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PDF file - 808K, Supplementary Figure S1: This figure depicts the impact of MALAT1 loss on gene expression, splicing and SR protein phosphorylation. Supplementary Figure S2: This figure describes the generation of MALAT1-Knockout A549 cells using an independent ZFN as well as their phenotypes regarding proliferation and MALAT1 target gene expression. Supplementary Figure S3: This figure displays the impact of loss of MALAT1 using an independent ZFN in A549 cells on cell migration. Supplementary Figure S4: This figure documents that Antisense Oligonucleotides (ASOs) are effective in silencing MALAT1 in vitro without affecting cell proliferation of EBC-1 cells. Supplementary Table S2: This table lists 23 genes whose differential expression (5 up / 18 down) in MALAT1 KO cells was validated via qRT-PCR and who had been previously identified with critical roles in lung cancer, migration, invasion, EMT or metastasis. Supplementary Table S3: This table lists the sequences of all primers used for qRT-PCR in 5'-3' direction. Supplementary Table S4: This table lists the sequences of all primers used for splicing analysis in 5'-3' direction. Supplementary Table S5: This table lists the sequences of all primers and probes used for TaqMan qRT-PCR in 5'-3' direction. Supplementary Table S6: This table lists the sequences of all Antisense Oligonucleotides (ASOs) in 5'-3' direction. Supplementary Methods: This chapter provides experimental details on the RNA isolation and DNase I digest, reverse transcription and quantitative PCR, exon microarray analysis, antisense oligonucleotides (ASOs), lung imaging by microCT scan, immunohistochemistry and in situ hybridization, statistical analysis, generation of ZFN-2 clones, cell proliferation assays, SDS-PAGE and Western blotting.

Distant metastasis is the predominant cause of death in early-stage non-small cell lung cancer (NSCLC). Currently, it is impossible to predict the occurrence of metastasis at early stages and thereby separate patients who could be cured by surgical resection alone from patients who would benefit from additional chemotherapy. In this study, we applied a comparative microarray approach to identify gene expression differences between early-stage NSCLC patients whose cancer ultimately did or did not metastasize during the course of their disease. Transcriptional profiling of 82 microarrays from two patient groups revealed differential expression of several gene families including known predictors of metastasis (e.g., matrix metalloproteinases). In addition, we found S100P, S100A2, trypsinogen C (TRY6), and trypsinogen IVb (PRSS3) to be overexpressed in tumors that metastasized during the course of the disease. In a third group of 42 patients, we confirmed the induction of S100 proteins and trypsinogens in metastasizing tumors and its significant correlation with survival by real-time quantitative reverse transcription-PCR. Overexpression of S100A2, S100P, or PRSS3 in NSCLC cell cultures led to increased transendothelial migration, corroborating the role of S100A2, S100P, and PRSS3 in the metastatic process. Taken together, we provide evidence that expression of S100 proteins and trypsinogens is associated with metastasis and predicts survival in early stages of NSCLC. For the first time, this implicates a role of S100 proteins and trypsinogens in the metastatic process of early-stage NSCLC.

Supplementary Table 3 from Epigenetically Deregulated microRNA-375 Is Involved in a Positive Feedback Loop with Estrogen Receptor α in Breast Cancer Cells

Supplementary Methods, Figures 1-2, Tables 1-2 from Epigenetically Deregulated microRNA-375 Is Involved in a Positive Feedback Loop with Estrogen Receptor α in Breast Cancer Cells

XLSX file - 564K, Supplementary Table S1: This table lists 459 genes that show a differential expression upon loss of MALAT1 in A549 lung cancer cells.

Figure, Table and Methods

Expression levels of microRNAs (miRNAs) are globally reduced in cancer compared with matched normal tissues, and miRNA function has recently been implicated in tumorigenesis. To test whether epigenetic silencing contributes to miRNA suppression in tumors, lung cancer cells were treated with inhibitors of DNA methylation or histone deacetylation. No significant alteration in miRNA expression was detected using microarray profiling. To search for tumor-associated mutations that could affect processing and expression of mature miRNAs, a panel of 91 cancer-derived cell lines was analyzed for sequence variations in 15 miRNAs implicated in tumorigenesis by virtue of their known target transcripts (let-7 family targeting oncogenic Ras) or their localization to sites of frequent chromosomal instability (miR-143, miR-145, miR-26a-1, and miR-21). No mutations were detected within any of the short mature miRNA sequences. In addition to previously reported polymorphisms, 1 sequence variant in a precursor miRNA and 15 variants in primary miRNA (pri-miRNA) transcripts were identified. Despite pri-miRNAs having dramatic changes in the predicted secondary folding structure flanking putative cleavage sites, processing and miRNA maturation were not affected in vivo. Thus, genetic variants in miRNA precursors are common in cancer cells but are unlikely to have physiologic significance. (Cancer Res 2006; 66(12): 6097-104)

Supplementary Tables 1-4 from Sequence Variations of MicroRNAs in Human Cancer: Alterations in Predicted Secondary Structure Do Not Affect Processing

Metastatic small cell lung cancer (SCLC) is not curable. While SCLC is initially sensitive to chemotherapy, remissions are short-lived. The relapse is induced by chemotherapy-selected tumor stem cells, which express the AC133 epitope of the CD133 stem cell marker. We studied the effectiveness of AC133-specific CAR T cells post-chemotherapy using human primary SCLC and an orthotopic xenograft mouse model. AC133-specific CAR T cells migrated to SCLC tumor lesions, reduced the tumor burden, and prolonged survival in a humanized orthotopic SCLC model, but were not able to entirely eliminate tumors. We identified CD73 and PD-L1 as immune-escape mechanisms and combined PD-1-inhibition and CD73-inhibition with CAR T cell treatment. This triple-immunotherapy induced cures in 25% of the mice, without signs of graft-versus-host disease or bone marrow failure. AC133+ cancer stem cells and PD-L1+CD73+ myeloid cells were detectable in primary human SCLC tissues, suggesting that patients may benefit from the triple-immunotherapy. We conclude that the combination of AC133-specific CAR T cells, anti-PD-1-antibody and CD73-inhibitor specifically eliminates chemo-resistant tumor stem cells, overcomes SCLC-mediated T cell inhibition, and might induce long-term complete remission in an otherwise incurable disease.

Cell migration is an essential process in health and in disease, including cancer metastasis. A comprehensive inventory of migration factors is nonetheless lacking—in part due to the difficulty in assessing migration using high‐throughput technologies. Hence, there are currently very few screens that systematically reveal factors controlling cell migration. Here, we introduce MigExpress as a platform for the ‘identification of Migration control genes by differential Expression’. MigExpress exploits the combination of in‐depth molecular profiling and the robust quantitative analysis of migration capacity in a broad panel of samples and identifies migration‐associated genes by their differential expression in slow‐ versus fast‐migrating cells. We applied MigExpress to investigate non‐small cell lung cancer (NSCLC), which is the most frequent cause of cancer mortality mainly due to metastasis. In 54 NSCLC cell lines, we comprehensively determined mRNA and protein expression. Correlating the transcriptome and proteome profiles with the quantified migration properties led to the discovery and validation of FLNC, DSE, CPA4, TUBB6, and BICC1 as migration control factors in NSCLC cells, which were also negatively correlated with patient survival. Notably, FLNC was the least expressed filamin in NSCLC, but the only one controlling cell migration and correlating with patient survival and metastatic disease stage. In our study, we present MigExpress as a new method for the systematic analysis of migration factors and provide a comprehensive resource of transcriptomic and proteomic data of NSCLC cell lines related to cell migration., Cell migration is an essential process in development and disease—prominently in cancer metastasis. We present MigExpress, a high‐throughput approach to systematically identify factors governing cell migration by combining migration quantification with molecular profiling. MigExpress on a panel of 54 NSCLC cell lines discovered differentially expressed mRNAs, circRNAs, and proteins between fast‐ and slow‐migrating cells, which controlled migration. ​

RNA–protein complexes have emerged as central players in numerous key cellular processes with significant relevance in health and disease. To further deepen our knowledge of RNA-binding proteins (RBPs), multiple proteome-wide strategies have been developed to identify RBPs in different species leading to a large number of studies contributing experimentally identified as well as predicted RBP candidate catalogs. However, the rapid evolution of the field led to an accumulation of isolated datasets, hampering the access and comparison of their valuable content. Moreover, tools to link RBPs to cellular pathways and functions were lacking. Here, to facilitate the efficient screening of the RBP resources, we provide RBP2GO (https://RBP2GO.DKFZ.de), a comprehensive database of all currently available proteome-wide datasets for RBPs across 13 species from 53 studies including 105 datasets identifying altogether 22 552 RBP candidates. These are combined with the information on RBP interaction partners and on the related biological processes, molecular functions and cellular compartments. RBP2GO offers a user-friendly web interface with an RBP scoring system and powerful advanced search tools allowing forward and reverse searches connecting functions and RBPs to stimulate new research directions., Graphical Abstract Graphical AbstractRBP2GO (https://RBP2GO.DKFZ.de) is a comprehensive database of all currently available proteome-wide datasets for RNA-binding proteins, their functions and interaction partners.Question: is it necessary to have a caption for the graphical abstract? I cannot see them on the website or PDFs of the articles.

Nonstop or stop-loss mutations convert a stop into a sense codon, resulting in translation into the 3′ untranslated region as a nonstop extension mutation to the next in-frame stop codon or as a readthrough mutation into the poly-A tail. Nonstop mutations have been characterized in hereditary diseases, but not in cancer genetics. In a pan-cancer analysis, we curated and analysed 3,412 nonstop mutations from 62 tumour entities, generating a comprehensive database at http://NonStopDB.dkfz.de. Six different nonstop extension mutations affected the tumour suppressor SMAD4, extending its carboxy terminus by 40 amino acids. These caused rapid degradation of the SMAD4 mutants via the ubiquitin–proteasome system. A hydrophobic degron signal sequence of ten amino acids within the carboxy-terminal extension was required to induce complete loss of the SMAD4 protein. Thus, we discovered that nonstop mutations can be functionally important in cancer and characterize their loss-of-function impact on the tumour suppressor SMAD4. Dhamija et al. profile 3,412 nonstop mutations from 62 tumour entities and as proof of concept demonstrate that six such mutations cause degradation and loss of the tumour suppressor SMAD4.

Since lung cancer remains the leading cause of cancer death globally, there is an urgent demand for novel therapeutic targets. We carried out a CRISPR interference (CRISPRi) loss-of-function screen for human lung adenocarcinoma (LUAD) targeting 2098 deregulated genes using a customized algorithm to comprehensively probe the functionality of every resolvable transcriptional start site (TSS). CASP8AP2 was identified as the only hit that significantly affected the viability of all eight screened LUAD cell lines while the viability of non-transformed lung cells was only moderately impacted. Knockdown (KD) of CASP8AP2 induced both autophagy and apoptotic cell death pathways. Systematic expression profiling linked the AP-1 transcription factor to the CASP8AP2 KD-induced cancer cell death. Furthermore, inhibition of AP-1 reverted the CASP8AP2 silencing-induced phenotype. Overall, the tailored CRISPRi screen profiled the impact of over 2000 genes on the survival of eight LUAD cell lines and identified the CASP8AP2 - AP-1 axis mediating lung cancer viability.

Following the concept of RNA dependence and exploiting its application in the R-DeeP screening approach, we have identified RNA-dependent proteins in A549 lung adenocarcinoma cells. RNA-dependent proteins are defined as proteins whose interactome depends on RNA and thus entails RNA-binding proteins (RBPs) as well as proteins in ribonucleoprotein complexes (RNPs) without direct RNA interaction. With this proteome-wide technique based on sucrose density gradient ultracentrifugation and fractionation followed by quantitative mass spectrometry and bioinformatic analysis, we have identified 1189 RNA-dependent proteins including 170 proteins which had never been linked to RNA before. R-DeeP provides quantitative information on the fraction of a protein being RNA-dependent as well as it allows the reconstruction of protein complexes based on co-segregation. The RNA dependence of three newly identified RNA-dependent proteins, DOCK5, ELMO2, also known as CED12A, and ABRAXAS1, also known as CCDC98, was validated using western blot analysis, and the direct RNA interaction was verified by iCLIP2 for the migration-related protein DOCK5 and the mitosis-related protein ABRAXAS1. The R-DeeP 2.0 database provides proteome-wide and cell line-specific information from A549 and HeLa S3 cells on proteins and their RNA dependence to contribute to understanding the functional role of RNA and RNA-binding proteins in cancer cells.

Synonymous mutations have been viewed as silent mutations, since they only affect the DNA and mRNA, but not the amino acid sequence of the resulting protein. Nonetheless, recent studies suggest their significant impact on splicing, RNA stability, RNA folding, translation or co-translational protein folding. Hence, we compile 659194 synonymous mutations found in human cancer and characterize their properties. We provide the user-friendly, comprehensive resource for synonymous mutations in cancer, SynMICdb (http://SynMICdb.dkfz.de), which also contains orthogonal information about gene annotation, recurrence, mutation loads, cancer association, conservation, alternative events, impact on mRNA structure and a SynMICdb score. Notably, synonymous and missense mutations are depleted at the 5'-end of the coding sequence as well as at the ends of internal exons independent of mutational signatures. For patient-derived synonymous mutations in the oncogene KRAS, we indicate that single point mutations can have a relevant impact on expression as well as on mRNA secondary structure., Synonymous mutations do not alter amino acid sequence but may exert oncogenic effects in other ways. Here, the authors present a catalogue of synonymous mutations in cancer and characterise their properties.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Following the publication of the above article, the Editor was notified that an error occurred in which all images were published with incorrect versions. The Editor has taken the decision that the manuscript is no longer acceptable in its current form, nor with a corrigendum, as the extensive changes to the figures and publication would lead to ambiguity for our readers. We have therefore made the decision to retract this manuscript from Cancer Letters with the possibility of resubmission and republication of the manuscript in its corrected form after peer review.

Metastatic small cell lung cancer (SCLC) is not curable. While SCLC is initially sensitive to chemotherapy, remissions are short-lived. The relapse is induced by chemotherapy-selected tumor stem cells, which express the AC133 epitope of the CD133 stem cell marker. We studied the effectiveness of AC133-specific CAR T cells post-chemotherapy using human primary SCLC and an orthotopic xenograft mouse model. AC133-specific CAR T cells migrated to SCLC tumor lesions, reduced the tumor burden, and prolonged survival in a humanized orthotopic SCLC model, but were not able to entirely eliminate tumors. We identified CD73 and PD-L1 as immune-escape mechanisms and combined PD-1-inhibition and CD73-inhibition with CAR T cell treatment. This triple-immunotherapy induced cures in 25% of the mice, without signs of graft-versus-host disease or bone marrow failure. AC133

The heat-shock response is critical for the survival of all organisms. Metastasis-associated long adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA localized in nuclear speckles, but its physiological role remains elusive. Here, we show that heat shock induces translocation of MALAT1 to a distinct nuclear body named the heat shock-inducible noncoding RNA-containing nuclear (HiNoCo) body in mammalian cells. MALAT1-knockout A549 cells showed reduced proliferation after heat shock. The HiNoCo body, which is formed adjacent to nuclear speckles, is distinct from any other known nuclear bodies, including the nuclear stress body, Cajal body, germs, paraspeckles, nucleoli and promyelocytic leukemia body. The formation of HiNoCo body is reversible and independent of heat shock factor 1, the master transcription regulator of the heat-shock response. Our results suggest the HiNoCo body participates in heat shock factor 1-independent heat-shock responses in mammalian cells.

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.

Circular RNAs (circRNAs) play critical roles in a broad spectrum of physiological and pathological processes, including cancer. Here, we provide a comprehensive database&mdash, circ2GO&mdash, systematically linking circRNAs to the functions and processes of their linear counterparts. circ2GO contains 148,811 circular human RNAs originating from 12,251 genes, which we derived from deep transcriptomics after rRNA depletion in a panel of 60 lung cancer and non-transformed cell lines. The broad circRNA expression dataset is mapped to all isoforms of the respective gene. The data are visualized in transcript maps and in heatmaps, to intuitively display a comprehensive portrait for the abundance of circRNAs across transcripts and cell lines. By integrating gene ontology (GO) information for all genes in our dataset, circ2GO builds a connection between circRNAs and their host genes&rsquo, biological functions and molecular mechanisms. Additionally, circ2GO offers target predictions for circRNA&mdash, microRNA (miRNA) pairs for 25,166 highly abundant circRNAs from 6578 genes and 897 high-confidence human miRNAs. Visualization, user-friendliness, intuitive and advanced forward and reverse search options, batch processing and download options make circ2GO a comprehensive source for circRNA information to build hypotheses on their function, processes, and miRNA targets.

The ability of cancer cells to adopt various migration modes (the plasticity of cancer cell invasiveness) is a substantive obstacle in the treatment of metastasis, yet still an incompletely understood process. We performed a comparison of publicly available transcriptomic datasets from various cell types undergoing a switch between the mesenchymal and amoeboid migration modes. Strikingly, lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was one of three genes that were found upregulated in all amoeboid cells analyzed. Accordingly, downregulation of MALAT1 in predominantly amoeboid cell lines A375m2 and A2058 resulted in decrease of active RhoA (Ras homolog family member A) and was accompanied by the amoeboid-mesenchymal transition in A375m2 cells. Moreover, MALAT1 downregulation in amoeboid cells led to increased cell proliferation. Our work is the first to address the role of MALAT1 in MAT/AMT (mesenchymal to amoeboid transition/amoeboid to mesenchymal transition) and suggests that increased MALAT1 expression is a common feature of amoeboid cells.

The class of circular RNA (circRNA) is characterized by head-to-tail bonds between exons formed by backsplicing. Here, we provide a resource of circRNA expression in a comprehensive panel of 60 lung cancer and non-transformed cell lines (FL3C dataset). RNA sequencing after depletion of ribosomal RNA quantified the expression of circRNA and linear RNA. We detected 148,811 circular RNAs quantified by 2.8 million backsplicing reads originating from 12,251 genes. The number of identified circRNAs was markedly higher using rRNA depletion compared to public polyA-enriched RNA-seq datasets. CircRNAs almost never started in the first exon nor ended in the last exon and started more frequently in earlier exons. Most circRNAs showed high cell line specificity and correlated positively with their linear RNA counterpart. Known cancer genes produced more circRNAs than non-cancer genes. Subsets of circRNAs correlated with cell proliferation, histological subtype or genotype. CircTNFRSF21 was translated crossing the backsplice site in two different reading frames. Overexpression of circPVT1, circERBB2, circHIPK3, circCCNB1, circSMAD2, circTNFRSF21 and circKIF5B significantly increased colony formation. In conclusion, our data provide a comprehensive map of circRNA expression in lung cancer cells and global patterns of circRNA production as a useful resource for future research into lung cancer circRNAs.

Although the number of documented noncoding RNAs (ncRNAs) is rapidly increasing, knowledge of their molecular function is lagging behind. The identification of specific RNA motifs that mediate transcript stability, interactions and localization may aid in the prediction of these features in new transcripts and may have potential implications for ncRNA function. Here, we review RNA motifs, focusing on four recent studies identifying nuclear-retention motifs, and discuss the limited specificity of short-RNA motifs and the resulting challenge for effective functional prediction. Future approaches may succeed by integrating combinatorial and cooperative effects of additional partially sequence-based properties.

The identification of viability-associated long noncoding RNAs (lncRNAs) might be a promising rationale for new therapeutic approaches in liver cancer. Here, we applied an RNA interference screening approach in hepatocellular carcinoma (HCC) cell lines to find viability-associated lncRNAs. Among the multiple identified lncRNAs with a significant impact on HCC cell viability, we selected cancer susceptibility 9 (CASC9) due to the strength of its phenotype, expression, and up-regulation in HCC versus normal liver. CASC9 regulated viability across multiple HCC cell lines as shown by clustered regularly interspaced short palindromic repeats interference and single small interfering RNA (siRNA)-mediated and siRNA pool-mediated depletion of CASC9. Further, CASC9 depletion caused an increase in apoptosis and a decrease of proliferation. We identified the RNA binding protein heterogeneous nuclear ribonucleoprotein L (HNRNPL) as a CASC9 interacting protein by RNA affinity purification and validated it by native RNA immunoprecipitation. Knockdown of HNRNPL mimicked the loss-of-viability phenotype observed upon CASC9 depletion. Analysis of the proteome (stable isotope labeling with amino acids in cell culture) of CASC9-depleted and HNRNPL-depleted cells revealed a set of coregulated genes which implied a role of the CASC9:HNRNPL complex in AKT signaling and DNA damage sensing. CASC9 expression levels were elevated in patient-derived tumor samples compared to normal control tissue and had a significant association with overall survival of HCC patients. In a xenograft chicken chorioallantoic membrane model, we measured decreased tumor size after knockdown of CASC9. Conclusion: Taken together, we provide a comprehensive list of viability-associated lncRNAs in HCC; we identified the CASC9:HNRNPL complex as a clinically relevant viability-associated lncRNA/protein complex which affects AKT signaling and DNA damage sensing in HCC.

Targeted modulation of gene expression represents a valuable approach to understand the mechanisms governing gene regulation. In a therapeutic context, it can be exploited to selectively modify the aberrant expression of a disease-causing gene or to provide the target cells with a new function. Here, we have established a novel platform for achieving precision epigenome editing using designer epigenome modifiers (DEMs). DEMs combine in a single molecule a DNA binding domain based on highly specific transcription activator-like effectors (TALEs) and several effector domains capable of inducing DNA methylation and locally altering the chromatin structure to silence target gene expression. We designed DEMs to target two human genes, CCR5 and CXCR4, with the aim of epigenetically silencing their expression in primary human T lymphocytes. We observed robust and sustained target gene silencing associated with reduced chromatin accessibility, increased promoter methylation at the target sites and undetectable changes in global gene expression. Our results demonstrate that DEMs can be successfully used to silence target gene expression in primary human cells with remarkably high specificity, paving the way for the establishment of a potential new class of therapeutics.

Objectives Distant metastasis arising from thyroid cancer is rare but has been associated with significantly reduced long-term survival, especially when refractory to radioactive iodine ablation. We provide one of the largest studies worldwide reporting the outcome after salvage pulmonary metastasectomy for this entity, aiming to identify prognostic factors and to analyse surgical indication. Methods We retrospectively analysed the medical records of 43 patients who had undergone pulmonary metastasectomy for radioactive iodine-refractory thyroid cancer from 1985 to 2016. Results The median follow-up period was 77 (95% confidence interval 41-113) months. Twenty-three (53%) patients were alive at the time of analysis. The majority of tumours were follicular thyroid cancer by histology, with 23% identified as Hurthle cell subtype. Five- and 10-year disease-specific (DS) survival was 84% and 59%, respectively. Thirty-one (72%) patients underwent R0-resection with a 5- and 10-year DS survival of 100% and 77%, respectively. This was significantly reduced to 62% and 22% (P = 0.013) in case of incomplete resection, respectively. Ten years after R0-metastasectomy, 17 (55%) patients were recurrence-free. Systematic mediastinal lymphadenectomy was performed in 16 (37%) patients and was associated with improved long-term DS survival (10 years 88% vs 46%, P = 0.034). Moreover, a reduction of > 80% in serum thyroglobulin levels post-metastasectomy correlates with better long-term DS survival (10 years 81% vs 36%, P = 0.007). Conclusions Pulmonary metastasectomy is associated with good survival for selected patients with radioactive iodine-refractory metastases of differentiated thyroid cancer, especially if R0-resection can be achieved. Moreover, it is worth considering whether a significant reduction of tumour load, as indicated by thyroglobulin serum levels, seems possible.

As a genetic disease, cancer is caused by the activation of oncogenes and the inhibition of tumor suppressor genes via genetic and epigenetic mechanisms. Given the important role of energy metabolism in tumors, we analyzed the cancer-derived mutations occurring in the DNA of the mitochondrion. Mutations in the mitochondrial DNA (mtDNA) compared to nuclear DNA are 62% decreased relative to the coding length per chromosome. We find that the majority of these mutations affects highly conserved nucleotides - significantly exceeding the conservation of the mtDNA - and are devoid of single nucleotide polymorphisms (SNPs). Surprisingly, the leading resources for tumor genetics information universally use the standard genetic code for translation of nucleotide into amino acid sequences in their online resources. However, the nuclear and mitochondrial genetic codes differ for four codons and the usage of incomplete STOP codons. Hence, we analyze and curate the consequences for all mutations in the mtDNA and comprehensively reclassify missense, nonsense and synonymous mutations accordingly. In total, 10% of the mutations are incorrectly translated leading to significant changes in the distribution of mutation types with tripling of nonsense and 69% loss of nonstop extension mutations. Lastly, we provide a curated dataset of coding and non-coding mitochondrial mutations in cancer merged, standardized, duplicate-free and aggregated from two databases as a resource including orthogonal data on their high conservation and SNPs. This study generally highlights the need to universally regard the important differences between the standard and mitochondrial genetic code in life science research.