Downregulation of TRIB3 enhances the sensitivity of lung cancer cells to amino acid deprivation by suppressing AKT activation.

Tribbles pseudokinase 3 (TRIB3), a member of the mammalian Tribbles family, is implicated in multiple biological processes. This study aimed to investigate the biological functions of TRIB3 in lung cancer and its effect on amino acid-deprived lung cancer cells. TRIB3 mRNA expression was elevated in lung cancer tissues and cell lines compared to normal lung tissues and cells. TRIB3 knockdown markedly reduced the viability and proliferation of H1299 lung cancer cells. Deprivation of amino acids, particularly arginine, glutamine, lysine, or methionine, strongly increased TRIB3 expression via ATF4 activation in H1299 lung cancer cells. Knockdown of TRIB3 led to transcriptional downregulation of ATF4 and reduced AKT activation induced by amino acid deprivation, ultimately increasing the sensitivity of H1299 lung cancer cells to amino acid deprivation. Additionally, TRIB3 knockdown enhanced the sensitivity of H1299 cells to V-9302, a competitive antagonist of transmembrane glutamine flux. These results suggest that TRIB3 is a pro-survival regulator of cell viability in amino acid-deficient tumor microenvironments and a promising therapeutic target for lung cancer treatment.

Duloxetine enhances the sensitivity of non-small cell lung cancer cells to EGFR inhibitors by REDD1-induced mTORC1/S6K1 suppression.

Although epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) have been effective targeted therapies for non-small cell lung cancer (NSCLC), most advanced NSCLC inevitably develop resistance to these therapies. Combination therapies emerge as valuable approach to preventing, delaying, or overcoming disease progression. Duloxetine, an antidepressant known as a serotonin-noradrenaline reuptake inhibitor, is commonly prescribed for the treatment of chemotherapy-induced peripheral neuropathy. In the present study, we investigated the combined effects of duloxetine and EGFR-TKIs and their possible mechanism in NSCLC cells. Compared with either monotherapy, the combination of duloxetine and EGFR-TKIs leads to synergistic cell death. Mechanistically, duloxetine suppresses 70-kDa ribosomal protein S6 kinase 1 (p70S6K1) activity through mechanistic target of rapamycin complex 1 (mTORC1), and this effect is associated with the synergistic induction of cell death of duloxetine combined with EGFR-TKIs. More importantly, activating transcription factor 4 (ATF4)-induced regulated in development and DNA damage response 1 (REDD1) is responsible for the suppression of mTORC1/S6K1 activation. Additionally, we found that the combination effect was significantly attenuated in REDD1 knockout NSCLC cells. Taken together, our findings reveal that the ATF4/REDD1/mTORC1/S6K1 signaling axis, as a novel mechanism, is responsible for the synergistic therapeutic effect of duloxetine with EGFR-TKIs. These results suggest that combining EGFR-TKIs with duloxetine appears to be a promising way to improve EGFR-TKI efficacy against NSCLC.

Amino acid deprivation induces TXNIP expression by NRF2 downregulation.

Thioredoxin-interacting protein (TXNIP) is sensitive to oxidative stress and is involved in the pathogenesis of various metabolic, cardiovascular, and neurodegenerative disorders. Therefore, several studies have suggested that TXNIP is a promising therapeutic target for several diseases, particularly cancer and diabetes. However, the regulation of TXNIP expression under amino acid (AA)-restricted conditions is not well understood. In the present study, we demonstrated that TXNIP expression was promoted by the deprivation of AAs, especially arginine, glutamine, lysine, and methionine, in non-small cell lung cancer (NSCLC) cells. Interestingly, we determined that increased TXNIP expression induced by AA deprivation was associated with nuclear factor erythroid 2-related factor 2 (NRF2) downregulation, but not with activating transcription factor 4 (ATF4) activation. Furthermore, N-acetyl-l-cysteine (NAC), a scavenger of reactive oxygen species (ROS), suppressed TXNIP expression in NSCLC cells deprived of AA. Collectively, the induction of TXNIP expression by AA deprivation was mediated by ROS production, potentially through NRF2 downregulation. Our findings suggest that TXNIP expression may be associated with the redox homeostasis of AA metabolism and provide a possible rationale for a therapeutic strategy to treat cancer with AA restriction.

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.

Inhibition of Glutamine Uptake Resensitizes Paclitaxel Resistance in SKOV3-TR Ovarian Cancer Cell via mTORC1/S6K Signaling Pathway.

Ovarian cancer is a carcinoma that affects women and that has a high mortality rate. Overcoming paclitaxel resistance is important for clinical application. However, the effect of amino acid metabolism regulation on paclitaxel-resistant ovarian cancer is still unknown. In this study, the effect of an amino acid-deprived condition on paclitaxel resistance in paclitaxel-resistant SKOV3-TR cells was analyzed. We analyzed the cell viability of SKOV3-TR in culture conditions in which each of the 20 amino acids were deprived. As a result, the cell viability of the SKOV3-TR was significantly reduced in cultures deprived of arginine, glutamine, and lysine. Furthermore, we showed that the glutamine-deprived condition inhibited mTORC1/S6K signaling. The decreased cell viability and mTORC1/S6K signaling under glutamine-deprived conditions could be restored by glutamine and α-KG supplementation. Treatment with PF-4708671, a selective S6K inhibitor, and the selective glutamine transporter ASCT2 inhibitor V-9302 downregulated mTOR/S6K signaling and resensitized SKOV3-TR to paclitaxel. Immunoblotting showed the upregulation of Bcl-2 phosphorylation and a decrease in Mcl-1 expression in SKOV3-TR via the cotreatment of paclitaxel with PF-4708671 and V-9302. Collectively, this study demonstrates that the inhibition of glutamine uptake can resensitize SKOV3-TR to paclitaxel and represents a promising therapeutic target for overcoming paclitaxel resistance in ovarian cancer.

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.

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.

Synergism of a novel MCL­1 downregulator, acriflavine, with navitoclax (ABT­263) in triple­negative breast cancer, lung adenocarcinoma and glioblastoma multiforme.

Myeloid cell leukemia sequence 1 (MCL­1), an anti­apoptotic B­cell lymphoma 2 (BCL­2) family molecule frequently amplified in various human cancer cells, is known to be critical for cancer cell survival. MCL­1 has been recognized as a target molecule for cancer treatment. While various agents have emerged as potential MCL­1 blockers, the present study presented acriflavine (ACF) as a novel MCL­1 inhibitor in triple­negative breast cancer (TNBC). Further evaluation of its treatment potential on lung adenocarcinoma and glioblastoma multiforme (GBM) was also investigated. The anticancer effect of ACF on TNBC cells was demonstrated when MDA­MB­231 and HS578T cells were treated with ACF. ACF significantly induced typical intrinsic apoptosis in TNBCs in a dose­ and time­dependent manner via MCL­1 downregulation. MCL­1 downregulation by ACF treatment was revealed at each phase of protein expression. Initially, transcriptional regulation via reverse transcription­quantitative PCR was validated. Then, post­translational regulation was explained by utilizing an inhibitor against protein biosynthesis and proteasome. Lastly, immunoprecipitation of ubiquitinated MCL­1 confirmed the post­translational downregulation of MCL­1. In addition, the synergistic treatment efficacy of ACF with the well­known MCL­1 inhibitor ABT­263 against the TNBC cells was explored [combination index (CI)<1]. Conjointly, the anticancer effect of ACF was assessed in GBM (U87, U251 and U343), and lung cancer (A549 and NCI­H69) cell lines as well, using immunoblotting, cytotoxicity assay and FACS. The effect of the combination treatment using ACF and ABT­263 was estimated in GBM (U87, U343 and U251), and non­small cell lung cancer (A549) cells likewise. The present study suggested a novel MCL­1 inhibitory function of ACF and the synergistic antitumor effect with ABT­263.

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.

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.

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.

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.

Hypoxia Suppresses Cysteine Deprivation-induced Cell Death Via ATF4 Regulation in MDA-MB-231 Breast Cancer Cells.

BACKGROUND/AIM: Cancer cells are frequently exposed to microenvironmental stresses, including amino acid deprivation and hypoxia, which are often targeted for cancer therapy. Here, we examined the effect of hypoxia in cysteine-deprived breast cancer cells and the mechanism to counteract the hypoxia effect. MATERIALS AND METHODS: Cell death was determined by annexin V-FITC and propidium iodide staining. Expression of mRNAs and proteins was determined by reverse transcription polymerase chain reaction and western blot analysis, respectively. RESULTS: Cysteine deprivation or sulfasalazine, a potent inhibitor of cysteine/glutamate transporter, induced cell death by activating transcription factor 4 (ATF4) up-regulation. Hypoxia significantly suppressed cell death and ATF4 up-regulation induced by cysteine deprived conditions. In addition, tumor necrosis factor-related apoptosis-inducing ligand reversed the effect of hypoxia on cysteine deprived conditions. CONCLUSION: Prevention of hypoxia may be a means for augmenting the effect of amino acid deprivation as a strategy for cancer therapy.

Knock-down of PSAT1 Enhances Sensitivity of NSCLC Cells to Glutamine-limiting Conditions.

BACKGROUND/AIM: Phosphoserine aminotransferase 1 (PSAT1) is an enzyme implicated in serine biosynthesis, and its overexpression has been linked to cancer cell proliferation. Therefore, targeting PSAT1 is considered to be an anticancer strategy. MATERIALS AND METHODS: The viability of non-small cell lung cancer (NSCLC) cells was measured by MTT assay. Protein and mRNA expression were determined by western blot and reverse transcription polymerase chain reaction, respectively. RESULTS: Glutamine-limiting conditions were generated through glutamine deprivation or CB-839 treatment, which induced PSAT1 expression in NSCLC cells. PSAT1 expression induced by glutamine-limiting conditions was regulated by activating transcription factor 4. Knock-down of PSAT1 enhanced the sensitivity of NSCLC cells to glutamine-limiting conditions. Interestingly, ionizing radiation induced PSAT1 expression, and knocking down PSAT1 increased cell sensitivity to ionizing radiation. CONCLUSION: Inhibiting PSAT1 might aid in the treatment of lung cancer, and PSAT1 may be a therapeutic target for lung cancer.

Increased Expression of FosB through Reactive Oxygen Species Accumulation Functions as Pro-Apoptotic Protein in Piperlongumine Treated MCF7 Breast Cancer Cells.

Piperlongumine (PL), a natural alkaloid compound isolated from long pepper (Piper longum), can selectively kill cancer cells, but not normal cells, by accumulation of reactive oxygen species (ROS). The objective of this study was to investigate functional roles of expression of SETDB1 and FosB during PL treatment in MCF7 breast cancer cells. PL downregulates SETDB1 expression, and decreased SETDB1 expression enhanced caspase 9 dependent-PARP cleavage during PL-induced cell death. PL treatment generated ROS. ROS inhibitor NAC (N-acetyl cysteine) recovered SETDB1 expression decreased by PL. Decreased SETDB1 expression induced transcriptional activity of FosB during PL treatment. PARP cleavage and positive annexin V level were increased during PL treatment with FosB overexpression whereas PARP cleavage and positive annexin V level were decreased during PL treatment with siFosB transfection, implying that FosB might be a pro-apoptotic protein for induction of cell death in PL-treated MCF7 breast cancer cells. PL induced cell death in A549 lung cancer cells, but molecular changes involved in the induction of these cell deaths might be different. These results suggest that SETDB1 mediated FosB expression may induce cell death in PL-treated MCF7 breast cancer cells.

Utilization of Archived Plasma to Detect Epidermal Growth Factor Receptor Mutation in Non-Small Cell Lung Cancer Patients.

Precision medicine has received increased attention as an effective approach for the treatment of cancer patients. Because of challenges associated with the availability of archived tissue, liquid biopsies are often performed to detect cancer-specific mutations. One of the major advantages of the liquid biopsy is that the treatment can be monitored longitudinally, even after the tumor tissue is no longer available. In a clinical setting, one component of precision medicine is the detection of cancer-specific mutations using archived samples. In this study, we evaluated the epidermal growth factor receptor (EGFR) mutation status of samples of lung cancer patients stored before introduction of the plasma EGFR test at our institution. The aim of this study was to validate the utility of archived plasma samples for detection of the EGFR mutation in nonsmall cell lung cancer (NSCLC) patients. The Cobas® EGFR Mutation Test v2 was the first liquid biopsy test approved as a companion diagnostic test for patients with NSCLC treated with tyrosine kinase inhibitors. We tested for the EGFR mutation in 116 plasma samples archived in the biobank, and the results were compared with those obtained in the tissue or cytology EGFR mutation test. The EGFR mutation-positive rate from archived plasma was lower than that determined from tissue or cytology at 19.0% and 53.4%, respectively, and the concordance rate between the two tests was 58.6%. Of interest, five (4.3%) samples showed the T790M mutation in the plasma test, whereas this mutation was only detected in two (1.7%) tissue/cytology samples. Five (4.3%) samples were additionally positive in the plasma test. Overall, these results indicate that archived plasma samples can serve as an alternative source for the plasma EGFR mutation test when tissue samples are not available, and can improve precision medicine and long-term follow-up in a noninvasive manner.

Induction of HSP27 and HSP70 by constitutive overexpression of Redd1 confers resistance of lung cancer cells to ionizing radiation.

Redd1 is a stress response protein that functions as a repressor of mTORC1, a central regulator of protein translation, resulting in the inhibition of cell growth and metabolism. However, paradoxically, high Redd1 expression favors cancer progression and generates resistance to cancer therapy. Herein, we revealed that constitutive overexpression of Redd1 induced HSP27 and HSP70 expression in lung cancer cells. The expression of Redd1, HSP27 and HSP70 was highly increased in lung cancer tissues compared with that in normal lung tissues. Inhibition of HSP27 or HSP70 suppressed AKT phosphorylation, which was induced by constitutive overexpression of Redd1 and enhanced the inhibitory effects on viability of Redd1­overexpressing cells. Inhibition of AKT phosphorylation resulted in a decrease of HSP27 and HSP70 expression in Redd1­overexpressing cells. These data indicated that HSPs and AKT in Redd1­overexpressing cells positively regulated the function and expression of each other and were involved in lung cancer cell survival. Knockdown of HSP27, HSP70 or AKT enhanced ionizing radiation (IR) sensitivity, particularly in lung cancer cells in which Redd1 was stably overexpressed. Collectively, constitutive overexpression of Redd1 led to HSP27 and HSP70 induction and AKT activation, which were involved in lung cancer cell survival and resistance to IR, suggesting that Redd1 may be used as a therapeutic target for lung cancer.

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.