Drug-resistant Cancer Cell Lines Research Articles

The novel SMYD3 inhibitor EM127 impairs DNA repair response to chemotherapy-induced DNA damage and reverses cancer chemoresistance

BackgroundSMYD3 has been found implicated in cancer progression. Its overexpression correlates with cancer growth and invasion, especially in gastrointestinal tumors. SMYD3 transactivates multiple oncogenic mechanisms, favoring cancer development. Moreover, it was recently shown that SMYD3 is required for DNA restoration by promoting homologous recombination (HR) repair.MethodsIn cellulo and in vivo models were employed to investigate the role of SMYD3 in cancer chemoresistance. Analyses of SMYD3-KO cells, drug-resistant cancer cell lines, patients’ residual gastric or rectal tumors that were resected after neoadjuvant therapy and mice models were performed. In addition, the novel SMYD3 covalent inhibitor EM127 was used to evaluate the impact of manipulating SMYD3 activity on the sensitization of cancer cell lines, tumorspheres and cancer murine models to chemotherapeutics (CHTs).ResultsHere we report that SMYD3 mediates cancer cell sensitivity to CHTs. Indeed, cancer cells lacking SMYD3 functions showed increased responsiveness to CHTs, while restoring its expression promoted chemoresistance. Specifically, SMYD3 is essential for the repair of CHT-induced double-strand breaks as it methylates the upstream sensor ATM and allows HR cascade propagation through CHK2 and p53 phosphorylation, thereby promoting cancer cell survival. SMYD3 inhibition with the novel compound EM127 showed a synergistic effect with CHTs in colorectal, gastric, and breast cancer cells, tumorspheres, and preclinical colorectal cancer models.ConclusionsOverall, our results show that targeting SMYD3 may be an effective therapeutic strategy to overcome chemoresistance.

Dissimilar effect of organometallic ruthenium complexes on the viability of MDR and non-MDR experimental models

Ruthenium complexes containing triphenylphosphine diamide ligands were prepared, characterized, and tested for their biological activity against various cancer cell lines and the malaria parasite, Plasmodium falciparum. The effect of M (mono-substituted) and B (bis-substituted) complexes on the human cervical carcinoma (HeLa) cell line was investigated using the MTT assay. Five (B2, B3, B5, B6, and B13) of the 24 synthesized ruthenium complexes showed significant effects with IC50 values ranging between 0.3 and 2.3 μM. Evaluation of the potential biomolecular targets of B2 and B13 by fluorescence spectroscopy revealed relevant interactions with BSA and only a weak affinity for ctDNA. Complexes M2, B2, M13 and B13 were selected for further biological characterization. Their effect on the viability of two ovarian cancer cell lines was compared to normal cell lines, denoting their selectivity. Upon treatment of four different drug-resistant gynaecological cancer cell lines, differing in their multidrug-resistant phenotypes, the efficacy of the bis-substituted complexes was shown to be greater than their mono-substituted counterparts. The non-MDR cells are sensitive to all the tested complexes, compared to MDR cells which are less sensitive. Upon investigation of complexes M2, M13, B2, and B13 against sensitive and multidrug-resistant parasite strains of P. falciparum, the bis-substituted complexes were again shown to be the most potent, with submicromolar activity against both strains. Furthermore, the resistance indexes for the complexes were approximately equal to 1, which is at least 5-fold lower than chloroquine diphosphate, suggesting the ability of these complexes to retain their activity in resistant forms of the parasite.

Hyperthermia/glutathione-triggered ferritin nanoparticles amplify the ferroptosis for synergistic tumor therapy

Breast cancer is the most diagnosed malignancy in women globally, and drug resistance is among the major obstacles to effective breast cancer treatment. Emerging evidence indicates that photothermal therapy and ferroptosis are both promising therapeutic techniques for the treatment of drug-resistant breast tumors. In this study, we proposed a thermal/ferroptosis/magnetic resonance imaging (MRI) triple functional nanoparticle (I@P-ss-FRT) in which ferritin, an iron storage material with excellent cellular uptake capacity, was attached via disulfide bonds onto polydopamine coated iron oxide nanoparticle (I@P) as photothermal transduction agent and MRI probe. I@P-ss-FRT converted the near-infrared light (NIR) into localized heat which accelerated the release of ferrous ions from ferritin accomplished by glutathione reduction and subsequently induced ferroptosis. The drug-resistant cancer cell lines exhibited a more significant uptake of I@P-ss-FRT and sensitivity to PTT/ferroptosis compared with normal cancer cell lines. In vivo, I@P-ss-FRT plus NIR displayed the best tumor-killing potential with inhibitory rate of 83.46 %, along with a decline in GSH/GPX-4 content and an increase in lipid peroxides generation at tumor sites. Therefore, I@P-ss-FRT can be applied to combat drug-resistant breast cancer.

Abstract LB053: A novel BRAF-specific allosteric inhibitor that effectively targets acquired RAFi-resistant BRAF(V600E) cancers and RAS-mutated cancers in vitro and in vivo

Abstract Targeting hyperactive RAS-RAF-MEK-ERK signaling for treating cancers has achieved a promising outcomes, and a number of RAS/RAF inhibitors have been applied to clinical cancer treatment. However, these inhibitors have only a short-term efficacy and their effectiveness is abrogated by acquired drug resistance in less than one year. For those resistant/refractory RAS/RAF-mutated cancers, there’s no effective therapeutics available in clinic. In this study, we developed a novel BRAF-specific inhibitor that docks on an allosteric site on BRAF kinase domain. This inhibitor effectively killed both primary and drug-resistant cancer cell lines with RAS mutation or BRAF(V600E) mutation in vitro and inhibited the growth of xenografted cancers derived from these cell lines in vivo without apparent toxicity. Mechanistically, we found that although this inhibitor blocked the catalytic activity of BRAF in vitro kinase assay, it triggered a super high ERK signaling through improving BRAF/CRAF heterodimerization and hence induced cellular apoptosis of cancer cells with RAS mutation or BRAF(V600E) mutation. Surprisingly, there’s no drug resistance occurring when treating cancer cells with RAS mutation or BRAF(V600E) mutation at a low concentration. To further evaluate the efficacy of this BRAF against RAS/RAF-mutated cancers in vivo, we constructed a series of pancreatic patient-derived xenograft cancer models with Kras G12D or G12V mutations, and found that our BRAF inhibitor exhibited a comparable or much better efficacy than Kras G12D-specific inhibitor on theses PDX models. Altogether, our data indicated that our BRAF-specific allosteric inhibitor may serve as a good therapeutic drug for treating BRAF(V600E)/RAS- cancers, particularly those resistant/refractory cancers, though it still need further evaluations via clinical trials. Citation Format: Jiancheng Hu. A novel BRAF-specific allosteric inhibitor that effectively targets acquired RAFi-resistant BRAF(V600E) cancers and RAS-mutated cancers in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB053.

Abstract 306: The role of exosomal UBE2R2 in colorectal cancer drug resistance

Abstract Background: Ctherapy resistance has been a major barrier against successful theratment. Exosome (EV), an important intercellular communication mediator, participates in a highly and complex network that drug resistance of cancer cells. EV has been reported to be able to transfer drug resistance from drug-resistant tumor cells to adjacent drug-sensitive cells. We identified a novel target, UBE2R2, based on our previous exosome proteomics analysis of resistant colorectal cancer cell lines. The main goal of this study is to validate the underlying mechanisms leading to chemotherapy resistance. Methods: We isolated EV from drug resistant cancer cell lines including coloractal cancer cell lines (HCT116-OXAR, DLD-1 OXA-R, DLD-1-OXA-R/SN38) its parental cell lines (HCT116, DLD-1) using the ExoLutE isolation kits based on a combination of multiple isolation techniques. Based on the existing data (proteomic analysis), we conducted experiments to elucidate the functions and mechanisms involved in cancer resistance of the identified UBE2R2. Cell viability assay and invasion asaay were performed to assess the effects of EVs drived from drug resistance colorectal cells on oxaliplatin and irinotecan resistance. In addition, cells transfected with siControl and siUBE2R2 were treated with oxaliplatin to access the relative UBE2R2 drug resistance effect. To explore the related mechanisms, we performed real time PCR and western blot to identify the UBE2R2-SMYD3-P53 pathway, and FACS analysis to confirm apoptosis. Additionally, TMA samples were utilized to confirm resistance according to patient stage, and UBE2R2 expression was validated by immunohistological methods. Results: UBE2R2, which is involved in ubiquitination, was most prominently expressed in resistant cell lines compared to controls in our previous proteomic analysis. Based on these results, we found that cell lines treated with exosomes which were derived from resistant cell lines showed lower sensitivity to the drug. Notably, treatment of resistant cell lines with siUBE2R2 resulted in increased sensitivity, confirming the involvement of UBE2R2 in resistance. As a possible mechanism in which UBE2R2 may be involved, western analysis targeting the SMYD-P53 mechanism showed that the combination of UBE2R2-SMYD3-p53 promoted the ubiquitination and degradation of p53. Analyzed using TMA patient samples, we found that the expression of UBE2R2 increased towards more advenced stage. Conclusion: Our results suggest a novel mechanism of chemotherapy resistance may be associated with UBE2R2. Exosome (including UBE2R2) which was drived from drug resistance cells may induce oxaliplatin and irinotecan resistance in parental colorectal cells via regulation of SMYD3-UBE2R2-P53 signaling pathway, which will be useful for investigating a potential clinical target in prediction drug resistance. Citation Format: Chi Hoon Maeng, Minji Choi. The role of exosomal UBE2R2 in colorectal cancer drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 306.

Abstract 1945: Mechanisms of acquired resistance to EGFR tyrosine kinase inhibitor osimertinib and KRAS-G12C inhibitor sotorasib in lung cancer

Abstract A series of molecular targeted drugs have been developed for the treatment of non-small cell lung cancer (NSCLC) harboring driver gene alterations. Although the use of these drugs has substantially improved the clinical outcomes of patients with the genetically altered NSCLC, these patients ultimately develop resistance to the drugs. Various molecular mechanisms of resistance to the molecular target therapies have been reported, it is still a crucial challenge to overcome the drug resistance. Osimertinib, the most used third-generation EGFR tyrosine kinase inhibitor to target both common EGFR mutations and secondary T790M mutation, and sotorasib, a recently developed KRAS inhibitor to target KRAS G12C mutation, are no exception. In this study, we aimed to investigate the mechanisms of acquired resistance to osimertinib and sotorasib in NSCLC by profiling drug-resistant lung cancer cell lines, which were established from EGFR-mutant HCC827(del E756_A750) and KRAS G12C-mutant H23 through exposure to each of the molecular targeted drugs. Phospho-RTK array and western blot analyses revealed MET overexpression and phosphorylation in the osimertinib-resistant HCC827 (HCC827OR) as previous studies reported. Notably in our study, HCC827OR cells were incompletely sensitive to a selective MET inhibitor savolitinib without osimertinib, suggesting that HCC827OR partially lost its dependency on mutant EGFR and got addiction to MET signaling. When sotolasib was administered to the sotolasib-resistant H23 (H23SR) cells, KRAS-GTP, and the downstream ERK and AKT phosphorylation were suppressed. To identify novel mechanisms of the resistance, we performed epigenomic profiling of active enhancer in those cells and found a candidate transcription factor, at whose gene locus H3K27 acetylation signal was increased in H23SR cells compared to parent H23 cells. Immunoblotting showed that protein expression of the gene was indeed higher in H23SR cells than the parent cells. To confirm the involvement of this gene to the resistance, further investigation is warranted. Citation Format: Yuri Yagami, Takashi Sato, Hiroki Yamamoto, Masayuki Shirasawa, Yoshiro Nakahara, Watanabe Hideo, Naoki Katsuhiko. Mechanisms of acquired resistance to EGFR tyrosine kinase inhibitor osimertinib and KRAS-G12C inhibitor sotorasib in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1945.

Rapid and automatic phenotyping of cells through their annexin-mediated enforced blebbing response

Cellular blebbing has been widely recognized as a hallmark of processes such as apoptosis and cell migration. Here, we developed a novel double-layer compression microfluidic device to trigger the enforced blebbing of cells in a programmable manner. It was found that the critical compression for inducing membrane bleb in highly invasive or drug-resistant breast and lung cancer cell lines could be several times higher than that of their non-invasive or drug-sensitive counterparts. Furthermore, we showed that knockdown of annexin-6, a protein known to be heavily involved in membrane and calcium dynamics in cells, led to a significantly reduced cellular volume, reflecting a lowered intracellular pressure, and an ∼twofold increase in the critical compressive strain for triggering blebbing. The fact that hundreds of cells can be tested and automatically analyzed in our device at the same time highlights the potential of this simple and label-free method in applications such as cell sorting and disease detection.

Mutation of rsmC gene from Serratia marcescens QBN using CRISPR-Cas9 to enhance prodigiosin synthesis

Prodigiosin is a bright red pigment, which is produced by various gram-negative and gram positive bacteria include S. marcescens, Hahella chejuensis, Vibrio psychroerythrus, Streptomyces coelicolor and many marine bacteria such as Pseudomonas sp, Vibrio sp. Prodigiosin has garnered considerable interest among the scientific community due to its immunosuppressive and anticancer properties against various drug-resistant cancer cell lines. In this study, we utilized the biosynthetic pathway of prodigiosin (Pg), using CRISPR/Cas9 gene editing techniques to eliminate the gene inhibiting Pg synthesis, known as rsmC, aiming to enhance the ability to synthesize Pg from S. marcescens strains. The rsmC gene of S. marcescens QBN was randomly mutated using the CRISPR-Cas9 system with the pCasPA vector (containing Cas9) and pCRISPR (carrying the spacer segment and sgRNA synthesis). A 20-nucleotide spacer segment was selected on rsmCpreceding the TGG triplet at the 3' end (PAM, the recognition site of Cas9) and inserted into pCRISPR between two BsaI sites. Following the binding of sgRNA to Cas9, mutations occurred in the nucleotides of the rsmC gene, resulting in the cleavage of the double-stranded DNA at the targeted site. A sequence homologous to the rsmC gene, lacking 100 nucleotides, was introduced into the cell and will involve the cleaved DNA's repair mechanism. The mutated rsmC strain of S. marcescens QBN synthesized Pg at 216 - 231 mg/L, higher than the original strain (180 mg/L). Furthermore, HPLC results and 1H spectral analysis showed that the synthesized red pigment from the mutated strain S. marcescens QBN was prodigiosin. These findings provide a basis for further improving the productivity of Pg synthesis in the S. marcescens QBN strain and serve as a basis for editing genes in other microorganisms using the CRISPR-Cas9 system.

Amphonal, a new polyene aldehyde from a deep-sea-derived Streptomyces amphotericinicus

A new polyene aldehyde, named amphonal (1), and two known (2 and 3) polyketides were isolated from the deep-sea-derived Streptomyces amphotericinicus OUCT16-38 strain. The structure of 1 was determined by extensive MS and NMR spectroscopic analysis. In the cytotoxicity evaluation, compound 2 showed significant growth inhibition against the drug-resistant human lung cancer cell line A549-Taxol with IC50 value of 0.44 μM, which was more potent than the positive control doxorubicin. Meanwhile, 2 showed considerable cytotoxic effect towards H1975, H1299 and HEL cell lines (IC50 = 0.93-4.73 μM) as well.

The TGFBI gene and protein expression in topotecan resistant ovarian cancer cell lines

PurposeThe primary limiting factor in achieving cures for patients with cancer, particularly ovarian cancer, is drug resistance. The mechanisms of drug resistance of cancer cells during chemotherapy may include compounds of the extracellular matrix, such as the transforming growth factor-beta-induced protein (TGFBI). In this study, we aimed to analyze the TGFBI gene and protein expression in different sensitive and drug-resistant ovarian cancer cell lines, as well as test if TGFBI can be involved in the response to topotecan (TOP) at the very early stages of treatment. Materials and methodsIn this study, we conducted a detailed analysis of TGFBI expression in different ovarian cancer cell lines (A2780, A2780TR1, A2780TR2, W1, W1TR, SKOV-3, PEA1, PEA2 and PEO23). The level of TGFBI mRNA (QPCR), intracellular and extracellular protein (Western blot analysis) were assessed in this study. ResultsWe observed upregulation of TGFBI mRNA in drug-resistant cell lines and estrogen-receptor positive cell lines, which was supported by overexpression of both intracellular and extracellular TGFBI protein. We also showed the TGFBI expression after a short period of treatment of sensitive ovarian cancer cell lines with TOP. ConclusionThe expression of TGFBI in ovarian cancer cell lines suggests its role in the development of drug resistance.

New copper(II) complex based-thiosemicarbazone and phenanthroline: DNA/BSA binding, antiproliferative activity, DFT and docking studies

A new copper(II) complex {Cu(HPP)}, bromo(1,10-phenanthroline)(triphenylphosphine)copper(I) with 2-hydroxy-5-methoxyacetophenone thiosemicarbazone (HMAT) was synthesized and characterized by experimental FT-IR, Raman, UV–vis, TGA and EPR spectral analysis. The Cu(HPP) was theoretically modeled using the B3LYP/VTZ method/basis set and the values of the spectroscopic parameters were calculated by the DFT method. The coordination geometry around the Cu(II) in the compound was predicted as an elongated octahedral. Spectroscopic investigations showed the intercalative binding mechanism of the Cu(HPP) with calf-thymus deoxyribonucleic acid (ct-DNA). Spectroscopic techniques were also used to evaluate the interaction of the Cu(HPP) with bovine serum albumin (BSA), which revealed that the Cu(HPP) could bind to BSA significantly. In addition, the molecular docking between the Cu(HPP) and BSA was investigated to support the bonding mechanism. Further, we studied the anticancer activity of the Cu(HPP) in drug resistant breast cancer cell line (MDA-MB-231), with a focus on the proliferative characteristics. Our findings emphasize that this newly synthetized complex has a significant cytotoxic effect on the cells and can be utilized as a candidate chemotherapeutic agent to effectively target breast cancer.

Molecular determinants of the response of cancer cells towards geldanamycin and its derivatives

Geldanamycin is an ansamycin-derivative of a benzoquinone isolated from Streptomyces hygroscopicus. It inhibits tyrosine kinases and heat shock protein 90 (HSP90). Geldanamycin and 11 derivatives were subjected to molecular docking to HSP90, and 17-desmethoxy-17-N,N-dimethylamino-geldanamycin (17-DMAG) was the compound with the highest binding affinity (−7.73 ± 0.12 kcal/mol) and the lowest inhibition constant (2.16 ± 0.49 μM). Therefore, 17-DMAG was selected for further experiments in comparison to geldanamycin. Multidrug resistance (MDR) represents a major problem for successful cancer therapy. We tested geldanamycin and 17-DMAG against various drug-resistant cancer cell lines. Although geldanamycin and 17-DMAG inhibited the proliferation in all cell lines tested, multidrug-resistant P-glycoprotein-overexpressing CEM/ADR5000 cells were cross-resistant, ΔEGFR-overexpressing tumor cells and p53 knockout cells were sensitive to these two compounds. COMPARE and hierarchical cluster analyses were performed, and 60 genes were identified to predict the sensitivity or resistance of 59 NCI tumor cell lines towards geldanamycin and 17-DMAG. The distribution of cell lines according to their mRNA expression profiles indicated sensitivity or resistance to both compounds with statistical significance. Moreover, bioinformatic tools were used to study possible mechanisms of action of geldanamycin and 17-DMAG. Galaxy Cistrome analyses were carried out to predict transcription factor binding motifs in the promoter regions of the candidate genes. Interestingly, the NF-ĸB DNA binding motif (Rel) was identified as the top transcription factor. Furthermore, these 60 genes were subjected to Ingenuity Pathway Analysis (IPA) to study the signaling pathway interactions of these genes. Interestingly, IPA also revealed the NF-ĸB pathway as the top network among these genes. Finally, NF-ĸB reporter assays confirmed the bioinformatic prediction, and both geldanamycin and 17-DMAG significantly inhibited NF-κB activity after exposure for 24 h. In conclusion, geldanamycin and 17-DMAG exhibited cytotoxic activity against different tumor cell lines. Their activity was not restricted to HSP90 but indicated an involvement of the NF-KB pathway.

Use of Enzymatically Activated Carbon Monoxide Donors for Sensitizing Drug-Resistant Tumor Cells.

The application of gaseous signaling molecules like NO, H2S or CO to overcome the multidrug resistance in cancer treatment has proven to be a viable therapeutic strategy. The development of CO-releasing molecules (CORMs) in a controlled manner and in targeted tissues remains a challenge in medicinal chemistry. In this paper, we describe the design, synthesis and chemical and enzymatic stability of a novel non-metal CORM (1) able to release intracellularly CO and, simultaneously, facilitate fluorescent degradation of products under the action of esterase. The toxicity of 1 against different human cancer cell lines and their drug-resistant counterparts, as well as the putative mechanism of toxicity were investigated. The drug-resistant cancer cell lines efficiently absorbed 1 and 1 was able to restore their sensitivity vs. chemotherapeutic drugs by causing a CO-dependent mitochondrial oxidative stress that culminated in mitochondrial-dependent apoptosis. These results demonstrate the importance of CORMs in cases where conventional chemotherapy fails and thus open the horizons towards new combinatorial strategies to overcome multidrug resistance.

Targeting microRNA-125b inhibited the metastasis of Alisertib resistance cells through mediating p53 pathway

Objective: To clarify the mechanisms involvement in Alisertib-resistant colorectal cells and explore a potential target to overcome Alisertib-resistance. Methods: Drug-resistant colon cancer cell line (named as HCT-8-7T cells) was established and transplanted into immunodeficient mice. The metastasis in vivo were observed. Proliferation and migration of HCT-8-7T cells and their parental cells were assessed by colony formation and Transwell assay, respectively. Glycolytic capacity and glutamine metabolism of cells were analyzed by metabolism assays. The protein and mRNA levels of critical factors which are involved in mediating glycolysis and epithelial-mesenchymal transition (EMT) were examined by western blot and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR), respectively. Results: In comparison with the mice transplanted with HCT-8 cells, which were survival with limited metastatic tumor cells in organs, aggressive metastases were observed in liver, lung, kidney and ovary of HCT-8-7T transplanted mice (P<0.05). The levels of ATP [(0.10±0.01) mmol/L], glycolysis [(81.77±8.21) mpH/min] and the capacity of glycolysis [(55.50±3.48) mpH/min] in HCT-8-7T cells were higher than those of HCT-8 cells [(0.04±0.01) mmol/L, (27.77±2.55) mpH/min and(14.00±1.19) mpH/min, respectively, P<0.05]. Meanwhile, the levels of p53 protein and mRNA in HCT-8-7T cells were potently decreased as compared to that in HCT-8 cells (P<0.05). However, the level of miRNA-125b (2.21±0.12) in HCT-8-7T cells was significantly elevated as compared to that in HCT-8 cells (1.00±0.00, P<0.001). In HCT-8-7T cells, forced-expression of p53 reduced the colon number (162.00±24.00) and the migration [(18.53±5.67)%] as compared with those in cells transfected with control vector [274.70±40.50 and (100.00±29.06)%, P<0.05, respectively]. Similarly, miR-125b mimic decreased the glycolysis [(25.28±9.51) mpH/min] in HCT-8-7T cells as compared with that [(54.38±12.70)mpH/min, P=0.003] in HCT-8-7T cells transfected with control. Meanwhile, in comparison with control transfected HCT-8-7T cells, miR-125b mimic also significantly led to an increase in the levels of p53 and β-catenin, in parallel with a decrease in the levels of PFK1 and HK1 in HCT-8-7T cells (P<0.05). Conclusions: Silencing of p53 by miR-125b could be one of the mechanisms that contributes to Alisertib resistance. Targeting miR-125b could be a strategy to overcome Alisertib resistance.

A cilia-related marker predicting suitability for immunotherapy in melanoma.

e21560 Background: Cilia exert tumor suppressor effects in several tumor types including melanoma, and scientists found that blocking cilia growth in drug-resistant cancer cell lines may restore cancer cells' sensitivity to immunotherapy. Therefore, we hypothesized that cilia might be used as a predictive biomarker for melanoma immunotherapy. Methods: Through literature search, a total of six sets of RNA-seq and WES data of melanoma samples receiving PD-1/CTLA-4 immunotherapy were collected. GSVA was used to obtain an enrichment score between the samples and a set of 112 ciliary genes, based on which ciliary signatures were screened for prognosis. The ciliary characteristic gene set was further optimized by LASSO regression, then a risk model was constructed by combing stepwise regression and COX regression. Log-rank test was used to verify survival differences between high and low risk groups in other independent data sets, and Wilcox test was used to explore differences in other clinical characteristics between high and low risk groups. Imaging mass spectrometry (IMC) and spatial position imaging (SPI) data were used to analyze cell states in tumor tissues. All analyses were performed by R 4.0.3. Results: A total of 10 cilia pathways related to prognosis were screened out in the training set, and 13 genes were picked out from these 10 pathways as selected features. Combining LASSO and COX regression, a risk model consisting of 5 genes were finally obtained. Patients in the high-risk (HR)group classified by this model had a significantly worse prognosis than the low-risk (LR) group in both the training and test sets (p &lt; 0.05, respectively). In addition, by analyzing immune infiltration and pathway enrichment, it was found that LR group was in the state of immune activation, and its immune score was significantly higher than that of HR group. Further analysis showed the model had no significant correlation with other clinical features, such as pathological stage, tumor mutation burden and NF1/KRAS/BRAF gene mutations, indicating that the model was relatively independent. By IMC data analysis, it was found that the number of tumor cells in LR and HR groups were similar (107082 vs 11928), but the number of lymphocytes in LR group was significantly higher than that in HR group (71578 vs 5313). Moreover, among the 35 protein markers related to immunotherapy, 16 (45.7%) proteins were up-regulated and 0 protein was down-regulated in LR group. By analyzing SPI of multiple regions of these samples, it was also observed that tumor tissues in LR group were mostly in a state of lymphocyte infiltration, while these in HR group were in a state of immune rejection or immune desert. Conclusions: The model constructed based on cilia-related features in this study can independently identify melanomas with high risk of recurrence or progression, and provide a theoretical basis for exploring whether targeted cilia structure therapy can be used as a treatment for melanoma.

Genome-directed discovery of antiproliferative bafilomycins from a deepsea-derived Streptomyces samsunensis

Genomic bioinformatics analysis identified a bafilomycin biosynthetic gene cluster (named bfl) in the deepsea-derived S. samsunensis OUCT16-12, from which two new (1 and 2, named bafilomycins R and S) along with four known (3–6) bafilomycins were targetly obtained. The structure of 3 was clearly identified for the first time, thus named bafilomycin T herein. Differ from the fumarate substitution at C-21 of known bafilomycins, its location on C-23 is a unique feature of 1 and 2. The stereochemistry of the compounds was established based on NOE correlations, ketoreductase (KR)-types in PKS modules of bfl, and ECD calculations. Moreover, a detailed biosynthetic model of 1–6 in S. samsunensis OUCT16-12 was provided based on the gene function prediction and sequence identity. Compared with the positive control doxorubicin, 1–6 showed more potent antiproliferative activities against drug-resistant lung cancer cell line A549-Taxol, with IC50 values ranging from 0.07 μM to 1.79 μM, which arrested cell cycle in G0/G1 phase to hinder proliferation.

Abstract 1668: Targeting drug-induced tolerant &amp; resistant cell populations by novel ferroptosis inducer

Abstract A subset of tumor cells escapes apoptotic and necroptotic death when subjected to chemo- or targeted therapy thereby entering a transient reversible less-proliferative “tolerant state.” Upon continuous drug treatment, these tumor cells become drug resistant, leading to metastases and cancer relapse. A long-standing goal in oncology has been to develop therapeutics to effectively target both cell states. Growing published evidence suggests that accumulation of reactive oxygen species and lipid remodeling make tolerant and resistant tumor cells more vulnerable to ferroptotic mode of cell death. To this end, we here characterize the activity of a proprietary inhibitor of GPX4, an enzyme that safeguards against ferroptosis, across a panel of tolerant and resistant cell line models. Drug tolerant and resistant cancer cell lines spanning three indications were generated by applying continuous treatments with relevant standard-of-cares (SOC) in vitro for short or long durations, respectively. Age-matched control cell lines were derived in parallel by treatment of parental cells with DMSO. Cells were subjected to high dose of SOC for nine days to generate tolerant lines, or to increased therapeutic doses of SOC over three months to generate resistant lines, after which resistance to SOCs was confirmed. In accordance with our hypothesis, we found that drug tolerant lines demonstrated enhanced sensitivity to GPX4 inhibition compared to age-matched control lines. Furthermore, we noted that resistant tumor cell lines retained exquisite sensitivity to GPX4 inhibition across all indications while demonstrating up to 1000-fold resistance to SOCs. In summary, we found that cell line models which evade cell death upon treatment with chemo- or targeted therapies continued to present vulnerability to ferroptosis induction across both cell states. Given the heterogeneity in responses to drug-induced cell-states, understanding the mechanisms of differential drug sensitivity to GPX4 inhibition may help in formulating successful strategies for targeting this relevant cell population in the clinic, thereby limiting cancer progression and/or improving curative potential. Citation Format: Taronish Dubash, Maria Cristina Munteanu, Shrouq Farah, Cristian Nunez, Laurence Jadin, Branko Radetich, Darby Schmidt, Nadia Gurvich. Targeting drug-induced tolerant &amp; resistant cell populations by novel ferroptosis inducer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1668.

Recent advances in the strategic incorporation of fluorine into new-generation taxoid anticancer agents

This account describes our recent progress on the strategic incorporation of fluorine and organofluorine moieties into new-generation taxoid anticancer agents for medicinal chemistry and chemical biology studies.In the case study 1, novel 3rd-generation fluorotaxoids bearing 3-OCF3 or 3-OCF2H group in the C2-benzoate moiety were designed, synthesized and examined for their anticancer activities. The potency of novel taxoids against drug-resistant cancer cell lines was 2–3 orders of magnitude higher than that of paclitaxel (PTX). Molecular modeling analysis indicated the favorable van der Waals interactions of OCF3 and OCHF2 groups in the binding site. Overall, taxoids bearing a OCHF2 group at the C2 benzoate position exhibited the highest potencies against multidrug-resistant (MDR) cancer cell lines and cancer stem cell (CSC)-enriched cell lines, indicating that the new 3rd-generation fluorotaxoids are promising candidates as chemotherapeutic agents.In the case study 2, novel 3rd-generation 3′-difluorovinyl (DFV)-taxoids, bearing 3-CF3O or 3-CHF2O group in the C2-benzoyl moiety, were designed, synthesized, and evaluated for their potencies and pharmacological properties. These new DFV-taxoids exhibited remarkable cytotoxicity against extremely drug-resistant cancer cell lines with subnanomolar IC50 values, indicating that these new DFV-taxoids can overcome MDR caused by the overexpression of Pgp and other ABC cassette transporters. The molecular docking analysis of new DFV-taxoids revealed that the 3′-DFV moiety and the 3-CF3O/3-CHF2O group of the C2-benzoate moiety are nicely accommodated to the deep hydrophobic pocket of the PTX/taxoid binding site in the β-tubulin, enabling an enhanced binding through unique attractive interactions between F/OCF3/OCHF2 and the protein. This enhancement in binding is reflected in the remarkable high potency of new 3rd-generation DFV-taxoids.In the case study 3.1, the therapeutic potential of new 3rd-generation DFV-taxoids in anaplastic thyroid cancer (ATC) cells was evaluated in vitro and in vivo. This study demonstrated that these new DFV-taxoids were more efficacious than PTX against ATC cell lines and tumor xenografts, as demonstrated by the efficient inhibition of cell proliferation and colony formation, induction of apoptosis via the mitotic arrest at the G2/M phase, as well as the suppression of tumorigenic potential in nude mice. Furthermore, tubulin polymerization assay and molecular docking analysis confirmed that these new DFV-taxoids promoted far more rapid polymerization of β-tubulin than PTX through stronger binding to tubulin/microtubules. Taken together, this study has indicated a promising therapeutic potential of these new DFV-taxoids against ATC.In the case study 3.2, DFV-OTX displayed potent cytotoxicity and effective induction of β-tubulin polymerization, as well as the G2/M phase arrest, leading to apoptosis in PTX-sensitive and PTX-resistant breast cancer cells. Furthermore, DFV-OTX clearly exhibited efficacy against MCF-7R and MDA-MB-231R tumor xenografts in mouse models. Thus, DFV-OTX effectively overcame PTX-resistance in MDA-MB-231R cells and tumor xenografts, wherein the drug resistance was attributed to ABCB1/ABCG2 upregulation. DFV-OTX was also effective against MCF-7R cells and tumor xenografts, which are PTX-resistant due to different MOA. Accordingly, DFV-OTX is a promising chemotherapeutic agent for the treatment of PTX-resistant cancers.Overall, these next-generation fluorotaxoids are promising candidates for highly potent chemotherapeutic agents, as well as payloads for tumor-targeting drug conjugates such as antibody-drug conjugates (ADCs).

Cytotoxicity of the methanol extracts and compounds of Brucea antidysenterica (Simaroubaceae) towards multifactorial drug-resistant human cancer cell lines

BackgroundCancer remains a global health concern and constitutes an important barrier to increasing life expectancy. Malignant cells rapidly develop drug resistance leading to many clinical therapeutic failures. The importance of medicinal plants as an alternative to classical drug discovery to fight cancer is well known. Brucea antidysenterica is an African medicinal plant traditionally used to treat cancer, dysentery, malaria, diarrhea, stomach aches, helminthic infections, fever, and asthma. The present work was designed to identify the cytotoxic constituents of Brucea antidysenterica on a broad range of cancer cell lines and to demonstrate the mode of induction of apoptosis of the most active samples.MethodsSeven phytochemicals were isolated from the leaves (BAL) and stem (BAS) extract of Brucea antidysenterica by column chromatography and structurally elucidated using spectroscopic techniques. The antiproliferative effects of the crude extracts and compounds against 9 human cancer cell lines were evaluated by the resazurin reduction assay (RRA). The activity in cell lines was assessed by the Caspase-Glo assay. The cell cycle distribution, apoptosis via propidium iodide (PI) staining, mitochondrial membrane potential (MMP) through 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, and the reactive oxygen species (ROS) via 2´,7´-dichlorodihydrofluoresceine diacetate (H2DCFH-DA) staining, were investigated by flow cytometry.ResultsPhytochemical studies of the botanicals (BAL and BAS) led to the isolation of seven compounds. BAL and its constituents 3, (3-(3-Methyl-1-oxo-2-butenyl))1H indole (1) and hydnocarpin (2), as well as the reference compound, doxorubicin, had antiproliferative activity against 9 cancer cell lines. The IC50 values varied from 17.42 µg/mL (against CCRF-CEM leukemia cells) to 38.70 µg/mL (against HCT116 p53−/− colon adenocarcinoma cells) for BAL, from 19.11 µM (against CCRF-CEM cells) to 47.50 µM (against MDA-MB-231-BCRP adenocarcinoma cells) for compound 1, and from 4.07 µM (against MDA-MB-231-pcDNA cells) to 11.44 µM (against HCT116 p53+/+ cells) for compound 2. Interestingly, hypersensitivity of resistant cancer cells to compound 2 was also observed. BAL and hydnocarpin induced apoptosis in CCRF-CEM cells mediated by caspase activation, the alteration of MMP, and increased ROS levels.ConclusionBAL and its constituents, mostly compound 2, are potential antiproliferative products from Brucea antidysenterica. Other studies will be necessary in the perspective of the discovery of new antiproliferative agents to fight against resistance to anticancer drugs.

A Pan-Cancer Landscape of ABCG2 across Human Cancers: Friend or Foe?

Emerging evidence from research or clinical studies reported that ABCG2 (ATP-binding cassette sub-family G member 2) interrelates with multidrug resistance (MDR) development in cancers. However, no comprehensive pan-cancer analysis is available at present. Therefore, we explore multiple databases, such as TCGA to investigate the potential therapeutic roles of ABCG2 across 33 different tumors. ABCG2 is expressed on a lower level in most cancers and shows a protective effect. For example, a lower expression level of ABCG2 was detrimental to the survival of adrenocortical carcinoma (TCGA-ACC), glioblastoma multiforme (GBM), and kidney renal clear cell carcinoma (KIRC) patients. Distinct associations exist between ABCG2 expression and stemness scores, microenvironmental scores, microsatellite instability (MSI), and tumor mutational burden (TMB) of tumor patients. We observed a significant positive correlation between the ABCG2 mutation site and prognosis in uterine corpus endometrial carcinoma (UCEC) patients. Moreover, transmembrane transporter activity and hormone biosynthetic-associated functions were found to be involved in the functionality of ABCG2 and its related genes. The cDNAs of cancer cell lines were collected to detect exon mutation sequences and to analyze ABCG2 mRNA expression. The mRNA expression level of ABCG2 showed a significant difference among spheres and drug-resistant cancer cell lines compared with their corresponding adherent cancer cell lines in six types of cancer. This pan-cancer study provides, for the first time, a comprehensive understanding of the multifunctionality of ABCG2 and unveils further details of the potential therapeutic role of ABCG2 in pan-cancer.