RESUMO
Mismatch repair (MMR) deficiencies are a hallmark of various cancers causing accumulation of DNA mutations and mismatches, which often results in chemotherapy resistance. Metalloinsertor complexes, including [Rh(chrysi)(phen)(PPO)]Cl2 (Rh-PPO), specifically target DNA mismatches and selectively induce cytotoxicity within MMR-deficient cells. Here, we present an in vivo analysis of Rh-PPO, our most potent metalloinsertor. Studies with HCT116 xenograft tumors revealed a 25% reduction in tumor volume and 12% increase in survival with metalloinsertor treatment (1 mg/kg; nine intraperitoneal doses over 20 d). When compared to oxaliplatin, Rh-PPO displays ninefold higher potency at tumor sites. Pharmacokinetic studies revealed rapid absorption of Rh-PPO in plasma with notable accumulation in the liver compared to tumors. Additionally, intratumoral metalloinsertor administration resulted in enhanced anticancer effects, pointing to a need for more selective delivery methods. Overall, these data show that Rh-PPO inhibits xenograft tumor growth, supporting the strategy of using Rh-PPO as a chemotherapeutic targeted to MMR-deficient cancers.
Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Ródio , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacocinética , Pareamento Incorreto de Bases/efeitos dos fármacos , Complexos de Coordenação/administração & dosagem , Complexos de Coordenação/química , Complexos de Coordenação/farmacocinética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Células HCT116 , Humanos , Camundongos , Estrutura Molecular , Ródio/química , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In 1943, Luria and Delbrück used a phage-resistance assay to establish spontaneous mutation as a driving force of microbial diversity. Mutation rates are still studied using such assays, but these can only be used to examine the small minority of mutations conferring survival in a particular condition. Newer approaches, such as long-term evolution followed by whole-genome sequencing, may be skewed by mutational 'hot' or 'cold' spots. Both approaches are affected by numerous caveats. Here we devise a method, maximum-depth sequencing (MDS), to detect extremely rare variants in a population of cells through error-corrected, high-throughput sequencing. We directly measure locus-specific mutation rates in Escherichia coli and show that they vary across the genome by at least an order of magnitude. Our data suggest that certain types of nucleotide misincorporation occur 10(4)-fold more frequently than the basal rate of mutations, but are repaired in vivo. Our data also suggest specific mechanisms of antibiotic-induced mutagenesis, including downregulation of mismatch repair via oxidative stress, transcriptionreplication conflicts, and, in the case of fluoroquinolones, direct damage to DNA.
Assuntos
Escherichia coli/genética , Evolução Molecular , Variação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutagênese/genética , Taxa de Mutação , Antibacterianos/farmacologia , Dano ao DNA/genética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Replicação do DNA/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Fluoroquinolonas/farmacologia , Loci Gênicos/efeitos dos fármacos , Loci Gênicos/genética , Variação Genética/efeitos dos fármacos , Genoma Bacteriano/efeitos dos fármacos , Genoma Bacteriano/genética , Mutação INDEL/genética , Mutagênese/efeitos dos fármacos , Nucleotídeos/genética , Nucleotídeos/metabolismo , Estresse Oxidativo/genética , Transcrição Gênica/genéticaRESUMO
Transcriptional regulation of DNA repair is of outmost importance for the restoration of DNA integrity upon genotoxic stress. Here we report that the potent environmental carcinogen benzo[a]pyrene (B[a]P) activates a cellular DNA damage response resulting in transcriptional repression of mismatch repair (MMR) genes (MSH2, MSH6, EXO1) and of RAD51, the central homologous recombination repair (HR) component, ultimately leading to downregulation of MMR and HR. B[a]P-induced gene repression is caused by abrogated E2F1 signalling. This occurs through proteasomal degradation of E2F1 in G2-arrested cells and downregulation of E2F1 mRNA expression in G1-arrested cells. Repression of E2F1-mediated transcription and silencing of repair genes is further mediated by the p21-dependent E2F4/DREAM complex. Notably, repression of DNA repair is also observed following exposure to the active B[a]P metabolite BPDE and upon ionizing radiation and occurs in response to a p53/p21-triggered, irreversible cell cycle arrest marking the onset of cellular senescence. Overall, our results suggest that repression of MMR and HR is an early event during genotoxic-stress induced senescence. We propose that persistent downregulation of DNA repair might play a role in the maintenance of the senescence phenotype, which is associated with an accumulation of unrepairable DNA lesions.
Assuntos
Benzo(a)pireno/toxicidade , Carcinógenos/toxicidade , Senescência Celular/genética , DNA/genética , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F4/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , DNA/metabolismo , Dano ao DNA , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos da radiação , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F4/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Raios gama , Humanos , Proteínas Interatuantes com Canais de Kv/genética , Proteínas Interatuantes com Canais de Kv/metabolismo , Células MCF-7 , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação/efeitos dos fármacos , Reparo de DNA por Recombinação/efeitos da radiação , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de SinaisRESUMO
Cancers with microsatellite instability (MSI), which include ≤20% of solid tumors, are characterized by resistance to chemotherapy due to deficiency in the DNA mismatch repair (MMR) pathway. Rhodium metalloinsertors make up a class of compounds that bind DNA mismatches with high specificity and show selective cytotoxicity in MSI cancer cells. We determined that rhodium complexes with an Nâ§O coordination showed significantly increased cell potency compared with that of Nâ§N-coordinated compounds, and we identified [Rh(chrysi)(phen)(PPO)]2+ (RhPPO) as the most potent, selective compound in this class. Using matched cell lines that are MMR-deficient (HCT116O) and MMR-proficient (HCT116N), we demonstrated that RhPPO preferentially activates the DNA damage response and inhibits DNA replication and cell proliferation in HCT116O cells, leading to cell death by necrosis. Using a fluorescent conjugate of RhPPO, we established that the metalloinsertor localizes to DNA mismatches in the cell nucleus and causes DNA double-strand breaks at or near the mismatch sites. Evaluation of RhPPO across MMR-deficient and MMR-proficient cell lines confirmed the broad potential for RhPPO to target MSI cancers, with cell potency significantly higher than that of platinum complexes used broadly as chemotherapeutics. Moreover, in a mouse xenograft model of MSI cancer, RhPPO shows promising antitumor activity and increased survival. Thus, our studies indicate that RhPPO is a novel DNA-targeted therapy with improved potency and selectivity over standard-of-care platinum-based chemotherapy and, importantly, that DNA mismatches offer a critical new target in the design of chemotherapeutics for MSI cancers.
Assuntos
Antineoplásicos/uso terapêutico , Complexos de Coordenação/uso terapêutico , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , DNA/metabolismo , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Complexos de Coordenação/farmacologia , Humanos , Estrutura Molecular , Necrose/induzido quimicamente , Ródio/químicaRESUMO
BACKGROUND: The advances in colorectal cancer (CRC) treatment include the identification of deficiencies in Mismatch Repair (MMR) pathway to predict the benefit of adjuvant 5-fluorouracil (5-FU) and oxaliplatin for stage II CRC and immunotherapy. Defective MMR contributes to chemoresistance in CRC. A growing body of evidence supports the role of Poly-(ADP-ribose) polymerase (PARP) inhibitors, such as Olaparib, in the treatment of different subsets of cancer beyond the tumors with homologous recombination deficiencies. In this work we evaluated the effect of Olaparib on 5-FU cytotoxicity in MMR-deficient and proficient CRC cells and the mechanisms involved. METHODS: Human colon cancer cell lines, proficient (HT29) and deficient (HCT116) in MMR, were treated with 5-FU and Olaparib. Cytotoxicity was assessed by MTT and clonogenic assays, apoptosis induction and cell cycle progression by flow cytometry, DNA damage by comet assay. Adhesion and transwell migration assays were also performed. RESULTS: Our results showed enhancement of the 5-FU citotoxicity by Olaparib in MMR-deficient HCT116 colon cancer cells. Moreover, the combined treatment with Olaparib and 5-FU induced G2/M arrest, apoptosis and polyploidy in these cells. In MMR proficient HT29 cells, the Olaparib alone reduced clonogenic survival, induced DNA damage accumulation and decreased the adhesion and migration capacities. CONCLUSION: Our results suggest benefits of Olaparib inclusion in CRC treatment, as combination with 5-FU for MMR deficient CRC and as monotherapy for MMR proficient CRC. Thus, combined therapy with Olaparib could be a strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC.
Assuntos
Antineoplásicos/farmacologia , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Fluoruracila/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/genética , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Sinergismo Farmacológico , Células HCT116 , HumanosRESUMO
Evolutionary processes are driven by diverse molecular mechanisms that act in the creation and prevention of mutations. It remains unclear how these mechanisms are regulated because limitations of existing mutation assays have precluded measuring how mutation rates vary over time in single cells. Toward this goal, I detected nascent DNA mismatches as a proxy for mutagenesis and simultaneously followed gene expression dynamics in single Escherichia coli cells using microfluidics. This general microscopy-based approach revealed the real-time dynamics of mutagenesis in response to DNA alkylation damage and antibiotic treatments. It also enabled relating the creation of DNA mismatches to the chronology of the underlying molecular processes. By avoiding population averaging, I discovered cell-to-cell variation in mutagenesis that correlated with heterogeneity in the expression of alternative responses to DNA damage. Pulses of mutagenesis are shown to arise from transient DNA repair deficiency. Constitutive expression of DNA repair pathways and induction of damage tolerance by the SOS response compensate for delays in the activation of inducible DNA repair mechanisms, together providing robustness against the toxic and mutagenic effects of DNA alkylation damage.
Assuntos
Antibacterianos/farmacologia , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , DNA Bacteriano , Escherichia coli , Técnicas Analíticas Microfluídicas , Mutagênese/efeitos dos fármacos , Resposta SOS em Genética/efeitos dos fármacos , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/citologia , Escherichia coli/genética , Escherichia coli/metabolismoRESUMO
BACKGROUND: Colorectal cancer (CRC) is one of the most common cancers worldwide. One of its subtypes is associated with defective mismatch repair (dMMR) genes. Saffron has many potentially protective roles against colon malignancy. However, these roles in the context of dMMR tumors have not been explored. In this study, we aimed to investigate the effects of saffron and its constituents in CRC cell lines with dMMR. METHODS: Saffron crude extracts and specific compounds (safranal and crocin) were used in the human colorectal cancer cell lines HCT116, HCT116+3 (inserted MLH1), HCT116+5 (inserted MSH3), and HCT116+3+5 (inserted MLH1 and MSH3). CDC25b, p-H2AX, TPDP1, and GAPDH were analyzed by Western blot. Proliferation and cytotoxicity were analyzed by MTT. The scratch wound assay was also performed. RESULTS: Saffron crude extracts restricted (up to 70%) the proliferation in colon cells with deficient MMR (HCT116) compared to proficient MMR. The wound healing assay indicates that deficient MMR cells are doing better (up to 90%) than proficient MMR cells when treated with saffron. CDC25b and TDP1 downregulated (up to 20-fold) in proficient MMR cells compared to deficient MMR cells, while p.H2AX was significantly upregulated in both cell types, particularly at >10 mg/mL saffron in a concentration-dependent manner. The reduction in cellular proliferation was accompanied with upregulation of caspase 3 and 7. The major active saffron compounds, safranal and crocin reproduced most of the saffron crude extracts' effects. CONCLUSIONS: Saffron's anti-proliferative effect is significant in cells with deficient MMR. This novel effect may have therapeutic implications and benefits for MSI CRC patients who are generally not recommended for the 5-fluorouracil-based treatment.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Crocus/química , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Instabilidade de Microssatélites/efeitos dos fármacos , Extratos Vegetais/farmacologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Células HCT116 , Humanos , Extratos Vegetais/químicaRESUMO
Up to 20% of solid tumors are characterized by DNA mismatch repair (MMR) deficiency and microsatellite instability that confer resistance to standard of care chemotherapy. MMR-deficient cancers have an increased mutation rate, and DNA mismatches accumulate as part of these cancers. We previously described a class of compounds, rhodium metalloinsertors, that bind DNA mismatches with high specificity and selectivity and have potential as targeted therapy. [Rh(chrysi)(phen)(PPO)]2+ (RhPPO) is the most potent, selective compound in this class and acts by targeting DNA mismatches, resulting in preferential cytotoxicity to MMR-deficient cancers. To explore further the cellular mechanism of action of RhPPO, we conjugated the metal complex to a fluorescent probe, cyanine 3 (Cy3). RhPPO-Cy3 binds DNA mismatches and retains the selectivity and potent cytotoxic activity of RhPPO for MMR-deficient cell lines. RhPPO-Cy3 forms discrete foci in the cell nucleus that overlap with sites of DNA damage, suggesting that the lesions occur at or near DNA mismatch sites. RhPPO-Cy3 foci persist over time, despite initial processing of the lesion and recruitment of repair proteins, consistent with the idea that the complex binding to a mismatch prevents repair. RhPPO-Cy3 binding does not lead to activation of p53 and the apoptotic pathway. Together, these findings support the idea that RhPPO-Cy3 binding leads to irreversible DNA damage at DNA mismatches that enables selective cytotoxicity to MMR-deficient cells.
Assuntos
Antineoplásicos/farmacologia , Carbocianinas/farmacologia , Complexos de Coordenação/farmacologia , Dano ao DNA , Corantes Fluorescentes/farmacologia , Ródio/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Carbocianinas/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Células HCT116 , Humanos , Estrutura Molecular , Imagem Óptica , Ródio/químicaRESUMO
Hexavalent chromium is recognized as a human carcinogen. Our previous studies revealed that lung cancer (LC) in chromate-exposed workers (chromate LC) had molecular features of frequent microsatellite instability (MSI), repression of MLH1 level, and aberrant DNA methylation of several tumor-suppressor genes, including MLH1. In the present study, we quantitatively investigated MLH1-promoter methylation status using bisulfite pyrosequencing of paired tumorous/nontumorous tissues from chromate and nonchromate LCs to determine the effect of chromate exposure on MLH1-promoter methylation. The methylation level of MLH1 promoter was significantly higher in chromate LC tumors (P < .001) than nonchromate LC tumors and, among chromate LC, significantly higher in tumorous tissue than nontumorous tissue (P = .004). Moreover, the methylation level of MLH1 promoter in normal lung tissue tended to be higher in chromate LC than nonchromate LC (P = .062). In addition, LC with reduced levels of MLH1 showed significantly higher methylation levels of MLH1 promoter than LC exhibiting normal MLH1 levels (P = .019). Moreover, immunohistochemical analyses determined that levels of SUV39H1, an H3K9me2-related methyltransferase, were higher in chromate LC than nonchromate LC (P = .076). Furthermore, we evaluated three DNA double-strand break-repair genes (MRE11, RAD50, and DNA-PKcs) as possible targets of MSI by fragment-length polymorphism analysis, revealing the mutation frequency of RAD50 as significantly higher in chromate LC than nonchromate LC (P = .047). These results suggest that chromate exposure might induce MLH1 hypermethylation in LC as a mechanism of chromate-induced carcinogenesis.
Assuntos
Cromatos/efeitos adversos , Metilação de DNA/efeitos dos fármacos , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Proteína 1 Homóloga a MutL/genética , Idoso , Carcinogênese/induzido quimicamente , Carcinogênese/genética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Humanos , Instabilidade de Microssatélites/efeitos dos fármacos , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
Mismatch repair (MMR) systems play important roles in maintaining the high fidelity of genomic DNA. It is well documented that a lack of MMR increases the mutation rate, including base exchanges and small insertion/deletion loops; however, it is unknown whether MMR deficiency affects the frequency of chromosomal recombination in somatic cells. To investigate the effects of MMR on chromosomal recombination, we used the Drosophila wing-spot test, which efficiently detects chromosomal recombination. We prepared MMR (MutS)-deficient flies (spel1(-/-)) using a fly line generated in this study. The spontaneous mutation rate as measured by the wing-spot test was slightly higher in MutS-deficient flies than in wild-type (spel1(+/-)) flies. Previously, we showed that N-nitrosodimethylamine (NDMA)-induced chromosomal recombination more frequently than N-nitrosodiethylamine (NDEA) in Drosophila. When the wing-spot test was performed using MMR-deficient flies, unexpectedly, the rate of NDMA-induced mutation was significantly lower in spel1(-/-) flies than in spel1(+/-) flies. In contrast, the rate of mutation induced by NDEA was higher in spel1(-/-) flies than in spel1(+/-) flies. These results suggest that in Drosophila, the MutS homologue protein recognises methylated DNA lesions more efficiently than ethylated ones, and that MMR might facilitate mutational chromosomal recombination due to DNA double-strand breaks via the futile cycle induced by MutS recognition of methylated lesions.
Assuntos
Aberrações Cromossômicas/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Drosophila melanogaster/genética , Recombinação Genética/efeitos dos fármacos , Animais , Cromossomos/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Reparo do DNA/efeitos dos fármacos , Dietilnitrosamina/farmacologia , Dimetilnitrosamina/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Mutagênese/efeitos dos fármacos , Recombinação Genética/genéticaRESUMO
The DNA mismatch repair (MMR) pathway recognizes and repairs errors in base pairing and acts to maintain genome stability. Cancers that have lost MMR function are common and comprise an important clinical subtype that is resistant to many standard of care chemotherapeutics such as cisplatin. We have identified a family of rhodium metalloinsertors that bind DNA mismatches with high specificity and are preferentially cytotoxic to MMR-deficient cells. Here, we characterize the cellular mechanism of action of the most potent and selective complex in this family, [Rh(chrysi)(phen)(PPO)]2+ (Rh-PPO). We find that Rh-PPO binding induces a lesion that triggers the DNA damage response (DDR). DDR activation results in cell-cycle blockade and inhibition of DNA replication and transcription. Significantly, the lesion induced by Rh-PPO is not repaired in MMR-deficient cells, resulting in selective cytotoxicity. The Rh-PPO mechanism is reminiscent of DNA repair enzymes that displace mismatched bases, and is differentiated from other DNA-targeted chemotherapeutics such as cisplatin by its potency, cellular mechanism, and selectivity for MMR-deficient cells.
Assuntos
Ciclo Celular/efeitos dos fármacos , Complexos de Coordenação/toxicidade , Citotoxinas/toxicidade , Dano ao DNA , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , DNA/metabolismo , Ródio/toxicidade , Linhagem Celular Tumoral , Complexos de Coordenação/química , Citotoxinas/química , Humanos , Ródio/químicaRESUMO
Colorectal cancer (CRC) is a common and lethal disease with a high therapeutic need. For most patients with metastatic CRC, chemotherapy is the only viable option. Currently, immunotherapy is restricted to the particular genetic subgroup of mismatch-repair deficient (MMRd)/microsatellite instable (MSI) CRC. Anti-PD1 therapy was recently FDA-approved as a second-line treatment in this subgroup. However, in a metastatic setting, these MMRd/MSI tumors are vastly outnumbered by mismatch-repair proficient (MMRp)/microsatellite stable (MSS) tumors. These MMRp/MSS tumors do not meaningfully respond to any traditional immunotherapy approach including checkpoint blockade, adoptive cell transfer and vaccination. This resistance to immunotherapy is due to a complex tumor microenvironment that counteracts antitumor immunity through a combination of poorly antigenic tumor cells and an immunosuppressive tumor microenvironment. To find ways of overcoming immunotherapy resistance in the majority of CRC patients, it is necessary to analyze the immunological makeup in an in-depth and personalized way and in the context of their tumor genetic makeup. Flexible, biomarker-guided early-phase immunotherapy trials are needed to optimize this workflow. In this review, we detail key mechanisms for immune evasion and emerging immune biomarkers for personalized immunotherapy in CRC. Also, we present a template for biomarker-guided clinical trials that are needed to move new immunotherapy approaches closer to clinical application.
Assuntos
Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Animais , Antígenos de Neoplasias/imunologia , Neoplasias Colorretais/imunologia , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Reparo de Erro de Pareamento de DNA/imunologia , Genômica/métodos , Humanos , Imunoterapia/métodos , Instabilidade de Microssatélites/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
BACKGROUND: Microsatellite instable/deficient mismatch repair (MSI/dMMR) metastatic colorectal cancers have been reported to have a poor prognosis. Frequent co-occurrence of MSI/dMMR and BRAFV600E complicates the association. PATIENTS AND METHODS: Patients with resected stage III colon cancer (CC) from seven adjuvant studies with available data for disease recurrence and MMR and BRAFV600E status were analyzed. The primary end point was survival after recurrence (SAR). Associations of markers with SAR were analyzed using Cox proportional hazards models adjusted for age, gender, performance status, T stage, N stage, primary tumor location, grade, KRAS status, and timing of recurrence. RESULTS: Among 2630 patients with cancer recurrence (1491 men [56.7%], mean age, 58.5 [19-85] years), multivariable analysis revealed that patients with MSI/dMMR tumors had significantly longer SAR than did patients with microsatellite stable/proficient MMR tumors (MSS/pMMR) (adjusted hazard ratio [aHR], 0.82; 95% CI [confidence interval], 0.69-0.98; P = 0.029). This finding remained when looking at patients treated with standard oxaliplatin-based adjuvant chemotherapy regimens only (aHR, 0.76; 95% CI, 0.58-1.00; P = 0.048). Same trends for SAR were observed when analyzing MSI/dMMR versus MSS/pMMR tumor subgroups lacking BRAFV600E (aHR, 0.84; P = 0.10) or those harboring BRAFV600E (aHR, 0.88; P = 0.43), without reaching statistical significance. Furthermore, SAR was significantly shorter in tumors with BRAFV600E versus those lacking this mutation (aHR, 2.06; 95% CI, 1.73-2.46; P < 0.0001), even in the subgroup of MSI/dMMR tumors (aHR, 2.65; 95% CI, 1.67-4.21; P < 0.0001). Other factors associated with a shorter SAR were as follows: older age, male gender, T4/N2, proximal primary tumor location, poorly differentiated adenocarcinoma, and early recurrence. CONCLUSIONS: In stage III CC patients recurring after adjuvant chemotherapy, and before the era of immunotherapy, the MSI/dMMR phenotype was associated with a better SAR compared with MSS/pMMR. BRAFV600E mutation was a poor prognostic factor for both MSI/dMMR and MSS/pMMR patients. TRIAL IDENTIFICATION NUMBERS: NCT00079274, NCT00265811, NCT00004931, NCT00004931, NCT00026273, NCT00096278, NCT00112918.
Assuntos
Quimioterapia Adjuvante , Neoplasias do Colo/tratamento farmacológico , Instabilidade de Microssatélites/efeitos dos fármacos , Recidiva Local de Neoplasia/tratamento farmacológico , Prognóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Intervalo Livre de Doença , Feminino , Fluoruracila/administração & dosagem , Humanos , Leucovorina/administração & dosagem , Masculino , Pessoa de Meia-Idade , Mutação/genética , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Resultado do Tratamento , Adulto JovemRESUMO
Defects in DNA mismatch repair (MMR) are commonly found in various cancers, especially in colorectal cancers. Despite the high prevalence of MMR-deficient cancers, mismatch-targeted therapeutics are limited and diagnostic tools are indirect. Here, we examine the cytotoxic properties of a rhodium metalloinsertor, [Rh(phen)(chrysi)(PPO)]2+ (RhPPO) in 27 diverse colorectal cancer cell lines. Despite the low frequency of genomic mismatches and the non-covalent nature of the RhPPO-DNA lesion, RhPPO is on average five times more potent than cisplatin. Importantly, the biological target and profile for RhPPO differs from that of cisplatin. A fluorescent metalloinsertor, RhCy3, was used to demonstrate that the cellular target of RhPPO is the DNA mismatch. RhCy3 represents a direct probe for MMR-deficiency and correlates directly with the cytotoxicity of RhPPO across different cell lines. Overall, our studies clearly indicate that RhPPO and RhCy3 are promising anticancer and diagnostic probes for MMR-deficient cancers, respectively.
Assuntos
Antineoplásicos/farmacologia , Pareamento Incorreto de Bases/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Compostos Organometálicos/farmacologia , Ródio/farmacologia , Antineoplásicos/química , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Humanos , Simulação de Acoplamento Molecular , Compostos Organometálicos/química , Ródio/químicaRESUMO
Despite advances in the treatment of castration-resistant prostate cancer (CRPC), options remain limited and non-curative; thus, prostate cancer remains one of the deadliest cancers in men. The discovery of novel therapeutic targets is needed to improve outcomes for men with metastatic CRPC. Precision/personalized medicine creates new opportunities to discover these targets. With an increase in the use of next-generation sequencing and tumor profiling, potentially clinically relevant tumor mutations are being identified. Here, we review the current use of and future direction for genetic testing and tumor profiling in patients with metastatic CRPC.
Assuntos
Antineoplásicos Imunológicos/uso terapêutico , Medicina de Precisão/métodos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/patologia , Anticorpos Monoclonais Humanizados/uso terapêutico , Ensaios Clínicos como Assunto , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Humanos , Masculino , Instabilidade de Microssatélites/efeitos dos fármacos , Gradação de Tumores , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Poli Adenosina Difosfato Ribose/antagonistas & inibidores , Neoplasias de Próstata Resistentes à Castração/genéticaRESUMO
A common strategy to overcome acquired chemotherapy resistance is the combination of a specific anticancer drug (e.g., topoisomerase I inhibitor irinotecan) together with a putative sensitizer. The purpose of this study was to analyze the cytostatic/cytotoxic response of colorectal carcinoma (CRC) cells to irinotecan, depending on the mismatch repair (MMR) and p53 status and to examine the impact of BV6, a bivalent antagonist of inhibitors of apoptosis c-IAP1/c-IAP2, alone or combined with irinotecan. Therefore, several MSH2- or MSH6-deficient cell lines were complemented for MMR deficiency, or MSH6 was knocked out/down in MMR-proficient cells. Upon irinotecan, MMR-deficient/p53-mutated lines repaired DNA double-strand breaks by homologous recombination less efficiently than MMR-proficient/p53-mutated lines and underwent elevated caspase-9-dependent apoptosis. Opposite, BV6-mediated sensitization was achieved only in MMR-proficient/p53-mutated cells. In those cells, c-IAP1 and c-IAP2 were effectively degraded by BV6, caspase-8 was fully activated, and both canonical and non-canonical NF-κB signaling were triggered. The results were confirmed ex vivo in tumor organoids from CRC patients. Therefore, the particular MMR+/p53mt signature, often found in non-metastasizing (stage II) CRC might be used as a prognostic factor for an adjuvant therapy using low-dose irinotecan combined with a bivalent IAP antagonist.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Reparo de Erro de Pareamento de DNA/genética , Irinotecano/farmacologia , Oligopeptídeos/farmacologia , Proteína Supressora de Tumor p53/genética , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Proteína 3 com Repetições IAP de Baculovírus/antagonistas & inibidores , Proteína 3 com Repetições IAP de Baculovírus/metabolismo , Caspase 8/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/metabolismo , Irinotecano/administração & dosagem , Proteína 2 Homóloga a MutS/genética , Oligopeptídeos/administração & dosagem , Tioléster Hidrolases/metabolismo , Inibidores da Topoisomerase I/farmacologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Dedifferentiated endometrial carcinoma (DDEC) is defined as an undifferentiated carcinoma admixed with differentiated endometrioid carcinoma (Grade 1 or 2). It has poor prognosis compared with Grade 3 endometrioid adenocarcinoma and is often associated with the loss of mismatch repair (MMR) proteins, which is seen in microsatellite instability (MSI)-type endometrial cancer. Recent studies have shown that the effectiveness of immune checkpoint inhibitor therapy is related to MMR deficiency; therefore, we analyzed the immunophenotype (MMR deficient and expression of PD-L1) of 17 DDEC cases. In the undifferentiated component, nine cases (53%) were deficient in MMR proteins and nine cases (53%) expressed PD-L1. PD-L1 expression was significantly associated with MMR deficiency (p = 0.026). In addition, the presence of tumor-infiltrating lymphocytes (CD8+) was significantly associated with MMR deficiency (p = 0.026). In contrast, none of the cases showed PD-L1 expression in the well-differentiated component. Our results show that DDEC could be a target for immune checkpoint inhibitors (anti PD-L1/PD-1 antibodies), especially in the undifferentiated component. As a treatment strategy for DDEC, conventional paclitaxel plus carboplatin and cisplatin plus doxorubicin therapies are effective for those with the well-differentiated component. However, by using immune checkpoint inhibitors in combination with other conventional treatments, it may be possible to control the undifferentiated component and improve prognosis.
Assuntos
Anticorpos Neutralizantes/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Endometrioide/tratamento farmacológico , Carcinoma/tratamento farmacológico , Neoplasias do Endométrio/tratamento farmacológico , Idoso , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Antígeno B7-H1/imunologia , Carboplatina/uso terapêutico , Carcinoma/genética , Carcinoma/imunologia , Carcinoma/patologia , Carcinoma Endometrioide/genética , Carcinoma Endometrioide/imunologia , Carcinoma Endometrioide/patologia , Cisplatino/uso terapêutico , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Doxorrubicina/uso terapêutico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/imunologia , Neoplasias do Endométrio/patologia , Feminino , Expressão Gênica , Humanos , Imunofenotipagem , Linfócitos do Interstício Tumoral , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Paclitaxel/uso terapêutico , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/genética , Receptor de Morte Celular Programada 1/imunologiaRESUMO
Rhodium metalloinsertors are a unique set of metal complexes that bind specifically to DNA base pair mismatches in vitro and kill mismatch repair (MMR)-deficient cells at lower concentrations than their MMR-proficient counterparts. A family of metalloinsertors containing rhodium-oxygen ligand coordination, termed "Rh-O" metalloinsertors, has been prepared and shown to have a significant increase in both overall potency and selectivity toward MMR-deficient cells regardless of structural changes in the ancillary ligands. Here we describe DNA-binding and cellular studies with the second generation of Rh-O metalloinsertors in which an ancillary ligand is varied in both steric bulk and lipophilicity. These complexes, of the form [Rh(L)(chrysi)(PPO)]2+, all include the O-containing PPO ligand (PPO = 2-(pyridine-2-yl)propan-2-ol) and the aromatic inserting ligand chrysi (5,6-chrysene quinone diimine) but differ in the identity of their ancillary ligand L, where L is a phenanthroline or bipyridyl derivative. The Rh-O metalloinsertors in this family all show micromolar binding affinities for a 29-mer DNA hairpin containing a single CC mismatch. The complexes display comparable lipophilic tendencies and p Ka values of 8.1-9.1 for dissociation of an imine proton on the chrysi ligand. In cellular proliferation and cytotoxicity assays with MMR-deficient cells (HCT116O) and MMR-proficient cells (HCT116N), the complexes containing the phenanthroline-derived ligands show highly selective cytotoxic preference for the MMR-deficient cells at nanomolar concentrations. Using mass spectral analyses, it is shown that the complexes are taken into cells through a passive mechanism and exhibit low accumulation in mitochondria, an off-target organelle that, when targeted by parent metalloinsertors, can lead to nonselective cytotoxicity. Overall, these Rh-O metalloinsertors have distinct and improved behavior compared to previous generations of parent metalloinsertors, making them ideal candidates for further therapeutic assessment.
Assuntos
Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Ródio/farmacologia , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Complexos de Coordenação/química , DNA/genética , DNA/metabolismo , Células HCT116 , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Ródio/químicaRESUMO
Sarcomas are a rare group of tumors of mesenchymal origin. Metastatic sarcomas are often difficult to treat and unresponsive to standard radio- and chemotherapy, resulting in a poor survival rate for patients. Novel treatments with immune checkpoint inhibitors have been proven to prolong survival of patients with a variety of cancers, including metastatic melanoma, lung, and renal cell carcinoma. Since immune checkpoint inhibitors could provide a novel treatment option for patients with sarcomas, clinical trials investigating their efficacy in these group of tumors are ongoing. However, the discrimination of patients that are the most likely to respond to these treatments is still an obstacle in the design of clinical trials. In this review, we provide a brief overview of the mechanisms of action of immune checkpoint inhibitors and discuss the proposed biomarkers of therapy response, such as lymphocytic infiltration, intratumoral PD-L1 expression, and mutational load in sarcomas.
Assuntos
Antineoplásicos/uso terapêutico , Receptores Coestimuladores e Inibidores de Linfócitos T/antagonistas & inibidores , Drogas em Investigação/uso terapêutico , Imunoterapia , Sarcoma/tratamento farmacológico , Animais , Anticorpos Bloqueadores/efeitos adversos , Anticorpos Bloqueadores/farmacologia , Anticorpos Bloqueadores/uso terapêutico , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Pesquisa Biomédica/métodos , Pesquisa Biomédica/tendências , Quimiotaxia de Leucócito/efeitos dos fármacos , Receptores Coestimuladores e Inibidores de Linfócitos T/metabolismo , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Drogas em Investigação/efeitos adversos , Drogas em Investigação/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoterapia/efeitos adversos , Imunoterapia/tendências , Ativação Linfocitária/efeitos dos fármacos , Sarcoma/imunologia , Sarcoma/metabolismo , Sarcoma/secundário , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
INTRODUCTION: Arsenic exposure and its adverse health outcome, including the association with cancer risk are well established from several studies across the globe. The present study aims to analyze the epigenetic regulation of key mismatch repair (MMR) genes in the arsenic-exposed population. METHOD: A case-control study was conducted involving two hundred twenty four (N=224) arsenic exposed [with skin lesion (WSL=110) and without skin lesion (WOSL=114)] and one hundred and two (N=102) unexposed individuals. The methylation status of key MMR genes i.e. MLH1, MSH2, and PMS2 were analyzed using methylation-specific PCR (MSP). The gene expression was studied by qRTPCR. The expression of H3K36me3, which was earlier reported to be an important regulator of MMR pathway, was assessed using ELISA. RESULTS: Arsenic-exposed individuals showed significant promoter hypermethylation (p < 0.0001) of MLH1 and MSH2 compared to those unexposed with consequent down-regulation in their gene expression [MLH1 (p=0.001) and MSH2 (p<0.05)]. However, no significant association was found in expression and methylation of PMS2 with arsenic exposure. We found significant down-regulation of H3K36me3 in the arsenic-exposed group, most significantly in the WSL group (p<0.0001). The expression of SETD2, the methyltransferase of an H3K36me3 moiety was found to be unaltered in arsenic exposure, suggesting the involvement of other regulatory factors yet to be identified. DISCUSSION: In summary, the epigenetic repression of DNA damage repair genes due to promoter hypermethylation of MLH1 and MSH2 and inefficient recruitment of MMR complex at the site of DNA damage owing to the reduced level of H3K36me3 impairs the mismatch repair pathway that might render the arsenic-exposed individuals more susceptible towards DNA damage and associated cancer risk.