mediated pathway. Pitti et al, (1998) discovered a decoy Fas receptor 3 (DcR3) which lacked an intracellular domain. This DcR3 receptor was able to bind its ligand FasL but could not transduce the apoptotic signal downstream into the cell to stimulate the assembly of the (DISC) and the activation of the caspase cascade. The DcR3 gene was found to be amplified in 35 primary lung tumours which were studied and therefore lung tumour cells can escape apoptosis by blocking the function of FasL using DcR. Inhibitors of apoptosis proteins-1 (IAP-1) are highly expressed in NSCLC, therefore inhibiting the tumour cell from going into apoptosis.
Brambillar and Gazdar 2009, mentioned that E2F1 involved in the G1/S transition is also involved in apoptosis. It functions by destabilising the alternative splicing of Flip-short (Flip-s) which is an inhibitor through MYC- induced and p53 dependent or independent pathways resulting in the downregulation of Flip-s. Flip-s is an inhibitor of Fas receptor (CD95) and when it becomes downregulated the tumour cells become able to evade apoptosis. Therefore high levels of E2F1 in SCLC might increase cell proliferation.
If cells lose suppressor gene functioning and gain some oncogenic activation they can proliferate uncontrollably and gain an unlimited replicative potential. Telomeres act by preventing the fusion of chromosomes but in DNA damage the chromosomes lose their telomeres. Lung cancer cells are able to overcome limited replication barrier by activating telomerase which is an enzyme responsible for the telomere generation. Therefore the lung tumour cells can continue to proliferate uncontrollably.
Lung carcinoma cells undergo the epithelial-mesenchymal transition (EMT) whereby they shed the polarised epithelial phenotype to a motile, fibroblastoid and mesenchymal phenotype. This enables them to acquire the ability to invade surrounding cells. E-cadherins form homophilic cell-cell interaction due to the interaction of their protruding ectodomains to maintain the adherens junctions which are vital in maintaining the epithelial cell structural integrity. In lung carcinoma tumours, the E-cadherin is lost leading to the loss of epithelial cell polarity. The loss of E-cadherin from the plasma membrane also release β-catenin which may act as an oncogene by translocating to the nucleus where it drives the transcription of genes such as Tcf/Lef transcription factors involved in promoting the EMT and proliferation.
According to Fong et al, 2003 lung cancer cells are often observed to have reduced expression of the laminin α3 and α5 chains. This can result in the fragmentation of the basal membrane, proliferation of some components of the stroma and also enhanced cell invasion. They also explained that LAMB3 a gene which codes for laminin 5 has been observed to be expressed in NSCLC. Laminin 5 specifically binds to its α6β4 integrin receptor to form hemidesmosomes resulting in stable adhesion to promote NSCL growth. The α6β4 is expressed in NSCLCs but not in SCLC and therefore laminin 5 expression appears to be vital in NSCLC metastasis. Investigations in patients with NSCLC have shown that overexpression of laminin-5 was associated with a decrease in the survival of the patients.
Angiogenesis is the sprouting of blood vessels from the existing ones and tumour growth is angiogenic dependent. In NSCLC, tumour-associated macrophages (TAMs) have been found to produce mainly matrix metalloproteinase-9 (MMP-9). Activated MMP-9 enhances angiogenesis by reorganising the structure of the tissue to create space for the expanding tumour cells. They also cleave off immobilised critical mitogens which are tethered onto the extracellular matrix (ECM) proteoglycans and angiogenic factors from the extracellular matrix which all promote angiogenesis.
The tumour-associated macrophages (TAMs) directly release angiogenic factors such as VEGF and interleukin-8 (IL-8). The lung cancer cells produce the vascular endothelial growth factor (VEGF) which results in the increase in neovasculature of the tumour cells. VEGF is divided into VEGFA,B,C,D and E with VEGFA the main subtype. VEGFA binds to its receptors VEGFR-1 and VEGFR-2 which are receptor tyrosine kinases, and they dimerise to create an intramolecular cross-talk required for optimum angiogenesis. Once activated, this promotes angiogenesis by stimulation of proliferation, migration and survival through the activation of the Ras, PLC-γ, FAK and the PI3-K pathways.
Therefore lung cancer pathogenesis involves a lot of molecular pathway abnormalities most of which are caused by genetic and epigenetic factors mainly stimulated by cigarette smoke. Studies in lung cancer have demonstrated that the progression of the two main types of lung cancer, the NSCLC and SCLC follows the 'hallmarks of cancer' or more specifically the 'hallmarks of lung cancer'. In lung cancer, the tumour cells acquire the ability to become self sufficient in growth signals, evade growth inhibitory signals and apoptosis, and become able to replicate indefinitely. The initiation of lung tumours invasion and metastasis begin when the epithelial cells lose their polarised epithelial phenotype to acquire the mesenchymal phenotype. The tumour cells then require angiogenesis to produce new blood supply for them to survive. Therefore these molecular pathways can be targeted for the treatment of lung cancers and hopefully in future therapies can be tailor made for individual patients based on genetic profile.