Mitochondria regulate the unfolded protein response leading to cancer cell survival under glucose deprivation conditions.

Cancer cells consume large amounts of glucose because of their specific metabolic pathway. However, cancer cells exist in tumor tissue where glucose is insufficient. To survive, cancer cells likely have the mechanism to elude their glucose addiction. Here we show that functional mitochondria are essential if cancer cells are to avoid glucose addiction. Cancer cells with dysfunctional mitochondria, such as mitochondrial DNA-deficient rho(0) cells and electron transport chain blocker-treated cells, were highly sensitive to glucose deprivation. Our data demonstrated that this sensitization was associated with failure of the unfolded protein response (UPR), an adaptive response mediated by the endoplasmic reticulum (ER). This study suggests a link between mitochondria and the ER during the UPR under glucose deprivation conditions and that mitochondria govern cell fate, not only through ATP production and apoptosis regulation, but also through modulating the UPR for cell survival.

TRF1 mediates mitotic abnormalities induced by Aurora-A overexpression.

Aurora-A, a conserved serine-threonine kinase, plays essential roles in mitosis. Aberrant upregulation of Aurora-A perturbs proper mitotic progression and results in a generation of multinucleated cells with centrosome amplification. The molecular mechanisms for these mitotic defects remain elusive. Here, we show that the overexpressed Aurora-A-induced mitotic defects depend on the telomeric protein TRF1. Live and fixed cell analyses revealed that Aurora-A overexpression in HeLa cells compromises chromosome biorientation, which leads to cytokinetic failure and tetraploidization with increased centrosome numbers. TRF1 depletion by small interfering RNAs or by tankyrase-1 overexpression suppresses Aurora-A-induced occurrence of unaligned chromosomes in metaphase, thus preventing the subsequent abnormalities. We found that Aurora-A binds and phosphorylates TRF1. When TRF1 knockdown cells are complemented with wild-type TRF1, Aurora-A-induced mitotic defects recur. By contrast, a TRF1 mutant that is not phosphorylatable by Aurora-A does not restore such Aurora-A-induced phenotype. We propose that TRF1 phosphorylation by excessive Aurora-A may provoke abnormal mitosis and chromosomal instability.

Evidence of carrier-mediated transport in the penetration of donepezil into the rat brain.

The objective of this study was to characterize the mechanism that controls the transport of donepezil into the brain. The apparent brain uptake clearance (CL(app,br)) was decreased as a function of the dose of donepezil, suggesting an involvement of a saturable transport process via transporter(s) in the penetration across the blood-brain barrier (BBB). Consistent with in vivo results, the uptake of substrates for organic cation transporters was significantly reduced by donepezil in both MBEC4 cells (i.e., a model for BBB) and HEK 293 cells expressing the transporters found in the brain, indicative of the involvement of organic cation transporters in the transport of the drug. Furthermore, donepezil transport was enhanced (p < 0.01) in HEK 293 cells expressing rOCNT1, rOCTN2, or rCHT1. The CL(app,br) was reduced up to 52.8% of the control in rats that had been pretreated with choline, while the CL(app,br) was unaffected with pretreatments with organic cations other than choline, suggesting that choline and donepezil share a common transport mechanism in the penetration across the BBB in vivo. Taken together, these observations suggest that the transport of donepezil across the BBB is mediated by organic cation transporters such as choline transport system(s).

Modulation of Wnt signaling by the nuclear localization of cellular FLIP-L.

Cellular FLIP (cFLIP) inhibits the apoptosis signaling initiated by death receptor ligation. We previously reported that a long form of cFLIP (cFLIP-L) enhances Wnt signaling via inhibition of beta-catenin ubiquitylation. In this report, we present evidence that cFLIP-L translocates into the nucleus, which could have a role in modulation of Wnt signaling. cFLIP-L has a functional bipartite nuclear localization signal (NLS) at the C-terminus. Wild-type cFLIP-L (wt-FLIP-L) localizes in both the nucleus and cytoplasm, whereas NLS-mutated cFLIP-L localizes predominantly in the cytoplasm. cFLIP-L also has a nuclear export signal (NES) near the NLS, and leptomycin B, an inhibitor of CRM1-dependent nuclear export, increases the nuclear accumulation of cFLIP-L, suggesting that it shuttles between the nucleus and cytoplasm. Expression of mutant cFLIP-L proteins with a deletion or mutations in the NLS and NES confers resistance to Fas-mediated apoptosis, as does wt-FLIP-L, but they do not enhance Wnt signaling, which suggests an important role of the C-terminus of cFLIP-L in Wnt-signaling modulation. When wt-FLIP-L is expressed in the cytoplasm by conjugation with exogenous NES (NES-FLIP-L), Wnt signaling is not enhanced, whereas the NES-FLIP-L increases cytoplasmic beta-catenin as efficiently as wt-FLIP-L. cFLIP-L physically interacts with the reporter plasmid for Wnt signaling, but not with the control plasmid. These results suggest a role for nuclear cFLIP-L in the modulation of Wnt signaling.

Prevention of carbon nanohorn agglomeration using a conjugate composed of comb-shaped polyethylene glycol and a peptide aptamer.

Assured dispersibility is a prerequisite for clinical application of nanomaterials. Carbon nanomaterials have hydrophobic surfaces and thus readily agglomerate under aqueous conditions. Various conjugates composed of a carbon surface-binding moiety and polyethylene glycol (PEG) have been examined as dispersants for carbon nanomaterials. Here we synthesized a conjugate composed of a comb-shaped PEG (cPEG) and carbon nanomaterial-binding peptide (NHBP-1). The resultant cPEG-NHBP3 conjugate displayed multiple units (2.4 on average) of NHBP-1 on a single cPEG molecule whose average molecular weight was 15-20 kDa. cPEG-NHBP3 endowed single-walled carbon nanohorns (SWNHs) with good dispersibility in vitro, which could not be achieved with 20PEG-NHBP, a conjugate composed of linear 20 kDa PEG and a single NHBP-1 peptide. Notably, cPEG-NHBP1, which was similar to 20PEG-NHBP but had a comb-shaped PEG backbone, functioned better as a dispersant than 20PEG-NHBP, suggesting a graft-type PEG formula is better-suited for dispersing nanomaterials. Finally, cPEG-NHBP3 treatment substantially suppressed formation of SWNH agglomerates in mouse lung, suggesting the potential utility of SWNHs as a carrier in drug delivery systems.

Induction of oncogene addiction shift to NF-kappaB by camptothecin in solid tumor cells.

The biological basis of the resistance of solid tumor cells to chemotherapy is not well understood. While addressing this problem, we found that gastric cancer cell line St-4/CPT, lung cancer cell line A549/CPT, and colon cancer cell line HT-29/CPT, all of which are resistant to camptothecin (CPT), showed strong and constitutive nuclear factor (NF)-kappaB activity driven by IkappaB kinase compared with their parental cell lines St-4, A549, and HT-29. A new NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), reduced viability and induced apoptosis in St-4/CPT, A549/CPT, and HT-29/CPT cell lines, while their parental cell lines were resistant to DHMEQ. The results in this study present an example of the shift in signals that support the survival of solid tumor cells to NF-kappaB during the acquisition of resistance to CPT. The results also indicate that solid tumor cells that become resistant to chemotherapy may be more easily treated by NF-kappaB inhibitors.

Dofequidar fumarate sensitizes cancer stem-like side population cells to chemotherapeutic drugs by inhibiting ABCG2/BCRP-mediated drug export.

The ATP-binding cassette (ABC) transporters (ABC-T) actively efflux structurally and mechanistically unrelated anticancer drugs from cells. As a consequence, they can confer multidrug resistance (MDR) to cancer cells. ABC-T are also reported to be phenotypic markers and functional regulators of cancer stem/initiating cells (CSC) and believed to be associated with tumor initiation, progression, and relapse. Dofequidar fumarate, an orally active quinoline compound, has been reported to overcome MDR by inhibiting ABCB1/P-gp, ABCC1/MDR-associated protein 1, or both. Phase III clinical trials suggested that dofequidar had efficacy in patients who had not received prior therapy. Here we show that dofequidar inhibits the efflux of chemotherapeutic drugs and increases the sensitivity to anticancer drugs in CSC-like side population (SP) cells isolated from various cancer cell lines. Dofequidar treatment greatly reduced the cell number in the SP fraction. Estimation of ABC-T expression revealed that ABCG2/breast cancer resistance protein (BCRP) mRNA level, but not the ABCB1/P-gp or ABCC1/MDR-associated protein 1 mRNA level, in all the tested SP cells was higher than that in non-SP cells. The in vitro vesicle transporter assay clarified that dofequidar had the ability to suppress ABCG2/BCRP function. Dofequidar treatment sensitized SP cells to anticancer agents in vitro. We compared the antitumor efficacy of irinotecan (CPT-11) alone with that of CPT-11 plus dofequidar in xenografted SP cells. Although xenografted SP tumors showed resistance to CPT-11, treatment with CPT-11 plus dofequidar greatly reduced the SP-derived tumor growth in vivo. Our results suggest the possibility of selective eradication of CSC by inhibiting ABCG2/BCRP.

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.

Biodistribution and ultrastructural localization of single-walled carbon nanohorns determined in vivo with embedded Gd2O3 labels.

Single-walled carbon nanohorns (SWNHs) are single-graphene tubules that have shown high potential for drug delivery systems. In drug delivery, it is essential to quantitatively determine biodistribution and ultrastructural localization. However, to date, these determinations have not been successfully achieved. In this report, we describe for the first time a method that can achieve these determinations. We embedded Gd(2)O(3) nanoparticles within SWNH aggregates (Gd(2)O(3)@SWNHag) to facilitate detection and quantification. Gd(2)O(3)@SWNHag was intravenously injected into mice, and the quantities of Gd in the internal organs were measured by inductively coupled plasma atomic emission spectroscopy: 70-80% of the total injected material accumulated in liver. The high electron scattering ability of Gd allows detection with energy dispersive X-ray spectroscopy and facilitates the ultrastructural localization of individual Gd(2)O(3)@SWNHag with transmission electron microscopy. In the liver, we found that the Gd(2)O(3)@SWNHag was localized in Kupffer cells but were not observed in hepatocytes. In the Kupffer cells, most of the Gd(2)O(3)@SWNHag was detected inside phagosomes, but some were in another cytoplasmic compartment that was most likely the phagolysosome.

Intestinal epithelial cancer cell anoikis resistance: EGFR-mediated sustained activation of Src overrides Fak-dependent signaling to MEK/Erk and/or PI3-K/Akt-1.

Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3-K/Akt-1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down-activation, cancer cells nevertheless exhibit sustained Fak-Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3-K/Akt-1 down-activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3-K/Akt-1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak-mediated signaling to MEK/Erk and PI3-K/Akt-1 in T84 cells, as in undifferentiated IECs, whereas PI3-K/Akt-1 is Src-independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak-Src interactions and down-activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3-K/Akt-1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR-mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak-Src interactions and MEK/Erk activation, thus not only overriding Fak-mediated signaling to MEK/Erk and/or PI3-K/Akt-1, but also the requirement of Fak and/or PI3-K/Akt-1 for survival.

Acyl-CoA synthetase as a cancer survival factor: its inhibition enhances the efficacy of etoposide.

Lipid metabolism is often elevated in cancer cells and plays an important role in their growth and malignancy. Acyl-CoA synthetase (ACS), which converts long-chain fatty acids to acyl-CoA, is overexpressed in various types of cancer. However, the role of ACS in cancer remains unknown. Here, we found that ACS enzyme activity is required for cancer cell survival. Namely, the ACS inhibitor Triacsin c induced massive apoptosis in glioma cells while this cell death was completely suppressed by overexpression of ACSL5, the Triacsin c-resistant ACS isozyme, but not by overexpression of a catalytically inactive ACSL5 mutant. ACS inhibition by Triacsin c markedly potentiated the Bax-induced intrinsic apoptotic pathway by promoting cytochrome c release and subsequent caspase activation. These effects were abrogated by ACSL5 overexpression. Correspondingly, ACS inhibition synergistically potentiated the glioma cell death induced by etoposide, a well-known activator of apoptosis. Furthermore, in a nude mouse xenograft model, Triacsin c at a non-toxic dose enhanced the antitumor efficacy of a low-dose chemotherapy with etoposide. These results indicate that ACS is an apoptosis suppressor and that ACS inhibition could be a rational strategy to amplify the antitumor effect of etoposide.

Chemical genomics identifies the unfolded protein response as a target for selective cancer cell killing during glucose deprivation.

Glucose deprivation, a cell condition that occurs in solid tumors, activates the unfolded protein response (UPR). A key feature of the UPR is the transcription program activation, which allows the cell to survive under stress conditions. Here, we show that the UPR transcription program is disrupted by the antidiabetic biguanides metformin, buformin, and phenformin depending on cellular glucose availability. These drugs inhibit production of the UPR transcription activators XBP1 and ATF4 and induce massive cell death during glucose deprivation as did the antitumor macrocyclic compound versipelostatin. Gene expression profiling shows remarkable similarity in the modes of action of biguanides and versipelostatin determined by the broad range of glucose deprivation-inducible genes. Importantly, during glucose deprivation, most of the biguanide suppression genes overlap with the genes induced by tunicamycin, a chemical UPR inducer. Gene expression profiling also identifies drug-driven signatures as a tool for discovering pharmacologic UPR modulators. Our findings show that disrupting the UPR during glucose deprivation could be an attractive approach for selective cancer cell killing and could provide a chemical genomic basis for developing UPR-targeting drugs against solid tumors.

PRMT5, a novel TRAIL receptor-binding protein, inhibits TRAIL-induced apoptosis via nuclear factor-kappaB activation.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily and has selective antitumor activity. Although TNF-alpha-induced intracellular signaling pathways have been well studied, TRAIL signaling is not fully understood. Here, we identified a novel TRAIL receptor-binding protein, protein arginine methyltransferase 5 (PRMT5), as a result of proteomic screening. PRMT5 selectively interacted with death receptor 4 and death receptor 5 but not with TNF receptor 1 or Fas. PRMT5 gene silencing sensitized various cancer cells to TRAIL without affecting TRAIL resistance in nontransformed cells. PRMT5 contributed to TRAIL-induced activation of inhibitor of kappaB kinase (IKK) and nuclear factor-kappaB (NF-kappaB), leading to induction of several NF-kappaB target genes. Although IKK inhibition increased sensitivity to both TRAIL and TNF-alpha, PRMT5 knockdown potentiated TRAIL-mediated cytotoxicity alone. PRMT5 had no effect on TNF-alpha-mediated NF-kappaB signaling. These results show the selectivity of PRMT5 for TRAIL signaling. The PRMT5 small interfering RNA-mediated susceptibility to TRAIL was rescued by ectopic expression of active IKKbeta, confirming the involvement of PRMT5 in TRAIL resistance by activating the NF-kappaB pathway. Collectively, our findings suggest the therapeutic potential of PRMT5 in TRAIL-based cancer treatments

Involvement of Fractin in TGF-beta-induced apoptosis in oligodendroglial progenitor cells.

Transforming growth factor-beta (TGF-beta) induces apoptotic cell death during the development of the nervous system. We recently identified that TGF-beta induced apoptosis in oligodendroglial progenitor cells (primary cells as well as oligodendroglial cell line OLI-neu) is characterized by down-regulation of Bcl-xl. In this report, we now focused on mechanisms that mediate TGF-beta dependent Bcl-xl down-regulation in oligodendroglial cells. We showed that the caspase-specific cleavage product Fractin is produced in oligodendroglial cells during TGF-beta-mediated apoptosis, which represents an early event of the cascade. Cleavage of actin into Fractin was dependent on functional actin and caspases, and occurred simultaneously with a Fractin-Bcl-xl-interaction. This Fractin-Bcl-xl interaction indicated a connection between Bcl-xl down-regulation and Fractin appearance, since Bcl-xl regulation was also dependent on caspases and functional actin, and an overexpression of Fractin induced a Bcl-xl protein down-regulation. Further analysis of Fractin-Bcl-xl interaction in other culture systems confirmed these data. In conclusion, we show that Fractin is not only an apoptotic marker, but has indeed a functional role in apoptotic signaling in oligodendrocytes.

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.

Platelet aggregation in the formation of tumor metastasis.

Metastasis is the major cause of death from cancer, yet the optimal strategy against it remains uncertain. The pathogenesis of hematogenous metastasis is dynamic and consists of the following steps: 1) detachment of tumor cells from the primary site, 2) invasion into the host's blood vessels, 3) migration in the host's blood stream, 4) transport along the circulation, 5) arrest in or adhesion to the capillary in a distant organ, 6) extravasation, and 7) proliferation within the foreign tissues. A key to successful hematogenous metastasis is tumor survival in the bloodstream because most circulating tumor cells are rapidly destroyed by the shear forces or are attacked by the immune system. Less than 0.01% of these cells result in metastasis. Tumor cell-induced platelet aggregation has been reported to facilitate hematogenous metastasis by increasing the arrest of tumor cell emboli in the microcirculation. Platelet aggregation is also believed to protect tumor cells from immunological assault in the circulation. We have identified Aggrus as a platelet-aggregating factor expressed on a number of human cancers. Because hematogenous metastasis is reduced when neutralizing antibodies or eliminating carbohydrates attenuates Aggrus function, Aggrus's main contribution to hematogenous metastasis of Aggrus-expressing cells, then, is by promoting platelet aggregation. Aggrus could serve as an ideal target for drug development to block metastasis.

IL12RB2 and ABCA1 genes are associated with susceptibility to radiation dermatitis.

PURPOSE: Severe acute radiation dermatitis is observed in approximately 5% to 10% of patients who receive whole-breast radiotherapy. Several factors, including treatment-related and patient-oriented factors, are involved in susceptibility to severe dermatitis. Genetic factors are also thought to be related to a patient's susceptibility to severe dermatitis. To elucidate genetic polymorphisms associated with a susceptibility to radiation-induced dermatitis, a large-scale single-nucleotide polymorphism (SNP) analysis using DNA samples from 156 patients with breast cancer was conducted. EXPERIMENTAL DESIGN: Patients were selected from more than 3,000 female patients with early breast cancer who received radiotherapy after undergoing breast-conserving surgery. The dermatitis group was defined as patients who developed dermatitis at a National Cancer Institute Common Toxicity Criteria grade of > or =2. For the SNP analysis, DNA samples from each patient were subjected to the genotyping of 3,144 SNPs covering 494 genes. RESULTS: SNPs that mapped to two genes, ABCA1 and IL12RB2, were associated with radiation-induced dermatitis. In the ABCA1 gene, one of these SNPs was a nonsynonymous coding SNP causing R219K (P = 0.0065). As for the IL12RB2 gene, the strongest association was observed at SNP-K (rs3790568; P = 0.0013). Using polymorphisms of both genes, the probability of severe dermatitis was estimated for each combination of genotypes. These analyses showed that individuals carrying a combination of genotypes accounting for 14.7% of the Japanese population have the highest probability of developing radiation-induced dermatitis. CONCLUSION: Our results shed light on the mechanisms responsible for radiation-induced dermatitis. These results may also contribute to the individualization of radiotherapy.

Involvement of the lysophosphatidic acid-generating enzyme autotaxin in lymphocyte-endothelial cell interactions.

Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.