Cisplatin-resistant Lung Adenocarcinoma Research Articles

Abstract 1096: Engineering a cisplatin-resistant lung cancer microenvironment to model recurrence of aggressive tumors following first line cytotoxic therapy

Abstract Aggressive lung cancers such as non-small cell lung carcinomas (NSCLC) commonly recur following first line therapy. Recurring tumors metastasize more rapidly. We hypothesize that drug-resistant phenotypes created by the selection pressure of first line treatments such as platinum-based cytotoxic agents contribute to the increased aggressivity of recurring NSCLC. We showed previously that an engineered model of cisplatin-resistant NSCLC tissues secretes more inflammatory cytokines compared to untreated controls and induces more severe muscle tissue injury in a microphysiological model of cachexia processes. Here, we report methods for the systematic generation of cisplatin-resistant A549 lung adenocarcinoma cell cultures and demonstrate their increased aggressivity in 3D spheroid culture and in 3D organotypic culture with patient-derived lung cancer-associated fibroblasts (CAF). We treated A549 cells with cisplatin for 96 hours at doses of 15-45µM, yielding cultures of 54.8-8.9% surviving cells as quantified via flow cytometric of cell size and permeability. Here we report characterization of spheroids and tissues composed of cells selected for resistance with 25 µM cisplatin (Cis-R) compared with untreated parent A549. This dose was selected because we were unable to recover proliferating cultures with lower percentages of surviving. Cis-R spheroids exhibited a significant reduction in circularity that is indicative of increased asymmetry. We measured a Ki67 index of 6.4 +/- 1.4% for parent A549 spheroids and 49.7 +/- 9.5% for Cis-R spheroids. The marked increase in cell proliferation of CisR spheroids correlated with increased distances of invasion and more mesenchymal-like cell morphologies of invading cells in 3D cultures of Cis-R spheroids without CAF. These data suggest that cisplatin resistant A549 created using our protocol are more proliferative and invasive in 3D culture environments due to cell intrinsic changes. Organotypic 3D cultures of Cis-R spheroids and CAF exhibited an increased density and alignment of myofibroblast-like CAF with smooth muscle actin cytoskeleton when compared to cultures of parent spheroids with the same CAF. Our results demonstrate proof of principle for modeling recurrent tumors with more aggressive phenotypes using this approach. Future experiments will translate these methods to patient-derived cultures and test the hypothesis that cisplatin-resistant recurrent NSCLC stimulates more severe local and distant stromal reactions as a driver of metastasis. Engineering validated models of drug-resistant recurring cancers will help accelerate the discovery of anti-metastasis therapies in human cell-based microphysiological systems. Citation Format: Omar M. Ahmad, Elisabet A. Olsen, Delia A. Carlino, Charles E. Byrne, Mark Mondrinos. Engineering a cisplatin-resistant lung cancer microenvironment to model recurrence of aggressive tumors following first line cytotoxic therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1096.

Polyphyllin I Inhibits Cisplatin Resistance of Cervical Cancer Cells by Inducing Mitochondrial Dysfunction and Endoplasmic Reticulum Stress

Resistance of cervical cancer to cisplatin chemotherapy compromises the therapeutic efficacy of the treatment. Polyphyllin I, a bioactive compound extracted from rhizoma of Paris polyphyllin, displays a selective inhibitory effect on a variety of tumor cells. Furthermore, it potentiates the chemosensitivity of cisplatin against cisplatin resistant gastric cancer cells and lung adenocarcinoma. However, its role in modulating chemosensitivity of cisplatin resistant cervical cancer cells remains unexplored. To this end, the repressive effect of polyphyllin I on proliferation, invasion and migration of cisplatin resistant cervical cancer cells compared to the parental cervical cancer cells as well as downregulation of mitochondrial membrane potential of cisplatin resistant cervical cancer cells, as well as upregulation of protein kinase R-like ER kinase/translation initiation factor 2-alpha kinase 3, protein-kinase and site-specific endoribonuclease/inositol-requiring enzyme 1, binding immunoglobulin protein, and C/EBP homologous transcription factor proteins. In conclusion, polyphyllin I stimulate mitochondrial dysfunction and endoplasmic reticulum stress to attenuate cisplatin resistance of cervical cancer.

LncRNA RP3-326I13.1 promotes cisplatin resistance in lung adenocarcinoma by binding to HSP90B and upregulating MMP13

ABSTRACT Cisplatin (DDP) resistance has become the major obstacle in the therapy of malignant tumors, including lung adenocarcinoma (LAD). Long non-coding RNAs (lncRNAs) were confirmed to be related to DDP-resistance. Studies have shown that RP3-326I13.1 (also known as PINCR) could promote the progression of colorectal cancer, and RP3-326I13.1 knockdown could induce hypersensitivity to chemotherapy drugs. While the function of RP3-326I13.1 in LAD is unclear, therefore, this study aimed to research the biological function and related molecular mechanisms of RP3-326I13.1 in DDP-resistance of LAD. QPCR analysis found that RP3-326I13.1 was highly expressed in A549/DDP cells and LAD tissues. Cytological assays found that RP3-326I13.1 pro-moted the proliferation, migration, invasion, and DDP-resistance of LAD cell lines. Moreover, knock-down of RP3-326I13.1 could induce G1 phase arrest. Nude mouse xenograft assay confirmed that RP3-326I13.1 could promote tumor growth and DDP-resistance in vivo. Mechanically, RNA pull-down and mass spectrometry analysis indicated that heat shock protein HSP 90-beta (HSP90B) could be combined with RP3-326I13.1. HSP90B knockdown inhibited the effect of RP3-326I13.1 on proliferation, invasion, and promoted LAD cell lines apoptosis. Transcriptome sequencing analysis found that MMP13 was the downstream mRNA of RP3-326I13.1. In conclusion, RP3-326I13.1 could promote DDP-resistance of LAD by binding to HSP90B and upregulating human matrix metalloproteinase-13 (MMP-13) and may serve as a therapeutic target, as well as a biomarker for predicting DDP-resistance in LAD. Abbreviations: DDP: Cisplatin; LAD: Lung adenocarcinoma; LncRNAs: Long non-coding RNAs; qPCR: real-time fluorescent quantitative PCR; HSP90B: Heat shock protein HSP 90-beta; RPMI: Roswell Park Memorial Institute; FBS: Fetal bovine serum; CT: computed tomography; MRI: magnetic resonance imaging; RECIST: Response evaluation criteria in solid tumors; NC: Negative control; OE: overexpression; shRNA: short hairpin RNA; siRNA: small interfering RNA; CCK-8: Cell Counting Kit-8; IC50: The half maximal inhibitory concentration; PBS: Phosphate buffer saline; PI: propidium iodide; SDS-PAGE: sodiumdodecylsulfate-polyacrylamide gel electrophoresis; ceRNA: Competing endogenous RNA; HE: hematoxylin-eosin; ns: no significance.

The Human Positive Cofactor 4 is a Promising Chemotherapeutic Target in Lung Adenocarcinoma.

Reduced sensitivity to chemotherapeutic drugs is almost inevitable in lung adenocarcinoma patients. Thus, understanding the relevant mechanisms is urgent. Positive cofactor 4 (PC4) was at first revealed to be a coactivator of basal transcription. Previous research has shown that PC4 participates in various cellular processes in normal and malignant cells. However, it is still unknown whether PC4 participates in altering the lung adenocarcinoma cell sensitivity to chemotherapy, and the relevant mechanisms remain to be explained. In this study, we discovered that PC4 was overexpressed in cisplatin-resistant lung adenocarcinoma cells. PC4 decreased cisplatin's cytotoxic effects on lung adenocarcinoma in vivo and in vitro. Furthermore, PC4 positively correlated with SOX9 in multiple cancers. PC4 was an upstream regulator of SOX9 in lung adenocarcinoma. Furthermore, PC4 mediated lung adenocarcinoma cell sensitivity to the HIF-PH inhibitor DMOG and the mTOR inhibitor rapamycin, and PC4 mediated the synergistic effect of DMOG and cisplatin. Finally, PC4 destabilized HIF-1α upon cisplatin treatment. Our research showed that PC4 participates in mediating lung adenocarcinoma cell sensitivity to multiple drugs. Mechanistically, PC4 governs multiple downstream pathways associated with chemotherapy resistance, including the SOX9 and HIF-1α pathways. Thus, PC4 is a promising chemotherapeutic target in lung adenocarcinoma.

Mechanistic study of lncRNA UCA1 promoting growth and cisplatin resistance in lung adenocarcinoma

AimThis study aimed to explore the mechanism of LncRNA urothelial carcinoma-associated 1 (UCA1) promoting cisplatin resistance in lung adenocarcinoma (LUAD).MethodThe UCA1 expression level in LUAD cell lines was detected by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). We overexpressed UCA1 in A549 cells and downregulated UCA1 in A549/DDP cells by the lentivirus‑mediated technique. Subsequently, in vitro, and in vivo functional experiments were performed to investigate the functional roles of UCA1 in the growth and metastasis of LUAD cell lines. Furthermore, RNA pulldown, mass spectrometry, and RNA immunoprecipitation technique were performed to analyze various downstream target factors regulated by UCA1.ResultsThe results revealed a higher UCA1 expression level in A549/DDP cells and LUAD tissues than in A549 cells and adjacent cancer tissues. UCA1 expression was significantly associated with distant metastasis, clinical stage, and survival time of patients with LUAD. UCA1 overexpression significantly increased the proliferation, invasion, clone formation, and cisplatin resistance ability and enhanced the expression levels of proliferating cell nuclear antigen and excision repair cross-complementing gene 1 in A549 cells. However, these trends were mostly reversed after the knockdown of UCA1 in A549/DDP cells. Tumorigenic assays in nude mice showed that UCA1 knockdown significantly inhibited tumor growth and reduced cisplatin resistance. Enolase 1 was the RNA-binding protein (RBP) of UCA1.ConclusionBased on the results, we concluded that UCA1 promoted LUAD progression and cisplatin resistance and hence could be a potential diagnostic marker and therapeutic target in patients with LUAD.

STAT3 contributes to cisplatin resistance, modulating EMT markers, and the mTOR signaling in lung adenocarcinoma.

Lung cancer is the second leading cause of cancer death worldwide and is strongly associated with cisplatin resistance. The transcription factor signal transducer and activator of transcription 3 (STAT3) is constitutively activated in cancer cells and coordinates critical cellular processes as survival, self-renewal, and inflammation. In several types of cancer, STAT3 controls the development, immunogenicity, and malignant behavior of tumor cells while it dictates the responsiveness to radio- and chemotherapy. It is known that STAT3 phosphorylation at Ser727 by mechanistic target of rapamycin (mTOR) is necessary for its maximal activation, but the crosstalk between STAT3 and mTOR signaling in cisplatin resistance remains elusive. In this study, using a proteomic approach, we revealed important targets and signaling pathways altered in cisplatin-resistant A549 lung adenocarcinoma cells. STAT3 had increased expression in a resistance context, which can be associated with a poor prognosis. STAT3 knockout (SKO) resulted in a decreased mesenchymal phenotype in A549 cells, observed by clonogenic potential and by the expression of epithelial-mesenchymal transition markers. Importantly, SKO cells did not acquire the mTOR pathway overactivation induced by cisplatin resistance. Consistently, SKO cells were more responsive to mTOR inhibition by rapamycin and presented impairment of the feedback activation loop in Akt. Therefore, rapamycin was even more potent in inhibiting the clonogenic potential in SKO cells and sensitized to cisplatin treatment. Mechanistically, STAT3 partially coordinated the cisplatin resistance phenotype via the mTOR pathway in non-small cell lung cancer. Thus, our findings reveal important targets and highlight the significance of the crosstalk between STAT3 and mTOR signaling in cisplatin resistance. The synergic inhibition of STAT3 and mTOR potentially unveil a potential mechanism of synthetic lethality to be explored for human lung cancer treatment.

A non-genetic, cell cycle-dependent mechanism of platinum resistance in lung adenocarcinoma.

We previously used a pulse-based in vitro assay to unveil targetable signalling pathways associated with innate cisplatin resistance in lung adenocarcinoma (Hastings et al., 2020). Here, we advanced this model system and identified a non-genetic mechanism of resistance that drives recovery and regrowth in a subset of cells. Using RNAseq and a suite of biosensors to track single-cell fates both in vitro and in vivo, we identified that early S phase cells have a greater ability to maintain proliferative capacity, which correlated with reduced DNA damage over multiple generations. In contrast, cells in G1, late S or those treated with PARP/RAD51 inhibitors, maintained higher levels of DNA damage and underwent prolonged S/G2 phase arrest and senescence. Combined with our previous work, these data indicate that there is a non-genetic mechanism of resistance in human lung adenocarcinoma that is dependent on the cell cycle stage at the time of cisplatin exposure.

Chaperonin containing TCP1 subunit 3 (CCT3) promotes cisplatin resistance of lung adenocarcinoma cells through targeting the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway

ABSTRACT Cisplatin resistance remains a major obstacle to effective chemotherapies for non-small cell lung cancer (NSCLC). Chaperonin containing TCP1 subunit 3 (CCT3) has been extensively investigated in various cancers, but not in the context of drug resistance. In the present study, we aimed to investigate the role of CCT3 in cisplatin resistance of lung adenocarcinoma (LUAD) cells. By surveying the Gene Expression Profiling Interactive Analysis (GEPIA) website, we found CCT3 expression to be up-regulated in NSCLCs, which correlated with the poor prognosis of LUAD patients. Furthermore, both mRNA and protein levels of CCT3 were upregulated in the cisplatin-resistant A549/DDP cells compared to the cisplatin-sensitive A549 cells. Importantly, upon cisplatin treatment, short hairpin RNA (shRNA)-mediated CCT3 knockdown significantly inhibited the proliferation, invasion and migration of A549/DDP cells, and induced significant G2/M cell cycle arrest and apoptosis in A549/DDP cells. Moreover, CCT3 knockdown significantly weakened the tumorigenicity of the cisplatin-treated A549/DDP cells in vitro and in vivo. Finally, CCT3 knockdown re-sensitized A549/DDP cells to cisplatin through inhibiting the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT3) pathway. In conclusion, our results demonstrated that CCT3 could promote cisplatin resistance of LUAD cells via activating the JAK2/STAT3 pathway, indicating that CCT3 may be a novel molecular target for overcoming cisplatin resistance in LUAD patients.

Overexpression of P4HA1 associates with poor prognosis and promotes cell proliferation and metastasis of lung adenocarcinoma.

Prolyl 4-hydroxylase subunit alpha 1 (P4HA1) is the core active catalytic portion of prolyl 4-hydroxylase, and has contributed to tumorigenesis in several cancers. In this study, we identified that P4HA1 mRNA and protein are both up-regulated in non-small cell lung cancer (NSCLC). Besides, overexpressed P4HA1 is correlated with poor clinical outcomes and serve as an independent prognosis biomarker in lung adenocarcinoma (LUAD), but not lung squamous cell carcinoma (LUSC). In vitro studies, decreased P4HA1 significantly inhibits proliferation and cell cycle, by regulating cyclin-dependent kinases (CDKs), cyclins and CDK inhibitor (CKI). Moreover, via inhibiting epithelial-mesenchymal transition (EMT) and matrix metalloprotease (MMPs), dysregulation of P4HA1 could restrain the tumor cell invasion and metastasis of lung adenocarcinoma. In addition, we found that P4HA1 could enhance cell stemness and cisplatin-resistance in lung adenocarcinoma. In summary, P4HA1 plays a crucial role in the development of NSCLC and may provide a brand-new target for lung cancer treatment.

Melittin inhibited glycolysis and induced cell apoptosis in cisplatinresistant lung adenocarcinoma cells via TRIM8

Chemotherapy is widely used for non-small cell lung cancer (NSCLC) patients at a late stage; however, NSCLC patients often acquire resistance to chemotherapeutic drugs, thus limiting the therapy efficacy. Melittin, a major component of bee venom, possesses anti-tumor activity in various cancer cells. Here, we examined the effects of melittin on A549/DDP cisplatin-resistant lung adenocarcinoma cells and xenografts formed from this cell line and investigated the possible target of melittin. Treatment with melittin resulted in the induction of cell apoptosis, glycolysis inhibition, and reduction of phosphorylated AKT (p-AKT) in A549/DDP cells. We also identified that tripartite motif-containing 8 (TRIM8) was a potential target of melittin. Moreover, we found that TRIM8 mRNA expression was elevated in NSCLC specimens as compared to adjacent normal tissues (N = 25) and that patients with high expression of TRIM8 had a poor prognosis for lung adenocarcinoma. The knockdown of TRIM8 had a similar effect of melittin, while overexpression of TRIM8 reversed the effects of melittin in A549/DDP cells. More importantly, we revealed that melittin enhanced cisplatin sensitivity in A549/DDP cells and tumor growth in vivo using a xenograft model of A549/DDP cells. In conclusion, melittin appears to be a potential chemotherapy sensitization agent in NSCLC.

Shenmai injection enhances cisplatin-induced apoptosis through regulation of Mfn2-dependent mitochondrial dynamics in lung adenocarcinoma A549/DDP cells.

Aim: Chemoresistance is the biggest obstacle in cancer treatment. Our previous study demonstrated that Shenmai injection (SMI), a Chinese herbal medicine, enhanced the antitumor effect of cisplatin via glucose metabolism reprogramming. This study aimed to further determine whether the SMI sensitizes the non-small cell lung cancer (NSCLC) cells to cisplatin through regulation mitochondrial dynamics. Methods: The Kaplan-Meier Plotter database was used to investigate the relationship between mRNA expression of mitofusin-2 (Mfn2) and the survival analysis of NSCLC patients. The protein expression of Mfn2 in a lung adenocarcinoma tissue chip was detected by immunohistochemistry staining. The expression of Mfn2 and ATAD3A were compared between cisplatin-sensitive A549 and cisplatin-resistant A549/DDP cells. Additionally, A549/DDP cells were co-treated with cisplatin and SMI to detect mitochondrial morphology by fluorescent staining, apoptosis-related protein expression with Western blotting, and mitochondrial membrane potential (ΔΨm) with flow cytometry analysis. Results: The mean survival time of the Mfn2low group was significantly lower than that of the Mfn2high group by Kaplan-Meier Plotter database analysis, and the Mfn2 protein expression level was lower in cancer tissues than in adjacent tissues. The combination of SMI and cisplatin induced dynamic changes in A549/DDP cells, with increased mitochondrial fusion followed by upregulation of Mfn2 and downregulation of ATAD3A and reduced mitochondrial mass and ΔΨm. Moreover, SMI significantly enhanced cisplatin-induced A549/DDP apoptosis, upregulated Bax and the active subunit of caspase-3, and downregulated Bcl-2 expression, as shown via Hoechst staining and flow cytometry analysis. Conclusion: Our findings suggest SMI enhances cisplatin-induced apoptosis through regulation of Mfn2-dependent mitochondrial dynamics in cisplatin-resistant lung adenocarcinoma cells.

PDK4 promotes tumorigenesis and cisplatin resistance in lung adenocarcinoma via transcriptional regulation of EPAS1.

The use of cisplatin for the treatment of non-small cell lung cancer has long been constrained by the rapid acquisition of tumor cell chemoresistance. In the present study, we sought to better elucidate the molecular mechanisms underlying this resistance phenotype. To that end, we assessed gene expression patterns in cisplatin-resistant lung adenocarcinoma cells, revealing pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4) to be the most up-regulated kinase in resistant cells. We further found PDK4 upregulation to be directly linked with the acquisition of chemoresistance, driving enhanced tumor cell growth in vitro and in vivo. In clinical samples, we also found that PDK4 upregulation was detectable in patients with lung adenocarcinoma and that it was correlated with a poorer prognosis for these patients. From a mechanistic perspective, we further determined that PDK4 was able to promote lung adenocarcinoma cell growth and cisplatin resistance at least in part via regulating endothelial PAS domain-containing protein 1 (EPAS1) expression, thus highlighting PDK4 as a potentially viable therapeutic target in efforts to treat lung adenocarcinoma patients that have become resistant to cisplatin.

LncRNA LINC01116 Contributes to Cisplatin Resistance in Lung Adenocarcinoma.

BackgroundLong non-coding RNAs (lncRNAs) have been found to contribute to cisplatin resistance in several cancers; however, the role of lncRNA LINC01116 in cisplatin resistance remains unknown in non-small-cell lung cancer. This study aimed to examine the contribution of LINC01116 to cisplatin resistance in lung adenocarcinoma (LAD).Materials and MethodsCisplatin-resistant A549/DDP cells were generated by treatment with cisplatin by dose escalation. LINC01116 expression was compared between A549 and A549/DDP cells, and between cisplatin-resistant and non-resistant LAD specimens. The cell viability, colony formation, proliferation, migration and invasion were measured using MTT and Transwell assays, and cell apoptosis and cell cycle were detected using flow cytometry. The expression of E-cadherin and Vimentin was quantified. LAD xenografts were modeled in nude mice to investigate the role of LINC01116 on the resistance of LAD to cisplatin.ResultsMTT assay measured the IC50 values of 13.49 ± 1.62 and 3.52 ± 1.33 μg/mL for A549/DDP and A549 cells, respectively. LINC01116 was overexpressed in cisplatin-resistant LAD specimens and A549/DDP cells (P < 0.05). Knockdown of LINC01116 inhibited cell viability, proliferation, migration and invasion, promoted apoptosis and enhanced the sensitivity to cisplatin in A549/DDP cells, while LINC01116 overexpression promoted cell viability, proliferation, migration and invasion, inhibited apoptosis and reduced the sensitivity to cisplatin in A549 cells. LINC01116 knockdown resulted in a 2.1-fold increase in E-cadherin expression and a 56% reduction in Vimentin expression in A549/DDP cells, and LINC01116 overexpression resulted in a 45% reduction in E-cadherin expression and a 1.82-fold increase in Vimentin expression in A549 cells.ConclusionDysregulation of lncRNA LINC01116 expression results in resistance of LAD to cisplatin via the EMT process. Our findings support the oncogenic role of LINC01116 to promote the development of cisplatin resistance in LAD, and LINC01116 may be a novel predictor of poor response to cisplatin.

Abstract 6329: Carfilzomib: A potential drug against cisplatin-resistant lung adenocarcinoma

Abstract Recent advances in the treatment of lung cancer, such as small molecule inhibitors and immunotherapy, have transformed care. However, platinum-based chemotherapy, such as cisplatin, is the recommended treatment option for those that do not express markers that respond to targeted therapy. In most cases, prolonged exposure to cisplatin eventually renders cancer cells resistant to therapy and patients progress on this line of care. Our study has shown that integrin beta 4 (ITGB4) and paxillin (PXN) in the focal adhesion complex are highly expressed in lung adenocarcinoma (LUAD) and their interaction plays an important role in mediating cisplatin resistance. Disrupting this axis with an FDA-approved compound can potentially alleviate cisplatin resistance in non-small cell lung cancer. Upon screening 10 LUAD cell lines, we determined one KRAS mutant (H2009) and one KRAS wild-type cell line (H1993) with highest resistance to cisplatin using a cell viability assay. Proximity ligation assay and co-immunoprecipitation results confirmed interaction between ITGB4 and PXN in H2009 cells. By virtually screening a library of 1440 FDA-approved compounds that bind to the N-terminal region of PXN, the top 40 hits were screened using a cell-based assay with H358 cells. These results determined the top 11 compounds with the lowest IC50 values (0.8 nM to 1.54 μM). Drug sensitivity to these 11 compounds was tested again using the cisplatin-resistant cell line H2009. Carfilzomib, a proteasome inhibitor, was identified to be the most effective (IC50 = 193.4 nM). This compound was further tested in 3D cell line-derived spheroids and patient cancer tissue-derived organoids that were allowed to form in multi-well ultra-low attachment plates. These cultures were monitored in real-time using the IncuCyte S3 Live-Cell Analysis System. In cisplatin-resistant cell lines H2009 and H1993, carfilzomib was the most effective (IC50 = 193.4 nM, 1.77 μM, respectively), irrespective of KRAS status. Wound healing was impeded by 20% in H2009 with a submicromolar dose (200 nM). In both H2009 and H1993 cells, a sublethal dose of carfilzomib decreased expression of ITGB4 and phospho-Rb (S807/811) and increased expression of p27, γH2AX, and cleaved PARP, indicating cell cycle inhibition and cell death. In H2009 spheroids, carfilzomib (600 nM) was highly effective in decreasing spheroid viability by 60% and inducing a 2.5-fold increase in caspase-3/7-mediated apoptosis. Also in patient-derived organoids, carfilzomib induced an increase in apoptosis by 5-fold in one case and 50-fold in a second case compared to cisplatin. Another proteasome inhibitor ixazomib was among the top 11 compounds tested in H2009 cells but had a higher IC50 (2.93 μM) compared to carfilzomib, indicating greater efficacy of carfilzomib against cisplatin-resistant LUAD. These results impart a potential therapeutic approach for overcoming chemoresistance in LUAD by targeting the focal adhesion complex through repurposing carfilzomib, an FDA-approved compound. Citation Format: Arin Nam, Atish Mohanty, Supriyo Bhattacharya, Saumya Srivastava, Rajendra Pangeni, Dan Raz, John Orban, Prakash Kulkarni, Ravi Salgia. Carfilzomib: A potential drug against cisplatin-resistant lung adenocarcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6329.

Low expression of PRKCDBP promoted cisplatin resistance in lung adenocarcinoma by DNMT1 and TNF‑α.

The aim of the present study was to explore the mechanism of protein kinase C delta binding protein (PRKCDBP) promoting cisplatin resistance in lung adenocarcinoma (LAD). The PRKCDBP expression level was herein detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We overexpressed PRKCDBP and tumor necrosis factor-α (TNF-α) in A549/DDP cell line, DNMT1 in A549 cells and siRNA TNF-α in A549 cells with lentivirus-mediated technique, and then, analyzed their biological diversification. The results showed a significantly lower expression level of PRKCDBP was lowly expressed in the A549/DDP cell line and LAD tissues than that in A549 cells and adjacent cancer tissues (P<0.05 and P<0.01), while the DNMT1 mRNA level was remarkably increased (P=0.000) and the promoter of PRKCDBP was hypermethylated in the A549/DDP cell line. Additionally, DNMT1 mRNA level in cisplatin-insensitive group was markedly higher than that in cisplatin-sensitive group (t=7.233, P<0.0001), while PRKCDBP mRNA level in cisplatin insensitive group was notably lower than that in cisplatin-sensitive group (t=8.784, P<0.0001). The results showed that PRKCDBP mRNA level was significantly elevated following treatment with 5 µM decitabine for 24 h (P<0.0001), while the DNMT1 mRNA level was notably reduced (P=0.000). When PRKCDBP was overexpressed, the DNMT1 mRNA level was markedly decreased (P=0.007), the rate of proliferation (P<0.05 or P<0.01), IC50 of cisplatin (P<0.001), G2/M phase and S phase cells were obviously reduced (P<0.001), while G0/G1 phase cells, apoptosis (P<0.001) distinctly increased, but migration ability did not significantly change. TNF-α overexpression resulted in an increase of PRKCDBP mRNA level (P<0.001), while TNF-α siRNA led to PRKCDBP mRNA level distinctly reduced (P<0.001). Overexpression of DNMT1 improved IC50 in A549 cells. Thus, findings of the present study ascertained the promoter of PRKCDBP was hypermethylated in A549/DDP cells. In conclusion, low expression of PRKCDBP promoted cisplatin resistance in LAD by DNMT1 and TNF-α.

CEACAM6 promotes cisplatin resistance in lung adenocarcinoma and is regulated by microRNA-146a and microRNA-26a.

BackgroundCarcinoembryonic antigen (CEA)‐related cell adhesion molecule 6 (CEACAM6) is a glycophosphoinositol‐anchored glycoprotein which mediates cell‐cell interactions. Here, we aimed to explore the specific functions and regulatory mechanisms of CEACAM6 on cisplatin (DDP) in lung adenocarcinoma (LUAD).MethodsRNA sequencing was performed in the DDP‐resistant A549/DDP cell line and parental A549 cell line; miRNA expression profiling of the two cell lines was analyzed using GEO data (GSE43249). Gain‐ and loss‐of‐function experiments were used to investigate the biological function of CEACAM6 in vitro. The expression status and prognostic value of CEACAM6 in LUAD were verified using The Cancer Genome Atlas (TCGA) database.ResultsCEACAM6 was first screened to be one of the most significantly upregulated genes in the DDP‐resistant A549/DDP cell line compared to the parental A549 cell line. Combined with computational prediction of candidate miRNAs that target CEACAM6, miR‐146a and miR‐26a were selected and verified by qPCR and luciferase reporter assay. The knockdown of CEACAM6 expression in A549/DDP cells inhibited cell proliferation, invasion and migration, decreased the IC50 values of DDP, and caused a significant downregulation of N‐cadherin, vimentin, Sox2, Oct4 and GTP‐RhoA and upregulation of E‐cadherin; while CEACAM6 overexpression in A549 cells resulted in the opposite effects. Of note, both miR‐146a and miR‐26a could counteract the biological effects of CEACAM6. Furthermore, CEACAM6 mRNA expression was significantly unregulated in DDP‐resistant LUAD tissues of TCGA database.ConclusionsCEACAM6 promotes DDP resistance in LUAD by affecting the epithelial‐mesenchymal transition (EMT) phenotype and stemness, which is post‐transcriptionally regulated by miR‐146a and miR‐26a.

Shenmai Injection Supresses Glycolysis and Enhances Cisplatin Cytotoxicity in Cisplatin-Resistant A549/DDP Cells via the AKT-mTOR-c-Myc Signaling Pathway.

Tumor cells, especially drug-resistant cells, predominately support growth by glycolysis even under the condition of adequate oxygen, which is known as the Warburg effect. Glucose metabolism reprogramming is one of the main factors causing tumor resistance. Previous studies on Shenmai injection (SMI), a Chinese herbal medicine, have shown enhanced efficacy in the treatment of tumors in combination with chemotherapy drugs, but the mechanism is not clear. In this study, we investigated the effect of SMI combined with cisplatin on cisplatin-resistant lung adenocarcinoma A549/DDP cells. Our results showed that cisplatin-resistant A549/DDP cells exhibited increased glucose consumption, lactate production, and expression levels of key glycolytic enzymes, including hexokinase 2 (HK2), pyruvate kinase M1/2 (PKM1/2), pyruvate kinase M2 (PKM2), glucose transporter 1 (GLUT1), and lactate dehydrogenase A (LDHA), compared with cisplatin-sensitive A549 cells. SMI combined with cisplatin in A549/DDP cells, led to significantly lower expression levels of key glycolytic enzymes, such as HK2, PKM1/2, GLUT1, and pyruvate dehydrogenase (PDH). In addition, we found that the combination of SMI and cisplatin could inhibit cell proliferation and promote apoptosis by reducing the expression levels of p-Akt, p-mTOR, and c-Myc, and then, it reduced the glycolysis level. These results suggest that SMI enhances the antitumor effect of cisplatin via glucose metabolism reprogramming. Therefore, the combination of SMI and cisplatin may be a potential therapeutic strategy to treat cisplatin-resistant nonsmall cell lung cancer.

MiR-320a is associated with cisplatin resistance in lung adenocarcinoma and its clinical value in non-small cell lung cancer: A comprehensive analysis based on microarray data

BackgroundCurrently, the main treatment for non-small cell lung cancer (NSCLC) is surgery and chemotherapy. Although major progress has been made in targeted treatment and immunotherapy, the survival rates for this disease are still low and associated with resistance to chemotherapy. Previous studies have shown that histone acetylation and microRNAs (miRNAs) might play an important role in chemotherapy resistance. The aim of this study was to identify candidate miRNAs related to cisplatin (DDP) resistance in lung adenocarcinoma. MethodsWe used 5-aza-2′-deoxycytidine and trichostatin A to reverse the drug resistance of A549/DDP cells in vitro, and miRNA expression profiling was performed by microarrays to identify candidate miRNAs. In addition, we investigated the correlations between miR-320a expression and clinical characteristics through data collected from Gene Expression Omnibus (GEO) microarrays, and The Cancer Genome Atlas (TCGA) to determine the clinical role of miR-320a in lung adenocarcinoma. Furthermore, we investigated the biological function of miR-320a. TargetScanHuman, PicTar2005 and miRanda v5.1. were used to predict the target genes of miR-320a; then, the function of these genes were suggested from the enrichment of GO categories items and KEGG analyses. ResultsTreatment with 5-Aza-dc significantly inhibited cellular proliferation, and increased apoptosis in the A549/DDP cells compared with the untreated cells. TSA did not reverse cisplatin resistance. MiR-320a was up-regulated during reversal of cisplatin resistance. The lung adenocarcinoma groups had a significantly lower level of miR-320a expression than the control groups. For the bioinformatics analyses, we found some target genes involved in cell cycle progression, tumor progression, the MAPK signaling pathway, and the ErbB signaling pathway. The promising target genes were highly enriched in various pathways in cancer. ConclusionsThe current study confirmed miR-320a was up-regulated during the revering of cisplatin resistance. The results of bioinformatics analyses may present a new method for investigating the pathogenesis of lung adenocarcinoma.

LncRNA RP11-838N2.3 Promoted Cisplatin Resistance in Lung Adenocarcinoma.

The mechanism of RP11-838N2.3 promoting cisplatin resistance in lung adenocarcinoma (LAD) was unclear. The RP11-838N2.3 expression level in cells and LAD tissues was detected by qPCR. We constructed lentivirus-mediated GV303 overexpression and GV248 shRNA vector targeting RP11-838N2.3, then infected A549 and A549/DDP cell and furtherly analyzed cell biology. High-throughput gene chip analysis showed that RP11-838N2.3 was significantly upregulated in A549/DDP (change fold = 66.056595). The qPCR results showed that the expression level of RP11-838N2.3 in A549/DDP cell was significantly higher than that in A549 cells (P < 0.05), and the expression level of RP11-838N2.3 in LAD tissues was also significantly higher than that in adjacent tissues (P < 0.05). The expression level of RP11-838N2.3 in cisplatin-insensitive LAD tissues was also significantly higher than that in cisplatin-sensitive LAD tissues (P < 0.05). Survival analysis showed that OS (overall survival) and DFS (progression-free survival) of high RP11-838N2.3 expression in the cisplatin-sensitive or cisplatin-insensitive LAD group were lower (P < 0.001 and P < 0.001) than those of low RP11-838N2.3 expression in the cisplatin-sensitive or cisplatin-insensitive LAD group. CCK8 showed that the OD450 value of RP11-838N2.3 overexpression increased significantly at 24 h, 48 h, and 72 h after transfection, while the knockdown of RP11-838N2.3 caused OD450 value at 24 h, 48 h, and 72 h after transfection significantly reduced, under the action of cisplatin that had the same trend (P < 0.05). The cell migration showed that the RP11-838N2.3 overexpression increased significantly migration activity and RP11-838N2.3 knockdown inhibited migration activity at 24 h, 48 h, and 72 h after transfection. The same trend was also observed under the action of cisplatin (P < 0.05). The cell invasion showed that the invasion rate of RP11-838N2.3 overexpression increased significantly, while the invasion rate of RP11-838N2.3 knockdown decreased significantly, and the same trend was observed under the action of cisplatin (P < 0.05). Apoptosis results showed that the apoptosis rate of RP11-838N2.3 overexpressed cells decreased significantly and the apoptosis rate of RP11-838N2.3 knockdown cells increased significantly, and the same trend was also observed under the action of cisplatin (P < 0.05). However, the results of cell cycle showed that there was no significant difference in the proportion of cells in each phase of the cell cycle after RP11-838N2.3 overexpression or knockdown (P > 0.05).RP11-838N2.3 was significantly upregulated in cisplatin-resistant cell and tissues of LAD. RP11-838N2.3 could enhance the proliferation, migration, and invasion and inhibit apoptosis of LAD cisplatin-resistant cell. So RP11-838N2.3 could enhance the cisplatin resistance of LAD cells and was a resistant lncRNA molecule.

Ubiquitin ligase CHAF1B induces cisplatin resistance in lung adenocarcinoma by promoting NCOR2 degradation

BackgroundLung cancer is the most common malignant tumor in the world. The Whole-proteome microarray showed that ubiquitin ligase chromatin assembly factor 1 subunit B (CHAF1B) expression in A549/DDP cells is higher than in A549 cells. Our study explored the molecular mechanism of CHAF1B affecting cisplatin resistance in lung adenocarcinoma (LUAD).MethodsProteome microarray quantify the differentially expressed proteins between LUAD cell line A549 and its cisplatin-resistant strain A549/DDP. Quantitative real-time quantitative polymerase chain reaction (qRT-PCR) and Western blot (WB) confirmed the CHAF1B expression. Public databases analyzed the prognosis of LUAD patients with varied LUAD expression followed by the substrates prediction of CHAF1B. Public databases showed that nuclear receptor corepressor 2 (NCOR2) may be substrates of CHAF1B. WB detected that CHAF1B expression affected the expression of NCOR2. Cell and animal experiments and clinical data detected function and integrating mechanism of CHAF1B compounds.ResultsProteome chips results indicated that CHAF1B, PPP1R13L, and CDC20 was higher than A549 in A549/DDP. Public databases showed that high expression of CHAF1B, PPP1R13L, and CDC20 was negatively correlated with prognosis in LUAD patients. PCR and WB results indicated higher CHAF1B expression in A549/DDP cells than that in A549 cells. NCOR2 and PPP5C were confirmed to be substrates of CHAF1B. CHAF1B knockdown significantly increased the sensitivity of cisplatin in A549/DDP cells and the upregulated NCOR2 expression. CHAF1B and NCOR2 are interacting proteins and the position of interaction between CHAF1B and NCOR2 was mainly in the nucleus. CHAF1B promotes ubiquitination degradation of NCOR2. Cells and animal experiments showed that under the action of cisplatin, after knockdown of CHAF1B and NCOR2 in A549/DDP group compared with CHAF1B knockdown alone, the cell proliferation and migratory ability increased and apoptotic rate decreased, and the growth rate and size of transplanted tumor increased significantly. Immunohistochemistry suggested that Ki-67 increased, while apoptosis-related indicators caspase-3 decreased significantly. Clinical data showed that patients with high expression of CHAF1B are more susceptible to cisplatin resistance.ConclusionUbiquitin ligase CAHF1B can induce cisplatin resistance in LUAD by promoting the ubiquitination degradation of NCOR2.