Single-cell RNA-seq analysis reveals the platinum resistance gene COX7B and the surrogate marker CD63.
ABSTRACT: Cancers acquire resistance to systemic treatment with platinum-based chemotherapy (eg, cisplatin [CDDP]) as a result of a dynamic intratumoral heterogeneity (ITH) and clonal repopulation. However, little is known about the influence of chemotherapy on ITH at the single-cell level. Here, mapping the transcriptome of cancers treated with CDDP by scRNA-seq, we uncovered a novel gene, COX7B, associated with platinum-resistance, and surrogate marker, CD63. Knockdown of COX7B in cancer cells decreased the sensitivity of CDDP whereas overexpression recovered the sensitivity of CDDP. Low COX7B levels correlated with higher mortality rates in patients with various types of cancer and were significantly associated with poor response to chemotherapy in urinary bladder cancer. Tumor samples from patients, who underwent CDDP therapy, showed decreased COX7B protein levels after the treatment. Analyzing scRNA-seq data from platinum-naïve cancer cells demonstrated a low-COX7B subclone that could be sorted out from bulk cancer cells by assaying CD63. This low-COX7B subclone behaved as cells with acquired platinum-resistance when challenged to CDDP. Our results offer a new transcriptome landscape of platinum-resistance that provides valuable insights into chemosensitivity and drug resistance in cancers, and we identify a novel platinum resistance gene, COX7B, and a surrogate marker, CD63.
Project description:Platinum-based antitumor agents are widely used in cancer chemotherapy. Drug resistance is a major obstacle to the successful use of these agents because once drug resistance develops, other effective treatment options are limited. Recently, we conducted a clinical trial using a copper-lowering agent to overcome platinum drug resistance in ovarian cancer patients and the preliminary results are encouraging. In supporting this clinical study, using three pairs of cisplatin (cDDP)-resistant cell lines and two ovarian cancer cell lines derived from patients who had failed in platinum-based chemotherapy, we showed that cDDP resistance associated with reduced expression of the high-affinity copper transporter (hCtr1), which is also a cDDP transporter, can be preferentially resensitized by copper-lowering agents because of enhanced hCtr1 expression, as compared with their drug-sensitive counterparts. Such a preferential induction of hCtr1 expression in cDDP-resistant variants by copper chelation can be explained by the mammalian copper homeostasis regulatory mechanism. Enhanced cell-killing efficacy by a copper-lowering agent was also observed in animal xenografts bearing cDDP-resistant cells. Finally, by analyzing a public gene expression dataset, we found that ovarian cancer patients with elevated levels of hCtr1 in their tumors, but not ATP7A and ATP7B, had more favorable outcomes after platinum drug treatment than those expressing low hCtr1 levels. This study reveals the mechanistic basis for using copper chelation to overcome cDDP resistance in clinical investigations.
Project description:Small cell lung cancer (SCLC) is an aggressive malignancy characterized by rapid onset of resistance to platinum chemotherapy. However, the mechanisms underlying platinum-resistance remain obscure in part due to scarcity of tissue samples, particularly from relapsed patients. Here, we generated circulating tumor cell (CTC)-derived xenograft (CDX) models from SCLC patients before or after relapse that faithfully recapitulate patient tumor genomics and platinum response. Platinum-sensitive models were relatively homogeneous, whereas transcriptomic and proteomic analyses revealed enrichment of multiple targetable pathways and intertumoral heterogeneity among resistant models. Single-cell RNAseq profiling further identified greater intratumoral heterogeneity (ITH) associated with platinum-resistance. This included a population of DLL3low cells in resistant CDX models that demonstrated greater chemoresistance, suggesting that subtle shifts in the proportion of DLL3-expressing cells could impact response. Similarly, longitudinal single-cell transcriptional profiling of CTCs from patient blood reveals emergence of molecular markers of resistance and significantly greater ITH after disease relapse. Together, these data suggest that platinum-resistance involves a heterogeneous process of transcriptional fluidity with contributions from both preexisting cellular subpopulations and outgrowth of resistant populations, yielding a diverse cellular composition refractory to single-agent therapeutics. Overall design: small cell lung cancer CTC derived xenograft RNA-seq
Project description:We previously reported that human squamous cell carcinoma (SCC) cell lines refractory to cis-diaminedichloro-platinum II (cisplatin [CDDP]) had significant upregulation of the phosphodiesterase 3B gene (PDE3B), suggesting that inhibiting PDE3B suppresses CDDP resistance. shRNA-mediated PDE3B depletion in CDDP-resistant cells derived from SCC cells and Hela cells and induced CDDP sensitivity and inhibited tumor growth with elevated cyclic GMP induction resulting in upregulation of the multidrug-resistant molecule, but this did not occur in the 5-fluorouracil-resistant hepatocellular carcinoma cell lines. Furthermore, the antitumor growth effect of the combination of a PDE3B inhibitor (cilostazol) and CDDP in vivo was also greater than with either cilostazol or CDDP alone, with a significant increase in the number of apoptotic and cell growth-suppressive cancer cells in CDDP-resistance cell lines. Our results provided novel information on which to base further mechanistic studies of CDDP sensitization by inhibiting PDE3B in human cancer cells and for developing strategies to improve outcomes with concurrent chemotherapy.
Project description:Resistance to platinum?based drugs, such as cisplatin (CDDP), has been one of the major factors adversely affecting the clinical prognosis of patients with advanced non?small cell lung cancer (NSCLC). While it has been demonstrated that dysregulation of microRNAs (miRNAs) may contribute to cisplatin resistance in NSCLC, the underlying mechanisms remain largely unclear. In the present study, the effect of exosomal miR?1273a on cisplatin sensitivity of NSCLC was investigated. Microarray analysis was conducted to analyze the miRNA expression profiles in exosomes isolated from A549 cells treated with or without CDDP, and miR?1273a was found to be the most prominently downregulated miRNA in CDDP?treated exosomes. Overexpression of miR?1273a significantly increased the cytotoxicity of CDDP and induced apoptosis in A549 cells. Syndecan binding protein (SDCBP) was predicted to be a direct target of miR?1273a by bioinformatics and was found to be downregulated by miR?1273a in A549 cells. Furthermore, decreased plasma exosomal miR?1273a and increased plasma SDCBP levels were found to be associated with worse therapeutic outcomes of patients with advanced NSCLC receiving platinum?based chemotherapy. These findings suggest that miR?1273a is closely associated with the development of cisplatin resistance and may serve as a potential prognostic biomarker and therapeutic target for NSCLC.
Project description:Background: Cisplatin (CDDP)-based chemotherapy is the gold standard treatment for many cancer types. However, the phenotypic hallmark of tumors often changes after CDDP, with the acquisition of Epithelial-to-mesenchymal transition (EMT) and platinum resistance. Yet, the mechanisms by which cancer cells acquire EMT under the control of CDDP remain unclear. Methods: Following an investigation of urothelial cancers (UCs) before and after the acquisition of platinum resistance, we offered the new target TNFAIP2, which led to EMT and tumor invasion. TNFAIP2 expression in cancers were examined at the protein and transcription levels. Results: Clinically, up-regulated TNFAIP2 expression was identified as a significant predictor of mortality in upper urinary tract UCs. TNFAIP2 knockdown resulted in up-regulated E-cadherin expression and down-regulated TWIST expression in cancers, which decreased motile function. TNFAIP2 overexpression led to down-regulated E-cadherin expression and up-regulated TWIST expression. Clinical investigations on matched pre- and post-CDDP-treated sections confirmed up-regulated TNFAIP2 expression in CDDP-treated tumors, but down-regulated E-cadherin expression. A global gene expression analysis following TNFAIP2 knockdown identified MTDH as a positive regulator of TNFAIP2-derived EMT acquisition. Conclusions: Our results suggest a relationship between TNFAIP2 and EMT in cancers, in which MTDH expression levels in cancer cells are vital for promoting TNFAIP2-derived EMT acquisition. Overall design: To investigate the relationship between TNFAIP2 and EMT, we compared changes in global gene expression between before and after the knockdown of TNFAIP2 in platinum-resistant 5637PR cells.
Project description:Proton beam therapy (PBT) combined with chemotherapy, such as cis-diamminedichloroplatinum (II) (CDDP) and 5-fluorouracil (5-FU), has been employed as an alternative approach to improve clinical outcomes. PBT has been reported to be effective against esophageal cancer. However, apart from 5-FU and CDDP, almost no other drug has been tested in combined chemotherapy with PBT. Therefore, we investigated the effects of a poly (ADP-ribose) polymerase inhibitor on enhancing proton beam effects using esophageal cancer cell lines that exhibit resistance to radiation and CDDP. Esophageal squamous cell carcinoma cell lines OE-21 and KYSE-450 were exposed to the drugs for 1 h prior to irradiation. The cell survival curve was obtained using a clonogenic assay and the sensitizing effect ratio (SER) was calculated. The clonogenic assay was used to compare the effect of multi-fractioned irradiation between 8 Gy/1 fraction (fr) and 8 Gy/4 fr. ?H2AX, Rad51, BRCA1, BRCA2 and 53BP1 foci were detected via immunofluorescence. Olaparib exhibited an SER of 1.5-1.7 on PBT. The same sensitizing effect was exhibited in multi-fractioned irradiation, and the combined use increased the expression of double-strand breaks and homologous recombination-related genes in an additive manner. Such additive effects were not observed on non-homologous end joining-related genes. We demonstrated that olaparib has a high sensitizing effect on PBT in platinum- and radiation-resistant esophageal cancer cells. Our results suggest a potential clinical application of olaparib-proton irradiation (PT) against platinum- and radiation-resistant esophageal cancer.
Project description:We developed subclone multiplicity allocation and somatic heterogeneity (SMASH), a new statistical method for intra-tumor heterogeneity (ITH) inference. SMASH is tailored to the purpose of large-scale association studies with one tumor sample per patient. In a pan-cancer study of 14 cancer types, we studied the associations between survival time and ITH quantified by SMASH, together with other features of somatic mutations. Our results show that ITH is associated with survival time in several cancer types and its effect can be modified by other covariates, such as mutation burden. SMASH is available at https://github.com/Sun-lab/SMASH .
Project description:De novo and acquired resistance to platinum therapy such as cisplatin (CDDP) is a clinical challenge in gastric cancer treatment. Aberrant expression and activation of aurora kinase A (AURKA) and eukaryotic translation initiation factor 4E (eIF4E) are detected in several cancer types. Herein, we investigated the role of AURKA in CDDP resistance in gastric cancer. Western blot analysis demonstrated overexpression of AURKA and phosphorylation of eIF4E in acquired and de novo CDDP-resistant gastric cancer models. Inhibition of AURKA with MLN8237 (alisertib) alone or in combination with CDDP significantly suppressed viability of CDDP-resistant cancer cells (P < 0.01). Additionally, inhibition or knockdown of AURKA decreased protein expression of p-eIF4E (S209), HDM2, and c-MYC in CDDP-resistant cell models. This was associated with a significant decrease in cap-dependent translation levels (P < 0.01). In vivo tumor xenografts data corroborated these results and confirmed that inhibition of AURKA was sufficient to overcome CDDP resistance in gastric cancer. Our data demonstrate that AURKA promotes acquired and de novo resistance to CDDP through regulation of p-eIF4E (S209), c-MYC, HDM2, and cap-dependent translation. Targeting AURKA could be an effective therapeutic approach to overcome CDDP resistance in refractory gastric cancer and possibly other cancer types.
Project description:Background:Epithelial ovarian cancer is the most lethal gynecological cancer and the high mortality is due to the frequent presentation at advanced stage, and to primary or acquired resistance to platinum-based therapy. Methods:We developed three new models of ovarian cancer patient-derived xenografts (ovarian PDXs) resistant to cisplatin (cDDP) after multiple in vivo drug treatments. By different and complementary approaches based on integrated metabolomics (both targeted and untargeted mass spectrometry-based techniques), gene expression, and functional assays (Seahorse technology) we analyzed and compared the tumor metabolic profile in each sensitive and their corresponding cDDP-resistant PDXs. Results:We found that cDDP-sensitive and -resistant PDXs have a different metabolic asset. In particular, we found, through metabolomic and gene expression approaches, that glycolysis, tricarboxylic acid cycle and urea cycle pathways were deregulated in resistant versus sensitive PDXs. In addition, we observed that oxygen consumption rate and mitochondrial respiration were higher in resistant PDXs than in sensitive PDXs under acute stress conditions. An increased oxidative phosphorylation in cDDP-resistant sublines led us to hypothesize that its interference could be of therapeutic value. Indeed, in vivo treatment of metformin and cDDP was able to partially reverse platinum resistance. Conclusions:Our data strongly reinforce the idea that the development of acquired cDDP resistance in ovarian cancer can bring about a rewiring of tumor metabolism, and that this might be exploited therapeutically.
Project description:Advanced ovarian cancers are initially responsive to chemotherapy with platinum drugs but develop drug resistance in most cases. We showed recently that hepatocyte growth factor (HGF) enhances death of human ovarian cancer cell lines treated with cisplatin (CDDP) and that this effect is mediated by the p38 mitogen-activated protein kinase. In this work, we integrated genome-wide expression profiling, in silico data survey, and functional assays to identify transcripts regulated in SK-OV-3 ovarian cancer cells made more responsive to CDDP by HGF. Using oligonucleotide microarrays, we found that HGF pretreatment changes the transcriptional response to CDDP. Quantitative reverse transcription-PCR not only validated all the 15 most differentially expressed genes but also confirmed that they were primarily modulated by the combined treatment with HGF and CDDP and reversed by suppressing p38 mitogen-activated protein kinase activity. Among the differentially expressed genes, we focused functional analysis on two regulatory subunits of the protein phosphatase 2A, which were down-modulated by HGF plus CDDP. Decrease of each subunit by RNA interference made ovarian cancer cells more responsive to CDDP, mimicking the effect of HGF. In conclusion, we show that HGF and CDDP modulate transcription in ovarian cancer cells and that this transcriptional response is involved in apoptosis regulation. We also provide the proof-of-concept that the identified genes might be targeted to either increase the efficacy of chemotherapeutics or revert chemotherapy resistance. Overall design: Two of the time-course experiments, done independently (TR5 and TR7), were analyzed. time 0 (h0) time 6 hours (h6) time 12 hours (h12) time 24 hours (h24) with (hgf1) and without addition of HGF (hgf0)