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Signal Transduction, Key Receptors and Apoptosis Mechanisms in Cancer


- Dr. James Meschino, DC, MS, ROHP

What Is Signal Transduction

Signal transduction refers to any process by which a cell converts one kind of signal or stimulus into another, most often involving ordered sequences of biochemical reactions inside the cell, that are carried out by enzymes and linked through second messengers resulting in a"second messenger pathway". Such processes are usually rapid, lasting in the order of milliseconds in the case of ion flux, to minutes for the activation of protein and lipid mediated kinase cascades. In many signal transduction processes, the number of proteins and other molecules participating in these events increases as the process eminates from the initial stimulus, resulting in a signal cascade and often results in a relatively small stimulus eliciting a large response.
Signal transduction usually involves the binding of small extracellular signaling molecules to receptors that face outwards from the plasma membrane and trigger events inside the cell. However, steroids (e.g. testosterone, estrogen, cortisone etc) as well as vitamin A and vitamin D represent an example of extracellular signalling molecules that may cross the plasma membrane due to their lipophilic or hydrophobic nature. Many steroids, but not all, have receptors within the cytoplasm and usually act by stimulating the binding of their receptors to the promoter region of steriod responsive genes.
Activation of genes, alterations in metabolism, the continued proliferation and death of the cell,and the stimulation or suppression of locomotion, are some of the cellular responses to extracellular stimulation that require signal transduction. Gene activation leads to further cellular effects, since the protein products of many of the responding genes include enzymes and transcription factors themselves. Transcription factors produced as a result of a signal transduction cascade can in turn activate yet more genes. Therefore an initial stimulus can trigger the expression of an entire cohort of genes, and this in turn can lead to the activation of any number of complex physiological events (Li E et al, 2006)
The total number of scientific papers related to signal transduction published since 1st Jan 1977 up to the 31st December 2007 was 48,377 of which only 11,211 were reviews of other papers.

Epidermal Growth Factor Receptors: Cell Proliferation and Cancer

Epidermal growth factor receptor (EGFR) consists of a family of four receptors, HER1 (ErbB1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4). These receptors exist singularly but form pairs when activated (EGFR-Info.com, 2003). Ligand pairing (dimerization) activates intracellular tyrosine kinase (Wells, 1999), which in turn, interacts with the ras protein. Ras protein plays a crucial role in regulating cell growth and has been associated with mutated cellular proliferation (Jackson, n.d.). The ras protein then activates the mitogen-activated protein kinase (MAPK). MAPK delivers the signal to the nucleus where it interacts with the cyclin D molecule (Wells). MAPK interference results in accumulation of cyclin D. This excess of cyclin D allows the nucleus to override its resting phase and move into uncontrolled growth (Lundberg & Weinberg, 1999). As such, over expression of EGFR results in excess accumulation of cyclin D, and a tendency for uncontrolled cellular proliferation.
Investigators have found overexpressed EGFR in a variety of solid tumors. Overexpressed EGFR is found in many human malignancies, including non-small cell lung cancer and breast, head and neck, gastric, colorectal, esophageal, prostate, bladder, renal, pancreatic, and ovarian cancers (Salomon et al., 1995). Overexpression of EGFR in these cancers is also associated with poor prognosis, including time to treatment failure and shorter overall and progression-free survival (Poon et al., 2001; Salomon et al.).
Overexpression of EGFR-mediated signaling is also responsible for other key cellular activities associated with malignancy. Although the exact mechanism remains under study, researcher suggests that EGFR-mediated signaling results in reduced apoptosis, the normal process by which dysfunctional cells are destroyed, thereby leading to increased volume of malignant cells (Karnes et al., 1998). Investigators also believe that EGFR-mediated signaling plays a role in angiogenesis, the creation of blood vessels in tumors, by increasing angiogenic factors such as vascular endothelial growth factor. The mechanism remains unknown, but overexpression of EGFR is associated with increased incidence of metastases (Salomon, Brandt, Ciardiello, & Normanno, 1995).

How The EGFR Receptors Work And Effect Downstream Proteins and Cellular Response

At present, ten ligands have been identified to bind HER-1, HER-3 and HER-4: epidermal growth factor (EGF), transforming growth factor alpha (TGF- ), amphiregulin, heparin-binding epidermal growth factor (HB-EGF), betacellulin, epiregulin and neuregulins 1 to 4 . HER-2 is considered to be an ‘orphan’ receptor, since no specific direct ligand has been identified as yet, and increasing evidence suggests that it acts mainly as a co-receptor, increasing the affinity of ligand binding to the dimeric receptor complex. Tyrosine kinase activity, which is required for receptor mediated cellular signaling, is present in all receptors except HER-3. Instead, HER-3 has six intracellular binding sites for phosphatidylinositol 3-kinase (PI3K), which is therefore a potent activator of this enzyme. The binding of ligands induces dimerization of two identical (homodimer) or different (heterodimer) receptors. The dimerization partner has an important impact on the type and number of downstream effectors activated and also on the downregulation mechanism of the ligand bound receptors. Signaling through HER-2 and -3 requires heterodimerization since, as stated, HER-2 has no known ligand and HER-3 lacks TK activity. Importantly, HER-2 is the preferred dimerization and signaling partner for all other HER receptors. Upon dimerization, intracellular TK domains are phosphorylated, which in turn provide docking sites for several adaptor proteins and signaling enzymes. These proteins link upstream membrane receptor kinases to downstream intracellular protein kinases, which control a wide variety of cellular processes, including cellular proliferation, apoptosis and angiogenesis. Not surprisingly, the involvement of multiple ligands, paired combinations of four receptors and plenty of intracellular downstream effectors (signal processing units) result in extensive diversification of EGFR signals mediated by the EGFR family of receptors. (Atalay G et al, 2003)

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SIGNAL TRANSDUCTION, KEY RECEPTORS AND APOPTOSIS MECHANISMS IN CANCER

Dr. James Meschino, 

DC, MS, ROHP

Monoclonal Antibodies and Small Molecule Agents Target EGFR: The New Frontier Of Cancer Treatment Drugs

Two new drug treatment methods for cancer are being developed, which target the EGFR and associated kinase second messengers involved in uncontrolled cellular proliferation:

  1. 1
    Monoclonal antibodies are made by fusing the spleen cells from a mouse that has been immunized with the desired antigen with human multiple myeloma cells. One problem in medical applications is that the standard procedure of producing monoclonal antibodies yields mouse antibodies. Although murine antibodies are very similar to human ones there are differences. The human immune system hence recognizes mouse antibodies as foreign, rapidly removing them from circulation and causing systemic inflammatory effects. Monoclonal antibodies have been generated and approved to treat: cancer, cardiovascular disease, inflammatory diseases, macular degeneration, transplant rejection, multiple sclerosis, and viral infection. (Carter P,2001). In August 2006 the Pharmaceutical Research and Manufacturers of America reported that U.S. companies had 160 different monoclonal antibodies in clinical trials or awaiting approval by the Food and Drug Administration. Herpectin is a monoclonal antibody drug that targets one of the EGFR’s in breast cancer treatment. It may also have application as preventive intervention in women who overexpress the Erb2 receptor, which carries a high risk for future breast cancer development.Monoclonal antibodies target the external ligand-binding domain. The monoclonal antibodies, Herceptin® for the treatment of breast cancer and Gleevec® (imatinib mesylate, STI-571) for the treatment of chronic myeloid leukemia have provided the most prominent initial proof that targeted cancer therapy can translate into improved clinical outcomes. Trastuzumab represents the only biological targeted therapy for breast cancer in routine clinical practice today. (Atalay G et al,2003). The establishment of the unique role of HER-2 in malignant transformation in preclinical models, coupled with the biological significance of HER-2 overexpression in breast cancer and the preclinical demonstration of the antitumor activity of monoclonal antibodies (mAbs) directed against HER-2, encouraged the development of this drug Other monoclonal antibodies that currently are in development and are directed specifically at EGFR include IMC-225, bevacizumab, and ABX-EGF. (Pharmaceutical Technology, August 24, 2006). The generation of active immune response to HER-2 represents an attractive alternative to passive immunity provided by monoclonal antibodies. Under development are vaccines that, for example, stimulates the patient’s own immune system to induce a strong killer cell (CTL) response against HER-2-overexpressing tumor cells. (Atalay G et al, 2003)
  2. 2
    Small molecules inhibit EGFR via the intracellular tyrosine kinase pathway. Some examples of EGFR tyrosine kinase inhibitor agents currently in development are GW572016, OSI-774, and CI-1033. They possess the unique ability to inhibit all members of the EGFR family (Rinehart et al., 2003).

    In studies with a variety of small molecule agents, investigators report rashes, most often maculopapular or acneiform in nature, as the most common adverse effect. These rashes also have been associated with pruritus. In studies with Iressa, investigators found skin rash to be dose related, whereas disease response was not (Gloss, 1995). In addition, some patients experienced disease responses without evidence of rash (Gloss). Other small molecule adverse events include nausea, vomiting, diarrhea, and asthenia. 

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SIGNAL TRANSDUCTION, KEY RECEPTORS AND APOPTOSIS MECHANISMS IN CANCER

Dr. James Meschino, 

DC, MS, ROHP

 REFERENCES

AstraZeneca Pharmaceuticals. (2003). About Iressa. First in a new class for NSCLC: An option where none existed before. Retrieved January 22, 2004, from http://www.iressa-us.com/content/prof/about

Atalay G, Cardoso F, Awada A and. Piccart M. J Novel therapeutic strategies targeting the epidermal growth factor receptor (EGFR) family and its downstream effectors in breast cancer. Annals of Oncology 14:1346-1363, 2003
Carter P: Improving the efficacy of antibody-based cancer therapies. Nat Rev Cancer 2001;1:118-129

EGFR-Info.com. (2003). The epidermal growth factor as a clinical target in cancer. Retrieved October 4, 2003, from http://www.egfr-info.com

Gloss, G. (1995). WCLC conference highlights: Growing wealth of clinical experience with EGFR inhibitors. Signal, 4(3), 13–18.

Li E, Hristova K. Role of receptor tyrosine kinase transmembrane domains in cell signaling and human pathologies.. Biochemistry 45 (20): 6241-51. 2006.

Jackson, J. (n.d.). Specificity of ras signaling in breast cancer. Retrieved October 4, 2003, from http://www.cbcrp.org/research/PageGrant.asp?grant_id=1703

Karnes, W.E., Weller, S.G., Adjei, P.N., Kottke, T.J., Glenn, K.S., Gores G.J., et al. (1998). Inhibition of epidermal growth factor receptor kinase induces protease-dependent apoptosis in human colon cancer cells. Gastroenterology, 114, 930–939.

Lundberg, A.S., & Weinberg, R.A. (1999). Control of the cell cycle and apoptosis. European Journal of Cancer, 35, 1886–1894.

Poon, T.C.W., Chan A.T.C., To, K.F., Teo, P.M.L., Chan, M.L., Mo, K.F., et al. (2001). Expression and prognostic significance of epidermal growth factor receptor and HER2 protein in nasopharyngeal carcinoma [Abstract 913]. Proceedings of the American Society of Clinical Oncology, 20, 229a.

PhRMA Reports Identifies More than 400 Biotech Drugs in Development. Pharmaceutical Technology, August 24, 2006

Rinehart, J.J., Wilding, G., Willson, J., Krishnamurthi, S., Natale, R., Dasse, K.D., et al. (2003). A phase I clinical and pharmacokinetic (PK)/food effect study of oral CI-1033, a pan-erb B tyrosine kinase inhibitor, in patients with advanced solid tumors [Abstract 821]. Proceedings of the American Society of Clinical Oncology, 22, 205.

Salomon, D.S., Brandt, R., Ciardiello, F., & Normanno, N. (1995). Epidermal growth factor-related peptides and their receptors in human malignancies. Critical Reviews in Oncology and Hematology, 19, 183–232.

Wells, A. (1999). Molecules in focus EGF receptor. International Journal of Biochemistry and Cell Biology, 31, 637–643.


G. Atalay, F. Cardoso, A. Awada and M. J. Piccart Novel therapeutic strategies targeting the epidermal growth factor receptor (EGFR) family and its downstream effectors in breast cancer. Annals of Oncology 14:1346-1363, 2003
Thompson, CB. Apoptosis in the pathogenesis and treatment of disease. Science 267. 1995.

Oncogenes, Proto-oncogenes and Cancer
Oncogenes and Cancer - An oncogene is a modified gene, or a set of nucleotides that codes for a protein and is believed to cause cancer. Genetic mutations resulting in the activation of oncogenes increase the chance that a normal cell will develop into a tumor cell. Oncogenes are figuratively thought to be in a perpetual tug-of-war with tumor suppressor genes (e.g, p53 and p21 tumor suppressor genes), which act to prevent DNA damage and keep the cell's activities under control. There is much evidence to support the notion that loss of tumor suppressors or gain of oncogenes can lead to cancer. Oncogenes are mutant forms of normal functional genes (called proto-oncogenes) that have a role in normal cell proliferation. Oncogenes are found in tumours and in retroviruses; in the latter case, having been picked up along with retroviral genes during retroviral replication in the host. Once incorporated as part of the retroviral genome, they are activated at inappropriate times and places by retroviral promoters, thereby becoming oncogenes.
Proto-oncogenes - A proto-oncogene is a normal gene that can become an oncogene due to mutations or increased expression. Proto-oncogenes code for proteins that help to regulate cell growth and differentiation. Proto-oncogenes are often involved in signal transductionand execution of mitogenic signals, usually through their protein products. Upon activation, a proto-oncogene (or its product) becomes a tumor inducing agent, an oncogene. Examples of proto-oncogenes include RAS, WNT, MYC, and ERK. For instance, proteins in the Ras family are very important molecular switches for a wide variety of signal pathways that control such processes as cytoskeletal integrity, proliferation, cell adhesion, apoptosis, and cell migration. Ras and ras-related proteins are often deregulated in cancers, leading to increased invasion and metastasis, and decreased apoptosis. RAS activates a number of pathways but an especially important one seems to be the mitogen-activated protein (MAP) kinases, which themselves transmit signals downstream to other protein kinases and gene regulatory proteins.
The proto-oncogene can become an oncogene by a relatively small modification of its original function. There are three basic activation types:
1.A mutation within a proto-oncogene can cause a change in the protein structure, causing an increase in protein (enzyme) activity or a loss of regulation.
2.An increase in protein concentration, caused by
a.an increase of protein expression (through misregulation)
b.an increase of protein stability, prolonging its existence and thus its activity in the cell
c.a gene duplication (one type of chromosome abnormality), resulting in an increased amount of protein in the cell.
3.Mutations in microRNAs can lead to activation of oncogenes. Research indicates that small RNAs 21-25 nucleotides in length called microRNAs (miRNAs) can control expression of these genes by down-regulating them, a function that is lost with microRNA mutation.

References:
1.Yokota J Tumor progression and metastasis. Carcinogenesis. 21 (3): 497-503. 2000.
2. Todd R, Wong DT. Oncogenes. Anticancer Res. 19 (6A): 4729-46. 1999

Tumor Suppressor Genes
Tumor suppressor genes, or more precisely, the proteins for which they code either have a dampening or repressive effect on the regulation of the cell cycle or promote apoptosis, and sometimes do both. The functions of tumor suppressor proteins fall into several categories:
1.Repression of genes that are essential for the continuing of the cell cycle. If these genes are not expressed, the cell cycle will not continue, effectively inhibiting cell division.
2.Coupling the cell cycle to DNA damage. As long as there is damaged DNA in the cell, it should not divide. If the damage can be repaired, the cell cycle can continue.
3.If the damage can not be repaired, the cell should initiate apoptosis, or programmed cell death, to remove the threat it poses for the greater good of the organism.
4.Some proteins involved in cell adhesion prevent tumor cells from dispersing, block loss of contact inhibition, and inhibit metastasis.

References:
Sherr C. Principles of tumor suppression. Cell 116 (2): 235-46. 2004
Hirohashi S, Kanai Y. Cell adhesion system and human cancer morphogenesis. Cancer Sci 94 (7): 575-81. 2003

Understanding Receptors and Second Messengers In Signal Transduction Pathways Related To Cell Proliferation, Apoptosis and Cancer

PI3 Kinase (Phosphatidylinositol 3-Kinase) PI3K is responsible for phosphorylation of the 3 position of the inositol ring of PI(4,5)P2, to generate PI(3,4,5)P3, a potent second messenger required for survival signaling, and insulin action. PI3 Kinase is also activated by tyrosine kinase, Ras, and by the β:γ subunits of heterotrimeric G-proteins. .Akt is the major known effector of the PI3 Kinase pathway. Generation of PIP3 results in the activation of PDK1, which phosphorylates Akt.


Akt ​Akt phosphorylates Bad, resulting in protection from apoptosis. Bad, or "Bcl-2 antagonist of cell death" is member of the Bcl-2 family and an important regulator of life versus death. Unphosphorylated Bad dimerizes with Bcl-2 and Bcl-XL, neutralizing their anti-apoptotic activity. Activation of the PI 3-Kinase pathway leads to activation of Akt which phosphorylates Bad.

Apoptosis/survival is regulated by the relative balance of pro-apoptotic (Bax, BclXS, Bak, Bad, Bar) and anti-apoptotic (Bcl2, BclXL, Mcl-1, Dad-1) family members, and their heterodimerization. Bcl2 is phosphorylated by the ASK1/MKK7/JNK1 pathway on Ser70 during progression through the cell cycle; whether this up-or down-regulates the anti-apoptotic effect of Bcl2 remains controversial

Cytochome c Cytochrome c, released from the mitochondria, along with APAF1 complex in the cytosol activates caspase 9 as part of the apoptotic pathway. Bak (Bcl2 homologous Antagonist/Killer) is closely associated with this event. Bak is a member of the Bcl2 family of proteins that are involved in the regulation of programmed cell death. Other members include Bax, Bad, Bcl-x and Mcl-1. Bak is a proapoptotic protein which forms mitochondrial pores by oligomerization. Recent studies suggest that pore formation leads to cytochrome c efflux from the mitochondria. tBID, the truncated form of the proapoptotic protein BID, has been implicated in an allosteric activation of Bak, resulting in the Bak pore formation.


Apaf -1 Apaf-1 (apoptosis protease-activating factor 1) binds to cytochrome c and caspase-9 which leads to caspase-9 activation. The release of cytochrome c from the mitochondria provides a mechanism for apoptosis through Apaf-1-induced activation of caspases as well as necrosis due to the cessation of electron chain-transport. It has been proposed that Bcl-2 may interfere with the mitochondria-dependent apoptotic pathways at two points; preventing cytochrome c release from the mitochondria and directly interfering with Apaf-1.


Caspase Enzymes and Apoptosis  Caspase - Cysteine aspartyl proteases related to the C. elegans CED-3 death protein comprise the caspase family. All are expressed as proenzymes which are activated by proteolysis. With respect to their roles in apoptosis, Caspases can be subdivided into initiator (Caspases 8, 9, 10) and effector (Caspases 3, 6, 7) caspases, depending on whether they are activated by receptor clustering (initiator) or by mitochondrial permeability transition (effector).
Effector caspases, most notably Caspase 3, cleave numerous substrates to effect the morphological changes associated with apoptosis. The significance of PARP cleavage is not clear, but it is an excellent marker for caspase activation and the presumption of ongoing apoptosis.
Mitochondria play an essential role in the apoptotic pathway of many cells by releasing apoptogenic proteins into the cytosol. The Bax protein, which shares highly conserved domains with Bcl-2, can form ion-conducting channels in the lipid bilayers of mitochondria. Bax presents an interesting therapeutic target for many diseases involving apoptosis such as cancer or neurodegenerative disorders.
Mitochondrial proteins known as SMACs (second mitochondria-derived activator of caspases) are released into the cytosol following an increase in permeability. SMAC binds to inhibitor of apoptosis proteins (IAPs) and inhibits them, preventing the IAPs from arresting the apoptotic process and therefore allowing apoptosis to proceed. IAP also normally suppresses the activity of a group of caspases, which carry out the degradation of the cell, therefore the actual degradation enzymes can be seen to be indirectly regulated by mitochondrial permeability.
Cytochrome c is also released from mitochondria due to increased permeability of the outer mitochondrial membrane, and serves a regulatory function as it precedes morphological change associated with apoptosis. Once cytochrome c is released it binds with Apaf-1 and ATP, which then bind to pro-caspase-9 to create a protein complex known as an apoptosome. The apoptosome cleaves the pro-caspase to its active form of caspase-9, which in turn activates the effector caspase-3, ultimately causing apoptosis to occur.


PARP PARP - Poly(ADP-Ribose) Polymerase 1 (PARP-1), when activated by breaks in DNA, poly ADP-ribosylates nuclear proteins, resulting in NAD+ depletion and cell death by apoptosis. During apoptosis, Caspase 3 cleaves PARP into signature 85 and 24 kDa fragments. The 24 kDa fragment irreversibly binds to the broken ends of DNA, preventing access of repair enzymes to DNA, and ensuring the irreversibility of apoptosis. PARP cleavage is commonly used as a marker to "prove" cell death by apoptosis versus necrosis. Western blot detection of PARP cleavage (appearance of the 85 kDa fragment) is widely used as a readout of apoptosis.


Between 50 billion and 70 billion cells die each day due to apoptosis in the average human adult. For an average child between the ages of 8 to 14, approximately 20 billion to 30 billion cells die a day. In a year, this amounts to the proliferation and subsequent destruction of a mass of cells equal to an individual's body weight. Excessive apoptosis causes hypotrophy, such as in ischemic damage, whereas an insufficient amount results in uncontrolled cell proliferation, such as cancer.


Apoptosis can occur when a cell is damaged beyond repair, infected with a virus, or undergoing stress conditions such as starvation. DNA damage from ionizing radiation or toxic chemicals can also induce apoptosis via the actions of the tumour-suppressing gene p53. The "decision" for apoptosis can come from the cell itself, from the surrounding tissue, or from a cell that is part of the immune system. In these cases apoptosis functions to remove the damaged cell, preventing it from sapping further nutrients from the organism, or to prevent the spread of viral infection.


Apoptosis also plays a role in preventing cancer; if a cell is unable to undergo apoptosis, due to mutation or biochemical inhibition, it can continue dividing and develop into a tumour. For example, infection by papillomaviruses causes a viral gene to interfere with the cell's p53 protein, an important member of the apoptotic pathway. This interference in the apoptotic capability of the cell plays a critical role in the development of cervical cancer.


In the adult organism, the number of cells is kept relatively constant through cell death and division. Cells must be replaced when they become diseased or malfunctioning; but proliferation must be compensated by cell death. This balancing process is part of the homeostasis required by living organisms to maintain their internal states within certain limits.


The Immune System and Apoptosis Cytotoxic T-cells are able to directly induce apoptosis in cells by opening up pores in the target's membrane and releasing chemicals which bypass the normal apoptotic pathway. The pores are created by the action of secreted perforin, and the granules contain granzyme B, a serine protease which activates a variety of caspases which induce apoptosis. Interferon, sometimes used in cancer therapy, is a protein made by immune cells that upregulates the function of cytotoxic T- cells (natural killer cells), helping them stimulate apoptosis of cancer cells.


FADD Death Receptors − FADD (Fas-Associated, Death Domain) binds to the cytoplasmic portion of Fas, and recruits Caspase 8 to the Fas receptor. As such, it acts as an adapter protein to transduce apoptotic signals from receptor into the cell.


Fas Death Receptor − Fas is a receptor of the TNF Receptor family involved in initiation of apoptosis of lymphoid cells. Fas ligand binds to Fas and causes clustering of Fas molecules. Adapter proteins such as FADD recruit Caspase 8 to Fas, which becomes activated and induces the apoptotic cascade. IgM against Fas is also able to induce apoptosis by clustering of receptor molecules.


Effects of Insulin − Insulin action stimulates the PI3 Kinase pathway, resulting in Akt activation, which phosphorylates and inactivates Glycogen Synthase Kinase 3 (GSK3). Glycogen Synthase is then rapidly dephosphorylated, and activated

Glycogen Synthase Kinase 3 is active in the absence of the action of signaling pathways. The function of GSK3 is to phosphorylate Glycogen Synthase and thereby inactivate it


Tissue Necrosis Factor Receptors − Tissue Necrosis Factor Receptors (TNFRs), which are members of a superfamily of proteins known as Death Receptors, resulting in receptor aggregation and recruitment of adapters (i.e., TRAF [TNF Receptor-Associated Factor] proteins) to the intracellular portion of the complex. TNFR complex formation activates Caspase 8, the SAP Kinase pathway (dependent on recruitment of Daxx), and the NFκB pathway (which controls apoptosis versus proliferation


Insuling-like Growth Factor − Insulin-like Growth Factor - The IGFs are involved in skeletal growth, and are essential for prevention of apoptosis. Serum levels of free IGFs are kept low by the action of IGF binding proteins (IGFBPs), which sequester the IGFs. Overexpression of IGFBPs may induce apoptosis, presumably by reduction of free IGF; IGFBP levels are also altered in some cancers. The IGF-I Receptor is not as mitogenic as some other growth factor receptors, but its ability to activate the PI3 Kinase pathway, through the Insulin Receptor Substrate (IRS) proteins, is critical for mediating cell survival


Jun N- Kinase − The stress-activated protein kinase 1 family is also referred to as the jun N-terminal kinase family. The stress-activated kinases are only weakly activated by mitogenic stimuli, but potently activated by stress stimuli, such as inflammatory cytokines, ischemia, chemotherapeutic agents, and irradiation.


JNK/SAPK1 kinases − The JNK/SAPK1 kinases like the other MAPK-like kinases, are thought to phosphorylate multiple substrates and regulate many processes, including proliferation (in some cell types) and apoptosis.


SAPK2/3 family − The SAPK2/3 family is most widely referred to as the p38 family. These kinases are also activated by stresses, most notably inflammatory cytokines, irradiation, and certain toxins such as anisomycin, and arsenite. The activating kinases of SAPK2/3 are SKK2/MEK3 for SAPK2a and 2b, and MKK6 for SAPK3. The targets of the SAPK2/3 family include the MAPKAP kinases 2 and 3/3pK. In addition, SKK4 is related to this family, exhibiting 60% identity, and is activated by MKK6.


p53 gene tumor suppressor gene − The p53 gene is mutated in approximately half of all human cancers. It is involved in the cellular response to cytotoxic stresses, and together with p19ARF, induces expression of p21Cip1, to cause cell cycle arrest.
In addition, p53 is able to induce apoptosis, both by transcriptional and non-transcriptional mechanisms.


Nuclear Factor – kappa beta (NFκB) − NFκB signaling is a critical regulator not only of immune function, but also of proliferation versus apoptosis in response to various stimuli. It is regulated by an inhibitory protein, IκB. IκB has a nuclear localization sequence, as well as an export sequence. When in complex with NFκB, the NLS is inactive (perhaps masked), and the complex is cytoplasmic. When I&kapp;B is free, the NLS is active, and at least partially dominant to the export sequence. IκB thus shuttles to and from the nucleus, and maintains NFκB in the cytoplasm, preventing NFκB mediated transcription. Various stimuli lead to activation of IKK (IκB Kinase), which phosphorylates IκB on serines 32 and 36, marking it for ubiquitination and degradation. Once IκB is degraded, NFκB is able to initiate transcription. Mutant IκB in which serines 32 and 36 are changed to alanines, is not phosphorylated, and therefore not degraded. Cells expressing this protein are not able to activate NFκB, providing a useful tool to study the role of NFκB in various pathways and processes


Interleukins (ILs) − Interleukins constitute a broad family of cytokines, primarily of hematopoietic cell origin. IL-1, IL-6 and IL-8 are pro-inflammatory cytokines, IL-2 is a T-cell growth factor, while IL-7 is a B-cell growth factor. IL-15 shares many of its properties with IL-2. In fact, IL-2, IL-4, IL-7 and IL-15 signaling involves a common IL-2Rγ subunit, a common theme in immune cell signaling, where receptors are multi-component, and some components are shared between multiple receptors.


T-cell Receptor (TCR) − The T-cell Receptor is found on the surface of T-cells and is responsible for recognition of foreign antigen. Antigen binding to the TCR results in T-cell activation and proliferation (clonal selection). The TCR is composed of an α and a β subunit, either CD4 (on Th cells) or CD8 (on Tc cells), and CD3, which is made up of six subunits arranged into three dimers derived from five proteins (δ, ε, γ, ζ and η). The TCR initiates a signaling cascade that begins with the CD3 complex and proceeds through Lck to LAT and so on to Ras and then ERK, or through Fyn and ZAP-70 and so on to Rac and then JNK.


FAK (Focal Adhesion Kinase) − FAK is a tyrosine kinase which localizes to focal adhesions. It autophosphorylates on tyrosine during adhesion and spreading, and is thought to be a critical transducer of adhesion-dependent growth and survival. FAK is a substrate of Src, and phosphorylation of FAK may be the mechanism by which Src-transformed cells achieve adhesion-independent growth. FAK is not known to be overexpressed in human cancer, but the activation state of FAK in cancer has not been thoroughly addressed.


Vascular Endothelial Growth Factor (VEGF) − VEGF is a dimeric ligand, and is among the most potent angiogenic mitogens. VEGF is secreted by tumor cells and other cells exposed to hypoxia. Expression of VEGF is stimulated by FGF-2 (basic FGF), and it activates Flt-1 and KDR, the two high-affinity receptors for VEGF. The VEGF receptors stimulate the Ras-MAP kinase pathway, suggesting that it signals as a conventional receptor tyrosine kinase.


Raf − Raf, in addition to its role in mitogenesis, Raf-1 may play a role in regulation of apoptosis and cell cycle progression. Activation of Raf-1 involves phosphorylation of Ser338/339 and Tyr340/341. Activating mutations of B-Raf that disrupt its auto-inhibition loop have been implicated in a number of cancers, including melanoma and colon cancer. In these cases the raf pathway remains activated prompting increase cellular proliferation and inhibiting apoptosis.


Ras–Raf–MAPK pathway − Ras proteins control a wide variety of cellular processes including growth, differentiation, apoptosis and cytoskeletal organization. Ras proteins are activated in response to a wide variety of intracellular and extracellular stimuli. Activated Ras is rapidly converted to its guanosine diphosphate (GDP)-bound inactive state by hydrolysis of the bound guanosine triphosphate (GTP) by an intrinsic GTPase activity. This mechanism of inactivation is thought to be impaired in mutant Ras, which remains in the activated form and conveys uncontrolled proliferative signals continuously, despite the absence of stimulation. Ras proteins are mutationally activated in 30% of human cancers, including BC.
Ras can be activated by receptor TKs. Tyrosine phosphorylated receptors provide binding sites for the adaptor protein Grb2 (growth factor receptor-bound protein 2), which recruits Ras activator protein SOS (son-of-sevenless guanine nucleotide exchange factor).
Upon binding to Grb2, SOS mediates the activation of Ras by facilitating its switch from the inactive GDP-bound from to the GTP-bound active form. Then, GTP-bound Ras can activate several downstream effector pathways.
Raf-1, another protein kinase, is one of these effector molecules. It phosphorylates MEK1 (MAPK/Erk kinase) and MEK2, which in turn phosphorylate the MAPKs, ERK1 and -2 (extracellular signal-regulated kinases). Once activated, Erk1 and -2 translocate into the nucleus, where they phosphorylate a variety of substrates, including nuclear transcription factors, and ultimately lead to transcription of target genes associated with cellular proliferation.
Other substrates for Ras signaling include Rac/Rho (small GTP-binding proteins), PI3K and mitogen-activated protein kinase kinase (MEKK). The Rac–rho pathway is involved in cytoskeletal organization, the PI3K pathway is associated with cell survival, and the MEKK and MAPK pathways mediate cellular proliferation signals. The MAPK pathway is activated preferentially by mitogens, growth factors and tumor promoters, and the other downstream pathways mediated by Ras signaling are stimulated by inflammatory cytokines, hormones and various forms of stress stimuli.
MEKK activates another MAPK family member JNK (Jun N-terminal kinase) via SEK (stress-activated protein kinase), and the activation of this pathway is associated with apoptosis and/or proliferation depending on the dynamic balance between different signal transduction pathways.
JNK activates nuclear transcription factor c-Jun, a major component of activator protein-1 (AP-1). The other component of AP-1, c-fos, is activated by the MAPK pathway mediated through EGFR signaling. The generation of AP-1 is thought to play an important role in the development of endocrine resistance in breast cancer as one of the critical intersection points between estrogen receptor (ER) and EGFR signaling pathways. Many signal transduction pathways impinge on Ras-mediated signaling at different levels, so that the net effect of a given stimulus reflects merely the balance of crosstalk among these pathways.


PI3K–Akt pathway − The PI3K (phoshpatidylinositol phosphate) family of enzymes has been linked to many aspects of malignant transformation, including increased proliferation, growth, motility, invasiveness, metastases, angiogenesis and cell survival and represent a good target for biological therapies (monoclonal antibodies and/or small molecules). PI3K is activated by a diverse set of stimuli including cell surface receptors (G-protein-coupled receptors, receptors with TK activity), tyrosine phosphorylated proteins (e.g. insulin-regulated substrate, IRS) and small G proteins (e.g. Ras). EGFR (ErbB-1), in contrast to other members of the HER family of receptors, induces activation of the PI3K pathway relatively weakly, since this receptor has no intracellular binding sites for PI3K.
Upon activation, PI3K recruit several proteins to the cell membrane such as PKB also known as Akt (protein kinase B, also known as Akt), PDK1 (3-phosphoinositide-dependent protein kinase) and PDK2, small proteins of the Ras family, enabling these signaling proteins to come into close contact with their substrates. PKB/Akt, is thought to be the major player in the PI3K pathway; however, it can be activated by other molecules independent of the PI3K pathway, such as PKA (protein kinase A), the calmodulin-dependent kinase pathway, and cellular stresses like heat shock and hyperosmolarity. Among three Akt proteins, Akt2 and Akt3 were shown to be activated in human breast cancer samples.
The binding of PKB to PI3K is followed by its phosphorylation and activation by PDKs. Once activated, Akt regulates a number of proteins involved in the regulation of the cell cycle (cyclin D and E2F), apoptosis and cell survival [p21, p27, nuclear factor B (NF- B), Forkhead proteins, caspases and BAD]. Akt also phosphorylates a number of other proteins, the functions of which are closely related to breast cancer, such as BRCA1 and ER. It has been shown that alpha estrogen receptors can bind PI3K and activate PI3K/Akt pathway in an estrogen-independent manner.
Many other signal transduction pathways converge on the PI3K pathway at the level of Akt. For example, Ras activates the PI3K pathway, while Akt also regulates the activity of the Ras–Raf–MAPK pathway through phosphorylation and inactivation of Raf.
PI3K signaling can be inhibited by protein phosphatases, the prototype of which is the tumor suppressor gene product PTEN (phosphatase and tensin homolog deleted on chromosome 10).
Loss of PTEN activity due to either mutations or gene deletions is associated with malignancies such as Cowden’s syndrome. mTOR (mammalian target of rapamycin) kinases are among the downstream targets of Akt, and they are thought to link mitogenic stimulation to protein synthesis and cell cycle regulation. The inhibition of mTOR blocks the progression of the cell cycle at the G1 phase. Tumor cells without PTEN activity are considered to be more sensitive to mTOR inhibitors due to their increased level of Akt activity.


Transforming Growth Factor – Beta (TGF-β) − TGF-β is a multifunctional peptide that controls proliferation, differentiation, and other functions in many cell types. TGF-β acts synergistically with TGF-α in inducing cellular transformation. It also acts as a negative autocrine growth factor. Specific receptors for TGF-β activation trigger apoptosis when activated. Many cells synthesize TGF-β and almost all of them have specific receptors for this peptide.TGF-β1, TGF-β2, and TGF-β3 all function through the same receptor signaling systems.
TGF beta induces apoptosis in numerous cell types. TGF beta can induce apoptosis in two ways: The SMAD pathway or the DAXX pathway.
The SMAD pathway is the classical signaling pathway that TGF beta family members signal through. In this pathway TGF beta dimers binds to a type II receptor which recruits and phosphorylates a type I receptor. The type I receptor then recruits and phosphorylates a receptor regulated SMAD (R-SMAD). SMAD3, an R-SMAD, is implicated in inducing apoptosis. The R-SMAD then binds to the common SMAD (coSMAD) SMAD4 and forms a heteromeric complex. This complex then enters the cell nucleus where it activates the Mitogen-activated protein kinase 8 pathway. In turn, this triggers apoptosis.
In the DAXX pathway, TGF beta may also trigger apoptosis via the death associated protein 6 (DAXX adapter protein). DAXX has been shown to associate with and bind to the type II TGF beta receptor kinase.


The EGFR signaling pathway in Breast Cancer

The EGFR family of receptors is involved in the regulation of normal breast development. Preclinical data suggest that all compartments of EGFR signaling network including ligands, receptors and downstream effectors are implicated in the development and progression of breast cancer. Overexpression of HER-2 in transgenic mouse mammary glands has been shown to promote oncogenic transformation and development of malignant phenotype. In line with this data, HER-2 activation has been reported to increase metastatic/invasive potential and to induce progression of cell cycle by disrupting the delicate balance between cyclins and the endogenous CDK inhibitors in BC cell lines.


As such, HER-2 overexpression is associated with aggressive disease biology and reduced survival in breast cancer patients. These preclinical findings are further supported clinically by the success of trastuzumab, a monoclonal antibody against HER-2, which led to improved survival, longer time to progression, higher response rate and longer duration of response in breast cancer patients with HER-2 overexpressing tumors.


Numerous studies have also reported that EGFR (HER-1) overexpression is a poor prognostic factor in BC, often associated with advanced disease. HER-2 is overexpressed in 20–25% of breast tumors and is associated with a poor prognosis (The EGFR expression rate in breast cancer is in the range of 14–91%, depending on the method of assessment, and is almost always caused by increased receptor synthesis). While HER-2 overexpression is shown to be an early event in breast cancer development, HER-1 seems to be involved in later stages . The expression of both receptors is inversely correlated with the ER status of the tumor and HER-1/2 heterodimers have been shown to increase metastatic potential of breast cancer cell lines. Accumulating data suggest that EGFR and HER-2 also predict for a poor response to endocrine therapy, and that the mutual interaction between ER and growth factor pathways seems to hotwire the events inducing endocrine resistance in ER-positive breast tumors. Acquisition of resistance to endocrine therapy was shown to be associated with upregulation of EGFR signaling in breast cancer cell lines. ErbB receptors enhance ER signaling either by directly activating ER or through activation of MAPK and Akt. So far, preclinical data have shown that agents blocking HER-1 or HER-2 driven signaling pathways can restore hormone sensitivity in endocrine-resistant, HER-2 overexpressing breast tumors, delay the development of endocrine resistance and enhance the antiproliferative effect of endocrine agents.
These effects are partly mediated by the inhibition of downstream signaling cascades involved in the development of endocrine resistance. The expression of MAPK or Akt has been reported to predict a worse clinical outcome in patients treated with endocrine therapy. Both MAPK and Akt are capable of activating ER in a ligand independent manner, and activated MAPK can also enhance ER signaling by recruiting nuclear receptor co-activators.


The impact of EGFR downstream effectors on cell cycle progression may also indirectly mediate endocrine resistance. For example, Akt and MAPK phosphorylate the cell cycle inhibitor p27, which is required for the G1 arrest mediated by anti-estrogens. In addition, phosphorylation by MAPK leads to destabilization and proteasome-mediated degradation of p27 in breast cancer cell lines, therefore disturbing its inhibitory effect on cell cycle progression. Importantly, Akt-induced cytoplasmic sequestration of p27 in human breast cancer specimens has recently been shown to be associated with reduced patient survival.


Endogenous Akt, either constitutively activated via PTEN mutations and overexpression of ErbB receptors or induced by therapeutic agents, has been shown to promote breast cancer cell survival. Induction of Akt activity in response to commonly used therapeutic modalities in breast cancer is considered to be a potential mechanism of therapeutic resistance. In fact, inhibition of Akt by a targeted agent has been shown to potentiate trastuzumab-, doxorubicin-, paclitaxel- and tamoxifen-mediated apoptosis in BC cell lines.


EGFR signaling also seems to be vital for the survival of ER-negative breast cancer. Akt-3 is overexpressed in ER-negative breast cell lines and in tissue samples taken from patients. In addition, EGF-induced activation of PKC (one of the downstream substrates of PI3K) and NF- B (nuclear factor – kappa beta) has been shown to mediate cellular proliferation in ER-negative BC cells.


Regarding the other receptors of the EGFR family, HER-3, the kinase deficient member, has also been shown to be expressed in breast cancer, while the expression of HER-4 is uncommon in breast carcinomas compared with normal breast epithelium and is associated with favorable prognostic factors . Of note, HER-2/HER-3 heterodimers have the highest mitogenic potential and are constitutively activated in breast cancer cells with HER-2 gene amplification.


Consequently, all these data render the EGFR family of receptors, their ligands and their downstream effectors rational targets for the development of novel antitumor strategies in breast cancer. (To this end natural substances such as curcumin represent interesting prophylactic measures in the prevention of breast cancer, as curcumin has been shown to be a pan Erb inhibitor, in that it induces inhibition of all EGFR receptors (1-4)). 


Some New Targeted Biological Therapies In Cancer Treatment and Prevention Based On Signal Transduction Pathways


Tyrosine kinase inhibitors  Another mechanism by which one can target HER-2 is the inhibition of its TK activity. The safety, tolerability and pharmacokinetics of a new selective HER-2 tyrosine kinase inhibitor (TKI), TAK-165, is currently being evaluated in a multicenter phase I trial in HER-2 expressing breast cancer patients.


Curcumin and emodin Inhibit HER-2 and Tyrosine Kinase and Enhance Effects of Chemotherpy DrugsAnother mechanism by which one can target HER-2 is the inhibition of its TK activity. The safety, tolerability and pharmacokinetics of a new selective HER-2 tyrosine kinase inhibitor (TKI), TAK-165, is currently being evaluated in a multicenter phase I trial in HER-2 expressing breast cancer patients.


Heat Shock Protein Inhibitors  Heat shock proteins have emerged as attractive targets for new anticancer drugs because these molecules modulate the signal transduction pathways controlling tumor cell growth and survival. These molecules are not mutated in cancer, but they facilitate malignant transformation by enhancing the activity of many oncogenic growth factor receptors, kinases and transcription factors. Heat Shock Protein 90 (HSP90) is required for the refolding of proteins in response to environmental stress and the conformational maturation of several signaling proteins. The function of Heat Shock Proteins can be inhibited by certain compounds, such as 17-AAG (17-allylamino-17-demathoxygeldanamycin), which is currently in phase I clinical trials in advanced cancer patients. HER-2 TK is one of the most sensitive targets for HSP90 inhibitors.


Ligand-based strategies  Toxin conjugated ligands are generated by conjugating a potent cellular toxin with one of the ligands of the HER family receptor. For example, the TGF- –pseudomonas exotoxin A conjugate has been evaluated in a phase I trial. The toxins conjugated to the ligand can destroy the receptor bringing about decreased concentrations of specific receptors on the cell surface and thus, down-regulating signal transduction from this class of receptors.
Agents targeting RAS–RAF–MAPK pathway - In approximately 30% of human cancers, mutated Ras genes produce abnormal proteins that remain locked in the activated state, thereby relaying uncontrolled proliferative signals. Ras mutations are not common in BC (<5%). However, the fact that Ras can be activated by multiple stimuli, including EGFR family TKs, renders Ras a promising therapeutic target in breast cancer and other cancers. Only a few agents targeting the Ras–Raf–MAPK pathway are in clinical stages of development in breast cancer, but many of these agents are in the late phases of clinical development in other cancer types


Targeting Ras. Farnesyl transferase (FTase)  Activation of this pathway catalyzes a critical step in Ras activation and represents an important target for drug development. However, the initial early clinical studies with farnesyl transferase inhibitors (FTIs) focusing on colorectal cancer (CRC), non small cell lung cancer (NSCLC) and pancreatic cancers, known to have a high incidence of K-ras mutations, did not satisfy the high expectations of therapeutic success.


Targeting MEK  

CI 1040 (PD 1843220), a small molecule MEK inhibitor, has shown low toxicity and moderate efficacy in phase I trials. Based on this promising phase I data, this agent is moving into phase II clinical trials.


Agents targeting PI3K/Akt pathway - Targeting mTOR. The mTOR pathway is a key growth factor-mediated signal transduction pathway that regulates cell growth, closely related to the PI3K/Akt pathway. mTOR-dependent growth factor signaling include estrogen, HER-2/neu and IGF-1, all of which can be inhibited by mTOR inhibition, resulting in G1 arrest of the cell cycle. CCI-779 is an m-TOR inhibitor with activity seen in breast cancer cells, both in vitro and in vivo, and is the only agent of this category in later stages of clinical development in breast cancer. Side-effects were generally mild and the most frequently occurring grade 3–4 toxicities were asthenia, depression, g glutamyl transpeptidase increase, hypercholestrolemia, leucopenia, mucositis and somnolence. Based on this preliminary clinical activity, further evaluation of this agent in combination with other treatment modalities is planned in breast cancer patients


Targeting Akt  17-AAG, an indirect inhibitor of Akt activity, is an ansamycin antibiotic which has shown antitumor effects in HER-2-overexpressing breast cancer cell lines in several preclinical experiments. It inhibits the chaperone function of Heat Shock Protein 90, which in turn depletes several key signaling proteins involved in cell cycle control, hormone signaling and growth factor pathways, including HER-2, Akt and Raf-1. The preliminary findings from phase I trials in advanced cancer patients reported disease stabilization with acceptable toxicity.


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SIGNAL TRANSDUCTION, KEY RECEPTORS AND APOPTOSIS MECHANISMS IN CANCER

Dr. James Meschino, 

DC, MS, ROHP

Global Integrative Medicine Academy

The Global Integrative Medicine Academy was created to satisfy a need, expressed by many health professionals, to establish credentials as experts in Nutritional Medicine. But, health professionals also needed to be able to complete the programme with a minimum impact on their career, family, and lifestyle. That is why the Advanced Nutritional Medicine and Sports Nutrition Certification Program was created.

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