Colorectal Cancer Xenograft Model Research Articles

ROS/JNK/C-Jun Pathway is Involved in Chaetocin Induced Colorectal Cancer Cells Apoptosis and Macrophage Phagocytosis Enhancement.

There is an urgent need for novel agents for colorectal cancer (CRC) due to the increasing number of cases and drug-resistance related to current treatments. In this study, we aim to uncover the potential of chaetocin, a natural product, as a chemotherapeutic for CRC treatment. We showed that, regardless of 5-FU-resistance, chaetocin induced proliferation inhibition by causing G2/M phase arrest and caspase-dependent apoptosis in CRC cells. Mechanically, our results indicated that chaetocin could induce reactive oxygen species (ROS) accumulation and activate c-Jun N-terminal kinase (JNK)/c-Jun pathway in CRC cells. This was confirmed by which the JNK inhibitor SP600125 partially rescued CRC cells from chaetocin induced apoptosis and the ROS scavenger N-acetyl-L-cysteine (NAC) reversed both the chaetocin induced apoptosis and the JNK/c-Jun pathway activation. Additionally, this study indicated that chaetocin could down-regulate the expression of CD47 at both mRNA and protein levels, and enhance macrophages phagocytosis of CRC cells. Chaetocin also inhibited tumor growth in CRC xenograft models. In all, our study reveals that chaetocin induces CRC cell apoptosis, irrelevant to 5-FU sensitivity, by causing ROS accumulation and activating JNK/c-Jun, and enhances macrophages phagocytosis, which suggests chaetocin as a candidate for CRC chemotherapy.

A nanotherapeutic strategy to overcome chemoresistance to irinotecan/7-ethyl-10-hydroxy-camptothecin in colorectal cancer

Clinical development of 7-ethyl-10‑hydroxy-camptothecin (SN38), the active metabolite of irinotecan (CPT-11), is hindered by its insolubility and poor stability. Another obstacle is that tumors could become resistant to SN38/CPT-11 through multiple mechanisms involving breast cancer resistance protein (BCRP). Herein one of the most potent and selective BCRP inhibitors, Ko143, is encapsulated into a recently constructed prodrug PEG-S-S-SN38 displaying a high and fixed drug loading, multiple intratumoral stimuli (oxidative stress, GSH and esterase)-responsive drug release and significant in vitro and in vivo superiorities over CPT-11. The obtained “combo” for simultaneous delivery of SN38 and Ko143, named as BI@PEG-SN38, has a high SN38 loading efficacy (14.85 wt.%) and a good Ko143 encapsulation efficacy (3.79%). Through generating panels of human colorectal cancer models expressing altered levels of BCRP via lentiviral transfection and CRISPR-Cas9, characteristics of different drug formulations are carefully evaluated. Impressively, BI@PEG-SN38 nanoparticles effectively reverse chemoresistance to CPT-11 (resistance index dropping from ∼274.00–456.00 to ∼1.70–4.68) and PEG-S-S-SN38 (resistance index dropping from ∼5.83–14.00 to ∼1.70–4.68) in three BCRP-overexpressing cancer cell lines. More importantly, reversal of BCRP-mediated chemoresistance to CPT-11 (P values lower than 0.001–0.0001) and PEG-S-S-SN38 (P values lower than 0.01–0.001) by BI@PEG-SN38 nanoparticles are further confirmed with two panels of colorectal cancer xenograft models in vivo. As the first nano-formulation of Ko143 and the first systemic co-delivery vehicle of SN38/CPT-11 and a BCRP inhibitor, BI@PEG-SN38 provides a new approach for resolving the bottlenecks for clinical translation of SN38 and numerous “chemosensitizers” like Ko143, and exhibits promising applicability in precision cancer medicine. Statement of significanceTo resolve the bottlenecks in clinical application of anticancer agents SN38/CPT-11 and the most potent breast cancer resistant protein (BCRP) inhibitor Ko143, a “combo” nanotherapeutic simultaneously delivering SN38 and Ko143 was constructed and named as BI@PEG-SN38. By generating panels of colorectal cancer models, we demonstrate that BI@PEG-SN38 nanoparticles effectively and selectively reversed BCRP-mediated tumor resistance to SN38/CPT-11 in vitro and in vivo. As the first nano-formulation of Ko143 and the first systemic co-delivery vehicle of SN38/CPT-11 and a BCRP inhibitor, BI@PEG-SN38 provides a new strategy for clinical development of SN38 and numerous “chemosensitizers”, and exhibits promising applicability in precision cancer medicine. Panels of cancer cell lines established here provides a useful platform for BCRP- and cancer-related research and technology development.

Discovery of AS-0141, a Potent and Selective Inhibitor of CDC7 Kinase for the Treatment of Solid Cancers.

CDC7, a serine-threonine kinase, plays conserved and important roles in regulation of DNA replication and has been recognized as a potential anticancer target. We report here the optimization of a series of furanone analogues starting from compound 1 with a focus on ADME properties suitable for clinical development. By replacing the 2-chlorobenzene moiety in 1 with various aliphatic groups, we identified compound 24 as a potent CDC7 inhibitor with excellent kinase selectivity and favorable oral bioavailability in multiple species. Oral administration of 24 demonstrated robust in vivo antitumor efficacy in a colorectal cancer xenograft model. Compound 24 (AS-0141) is currently in phase I clinical trials for the treatment of solid cancers.

FBXW11 contributes to stem-cell-like features and liver metastasis through regulating HIC1-mediated SIRT1 transcription in colorectal cancer

Colorectal tumorigenesis is a heterogeneous disease driven by multiple genetic and epigenetic alterations. F-box and WD repeat domain containing 11 (FBXW11) is a member of the F-box protein family that regulates the ubiquitination of key factors associated with tumor growth and aggressiveness. Our study aimed to explore the role of FBXW11 in the development and metastasis of colorectal cancer (CRC). FBXW11 was overexpressed in colorectal tumor tissues and its overexpression was associated with a poor prognosis of CRC patients. The upregulation of FBXW11 not only promoted cell proliferation, invasion, and migration, but also contributed to maintaining stem-cell features in colorectal tumor cells. Further analysis revealed that FBXW11 targeted hypermethylated in cancer 1 (HIC1) and reduced its stability in CRC cells through ubiquitination. Moreover, the expression of sirtuin 1 (SIRT1), a deacetylase in tumor cells was upregulated by FBXW11 via regulating HIC1 expression. The mouse xenograft models of CRC confirmed that FBXW11 knockdown impeded colorectal tumor growth and liver metastasis in vivo. In summary, our study identified FBXW11 as an oncogenic factor that contributed to stem-cell-like properties and liver metastasis in CRC via regulating HIC1-mediated SIRT1 expression. These results provide a rationale for the development of FBXW11-targeting drugs for CRC patients.

LDH-A negatively regulates dMMR in colorectal cancer.

Although immune checkpoint inhibitors (ICIs) have achieved unprecedented success in dMMR tumors, pMMR tumors accounting for 85% of colorectal cancer (CRC) cases remain unresponsive. Lactate dehydrogenase A (LDH‐A) is the rate‐limiting enzyme that catalyzes the transformation of pyruvate to lactate in the process of glycolysis. We investigated the relationship between LDH‐A and dMMR with the purpose of exploring the treatment strategy for pMMR CRC patients. We here show that LDH‐A can promote the proliferation of dMMR and pMMR CRC cells by positively regulating MMR proteins both in vitro and in vivo. LDH‐A inhibition can improve the efficacy of PD‐1 blockade in a pMMR CRC xenograft model. A statistical analysis of 186 CRC specimens showed a significant correlation between LDH‐A and dMMR status. Moreover, patients with both low LDH‐A expression and dMMR exhibited better disease‐free survival compared with patients with other combinations. The close correlation of LDH‐A and dMMR may offer a promising therapeutic strategy in which the combination of LDH‐A inhibitor and ICIs may improve the clinical benefit for pMMR CRC patients.

Abstract 1454: Antitumor activity of a selective FAK/Pyk2 inhibitor, SJP1602 against TNBC as well as CRC

Abstract Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that serves as a key mediator in response to integrin and growth factor signaling to control multiple aspects of cellular behavior. High expression and/or aberrant activation of FAK is observed in a broad range of human cancers, contributing to malignant phenotype in cancer and development of tumor resistance. FAK, therefore, has been a prominent target in oncology and a small number of FAK inhibitors are currently in clinical development. However, selective FAK inhibitors have shown insufficient antitumor responses due to a potential compensatory role of proline-rich tyrosine kinase 2 (Pyk2), a homologue of FAK, and require dual specific FAK/Pyk2 inhibition. SJP1602 is a novel dual inhibitor of FAK and Pyk2 without multi-kinase inhibition. We have previously reported that SJP1602 is highly specific against FAK and Pyk2 and its antitumor efficacy in triple negative breast cancer (TNBC) is superior over the clinically active FAK inhibitors. In this study, we further assessed pharmacological efficacy of SJP1602 in another type of cancer, colorectal cancer (CRC). As evaluated by cell-based ELISA, SJP1602 has nanomolar potency against FAK and Pyk2 in a panel of CRC cell lines. Validation through immunoblotting analyses revealed significant reduction of FAK/Pyk2 activation by treatment of SJP1602 in CRC cell lines, consistent with previous observations in TNBC cell lines. We then investigated the inhibition efficiency of SJP1602 in three-dimensional (3D) spheroid models using the CRC cell lines. As determined by CellTiter-Glo 3D cell viability assay, SJP1602 showed the significantly lower IC50 values than other FAK inhibitors. Next, to investigate the mechanism of action (MOA) of SJP1602, we examined changes in activation of several FAK/Pyk2-mediated signaling pathways. As shown by immunoblotting analyses, SJP1602 decreased JAK2 and Akt activity and increased the expression levels of cleaved caspase 3, a critical marker of apoptosis. Further assessment of SJP1602 in vivo showed significant antitumor activities in different human CRC xenograft models containing BRAF-mutant or KRAS-mutant tumors. In addition, the specificity and MOA of SJP1602 were confirmed using xenograft tumors. In consistent with in vitro results, SJP1602 reduced JAK2 and Akt activity in tumors from mice with SJP1602 administration. Therefore, our results demonstrate that SJP1602 has the strong antitumor efficacy in vitro and in vivo via downregulation of JAK2 and PI3K/Akt signaling pathways. SJP1602 is currently undergoing preclinical toxicology studies and in vitro safety evaluation. Taken together, our data provides promising preclinical results for the further development of SJP1602 as a potent anti-cancer agent in TNBC, CRC and resistant cancer involving FAK activation. Citation Format: Kug Hwa Lee, Hyoung Min Cho, Yongbin Park, Sungpyo Hong, Soon Kil Ahn, Min-Hyo Ki. Antitumor activity of a selective FAK/Pyk2 inhibitor, SJP1602 against TNBC as well as CRC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1454.

LncRNA NEAT1 facilitates the progression of colorectal cancer via the KDM5A/Cul4A and Wnt signaling pathway.

Colorectal cancer (CRC) is a major cause of cancer‑related mortality. The aberrant expression of long non‑coding RNAs (lncRNAs) is implicated in the pathogenesis of CRC. The present study investigated the role of lncRNA nuclear paraspeckle assembly transcript1 (NEAT1) in CRC. lncRNA NEAT1 expression was detected in CRC tissues and cell lines. HCT116 cells were transfected with si‑NEAT1, and the malignant behavior of the cells was detected. The binding associations between NEAT1 and E2F1, as well as between E2F1 and KDM5A were verified. si‑NEAT1‑transfected cells were also transfected with si‑KDM5A. H3K4me3 methylation and cullin4A (Cul4A) expression in HCT116 cells were detected. The si‑NEAT1‑transfected cells were also transfected with pc‑Cul4A. Proteins related to the Wnt pathway were detected. A xenograft model of CRC using nude mice was established and the mice were injected with si‑NEAT1‑transfected HCT116 cells. lncRNA NEAT1 was found to be upregulated in CRC tissues and cells. NEAT1 silencing inhibited the malignant behaviors of the HCT116 cells. lncRNA NEAT1 inhibited KDM5A expression by binding to E2F1. The downregulation of KDM5A reversed the inhibitory effects of NEAT1 silencing on the malignant behavior of the cells. KDM5A inhibited Cul4A expression via the demethylation of H3K4me3. The overexpression of Cul4A promoted the malignant behavior of the si‑NEAT1‑transfected HCT116 cells. lncRNA NEAT1 activated the Wnt pathway via KDM5A/Cul4A. Invivo experiments confirmed the role of NEAT1 in CRC. On the whole, the present study demonstrates that lncRNA NEAT1 facilitates the progression of CRC via the KDM5A/Cul4A/Wnt axis.

Therapeutic Uses of Bacterial Subunit Toxins.

The B subunit pentamer verotoxin (VT aka Shiga toxin-Stx) binding to its cellular glycosphingolipid (GSL) receptor, globotriaosyl ceramide (Gb3) mediates internalization and the subsequent receptor mediated retrograde intracellular traffic of the AB5 subunit holotoxin to the endoplasmic reticulum. Subunit separation and cytosolic A subunit transit via the ER retrotranslocon as a misfolded protein mimic, then inhibits protein synthesis to kill cells, which can cause hemolytic uremic syndrome clinically. This represents one of the most studied systems of prokaryotic hijacking of eukaryotic biology. Similarly, the interaction of cholera AB5 toxin with its GSL receptor, GM1 ganglioside, is the key component of the gastrointestinal pathogenesis of cholera and follows the same retrograde transport pathway for A subunit cytosol access. Although both VT and CT are the cause of major pathology worldwide, the toxin–receptor interaction is itself being manipulated to generate new approaches to control, rather than cause, disease. This arena comprises two areas: anti neoplasia, and protein misfolding diseases. CT/CTB subunit immunomodulatory function and anti-cancer toxin immunoconjugates will not be considered here. In the verotoxin case, it is clear that Gb3 (and VT targeting) is upregulated in many human cancers and that there is a relationship between GSL expression and cancer drug resistance. While both verotoxin and cholera toxin similarly hijack the intracellular ERAD quality control system of nascent protein folding, the more widespread cell expression of GM1 makes cholera the toxin of choice as the means to more widely utilise ERAD targeting to ameliorate genetic diseases of protein misfolding. Gb3 is primarily expressed in human renal tissue. Glomerular endothelial cells are the primary VT target but Gb3 is expressed in other endothelial beds, notably brain endothelial cells which can mediate the encephalopathy primarily associated with VT2-producing E. coli infection. The Gb3 levels can be regulated by cytokines released during EHEC infection, which complicate pathogenesis. Significantly Gb3 is upregulated in the neovasculature of many tumours, irrespective of tumour Gb3 status. Gb3 is markedly increased in pancreatic, ovarian, breast, testicular, renal, astrocytic, gastric, colorectal, cervical, sarcoma and meningeal cancer relative to the normal tissue. VT has been shown to be effective in mouse xenograft models of renal, astrocytoma, ovarian, colorectal, meningioma, and breast cancer. These studies are herein reviewed. Both CT and VT (and several other bacterial toxins) access the cell cytosol via cell surface ->ER transport. Once in the ER they interface with the protein folding homeostatic quality control pathway of the cell -ERAD, (ER associated degradation), which ensures that only correctly folded nascent proteins are allowed to progress to their cellular destinations. Misfolded proteins are translocated through the ER membrane and degraded by cytosolic proteosome. VT and CT A subunits have a C terminal misfolded protein mimic sequence to hijack this transporter to enter the cytosol. This interface between exogenous toxin and genetically encoded endogenous mutant misfolded proteins, provides a new therapeutic basis for the treatment of such genetic diseases, e.g., Cystic fibrosis, Gaucher disease, Krabbe disease, Fabry disease, Tay-Sachs disease and many more. Studies showing the efficacy of this approach in animal models of such diseases are presented.

Helicase-like transcription factor-deletion from the tumor microenvironment in a cell line-derived xenograft model of colorectal cancer reprogrammed the human transcriptome-S-nitroso-proteome to promote inflammation and redirect metastasis.

Methylation of the HLTF gene in colorectal cancer (CRC) cells occurs more frequently in men than women. Progressive epigenetic silencing of HLTF in tumor cells is accompanied by negligible expression in the tumor microenvironment (TME). Cell line-derived xenografts (CDX) were established in control (Hltf+/+) and Hltf-deleted male Rag2-/-IL2rg-/- mice by direct orthotopic cell microinjection (OCMI) of HLTF+/+HCT116 Red-FLuc cells into the submucosa of the cecum. Combinatorial induction of IL6 and S100A8/A9 in the Hltf-deleted TME with ICAM-1 and IL8 in the primary tumor activated a positive feedback loop. The proinflammatory niche produced a major shift in CDX metastasis to peritoneal dissemination compared to controls. Inducible nitric oxide (iNOS) gene expression and transactivation of the iNOS-S100A8/A9 signaling complex in Hltf-deleted TME reprogrammed the human S-nitroso-proteome. POTEE, TRIM52 and UN45B were S-nitrosylated on the conserved I/L-X-C-X2-D/E motif indicative of iNOS-S100A8/A9-mediated S-nitrosylation. 2D-DIGE and protein identification by MALDI-TOF/TOF mass spectrometry authenticated S-nitrosylation of 53 individual cysteines in half-site motifs (I/L-X-C or C-X-X-D/E) in CDX tumors. POTEE in CDX tumors is both a general S-nitrosylation target and an iNOS-S100A8/A9 site-specific (Cys638) target in the Hltf-deleted TME. REL is an example of convergence of transcriptomic-S-nitroso-proteomic signaling. The gene is transcriptionally activated in CDX tumors with an Hltf-deleted TME, and REL-SNO (Cys143) was found in primary CDX tumors and all metastatic sites. Primary CDX tumors from Hltf-deleted TME shared 60% of their S-nitroso-proteome with all metastatic sites. Forty percent of SNO-proteins from primary CDX tumors were variably expressed at metastatic sites. Global S-nitrosylation of proteins in pathways related to cytoskeleton and motility was strongly implicated in the metastatic dissemination of CDX tumors. Hltf-deletion from the TME played a major role in the pathogenesis of inflammation and linked protein S-nitrosylation in primary CDX tumors with spatiotemporal continuity in metastatic progression when the tumor cells expressed HLTF.

SOX2 promotes chemoresistance, cancer stem cells properties, and epithelial\u2013mesenchymal transition by \u03b2-catenin and Beclin1/autophagy signaling in colorectal cancer

Sex-determining region Y-box2 (SOX2), a master regulator of embryonic and induced pluripotent stem cells, drives cancer stem cells (CSCs) properties, fuels tumor initiation, and contributes to tumor aggressiveness. Our previous study has demonstrated the oncogenic role of SOX2 in colorectal cancer (CRC). In this study, we sought to elucidate the underlying mechanisms. Cell function experiments were performed to detect chemoresistance, proliferation, stemness, migration, and invasion in vitro. Chromatin immunoprecipitation, co-immunoprecipitation, luciferase reporter assay, and immunofluorescence were performed to explore the regulation of ABCC2, β-catenin, and Beclin1 by SOX2. The carcinogenic role of SOX2-β-catenin/Beclin1-ABCC2 axis in vivo was analyzed by CRC tissues and xenograft models. Here, we reported that SOX2 sustained chemoresistance by transcriptional activation of ABCC2 expression. Suppressing either β-catenin or autophagy signaling curbed SOX2-driven chemoresistance, stemness, and epithelial–mesenchymal transition (EMT). Mechanistically, SOX2 combined with β-catenin and increased its nuclear expression and transcriptional activity. Transcriptional activation of Beclin1 expression by SOX2 consequently activating autophagy and inducing malignant phenotype. Furthermore, overexpression of β-catenin or Beclin1 facilitated ABCC2 expression. The clinical analyses showed that high expression of ABCC2 and Beclin1 were positively correlated with SOX2 and were associated with poor prognosis in CRC patients. Finally, xenograft models revealed that inhibition of SOX2 expression and autophagy restrained tumor growth and chemoresistance in vivo. Conclusively, we demonstrated a novel mechanism by which the SOX2-β-catenin/Beclin1/autophagy signaling axis regulates chemoresistance, stemness, and EMT in CRC. Our findings provide novel insights into CRC carcinogenesis and may help develop potential therapeutic candidates for CRC.

Targeted microbubbles carrying lipid-oil-nanodroplets for ultrasound-triggered delivery of the hydrophobic drug, combretastatin A4

The hydrophobicity of a drug can be a major challenge in its development and prevents the clinical translation of highly potent anti-cancer agents. We have used a lipid-based nanoemulsion termed Lipid-Oil-Nanodroplets (LONDs) for the encapsulation and in vivo delivery of the poorly bioavailable combretastatin A4 (CA4). Drug delivery with CA4 LONDs was assessed in a xenograft model of colorectal cancer. LC–MS/MS analysis revealed that CA4 LONDs, administered at a drug dose four times lower than drug control, achieved equivalent concentrations of CA4 intratumorally. We then attached CA4 LONDs to microbubbles (MBs) and targeted this construct to VEGFR2. A reduction in tumor perfusion was observed in CA4 LONDs-MBs treated tumors. A combination study with irinotecan demonstrated a greater reduction in tumor growth and perfusion (P = 0.01) compared to irinotecan alone. This study suggests that LONDs, either alone or attached to targeted MBs, have the potential to significantly enhance tumor-specific hydrophobic drug delivery.

Utidelone inhibits growth of colorectal cancer cells through ROS/JNK signaling pathway

Utidelone (UTD1), a novel microtubule stabilizing agent, is an epothilone B analogue which was produced by genetic engineering. UTD1 has exhibited broad antitumor activity in multiple solid tumors. However, its activity and mechanism in colorectal cancer (CRC) remain to be studied. In this study, UTD1 dramatically inhibited CRC cell proliferation (with 0.38 µg/ml, 0.77 µg/ml IC50 in RKO and HCT116, respectively) in vitro. Immunofluorescence staining showed that UTD1 induced the formation of microtubule bundling and asters in RKO cells. Flow cytometry analysis demonstrated that UTD1 induced cell cycle to arrest in G2/M phase, subsequent apoptosis. Significantly, UTD1 exhibited stronger effect on inducing apoptosis than paclitaxel and 5-FU, especially in HCT15 cells which is ABCB1 high-expression. UTD1 exposure cleaved caspase-3 and poly ADP-ribose polymerase (PARP), decreased mitochondrial membrane potential, released cytochrome c, increased the production of active oxygen and activated c-Jun N-terminal kinase (JNK), suggesting ROS/JNK pathway was involved in this process. Moreover, UTD1 inhibited tumor growth and was more effective and safer compared with paclitaxel and 5-FU in RKO xenograft in nude mice. Taken together, our findings first indicate that UDT1 inhibits tumor growth in CRC xenograft model and may be a promising agent for CRC treatment.

A Novel Fluorescent c-Met Targeted Imaging Agent for Intra-Operative Colonic Tumour Mapping: Translation from the Laboratory into a Clinical Trial

Background: Curative resection of colon cancer requires accurate intraoperative identification of tumour margins and lymph node (LN) burden. The c-Met protein is important for cell differentiation and migration and is overexpressed in many gastrointestinal cancers. We explored EMI-137, a novel c-Met targeting fluorescent probe, for application in fluorescence-guided colon surgery, in HT-29 colorectal cancer (CRC) cell line and an in vivo murine model. This informed a first-in-man clinical trial of EMI-137. Methods: HT-29 cells were used as a representative CRC model. SiRNA was used to demonstrate the specificity of EMI-137 for c-Met. A HT-29 CRC xenograft model was developed in female BALB/c mice. Tumours were cultured for 14 days to 10mm diameter. EMI-137 (0.18mg/kg) was injected into tail veins and biodistribution analysed through in vivo fluorescent imaging. Nine patients, aged 67-77 years, received a single intravenous dose of EMI-137 (0.13mg/kg), 1-3 hours before laparoscopic-assisted colon cancer resection surgery (NCT03360461). Tumour and LN fluorescence was assessed with a modified Karl Storz® laparoscope. Immunohistochemistry with a monoclonal antibody analysed c-Met expression in eight resected samples. Findings: c-Met expression in HT-29 cells was effectively silenced and imaged with EMI-137. Strong EMI-137 uptake in tumour xenografts was observed for up to 6 hours post-administration. At clinical trial, no serious adverse events related to EMI-137 were reported. Marked background fluorescence was observed in all trial participants, 4/9 showed an increase in tumour fluorescence over background; 5/9 had histological LN metastases; no fluorescent LN were detected intraoperatively. All primary tumours (8/8) and malignant LN (15/15) exhibited moderate to high c-Met protein expression. Interpretation: EMI-137, binds specifically to the human c-Met transmembrane protein, is safe in patients and with further refinement, shows potential for long-term application in fluorescence-guided colon surgery. Trial Registration: The trial was registered with ClinicalTrials.gov identifier NCT03360461. Funding: Research was supported by internal University of Leeds funding. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Ethical approval was granted by the Yorkshire and Humber Leeds West Research and Ethics Committee (Ref 17/YH/0263), on 19th October 2017 and Regulatory approval was granted by the Medicines and Healthcare products Regulatory Agency (MHRA), under The Medicines for Human Use (Clinical Trials) regulations on 29th August 2017.

Establishment of a Patient-Derived Xenograft Model of Colorectal Cancer in CIEA NOG Mice and Exploring Smartfish Liquid Diet as a Source of Omega-3 Fatty Acids.

Cancer patient-derived xenografts (PDXs) better preserve tumor characteristics and microenvironment than traditional cancer cell line derived xenografts and are becoming a valuable model in translational cancer research and personalized medicine. We have established a PDX model for colorectal cancer (CRC) in CIEA NOG mice with a 50% engraftment rate. Tumor fragments from patients with CRC (n = 5) were engrafted in four mice per tumor (n = 20). Mice with established PDXs received a liquid diet enriched with fish oil or placebo, and fatty acid profiling was performed to measure fatty acid content in whole blood. Moreover, a biobank consisting of tissue and blood samples from patients was established. Histology, immunohistochemistry and in situ hybridization procedures were used for staining of tumor and xenograft tissue slides. Results demonstrate that key histological characteristics of the patients’ tumors were retained in the established PDXs, and the liquid diets were consumed as intended by the mice. Some of the older mice developed lymphomas that originated from human Ki67+, CD45+, and EBV+ lymphoid cells. We present a detailed description of the process and methodology, as well as possible issues that may arise, to refine the method and improve PDX engraftment rate for future studies. The established PDX model for CRC can be used for exploring different cancer treatment regimes, and liquid diets enriched with fish oil may be successfully delivered to the mice through the drinking flasks.

Inhibition of nucleophosmin 1 suppresses colorectal cancer tumor growth of patient -derived xenografts via activation of p53 and inhibition of AKT

ABSTRACT The nucleophosmin 1 (NPM1) protein is frequently overexpressed in various cancers compared to normal tissues and represents a potential biomarker for maliganancy. However, its role in colorectal cancer (CRC) is still not fully understood. In this report, we show that NPM1 levels in CRC correlate with prognosis and sensitivity to chemotherapy. NPM1 expression was found to be significantly increased in CRC tumors (P < .001) and was associated with poor overall 5-year survival (P < .05). For individuals with Stage IV disease, this represented a reduction in survival by 11 months (P < .01; HR = 0.38, CI [0.21, 0.69]. In vitro, we show that NPM1 gene silencing enhanced the chemosensitivity of CRC cells and that pharmacological inhibition of NPM1 by NSC348884 triggered the onset of programmed cell death. Our immunofluorescence microscopy and immunoblot analyses also revealed that blocking NPM1 function sensitized CRC cells to chemotherapy-induced apoptosis through a mechanism that involves proteins in the AKT pathway. Consistent with the in vitro data, our patient-derived CRC xenograft model showed that inhibition of NPM1 suppressed tumor growth and attenuated AKT signaling in vivo. Moreover, LY294002, an inhibitor of the PI3K/AKT pathway, restored the chemosensitivity of CRC cells expressing high levels of NPM1. The findings that NPM1’s expression in CRC tissue correlates with prognosis and supports anti-apoptotic activity mediated by AKT signaling, further our understanding of the role of NPM1 in CRC.

Continuous formation of small clusters with LGR5-positive cells contributes to tumor growth in a colorectal cancer xenograft model

AbstractNew cancer characteristics can be discovered by focusing on the process of tumor formation. Cancer stem cells (CSCs) are a key subpopulation, as they are theorized to be at the apex of the tumor hierarchy. We can better understand their function in the tumor hierarchy by using sectioned samples to observe the growth of tumors from their origins as CSCs. In this study, we evaluated the growth of moderate differentiated colorectal cancer from LGR5-positive cells, which is a CSC marker of colorectal cancer, using xenograft and three-dimensional culture models spatiotemporally. These cells express LGR5 at high levels and show CSC phenotypes. To detect them, we used a previously generated antibody that specifically targets LGR5, and were therefore able to observe LGR5-positive cells aggregating into small clusters (sCLs) over the course of tumor growth. Because these LGR5-expressing sCLs formed continuously during growth mainly in the invasive front, we concluded that the structure must contribute significantly to the expansion of CSCs and to tumor growth overall. We confirmed the formation of sCLs from gland structures using a three-dimensional culture model. In addition, sCLs exhibited upregulated genes related to stress response and partial/hybrid epithelial–mesenchymal transition (EMT), as well as genes reported to be prognosis factors. Finally, sCLs with high LGR5 expression were identified in clinical samples. Based on these results, we elucidate how sCLs are an important contributors to tumor growth and the expansion of CSCs.This study demonstrates that small clusters (sCLs) of tumor cells with high expression of LGR5 continuously form in the invasive front in a colorectal cancer xenograft model. This structure is characterized by stress response and partial/hybrid epithelial-mesenchymal transition. These sCLs are an important contributor to tumor growth and the expansion of cancer stem cells.

Long non-coding RNA COL4A2-AS1 facilitates cell proliferation and glycolysis of colorectal cancer cells via miR-20b-5p/hypoxia inducible factor 1 alpha subunit axis

ABSTRACT Long non-coding RNAs (lncRNAs) have critical functions in tumorigenesis and progression of colorectal cancer (CRC). The role of lncRNA COL4A2-AS1 (COL4A2-AS1) lacks system investigation. The current study comprehensively analyzed the expression, biological functions, and mechanism of COL4A2-AS1 in CRC through performing real-time quantitative PCR (RT-qPCR), Western blot, cell transfection, cell colony assay, MTT assay, flow cytometry and dual-luciferase reporter system assays. A xenograft model of CRC was constructed to further verify the function of COL4A2-AS1 in CRC progression in vivo. The data revealed an upregulated expression of COL4A2-AS1 in CRC tissues and cell lines than paired adjacent tissues and normal cell line. Silencing COL4A2-AS1 inhibited proliferation, aerobic glycolysis, and promoted apoptosis of CRC cells in vivo and in vitro. However, overexpression of COL4A2-AS1 significantly promoted CRC cell proliferation and aerobic glycolysis. In CRC cells, miR-20b-5p was sponged by COL4A2-AS1 and hypoxia-inducible factor 1 alpha subunit (HIF1A). Restoration of HIF1A expression reversed the inhibitory effects of silencing COL4A2-AS1 on aerobic glycolysis and proliferation of CRC cells. The current findings showed that COL4A2-AS1 promoted the proliferation, and aerobic glycolysis of CRC cells potentially through modulating the miR-20b-5p/HIF1A axis.

Aberrant SLC6A14 Expression Promotes Proliferation and Metastasis of Colorectal Cancer via Enhancing the JAK2/STAT3 Pathway.

BackgroundSolute carrier family 6 member 14 (SLC6A14) is a high-capacity amino acid transporter in mammalian cells. It has gained increasing attention for its potential involvement in the progression and metabolic reprogramming of various malignant tumors. However, the role of SLC6A14 in colorectal cancer (CRC) remains unclear.MethodsReal-time polymerase chain reaction (qRT-PCR), immunoblotting and immunohistochemistry were carried out to detect the expression level of SLC6A14 in human CRC tissues and CRC-derived cell lines. HCT-116 and Caco-2 cell lines were selected to conduct in vitro functional studies. Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, cell migration and invasion assays were performed to investigate the role of SLC6A14 in CRC cells. Besides, azoxymethane/dextran sulfate sodium salt (AOM/DSS)-induced CRC and tumor xenograft models were constructed to explore the effects of SLC6A14 blockade or overexpression during tumor progression in vivo.ResultsSLC6A14 was substantially increased in human CRC samples and higher levels of SLC6A14 was correlated with advanced tumor stage, lymph node metastasis and dismal survival of CRC patients. SLC6A14 markedly promoted cell growth, inhibited cell apoptosis, and exacerbated migration and invasion of CRC cells in vitro. Mechanistically, SLC6A14 aggravated these malignant phenotypes through activating JAK2/STAT3 signaling pathway, and inhibiting JAK2/STAT3 signaling with specific inhibitors could reverse SLC6A14-mediated tumorigenic effects. Besides, two different animal studies verified the tumor-promoting effect of SLC6A14 in CRC.ConclusionOur data illustrated the crucial function of SLC6A14 during CRC progression, suggesting SLC6A14/JAK2/STAT3 axis may serve as novel therapeutic targets for patients with CRC.

LncRNA-PACER upregulates COX-2 and PGE2 through the NF-κB pathway to promote the proliferation and invasion of colorectal-cancer cells.

Backgroundp50-associated cyclooxygenase-2 extragenic RNA (PACER) is a recently identified antisense long non-coding RNA (lncRNA) located on the upstream of the promoter region of cyclooxygenase-2 (COX-2). Preliminary studies have suggested that PACER is involved in the regulation of COX-2 expression in macrophagocyte and osteosarcoma cells. However, the role of this lncRNA in colorectal cancer (CRC) remains elusive. Here, we investigated the expression of PACER and its effect on cell proliferation and invasion to explore the role of PACER in CRC.MethodsReal-time quantitative PCR (RT-qPCR) analysis was used to evaluate the expression of PACER in CRC tissues and cells. Methyl thiazolyl tetrazolium (MTT) analysis was then used to investigate the inhibition effect of PACER knock-down in cell proliferation. The promoting role of this lncRNA on invasion by CRC cells was analysed by wound-healing assays, colony-formation assay, and transwell assays. We then used fluorescence in situ hybridization (FISH) to establish the subcellular localization of PACER. COX-2 protein levels were quantified by Western blot analysis and grayscale scanning analysis following the knock-down of PACER. Luciferase assay was carried out to monitor the modulation of the COX-2 promoter region by PACER. Tumor xenografts models were used to investigate the impact of PACER on the tumorigenesis of CRC cells in vivo. Enzyme-linked immunosorbent assay (ELISA) was then used to quantify prostaglandin E2 (PGE2) production upon knock-down of PACER.ResultsRT-qPCR analysis revealed that PACER was highly expressed in CRC tissues and cells, and a high PACER-expression level was associated with poor prognosis. MTT assay, wound-healing assay, colony-formation assay, and transwell assay revealed that PACER enhanced CRC-cell proliferation, invasion, and metastasis in vitro. Analysis of lncRNA localization by FISH showed that it mainly resided in the nucleus. RT-qPCR showed that PACER increased mRNA levels of COX-2. Western blot analysis demonstrated, under normal circumstances, that knock-down of PACER decreased the COX-2 protein level. In the case of p50 absence, COX-2 protein increased rapidly and remained highly expressed after knocking down PACER. Luciferase assay revealed that PACER modulated the COX-2 promoter region. Mouse xenograft models of CRC revealed that PACER promoted colorectal tumorigenesis in vivo. ELISA revealed that PACER knock-down inhibited PGE2 production.ConclusionsPACER modulates COX-2 expression through the nuclear factor kappa B (NF-κB) pathway in CRC. An increased level of PACER enhances proliferation, migration, and invasion of tumor cells by increasing COX-2 and PGE2 synthesis.

Differential Ser phosphorylation of vasodilator-stimulated phosphoprotein regulates colon tumor formation and growth

AimsVasodilator-stimulated phosphoprotein (VASP) controls actin dynamics associated with the malignant phenotype of colorectal tumors. Oncogenic VASP function, in turn, is finely regulated by cyclic nucleotide-dependent phosphorylation of serine (Ser) residues 157 and 239, whose differential expression determines cell survival behavior in colon cancer. However, the role of differential VASP Ser phosphorylation in colorectal carcinogenesis remains unclear. Main methodsSpecific VASP phosphomutant constructs were employed to selectively silence Ser157 or Ser239 phosphorylation in human colon carcinoma cells. Cyclic nucleotide-dependent manipulation of VASP Ser phosphorylation was performed with 8-bromoadenosine 3′,5′-cyclic adenosine monophosphate (8-Br-cAMP) or 8-chlorophenylthio 3′,5′-cyclic guanosine monophosphate (8-CPT-cGMP). Tumorigenic and locomotory phenotypes were examined in vitro with clonogenic and wound healing assays, respectively. Finally, tumor formation and growth were investigated in vivo employing two distinct xenograft models of colorectal cancer. Key findingsDisruption of VASP Ser157 phosphorylation weakened the clonogenic and migratory abilities of human colon cancer cells, effects mimicked by 8-CPT-cGMP-dependent regulation of VASP Ser239. In contrast, inhibition of VASP Ser239 phosphorylation enhanced cell clonogenicity and migration and was phenocopied by 8-Br-cAMP-dependent regulation of VASP Ser157. Importantly, cancer cells bearing the phosphomutant construct targeting VASP Ser157 decreased, while those with the phosphomutation at Ser239 improved their abilities to establish productive tumor colonies and grow in the peritoneal cavity or subcutaneous tissues of nude mice. SignificanceTogether, present observations suggest differential VASP Ser phosphorylation is a relevant, targetable molecular event underlying tumor formation and progression in colon cancer.