Construction of an Oxidative Stress Risk Model to Analyze the Correlation Between Liver Cancer and Tumor Immunity.

BACKGROUND: Hepatocellular carcinoma (HCC) remains one of the most lethal cancers globally. Despite advancements in immunotherapy, the prognosis for patients with HCC continues to be poor. As oxidative stress plays a significant role in the onset and progression of various diseases, including metabolism-related HCC, comprehending its mechanism in HCC is critical for effective diagnosis and treatment. METHODS: This study utilized the TCGA dataset and a collection of oxidative stress genes to determine the expression of oxidative stress-related genes in HCC and their association with overall survival using diverse bioinformatics methods. A novel prognostic risk model was developed, and the TCGA cohort was divided into high-risk and low-risk groups based on each tumor sample's risk score. Levels of immune cell infiltration and the expression of immune checkpoint-related genes in different risk subgroups were analyzed to investigate the potential link between tumor immunity and oxidative stress-related features. The expression of model genes in actual samples was validated through immunohistochemistry, and their mRNA and protein expression levels were measured in cell cultures. RESULTS: Four oxidative stress-related genes (EZH2, ANKZF1, G6PD, and HMOX1) were identified and utilized to create a predictive risk model for HCC patient overall survival, which was subsequently validated in an independent cohort. A significant correlation was found between the expression of these prognostic genes and the infiltration of tumor immune cells. Elevated expression of EZH2, ANKZF1, G6PD, and HMOX1 was observed in both HCC tissues and cell lines. CONCLUSION: The combined assessment of EZH2, ANKZF1, G6PD, and HMOX1 gene expression can serve as a model to evaluate the risk of oxidative stress in HCC. Furthermore, there is a notable correlation between the expression of these risk model genes and tumor immunity.

SUMO1 degrader induces ER stress and ROS accumulation through deSUMOylation of TCF4 and inhibition of its transcription of StarD7 in colon cancer.

Small molecule degraders of small ubiquitin-related modifier 1 (SUMO1) induce SUMO1 degradation in colon cancer cells and inhibits the cancer cell growth; however, it is unclear how SUMO1 degradation leads to the anticancer activity of the degraders. Genome-wide CRISPR-Cas9 knockout screen has identified StAR-related lipid transfer domain containing 7 (StarD7) as a critical gene for the degrader's anticancer activity. Here, we show that both StarD7 mRNA and protein are overexpressed in human colon cancer and its knockout significantly reduces colon cancer cell growth and xenograft progression. The treatment with the SUMO1 degrader lead compound HB007 reduces StarD7 mRNA and protein levels and increases endoplasmic reticulum (ER) stress and reactive oxygen species (ROS) production in colon cancer cells and three-dimensional (3D) organoids. The study further provides a novel mechanism of the compound anticancer activity that SUMO1 degrader-induced decrease of StarD7 occur through degradation of SUMO1, deSUMOylation and degradation of T cell-specific transcription 4 (TCF4) and thereby inhibition of its transcription of StarD7 in colon cancer cells, 3D organoids and patient-derived xenografts (PDX).

The Role of Oxidative Stress in the Development and Therapeutic Intervention of Hepatocellular Carcinoma.

Oxidative stress (OS) is a condition in which the body has an unbalanced oxidative and antioxidant effect. Oxidative stress has emerged as a critical component in the onset and progression of numerous diseases, including liver cancer and chronic liver disease caused by the hepatitis C virus and hepatitis B virus. Reactive oxygen species (ROS) are the most prevalent reactive chemical species involved in the oxidative stress response during the progression of the disease. Oxidative stress has a unique role in the development of hepatocellular carcinoma (HCC), and excessive ROS production is a common occurrence in liver illnesses of various etiologies. In response to various deleterious stimuli, the liver shows manifestations of lipid accumulation, oxidative damage, inflammatory infiltration, and immune response, which interact with each other in a mutually reinforcing manner, collectively exacerbating liver damage and malignant transformation. The intracellular buildup of ROS is a two-edged sword for tumor advancement. ROS are tumorigenic, and low amounts of ROS can trigger different signaling pathways that promote proliferation, survival, and migration, among other aspects. However, excessive oxidative stress can induce tumor cell death. Understanding the mechanisms of oxidative stress in hepatocellular carcinogenesis is beneficial for the prevention and surveillance of hepatocellular carcinoma in humans. An improved knowledge of the impacts and potential implications of oxidative stress regulation in therapeutic strategies will likely allow us to find new therapeutic targets for cancer. Oxidative stress also plays a significant role in the treatment of hepatocellular carcinoma and the mechanisms of drug resistance involved. This paper reviews recent studies on oxidative stress in HCC that are more reliable and important, and provides a more comprehensive view of the development of the treatment of HCC based on the relevant summaries of the effect of oxidative stress on the treatment.

PD-1 independent of PD-L1 ligation promotes glioblastoma growth through the NFκB pathway.

Brain tumor­initiating cells (BTICs) drive glioblastoma growth through not fully understood mechanisms. Here, we found that about 8% of cells within the human glioblastoma microenvironment coexpress programmed cell death 1 (PD-1) and BTIC marker. Gain- or loss-of-function studies revealed that tumor-intrinsic PD-1 promoted proliferation and self-renewal of BTICs. Phosphorylation of tyrosines within the cytoplasmic tail of PD-1 recruited Src homology 2­containing phosphatase 2 and activated the nuclear factor kB in BTICs. Notably, the tumor-intrinsic promoting effects of PD-1 did not require programmed cell death ligand 1(PD-L1) ligation; thus, the therapeutic antibodies inhibiting PD-1/PD-L1 interaction could not overcome the growth advantage of PD-1 in BTICs. Last, BTIC-intrinsic PD-1 accelerated intracranial tumor growth, and this occurred in mice lacking T and B cells. These findings point to a critical role for PD-1 in BTICs and uncover a nonimmune resistance mechanism of patients with glioblastoma to PD-1­ or PD-L1­blocking therapies.

Ubiquitination and degradation of SUMO1 by small-molecule degraders extends survival of mice with patient-derived tumors.

Discovery of small-molecule degraders that activate ubiquitin ligase­mediated ubiquitination and degradation of targeted oncoproteins in cancer cells has been an elusive therapeutic strategy. Here, we report a cancer cell­based drug screen of the NCI drug-like compounds library that enabled identification of small-molecule degraders of the small ubiquitin-related modifier 1 (SUMO1). Structure-activity relationship studies of analogs of the hit compound CPD1 led to identification of a lead compound HB007 with improved properties and anticancer potency in vitro and in vivo. A genome-scale CRISPR-Cas9 knockout screen identified the substrate receptor F-box protein 42 (FBXO42) of cullin 1 (CUL1) E3 ubiquitin ligase as required for HB007 activity. Using HB007 pull-down proteomics assays, we pinpointed HB007's binding protein as the cytoplasmic activation/proliferation-associated protein 1 (CAPRIN1). Biolayer interferometry and compound competitive immunoblot assays confirmed the selectivity of HB007's binding to CAPRIN1. When bound to CAPRIN1, HB007 induced the interaction of CAPRIN1 with FBXO42. FBXO42 then recruited SUMO1 to the CAPRIN1-CUL1-FBXO42 ubiquitin ligase complex, where SUMO1 was ubiquitinated in several of human cancer cells. HB007 selectively degraded SUMO1 in patient tumor­derived xenografts implanted into mice. Systemic administration of HB007 inhibited the progression of patient-derived brain, breast, colon, and lung cancers in mice and increased survival of the animals. This cancer cell­based screening approach enabled discovery of a small-molecule degrader of SUMO1 and may be useful for identifying other small-molecule degraders of oncoproteins.

LSD1 and Aberrant DNA Methylation Mediate Persistence of Enteroendocrine Progenitors That Support BRAF-Mutant Colorectal Cancer.

Despite the connection of secretory cells, including goblet and enteroendocrine (EEC) cells, to distinct mucus-containing colorectal cancer histologic subtypes, their role in colorectal cancer progression has been underexplored. Here, our analysis of The Cancer Genome Atlas (TCGA) and single-cell RNA-sequencing data demonstrates that EEC progenitor cells are enriched in BRAF-mutant colorectal cancer patient tumors, cell lines, and patient-derived organoids. In BRAF-mutant colorectal cancer, EEC progenitors were blocked from differentiating further by DNA methylation and silencing of NEUROD1, a key gene required for differentiation of intermediate EECs. Mechanistically, secretory cells and the factors they secrete, such as trefoil factor 3, promoted colony formation and activation of cell survival pathways in the entire cell population. Lysine-specific demethylase 1 (LSD1) was identified as a critical regulator of secretory cell specification in vitro and in a colon orthotopic xenograft model, where LSD1 loss blocks formation of EEC progenitors and reduces tumor growth and metastasis. These findings reveal an important role for EEC progenitors in supporting colorectal cancer. SIGNIFICANCE: This study establishes enteroendocrine progenitors as a targetable population that promotes BRAF-mutant colorectal cancer and can be blocked by LSD1 inhibition to suppress tumor growth.

Fibrinogen in the glioblastoma microenvironment contributes to the invasiveness of brain tumor-initiating cells.

Glioblastomas (GBMs) are highly aggressive, recurrent, and lethal brain tumors that are maintained via brain tumor-initiating cells (BTICs). The aggressiveness of BTICs may be dependent on the extracellular matrix (ECM) molecules that are highly enriched within the GBM microenvironment. Here, we investigated the expression of ECM molecules in GBM patients by mining the transcriptomic databases and also staining human GBM specimens. RNA levels for fibronectin, brevican, versican, heparan sulfate proteoglycan 2 (HSPG2), and several laminins were high in GBMs compared to normal brain, and this was corroborated by immunohistochemistry. While fibrinogen transcript was at normal level in GBM, its protein immunoreactivity was prominent within GBM tissues. These ECM molecules in tumor specimens were in proximity to, and surrounding BTICs. In culture, fibronectin and pan-laminin induced the adhesion of BTICs onto the plastic substratum. However, fibrinogen increased the size of the BTIC spheres by facilitating the adhesive property, motility, and invasiveness of BTICs. These features of elevated invasiveness were corroborated in resected GBM specimens by the close proximity of fibrinogen with matrix metalloproteinase (MMP)-2 and-9, which are proteases implicated in metastasis. Moreover, the effect of fibrinogen-induced invasiveness was attenuated in BTICs where MMP-2 and -9 have been inhibited with siRNAs or pharmacological inhibitors. Our results implicate fibrinogen in GBM as a mediator of the invasive properties of BTICs, and as a target for therapy to reduce BTIC tumorigenecity.

SUMO1 modification stabilizes CDK6 protein and drives the cell cycle and glioblastoma progression.

Ubiquitination governs oscillation of cyclin-dependent kinase (CDK) activity through a periodic degradation of cyclins for orderly cell cycle progression; however, the mechanism that maintains the constant CDK protein levels throughout the cell cycle remains unclear. Here we show that CDK6 is modified by small ubiquitin-like modifier-1 (SUMO1) in glioblastoma, and that CDK6 SUMOylation stabilizes the protein and drives the cell cycle for the cancer development and progression. CDK6 is also a substrate of ubiquitin; however, CDK6 SUMOylation at Lys 216 blocks its ubiquitination at Lys 147 and inhibits the ubiquitin-mediated CDK6 degradation. Throughout the cell cycle, CDK1 phosphorylates the SUMO-specific enzyme, ubiquitin-conjugating enzyme9 (UBC9) that in turn mediates CDK6 SUMOylation during mitosis; CDK6 remains SUMOylated in G1 phase and drives the cell cycle through G1/S transition. Thus, SUMO1-CDK6 conjugation constitutes a mechanism of cell cycle control and inhibition of this SUMOylation pathway may provide a strategy for treatment of glioblastoma.

PD-0332991 induces G1 arrest of colorectal carcinoma cells through inhibition of the cyclin-dependent kinase-6 and retinoblastoma protein axis.

Preclinical and clinical studies have demonstrated the anticancer activity of PD-0332991, a selective cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, in the treatment of various types of cancer in a retinoblastoma protein (RB)-dependent manner. However, it remains unclear whether CDK4, CDK6 or both are required for RB phosphorylation in colorectal carcinoma and thus PD-0332991 can be used to target this CDK-RB axis for the cancer therapy. The aim of this study was to determine whether CDK4, CDK6 and phosphorylated RB proteins were overexpressed in colorectal carcinoma tissues as compared to matched normal colorectal tissues. The results showed that knockdown of CDK6 but not CDK4 reduced RB phosphorylation and inhibited carcinoma cell growth. Thus, CDK6 plays a critical role in RB phosphorylation and cancer growth. PD-0332991 treatment blocked RB phosphorylation and inhibited cell growth through the induction of G1 arrest of colorectal carcinoma cells. The results demonstrated that, by targeting of CDK6-RB axis, PD-0332991 may prove to be a novel therapeutic agent in treating colorectal carcinoma.

Real-time magnetic resonance-guided stereotactic laser amygdalohippocampotomy for mesial temporal lobe epilepsy.

BACKGROUND: Open surgery effectively treats mesial temporal lobe epilepsy, but carries the risk of neurocognitive deficits, which may be reduced with minimally invasive alternatives. OBJECTIVE: To describe technical and clinical outcomes of stereotactic laser amygdalohippocampotomy with real-time magnetic resonance thermal imaging guidance. METHODS: With patients under general anesthesia and using standard stereotactic methods, 13 adult patients with intractable mesial temporal lobe epilepsy (with and without mesial temporal sclerosis [MTS]) prospectively underwent insertion of a saline-cooled fiberoptic laser applicator in amygdalohippocampal structures from an occipital trajectory. Computer-controlled laser ablation was performed during continuous magnetic resonance thermal imaging followed by confirmatory contrast-enhanced anatomic imaging and volumetric reconstruction. Clinical outcomes were determined from seizure diaries. RESULTS: A mean 60% volume of the amygdalohippocampal complex was ablated in 13 patients (9 with MTS) undergoing 15 procedures. Median hospitalization was 1 day. With follow-up ranging from 5 to 26 months (median, 14 months), 77% (10/13) of patients achieved meaningful seizure reduction, of whom 54% (7/13) were free of disabling seizures. Of patients with preoperative MTS, 67% (6/9) achieved seizure freedom. All recurrences were observed before 6 months. Variances in ablation volume and length did not account for individual clinical outcomes. Although no complications of laser therapy itself were observed, 1 significant complication, a visual field defect, resulted from deviated insertion of a stereotactic aligning rod, which was corrected before ablation. CONCLUSION: Real-time magnetic resonance-guided stereotactic laser amygdalohippocampotomy is a technically novel, safe, and effective alternative to open surgery. Further evaluation with larger cohorts over time is warranted.

Immunohistochemical demonstration of isocitrate dehydrogenase 1 (IDH1) mutation in a small subset of prostatic carcinomas.

Recent advances in genomic sequencing have resulted in the discovery of the somatic mutations of cytoplasmic isocitrate dehydrogenase 1 (IDH1) in human solid tumors such as gliomas. The most common IDH1 mutation affects codon 132 and results in the conversion of amino acid residue arginine (R) to histidine (H). This IDH1 mutation is associated with a genetic and clinical characteristic group of gliomas in terms of grade and prognosis. We investigated whether immunohistochemistry (IHC) using a monoclonal antibody against the IDH1 mutant protein could be used in routine surgical pathology for identification of the mutation in solid human tumors. A total of 549 solid human tumors were examined in tissue microarrays, including prostate, thyroid, renal cell, ovarian, endometrial, breast, colorectal, non-small cell lung carcinoma, melanomas, and gliomas. IHC detected the IDH1 mutation in 72% (13/18) anaplastic astrocytomas and 30% (3/10) astrocytomas; however, it failed to detect the mutation in 258 thyroid, 11 renal cell, 10 ovarian, 18 endometrial, 20 breast, 25 colorectal, 22 non-small cell lung carcinoma, 25 melanomas, and 8 thyroid follicular adenomas. In contrast, expression of the IDH1 mutation was noted in 3 of 118 (2.5%) prostate carcinomas. Western blotting and polymerase chain reaction-based sequencing confirmed the mutation in 2 prostate carcinomas. This study indicates that IHC is a reliable method for the pathologic identification of the IDH1 mutation in solid human cancers such as prostate carcinomas.

The association of TP53 mutations with the resistance of colorectal carcinoma to the insulin-like growth factor-1 receptor inhibitor picropodophyllin.

BACKGROUND: There is growing evidence indicating the insulin-like growth factor 1 receptor (IGF-1R) plays a critical role in the progression of human colorectal carcinomas. IGF-1R is an attractive drug target for the treatment of colon cancer. Picropodophyllin (PPP), of the cyclolignan family, has recently been identified as an IGF-1R inhibitor. The aim of this study is to determine the therapeutic response and mechanism after colorectal carcinoma treatment with PPP. METHODS: Seven colorectal carcinoma cell lines were treated with PPP. Following treatment, cells were analyzed for growth by a cell viability assay, sub-G1 apoptosis by flow cytometry, caspase cleavage and activation of AKT and extracellular signal-regulated kinase (ERK) by western blot analysis. To examine the in vivo therapeutic efficacy of PPP, mice implanted with human colorectal carcinoma xenografts underwent PPP treatment. RESULTS: PPP treatment blocked the phosphorylation of IGF-1R, AKT and ERK and inhibited the growth of TP53 wild-type but not mutated colorectal carcinoma cell lines. The treatment of PPP also induced apoptosis in TP53 wild-type cells as evident by the presence of sub-G1 cells and the cleavage of caspase-9, caspase-3, DNA fragmentation factor-45 (DFF45), poly (ADP-ribose) polymerase (PARP), and X-linked inhibitor of apoptosis protein (XIAP). The loss of BAD phosphorylation in the PPP-treated TP53 wild type cells further suggested that the treatment induced apoptosis through the BAD-mediated mitochondrial pathway. In contrast, PPP treatment failed to induce the phosphorylation of AKT and ERK and caspase cleavage in TP53 mutated colorectal carcinoma cell lines. Finally, PPP treatment suppressed the growth of xenografts derived from TP53 wild type but not mutated colorectal carcinoma cells. CONCLUSIONS: We report the association of TP53 mutations with the resistance of treatment of colorectal carcinoma cells in culture and in a xenograft mouse model with the IGF-1R inhibitor PPP. TP53 mutations often occur in colorectal carcinomas and could be used as a biomarker to predict the resistance of colorectal carcinomas to the treatment by this IGF-1R inhibitor.

Inhibition of the CXCL12/CXCR4-axis as preventive therapy for radiation-induced pulmonary fibrosis.

BACKGROUND: A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process. METHODOLOGY/PRINCIPAL FINDINGS: The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process. CONCLUSIONS/SIGNIFICANCE: CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation.

Combination of mTOR and EGFR kinase inhibitors blocks mTORC1 and mTORC2 kinase activity and suppresses the progression of colorectal carcinoma.

Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT(S473)) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR(T1068)). The parallel increase of AKT(S473) and EGFR(T1068) in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.

Glioblastoma with oligodendroglioma component (GBM-O): molecular genetic and clinical characteristics.

Glioblastoma (GBM) is an aggressive primary brain tumor with an average survival of approximately 1 year. A recently recognized subtype, glioblastoma with oligodendroglioma component (GBM-O), was designated by the World Health Organization (WHO) in 2007. We investigated GBM-Os for their clinical and molecular characteristics as compared to other forms of GBM. Tissue samples were used to determine EGFR, PTEN, and 1p and 19q status by fluorescence in situ hybridization (FISH); p53 and mutant IDH1 protein expression by immunohistochemistry (IHC); and MGMT promoter status by methylation-specific polymerase chain reaction (PCR). GBM-Os accounted for 11.9% of all GBMs. GBM-Os arose in younger patients compared to other forms of GBMs (50.7 years vs. 58.7 years, respectively), were more frequently secondary neoplasms, had a higher frequency of IDH1 mutations and had a lower frequency of PTEN deletions. Survival was longer in patients with GBM-Os compared to those with other GBMs, with median survivals of 16.2 and 8.1 months, respectively. Most of the survival advantage for GBM-O appeared to be associated with a younger age at presentation. Among patients with GBM-O, younger age at presentation and 1p deletion were most significant in conferring prolonged survival. Thus, GBM-O represents a subset of GBMs with distinctive morphologic, clinical and molecular characteristics.

A20 ubiquitin ligase-mediated polyubiquitination of RIP1 inhibits caspase-8 cleavage and TRAIL-induced apoptosis in glioblastoma.

UNLABELLED: The TNF-related apoptosis-inducing ligand (TRAIL) apoptotic pathway has emerged as a therapeutic target for the treatment of cancer. However, clinical trials have proven that the vast majority of human cancers are resistant to TRAIL apoptotic pathway-targeted therapies. We show that A20-mediated ubiquitination inhibits caspase-8 cleavage and TRAIL-induced apoptosis in glioblastoma through 2 signaling complexes. A20 is highly expressed in glioblastomas and, together with the death receptor 5 and receptor-interacting protein 1, forms a plasma membrane-bound preligand assembly complex under physiologic conditions. Treatment with TRAIL leads to the recruitment of caspase-8 to the plasma membrane-bound preligand assembly complex for the assembly of a death-inducing signaling complex. In the death-inducing signaling complex, the C-terminal zinc finger (Znf) domain of the A20 ubiquitin ligase mediates receptor-interacting protein 1 polyubiquitination through lysine-63-linked polyubiquitin chains, which bind to the caspase-8 protease domain and inhibit caspase-8 dimerization, cleavage, and the initiation of TRAIL-induced apoptosis in glioblastoma-derived cell lines and tumor-initiating cells. SIGNIFICANCE: These results identify A20 E3 ligase as a therapeutic target whose inhibition can overcome TNF-related apoptosis-inducing ligand resistance in glioblastoma and thus have an impact on ongoing clinical trials of TNF-related apoptosis-inducing ligand-targeted combination cancer therapies.

Simultaneous targeting of EGFR and mTOR inhibits the growth of colorectal carcinoma cells.

Epidermal growth factor receptor (EGFR) is highly expressed in colorectal carcinomas and, as a result, it leads to the activation of downstream mammalian target of rapamycin (mTOR) kinase pathways for cancer growth and progression. Clinical and preclinical studies, however, have shown that inhibition of epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) alone is not sufficient to treat colorectal carcinomas. In search of effective combination therapies, we show here that simultaneous targeting of EGFR with its inhibitor, erlotinib and mTOR with its inhibitor, rapamycin inhibits the phosphorylation and activation of downstream phosphatidylinositol 3-kinase (PI3K), Akt, mTOR and extracellular-signal-regulated kinase 1/2 (Erk1/2) pathways, resulting in the inhibition of cell cycle progression and the growth of both KRAS wild-type and mutated colorectal carcinoma cells. This study has demonstrated the principle that the combination of erlotinib and rapamycin may provide an effective therapy for colorectal carcinomas.

K-Ras mutation-mediated IGF-1-induced feedback ERK activation contributes to the rapalog resistance in pancreatic ductal adenocarcinomas.

Mammalian target of rapamycin complex 1 (mTORC1) is frequently activated in human cancers; however, clinical trials of rapalog (the mTORC1 inhibitors) have shown that pancreatic ductal adenocarcinomas (PDACs) resist to the treatment. Rapalog treatment activated the extracellular signal-regulated kinase (ERK) pathway in K-Ras mt PDAC cells. K-Ras knockdown abolished the insulin-like growth factor-1 (IGF-1)-induced ERK pathway in the K-Ras mt PDAC cells and enhanced the therapeutic efficacy of everolimus in treating K-Ras mt PDAC cells-derived mouse xenografts. The results indicate that targeting of K-Ras mutation may lead to the development of therapies that overcome rapalog resistance in PDAC.

Targeting A20 enhances TRAIL-induced apoptosis in hepatocellular carcinoma cells.

A20 was initially identified as a primary gene product following TNF α treatment in human umbilical vein endothelial cells. Increased A20 expression is associated with tumorigenesis in many cancers, whereas the loss of A20 function is linked to lymphoma. It has been reported that A20 protects cells from TRAIL-induced apoptosis; however, the mechanism by which A20 is involved is still largely unknown. Our results indicate that TRAIL induces the hepatocellular carcinoma apoptosis associated with A20 knockdown in a concentration-dependent manner. TRAIL-induced apoptosis requires p18 caspase-8 activation, and, the activation of caspase-8 is at least in part, due to the direct cleavage of RIP1 by A20 knockdown. These findings suggest that A20 modulates the sensitivity to TRAIL by RIP1 ubiquitination, thereby repressing the recruitment and activation of pro-caspase-8 into the active form caspase-8. Thus, our study suggests that A20 protects against TRAIL-induced apoptosis through the regulation of RIP1 ubiquitination.