Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway

Adipose tissue is now considered as an endocrine organ involved in metabolic and inflammatory reactions. Adiponectin, a 244–amino acid peptide hormone, is associated with insulin resistance and carcinogenesis. Curcumin (diferuloylmethane) is the principal curcuminoid of the popular Indian spice, turmeric. Curcumin possesses antitumor effects, including the inhibition of neovascularization and regulation of cell cycle and apoptosis. However, the effects of adiponectin and curcumin on non-small cell lung cancer (NSCLC) remain unclear. In this study, we evaluated the expression of adiponectin in paired tumors and normal lung tissues from 77 patients with NSCLC using real-time polymerase chain reaction, western blotting, and immunohistochemistry. Kaplan–Meier survival analysis showed that patients with low adiponectin expression ratio (<1) had significantly longer survival time than those with high expression ratio (>1) (p = 0.015). Curcumin inhibited the migratory and invasive ability of A549 cells via the inhibition of adiponectin expression by blocking the adiponectin receptor 1. Curcumin treatment also inhibited the in vivo tumor growth of A549 cells and adiponectin expression. These results suggest that adiponectin can be a prognostic indicator of NSCLC. The effect of curcumin in decreasing the migratory and invasive ability of A549 cells by inhibiting adiponectin expression is probably mediated through NF-κB/MMP pathways. Curcumin could be an important potential adjuvant therapeutic agent for lung cancer in the future.     

Curcumin induces EGFR degradation in lung adenocarcinoma and modulates p38 activation in intestine: the versatile adjuvant for gefitinib therapy

Background

Non-small cell lung cancer (NSCLC) patients with L858R or exon 19 deletion mutations in epidermal growth factor receptor (EGFR) have good responses to the tyrosine kinase inhibitor (TKI), gefitinib. However, patients with wild-type EGFR and acquired mutation in EGFR T790M are resistant to gefitinib treatment. Here, we showed that curcumin can improve the efficiency of gefitinib in the resistant NSCLC cells both in vitro and in vivo models.

Methods/Principal Findings

After screening 598 herbal and natural compounds, we found curcumin could inhibit cell proliferation in different gefitinib-resistant NSCLC cell lines; concentration-dependently down-regulate EGFR phosphorylation through promoting EGFR degradation in NSCLC cell lines with wild-type EGFR or T790M EGFR. In addition, the anti-tumor activity of gefitinib was potentiated via curcumin through blocking EGFR activation and inducing apoptosis in gefitinib-resistant NSCLC cell lines; also the combined treatment with curcumin and gefitinib exhibited significant inhibition in the CL1-5, A549 and H1975 xenografts tumor growth in SCID mice through reducing EGFR, c-MET, cyclin D1 expression, and inducing apoptosis activation through caspases-8, 9 and PARP. Interestingly, we observed that the combined treatment group represented better survival rate and less intestinal mucosal damage compare to gefitinib-alone therapy. We showed that curcumin attenuated the gefitinib-induced cell proliferation inhibition and apoptosis through altering p38 mitogen-activated protein kinase (MAPK) activation in intestinal epithelia cell.

Conclusions/Significance

Curcumin potentiates antitumor activity of gefitinib in cell lines and xenograft mice model of NSCLC through inhibition of proliferation, EGFR phosphorylation, and induction EGFR ubiquitination and apoptosis. In addition, curcumin attenuates gefitinib-induced gastrointestinal adverse effects via altering p38 activation. These findings provide a novel treatment strategy that curcumin as an adjuvant to increase the spectrum of the usage of gefitinib and overcome the gefitinib inefficiency in NSCLC patients.     

Curcumin inhibits lung cancer cell migration and invasion through Rac1-dependent signaling pathway

Curcumin, a natural and crystalline compound isolated from the plant Curcuma longa with low toxicity in normal cells, has been shown to protect against carcinogenesis and prevent tumor development. However, little is known about antimetastasis effects and mechanism of curcumin in lung cancer. Rac1 is an important small Rho GTPases family protein and has been widely implicated in cytoskeleton rearrangements and cancer cell migration, invasion and metastasis. In this study, we examined the influence of curcumin on in vitro invasiveness of human lung cancer cells and the expressions of Rac1. The results indicate that curcumin at 10 μM slightly reduced the proliferation of 801D lung cancer cells but showed an obvious inhibitory effect on epidermal growth factor or transforming growth factor β1-induced lung cancer cell migration and invasion. Meanwhile, we demonstrated that the suppression of invasiveness correlated with inhibition of Rac1/PAK1 signaling pathways and matrix metalloproteinase (MMP) 2 and 9 protein expression by combining curcumin treatment with the methods of Rac1 gene silence and overexpression in lung cancer cells. Laser confocal microscope also showed that Rac1-regulated actin cytoskeleton rearrangement may be involved in anti-invasion effect of curcumin on lung cancer cell. At last, through xenograft experiments, we confirmed the connection between Rac1 and the growth and metastasis inhibitory effect of curcumin in vivo. In summary, these data demonstrated that low-toxic levels of curcumin could efficiently inhibit migration and invasion of lung cancer cells through inhibition of Rac1/PAK1 signaling pathway and MMP-2 and MMP-9 expression, which provided a novel insight into the molecular mechanism of curcumin against lung cancer.     

Systems pharmacology unravels the synergic target space and therapeutic potential of Rhodiola rosea L. for non-small cell lung cancer

BackgroundLung cancer is the most common and mortal cancer worldwide. Rhodiola rosea L. (RR), a well-known traditional Chinese medicine (TCM), has been turned out to be effective in anti-lung cancer therapy, but its molecular mechanism of action has not been clearly understood.PurposeIn this study, we aimed to elucidate the possible molecular mechanism underlying the effect of RR against non-small cell lung cancer (NSCLC) by systems pharmacology.MethodsThe effects of RR on NSCLC were examined in Lewis lung carcinoma (LLC) tumor-bearing mice models. The possible molecular mechanism was unraveled by systems pharmacology, which includes pharmacokinetics evaluation, active compounds screening, target prediction and network analysis. Cell proliferation was examined by cell counting kit-8 (CCK-8) assay; cell apoptosis was detected by flow cytometry; protein and proinflammatory cytokines expression were evaluated by Western blot and qRT-PCR.ResultsIn vivo, RR significantly inhibited the tumor growth and prolonged the survival of the tumor bearing mice. In silico, we identified 19 potential active molecules (e.g., salidroside and rhodiosin), 112 targets (e.g., COX-2 and AKT) and 27 pathways (e.g., PI3K/AKT signaling pathway and NF-κB signaling pathway) for RR. Additionally, targets analysis and networks construction further revealed that RR exerted anti-cancer effects by regulating apoptosis, angiogenesis and inflammation. In vitro, salidroside could significantly decrease expression of pro-angiogenic factors (e.g., VEGF and eNOS) and proinflammatory cytokines (e.g., COX-2, iNOS and TNF-α). Also, Bcl-2, an anti-apoptotic protein was decreased whereas Bax, a pro-apoptotic protein, was increased. Further flow cytometry analysis showed that salidroside could induce apoptosis in H1975 cells.ConclusionsMechanistically, the antitumor effect of RR on NSCLC was responsible for the synergy among anti-inflammatory, anti-angiogenic and pro-apoptotic.     

Curcumin induces apoptosis and inhibits growth of human Burkitt’s lymphoma in xenograft mouse model

Curcumin, a natural compound extracted from rhizomes of curcuma Curcuma species, has been shown to possess potent anti-inflammatory, anti-tumor and anti-oxidative properties. However, the mechanism of action of the compound remains poorly understood. In this report, we have analyzed the effects of curcumin on the cell proliferation of Burkitt’s lymphoma Raji cells. The results demonstrated that curcumin could effectively inhibit the growth of Raji cells in a dose- and time-dependent manner. Further studies indicated that curcumin treatment resulted in apoptosis of cells. Biochemical analysis showed that the expression of Bax, Bid and cytochrome C were up-regulated, while the expression of oncogene c-Myc was down regulated after curcumin treatment. Furthermore, poly (ADP-ribose) polymerase (PARP) cleavage was induced by the compound. Interestingly, the antiapoptotic Bcl-2 expression was not significantly changed in Raji cells after curcumin treatment. These results suggested that the mechanism of action of curcumin was to induce mitochondrial damage and therefore led to Raji cell apoptosis. We further investigated the in vivo effects of curcumin on the growth of xenograft tumors in nude mice. The results showed that curcumin could effectively inhibit tumor growth in the xenograft mouse model. The overall results showed that curcumin could suppress the growth of Burkitt’s lymphoma cells in both in vitro and in vitro systems.     

SIRT1 Expression Is Associated with the Chemotherapy Response and Prognosis of Patients with Advanced NSCLC

Aim

The role of Sirtuin 1 (SIRT 1) in carcinogenesis is controversial. This study was to explore the association between the SIRT1 expression and the clinical characteristics, the responsiveness to chemotherapy and prognosis in Non-small cell lung cancer (NSCLC).

Methods

We enrolled 295 patients with inoperable advanced stage of NSCLC, namely, stage III (A+B) and IV NSCLC. All patients had received platinum-based chemotherapy after diagnosis and the chemotherapy response were evaluated. All patients were followed up for overall survival (OS) and progression free survival (PFS). In vitro, H292 cells were tranfected with SIRT1 small interfering RNA (siRNA). The cell biological behaviors and chemosensitivity to cisplatin treatment were studied. The in vivo tumorgenesis and metastasis assays were performed in nude mice.

Results

We found that the SIRT1 expressions were significantly associated with the tumor stage, tumor size and differentiation status. Patients with high SIRT 1 expressions had a significantly higher chance to be resistant to chemotherapy than those with low SIRT 1 expression. Patients with high expression of SIRT1 had significantly shorter OS and DFS than those with low expression. Cox analyses confirmed that the SIRT 1 expression was a strong predictor for a poor OS and PFS in NSCLC patients underwent Platinum-based chemotherapy. In vitro studies revealed that the reduced expression SIRT 1 by siRNA technique significantly inhibited cell proliferation, migration and invasion. More importantly, SIRT1 si-RNA significantly enhanced the chemosensitivity of H292 cells to cisplatin treatment. The in vivo tumorgenesis and metastasis assays showed that SIRT1 knockdown dramatically reduced the tumor volume and the metastatic ability in nude mice.

Conclusion

Collectively, our data suggest that the SIRT1 expression may be a molecular marker associated with the NSLCLC clinical features, treatment responsiveness and prognosis of advanced NSCLC.     

The Curcumin Analog EF24 Targets NF-κB and miRNA-21, and Has Potent Anticancer Activity In Vitro and In Vivo

EF24 is a curcumin analog that has improved anticancer activity over curcumin, but its therapeutic potential and mechanism of action is unknown, which is important to address as curcumin targets multiple signaling pathways. EF24 inhibits the NF-κB but not the JAK-STAT signaling pathway in DU145 human prostate cancer cells and B16 murine melanoma cells. EF24 induces apoptosis in these cells apparently by inhibiting miR-21 expression, and also enhances the expression of several miR-21 target genes, PTEN and PDCD4. EF24 treatment significantly suppressed the growth of DU145 prostate cancer xenografts in immunocompromised mice and resulted in tumor regression. EF24 enhanced the expression of the miR-21 target PTEN in DU145 tumor tissue, but suppressed the expression of markers of proliferating cells (cyclin D1 and Ki67). In syngeneic mice injected with B16 cells, EF24 treatment inhibited the formation of lung metastasis, prolonged animal survival, inhibited miR-21 expression and increased the expression of miR-21 target genes. Expression profiling of miRNAs regulated by EF24 in vitro and in vivo showed that the antitumor activity of EF24 reflected the enhanced expression of potential tumor suppressor miRNAs as well as the suppressed expression of oncogenic miRNAs, including miR-21. Taken together, our data suggest that EF24 is a potent anticancer agent and selectively targets NF-κB signaling and miRNA expression, indicating that EF24 has significant potential as a therapeutic agent in various cancers.     

Luteolin inhibits the Nrf2 signaling pathway and tumor growth in vivo

Nuclear factor erythroid 2-related factor 2 (Nrf2) is over-expressed in many types of tumor, promotes tumor growth, and confers resistance to anticancer therapy. Hence, Nrf2 is regarded as a novel therapeutic target in cancer. Previously, we reported that luteolin is a strong inhibitor of Nrf2 in vitro. Here, we showed that luteolin reduced the constitutive expression of NAD(P)H quinone oxidoreductase 1 in mouse liver in a time- and dose-dependent manner. Further, luteolin inhibited the expression of antioxidant enzymes and glutathione transferases, decreasing the reduced glutathione in the liver of wild-type mice under both constitutive and butylated hydroxyanisole-induced conditions. In contrast, such distinct responses were not detected in Nrf2^−/− mice. In addition, oral administration of luteolin, either alone or combined with intraperitoneal injection of the cytotoxic drug cisplatin, greatly inhibited the growth of xenograft tumors from non-small-cell lung cancer (NSCLC) cell line A549 cells grown subcutaneously in athymic nude mice. Cell proliferation, the expression of Nrf2, and antioxidant enzymes were all reduced in tumor xenograft tissues. Furthermore, luteolin enhanced the anti-cancer effect of cisplatin. Together, our findings demonstrated that luteolin inhibits the Nrf2 pathway in vivo and can serve as an adjuvant in the chemotherapy of NSCLC.     

Curcumin sensitizes human lung cancer cells to apoptosis and metastasis synergistically combined with carboplatin

Although carboplatin is one of the standard chemotherapeutic agents for non-small cell lung cancer (NSCLC), it has limited therapeutic efficacy due to activation of a survival signaling pathway and the induction of multidrug resistance. Curcumin, a natural compound isolated from the plant Curcuma longa, is known to sensitize tumors to different chemotherapeutic agents. The aim of this study is to evaluate whether curcumin can chemosensitize lung cancer cells to carboplatin and to analyze the signaling pathway underlying this synergism. We investigated the synergistic effect of both agents on cell proliferation, apoptosis, invasion, migration, and expression of related signaling proteins using the human NSCLC cell line, A549. A549 cell was treated with different concentrations of curcumin and carboplatin alone and in combination. Combined treatment with curcumin and carboplatin inhibited tumor cell growth, migration, and invasion compared with either drug alone. Matrix metalloproteinase (MMP)-2 and MMP-9 were more efficiently downregulated by co-treatment than by each treatment alone. mRNA and protein expression of caspase-3 and caspase-9 and proapoptotic genes was increased in cells treated with a combination of curcumin and carboplatin, whereas expression of the antiapoptotic Bcl-2 gene was suppressed. Co-treatment of both agents substantially suppressed NF-κB activation and increased expression of p53. Phosphorylation of Akt, a protein upstream of NF-κB, was reduced, resulting in inhibition of the degradation of inhibitor of κB(IκBα), whereas the activity of extracellular signal-regulated kinase (ERK1/2) was enhanced. Our study demonstrated that the synergistic antitumor activity of curcumin combined with carboplatin is mediated by multiple mechanisms involving suppression of NF-κB via inhibition of the Akt/IKKα pathway and enhanced ERK1/2 activity. Based on this mechanism, curcumin has potential as a chemosensitizer for carboplatin in the treatment of patients with NSCLC.     

Neuropilin 1 expression correlates with the Radio‐resistance of human non‐small‐cell lung cancer cells

The purpose of this study was to determine the correlation between over‐expression of the neuropilin 1 (NRP1) gene and growth, survival, and radio‐sensitivity of non‐small cell lung carcinoma (NSCLC) cells. 3‐[4,5‐dimethylthylthiazol‐2‐yl]‐2,5 diphenyltetrazolium broide (MTT) and colony assays were then performed to determine the effect of NRP1 inhibition on the in vitro growth of NSCLC cells. The Annexin V‐Fluorescein Isothiocyanate (FITC) apoptosis detection assay was performed to analyse the effect of NRP1 enhancement on apoptosis of NSCLC cells. Transwell invasion and migration assays were employed to examine the metastatic ability of A549 cells post X‐ray irradiation. In addition, Western blot assays were carried out to detect the protein level of VEGFR2, PI3K and NF‐κB. Finally, to examine the effect of shNRP1 on proliferation and radio‐sensitivity in vivo, a subcutaneous tumour formation assay in nude mice was performed. Microvessel density in tumour tissues was assessed by immunohistochemistry. The stable transfected cell line (shNRP1‐A549) showed a significant reduction in colony‐forming ability and proliferation not only in vitro, but also in vivo. Moreover, shRNA‐mediated NRP1 inhibition also significantly enhanced the radio‐sensitivity of NSCLC cells both in vitro and in vivo. The over‐expression of NRP1 was correlated with growth, survival and radio‐resistance of NSCLC cells via the VEGF‐PI3K‐ NF‐κB pathway, and NRP1 may be a molecular therapeutic target for gene therapy or radio‐sensitization of NSCLC.     

Differential regulation of CD4+ T helper cell responses by curcumin in experimental autoimmune encephalomyelitis

Nutraceuticals and phytochemicals are important regulators of human health and diseases. Curcumin is a polyphenolic phytochemical isolated from the rhizome of the plant Curcuma longa (turmeric) that has been traditionally used for the treatment of inflammation and wound healing for centuries. Systematic analyses have shown that curcumin exerts its beneficial effects through antioxidant, antiproliferative and anti-inflammatory properties. We and others have shown earlier that curcumin ameliorates experimental autoimmune encephalomyelitis (EAE) model for multiple sclerosis. In this study, we show that C57BL/6 mice induced to develop EAE express elevated levels of interferon (IFN) γ and interleukin (IL)-17 in the central nervous system (CNS) and lymphoid organs that decreased significantly following in vivo treatment with curcumin. The EAE mice also showed elevated expression of IL-12 and IL-23 that decreased after treatment with curcumin. Ex vivo and in vitro treatment with curcumin resulted in a dose-dependent decrease in the secretion of IFNγ, IL-17, IL-12 and IL-23 in culture. The inhibition of EAE by curcumin was also associated with an up-regulation of IL-10, peroxisome proliferator activated receptor γ and CD4⁺CD25⁺Foxp3⁺ Treg cells in the CNS and lymphoid organs. These findings highlight that curcumin differentially regulates CD4⁺ T helper cell responses in EAE.     

Inhibition of HDAC6 Attenuates Tumor Growth of Non-Small Cell Lung Cancer

Histone deacetylase 6 (HDAC6) regulates cytoplasmic signaling networks through deacetylation of various cytoplasmic substrates and serves as a key member of the ubiquitin proteasome system (UPS). This study is focused on HDAC6 regulation of the Notch1 receptor that plays a crucial role in tumor growth in NSCLC. A series of cell culture experiments were employed using A549, Lewis lung carcinoma 2 (LL2), and H1299 NSCLC cell lines to investigate HDAC6-mediated regulation of the Notch1 receptor through the UPS. HDAC6 was inhibited with small molecule inhibitors tubacin and ACY1215 in vitro and in vivo. Inhibition of HDAC6 led to reduced levels of Notch1 receptor in a dose-dependent manner in all three NSCLC cell lines tested. HDAC6 inhibition with ACY1215 led to G2 arrest, increased apoptosis, and increased levels of cleaved PARP1 in A549, LL2, and H1299 cell lines. In vivo inhibition of HDAC6 with ACY1215 significantly reduced LL2 tumor growth rate. Our data show that HDAC6 in NSCLC cells supports Notch1 signaling and promotes cell survival and proliferation. Our results support clinical investigation of HDAC6 inhibitors as a potential therapeutic option for treatment of NSCLC patients.     

Bioluminescent orthotopic mouse models of human localized non-small cell lung cancer: feasibility and identification of circulating tumour cells

Background

Preclinical models of non-small cell lung cancer (NSCLC) require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC.

Methods

Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models.

Results

Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours.

Conclusions

Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.     

Curcumin ameliorates CKD-induced mitochondrial dysfunction and oxidative stress through inhibiting GSK-3β activity

Curcumin has been reported to attenuate muscle atrophy. However, the underling mechanism remains unclear. The aim of this study was to investigate whether curcumin could improve chronic kidney disease (CKD)-induced muscle atrophy and mitochondrial dysfunction by inhibiting glycogen synthase kinase-3β (GSK-3β) activity. The sham and CKD mice were fed either a control diet or an identical diet containing 0.04% curcumin for 12 weeks. The C2C12 myotubes were treated with H₂O₂ in the presence or absence of curcumin. In addition, wild-type and muscle-specific GSK-3β knockout (KO) CKD model mice were made by 5/6 nephrectomy, and the sham was regarded as control. Curcumin could exert beneficial effects, including weight maintenance and improved muscle function, increased mitochondrial biogenesis, alleviated mitochondrial dysfunction by increasing adenosine triphosphate levels, activities of mitochondrial electron transport chain complexes and basal mitochondrial respiration and suppressing mitochondrial membrane potential. In addition, curcumin modulated redox homeostasis by increasing antioxidant activity and suppressed mitochondrial oxidative stress. Moreover, the protective effects of curcumin had been found to be mediated via inhibiting GSK-3β activity in vitro and in vivo. Importantly, GSK-3β KO contributed to improved mitochondrial function, attenuated mitochondrial oxidative damage and augmented mitochondrial biogenesis in muscle of CKD. Overall, this study suggested that curcumin alleviated CKD-induced mitochondrial oxidative damage and mitochondrial dysfunction via inhibiting GSK-3β activity in skeletal muscle.     

Integrin αvβ6 Promotes Lung Cancer Proliferation and Metastasis through Upregulation of IL-8–Mediated MAPK/ERK Signaling

Lung cancer is notorious for high morbidity and mortality around the world. Interleukin (IL)-8, a proinflammatory chemokine with tumorigenic and proangiogenic effects, promotes lung cancer cells growth and migration and contributes to cell aggressive phenotypes. Integrin αvβ6 is a receptor of transmembrane heterodimeric cell surface adhesion, and its overexpression correlates with poor survival from non–small cell lung cancer. However, the cross talk between αvβ6 and IL-8 in lung cancer has not been characterized so far. Herein, human lung cancer samples were analyzed, and it revealed that the immunohistochemical and mRNA expression of integrin αvβ6 was significantly correlated with the expression of IL-8. Furthermore, in vitro, integrin αvβ6 increased cell proliferation, migration, and invasion by impairing the expressions of MMP-2 and MMP-9 and inhibited cell apoptosis in human lung cancer cells A549 and H460. In addition, integrin αvβ6 upregulated IL-8 expression through activating MAPK/ERK signaling. The in vivo experiment showed that integrin αvβ6 promoted tumor growth in xenograft model mice by accelerating tumor volume and reducing apoptosis. Meanwhile, lung metastasis model experiment suggested that integrin αvβ6 stimulated tumor metastasis with the increase of lung/total weight and tumor nodules. Simultaneously, integrin αvβ6 upregulated IL-8 expression detected by both Western blots and immunohistochemistry, along with the activation of MAPK/ERK signaling. Overall, these data suggested that, in vitro and in vivo, integrin αvβ6 promoted lung cancer proliferation and metastasis, at least in part, through upregulation of IL-8–mediated MAPK/ERK signaling. Thus, the inhibition of integrin αvβ6 and IL-8 may be the key for the treatment of lung cancer.     

The Tumor Suppressor Gene TUSC2 (FUS1) Sensitizes NSCLC to the AKT Inhibitor MK2206 in LKB1-dependent Manner

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.