Colorectal Cancer Tumor Growth Research Articles

SHARPIN is a novel gene of colorectal cancer that promotes tumor growth potentially via inhibition of p53 expression.

Colorectal cancer (CRC) is widely prevalent and represents a significant contributor to global cancer‑related mortality. There remains a pressing demand for advancements in CRC treatment modalities. The E3 ubiquitin ligase is a critical enzyme involved in modulating protein expression levels via posttranslational ubiquitin‑mediated proteolysis, and it is reportedly involved in the progression of various cancers, making it a target of recent interest in anticancer therapy. In the present study, using comprehensive expression analysis involving spatial transcriptomic analysis with single‑cell RNA sequencing in clinical CRC datasets, the ubiquitin‑associated protein Shank‑associated RH domain interactor (SHARPIN) was identified, located on amplified chromosome 8q, which could promote CRC progression. SHARPIN was found to be upregulated in tumor cells, with elevated expression observed in tumor tissues. This heightened expression of SHARPIN was positively associated with lymphatic invasion and served as an independent predictor of a poor prognosis in patients with CRC. In vitro and in vivo analyses using SHARPIN‑overexpressing or ‑knockout CRC cells revealed that SHARPIN overexpression upregulated MDM2, resulting in the downregulation of p53, while SHARPIN silencing or knockout downregulated MDM2, leading to p53 upregulation, which affects cell cycle progression, tumor cell apoptosis and tumor growth in CRC. Furthermore, SHARPIN was found to be overexpressed in several cancer types, exerting significant effects on survival outcomes. In conclusion, SHARPIN represents a newly identified novel gene with the potential to promote tumor growth following apoptosis inhibition and cell cycle progression in part by inhibiting p53 expression via MDM2 upregulation; therefore, SHARPIN represents a potential therapeutic target for CRC.

DST regulates cisplatin resistance in colorectal cancer via PI3K/Akt pathway.

Dystonin (DST), a potential tumor suppressor gene, plays a crucial role in regulating cancer cell proliferation and resistance to chemotherapy. However, DST's specific role in colorectal cancer (CRC) has not been thoroughly investigated, and this study aims to elucidate its molecular role in modulating cisplatin (DDP) resistance in CRC. DST expression was analyzed in CRC tumors, DDP-resistant CRC tissues, paracancer tissues, and normal tissues. Lentiviral overexpression and shRNA knockdown were conducted in advanced CRC and DDP-resistant cell lines to assess cell viability, apoptosis, invasion, migration, proliferation, and angiogenesis. Xenograft mouse models studied DST's impact on CRC tumor growth and DDP resistance in vivo. DST expression was significantly reduced in CRC tumor and DDP-resistant CRC tissues compared to paracancer and normal tissues (P < .001). Upregulating DST inhibited CRC and DDP-resistant cell viability, proliferation, invasion, and migration while promoting apoptosis. DST overexpression also reduced angiogenesis and attenuated DDP-induced cytotoxicity in CRC cells. Mechanistically, DST upregulation suppressed DDP resistance in CRC cells via the PI3K/Akt signaling pathway. DST upregulation reduced CRC tumor growth and mitigated DDP resistance, in vivo. DST plays a crucial role in limiting CRC progression and overcoming DDP resistance, suggesting potential for targeted CRC therapies.

Novel Celecoxib Derivative, RF26, Blocks Colon Cancer Cell Growth by Inhibiting PDE5, Activating cGMP/PKG Signaling, and Suppressing β-catenin-dependent Transcription.

Previous studies have reported that the cGMP-specific PDE5 isozyme is overexpressed in colon adenomas and adenocarcinomas and essential for colon cancer cell proliferation, while PDE5 selective inhibitors (e.g., sildenafil) have been reported to have cancer chemopreventive activity. This study aimed to determine the anticancer activity of a novel PDE5 inhibitor, RF26, using colorectal cancer (CRC) cells and the role of PDE5 in CRC tumor growth in vivo. The objective of this study was to characterize the anticancer activity of a novel celecoxib derivative, RF26, in CRC cells previously reported to lack COX-2 inhibition but have potent PDE5 inhibitory activity. Anticancer activity of RF26 was studied using human CRC cell lines. Its effects on intracellular cGMP levels, cGMP-dependent protein kinase (PKG) activity, β-catenin levels, TCF/LEF transcriptional activity, cell cycle distribution, and apoptosis were measured. CRISPR/cas9 PDE5 knockout techniques were used to determine if PDE5 mediates the anticancer activity of RF26 and validate PDE5 as a cancer target. RF26 was appreciably more potent than celecoxib and sildenafil to suppress CRC cell growth and was effective at concentrations that increased intracellular cGMP levels and activated PKG signaling. RF26 suppressed β-catenin levels and TCF/LEF transcriptional activity and induced G1 cell cycle arrest and apoptosis within the same concentration range. CRISPR/cas9 PDE5 knockout CRC cells displayed reduced sensitivity to RF26, proliferated slower than parental cells, and failed to establish tumors in mice. Further evaluation of RF26 for the prevention or treatment of cancer and studying the role of PDE5 in tumorigenesis are warranted.

Telomere transcripts act as tumor suppressor and are associated with favorable prognosis in colorectal cancer with low proliferating cell nuclear antigen expression.

Telomeric repeat-containing RNAs (TERRA) and telomerase RNA component (TERC) regulate telomerase activity (TA) and thereby contribute to telomere homeostasis by influencing telomere length (TL) and the cell immortality hallmark of cancer cells. Additionally, the non-canonical functions of telomerase reverse transcriptase (TERT) and TERRA appear to be involved in the epithelial-mesenchymal transition (EMT), which is important for cancer progression. However, the relationship between TERRA and patient prognosis has not been fully characterized. In this small-scale study, 68 patients with colorectal cancer (CRC) were evaluated for correlations between telomere biology, proliferation, and EMT gene transcripts and disease outcome. The proliferating cell nuclear antigen (PCNA) and the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2) showed a positive correlation with TERRA, while TA and TERRA exhibited an inverse correlation. Consistent with previous findings, the present study revealed higher expression levels of TERT and TERC, and increased TA and TL in CRC tumor tissue compared to adjacent non-tumor tissue. In contrast, lower expression levels of TERRA were observed in tumor tissue. Patients with high TERRA expression and low PCNA levels exhibited favorable overall survival rates compared to individuals with the inverse pattern. Furthermore, TERRA suppressed CRC tumor growth in severe combined immunodeficiency disease (SCID) mice. In conclusion, our study extends previously published research on TERRA suggesting its potential therapeutic role in telomerase-positive CRC.

Loss of Trefoil Factor 1 Accelerates the Immune Response to Colorectal Cancer.

Recent studies suggest that PD-L1 expression in immune cells, rather than tumor cells, plays a key role in tumor immunity. Trefoil factor family 1 (TFF1) is a secreted protein expressed mainly by the gastrointestinal epithelium and is related to the development of malignant disease. This study investigated the effects of TFF1 on tumor immunity in a xenograft mouse model of colorectal cancer (CRC). MC38 cells were implanted in wild-type (WT) and TFF1KO mice, and the tumor micro-environment was investigated using immunohistochemistry. The circulating immune cells were analyzed using flow cytometry. Tumor growth was suppressed in TFF1KO mice. In the tumor microenvironment, CD8- and CD4-positive T cells and CD11c-positive dendritic cells (DCs) were frequently found in TFF1KO mice. When an immune checkpoint inhibitor was administered to these mice, almost half of the tumors in TFF1KO mice showed a complete response. The number of circulating PD-L1/DCs was markedly associated with tumor volume, with TFF1 deletion accelerating this effect and its injection decreasing it. These findings indicate that loss of TFF1 activates tumor immunity via frequent T-cell priming by DCs, and eventually suppresses tumor growth in CRC. In addition, the number of circulating PD-L1/DCs was identified as a predictive marker of checkpoint-inhibiting therapy efficacy. Loss of TFF1 resulted in accelerated immune response to colorectal cancer. Further studies are needed to investigate the precise mechanisms of TFF1 in immunotolerance and develop a novel TFF1-inhibiting immunotherapeutic strategy for CRC.

Inhibition of IRP2-dependent reprogramming of iron metabolism suppresses tumor growth in colorectal cancer

BackgroundDysregulation of iron metabolism is implicated in malignant transformation, cancer progression, and therapeutic resistance. Here, we demonstrate that iron regulatory protein 2 (IRP2) preferentially regulates iron metabolism and promotes tumor growth in colorectal cancer (CRC).MethodsIRP2 knockdown and knockout cells were generated using RNA interference and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 methodologies, respectively. Cell viability was evaluated using both CCK-8 assay and cell counting techniques. Furthermore, IRP2 inhibition was determined by surface plasmon resonance (SPR) and RNA immunoprecipitation (IP). The suppressive effects of IRP2 were also corroborated in both organoid and mouse xenograft models, providing a comprehensive validation of IRP2’s role.ResultsWe have elucidated the role of IRP2 as a preferential regulator of iron metabolism, actively promoting tumorigenesis within CRC. Elevated levels of IRP2 expression in patient samples are correlated with diminished overall survival, thereby reinforcing its potential role as a prognostic biomarker. The functional suppression of IRP2 resulted in a pronounced delay in tumor growth. Building on this proof of concept, we have developed IRP2 inhibitors that significantly reduce IRP2 expression and hinder its interaction with iron-responsive elements in key iron-regulating proteins, such as ferritin heavy chain 1 (FTH1) and transferrin receptor (TFRC), culminating in iron depletion and a marked reduction in CRC cell proliferation. Furthermore, these inhibitors are shown to activate the AMPK-ULK1-Beclin1 signaling cascade, leading to cell death in CRC models.ConclusionsCollectively, these findings highlight the therapeutic potential of targeting IRP2 to exploit the disruption of iron metabolism in CRC, presenting a strategic advancement in addressing a critical area of unmet clinical need.

Novel ST1926 Nanoparticle Drug Formulation Enhances Drug Therapeutic Efficiency in Colorectal Cancer Xenografted Mice.

Cancer is a major public health problem that ranks as the second leading cause of death. Anti-cancer drug development presents with various hurdles faced throughout the process. Nanoparticle (NP) formulations have emerged as a promising strategy for enhancing drug delivery efficiency, improving stability, and reducing drug toxicity. Previous studies have shown that the adamantyl retinoid ST1926 displays potent anti-tumor activities in several types of tumors, particularly in colorectal cancer (CRC). However, phase I clinical trials in cancer patients using ST1926 are halted due to its low bioavailability. In this manuscript, we developed ST1926-NPs using flash nanoprecipitation with polystyrene-b-poly (ethyleneoxide) as an amphiphilic stabilizer and cholesterol as a co-stabilizer. Dynamic light scattering revealed that the resulting ST1926-NPs Contin diameter was 97 nm, with a polydispersity index of 0.206. Using cell viability, cell cycle analysis, and cell death assays, we showed that ST1926-NP exhibited potent anti-tumor activities in human CRC HCT116 cells. In a CRC xenograft model, mice treated with ST1926-NP exhibited significantly lowered tumor volumes compared to controls at low drug concentrations and enhanced the delivery of ST1926 to the tumors. These findings highlight the potential of ST1926-NPs in attenuating CRC tumor growth, facilitating its further development in clinical settings.

Circ_0006174 Upregulates IGF1R to Enhance Radioresistance and Tumorigenesis in Colorectal Cancer via miR-940 Suppression.

Colorectal cancer (CRC) is one of the most common malignancies all over the world. Increasing evidence has revealed that circular RNAs (circRNAs) are involved in the progression of CRC. In this study, we aimed to investigate the role and underlying mechanism of circ_0006174 in the development and radiosensitivity of CRC. Circ_0006174, microRNA-940 (miR-940), and insulin-like growth factor 1 receptor (IGF1R) expression levels were evaluated by real-time quantitative polymerase chain reaction (RT-qPCR). Theradiosensitivityofcellsalsowasassessedusingcolonyformationassay. Besides, cell proliferation, apoptosis, migration, and invasion were detected by cell counting kit-8 (CCK-8), flow cytometry, and transwell assays. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were performed to verify the relationship between miR-940 and circ_0006174 or IGF1R. IGF1R protein level was examined using western blot. A xenograft tumor model was used to verify the function of circ_0006174 in CRC tumor growth in vivo. Circ_0006174 and IGF1R levels were elevated and miR-940 expression was decreased in CRC tissues and cells. Circ_0006174 knockdown enhanced the radiosensitivity of CRC cells by regulating cell proliferation, apoptosis, migration, and invasion in vitro. In mechanism, circ_0006174 served as a sponge for miR-940 to upregulate IGF1R expression. Moreover, circ_0006174 silencing suppressed CRC growth in vivo. Circ_0006174 boosts radioresistance of CRC cells at least partly through upregulating IGF1R expression by sponging miR-940, providing a novel theoretical basis for CRC therapy.

Linear polyubiquitylation of Gli protein regulates its protein stability and facilitates tumor growth in colorectal cancer

The linear ubiquitin chain assembly complex (LUBAC) mediates the linear ubiquitination of various proteins and is involved in NF-κB signaling and immune regulation. However, the function and mechanism of linear ubiquitination in regulating oncogenic signaling and tumor growth have remained poorly understood. Herein, we identified Gli proteins, key transcription factors in the Hedgehog (Hh) signaling pathway, as novel substrates of LUBAC. Linear ubiquitination stabilizes Gli proteins, leading to the noncanonical activation of Hh signaling in CRC cells. Furthermore, LUBAC facilitates tumor growth in CRC cells. Additionally, elevated expression of LUBAC components in CRC tissues was observed, and higher expression levels of these components correlated with poor prognosis in CRC patients. Interestingly, inhibition of LUBAC using either a small molecule agonist or RNA silencing specifically suppressed cell growth in CRC cells but had no effect on normal intestinal cells. Taken together, aberrant expression of LUBAC components activates Hh signaling noncanonically by mediating linear ubiquitination, promoting tumor growth in CRC, demonstrating the novel function of linear ubiquitination in regulating the protein stability of its substrates and highlighting the potential of targeting LUBAC as a therapeutic strategy in CRC.

Serotonylation in tumor-associated fibroblasts contributes to the tumor-promoting roles of serotonin in colorectal cancer

Accumulated studies have highlighted the diverse roles of 5-hydroxytryptamine (5-HT), or serotonin, in cancer biology, particularly in colorectal cancer (CRC). While 5-HT primarily exerts its effects through binding to various 5-HT receptors, receptor-independent mechanisms such as serotonylation remain unclear. This study revealed that depleting 5-HT, either through genetic silencing of Tph1 or using a selective TPH1 inhibitor, effectively reduced the growth of CRC tumors. Interestingly, although intrinsic 5-HT synthesis exists in CRC, it is circulating 5-HT that mediates the cancer-promoting function of 5-HT. Blocking the function of 5-HT receptors showed that the oncogenic roles of 5-HT in CRC operate through a mechanism that is separate from its receptor. Instead, serotonylation of histone H3Q5 (H3Q5ser) was found in CRC cells and cancer-associated fibroblasts (CAFs). H3Q5ser triggers a phenotypic switch of CAFs towards an inflammatory-like CAF (iCAF) subtype, which further enhances CRC cell proliferation, invasive characteristics, and macrophage polarization. Knockdown of the 5-HT transporter SLC22A3 or inhibition of TGM2 reduces H3Q5ser levels and reverses the tumor-promoting phenotypes of CAFs in CRC. Collectively, this study sheds light on the serotonylation-dependent mechanisms of 5-HT in CRC progression, offering insights into potential therapeutic strategies targeting the serotonin pathway for CRC treatment.

Miltirone induces GSDME-dependent pyroptosis in colorectal cancer by activating caspase 3

Colorectal cancer (CRC) is a common and malignant tumor, ranking as the third most common cancer in men and the second most common cancer in women. Pyroptosis, a recently described programmed cell death mechanism mediated by the GSDM family, has emerged as an immunogenic mechanism for chemotherapy drugs in tumor treatment. In this study, we discovered that Miltirone has the ability to reduce the viability of CRC cells (SW620 and HCT116) and cause the proteolytic cleavage of gasdermin E (GSDME) in CRC cells. It was also observed that inhibiting GSDME prevented pyroptotic cell death induced by Miltirone in SW620 and HCT116 cells. Furthermore, the main active component of Miltirone was found to effectively bind with caspase 3. SiRNA-mediated caspase 3 silencing and specific caspase 3 inhibitor Z-DEVD-FMK were shown to weaken Miltirone-induced GSDME-dependent cell death. The findings of the study suggest that Miltirone has the potential to inhibit the growth of CRC tumors in vivo by inducing pyroptotic cell death. This indicates that Miltirone could be a viable therapeutic agent for the treatment of CRC through GSDME-dependent pyroptosis. These results offer a promising new option for the clinical treatment of CRC.

Circ_0000395 promotes cell growth, metastasis and oxaliplatin resistance by regulating miR-153–5p/MYO6 in colorectal cancer

BackgroundCircular RNAs (circRNAs) are involved in the regulation of colorectal cancer (CRC) progression and chemoresistence. Here, we attempted to reveal the function and mechanism of circ_0000395 in CRC chemoresistence. MethodsThe expression levels of circ_0000395, microRNA (miR)-153–5p, and myosin VI (MYO6) were determined by quantitative real-time PCR. Cell growth, metastasis and oxaliplatin resistance were evaluated via EdU assay, colony formation assay, flow cytometry, transwell assay, and cell counting kit 8 assay. Xenograft tumor model was adopted to evaluate the role of circ_0000395 on CRC tumor growth and oxaliplatin sensitivity. Protein expression of drug-resistance markers and MYO6 was analyzed by western blot. The target relationship between miR-153–5p and circ_0000395 or MYO6 was validated via dual-luciferase reporter assay and RIP assay. ResultsCirc_0000395 expression was enhanced in CRC tissues and cells. Silencing of circ_0000395 repressed CRC cell proliferation, migration and invasion, while promoted apoptosis and oxaliplatin sensitivity. Besides, circ_0000395 knockdown also reduced CRC tumor growth and enhanced the sensitivity of tumor to oxaliplatin. Additionally, circ_0000395 acted as a sponge for miR-153–5p, and miR-153–5p targeted MYO6. Functional experiments suggested that miR-153–5p inhibitor or MYO6 overexpression could reverse the suppressive effect of circ_0000395 knockdown on CRC cell growth, metastasis and oxaliplatin resistance. ConclusionCirc_0000395 promoted CRC cell growth, metastasis and oxaliplatin resistance via the miR-153–5p/MYO6 axis, which might provide new insights into the treatment of CRC.

ATP6V0A1-dependent cholesterol absorption in colorectal cancer cells triggers immunosuppressive signaling to inactivate memory CD8+ T cells

Obesity shapes anti-tumor immunity through lipid metabolism; however, the mechanisms underlying how colorectal cancer (CRC) cells utilize lipids to suppress anti-tumor immunity remain unclear. Here, we show that tumor cell-intrinsic ATP6V0A1 drives exogenous cholesterol-induced immunosuppression in CRC. ATP6V0A1 facilitates cholesterol absorption in CRC cells through RAB guanine nucleotide exchange factor 1 (RABGEF1)-dependent endosome maturation, leading to cholesterol accumulation within the endoplasmic reticulum and elevated production of 24-hydroxycholesterol (24-OHC). ATP6V0A1-induced 24-OHC upregulates TGF-β1 by activating the liver X receptor (LXR) signaling. Subsequently, the release of TGF-β1 into the tumor microenvironment by CRC cells activates the SMAD3 pathway in memory CD8+ T cells, ultimately suppressing their anti-tumor activities. Moreover, we identify daclatasvir, a clinically used anti-hepatitis C virus (HCV) drug, as an ATP6V0A1 inhibitor that can effectively enhance the memory CD8+ T cell activity and suppress tumor growth in CRC. These findings shed light on the potential for ATP6V0A1-targeted immunotherapy in CRC.

TRIP13 Activates Glycolysis to Promote Cell Stemness and Strengthen Doxorubicin Resistance of Colorectal Cancer Cells.

Chemotherapy resistance is one of the main causes of clinical chemotherapy failure. Current cancer research explores the drug resistance mechanism and new therapeutic targets. This work aims to elucidate the mechanism of thyroid hormone receptor interactor 13 (TRIP13) affecting doxorubicin (DOX) resistance in colorectal cancer (CRC). Bioinformatics analyses were employed to clarify TRIP13 expression in CRC tissues and predict the correlation of the TRIP13 enrichment pathway with glycolysis-related genes and stemness index mRNAsi. Quantitative real-time polymerase chain reaction and western blot were adopted to analyze the expression of TRIP13 and glycolysis- related genes. Cell Counting Kit-8 was utilized to determine the cell viability and IC50 value. Western blot was employed to measure the expression of stemness-related factors. Cell function assays were performed to detect cells' sphere-forming ability and glycolysis level. Animal models were constructed to determine the effects of TRIP13 expression on CRC tumor growth. TRIP13 was significantly overexpressed in CRC, concentrated in the glycolysis signaling pathway, and positively correlated with stemness index mRNAsi. High expression of TRIP13 facilitated DOX resistance in CRC. Further mechanistic studies revealed that overexpression of TRIP13 could promote cell stemness through glycolysis, which was also confirmed in animal experiments. TRIP13 was highly expressed in CRC, which enhanced the DOX resistance of CRC cells by activating glycolysis to promote cell stemness. These findings offer new insights into the pathogenesis of DOX resistance in CRC and suggest that TRIP13 may be a new target for reversing DOX resistance in CRC.

Investigating the Effects of AL049796.1 Silencing in Inhibiting High Glucose-Induced Colorectal Cancer Progression.

Patients with colorectal cancer (CRC) and diabetes share many risk factors. Despite a strong association between diabetes and CRC being widely studied and confirmed, further genetic research is needed. This study found higher AL049796.1 and TEA domain transcription factor 1 (TEAD1) levels (both mRNA and protein) in CRC tissues of diabetic patients compared with nondiabetics, but no significant difference in miR-200b-3p levels. A positive correlation between AL049796.1 and TEAD1 protein existed regardless of diabetes status, whereas miR-200b-3p was only negatively correlated with TEAD1 protein in nondiabetic CRC tissues. In vitro experiments have shown that high glucose (HG) treatment increased AL049796.1 in CRC cells, and AL049796.1 silencing reduced HG-induced proliferation, migration and invasion, as well as connective tissue growth factor, cysteine-rich angiogenic inducer 61, and epidermal growth factor receptor protein expression. Mechanistic investigations indicated that AL049796.1 could mitigate suppression of miR-200b-3p on TEAD1 posttranscriptionally by acting as a competitive binder. In vivo, subcutaneous CRC tumors in streptozotocin (STZ)-induced mice grew significantly faster; AL049796.1 silencing did not affect the growth of subcutaneous CRC tumors but significantly reduced that of STZ-induced mice. Our study suggests that AL049796.1 independently contributes to the risk of CRC in diabetic patients, highlighting its potential as both a therapeutic target and a novel biomarker for CRC among individuals with diabetes.

Death receptors 4/5 mediate tumour sensitivity to natural killer cell-mediated cytotoxicity in mismatch repair deficient colorectal cancer.

Identifying the target of natural killer (NK) cells in colorectal cancer (CRC) is critical for optimising the clinical use of NK cell-mediated immunotherapy. Mismatch repair deficiency (dMMR) is associated with high immune cell infiltration and MHC Class I defects. Whether dMMR CRC responses to NK cell therapy remains unclear. MLH1, DR4, and DR5 knockout cell lines were established using CRISPR-Cas9 system. NK92-MI or NK cell isolated from BABL/C mice were used as effector cells against tumour cells. Inflammatory cytokines secretion by CRC cells was assessed via cytokine analysis. NK-cell-deficient/proficient animal models were used to validate the NK cell sensitivity. We observed that dMMR CRC cells were more sensitive to NK cell-mediated cytotoxicity than were mismatch-repair-proficient (pMMR) CRC cells. In dMMR CRC, Death receptor (DR)4/5 was upregulated and mediated sensitivity to NK cell-mediated cytotoxicity. DR4/5-mediated secretion of interleukin -12 sustained NK cell viability in dMMR CRC. NK cell depletion induced dMMR CRC tumour growth, and NK cell transfer inhibited lung metastasis of dMMR CRC with DR4/5 expression in vivo. TP53 upregulated DR4/DR5 expression in dMMR CRC. dMMR associated with increased sensitivity to NK cell-mediated cytotoxicity in CRC. DR4/DR5 sensitise dMMR CRC to NK cell-mediated cytotoxicity.

METTL5 promotes cell proliferation, invasion, and migration by up-regulating Toll-like receptor 8 expression in colorectal cancer.

N6-methyladenosine (m6A) modification represents the predominant alteration found in eukaryotic messenger RNA and plays a crucial role in the progression of various tumors. However, despite its significance, the comprehensive investigation of METTL5, a key m6A methyltransferase, in colorectal cancer (CRC) remains limited. To investigate the role of METTL5 in CRC. We assessed METTL5 expression levels in clinical samples obtained from CRC patients as well as in CRC cell lines. To elucidate the downstream targets of METTL5, we performed RNA-sequencing analysis coupled with correlation analysis, leading us to identify Toll-like receptor 8 (TLR8) as a potential downstream target. In vitro functional assessments of METTL5 and TLR8 were conducted using CCK-8 assays, scratch assays, as well as assays measuring cell migration and invasion. Our findings reveal a pronounced upregulation of METTL5 expression in both CRC cells and tissues, which correlated significantly with an unfavorable prognosis. In vitro experiments unequivocally demonstrated the oncogenic role of METTL5, as evidenced by its promotion of CRC cell proliferation, invasion, and migration. Notably, we identified TLR8 as a downstream target of METTL5, and subsequent down-regulation of TLR8 led to a significant inhibition of CRC cell proliferation, invasion, and tumor growth. The heightened expression of METTL5 in CRC is strongly associated with clinicopathological features and a poor prognosis, thereby underscoring its potential utility as a critical marker for facilitating early diagnosis and prognostication in CRC.

Isthmin-1 promotes growth and progression of colorectal cancer through the interaction with EGFR and YBX-1

While previous studies have indicated the involvement of Isthmin 1 (ISM1), a secreted protein, in cancer development, the precise mechanisms have remained elusive. In this study, we unveiled that ISM1 is significantly overexpressed in both the blood and tissue samples of colorectal cancer (CRC) patients, correlating with their poor prognosis. Functional experiments demonstrated that enforced ISM1 expression significantly enhances CRC proliferation, migration, invasion and tumor growth. Notably, our investigation reveals an interaction of ISM1 with epidermal growth factor receptor (EGFR), a member of the receptor tyrosine kinase (RTK) family of CRC cells. The binding of ISM1 triggered EGFR activation and initiate downstream signaling pathways. Meanwhile, intracellular ISM1 interacted with Y-box binding protein 1 (YBX1), enhancing its transcriptional regulation on EGFR. Furthermore, our research uncovered the regulation of ISM1 expression by the hypoxia-inducible transcription factor HIF-1α in CRC cells. Mechanistically, we identified HIF-1α as a direct regulator of ISM1, binding to a hypoxia response element on its promoter. This novel mechanism illuminated potential therapeutic targets, offering insights into restraining HIF-1α/ISM1/EGFR-driven CRC progression and metastasis.

Abstract 5937: KS20226, a first-in-class IRP2 inhibitor, preferentially induces reprogramming of iron metabolism and suppresses tumor growth in colorectal cancer

Abstract Background: Iron homeostasis plays a role in redox activity, mitochondrial function, and cell cycle regulation. Specifically, the dysregulation of iron metabolism is implicated in malignant transformation and cancer progression. Iron regulatory protein 2 (IRP2) is crucial for maintaining balance within iron levels and primarily controls genes associated with iron utilization. Our discovery of KS20226, a first-in-class IRP2 inhibitor, reveals remarkable potential as an effective anti-cancer drug candidate. We have elucidated the mechanisms underlying the therapeutic effects of KS20226, thereby highlighting the feasibility of targeting IRP2 in colorectal cancer (CRC). Methods: The proliferation inhibitory activity of KS20226 was assessed the CCK-8 and 3D Spheroid models. To investigate mitochondrial function, we conducted measurement of oxidative phosphorylation (OCR) and glycoPER. Furthermore, gene expression analysis was performed through RNA sequencing (RNA-seq) using the Illumina HiSeq 2500 system platform (Macrogen Inc.). Differential expressed gene (DEG) analysis was carried out using the R package DESeq2, and pathway enrichment analysis was analyzed using GSEA (v_4.2.3.). For the observation of autophagic morphology, we utilized transmission electron microscopy (TEM), immunofluorescence staining targeting the autophagy marker LC3B, and immunoblotting. Results: KS20226 exhibited a remarkable growth inhibitory effect in 10 different CRC cell lines, with GI50 values ranging from 1 to 10 μM. KS20226 suppressed IRP2 expression and the corresponding occupancy of the iron-responsive elements of ferritin H and transferrin receptor 1, resulting in iron deprivation, and efficiently controlled CRC growth. Pharmacologic inhibition of IRP2 induced reprogramming of mitochondrial respiration, reducing reliance on oxidative phosphorylation. The observed mitochondrial dysfunction induced by KS20226 was correlated with pathway analysis, revealing a decrease in the gene-set for HALLMARK_OXIDATIVE_PHOSPHORYLATION (NES = -1.38, p-value = 0.008) and an increase in the gene-set for POSITIVE_REGULATION_OF_AUTOPHAGY (NES=1.43, p-value=0.016). Immunoblotting demonstrated that KS20226 activated the phosphorylation of AMPK, ULK, and Beclin1 cascade, leading to the autophagic cell death of the CRC cells. Conclusions: The reliance of cancer cells on iron metabolism to drive proliferation has led to the development of therapeutic strategies targeting the inhibition of IRP2. KS20226, a first-in-class IPR2 inhibitor, exhibits distinct therapeutic properties, such as perturbation of mitochondrial iron metabolism and induction of autophagic cell death. Our findings represent important progress toward exploiting IRP2-mediated abnormal iron metabolism in cancer, an unexplored area of research with substantial unmet needs. Citation Format: Jieon Hwang, Areum Park, Chinwoo Kim, Chang Gon Kim, Jaesung Kwak, Byungil Kim, Hyunjin Shin, Minhee Ku, Jaemoon Yang, Ayoung Baek, Jiwon Choi, Hocheol Lim, Kyoung Tai No, Xianghua Zhao, Uyeong Choi, Tae Il Kim, Kyu-Sung Jeong, Hyuk Lee, Sang Joon Shin. KS20226, a first-in-class IRP2 inhibitor, preferentially induces reprogramming of iron metabolism and suppresses tumor growth in colorectal cancer [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 5937.

Abstract 6832: Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC

Abstract Background: Lemur tail kinase 3 (LMTK3) is a serine/threonine protein kinase with roles in multiple cellular pathways, including Wnt signaling, KIT modulation, and the estrogen receptor pathway; identifying it as a potential target in various cancers. LMTK3 is highly expressed within the central nervous system (CNS) and has been demonstrated to influence neuronal signaling. This study aimed to investigate the potential connection between LMTK3 and neuronal signaling pathways in colorectal cancer (CRC). Methods: Using the Cre/loxP system, we generated a C57BL/6 mouse strain that lacks the expression of LMTK3. 1 × 106 MC38 cells were subcutaneously implanted into 8 weeks old male and female wildtype (WT) and knockout (KO) mice. Tumor growth was measured until endpoint was reached. Tumors were then processed for RNA sequencing. Differentially expressed genes (DEGs) for each comparison group: WT Female vs WT Male, WT Female vs KO Female, WT Male vs KO Male, and KO Female vs KO Male were analyzed. Ingenuity pathway analysis (IPA) was done for identification of upstream regulators involved and affected cellular pathways. Expression levels of DEGs for glutamate receptor subunits were validated using qRT-PCR. Results: Both male and female LMTK3 KO mice showed highly significant (p&amp;lt;0.01) reduced tumor size when compared to WT mice of the same sex. Additionally, the KO female tumors were significantly (p&amp;lt;0.05) smaller when compared to the KO males. RNA sequencing revealed 761 DEGs in KO and WT female and 133 DEGs in KO female and male cohort comparisons (q&amp;lt;0.05). IPA results showed significant (p&amp;lt;0.01, Z-score←2) alterations in pathways including glutaminergic receptor signaling, neurovascular coupling, CREB signaling in neurons, and synaptic long-term depression in the WT vs KO female tumor cohort comparison. Within these pathways DEGs: CACNA1E, CACNA2DA, CACNG2, GRIA1, GRIA2, GRIN1, and GRIN2B, were downregulated in KO females. Upstream regulator analysis identified transcription regulators such as OTX2 and NEUROD1 as well as beta-estradiol to be significantly inhibited (p&amp;lt;0.01, Z-score←2) in KO females. qRT-PCR analysis showed significant (p&amp;lt;0.05) decreased expression in GRIA1, GRIN1, and GRIN2B as well as GRIN2A and GRIN2C in KO females when compared to WT females or KO males, validating RNA seq results. Conclusions: These results showed the involvement of LMTK3 in the gut-brain axis and its effect on CRC tumor growth. Downregulation of multiple neuronal pathways and genes suggest that LMTK3 may influence neuron signaling and neurotransmitter release to produce stimulatory or inhibitory effects within the tumor environment. The concurrent inhibition of beta-estradiol in LMTK3 KO tumors allude to possible sex differences the kinase’s effect on the observed tumor growth. Further studies are needed to confirm the mechanism of interaction of LMTK3 within these pathways. Citation Format: Lesly Torres-Gonzalez, Shivani Soni, Yan Yang, Goar Smbatyan, Jae Ho Lo, Pooja Mittal, Francesca Battaglin, Indrakant K. Singh, Priya Jayachandran, Sandra Algaze, Alexandra Wong, Karam Ashouri, Wu Zhang, Georgios Giamas, Joshua Millstein, Heinz-Josef Lenz. Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC [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 6832.