| Background and AimsLung cancer, the rates of which have risen more quickly over the past three decades, is the leading cause of incidence and mortality among malignant tumors patients in China according to the Third National Retrospective Sampling Death Survey. Non-small cell lung carcinoma(NSCLC), the most common type of lung cancer, is responsible for more than 85% of cases. The 5-year overall survival rate of NSCLC remains less than 15% despite integrative therapies that include surgery, chemotherapy and radiotherapy. In recent years, molecularly targeted therapy, represented with epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs), was widely used as the option for the second- and third-line treatment of NSCLC because of its accurate, effective and less side effect. However, patients with EGFR mutations who initially respond to EGFR-TKIs Seventually acquire resistance after 8- 12 months. The study of mechanisms underlying drug-resistance to EGFR-TKIsis a critical problem in the treatment of patients with advanced NSCLC.MicroRNAs(miRNAs), a class of noncoding single-stranded RNAs of 19-25 nucleotides in length, are involved in cell growth and apoptosis by regulating almost 30% of genes expression at the post-transcriptional level. Studies have shown that miR-223 affects cell function via IGF-1R/P13K/Akt signal pathway. The abnormal activation of IGF-1R/P13K/Akt signal may be involved in the resistance of NSCLC cells to EGFR-TKIs. In this study, we first established the Erlotinib-resistant cell lines HCC827/ER and PC9/ER by exposing the parent cells to high-dose pulses combined with continuous low-dose administration of Erlotinib. miR-223 expression was detected in HCC827, HCC827/ER, PC9 and PC9/ER cells. The key molecules of the IGF-1R/PI3K/Akt signaling pathway were analyzed via western-blot in vitro and in vivo. In PC9/ER cells, downregulated mi R-223 expression was observed due to the overexpression of insulin-like growth factor 1 receptor(IGF-1R) based on qRT-PCR analysis, and the expression levels of miR-223 and IGF-1R were inversely correlated in PC9/ER cells. Next, following lentiviral packaging, a PC9/ER cell line in which mi R-223 expression was stably upregulated was established. The expression of IGF-1R and p-Akt was reduced in these cells, and the sensitivity of these cells to Erlotinib was partially restored. We obtained similar results in vivo nude mice xenografts. In our study, we found an interesting result. In PC9 cells, there were 3.05% CD133 positive cells, which increased 4.96 times in continuing resistant PC9 cells. So we isolated and cultured stem like PC9/CD133+ cells and detected the expression of miR-223 in PC9 and PC9/CD133+ cells. The expression of IGF-1R, p-IGF-1R, Akt, p-Akt was detected. We found the expression of miR-223 decreased in continuing resistant PC9 cells compared with the parental cells. miR-223 negatively regulated its target molecule IGF-1R and activated downstream PI3K/Akt pathway. Our results suggested that the expression of mi R-223 was lower in PC9/ER cells compared to PC9 cells. The downregulation of miR-223, which activated the IGF-1R/PI3K/Akt pathway signaling pathway in PC9/ER cells, is one mechanism that is responsible for the resistance of PC9/ER cells to Erlotinib.Materials and Methods:1. Establishment of Erlotinib-resistant PC9 and HCC827 CellsTwo EGFR-TKI-resistant cell lines, PC9/ER and HCC827/ER, were established from parental cells by administering high-dose(1-5 μM) pulses combined with continuous low-dose(0.01 μM) applications of Erlotinib for more than 8 months. EGFR mutations(exons 18, 19, 20 and 21, T790 M and S768I) were performed by ARMS-PCR analysis. The effects of increasing concentrations of Erlotinib on cell viability were determined via the CCK8 assay. The mRNA levels of miR-223, PTEN and c-Met were detected in HCC827, HCC827/ER, PC9 and PC9/ER cells by Real-time RT-PCR. The protein levels of PTEN, Akt and p-Akt were examined by western-blot.2. Culture and identification of stem-like PC9/CD133+ cells:Tumor initiating cells were induced by serum-free medium suspension culture in combination with chemotherapy drugs paclitaxel(0.1 μmol / L). CD133-positive cells(PC9/CD133+ cells) were isolated by FACS. The rate of CD133 positive cells were detected by Flow Cytometry in the forth generation cells treated by paclitaxel. The inhibition rate of Erlotinib on PC9 and PC9/CD133+ cells(IC50) was detected by CCK8 assay. Xenografts in nude mice was used to detect the tumor capacity of stem-like PC9/CD133+ cells.3. miR-223 partially reversed the acquired resistance to Erlotinib by inhibiting the IGF-1R/PI3K/Akt pathway in vitroNSCLC cell lines PC9, PC9/ER and PC9/CD133+ were cultured. PC9/ER and PC9/CD133+ cells were infected with the lentivirus carrying miR-223 to generate the PC9/ER-miR-223-upregulated cell lines. The effects of increasing concentrations of Erlotinib on cell viability of PC9, PC9/ER, PC9/ER-miR-223、PC9/ER-EV were determined via the CCK8 assay. The mRNA expression of IGF-1R in the acquired resistance of PC9/ER cells was investigated by Real-time RT-PCR. The protein levels of IGF-1R, p-IGF-1R, Akt, p-Akt, p70S6 K and p-p70S6 K were examined by western-blot in PC9, PC9/ER, PC9/EREV and PC9/ER-miR-223 cells. PC9 and PC9/ER cells were treated with Erlotinib(0, 0.001, or 0.01 μM) for 72 h. After that, the protein expression of EGFR, p-EGFR, IGF-1R, pIGF-1R, Akt, p-Akt, p70S6 K and p-p70S6 K was detected by western-blot. After treated by 10 ng/mL IGF-1, the IGF-1R agonists, the above parameters were examined again.4. miR-223 partially reversed the acquired resistance to Erlotinib by inhibiting the IGF-1R/PI3K/Akt pathway in vivoMale BALB/c nude mice(SPF, 6- 8 weeks old), purchased from the Animal Research Center of Xinqiao Hospital of The Third Military Medical University, were given water and chow ad libitum at all times. Nude mice were randomized into four groups(n = 5 per group) and subcutaneously injected into the left front leg for the tumorigenesis assay with a total of 5×106 PC9, PC9/ER, PC9/ER cells transfected with the control lentivirus(PC9/ER-EV) and PC9/ER cells transfected with the miR-223-overexpressing lentivirus(PC9/ER-mi R-223), respectively. The tumor volume(V) was calculated according to the formula V =(length × width2)/2. When the tumors reached a mean volume of approximately 100 mm3, the mice began to receive the Erlotinib treatment(30 mg/kg/d, n=5) via gavage for two weeks. Tumor size was measured every 3 days until the tumors were removed. Nude mice were sacrificed with cervical dislocation under ether inhalation anesthesia at the experimental endpoint. The mRNA levels of miR-223 and IGF-1R in xenografts were detected by Real-time RT-PCR. The protein levels of IGF-1R, p-IGF-1R, Akt, p-Akt, p70S6 K and p-p70S6 K were determined by western-blot.Results:1. Two EGFR-TKI-resistant cell lines, PC9/ER and HCC827/ER, were established from parental cells by administering high-dose(1-5 μM) pulses combined with continuous low-dose(0.01 μM) applications of Erlotinib for more than 8 months. The IC50 values for Erlotinib in PC9/ER and HCC827/ER cells were 734.84 ± 69.02 nM and 3103 ± 601.16 nM individually, approximately 37-fold higher in PC9/ER cells and 155.4-fold higher in HCC827/ER cells than in the corresponding parental cells. EGFR-TKI-resistant cell lines were fusiform and smaller than parent cells. The secondary T790 M EGFR mutation was not detected in PC9/ER or HCC827/ER cells based on the ARMS assay. c-Met gene amplification was not detected in PC9/ER or HCC827/ER cells based on real-time RT-PCR. PTEN mRNA expression was decreased by 50% in HCC827/ER cells compared with HCC827 cells. The protein level of PTEN was also downregulated in HCC827/ER cells. No difference in the expression of PTEN was observed between PC9 and PC9/ER cells. The level of mi R-223 in PC9/ER cells was decreased by 88% compared with parent cells. No difference in the expression of miR-223 was observed between HCC827 and HCC827/ER cells.2. The rate of CD133 positive cells was 91.74% in the forth generation of PC9 cells treated by paclitaxel. The cells grew into spheres in the stem cells growth medium. The inhibition rate of Erlotinib on PC9/CD133+ cells(IC50) was 653.73 ± 15.45 nM and resistance index is 33.28 times higher than that of PC9 cells. Both CD133+ cells(5 × 104 cells) and PC9 cells(5 × 106 cells) successfully developed into tumors in nude mice. HE staining showed a typical adenocarcinoma structure. On the contrary, PC9 cells(5 × 104 cells) failed to develop into tumors.3. In the PC9/ER cells, miR-223 expression decreased(p < 0.05), while the level of IGF-1R mRNA increased. IGF-1R mRNA expression was reduced by 50% in PC9/ERmi R- 223 cells compared with PC9/ER cells. The overexpression of miR-223 inhibited the protein expression of p-IGF-1R, p-Akt and p-70S6 K. Our results demonstrated that both IGF-1R and its downstream PI3K/Akt/mTOR/p70S6 K signal pathway were inhibited by mi R-223. The downregulation of miR-223, which activated the IGF-1R/PI3K/Akt pathway signaling pathway in PC9/ER cells, is one mechanism that is responsible for the resistance of PC9/ER cells to Erlotinib. After treated by IGF-1, which mimicking the downregulation of miR-223, the expression of IGF-1R, p-IGF-1R and p-Akt in PC9/ER-miR-223-IGF-1 cells increased. IGF-1 reversed the inhibition of miR-223 on IGF-1R/PI3K/Akt pathway. The expression of miR-223 in stem-like PC9/CD133+ cells was reduced by 72.6% than in PC9 cells. The expression of p-IGF-1R and p-Akt was significantly decreased in stem-like PC9/CD133+ cells.4. A total of 5×106 PC9, PC9/ER, PC9/ER-EV or PC9/ER-miR-223 cells were subcutaneously injected into nude mice(6-8 weeks old) under the front left leg for the tumorigenesis assay, which was analyzed via confocal laser scanning microscopy. The stable expression of miR-223 in the Xenografts was confirmed by Real-time PCR. IGF-1R mRNA and p-IGF-lR protein expression in the xenografted tumors from PC9/ER-miR-223 cells were lower than that from PC9/ER and PC9/ER-EV cells. The expression of p-Akt and p-p70S6 K was also inhibited in the xenografted tumors derived from PC9/ER-mi R-223 cells. These results were consistent with the in vitro results.Conclusions:1.We established the Erlotinib-resistant cell lines HCC827/ER and PC9/ER by exposing the parent cells to high-dose pulses combined with continuous low-dose administration of Erlotinib and revealed that different resistance mechanisms might be in involved in the sensitivity of mutant lung adenocarcinoma cells to EGFR-TKIs, despite the establishment of these cell lines using the same culture conditions and methods. The mechanisms underlying the secondary resistance to EGFR-TKIs may be more complex.2.In PC9/ER cells, CD133-positive cells increased. In PC9/ER cells, the expression of mi R-223 was decreased and IGF-1R, the negative regulatory target gene of miR-223, increased and activated downstream PI3K/Akt signal pathway, which is one mechanism that is responsible for the resistance of PC9/ER cells to Erlotinib. Besides, miR-223 may be involved in the regulation of tumor cell proliferation by inhibiting mTOR/p70S6 K signal pathway, which is required further study. The results in vivo were consistent with the in vitro results.3.The downregulation of miR-223 in stem-like PC9/CD133+ cells induced the upregulation of IGF-1R and activated PI3K/Akt pathway. Upregulation of miR-223 inhibited IGF-1R/PI3K/Akt pathway in stem-like PC9/CD133+ cells. As a result, we speculated that the activation of IGF-1R/PI3K/Akt pathway may be one mechanism that is responsible for the resistance of stem-like PC9/CD133+ cells to Erlotinib. Erlotinib is not able to eliminate the stem cells in PC9 cells, which may be one mechanism of the secondary resistance. |
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