RNA Activation Reverses TKI Resistance In EGFR-mutant NSCLC Patients By Upregulating PTEN Expression

BackgroundLung cancer is the most common malignant tumors with the highest mortality in the world at present. With the development and Application of genomics and targeted drugs, targeted therapy has taken the place of traditional chemotherapy as the first line treatment of advanced non-small cell lung cancer.Tyrosine kinase inhibitors of epidermal growth factor receptor has been widely used in the treatment of NSCLC.Asian women with no smoking are particularly sensitive to TKI treatment. A number of studies,including IPASS、NEJ002、EURTAC、WJTOG3405, have confirmed that EGFR-TKI as first-line treatment of EGFR mutation in patients with advanced NSCLC curative effect is better than that of chemotherapy. However, some NSCLC patients have primary resistance to EGFR-TKI. In addition, even if EGFR-TKI treatment is effective for patients, most patients will appear acquired drug resistance within a year. Therefore, no matter EGFR-TKI therapy is effective or not, NSCLC patients have to face the disease recurrence and progress.The resistance of EGFR-TKI has become the main bottleneck of NSCLC targeted therapy, thus clarifying the mechanisms of resistance to EGFR-TKI and finding a method to overcome the resistance will make patients with NSCLC benefit from the treatment. According to the present studies, T790M mutation and amplification of MET are most common mechanisms of acquired drug resistance, accounted for about 60%. The other 40% mechanisms have not been fully elucidated. Besides these, loss of PTEN is the most widely studied factor to the TKI resistance. PTEN is the first found tumor suppressor gene with the phosphatase activity so far. The expression product has the inhibitory effect on tumor. It can negatively regulate Akt activiton and plays an important role in the PI3K/Akt pathway which control the proliferation and survival of cells. Researches show that inactive expression of PTEN was found in variety of malignant tumors including lung cancer, esophageal cancer, liver cancer, and it plays an important role in these tumors. A study by Endoh et al shows that patients receive the TKI treatment with high expression of PTEN have a long survial period. And to the patients wih PTEN lost, the treatment effect is poor. Sos et al. Showed that PTEN can activate Akt and lead to EGFR-TKI resistance. Zhuang et al enhanced the sensitivity of tumor cells to TKI by up-ragulating expression of PTEN using X-ray irradiation. The above studies show that PTEN plays a very important role in the mechanism of EGFR-TKI drug resistance excluding T790M mutation and amplification of MET.In 2006, Li et al found dsRNA targeting the non CpG Island region of the promoter can induce increase of the expression of some silent gene in mRNA and protein level. He called this phenomenon RNA activation. And he named the dsRNA with activing function as small activiting RNA(saRNA). Effect of RNAa is enhanceing expression of genes and effect of RNA interference is inhibiting the expression of genes. Both of them have the characteristics of high efficiency, specificity and relative easy operation. RNAa provides a new strategy for tumor gene therapy. Compared with RNAi, RNAa does not need to consider whether there is a tumor specific oncogenes as target genes in the treatment of cancer or not, it can specifically activate existing gene with low expression in cells. saRNA does not need to import a new gene, so that people can increase a gene more easily. So saRNA has its unique advantages. We can use RNAa to activate tumor suppressor genes, apoptosis suppressor gene to treat tumors.Through the analysis of human cadherin E and p21Waf1/Cip1 (p21) gene promoter structure, Li selected the saRNA target site which can activate downstream gene transcription from this sequence. He synthesised saRNA with 21 nucleotides in length, then he transfected this saRNA into human bladder cancer, prostate cancer, breast cancer and cervical cancer and other tumor cells in vitro. The results showed that compared with the control cells, expressions of cadherin E and p21 in these cells were increased more than 2-13 times respectively in the RNA and protein level. Although the majority of these studies are in vitro, but has achieved good results, indicating that RNAa technology has a good prospect in tumor gene therapy.The loss and downragulation of PTEN expression are related with TKI resistance. There have been no studies using RNAa technology to activate expression of PTEN in lung cancer. In view of this study selected PTEN as the target gene, using RNAa to upragulate the expression of PTEN to reverse the TKI resistance.Part1. The relation between the PTEN expression and TKI resistance.Objective:Analyse of the expression of PTEN and pAkt in H-157, H-1650, H-1355 lung cancer cell lines and sensitivity of these cell lines to TKI. To investigate the relation between PTEN expression and TKI resistance and to study the possible mechanism of drug resistance.Methods:Analyse the expression of PTEN in H-157, H-1650, H-1355 lung cancer cell lines from RNA and protein level by RT-PCR and Western blot. Draw the growth curves of three kinds of cells before and after TKI treatment and detect cell apoptosis by flow cytometry. Then evaluate TKI treatment effect to these cell lines.Results:Expression of PTEN in the H-1355 cell line was high, expression of PTEN in the H-157 cell line was low and expression of PTEN in the H-1650 was lost. After TKI treatment the growth curve of H-1355 cells was significantly inhibited and cell optosis is obvious (P< 0.05). The growth curve of H-1650 cells slightly inhibited, there was no significant apoptosis (P> 0.05). The growth curve of H-157 cells did not change significantly, there was no significant apoptosis (P> 0.05). After treatment with TKI, pAkt in H-1355 cells showed low expression, pAkt in H-157 and H-1650 showed high expression.Conclusion:There is relationship between the expression of PTEN and TKI resistance. Cancer cells with normal PTEN expression is more sensitive to TKI treatment than the cells with low PTEN expressoin or loss of PTEN. Therefore, the low expression of PTEN may lead to the resistance of TKI.The sustained activation of Akt/PI3K pathway is the reason why PTEN loss cause TKI resistance.Part2. Construction and screening of saRNA targeting PTENObjective Designed and screened out saRNA targeting PTEN with obvious activation functionMethods:According to the design principle of saRNA of the existing literature, we designed 5 dsRNAs as the candidate saRNA. These dsRNAs were synthesised by Sagon company Shanghai. Then were transfected into H-157 cells. We detected the PTEN expression by RT-PCR to screen out functional saRNA. We transfected this functional saRNA to H-157 cells to evaluate whether it can activiate the PTEN expression in lung tissue by Western blot.Results:Three of the saRNA we designed out can upragulate the expression of PTEN in H-157 cell. One of this saRNA can enhanc the expression more than twice. After transfecing this functional saRNA the PTEN expression was increased from both RNA and protein level.Conclusion:RNA activation phenomenon also exists in the lung tissue cells, through transfecting functional saRNA targeting PTEN to lung cancer cells, we can specifically enhance PTEN expression.Part3. Relationship between the upragulation of PTEN by saRNA and TKI resistanceObjective:To evaluate whether the upragulation of PTEN by saRNA can reverse TKI resistance.Methods:Using RT-PCR and Western blot to detect the expression of PTEN in H-157 cells after transfected into saRNA.And evaluate sensitivity of H-157 cell to TKI by cell growth curves and aptosis anylasis.Results:After transfecting into saRNA, PTEN expression in H-157 cell was obviously enhanced(p<0.05), and the growth curve was significally inhibited (p< 0.05).the aptosis rate of saRNA group was%, dsControl group was% and mock group was%(p<0.05).Conclusion:The low expression of PTEN is one of the mechanisms causing TKI resistance, the cause of drug resistance is hyperphosphorylation of Akt caused by low expression of PTEN.Then the activation of Akt/PI3K pathway induced TKI resistance. We can reverse the TKI resistance by upragulate the expression of PTEN using RNAa tecnology.