KRAS and EGFR Mutations Differentially Alter ABC Drug Transporter Expression in Cisplatin-Resistant Non-Small Cell Lung Cancer

Lung carcinoma is still the most common malignancy worldwide. One of the major subtypes of non-small cell lung cancer (NSCLC) is adenocarcinoma (AC). As driver mutations and hence therapies differ in AC subtypes, we theorized that the expression and function of ABC drug transporters important in multidrug resistance (MDR) would correlate with characteristic driver mutations KRAS or EGFR. Cisplatin resistance (CR) was generated in A549 (KRAS) and PC9 (EGFR) cell lines and gene expression was tested. In three-dimensional (3D) multicellular aggregate cultures, both ABCB1 and ABCG2 transporters, as well as the WNT microenvironment, were investigated. ABCB1 and ABCG2 gene expression levels were different in primary AC samples and correlated with specific driver mutations. The drug transporter expression pattern of parental A549 and PC9, as well as A549-CR and PC9-CR, cell lines differed. Increased mRNA levels of ABCB1 and ABCG2 were detected in A549-CR cells, compared to parental A549, while the trend observed in the case of PC9 cells was different. Dominant alterations were observed in LEF1, RHOU and DACT1 genes of the WNT signalling pathway in a mutation-dependent manner. The study confirmed that, in lung AC-s, KRAS and EGFR driver mutations differentially affect both drug transporter expression and the cisplatin-induced WNT signalling microenvironment.     

非含铂双药和非含铂单药治疗老年和/或PS差的非小细胞肺癌病人疗效:荟萃分析

Objective:The aim of the study was to compare the efficacies and toxicities of non-platinum doublets (doublets group) with a non-platinum single agent (single-agent group) in previously untreated advanced non-small cell lung cancer (NSCLC) patients with elderly age and/or poor performance status (PS).Methods:The PubMed database was screened.Subsequently,the hazard ratios (HRs) for overall survival (OS) and progression-free survival (PFS),relative risks (RRs) for overall response rate (ORR) and one-year survival,and odds ratios (ORs) for the different types of toxicities were pooled using the Review Manager 5.0 package.Results:This study comprised of 1427 patients enrolled in four randomized controlled trials.The pooled HR showed that the doublet group could increase ORR (P = 0.002) with no heterogeneity (P = 0.64),and might improve OS (P = 0.01 / P = 0.06) with heterogeneity (P < 0.001).There was no significant difference in PFS (P = 0.16) and one-year survival (P = 0.25) between two treatment groups.The doublet group led to more grade 3/4 neutropenia and thrombocytopenia than the single-agent group (P = 0.02 and P = 0.000,respectively).The incidences of grade 3/4 anemia,vomiting,mucositis,constipation,diarrhea,neurotoxicity,allergy,and fatigue between the two treatment groups were insignificant.Conclusion:Except for neutropenia and thrombocytopenia,the non-platinum doublets could increase ORR,and might improve OS for NSCLC patients with elderly age and/or poor PS without addition of more side effects;however,the doublets showed an increased rate of neutropenia and thrombocytopenia.The addition of doublets may not improve PFS and one-year survival.     

Overcoming AZD9291 Resistance and Metastasis of NSCLC via Ferroptosis and Multitarget Interference by Nanocatalytic Sensitizer Plus AHP-DRI-12

The acquired resistance to Osimertinib (AZD9291) greatly limits the clinical benefit of patients with non-small cell lung cancer (NSCLC), whereas AZD9291-resistant NSCLCs are prone to metastasis. It's challenging to overcome AZD9291 resistance and suppress metastasis of NSCLC simultaneously. Here, a nanocatalytic sensitizer (VF/S/A@CaP) is proposed to deliver Vitamin c (Vc)–Fe(II), si-OTUB2, ASO-MALAT1, resulting in efficient inhibition of tumor growth and metastasis of NSCLC by synergizing with AHP-DRI-12, an anti-hematogenous metastasis inhibitor by blocking the amyloid precursor protein (APP)/death receptor 6 (DR6) interaction designed by our lab. Fe²⁺ released from Vc–Fe(II) generates cytotoxic hydroxyl radicals (•OH) through Fenton reaction. Subsequently, glutathione peroxidase 4 (GPX4) is consumed to sensitize AZD9291-resistant NSCLCs with high mesenchymal state to ferroptosis due to the glutathione (GSH) depletion caused by Vc/dehydroascorbic acid (DHA) conversion. By screening NSCLC patients’ samples, metastasis-related targets (OTUB2, LncRNA MALAT1) are confirmed. Accordingly, the dual-target knockdown plus AHP-DRI-12 significantly suppresses the metastasis of AZD9291-resistant NSCLC. Such modality leads to 91.39% tumor inhibition rate in patient-derived xenograft (PDX) models. Collectively, this study highlights the vulnerability to ferroptosis of AZD9291-resistant tumors and confirms the potential of this nanocatalytic-medicine-based modality to overcome critical AZD9291 resistance and inhibit metastasis of NSCLC simultaneously.     

MALDI质谱成像技术在非小细胞肺

采用基质辅助激光解吸电离时间飞行质谱成像技术分析组织切片已在基础与临床医学研究中迅速发展。质谱成像技术通过对冰冻组织切片表面的质谱扫描可以快速直观地得到组织中的分子,如蛋白、多肽等分布信息。采用质谱成像技术对人的非小细胞肺癌组织切片及癌旁组织切片进行研究,对基质覆盖方式和质谱条件进行了优化,初步建立了以质谱成像法寻找非小细胞肺癌中生物标志物的方法。在-20℃制备10μm的冰冻组织切片,用15g.L-1的α-腈基-4-羟基肉桂酸(含0.1%三氟乙酸)的50%乙腈水溶液,以气流辅助喷洒的方式覆盖在组织切片表面,循环10次,置于干燥器中待干后,用正离子反射式和线性模式分别进行质谱扫描。通过对来自3位患者的10对肺癌/癌旁组织研究,结果表明,癌组织中有m/z3000~3500范围内的特征性簇峰出现。质谱成像技术用于非小细胞肺癌能够直观地在分子水平上反映出癌组织和正常组织间的差异,有望进一步提高肺癌临床诊断的准确度。     

Hiltonol Cocktail Kills Lung Cancer Cells by Activating Cancer-Suppressors,PKR/OAS,and Restraining the Tumor Microenvironment

NSCLC (non-small cell lung cancer) is a leading cause of cancer-related deaths worldwide. Clinical trials showed that Hiltonol, a stable dsRNA representing an advanced form of polyI:C (polyinosinic-polycytidilic acid), is an adjuvant cancer-immunomodulator. However, its mechanisms of action and effect on lung cancer have not been explored pre-clinically. Here, we examined, for the first time, how a novel Hiltonol cocktail kills NSCLC cells. By retrospective analysis of NSCLC patient tissues obtained from the tumor biobank; pre-clinical studies with Hiltonol alone or Hiltonol⁺⁺⁺ cocktail [Hiltonol+anti-IL6+AG490 (JAK2 inhibitor)+Stattic (STAT3 inhibitor)]; cytokine analysis; gene knockdown and gain/loss-of-function studies, we uncovered the mechanisms of action of Hiltonol⁺⁺⁺. We demonstrated that Hiltonol⁺⁺⁺ kills the cancer cells and suppresses the metastatic potential of NSCLC through: (i) upregulation of pro-apoptotic Caspase-9 and Caspase-3, (ii) induction of cytosolic cytochrome c, (iii) modulation of pro-inflammatory cytokines (GRO, MCP-1, IL-8, and IL-6) and anticancer IL-24 in NSCLC subtypes, and (iv) upregulation of tumor suppressors, PKR (protein kinase R) and OAS (2′5′ oligoadenylate synthetase). In silico analysis showed that Lys296 of PKR and Lys66 of OAS interact with Hiltonol. These Lys residues are purportedly involved in the catalytic/signaling activity of the tumor suppressors. Furthermore, knockdown of PKR/OAS abrogated the anticancer action of Hiltonol, provoking survival of cancer cells. Ex vivo analysis of NSCLC patient tissues corroborated that loss of PKR and OAS is associated with cancer advancement. Altogether, our findings unraveled the significance of studying tumor biobank tissues, which suggests PKR and OAS as precision oncological suppressor candidates to be targeted by this novel Hiltonol⁺⁺⁺ cocktail which represents a prospective drug for development into a potent and tailored therapy for NSCLC subtypes.     

非小细胞肺癌驱动基因检测研究进展

对非小细胞肺癌分子病理学的深入理解,有助于开展个性化临床治疗和预后。基因检测可以指导非小细胞肺癌靶向药物治疗,提升药物疗效,延长患者生存期。本文简要介绍了与非小细胞肺癌相关的EGFR基因和EML4-ALK融合基因检测方法的研究进展。     

Targeting the ERβ/HER Oncogenic Network in KRAS Mutant Lung Cancer Modulates the Tumor Microenvironment and Is Synergistic with Sequential Immunotherapy

High ERβ/HER oncogenic signaling defines lung tumors with an aggressive biology. We previously showed that combining the anti-estrogen fulvestrant with the pan-HER inhibitor dacomitinib reduced ER/HER crosstalk and produced synergistic anti-tumor effects in immunocompromised lung cancer models, including KRAS mutant adenocarcinoma. How this combination affects the tumor microenvironment (TME) is not known. We evaluated the effects of fulvestrant and dacomitinib on murine bone marrow-derived macrophages (BMDMs) and CD8+ T cells, and tested the efficacy of the combination in vivo, using the KRAS mutant syngeneic lung adenocarcinoma model, FVBW-17. While this combination synergistically inhibited proliferation of FVBW-17 cells, it had unwanted effects on immune cells, by reducing CD8+ T cell activity and phagocytosis in BMDMs and inducing PD-1. The effects were largely attributed to dacomitinib, which caused downregulation of Src family kinases and Syk in immune cells. In a subcutaneous flank model, the combination induced an inflamed TME with increased myeloid cells and CD8+ T cells and enhanced PD-1 expression in the splenic compartment. Concomitant administration of anti-PD-1 antibody with fulvestrant and dacomitinib was more efficacious than fulvestrant plus dacomitinib alone. Administering anti-PD-1 sequentially after fulvestrant plus dacomitinib was synergistic, with a two-fold greater tumor inhibitory effect compared to concomitant therapy, in both the flank model and in a lung metastasis model. Sequential triple therapy has potential for treating lung cancer that shows limited response to current therapies, such as KRAS mutant lung adenocarcinoma.     

Downregulation of miR-506-3p Facilitates EGFR-TKI Resistance through Induction of Sonic Hedgehog Signaling in Non-Small-Cell Lung Cancer Cell Lines

Non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation eventually develop resistance to EGFR-targeted tyrosine kinase inhibitors (TKIs). Treatment resistance remains the primary obstacle to the successful treatment of NSCLC. Although drug resistance mechanisms have been studied extensively in NSCLC, the regulation of these mechanisms has not been completely understood. Recently, increasing numbers of microRNAs (miRNAs) are implicated in EGFR-TKI resistance, indicating that miRNAs may serve as novel targets and may hold promise as predictive biomarkers for anti-EGFR therapy. MicroRNA-506 (miR-506) has been identified as a tumor suppressor in many cancers, including lung cancer; however, the role of miR-506 in lung cancer chemoresistance has not yet been addressed. Here we report that miR-506-3p expression was markedly reduced in erlotinib-resistant (ER) cells. We identified Sonic Hedgehog (SHH) as a novel target of miR-506-3p, aberrantly activated in ER cells. The ectopic overexpression of miR-506-3p in ER cells downregulates SHH signaling, increases E-cadherin expression, and inhibits the expression of vimentin, thus counteracting the epithelial–mesenchymal transition (EMT)-mediated chemoresistance. Our results advanced our understanding of the molecular mechanisms underlying EGFR-TKI resistance and indicated that the miR-506/SHH axis might represent a novel therapeutic target for future EGFR mutated lung cancer treatment.     

Epithelial Membrane Protein 2 Suppresses Non-Small Cell Lung Cancer Cell Growth by Inhibition of MAPK Pathway

Epithelial membrane proteins (EMP1-3) are involved in epithelial differentiation and carcinogenesis. Dysregulated expression of EMP2 was observed in various cancers, but its role in human lung cancer is not yet clarified. In this study, we analyzed the expression of EMP1-3 and investigated the biological function of EMP2 in non-small cell lung cancer (NSCLC). The results showed that lower expression of EMP1 was significantly correlated with tumor size in primary lung tumors (p = 0.004). Overexpression of EMP2 suppressed tumor cell growth, migration, and invasion, resulting in a G1 cell cycle arrest, with knockdown of EMP2 leading to enhanced cell migration, related to MAPK pathway alterations and disruption of cell cycle regulatory genes. Exosomes isolated from transfected cells were taken up by tumor cells, carrying EMP2-downregulated microRNAs (miRNAs) which participated in regulation of the tumor microenvironment. Our data suggest that decreased EMP1 expression is significantly related to increased tumor size in NSCLC. EMP2 suppresses NSCLC cell growth mainly by inhibiting the MAPK pathway. EMP2 might further affect the tumor microenvironment by regulating tumor microenvironment-associated miRNAs.