Induction of stearoyl-CoA desaturase confers cell density-dependent ferroptosis resistance in melanoma.

Ferroptosis is a form of regulated cell death that is induced by inhibiting glutathione peroxidase 4 (GPX4), which eliminates lipid peroxidation. Ferroptosis induction is influenced by the cell environment. However, the cellular states altering ferroptosis susceptibility remain largely unknown. We found that melanoma cell lines became resistant to ferroptosis as cell density increased. Comparative transcriptome and metabolome analyses revealed that cell density-dependent ferroptosis resistance was coupled with a shift toward a lipogenic phenotype accompanied by strong induction of stearoyl-CoA desaturase (SCD). Database analysis of gene dependency across hundreds of cancer cell lines uncovered a negative correlation between GPX4 and SCD dependency. Importantly, SCD inhibition, either pharmacologically or through genetic knockout, sensitized melanoma cells to GPX4 inhibition, thereby attenuating ferroptosis resistance in cells at high density. Our findings indicate that transition to an SCD-inducing, lipogenic cell state produces density-dependent resistance to ferroptosis, which may provide a therapeutic strategy against melanoma.

Duplication of the appendix masquerading as appendiceal tumor: a case report.

BACKGROUND: This case report highlights the exceptional rarity of appendix duplication in adults, a condition that closely mimics appendiceal tumors, posing diagnostic challenges. The novelty of this case lies in its presentation of a Type A duplication, emphasizing the diagnostic intricacies involved in distinguishing it from other pathologies. CASE PRESENTATION: We present the case of a 69-year-old male with a history of hypertension, hyperuricemia, and duodenal gastric ulcer, who presented with a positive occult blood test. Lower gastrointestinal endoscopy revealed an appendiceal orifice with atypical hyperemia and edema. Subsequent imaging and biopsy results suggested an appendiceal tumor, prompting laparoscopic ileocecal resection. Intraoperative findings revealed an unremarkable appendix, but histopathological analysis unveiled appendiceal duplication, characterized by bifurcation into two lumens within a thick serosal wall. The patient was discharged without complications. CONCLUSIONS: This case underscores the importance of recognizing appendix duplication as a rare differential diagnosis for appendiceal tumors. Surgeons should remain vigilant, especially in cases of Type A duplication, where preoperative diagnosis remains challenging. Early identification can avert potential complications and missed congenital anomalies.

Activating mutations in EGFR and PI3K promote ATF4 induction for NSCLC cell survival during amino acid deprivation.

Some oncoproteins along with stress kinase general control non-derepressible 2 (GCN2) can ensure the induction of activating transcription factor 4 (ATF4) to counteract amino acid deprivation; however, little is known regarding the role of the oncogenic EGFR-PI3K pathway. In this study, we demonstrate that both mutated EGFR and PIK3CA contribute to ATF4 induction following GCN2 activation in NSCLC cells. The inhibition of EGFR or PI3K mutant proteins, pharmacologically or through genetic knockdown, inhibited ATF4 induction without affecting GCN2 activation. A downstream analysis revealed that the oncogenic EGFR-PI3K pathway may utilize mTOR-mediated translation control mechanisms for ATF4 induction. Furthermore, in NSCLC cells harboring co-mutations in EGFR and PIK3CA, the combined inhibition of these oncoproteins markedly suppressed ATF4 induction and the subsequent gene expression program as well as cell viability during amino acid deprivation. Our findings establish a role for the oncogenic EGFR-PI3K pathway in the adaptive stress response and provide a strategy to improve EGFR-targeted NSCLC therapy.

Spautin-1 inhibits mitochondrial complex I and leads to suppression of the unfolded protein response and cell survival during glucose starvation.

The unfolded protein response (UPR) is an adaptive stress response pathway that is essential for cancer cell survival under endoplasmic reticulum stress such as during glucose starvation. In this study, we identified spautin-1, an autophagy inhibitor that suppresses ubiquitin-specific peptidase 10 (USP10) and USP13, as a novel UPR inhibitor under glucose starvation conditions. Spautin-1 prevented the induction of UPR-associated proteins, including glucose-regulated protein 78, activating transcription factor 4, and a splicing variant of x-box-binding protein-1, and showed preferential cytotoxicity in glucose-starved cancer cells. However, USP10 and USP13 silencing and treatment with other autophagy inhibitors failed to result in UPR inhibition and preferential cytotoxicity during glucose starvation. Using transcriptome and chemosensitivity-based COMPARE analyses, we identified a similarity between spautin-1 and mitochondrial complex I inhibitors and found that spautin-1 suppressed the activity of complex I extracted from isolated mitochondria. Our results indicated that spautin-1 may represent an attractive mitochondria-targeted seed compound that inhibits the UPR and cancer cell survival during glucose starvation.

eIF4B enhances ATF4 expression and contributes to cellular adaptation to asparagine limitation in BRAF-mutated A375 melanoma.

ATF4 is a crucial transcription factor in the integrated stress response, a major adaptive signaling pathway activated by tumor microenvironment and therapeutic stresses. BRAF inhibitors, such as vemurafenib, induce ATF4 in BRAF-mutated melanoma cells, but the mechanisms of ATF4 induction are not fully elucidated. Here, we show that ATF4 expression can be upregulated by eukaryotic initiation factor 4B (eIF4B) in BRAF-mutated A375 cells. Indeed, eIF4B knockout (KO) prevented ATF4 induction and activation of the uORF-mediated ATF4 translation mechanism during vemurafenib treatment, which were effectively recovered by the rescue of eIF4B. Transcriptome analysis revealed that eIF4B KO selectively influenced ATF4-target gene expression among the overall gene expression changed by vemurafenib. Interestingly, eIF4B supported cellular proliferation under asparagine-limited conditions, possibly through the eIF4B-ATF4 pathway. Our findings indicate that eIF4B can regulate ATF4 expression, thereby contributing to cellular stress adaptation, which could be targeted as a therapeutic approach against malignancies, including melanoma.

Lamellarin 14, a derivative of marine alkaloids, inhibits the T790M/C797S mutant epidermal growth factor receptor.

The emergence of acquired resistance is a major concern associated with molecularly targeted kinase inhibitors. The C797S mutation in the epidermal growth factor receptor (EGFR) confers resistance to osimertinib, a third-generation EGFR-tyrosine kinase inhibitor (EGFR-TKI). We report that the derivatization of the marine alkaloid topoisomerase inhibitor lamellarin N provides a structurally new class of EGFR-TKIs. One of these, lamellarin 14, is effective against the C797S mutant EGFR. Bioinformatic analyses revealed that the derivatization transformed the topoisomerase inhibitor-like biological activity of lamellarin N into kinase inhibitor-like activity. Ba/F3 and PC-9 cells expressing the EGFR in-frame deletion within exon 19 (del ex19)/T790M/C797S triple-mutant were sensitive to lamellarin 14 in a dose range similar to the effective dose for cells expressing EGFR del ex19 or del ex19/T790M. Lamellarin 14 decreased the autophosphorylation of EGFR and the downstream signaling in the triple-mutant EGFR PC-9 cells. Furthermore, intraperitoneal administration of 10 mg/kg lamellarin 14 for 17 days suppressed tumor growth of the triple-mutant EGFR PC-9 cells in a mouse xenograft model using BALB/c nu/nu mice. Thus, lamellarin 14 serves as a novel structural backbone for an EGFR-TKI that prevents the development of cross-resistance against known drugs in this class.

GZD824 Inhibits GCN2 and Sensitizes Cancer Cells to Amino Acid Starvation Stress.

Eukaryotic initiation factor 2α (eIF2α) kinase general control nonderepressible 2 (GCN2) drives cellular adaptation to amino acid limitation by activating the integrated stress response that induces activating transcription factor 4 (ATF4). Here, we found that a multikinase inhibitor, GZD824, which we identified using a cell-based assay with ATF4 immunostaining, inhibited the GCN2 pathway in cancer cells. Indeed, GZD824 suppressed GCN2 activation, eIF2α phosphorylation, and ATF4 induction during amino acid starvation stress. However, at lower nonsuppressive concentrations, GZD824 paradoxically stimulated eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under unstressed conditions. Such dual properties conceivably arose from a direct effect on GCN2, as also observed in a cell-free GCN2 kinase assay and shared by a selective GCN2 inhibitor. Consistent with the GCN2 pathway inhibition, GZD824 sensitized certain cancer cells to amino acid starvation stress similarly to ATF4 knockdown. These results establish GZD824 as a multikinase GCN2 inhibitor and may enhance its utility as a drug under development. SIGNIFICANCE STATEMENT: GZD824, as a direct general control nonderepressible 2 (GCN2) inhibitor, suppresses activation of the integrated stress response during amino acid limitation, whereas it paradoxically stimulates this stress-signaling pathway at lower nonsuppressive concentrations. The pharmacological activity we identify herein will provide the basis for the use of GZD824 to elucidate the regulatory mechanisms of GCN2 and to evaluate the potential of the GCN2-activating transcription factor 4 pathway as a target for cancer therapy.

Disrupting ATF4 Expression Mechanisms Provides an Effective Strategy for BRAF-Targeted Melanoma Therapy.

BRAF V600 mutation influences cellular signaling pathways for melanoma development. However, the role of oncogenic BRAF in adaptive stress response pathways is not fully understood. Here, we show that oncogenic BRAF plays an essential role in the induction of ATF4 following the activation of general control non-derepressible 2 (GCN2) kinase during nutrient stress and BRAF-targeted, therapeutic stress. Under GCN2 activation, BRAF ensures ATF4 induction by utilizing mTOR and eIF4B as downstream regulators. In contrast to the MEK-ERK pathway, this signaling pathway remains temporarily active even during treatment with BRAF inhibitors, thereby enabling the transient induction of ATF4. We also identify a chemical compound that prevents BRAF inhibitor-induced activation of the GCN2-ATF4 pathway and produces synergistic cell killing with BRAF inhibitors. Our findings establish a collaborative relationship between oncogenic BRAF and the GCN2-ATF4 signaling pathway, which may provide a novel therapeutic approach to target the adaptive stress response.

The kinase PERK represses translation of the G-protein-coupled receptor LGR5 and receptor tyrosine kinase ERBB3 during ER stress in cancer cells.

As a branch of the unfolded protein response, protein kinase R-like endoplasmic reticulum kinase (PERK) represses global translation in response to endoplasmic reticulum (ER) stress. This pathophysiological condition is associated with the tumor microenvironment in cancer. Previous findings in our lab have suggested that PERK selectively represses translation of some mRNAs, but this possibility awaits additional investigation. In this study, we show that a stem-cell marker protein, leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), is rapidly depleted in colon cancer cells during ER stress, an effect that depended on the PERK-mediated translational repression. Indeed, the PERK inhibition led to the accumulation of premature, underglycosylated forms of LGR5, which were produced only at low levels during proper PERK activation. Unlike the mature LGR5 form, which is constitutively degraded regardless of PERK activation, the underglycosylated LGR5 exhibited a prolonged half-life and accumulated inside the cells without being expressed on the cell surface. We also found that Erb-B2 receptor tyrosine kinase 3 (ERBB3) is subjected to a similarly-regulated depletion by PERK, whereas the epidermal growth factor receptor (EGFR), stress-inducible heat-shock protein family A (Hsp70) member 5 (HSPA5), and anterior gradient 2 protein-disulfide isomerase family member (AGR2) were relatively. insensitive to the PERK-mediated repression of translation. These results indicate that LGR5 and ERBB3 are targets for PERK-mediated translational repression during ER stress.

Neoadjuvant CAPOX and bevacizumab alone for locally advanced rectal cancer: long-term results from the N-SOG 03 trial.

BACKGROUND: Neoadjuvant chemotherapy (NAC) alone for locally advanced rectal cancer (LARC) remains an experimental treatment, and the efficacy in terms of long-term outcome has not been fully elucidated. The N-SOG 03 trial examined the safety and efficacy of neoadjuvant CAPOX and bevacizumab (Bev) without radiotherapy in patients with poor-risk LARC. METHODS: Thirty-two patients with MRI-defined LARC received neoadjuvant CAPOX and Bev followed by curative resection between 2010 and 2011. The overall survival (OS), progression-free survival (PFS), and local-relapse rate (LRR) were calculated using the Kaplan-Meier method, and the risk factors were evaluated by multivariate analysis using the Cox proportional hazard models. This trial is registered with UMIN, number 000003507. RESULTS: In the entire cohort, the 5-year OS was 81.3%. Because of disease progression during chemotherapy, 3 patients ultimately did not undergo curative surgery. As a result, 29 patients underwent R0/1 resection. Among these 29 patients, the 5-year OS, PFS, and LRR were 89.7%, 72.4% and 13.9%, respectively. In multivariate analysis, cT4b tumor was an independent poor prognostic factor for OS and LRR, and ypT4b tumor and absence of N down-staging were independent poor prognostic factors for PFS. CONCLUSIONS: Patients with cT4b tumor were not suitable for NAC alone. However, the long-term outcomes of the other patients were satisfactory, and NAC alone might be an option for treatment of LARC. N down-staging was likely to bring favorable PFS, even in patients with cStage III.

BCR-ABL tyrosine kinase inhibition induces metabolic vulnerability by preventing the integrated stress response in K562 cells.

The integrated stress response (ISR) is a cellular process that is characterized by activation of eukaryotic initiation factor (eIF)2α kinases and subsequent induction of activating transcription factor (ATF)4. The ISR plays an important role in protecting cells from tumor-related metabolic stresses, such as nutrient deprivation and perturbed proteostasis. Here, we showed that disruption of the ISR, together with increased cellular stress vulnerability, was produced by pharmacological inhibition of BCR-ABL, the oncogenic driver in chronic myeloid leukemia (CML). Treatment of CML-derived K562 cells with BCR-ABL tyrosine kinase inhibitors, including imatinib, dasatinib, nilotinib and ponatinib, prevented activation of eIF2α kinases, protein kinase-like endoplasmic reticulum kinase (PERK) and general control nonderepressible 2, and downstream ATF4 induction during metabolic stress. Prevention of ATF4 induction likely occurred as a result of the combinatorial suppression of the eIF2α kinase and phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathways. In addition, we found that pharmacological inhibition of PERK mimicked BCR-ABL inhibition to enhance apoptosis induction under stress conditions. These findings indicate that the ISR is under the control of BCR-ABL and may foster adaptation to tumorigenic stresses in CML cells.

InDePTH: detection of hub genes for developing gene expression networks under anticancer drug treatment.

It has been difficult to elucidate the structure of gene regulatory networks under anticancer drug treatment. Here, we developed an algorithm to highlight the hub genes that play a major role in creating the upstream and downstream relationships within a given set of differentially expressed genes. The directionality of the relationships between genes was defined using information from comprehensive collections of transcriptome profiles after gene knockdown and overexpression. As expected, among the drug-perturbed genes, our algorithm tended to derive plausible hub genes, such as transcription factors. Our validation experiments successfully showed the anticipated activity of certain hub gene in establishing the gene regulatory network that was associated with cell growth inhibition. Notably, giving such top priority to the hub gene was not achieved by ranking fold change in expression and by the conventional gene set enrichment analysis of drug-induced transcriptome data. Thus, our data-driven approach can facilitate to understand drug-induced gene regulatory networks for finding potential functional genes.

Mitochondrial deficiency impairs hypoxic induction of HIF-1 transcriptional activity and retards tumor growth.

Mitochondria can be involved in regulating cellular stress response to hypoxia and tumor growth, but little is known about that mechanistic relationship. Here, we show that mitochondrial deficiency severely retards tumor xenograft growth with impairing hypoxic induction of HIF-1 transcriptional activity. Using mtDNA-deficient ρ0 cells, we found that HIF-1 pathway activation was comparable in slow-growing ρ0 xenografts and rapid-growing parental xenografts. Interestingly, we found that ex vivo ρ0 cells derived from ρ0 xenografts exhibited slightly increased HIF-1α expression and modest HIF-1 pathway activation regardless of oxygen concentration. Surprisingly, ρ0 cells, as well as parental cells treated with oxidative phosphorylation inhibitors, were unable to boost HIF-1 transcriptional activity during hypoxia, although HIF-1α protein levels were ordinarily increased in these cells under hypoxic conditions. These findings indicate that mitochondrial deficiency causes loss of hypoxia-induced HIF-1 transcriptional activity and thereby might lead to a constitutive HIF-1 pathway activation as a cellular adaptation mechanism in tumor microenvironment.

BRAF-mutated cells activate GCN2-mediated integrated stress response as a cytoprotective mechanism in response to vemurafenib.

In BRAF-mutated melanoma cells, the BRAF inhibitor, vemurafenib, induces phosphorylation of eukaryotic initiation factor 2α (eIF2α) and subsequent induction of activating transcription factor 4 (ATF4), the central regulation node of the integrated stress response (ISR). While the ISR supports cellular adaptation to various stresses, the role of vemurafenib-triggered ISR has not been fully characterized. Here, we showed that in response to vemurafenib, BRAF-mutated melanoma and colorectal cancer cells rapidly induced the ISR as a cytoprotective mechanism through activation of general control nonderepressible 2 (GCN2), an eIF2α kinase sensing amino acid levels. The vemurafenib-triggered ISR, an event independent of downstream MEK inhibition, was specifically prevented by silencing GCN2, but not other eIF2α kinases, including protein kinase-like endoplasmic reticulum kinase, which transmits endoplasmic reticulum (ER) stress. Consistently, the ER stress gatekeeper, GRP78, was not induced by vemurafenib. Interestingly, ATF4 silencing by siRNA rendered BRAF-mutated melanoma cells sensitive to vemurafenib. Thus, the GCN2-mediated ISR can promote cellular adaptation to vemurafenib-induced stress, providing an insight into the development of drug resistance.

PMEPA1, a TGF-ß- and hypoxia-inducible gene that participates in hypoxic gene expression networks in solid tumors.

Prostate transmembrane protein, androgen induced 1 (PMEPA1) is highly expressed in various solid tumors and is known to play important roles in the transforming growth factor-ß (TGF-ß) signaling pathway. Here, we demonstrate a novel relationship between PMEPA1 and hypoxia, a common microenvironmental stress condition in solid tumors. We showed that induction of PMEPA1 expression occurred during hypoxia in a manner dependent on both TGF-ß signaling and hypoxia-inducible factor-1 (HIF-1) pathways. Furthermore, overexpression and knockdown experiments revealed that PMEPA1 enhanced HIF-1 transcription activity. Bioinformatics analyses of PMEPA1-correlated genes using a gene expression database in clinical settings showed significant enrichment of gene sets defined by TGF-ß and hypoxia and these two signaling pathways-related angiogenesis and epithelial-mesenchymal transition in many types of solid tumors. Collectively, our findings indicated that PMEPA1 participates in TGF-ß- and hypoxia-regulated gene expression networks in solid tumors and thereby may contribute to tumor progression.

TBL2 Associates With ATF4 mRNA Via Its WD40 Domain and Regulates Its Translation During ER Stress.

PKR-like ER-resident kinase (PERK) phosphorylates eukaryotic translation initiation factor 2 α (eIF2α) under endoplasmic reticulum (ER) stress; this results in repression of general translation and induction of specific gene expression, such as activating transcription factor 4 (ATF4). We previously showed that, upon ER stress, transducin (ß)-like 2 (TBL2) was an ER-localized transmembrane protein and interacted with PERK and that TBL2 was involved in ATF4 expression and cell survival. Here, we show that TBL2 is able to associate with ATF4 mRNA and regulate its translation. The RNA-immunoprecipitation analysis using several TBL2 deletion mutants revealed that the WD40 domain was essential for association with ATF4 mRNA. Importantly, suppression of TBL2 by knockdown or overexpression of the TBL2 mutant with a defective WD40 domain diminished ATF4 induction at the translational level. Thus, our findings indicate that, under ER stress, TBL2 participates in ATF4 translation through its association with the mRNA.

The endoplasmic reticulum-localized protein TBL2 interacts with the 60S ribosomal subunit.

Transducin (beta)-like 2 (TBL2) is a poorly characterized protein comprising the N-terminal transmembrane region and the C-terminal WD40 domain. We previously showed that TBL2 is an endoplasmic reticulum (ER)-localized protein that interacts with PKR-like ER-resident kinase (PERK), and under ER stress, it mediates protein expression of activating transcription factor 4 (ATF4). However, further molecular characterization of TBL2 is useful to better understand the function of this molecule. Here, we show that TBL2 associates with the eukaryotic 60S ribosomal subunit but not with the 40S subunit. The association of TBL2 with the 60S subunit was ER stress independent while the TBL2-PERK interaction occurred upon ER stress. Immunoprecipitation analysis using TBL2 deletion mutants revealed that the WD40 domain was essential for the 60S subunit association. These results could provide an important clue to understanding how TBL2 is involved in the expression of specific proteins under ER stress conditions.

Comprehensive transcriptomic analysis of molecularly targeted drugs in cancer for target pathway evaluation.

Targeted therapy is a rational and promising strategy for the treatment of advanced cancer. For the development of clinical agents targeting oncogenic signaling pathways, it is important to define the specificity of compounds to the target molecular pathway. Genome-wide transcriptomic analysis is an unbiased approach to evaluate the compound mode of action, but it is still unknown whether the analysis could be widely applicable to classify molecularly targeted anticancer agents. We comprehensively obtained and analyzed 129 transcriptomic datasets of cancer cells treated with 83 anticancer drugs or related agents, covering most clinically used, molecularly targeted drugs alongside promising inhibitors of molecular cancer targets. Hierarchical clustering and principal component analysis revealed that compounds targeting similar target molecules or pathways were clustered together. These results confirmed that the gene signatures of these drugs reflected their modes of action. Of note, inhibitors of oncogenic kinase pathways formed a large unique cluster, showing that these agents affect a shared molecular pathway distinct from classical antitumor agents and other classes of agents. The gene signature analysis further classified kinome-targeting agents depending on their target signaling pathways, and we identified target pathway-selective signature gene sets. The gene expression analysis was also valuable in uncovering unexpected target pathways of some anticancer agents. These results indicate that comprehensive transcriptomic analysis with our database (http://scads.jfcr.or.jp/db/cs/) is a powerful strategy to validate and re-evaluate the target pathways of anticancer compounds.

TBL2 is a novel PERK-binding protein that modulates stress-signaling and cell survival during endoplasmic reticulum stress.

Under ER stress, PKR-like ER-resident kinase (PERK) phosphorylates translation initiation factor eIF2α, resulting in repression of global protein synthesis and concomitant upregulation of the translation of specific mRNAs such as activating transcription factor 4 (ATF4). This PERK function is important for cell survival under ER stress and poor nutrient conditions. However, mechanisms of the PERK signaling pathway are not thoroughly understood. Here we identify transducin (beta)-like 2 (TBL2) as a novel PERK-binding protein. We found that TBL2 is an ER-localized type-I transmembrane protein and preferentially binds to the phosphorylated form of PERK, but not another eIF2α kinase GCN2 or ER-resident kinase IRE1, under ER stress. Immunoprecipitation analysis using various deletion mutants revealed that TBL2 interacts with PERK via the N-terminus proximal region and also associates with eIF2α via the WD40 domain. In addition, TBL2 knockdown can lead to impaired ATF4 induction under ER stress or poor nutrient conditions such as glucose and oxygen deprivation. Consistently, TBL2 knockdown rendered cells vulnerable to stresses similarly to PERK knockdown. Thus, TBL2 serves as a potential regulator of the PERK pathway.

Ultrastructural localization of intravenously injected carbon nanohorns in tumor.

Nanocarbons have many potential medical applications. Drug delivery, diagnostic imaging, and photohyperthermia therapy, especially in the treatment of tumors, have attracted interest. For the further advancement of these application studies, the microscopic localization of nanocarbons in tumor tissues and cells is a prerequisite. In this study, carbon nanohorns (CNHs) with sizes of about 100 nm were intravenously injected into mice having subcutaneously transplanted tumors, and the CNHs in tumor tissue were observed with optical and electron microscopy. In the tumor tissue, the CNHs were found in macrophages and endothelial cells within the blood vessels. Few CNHs were found in tumor cells or in the region away from blood vessels, suggesting that, under these study conditions, the enhanced permeability of tumor blood vessels was not effective for the movement of CNHs through the vessel walls. The CNHs in normal skin tissue were similarly observed. The extravasation of CNHs was not so obvious in tumor but was easily found in normal skin, which was probably due to their vessel wall structure difference. Proper understanding of the location of CNHs in tissues is helpful in the development of the medical uses of CNHs.