Sensitivity Of Colorectal Cancer Cells Research Articles

UGT8 mediated sulfatide synthesis modulates BAX localization and dictates apoptosis sensitivity of colorectal cancer.

Elevated de novo lipid synthesis is a remarkable adaptation of cancer cells that can be exploited for therapy. However, the role of altered lipid metabolism in the regulation of apoptosis is still poorly understood. Using thermal proteome profiling, we identified Manidipine-2HCl, targeting UGT8, a key enzyme in the synthesis of sulfatides. In agreement, lipidomic analysis indicated that sulfatides are strongly reduced in colorectal cancer cells upon treatment with Manidipine-2HCl. Intriguingly, this reduction led to severe mitochondrial swelling and a strong synergism with BH3 mimetics targeting BCL-XL, leading to the activation of mitochondria-dependent apoptosis. Mechanistically, Manidipine-2HCl enhanced mitochondrial BAX localization in a sulfatide-dependent fashion, facilitating its activation by BH3 mimetics. In conclusion, our data indicates that UGT8 mediated synthesis of sulfatides controls mitochondrial homeostasis and BAX localization, dictating apoptosis sensitivity of colorectal cancer cells.

In vivo delivery of PBAE/ZIF-8 enhances the sensitivity of colorectal cancer to doxorubicin through sh-LncRNA ASB16-AS1.

The aim of this study is to investigate the impact of sh-LncRNA ASB16-AS1 on doxorubicin (DOX) resistance in colorectal cancer (CRC). First, an in vitro study was conducted to investigate the effects of LncRNA ASB16-AS1, miR-185-5p, and TEAD1 on drug resistance in CRC cells. Subsequently, utilizing nanotechnology, poly(beta amino esters) (PBAE)/zeolitic imidazolate framework-8 (ZIF-8)@sh-LncRNA ASB16-AS1 nanoparticles (PZSNP) were synthesized and characterized, evaluating their cellular toxicity and hemolytic activity. Finally, a mouse subcutaneous tumor model was established by subcutaneous injection of SW480/DOX cell suspension to investigate the impact of PZSNP on the tumor. Under DOX treatment, downregulation of LncRNA ASB16-AS1, overexpression of miR-185-5p, or downregulation of TEAD1 suppressed the viability and proliferation of drug-resistant CRC cells while promoting apoptosis. Conversely, overexpression of LncRNA ASB16-AS1, inhibition of miR-185-5p, or overexpression of TEAD1 enhanced the viability and proliferation of drug-resistant CRC cells while inhibiting apoptosis. The synthesized PZSNP exhibited a spherical shape with an average particle size of 123.6 nm, possessed positive charge, displayed good stability. It effectively encapsulated shRNA and displayed low cellular toxicity and hemolytic activity. Under DOX treatment, significant tumor necrosis was observed in the PZSNP group, and tumor growth was suppressed without causing weight loss. LncRNA ASB16-AS1, miR-185-5p, and TEAD1 are involved in regulating cell viability, proliferation, and apoptosis, contributing to drug resistance in CRC cells. sh-LncRNA ASB16-AS1 enhances the sensitivity of CRC cells to DOX during treatment, and in vivo delivery of PZSNP may serve as an effective strategy to overcome chemotherapy resistance in CRC.

DNA methylation-driven gene FAM3D promotes colorectal cancer growth via the ATF4-SESN2-mTORC1 pathway.

Globally, colorectal cancer (CRC) is the malignant tumor with the highest mortality rate after lung cancer. Abnormal DNA methylation drives dysregulated gene expression, thereby promoting CRC progression and leading to poor prognosis. We identified a 3-CpG methylation signature that is independently associated with CRC prognosis. The model consists of three methylation-driven genes: FAM3 Metabolism Regulating Signaling Molecule D (FAM3D), DAPP1, and PIGR. However, the prognostic significance, biological function, and related mechanisms of the individual methylation-driven gene FAM3D in CRC have not been studied. Here, we discovered that FAM3D expression was reduced in CRC tissues and cells, and that high methylation and low expression of FAM3D were independent prognostic risk factors for CRC. In addition, FAM3D promoted the growth and movement of CRC cells in vitro and the proliferation in nude mice, mainly by inhibiting ATF4 transcription and downregulating SESN2 expression, and ultimately activating mTORC1. Furthermore, FAM3D resulted in reduced sensitivity of CRC cells to oxaliplatin, cisplatin, and 5-fluorouracil. Our study showed that FAM3D activates the mTORC1 pathway through the ATF4-SESN2 axis and promotes the malignant progression of CRC, which contributes to predict CRC prognosis and guide individualized treatment.

MYC upstream region orchestrates resistance to PI3K inhibitors in cancer cells through FOXO3a-mediated autophagic adaptation.

The MYC oncogene is frequently overexpressed in tumors and inhibition of its translation is considered an attractive therapeutic opportunity. Despite numerous reports proposing an internal ribosome entry site (IRES) within the MYC Upstream Region (MYC UR) to sustain MYC translation during cellular stress or chemotherapy, conflicting evidence remains regarding the validity of such a mechanism. Through comprehensive investigations in MYC-driven Colorectal Cancer (CRC) and Burkitt Lymphoma (BL) cells, we demonstrate that MYC UR does not facilitate cap-independent translation, but instead orchestrates resistance to PI3K inhibitors. Genomic deletion of MYC UR neither impacts MYC protein levels nor viability in CRC cells, either untreated or exposed to cellular stress. However, in response to PI3K inhibitors, MYC UR drives a FOXO3a-dependent transcriptional upregulation of MYC, conferring drug resistance. This resistance is mediated by enhanced autophagic flux, governed by MYC, and blockade of autophagy sensitizes CRC cells to PI3K inhibition in vitro and in vivo. Remarkably, BL cells lacking the translocation of MYC UR exhibit sensitivity to PI3K inhibitors, whereas MYC UR-translocated cells respond to these drugs only when autophagy is inhibited. These findings challenge previous notions regarding IRES-mediated translation and highlight a promising strategy to overcome resistance to PI3K inhibitors in MYC-driven malignancies, offering potential clinical implications for CRC and BL treatment. In response to BKM120, the upstream region of MYC (UR) enhances MYC expression, via FOXO3a, leading to increased autophagic flux and resistance to PI3K inhibitors (left). Pharmacological blockade of autophagy (center) or lack of translocated MYC UR along with MYC CDS in BL (right) overcome resistance and induces cells death. Image created in BioRender.

LncRNA-HMG incites colorectal cancer cells to chemoresistance via repressing p53-mediated ferroptosis

Upon chemotherapy, excessive reactive oxygen species (ROS) often lead to the production of massive lipid peroxides in cancer cells and induce cell death, namely ferroptosis. The elimination of ROS is pivotal for tumor cells to escape from ferroptosis and acquire drug resistance. Nevertheless, the precise functions of long non-coding RNAs (lncRNAs) in ROS metabolism and tumor drug-resistance remain elusive. In this study, we identify LncRNA-HMG as a chemoresistance-related lncRNA in colorectal cancer (CRC) by high-throughput screening. Abnormally high expression of LncRNA-HMG predicts poorer prognosis in CRC patients. Concurrently, we found that LncRNA-HMG protects CRC cells from ferroptosis upon chemotherapy, thus enhancing drug resistance of CRC cells. LncRNA-HMG binds to p53 and facilitates MDM2-mediated degradation of p53. Decreased p53 induces upregulation of SLC7A11 and VKORC1L1, which contribute to increase the supply of reducing agents and eliminate excessive ROS. Consequently, CRC cells escape from ferroptosis and acquire chemoresistance. Importantly, inhibition of LncRNA-HMG by anti-sense oligo (ASO) dramatically sensitizes CRC cells to chemotherapy in patient-derived xenograft (PDX) model. LncRNA-HMG is also a transcriptional target of β-catenin/TCF and activated Wnt signals trigger the marked upregulation of LncRNA-HMG. Collectively, these findings demonstrate that LncRNA-HMG promotes CRC chemoresistance and might be a prognostic or therapeutic target for CRC.

CEBPB dampens the cuproptosis sensitivity of colorectal cancer cells by facilitating the PI3K/AKT/mTOR signaling pathway.

Cuproptosis is a novel pathway that differs from other forms of cell death and has been confirmed to be applicable for predicting tumor prognosis and clinical treatment response. However, the mechanism underlying the resistance of colorectal cancer (CRC) to cuproptosis at the molecular level has not been elucidated. Using bioinformatics analysis, the expression of CCAAT/enhancer-binding protein beta (CEBPB) in CRC tissues and its enrichment in biological processes were detected. Quantitative reverse transcription polymerase chain reaction and western blotting (WB) were employed to test the expression of CEBPB in CRC cells. WB was utilized to assess the levels of proteins related to cuproptosis and the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. The MTT assay was used to test cell viability. Cell proliferation was assessed by a colony formation assay. Transwell assays were used to measure cell migration and invasion ability. DLAT-aggregate formation was determined by immunofluorescence. CEBPB was highly upregulated in CRC cells to enhance cell viability, proliferation, migration, and invasion. CEBPB was strongly implicated in copper ion homeostasis and the mTOR signaling pathway in CRC. In a CRC cuproptosis cell model, rescue experiments revealed that a PI3K/AKT/mTOR pathway inhibitor attenuated the promoting effect of CEBPB overexpression on the PI3K/AKT/mTOR pathway and rescued the sensitivity of CRC to cuproptosis. This work demonstrated that CEBPB can activate the PI3K/AKT/mTOR signaling pathway, thereby decreasing the sensitivity of CRC to cuproptosis. These data suggested that targeting CEBPB or the PI3K/AKT/mTOR pathway may enhance the sensitivity of CRC patients to cuproptosis, providing a combined therapeutic strategy for cuproptosis-induced therapy.

Mitochondrial Deoxyguanosine Kinase Induces 5-Fluorouracil Chemotherapy Sensitivity through Autophagy.

The purpose of this study was to investigate the role of DGUOK in the pro-gression of colorectal cancer (CRC) and its impact on the sensitivity of CRC cells to 5-FU treatment. We conducted bioinformatics analysis and qRT-PCR to evaluate DGUOK expression in CRC tissues/cells. Cell viability of CRC cells treated with 5-FU was assessed using CCK-8 and colony formation assays. Autophagy levels were determined through immunofluorescence assays and Western blot analysis. Additionally, the influence of p-p38 on autophagy was inves-tigated via Western blotting. A rescue assay was performed to confirm whether DGUOK/p38 affects 5-FU sensitivity in CRC cells through autophagy. Our findings indicate that DGUOK is upregulated in CRC tissues compared to normal tissues, correlating with increased cell proliferation and migration. Functionally, inhibition of DGUOK enhances autophagy, thereby decreasing the sensitivity of CRC cells to 5-FU. This ef-fect is partly mediated by DGUOK's impact on the mitogen-activated protein kinase (MAPK) pathway, specifically promoting the phosphorylation of p38 MAPK, a crucial regulator in au-tophagy pathways. These results suggest that DGUOK could serve as a novel marker for predicting the efficacy of 5-FU in CRC treatment.

Ropivacaine prompts ferroptosis to enhance the cisplatin-sensitivity of human colorectal cancer through SIRT1/Nrf2 signaling pathway

The ineffectiveness of cisplatin therapy in treating colorectal cancer (CRC) is attributed to an increase of resistance. It's necessary to investigate adjunctive agents capable of enhancing drug efficacy. Previous studies have shown that ropivacaine inhibit the growth of various cancer cells, but its impact on cisplatin resistance in tumors is not well understood. This study was to illustrate the impact and mechanism of ropivacaine enhanced cisplatin-sensitivity of CRC. Cisplatin alone treatment resulted in the elevation of reactive oxygen species (ROS) and intracellular Fe2+ levels, as well as a reduction in mitochondrial membrane potential (MMP) in cisplatin-sensitive LOVO cells, while these effects were mitigated in the cisplatin-resistant LOVO/DDP cells. The co-administration of ropivacaine with cisplatin inhibited cell viability and cell migration, decreased MMP, and promoted ROS accumulation and apoptosis in both LOVO cells and LOVO/DDP cells. And they upregulated the levels of ferroptosis makers and downregulated the expression of anti-ferroptosis proteins. However, this effect was reversed by ferroptosis inhibitor ferrostatin-1 or liproxstatin-1. Furthermore, we o demonstrated that the co-administration of ropivacaine and cisplatin resulted in a decrease in SIRT1 expression, and SIRT1 knockdown in LOVO/DDP cells enhanced the ferroptosis and the anti-tumor properties of ropivacaine, while also inhibiting the activation of the Nrf2/Keap1 pathway. The above results suggested that ropivacaine increased the sensitivity of CRC cells to cisplatin by promoting ferroptosis through the inhibition of SIRT1 expression, which proposes a therapeutic approach for overcoming cisplatin resistance in CRC.

SLC12A8 upregulation promotes colorectal cancer progression and chemoresistance.

Colorectal cancer (CRC), a prevalent gastrointestinal malignant disease, causes substantial morbidity and mortality. Identification of novel prognostic biomarkers and therapeutic targets is critically needed to improve patient outcomes. Although solute carrier family 12 member 8 (SLC12A8) has high expression in various tumors and affects tumor progression, its role in CRC remains unclear. The aim of this study was to investigate the functions of SLC12A8 in CRC. SLC12A8 expression and its association with clinical significance in CRC patients were explored via multiple public databases, including The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), The Human Protein Atlas (HPA), The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN), and Kaplan-Meier plotter. The effects of SLC12A8 on the CRC cell apoptosis, epithelial-mesenchymal transition (EMT), reactive oxygen species (ROS) production, and sensitivity to oxaliplatin were verified by in vitro experiments. SLC12A8 expression was upregulated in CRC tissues compared with normal colorectal tissues. Furthermore, high expression of SLC12A8 was associated with poorer prognosis in CRC patients. Pathway enrichment analyses revealed SLC12A8 involvement in oxidative stress and transforming growth factor-beta (TGF-β) signaling. Experiments in CRC cells showed that SLC12A8 upregulation promoted apoptosis resistance, EMT, and inhibited ROS production. Moreover, SLC12A8 knockdown enhanced the sensitivity of CRC cells to oxaliplatin chemotherapy. Our integrative analyses identify SLC12A8 as a candidate biomarker for CRC progression. Targeting SLC12A8 may improve patient responses to oxaliplatin-based treatment regimens.

TOPK Inhibition Enhances the Sensitivity of Colorectal Cancer Cells to Radiotherapy by Reducing the DNA Damage Response.

Abnormal expression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) was reported to be closely related to the resistance of prostate cancer to radiotherapy and to targeted drug resistance in lung cancer. However, the role of TOPK inhibition in enhancing radiosensitivity of colorectal cancer (CRC) cells is unclear. This study aimed to evaluate the radiosensitization of TOPK knockdown in CRC cells. The expression of TOPK was detected in CRC tissues by immunohistochemistry, and the effect of TOPK knockdown was detected in CRC cells by Western blotting. CCK-8 and clonogenic assays were used to detect the growth and clonogenic ability of CRC cells after TOPK knockdown combined with radiotherapy in CRC cells. Furthermore, proteomic analysis showed that the phosphorylation of TOPK downstream proteins changed after radiotherapy. DNA damage was detected by the comet assay. Changes in the DNA damage response signaling pathway were analyzed by Western blotting, and apoptosis was detected by flow cytometry. The expression of TOPK was significantly greater in CRC tissues at grades 2-4 than in those at grade 1. After irradiation, CRC cells with genetically silenced TOPK had shorter comet tails and reduced expression levels of DNA damage response-associated proteins, including phospho-cyclin-dependent kinase 1 (p-CDK1), phospho-ataxia telangiectasia-mutated (p-ATM), poly ADP-ribose polymerase (PARP), and meiotic recombination 11 homolog 1 (MRE11). TOPK was overexpressed in patients with moderately to poorly differentiated CRC. Moreover, TOPK knockdown significantly enhanced the radiosensitivity of CRC cells by reducing the DNA damage response.

Clinicopathological characteristics and survival in metastatic colorectal cancer with short-form APC mutations.

3555 Background: Wnt/β-catenin signaling is activated by loss-of-function mutations in the adenomatous polyposis coli ( APC) gene and plays a key role in tumorigenesis in colorectal cancer (CRC). Tankyrase inhibitors, which cause degradation of β-catenin, are expected to have a promising therapeutic effect for CRC, and the short-form APC mutations predict the sensitivity of CRC cells to tankyrase inhibitors (Tanaka et al. Mol Cancer Ther 2017; Chen et al. Br J Cancer, in press).However, clinicopathological characteristics and survival in metastatic CRC (mCRC) with short-form APCmutations remain unclear. Methods: Patients with mCRC who were treated with chemotherapy between 2010 and 2021 and have tissue or circulating tumor DNA-based next-generation sequencing results were enrolled. Short-form APC mutations were defined as those truncated at amino acid 1,256 or shorter, lacking all seven 20-amino acid repeat (20-AAR) regions. Non-short-form APC mutations were defined as those possess more than one 20-AAR. Short-form and non-short-form heterozygous mutations were classified as non-short-form APC mutations. We confirmed incidence of mCRC with short-form APC mutations and compared clinicopathological characteristics and overall survival (OS) between mCRC patients with short-form and non-short-form APCmutations. Results: Of 177 patients (median age, 56.0 years) included in this study, 44 (25%) and 79 (45%) had short-form and non-short-form APC mutations, respectively. Patients with short-form APC mutations were not likely to have lung metastasis (P = 0.15). Logistic regression multivariate analysis, lung metastasis was related to mCRC patients with short-form APC mutations (Odds ratio: 0.46, P = 0.047). Patients with short-form APC mutations had significantly shorter overall survival (OS) than did those without (n = 133), with a median OS of 34.7 vs 50.5 months ( P Log-rank= 0.035, hazard ratio (HR), 1.59; 95% confidence interval (CI), 1.03-2.45). In multivariate analysis, short-form APC mutation was independent factor related to OS (hazard ratio (HR): 1.68, P = 0.02), as well as the BRAF V600E mutation, liver metastasis and peritoneal metastasis. Conclusions: Short-form APC mutation is more common in mCRC patients without lung metastases and is independently associated with a short prognosis.

Correlation of gasdermin B staining patterns with prognosis, progression, and immune response in colorectal cancer

BackgroundPyroptosis is a type of programmed cell death mediated by the gasdermin family. Gasdermin B (GSDMB), as a member of gasdermin family, can promote the occurrence of cell pyroptosis. However, the correlations of the GSDMB expression in colorectal cancer with clinicopathological predictors, immune microenvironment, and prognosis are unclear.MethodsSpecimens from 267 colorectal cancer cases were analyzed by immunohistochemistry to determine GSDMB expression, CD3+, CD4+, and CD8+ T lymphocytes, CD20+ B lymphocytes, CD68+ macrophages, and S100A8+ immune cells. GSDMB expression in cancer cells was scored in the membrane, cytoplasm, and nucleus respectively. GSDMB+ immune cell density was calculated. Univariate and multivariate survival analyses were performed. The association of GSDMB expression with other clinicopathological variables and immune cells were also analyzed. Double immunofluorescence was used to identify the nature of GSDMB+ immune cells. Cytotoxicity assays and sensitivity assays were performed to detect the sensitivity of cells to 5-fluorouracil.ResultsMultivariate survival analysis showed that cytoplasmic GSDMB expression was an independent favorable prognostic indicator. Patients with positive cytoplasmic or nuclear GSDMB expression would benefit from 5-fluorouracil based chemotherapy. The assays in vitro showed that high GSDMB expression enhanced the sensitivity of colorectal cancer cells to 5-fluorouracil. Patients with positive membranous or nuclear GSDMB expression had more abundant S100A8+ immune cells in the tumor invasive front. Positive nuclear GSDMB expression indicated more CD68+ macrophages in the tumor microenvironment. Moreover, GSDMB+ immune cell density in the stroma was associated with a higher neutrophil percentage but a lower lymphocyte counts and monocyte percentage in peripheral blood. Furthermore, the results of double immunofluorescence showed that GSDMB co-expressed with CD68 or S100A8 in stroma cells.ConclusionThe GSDMB staining patterns are linked to its role in cancer progression, the immune microenvironment, systemic inflammatory response, chemotherapeutic efficacy, and prognosis. Colorectal cancer cells with high GSDMB expression are more sensitive to 5-fluorouracil. However, GSDMB expression in immune cells has different effects on cancer progression from that in cancer cells.

Ketogenesis attenuated KLF5 disrupts iron homeostasis via LIF to confer oxaliplatin vulnerability in colorectal cancer

Oxaliplatin has been included as a basal drug in various chemotherapy regimens for colorectal cancer (CRC), a global health concern. However, acquired resistance to oxaliplatin affects the prognosis. This study aimed to determine whether the consumption of a KD increases the sensitivity of CRC cells to oxaliplatin via the inhibition of a classical stem cell marker, Krupple-like factor 5 (KLF5). KLF5 functions as a transcription factor for the leukemia inhibitory factor (LIF) and directly binds to its promoter region. LIF upregulation induces dephosphorylation of metal regulatory transcription factor 1 (MTF1), which is recruited to the promoter area of Ferroportin (FPN1), the only cellular iron exporter. FPN1 upregulation reduces the labile iron pool (LIP) and ferroptosis in CRC cells. KLF5 knockdown inhibits the LIF/MTF1/FPN1 axis and induces iron overload, thereby conferring sensitivity to oxaliplatin to CRC cells. KD mimicked KLF5 silencing and sensitized CRC cells to oxaliplatin via a similar mechanism. Thus, potential correlations were observed among ketogenesis, stemness, and iron homeostasis. This finding can be used to formulate a new strategy for overcoming oxaliplatin resistance in patients with CRC.

Establishment of a novel microfluidic co-culture system for simultaneous analysis of multiple indicators of gefitinib sensitivity in colorectal cancer cells.

The therapeutic effect of gefitinib on colorectal cancer (CRC) is unclear, but it has been reported that stromal cells in the tumor microenvironment may have an impact on drug sensitivity. Herein, we established a microfluidic co-culture system and explored the sensitivity of CRC cells co-cultured with cancer-associated fibroblasts (CAFs) to gefitinib. The system consisted of a multichannel chip and a Petri dish. The chambers in the chip and dish were designed to continuously supply nutrients for long-term cell survival and create chemokine gradients for driving cell invasion without any external equipment. Using this system, the proliferation and invasiveness of cells were simultaneously evaluated by quantifying the area of cells and the migration distance of cells. In addition, the system combined with live cell workstation could evaluate the dynamic drug response of co-cultured cells and track individual cell trajectories in real-time. When CRC cells were co-cultured with CAFs, CAFs promoted CRC cell proliferation and invasion and reduced the sensitivity of cells to gefitinib through the exosomes secreted by CAFs. Furthermore, the cells that migrated out of the chip were collected, and EMT-related markers were determined by immunofluorescent and western blot assays. The results demonstrated that CAFs affected the response of CRC cells to gefitinib by inducing EMT, providing new ideas for further research on the resistance mechanism of gefitinib. This suggests that targeting CAFs or exosomes might be a new approach to enhance CRC sensitivity to gefitinib, and our system could be a novel platform for investigating the crosstalk between tumor cells and CAFs and understanding multiple biological changes of the tumor cells in the tumor microenvironment.

Upregulated lncRNA PRNT promotes progression and oxaliplatin resistance of colorectal cancer cells by regulating HIPK2 transcription.

Colorectal cancer (CRC) is the third most common cancer and a significant cause of cancer-related mortality globally. Resistance to chemotherapy, especially during CRC treatment, leads to reduced effectiveness of drugs and poor patient outcomes. Long noncoding RNAs (lncRNAs) have been implicated in various pathophysiological processes of tumor cells, including chemotherapy resistance, yet the roles of many lncRNAs in CRC remain unclear. To identify and analyze the lncRNAs involved in oxaliplatin resistance in CRC and to understand the underlying molecular mechanisms influencing this resistance. Gene Expression Omnibus datasets GSE42387 and GSE30011 were reanalyzed to identify lncRNAs and mRNAs associated with oxaliplatin resistance. Various bioinformatics tools were employed to elucidate molecular mechanisms. The expression levels of lncRNAs and mRNAs were assessed via quantitative reverse transcription-polymerase chain reaction. Functional assays, including MTT, wound healing, and Transwell, were conducted to investigate the functional implications of lncRNA alterations. Interactions between lncRNAs and transcription factors were examined using RIP and luciferase reporter assays, while Western blotting was used to confirm downstream pathways. Additionally, a xenograft mouse model was utilized to study the in vivo effects of lncRNAs on chemotherapy resistance. LncRNA prion protein testis specific (PRNT) was found to be upregulated in oxaliplatin-resistant CRC cell lines and negatively correlated with homeodomain interacting protein kinase 2 (HIPK2) expression. PRNT was demonstrated to sponge transcription factor zinc finger protein 184 (ZNF184), which in turn could regulate HIPK2 expression. Altered expression of PRNT influenced CRC cell sensitivity to oxaliplatin, with overexpression leading to decreased sensitivity and decreased expression reducing resistance. Both RIP and luciferase reporter assays indicated that ZNF184 and HIPK2 are targets of PRNT. The PRNT/ZNF184/HIPK2 axis was implicated in promoting CRC progression and oxaliplatin resistance both in vitro and in vivo. The study concludes that PRNT is upregulated in oxaliplatin-resistant CRC cells and modulates the expression of HIPK2 by sponging ZNF184. This regulatory mechanism enhances CRC progression and resistance to oxaliplatin, positioning PRNT as a promising therapeutic target for CRC patients undergoing oxaliplatin-based chemotherapy.

Nanoparticle delivery of si-Notch1 modulates metabolic reprogramming to affect 5-FU resistance and cell pyroptosis in colorectal cancer

BackgroundNanocarrier delivery of small interfering RNAs (siRNAs) to silence cancer-associated genes is a promising method for cancer treatment. Here, we explored the role and mechanisms of PLAG NPs-delivered si-Notch1 in colorectal cancer (CRC).ResultsHigh Notch1 expression was observed in both sensitive and resistant CRC tissues and cells. Notch1 silencing repressed proliferation and facilitates apoptosis of resistant CRC cells, and suppressed glycolysis and promoted pyroptosis in resistant CRC cells. Notch1 directly interacts with PCAF. Notch1 knockdown’s suppressive effect on glycolysis was reversed by overexpression of PCAF. Moreover, a nanocarrier called PLAG NPs was built with a higher delivery efficiency compared with lipo2000. Si-Notch1 delivered by PLAG NPs efficiently overcame the CRC cells’ 5-FU resistance and facilitated pyroptosis in a CRC mouse model.ConclusionsPLAG NPs carrying si-Notch1 had a great advantage in the extension of half-life circulation and targeting ability, providing a theoretical foundation for precise clinical treatment of CRC.

Targeting FTO induces colorectal cancer ferroptotic cell death by decreasing SLC7A11/GPX4 expression

Ferroptosis is a newly identified iron-dependent form of death that is becoming increasingly recognized as a promising avenue for cancer therapy. N6-methyladenosine (m6A) is the most abundant reversible methylation modification in mRNA contributing to tumorigenesis. However, the crucial role of m6A modification in regulating ferroptosis during colorectal cancer (CRC) tumorigenesis remains elusive. Herein, we find that m6A modification is increased during ferroptotic cell death and correlates with the decreased m6A demethylase fat mass and obesity-associated protein (FTO) expression. Functionally, we demonstrate that suppressing FTO significantly induces CRC ferroptotic cell death, as well as enhancing CRC cell sensitivity to ferroptosis inducer (Erastin and RSL3) treatment. Mechanistically, high FTO expression increased solute carrier family 7 member 11 (SLC7A11) or glutathione peroxidase 4 (GPX4) expressions in an m6A-YTHDF2 dependent manner, thereby counteracting ferroptotic cell death stress. In addition, we identify Mupirocin as a novel inhibitor of FTO, and Mupirocin induces CRC ferroptosis and inhibits tumor growth. Clinically, the levels of FTO, SLC7A11, and GPX4, are highly correlated expression in CRC tissues. Our findings reveal that FTO protects CRC from ferroptotic cell death in promoting CRC tumorigenesis through triggering SLC7A11/GPX4 expression.

Quercetin Enhances 5-fluorouracil Sensitivity by Regulating the Autophagic Flux and Inducing Drp-1 Mediated Mitochondrial Fragmentation in Colorectal Cancer Cells.

While chemotherapy treatment demonstrates its initial effectiveness in eliminating the majority of the tumor cell population, nevertheless, most patients relapse and eventually succumb to the disease upon its recurrence. One promising approach is to explore novel, effective chemotherapeutic adjuvants to enhance the sensitivity of cancer cells to conventional chemotherapeutic agents. In the present study, we explored the effect of quercetin on the sensitivity of colorectal cancer (CRC) cells to conventional chemotherapeutic agent 5-fluorouracil (5-FU) and the molecular mechanisms. MTT assay, colony formation assay and Hoechst staining were performed to investigate the growth inhibition effect of quercetin alone or combined with 5-FU. The expression levels of apoptosis- and autophagy-related proteins were assessed by western blotting. Intracellular ROS was detected using DCFH-DA. The change in the mitochondrial membrane potential was measured by a JC-1 probe. The effect of quercetin on mitochondrial morphology was examined using a mitochondrial-specific fluorescence probe, Mito-Tracker red. The results demonstrated quercetin-induced apoptosis and autophagy, as well as imbalanced ROS, decreased mitochondrial membrane potential, and Drp-1-mediated mitochondrial fission in CRC cells. Autophagy blockage with autophagy inhibitor chloroquine (CQ) enhanced quercetininduced cytotoxicity, indicating that quercetin-induced cytoprotective autophagy. Meanwhile, quercetin enhanced the sensitivity of CRC cells to 5- FU via the induction of mitochondrial fragmentation, which could be further enhanced when the quercetin-induced protective autophagy was blocked by CQ. Our findings suggested that quercetin could induce protective autophagy and Drp-1-mediated mitochondrial fragmentation and enhance the sensitivity of CRC cells to conventional agent 5-FU, which not only suggests that quercetin may act as a chemotherapeutic adjuvant but also implies that the regulation of autophagic flux may be a potential therapeutic strategy for colorectal cancer.

Depletion of CPNE7 sensitizes colorectal cancer to 5-fluorouracil by downregulating ATG9B expression.

We aimed to explore the biological function of CPNE7 and determine the impact of CPNE7 on chemotherapy resistance in colorectal cancer (CRC) patients. According to the Gene Expression Profiling Interactive Analysis database and previously published data, CPNE7 was identified as a potential oncogene in CRC. RT-qPCR and Western blotting were performed to verify the expression of CPNE7. Chi-square test was used to evaluate the associations between CPNE7 and clinical features. Cell proliferation, colony formation, cell migration and invasion, cell cycle and apoptosis were assessed to determine the effects of CPNE7. Transcriptome sequencing was used to identify potential downstream regulatory genes, and gene set enrichment analysis was performed to investigate downstream pathways. The effect of CPNE7 on 5-fluorouracil chemosensitivity was verified by half maximal inhibitory concentration (IC50). Subcutaneous tumorigenesis assay was used to examine the role of CPNE7 in sensitivity of CRC to chemotherapy invivo. Transmission electron microscopy was used to detect autophagosomes. CPNE7 was highly expressed in CRC tissues, and its expression was correlated with T stage and tumour site. Knockdown of CPNE7 inhibited the proliferation and colony formation of CRC cells and promoted apoptosis. Knockdown of CPNE7 suppressed the expression of ATG9B and enhanced the sensitivity of CRC cells to 5-fluorouracil invitro and invivo. Knockdown of CPNE7 reversed the induction of the autophagy pathway by rapamycin and reduced the number of autophagosomes. Depletion of CPNE7 attenuated the malignant proliferation of CRC cells and enhanced the chemosensitivity of CRC cells to 5-fluorouracil.

Abstract 520: circBRIP1 RNA as a non-invasive target engagement pharmacodynamic biomarker for DHX9 inhibition

Abstract DHX9 is a multifunctional DEAH-box ATP-independent RNA helicase which has been reported to play important roles in replication, transcription, translation, RNA splicing and RNA processing which contribute to DHX9’s role in maintenance of genomic stability. DHX9 can bind specifically to inverted repeat Alu elements, preventing back-splicing events that lead to circular RNA and exon skipping of the linear transcript. Upon DHX9 inhibition, a robust induction of Alu-mediated circular RNA is observed. For example, circBRIP1 is an Alu-mediated circular RNA that is robustly elevated upon DHX9 loss, with no observed change to levels of linear BRIP1. Here we describe data demonstrating circBRIP1 as a DHX9 specific target engagement pharmacodynamic (PD) biomarker and its potential utility as a non-invasive clinical PD biomarker. We have previously demonstrated that DHX9 inhibition by novel small molecule inhibitors is efficacious in microsatellite instable (MSI) colorectal cancer (CRC) with defective mismatch repair (dMMR), both in vitro and in vivo. DHX9 inhibition in the HCT116 dMMR CRC cell line results in a robust dose dependent induction of the Alu-mediated circular RNA, circBRIP1, as early as 6 hours after compound treatment. The development of lead series DHX9 inhibitors exhibited a positive correlation between circBRIP1 EC50 and DHX9 biochemical activity, as well as proliferation inhibition in a dMMR CRC cell line. However, dose dependent induction of circBRIP1 following DHX9 inhibition occurs with similar potencies across DHX9i sensitive and insensitive CRC cell lines. This suggests that elevation of circBRIP1 is a unique proximal target engagement PD biomarker of DHX9 inhibition, but not a predictor of DHX9 sensitivity. Importantly, induction of circBRIP1 is observed intratumorally in human xenograft in vivo efficacy studies in mice. Induction in each individual tumor correlates with observed tool compound exposure, providing evidence of a predictable PK/PD relationship. We also observe dose dependent circBRIP1 induction in mouse peripheral blood mononuclear cells (PBMC). We have extended these studies to primary human whole blood, where we see robust dose dependent induction of circBRIP1 after ex vivo treatment with DHX9 inhibitor for 24 hours. Altogether, these studies show evidence for the utility of circBRIP1 as a non-invasive target engagement biomarker for DHX9 inhibitors in the clinic. Citation Format: David Brennan, Jennifer Castro, Matthew H. Daniels, Monique Laidlaw, Chuang Lu, Stephen J. Blakemore, Serena J. Silver, P. Ann Boriack-Sjodin, Kenneth W. Duncan, Jason A. Sager, Robert A. Copeland. circBRIP1 RNA as a non-invasive target engagement pharmacodynamic biomarker for DHX9 inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 520.