piR-39980 mediates doxorubicin resistance in fibrosarcoma by regulating drug accumulation and DNA repair

Resistance to doxorubicin (DOX) is an obstacle to successful sarcoma treatment and a cause of tumor relapse, with the underlying molecular mechanism still unknown. PIWI-interacting RNAs (piRNAs) have been shown to enhance patient outcomes in cancers. However, there are few or no reports on piRNAs affecting chemotherapy in cancers, including fibrosarcoma. The current study aims to investigate the relationship between piR-39980 and DOX resistance and the underlying mechanisms. We reveal that piR-39980 is less expressed in DOX-resistant HT1080 (HT1080/DOX) fibrosarcoma cells. Our results show that inhibition of piR-39980 in parental HT1080 cells induces DOX resistance by attenuating intracellular DOX accumulation, DOX-induced apoptosis, and anti-proliferative effects. Its overexpression in HT1080/DOX cells, on the other hand, increases DOX sensitivity by promoting intracellular DOX accumulation, DNA damage, and apoptosis. The dual-luciferase reporter assay indicates that piR-39980 negatively regulates RRM2 and CYP1A2 via direct binding to their 3′UTRs. Furthermore, overexpressing RRM2 induces DOX resistance of HT1080 cells by rescuing DOX-induced DNA damage by promoting DNA repair, whereas CYP1A2 confers resistance by decreasing intracellular DOX accumulation, which piR-39980 restores. This study reveals that piR-39980 could reduce fibrosarcoma resistance to DOX by modulating RRM2 and CYP1A2, implying that piRNA can be used in combination with DOX.

Doxorubicin-Resistant TNBC Cells Exhibit Rapid Growth with Cancer Stem Cell-like Properties and EMT Phenotype, Which Can Be Transferred to Parental Cells through Autocrine Signaling

Emerging evidence suggests that breast cancer stem cells (BCSCs), and epithelial–mesenchymal transition (EMT) may be involved in resistance to doxorubicin. However, it is unlear whether the doxorubicin-induced EMT and expansion of BCSCs is related to cancer dormancy, or outgrowing cancer cells with maintaining resistance to doxorubicin, or whether the phenotypes can be transferred to other doxorubicin-sensitive cells. Here, we characterized the phenotype of doxorubicin-resistant TNBC cells while monitoring the EMT process and expansion of CSCs during the establishment of doxorubicin-resistant MDA-MB-231 human breast cancer cells (DRM cells). In addition, we assessed the potential signaling associated with the EMT process and expansion of CSCs in doxorubicin-resistance of DRM cells. DRM cells exhibited morphological changes from spindle-shaped MDA-MB-231 cells into round-shaped giant cells. They exhibited highly proliferative, EMT, adhesive, and invasive phenotypes. Molecularly, they showed up-regulation of Cyclin D1, mesenchymal markers (β-catenin, and N-cadherin), MMP-2, MMP-9, ICAM-1 and down-regulation of E-cadherin. As the molecular mechanisms responsible for the resistance to doxorubicin, up-regulation of EGFR and its downstream signaling, were suggested. AKT and ERK1/2 expression were also increased in DRM cells with the advancement of resistance to doxorubicin. Furthermore, doxorubicin resistance of DRM cells can be transferred by autocrine signaling. In conclusion, DRM cells harbored EMT features with CSC properties possessing increased proliferation, invasion, migration, and adhesion ability. The doxorubicin resistance, and doxorubicin-induced EMT and CSC properties of DRM cells, can be transferred to parental cells through autocrine signaling. Lastly, this feature of DRM cells might be associated with the up-regulation of EGFR.

USP47-Mediated Deubiquitination and Stabilization of TCEA3 Attenuates Pyroptosis and Apoptosis of Colorectal Cancer Cells Induced by Chemotherapeutic Doxorubicin

The ubiquitin–proteasome system regulates a variety of cellular processes including growth, differentiation and apoptosis. While E1, E2, and E3 are responsible for the conjugation of ubiquitin to substrates, deubiquitinating enzymes (DUBs) reverse the process to remove ubiquitin and edit ubiquitin chains, which have profound effects on substrates’ degradation, localization, and activities. In the present study, we found that the deubiquitinating enzyme USP47 was markedly decreased in primary colorectal cancers (CRC). Its reduced expression was associated with shorter disease-free survival of CRC patients. In cultured CRC cells, knockdown of USP47 increased pyroptosis and apoptosis induced by chemotherapeutic doxorubicin. We found that USP47 was able to bind with transcription elongation factor a3 (TCEA3) and regulated its deubiquitination and intracellular level. While ectopic expression of USP47 increased cellular TCEA3 and resistance to doxorubicin, the effect was markedly attenuated by TCEA3 knockdown. Further analysis showed that the level of pro-apoptotic Bax was regulated by TCEA3. These results indicated that the USP47-TCEA3 axis modulates cell pyroptosis and apoptosis and may serve as a target for therapeutic intervention in CRC.

CK2α/CSNK2A1 Induces Resistance to Doxorubicin through SIRT6-Mediated Activation of the DNA Damage Repair Pathway

CK2α/CSNK2A1 is involved in cancer progression by phosphorylating various signaling molecules. Considering the role of CSNK2A1 in cancer progression and the phosphorylation of SIRT6 and the role of SIRT6 in chemoresistance through the DNA damage repair pathway, CSNK2A1 and SIRT6 might be involved in resistance to conventional anti-cancer therapies. We evaluated the expression of CSNK2A1 and phosphorylated SIRT6 in the 37 osteosarcoma patients and investigated the effects of CSNK2A1 and the phosphorylation of SIRT6 on Ser338 on resistance to the anti-cancer effects of doxorubicin. Higher expression of CSNK2A1 and phosphorylated SIRT6 was associated with shorter survival in osteosarcoma patients. U2OS and KHOS/NP osteosarcoma cells with induced overexpression of CSNK2A1 were resistant to the cytotoxic effects of doxorubicin, and the knock-down of CSNK2A1 potentiated the cytotoxic effects of doxorubicin. CSNK2A1 overexpression-mediated resistance to doxorubicin was associated with SIRT6 phosphorylation and the induction of the DNA damage repair pathway molecules. CSNK2A1- and SIRT6-mediated resistance to doxorubicin in vivo was attenuated via mutation of SIRT6 at the Ser338 phosphorylation site. Emodin, a CSNK2A1 inhibitor, potentiated the cytotoxic effects of doxorubicin in osteosarcoma cells. This study suggests that blocking the CSNK2A1-SIRT6-DNA damage repair pathway might be a new therapeutic stratagem for osteosarcomas.

CK2α/CSNK2A1 Induces Resistance to Doxorubicin Through SIRT6 Mediated Activation of the DNA Damage Repair Pathway

BackgroundCK2α/CSNK2A1 is involved in cancer progression by phosphorylating various signaling molecules. Considering the role of CSNK2A1 in cancer progression and phosphorylation of SIRT6 and the role of SIRT6 in chemoresistance through the DNA damage repair pathway, CSNK2A1 and SIRT6 might be involved in resistance to the conventional anti-cancer therapies.MethodsWe evaluated the expression of CSNK2A1 in the 37 osteosarcoma patients and investigated the effects of CSNK2A1 and phosphorylation of SIRT6 on Ser338 on the resistance to the anti-cancer effects of doxorubicin. Results Higher expression of CSNK2A1 predicted shorter overall survival and relapse-free survival in both general osteosarcoma patients and sub-population of patients who received postoperative chemotherapies. U2OS and KHOS/NP osteosarcoma cells with induced overexpression of CSNK2A1 were resistant to cytotoxic effects of doxorubicin, and knock-down of CSNK2A1 potentiated the cytotoxic effects of doxorubicin. CSNK2A1 overexpression-mediated resistance to doxorubicin was associated with SIRT6 phosphorylation and induction of the DNA damage repair pathway molecules ATM and Chk2. CSNK2A1 and SIRT6 mediated resistance to doxorubicin in vivo was attenuated via mutation of SIRT6 at the Ser338 phosphorylation site. Emodin, a CSNK2A1 inhibitor, potentiated the cytotoxic effects of doxorubicin in osteosarcoma cells in vitro. ConclusionsThis study demonstrates that the expression of CSNK2A1 might be used as a prognostic indicator of osteosarcoma. In addition, it suggests that CSNK2A1 induces resistance to doxorubicin through phosphorylation of SIRT6-mediated activation of the DNA damage repair pathway. Therefore, blocking the CSNK2A1-SIRT6-DNA damage repair pathway might be a new therapeutic stratagem for the poor prognostic subgroup of osteosarcomas with high expression of CSNK2A1.

MiR-194-5p enhances the sensitivity of nonsmall-cell lung cancer to doxorubicin through targeted inhibition of hypoxia-inducible factor-1

Background Despite chemotherapy being a common treatment, an increase in chemoresistance over time is unavoidable. We therefore investigated the role of miR-194-5p in regulating chordoma cell behavior and examined the downstream effectors of miR-194-5p. Methods In this study, NSCLC cell lines A549 and H460 were cultured under hypoxic conditions for 1 week to induce drug resistance to doxorubicin (DOX). The connection between miR-194-5p and HIF-1 was revealed by reverse transcription and real-time polymerase chain reaction (RT-qPCR), western blot, and dual-luciferase assays. We used TUNEL staining and the CCK-8 test to assess the sensitivity of NSCLC cells to DOX. Results We found that hypoxia-induced NSCLC cells enhanced resistance to DOX. MiR-194-5p was substantially reduced, and HIF-1 was increased in hypoxia-induced drug-resistant NSCLC cells. Moreover, miR-194-5p successfully induced NSCLC cell apoptosis by directly inhibiting HIF-1, thereby enhancing DOX sensitivity. Conclusions MiR-194-5p enhanced the sensitivity of NSCLC cells to DOX by directly inhibiting HIF-1. This work provides insights into underlying treatments for drug-resistant NSCLC.

miR-503-5p induces doxorubicin resistance in triple-negative breast cancer.

1083 Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer (BC) subtype comprising approximately 15% of BC. Conventional cytotoxic chemotherapies continue to be the mainstay for treatment of this BC, which lacks targetable markers. In this context, microRNAs have been described to have an important role. The aim of this work was to elucidate the function of miR-503-5p in doxorubicin resistance in TNBC. Methods: miR-503-5p expression was evaluated in the TNBC cell line with acquired resistance to doxorubicin (MDA-MB-231R) and its parental cell line (MDA-MB-231), by qRT-PCR. Studies of gain/loss of function of miR-503-5p were carried out in MDA-MB-231 and MDA-MB-231R cells by transient transfection of mimics and inhibitors. Cells were treated with doxorubicin, and viability was measured by flow cytometry and MTT assay. The role of miR-503-5p was also evaluated in vivo by Chicken Chorioallantoic Membrane (CAM) assay. MDA-MB-231 cells transfected with miR-503-5p mimic or scramble miRNA were inoculated onto the CAM of fertilized chicken eggs. After 48 hours, tumours were treated with doxorubicin or supplemented media for 48 hours and tumour growth was measured. miR-503-5p expression was quantified by qRT-PCR in a retrospective cohort of 74 TNBC patients treated with anthracycline + taxane regimens. Overall survival analysis for miR-503-5p in TNBC patients from METABRIC dataset was evaluated by the KM plotter online tool. Results: miR-503-5p was significantly upregulated in the resistant MDA-MB-231R TNBC cell line when compared to its parental cell line MDA-MB-231 (̃3.5-fold; p< 0.0001). Then, gain/loss function assays showed that upregulation of miR-503-5p in MDA-MB-231 cells increased resistance to doxorubicin ( p< 0.0001) and its downregulation in MDA-MB-231R cells had the opposite effect ( p< 0.0001). Moreover, the role of miR-503-5p was also confirmed in the CAM assay in vivo model, where miR-503-5p overexpression inhibited the effect of doxorubicin. In our cohort of patients, miR-503-5p expression levels in core biopsies sampled before preoperative chemotherapy were associated with residual cancer burden (RCB). miR-503-5p expression was significantly higher in patients with poor response to chemotherapy (RCB II and III; median, 95% CI: 0.00055, 0.00024 - 0.00136) than in patients with good response (RCB 0 and I; median, 95% CI: 0.00018, 0.00011 - 0.00034; p = 0.036). Moreover, we confirmed that TNBC patients with high expression of miR-503-5p had worse overall survival than patients with low expression ( p= 0.016). Conclusions: We identified miR-503-5p as a modulator of doxorubicin resistance in TNBC. Our in vitro findings are supported by the clinical data of TNBC patients and in vivo assays. Hence, the inhibition of miR-503-5p may be a promising strategy to improve chemotherapeutic efficacy. Moreover, the expression levels of miR-503-5p may be used as a biomarker for therapy response in TNBC.