M6A RNA Methylation-Mediated Dysregulation of AGAP2-AS1 Promotes Trastuzumab Resistance of Breast Cancer.

INTRODUCTION: Trastuzumab is commonly used to treat human epidermal growth factor receptor-2-positive (HER2+) breast cancer, but its efficacy is often limited by chemotherapy resistance. Recent studies have indicated that long non-coding RNAs (lncRNAs) play important roles in tumor progression and response to therapy. However, the regulatory mechanisms associating lncRNAs and trastuzumab resistance remain unknown. METHODS: Quantitative polymerase chain reaction was performed to detect the expression of related genes. Western blot and immunofluorescence assays were used to evaluate protein expression levels. A series of gain- or loss-of-function assays confirmed the function of AGAP2-AS1 in trastuzumab resistance, both in vitro and in vivo. RNA immunoprecipitation and pull-down analyses were conducted to verify the interaction between METTL3/YTHDF2 and lncRNA AGAP2-AS1. RESULTS: AGAP2-AS1 was upregulated in trastuzumab-resistant cells and SKBR-3R-generated xenografts in nude mice. Silencing AGAP2-AS1 significantly decreased trastuzumab-induced cytotoxicity both in vitro and in vivo. Furthermore, m6A methylation of AGAP2-AS1 was reduced in trastuzumab-resistant cells compared to that in parental cells. In addition, METTL3 increased m6A methylation of AGAP2-AS1, which finally induced the suppressed AGAP2-AS1 expression. Moreover, YTHDF2 was essential for METTL3-mediated m6A methylation of AGAP2-AS1. Functionally, AGAP2-AS1 regulated trastuzumab resistance by inducing autophagy and increasing ATG5 expression. CONCLUSION: we demonstrated that METTL3/YTHDF2-mediated m6A methylation increased the expression of AGAP2-AS1, which could promote trastuzumab resistance in breast cancer. AGAP2-AS1 regulates trastuzumab resistance by inducing autophagy. Therefore, AGAP2-AS1 may be a promising predictive biomarker and therapeutic target in patients with breast cancer.

Increased Expression of Exosomal AGAP2-AS1 (AGAP2 Antisense RNA 1) In Breast Cancer Cells Inhibits Trastuzumab-Induced Cell Cytotoxicity.

BACKGROUND Trastuzumab therapy is important for patients with HER2-positive breast cancer, but more and more patients have experienced trastuzumab resistance during recent years. Accumulating evidence from recent studies showed that long non-coding RNAs (lncRNAs) play essential roles in chemoresistance of various cancer types, but the precise role of lncRNAs in trastuzumab resistance is unclear. In the present study, we aimed to identify the biofunction of lncRNA APAP2-AS1 in tranastuzumab resistance and to reveal the underlying regulatory mechanism. MATERIAL AND METHODS By culturing HER2-positive SKBR-3 and BT474 cells with transtuzumab-containing medium, we built trastuzumab-resistant cells. Quantitative real-time PCR was used to test the expression of AGAP2-AS1 in the built trastuzumab-resistant cells. Cell viability assay and TUNEL assay were used to test the cell viability and apoptosis in each group. Exosomes were purified from cells cultured in exosomes-depleted FBS and identified by transmission electron microscopy. RESULTS qRT-PCR assay suggested that AGAP2-AS1 was upregulated in the built trastuzumab-resistant cells when compared with parental sensitive cells. Cell viability assay showed that silencing of AGAP2-AS1 enhanced the cytotoxicity induced by trastuzumab treatment. Mechanistically, we revealed that AGAP2-AS1 was secreted outside cells by incorporation into exosomes in an hnRNPA2B1-dependent manner. More importantly, co-culture AGAP2-AS1-containing exosomes with sensitive cells reduced the trastuzumab-induced cell death, and silencing of AGAP2-AS1 from exosomes reversed this effect. In summary, AGAP2-AS1 promotes trastuzumab resistance of breast cancer cells through packaging into exosomes. CONCLUSIONS Knockdown of AGAP2-AS1 may be helpful for improving the clinical outcome for HER2+ breast cancer patients and could serve as a therapeutic target.

The utility of sentinel Lymph node biopsy in the lateral neck in papillary thyroid carcinoma.

Background: Regional lymph node metastases (LNMs) are very common in papillary thyroid carcinoma (PTC) and associate with locoregional recurrence. The appropriate management of cervical lymph nodes is very important. Therefore, this study evaluated the application of sentinel lymph node biopsy (SLNB) in the lateral neck in PTC patients. Methods: This prospective study was conducted from 1 November 2015 to 31 December 2017 and recruited 78 PTC patients treated with SLNB in the lateral neck and prophylactic lateral neck dissection (compartments II-IV) followed by thyroidectomy or lobectomy and central neck dissection. Results: There were 78 PTC patients enrolled and sentinel lymph nodes (SLNs) were detected among 77 patients. A total of 30 patients were diagnosed with SLN metastases (SLNMs). The remaining 47 patients were pathologically negative of SLN, whereas 4 patients were found with metastases in the non-SLN samples. The detection rate, sensitivity, specificity, and accuracy rate of SLNB in the lateral neck were 98.7%, 87.1%, 98.7%, and 93.6%, respectively. However, the values varied greatly in each specific compartment of the lateral neck, and all of them were no more than 80%. These 34 PTC patients diagnosed with lateral compartment LNM (LLNM) were more likely to be younger (41.38 vs. 48.95 years old, p = 0.002) and exhibit extrathyroidal extension (56.8% vs. 31.7%, p = 0.026) and central compartment LNM (66.7% vs. 12.1%, p < 0.001). Tumors located in the upper third of the thyroid lobe also had a significantly higher probability of LLNM compared with those in middle or inferior location (66.7% vs. 35.3% vs. 34.8%, p = 0.044). At last, age (OR=0.912, p = 0.026), tumor location (upper vs inferior, OR=17.478, p = 0.011), and central compartment LNM (OR=25.364, p < 0.001) were independently predictive of LLNM. Conclusions: SLNB can help surgeons to identify some PTC patients who may benefit from therapeutic lateral neck dissection and protect some patients from prophylactic lateral neck dissection. However, it cannot accurately indicate specific lateral compartment-oriented neck dissection. Meanwhile, LLNM is more likely to occur in PTC patients with younger age or upper pole tumors or central compartment LNM.

Polymorphisms of the FAS and FASL genes and risk of breast cancer.

FAS and its ligand FASL are crucial in apoptotic cell death. Loss of FAS and gain of aberrant FASL expression are common features of malignant transformation. This study was designed to investigate whether the functional polymorphisms of FAS -1377G/A (rs2234767) and FASL -844T/C (rs763110) affect the risk of developing breast cancer. Genotypes were analyzed by a polymerase chain reaction-restriction fragment length polymorphism assay in 436 breast cancer patients and 496 healthy controls. In this study, as compared to the wild-type homozygote and heterozygote, the distribution of the FAS -1377GG, GA and AA genotypes among breast cancer patients were significantly different from those among healthy controls (P=0.011), with the AA genotype being more prevalent among patients than the controls (P=0.003). Similarly, the frequencies of the FASL -844TT, TC and CC genotypes also significantly differed among breast cancer patients and healthy controls (P<0.001), with the CC genotype being significantly over-represented in breast cancer patients compared with the controls (P<0.001). In the unconditional logistic regression model following adjustment for age, the subjects carrying the FAS -1377AA genotype had a 1.75-fold increased risk [95% confidence interval (CI), 1.13-2.69] for development of breast cancer compared with patients carrying the GG genotype. Similarly, in the recessive model, the FASL -844CC genotype significantly increased the risk of breast cancer with an odds ratio (OR) of 1.92 (95% CI 1.46-2.54) compared with the TT or TT + TC genotypes. Our results suggest that functional polymorphisms in the death pathway genes FAS and FASL significantly contribute to the occurrence of breast cancer.