A novel FAK-degrading PROTAC molecule exhibited both anti-tumor activities and efficient MDR reversal effects.

FAK (focal adhesion kinase) is widely involved in cancer growth and drug resistance development. Thus, FAK inhibition has emerged as an effective strategy for tumor treatment both as a monotherapy or in combination with other treatments. But the current FAK inhibitors mainly concentrate on its kinase activity, overlooking the potential significance of FAK scaffold proteins. In this study we employed the PROTAC technology, and designed a novel PROTAC molecule F2 targeting FAK based on the FAK inhibitor IN10018. F2 exhibited potent inhibitory activities against 4T1, MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells with IC50 values of 0.73, 1.09, 5.84 and 3.05 µM, respectively. On the other hand, F2 also remarkably reversed the multidrug resistance (MDR) in HCT8/T, A549/T and MCF-7/ADR cells. Both the effects of F2 were stronger than the FAK inhibitor IN10018. To our knowledge, F2 was the first reported FAK-targeted PROTAC molecule exhibiting reversing effects on chemotherapeutic drug resistance, and its highest reversal fold could reach 158 times. The anti-tumor and MDR-reversing effects of F2 might be based on its inhibition on AKT (protein kinase B, PKB) and ERK (extracellular signal-regulated kinase) signaling pathways, as well as its impact on EMT (epithelial-mesenchymal transition). Furthermore, we found that F2 could reduce the protein level of P-gp in HCT8/T cells, thereby contributing to reverse drug resistance from another perspective. Our results will boost confidence in future research focusing on targeting FAK and encourage further investigation of PROTAC with potent in vivo effects.

miR-92b-3p Regulates Cell Cycle and Apoptosis by Targeting CDKN1C, Thereby Affecting the Sensitivity of Colorectal Cancer Cells to Chemotherapeutic Drugs.

Colorectal cancer (CRC) is the third most common malignant tumor in the world and the second leading cause of cancer death. Multidrug resistance (MDR) has become a major obstacle in the clinical treatment of CRC. The clear molecular mechanism of MDR is complex, and miRNAs play an important role in drug resistance. This study used small RNAomic screens to analyze the expression profiles of miRNAs in CRC HCT8 cell line and its chemoresistant counterpart HCT8/T cell line. It was found that miR-92b-3p was highly expressed in HCT8/T cells. Knockdown of miR-92b-3p reversed the resistance of MDR HCT8/T cells to chemotherapeutic drugs in vitro and in vivo. Paclitaxel (PTX, a chemotherapy medication) could stimulate CRC cells to up-regulate miR-92b-3p expression and conferred cellular resistance to chemotherapeutic drugs. In studies on downstream molecules, results suggested that miR-92b-3p directly targeted Cyclin Dependent Kinase Inhibitor 1C (CDKN1C, which encodes a cell cycle inhibitor p57Kip2) to inhibit its expression and regulate the sensitivity of CRC cells to chemotherapeutic drugs. Mechanism study revealed that the miR-92b-3p/CDKN1C axis exerted a regulatory effect on the sensitivity of CRC cells via the regulation of cell cycle and apoptosis. In conclusion, these findings showed that miR-92b-3p/CDKN1C was an important regulator in the development of drug resistance in CRC cells, suggesting its potential application in drug resistance prediction and treatment.

Autophagy inhibition and microRNA­199a­5p upregulation in paclitaxel­resistant A549/T lung cancer cells.

Multidrug resistance (MDR) is one of the major reasons for the clinical failure of cancer chemotherapy. Autophagy activation serves a crucial role in MDR. However, the specific molecular mechanism linking autophagy with MDR remains unknown. The results of the present study demonstrated that autophagy was inhibited and microRNA (miR)­199a­5p levels were upregulated in MDR model lung cancer cells (A549/T and H1299/T) compared with those in the parental cell lines. Paclitaxel (PTX) treatment increased the expression levels of miR­199a­5p in parental lung cancer cells compared with those in PTX­untreated cells, and these expression levels were negatively correlated with PTX sensitivity of the cells. miR­199a­5p knockdown in A549/T cells induced autophagy and resensitized cells to multiple chemotherapeutic drugs including PTX, taxotere, topotecan, SN38, oxaliplatin and vinorelbine. By contrast, miR­199a­5p overexpression in A549 cells suppressed autophagy and desensitized cells to these chemotherapeutic drugs. Mechanistically, the results of the present study demonstrated that miR­199a­5p blocked autophagy by activating the PI3K/Akt/mTOR signaling pathway and inhibiting the protein expression of autophagy­related 5. Furthermore, p62 protein was identified as a direct target of miR­199a­5p; miR­199a­5p bound to p62 mRNA to decrease its mRNA and protein expression levels. In conclusion, the results of the present study suggested that miR­199a­5p may contribute to MDR development in lung cancer cells by inhibiting autophagy and targeting p62. The regulatory effect of miR­199a­5p on autophagy may provide novel insights for future multidrug­resistant lung cancer chemotherapy.

Binding of RNA m6A by IGF2BP3 triggers chemoresistance of HCT8 cells via upregulation of ABCB1.

The overexpression of ATP-binding cassette transporters subfamily B member 1 (ABCB1) is known to be the primary trigger of multidrug resistance (MDR) in colorectal cancer (CRC), leading to chemotherapy failure. However, factors that regulate chemoresistance in CRC cells are largely unknown. To identify proteins involved in MDR in CRC, we used proteomics and transcriptomics approaches to analyze HCT8/T cells and parental HCT8 cells. Results showed that the expression of insulin-like growth factor-2 mRNA-binding protein 3 (IGF2BP3) was upregulated in HCT8/T cells, and siIGF2BP3 remarkably elevated the sensitivity of HCT8/T cells to DOX. Overexpression of IGF2BP3 promoted ABCB1 expression, and reduced the sensitivity to ABCB1 substrates. Conversely, knockdown of IGF2BP3 reduced ABCB1 expression, and increased the sensitivity to ABCB1 substrates in vitro and in vivo. This phenomeon was further confirmed by the strong association of IGF2BP3 and ABCB1 expression with DOX sensitivity. Mechanistically, IGF2BP3, as a N6-methyladenosine (m6A) reader, directly bound to the m6A-modified region of ABCB1 mRNA, thereby promoting the stability and expression of ABCB1 mRNA. Overall, the results showed that IGF2BP3 bound to the m6A modification region of ABCB1 mRNA, and conferred chemoresistance in CRC cells via upregulation of ABCB1. These findings suggest that IGF2BP3 might be a potential biomarker for predicting the development of MDR in CRC. Targeting IGF2BP3 might be an important chemotherapeutic strategy for preventing MDR development in CRC.