Tumor promoting effect of PDLIM2 downregulation involves mitochondrial ROS, oncometabolite accumulations and HIF-1α activation.

BACKGROUND: Cancer is characterized by dysregulated cellular metabolism. Thus, understanding the mechanisms underlying these metabolic alterations is important for developing targeted therapies. In this study, we investigated the pro-tumoral effect of PDZ and LIM domain 2 (PDLIM2) downregulation in lung cancer growth and its association with the accumulation of mitochondrial ROS, oncometabolites and the activation of hypoxia-inducible factor-1 (HIF-1) α in the process. METHODS: Databases and human cancer tissue samples were analyzed to investigate the roles of PDLIM2 and HIF-1α in cancer growth. DNA microarray and gene ontology enrichment analyses were performed to determine the cellular functions of PDLIM2. Seahorse assay, flow cytometric analysis, and confocal microscopic analysis were employed to study mitochondrial functions. Oncometabolites were analyzed using liquid chromatography-mass spectrometry (LC-MS). A Lewis lung carcinoma (LLC) mouse model was established to assess the in vivo function of PDLIM2 and HIF-1α. RESULTS: The expression of PDLIM2 was downregulated in lung cancer, and this downregulation correlated with poor prognosis in patients. PDLIM2 highly regulated genes associated with mitochondrial functions. Mechanistically, PDLIM2 downregulation resulted in NF-κB activation, impaired expression of tricarboxylic acid (TCA) cycle genes particularly the succinate dehydrogenase (SDH) genes, and mitochondrial dysfunction. This disturbance contributed to the accumulation of succinate and other oncometabolites, as well as the buildup of mitochondrial reactive oxygen species (mtROS), leading to the activation of hypoxia-inducible factor 1α (HIF-1α). Furthermore, the expression of HIF-1α was increased in all stages of lung cancer. The expression of PDLIM2 and HIF-1α was reversely correlated in lung cancer patients. In the animal study, the orally administered HIF-1α inhibitor, PX-478, significantly reduces PDLIM2 knockdown-promoted tumor growth. CONCLUSION: These findings shed light on the complex action of PDLIM2 on mitochondria and HIF-1α activities in lung cancer, emphasizing the role of HIF-1α in the tumor-promoting effect of PDLIM2 downregulation. Additionally, they provide new insights into a strategy for precise targeted treatment by suggesting that HIF-1α inhibitors may serve as therapy for lung cancer patients with PDLIM2 downregulation.

The mystery of phospho-Drp1 with four adaptors in cell cycle: when mitochondrial fission couples to cell fate decisions.

Recent study had deepened our knowledge of the mitochondrial dynamics to classify mitochondrial fission into two types. To further clarify the relationship between the two distinct fission machinery and the four major adaptors of Drp1, we propose a model of mechanism elucidating the multiple functions of phospho-Drp1 with its adaptors during cell cycle and providing in-depth insights into the molecular basis and evolutionary implications in depth. The model highlights not only the clustering characteristics of different phospho-Drp1 with respective subsets of mitochondrial pro-fission adaptors but also the correlation, crosstalk and shifting between different clustering of phosphorylated Drp1-adaptors during different key fission situations. Particularly, phospho-Drp1 (Ser616) couples with Mff/MiD51 to exert mitochondrial division and phospho-Drp1 (Ser637) couples with MiD49/Fis1 to execute mitophagy in M-phase. We then apply the model to address the relationship of mitochondrial dynamics to Parkinson's disease (PD) and carcinogenesis. Our proposed model is indeed compatible with current research results and pathological observations, providing promising directions for future treatment design.

Repurposing thioridazine for inducing immunogenic cell death in colorectal cancer via eIF2α/ATF4/CHOP and secretory autophagy pathways.

BACKGROUND: Colorectal cancer (CRC) is a highly prevalent cancer type with limited targeted therapies available and 5-year survival rate, particularly for late-stage patients. There have been numerous attempts to repurpose drugs to tackle this problem. It has been reported that autophagy inducers could augment the effect of certain chemotherapeutic agents by enhancing immunogenic cell death (ICD). METHODS: In this study, we employed bioinformatics tools to identify thioridazine (THD), an antipsychotic drug, and found that it could induce autophagy and ICD in CRC. Then in vitro and in vivo experiments were performed to further elucidate the molecular mechanism of THD in CRC. RESULTS: THD was found to induce endoplasmic reticulum (ER) stress in CRC cells by activating the eIF2α/ATF4/CHOP axis and facilitating the accumulation of secretory autophagosomes, leading to ICD. In addition, THD showed a remarkable ICD-activating effect when combined with oxaliplatin (OXA) to prevent tumor progression in the mouse model. CONCLUSIONS: Together, our findings suggest that the repurposed function of THD in inhibiting CRC involves the upregulation of autophagosomes and ER stress signals, promoting the release of ICD markers, and providing a potential candidate to enhance the clinical outcome for CRC treatment. Video Abstract.

Induction of Immune Responses and Phosphatidylserine Exposure by TLR9 Activation Results in a Cooperative Antitumor Effect with a Phosphatidylserine-targeting Prodrug.

Head and neck cancer is a major cancer type, with high motility rates that reduce the quality of life of patients. Herein, we investigated the effectiveness and mechanism of a combination therapy involving TLR9 activator (CpG-2722) and phosphatidylserine (PS)-targeting prodrug of SN38 (BPRDP056) in a syngeneic orthotopic head and neck cancer animal model. The results showed a cooperative antitumor effect of CpG-2722 and BPRDP056 owing to their distinct and complementary antitumor functions. CpG-2722 induced antitumor immune responses, including dendritic cell maturation, cytokine production, and immune cell accumulation in tumors, whereas BPRDP056 directly exerted cytotoxicity toward cancer cells. We also discovered a novel function and mechanism of TLR9 activation, which increased PS exposure on cancer cells, thereby attracting more BPRDP056 to the tumor site for cancer cell killing. Killed cells expose more PS in tumor for BPRDP056 targeting. Tumor antigens released from the dead cells were taken up by antigen-presenting cells, which enhanced the CpG-272-promoted T cell-mediated tumor-killing effect. These form a positive feed-forward antitumor effect between the actions of CpG-2722 and BPRDP056. Thus, the study findings suggest a novel strategy of utilizing the PS-inducing function of TLR9 agonists to develop combinational cancer treatments using PS-targeting drugs.

GSKIP modulates cell aggregation through EMT/MET signaling rather than differentiation in SH-SY5Y human neuroblastoma cells.

GSK3ß interacting protein (GSKIP) is a small A-kinase anchor protein previously reported to mediate the N-cadherin/ß-catenin pool for differentiation in SH-SY5Y cells through overexpression of GSKIP to present the neuron outgrowth phenotype. To further investigate how GSKIP functions in neurons, CRISPR/Cas9 technology was utilized to knock out GSKIP (GSKIP-KO) in SH-SY5Y. Several GSKIP-KO clones resulted in an aggregation phenotype and reduced cell growth without retinoic acid (RA) treatment. However, neuron outgrowth was still observed in GSKIP-KO clones treated with RA. The GSKIP-KO clones exhibited an aggregation phenotype through suppression of GSK3ß/ß-catenin pathways and cell cycle progression rather than cell differentiation. Gene set enrichment analysis indicated that GSKIP-KO was related to epithelial mesenchymal transition/mesenchymal epithelial transition (EMT/MET) and Wnt/ß-catenin/cadherin signaling pathways, suppressing cell migration and tumorigenesis through the inhibition of Wnt/ß-catenin mediated EMT/MET. Conversely, reintroduction of GSKIP into GSKIP-KO clones restored cell migration and tumorigenesis. Notably, phosphor-ß-catenin (S675) and ß-catenin (S552) but not phosphor-ß-catenin (S33/S37/T41) translocated into the nucleus for further gene activation. Collectively, these results suggested that GSKIP may function as an oncogene to form an aggregation phenotype for cell survival in harsh environments through EMT/MET rather than differentiation in the GSKIP-KO of SH-SY5Y cells. GSKIP Implication in Signaling Pathways with Potential Impact on SHSY-5Y Cell Aggregation.

Identifying essential genes in genome-scale metabolic models of consensus molecular subtypes of colorectal cancer.

Identifying essential targets in the genome-scale metabolic networks of cancer cells is a time-consuming process. The present study proposed a fuzzy hierarchical optimization framework for identifying essential genes, metabolites and reactions. On the basis of four objectives, the present study developed a framework for identifying essential targets that lead to cancer cell death and evaluating metabolic flux perturbations in normal cells that have been caused by cancer treatment. Through fuzzy set theory, a multiobjective optimization problem was converted into a trilevel maximizing decision-making (MDM) problem. We applied nested hybrid differential evolution to solve the trilevel MDM problem to identify essential targets in genome-scale metabolic models for five consensus molecular subtypes (CMSs) of colorectal cancer. We used various media to identify essential targets for each CMS and discovered that most targets affected all five CMSs and that some genes were CMS-specific. We obtained experimental data on the lethality of cancer cell lines from the DepMap database to validate the identified essential genes. The results reveal that most of the identified essential genes were compatible with the colorectal cancer cell lines obtained from DepMap and that these genes, with the exception of EBP, LSS, and SLC7A6, could generate a high level of cell death when knocked out. The identified essential genes were mostly involved in cholesterol biosynthesis, nucleotide metabolisms, and the glycerophospholipid biosynthetic pathway. The genes involved in the cholesterol biosynthetic pathway were also revealed to be determinable, if a cholesterol uptake reaction was not induced when the cells were in the culture medium. However, the genes involved in the cholesterol biosynthetic pathway became non-essential if such a reaction was induced. Furthermore, the essential gene CRLS1 was revealed as a medium-independent target for all CMSs.

BMX, a specific HDAC8 inhibitor, with TMZ for advanced CRC therapy: a novel synergic effect to elicit p53-, ß-catenin- and MGMT-dependent apoptotic cell death.

BACKGROUND: Despite advances in treatment, patients with refractory colorectal cancer (CRC) still have poor long-term survival, so there is a need for more effective therapeutic options. METHODS: To evaluate the HDAC8 inhibition efficacy as a CRC treatment, we examined the effects of various HDAC8 inhibitors (HDAC8i), including BMX (NBM-T-L-BMX-OS01) in combination with temozolomide (TMZ) or other standard CRC drugs on p53 mutated HT29 cells, as well as wild-type p53 HCT116 and RKO cells. RESULTS: We showed that HDAC8i with TMZ cotreatment resulted in HT29 arrest in the S and G2/M phase, whereas HCT116 and RKO arrest in the G0/G1 phase was accompanied by high sub-G1. Subsequently, this combination approach upregulated p53-mediated MGMT inhibition, leading to apoptosis. Furthermore, we observed the cotreatment also enabled triggering of cell senescence and decreased expression of stem cell biomarkers. Mechanistically, we found down-expression levels of ß-catenin, cyclin D1 and c-Myc via GSK3ß/ß-catenin signaling. Intriguingly, autophagy also contributes to cell death under the opposite status of ß-catenin/p62 axis, suggesting that there exists a negative feedback regulation between Wnt/ß-catenin and autophagy. Consistently, the Gene Set Enrichment Analysis (GSEA) indicated both apoptotic and autophagy biomarkers in HT29 and RKO were upregulated after treating with BMX. CONCLUSIONS: BMX may act as a HDAC8 eraser and in combination with reframed-TMZ generates a remarkable synergic effect, providing a novel therapeutic target for various CRCs. Video Abstract.

Severe cellular stress drives apoptosis through a dual control mechanism independently of p53.

For past two decades, p53 has been claimed as the primary sensor initiating apoptosis. Under severe cellular stress, p53 transcriptional activity activates BH3-only proteins such as Bim, Puma, or Noxa to nullify the inhibitory effects of anti-apoptotic proteins on pro-apoptotic proteins for mitochondrial outer membrane permeabilization. Cellular stress determines the expression level of p53, and the amount of p53 corresponds to the magnitude of apoptosis. However, our studies indicated that Bim and Puma are not the target genes of p53 in three cancer models, prostate cancer, glioblastoma, and osteosarcoma. Bim counteracted with Bcl-xl to activate apoptosis independently of p53 in response to doxorubicin-induced severe DNA damage in prostate cancer. Moreover, the transcriptional activity of p53 was more related to cell cycle arrest other than apoptosis for responding to DNA damage stress generated by doxorubicin in prostate cancer and glioblastoma. A proteasome inhibitor that causes protein turnover dysfunction, bortezomib, produced apoptosis in a p53-independent manner in glioblastoma and osteosarcoma. p53 in terms of both protein level and nuclear localization in combining doxorubicin with bortezomib treatment was obviously lower than when using DOX alone, inversely correlated with the magnitude of apoptosis in glioblastoma. Using a BH3-mimetic, ABT-263, to treat doxorubicin-sensitive p53-wild type and doxorubicin-resistant p53-null osteosarcoma cells demonstrated only limited apoptotic response. The combination of doxorubicin or bortezomib with ABT-263 generated a synergistic outcome of apoptosis in both p53-wild type and p53-null osteosarcoma cells. Together, this suggested that p53 might have no role in doxorubicin-induced apoptosis in prostate cancer, glioblastoma and osteosarcoma. The effects of ABT-263 in single and combination treatment of osteosarcoma or prostate cancer indicated a dual control to regulate apoptosis in response to severe cellular stress. Whether our findings only apply in these three types of cancers or extend to other cancer types remains to be explored.

Many faces and functions of GSKIP: a temporospatial regulation view.

Glycogen synthase kinase 3 (GSK3)-ß (GSK3ß) interaction protein (GSKIP) is one of the smallest A-kinase anchoring proteins that possesses a binding site for GSK3ß. Recently, our group identified the protein kinase A (PKA)-GSKIP-GSK3ß-X axis; knowledge of this axis may help us decipher the many roles of GSKIP and perhaps help explain the evolutionary reason behind the interaction between GSK3ß and PKA. In this review, we highlight the critical and multifaceted role of GSKIP in facilitating PKA kinase activity and its function as a scaffolding protein in signaling pathways. We also highlight how these pivotal PKA and GSK3 kinases can control context-specific functions and interact with multiple target proteins, such as ß-catenin, Drp1, Tau, and other proteins. GSKIP is a key regulator of multiple mechanisms because of not only its location at certain subcellular compartments but also its serial changes during the developmental process. Moreover, the involvement of critical upstream regulatory signaling pathways in GSKIP signaling in various cancers, such as miRNA (microRNA) and lncRNA (long noncoding RNA), may help in the identification of therapeutic targets in the era of precision medicine and personalized therapy. Finally, we emphasize on the model of the early stage of pathogenesis of Alzheimer Disease (AD). Although the model requires validation, it can serve as a basis for diagnostic biomarkers development and drug discovery for early-stage AD.

Compound cellular stress maximizes apoptosis independently of p53 in glioblastoma.

We examined the apoptotic response of two glioblastoma cells, p53 wild type U87 and p53 mutated T98G, to doxorubicin, bortezomib, and vorinostat, which respectively target DNA, 26S proteasome and histone deacetylase, to clarify p53's function in apoptosis. We demonstrated that doxorubicin induced apoptosis in U87 cells but not in T98G cells. The level of p53 was definitively correlated to the extent of DNA damage and apoptosis initiation. Dominant-negative p53 reduced p21 expression, but did not affect doxorubicin-induced apoptosis, so the transcriptional activity of p53 seemed not to participate in doxorubicin-induced apoptosis. However, p53 concentrated into the nucleus during heavy apoptosis. Bortezomib could induce apoptosis in U87 with high sensitivity and T98G cells with low sensitivity. In contrast, vorinostat promoted apoptosis in both U87 and T98G cells and reduced the basal level of p53 in U87 cells, indicating that p53 played no role in the vorinostat-induced apoptosis. To clearly define the role of p53 in bortezomib- and doxorubicin-induced apoptosis, we combined doxorubicin with bortezomib to treat U87 cells to assess this combination's effect on apoptosis and p53 status. Interestingly, the combination of doxorubicin with bortezomib engendered compound stress, resulting in a synergistic outcome for apoptosis in U87 cells. However, the amounts of p53 in the total count and in the nucleus were much lower with the combination than with doxorubicin alone, suggesting that p53 played no role in either the compound stress, doxorubicin-only or bortezomib-induced apoptosis.

Sharpening up tumor microenvironment to enhance the efficacy of immune checkpoint blockade on head and neck cancer using a CpG-oligodeoxynucleotide.

Head and neck cancers are a type of life-threatening cancers characterized by an immunosuppressive tumor microenvironment. Only less than 20% of the patients respond to immune checkpoint blockade therapy, indicating the need for a strategy to increase the efficacy of immunotherapy for this type of cancers. Previously, we identified a type B CpG-oligodeoxynucleotide (CpG-ODN) called CpG-2722, which has the universal activity of eliciting an immune response in grouper, mouse, and human cells. In this study, we further characterized and compared its cytokine-inducing profiles with different types of CpG-ODNs. The antitumor effect of CpG-2722 was further investigated alone and in combination with an immune checkpoint inhibitor in a newly developed syngeneic orthotopic head and neck cancer animal model. Along with other inflammatory cytokines, CpG-2722 induces the gene expressions of interleukin-12 and different types of interferons, which are critical for the antitumor response. Both CpG-2722 and anti-programmed death (PD)-1 alone suppressed tumor growth. Their tumor suppression efficacies were further enhanced when CpG-2722 and anti-PD-1 were used in combination. Mechanistically, CpG-2722 shaped a tumor microenvironment that is favorable for the action of anti-PD-1, which included promoting the expression of different cytokines such as IL-12, IFN-ß, and IFN-γ, and increasing the presence of plasmacytoid dendritic cells, M1 macrophages, and CD8 positive T cells. Overall, CpG-2722 provided a priming effect for CD8 positive T cells by sharpening the tumor microenvironment, whereas anti-PD-1 released the brake for their tumor-killing effect, resulting in an enhanced efficacy of the combined CpG-2722 and anti-PD-1.

Computer-Aided Design for Identifying Anticancer Targets in Genome-Scale Metabolic Models of Colon Cancer.

The efficient discovery of anticancer targets with minimal side effects is a major challenge in drug discovery and development. Early prediction of side effects is key for reducing development costs, increasing drug efficacy, and increasing drug safety. This study developed a fuzzy optimization framework for Identifying AntiCancer Targets (IACT) using constraint-based models. Four objectives were established to evaluate the mortality of treated cancer cells and to minimize side effects causing toxicity-induced tumorigenesis on normal cells and smaller metabolic perturbations. Fuzzy set theory was applied to evaluate potential side effects and investigate the magnitude of metabolic deviations in perturbed cells compared with their normal counterparts. The framework was applied to identify not only gene regulator targets but also metabolite- and reaction-centric targets. A nested hybrid differential evolution algorithm with a hierarchical fitness function was applied to solve multilevel IACT problems. The results show that the combination of a carbon metabolism target and any one-target gene that participates in the sphingolipid, glycerophospholipid, nucleotide, cholesterol biosynthesis, or pentose phosphate pathways is more effective for treatment than one-target inhibition is. A clinical antimetabolite drug 5-fluorouracil (5-FU) has been used to inhibit synthesis of deoxythymidine-5'-triphosphate for treatment of colorectal cancer. The computational results reveal that a two-target combination of 5-FU and a folate supplement can improve cell viability, reduce metabolic deviation, and reduce side effects of normal cells.

NBM-BMX, an HDAC8 Inhibitor, Overcomes Temozolomide Resistance in Glioblastoma Multiforme by Downregulating the ß-Catenin/c-Myc/SOX2 Pathway and Upregulating p53-Mediated MGMT Inhibition.

Although histone deacetylase 8 (HDAC8) plays a role in glioblastoma multiforme (GBM), whether its inhibition facilitates the treatment of temozolomide (TMZ)-resistant GBM (GBM-R) remains unclear. By assessing the gene expression profiles from short hairpin RNA of HDAC8 in the new version of Connectivity Map (CLUE) and cells treated by NBM-BMX (BMX)-, an HDAC8 inhibitor, data analysis reveals that the Wnt signaling pathway and apoptosis might be the underlying mechanisms in BMX-elicited treatment. This study evaluated the efficacy of cotreatment with BMX and TMZ in GBM-R cells. We observed that cotreatment with BMX and TMZ could overcome resistance in GBM-R cells and inhibit cell viability, markedly inhibit cell proliferation, and then induce cell cycle arrest and apoptosis. In addition, the expression level of ß-catenin was reversed by proteasome inhibitor via the ß-catenin/ GSK3ß signaling pathway to reduce the expression level of c-Myc and cyclin D1 in GBM-R cells. BMX and TMZ cotreatment also upregulated WT-p53 mediated MGMT inhibition, thereby triggering the activation of caspase-3 and eventually leading to apoptosis in GBM-R cells. Moreover, BMX and TMZ attenuated the expression of CD133, CD44, and SOX2 in GBM-R cells. In conclusion, BMX overcomes TMZ resistance by enhancing TMZ-mediated cytotoxic effect by downregulating the ß-catenin/c-Myc/SOX2 signaling pathway and upregulating WT-p53 mediated MGMT inhibition. These findings indicate a promising drug combination for precision personal treating of TMZ-resistant WT-p53 GBM cells.

Ionizing Radiation Induces Resistant Glioblastoma Stem-Like Cells by Promoting Autophagy via the Wnt/ß-Catenin Pathway.

Therapeutic resistance in recurrent glioblastoma multiforme (GBM) after concurrent chemoradiotherapy (CCRT) is a challenging issue. Although standard fractionated radiation is essential to treat GBM, it has led to local recurrence along with therapy-resistant cells in the ionizing radiation (IR) field. Lines of evidence showed cancer stem cells (CSCs) play a vital role in therapy resistance in many cancer types, including GBM. However, the molecular mechanism is poorly understood. Here, we proposed that autophagy could be involved in GSC induction for radioresistance. In a clinical setting, patients who received radiation/chemotherapy had higher LC3II expression and showed poor overall survival compared with those with low LC3 II. In a cell model, U87MG and GBM8401 expressed high level of stemness markers CD133, CD44, Nestin, and autophagy marker P62/LC3II after receiving standard fractionated IR. Furthermore, Wnt/ß-catenin proved to be a potential pathway and related to P62 by using proteasome inhibitor (MG132). Moreover, pharmacological inhibition of autophagy with BAF and CQ inhibit GSC cell growth by impairing autophagy flux as demonstrated by decrease Nestin, CD133, and SOX-2 levels. In conclusion, we demonstrated that fractionated IR could induce GSCs with the stemness phenotype by P62-mediated autophagy through the Wnt/ß-catenin for radioresistance. This study offers a new therapeutic strategy for targeting GBM in the future.

The Phosphorylation Status of Drp1-Ser637 by PKA in Mitochondrial Fission Modulates Mitophagy via PINK1/Parkin to Exert Multipolar Spindles Assembly during Mitosis.

Mitochondrial fission and fusion cycles are integrated with cell cycle progression. Here we first re-visited how mitochondrial ETC inhibition disturbed mitosis progression, resulting in multipolar spindles formation in HeLa cells. Inhibitors of ETC complex I (rotenone, ROT) and complex III (antimycin A, AA) decreased the phosphorylation of Plk1 T210 and Aurora A T288 in the mitotic phase (M-phase), especially ROT, affecting the dynamic phosphorylation status of fission protein dynamin-related protein 1 (Drp1) and the Ser637/Ser616 ratio. We then tested whether specific Drp1 inhibitors, Mdivi-1 or Dynasore, affected the dynamic phosphorylation status of Drp1. Similar to the effects of ROT and AA, our results showed that Mdivi-1 but not Dynasore influenced the dynamic phosphorylation status of Ser637 and Ser616 in Drp1, which converged with mitotic kinases (Cdk1, Plk1, Aurora A) and centrosome-associated proteins to significantly accelerate mitotic defects. Moreover, our data also indicated that evoking mito-Drp1-Ser637 by protein kinase A (PKA) rather than Drp1-Ser616 by Cdk1/Cyclin B resulted in mitochondrial fission via the PINK1/Parkin pathway to promote more efficient mitophagy and simultaneously caused multipolar spindles. Collectively, this study is the first to uncover that mito-Drp1-Ser637 by PKA, but not Drp1-Ser616, drives mitophagy to exert multipolar spindles formation during M-phase.

Curcumin functions as a MEK inhibitor to induce a synthetic lethal effect on KRAS mutant colorectal cancer cells receiving targeted drug regorafenib.

Curcumin, a major yellow pigment and spice in turmeric and curry, has been demonstrated to have an anticancer effect in human clinical trials. Mutation of KRAS has been shown in 35%-45% of colorectal cancer, and regorafenib has been approved by the US FDA to treat patients with colorectal cancer. Synthetic lethality is a type of genetic interaction between two genes such that simultaneous perturbations of the two genes result in cell death or a dramatic decrease of cell viability, while a perturbation of either gene alone is not lethal. Here, we reveal that curcumin significantly enhanced the growth inhibition of regorafenib in human colorectal cancer HCT 116 cells (KRAS mutant) to a greater extent than in human colorectal cancer HT-29 cells (KRAS wild-type), producing an additive or synergistic effect in HCT 116 cells and causing an antagonistic effect in HT-29 cells. Flow cytometric analysis showed that the addition of curcumin elevated apoptosis and greatly increased autophagy in HCT 116 cells but not in HT-29 cells. Mechanistically, curcumin behaved like MEK-specific inhibitor (U0126) to enhance regorafenib-induced growth inhibition, apoptosis and autophagy in HCT 116 cells. Our data suggest that curcumin may target one more gene other than mutant KRAS to enhance regorafenib-induced growth inhibition (synthetic lethality) in colorectal cancer HCT 116 cells, indicating a possible role of curcumin in regorafenib-treated KRAS mutant colorectal cancer.

Thioridazine Enhances P62-Mediated Autophagy and Apoptosis Through Wnt/ß-Catenin Signaling Pathway in Glioma Cells.

Thioridazine (THD) is a common phenothiazine antipsychotic drug reported to suppress growth in several types of cancer cells. We previously showed that THD acts as an antiglioblastoma and anticancer stem-like cell agent. However, the signaling pathway underlying autophagy and apoptosis induction remains unclear. THD treatment significantly induced autophagy with upregulated AMPK activity and engendered cell death with increased sub-G1 in glioblastoma multiform (GBM) cell lines. Notably, through whole gene expression screening with THD treatment, frizzled (Fzd) proteins, a family of G-protein-coupled receptors, were found, suggesting the participation of Wnt/ß-catenin signaling. After THD treatment, Fzd-1 and GSK3ß-S9 phosphorylation (inactivated form) was reduced to promote ß-catenin degradation, which attenuated P62 inhibition. The autophagy marker LC3-II markedly increased when P62 was released from ß-catenin inhibition. Additionally, the P62-dependent caspase-8 activation that induced P53-independent apoptosis was confirmed by inhibiting T-cell factor/ß-catenin and autophagy flux. Moreover, treatment with THD combined with temozolomide (TMZ) engendered increased LC3-II expression and caspase-3 activity, indicating promising drug synergism. In conclusion, THD induces autophagy in GBM cells by not only upregulating AMPK activity, but also enhancing P62-mediated autophagy and apoptosis through Wnt/ß-catenin signaling. Therefore, THD is a potential alternative therapeutic agent for drug repositioning in GBM.

GSK3ß­mediated Ser156 phosphorylation modulates a BH3­like domain in BCL2L12 during TMZ­induced apoptosis and autophagy in glioma cells.

BH3 domains, classified initially as BCL2 homology domains, participate in both apoptosis and autophagy. Beclin­1 contains a BH3 domain, which is required for binding to antiapoptotic BCL2 homologs and BCL2­mediated inhibition of autophagy. BCL2­like 12 (BCL2L12) also harbors a BH3­like domain, which is 12 residues long and contains a LXXXAE/D motif. In a yeast two­hybrid system performed in the present study, BCL2L12 shared similar binding partnerships to antiapoptotic BCL2 homologs, such as Beclin­1. In addition, this BH3­like domain was involved in anti­apoptosis and drug­induced autophagy in glioma cell lines. Mutations in S156 and hydrophobic L213 to alanine counteracted the antiapoptotic properties of BCL2L12 and downregulated the activation of microtubule associated protein 1 light chain 3B (LC3B), autophagy­related (ATG)12­ATG5 conjugates and Beclin­1, compared with a BCL2L12 wild­type group. Molecular dynamics simulations revealed that phosphorylation at Ser156 of BCL2L12 (within α­6 and α­7 helices) influenced the BH3­like domain conformation (α­9 helix), indicating that glycogen synthase kinase (GSK) 3ß­mediated Ser156 phosphorylation modulated a BH3­like domain in BCL2L12. Altogether, the present findings indicated that BCL2L12 may participate in anti­apoptosis and autophagy via a BH3­like domain and GSK3ß­mediated phosphorylation at Ser156. Furthermore, blockade of temozolomide (TMZ)­induced autophagy by 3­methyladenine (3­MA) resulted in enhanced activation of apoptotic markers, as well as tumor suppresor protein p53 (p53) expression in U87MG cells. The present results suggested that p53 and O6­methylguanine DNA methyltransferase activation, and BCL2, BCL­extra large, Beclin­1 and BCL2L12 expression may be used as a detection panel to determine which patients can benefit from TMZ and ABT­737 combination treatment.

The origin of GSKIP, a multifaceted regulatory factor in the mammalian Wnt pathway.

GSK3ß interacting protein (GSKIP) is a naturally occurring negative regulator of GSK3ß and retains both the Protein Kinase A Regulatory subunit binding (PKA-RII) domain and GSK3ß interacting domain. Of these two domains, we found that PKA-RII is required for forming a working complex comprising PKA/GSKIP/GSK3ß/Drp1 to influence phosphorylation of Drp1 Ser637. In this study, bioinformatics and experimental explorations re-analyzing GSKIP's biofunctions suggest that the evolutionarily conserved Domain of Unknown Function (DUF727) is an ancestral prototype of GSKIP in prokaryotes, and acquired the C-terminal GSK3ß binding site (tail) in invertebrates except for Saccharomyces spp., after which the N-terminal PKA-RII binding region (head) evolved in vertebrates. These two regions mutually influence each other and modulate GSKIP binding to GSK3ß in yeast two-hybrid assays and co-immunoprecipitation. Molecular modeling showed that mammalian GSKIP could form a dimer through the L130 residue (GSK3ß binding site) rather than V41/L45 residues. In contrast, V41/L45P mutant facilitated a gain-of-function effect on GSKIP dimerization, further influencing binding behavior to GSK3ß compared to GSKIP wild-type (wt). The V41/L45 residues are not only responsible for PKA RII binding that controls GSK3ß activity, but also affect dimerization of GSKIP monomer, with net results of gain-of-function in GSKIP-GSK3ß interaction. In addition to its reported role in modulating Drp1, Ser637 phosphorylation caused mitochondrial elongation; we postulated that GSKIP might be involved in the Wnt signaling pathway as a scavenger to recruit GSK3ß away from the ß-catenin destruction complex and as a competitor to compete for GSK3ß binding, resulting in accumulation of S675 phosphorylated ß-catenin.

Downregulation of nuclear and cytoplasmic Chibby is associated with advanced cervical cancer.

Chibby has been identified as a putative tumor suppressor and antagonist to ß-catenin, thereby controlling the Wnt signaling pathway. Chibby is typically downregulated in numerous types of cancer and may be associated with tumorigenesis. The present study aimed at clarifying the following: i) Whether Chibby antagonizes ß-catenin in cervical cancer; ii) whether Chibby and ß-catenin mRNA expression is associated with cancer progression; and iii) whether Chibby and ß-catenin expression may be used as a biomarker. A total of 87 paraffin-embedded cervical sections with distinct cervical intraepithelial neoplasia (CIN) stages (chronic cervicitis, CIN 1, CIN 2, CIN 3 and invasive squamous cell carcinoma) were collected between June 2004 and October 2012 The mRNA expression level of Chibby and ß-catenin was determined using the polymerase chain reaction. Protein expression and cellular localization of Chibby and ß-catenin were determined using immunohistochemistry. Chibby and ß-catenin were analyzed for possible association with the progression of cervical cancer. Chibby mRNA expression and the Chibby/ß-catenin ratio were identified to be downregulated in invasive tumors. Positive cytoplasmic and nuclear staining for Chibby was associated with CIN staging and decreased as the CIN stage increased. In addition, the cytoplasmic and membrane intensity of ß-catenin was associated with invasive tumors, in which a significantly increased level of protein expression was detected. Chibby may be a tumor suppressor in cervical cancer, since the dysregulation of Chibby expression is associated with tumorigenesis in cervical cancer. Chibby and ß-catenin expression together may potentially to a biomarker for disease progression in cervical cancer.