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.

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.

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.

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.

Development of a gene expression database and related analysis programs for evaluation of anticancer compounds.

Genome-wide transcriptional expression analysis is a powerful strategy for characterizing the biological activity of anticancer compounds. It is often instructive to identify gene sets involved in the activity of a given drug compound for comparison with different compounds. Currently, however, there is no comprehensive gene expression database and related application system that is; (i) specialized in anticancer agents; (ii) easy to use; and (iii) open to the public. To develop a public gene expression database of antitumor agents, we first examined gene expression profiles in human cancer cells after exposure to 35 compounds including 25 clinically used anticancer agents. Gene signatures were extracted that were classified as upregulated or downregulated after exposure to the drug. Hierarchical clustering showed that drugs with similar mechanisms of action, such as genotoxic drugs, were clustered. Connectivity map analysis further revealed that our gene signature data reflected modes of action of the respective agents. Together with the database, we developed analysis programs that calculate scores for ranking changes in gene expression and for searching statistically significant pathways from the Kyoto Encyclopedia of Genes and Genomes database in order to analyze the datasets more easily. Our database and the analysis programs are available online at our website (http://scads.jfcr.or.jp/db/cs/). Using these systems, we successfully showed that proteasome inhibitors are selectively classified as endoplasmic reticulum stress inducers and induce atypical endoplasmic reticulum stress. Thus, our public access database and related analysis programs constitute a set of efficient tools to evaluate the mode of action of novel compounds and identify promising anticancer lead compounds.

Hyperactivation of 4E-binding protein 1 as a mediator of biguanide-induced cytotoxicity during glucose deprivation.

Biguanides, including metformin, buformin, and phenformin, are potential antitumorigenic agents and induce cell death during glucose deprivation, a cell condition that occurs in the tumor microenvironment. Here, we show that this selective killing of glucose-deprived cells is coupled with hyperactivation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation initiation. We found, in fact, that the 4E-BP1 hyperactivation led to failure of the unfolded protein response (UPR), an endoplasmic reticulum-originated stress signaling pathway for cell survival. We also found that the 4E-BP1-mediated UPR inhibition occurred through a strong inhibition of the mTOR signaling pathway, a proven antitumor target. Importantly, the 4E-BP1 hyperactivation can be also seen in xenografted cancer cells through an in vivo biguanide treatment. Our findings indicate that antitumor action of biguanides can be mediated by 4E-BP1 hyperactivation, which results in UPR inhibition and selective cell killing when glucose is withdrawn.

In vivo expansion of MDR1-transduced cells accompanied by a post-transplantation chemotherapy regimen with mitomycin C and methotrexate.

BACKGROUND: A recurrent breast cancer patient received high-dose chemotherapy, a transplant of multidrug resistance 1 (MDR1)-transduced cells and four different protocols of post-transplantation chemotherapy. We report the analysis of MDR1-transduced cells in this patient. METHODS: MDR1 transgene levels in the peripheral blood mononuclear cells of the patient were evaluated by polymerase chain reaction (PCR). Retroviral integration sites of the MDR1-transduced cells were identified by linear amplification-mediated (LAM)-PCR. RESULTS: Twelve days after transplantation, approximately 1% of the peripheral blood mononuclear cells were MDR1 transgene-positive. The transgene levels decreased quickly, and were at low levels until day 504. A remarkable increase in MDR1 transgene-positive cells was observed on day 532, during combination chemotherapy with mitomycin C and methotrexate. Using LAM-PCR, 31 MDR1-transduced clones were identified, and eight of these were long-life clones that survived for more than 500 days. Among the 31 clones, ten had a retroviral integration site near genes listed in the Retroviral Tagged Cancer Gene (RTCG) Database. Two long-life clones, N-30 and N-31, had retroviral integration sites within the MDS1-EVI1 locus. Another two long-life clones had integration sites close to PRDM16 or CUEDC1. CONCLUSIONS: These results suggest that MDR1-transduced cells were enriched in vivo by an MDR1 substrate, mitomycin C. The possible activation of EVI1 or other RTCGs by retroviral insertion may have affected the survival and persistence of a proportion of the transduced cells.

A novel endoplasmic reticulum export signal: proline at the +2-position from the signal peptide cleavage site.

NUCB1 (nucleobindin 1) is a Golgi-localized soluble protein with a signal peptide and multiple functional domains. We reported recently that NUCB1 is a negative regulator of the unfolded protein response that activates various endoplasmic reticulum (ER)-originating signaling pathways. In that report, we also showed that Golgi localization of NUCB1 was essential to regulate the unfolded protein response. However, the localization mechanism of NUCB1 is still unknown. Here, we report that the proline residue at the +2-position (Pro(+2)) from the signal peptide cleavage site is the determinant of NUCB1 protein export from the ER and subsequent transport to the Golgi. Fusion of the N-terminal amino acids 1-35 peptide region, including both signal peptide (amino acids 1-26) and Pro(+2), was sufficient for enhanced green fluorescent protein to localize in the Golgi, whereas single amino acid mutation of Pro(+2) resulted in defective export from the ER without affecting the protein maturation process. Furthermore, we demonstrated that Pro(+2) was important for the enhanced green fluorescent protein fusion protein to concentrate at a transport vesicle formation site within the ER, often termed the ER exit site. Interestingly, such a Pro(+2) has also been functionally conserved in other Golgi-localized soluble proteins, Cab45 (Ca(2+)-binding protein of 45 kDa), reticulocalbin 1, and calumenin. Our findings indicate that Pro(+2) can function as a novel ER export signal of some Golgi proteins.

Preventing the unfolded protein response via aberrant activation of 4E-binding protein 1 by versipelostatin.

We recently isolated a macrocyclic compound, versipelostatin (VST), that exerts in vivo antitumor activity. VST shows unique, selective cytotoxicity to glucose-deprived tumor cells by preventing the unfolded protein response (UPR). Here we show that eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of eukaryotic initiation factor 4E-mediated protein translation, plays a role in the UPR-inhibitory action of VST. Indeed, 4E-BP1 is aberrantly activated by VST. This activation occurs specifically during glucose deprivation and results in profound translation repression and prevents induction of the typical UPR markers glucose-regulated protein (GRP) 78 and activating transcription factor (ATF) 4. Our overexpression and knockdown experiments showed that 4E-BP1 can regulate GRP78 and ATF4 expression. These mechanisms appear to be specific for VST. By contrast, rapamycin, which activates 4E-BP1 regardless of cellular glucose availability, has only marginal effects on the expression of GRP78 and ATF4. Our present findings demonstrate that aberrant 4E-BP1 activation can contribute to UPR preventing by VST, possibly through a mechanism that does not operate in rapamycin-treated cells.

Retroviral integration site analysis and the fate of transduced clones in an MDR1 gene therapy protocol targeting metastatic breast cancer.

A clinical study of an MDR1 gene therapy protocol targeting metastatic breast cancer has been conducted in which the patients received high-dose chemotherapy, a transplant of MDR1-transduced autologous CD34(+) cells, and docetaxel. We herein report the molecular results of a 6-year follow-up of an individual in this study (patient 1). HaMDR-transduced cells, which had been initially detected in the peripheral blood of this individual, were found to have gradually decreased. After 10 cycles of docetaxel (days 71-316), MDR1 transgene levels were found to have increased, and then decreased to undetectable levels by day 1461. Thirty-eight MDR1-transduced clones were identified in patient 1, of which 11 showed a retroviral integration in close proximity to genes listed in the Retrovirus Tagged Cancer Gene Database (RTCGD). Four short-life clones in this group were found to harbor retroviral integrations close to the ZFHX1B, NOTCH1, BMI1, or HHEX gene; these genes have been frequently reported in the RTCGD. In addition, a long-lived RTCGD-hit clone, L-34, had a retroviral integration at a position 179 kb upstream of the EVI1 gene. L-34 was detectable on days 327-1154, but became undetectable 3 years after the docetaxel treatments had ceased. An additional three docetaxel-induced long-life clones showed comparable polymerase chain reaction profiles, which were also similar to that of the total MDR1-transduced cells. Our results thus show that docetaxel may have been effective in promoting the expansion of several MDR1-transduced clones in patient 1, but that they persist in the peripheral blood for only a few years.

Pilot study of MDR1 gene transfer into hematopoietic stem cells and chemoprotection in metastatic breast cancer patients.

A major problem in high-dose chemotherapy with autologous hematopoietic stem cell transplantation is insufficient function of reconstituted bone marrow that limits the efficacy of post-transplantation chemotherapy. Because transduction of hematopoietic stem cells with the multidrug resistance 1 (MDR1) gene might circumvent this problem, we conducted a pilot study of MDR1 gene therapy against metastatic breast cancer. Peripheral blood stem cells were harvested, and one-third of the cells were transduced with MDR1 retrovirus. After the reconstitution of bone marrow function, the patients received high-dose chemotherapy with transplantation of both MDR1-transduced and unprocessed peripheral blood stem cells. The patients then received docetaxel chemotherapy. Two patients received transplantation of the MDR1-transduced cells in 2001. Peripheral blood MDR1-transduced leukocytes were 3-5% of the total cells after transplantation, but decreased gradually. During docetaxel chemotherapy, an increase in the rate of MDR1-transduced leukocytes (up to 10%) was observed. Comparison of docetaxel-induced granulocytopenia in the two patients suggested a bone marrow-protective effect of the MDR1-transduced cells. No serious side-effect was observed, and the patients were in complete remission for more than 3 years. The MDR1-transduced cells gradually decreased and disappeared almost entirely by the end of 2004. Results of linear amplification-mediated polymerase chain reaction of the MDR1-transduced leukocytes suggested no sign of abnormal amplification of the transduced cells. A third patient received transplantation of the MDR1-transduced cells in 2004. These results suggest the feasibility of our MDR1 gene therapy against metastatic breast cancer, and follow-up is ongoing.