The fitness trade-off between growth and stress resistance determines the phenotypic landscape.

BACKGROUND: A central challenge in biology is to discover a principle that determines individual phenotypic differences within a species. The growth rate is particularly important for a unicellular organism, and the growth rate under a certain condition is negatively associated with that of another condition, termed fitness trade-off. Therefore, there should exist a common molecular mechanism that regulates multiple growth rates under various conditions, but most studies so far have focused on discovering those genes associated with growth rates under a specific condition. RESULTS: In this study, we found that there exists a recurrent gene expression signature whose expression levels are related to the fitness trade-off between growth preference and stress resistance across various yeast strains and multiple conditions. We further found that the genomic variation of stress-response, ribosomal, and cell cycle regulators are potential causal genes that determine the sensitivity between growth and survival. Intriguingly, we further observed that the same principle holds for human cells using anticancer drug sensitivities across multiple cancer cell lines. CONCLUSIONS: Together, we suggest that the fitness trade-off is an evolutionary trait that determines individual growth phenotype within a species. By using this trait, we can possibly overcome anticancer drug resistance in cancer cells.

A Cell-Fate Reprogramming Strategy Reverses Epithelial-to-Mesenchymal Transition of Lung Cancer Cells While Avoiding Hybrid States.

The epithelial-to-mesenchymal transition (EMT) of primary cancer contributes to the acquisition of lethal properties, including metastasis and drug resistance. Blocking or reversing EMT could be an effective strategy to improve cancer treatment. However, it is still unclear how to achieve complete EMT reversal (rEMT), as cancer cells often transition to hybrid EMT states with high metastatic potential. To tackle this problem, we employed a systems biology approach and identified a core-regulatory circuit that plays the primary role in driving rEMT without hybrid properties. Perturbation of any single node was not sufficient to completely revert EMT. Inhibition of both SMAD4 and ERK signaling along with p53 activation could induce rEMT in cancer cells even with TGFß stimulation, a primary inducer of EMT. Induction of rEMT in lung cancer cells with the triple combination approach restored chemosensitivity. This cell-fate reprogramming strategy based on attractor landscapes revealed potential therapeutic targets that can eradicate metastatic potential by subverting EMT while avoiding hybrid states. SIGNIFICANCE: Network modeling unravels the highly complex and plastic process regulating epithelial and mesenchymal states in cancer cells and discovers therapeutic interventions for reversing epithelial-to-mesenchymal transition and enhancing chemosensitivity.

Systems analysis identifies endothelin 1 axis blockade for enhancing the anti-tumor effect of multikinase inhibitor.

Multikinase inhibitors, such as sorafenib, are used for the treatment of advanced carcinomas but the response shows limited efficacy or varies a lot with patients. Here we adopted the systems approach combined with high-throughput data analysis to discover key mechanism embedded in the drug response. When analyzing the transcriptomic data from the Cancer Cell Line Encyclopedia (CCLE) database, endothelin 1 (EDN1) was enriched in cancer cells with low responsiveness to sorafenib. We found that the level of EDN1 is higher in the tissue and blood of hepatocellular carcinoma (HCC) patients showing poor response to sorafenib. In vitro experiment showed that EDN1 not only induces activation of angiogenic-promoting pathways in HCC cells but also stimulates proliferation and migration. Moreover, EDN1 is related with poor responsiveness to sorafenib by mitigating unfolded protein response (UPR), which was validated in both transcriptomic data analysis and in silico simulation. Finally, we found that endothelin receptor B (EDNRB) antagonists can enhance the efficacy of sorafenib in both HCC cells and xenograft mouse models. Our findings provide that EDN1 is a novel diagnostic marker for sorafenib responsiveness in HCC and a basis for testing macitentan, which is currently used for pulmonary artery hypertension, in combination with sorafenib in advanced HCC patients.

Network Analysis Identifies Regulators of Basal-Like Breast Cancer Reprogramming and Endocrine Therapy Vulnerability.

Basal-like breast cancer is the most aggressive breast cancer subtype with the worst prognosis. Despite its high recurrence rate, chemotherapy is the only treatment for basal-like breast cancer, which lacks expression of hormone receptors. In contrast, luminal A tumors express ERα and can undergo endocrine therapy for treatment. Previous studies have tried to develop effective treatments for basal-like patients using various therapeutics but failed due to the complex and dynamic nature of the disease. In this study, we performed a transcriptomic analysis of patients with breast cancer to construct a simplified but essential molecular regulatory network model. Network control analysis identified potential targets and elucidated the underlying mechanisms of reprogramming basal-like cancer cells into luminal A cells. Inhibition of BCL11A and HDAC1/2 effectively drove basal-like cells to transition to luminal A cells and increased ERα expression, leading to increased tamoxifen sensitivity. High expression of BCL11A and HDAC1/2 correlated with poor prognosis in patients with breast cancer. These findings identify mechanisms regulating breast cancer phenotypes and suggest the potential to reprogram basal-like breast cancer cells to enhance their targetability. SIGNIFICANCE: A network model enables investigation of mechanisms regulating the basal-to-luminal transition in breast cancer, identifying BCL11A and HDAC1/2 as optimal targets that can induce basal-like breast cancer reprogramming and endocrine therapy sensitivity.

Sestrin2 Attenuates Cellular Senescence by Inhibiting NADPH Oxidase 4 Expression.

BACKGROUND: Sestrin2 (Sesn2) is involved in the maintenance of metabolic homeostasis and aging via modulation of the 5' AMP-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) pathway. METHODS: Wild-type and Sesn2 knockout (KO) mice of the 129/SvJ background were maintained in a pathogen-free authorized facility under a 12-hour dark/light cycle at 20°C-22°C and 50%-60% humidity. Mouse embryonic fibroblasts (MEFs) were prepared from 13.5-day-old embryos derived from Sesn2-KO mice mated with each other. RESULTS: The MEFs from Sesn2-KO mice showed enlarged and flattened morphologies and senescence-associated ß-galactosidase activity, accompanied by an elevated level of reactive oxygen species. These senescence phenotypes recovered following treatment with N-acetyl-cysteine. Notably, the mRNA levels of NADPH oxidase 4 (NOX4) and transforming growth factor (TGF)-ß were markedly increased in Sesn2-KO MEFs. Treatment of Sesn2-KO MEFs with the NOX inhibitor diphenyleneiodonium and the TGF-ß inhibitor SB431542 restored cell growth inhibited by Sesn2-KO. CONCLUSION: Sesn2 attenuates cellular senescence via suppression of TGF-ß- and NOX4-induced reactive oxygen species generation and subsequent inhibition of AMPK.

A Systems Biology Approach to Identifying a Master Regulator That Can Transform the Fast Growing Cellular State to a Slowly Growing One in Early Colorectal Cancer Development Model.

Colorectal cancer (CRC) has been most extensively studied for characterizing genetic mutations along its development. However, we still have a poor understanding of CRC initiation due to limited measures of its observation and analysis. If we can unveil CRC initiation events, we might identify novel prognostic markers and therapeutic targets for early cancer detection and prevention. To tackle this problem, we establish the early CRC development model and perform transcriptome analysis of its single cell RNA-sequencing data. Interestingly, we find two subtypes, fast growing vs. slowly growing populations of distinct growth rate and gene signatures, and identify CCDC85B as a master regulator that can transform the cellular state of fast growing subtype cells into that of slowly growing subtype cells. We further validate this by in vitro experiments and suggest CCDC85B as a novel potential therapeutic target that may prevent malignant CRC development by suppressing stemness and uncontrolled cell proliferation.

Feedback analysis identifies a combination target for overcoming adaptive resistance to targeted cancer therapy.

Targeted drugs aim to treat cancer by directly inhibiting oncogene activity or oncogenic pathways, but drug resistance frequently emerges. Due to the intricate dynamics of cancer signaling networks, which contain complex feedback regulations, cancer cells can rewire these networks to adapt to and counter the cytotoxic effects of a drug, thereby limiting the efficacy of targeted therapies. To identify a combinatorial drug target that can overcome such a limitation, we developed a Boolean network simulation and analysis framework and applied this approach to a large-scale signaling network of colorectal cancer with integrated genomic information. We discovered Src as a critical combination drug target that can overcome the adaptive resistance to the targeted inhibition of mitogen-activated protein kinase pathway by blocking the essential feedback regulation responsible for resistance. The proposed framework is generic and can be widely used to identify drug targets that can overcome adaptive resistance to targeted therapies.

Network Inference Analysis Identifies SETDB1 as a Key Regulator for Reverting Colorectal Cancer Cells into Differentiated Normal-Like Cells.

Cancer cells exhibit properties of cells in a less differentiated state than the adjacent normal cells in the tissue. We explored whether cancer cells can be converted to a differentiated normal-like state by restoring the gene regulatory network (GRN) of normal cells. Here, we report that colorectal cancer cells exhibit a range of developmental states from embryonic and intestinal stem-like cells to differentiated normal-like cells. To identify the transcription factors (TF) that commit stem-like colorectal cancer cells into a differentiated normal-like state, we reconstructed GRNs of normal colon mucosa and identified core TFs (CDX2, ELF3, HNF4G, PPARG, and VDR) that govern the cellular state. We further found that SET Domain Bifurcated 1 (SETDB1), a histone H3 lysine 9-specific methyltransferase, hinders the function of the identified TFs. SETDB1 depletion effectively converts stem-like colorectal cancer cells into postmitotic cells and restores normal morphology in patient-derived colorectal cancer organoids. RNA-sequencing analyses revealed that SETDB1 depletion recapitulates global gene expression profiles of normal differentiated cells by restoring the transcriptional activity of core TFs on their target genes. IMPLICATIONS: Our study provides insights into the molecular regulatory mechanism underlying the developmental hierarchy of colorectal cancer and suggests that induction of a postmitotic state may be a therapeutic alternative to destruction of cancer cells.

Systems analysis identifies potential target genes to overcome cetuximab resistance in colorectal cancer cells.

Cetuximab (CTX), a monoclonal antibody against epidermal growth factor receptor, is being widely used for colorectal cancer (CRC) with wild-type (WT) KRAS. However, its responsiveness is still very limited and WT KRAS is not enough to indicate such responsiveness. Here, by analyzing the gene expression data of CRC patients treated with CTX monotherapy, we have identified DUSP4, ETV5, GNB5, NT5E, and PHLDA1 as potential targets to overcome CTX resistance. We found that knockdown of any of these five genes can increase CTX sensitivity in KRAS WT cells. Interestingly, we further found that GNB5 knockdown can increase CTX sensitivity even for KRAS mutant cells. We unraveled that GNB5 overexpression contributes to CTX resistance by modulating the Akt signaling pathway from experiments and mathematical simulation. Overall, these results indicate that GNB5 might be a promising target for combination therapy with CTX irrespective of KRAS mutation.

Protein disulfide isomerase inhibition synergistically enhances the efficacy of sorafenib for hepatocellular carcinoma.

Sorafenib is the only approved targeted drug for hepatocellular carcinoma (HCC), but its effect on patients' survival gain is limited and varies over a wide range depending on pathogenetic conditions. Thus, enhancing the efficacy of sorafenib and finding a reliable predictive biomarker are crucial to achieve efficient control of HCCs. In this study, we utilized a systems approach by combining transcriptome analysis of the mRNA changes in HCC cell lines in response to sorafenib with network analysis to investigate the action and resistance mechanism of sorafenib. Gene list functional enrichment analysis and gene set enrichment analysis revealed that proteotoxic stress and apoptosis modules are activated in the presence of sorafenib. Further analysis of the endoplasmic reticulum stress network model, combined with in vitro experiments, showed that introducing an additional stress by treating the orally active protein disulfide isomerase (PDI) inhibitor (PACMA 31) can synergistically increase the efficacy of sorafenib in vitro and in vivo, which was confirmed using a mouse xenograft model. We also found that HCC patients with high PDI expression show resistance to sorafenib and poor clinical outcomes, compared to the low-PDI-expression group. CONCLUSION: These results suggest that PDI is a promising therapeutic target for enhancing the efficacy of sorafenib and can also be a biomarker for predicting sorafenib responsiveness. (Hepatology 2017;66:855-868).

CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species.

Reactive oxygen species modulate cell fate in a context-dependent manner. Sublethal doses of H(2)O(2) decreased the level of proliferating cell nuclear antigen (PCNA) in normal cells (including primary human dermal fibroblasts and IMR-90 cells) without affecting cyclin-dependent kinase 2 (CDK2) activity, leading to cell cycle arrest and subsequent senescence. In contrast, exposure of cancer cells (such as HeLa and MCF7 cells) to H(2)O(2) increased CDK2 activity with no accompanying change in the PCNA level, leading to cell proliferation. A CDK2 inhibitor, CVT-313, prevented H(2)O(2)-induced cancer cell proliferation. These results support the notion that the cyclin/CDK2/p21(Cip1)/PCNA complex plays an important role as a regulator of cell fate decisions.

TRIM32 protein sensitizes cells to tumor necrosis factor (TNFα)-induced apoptosis via its RING domain-dependent E3 ligase activity against X-linked inhibitor of apoptosis (XIAP).

TRIM32, which belongs to the tripartite motif (TRIM) protein family, has the RING finger, B-box, and coiled-coil domain structures common to this protein family, along with an additional NHL domain at the C terminus. TRIM32 reportedly functions as an E3 ligase for actin, a protein inhibitor of activated STAT y (PIASy), dysbindin, and c-Myc, and it has been associated with diseases such as muscular dystrophy and epithelial carcinogenesis. Here, we identify a new substrate of TRIM32 and propose a mechanism through which TRIM32 might regulate apoptosis. Our overexpression and knockdown experiments demonstrate that TRIM32 sensitizes cells to TNFα-induced apoptosis. The RING domain is necessary for this pro-apoptotic function of TRM32 as well as being responsible for its E3 ligase activity. TRIM32 colocalizes and directly interacts with X-linked inhibitor of apoptosis (XIAP), a well known cancer therapeutic target, through its coiled-coil and NHL domains. TRIM32 overexpression enhances XIAP ubiquitination and subsequent proteasome-mediated degradation, whereas TRIM32 knockdown has the opposite effect, indicating that XIAP is a substrate of TRIM32. In vitro reconstitution assay reveals that XIAP is directly ubiquitinated by TRIM32. Our novel results collectively suggest that TRIM32 sensitizes TNFα-induced apoptosis by antagonizing XIAP, an anti-apoptotic downstream effector of TNFα signaling. This function may be associated with TRIM32-mediated tumor suppressive mechanism.

Hsp70 interacts with the retroviral restriction factor TRIM5alpha and assists the folding of TRIM5alpha.

Tripartite motif (TRIM) protein TRIM5alpha has been shown to restrict human immunodeficiency virus, type 1 infection in Old World monkey cells at the early post-entry step by poorly understood mechanisms. Currently, the physiological function of TRIM5alpha is not known. In this study, we showed that transiently overexpressed TRIM5alpha causes a morphological change in HEK293T cells. A proteomics analysis of the protein complexes that were pulled down with hemagglutinin-tagged TRIM5alpha suggested that the heat shock protein 70 (Hsp70) may serve as a TRIM5alpha-binding partner. The interaction between Hsp70 and TRIM5alpha was confirmed by co-localization and co-immunoprecipitation assays. Co-expression of Hsp70 reversed the TRIM5alpha-induced morphological change in HEK293T cells. Another heat shock protein Hsc70 also bound to TRIM5alpha, but unlike Hsp70, Hsc70 was not able to reverse the TRIM5alpha-induced morphological change, suggesting that Hsp70 specifically reverses the morphological change caused by TRIM5alpha. Studies using a series of TRIM5alpha deletion mutants demonstrate that, although the PRYSPRY domain is critical for binding to Hsp70, the entire TRIM5alpha structure is necessary to induce the morphological change of cells. When the ATPase domain of Hsp70 was mutated, the mutated Hsp70 could not counteract the morphological change induced by TRIM5alpha, indicating that the catalytic activity of Hsp70 protein is important for this function. Co-expression of Hsp70 elevated the levels of TRIM5alpha in the detergent-soluble fraction with a concomitant decrease in the detergent-insoluble fraction. Together these results suggest that Hsp70 plays critical roles in the cellular management against the TRIM5alpha-induced cellular insults.

Extracellular signal-regulated kinase 2-dependent phosphorylation induces cytoplasmic localization and degradation of p21Cip1.

p21(Cip1) is an inhibitor of cell cycle progression that promotes G(1)-phase arrest by direct binding to cyclin-dependent kinase and proliferating cell nuclear antigen. Here we demonstrate that mitogenic stimuli, such as epidermal growth factor treatment and oncogenic Ras transformation, induce p21(Cip1) downregulation at the posttranslational level. This downregulation requires the sustained activation of extracellular signal-regulated kinase 2 (ERK2), which directly interacts with and phosphorylates p21(Cip1), promoting p21(Cip1) nucleocytoplasmic translocation and ubiquitin-dependent degradation, thereby resulting in cell cycle progression. ERK1 is not likely involved in this process. Phosphopeptide analysis of in vitro ERK2-phosphorylated p21(Cip1) revealed two phosphorylation sites, Thr57 and Ser130. Double mutation of these sites abolished ERK2-mediated p21(Cip1) translocation and degradation, thereby impairing ERK2-dependent cell cycle progression at the G(1)/S transition. These results indicate that ERK2 activation transduces mitogenic signals, at least in part, by downregulating the cell cycle inhibitory protein p21(Cip1).

The tumour suppressor PTEN mediates a negative regulation of the E3 ubiquitin-protein ligase Nedd4.

The tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10; a phosphatidylinositol 3-phosphatase) is a multifunctional protein deregulated in many types of cancer. It is suggested that a number of proteins that relate with PTEN functionally or physically have not yet been found. In order to search for PTEN-interacting proteins that might be crucial in the regulation of PTEN, we exploited a proteomics-based approach. PTEN-expressing NIH 3T3 cell lysates were used in affinity chromatography and then analysed by LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem MS). A total of 93 proteins were identified. Among the proteins identified, we concentrated on the E3 ubiquitin-protein ligase Nedd4 (neural-precursor-cell-expressed, developmentally down-regulated gene 4), and performed subsequent validation experiments using HeLa cells. Nedd4 inhibited PTEN-induced apoptotic cell death and, conversely, the Nedd4 level was down-regulated by PTEN. The down-regulation effect was diminished by a mutation (C124S) in the catalytic site of PTEN. Nedd4 expression was also decreased by a PI3K (phosphoinositide 3-kinase) inhibitor, LY294002, suggesting that the regulation is dependent on the phosphatase-kinase activity of the PTEN-PI3K/Akt pathway. Semi-quantitative real-time PCR analysis revealed that Nedd4 was transcriptionally regulated by PTEN. Thus our results have important implications regarding the roles of PTEN upon the E3 ubquitin ligase Nedd4 as a negative feedback regulator as well as a substrate.

Cytoplasmic localization and ubiquitination of p21(Cip1) by reactive oxygen species.

Reactive oxygen species were previously shown to trigger p21(Cip1) protein degradation through a proteasome-dependent pathway, however the detailed mechanism of degradation remains to be elucidated. In this report, we showed that p21(Cip1) was degraded at an early phase after low dose H(2)O(2) treatment of a variety of cell types and that preincubation of cells with the antioxidant, N-acetylcysteine, prolonged p21(Cip1) half-life. A mutant p21(Cip1) in which all six lysines were changed to arginines was protected against H(2)O(2) treatment. Direct interaction between p21(Cip1) and Skp2 was elevated in the H(2)O(2)-treated cells. Disruption of the two nuclear export signal (NES) sequences in p21(Cip1), or treatment with leptomycin B blocked H(2)O(2)-induced p21(Cip1) degradation. Altogether, these results demonstrate that reactive oxygen species induce p21(Cip1) degradation through an NES-, Skp2-, and ubiquitin-dependent pathway.

Redox-regulated cochaperone activity of the human DnaJ homolog Hdj2.

The human DnaJ homolog Hdj2 is a cochaperone containing a cysteine-rich zinc finger domain. We identified a specific interaction of Hdj2 with the cellular redox enzyme thioredoxin using a yeast two-hybrid assay and a coimmunoprecipitation assay, thereby investigating how the redox environment of the cell regulates Hdj2 function. In reconstitution experiments with Hsc70, we found that treatment with H2O2 caused the oxidative inactivation of Hdj2 cochaperone activity. Hdj2 inactivation paralleled the oxidation of cysteine thiols and concomitant release of coordinated zinc, suggesting a role of cysteine residues in the zinc finger domain of Hdj2 as a redox sensor of chaperone-mediated protein-folding machinery. H2O2-induced negative regulation of Hdj2 cochaperone activity was also confirmed in mammalian cells using luciferase as a foreign reporter cotransfected with Hsc70 and Hdj2. The in vivo oxidation of cysteine residues in Hdj2 was detected only in thioredoxin-knockdown cells, implying that thioredoxin is involved in the in vivo reduction. The oxidative inactivation of Hdj2 was reversible. Wild-type thioredoxin notably recovered the oxidatively inactivated Hdj2 activity accompanied by the reincorporation of zinc, whereas the catalytically inactive mutant thioredoxin (Cys32Ser/Cys35Ser) did not. Taken together, we propose that oxidation and reduction reversibly regulate Hdj2 function in response to the redox states of the cell.

Gender-dependent hepatic alterations in H-ras12V transgenic mice.

BACKGROUND/AIMS: Although it has been proposed that Ras and related signal pathways play important roles in hepatocarcinogenesis, appropriate in vivo models are lacking. METHODS: Two hepatocellular carcinoma lines were established using pronuclear microinjection techniques to create an insertion of the H-ras12V transgene under the control of the albumin enhancer/promoter. The resulting phenotypes and related molecular events were then examined. RESULTS: Male (but not female) transgenic mice older than 2 months showed hepatic alterations with a high degree of reproducibility, as compared to the wild-type mice. The liver/body-weight ratios were lower for the females than for the males. The transgene-carrying line 28 was investigated extensively with respect to molecular differences between the genders. Male hepatocytes showed higher Ras activity and higher reactive oxygen species (ROS) levels than female hepatocytes. The female hepatocytes showed higher expression levels of p53 and p21Waf1/Cip1, enhanced cytochrome c release, which correlated with cell cycle arrest, and higher levels of hypodiploid cell formation, as compared to the male hepatocytes. CONCLUSIONS: The gender-related differences in molecular responses to activated Ras may have implications for the prevalence of hepatic alterations in males. Our transgenic mice represent a potentially valuable animal model for future investigations.

Tumor suppressor VDUP1 increases p27(kip1) stability by inhibiting JAB1.

Vitamin D3 up-regulated protein 1 (VDUP1) is a stress-response gene that is up-regulated by 1,25(OH)2D3 in many cells. It has been reported that VDUP1 expression is reduced in many tumor cells and the enforced expression of VDUP1 inhibits cell proliferation by arresting cell cycle progression. Here, we found that VDUP1-/- fibroblast cells proliferated more rapidly compared with wild-type cells with reduced expression of p27(kip1), a cyclin-dependent kinase inhibitor. JAB1 is known to interact with p27(kip1) and to decrease the stability of p27(kip1). VDUP1 interacted with JAB1 and restored JAB1-induced suppression of p27(kip1) stability. In this process, VDUP1 blocked the JAB1-mediated translocation of p27(kip1) from the nucleus to the cytoplasm. In addition, VDUP1 inhibited JAB1-mediated activator protein-1 activation and cell proliferation. Taken together, these results indicate that VDUP1 is a novel factor of p27(kip1) stability via regulating JAB1.