Analysis and verification of the HMGB1 signaling pathway
© Gong etal; licensee BioMed Central Ltd. 2010
Published: 15 October 2010
Recent studies have found that overexpression of the High-mobility group box-1 (HMGB1) protein, in conjunction with its receptors for advanced glycation end products (RAGEs) and toll-like receptors (TLRs), is associated with proliferation of various cancer types, including that of the breast and pancreatic.
We have developed a rule-based model of crosstalk between the HMGB1 signaling pathway and other key cancer signaling pathways. The model has been simulated using both ordinary differential equations (ODEs) and discrete stochastic simulation. We have applied an automated verification technique, Statistical Model Checking, to validate interesting temporal properties of our model.
Our simulations show that, if HMGB1 is overexpressed, then the oncoproteins CyclinD/E, which regulate cell proliferation, are overexpressed, while tumor suppressor proteins that regulate cell apoptosis (programmed cell death), such as p53, are repressed. Discrete, stochastic simulations show that p53 and MDM2 oscillations continue even after 10 hours, as observed by experiments. This property is not exhibited by the deterministic ODE simulation, for the chosen parameters. Moreover, the models also predict that mutations of RAS, ARF and P21 in the context of HMGB1 signaling can influence the cancer cell's fate - apoptosis or survival - through the crosstalk of different pathways.
The cell cycle is strictly regulated and controlled by a complex network of signaling pathways , comprised of hundreds of proteins. If some important proteins are mutated or there are defects in the signaling mechanisms, normal cell growth regulation will break down, possibly leading to the occurrence of cancer in the future. Moreover, a number of extracellular proteins can bind to their receptors and activate signaling pathways that promote the proliferation of cancer cells.
The high-mobility group box-1 (HMGB1) protein is a DNA-binding nuclear protein, released actively in response to cytokine stimulation, or passively during cell death , and it is present in almost all eukaryotic cells [3–6]. HMGB1 can activate a series of signaling components, including mitogen-activated protein kinases (MAPKs) and AKT, which play an important role in tumor growth and inflammation, through binding to different surface receptors, such as RAGE and TLR2/4. Several studies have shown that elevated expression of HMGB1 occurs in many tumors [7–10] and accelerates cell-cycle progression. Recent in vitro studies with pancreatic cancer cells  revealed that the targeted knockout or inhibition of HMGB1 and RAGE could increase apoptosis and suppress pancreatic cancer cell growth. This phenomenon has been also observed with lung cancer and other types of cancer cells [8, 12].
The HMGB1 signal transduction can influence the cell's fate by two important processes - apoptosis and cell proliferation - which are regulated respectively by the proteins p53 and CyclinE, acting in two different signaling pathways. The protein p53 is one of the most important tumor suppressor proteins: its activation can lead to cell cycle arrest, DNA repair, or apoptosis. Mutations of p53 occur at a frequency of 50% or higher in many different cancer types . CyclinE is a cell cycle regulatory protein which regulates the G1-S phase transition during cell proliferation. Cancer cells often exhibit high expression levels of CyclinE and aberrant CyclinE activity . Many studies have found evidence of crosstalk between the two signaling pathways involving p53 and CyclinE . The crosstalk is regulated by tumor suppressor proteins, including ARF, P21 and FBXW7, which are also frequently mutated in many cancers. In this paper, we ask the following questions: How do these proteins and their mutations change the cell's fate - apoptosis or survival - when HMGB1 signal transduction is activated? Which signaling pathways are fundamental for describing HMGB1 signal transduction, and what mechanisms are responsible to explain recent results linking overexpression of HMGB1 with decrease of apoptosis (and increased cancer cell survival)?
To the best of the authors' knowledge, no computational model has been proposed to investigate the importance of HMGB1 in tumor proliferation. In this work, we construct a simple model of HMGB1 signal transduction to investigate tumorigenesis on the basis of known signaling pathway studies [16–21]. We also constructed a crosstalk network between these known pathways based on hypothetical mechanisms suggested by recent experiments. The HMGB1 pathway is not well understood at the mechanistic level, so our model can provide some insights into the study of HMGB1's roles in tumor proliferation. A series of deterministic and stochastic simulation experiments was conducted to investigate the properties of the HMGB1 pathway.
Finally, we analyze our pathway model against interesting behavorial properties by means of Model Checking techniques. Model Checking is an automated verification technique for hardware and software systems . Recently, there has been growing interest in formal verification of stochastic systems, and, which has recently seen a growing number of applications to biological systems [23–25], by means of Model Checking techniques. The Methods section introduces statistical model checking, which we then apply to validate our pathway model against experimental results from the literature.
HMGB1 signaling pathway
The p53-MDM2 pathway is regulated by a negative feedback loop : PI3K → PIP3 → AKT → MDM2 ⊣ p53 → MDM2, and a positive feedback loop: p53 → PTEN ⊣ PIP3 → AKT → MDM2 ⊣ p53. The protein PI3K is activated by the toll-like receptors (TLR2/4) within several minutes after TLR2/4 activation by HMGB1 . In turn, PI3K phosphorylates the phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol (3,4,5)-trisphosphate (PIP3), leading to phosphorylation of AKT. The unphosphorylated oncoprotein MDM2, which is one of p53's transcription targets , resides in the cytoplasm, and cannot enter the nucleus until it is phosphorylated by activated AKT. The phosphorylated MDM2 translocates into the nucleus to bind with p53, inhibiting p53's transcription activity and initializing p53 polyubiquitination , which targets it for degradation. Also, p53 can regulate the transcription of PTEN , a tumor suppressor protein, which can hydrolyze PIP3 to PIP2, thereby inhibiting the activation of AKT and MDM2.
The RAS-ERK pathway is the activation sequence: RAS → RAF → MEK → ERK → CyclinD. Activation of RAGE by HMGB1 leads to RAS activation, which in turn activates its effector protein RAF. Activated RAF will phosphorylate the MEK proteins (mitogen-activated protein kinase kinases (MAPKK)), leading to the phosphorylation of ERK1/2 (also called MAPKs). Activated ERK can phosphorylate some transcription factors which activate the expression of the regulatory protein CyclinD and Myc, enabling progression of the cell cycle through the G1 phase. K-RAS, a member of the RAS protein family, is found to be mutated in over 90% of pancreatic cancers .
The Rb-E2F pathway is composed of the interactions: CyclinD ⊣ Rb ⊣ E2F → CyclinE ⊣ Rb. The Rb-E2F pathway regulates the G1-S phase transition in the cell cycle during cell proliferation. E2F is a transcription factor that can activate the transcription of many proteins involved in DNA replication and cell-cycle progression . In quiescent cells, E2F is bound by unphosphorylated Rb - a tumor suppressor protein - forming an Rb-E2F complex which inhibits E2F's transcription activity. E2F will be activated and released when its inhibitor Rb is phosphorylated by some oncoproteins (CyclinD and Myc in Fig. 1), leading to the transcription of CyclinE and Cyclin-dependent protein kinase 2 (CDK2) which promote cell-cycle progression. CyclinE, in turn, continues to inhibit the activity of Rb, leading to a positive feedback loop [33–35]. Fig. 1 shows that the activity of CyclinD-CDK4/6 (only CyclinD is shown in Fig. 1) is inhibited by the tumor suppressor protein INK4A, which is inactivated in up to 90% pancreatic cancers .
The crosstalk between these pathways can influence the cell's fate since the three signaling pathways in HMGB1 signal transduction are not independent. As shown in Fig. 1, the oncoprotein RAS can also activate the PI3K-AKT signaling pathway; the tumor suppressor ARF protein induced by E2F can bind to MDM2 to promote its rapid degradation and stabilize p53. Furthermore, it has been experimentally observed  that the p53-dependent tumor suppressor proteins P21 and FBXW7 can inhibit the activity of cyclin dependent kinases (In Fig. 1, we use P21 to represent both P21 and FBXW7's contribution). Mutations of RAS, ARF, P21 and FBXW7 have been found in many cancers [31, 36, 37]. One of our aims is to investigate how these mutations might influence the cell's fate.
In the HMGB1 model, all substrates are expressed in the number of molecules; proteins with the subscript "a" or "p" correspond respectively to active or phosphorylated forms of the proteins. For example,
RAGE (RAGE a ) - inactive (active) form of HMGB1's receptor
MDM2 (MDM2 p ) - unphosphorylated (phosphorylated) MDM2.
We denote the mRNA transcript of MDM2 by mdm 2. We assume that the total number of active and inactive forms of the RAGE, PI3K, PIP, AKT, RAS, RAF, MEK, and ERK molecules is constant. For example, AKT + AKT p = AKT tot , PIP2 + PIP3 = PIP tot . We sometimes use CD to stand for the CyclinD-CDK4/6 complex, CE for CyclinE, and RE for the Rb-E2F complex.
The p53-MDM2 and RAS-ERK pathways have been studied individually using deterministic ODE methods [16–19, 32]. We instead formulated a reaction model corresponding to the reactions illustrated in Fig. 1 in the form of rules specified in the BioNetGen language . We used Hill functions to describe the rate laws governing protein synthesis, including PTEN, MDM2, CyclinD (CD), Myc, E2F and CyclinE (CE). Our choice was motivated by several studies [19, 39–41], which showed that transcription rates of these proteins are sigmoidal functions of transcription factor (TF) concentrations with positive cooperative Hill coefficients. We used mass action rules for other types of chemical reactions. Both ODEs and Gillespie's stochastic simulation algorithm (SSA)  are used to simulate the model with BioNetGen . Stochastic simulation is important because when the number of molecules involved in the reactions is small, stochasticity and discretization effects become more prominent [43–45]. In the online Additional file 1, we list 23 ordinary differential equations which describe the deterministic HMGB1 model and all the input parameters. The BioNetGen code which implements SSA and ODE models is available at .
Since our understanding of many chemical reactions at the mechanistic level is not clear, a large number of parameters involved in these reactions are difficult to estimate based on existing data. We emphasize that in our HMGB1 model the values for some undetermined parameters were chosen in order to produce a qualitative agreement with previous experiments.
Model Checking [22, 47] is one of the leading techniques for the automated verification and analysis of hardware and software systems. Given a high-level behavior specification, a model checker verifies whether a system (or model) satisfies it. A specification might be satisfied by many different models. Thus, model checking is the process of determining whether or not a given system model satisfies (is a model of) a property describing the desired behavior of the system. Mathematically, system models take the form of state-transition diagrams, while some version of temporal logic  is used to describe the desired properties (specifications) of system executions. A typical property stated in temporal logic is G(grant_req→ F ack), meaning that it is always (G = globally) true that a grant request eventually (F = future) triggers an acknowledgment. One important aspect of Model Checking is that it can be performed algorithmically - user intervention is limited to providing a system model and a property to check.
The Probabilistic Model Checking problem (PMC) is to decide whether a stochastic model satisfies a temporal logic property with a probability greater than or equal to a certain threshold. To express temporal properties, we use a logic in which the temporal operators are equipped with bounds. For example, the property "CyclinD will always stay below 10 in the next fifty time units " is written as G50(CyclinD < 10). We now ask whether our stochastic system M satisfies that formula with a probability greater than or equal to a fixed threshold (say 0.9), and we write M |= Pr≥ 0.9[G50(CyclinD < 10)]. In the next section, we formally define the temporal logic used in this work, Bounded Linear Temporal Logic .
Bounded Linear Temporal Logic (BLTL)
The bounded until operator ϕ1 U t ϕ2 requires that, within time t, ϕ2 will be true and ϕ1 will hold until then. Bounded versions of the F and G operators can be easily defined: F t ϕ = true U t ϕ requires ϕ to hold true within time t;G t ϕ = ¬F t ¬ ϕ requires ϕ to hold true up to time t.
The semantics of BLTL is defined with respect to traces (or executions) of a system. In our case, a trace will be the output of a simulation of a BioNetGen stochastic model. Formally, a trace is a sequence of time-stamped state transitions of the form σ = (s0,t0), (s1,t1),..., which means that the system moved to state si+1after having sojourned for time t i in state s i . The fact that a trace σ satisfies the BLTL property ϕ is written as σ |= ϕ. We denote the trace suffix starting at step k by σ k . We have the following semantics of BLTL:
σ k ⊨ AP if and only if AP holds true in state s k ;
σ k ⊨ ϕ1 ∧ ϕ2 if and only if σ k ⊨ ϕ1 and σ k ⊨ ϕ2;
σ k ⊨ ϕ1 ∨ ϕ2 if and only if σ k ⊨ ϕ1 or σ k ⊨ ϕ2;
σ k ⊨ ¬ϕ1 if and only if σ k ⊨ ϕ1 does not hold;
σ k ⊨ ϕ1U t ϕ2 if and only if there exists i ∈ N such that, (a)∑0 ≤ l <itk+l≤ t, (b) σk+i⊨ ϕ2 and (c) for each 0 ≤ j <i, σk+j⊨ ϕ1.
The semantics of BLTL are defined over infinite traces, but it can be shown that traces of an appropriate (finite) length are sufficient to decide BLTL properties .
Statistical Model Checking
We briefly explain Statistical Model Checking [50, 51], the technique we use for verifying BioNetGen models simulated by Gillespie's algorithm. Statistical Model Checking treats the Probabilistic Model Checking problem as a statistical inference problem, and solves it by randomized sampling of the traces (simulations) from the model. In particular, the PMC problem is naturally phrased as a hypothesis testing problem, i.e., deciding between two hypotheses - M ⊨ Pr≥θ[ϕ] versus M ⊨ Pr < θ [ϕ]. In other words, to determine whether a stochastic system M satisfies ϕ with a probability p ≥ θ, we test the hypothesis H0 : p ≥ θ against H1 : p < θ. Sampled traces are model checked individually to determine whether a given property ϕ holds, and the number of satisfying traces is used by a hypothesis testing procedure to decide between H0 and H1. Note that Statistical Model Checking cannot guarantee a correct answer to the PMC problem. However, the probability of giving a wrong answer can be made arbitrarily small.
We have introduced a Bayesian sequential hypothesis testing approach and applied it to the verification of rule-based models of signaling pathways and other stochastic systems [23, 49]. Sequential sampling means that the number of sampled traces is not fixed a priori, but is instead determined at "run-time ", depending on the evidence gathered by the samples seen so far. This often leads to a significantly smaller number of sampled traces.
Therefore, by running a system simulation (i.e., a BioNetGen stochastic simulation) and by checking ϕ on the resulting trace we can obtain a sample from random variable X. When a sample of X evaluates to 1 we call it a success, otherwise, a failure.
The Bayesian approach assumes that p is given by a random variable whose distribution is called the prior distribution. The prior is usually based on our previous experiences and knowledge about the system.
The following Proposition shows that, in our special case of Bernoulli samples, the computation of the Bayes Factor is straightforward.
where is the number of successes in(x1,..., x n ) and F(s,t)(·) is the Beta distribution function of parameters s, t.
The Beta distribution function can be efficiently computed by standard software packages. Thus, no numerical integration is required for the evaluation of the Bayes Factor.
Finally, we must show that the error probability of our decision procedure, i.e., the probability that we reject (accept) the null hypothesis although it is true (false), can be bounded.
Theorem. The error probability for the sequential Bayesian hypothesis testing algorithm is bounded above by where T is the Bayes Factor threshold given as input.
Results and discussion
We first conducted a series of deterministic and stochastic simulation experiments to study the properties of our HMGB1 signaling pathway model. Then, we applied the statistical model checking technique to validate some important temporal properties of our HMGB1 model.
Initial values for the model
MDM 2 p
2 × 104
2 × 104
Fig. 4(B, E) shows that the cell cycle regulatory proteins CyclinD/E will increase with the elevated expression of HMGB1, a behavior which could be verified by future experiments. Fig. 4(A-B, D-E) explains the experimental discovery that the overexpression of HMGB1 decreases apoptosis and promotes DNA replication and proliferation in cancer cells.
The oncoprotein AKT is overexpressed in many cancers . In Fig. 4(C, F), we first increase the number of unphosphorylated AKT molecules and fix the other proteins' concentration, then measure p53 and MDM2 p 's expression levels at 10 hours in phase G1 after HMGB1 activates its receptor RAGE. Fig. 4(C, F) shows that with the increase of AKT's expression level, p53 is repressed due to the ubiquitination initiated by the overexpressed MDM2 p , which is promoted by the activated and overexpressed AKT protein. The results in Fig. 4(C, F) suggest a way to inhibit tumor cell proliferation and induce tumor cell apoptosis through the inhibition of protein phosporylation events downstream from AKT kinases in the PI3K/AKT pathway, using an AKT kinase inhibitor (such as the drug GSK-690693 ).
K-RAS is a member of the RAS protein family. K-RAS mutation and ARF loss occur in more than 80% of pancreatic cancers [36, 53]. The P21 and FBXW7 proteins are also frequently mutated in many cancers . ARF and P21 play an important role in the crosstalk between the p53 and Rb pathways. ARF is able to reroute cells with oncogenic damage to p53-dependent fates through binding to MDM2 and targeting its degradation. The p53-dependent tumor suppressor proteins P21 and FBXW7 can inhibit CyclinD/E's activity to prevent the proliferation of cancer cells.
Fig. 5(A, D) shows that wild-type ARF (large d ARF ) can decrease the number of MDM2 p molecules and increase p53's expression level to initiate apoptosis even if the cell proceeds to the S phase. Moreover, mutated ARF (smaller d ARF ) can not stabilize p53 expression and prevent the proliferation of cancer cells if HMGB1 is overexpressed. This could explain the phenomenon that ARF loss exists in over 80% of pancreatic cancers . Fig. 5(B, E) demonstrates that CyclinD/E proteins will increase if P21 is mutated (smaller dP 21), thereby accelerating cell cycle progression.
K-RAS is mutated in most cancers, especially in pancreatic cancer . The activation of RAS is initiated by HMGB1 and its receptors, and the wild-type RAS can be deactivated by some kinases. Studies have found that the mutated K-RAS can not be deactivated , even if HMGB1 is knocked out, so it will continuously activate the downstream signaling pathways which promote cell proliferation. Fig. 5(C, F) shows that with the increase of RAS deactivation rate d RAS (b1 in the ODE model), the synthesis of CyclinD/E will be inhibited, but a small deactivation rate of RAS will lead to overexpression of CyclinD/E. The results visualized in Fig. 5 suggest some ways to inhibit cancer cell proliferation through inhibition or deactivation of the signaling pathways involving RAS, Cyclin, and Cyclin-dependent kinases (CDK). Recently, CDK and RAS inhibitor drugs [57–59] have been developed to inhibit tumor growth.
Verification of the HMGB1 model
We use Statistical Model Checking (SMC) to verify some fundamental properties that our model should satisfy. We test whether the model satisfies a given BLTL property with probability p ≥ 0.9. We set the threshold T = 1000 for the verification, so the probability of a wrong answer is smaller than 10-3.
Verification of property 1
Property 1: Pr≥ 0.9[F t (G900(P 53 < 3.3 × 104))]
# of Samples
# of Successes
that is, within 100 minutes p53's level will eventually be larger than 5.3 × 104. SMC accepts this property as true, after sampling 38 traces (of which 37 satisfying traces).
Verification of property 3
Property 3: Pr≥ 0.9[F20(PI 3Ka/PI 3Ktot > 0.5)]
# of Samples
# of Successes
9 × 103
Verification of property 4 and 5
Property 4: Pr≥ 0.9[F600(CyclinE> 900)]
Property 5: Pr≥ 0.9[F600(CyclinD> 900)]
# of Samples
# of Success
# of Samples
# of Success
with different RAS deactivation rates (d RAS ). The results are presented in Table 4. Properties 4 and 5 show that the overexpression of HMGB1 and mutation of RAS (small d RAS value) will accelerate the expression of cell regulatory protein CyclinD/E to promote cell proliferation. However, inhibition of HMGB1 and an increase of RAS deactivation rate will prevent tumor growth.
SMC accepted this property as true (22 satisfying traces).
Verification of property 7
Property 7: Pr≥ 0.9[F100(P 53/10000 ≥ a∧ F100(P 53/10000 ≤ 0.4))]
# of Samples
# of Successes
We have presented a reaction network model of the signaling transduction initiated by HMGB1. The model incorporates the contributions from the most important known signaling components of the HMGB1 signal transduction network. The model is expressed in the form of BioNetGen rules, and simulated using ODEs and Gillespie's algorithm under a range of conditions. We used Statistical Model Checking to automatically validate our model with respect to known experimental results.
Our simulations demonstrate a dose-dependent p53 and CyclinE response curve to increasing HMGB1 stimulus. This hypothesis could be tested by future experiments. In particular, overexpression of HMGB1 promotes the cell cycle regulatory proteins E2F and CyclinD/E and inhibits the pro-apoptotic protein p53, leading to increased cancer cell survival and decreased apoptosis. This is consistent with experimental observations in recent studies of cancer cells . We also investigated the roles of different components in the pathway and predicted their activity in response to various conditions. We investigated how mutations of the RAS, ARF and P21 proteins influence the fate of the cancer cell. In particular, parameter variation showed that the mutated RAS increases the expression level of CyclinE, leading to cancer cell proliferation. Mutation or loss of the ARF protein leads to high MDM2 activity and loss of p53 expression in the face of HMGB1 overexpression, resulting in decreased apoptosis. Our model shows that the inhibition (or deactivation) of RAS, Cyclin, and Cyclin-dependent kinases (CDK) might inhibit tumor growth.
Since our proposed model is based on just three signaling pathways, we are far from capturing the entire HMGB1 network dynamics. Studies have found that HMGB1 can not only activate the PI3K-AKT and RAS-ERK pathways, but can also activate the NFκ B signaling pathway , which regulates many pro-apoptotic and anti-apoptotic proteins' transcription . Since HMGB1 could be released passively during necrosis, there might exist crosstalk between the tumor necrosis factor (TNF) pathway and the HMGB1 pathway. Besides the incorporation of new pathways, recent work has demonstrated that HMGB1 can bind to p53 directly to influence p53-mediated transcriptional activity . A larger network for HMGB1 signal transduction will be explored in our future work.
It has been recently observed that pancreatic tumor cells increase autophagy  and release HMGB1  in response to chemotherapy, radiation, and hypoxia, which may promote tumor cell survival. It has been hypothesized that direct inhibition of autophagy may be another way to inhibit tumor growth and enhance the efficacy of cancer therapies . The incorporation of autophagic proteins into the HMGB1 signaling pathway is worth considering in future work.
Although our current model can only qualitatively compare with the experimental behavior, it still provides valuable information about the behavior of HMGB1 signal transduction in response to different stimuli. Future experiments will enable the development of more realistic models. We anticipate that the application of model checking techniques, such as those explored in this work, will facilitate the development of targeted and effective anti-cancer therapies.
This work was supported by a grant from the U.S. National Science Foundation's Expeditions in Computing Program (award ID 0926181). The authors thank Michael T. Lotze (University of Pittsburgh) for calling their attention to HMGB1 and for helpful discussions of the topic. H.G. would like to thank Marco E. Bianchi (San Raffaele University) for email discussions on HMGB1. We would also like to thank Ilya Korsunsky and Màtè L. Nagy for their comments on this paper.
This article has been published as part of BMC Bioinformatics Volume 11 Supplement 7, 2010: Ninth International Conference on Bioinformatics (InCoB2010): Bioinformatics. The full contents of the supplement are available online at http://www.biomedcentral.com/1471-2105/11?issue=S7.
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