Cisplatin-enriching cancer stem cells confer multidrug resistance in non-small cell lung cancer via enhancing TRIB1/HDAC activity.
ABSTRACT: Chemotherapeutic agents are generally used as a frontline therapy for non-small cell lung cancer (NSCLC). However, resistance to chemotherapy arises rapidly in NSCLC, and the reasons for chemotherapy resistance have not been fully determined. Here, we found cisplatin, but not paclitaxel and doxorubicin, induced the enrichment of cancer stem cell (CSC) and conferred multidrug resistance in NSCLC cell lines. In vivo study confirmed drug-resistant tumors displayed the enhanced expressions of CSC transcription factors. Mechanistically, cisplatin treatment resulted in C/EBP-?-dependent increasing of TRIB1. The crucial role of TRIB1 in cisplatin-induced enrichment of CSC and drug resistance was verified by knockdown TRIB1. Interestingly, cisplatin treatment also contributed to the increasement of HDAC, the interaction of TRIB1 with HDAC, and inactivation of p53. Similarly, the silencing of HDAC led to reduction of cisplatin-induced CSC, and combined knockdown of HDAC and TRIB1 exhibited enhanced effect. Additionally, the combination of HDAC inhibitor and cisplatin showed a reinforced antitumor action in NSCLC cell lines with TRIB1-dependent manner and remarkably shrink tumors in xenograft models. Moreover, cisplatin-treated NSCLC patients with high levels of TRIB1 exhibited a significantly poorer prognosis. Our findings illustrate a novel perspective in the evolution of chemotherapy resistance and provide a promising approach for the treatment of patients with NSCLC.
Project description:BACKGROUND:Cisplatin is a first-line drug for the treatment of human non-small cell lung cancer (NSCLC); however, the majority of patients will develop drug resistance after treatment. In order to overcome cisplatin resistance, it is important to understand the mechanisms underlying the resistance. METHODS:A gene microarray was used to screen for genes related to cisplatin resistance in NSCLC cell lines. Subsequently, the correlation between the HDAC, RXR and HtrA1 genes, in NSCLC, were verified using gene manipulation. Immunohistochemical staining was used to detect HDAC, RXR and HtrA1 expression in NSCLC specimens. Proliferation, migration and invasion assays were performed in vitro and in vivo to determine the role of the HDAC/RXR/HtrA1 signaling axis in cisplatin resistance, and luciferase reporter analysis and ChIP assays were performed to ascertain the mechanisms by which HDAC and RXR regulate the expression of HtrA1. Furthermore, in vitro and in vivo experiments were conducted in NSCLC cisplatin-resistant NSCLC to elucidate the effect of the low molecular weight compound, DW22, which targets the NSCLC cisplatin resistance HDAC/RXR/HtrA1 signaling pathway. RESULTS:HtrA1 was identified as a cisplatin resistance-related gene in NSCLC cells. The regulation of HtrA1 by HDAC and RXR significantly decreased the efficacy of cisplatin in NSCLC cells resistant to cisplatin. Immunohistochemistry results showed a negative relationship between HDAC1 and HtrA1, and a positive relationship between RXR? and HtrA1 in NSCLC patients' tissues. Notably, the expression of HDAC1 and HtrA1 can be considered as biomarkers for the efficacy of platinum-based drugs and prognosis in NSCLC patients. Mechanistically, the heterodimers of the nuclear receptor RXR, in combination with the enzyme, HDAC, regulate the transcription of HtrA1 in NSCLC cells. The rescue of HtrA1 expression by dual targeting of HDAC and RXR with the compound, DW22, significantly inhibited the proliferation, migration and invasion of NSCLC cells resistant to cisplatin, and induced NSCLC cell apoptosis. CONCLUSION:Our results indicate that HtrA1, a cisplatin resistance-related gene, is synergistically regulated by HDAC and RXR in NSCLC. Targeting the HDAC/RXR/HtrA1 signaling axis can rescue HtrA1 expression and reverse cisplatin resistance in NSCLC.
Project description:Cisplatin yields significant efficacy and is generally used as a frontline therapy for non-small cell lung cancer (NSCLC). However, acquired resistance strongly limits its application. Here, we identified that a novel histone deacetylase (HDAC) inhibitor S11, with P-glycoprotein inhibitory activity, could obviously suppress cell growth in cisplatin-resistant NSCLC cell lines. In addition, S11 could increase the expression of Ac-H4 and p21, which confirmed its HDAC inhibitory action, suppress colony formation, and block cell migration of cisplatin-resistant NSCLC cells. Notably, co-treatment with S11 and cisplatin exhibited synergistically inhibitory efficacy in cisplatin-resistant NSCLC cells. Gene microarray data showed that OAZ1 was downregulated in resistant cells but upregulated after S11 treatment. Further study indicated that knockdown of OAZ1 by siRNA resulted in the decrease of sensitivity of resistant cells to cisplatin treatment and contributed to the increase of resistant cell migration. Additionally, ChIP assay data demonstrated that HDAC inhibitor S11 could increase the accumulation of Ac-H4 in OAZ1 promoter region, suggesting the direct regulation of OAZ1 by HDAC. Importantly, the combination of S11 and cisplatin overcome resistance through inhibiting HDAC activity and subsequently increasing the OAZ1 expression in preclinical model. Moreover, we observed that positive expression of HDAC1 was associated with the downregulation of OAZ1 in NSCLC patients with platinum-based treatment, and predicted drug resistance and poor prognosis. In summary, we demonstrated a role of HDAC/OAZ1 axis in cisplatin-resistant NSCLC and identified a promising compound to overcome cisplatin resistance.
Project description:Chemotherapy resistance and tumor relapse are the major contributors to low patient survival, and both have been largely attributed to cancer stem-like cells (CSCs) or tumor-initiating cells (TICs). Moreover, most conventional therapies are not effective against CSCs, which necessitates the discovery of CSC-specific biomarkers and drug targets. Here, we demonstrated that the embryonic transcription factor SOX9 is an important regulator of acquired chemoresistance in non-small-cell lung cancer (NSCLC). Our results show that SOX9 expression is elevated in NSCLC cells after treatment with the chemotherapeutic cisplatin and that overexpression of SOX9 correlates with worse overall survival in lung cancer patients. We further demonstrated that SOX9 knockdown increases cellular sensitivity to cisplatin, whereas its overexpression promotes drug resistance. Moreover, this transcription factor promotes the stem-like properties of NSCLC cells and increases their aldehyde dehydrogenase (ALDH) activity, which was identified to be the key mechanism of SOX9-induced chemoresistance. Finally, we showed that ALDH1A1 is a direct transcriptional target of SOX9, based on chromatin immunoprecipitation and luciferase reporter assays. Taken together, our novel findings on the role of the SOX9-ALDH axis support the use of this CSC regulator as a prognostic marker of cancer chemoresistance and as a potential drug target for CSC therapy.
Project description:Non-small cell lung cancer (NSCLC) accounts for a large proportion of cancer deaths and is characterized by low treatment response rates and poor overall prognosis. In the absence of specific treatable mutations, cisplatin-based chemotherapy plays an important role in the treatment of this disease. Unfortunately, the development of resistance has become a major therapeutic challenge in the use of this cytotoxic drug. Elucidating the mechanisms underlying this resistance phenotype, may result in the development of novel agents that enhance sensitivity to cisplatin in lung cancer patients. In this study, targeting the cancer stem cell activity of aldehyde dehydrogenase 1 (ALDH1) was investigated as a strategy to overcome chemoresistance in NSCLC. Tumors from NSCLC patients showed an increase in their profile of pluripotent stemness genes. Cisplatin exposure induced the emergence or expansion of an ALDH1-positive subpopulation in cisplatin sensitive and resistant NSCLC cell lines, respectively, further enhancing cisplatin resistance. Using the Aldefluor assay and FACS analysis, ALDH1 subpopulations were isolated and evaluated in terms of stem cell characteristics. Only ALDH1-positive cells exhibited asymmetric division, cisplatin resistance and increased expression of stem cell factors in vitro. Xenograft studies in NOD/SCID mice demonstrated efficient tumorigenesis from low cell numbers of ALDH1-positive and ALDH1-negative subpopulations. Targeting ALDH1 with Diethylaminobenzaldehyde (DEAB) and Disulfiram, significantly re-sensitized resistant lung cancer cells to the cytotoxic effects of cisplatin. Our data demonstrate the existence of a lung CSC population and suggest a role for targeting ALDH1 as a potential therapeutic strategy in re-sensitizing NSCLC cells to the cytotoxic effects of cisplatin.
Project description:Cancer stem cells (CSC) are thought to be responsible for tumor initiation and tumor regeneration after chemotherapy. Previously, we showed that chemotherapy of non-small cell lung cancer (NSCLC) cells lines can select for outgrowth of highly tumorigenic and metastatic CSCs. The high malignancy of lung CSCs was associated with an efficient cytokine network. In this study, we provide evidence that blocking stem cell factor (SCF)-c-kit signaling is sufficient to inhibit CSC proliferation and survival promoted by chemotherapy. CSCs were isolated from NSCLC cell lines as tumor spheres under CSC-selective conditions and their stem properties were confirmed. In contrast to other tumor cells, CSCs expressed c-kit receptors and produced SCF. Proliferation of CSCs was inhibited by SCF-neutralizing antibodies or by imatinib (Gleevec), an inhibitor of c-kit. Although cisplatin treatment eliminated the majority of tumor cells, it did not eliminate CSCs, whereas imatinib or anti-SCF antibody destroyed CSCs. Significantly, combining cisplatin with imatinib or anti-SCF antibody prevented the growth of both tumor cell subpopulations. Our findings reveal an important role for the SCF-c-kit signaling axis in self-renewal and proliferation of lung CSCs, and they suggest that SCF-c-kit signaling blockade could improve the antitumor efficacy of chemotherapy of human NSCLC.
Project description:Chemotherapy paclitaxel yields significant reductions in tumor burden in the majority of advanced non-small cell lung cancer (NSCLC) patients. However, acquired resistance limits its clinical use. Here we demonstrated that the histone deacetylase (HDAC) was activated in paclitaxel-resistant NSCLC cells, and its activation promoted proliferation and tumorigenesis of paclitaxel-resistant NSCLC cells in vitro and in vivo. By contrast, knockdown of HDAC1, a primary isoform of HDAC, sensitized resistant cells to paclitaxel in vitro. Furthermore, we observed that overexpression of HDAC1 was associated with the downregulation of p21, a known HDAC target, in advanced NSCLC patients with paclitaxel treatment, and predicted chemotherapy resistance and bad outcome. In addition, we also identified a novel HDACs inhibitor, SNOH-3, which inhibited HDAC expression and activity, induced cell apoptosis, and suppressed cell migration, invasion and angiogenesis. Notably, co-treatment with SNOH-3 and paclitaxel overcome paclitaxel resistance through inhibiting HDAC activity, leading to the induction of apoptosis and suppression of angiogenesis in vitro and in preclinical model. In summary, our data demonstrate a role of HDAC in paclitaxel-resistant NSCLC and provide a promising therapeutic strategy to overcome paclitaxel-acquired resistance.
Project description:The underlying molecular mechanisms of cisplatin resistance in non?small cell lung cancer (NSCLC) are unclear. In this study, a novel differential methylation region located in the upstream regulatory region of the forkhead box F1 (FOXF1) gene was identified. The abnormal hypomethylation of FOXF1 increased the expression of FOXF1, and the high expression of FOXF1 promoted cell proliferation and inhibited cell apoptosis induced by cisplatin, which resulted in cisplatin resistance in NSCLC cells. In addition, FOXF1 promoted the expression of stem cell markers and self?renewal capability, indicating that FOXF1 regulated cisplatin resistance by promoting cancer stem cell properties in NSCLC cells. Moreover, a strong association was observed between FOXF1 upregulation and the presence of platinum?based chemotherapy resistance in patients with NSCLC. On the whole, the findings of this study indicate the regulatory mechanisms of cisplatin resistance by FOXF1 in NSCLC, and suggest that FOXF1 may be used as a prognostic biomarker of platinum?based chemotherapy resistance in NSCLC.
Project description:Chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) is a recently identified gene associated with malignant tumor progression and patient chemotherapy resistance in human hepatocellular carcinoma (HCC). Previously, we found an association between CHD1L overexpression and poor patient survival in non-small-cell lung cancer (NSCLC). However, little is known about the relationship between CHD1L expression and chemotherapy resistance of NSCLC. By employing immunohistochemistry, we analyzed the expression of CHD1L in NSCLC samples and elucidated the roles and mechanism of CHD1L in NSCLC chemoresistance. We found that the increased expression of CHD1L is positively correlated with a shorter survival time of patients who had received chemotherapy after surgery. We also found that the expression of CHD1L was increased after cisplatin treatment in A549 cells. Conversely, the depletion of CHD1L in cisplatin-resistance cells increased the cell sensitivity to cisplatin, indicating that CHD1L plays a critical role in cisplatin resistance of NSCLC cells. Importantly, we identified the ATP-Binding Cassette Sub-Family B Member (ABCB1) gene as a potential downstream target of CHD1L in NSCLC cells. Knocking down ABCB1 coupled with ectopic expression of CHD1L enhanced the effect of cisplatin on NSCLC cells apoptosis. In addition, overexpressed CHD1L increase the transcription of c-Jun which targeted directly to the promoter of ABCB1. Our data demonstrate that CHD1L could induce cisplatin resistance in NSCLC via c-Jun-ABCB1-NF-?B axis, and may serve as a novel predictive marker and the potential therapeutic target for cisplatin resistance in NSCLC.
Project description:PAK1 confers resistance to the estrogen antagonist tamoxifen in breast cancer. However, a role for PAK1 remains to be elucidated for chemoresistance and prognosis in non-small cell lung cancer (NSCLC). We provide evidence that PAK1 confers cisplatin resistance by increasing ?-catenin expression through ERK/GSK3? signaling. The increased ?-catenin expression promotes sphere cell formation and expression of stemness markers and this ?-catenin-induced stemness is responsible for PAK1-mediated cisplatin resistance. We enrolled 87 NSCLC patients who had received cisplatin-based chemotherapy to confirm the association between PAK1 expression and response to chemotherapy and outcomes. PAK1 expression, evaluated by immunohistochemistry, was positively correlated with pERK and ?-catenin expression in lung tumors. Patients with high-PAK1, high-pERK, and high-nuclear ?-catenin tumors more frequently showed an unfavorable response to cisplatin-based chemotherapy when compared to their counterparts. Kaplan-Meier and Cox regression analysis also indicated a poorer overall survival (OS) and relapse free survival (RFS) in patients with high-PAK1, high-pERK, and high-nuclear ?-catenin tumors. In conclusion, PAK1 confers cisplatin resistance in NSCLC via ?-catenin-mediated stemness. Therefore, we suggest that clinical use of a combination of the MEK/ERK inhibitor AZD6244 and cisplatin might improve sensitivity to cisplatin-based chemotherapy and outcomes in NSCLC patients who harbor high-PAK1-expressing tumors.
Project description:The TRIB1 gene has been associated with multiple malignancies, plasma triglycerides and coronary artery disease (CAD). Despite the clinical significance of this pseudo-kinase, there is little information on the regulation of TRIB1. Previous studies reported TRIB1 mRNA to be unstable, hinting that TRIB1 might be subject to post-transcriptional regulation. This work explores TRIB1 regulation, focusing on its post-transcriptional aspects. In 3 distinct model systems (HEK293T, HeLa and arterial smooth muscle cells) TRIB1 was undetectable as assessed by western blot. Using recombinant TRIB1 as a proxy, we demonstrate TRIB1 to be highly unstable at the protein and RNA levels. By contrast, recombinant TRIB1 was stable in cellular extracts. Blocking proteasome function led to increased protein steady state levels but failed to rescue protein instability, demonstrating that the 2 processes are uncoupled. Unlike as shown for TRIB2, CUL1 and TRCP? did not play a role in mediating TRIB1 instability although TRCP? suppression increased TRIB1 expression. Lastly, we demonstrate that protein instability is independent of TRIB1 subcellular localization. Following the identification of TRIB1 nuclear localization signal, a cytosolic form was engineered. Despite being confined to the cytosol, TRIB1 remained unstable, suggesting that instability occurs at a stage that precedes its nuclear translocation and downstream nuclear function. These results uncover possible avenues of intervention to regulate TRIB1 function by identifying two distinct regulatory axes that control TRIB1 at the post-transcriptional level.