6.1.1. Growth Factors
Growth Factor Receptors are Receptors tyrosine kinase pathway. Growth factors are required by all cell to undergo proliferation. Growth Factors has paracrine action. However many cancer cells start expressing the growth factor to which they are responsive, generating an autocrine loop. Growth factor receptors can be constitutively activated in tumors by various mechanisms, including mutations, gene rearrangements, and overexpression.
- Glioblastomas secrete platelet-derived growth factor (PDGF) and express the PDGF receptor.
- Sarcomas secrete transforming growth factor α (TGF-α) and express the receptor. Some times the growth factor is not mutated but a transcription factor like RAS is mutated. Mutated RAS containing cell express large amount of growth factors, such as TGF-α.
- Breast and ovarian cancers FLT3 receptor (FMS-like tyrosine kinase 3) is amplified on cell surface.
- Leukemia cancer is due to Point mutation in RET receptor (receptor for neurotrophic factors).
- Squamous cell carcinoma of lungs, gliomas is due to overexpression of ErbB1 (EGFR), ErbB2. These proteins are EGF-receptor.
- Gastrointestinal stromal tumors, seminomas are because of point mutation in KIT. KIT is a receptor for stem cell factor.
- RET proto-oncogene, a receptor tyrosine kinase for glial cell line it promote cell survival during neural development. RET loss of function mutation are associated with the development of Hirschsprung’s Disease. While gain of function mutation are associated with the development of various types of Human cancer.
6.1.2. Signal-Transducing Proteins
The most well-studied example of a signal-transducing oncoprotein is the RAS family of guanine triphosphate (GTP)-binding proteins (G proteins).
6.1.3. The RAS Oncogene
Three types of RAS genes are associated with cancer in human genome (HRAS, KRAS, NRAS) Ras oncogenes is originally discovered as reteroviral oncogene and its constitutively activating RAS.
Mutation have been identified in human tumors. The RAS genes, of which there are three in the human genome (HRAS, KRAS, NRAS), were discovered initially in transforming retroviruses. The single most common abnormality of proto-oncogenes in human tumors is Point mutation of RAS family genes. RAS is the most important downstream signaling of growth factors. RAS is a G-protein. When RAS is bounded with GDP it is inactive and when it is bounded with GTP it is active. The activated RAS stimulates downstream regulators of proliferation, such as the mitogen-activated protein (MAP) kinase cascade.
The orderly cycling of the RAS protein depends on two reactions:
- Nucleotide exchange (GDP by GTP), which activates RAS protein, and
- GTP hydrolysis, which converts the GTP-bound, active RAS to the GDP-bound, inactive form.
GEFs and GAPs: critical elements in the control of small G proteins.
Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) regulate the activity of small guanine nucleotide-binding (G) proteins to control cellular functions. In general, GEFs turn on signaling by catalyzing the exchange from G-protein-bound GDP to GTP, whereas GAPs terminate signaling by inducing GTP hydrolysis.
Several different point mutations of RAS have been identified in cancer cells. The point mutation reduce the GTPase activity of the RAS protein, thus cell is forced into a continuously proliferating state.
In Colon, lung and pancreatic tumors point mutation is found in KRAS.
In Bladder and Kidney tumors point mutation is found in HRAS.
In Melanomas tumors point mutation is found in NRAS and also in BRAF.
In Hepatoblastomas, hepatocellular carcinoma tumors point mutation causes overexpression of b-catenin. b-catenin is involved in WNT signal transduction .
In Acute lymphoblastic leukemia ABL is translocated. ABL is a Nonreceptor tyrosine kinase.
All the cancer therapies are targeted toward anti – RAS modalities but unfortunately none of these strategies has so far proven to be successful for clinical use.
6.1.4. Transcription Factors
Some protein work as transcription factor and involve in tumour formation.
The MYC Oncogene
Myc (c-Myc) is a regulator gene that codes for a transcription factor. The transcription factor plays a role in cell cycle progression, apoptosis and cellular transformation. Overexpression of the MYC protein are commonly found in tumors. In Burkitt lymphoma, a B-cell tumor the Myc gene is translocate to Burkitt lymphoma, a reciprocal translocation between chromosomes 14 and 18 is also extremely common in follicular B cell lymphoma. This translocation leads to the fusion of the BCL-2 gene with the IGH locus, resulting in anti-apoptotic protein BCL-2 overexpression.
Dysregulation of MYC expression resulting from translocation of the gene occurs in Burkitt lymphoma, a B-cell tumor. MYC is amplified in some cases of breast, colon, lung, and many other carcinomas. The related N-MYC and L-MYC genes are amplified in neuroblastomas and small-cell cancers of the lung, respectively.
6.1.5. Alterations in Nonreceptor Tyrosine Kinases
Mutations in Nonreceptor Tyrosine Kinases are also associates with cancer. Normally the type of mutation is chromosomal translocation or rearrangements that create fusion genes encoding constitutively active tyrosine kinases.
An important example of this oncogenic mechanism involves the chromosomal translocation. The Chromosomal translocation cause a proto-oncogene to move to a different chromosomal site associated with increased expression. The translocation also cause a proto-oncogene to fuse another gene to produce a protein that has oncogenic activity.
Example: Retinoic acid receptor/acute promyelocytic leukemia (RARA/APL): APL is a leukemia in which differentiation is blocked at the cell stage. It is a reciprocal chromosome translocation. The RARA receptor binds with trans retinoic acid forms a functional transcription factor by heterodimerizing with retinoic acid X receptor(RXR) Plus its ligand cis-retinoic acid. The PML-RARA fusion protein will heterodimerize with retinoic acid X receptor(RXR) and interfere with RXR functions. The RARA gene from chromosome 17 is translocated next to PML gene on chromosome 15.this translocation generate two different PML-RARA fusion proteins. Also, loss of part of the RARA transactivation domain interferes with trans retinoic acid function.
Example is c-ABL tyrosine kinase. The ABL1 is a proto-oncogene encodes a cytoplasmic and nuclear protein tyrosine kinase which involve in cell differentiation, cell division, cell adhesion, and stress response. In CML the ABL gene is translocated from its chromosome 9 to chromosome 22, where it fuses with the BCR gene.
Activity of ABL1 protein is negatively regulated by its SH3 domain and Mutation or deletion of this SH3 domain convert ABL1 gene into oncogene. The resultant chimeric gene product is constitutively active, oncogenic BCR-ABL tyrosine kinase. BCR –ABL protein can be inhibited by various inhibitor.
6.2. Viral Carcinogenesis
Carcinogenesis is the mechanisms whereby viruses cause cancer that virus carried out a gene altered the cell regulation. Example of viruses that contribute to carcinogenesis.
There are two classes of tumour viruses: RNA viruses and DNA viruses.
6.2.1. RNA viruses
RNA virus infect competent cells. Their RNA is converted into DNA that incorporated into the host genome. Hence, that is classification as retroviruses. Example of retrovirus: human T-lymphotropic virus(HTLV-1) and Rous sarcoma virus(RSV). RNA viruses show two general ways: provision of an oncogene or insertional mutagenesis in which regulatory sequences alter host gene activity.
Provision of an oncogene: Many oncogenic RNA viruses contain an oncogene additional to the sequences for viral replication. The Rous sarcoma virus, in which the oncogene v-src codes for a 60 kDa phoshoprotein(pp60src) that has tyrosine kinase activity.
Insertional mutagenesis: Mouse mammary tumour virue(MMTV) has regulatory sequences that can stimulate host genes. The regulatory sequences are repeated at each end of viral genme(long terminal repeats, LTRs). The LTRs contain enhancer sequences capable of increasing transcription from nearby genes(int) that codes for fibroblast growth factor. HTLV code for transcription factors that regulate host genes such as fos.
6.2.2. DNA viruses
Several DNA viruses are specific in human cancers: human papilloma virus(HPV) , hepatitis B virus(HBV), Epstein-barr virus(EBV), Kaposi sarcoma virus(KSV). The cellular responses of these viruses derive from the proteins E6 and E7. The E6 and E7 mRNAs are generated by cleavage of larger RNA. Carcinogenesis is time-dependent, multistage process; inactivation of Rb and p53 by viral E6 and E7 proteins. The progress of the disease is favoured by various cofactors such as tobacco, smoke and oral contraceptives.
If a protooncogene under goes a somatic mutation, control of cell growth is lost in the cell in which the mutation occurs and cancer can occur. Those protooncogenes which have been shown to mutate in any individual are called cellular oncogenes and are designated by the prefix "c" (i.e. c-myc, c-abl) to distinguish them from the viral oncogenes. Those protooncogenes that have not been found to mutate are called normal oncogenes and are designated "n" (i.e. n-ras).
6.2.3. Reterovirus Associated Oncogenes
The oncogene of those viruses transform a cell to unproliferated growth carry an oncogene in addition to the three primary genes required of all retroviruses. The figure below is the generalized structure of a retrovirus.
Example of Reterovirus
Rous Sarcoma Virus.
The oncogene found in this retrovirus is src. Src is a receptor Tyrosine kinase and involve in cell division and cell proliferation. After the infection of Rous Sarcoma Virus the cell start expressing more src which leads to tumor formation.
Avian erythroblastosis Virus
The oncogene found in this virus is V-erbB. V-erbB is epidermal growth factor receptor. V-erbB is a receptor Tyrosine kinase and involve in cell division and cell proliferation. After the infection of Avian erythroblastosis Virus the cell start expressing more V-erbB receptor on cell surface which leads to tumor formation.
Harvey Murine Sarcoma
The oncogene found in this virus is V-H-Ras. V-H-Ras binds to the GTP. V-H-Ras involve in cell division and cell proliferation. After the infection of Harvey Murine Virus the cell start expressing more V-H-Ras which leads to tumour formation.
6.3. Tumour suppressor genes
Tumor suppressor genes are genes that slow down cell division, involve in DNA Repair and apoptosis. When tumor suppressor genes don't work properly, cells can grow out of control, which can lead to cancer that is created by loss of function mutation. In contrast activate mutation inactivated oncogene from protooncogenes, tumor suppressor genes and proteins that are encode. Tumor suppressor gene suppress the tumor. Tumor suppressor gene control the processes of genetic of integration, the progression of cell cycle, differentiation , cell-cell interactions and apoptosis.
Tumor suppressor genes are inactivated by mutations contribute to the loss of tissue homeostasis –the hallmark of a developing neoplasm. The open reading frames of tumor suppressor genes are commonly truncated by nonsense mutations, frameshift mutations, small addition or deletions, or splice site mutations. Sometime exon removed by larger deletions. Tumor suppressor gene also inactivated by alteration of single nucleotide residues in tumor suppressor protein, there by encoded protein become non-functional.
TSG can be grouped into categories including caretaker genes, gatekeeper gene and landscaper genes Retinoblastoma.
It is caused by loss of both alleles of a gene. Rb is tumor suppressor protein. Mutation in Rb results in cancer. Normal cell express suppressor protein that inhibits the cancer.
6.3.1. Ratinoblastoins :
Rb is a large gene (300kb), although mostly mutations are in the 3kb coding region and mostly involve chromosomal changes. Several retinoblastoma can arise in one eye, each with a different Rb mutation. The Rb protein has about 10 phosphorylation sites. Rb has the ability to interact with other proteins. Over 25 Rb-binding protein have been identify with functions such as nucleosome structure(Brm), Tyrosine phosphorylation (abl), oncogenes (Mdm2) and transcription factors (E2F, DP) are responsible for proliferation. The Rb proiein has more than 10 phosphorylation (ser/thr) sites. Conversion from hypo- to hyperphosphorylated states changes the interaction of Rb with other proteins. Rb binds with transcription factor E2F. It is regulated by phosphorylation. In hypophosphorylated state of Rb the E2f is bounded this makes E2f inactive. The proliferation of cell cycle is regulated by cyclin dependent kinases (CDKs). These cyclin-dependent kinases phosphorylate and inactivate Rb, there by relieving the cycle block.
The released E2F stimulates the transcription of gene that regulate growth , such as cdc2, myc and DNA polymerase.
Rb also inhibits transcription from rRNA and tRNA genes by binding of UBF (upstream binding factor) and TF-IIIB(transcription factor IIIB) . Rb thus influences the mass of a cell(protein content). Rb suppression occurs normally by hyperphosphorylation and abnormally by Rb mutation or binding to other proteins.
The p53 tumor suppressor gene is activated in response to a wide variety of cellular stresses including DNA damage, ribonucleotide depletion, redox modulation, hypoxia, changes in cell adhesion, and the stresses created by activated oncogenes. The p53 protein work as transcription factors and activated the genes which is involved in DNA repair, apoptosis and growth arrest. These activities of P53 help in maintaining the genomic stability. Hence P53 is called as guardian of genome.
If normal p53 is mutated and non-functional by binding of other protens.p53 has four functional domains involved transcription activation domain(TAD), DNA binding domain(DBD), oligomerization domain(OD), autoinhibitory domain(AID). Each domains binds to several proteins that regulate p53 function. P53 binds to its response element present upstream to the gene to transcribe the gene. P53 binds in tetrameric form. p53 increase the expression of Bax, p21, insulin-like growth factor binding protein 3(IGFB3), GADD45 and thrombospondin. Expression of genes such as Bcl2, Fos and jun can be inhibited by p53. Thus p53 inhibits cell proliferation.
The gene p21 that code for cyclin-dependent kinase inhibitor. p21 inactivates CDK that is essential for DNA synthesis. GADD binds to a protein proliferating cell nuclear antigen(PCNA) that needed for both DNA synthesis and repair. Hence, p53 inhibit DNA synthesis while allowing repair to continue. DNA damage activates p53 function by post –transcriptional and cell-type specific mechanisms.
Normal p53 show inactivation by protein binding. For example- Adenovirus codes for E18 that binds to TAD of normal p53 and block its transcription. Some human sarcomas have mdm2 gene that have same end results. Human papilloma virus have protein E6 that binds to OD and prevents dimerisation, while the HBX protein of hepatitis B virus binds and inactivates p53.
P53 transcribe the thrombospondin, PAI, KAI, BAI and they block the angiogenesis. Mutation in p53 thrombospondin, PAI, KAI, BAI.