Cytotoxic Efficacy and Resistance Mechanism of a TRAIL and VEGFA-Peptide Fusion Protein in Colorectal Cancer Models

TNF-related apoptosis-inducing ligand (TRAIL) is a type II transmembrane protein capable of selectively inducing apoptosis in cancer cells by binding to its cognate receptors. Here, we examined the anticancer efficacy of a recently developed chimeric AD-O51.4 protein, a TRAIL fused to the VEGFA-originating peptide. We tested AD-O51.4 protein activity against human colorectal cancer (CRC) models and investigated the resistance mechanism in the non-responsive CRC models. The quantitative comparison of apoptotic activity between AD-O51.4 and the native TRAIL in nine human colorectal cancer cell lines revealed dose-dependent toxicity in seven of them; the immunofluorescence-captured receptor abundance correlated with the extent of apoptosis. AD-O51.4 reduced the growth of CRC patient-derived xenografts (PDXs) with good efficacy. Cell lines that acquired AD-O51.4 resistance showed a significant decrease in surface TRAIL receptor expression and apoptosis-related proteins, including Caspase-8, HSP60, and p53. These results demonstrate the effectiveness of AD-O51.4 protein in CRC preclinical models and identify the potential mechanism underlying acquired resistance. Progression of AD-O51.4 to clinical trials is expected.     

Circulating miRNA Expression Profiles and Machine Learning Models in Association with Response to Irinotecan-Based Treatment in Metastatic Colorectal Cancer

Colorectal cancer represents a leading cause of cancer-related morbidity and mortality. Despite improvements, chemotherapy remains the backbone of colorectal cancer treatment. The aim of this study is to investigate the variation of circulating microRNA expression profiles and the response to irinotecan-based treatment in metastatic colorectal cancer and to identify relevant target genes and molecular functions. Serum samples from 95 metastatic colorectal cancer patients were analyzed. The microRNA expression was tested with a NucleoSpin miRNA kit (Machnery-Nagel, Germany), and a machine learning approach was subsequently applied for microRNA profiling. The top 10 upregulated microRNAs in the non-responders group were hsa-miR-181b-5p, hsa-miR-10b-5p, hsa-let-7f-5p, hsa-miR-181a-5p, hsa-miR-181d-5p, hsa-miR-301a-3p, hsa-miR-92a-3p, hsa-miR-155-5p, hsa-miR-30c-5p, and hsa-let-7i-5p. Similarly, the top 10 downregulated microRNAs were hsa-let-7d-5p, hsa-let-7c-5p, hsa-miR-215-5p, hsa-miR-143-3p, hsa-let-7a-5p, hsa-miR-10a-5p, hsa-miR-142-5p, hsa-miR-148a-3p, hsa-miR-122-5p, and hsa-miR-17-5p. The upregulation of microRNAs in the miR-181 family and the downregulation of those in the let-7 family appear to be mostly involved with non-responsiveness to irinotecan-based treatment.     

4-Acetyl-Antroquinonol B Improves the Sensitization of Cetuximab on Both Kras Mutant and Wild Type Colorectal Cancer by Modulating the Expression of Ras/Raf/miR-193a-3p Signaling Axis

The KRAS mutation is one of the leading driver mutations in colorectal cancer (CRC), and it is usually associated with poor prognosis and drug resistance. Therapies targeting the epidermal growth factor receptor (EFGR) are widely used for end-stage CRC. However, patients with KRAS mutant genes cannot benefit from this therapy because of Ras signaling activation by KRAS mutant genes. Our previous study revealed the anti-proliferative effect of 4-acetyl-antroquinonol B (4-AAQB) on CRC cells, but whether the drug is effective in KRAS-mutant CRC remains unknown. We screened CRC cell lines harboring the KRAS mutation, namely G12A, G12C, G12V and G13D, with one wild type cell line as the control; SW1463 and Caco-2 cell lines were used for further experiments. Sulforhodamine B assays, together with the clonogenicity and invasion assay, revealed that KRAS-mutant SW1463 cells were resistant to cetuximab; however, 4-AAQB treatment effectively resensitized CRC cells to cetuximab through the reduction of colony formation, invasion, and tumorsphere generation and of oncogenic KRAS signaling cascade of CRC cells. Thus, inducing cells with 4-AAQB before cetuximab therapy could resensitize KRAS-mutant, but not wild-type, cells to cetuximab. Therefore, we hypothesized that 4-AAQB can inhibit KRAS. In silico analysis of the publicly available GEO ({"type":"entrez-geo","attrs":{"text":"GSE66548","term_id":"66548"}}GSE66548) dataset of KRAS-mutated versus KRAS wild-type CRC patients confirmed that miR-193a-3p was significantly downregulated in the former compared with the latter patient population. Overexpression of miR-193a-3p considerably reduced the oncogenicity of both CRC cells. Furthermore, KRAS is a key target of miR-193a-3p. In vivo treatment with the combination of 4-AAQB and cetuximab significantly reduced the tumor burden of a xenograft mice model through the reduction of the expression of oncogenic markers (EGFR) and p-MEK, p-ERK, and c-RAF/p-c-RAF signaling, with the simultaneous induction of miR-193a-3p expression in the plasma. In summary, our findings provide strong evidence regarding the therapeutic effect of 4-AAQB on KRAS-mutant CRC cells. Furthermore, 4-AAQB effectively inhibits Ras singling in CRC cells, through which KRAS-mutant CRC can be resensitized to cetuximab.     

Photoaffinity Labeling-Based Chemoproteomic Strategy Reveals RBBP4 as a Cellular Target of Protopanaxadiol against Colorectal Cancer Cells

Protopanaxadiol (PPD), a main ginseng metabolite, exerts powerful anticancer effects against multiple types of cancer; however, its cellular targets remain elusive. Here, we synthesized a cell-permeable PPD probe via introducing a bifunctional alkyne-containing diazirine photo-crosslinker and performed a photoaffinity labeling-based chemoproteomic study. We identified retinoblastoma binding protein 4 (RBBP4), a chromatin remodeling factor, as an essential cellular target of PPD in HCT116 colorectal cancer cells. PPD significantly decreased RBBP4-dependent trimethylation at lysine 27 of histone H3 (H3K27me3), a crucial epigenetic marker that correlates with histologic signs of colorectal cancer aggressiveness, and PPD inhibition of proliferation and migration of HCT116 cells was antagonized by RBBP4 RNA silencing. Collectively, our study highlights a previously undisclosed anti-colorectal cancer cellular target of the ginseng metabolite and advances the fundamental understanding of RBBP4 functions via a chemical biology strategy.     

p130Cas Is Correlated with EREG Expression and a Prognostic Factor Depending on Colorectal Cancer Stage and Localization Reducing FOLFIRI Efficacy

p130 Crk-associated substrate (p130Cas) is associated with poor prognosis and treatment resistance in breast and lung cancers. To elucidate p130Cas functional and clinical role in colorectal cancer (CRC) progression/therapy resistance, we performed cell culture experiments and bioinformatic/statistical analyses of clinical data sets. p130Cas expression was associated with poor survival in the cancer genome atlas (TCGA) data set. Knockdown/reconstitution experiments showed that p130Cas drives migration but, unexpectedly, inhibits proliferation in CRC cells. TCGA data analyses identified the growth factor epiregulin (EREG) as inversely correlated with p130Cas. p130Cas knockdown and simultaneous EREG treatment further enhanced proliferation. RNA interference and EREG treatment experiments suggested that p130Cas/EREG limit each other’s expression/activity. Inverse p130Cas/EREG Spearman correlations were prominent in right-sided and earlier stage CRC. p130Cas was inducible by 5-fluorouracil (5-FU) and FOLFIRI (folinic acid, 5-FU, irinotecan), and p130Cas and EREG were upregulated in distant metastases ({"type":"entrez-geo","attrs":{"text":"GSE121418","term_id":"121418"}}GSE121418). Positive p130Cas/EREG correlations were observed in metastases, preferentially in post-treatment samples (especially pulmonary metastases). p130Cas knockdown sensitized CRC cells to FOLFIRI independent of EREG treatment. RNA sequencing and gene ontology analyses revealed that p130Cas is involved in cytochrome P450 drug metabolism and epithelial-mesenchymal transition. p130Cas expression was associated with poor survival in right-sided, stage I/II, MSS (microsatellite stable), or BRAF-mutated CRC. In summary, p130Cas represents a prognostic factor and potential therapeutic target in CRC.     

Significance of Hypermethylation of Tumor-Suppressor Genes PTGER4 and ZNF43 at CpG Sites in the Prognosis of Colorectal Cancer

The status of DNA methylation in primary tumor tissue and adjacent tumor-free tissue is associated with the occurrence of aggressive colorectal cancer (CRC) and can aid personalized cancer treatments at early stages. Tumor tissue and matched adjacent nontumorous tissue were extracted from 208 patients with CRC, and the correlation between the methylation levels of PTGER4 and ZNF43 at certain CpG loci and the prognostic factors of CRC was determined using the MassARRAY System testing platform. The Wilcoxon signed-rank test, a Chi-square test, and McNemar’s test were used for group comparisons, and Kaplan–Meier curves and a log-rank test were used for prediction. The hypermethylation of PTGER4 at the CpG_4, CpG_5, CpG_15, and CpG_17 tumor tissue sites was strongly correlated with shorter recurrence-free survival (RFS), progression-free survival (PFS), and overall survival (OS) [hazard ratio (HR) = 3.26, 95% confidence interval (CI) = 1.38–7.73 for RFS, HR = 2.35 and 95% CI = 1.17–4.71 for PFS, HR = 4.32 and 95% CI = 1.8–10.5 for OS]. By contrast, RFS and PFS were significantly longer in the case of increased methylation of ZNF43 at the CpG_5 site of normal tissue [HR = 2.33, 95% CI = 1.07–5.08 for RFS, HR = 2.42 and 95% CI = 1.19–4.91 for PFS]. Aberrant methylation at specific CpG sites indicates tissue with aggressive behavior. Therefore, the differential methylation of PTGER4 and ZNF43 at specific loci can be employed for the prognosis of patients with CRC.