Primary Colorectal Cancer Cells Research Articles

Nuclear Softness Promotes the Metastatic Potential of Large-Nucleated Colorectal Cancer Cells via the ErbB4-Akt1-Lamin A/C Signaling Pathway.

Abnormal nuclear enlargement is a diagnostic and physical hallmark of malignant tumors. Large nuclei are positively associated with an increased risk of developing metastasis; however, a large nucleus is inevitably more resistant to cell migration due to its size. The present study demonstrated that the nuclear size of primary colorectal cancer (CRC) cells at an advanced stage was larger than cells at an early stage. In addition, the nuclei of CRC liver metastases were larger than those of the corresponding primary CRC tissues. CRC cells were sorted into large-nucleated cells (LNCs) and small-nucleated cells (SNCs). Purified LNCs exhibited greater constricted migratory and metastatic capacity than SNCs in vitro and in vivo. Mechanistically, ErbB4 was highly expressed in LNCs, which phosphorylated lamin A/C at serine 22 via the ErbB4-Akt1 signaling pathway. Furthermore, the level of phosphorylated lamin A/C was a negative determinant of nuclear stiffness. Taken together, CRC LNCs possessed greater constricted migratory and metastatic potential than SNCs due to ErbB4-Akt1-mediated lamin A/C phosphorylation and nuclear softening. These results may provide a potential treatment strategy for tumor metastasis by targeting nuclear stiffness in patients with cancer, particularly CRC.

Selective pks+ Escherichia coli strains induce cell cycle arrest and apoptosis in colon cancer cell line.

pks+ Escherichia coli (E. coli) triggers genomic instability in normal colon cells which leads to colorectal cancer (CRC) tumorigenesis. Previously, we reported a significant presentation of pks+ E. coli strains in CRC patients' biopsies as compared to healthy cohorts. In this work, using anin vitro infection model, we further explored the ability of these strains in modulating cell cycle arrest and activation of apoptotic mediators in both primary colon epithelial cells (PCE) and CRC cells (HCT-116). Sixteen strains, of which eight tumours and the matching non-malignant tissues, respectively, from eight pks+ E. coli CRC patients were subjected to BrDU staining and cell cycle analysis via flow cytometry, while a subset of these strains underwent analysis of apoptotic mediators including caspase proteins, cellular reactive oxygen species (cROS) and mitochondrial membrane potential (MMP) via spectrophotometry as well as proinflammatory cytokines via flow cytometry. Data revealed that all strains exerted S-phase cell cycle blockade in both cells and G2/M phase in PCE cells only. Moreover, more significant upregulation of Caspase 9, cROS, proinflammatory cytokines and prominent downregulation of MMP were detected in HCT-116 cells indicating the potential role of pks related bacterial toxin as anticancer agent as compared to PCE cells which undergo cellular senescence leading to cell death without apparent upregulation of apoptotic mediators. These findings suggest the existence of discrepancies underlying the mechanism of action of pks+ E. coli on both cancer and normal cell lines. This work propounds the rationale to further understand the mechanism underlying pks+ E. coli-mediated CRC tumorigenesis and cancer killing.

Inhibiting BRAF/EGFR/MEK suppresses cancer stemness and drug resistance of primary colorectal cancer cells.

Drug resistance is a major barrier against successful treatments of cancer patients. Gain of stemness under drug pressure is a major mechanism that renders treatments ineffective. Identifying approaches to target cancer stem cells (CSCs) is expected to improve treatment outcomes for patients. To elucidate the role of cancer stemness in resistance of colorectal cancer cells to targeted therapies, we developed spheroid cultures of patient-derived BRAFmut and KRASmut tumor cells and studied resistance mechanisms to inhibition of MAPK pathway through phenotypic and gene and protein expression analysis. We found that treatments enriched the expression of CSC markers CD166, ALDH1A3, CD133, and LGR5 and activated PI3K/Akt pathway in cancer cells. We examined various combination treatments to block these activities and found that a triple combination against BRAF, EGFR, and MEK significantly reduced stemness and activities of oncogenic signaling pathways. This study demonstrates the feasibility of blocking stemness-mediated drug resistance and tumorigenic activities in colorectal cancer.

Epigenomic analysis reveals a unique DNA methylation program of metastasis-competent circulating tumor cells in colorectal cancer

Circulating tumor cells (CTCs) and epigenetic alterations are involved in the development of metastasis from solid tumors, such as colorectal cancer (CRC). The aim of this study was to characterize the DNA methylation profile of metastasis-competent CTCs in CRC. The DNA methylome of the human CRC-derived cell line CTC-MCC-41 was analyzed and compared with primary (HT29, Caco2, HCT116, RKO) and metastatic (SW620 and COLO205) CRC cells. The association between methylation and the transcriptional profile of CTC-MCC-41 was also evaluated. Differentially methylated CpGs were validated with pyrosequencing and qMSP. Compared to primary and metastatic CRC cells, the methylation profile of CTC-MCC-41 was globally different and characterized by a slight predominance of hypomethylated CpGs mainly distributed in CpG-poor regions. Promoter CpG islands and shore regions of CTC-MCC-41 displayed a unique methylation profile that was associated with the transcriptional program and relevant cancer pathways, mainly Wnt signaling. The epigenetic regulation of relevant genes in CTC-MCC-41 was validated. This study provides new insights into the epigenomic landscape of metastasis-competent CTCs, revealing biological information for metastasis development, as well as new potential biomarkers and therapeutic targets for CRC patients.

Nuclear localization of TET2 requires β-catenin activation and correlates with favourable prognosis in colorectal cancer.

Mutation-induced malfunction of ten-eleven translocation methylcytosine dioxygenase 2 (TET2) is widely reported in haematological malignancies. However, the role of TET2 in solid cancers, including colorectal cancer (CRC), is unclear. Here, we found that TET2 malfunction in CRC is mostly due to decreased nuclear localization and that nuclear localization of TET2 is correlated with better survival of patients. To explore the underlying mechanisms, 14 immortalized solid tumour cell lines and 12 primary CRC cell lines were used. TET2 was mostly detected in the nucleus, and it induced significant DNA demethylation and suppressed cell growth by demethylating RORA and SPARC in cell lines like SW480. While in cell lines like SW620, TET2 was observed in the cytosol and did not affect DNA methylation or cell growth. Further examination with immunoprecipitation-mass spectrometry illustrated that β-catenin activation was indispensable for the nuclear localization and tumour suppression effects of TET2. In addition, the β-catenin pathway activator IM12 and the TET2 activator vitamin C were used simultaneously to enhance the effects of TET2 under low-expression conditions, and synergistic inhibitory effects on the growth of cancer were observed both in vitro and in vivo. Collectively, these data suggest that β-catenin-mediated nuclear localization of TET2 is an important therapeutic target for solid tumours.

Comparative proteomic analysis of three major extracellular vesicle classes secreted from human primary and metastatic colorectal cancer cells: Exosomes, microparticles, and shed midbody remnants.

Cell-derived extracellular vesicles (EVs) are evolutionary-conserved secretory organelles that, based on their molecular composition, are important intercellular signaling regulators. At least three classes of circulating EVs are known based on mechanism of biogenesis: exosomes (sEVs/Exos), microparticles (lEVs/MPs), and shed midbody remnants (lEVs/sMB-Rs). sEVs/Exos are of endosomal pathway origin, microparticles (lEVs/MPs) from plasma membrane blebbing and shed midbody remnants (lEVs/sMB-Rs) arise from symmetric cytokinetic abscission. Here, we isolate sEVs/Exos, lEVs/MPs, and lEVs/sMB-Rs secreted from human isogenic primary (SW480) and metastatic (SW620) colorectal cancer (CRC) cell lines in milligram quantities for label-free MS/MS-based proteomic profiling. Purified EVs revealed selective composition packaging of exosomal protein markers in SW480/SW620-sEVs/Exos, metabolic enzymes in SW480/SW620-lEVs/MPs, while centralspindlin complex proteins, nucleoproteins, splicing factors, RNA granule proteins, translation-initiation factors, and mitochondrial proteins selectively traffic to SW480/SW620- lEVs/sMB-Rs. Collectively, we identify 39 human cancer-associated genes in EVs; 17 associated with SW480-EVs, 22 with SW620-EVs. We highlight oncogenic receptors/transporters selectively enriched in sEVs/Exos (EGFR/FAS in SW480-sEVs/Exos and MET, TGFBR2, ABCB1 in SW620-sEVs/Exos). Interestingly, MDK, STAT1, and TGM2 are selectively enriched in SW480-lEVs/sMB-Rs, and ADAM15 to SW620-lEVs/sMB-Rs. Our study reveals sEVs/Exos, lEVs/MPs, and lEVs/sMB-Rs have distinct protein signatures that open potential diagnostic avenues of distinct types of EVs for clinical utility.

Daphnanes diterpenes from the latex of Hura crepitans L. and their PKCζ-dependent anti-proliferative activity on colorectal cancer cells

Hura crepitans L. (Euphorbiaceae) is a thorn-covered tree widespread in South America, Africa and Asia which produces an irritating milky latex containing numerous secondary metabolites, notably daphnane-type diterpenes known as Protein Kinase C activators. Fractionation of a dichloromethane extract of the latex led to the isolation of five new daphnane diterpenes (1–5), along with two known analogs (6–7) including huratoxin. Huratoxin (6) and 4′,5′-epoxyhuratoxin (4) were found to exhibit significant and selective cell growth inhibition against colorectal cancer cell line Caco-2 and primary colorectal cancer cells cultured as colonoids. The underlying mechanism of 4 and 6 was further investigated revealing the involvement of PKCζ in the cytostatic activity.

Abstract 6178: A novel second-generation nano-fluoropyrimidine to treat metastatic colorectal cancer and overcome 5-fluorouracil resistance

Abstract Colorectal cancer (CRC) is the 3rd leading cause of cancer-related mortality. The mortality associated with colon cancer results notably from metastatic disease (mCRC), with a 5-year survival rate for patients with stage IV CRC being < 14%. Despite extensive efforts towards developing personalized therapies, limited success has been achieved and chemotherapeutic regimens that include the fluoropyrimidine (FP) drug 5-fluorouracil (5-FU) are central to patient management. While using 5-FU-based regimens such as FOLFOX and FOLFIRI in combination with biologics confer a survival benefit, their ineffectiveness at promoting long-term survival in a substantial fraction of mCRC patients and associated toxicities limits their use. This underscores the pressing need to develop improved FP drugs. To overcome the limitations of 5-FU that include resistance due to elevated expression of thymidylate synthase (TS) and inefficient anabolic metabolism to FdUMP, the TS-inhibitory/active metabolite, we are developing FP polymers consisting of single-stranded DNA with FdUMP (only active metabolite) as the repeating nucleotide unit. Our therapeutic objective is to initiate a Phase I clinical trial with a new 2nd generation nanoscale FP polymer (CF10) that demonstrates promising anticancer effects in several preclinical models and significantly lower systemic toxicity. We observed that CF10 displayed significantly higher anti-tumor activity than 5-FU in flank and syngeneic tumor models. Additionally, CF10 treatment improved survival (84.5 days vs 32 days; P < 0.0001) relative to 5-FU in an orthotopic HCT-116-luc colorectal cancer mice model that spontaneously metastasized to the liver. Interestingly, a reduction in metastatic tumor burden in the CF10 treatment group led us to hypothesize that CF10 could inhibit the TS-mediated EMT phenotype and pro-metastatic activities in colorectal cancer cells (CRCs). This was supported by increased vimentin, decreased E-cadherin expression, and increased cell migration and invasion in TS-overexpressing CRC cells (“Tet-on” system). Alkaline comet assay, immunofluorescence imaging, and immunoblot identified significantly higher replication stress with CF10 treatment than 5-FU in mouse, rat, and human-established CRC cell lines and primary CRC cells. Strikingly, we also discovered that CF10 attenuates the growth of TS-overexpressing primary colonoids. In conclusion, CF10 induces increased replication stress compared to a similar concentration of conventional drugs. Our findings suggest the significance of elevated TS in CRC metastatic progression and 5-FU resistance and demonstrate that CF10 may be effective at inhibiting CRC metastatic progression. CF10 may be a successful candidate for early-phase clinical trials to treat mCRC. Citation Format: Naresh Sah, Pamela Luna, Chinnadurai Mani, William Gmeiner, Komaraiah Palle. A novel second-generation nano-fluoropyrimidine to treat metastatic colorectal cancer and overcome 5-fluorouracil resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6178.

Post‐translational modification and phenotype

Post‐translational modification and phenotype

Alternatively Spliced Isoforms of MUC4 and ADAM12 as Biomarkers for Colorectal Cancer Metastasis.

There is a pertinent need to develop prognostic biomarkers for practicing predictive, preventive and personalized medicine (PPPM) in colorectal cancer metastasis. The analysis of isoform expression data governed by alternative splicing provides a high-resolution picture of mRNAs in a defined condition. This information would not be available by studying gene expression changes alone. Hence, we utilized our prior data from an exon microarray and found ADAM12 and MUC4 to be strong biomarker candidates based on their alternative splicing scores and pattern. In this study, we characterized their isoform expression in a cell line model of metastatic colorectal cancer (SW480 & SW620). These two genes were found to be good prognostic indicators in two cohorts from The Cancer Genome Atlas database. We studied their exon structure using sequence information in the NCBI and ENSEMBL genome databases to amplify and validate six isoforms each for the ADAM12 and MUC4 genes. The differential expression of these isoforms was observed between normal, primary and metastatic colorectal cancer cell lines. RNA-Seq analysis further proved the differential expression of the gene isoforms. The isoforms of MUC4 and ADAM12 were found to change expression levels in response to 5-Fluorouracil (5-FU) treatment in a dose-, time- and cell line-dependent manner. Furthermore, we successfully detected the protein isoforms of ADAM12 and MUC4 in cell lysates, reflecting the differential expression at the protein level. The change in the mRNA and protein expression of MUC4 and ADAM12 in primary and metastatic cells and in response to 5-FU qualifies them to be studied as potential biomarkers. This comprehensive study underscores the importance of studying alternatively spliced isoforms and their potential use as prognostic and/or predictive biomarkers in the PPPM approach towards cancer.

The sodium channel subunit SCNN1B suppresses colorectal cancer via suppression of active c-Raf and MAPK signaling cascade

The incidence of colorectal cancer (CRC) is rising worldwide. Here, we identified SCNN1B as an outlier down-regulated in CRC and it functions as a tumor suppressor. SCNN1B mRNA and protein expression were down-regulated in primary CRC and CRC cells. In a tissue microarray cohort (N = 153), SCNN1B protein was an independent prognostic factor for favorable outcomes in CRC. Ectopic expression of SCNN1B in CRC cell lines suppressed cell proliferation, induced apoptosis, and cell cycle arrest, and suppressed cell migration in vitro. Xenograft models validated tumor suppressive function of SCNN1B in vivo. Mechanistically, Gene Set Enrichment Analysis (GSEA) showed that SCNN1B correlates with KRAS signaling. Consistently, MAPK qPCR and kinase arrays revealed that SCNN1B suppressed MAPK signaling. In particular, SCNN1B overexpression suppressed p-MEK/p-ERK expression and SRE-mediated transcription activities, confirming blockade of Ras-Raf-MEK-ERK cascade. Mechanistically, SCNN1B did not affect KRAS activation, instead impairing activation of c-Raf by inducing its inhibitory phosphorylation and targeting active c-Raf for degradation. The ectopic expression of c-Raf fully rescued cell proliferation and colony formation in SCNN1B-overexpressing CRC cells, confirming c-Raf as the principal molecular target of SCNN1B. In summary, we identified SCNN1B as a tumor suppressor by functioning as a c-Raf antagonist, which in turn suppressed oncogenic MEK-ERK signaling.

Hepcidin Upregulation in Colorectal Cancer Associates with Accumulation of Regulatory Macrophages and Epithelial-Mesenchymal Transition and Correlates with Progression of the Disease.

Advanced, metastatic colorectal cancer (CRC) is associated with high rate of mortality because of its poor responsiveness to chemotherapy/immunotherapy. Recent studies have shown that hepcidin, a peptide hormone produced mainly by hepatocytes, is expressed by and enhances the growth of tumor cells. We here assessed whether hepcidin expression helps identify subsets of CRC with advanced and aggressive course. By integrating results of in vitro/ex vivo studies with data of bioinformatics databases, we initially showed that hepcidin RNA and protein expression was more pronounced in tissue samples taken from the tumor area, as compared to the macroscopically unaffected, adjacent, colonic mucosa of CRC patients. The induction of hepcidin in the colonic epithelial cell line HCEC-1ct by interleukin (IL)-6, IL-21 and IL-23 occurred via a Stat3-dependent mechanism and, in primary CRC cells, hepcidin co-localized with active Stat3. In CRC tissue, hepcidin content correlated mainly with macrophage accumulation and IL-10 and CD206 expression, two markers of regulatory macrophages. Consistently, both IL-10 and CD206 were up-regulated by hepcidin in blood mononuclear cells. The highest levels of hepcidin were found in metastatic CRC and survival analysis showed that high expression of hepcidin associated with poor prognosis. Moreover, hepcidin expression correlated with markers of epithelial-to-mesenchymal transition and the silencing of hepcidin in CRC cells reduced epithelial-to-mesenchymal transition markers. These findings indicate that hepcidin is markedly induced in the advanced stages of CRC and suggest that it could serve as a prognostic biomarker in CRC.

Expression profiling of anticancer genes in colorectal cancer patients and their in vitro induction by riproximin, a ribosomal inactivating plant protein.

Ectopic expression of anticancer genes (ACGs) imposes antineoplastic effects on transformed cells. Clinically, reduced expression of these genes has been linked with poor prognosis, metastasis and chemo/radiotherapy resistance in cancers. Identifying expression pattern of ACGs is crucial to establish their prognostic and therapeutic relevance in colorectal cancer (CRC). In addition to the clinical perspective, naturally occurring compounds can be explored in parallel for inducing ACGs to achieve cancer cell-specific death. Expression profiles of three ACGs (NOXA, PAR-4, TRAIL) were identified via real-time PCR in CRC clinical isolates. Time lapse-based expression modifications in ACGs were studied in a CRC liver metastasis animal model using microarray methodology. Effects of a purified plant protein (riproximin) on selected ACGs were identified in three primary and metastatic CRC cell lines by real-time PCR. Lastly, importance of the ACGs in a cellular environment was highlighted via bioinformatic analysis. ACGs (except NOXA) were persistently downregulated in clinical isolates when comparing the overall mean expression values with normal mucosa levels. In vivo studies showed a prominent inhibition of NOXA and PAR-4 genes in implanted CRC cells during rat liver colonization. TRAIL showed deviation from this theme while showing marked induction during the early period of liver colonization (days 3 and 6 after CRC cell implantation). Riproximin exhibited substantial potential of inducing ACGs at transcriptome levels in selected CRC cell lines. Bioinformatic analysis showed that vital molecular/functional aspects of a cell are associated with the presence of ACGs. ACGs are downregulated in primary and metastatic phase of CRC. Riproximin effectively induces ACGs in CRC cells and can be exploited for clinical investigations over time.

Smad7 Sustains Stat3 Expression and Signaling in Colon Cancer Cells.

Simple SummaryColorectal cancer (CRC) cells contain elevated levels of Stat3 and Smad7, two proteins involved in the growth and survival of neoplastic cells. This study was aimed at examining whether Smad7 positively controls the expression of Stat3 in CRC cells. By employing antisense technology, which specifically inhibits Smad7 expression in selected cells, we hereby show that the reduction of Smad7 in CRC cells is paralleled by the marked suppression of the levels of Stat3 and of Stat3-related genes. Finally, we provide evidence that in human CRC tissue samples, there is a positive correlation between Smad7 expression and Stat3 content. Overall, these findings delineate a novel positive feedback loop that sustains CRC cell behavior and suggest that Smad7 is a target for therapeutic intervention in patients with such a disease.Colorectal cancer (CRC) cells contain elevated levels of active signal transducer and the activator of transcription (Stat)-3, which exerts proliferative and anti-apoptotic effects. Various molecules produced in the CRC tissue can activate Stat3, but the mechanisms that amplify such an activation are yet to be determined. In this paper, we assessed whether Smad7, an inhibitor of Transforiming Growth Factor (TGF)-β1 activity, sustains Stat3 expression/activation in CRC cells. Both Smad7 and phosphorylated (p)/activated-Stat3 were more expressed in the tumoral areas of CRC patients, compared to the normal adjacent colonic mucosa of the same patients, and were co-localized in primary CRC cells and CRC cell lines. The knockdown of Smad7 with a Smad7 antisense oligonucleotide (AS) reduced p-Stat3 in both unstimulated and interleukin (IL)-6- and IL-22-stimulated DLD-1 and HCT116 cells. Consistently, reduced levels of BCL-xL and survivin, two downstream signaling targets of Stat3 activation, were seen in Smad7 AS-treated cells. An analysis of the mechanisms underlying Smad7 AS-induced Stat3 inactivation revealed that Smad7 AS reduced Stat3 RNA and protein expression. A chromatin immunoprecipitation assay showed the direct regulatory effect of Smad7 on the Stat3 promoter. RNA-sequencing data from the Tumor, Normal and Metastatic (TNM) plot database showed a positive correlation between Smad7 and Stat3 in 1450 CRC samples. To our knowledge, this is the first evidence supporting the theory that Smad7 positively regulates Stat3 function in CRC.

Exosomal lncRNA PCAT1 Promotes Tumor Circulating Cell-Mediated Colorectal Cancer Liver Metastasis by Regulating the Activity of the miR-329-3p/Netrin-1-CD146 Complex.

Objective This study explored the colorectal cancer exosome lncRNA prostate cancer associated transcript 1– (PCAT1) mediated circulating tumors and the mechanism of cell colorectal cancer liver metastasis. Methods Exosomes were extracted from the primary colorectal cancer (CRC) cell lines HCT116 and SW480 and cultured with T84 and human umbilical vein endothelial (HUVE) cells. The expression of PCAT1 and miR-329-3p was detected by real-time quantitative polymerase chain reaction (RT-qPCR), the expression of Netrin-1, CD146, and epithelial mesenchymal transition (EMT) related proteins was detected by Western blot, the proliferation activity of T84 cells was detected by cell counting kit 8 (CCK-8), and cell migration was detected by Transwell. The expression of the F-actin signal was detected by immunofluorescence after coculture of exosomes with human umbilical vein endothelial cells (HUVECs). Changes in subcutaneous tumor and liver nodule size after PCAT1 deletion were observed in a mouse model of liver metastasis from rectal cancer. Results PCAT1 expression was upregulated in primary cell lines and their exosomes. After exosomes were cocultured with colorectal cancer tumor circulating T84 cells, the expression of Netrin-1 and CD146 was upregulated, the expression of miR-329-3p was downregulated, the proliferation and migration ability of T84 cells were enhanced, and EMT occurred. After knocking down PCAT1, the above phenomenon was reversed. Similarly, after exosomes were cocultured with HUVECs, the expression of the F-actin signal increased, and after PCAT1 was knocked down, the F-actin signal also decreased. PCAT1 regulates miR-329-3p/Netrin-1 and affects the biological behavior of T84 and F-actin signal expression in HUVECs. In a mouse model of colorectal cancer liver metastasis, knocking down PCAT1 significantly reduced the nodules formed by liver metastasis in mice. Conclusions LncRNA PCAT1 derived from colorectal cancer exosomes regulates the activity of the Netrin-1-CD146 complex in circulating tumor cells (CTCs) to promote the occurrence of colorectal cancer EMT and liver metastasis and provides new molecular targets for the treatment of colorectal cancer liver metastasis.

Global metabolic alterations in colorectal cancer cells during irinotecan-induced DNA replication stress

BackgroundMetabolic adaptations can allow cancer cells to survive DNA-damaging chemotherapy. This unmet clinical challenge is a potential vulnerability of cancer. Accordingly, there is an intense search for mechanisms that modulate cell metabolism during anti-tumor therapy. We set out to define how colorectal cancer CRC cells alter their metabolism upon DNA replication stress and whether this provides opportunities to eliminate such cells more efficiently.MethodsWe incubated p53-positive and p53-negative permanent CRC cells and short-term cultured primary CRC cells with the topoisomerase-1 inhibitor irinotecan and other drugs that cause DNA replication stress and consequently DNA damage. We analyzed pro-apoptotic mitochondrial membrane depolarization and cell death with flow cytometry. We evaluated cellular metabolism with immunoblotting of electron transport chain (ETC) complex subunits, analysis of mitochondrial mRNA expression by qPCR, MTT assay, measurements of oxygen consumption and reactive oxygen species (ROS), and metabolic flux analysis with the Seahorse platform. Global metabolic alterations were assessed using targeted mass spectrometric analysis of extra- and intracellular metabolites.ResultsChemotherapeutics that cause DNA replication stress induce metabolic changes in p53-positive and p53-negative CRC cells. Irinotecan enhances glycolysis, oxygen consumption, mitochondrial ETC activation, and ROS production in CRC cells. This is connected to increased levels of electron transport chain complexes involving mitochondrial translation. Mass spectrometric analysis reveals global metabolic adaptations of CRC cells to irinotecan, including the glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways. P53-proficient CRC cells, however, have a more active metabolism upon DNA replication stress than their p53-deficient counterparts. This metabolic switch is a vulnerability of p53-positive cells to irinotecan-induced apoptosis under glucose-restricted conditions.ConclusionDrugs that cause DNA replication stress increase the metabolism of CRC cells. Glucose restriction might improve the effectiveness of classical chemotherapy against p53-positive CRC cells.Graphical The topoisomerase-1 inhibitor irinotecan and other chemotherapeutics that cause DNA damage induce metabolic adaptations in colorectal cancer (CRC) cells irrespective of their p53 status. Irinotecan enhances the glycolysis and oxygen consumption in CRC cells to deliver energy and biomolecules necessary for DNA repair and their survival. Compared to p53-deficient cells, p53-proficient CRC cells have a more active metabolism and use their intracellular metabolites more extensively. This metabolic switch creates a vulnerability to chemotherapy under glucose-restricted conditions for p53-positive cells.

Implication of Different Tumor Biomarkers in Drug Resistance and Invasiveness in Primary and Metastatic Colorectal Cancer Cell Lines.

Protein expression profiles are directly related to the different properties of cells and are conditioned by the cellular niche. As an example, they are the cause of the characteristic cell plasticity, epithelium–mesenchymal transition (EMT), and drug resistance of cancer cells. This article characterizes ten biomarkers related to these features in three human colorectal cancer cell lines: SW-480, SW-620, and DLD-1, evaluated by flow cytometry; and in turn, resistance to oxaliplatin is studied through dose–response trials. The main biomarkers present in the three studied lines correspond to EpCAM, CD-133, and AC-133, with the latter two in low proportions in the DLD-1 line. The biomarker CD166 is present in greater amounts in SW-620 and DLD-1 compared to SW-480. Finally, DLD-1 shows high values of Trop2, which may explain the aggressiveness and resistance of these cells to oxaliplatin treatments, as EpCAM is also highly expressed. Exposure to oxaliplatin slows cell growth but also helps generate resistance to the treatment. In conclusion, the response of the cell lines is variable, due to their genetic variability, which will condition protein expression and cell growth. Further analyses in this area will provide important information for better understanding of patients’ cellular response and how to prevent resistance.

Colorectal cancer on a dish: exploring the 3D-sphere culture of primary colorectal cancer cells from an Indonesian perspective

Background : Colorectal Cancer (CRC) is one of the deadliest types of cancer and has emerged as one of Indonesia's most devastating diseases. The growing number of colorectal cancer cases is frequently undiagnosed until the disease has progressed to a metastatic stage. This issue has lasted for years, limiting therapy options and resulting in a bad prognosis for the majority of patients. Thus, the purpose of this work is to develop a CRC detection method for Indonesia and other low-middle income nations that integrates in vitro 3D culture, molecular analysis, and in silico analysis. Methods : Colorectal cancer biopsies were transported to the lab and underwent mechanical disaggregation and centrifuged at 300 x g for five minutes. Approximately 10,000 cells were seeded in each Nunc-Sphera 96-well plate (u-bottom) for the following 7 days in standard culture medium. The 3D-sphere was harvested and RNA was extracted afterwards. Molecular analysis was performed using qPCR and the Human Cancer Pathway Profiler. Protein interaction and pathway analysis were conducted using STRING and Reactome online tools. Results : Following initial seeding, primary CRC 3D-spheres were grown for 14–16 days. Gene profiling and in silico analyses suggest that CDC20, AURKA, and ACLY are expressed at lower levels than the positive control in the 3D-sphere. These markers have been implicated in metastasis, CRC proliferation, and as a drug target ligand. Conclusion : A combination of 3D culture, gene profiling, and in silico analysis is feasible to detect CRC for Indonesia and other low- and middle-income countries. A future possibility is to use minicolorectal cancer in a dish for ex vivo cancer modeling and pharmacological testing.

Identification of histone methyltransferase NSD2 as an important oncogenic gene in colorectal cancer

Colorectal cancer (CRC) is the second common cause of cancer-related human mortalities. Dysregulation of histone 3 (H3) methylation could lead to transcriptional activation of multiple oncogenes, which is closely associated with CRC tumorigenesis and progression. Nuclear receptor-binding SET Domain protein 2 (NSD2) is a key histone methyltransferase catalyzing histone H3 lysine 36 dimethylation (H3K36me2). Its expression, the potential functions, and molecular mechanisms in CRC are studied here. Gene Expression Profiling Interactive Analysis (GEPIA) bioinformatics results showed that the NSD2 mRNA expression is elevated in both colon cancers and rectal cancers. Furthermore, NSD2 mRNA and protein expression levels in local colon cancer tissues are significantly higher than those in matched surrounding normal tissues. In primary human colon cancer cells and established CRC cell lines, shRNA-induced silencing or CRISPR/Cas9-induced knockout of NSD2 inhibited cell viability, proliferation, cell cycle progression, migration, and invasion. Furthermore, NSD2 shRNA or knockout induced mitochondrial depolarization, DNA damage, and apoptosis in the primary and established CRC cells. Contrarily, ectopic NSD2 overexpression in primary colon cancer cells further enhanced cell proliferation, migration, and invasion. H3K36me2, expressions of multiple oncogenes (ADAM9, EGFR, Sox2, Bcl-2, SYK, and MET) and Akt activation were significantly decreased after NSD2 silencing or knockout in primary colon cancer cells. Their levels were however increased after ectopic NSD2 overexpression. A catalytic inactive NSD2 (Y1179A) also inhibited H3K36me2, multiple oncogenes expression, and Akt activation, as well as cell proliferation and migration in primary colon cancer cells. In vivo, intratumoral injection of adeno-associated virus (AAV)-packed NSD2 shRNA largely inhibited primary colon cancer cell xenograft growth in nude mice. Together, NSD2 exerted oncogenic functions in CRC and could be a promising therapeutic target.

The Ultrastructural Analysis of Human Colorectal Cancer Stem Cell-Derived Spheroids and Their Mouse Xenograft Shows That the Same Cells Types Have Different Ratios.

Simple SummaryA comparative ultrastructural and flow cytometric analysis of colorectal cancer-derived spheroids and their mouse xenografts showed that they both contain the same cell types but with different ratios, reflecting the interaction of cancer cells, respectively, with the in vitro and in vivo microenvironment.Spheroids from primary colorectal cancer cells and their mice xenografts have emerged as useful preclinical models for cancer research as they replicate tumor features more faithfully as compared to cell lines. While 3D models provide a reliable system for drug discovery and testing, their structural complexity represents a challenge and their structure-function relationships are only partly understood. Here, we present a comparative ultrastructural and flow citometric analysis of patient colorectal cancer-derived spheroids and their mice xenografts. Ultrastructural observations highlighted that multicellular spheroids and their xenografts contain the same cancer cell types but with different ratios, specifically multicellular spheroids were enriched in cells with a stem-like phenotype, while xenografts had an increased amount of lipid droplets-containing cells. The flow cytometric analysis for stem cell marker and activity showed enrichment of stem-like cells presence and activity in spheroids while xenografts had the inverse response. Our results evidence the effects on cancer cells of different in vitro and in vivo microenvironments. Those differences have to be paid into account in designing innovative experimental models for personalized drug testing.