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BACKGROUND: The metastasis inducing gene MACC1 is a prognostic and predictive biomarker for metastasis in several cancers. Its mechanism of inducing metastasis includes the transcriptional control of other cancer-related target genes. Here, we investigate the interplay with the metastasis driver S100P in CRC progression. METHODS: MACC1-dependent S100P expression was analysed by qRT-PCR. The binding of MACC1 to the S100P promoter was determined by ChIP. Alterations in cell proliferation and motility were determined by functional in vitro assays. In vivo metastasis after intrasplenic transplantation was assessed by bioluminescence imaging and evaluation of tumour growth and liver metastasis. The prognostic value of S100P was determined in CRC patients by ROC-based Kaplan-Meier analyses. RESULTS: Expression of S100P and MACC1 correlated positively in CRC cells and colorectal tumours. MACC1 was found binding to the S100P promoter and induces its expression. The overexpression of S100P increased proliferation, migration and invasion in vitro and significantly induced liver metastasis in vivo. S100P expression was significantly elevated in metachronously metastasising CRC and was associated with shorter metastasis-free survival. CONCLUSIONS: We identified S100P as a transcriptional target gene of MACC1. Expression of S100P increases the metastatic potential of CRC cells in vitro and in vivo, and serves as a prognostic biomarker for metastasis-free survival of CRC patients, emphasising novel therapeutic interventions targeting S100P.
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Neoplasias Colorretais , Neoplasias Hepáticas , Proteínas de Ligação ao Cálcio/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/genética , Proteínas de Neoplasias/genética , Prognóstico , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Tumor cell crosstalk with platelets and, subsequently, their activation are key steps in hematogenous tumor metastasis. MACC1 is an oncogene involved in molecular pathogenesis of colorectal cancer (CRC) and other solid tumor entities, mediating motility and metastasis, making MACC1 an accepted prognostic biomarker. However, the impact of MACC1 on platelet activation has not yet been addressed. Here, we investigated the activation of platelets by human CRC cells upon MACC1 modulation, indicated by platelet aggregation and granule release. These approaches led to the identification of insulin-like growth factor binding protein-2 (IGFBP2) as a functional downstream molecule of MACC1, affecting communication with platelets. This was confirmed by an shRNA-mediated IGFBP2 knockdown, while maintaining MACC1 activity. Although IGFBP2 displayed an attenuated platelet activation potential, obviously by scavenging IGF-I as a platelet costimulatory mediator, the MACC1/IGFBP2 axis did not affect the thrombin formation potential of the cells. Furthermore, the IGFBP2/MACC1-driven cell migration and invasiveness was further accelerated by platelets. The key role of IGFBP2 for the metastatic spread in vivo was confirmed in a xenograft mouse model. Data provide evidence for IGFBP2 as a downstream functional component of MACC1-driven metastasis, linking these two accepted oncogenic biomarkers for the first time in a platelet context.
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Plaquetas/metabolismo , Comunicação Celular , Neoplasias Colorretais/metabolismo , Proteína 2 de Ligação a Fator de Crescimento Semelhante à Insulina/metabolismo , Proteínas de Neoplasias/metabolismo , Transativadores/metabolismo , Animais , Plaquetas/patologia , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase NeoplásicaRESUMO
BACKGROUND: Metastasis-associated in colon cancer 1 (MACC1) is an established marker for metastasis and tumor cell migration in a multitude of tumor entities, including glioblastoma (GBM). Nevertheless, the mechanism underlying the increased migratory capacity in GBM is not comprehensively explored. METHODS: We performed live cell and atomic force microscopy measurements to assess cell migration and mechanical properties of MACC1 overexpressing GBM cells. We quantified MACC1 dependent dynamics of 3D aggregate formation. For mechanistic studies we measured the expression of key adhesion molecules using qRT-PCR, and MACC1 dependent changes in short term adhesion to fibronectin and laminin. We then determined changes in sub-cellular distribution of integrins and actin in dependence of MACC1, but also in microtubule and intermediate filament organization. RESULTS: MACC1 increased the migratory speed and elastic modulus of GBM cells, but decreased cell-cell adhesion and inhibited the formation of 3D aggregates. These effects were not associated with altered mRNA expression of several key adhesion molecules or altered short-term affinity to laminin and fibronectin. MACC1 did neither change the organization of the microtubule nor intermediate filament cytoskeleton, but resulted in increased amounts of protrusive actin on laminin. CONCLUSION: MACC1 overexpression increases elastic modulus and migration and reduces adhesion of GBM cells thereby impeding 3D aggregate formation. The underlying molecular mechanism is independent on the organization of microtubules, intermediate filaments and several key adhesion molecules, but depends on adhesion to laminin. Thus, targeting re-organization of the cytoskeleton and cell motility via MACC1 may offer a treatment option to impede GBM spreading. Video Abstract.
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Movimento Celular , Regulação Neoplásica da Expressão Gênica , Glioblastoma , Transativadores/fisiologia , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Glioblastoma/metabolismo , Glioblastoma/patologia , HumanosRESUMO
MACC1 (Metastasis Associated in Colon Cancer 1) is a key driver and prognostic biomarker for cancer progression and metastasis in a large variety of solid tumor types, particularly colorectal cancer (CRC). However, no MACC1 inhibitors have been identified yet. Therefore, we aimed to target MACC1 expression using a luciferase reporter-based high-throughput screening with the ChemBioNet library of more than 30,000 compounds. The small molecules lovastatin and rottlerin emerged as the most potent MACC1 transcriptional inhibitors. They remarkably inhibited MACC1 promoter activity and expression, resulting in reduced cell motility. Lovastatin impaired the binding of the transcription factors c-Jun and Sp1 to the MACC1 promoter, thereby inhibiting MACC1 transcription. Most importantly, in CRC-xenografted mice, lovastatin and rottlerin restricted MACC1 expression and liver metastasis. This is-to the best of our knowledge-the first identification of inhibitors restricting cancer progression and metastasis via the novel target MACC1. This drug repositioning might be of therapeutic value for CRC patients.
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Acetofenonas/uso terapêutico , Antineoplásicos/uso terapêutico , Benzopiranos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Fatores de Transcrição/antagonistas & inibidores , Desacopladores/uso terapêutico , Acetofenonas/efeitos adversos , Acetofenonas/química , Acetofenonas/farmacologia , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Antineoplásicos/farmacologia , Benzopiranos/efeitos adversos , Benzopiranos/química , Benzopiranos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Genes Reporter/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neoplasias Hepáticas Experimentais/prevenção & controle , Neoplasias Hepáticas Experimentais/secundário , Camundongos SCID , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Distribuição Aleatória , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Bibliotecas de Moléculas Pequenas , Transativadores , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Carga Tumoral/efeitos dos fármacos , Desacopladores/efeitos adversos , Desacopladores/química , Desacopladores/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Deciphering the paths to metastasis and identifying key molecules driving this process is one important issue for understanding and treatment of cancer. Such a key driver molecule is Metastasis Associated in Colon Cancer 1 (MACC1). A decade long research on this evolutionarily conserved molecule with features of a transcription factor as well as an adapter protein for versatile protein-protein interactions has shown that it has manifold properties driving tumors to their metastatic stage. MACC1 transcriptionally regulates genes involved in epithelial-mesenchymal transition (EMT), including those which are able to directly induce metastasis like c-MET, impacts tumor cell migration and invasion, and induces metastasis in solid cancers. MACC1 has proven as a valuable biomarker for prognosis of metastasis formation linked to patient survival and gives promise to also act as a predictive marker for individualized therapies in a broad variety of cancers. This review discusses the many features of MACC1 in the context of the hallmarks of cancer and the potential of this molecule as biomarker and novel therapeutic target for restriction and prevention of metastasis.
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Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Fatores de Transcrição/genética , Animais , Biomarcadores Tumorais/genética , Humanos , Metástase Neoplásica , Neoplasias/patologia , TransativadoresRESUMO
The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refinement of gene delivery techniques. We set out to improve SB gene transfer into hard-to-transfect human CD34+ cells by vectorizing the SB system components in the form of minicircles that are devoid of plasmid backbone sequences and are, therefore, significantly reduced in size. As compared to conventional plasmids, delivery of the SB transposon system as minicircle DNA is â¼20 times more efficient, and it is associated with up to a 50% reduction in cellular toxicity in human CD34+ cells. Moreover, providing the SB transposase in the form of synthetic mRNA enabled us to further increase the efficacy and biosafety of stable gene delivery into hematopoietic progenitors ex vivo. Genome-wide insertion site profiling revealed a close-to-random distribution of SB transposon integrants, which is characteristically different from gammaretroviral and lentiviral integrations in HSPCs. Transplantation of gene-marked CD34+ cells in immunodeficient mice resulted in long-term engraftment and hematopoietic reconstitution, which was most efficient when the SB transposase was supplied as mRNA and nucleofected cells were maintained for 4-8 days in culture before transplantation. Collectively, implementation of minicircle and mRNA technologies allowed us to further refine the SB transposon system in the context of HSPC gene delivery to ultimately meet clinical demands of an efficient and safe non-viral gene therapy protocol.
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Elementos de DNA Transponíveis , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Animais , Sobrevivência Celular , Citometria de Fluxo , Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Retroviridae/genética , Transfecção , TransgenesRESUMO
BACKGROUND: Bacterial toxins have evolved to an effective therapeutic option for cancer therapy. The Clostridium perfringens enterotoxin (CPE) is a pore-forming toxin with selective cytotoxicity. The transmembrane tight junction proteins claudin-3 and -4 are known high affinity CPE receptors. Their expression is highly upregulated in human cancers, including breast, ovarian and colon carcinoma. CPE binding to claudins triggers membrane pore complex formation, which leads to rapid cell death. Previous studies demonstrated the anti-tumoral effect of treatment with recombinant CPE-protein. Our approach aimed at evaluation of a selective and targeted cancer gene therapy of claudin-3- and/or claudin-4- expressing colon carcinoma in vitro and in vivo by using translation optimized CPE expressing vector. METHODS: In this study the recombinant CPE and a translation optimized CPE expressing vector (optCPE) was used for targeted gene therapy of claudin-3 and/or -4 overexpressing colon cancer cell lines. All experiments were performed in the human SW480, SW620, HCT116, CaCo-2 and HT-29 colon cancer and the isogenic Sk-Mel5 and Sk-Mel5 Cldn-3-YFP melanoma cell lines. Claudin expression analysis was done at protein and mRNA level, which was confirmed by immunohistochemistry. The CPE induced cytotoxicity was analyzed by the MTT cytotoxicity assay. In addition patient derived colon carcinoma xenografts (PDX) were characterized and used for the intratumoral in vivo gene transfer of the optCPE expressing vector in PDX bearing nude mice. RESULTS: Claudin-3 and -4 overexpressing colon carcinoma lines showed high sensitivity towards both recCPE application and optCPE gene transfer. The positive correlation between CPE cytotoxicity and level of claudin expression was demonstrated. Transfection of optCPE led to targeted, rapid cytotoxic effects such as membrane disruption and necrosis in claudin overexpressing cells. The intratumoral optCPE in vivo gene transfer led to tumor growth inhibition in colon carcinoma PDX bearing mice in association with massive necrosis due to the intratumoral optCPE expression. CONCLUSIONS: This novel approach demonstrates that optCPE gene transfer represents a promising and efficient therapeutic option for a targeted suicide gene therapy of claudin-3 and/or claudin-4 overexpressing colon carcinomas, leading to rapid and effective tumor cell killing in vitro and in vivo.
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Claudina-3/genética , Claudina-4/genética , Neoplasias do Colo/terapia , Enterotoxinas/uso terapêutico , Genes Transgênicos Suicidas , Terapia Genética , Animais , Efeito Espectador , Clostridium perfringens , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Feminino , Humanos , Camundongos , Camundongos Nus , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The increasing unraveling of the molecular basis of cancer offers manifold novel options for intervention strategies. However, the discovery and development of new drugs for potential clinical applications is a tremendously time-consuming and costly process. Translating a novel lead candidate compound into an approved clinical drug takes often more than a decade, and the success rate is very low due to versatile efforts including defining its pharmacokinetics, pharmacodynamics, side effects as well as lack of sufficient efficacy. Thus, strategies are needed to minimize time and costs, while maximizing success rates. A very attractive strategy for novel cancer therapeutic options is the repositioning of already approved drugs. These medicines, approved for the treatment of non-malignant disorders, have already passed some early costs and time, have been tested in humans and are ready for clinical trials as anti-cancer drugs. Here we discuss the repositioning of nonsteroidal anti-inflammatory drugs (NSAID), statins, anti-psychotic drugs, anti-helminthic drugs and vitamin D as anti-tumor agents. We focus on their novel actions and potential for inhibition of cancer growth and metastasis by interfering with target molecules and pathways, which drive these malignant processes. Furthermore, important pre-clinical and clinical data are reviewed herein, which elucidate their therapeutic mechanisms which enable their repositioning for cancer therapy and disruption of metastasis.
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Antineoplásicos/farmacologia , Reposicionamento de Medicamentos , Neoplasias/tratamento farmacológico , Animais , Progressão da Doença , Humanos , Terapia de Alvo Molecular , Metástase Neoplásica , Neoplasias/patologiaRESUMO
Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease, which is thought to be initiated by colon cancer stem cells, according to the cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcomes. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our MACC1 transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and the CSC-associated invasive phenotype based on patient-derived tumor organoids (PDOs), patient-derived xenografts (PDXs) and human CRC cell lines. We showed that CD44-enriched CSCs from PDO models express significantly higher levels of MACC1 and LGR5 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PDO and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with the CSC markers CD44, NANOG and LGR5 in PDO models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter, uncovering the long-known CSC marker LGR5 as a novel essential signaling mediator employed by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by a significant positive correlation of MACC1 with LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis inducer MACC1 acts as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs and prevent cancer recurrence and distant metastasis formation.
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Background: Identification of cancer metastasis-relevant molecular networks is desired to provide the basis for understanding and developing intervention strategies. Here we address the role of GIPC1 in the process of MACC1-driven metastasis. MACC1 is a prognostic indicator for patient metastasis formation and metastasis-free survival. MACC1 controls gene transcription, promotes motility, invasion and proliferation of colon cancer cells in vitro, and causes tumor growth and metastasis in mice. Methods: By using yeast-two-hybrid assay, mass spectrometry, co-immunoprecipitation and peptide array we analyzed GIPC1 protein binding partners, by using the MACC1 gene promoter and chromatin immunoprecipitation and electrophoretic mobility shift assay we probed for GIPC1 as transcription factor. We employed GIPC1/MACC1-manipulated cell lines for in vitro and in vivo analyses, and we probed the GIPC1/MACC1 impact using human primary colorectal cancer (CRC) tissue. Results: We identified MACC1 and its paralogue SH3BP4 as protein binding partners of the protein GIPC1, and we also demonstrated the binding of GIPC1 as transcription factor to the MACC1 promoter (TSS to -60 bp). GIPC1 knockdown reduced endogenous, but not CMV promoter-driven MACC1 expression, and diminished MACC1-induced cell migration and invasion. GIPC1 suppression reduced tumor growth and metastasis in mice intrasplenically transplanted with MACC1-overexpressing CRC cells. In human primary CRC specimens, GIPC1 correlates with MACC1 expression and is of prognostic value for metastasis formation and metastasis-free survival. Combination of MACC1 and GIPC1 expression improved patient survival prognosis, whereas SH3BP4 expression did not show any prognostic value. Conclusions: We identified an important, dual function of GIPC1 - as protein interaction partner and as transcription factor of MACC1 - for tumor progression and cancer metastasis.
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Metastasis is directly linked to poor prognosis of cancer patients and warrants search for effective anti-metastatic drugs. MACC1 is a causal key molecule for metastasis. High MACC1 expression is prognostic for metastasis and poor survival. Here, we developed novel small molecule inhibitors targeting MACC1 expression to impede metastasis formation. We performed a human MACC1 promoter-driven luciferase reporter-based high-throughput screen (HTS; 118.500 compound library) to identify MACC1 transcriptional inhibitors. HTS revealed 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds as efficient transcriptional inhibitors of MACC1 expression, able to decrease MACC1-induced cancer cell motility in vitro. Structure-activity relationships identified the essential inhibitory core structure. Best candidates were evaluated for metastasis inhibition in xenografted mouse models demonstrating metastasis restriction. ADMET showed high drug-likeness of these new candidates for cancer therapy. The NFκB pathway was identified as one mode of action targeted by these compounds. Taken together, 1,2,3,4-tetrazolo[1,5-b]pyridazine-based compounds are effective MACC1 inhibitors and pose promising candidates for anti-metastatic therapies particularly for patients with MACC1-overexpressing cancers, that are at high risk to develop metastases. Although further preclinical and clinical development is necessary, these compounds represent important building blocks for an individualized anti-metastatic therapy for solid cancers.
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Neoplasias , Transativadores , Animais , Humanos , Camundongos , Regulação Neoplásica da Expressão Gênica , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Regiões Promotoras Genéticas , Transativadores/antagonistas & inibidoresRESUMO
Gene therapy has started in the late 1980s as novel, clinically applicable therapeutic option. It revolutionized the treatment of genetic diseases with the initial intent to repair or replace defective genes. Gene therapy has been adapted for treatment of malignant diseases to improve the outcome of cancer patients. In fact, cancer gene therapy has rapidly gained great interest and evolved into a research field with highest proportion of research activities in gene therapy. In this context, cancer gene therapy has long entered translation into clinical trials and therefore more than two-thirds of all gene therapy trials worldwide are aiming at the treatment of cancer disease using different therapeutic strategies. During the decades in cancer gene therapy, tremendous knowledge has accumulated. This led to significant improvements in vector design, transgene repertoire, more targeted interventions, use of novel gene therapeutic technologies such as CRISPR/Cas, sleeping beauty vectors, and development of effective cancer immunogene therapies. In this chapter, a brief overview of current key developments in cancer gene therapy is provided to gain insights into the recent directions in research as well as in clinical application of cancer gene therapy.
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Vetores Genéticos , Neoplasias , Técnicas de Transferência de Genes , Genes Neoplásicos , Terapia Genética , Vetores Genéticos/genética , Humanos , Neoplasias/genética , Neoplasias/terapia , TransgenesRESUMO
Plasmid DNA in any form (plasmid DNA, minicircle, miniplasmid) does experience renewed and increasing attention for use in gene therapy and DNA vaccination. For such applications, stability analyses and quality control are essential prerequisites for clinical use. In this context we analyzed the stability of good manufacturing practice (GMP)-grade pCMVß reporter plasmid DNA by capillary gel electrophoresis. The plasmid DNA was produced for a clinical gene transfer study for treatment of malignant melanoma. The pCMVß plasmid DNA was stored at -20 °C for 20 years under continuous, controlled monitoring. Another plasmid., pCMV-Luc, stored for 15 years, served as reference. The stability of plasmid DNA was analyzed by capillary gel electrophoresis (CGE) and functionally tested in vitro by LacZ functional assay. In this chapter we provide the detailed description of CGE and functional analysis of the GMP-grade pCMVß and also pCMV-Luc plasmid DNA. By this the proportion of open circular and supercoiled or covalently closed circular forms of plasmid DNA is analyzed. Functionality of the plasmid was tested by in vitro transfection and LacZ functional assay. In result of this, the 20-year-old plasmid DNA showed topology and expression performance, which revealed significant alterations in topology while maintaining functionality regarding transgene expression. Therefore, stable storage conditions are effective to mainly preserve the integrity of the plasmid DNA as important parameter for long-term storage of, for example, reference samples.
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Eletroforese Capilar , Terapia Genética , DNA/genética , Eletroforese Capilar/métodos , Terapia Genética/métodos , Plasmídeos/genética , Controle de QualidadeRESUMO
Metastasis-associated in colon cancer 1 (MACC1) is a marker for metastasis, tumor cell migration, and increased proliferation in colorectal cancer (CRC). Tumors with high MACC1 expression show a worse prognosis and higher invasion into neighboring structures. Yet, many facets of the pro-migratory effects are not fully understood. Atomic force microscopy and single cell live imaging were used to quantify biomechanical and migratory properties in low- and high-MACC1-expressing CRC cells. Furthermore, collective migration and expansion of small, cohesive cell colonies were analyzed using live cell imaging and particle image velocimetry. Lastly, the impact of proliferation on collective migration was determined by inhibition of proliferation using mitomycin. MACC1 did not affect elasticity, cortex tension, and single cell migration of CRC cells but promoted collective migration and colony expansion in vitro. Measurements of the local velocities in the dense cell layers revealed proliferation events as regions of high local speeds. Inhibition of proliferation via mitomycin abrogated the MACC1-associated effects on the collective migration speeds. A simple simulation revealed that the expansion of cell clusters without proliferation appeared to be determined mostly by single cell properties. MACC1 overexpression does not influence single cell biomechanics and migration but only collective migration in a proliferation-dependent manner. Thus, targeting proliferation in high-MACC1-expressing tumors may offer additional effects on cell migration.
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Bacterial toxins gain growing attention as potential cancer treatment due to their potent cytotoxic effects. Among the very different toxins with diverse modes of action, the Clostridium perfringens enterotoxin (CPE) is in focus to treat solid cancers. This toxin targets the tight junction proteins claudin-3 and -4 (Cldn-3/4), which are frequently overexpressed in solid cancers. Binding to these claudins induces pore formation in the host cell plasma membrane leading to rapid oncoleaking cell death of tumor cells. Based on this, extending the targeting of CPE beyond Cldn-3/4 is of interest, since other claudins, such as claudin-1 or -5 are often overexpressed in various cancer entities such as non-small-cell lung cancer (NSCLC) or papillary thyroid carcinoma. In this chapter we describe the modification of a CPE-encoding vector by structure-directed mutagenesis to either preferentially target Cldn-1 and -5 or to expand targeting to Cldn1-9 for improved broadened cytotoxic targeting of claudin-overexpressing tumors such as but not limited to lung cancer via CPE gene transfer.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Claudinas/genética , Claudinas/metabolismo , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Enterotoxinas/genética , Enterotoxinas/metabolismo , Terapia Genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapiaRESUMO
Obesity is a major and increasing public health concern, associated with an increased risk of and mortality from several types of cancer including colorectal cancer (CRC), being associated with cancer progression, metastasis and resistance to therapy. It was hypothesized that the expression of cancer/metastasisinducing gene metastasisassociated in colon cancer 1 (MACC1) is increased in obesity, which may constitute a link to obesityinduced cancer. The present study thus analyzed circulating cellfree plasma MACC1 expression levels in human obese (vs. normal weight) adult individuals from independent studies, namely the Martin Luther University (MLU) study (n=32) and the Metabolic syndrome study (MetScan, Berlin) (n=191). Higher plasma MACC1 levels were found in obese individuals, increasing with a greater body fat mass and body mass index; these levels were predominantly observed in male and to a lesser extent in female individuals, although the results were not significant. A reduction in body fat mass following dietary intervention and physical exercise decreased the MACC1 expression levels in the MLU study. Furthermore, Wistar rats with dietinduced obesity exhibited slightly increased plasma MACC1 levels compared with rats of normal weight. The obese Wistar rats exposed to azoxymethane to induce colon cancer exhibited a more severe colon tumor outcome, which was associated with significantly increased MACC1 levels compared with their nonobese littermates. On the whole, the findings of the present study suggest an association between MACC1 and obesity, as well as with obesityinduced CRC.
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Neoplasias Colorretais/genética , Obesidade/genética , Transativadores/metabolismo , Adiposidade/genética , Adulto , Idoso , Animais , Índice de Massa Corporal , Movimento Celular/genética , Neoplasias Colorretais/epidemiologia , Feminino , Expressão Gênica/genética , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/epidemiologia , Ratos , Ratos Wistar , Transativadores/genéticaRESUMO
Conventional eukaryotic expression plasmids contain a DNA backbone that is dispensable for the cellular expression of the transgene. In order to reduce the vector size, minicircle DNA technology was introduced. A drawback of the minicircle technology are considerable production costs. Nanoplasmids are a relatively new class of mini-DNA constructs that are of tremendous potential for pharmaceutical applications. In this study we have designed novel suicide nanoplasmid constructs coding for plant derived ribosome-inactivating proteins. The suicide-nanoplasmids were formulated with a targeted K16-lysine domain, analyzed for size, and characterized by electron microscopy. The anti-proliferative activity of the suicide-nanoplasmids was investigated in vitro by real time microscopy and the expression kinetic was determined using an enhanced green fluorescent protein nanoplasmid variant. In an aggressive in vivo neuroblastoma tumor model, treated mice showed a reduced tumor growth whereby the therapy was well tolerated.
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Vetores Genéticos , Proteínas Inativadoras de Ribossomos , Animais , Camundongos , Plasmídeos , RibossomosRESUMO
Metastasis-Associated in Colon Cancer 1 (MACC1) is a strong prognostic biomarker inducing proliferation, migration, invasiveness, and metastasis of cancer cells. The context of MACC1 dysregulation in cancers is, however, still poorly understood. Here, we investigated whether chromosomal instability and somatic copy number alterations (SCNA) frequently occurring in CRC contribute to MACC1 dysregulation, with prognostic and predictive impacts. Using the Oncotrack and Charité CRC cohorts of CRC patients, we showed that elevated MACC1 mRNA expression was tightly dependent on increased MACC1 gene SCNA and was associated with metastasis and shorter metastasis free survival. Deep analysis of the COAD-READ TCGA cohort revealed elevated MACC1 expression due to SCNA for advanced tumors exhibiting high chromosomal instability (CIN), and predominantly classified as CMS2 and CMS4 transcriptomic subtypes. For that cohort, we validated that elevated MACC1 mRNA expression correlated with reduced disease-free and overall survival. In conclusion, this study gives insights into the context of MACC1 expression in CRC. Increased MACC1 expression is largely driven by CIN, SCNA gains, and molecular subtypes, potentially determining the molecular risk for metastasis that might serve as a basis for patient-tailored treatment decisions.
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Colorectal cancer (CRC) is the second-most common malignant disease worldwide, and metastasis is the main culprit of CRC-related death. Metachronous metastases remain to be an unpredictable, unpreventable, and fatal complication, and tracing the molecular chain of events that lead to metastasis would provide mechanistically linked biomarkers for the maintenance of remission in CRC patients after curative treatment. We hypothesized, that Metastasis-associated in colorectal cancer-1 (MACC1) induces a secretory phenotype to enforce metastasis in a paracrine manner, and found, that the cell-free culture medium of MACC1-expressing CRC cells induces migration. Stable isotope labeling by amino acids in cell culture mass spectrometry (SILAC-MS) of the medium revealed, that S100A4 is significantly enriched in the MACC1-specific secretome. Remarkably, both biomarkers correlate in expression data of independent cohorts as well as within CRC tumor sections. Furthermore, combined elevated transcript levels of the metastasis genes MACC1 and S100A4 in primary tumors and in blood plasma robustly identifies CRC patients at high risk for poor metastasis-free (MFS) and overall survival (OS). Mechanistically, MACC1 strengthens the interaction of ß-catenin with TCF4, thus inducing S100A4 synthesis transcriptionally, resulting in elevated secretion to enforce cell motility and metastasis. In cell motility assays, S100A4 was indispensable for MACC1-induced migration, as shown via knock-out and pharmacological inhibition of S100A4. The direct transcriptional and functional relationship of MACC1 and S100A4 was probed by combined targeting with repositioned drugs. In fact, the MACC1-ß-catenin-S100A4 axis by statins (MACC1) and niclosamide (S100A4) synergized in inhibiting cancer cell motility in vitro and metastasis in vivo. The MACC1-ß-catenin-S100A4 signaling axis is causal for CRC metastasis. Selectively repositioned drugs synergize in restricting MACC1/S100A4-driven metastasis with cross-entity potential.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Inibidores de Hidroximetilglutaril-CoA Redutases , Neoplasias Retais , Aminoácidos/metabolismo , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Niclosamida/farmacologia , Niclosamida/uso terapêutico , Neoplasias Retais/genética , Proteína A4 de Ligação a Cálcio da Família S100/genética , Proteína A4 de Ligação a Cálcio da Família S100/metabolismo , Transativadores/genética , beta Catenina/metabolismoRESUMO
Esophageal and Gastric Adenocarcinomas (AGE/S) are characterized by early metastasis and poor survival. MACC1 (Metastasis Associated in Colon Cancer 1) acts in colon cancer as a metastasis inducer and is linked to reduced survival. This project illuminates the role and potential for the inhibition of MACC1 in AGE/S. Using 266 of 360 TMAs and survival data of AGE/S patients, we confirm the value of MACC1 as an independent negative prognostic marker in AGE/S patients. MACC1 gene expression is correlated with survival and morphological characteristics. In vitro analysis of lentivirally MACC1-manipulated subclones of FLO-1 and OE33 showed enhanced migration induced by MACC1 in both cell line models, which could be inhibited by the MEK1 inhibitor selumetinib. In vivo, the efficacy of selumetinib on tumor growths and metastases of MACC1-overexpressing FLO-1 cells xenografted intrasplenically in NOG mice was tested. Mice with high-MACC1-expressing cells developed faster and larger distant metastases. Treatment with selumetinib led to a significant reduction in metastasis exclusively in the MACC1-positive xenografts. MACC1 is an enhancer of tumor aggressiveness and a predictor of poor survival in AGE/S. This effect can be inhibited by selumetinib.