PTEN expression can be used as a switch between senescence and apoptosis in breast cancer cells according to a logical model of the G2/M checkpoint.

Worldwide, the second-highest mortality rate is caused by breast cancer (BC). The most studied BC cell line is MCF-7 because it exhibits strong consistency with clinical cases and is a good system for analyzing tumors with functional estrogen receptors (ER-positive cancers). In this paper, we introduce the first theoretical method for describing PTEN-loss-induced cellular senescence (PICS), which is an increase in cellular senescence caused by PTEN knockout, utilizing a logical model of the G2/M checkpoint. We predict that PTEN expression acts as a switch between cell phenotypes associated with senescence and apoptosis. We show that PICS is induced by the activity of the positive feedback between AKT and mTORC2, and that overexpression of PTEN will disrupt the feedback, abrogating senescence and only leading to arrest or apoptosis. Furthermore, we demonstrate that miR-21 can be used as a target against proliferation control because its knockout is equivalent to PTEN overexpression. We think the findings can be used to motivate new strategies for MCF-7 strain proliferation control.

A dynamic Boolean network reveals that the BMI1 and MALAT1 axis is associated with drug resistance by limiting miR-145-5p in non-small cell lung cancer.

Patients with non-small cell lung cancer (NSCLC) are often treated with chemotherapy. Poor clinical response and the onset of chemoresistance limit the anti-tumor benefits of drugs such as cisplatin. According to recent research, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is a long non-coding RNA related to cisplatin resistance in NSCLC. Furthermore, MALAT1 targets microRNA-145-5p (miR-145), which activates Krüppel-like factor 4 (KLF4) in associated cell lines. B lymphoma Mo-MLV insertion region 1 homolog (BMI1), on the other hand, inhibits miR-145 expression, which stimulates Specificity protein 1 (Sp1) to trigger the epithelial-mesenchymal transition (EMT) process in pemetrexed-resistant NSCLC cells. The interplay between these molecules in drug resistance is still unclear. Therefore, we propose a dynamic Boolean network that can encapsulate the complexity of these drug-resistant molecules. Using published clinical data for gain or loss-of-function perturbations, our network demonstrates reasonable agreement with experimental observations. We identify four new positive circuits: miR-145/Sp1/MALAT1, BMI1/miR-145/Myc, KLF4/p53/miR-145, and miR-145/Wip1/p38MAPK/p53. Notably, miR-145 emerges as a central player in these regulatory circuits, underscoring its pivotal role in NSCLC drug resistance. Our circuit perturbation analysis further emphasizes the critical involvement of these new circuits in drug resistance for NSCLC. In conclusion, targeting MALAT1 and BMI1 holds promise for overcoming drug resistance, while activating miR-145 represents a potential strategy to significantly reduce drug resistance in NSCLC.

The lncRNA DLX6-AS1/miR-16-5p axis regulates autophagy and apoptosis in non-small cell lung cancer: A Boolean model of cell death.

Long non-coding RNA (lncRNA) distal-less homeobox 6 antisense RNA 1 (DLX6-AS1) is elevated in a variety of cancers, including non-small cell lung cancer (NSCLC) and cervical cancer. Although it was found that the microRNA-16-5p (miR-16), which is known to regulate autophagy and apoptosis, had been downregulated in similar cancers. Recent research has shown that in tumors with similar characteristics, DLX6-AS1 acts as a sponge for miR-16 expression. However, the cell death-related molecular mechanism of the DLX6-AS1/miR-16 axis has yet to be investigated. Therefore, we propose a dynamic Boolean model to investigate gene regulation in cell death processes via the DLX6-AS1/miR-16 axis. We found the finest concordance when we compared our model to many experimental investigations including gain-of-function genes in NSCLC and cervical cancer. A unique positive circuit involving BMI1/ATM/miR-16 is also something we predict. Our results suggest that this circuit is essential for regulating autophagy and apoptosis under stress signals. Thus, our Boolean network enables an evident cell-death process coupled with NSCLC and cervical cancer. Therefore, our results suggest that DLX6-AS1 targeting may boost miR-16 activity and thereby restrict tumor growth in these cancers by triggering autophagy and apoptosis.

A Boolean Model of the Proliferative Role of the lncRNA XIST in Non-Small Cell Lung Cancer Cells.

The long non-coding RNA X inactivate-specific transcript (lncRNA XIST) has been verified as an oncogenic gene in non-small cell lung cancer (NSCLC) whose regulatory role is largely unknown. The important tumor suppressors, microRNAs: miR-449a and miR-16 are regulated by lncRNA XIST in NSCLC, these miRNAs share numerous common targets and experimental evidence suggests that they synergistically regulate the cell-fate regulation of NSCLC. LncRNA XIST is known to sponge miR-449a and miR-34a, however, the regulatory network connecting all these non-coding RNAs is still unknown. Here we propose a Boolean regulatory network for the G1/S cell cycle checkpoint in NSCLC contemplating the involvement of these non-coding RNAs. Model verification was conducted by comparison with experimental knowledge from NSCLC showing good agreement. The results suggest that miR-449a regulates miR-16 and p21 activity by targeting HDAC1, c-Myc, and the lncRNA XIST. Furthermore, our circuit perturbation simulations show that five circuits are involved in cell fate determination between senescence and apoptosis. The model thus allows pinpointing the direct cell fate mechanisms of NSCLC. Therefore, our results support that lncRNA XIST is an attractive target of drug development in tumor growth and aggressive proliferation of NSCLC, and promising results can be achieved through tumor suppressor miRNAs.

Systems biology approach suggests new miRNAs as phenotypic stability factors in the epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is a cellular programme on which epithelial cells undergo a phenotypic transition to mesenchymal ones acquiring metastatic properties such as mobility and invasion. TGF-ß signalling can promote the EMT process. However, the dynamics of the concentration response of TGF-ß-induced EMT is not well explained. In this work, we propose a logical model of TGF-ß dose dependence of EMT in MCF10A breast cells. The model outcomes agree with experimentally observed phenotypes for the wild-type and perturbed/mutated cases. As important findings of the model, it predicts the coexistence of more than one hybrid state and that the circuit between TWIST1 and miR-129 is involved in their stabilization. Thus, miR-129 should be considered as a phenotypic stability factor. The circuit TWIST1/miR-129 associates with ZEB1-mediated circuits involving miRNAs 200, 1199, 340, and the protein GRHL2 to stabilize the hybrid state. Additionally, we found a possible new autocrine mechanism composed of the circuit involving TGF-ß, miR-200, and SNAIL1 that contributes to the stabilization of the mesenchymal state. Therefore, our work can extend our comprehension of TGF-ß-induced EMT in MCF10A cells to potentially improve the strategies for breast cancer treatment.

Towards DNA-damage induced autophagy: A Boolean model of p53-induced cell fate mechanisms.

How a cell determines a given phenotype upon damaged DNA is an open problem. Cell fate decisions happen at cell cycle checkpoints and it is becoming clearer that the p53 pathway is a major regulator of cell fate decisions involving apoptosis or senescence upon DNA damage, especially at G1/S. However, recent results suggest that this pathway is also involved in autophagy induction upon DNA damage. To our knowledge, in this work we propose the first model of the DNA damage-induced G1/S checkpoint contemplating the decision between three phenotypes: apoptosis, senescence, and autophagy. The Boolean model is proposed based on experiments with U87 glioblastoma cells using the transfection of miR-16 that can induce a DNA damage response. The wild-type case of the model shows that DNA damage induces the checkpoint and the coexistence of the three phenotypes (tristable dynamics), each with a different probability. We also predict that the positive feedback involving ATM, miR-16, and Wip1 has an influence on the tristable state. The model predictions were compared to experiments of gain and loss of function in other three different cell lines (MCF-7, A549, and U2OS) presenting agreement. For p53-deficient cell lines such as HeLa, H1299, and PC-3, our model contemplates the experimental observation that the alternative AMPK pathway can compensate this deficiency. We conclude that at the G1/S checkpoint the p53 pathway (or, in its absence, the AMPK pathway) can regulate the induction of different phenotypes in a stochastic manner in the U87 cell line and others.

Integrative data modeling from lung and lymphatic cancer predicts functional roles for miR-34a and miR-16 in cell fate regulation.

MiR-34a and miR-16 coordinately control cell cycle checkpoint in non-small cell lung cancer (NSCLC) cells. In cutaneous T-cell lymphoma (CTCL) cells miR-16 regulates a switch between apoptosis and senescence, however the role of miR-34a in this process is unclear. Both miRNAs share many common targets and experimental evidences suggest that they synergistically control the cell-fate regulation of NSCLC. In this work we investigate whether the coordinate action between miR-34a and miR-16 can explain experimental results in multiple cell lines of NSCLC and CTCL. For that we propose a Boolean model of the G1/S checkpoint regulation contemplating the regulatory influences of both miRNAs. Model validation was performed by comparisons with experimental information from the following cell lines: A549, H460, H1299, MyLa and MJ presenting excellent agreement. The model integrates in a single logical framework the mechanisms responsible for cell fate decision in NSCLC and CTCL cells. From the model analysis we suggest that miR-34a is the main controller of miR-16 activity in these cells. The model also allows to investigate perturbations of single or more molecules with the purpose to intervene in cell fate mechanisms of NSCLC and CTCL cells.

ATM/miR-34a-5p axis regulates a p21-dependent senescence-apoptosis switch in non-small cell lung cancer: a Boolean model of G1/S checkpoint regulation.

MicroRNA-34a-5p regulates the G1/S checkpoint in non-small cell lung cancer (NSCLC) cells. Forced expression of miR-34a-5p enhances p21 expression and promotes cellular senescence, whereas knockout of miR-34a-5p decreases senescence and increases apoptosis. This suggests that p21 is the main effector of a senescence-apoptosis switch in NSCLC cells; however, the molecular mechanisms controlling this switch are unclear. In this work, we propose a Boolean model of G1/S checkpoint regulation, contemplating the regulatory influences of p21 by miR-34a-5p. The predicted probabilities of our model are in excellent agreement with experimental data. Our model supports that p21 is the main effector of a senescence/apoptosis switch and that the disruption of the positive feedback involving ATM, miR-34a-5p, and the histone deacetylase HDAC1 abrogates senescence.

Dynamics of the feedback loops required for the phenotypic stabilization in the epithelial-mesenchymal transition.

The epithelial-mesenchymal transition (EMT) is a complex mechanism in which cells undergo a transition from epithelial to mesenchymal phenotypes (there is also an intermediary hybrid state) in response to microenvironmental alterations and aberrant stimuli triggered by molecules such as TGF-ß. Recent studies in breast cancer progression reported new feedback loops and new participant molecules such as microRNAs 340 and 1199. In this work, we propose a logical model of EMT contemplating the influence of these new published molecules on the regulatory core of EMT. The model results were compared with theoretical and experimental data for the human breast epithelial cell line MCF10A presenting excellent agreement. We propose that the miRNAs 340 and 1199 should be considered phenotypic stability factors of the hybrid state based on the positive feedback loops they form with ZEB1. In addition, the model allows the prediction of phenotype probabilities at the coexistence region. For the tristable dynamics when epithelial, hybrid, and mesenchymal phenotypes coexist, we found that the hybrid state is the most probable, agreeing with experiments. Our results highlight new mechanisms related to the EMT dynamics in response to TGF-ß stimulus in epithelial breast cells and might help the design of therapeutic strategies for breast cancer.

Towards the contribution of the p38MAPK pathway to the dual role of TGFß in cancer: A boolean model approach.

The transforming growth factor-beta (TGF-ß) pathway is involved in the regulation of cell growth and differentiation. In normal cells or in the early stages of cancer, this pathway can control proliferation stimuli by inducing cell cycle arrest or apoptosis (through the MAP-kinase protein p38MAPK), while in late stages it seems to act as a tumor promoter. This feature is known as the TGF-ß dual role in cancer and it is not completely explained. This seems to arise through the accumulation of mutations in cancer development that affect the normal function of these pathways. In this work we propose a Boolean model of the crosstalk between the TGF-ß, p38 MAPK and cell cycle checkpoint pathways which qualitatively describes this dual behavior. The model shows that for the wild type case, TGF-ß acts as tumor supressor by inducing cell cycle arrest or apoptosis, as expected. However, the loss of function (LoF) of its two signaling proteins: SMAD2 and SMAD3 has immortalization effects due to the activation of the PI3K/AKT pathway that contributes to inhibit apoptosis. In silico mutations of the model elements were compared with cell phenotypes in experiments presenting agreement. In addition, we performed a series of double gene perturbations (that simulate random deleterious mutations) to determine the main regulators of the network. The results suggest that SMAD2/3 and p38MAPK are key players in processing the network input. In addition, when the LoF of SMAD2/3 is combined with the LoF of p38MAPK and p53, cell cycle arrest is completely abrogated. In conclusion, the model allows to visualize, through in silico mutations, the dual role of TGF-ß: for the wild-type case TGF-ß is able to block proliferation, however deleterious mutations can impair cell cycle arrest promoting cellular proliferation.

Modeling the role of microRNA-449a in the regulation of the G2/M cell cycle checkpoint in prostate LNCaP cells under ionizing radiation.

Recent studies showed that induced microRNA-449a (miR-449a) enhances a G2/M cell cycle checkpoint arrest in prostate cancer (LNCaP) and lung adenocarcinoma cell lines. In the case of LNCaP cells, upregulated miR-449a directly downregulates c-Myc that is required to induce the cell cycle regulators Cdc25A and Cdc2/CyclinB whose inactivation blocks G2 to M phase transition. However, the molecular mechanisms involved are yet unclear, although in other prostate cancer cells the interactions among p53, miR-449a and Sirt-1 can affect the induction of the G2/M arrest. In order to clarify these molecular mechanisms, in this work we propose a boolean model of the G2/M checkpoint arrest regulation contemplating the influence of miR-449a. The model shows that the cell fate determination between two cellular phenotypes: G2/M-Arrest for DNA repair and G2/M-induced apoptosis is stochastic and influenced by miR-449a state of activation. The results were compared with experimental data available presenting agreement. We also found that several feedback loops are involved in this cell fate regulation and we indicate, through in silico gain or loss of function perturbations of genes, which of these feedback loops are more efficient to favor a specific phenotype.

MicroRNA-16 feedback loop with p53 and Wip1 can regulate cell fate determination between apoptosis and senescence in DNA damage response.

Cell fate regulation is an open problem whose comprehension impacts several areas of the biosciences. DNA damage induces cell cycle checkpoints that activate the p53 pathway to regulate cell fate mechanisms such as apoptosis or senescence. Experiments with different cell types show that the p53 pathway regulates cell fate through a switch behavior in its dynamics. For low DNA damage the pathway presents an oscillatory pattern associated with intense DNA damage repair while for high damage there are no oscillations and either p53 concentration increases inducing apoptosis or the cell enters a senescence state. Apoptosis and senescence phenotypes seem to have compensatory functions in tissues and the microRNA 16-1 (miR-16) is involved in the regulation of the fate between both phenotypes in cancer cells. To investigate the regulation of cell fate we developed a logical model of the G1/S checkpoint in DNA damage response that takes into account different levels of damage and contemplates the influence of miR-16 through its positive feedback loop formed with p53 and Wip1. The model reproduces the observed cellular phenotypes in experiments: oscillatory (for low DNA damage) regulated by negative feedback loops involving mainly p53 and Mdm2 and apoptotic or senescent (for high DNA damage) regulated by the positive p53/Wip1/miR-16 feedback loop. We find good agreement between the level of DNA damage and the probability of the phenotype produced according to experiments. We also find that this positive feedback makes senescent and apoptotic phenotypes to be determined stochastically (bistable), however controlling the expression level of miR-16 allows the control of fate determination as observed experimentally.