Transcriptome-based variant calling and aberrant mRNA discovery enhance diagnostic efficiency for neuromuscular diseases.

BACKGROUND: Whole-exome sequencing-based diagnosis of rare diseases typically yields 40%-50% of success rate. Precise diagnosis of the patients with neuromuscular disorders (NMDs) has been hampered by locus heterogeneity or phenotypic heterogeneity. We evaluated the utility of transcriptome sequencing as an independent approach in diagnosing NMDs. METHODS: The RNA sequencing (RNA-Seq) of muscle tissues from 117 Korean patients with suspected Mendelian NMD was performed to evaluate the ability to detect pathogenic variants. Aberrant splicing and CNVs were inspected to identify additional causal genetic factors for NMD. Aberrant splicing events in Dystrophin (DMD) were investigated by using antisense oligonucleotides (ASOs). A non-negative matrix factorisation analysis of the transcriptome data followed by cell type deconvolution was performed to cluster samples by expression-based signatures and identify cluster-specific gene ontologies. RESULTS: Our pipeline called 38.1% of pathogenic variants exclusively from the muscle transcriptomes, demonstrating a higher diagnostic rate than that achieved via exome analysis (34.9%). The discovery of variants causing aberrant splicing allowed the application of ASOs to the patient-derived cells, providing a therapeutic approach tailored to individual patients. RNA-Seq data further enabled sample clustering by distinct gene expression profiles that corresponded to clinical parameters, conferring additional advantages over exome sequencing. CONCLUSION: The RNA-Seq-based diagnosis of NMDs achieves an increased diagnostic rate and provided pathogenic status information, which is not easily accessible through exome analysis.

Knockdown of YAP/TAZ sensitizes tamoxifen-resistant MCF7 breast cancer cells.

Although endocrine therapy with tamoxifen has improved survival in breast cancer patients, resistance to this therapy remains one of the major causes of breast cancer mortality. In the present study, we found that the expression level of YAP/TAZ in tamoxifen-resistant MCF7 (MCF7-TR) breast cancer cells was significantly increased compared with that in MCF7 cells. Knockdown of YAP/TAZ with siRNA sensitized MCF7-TR cells to tamoxifen. Furthermore, siRNA targeting PSAT1, a downstream effector of YAP/TAZ, enhanced sensitivity to tamoxifen in MCF7-TR cells. Additionally, mTORC1 activity and survivin expression were significantly decreased during cell death induced by combination treatment with YAP/TAZ or PSAT1 siRNA and tamoxifen. In conclusion, targeting the YAP/TAZ-PSAT1 axis could sensitize tamoxifen-resistant MCF7 breast cancer cells by modulating the mTORC1-survivin axis.

Inhibition of mTORC1 through ATF4-induced REDD1 and Sestrin2 expression by Metformin.

BACKGROUND: Although the major anticancer effect of metformin involves AMPK-dependent or AMPK-independent mTORC1 inhibition, the mechanisms of action are still not fully understood. METHODS: To investigate the molecular mechanisms underlying the effect of metformin on the mTORC1 inhibition, MTT assay, RT-PCR, and western blot analysis were performed. RESULTS: Metformin induced the expression of ATF4, REDD1, and Sestrin2 concomitant with its inhibition of mTORC1 activity. Treatment with REDD1 or Sestrin2 siRNA reversed the mTORC1 inhibition induced by metformin, indicating that REDD1 and Sestrin2 are important for the inhibition of mTORC1 triggered by metformin treatment. Moreover, REDD1- and Sestrin2-mediated mTORC1 inhibition in response to metformin was independent of AMPK activation. Additionally, lapatinib enhances cell sensitivity to metformin, and knockdown of REDD1 and Sestrin2 decreased cell sensitivity to metformin and lapatinib. CONCLUSIONS: ATF4-induced REDD1 and Sestrin2 expression in response to metformin plays an important role in mTORC1 inhibition independent of AMPK activation, and this signalling pathway could have therapeutic value.

Lysine is required for growth factor-induced mTORC1 activation.

Mechanistic target of rapamycincomplex 1 (mTORC1) integrates various environmental signals to regulate cell growth and metabolism. mTORC1 activity is sensitive to changes in amino acid levels. Here, we investigated the effect of lysine on mTORC1 activity in non-small cell lung cancer (NSCLC) cells. Lysine deprivation suppressed mTORC1 activity and lysine replenishment restored the decreased mTORC1 activity in lysine-deprived cells. Supplementing growth factors, such as insulin growth factor-1 or insulin restored the decreased mTORC1 activity in serum-deprived cells. However, in serum/lysine-deprived cells, supplementing growth factors was not sufficient to restore mTORC1 activity, suggesting thatgrowth factors could not activate mTORC1 efficiently in the absence of lysine. General control nonderepressible 2 and AMP-activated protein kinase were involved in lysine deprivation-mediated inhibition of mTORC1. Taken together, these results suggest that lysine might play role in the regulation of mTORC1 activation in NSCLC cells.

Silencing of secretory clusterin sensitizes NSCLC cells to V-ATPase inhibitors by downregulating survivin.

Secretory clusterin (sCLU) is a stress-associated protein that confers resistance to therapy when overexpressed. In this study, we observed that the V-ATPase inhibitors bafilomycin A1 and concanamycin A significantly stimulated sCLU protein expression. Knockdown of sCLU with siRNA sensitized non-small cell lung cancer (NSCLC) cells to bafilomycin A1, suggesting that sCLU expression renders cells resistant to V-ATPase inhibitors. The dual PI3K/AKT and mTOR inhibitor BEZ235 suppressed sCLU expression and enhanced cell sensitivity induced by bafilomycin A1. Notably, sCLU knockdown further decreased the expression of the survivin protein by bafilomycin A1, and the ectopic expression of survivin alleviated the cell sensitivity by bafilomycin A1 and sCLU depletion, suggesting that increased sensitivity to sCLU depletion in the cells with V-ATPase inhibitors is due, at least in part, to the down-regulation of survivin. Taken together, we demonstrated that the depletion of sCLU expression enhances the sensitivity of NSCLC cells to V-ATPase inhibitors by decreasing survivin expression. Inhibition of the PI3K/AKT/mTOR pathway enhances the sensitivity of NSCLC cells to V-ATPase inhibitors, leading to decreased sCLU and survivin expression. Thus, we suggest that a combination of PI3K/AKT/mTOR inhibitors with V-ATPase inhibitors might be an effective approach for NSCLC treatment.

Targeting HIF-1α is a prerequisite for cell sensitivity to dichloroacetate (DCA) and metformin.

Recently, targeting deregulated energy metabolism is an emerging strategy for cancer therapy. In the present study, combination of DCA and metformin markedly induced cell death, compared with each drug alone. Furthermore, the expression levels of glycolytic enzymes including HK2, LDHA and ENO1 were downregulated by two drugs. Interestingly, HIF-1α activation markedly suppressed DCA/metformin-induced cell death and recovered the expressions of glycolytic enzymes that were decreased by two drugs. Based on these findings, we propose that targeting HIF-1α is necessary for cancer metabolism targeted therapy.

TRAIL restores DCA/metformin-mediated cell death in hypoxia.

Previous studies have shown that hypoxia can reverse DCA/metformin-induced cell death in breast cancer cells. Therefore, targeting hypoxia is necessary for therapies targeting cancer metabolism. In the present study, we found that TRAIL can overcome the effect of hypoxia on the cell death induced by treatment of DCA and metformin in breast cancer cells. Unexpectedly, DR5 is upregulated in the cells treated with DCA/metformin, and sustained under hypoxia. Blocking DR5 by siRNA inhibited DCA/metformin/TRAIL-induced cell death, indicating that DR5 upregulation plays an important role in sensitizing cancer cells to TRAIL-induced cell death. Furthermore, we found that activation of JNK and c-Jun is responsible for upregulation of DR5 induced by DCA/metformin. These findings support the potential application of combining TRAIL and metabolism-targeting drugs in the treatment of cancers under hypoxia.

Combined effects of EGFR tyrosine kinase inhibitors and vATPase inhibitors in NSCLC cells.

Despite excellent initial clinical responses of non-small cell lung cancer (NSCLC) patients to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), many patients eventually develop resistance. According to a recent report, vacuolar H+ ATPase (vATPase) is overexpressed and is associated with chemotherapy drug resistance in NSCLC. We investigated the combined effects of EGFR TKIs and vATPase inhibitors and their underlying mechanisms in the regulation of NSCLC cell death. We found that combined treatment with EGFR TKIs (erlotinib, gefitinib, or lapatinib) and vATPase inhibitors (bafilomycin A1 or concanamycin A) enhanced synergistic cell death compared to treatments with each drug alone. Treatment with bafilomycin A1 or concanamycin A led to the induction of Bnip3 expression in an Hif-1α dependent manner. Knock-down of Hif-1α or Bnip3 by siRNA further enhanced cell death induced by bafilomycin A1, suggesting that Hif-1α/Bnip3 induction promoted resistance to cell death induced by the vATPase inhibitors. EGFR TKIs suppressed Hif-1α and Bnip3 expression induced by the vATPase inhibitors, suggesting that they enhanced the sensitivity of the cells to these inhibitors by decreasing Hif-1α/Bnip3 expression. Taken together, we conclude that EGFR TKIs enhance the sensitivity of NSCLC cells to vATPase inhibitors by decreasing Hif-1α/Bnip3 expression. We suggest that combined treatment with EGFR TKIs and vATPase inhibitors is potentially effective for the treatment of NSCLC.

Blockage of Stat3 enhances the sensitivity of NSCLC cells to PI3K/mTOR inhibition.

The PI3K/Akt/mTOR axis in lung cancer is frequently activated and implicated in tumorigenesis. Specific targeting of this pathway is therefore an attractive therapeutic approach for lung cancer. However, non-small cell lung cancer cells are resistant to BEZ235, a dual inhibitor of PI3K and mTOR. Interestingly, blockage of Stat3 with a selective inhibitor, S3I-201, or siRNA dramatically sensitized the BEZ235-induced cell death, as evident from increased PARP cleavage. Furthermore, inhibition of Stat3 led to enhancement of cell death induced by LY294002, a PI3K inhibitor. Treatment of cells with a combination of BEZ235 and S3I-201 significantly induced the proapoptotic transcription factor, CHOP, and its targets, Bim and DR4. Knockdown of CHOP or Bim suppressed cell death stimulated by the combination treatment, implicating the involvement of these BEZ235/S3I-201-induced factors in pronounced cell death. Moreover, the BEZ235/S3I-201 combination enhanced TRAIL-induced cell death. Our results collectively suggest that blockage of Stat3 presents an effective strategy to overcome resistance to PI3K/Akt/mTOR inhibition.

Inhibition of S6K1 enhances glucose deprivation-induced cell death via downregulation of anti-apoptotic proteins in MCF-7 breast cancer cells.

Nutrient-limiting conditions are frequently encountered by tumor cells in poorly vascularized microenvironments. These stress conditions may facilitate the selection of tumor cells with an inherent ability to decrease apoptotic potential. Therefore, selective targeting of tumor cells under glucose deprivation conditions may provide an effective alternative strategy for cancer therapy. In the present study, we investigated the effects of S6 kinase 1 (S6K1) inhibition on glucose deprivation-induced cell death and the underlying mechanisms in MCF-7 breast cancer cells. PF4708671, a selective inhibitor of S6K1, and knockdown of S6K1 with specific siRNA enhanced cell death induced under glucose deprivation conditions. Moreover, inhibition of S6K1 led to apoptosis in glucose-starved MCF-7 cells via downregulation of the anti-apoptotic proteins, Mcl-1 and survivin. Further experiments revealed that sorafenib, shown to be involved in Mcl-1 and survivin downregulation via mTOR/S6K1 inhibition significantly promotes cell death under glucose deprivation conditions. These findings collectively suggest that S6K1 plays an important role in tumor cell survival under stress conditions, and thus inhibition of S6K1 may be an effective strategy for sensitizing cells to glucose deprivation.

S6K1 inhibition enhances tamoxifen-induced cell death in MCF-7 cells through translational inhibition of Mcl-1 and survivin.

S6 kinase 1 (S6K1) was suggested to be a marker for endocrine therapy resistance in breast cancer. We examined whether tamoxifen's effect can be modulated by S6K1 inhibition. S6K1 inhibition by PF4708671, a selective inhibitor of S6K1, acts synergistically with tamoxifen in S6K1-high MCF-7 cells. Similarly, the knockdown of S6K1 with small interfering RNA (siRNA) significantly sensitized MCF-7 cells to tamoxifen. Inhibition of S6K1 by PF4708671 led to a marked decrease in the expression levels of the anti-apoptotic proteins Mcl-1 and survivin, which was not related to mRNA levels. In addition, suppression of Mcl-1 or survivin, using specific siRNA, further enhanced cell sensitivity to tamoxifen. These results showed that inhibition of S6K1 acts synergistically with tamoxifen, via translational modulation of Mcl-1 and survivin. Based on these findings, we propose that targeting S6K1 may be an effective strategy to overcome tamoxifen resistance in breast cancer.

Nebivolol Sensitizes BT-474 Breast Cancer Cells to FGFR Inhibitors.

BACKGROUND/AIM: The fibroblast growth factor receptor (FGFR) signaling pathway is abnormally activated in human cancers, including breast cancer. Therefore, targeting the FGFR signaling pathway is a potent strategy to treat breast cancer. The purpose of this study was to find drugs that could increase sensitivity to FGFR inhibitor effects in BT-474 breast cancer cells, and to investigate the combined effects and underlying mechanisms of these combinations for BT-474 breast cancer cell survival. MATERIALS AND METHODS: Cell viability was measured by MTT assay. Protein expression was determined by western blot analysis. mRNA expression was detected by Real-time PCR. Drug synergy effect was determined by isobologram analysis. RESULTS: Nebivolol, a third generation ß1-blocker, synergistically increased the sensitivity of BT-474 breast cancer cells to the potent and selective FGFR inhibitors erdafitinib (JNJ-42756493) and AZD4547. A combination of nebivolol and erdafitinib markedly reduced AKT activation. Suppression of AKT activation using specific siRNA and a selective inhibitor further enhanced cell sensitivity to combined treatment with nebivolol and erdafitinib, whereas SC79, a potent activator of AKT, reduced cell sensitivity to nebivolol and erdafitinib. CONCLUSION: Enhanced sensitivity of BT-474 breast cancer cells to nebivolol and erdafitinib was probably associated with down-regulation of AKT activation. Combined treatment with nebivolol and erdafitinib is a promising strategy for breast cancer treatment.

Knockdown of NUPR1 Enhances the Sensitivity of Non-small-cell Lung Cancer Cells to Metformin by AKT Inhibition.

BACKGROUND/AIM: Metformin is a widely used drug for type 2 diabetes mellitus and has recently attracted broad attention for its therapeutic effects on many cancers. This study aimed to investigate the molecular mechanism of metformin's anticancer activity. MATERIALS AND METHODS: Cell viability was measured by MTT assay. Gene and protein expression levels were determined by reverse transcription-polymerase chain reaction and western blot analyses, respectively. RESULTS: Metformin and phenformin markedly induced NUPR1 expression in a dose- and time-dependent manner in H1299 non-small-cell lung cancer (NSCLC) cells. The silencing of NUPR1 in H1299 NSCLC cells enhanced cell sensitivity to metformin or ionizing radiation. Our previous report showed that metformin induces AKT serine/threonine kinase (AKT) activation in an activating transcription factor 4 (ATF4)-dependent manner and that the inhibition of AKT promotes cell sensitivity to metformin in H1299 NSCLC cells. Interestingly, ATF4-induced AKT activation in H1299 NSCLC cells treated with metformin was suppressed by the knockdown of NUPR1. CONCLUSION: Targeting NUPR1 could enhance the sensitivity of H1299 NSCLC cells to metformin by AKT inhibition.

Systematic analysis of inheritance pattern determination in genes that cause rare neurodevelopmental diseases.

Despite recent advancements in our understanding of genetic etiology and its molecular and physiological consequences, it is not yet clear what genetic features determine the inheritance pattern of a disease. To address this issue, we conducted whole exome sequencing analysis to characterize genetic variants in 1,180 Korean patients with neurological symptoms. The diagnostic yield for definitive pathogenic variant findings was 50.8%, after including 33 cases (5.9%) additionally diagnosed by reanalysis. Of diagnosed patients, 33.4% carried inherited variants. At the genetic level, autosomal recessive-inherited genes were characterized by enrichments in metabolic process, muscle organization and metal ion homeostasis pathways. Transcriptome and interactome profiling analyses revealed less brain-centered expression and fewer protein-protein interactions for recessive genes. The majority of autosomal recessive genes were more tolerant of variation, and functional prediction scores of recessively-inherited variants tended to be lower than those of dominantly-inherited variants. Additionally, we were able to predict the rates of carriers for recessive variants. Our results showed that genes responsible for neurodevelopmental disorders harbor different molecular mechanisms and expression patterns according to their inheritance patterns. Also, calculated frequency rates for recessive variants could be utilized to pre-screen rare neurodevelopmental disorder carriers.

Redd1 inhibits the invasiveness of non-small cell lung cancer cells.

Redd1 acts as a negative regulator of mTOR in response to various stress conditions, but its specific physiological role is currently unclear. In the present study, we showed that Redd1 inhibits the invasive activity of non-small cell lung cancer (NSCLC) cells. Interestingly, expression of Redd1 was extremely low in H1299 cells displaying high invasiveness, compared with that in H460 cells with lower invasive activity. Overexpression of Redd1 inhibited the invasive activity of H1299 cells, while suppression with specific siRNAs enhanced the invasiveness of H460 cells. Knockdown of the mTOR downstream substrate, S6K, resulted in a decrease in the invasive property of H1299 cells. Our results provide preliminary evidence that Redd1 inhibits the invasive activity of NSCLC cells via suppression of the mTOR downstream pathway.

Amino acid deprivation induces AKT activation by inducing GCN2/ATF4/REDD1 axis.

Amino acid availability is sensed by various signaling molecules, including general control nonderepressible 2 (GCN2) and mechanistic target of rapamycin complex 1 (mTORC1). However, it is unclear how these sensors are associated with cancer cell survival under low amino acid availability. In the present study, we investigated AKT activation in non-small cell lung cancer (NSCLC) cells deprived of each one of 20 amino acids. Among the 20 amino acids, deprivation of glutamine, arginine, methionine, and lysine induced AKT activation. AKT activation was induced by GCN2/ATF4/REDD1 axis-mediated mTORC2 activation under amino acid deprivation. In CRISPR-Cas9-mediated REDD1-knockout cells, AKT activation was not induced by amino acid deprivation, indicating that REDD1 plays a major role in AKT activation under amino acid deprivation. Knockout of REDD1 sensitized cells cultured under glutamine deprivation conditions to radiotherapy. Taken together, GCN2/ATF4/REDD1 axis induced by amino acid deprivation promotes cell survival signal, which might be a potential target for cancer therapy.

Preparation and Characterization of Planar-Type ZnO Powder with High Aspect Ratio for Application in Ultraviolet- and Heat-Shield Cosmetics.

In this study, a [0001]-plane planar-type ZnO ceramic powder material with a high aspect ratio ranging from 20:1-50:1 is synthesized using the electrolyte collected from zinc air battery power generation. This high aspect ratio may be due to the Zn(OH)2-4 anion dissolved in the electrolyte. The obtained planar-type ZnO exhibits excellent formulation stability and applicability, even when formulated as a cosmetic with a single inorganic ingredient. Compared to commercial ZnO or TiO2 powders, relatively better protection against infrared and ultraviolet (UV) radiation is realized due to its asymmetric characteristics, with a width of approximately 1 µm and thickness of tens of nm. The synthesized planar-type ZnO is mixed with nanosized ZnO or TiO2 commercial powders and formulated into various combinations to achieve a high UV protection rate and heat-blocking effect. In particular, the addition of planar-type ZnO to nanosized TiO2 powders increases the heat-blocking effect, and improves the applicability and formulation stability of the cosmetic formulation, despite the decrease in turbidity. Among all the ceramic powder combinations examined in this study, the best UV protection rate and heat-blocking effect are obtained when the synthesized planar-type ZnO is mixed with microsized and nanosized TiO2.

Inhibition of AKT Enhances the Sensitivity of NSCLC Cells to Metformin.

BACKGROUND/AIM: Metformin is an antidiabetic drug that has been reported to have antitumor activity in many cancer types. This study investigated the molecular mechanisms underlying the antitumor effect of metformin. MATERIALS AND METHODS: We investigated the molecular mechanism of the antitumor effect of metformin alone and in combination with AKT serine/threonine kinase (AKT) inhibition via cell viability and western blot analyses. RESULTS: Notably, metformin increased the phosphorylation of AKT at serine 473 using protein array screening. Metformin-induced AKT activation was markedly suppressed by siRNA targeting activating transcription factor 4 (ATF4) but not AMP-activated protein kinase α. These results indicate that AKT activation by metformin was induced in an ATF4-dependent and AMPKα-independent manner. Treatment using metformin combined with MK-2206, an AKT inhibitor, or a siRNA for AKT markedly reduced the viability of cells compared with those cells treated with these agents alone. In addition, MK-2206 increased cell sensitivity to the combination of metformin with ionizing radiation or cisplatin. CONCLUSION: Inhibition of AKT can enhance the antitumor effect of metformin and would be a promising strategy to sensitize non-small-cell lung cancer to a combination of metformin with radiation or cisplatin.

miR-3188 Enhances Sensitivity of Breast Cancer Cells to Ionizing Radiation by Down-regulating Rictor.

BACKGROUND/AIM: Rictor is an adaptor protein essential for mTORC2, which regulates cell growth and survival. The aim of this study was to identify microRNAs (miR) that down-regulate Rictor and investigate their function on breast cancer cell survival. MATERIALS AND METHODS: Trypan blue assay, MTT assay, polymerase chain reaction analysis, luciferase reporter assay and western blot analysis were carried out in breast cancer cell lines HCC1954, MDA-MB-231, SK-BR-3, and BT474. RESULTS: miR-3188 overexpression suppressed the expression of Rictor and inhibited cell viability in HCC1954 and MDA-MB-231, highly Rictor-expressing breast cancer cells. In addition, miR-3188 overexpression decreased the protein level of p-AKT at Ser473, a substrate of mTORC2. Moreover, miRNA-3188 overexpression sensitized breast cancer cells to ionizing radiation (IR) by down-regulating Rictor and p-AKT. CONCLUSION: miR-3188 enhances IR sensitivity by affecting the mTORC2/AKT signalling pathway by altering the expression of Rictor, which could be a promising therapeutic strategy for the future treatment of breast cancer.