The Cisplatin Resisting Mechanisms For Poorly-differentiated Non-small-cell Lung Adenocarcinoma

Lung cancer has one of the highest incidence and mortality rates of all malignant tumors globally,and accounts for approximately one third of all cancer-related deaths.Approximately 80%of lung carcinomas are classified as non-small cell lung carcinomas(NSCLCs),which have a 5-year survival rate that is less than 5%when accompanied by metastasis.The early symptoms of NSCLCs are not obvious and many patients with non-small cell lung cancer(NSCLC)receive a diagnosis of stage III disease.At present,chemotherapy is one of the most effective treatments for NSCLCs,and cisplatin is the standard first-line chemotherapy drug for NSCLCs.Increasing evidence demonstrates that cases lung adenocarcinomas at different stages of differentiation displayed varied sensitivity towards chemotherapeutic interventions.In general,poorly-differentiated lung adenocarcinomas are more resisting towards platinum drugs than that of well-differentiated lung adenocarcinomas,which results in frequent tumor recurrence and poor prognosis for patients with poorly-differentiated lung adenocarcinomas,but the mechanisms remain poorly understood.Thus,understanding the molecular underpinninggs of platinum resistance is becoming increasingly significant for the improvement of chemotherapy effect and the quality of patients'life and for the discovery of new strategies and drugs towards lung adenocarcinomas.Here using methods of clinical tumor pathology,biochemistry and molecular pharmacology,we identified GCN2 as a regulator of multidrug resistance(MDR)and RCD91 as a regulator of cancer stemness in poorly-differentiated NSCLC adenocarcinomas.This work has provided solid theoretical evidence and strategies for treatment of NSCLC adenocarcinomas.1:GCN2 mediates the sensitivity towards platinum drugs in poorly-differentiated NSCLC adenocarcinomas.In this first part,it was found that the expression level of GCN2,one upstream kinase for eIF2a,was negatively correlated with the clinical differentiation status of NSCLC adenocarcinomas,which was highly expressed in poorly-differentiated NSCLCs with increased resistance towards cisplatin(DDP).GCN2 depletion in poorly-differentiated NSCLC adenocarcinomas increased their apoptotic sensitivity towards DDP.These results demonstrated that GCN2 could be key regulator for DDP resistance in poorly-differentiated NSCLC adenocarcinomas.Further experiments demonstrated that GCN2 activated the transcription of multidrug resistance(MDR)-related genes including P-gp,MRP-1,and MRP-2 via the CHOP activation.Interestingly,although GCN2 level determined cisplatin sensitivity,DDP promoted GCN2 ubiquitination mediated by arrestins and NEDD4L in DDP-sensitive NSCLCs,which was compromised in DDP-resistant cells because of an elevated level of the phosphorylated GCN2 at Thr 899 site.The phosphorylation of GCN2 at Thr899 blocked the interaction of GCN2 with its ubiquitin complex and inhibited its degradation.Furthermore,the level of the phosphorylated GCN2 at the Thr 899 site was high in poorly-differentiated lung adenocarcinomas.Demonstrated by the results of medical intervention using small molecues,it was found that upregulation of the phosphorylated GCN2 at the Thr 899 site resulted in increased resistance towards DDP and decreased DDP uptake.On the contrary,downregulation of phosphorylated GCN2 at the Thr 899 site resulted in decreased DDP resistance and its increased cellular uptake in DDP-resistant lung adenocarcinoma cells.Therefore this work supported a role for the phorsphorylated GCN2 in regulating DDP resistance in poorly-differentiated NSCLC adenocarcinomas.DDP failed to promote GCN2 degradation and was subsequently pumped out of poorly-differentiated lung adenocarcinoma cells because of the elevated GCN2 phosphorylation at Thr 899 site and the subsequent activation of CHOP and its downstream drug-pumping genes.Inhibition of GCN2 phosphorylation at Thr 899 site reversed the DDP resistance in poorly-differentiated NSCLC adenocarcinomas.In conclusion,we described here for the first time that GCN2 played a very important role in determining the sensitivity of poorly-differentiated lung adenocarcinomas towards platinum-based therapy.2:RCD91 mediates cancer stemness and cisplatin resistance in poorly-differentiated NSCLC adenocarcinomas.There is a small group of cells named as cancer stem-like cells(CSCs)possessing the ability of self-renewal and higher proliferation rate,the increased capacity of invasion,metastasis and tumor formation.Increasing evidence confirms the existence of the CSCs as the "arch-criminal" which finally leads to the drug resistance and tumor recurrence after therapy.Therefore,elucidation of the biochemical mechanisms for CSCs and identification of the key regulator of cancer stemness are very meaningful to combat lung cancer in clinic.In this study,we found that RCD91,a RNA binding protein,was significantly downregulated in poorly-differentiated NSCLC adenocarcinomas and was highly expressed in well-differentiated NSCLC adenocarcinomas.Silencing of RCD91 expression in NSCLC cells promoted the growth of xenograft tumors derived from lung adenocarcinomas in nude mice,indicating that RCD91 could be one tumor suppressor gene.By exploring the underlied molecular mechanisms,it was found that that the expression of RCD91 was negatively correlated with the expressions of well-established tumor stem cells makers including CD 133 and the tumor or sphere initiating rate as well as the propotions of side population cells.Meanwhile,overexpression of RCD91 inhibited cancer stemness and overcame cisplatin resistance in poorly-differentiated lung adenocarcinomas.Thus RCD91 was identified as a negative regulator of cancer stemness in NSCLC adenocarcinomas.Previous reports have demonstrated that activation of WNT/?-catenin signaling accounted for the maintenance of CSCs in lung adenocarcinomas.Here we found that RCD91 interacted with the TCF4 binding region of ?-catenin via its RRM3 domain,suppressed the activation of WNT pathway and downregulated the transcriptional activity of TCF4-targeted genes including CCND1 and survivin.Interestingly RCD91 downregulated the level of c-Myc by interfering with the WNT pathway.However,c-Myc could downregulate the transcriptional activity of RCD91 in coordination with the transcriptional factor Spl.Thus regulation of cancer stemness in NSCLC adenocarcinomas was under precise control in the presence of the RCD91-c-Myc feedback loop.Considering the importance of RCD91 in regulating stemness in NSCLC adenocarcinomas,a drug screening system based on the transcription regulation of RCD91 was estabulished.It was found that resveratrol,emodin,wogonin,quercetin and the c-Myc inhibitor JQ-1 could all suppress the transcriptional activity of RCD91 mRNA and negatively regulate cancer stemness in poorly-differentiated lung adenocacinomas.Thus,this work demonstrated the new function and molecular mechanism of RCD91 in regulating cancer stemness in NSCLC adenocarcinomas,indicating that RCD91 could be a promising target therapeutic intervention of cancer stemness in treatment of pooyly-differentiated NSCLC adenocarcinomas.Conclusion:As the first-line chemotherapy drug for NSCLC adenocarcinomas,cisplatin therapy is of great therapeutic value in prolonging the survival and improving the life quality of patients with NSCLC adenocarcinomas.This thesis focused on the DDP resistance in poorly-differentiated NSCLCs and demonstrated the new roles and mechanisms of GCN2 phosphorylation and RCD91 in regulating DDP resistance.The elucidation of the mechanisms will provide an important theratical basis for the clinical correction of cisplatin resistance in poorly-differentiated NSCLC adenocarcinomas and will lay the foundation for making strategies to reverse cisplatin resistance in poorly-differentiated NSCLC adenocarcinomas.