Induction of circulating ABCB1 transcripts under platinum-based chemotherapy indicates poor prognosis and a bone micrometastatic phenotype in ovarian cancer patients.

The drug efflux transporter P-glycoprotein, encoded by the ABCB1 gene, promotes acquired chemoresistance. We explored the presence and clinical relevance of circulating cell-free ABCB1 transcripts (cfABCB1tx) in ovarian cancer patients (173 longitudinal serum samples from 79 cancer patients) using digital droplet PCR. cfABCB1tx were readily detectable at primary diagnosis (median 354 mRNA copies/20 µl serum), paralleled FIGO-stage and predicted surgical outcome (p = 0.023, p=0.022, respectively). Increased cfABCB1tx levels at primary diagnosis indicated poor PFS (HR = 2.329, 95%CI:1.374-3.947, p = 0.0017) and OS (HR = 2.074, 95%CI:1.194-3.601, p = 0.0096). cfABCB1tx induction under platinum-based chemotherapy was an independent predictor for poor OS (HR = 2.597, 95%CI: 1.218-5.538, p = 0.013) and paralelled a micrometastatic phenotype, shaped by the presence of disseminated tumor cells in the bone marrow. A strong correlation was observed between cfABCB1tx and circulating transcripts of the metastasis-inducer MACC1, which is the transcriptional activator of ABCB1. Combined assessment of cfABCB1tx and circulating cell-free MACC1 transcripts (cfMACC1tx) resulted in an improved prognostic prediction, with  the cfABCB1tx-high/cfMACC1tx-high phenotype bearing the highest risk for relapse and death. Conclusively, we provide proof of principle, that ABCB1 transcripts are readily traceable in the liquid-biopsy of ovarian cancer patients, advancing a new dimension for systemic monitoring of ABCB1/P-glycoprotein expression dynamics.

Cantharidin and Its Analogue Norcantharidin Inhibit Metastasis-Inducing Genes S100A4 and MACC1.

Colorectal cancer (CRC) is the third most prevalent and second deadliest cancer worldwide. In addition, metastasis directly causes up to 90% of all CRC deaths, highlighting the metastatic burden of the disease. Biomarkers such as S100A4 and MACC1 aid in identifying patients with a high risk of metastasis formation. High expression of S100A4 or MACC1 and to a greater extent the combination of both biomarkers is a predictor for metastasis and poor patient survival in CRC. MACC1 is a tumor-initiating and metastasis-promoting oncogene, whereas S100A4 has not been shown to initiate tumor formation but can, nevertheless, promote malignant tumor growth and metastasis formation. Cantharidin is a natural drug extracted from various blister beetle species, and its demethylated analogue norcantharidin has been shown in several studies to have an anti-cancer and anti-metastatic effect in different cancer entities such as CRC, breast cancer, and lung cancer. The impact of the natural compound cantharidin and norcantharidin on S100A4 and MACC1 gene expression, cancer cell migration, motility, and colony formation in vitro was tested. Here, for the first time, we have demonstrated that cantharidin and norcantharidin are transcriptional inhibitors of S100A4 and MACC1 mRNA expression, protein expression, and motility in CRC cells. Our results clearly indicate that cantharidin and, to a lesser extent, its analogue norcantharidin are promising compounds for efficient anti-metastatic therapy targeting the metastasis-inducing genes S100A4 and MACC1 for personalized medicine for cancer patients.

Calcium-binding protein S100P is a new target gene of MACC1, drives colorectal cancer metastasis and serves as a prognostic biomarker.

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.

MACC1 regulates clathrin-mediated endocytosis and receptor recycling of transferrin receptor and EGFR in colorectal cancer.

Metastasis Associated in Colon Cancer 1 (MACC1) is a novel prognostic, predictive and causal biomarker for tumor progression and metastasis in many cancer types, including colorectal cancer. Besides its clinical value, little is known about its molecular function. Its similarity to SH3BP4, involved in regulating uptake and recycling of transmembrane receptors, suggests a role of MACC1 in endocytosis. By exploring the MACC1 interactome, we identified the clathrin-mediated endocytosis (CME)-associated proteins CLTC, DNM2 and AP-2 as MACC1 binding partners. We unveiled a MACC1-dependent routing of internalized transferrin receptor towards recycling. Elevated MACC1 expression caused also the activation and internalization of EGFR, a higher rate of receptor recycling, as well as earlier and stronger receptor activation and downstream signaling. These effects are limited by deletion of CME-related protein interaction sites in MACC1. Thus, MACC1 regulates CME and receptor recycling, causing increased growth factor-mediated downstream signaling and cell proliferation. This novel mechanism unveils potential therapeutic intervention points restricting MACC1-driven metastasis.

Decoding and targeting the molecular basis of MACC1-driven metastatic spread: Lessons from big data mining and clinical-experimental approaches.

Metastasis remains the key issue impacting cancer patient survival and failure or success of cancer therapies. Metastatic spread is a complex process including dissemination of single cells or collective cell migration, penetration of the blood or lymphatic vessels and seeding at a distant organ site. Hundreds of genes involved in metastasis have been identified in studies across numerous cancer types. Here, we analyzed how the metastasis-associated gene MACC1 cooperates with other genes in metastatic spread and how these coactions could be exploited by combination therapies: We performed (i) a MACC1 correlation analysis across 33 cancer types in the mRNA expression data of TCGA and (ii) a comprehensive literature search on reported MACC1 combinations and regulation mechanisms. The key genes MET, HGF and MMP7 reported together with MACC1 showed significant positive correlations with MACC1 in more than half of the cancer types included in the big data analysis. However, ten other genes also reported together with MACC1 in the literature showed significant positive correlations with MACC1 in only a minority of 5 to 15 cancer types. To uncover transcriptional regulation mechanisms that are activated simultaneously with MACC1, we isolated pan-cancer consensus lists of 1306 positively and 590 negatively MACC1-correlating genes from the TCGA data and analyzed each of these lists for sharing transcription factor binding motifs in the promotor region. In these lists, binding sites for the transcription factors TELF1, ETS2, ETV4, TEAD1, FOXO4, NFE2L1, ELK1, SP1 and NFE2L2 were significantly enriched, but none of them except SP1 was reported in combination with MACC1 in the literature. Thus, while some of the results of the big data analysis were in line with the reported experimental results, hypotheses on new genes involved in MACC1-driven metastasis formation could be generated and warrant experimental validation. Furthermore, the results of the big data analysis can help to prioritize cancer types for experimental studies and testing of combination therapies.

Statin and rottlerin small-molecule inhibitors restrict colon cancer progression and metastasis via MACC1.

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.

In-vitro and in-vivo investigations into the carbene-gold anticancer drug candidates NHC*-Au-SCSNMe2 and NHC*-Au-S-GLUC against advanced prostate cancer PC3.

The anticancer drug candidates 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative exhibited nanomolar in-vitro activity against prostate cancer cells advanced prostate cancer (PC3) and micromolar inhibition of mammalian thioredoxin reductase. Encouraging maximum tolerable dose experiments led to human prostate cancer subcutaneous xenograft experiments; 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative were applied twelve times at two doses in groups of n = 5 PC3 to tumor-bearing NMRI:nu/nu mice. 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate and 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative at the dose of 10 and 20 mg/kg showed good tolerability, while no significant body weight loss was seen in both groups. In particular, for the drug 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene gold(I) dimethylamino dithiocarbamate the tumor growth inhibition suggested to be dose dependent, reflected by the respective optimal T/C values of 0.45 at the dose of 10 mg/kg and of 0.31 at the dose of 20 mg/kg. By contrast, the 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-1-thiolate derivative treated groups showed no indication for dose-dependent antitumoral activity, as reflected by the optimal T/C values of 0.44 for the 10 mg/kg and for the 20 mg/kg treated mice. Immunohistochemical experiments involving Ki67 staining of tumor tissue showed that both compounds reduced PC3 cell proliferation against the difficult to treat advanced human prostate tumors derived from PC3.

MACC1-the first decade of a key metastasis molecule from gene discovery to clinical translation.

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.

Efficient Non-viral Gene Delivery into Human Hematopoietic Stem Cells by Minicircle Sleeping Beauty Transposon Vectors.

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 ∼20 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.

Phase II trial to investigate the safety and efficacy of orally applied niclosamide in patients with metachronous or sychronous metastases of a colorectal cancer progressing after therapy: the NIKOLO trial.

BACKGROUND: Colorectal cancer (CRC) is the second most common cause of all cancer deaths in Europe and the Western world with a lifetime risk of approximately 5%. Despite several improvements in the treatment of patients with unresectable CRC prognosis is poor and there is the need of developing new treatment strategies for patients with metastatic chemorefractory disease. The S100 calcium binding protein A4 (S100A4) predicts metastasis formation and reduced CRC patient survival. S100A4 was previously identified as transcriptional target of the Wnt/ß-catenin signaling pathway. The Food and Drug Administration (FDA)-approved anti-helminthic drug niclosamide is known to intervene in the Wnt/ß-catenin pathway signaling, leading to reduced expression of S100A4 linked to restricted in vivo metastasis formation. Thus, we aim at translation of our findings on restricting S100A4-driven metastasis into clinical practice for treating metastasized CRC patients progressing after standard therapy. METHODS/DESIGN: NIKOLO is a phase II, single center, one-arm open-label clinical trial to investigate the safety and efficacy of niclosamide tablets in patients with metastasized CRC progressing under standard therapy. Eligible patients will receive 2 g of orally applied niclosamide once a day and will continue with the treatment once daily till disease progression or toxicity. Toxicities will be graded according to National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) v4.03. The primary objective of this trial is to assess the progression free survival after 4 months, secondary objectives are overall survival, time to progression, disease control rate (remission + partial remission + stable disease), and safety. Furthermore, pharmacokinetic analysis will be conducted to evaluate niclosamide plasma concentration. DISCUSSION: This study is expected to provide evidence of the feasibility, toxicity and efficacy of niclosamide in the treatment of patients with metastasized CRC and could help to establish a new treatment option. TRIAL REGISTRATION: The study is registered with ClinicalTrials.gov (NCT02519582) and the European Clinical Trials Database (EudraCT 2014-005151-20).

Mechanisms of Targeting the MDM2-p53-FOXM1 Axis in Well-Differentiated Intestinal Neuroendocrine Tumors.

BACKGROUND/AIMS: The tumor suppressor p53 is rarely mutated in gastroenteropancreatic neuroendocrine neoplasms (GEP-NEN) but they frequently show a strong expression of negative regulators of p53, rendering these tumors excellent targets for a p53 recovery therapy. Therefore, we analyzed the mechanisms of a p53 recovery therapy on intestinal neuroendocrine tumors in vitro and in vivo. METHODS: By Western blot and immunohistochemistry, we found that in GEP-NEN biopsy material overexpression of MDM2 was present in intestinal NEN. Therefore, we analyzed the effect of a small-molecule inhibitor, nutlin-3a, in p53 wild-type and mutant GEP-NEN cell lines by proliferation assay, flow cytometry, immunofluorescence, Western blot, and by multiplex gene expression analysis. Finally, we analyzed the antitumor effect of nutlin-3a in a xenograft mouse model in vivo. During the study, the tumor volume was determined. RESULTS: The midgut wild-type cell line KRJ-I responded to the treatment with cell cycle arrest and apoptosis. By gene expression analysis, we could demonstrate that nutlins reactivated an antiproliferative p53 response. KRJ-I-derived xenograft tumors showed a significantly decreased tumor growth upon treatment with nutlin-3a in vivo. Furthermore, our data suggest that MDM2 also influences the expression of the oncogene FOXM1 in a p53-independent manner. Subsequently, a combined treatment of nutlin-3a and cisplatin (as chemoresistance model) resulted in synergistically enhanced antiproliferative effects. CONCLUSION: In summary, MDM2 overexpression is a frequent event in p53 wild-type intestinal neuroendocrine neoplasms and therefore recovery of a p53 response might be a novel personalized treatment approach in these tumors.

In Colon Epithelia, Clostridium perfringens Enterotoxin Causes Focal Leaks by Targeting Claudins Which are Apically Accessible Due to Tight Junction Derangement.

Clostridium perfringens enterotoxin (CPE) causes food poisoning and antibiotic-associated diarrhea. It uses some claudin tight junction proteins (eg, claudin-4) as receptors to form Ca2+-permeable pores in the membrane, damaging epithelial cells in small intestine and colon. We demonstrate that only a subpopulation of colonic enterocytes which are characterized by apical dislocation of claudins are CPE-susceptible. CPE-mediated damage was enhanced if paracellular barrier was impaired by Ca2+ depletion, proinflammatory cytokine tumor necrosis factor α, or dedifferentiation. Microscopy, Ca2+ monitoring, and electrophysiological data showed that CPE-mediated cytotoxicity and barrier disruption was limited by extent of CPE-binding. The latter was restricted by accessibility of non-junctional claudin molecules such as claudin-4 at apical membranes. Focal-leaks detected in HT-29/B6 colonic monolayers were verified for native tissue using colon biopsies. These mechanistic findings indicate how CPE-mediated effects may turn from self-limiting diarrhea into severe clinical manifestation such as colonic necrosis-if intestinal barrier dysfunction, eg, during inflammation facilitates claudin accessibility.

Rapid eradication of colon carcinoma by Clostridium perfringens Enterotoxin suicidal gene therapy.

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.

Repositioning of drugs for intervention in tumor progression and metastasis: Old drugs for new targets.

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.

Bacterial Toxins for Oncoleaking Suicidal Cancer Gene Therapy.

For suicide gene therapy, initially prodrug-converting enzymes (gene-directed enzyme-producing therapy, GDEPT) were employed to intracellularly metabolize non-toxic prodrugs into toxic compounds, leading to the effective suicidal killing of the transfected tumor cells. In this regard, the suicide gene therapy has demonstrated its potential for efficient tumor eradication. Numerous suicide genes of viral or bacterial origin were isolated, characterized, and extensively tested in vitro and in vivo, demonstrating their therapeutic potential even in clinical trials to treat cancers of different entities. Apart from this, growing efforts are made to generate more targeted and more effective suicide gene systems for cancer gene therapy. In this regard, bacterial toxins are an alternative to the classical GDEPT strategy, which add to the broad spectrum of different suicide approaches. In this context, lytic bacterial toxins, such as streptolysin O (SLO) or the claudin-targeted Clostridium perfringens enterotoxin (CPE) represent attractive new types of suicide oncoleaking genes. They permit as pore-forming proteins rapid and also selective toxicity toward a broad range of cancers. In this chapter, we describe the generation and use of SLO as well as of CPE-based gene therapies for the effective tumor cell eradication as promising, novel suicide gene approach particularly for treatment of therapy refractory tumors.

Immune Response and Metastasis-Links between the Metastasis Driver MACC1 and Cancer Immune Escape Strategies.

Metastasis remains the most critical factor limiting patient survival and the most challenging part of cancer-targeted therapy. Identifying the causal drivers of metastasis and characterizing their properties in various key aspects of cancer biology is essential for the development of novel metastasis-targeting approaches. Metastasis-associated in colon cancer 1 (MACC1) is a prognostic and predictive biomarker that is now recognized in more than 20 cancer entities. Although MACC1 can already be linked with many hallmarks of cancer, one key process-the facilitation of immune evasion-remains poorly understood. In this review, we explore the direct and indirect links between MACC1 and the mechanisms of immune escape. Therein, we highlight the signaling pathways and secreted factors influenced by MACC1 as well as their effects on the infiltration and anti-tumor function of immune cells.

Broadening the Scope of Sapofection: Cationic Peptide-Saponin Conjugates Improve Gene Delivery In Vitro and In Vivo.

Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo. In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.

MACC1 Regulates LGR5 to Promote Cancer Stem Cell Properties in Colorectal Cancer.

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.

Muscarinic receptor drug trihexyphenidyl can alter growth of mesenchymal glioblastoma in vivo.

Glioblastoma (GBM) is the most commonly occurring and most aggressive primary brain tumor. Transcriptomics-based tumor subtype classification has established the mesenchymal lineage of GBM (MES-GBM) as cancers with particular aggressive behavior and high levels of therapy resistance. Previously it was show that Trihexyphenidyl (THP), a market approved M1 muscarinic receptor-targeting oral drug can suppress proliferation and survival of GBM stem cells from the classical transcriptomic subtype. In a series of in vitro experiments, this study confirms the therapeutic potential of THP, by effectively suppressing the growth, proliferation and survival of MES-GBM cells with limited effects on non-tumor cells. Transcriptomic profiling of treated cancer cells identified genes and associated metabolic signaling pathways as possible underlying molecular mechanisms responsible for THP-induced effects. In vivo trials of THP in immunocompromised mice carry orthotopic MES-GBMs showed moderate response to the drug. This study further highlights the potential of THP repurposing as an anti-cancer treatment regimen but mode of action and d optimal treatment procedures for in vivo regimens need to be investigated further.