An Examination of P13K-mTOR and RAS-RAF-MEK-ERK Pathways and the Entropy-Generation Modeling of Cancer
by Nikhil S. Babu
An undergraduate thesis submitted in partial completion of the Metropolitan State University of Denver Honors Program
May 5, 2017
Dr. Colin Mant
Dr. Vida Melvin Dr. Megan Hughes-Zarzo
Honors Program Director
An Examination of PI3K-mTOR and RAS-RAF-MEK-ERK Pathways and the Entropy-Generation Modeling of Cancer
Nikhil S. Babu*
Department of Chemistry, Metropolitan State University of Denver, Denver, Colorado S0204
In 2016, there were an estimated 1,685,210 new cases of cancer of any site and 595,690 resulting deaths8. As cancer occurs when inherent growth regulation mechanismsin particular signaling mechanismswithin our body malfunction and as RAS proteins are key regulators in normal cell growth and malignant transformation, modern cancer therapies focus on targeting RAS signaling pathways. Such therapies targeting RAS usually seek to inhibit the following: farnesyl transferase activity, MAPK/ERK (mitogen-activated protein kinases/extracellular signal-regulated kinases kinase) pathway, epidermal growth factor (EGF) receptor and AKT/PKB (phosphatidylinositol 3-kinase (PI3K) and Akt/ Protein Kinase B) kinase activity2. While such therapies aggressively treat parts of cancer, they still fail to stop the progression of cancer as a whole. This paper serves to suggest an entropy-generation approach as a potential tool to analyze and treat cancer as a whole unit.
Cancer arises when certain cells within the body undergo uncontrolled cell division. Cancer is not a single disease, but rather a term now used to describe a variety of different diseases occurring all over the body. These diseases are defined by their anatomical position of occurrence, the cells undergoing the transformation, and the oncogenic mutations leading to ab-nonnality. Cancer is nothing newthe Egyptians have documented it as early as 1600 BC2, but we have come a long way in the treatment of cancer in the past century. The availability of various surgical, radiological and chemotherapeutic interventions has allowed us to effectively hinder the progress of various cancers, especially ones seen in pediatric patients. However, increasing industrialization, obesity and increased life expectancies have all led to the emergence to new types of cancers at an increased rate. Additionally, the suppression of secondary cancers and side effects occurring after initial intervention has become a monumental clinical task. This paper will examine two crucial cancer pathways (PI3K-mTOR/RAS-ERK), the role of entropy in protein folding, and finally the application of maximum entropy production principle (MEPP) to analyzing cancer.
Overview of Signaling and Crosstalk
Two of the key pathways involved in oncogenesis are the PI3K-mTOR (Phosphatidylinositol 3-Kinase mechanistic Target Of Rapamycin) and RAS-RAF-MEK-ERK (RAt Sarcoma Rapidly Accelerated Fibrosarcoma and MAPK/ERK Kinase Extracellular signal-Regulated Kinase) signaling cascades. The PI3K-mTOR pathway is in tight loop with the RAS-RAF-MEK-ERK pathway and allows for the anabolic proliferation of cells. The PI3K-mTOR pathvvay is responsible for three key cellular functions10:
1. Receiving signals from EGF (epidermal growth factor), GPCRs (G-protein-coupled receptors), cytokines and steroids
2. Sustaining continuous anabolic cell proliferation via the detection of available energy
3. Inhibiting mitochondrial and extra-mitochondrial apoptosis signals
The question that has puzzled researchers for decades was how this pathway somehow manages to carry out all three of these functions with a much higher efficiency than expected. The answer to this question is revealed when we take a step back and consider all known signaling cascades as a whole. When doing so, we find signaling redundancy and connections in both RAS-ERK and P13K-mTOR with both upstream and downstream effectors via extensive cross-talk and bidirectional feedback loops.
The RAS-RAF-MEK-ERK pathway begins with the binding of EGF to RTK (receptor tyrosine kinase) leading to dimerization (Figure l)26. GRB2 (growth factor receptor-bound protein 2) then binds to RTK and recruits SOS (Son of Sevenless), which binds to RAS-GDP, resulting in RAS-GTP. This activates B-Raf, which in turn activates ERK and MEK, leading to activation of AP-l-fos-jun and various growth factors. GAP (GTPase-Activating Proteins) then inactivates RAS-GTP-B-Raf by converting RAS-GTP to RAS-GDP. Although the mutated RAS allows GAP to bind, RAS-GTP is not able to convert to RAS-GDP. This results in constitutively activated GAP-RAS-GTP-B-Raf.
RAS is the key player in this pathway. RAS consists of four small GTPases: HRAS, NRAS, KRAS4A and KRAS4B. Posttranslational modification of RAS, for example via fame-sylation, geranylgeranylation or palmitoylation, results in these RAS proteins (with the help of Grb2/SOS) being recmited to the ligand-bound plasma membrane receptors. Activation of
RAS then allows for the recruitment of serine/threonine RAF to the plasma membranethis leads to cell survival, proliferation and motility via activation of MEK and ERK (Figure la)22.
Like with RAS, the PI3K cascade is also triggered by RTKs and GPCRs on the plasma membrane. When these receptors are activated via binding of extracellular ligands, the catalytic subunit of PI3K converts PIP2 (phosphatidylinositol 4,5-bisphosphate) to PIP3 (Figure lb). PIP3 production is antagonized by PTEN (phosphatase and tensin homolog) PTEN was found to be silenced in various cancers. PDK1 (3-phosphoinosi-tide dependent protein kinase-1) then binds PIP3 at the plasma membrane and phosphorylates AKT (protein kinase B), which in turn goes on to phosphorylate a multitude of targets. For example, AKT promotes cell survival via the phosphorylation of MDM2 (mouse double minute 2 homolog) and the negative regulation of BAD (bcl-2-associated death promoter), BAX (bcl-2-like protein 4), and forkhead transcription factors like FOXO (forkhead box protein 01). It also negatively regulates TSC 1 and 2 (tuberous sclerosis 1 and 2) leading to the activation of mTORCl (mammalian target of rapamycin complex 1), a key regulator of cellular growth22.
There are four main mechanisms of pathway crosstalk: positive feedforward loop, negative feedback loop, cross-inhibition, cross-activation, and pathway convergence. In positive feedforward loops, a member downstream in a given pathway positively regulates its upstream activator. In negative
feedback loops, a member downstream in a given pathway negatively regulates its upstream activator. In cross-inhibition, a member in one pathway inhibits a member upstream in a different pathway. In cross-activation, a member in one pathway activates a member upstream in a different pathway. In pathway convergence, signaling resulting from two separate pathways converge onto the activation or inhibition of a single member downstream.
In an example of positive feedforward loop, GAB (GRB2-associated-binding) docking proteins can bind GRB2-S0S complex on activated RTKs. GABs can then bind RasGAP, SHP2 (src homology region 2), PI3K and P1P3. SHP2 then dephosphorylates Ras-GAP docking sites on GAB1 (GRB2-associated-binding protein 1), resulting in reduced Ras inactivation and increased Ras-ERK signaling. Binding of GABs to PI3K creates local PIP3, resulting in the recruitment of additional GAB1 to the plasma membrane, which in turn increases PI3K signaling22.
On the other hand, negative feedback loops seek to hinder signaling. For example, ERK phosphorylates and inhibits SOS, Raf and MEK1resulting in ERK downregulation. ERK, through the induction of genes coding for sprouty proteins, also interferes with Raf-mediated MEK activation and MAPK phosphatases. This intervention by ERK results in its inactivation22. In cross-inhibition (Figure 1), PI3K-AKT and RAS-ERK pathways negatively regulate each others activity. For example,
Figure 1. Crosstalk between RAS-ERK and PI3K-mTOR pathways. Green arrows indicate cross-activation, and red arrows indicate cross-inhibition, (a) RAS-ERK pathway. Activation of receptor tyrosine kinase (RTK) activates the G protein Ras, which in turn recruits Raf to the inner plasma membrane. Raf then phosphorylates MEK, which then phosphorylates ERK. Activated ERK then goes on to regulate gene expression in the nucleus via interaction with various transcription factors, (b) PI3K-mTOR pathway. Upon RTK activation, PI3K converts PIP2 (phosphatidylinositol 4,5-bisphosphate) to PIP3 (phosphatidylinositol 3,4,5-tri-phosphate). This leads to the activation of Akt at the plasma membrane, leading to cell survival, proliferation and growth.
Cell survival Cell proliferation Cell motility
Cell survival Cell proliferation Cell motility
Ribosome biogenesis and translation
Source: Figure adapted from Mendoza, M. C.; Er, E. E.; BLenis, J. Trends in Biochemical Sciences. 2011,36(6), 320-328. Copyright 2011 Cell Press.
when MEK is inhibited, AKT activity due to EGF induction was seen to increase10. Inhibition of MEK, releases the cross-inhibition mechanism between PI3K-AKT and RAS-ERK, thereby allowing for AKT activation. One plausible mechanism for this might be initiated through the phosphorylation of GAB1 by ERK, preventing the recruitment of PI3K to EGFR.
In cross-inhibition between AKT and Raf, strong IGF1 stimulation causes ERK activation to be negatively regulated via phosphorylation of Raf N-terminus inhibitory sites. The auto-inhibited Rafis then sequestered away front Ras and MEK. PP1 then removes inhibitory phosphorylations by AKT on RAF. One might think that this cross-inhibition is required for the suppression of cancer. However, contrary to our intuition, this cross-inhibition is actually required for the progression of cancer. Activating mutations in RAS/RAF result in very high levels of consistent activation of Ras-ERK signaling that senescence occurs22.
However, there are some problems in targeting this pathway. For example, there are no strategies that can deactivate RAS. MAPK (Mitogen-activated protein kinase)/ERK is complex pathway and not linearthere is extensive crosstalk. While modem cancer drugs inhibiting multiple pathways alter the natural course of the disease, they do not cure it. As we saw earlier, RAS is a key target, but extremely difficult to tackle due to extensive cross-talk. Currently, we only have drugs that target the downstream effectors of RAS: MEK, RAF and ERKthese drugs are ineffective in stopping the progression of many cancers6. Additionally, one ofthe biggest challenges in chemotherapy is to just target the tumor cells and not healthy cells.
PI3K, mTOR, RAS, RAF, MEK and ERK are all proteins. As such, their behavior can be described using thermody-namic equations. Proteins in their folded (native) state are very rigid, and held together in their specific conformation mostly by weak interactions such as hydrogen bonding, ionic interactions, and van der Waals interactions. When most proteins are considered in a polar solvent such as water, non-polar amino acid residues tend to be forced into the interior of the protein via hydro-phobic exclusionthe exclusion of hydrophobic regions of the protein by water to achieve a more favorable state. This results in a compact, folded protein that exhibits very little fluctuation of structure or bond rotation. On the other hand, proteins in their unfolded state show a much greater degree of freedomthe residue side chains are solvated, able to rotate freely and occupy a much greater volume.32
The non-covalent interactions holding a native protein in place can be easily disrupted via heat or changes in pH. For example, when heat is added, many non-covalent interactions are broken simultaneously. This influx of heat allows a protein in its native state to transition into an unfolded state via the co-operativity of transition, much like the latent heat involved in the melting of ice. The enthalpy of denaturation, AHd, is the difference in enthalpies between the folded state and the unfolded state AHu AHf. The melting temperature, Tm, the temperature at which a protein unfolds, is highly dependent on not just protein composition but also its interaction with the environment. Tire entropy of denaturation of the protein system, ASj,
could then defined as, AHj divided by T,. At constant external pressure, AHd=, qp, where qp is the heat transferred to a protein system at constant pressure. As AH<] is also equal to CPAT, we get:
Summing up all entropic contributions over a given temperature range (Ti to T2) and assuming ACP (change in heat capacity at constant pressure) is constant from Ti to T2, we obtain:27
Thus, using equation 2 we can calculate the entropic change at a given temperature from the AS value at a baseline temperature, the heat capacity, and the two temperatures Ti and T2.
Now, why do we bother calculating the entropic change involved in protein folding/unfolding? Consider lowering the pH of a solution containing a native protein. This proto-nates residues and lowers the transition/melting temperature, Tra. This lowering of Tm can be explained by changes in enthalpy with respect to the change in entropy of the protein system. For example, during protein unfolding, the entropic difference between the folded and unfolded states changes much more rapidly than the change in enthalpy and this allows AH / AS to decrease, which in turn reduces Tm (Tm= AHj/ ASd).7 This dramatic shift in entropy during pH reduction is due to the change in ionization states of the acidic side chains of the protein. As the concentration of H+ increases, the net positive charge of the protein increases. This increases the frequency and intensity of repulsions among residues, destabilizing the native structure of the protein. This transition from the native state to the unfolded state can be represented as follows:
Pnalivc nH+ P* -> Prided, where P* is the destabilized folded conformation.27
As we can see, the entropic force is the primary motivating factor for proteins to unfold or for hydrophobic residues to congregate when in a polar solvent. Any environmental disturbance to the native protein forces it to undergo conformational changes to maximize the number of microstates within a given macrostate (increase in ASa). Through this transition, the rigidity of native proteins is removed, causing the protein to unfold. In protein folding, hydrophobic exclusion to maximize favorable interactions with water results in the removal of hydration spheres; this contributes to an entropic increase among water molecules. As a result, while there is an entropic reduction within the protein system, the entropy of the surrounding increases to a greater degree. Titus, in both protein folding and unfolding there is a net increase in entropy.
Figure 2. Jmol image representing a conformational shift from RAS-GTP (left) to RAS-GDP (right)6. RAS-GTP is the active conformation and RAS-GDP is the inactive conformation. Both models use a non-cleavable analogue of GTP in the binding site. The protein is colored blue, with the switch regions colored green. The base-sugar backbone is shown in white, and the phosphates in pink and red. The magnesium ion is shown in magenta. Going from left to right, we see a confonnational shift in the switch regions of the protein following the removal of the terminal phosphate (red).
Cancer and Maximum Entropy Production Principle (MEPP)
From our review of protein thermodynamics, entropy was shown to be a crucial factor in effecting confonnational changes within a proteina key determinant in protein function. As such, a rational approach to targeting cancer cells may involve the application of fundamental entropy principles, specifically the second law of thennodynamics (mathematically defined as q/T). Delineation of the second law leads us to the maximum entropy production principle (MEPP): the system will select the path, or assembly of paths, out of otherwise available paths, that minimize the potential or maximize the entropy at the fastest rate given the constraints24. This principle suggests a maximization of entropy production during nonequilibrium processes.
Signaling cascades in cancer may be thought of as nonequilibrium dissipative processes. Such processes are consistent with MEPPthey couple extended regions of high order (e.g. bulk transport pathways) with localized regions of high dissipation (increased AS) such as the plasma membrane16. The high ordered structures tend to export the entropy generated by localized structures at the fastest rate plausible given particular constraintsfor example the stationarity, local energy and mass balance. This shipping of entropy by cells to their environment may then be quantitatively measured.
Consider the active to inactive confonnational shift involved in one of the key players in carcinogenesisRAS (Figure 2). One can, for example using nuclear magnetic resonance (NMR), measure the confonnational entropy involved in going from RAS-GTP to RAS-GDP12. Measurements of RAS confonnational entropy can then provide us information about the overall binding entropy. The overall binding entropy value
can then be used to better understand interaction of RAS with other proteins/ligands, which in turn can be used to aid in the development of specific drugs targeting just RAS.
Cancer, in its essence, is an open system from a thermodynamic perspective. It is a complex, self-organizing, irreversible system allowing for the exchange of both energy and matter. As such, we may use thermodynamic models to analyze the progression of cancer. In accordance MEPP, changes in cancer entropy may be modeled within the bounds of a given range of entropy values and this range of values then used to better design drugs. Moreover, outside of this range cancer progression will theoretically be halted. Therefore, another plausible therapy based on the entropy-generation model may involve altering the value of entropy generation to fall outside this range, for example via electromagnetic and ultraviolent radiation.
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Dr. Colin Mant (Department of Chemistry, Metropolitan State University of Denver), Dr. Vida Melvin (Department of Biology, Metropolitan State University of Denver) and Dr. Megan Hughes-Zarzo (Honors Program, Metropolitan State University of Denver)