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Growth Factors and Cytokines
Prospects for Cytokine Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand and Ovarian Cancer
Among gynecological cancers , ovarian cancer causes more deaths than the others. One of the problems is that ovarian cancer is often diagnosed when it has already progressed to a late stage. Even when initial treatment seems successful, the cancer can reoccur and when this happens, the cancer is usually not curable. During a lifetime, 1 female in 72 will be diagnosed with this disease. Most deaths occur when women are diagnosed in the age range of 60–70. For the year 2013 it is estimated that there were more than 14,000 deaths from ovarian cancer of more than 22,000 women diagnosed. About 5%–10% of these cancers are hereditary. The cure rate of ovarian cancer can be as high as 95% if the disease is diagnosed early. At present, of the women diagnosed with ovarian cancer, only 46% will survive for 5 years or longer.
Work is proceeding on tests for the early diagnosis , but there is little in the way of early diagnosis available in practice. Recently, researchers are using cells shed into the female gynecological tract from ovarian cancers to detect mutations in the DNA of those cells using automated systems. It is hoped to combine this approach with the frequently used Pap test. So far, experiments show that 40% of ovarian cancers can be detected, with no false positives and this test can be used to detect early stage disease.
Increased risk of developing ovarian cancer is associated with a family history of this disease or of breast cancer. Other possible conditions are early menopause and no history of pregnancy. In general, the more children a woman has the lower the risk of developing ovarian cancer. The presence of the BRCA1 gene (on chromosome 17) and the BRCA2 gene (on chromosome 13), especially hypermethylated BRCA1 ( mutated BRCA1 ) is related more to ovarian cancer than mutated BRCA2. When they are mutated, BRCA1 and BRCA2 genes become oncogenes , predisposing to cancers. In particular, BRCA1, when it is mutated, increases the risk of developing breast or ovarian cancer. There are many types of mutations that BRCA1 can undergo and some of these, in addition to hypermethylation , are 11-base pair deletion , 1-base pair insertion , a misplaced stop codon , or a missense substitution . These genes are carried in the germline and are expressed in many tissues in addition to breast and ovary. Normally, the BRCA1 gene encodes [through its messenger RNA (mRNA)] for a zinc finger–containing protein of 1863 amino acids, a fairly large protein. Both BRCA1 and BRCA2 normally function in the repair of DNA damage and in the regulation of transcription . If there is DNA damage and it cannot be repaired, the BRCA1 gene will aid in the destruction of the cell in which the damage has occurred. Because of high densities of repetitive elements in both genes, there is the risk of gene instability . Consequently, there can be large genomic rearrangements generating inherited and somatic mutations. In the face of the proliferative actions of estrogen in both breast and ovarian tissues, the normal BRCA1 gene is needed to control tissue proliferation. Loss of heterozygosity ( LOH ) is a further indication and may be a factor in tumor spread. LOH occurs when a somatic cell contains only one copy of an allele. This can be the result of segregation during recombination, deletion of a chromosomal segment or due to nondisjunction during mitosis. This occurrence becomes critical when the surviving allele contains a mutation that results in an inactive gene . This frequently is the case when a tumor suppressor gene is mutated.
For the formation of ovarian tumors, the oncogenes human epidermal growth factor (EGF) receptor-2/neu , K-ras , p53 , BRCA1, and some tumor suppressor genes on chromosome 17 may be involved in a complex pathway. Tumors at grades 2 or 3 have induced expression of genes associated with the cell cycle: signal transducer and activator of transcription ( STAT ) 1 or 3 or Janus tyrosine kinase ( JAK ) 1 or 2.
Hereditary ovarian cancer involves the mutation of the BRCA1 tumor suppressor gene (produces a protein through an mRNA that is a tumor suppressor). Mutations in BRCA1 and BRCA2 genes account for 5%–10% of all breast cancers in females and mutations in either gene result in a fivefold higher risk than normal for developing breast cancer and a 10- to 30-fold risk for developing ovarian cancer . Mutations in these genes can be inherited from either parent and being a dominant mutation , can be passed on to both male and female children, so that half the population with BRCA mutations are males meaning that each child has a 50% chance of inheriting the mutated gene from the one parent who carries it. In the male offspring with the mutation, there is only a slightly higher risk of developing breast cancer but there is an increased risk of developing other cancers, such as colon cancer and cancers of the prostate and pancreas.
Symptoms of ovarian cancer are somewhat unspecific: vaginal bleeding, abnormal intestinal gas, and nausea. An enlarged ovary is probably the first sign but the ovaries are located deep in the pelvic cavity so that it may not be detected for some time and when enlargement is detected, it can also be due to benign fibrosis and not cancer. In advanced ovarian cancer, there is a swollen abdomen with lower abdominal and leg pain and a sudden change in body weight (increase or decrease), change in bowel and bladder function, nausea, and swelling of the legs. An ovarian cancer can shed cells that can grow in other tissues, including the uterus, bladder, and bowel and these secondary tumors can develop even before the primary cancer is diagnosed. Treatment of advanced ovarian cancer involves surgery to reduce the tumor bulk and administration of Taxol and carboplatin for chemotherapy. Unfortunately, drug resistance develops in a majority of patients and they may die as a result. Expression of STAT1 by the tumor leads to resistance to carboplatin (or cisplatin). The location of the ovaries and the appearance of an ovarian carcinoma are shown in Fig. 17.1 .
There are four stages of ovarian cancer . In stage 1 the cancer is limited to one or both ovaries. Précis, 90% of women with this stage cancer have a 5-year survival rate. Stage 2 is characterized by spread of the tumor into the pelvic region (uterus, fallopian tubes, sigmoid colon, or rectum) but not to the abdomen. So far, there has been a poor record of diagnosis at this stage. Of the patients with stage 2 ovarian cancer, 80% will survive for 5 years. In stage 3 the cancer will have spread beyond the pelvis to the abdomen, to the abdominal wall, small bowel, lymph nodes, or surface of the liver. Of women with this stage cancer, 20%–50% will survive for 5 years. The most advanced condition of the disease is stage 4 in which the cancer has metastasized to the liver, spleen or lung. Only 10%–20% of these patients will survive for 5 years. The grade of the tumor corresponds to the stage of the cancer and this assignment occurs when surgery takes place and then treatment is adjusted to the stage. The grade 1 tumor is well differentiated and appears much like normal tissue. Grade 2 is somewhat differentiated and grade 3 is poorly differentiated and is clearly abnormal. After completion of therapy for a given grade of the tumor the ovarian cancer can reoccur in its original stage. Many clinical trials have been completed with the administration of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) as a form of treatment.
Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand and Its Mechanism
TRAIL is a protein related to the TNF family of cytokines that can stimulate the growth of certain cells [e.g., vascular smooth muscle cells via activation of nuclear factor kappa B ( NF κB) and the induction of insulin-like growth factor-1 (IGF-I) ; human glioma cells via cFLIPL -mediated activation of extracellular signal-regulated kinase (ERK) 1 and 2 kinases (cFLIP L is an inhibitor of caspase-8 )]and kills most types of tumor cells without, seemingly, affecting normal cells. Normal cells may have low levels of the TRAIL receptors or high levels of TRAIL decoy receptors (DcRs) to buffer the effects of the cytokine. TRAIL is effective in killing ovarian cancer cells in culture but the cancer cells secrete interleukin-8 ( IL-8) that inhibits the killing effects of TRAIL. Inhibition by IL-8 is mediated by the p38-mitogen-activated protein kinase (MAPK) pathway. Patients with ovarian cancer form ascites in which the tumor cells float in peritoneal fluid . This forms a microenvironment in which the cancer cells may synthesize and secrete factors that affect the killing process and the peritoneal fluid also may contain factors that affect the action of TRAIL [this would pertain to clinical trials in which soluble TRAIL is administered (it is possible that insoluble TRAIL might be more effective than the soluble form)]. Treatment of ovarian cancer with TRAIL is augmented by combining TRAIL with paclitaxel (Taxol relative).
There are five cellular receptors for TRAIL : R1–R5. R1 is death receptor 4 (Dr4) and R2 (Dr5) are TRAIL receptors that have transmembrane domains and cytoplasmic domains. The TRAIL ligand binds to these receptors to generate a signaling mechanism from the membrane to the cytoplasm that results in the programmed death of the cell. R3 (DcR1) and R4 (DcR2) are DcRs that bind TRAIL. R3 is attached to the cell membrane by a glycophospholipid anchor but lacks a transmembrane domain so that the death signal cannot reach the cytoplasm. Although R4 does have a cytoplasmic domain, it lacks the death domain ( DD ). Thus R3 and R4 can bind TRAIL but such complexes are unproductive and for that reason these receptors are named DcRs. Osteoprotegerin is a secreted receptor for TRAIL that lacks a transmembrane domain, a membrane anchor, and a cytoplasmic domain, so this soluble receptor also is a decoy. If the levels of the DcRs in a TRAIL target cell exceed the levels of Dr4 and Dr5, TRAIL resistance will occur. Conversely, if the productive receptors for TRAIL exceed the numbers of DcRs, the TRAIL program will be activated. The types of TRAIL receptors are summarized in Fig. 17.2 .
TRAIL initiates a signal transduction system leading to apoptosis (programmed cell death) when the TRAIL trimer binds to a productive cell membrane receptor. Following this binding, cytoplasmic DDs form an aggregate, called a DISC (death-inducing signaling complex). DISC reacts with another intracellular protein, fas-associated DD protein that causes the activation of procaspase-8 to the activated enzyme (caspase-8). Active caspase-8 cleaves proapoptotic Bid to tBID ; tBID translocates to the mitochondria and promotes the assembly of Bax-Bak ( Bax , B cl-2- a ssociated X protein; Bak , B cl-2 homologous a ntagonist/ k iller) oligomers (both are proapoptotic members of the Bcl-2 family of proteins) and permeability alterations in the mitochondrial outer membrane. Bax-Bak causes the release of cytochrome c from mitochondria and Smac/DIABLO (mitochondrial protein that neutralizes the activity of inhibitor of apoptosis proteins , i nhibitors of ap optosis) from the mitochondria into the soluble cytoplasm. With cytochrome c and a poptotic p rotease– a ctivating f actor-1 in the cytoplasm, the apoptosome is formed and facilitates the activation of caspase-9. Caspase-9 promotes the activation of the executioner caspases (caspases-3, -6, and -7) that cleave key cellular proteins resulting in DNA cleavage and cell death. DNA fragmentation in apoptosis occurs when caspase-3 activates a nuclear DNase of about 40 kDa. DNA fragmentation in apoptosis can be visualized on an agarose gel that separates nucleic acids based on size ( Fig. 17.3 ).
The overall extrinsic pathway and intrinsic pathway of apoptosis are summarized in Fig. 17.4 .
Active caspase-9 leads to the activation of procaspase-3 and other executioner caspases and these cleave key proteins in the cell that precipitate DNA fragmentation and apoptosis. The extrinsic pathway of apoptosis does not involve the mitochondria whereas the intrinsic pathway does involve the mitochondria.
The Tumor Necrosis Factor Superfamily
There are an extensive number of ligands related to the TNF and these form a superfamily (TNFSF) of which TRAIL is a member. Correspondingly, there are a number of receptors for these ligands called the TNF receptor superfamily . This combination of ligands and receptors is involved in the regulation of the immune system , inflammation , and, in some cases, antitumor activity as the name “tumor necrosis factor” implies. Spontaneous regression of tumors sometimes occurs and the activities of these factors may be responsible. Some of the TNF family of ligands and receptors is listed in Table 17.1 .
Table 17.1
The TNF Superfamily (TNFSF) and the TNF Receptor Superfamily (TNFRSF).
Source: Much of the information in this table was taken from http://www.rndsystems.com/asp/g_sitebuilder.asp?bodyid=227#top (no longer retrievable on the internet).
| Ligands/Coreceptors a | AAs; Molecular Weight | Comments and Characteristics |
|---|---|---|
| NGF | 120AA; 12.5 kDa | From a propeptide with signal sequence; homodimer 1525 kDa; binds to LNGFR member of the TNFRSF |
| CD40L | 261AA; 39 kDa; 22AA cytoplasmic domain; 215AA extracellular domain | Membranes of B cells, CD4 + & CD8 + T cells, mast cells, basophils, eosinophils, dendritic cells, monocytes, NK cells, and gd cells. Also as proteolytically cleaved, cytoplasmic form of 15–18 kDa with biological activity; forms trimeric structures like TNFα |
| CD 137 L/4–1BBL | 309AA; 50 kDa; 82AA and 34-kDa cytoplasmic region; 21AA transmembrane segment; 206AA extracellular domain | Expressed in B cells, dendritic cells, and macrophages |
| TNFα | 233AA; 26 kDa in membrane with 29AA cytoplasmic domain; 28AA transmembrane domain; 176AA extracellular domain | Either transmembrane or soluble protein is biologically active; expressed in many cell types, including macrophages, CD4 + and CD8 + T cells, adipocytes, keratinocytes, mammary and colon epithelia, osteoblasts, mast cells, dendritic cells, pancreatic β cells, astrocytes, neurons, monocytes, and steroid-producing cells of zona reticularis |
| CD134/OX40L | 183AA; 21AA cytoplasmic domain; 23AA transmembrane segment; 139AA extracellular domain | OX40L exists as a trimer; limited expression: activated CD4 + and CD8 + T cells, B cells, and vascular epithelial cells |
| CD27L/CD70 | 50 kDa; 193AA transmembrane glycoprotein; 20AA cytoplasmic segment; 18AA transmembrane segment; 155AA extracellular domain | Expressed by NK cells, B cells, CD45RO + , CD4 + , and CD8 + T cells, gd T cells, and some leukemic cells; may be involved in antibody production in B cells |
| FasL | 40-kDa transmembrane protein 281AA; 80AA cytoplasmic domain; 179AA extracellular domain | Can occur as circulating trimer; can be cleaved by a protease to give an active 70-kDa trimer of 26-kDa monomers; F273L mutation results in gld/gld generalized lymphoproliferative disease; expressed by: type II pneumocytes and bronchial epithelium, monocytes, LAK cells, NK cells, dendritic cells, B cells, macrophages, CD4 + and CD8 + T cells, colon, and lung carcinoma cells |
| CD30L | 40-kDa; 234AA transmembrane glycoprotein: 46AA cytoplasmic domain; 21AA transmembrane segment; 172AA extracellular domain | Expressed by monocytes and macrophages, B cells, activated CD4 + and CD8 + T cells, neutrophils, megakaryocytes, resting CD2 + T cells, erythroid precursors, and eosinophils |
| TNFβ/LT-α | Circulates as 171AA, 25-kDa glycosylated polypeptide; a larger form (205AA) exists, suggesting proteolytic processing; no transmembrane form, but it can be membrane associated as it can bind to membrane-anchored LTβ, forming a heterotrimer | Circulating TNFβ is ~150 pg/mL heterotrimer binds to LTβR and TNFRI receptor, but TNFRI activation will not occur |
| LTβ | 33-kDa type II transmembrane glycoprotein; 244AA; 16AA cytoplasmic segment; 31AA transmembrane domain; 197AA extracellular region | LTβ forms a heterotrimer with TNFβ on membrane; LTβ is not secreted |
| TRAIL | 32 kDa, 281AA; 17AA cytoplasmic domain; 21AA transmembrane segment; 243AA extracellular domain | Homotrimer in membrane; many tissues express TRAIL, including lymphocytes; may have anticancer cell activity |
| Receptors (TNFRSF) b | ||
| LNGFR/p75 human low-affinity nerve growth factor receptor | 75 kDa; 427AA with extracellular N-terminus; 25AA signal sequence; 225AA extracellular domain; 23AA transmembrane segment; 154AA cytoplasmic domain | Transmembrane glycoprotein; can appear as a 200-kDa disulfide-linked homodimer; neurotrophins bind to LNGFR with KD ~l–3 mM, no inherent tyrosine kinase activity; death domain in cytoplasmic domain; protease cleavage-35–45-kDa LNGFR; cells expressing LNGFR: oligodendrocytes, B cells, bone marrow fibroblasts, autonomic and sensory neurons, Schwann cells, follicular, dendritic cells, select astrocytes, and mesenchymal cells |
| CD40 | 50 kDa; 277AA transmembrane glycoprotein (B-cell proliferation and differentiation); 20AA signal sequence; 173AA extracellular domain; 22AA transmembrane segment: 62AA cytoplasmic domain | Four Cys-rich motifs in extracellular region with juxtamembrane sequence rich in SER and THR: CD40 upregulates FAS to prime cells for subsequent FAS-mediated apoptosis; CD40 pathway involves NFκβ and protein kinase (LYN) activation: cells expressing CD40: monocytes, basophils (not mast cells), eosinophils, endothelial cells, interdigitating dendritic cells, Langerhans cells, blood dendritic cells, fibroblasts, keratinocytes, and Reed-Sternberg cells of Hodgkin’s disease and Kaposi’s sarcoma cells |
| CD137/4-IBB/ILA | 30–35 kDa; monomer and dimer on cell surface 255AA; 17AA signal sequence, 169AA extracellular region, 27AA transmembrane segment; 42AA cytoplasmic domain | Cys-rich motif in extracellular domain CD 137 binds its ligand, CD137L, at KD of ~30 pM; alternative splicing event can give rise to soluble form; CD137 ligation can interrupt cell apoptotic program associated with activation-induced cell death; cell expressing CD 137: fibroblasts, thymocytes, monocytes, and CD4 + and CD8 + T cells |
| TNFR1/p55/CD120a | 55 kDa; 455AA transmembrane glycoprotein; 190AA extracellular domain; 25AA transmembrane segment; 220AA cytoplasmic domain | Expressed in all nucleated mammalian cells; four Cys-rich motifs in extracellular region; first Cys-rich motif required for binding; 80AA death domain in cytoplasmic region; NFκB is activated by TNFR1; TNFR1 binds both TNFα and TNFβ; KD ~20–60 pM; for soluble TNFα; KD for TNFβ=650 pM; TNFR1 most important for circulating TNFα; membrane-bound TNFα associates with TNFR2; soluble TNFR1 blocks TNFα activity (decoy) and occurs in blood and urine at 1–3 ng/mL; protease activity gives soluble forms of 32 and 48 kDa; cells expressing TNFR1: hepatocytes, monocytes, and neutrophils; cardiac muscle cells; endothelial cells; and CD34 + hematopoietic progenitors |
| TNFR2/p75/CD120b | 75 kDa; 461AA transmembrane glycoprotein; 240AA extracellular region; 27AA transmembrane: segment; 173AA cytoplasmic domain | TNFR2 binds TNFα and transfers it to TNFR1, which becomes activated; TNFα binding to TNFR2 induces apoptosis in rhabdomyosarcoma cells and cell migration in Langerhans cells; soluble TNFα binds TNFR2 with A KD of 300 pM; TNFα levels are usually at 100 pM so that it should normally bind to TNFR1; therefore TNFR2 acts as a decoy; cells expressing TNFR2: monocytes, endothelial cells, Langerhans cells, and macrophages |
| CD134/OX40/ACT35 | 48 kDa; 250AA; 188AA in extracellular region; 26AA transmembrane segment; 36AA cytoplasmic domain | Expressed in CD4 + and CD8 + T cells only |
| CD27 | 50–55 kDa; mature CD27 is 27 kDa, 242AA; 175AA in extracellular domain; 21AA transmembrane segment; 46AA cytoplasmic domain | Expressed as homodimer on cell surface; it has no death domain but induces apoptosis by associating with Siva cytoplasmic protein, which has a death domain; blood and urine contain a soluble 32-kDa CD27 (probably from proteolysis) cells expressing CD27: NK cells, B cells, CD4 + and CD8 + T cells, and thymocytes |
| FAS/CD95/APO-1 | 43 kDa; 335AA; 156AA extracellular region; 20AA transmembrane segment; 144AA cytoplasmic domain | On fibroblasts FAS ligation can lead to either proliferation or apoptosis depending on number of expressed FAS molecules; three Cys-rich motifs; 68AA death domain in cytoplasmic region identical to one found in TNFR1 cytoplasmic domain; death domain associates FADD protein (with FAS) or TRADD protein with TFNR1; both transmit apoptotic signals; alternative gene splicing produces soluble forms of FAS |
| Soluble blood FAS circulates as dimer and trimer at low mg/mL concentrations; cells expressing FAS: CD34 + stem cells, fibroblasts, NK cells, keratinocytes, hepatocytes, B cells and B-cell precursors, monocytes CD4 + and CD8 + T cells, CD45RO + gd T cells eosinophils and thymocytes | ||
| CD30/Ki-1 | 105–120-kDa transmembrane glycoprotein; mature CD30 is 577AA; 18AA signal sequence, 365 extracellular region, 24AA transmembrane segment; 188AA cytoplasmic domain | Six Cys-rich motifs in extracellular region- patients with CD30 + lymphomas have 85-kDa soluble CD30 in blood; cells expressing CD30 Reed-Sternberg cells, CD8 + T cells and CD4 + T cells |
| LT-βR | 75-kDa transmembrane glycoprotein; 201AA extracellular domain; 26AA transmembrane segment; 187AA cytoplasmic domain | Four Cys-rich motifs in extracellular domain; LT-βR binds heterotrimers (1 TNFβ+2 LT-β) over LT-β homotrimers: first two Cys-rich motifs resemble TNFR1, and third & fourth Cys-rich motifs resemble TNFR2; LT-βR activates NFκB and induces cell death via TRAF-3; LT-βR activates genes for IL-8 and Rantes; LTβR expressed by monocytes, fibroblasts, smooth muscle, and skeletal muscle cells |
| Dr3/WSL-1/TRAMP/APO-3/LARD (death receptor 3) | 54 kDa; 417AA; 24AA signal sequence; 178AA extracellular domain; 23AA transmembrane segment; 192AA cytoplasmic domain | Can activate both NFκB and induce apoptosis-like TNFR1; four Cys-rich motifs in extracellular region, many alternate splice forms of Dr3, some of which may be soluble, cells expressing Dr3: T and B cells and human umbilical vein endothelial cells |
| Dr4 (death receptor 4) | 468AA; 23AA signal sequence; 226AA extracellular domain; 19AA transmembrane segment, 220AA cytoplasmic domain | One of the three known receptors for TRAIL; two Cys-rich motifs in extracellular domain; expressed by activated T cells |
| Dr5 (death receptor 5) | 411AA; 51AA signal sequence; 132AA extracellular domain; 22AA transmembrane segment; 206AA cytoplasmic domain | Second receptor for TRAIL; triggers apoptotic program like Dr4 without FADD participation; two Cys-rich motifs in extracellular domain |
| DcR1/TRID (decoy receptor 1) (TRAIL receptor without an intracellular domain) | 259AA; 23AA signal sequence; 217AA extracellular domain; 19AA transmembrane domain | Membrane receptor for TRAIL with no intracellular domain two Cys-rich motifs in extracellular region 50%–60% identical to AA sequences in same regions of Dr4 and Dr5; inhibits responsiveness to TRAIL (decoy receptor) |
| TR2 | 32 kDa; 283AA; 36AA signal sequence; 165AA extracellular region; 23AA transmembrane segment; 59AA cytoplasmic domain | No known ligand as yet; found in T cells, B cells, monocytes, and endothelium; four Cys-rich motifs in extracellular domain |
| GITR | 228AA; 19AA signal sequence; 134AA extracellular domain; 23AA transmembrane segment; 52AA cytoplasmic domain | Inducible during T-cell activation; three Cys-rich motifs in extracellular region; ligation interrupts TCR-DC3-induced apoptosis in T cells |
| OPG | 55 kDa; 380AA | Inhibits osteoclasts and protects bone from breakdown; secreted member of TNFRSF; similar to TNFR2 and CD40; no transmembrane segment circulates as a disulfide-linked homodimer; ligand unknown |
| TL1A | TL1A is a ligand for the Dr3 receptor. Dr3 (TNFRSF25) is a TNFR receptor expressed primarily on lymphocytes and is the receptor for TL1A (TNFSF15). Dr3 is a costimulator of T-cell activation (which produces a variety of cytokines) signaling through an intracytoplasmic DD and the adapter protein TRADD. Dr3 stimulates T-cell proliferation and inflammation of tissue sites | |
| TWEAK | 102 amino acids plus six cysteine residues in its extracellular domain | TWEAK is a member of the TNF family of ligands. The TREAKR cytoplasmic domains bind TRAFs-1, -2, and -3 (TRAF, TNFR-associated factor, that is a scaffold or adapter protein linking the IL1R/Toll to TNFR). The TWEAKR inhibits endothelial cell migration in vitro and inhibits corneal angiogenesis in vivo |
AAs , Amino acid residues; DD , death domain; FADD , Fas-associating protein with the death domain; FAS , fatty acid synthase; FasL , Fas Ligand; GITR , glucocorticoid-Induced TNFR family-related; LAK , lymphokine activated killer; LYN , member of the Src family of tyrosine protein kinases; NGF , nerve growth factor; NK , natural killer; OPG , osteoprotegerin; SER , smooth endoplasmic reticulum; TL1A , TNF-like ligand 1A; TNF , tumor necrosis factor; TNFR , tumor necrosis factor receptor; TRADD , TNFR-associated death domain; TRAIL , tumor necrosis factor-related apoptosis-inducing ligand; TWEAK , TNF-like weak inducer of apoptosis.
a TNFSF usually forms trimeric structures, ligands, and receptors of the TNSF and TNFRSF undergo clustering during signal transduction, monomers of TNFSF are two-sheet structures composed of β-strands.
b TNFRSF is usually trimeric or multimeric, stabilized by intracysteine disulfide bonds; it exists in both membrane-bound and soluble forms; many forms transduce apoptotic signals in a variety of cells.
At present, there are 19 soluble and membrane-bound ligands and 32 receptors in the TNFSF ( Fig. 17.5 ).
The gene for TRAIL is tetrahydrofolate-like-2 or TL2 that is located on chromosome 3. TRAIL is considered the 10th member of the TNF ligand superfamily ( TNFSF10 ). It occurs in the cell in two forms, soluble and insoluble (membrane bound). It can kill a number of cancer cells and transformed lines of cells and, in these cells, TRAIL induces DNA fragmentation and apoptosis. Some investigators posit that the insoluble form is the more active form of the two. TRAIL is expressed in many tissues, including the spleen, thymus, prostate, lung, kidney, and intestine. There are alternative splice variants that occur in both neoplastic and normal tissues and these are designated TRAIL β (lacks exon 3 of the TRAIL gene) and TRAIL γ (lacks exons 2 and 3 of the TRAIL gene). Both tumor cells and normal cells express the TRAIL receptor, Dr4; tumor cells (transformed cells) suffer apoptosis by TRAIL but most normal cells (untransformed) are resistant to TRAIL. As mentioned previously, the situation in normal cells may be an excess of DcRs over normal receptors. Some tumor cells may become partially resistant to TRAIL by producing IL-8 (or some other factor) either from the tumor cell or from the medium (in the case of ovarian tumors, they form ascites so that the medium is an important factor in vivo).
There is an insertion of 12–16 amino acids in the sequence of TRAIL that creates an elongated loop that other members of the TNF family do not have. It turns out that the extracellular region of Dr4 has extensive interactions with this extended loop of TRAIL accounting for the specificity for TRAIL.
Trimers of TNFα, like TRAIL, bind to the TNF receptor (TNFR1) forming an aggregate on the cell membrane. The form of a trimer may amplify the signal or facilitate the clearance of the aggregate from the membrane after functional completion, or both ( Fig. 17.6A and B ).
TNF-related cytokines , such as TNF α and TRAIL , appear as monomers, dimers, and trimers. In the soluble form the trimer seems to be the most active specie. An aggregate of TNF trimers binds to parallel receptor dimers (sTNFR1) as shown in Fig. 17.7 .
Many other cytokines and their receptors are summarized briefly in Table 17.1 and in Fig. 17.5 .
Growth Factors
Growth factors and cytokines are somewhat similar. Cytokines can have negative effects, such as the production of inflammation . Here, it is emphasized that some cytokines, like TRAIL and TNF, are able to kill cells by activating apoptosis , although certain cytokines, in the right conditions, can also stimulate the growth of cells. In general, growth factors inherently cause cells to grow and divide. Like cytokines, growth factors interact with cell membrane receptors and activate them but, invariably, they induce a signal induction process that leads to cell division and proliferation. Some of these proteins are like cytokines but produce growth effects, for example, many of the ILs . However, here the ILs are considered along with the growth factors. A great many growth factors have been identified. For convenience a few of the most common growth factors will be considered first and they are listed in Table 17.2 .
Table 17.2
Some Common Growth Factors and Their Properties.
Source: Reproduced from http://web.indstate.claytheme/mwking/growthfactors.html .
| Factor | Principal Source | Primary Activity | Comments |
|---|---|---|---|
| PDGF | Platelets, endothelial cells, placenta | Promotes proliferation of connective tissue, glial and other cells | Two different protein chains form three distinct dimer forms; AA, AB, and BB |
| EGF | Submaxillary gland, Brunners gland | Promotes proliferation of mesenchymal glial, and epithelial cells | |
| TGF-α | Common in transformed cells | May be important for normal wound healing | Related to EGF |
| FGF | Wide range of cells; protein is associated with the extracellular matrix | Promotes proliferation of many cells; inhibits some stem cells; induces mesoderm to form in early embryos | At least 19 family members, 4 distinct receptors |
| NGF | Promotes neurite outgrowth and neural cell survival | Several related proteins first identified as protooncogenes, trkA, trkB, trkC | |
| Erythropoietin | Kidney | Promotes proliferation and differentiation of erythrocytes | |
| TCF-β | Activated TH1, cells, and NK cells | Ami-inflammatory (suppresses cytokine production and class II MHC expression), promotes wound healing, inhibits macrophage and lymphocyte proliferation | At least 100 different family members |
| IGF-I | Primarily liver | Promotes proliferation of many cell types | Related to IGF-II and proinsulin, also called somatomedin C |
| IGF-II | Cells | Promotes proliferation of many cell types primarily of fetal origin | Related to IGF-I and proinsulin |
EGE , Epidermal growth factor; FGF , fibroblast growth factor; IGF-I , insulin-like growth factor I; IGF-II , insulin-like growth factor II; IGF-α , insulin growth factor α; MHC , major histo-compatability complex; NGF , nerve growth factor; NK , natural killer; PDGF , platelet-derived growth factor; TCF-β , T-cell factor beta; TGF-β , transforming growth factor β; TH1 , T-helper 1; trk , track.
Platelet-Derived Growth Factor
Platelet-derived growth factor (PDGF) is a stimulator of cell division ( mitogen ) for different cell types, such as cells in connective tissue and developing nervous system . It has five different polypeptide chains (subunits), labeled AA, AB, BB, CC, and DD. The AA and BB chains are somewhat similar (AA chain, 211 amino acids; BB chain, 241 amino acids). The dimerized subunits are stabilized by disulfide bonds. There is some specificity for either chain at the receptor level. There also exist two distinct subunits of the PDGF receptor (PDGFR) that also occurs in the cell membrane as a homodimer (αα, ββ) or a heterodimer (αβ). The αα-receptor [PDGFRα] recognizes the AA, CC, AB, and BB subunits of PDGF. The ββ-receptor (molecular weight about 180 kDa) recognizes subunits BB and DD of PDGF. The binding of PDGF by the receptor requires that the receptor also exists in the cell membrane as a dimer. PDGFR αβ binds the AB and the BB forms of PDGF. PDGFR ββ binds the BB and DD forms of PDGF ( Figs. 17.8 and 17.9 ).
The three different PDGFRs and the five different forms of PDGF are shown in Fig. 17.9 .
Activation of these receptors by the binding of the PDGF dimer generates cell growth. The effects of PDGF can change the shape of the cell and affect its motility. PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD all act through the PDGFR α- and β-tyrosine kinases. PDGF-AA and PDGF-BB undergo intracellular activation during transport in the exocytotic pathway for subsequent secretion, whereas PDGF-CC and PDGF-DD are secreted as latent products that can become activated by extracellular proteases . PDGF-AA and PDGF-BB are active in many physiological and disease processes in target cells, especially those that originate from mesenchymal or neuroectodermal tissues. PDGF also mediates interactions between glial cells and it may play a role in the communication between neurons and glial cells and also between neurons. PDGFRs are present on neurons of the developing nervous system and, therefore, PDGF is a mediator of intercellular signaling during the process of neuronal development. PDGFR may be present transiently as in the case of ganglion cells , for example, that possess PDGFR only when active outgrowth is occurring.
In hepatic stellate cells (versatile mesenchymal cells of the liver that are active in fibrosis and repair and contain large amounts of vitamin An in lipid droplets), PDGF activates its receptor that, in addition to stimulating growth through Ras-ERK , can induce cyclooxygenase-2 ( COX-2 ) giving rise to increased prostaglandin synthesis and the elevation of cyclic adenosine monophosphate that inhibits the effects of Ras -ERK on cell proliferation . The activated PDGFR can also act through phosphatidylinositol 3-kinase and protein kinase B to stimulate cell survival as well as proliferation. These activities are enhanced by intracellular sodium and calcium ions, the cellular transporters of which are also activated by signaling of the phosphorylated PDGFR.
In addition to its receptors, PDGF binds to other soluble proteins. α 2-Macroglobulin binds PDGF-BB but not PDGF-AA. This is a regulatory mechanism for controlling the amount of PDGF available to its receptor (much like the DcRs that bind TRAIL). PDGF-associated protein in the neural retinal cell binds PDGF with low affinity and enhances the activity of PDGF-AA but lowers the activity of PDGF-BB.
As PDGF is a dimer ( Fig. 17.9 ), it binds two receptor monomers at once forming a bridge between them. The dimeric receptor complex is further stabilized, in addition to disulfide bonds, by one of the Ig (immunoglobulin) domains (domain 4) of the receptors. PDGF-BB activates protein kinase C (PKC) that leads to the activation of the MAPK pathway.
In some cells, PDGF signaling can lead to the phosphorylation of ERK 1/2 that can further phosphorylate cytoplasmic phospholipase A2 ( cPLA2 ). The activated cPLA 2 releases arachidonic acid from the cell membrane phospholipids and stimulated PKC (by PDGF-BB) activates membrane NADPH oxidase that can affect reactive oxygen species ( ROS ) and ROS activates the p38 MAPK pathway leading to cell division and proliferation.
Some recent evidence suggests that, at least in certain cancer cells, PDGF-regulated gene transcription involves alterations in micro RNA (noncoding RNA).
Termination of the PDGF signal occurs with the recycling of the PDGFRs and this seems to be mediated by protein tyrosine phosphatases that remove the phosphate groups from the activated receptors thus inactivating them. This may not occur with all forms of the receptors. It is not completely clear how PDGF itself is removed from the surface of the target cell but, clearly, the action of PDGF is initiated once its receptor has been activated .
Epidermal Growth Factor
There exist high-affinity cell surface receptors for EGF having tyrosine kinase catalytic centers in the cytoplasmic domain. When EGF binds to and activates the receptor, its tyrosine kinase becomes activated and autophosphorylates the receptor and other proteins in the signaling pathway. Cells that are derived from mesoderm and ectoderm respond to EGF and proliferate. In the case of PDGF , it becomes cleared away from the target cell on its surface. EGF, on the other hand, is absorbed into the cell by endocytosis . This uptake process permits the downregulation of the ligand–receptor complex after the signal for mitosis has been transmitted through the signal pathway. The cellular uptake process is mediated by the protein clathrin . This process involves the formation of a clathrin-coated vesicle. Different proteins interact with the vesicle that interacts with the cell membrane to form a clathrin-coated pit that becomes the internalized endosome ( Figs. 17.10 and 17.11 ).
The internalized endosome is either degraded by transport into the lysosome or it is recycled to the cell surface via the Golgi network ( Fig. 17.11 ). The clathrin molecule forms a triskelion as shown in Fig. 17.12 . This figure also illustrates the clathrin-coated pit.
At first, the clathrin network is developed as an outline structure that is subsequently filled in to form a coat surrounding the complete vesicle. The network contains 36 triskelia in a structure composed of pentagons and hexagons ( Fig. 17.12C ). Ultimately, the removal of clathrin is accomplished by uncoating adenosine triphosphate (ATP)ase ( hsc70 , heat shock cognate 70). Three of these ATPases bind to one triskelion when ATP is absent but not when ATP is present. In the presence of ATP, the ATPase is activated and the clathrin molecules disassemble.
The signal transduction pathway initiated by the activation of EGF receptors (EGFRs) upon binding EGF is shown in Fig. 17.13 .
Two molecules of EGF bind to EGFRs that dimerize on the surface of the target cell. The activated receptor dimer autophosphorylates in the receptor cytoplasmic domains and the activated receptor phosphorylates other proteins as well. A number of pathways become activated, including mTOR ( mechanistic target of rapamycin ), STAT , and the proliferation pathway through ERK or MAPK . These activated signaling pathways lead to cell proliferation and to other effects as shown in the figure.
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