Restoring Treatment Response in Colorectal Cancer Cells by Targeting MACC1-Dependent ABCB1 Expression in Combination Therapy

Treatment failure of solid cancers, represented by the development of drug resistance in the primary tumor or later outgrowth of drug resistant metastases, is the major cause of death for cancer patients. It represents an urgent clinical need for predictive biomarkers which indicate the success or failure of standard treatment regimens. Besides treatment prediction, interfering with cellular processes associated with drug resistance might improve treatment response by applying combination therapies. Metastasis-associated in colon cancer (MACC) 1 was identified in our group as a prognostic biomarker in human colorectal cancer, and has been established as key player, prognostic, and predictive biomarker for tumor progression and metastasis in a variety of solid cancers. Besides increased cell proliferation and motility, subsequently contributing to growth and metastatic spread of the primary tumor, MACC1 has also been shown to dysregulate apoptosis and is contributing to treatment resistance. Here we report the MACC1 dependent treatment resistance of colorectal cancer (CRC) cells to standard therapeutics like doxorubicin by upregulating ATP-binding cassette subfamily B member 1 (ABCB1) protein. Overexpression of MACC1 in CRC cells increased both its presence on the ABCB1 promoter and its transcriptional activity, resulting in elevated ABCB1 expression and thus treatment resistance to standard therapeutics. In contrast, depleting MACC1 increased intracellular drug concentrations, leading to better treatment response. We already identified the first MACC1 transcriptional inhibitors, such as lovastatin, by high-throughput screening of clinically approved small molecule drugs. These compounds inhibited cell motility in vitro but also restricted metastasis development in xenograft mouse models by reducing MACC1 expression. Here we report, that treating high MACC1 expressing CRC cells with a combination of statins and standard therapeutics increased the rate of cytotoxicity and resulted in higher treatment response

Non-thermal effects of radiofrequency electromagnetic fields

We explored the non-thermal effects of radiofrequency (RF) electromagnetic fields and established a theoretical framework to elucidate their electrophysiological mechanisms. In experiments, we used a preclinical treatment device to treat the human colon cancer cell lines HT-29 and SW480 with either water bath heating (WB-HT) or 13.56 MHz RF hyperthermia (RF-HT) at 42 degrees C for 60 min and analyzed the proliferation and clonogenicity. We elaborated an electrical model for cell membranes and ion channels and estimated the resulting ion fluxes. The results showed that, for both cell lines, using RF-HT significantly reduced proliferation and clonogenicity compared to WB-HT. According to our model, the RF electric field component was rectified and smoothed in the direction of the channel, which resulted in a DC voltage of similar to 1 mu V. This may induce ion fluxes that can potentially cause relevant disequilibrium of most ions. Therefore, RF-HT creates additional non-thermal effects in association with significant ion fluxes. Increasing the understanding of these effects can help improve cancer therapy

Big data simulations for capacity improvement in a general ophthalmology clinic

PURPOSE Long total waiting times (TWT) experienced by patients during a clinic visit have a significant adverse effect on patient's satisfaction. Our aim was to use big data simulations of a patient scheduling calendar and its effect on TWT in a general ophthalmology clinic. Based on the simulation, we implemented changes to the calendar and verified their effect on TWT in clinical practice. DESIGN AND METHODS For this retrospective simulation study, we generated a discrete event simulation (DES) model based on clinical timepoints of 4.401 visits to our clinic. All data points were exported from our clinical warehouse for further processing. If not available from the electronic health record, manual time measurements of the process were used. Various patient scheduling models were simulated and evaluated based on their reduction of TWT. The most promising model was implemented into clinical practice in 2017. RESULTS During validation of our simulation model, we achieved a high agreement of mean TWT between the real data (229 ± 100 min) and the corresponding simulated data (225 ± 112 min). This indicates a high quality of the simulation model. Following the simulations, a patient scheduling calendar was introduced, which, compared with the old calendar, provided block intervals and extended time windows for patients. The simulated TWT of this model was 153 min. After implementation in clinical practice, TWT per patient in our general ophthalmology clinic has been reduced from 229 ± 100 to 183 ± 89 min. CONCLUSION By implementing a big data simulation model, we have achieved a cost-neutral reduction of the mean TWT by 21%. Big data simulation enables users to evaluate variations to an existing system before implementation into clinical practice. Various models for improving patient flow or reducing capacity loads can be evaluated cost-effectively

Response of neovascular central serous chorioretinopathy to an extended upload of anti-VEGF agents

Purpose To determine the anatomical and functional outcomes of an extended 6-month intravitreal anti-vascular endothelial growth factor (anti-VEGF) upload in choroidal neovascularization (CNV) secondary to chronic central serous chorioretinopathy (CSCR). Methods A retrospective database analysis was performed applying the following inclusion criteria: (1) diagnosis of CSCR, (2) diagnosis of secondary CNV, and (3) treatment of at least six consecutive injections of anti-VEGF. Outcome measures included the change of central retinal subfield thickness, remodeling of the pigment epithelium detachments, and change in visual function. Results Twenty-one eyes of 21 patients were included. Mean patient age was 65 ± 8.3 years, and 35% of the patients (n = 8) were female. Mean disease duration before diagnosis of CNV was 48 ± 25.3 months. Mean central retinal thickness decreased from 346 ± 61 to 257 ± 57 μm (p < 0.01) after the sixth injection while mean visual acuity improved from 0.65 ± 0.35 to 0.49 ± 0.29 (logMAR; p < 0.01). Of note, an extended upload of six as opposed to three injections yielded an additional mean central retinal thickness reduction (280 ± 46 μm vs. 257 ± 57 μm, p = 0.038). Significant CNV remodeling was observed as a decrease in pigment epithelium detachment (PED) vertical (p = 0.021) and horizontal diameter (p = 0.024) as well as PED height (p < 0.01). Conclusion An extended anti-VEGF upload of six consecutive injections seems to be effective in inducing CNV remodeling and fluid resorption in CNV complicating chronic CSCR

Repositionierte Arzneistoffe gegen die MACC1-β-Catenin-S100A4-Achse der Metastasierung unterdrücken Darmkrebsmetastasen in synergistischer Weise

Einleitung: Metastasen sind Haupttodesursache des kolorektalen Karzinoms (KRK). Trotz kurativer Therapie entwickeln 50% der nicht-metastasierten KRK-Fälle metachro-ne Metastasen. Das Sekretom des Primärtumors könnte eine frühe Metastasierung fördern. Metastasis-associated in colon cancer-1 (MACC1) ist ein Treiber der Tumorprogression, aber seine Rolle im Tumorsekretom ist unbekannt. Hier wurde versucht durch Hemmung der MACC1-abhängigen Tumorzellmotilität die metachrone Metasta-sierung zu verhindern. Methoden: MACC1-konditioniertes Medium wurde funktionell und in seiner Peptid-Zusammensetzung analysiert. Kaplan-Meier Schätzer bewertete das KRK-Risiko anhand der RNA-Expression von MACC1 und S100A4 in Tumor- und Blutproben. Korrelation von MACC1 und S1000A4 wurde in drei Kohorten von KRK-Tumorproben analysiert. S100A4-Regulation wurde auf Promoter-, RNA- und Proteinebene gemessen. In funktionellen Versuchen wurde S100A4 mittels CRISPR-Cas9 oder mit pharmakologischen Inhibitoren unterdrückt. Proteininteraktionen wurden mit Massen-Spektrometrie und Ko-Immunopräzipitation untersucht. Wundheilungsversuche ermittelten die KRK-Zellmigration unter Gabe von Statinen (Atorvastatin, Fluvastatin, Lovastatin) und Niclosamid. Metastasen wurden modelliert durch Injektion von KRK-Zellen in die Milz von SCID bg/bg Mäusen und unter oraler Gabe von Statinen und Niclosamid monitiert. Mikrometastasen wurden anhand der Last an humaner Satelliten-DNA in tumorfrei erscheinendem Lebergewebe gemessen. Ergebnisse: MACC1 induzierte KRK-Zellmotilität und S100A4-Sekretion in das Kul-turmedium. Hohe MACC1- und S100A4-Expression im Tumorgewebe und in Patientenblut sagte ein schlechtes metastasenfreies und gesamtes Überleben voraus. MACC1 stimulierte die Promoteraktivität, die RNA- und Proteinexpression von S100A4 in Zellkultur und in Tumoren von ApcMin-Mäusen mit ektopischem MACC1. MACC1 steigerte die Motilität nur in KRK-Zellen mit intakter S100A4-Expression, aber nicht unter S100A4-Depletion. Wnt/β-Catenin-Inhibitoren unterdrückten MACC1-abhängige Hochregulation von S100A4. MACC1 interagierte mit β-Catenin und verstärkte dessen Phosphorylierung und Interaktion mit TCF4. Kombinierte Gabe von Statinen und Niclosamid unterdrückte die Motilität von KRK-Zellen. Orale Gabe von Statin und Niclosamid verhinderte nicht das Auswachsen von Metastasen, aber unterdrückte die Absiedelung von Mikrometastasen in Mauslebern. Schlussfolgerungen: MACC1 treibt die Krebsprogression durch sekretorisches S100A4, und Überexpression beider Biomarker zeichnet Hochrisiko-KRK aus. MACC1 induziert S100A4 via Wnt/β-Catenin durch Interaktion mit β-Catenin und Stimulation seiner transkriptionellen Aktivität. Die Kombination transkriptioneller Inhibitoren von MACC1 und S100A4 unterdrücken synergistisch das metastatische Potential von KRK-Zellen in vitro und in vivo. MACC1 und S100A4 kooperieren in der KRK-Progression als Induktor und Effektor innerhalb einer MACC1-β-Catenin-S100A4-Achse der Metastasierung.Introduction: Metastasis is the main cause of colorectal cancer CRC death. Despite curative therapy, 50% of non-metastasized CRC cases will develop metachronous metastasis. Factors secreted by the primary tumor might facilitate early metastasis into distant organs. Metastasis-associated in colon cancer-1 (MACC1) drives cancer progression, yet it’s involvement in the tumor secretome is unknown. This project aimed to tar-get MACC1-driven motility of cancer cells to prevent metachronous metastasis. Methodology: MACC1-conditioned medium was analyzed functionally and for peptide composition. CRC risk assessment employed Kaplan-Meier estimation on MACC1 and S100A4 RNA expression in patient-derived tumor and blood samples. Correlation of MACC1 and S1000A4 expression was analyzed in three cohorts of CRC tumor specimens. Regulation of S100A4 was measured with promoter reporters, and on RNA and protein level. CRISPR-Cas9 knock-out and pharmacological inhibitors were employed to inhibit S100A4 in functional assays. Protein-protein interactions were examined via Mass-spectrometry and Co-Immunoprecipitation. Wound healing experiments as-sessed CRC cell migration under statins (atorvastatin, fluvastatin or lovastatin) and niclosamide. Metastasis was modelled by injection of CRC cell into the spleen of SCID bg/bg mice and monitored non-invasively under oral administration of statins and niclosamide. Micrometastases were quantified by measuring loads of human satellite DNA in tumor-free appearing liver tissue. Results: MACC1 induced CRC cell motility and S100A4 secretion into culture medium. High MACC1 and S100A4 expression in tumor tissue and in patient blood predicted dismal metastasis-free and overall survival. MACC1 stimulated promoter activity, RNA, and protein expression of S100A4 in cell culture and in tumors of ApcMin mice with ectopic MACC1. MACC1 enhanced motility only in CRC cells with intact S100A4 expression, but not in S100A4-depleted cells. Wnt/β-catenin signaling inhibitors sup-pressed S100A4 upregulation by MACC1. MACC1 interacted with β-catenin and enforced its phosphorylation and interaction with TCF4. Combined administration of statins and niclosamide synergistically suppressed the motility of CRC cells. Oral com-binations of statin and niclosamide did not prevent metastatic outgrowth but suppressed the abundance of micrometastases in mouse liver. Conclusion: MACC1 drives cancer progression via secretory S100A4, and combined overexpression of these biomarkers hallmarks high-risk CRC tumors. MACC1 induces S100A4 via Wnt/β-catenin by interacting with β-catenin and stimulating its transcrip-tional activity. Combination of respective transcriptional inhibitors synergistically suppress the metastatic potential of CRC cells in vitro and in vivo. MACC1 and S100A4 cooperate in CRC progression as inducer and effector within a druggable MACC1-β-catenin-S100A4 axis of metastasis

Mesalazine and thymoquinone attenuate intestinal tumour development in Msh2loxP/loxP Villin-Cre mice

Objective: Lynch syndrome is caused by germline mutations in DNA mismatch repair genes leading to microsatellite instability (MSI) and colorectal cancer. Mesalazine, commonly used for the treatment of UC, reduces MSI in vitro. Here, we tested natural compounds for such activity and applied mesalazine and thymoquinone in a Msh2loxP/loxP Villin-Cre mouse model for Lynch syndrome. Design: Flow cytometry was used for quantitation of mutation rates at a CA13 microsatellite in human colon cancer (HCT116) cells that had been stably transfected with pIREShyg2-enhanced green fluorescent protein/CA13, a reporter for frameshift mutations. Mice were treated for 43 weeks with mesalazine, thymoquinone or control chow. Intestines were analysed for tumour incidence, tumour multiplicity and size. MSI testing was performed from microdissected normal intestinal or tumour tissue, compared with mouse tails and quantified by the number of mutations per marker (NMPM). Results: Besides mesalazine, thymoquinone significantly improved replication fidelity at 1.25 and 2.5 µM in HCT116 cells. In Msh2loxP/loxP Villin-Cre mice, tumour incidence was reduced by mesalazine from 94% to 69% (p=0.04) and to 56% (p=0.003) by thymoquinone. The mean number of tumours was reduced from 3.1 to 1.4 by mesalazine (p=0.004) and to 1.1 by thymoquinone (p<0.001). Interestingly, MSI was reduced in normal intestinal tissue from 1.5 to 1.2 NMPM (p=0.006) and to 1.1 NMPM (p=0.01) by mesalazine and thymoquinone, respectively. Thymoquinone, but not mesalazine, reduced MSI in tumours. Conclusions: Mesalazine and thymoquinone reduce tumour incidence and multiplicity in Msh2loxP/loxP Villin-Cre mice by reduction of MSI independent of a functional mismatch repair system. Both substances are candidate compounds for chemoprevention in Lynch syndrome mutation carriers

MSH3-Deficiency Initiates EMAST without Oncogenic Transformation of Human Colon Epithelial Cells

<div><h3>Background/Aim</h3><p>Elevated microsatellite instability at selected tetranucleotide repeats (EMAST) is a genetic signature in certain cases of sporadic colorectal cancer and has been linked to MSH3-deficiency. It is currently controversial whether EMAST is associated with oncogenic properties in humans, specifically as cancer development in Msh3-deficient mice is not enhanced. However, a mutator phenotype is different between species as the genetic positions of repetitive sequences are not conserved. Here we studied the molecular effects of human MSH3-deficiency.</p> <h3>Methods</h3><p>HCT116 and HCT116+chr3 (both MSH3-deficient) and primary human colon epithelial cells (HCEC, MSH3-wildtype) were stably transfected with an EGFP-based reporter plasmid for the detection of frameshift mutations within an [AAAG]17 repeat. MSH3 was silenced by shRNA and changes in protein expression were analyzed by shotgun proteomics. Colony forming assay was used to determine oncogenic transformation and double strand breaks (DSBs) were assessed by Comet assay.</p> <h3>Results</h3><p>Despite differential MLH1 expression, both HCT116 and HCT116+chr3 cells displayed comparable high mutation rates (about 4×10⁻⁴) at [AAAG]17 repeats. Silencing of MSH3 in HCECs leads to a remarkable increased frameshift mutations in [AAAG]17 repeats whereas [CA]13 repeats were less affected. Upon MSH3-silencing, significant changes in the expression of 202 proteins were detected. Pathway analysis revealed overexpression of proteins involved in double strand break repair (MRE11 and RAD50), apoptosis, L1 recycling, and repression of proteins involved in metabolism, tRNA aminoacylation, and gene expression. MSH3-silencing did not induce oncogenic transformation and DSBs increased 2-fold.</p> <h3>Conclusions</h3><p>MSH3-deficiency in human colon epithelial cells results in EMAST, formation of DSBs and significant changes of the proteome but lacks oncogenic transformation. Thus, MSH3-deficiency alone is unlikely to drive human colon carcinogenesis.</p> </div

Characterization of frame-shift reporter-plasmid and -cell lines.

<p>(A) Sequence analysis of pIREShyg2-EGFP-[AAAG]17 plasmid. Genomic DNA was isolated, the EGFP region containing the [AAAG]17-repeat was amplified by PCR and sequenced. (B) Verification of plasmid insertion number by Southern blot analysis. 20µg of total DNA was digested with BamHI, EcoRV, or both (B/E), resolved on a 0.8% agarose gel, and transferred onto a nylon membrane. Complementary EGFP-DNA was labeled with [P-32]-dCTP, hybridized, and the blots were analyzed by autoradiography. The 811bp fragment (harboring the coding region for EGFP with the [AAAG]17 microsatellite] is generated by restriction with BamHI (position 957) and EcoRV (position 1768). (C) Flow cytometric analysis of unsorted HCT116-[AAAG]17 and HCT116+chr3-[AAAG]17 cell clones showing similar accumulation of EGFP-positive (mutated) cells and fluorescence intensities (the geometric mean of the FL1 intensity was expressed as mean +SD for the M1 and the M2 fractions).</p