Protein Expression In Tumor Tissue Research Articles

Significance of NKX2-1 as a biomarker for clinical prognosis, immune infiltration, and drug therapy in lung squamous cell carcinoma.

This study was performed to determine the biological processes in which NKX2-1 is involved and thus its role in the development of lung squamous cell carcinoma (LUSC) toward improving the prognosis and treatment of LUSC. Raw RNA sequencing (RNA-seq) data of LUSC from The Cancer Genome Atlas (TCGA) were used in bioinformatics analysis to characterize NKX2-1 expression levels in tumor and normal tissues. Survival analysis of Kaplan-Meier curve, the time-dependent receiver operating characteristic (ROC) curve, and a nomogram were used to analyze the prognosis value of NKX2-1 for LUSC in terms of overall survival (OS) and progression-free survival (PFS). Then, differentially expressed genes (DEGs) were identified, and Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Gene Set Enrichment Analysis (GSEA) were used to clarify the biological mechanisms potentially involved in the development of LUSC. Moreover, the correlation between the NKX2-1 expression level and tumor mutation burden (TMB), tumor microenvironment (TME), and immune cell infiltration revealed that NKX2-1 participates in the development of LUSC. Finally, we studied the effects of NKX2-1 on drug therapy. To validate the protein and gene expression levels of NKX2-1 in LUSC, we employed immunohistochemistry(IHC) datasets, The Gene Expression Omnibus (GEO) database, and qRT-PCR analysis. NKX2-1 expression levels were significantly lower in LUSC than in normal lung tissue. It significantly differed in gender, stage and N classification. The survival analysis revealed that high expression of NKX2-1 had shorter OS and PFS in LUSC. The multivariate Cox regression hazard model showed the NKX2-1 expression as an independent prognostic factor. Then, the nomogram predicted LUSC prognosis. There are 51 upregulated DEGs and 49 downregulated DEGs in the NKX2-1 high-level groups. GO, KEGG and GSEA analysis revealed that DEGs were enriched in cell cycle and DNA replication.The TME results show that NKX2-1 expression was positively associated with mast cells resting, neutrophils, monocytes, T cells CD4 memory resting, and M2 macrophages but negatively associated with M1 macrophages. The TMB correlated negatively with NKX2-1 expression. The pharmacotherapy had great sensitivity in the NKX2-1 low-level group, the immunotherapy is no significant difference in the NKX2-1 low-level and high-level groups. The analysis of GEO data demonstrated concurrence with TCGA results. IHC revealed NKX2-1 protein expression in tumor tissues of both LUAD and LUSC. Meanwhile qRT-PCR analysis indicated a significantly lower NKX2-1 expression level in LUSC compared to LUAD. These qRT-PCR findings were consistent with co-expression analysis of NKX2-1. We conclude that NKX2-1 is a potential biomarker for prognosis and treatment LUSC. A new insights of NKX2-1 in LUSC is still needed further research.

Spatial intratumor heterogeneity of programmed death-ligand 1 expression predicts poor prognosis in resected non-small cell lung cancer.

We quantified the pathological spatial intratumor heterogeneity (ITH) of programmed death-ligand 1 (PD-L1) expression and investigated its relevance to patient outcomes in surgically resected non-small cell lung carcinoma (NSCLC). This study enrolled 239 consecutive surgically resected NSCLC specimens of pathological stage IIA-IIIB. To characterize the spatial ITH of PD-L1 expression in NSCLC tissues, we developed a mathematical model based on texture image analysis and determined the spatial heterogeneity index of PD-L1 (SHIP) for each tumor. The correlation between the SHIP values and clinicopathological characteristics, including prognosis, was analyzed. Furthermore, an independent cohort of 70 cases was analyzed for model validation. Clinicopathological analysis showed correlations between high SHIP values and histological subtype (squamous cell carcinoma, p < .001) and vascular invasion (p = .004). Survival analysis revealed that patients with high SHIP values presented a significantly worse recurrence-free rate than those with low SHIP values (5-year RFS 26.3% vs 47.1%, p < .005). The impact of SHIP on cancer survival rates was verified through validation in an independent cohort. Moreover, high SHIP values were significantly associated with tumor recurrence in squamous cell carcinoma (5-year RFS 29.2% vs 52.8%, p < .05) and adenocarcinoma (5-year RFS 19.6% vs 43.0%, p < .01). Moreover, we demonstrated that a high SHIP value was an independent risk factor for tumor recurrence. We presented an image analysis model to quantify the spatial ITH of protein expression in tumor tissues. This model demonstrated that the spatial ITH of PD-L1 expression in surgically resected NSCLC predicts poor patient outcomes.

DUSP4 maintains the survival and LSD1 protein stability in esophageal squamous cell carcinoma cells by inhibiting JNK signaling-dependent autophagy.

DUSP4 is a biomarker of esophageal squamous cell carcinoma (ESCC), which is responsible for the prognosis in ESCC. However, the underlying mechanism of DUSP4-regulated ESCC carcinogenesis is unknown. As a negative regulator of JNK, DUSP4 can inhibit autophagy, which contributes to tumorigenesis. This study aimed to explore the role of autophagy in DUSP4-regulated ESCC carcinogenesis. Our results showed that DUSP4 overexpression inhibited autophagy and promoted LSD1 protein expression in ESCC cells, while DUSP4 silencing showed the opposite effects. However, DUSP4 overexpression and silencing did not affect LSD1 mRNA expression. But the regulatory ability of DUSP4 overexpression on autophagy, death level, and LSD1 protein was reversed by rapamycin. In addition, DUSP4 overexpression inhibited JNK and Bcl2 phosphorylation and the dissociation of Bcl2-Beclin1 complex, while DUSP4 silencing promoted JNK and Bcl2 phosphorylation. Moreover, the regulatory ability of DUSP4 overexpression on autophagy, death, and LSD1 protein was reversed by JNK activator anisomycin. The xenograft assays also showed that DUSP4 overexpression-promoted ESCC tumor growth in vivo and LC3II and LSD1 protein expression in tumor tissues were reversed by rapamycin or anisomycin. Overall, DUSP4 inhibits Bcl2-Beclin1-autophagy signal transduction through the negative regulation of JNK, thus suppressing autophagic death and the autophagic degradation of LSD1 in ESCC, by which DUSP4 promotes ESCC carcinogenesis.

FEATURES OF COL1A1 EXPRESSION IN BREAST CANCER TISSUE OF YOUNG PATIENTS.

In the last decades, the incidence of breast cancer (BCa) in young women has been increasing steadily. The quantitative indicators of expression of collagen, which play important role in stromal microenvironment, and their association with the age and survival rates of BCa patients have not been yet definitively clarified. To investigate the relationship between the COL1A1 gene expression at the mRNA and protein levels in BCa tissue and the clinicopatological features and survival rates of BCa patients of different age groups. The study was conducted on the clinical material of 50 patients with stage I-III BCa. COL1A1 gene expression at the mRNA and protein levels in BCa tissue were studied using the real-time PCR and immunohistochemical methods, as well as the bioinformatic analysis (UALCAN and Kaplan - Meier Plotter databases). The bioinformatic analysis showed that BCa tissue is characterized by 6.0 times (p < 0.05) higher level of COL1A1 mRNA compared to normal breast tissue. The correlation of COL1A1 expression at the mRNA and protein levels with the molecular subtype of neoplasms was demonstrated. According to Kaplan - Meier Plotter database, a low level of expression of COL1A1 protein level in BCa tissue is associated with lower rates of relapse-free survival of patients. The ex vivo study of the clinical material revealed a decrease in COL1A1 protein expression in tumor tissue of young patients with BCa of T3 category (p < 0.0374), low differentiation grade (p < 0.0163) and basal molecular subtype (p < 0.0001). A correlation between the expression of COL1A1 at the mRNA and protein levels and the expression status of estrogen receptors (p < 0.0001) and progesterone receptors (p < 0.0040) was established. The relapse-free 3-year survival rate of young BCa patients is significantly lower in the presence of a low COL1A1 optical density index in the tumor tissue. The identified relationship between COL1A1 expression and such indicators of BCa malignancy as tumor size, differentiation grade, molecular subtype, receptor status, and the recurrencefree survival of patients indicates the prospects of its use to predict the aggressiveness of the BCa course in young patients.

Secreted Protein Acidic and Rich in Cysteine (SPARC) Polymorphisms in Response to Neoadjuvant Chemotherapy in HER2-Negative Breast Cancer Patients.

Secreted protein acidic and rich in cysteine (SPARC) expression has been proposed as a prognostic and predictive biomarker for some cancer types, but knowledge about the predictive value of SPARC polymorphisms in the context of neoadjuvant therapy for breast cancer (BC) is lacking. In 132 HER2-negative BC patients treated with neoadjuvant chemotherapy, we determined polymorphisms in the SPARC gene and analyzed their association with outcome. We also determined SPARC protein expression in tumor tissue. SPARC rs19789707 was significantly associated with response to treatment according to the Miller and Payne system in the breast (multivariate: odds ratio (OR), 3.81; p = 0.028). This association was significant in the subgroup of patients with luminal tumors (univariate: p = 0.047). Regarding survival, two SPARC variants showed significant associations with event-free survival: the rs19789707 variant in the subgroup of luminal A tumors (univariate: p = 0.006), and the rs4958487 variant in the subgroup of luminal B tumors (univariate: p = 0.022). In addition, SPARC rs4958487, rs10065756, and rs12153644 were significantly correlated with SPARC protein expression. Our findings suggest that SPARC polymorphisms could be good predictors of treatment response and survival in BC patients treated with neoadjuvant chemotherapy, especially those with luminal tumors.

Abstract C013: Clinical biomarkers based on PK/PD modeling to guide the development for a first-in-class, highly selective SMARCA2 (BRM) degrader, PRT3789

Abstract Background: The SWI/SNF family of chromatin-remodeling complexes is frequently dysregulated in multiple tumor types, resulting in aberrant expression of genes. Damaging mutations resulting in loss of function of one of its core catalytic subunits SMARCA4 (BRG1) occur in multiple tumor types, including in 5-10% of non-small cell lung cancer (NSCLC). SMARCA4-deleted cells become highly dependent on the other catalytic subunit, SMARCA2 (BRM), for their survival, therefore selective degradation of SMARCA2 has therapeutic potential in these cancer types. PRT3789 is a first-in-class, potent and selective SMARCA2 degrader that is currently under evaluation in a Phase 1 study in patients with advanced solid tumors with loss of SMARCA4 (NCT05639751). We deeply characterized the degradation kinetic parameters of PRT3789 based on preclinical PK/PD studies, which enabled dose-selection. Based on this modeling, we developed a suite of biomarker assays that can be used to assess target engagement in this Phase 1 study. Methods: A sensitive and quantitative plate-based immunoassay utilizing the MSD® platform was developed for the measurement of SMARCA2 protein in human PBMCs following treatment with PRT3789. A secondary, qPCR-based assay was developed for assessment of SMARCA2 transcriptional targets in peripheral blood that may be associated with response to SMARCA2 degradation by PRT3789. Additionally, an immunohistochemistry (IHC) assay for the assessment of SMARCA2 and SMARCA4 protein expression in tumor tissue was developed on the Leica BOND III platform using commercially available antibodies. Results: Detailed in vitro enzymology characterized the degradation kinetic parameters of PRT3789 and was successfully used to predict the PRT3789 effects observed in PK/PD and xenograft efficacy studies in mice. The MSD® assay demonstrated a wide dynamic range of detection for SMARCA2 with a lower level of quantification of 23 pg/mL, as well as exhibiting acceptable spike recovery and minimal cross-reactivity to SMARCA4. Based on the qPCR assay, we developed a differential gene expression (DGE) score based on expression of an 8-gene panel that correlates with SMARCA2 protein degradation in PBMCs. The IHC assay demonstrated discrete and robust nuclear tumor staining with a dynamic intensity range and limited cytoplasmic background for both SMARCA2 and SMARCA4. A panel of NSCLC tumor samples with known SMARCA4 mutations were further evaluated in this assay. Loss of SMARCA4 protein expression in tumor cells by IHC was associated with nonsense and frameshift mutations in the SMARCA4 gene. Conclusions: We demonstrate the sensitive assessment of SMARAC2 protein degradation and effects on downstream gene expression in PBMCs, which will be used to assess target engagement following treatment with PRT3789 in a Phase 1 clinical study. Citation Format: Min Wang, Andrew Moore, Alexander Grego, Miles Cowart, Joseph Rager, Gina Paris, Koichi Ito, Naveen Babbar, Sandy Geeganage, Peegy Scherle, Neha Bhagwat. Clinical biomarkers based on PK/PD modeling to guide the development for a first-in-class, highly selective SMARCA2 (BRM) degrader, PRT3789 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C013.

Silencing of PD-1 combined with EBV-specific killer T cells for the treatment of EBV-associated B lymphoma

Epstein-Barr Virus (EBV) infection is closely associated with the development of lymphoma, as it plays a significant role in the malignant transformation of lymphocytes. The expression of programmed death-1 (PD-1), which binds to PD-L1 in tumor cells, can lead to immune evasion by lymphoma cells and promote tumor progression. In this study, immortalized B lymphoblastoid cell lines (B-LCLs) positive for EBV-specific proteins were established from human peripheral mononuclear cells (PBMCs) using EBV induction along with CpG-ODN 2006 and cyclosporin A. EBV-specific T cells (EBVST) were generated by multiple immunizations of CD3+ T lymphocytes using irradiated B-LCLs. Flow cytometry analysis confirmed the activation of EBVST through the detection of CD3+, CD4+, and CD8+ markers. Co-incubation of EBVST with EBV-positive B lymphocyte cell lines resulted in the secretion of perforin by EBVST, leading to granzyme B-mediated cell death and an increase in LDH levels. Silencing PD-1 in EBVST cells enhanced perforin production, increased granzyme B release, and upregulated cell death in co-incubated B lymphocytes. In a nude mice tumor transplantation model, silencing PD-1 in combination with EBV-specific killer T cells exhibited the maximum inhibition of B-lymphoblastoma. This treatment upregulated the expression of proteins associated with apoptosis and immune response, while inhibiting anti-apoptotic protein expression in tumor tissues. Silencing PD-1 also increased the infiltration of EBV-specific killer T cells in the tumor tissues. Overall, PD-1 silencing enhanced the tumor targeting effect of EBV-specific killer T cells on EBV-infected B lymphocytes and attenuated the immune escape effect mediated by the PD-1 pathway.

RNF183 Promotes Colon Cancer Cell Stemness through Fatty Acid Oxidation

Colon cancer (COAD) is a prevalent gastrointestinal tumor, composed of a few cancer stem cells (CSCs). High expression of RNF183 drives colorectal cancer metastasis, but its role in COAD cell stemness is still unclear. Bioinformatics analyzed expression and enriched pathway of RNF183 in COAD tissue. IHC analyzed RNF183 protein expression in tumor tissue. CD133 + CD44+ CSCs were sorted by flow cytometry, and RNF183 expression in COAD cells or CSCs was detected by qPCR, western blot and immunofluorescence. CCK-8 assay assessed cell viability, and sphere formation assay tested cell sphere-forming ability. Western blot measured protein expression of stem cell markers. qPCR assayed expression of fatty acid oxidation genes. The ability of fatty acid oxidation was analyzed by detecting fatty acid metabolism. RNF183 was highly expressed in COAD and CD133 + CD44+ CSCs, and was enriched in fatty acid metabolism pathway. RNF183 expression was positively correlated with enzymes involved in fatty acid oxidation. RNF183 could promote COAD stemness and fatty acid oxidation. Rescue experiments showed that Orlistat (a fatty acid oxidation inhibitor) reversed stimulative impact of RNF183 overexpression on COAD stemness. RNF183 promoted COAD stemness by affecting fatty acid oxidation, which may be a new therapeutic target for inhibiting COAD development.

PD-L1 (CD274) promoter hypomethylation predicts immunotherapy response in metastatic urothelial carcinoma

ABSTRACT PD-L1 status assessed by immunohistochemistry (IHC) has failed to reliably predict outcomes for patients with metastatic urothelial carcinoma (mUC) on immune checkpoint blockade (ICB). PD-L1 promoter methylation is an epigenetic mechanism that has been shown to regulate PD-L1 mRNA expression in various malignancies. The aim of our present study was to evaluate the predictive potential of PD-L1 promoter methylation status (mPD-L1) in ICB-treated mUC compared to conventional IHC-based PD-L1 assessment. We quantified mPD-L1 in formalin-fixed and paraffin-embedded tissue sections using an established quantitative methylation-specific PCR assay (qMSP) in a well-characterized multicenter ICB-treated cohort comprising N = 107 patients with mUC. Additionally, PD-L1 protein expression in tumor tissues was assessed using regulatory approved IHC protocols. The effect of pharmacological hypomethylation by the DNA methyltransferase inhibitor decitabine in combination with interferon-γ stimulation in urothelial carcinoma cell lines was investigated by IHC and FACS. mPD-L1 hypomethylation predicted objective response rate at the first staging on ICB. Patients with tumors categorized as PD-L1 hypomethylated (lower quartile) showed significantly prolonged progression-free (PFS) and overall survival (OS) after ICB initiation. In contrast, PD-L1 protein expression status neither correlated with response nor survival. In multivariable Cox regression analyses, PD-L1 promoter hypermethylation remained an independent predictor of unfavorable PFS and OS. In urothelial carcinoma cell lines, pharmacological demethylation led to an upregulation of membranous PD-L1 expression and an enhanced inducibility of PD-L1 expression by interferon γ. Hypomethylation of the PD-L1 promoter is a promising predictive biomarker for response to ICB in patients with mUC.

A Chinese Medicine, Huajie Xiaoliu Formula, Enhances the Anti-Colon Cancer Effect of Tegafur, Gimeracil, and Oteracil Potassium Capsules Through Bcl-2/Bax Signaling Pathway

Research Objective This study was aimed to determine the main ingredients of Huajie Xiaoliu Formula (HJF) and investigate the anti-colon cancer effect of its combination with Tegafur, Gimeracil, and Oteracil Potassium Capsules (S-1) in vivo as well as to preliminarily explore its possible mechanism. Materials and Methods The chemical composition of HJF was determined by ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). The murine colon carcinoma cell (CT 26.WT) subcutaneous xenograft model of colon cancer mice was established to evaluate the in vivo anti-colon cancer effect of HJF. Evaluation of pathological changes in tumor tissue was evaluated by hematoxylin–eosin staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expressions of interleukin-1β (IL-1β), inflammatory cytokines interleukin (IL-6), and tumor necrosis factor-α (TNF-α) in mice serum. Immunohistochemistry was used to evaluate B cell lymphoma-2 (Bcl-2), Bax, and Ki67 protein expression in tumor tissue. Results Twenty chemicals in HJF were identified by UPLC-QTOF-MS. In vivo experiments showed that HJF has obvious inhibitory effects on the subcutaneous xenograft model of colon cancer mice. Results of ELISA showed HJF is able to increase mice body weight, inhibit tumor growth, and reduce the serum levels of IL-1β and IL-6 along with TNF-α. Immunohistochemistry results demonstrated that HJF can significantly inhibit the levels of Bax, Bcl-2, and Ki67 in mice with colon cancer transplant tumors. Furthermore, the combination of HJF and S-1 demonstrated a more significant antitumor effect than any other group in vivo. Conclusions Huajie Xiaoliu Formula is effective in CT 26.WT subcutaneous xenograft model of colon cancer mice by inhibiting tumorigenesis, metastasis, and angiogenesis through the Bcl-2/Bax signaling pathway, resulting in anti-colorectal tumor effects. Huajie Xiaoliu Formula provides a strong theoretical basis for its clinical use and also provides a basis for further research.

Aidi injection enhances the anti-tumor impact of doxorubicin in H22 tumor-containing mice

Ethnopharmacological relevanceAidi injection (AD) is a traditional medical preparation that has a Chinese origin. It is extensively used particularly in combination with doxorubicin (DOX) for the management of hepatocellular carcinoma (HCC). However, the combination's synergistic mechanism has not yet been clarified. Aim of the studyTo investigate the anti-tumor impact of AD in combination with DOX and their synergistic mechanism in HCC. Materials and methodsAn H22 mouse xenograft model was utilized to study the impact of AD, DOX, and their combination on HCC in vivo. Their effects on cell vitality, apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) production, caspase-3, and cleaved caspase-3 protein expression were also investigated in H22 cells in vitro. Subsequently, human umbilical vein endothelial cells (HUVECs) were utilized to investigate the impacts of AD, DOX, and their combination on cell viability, migration, invasion, tube formation, and vascular endothelial growth factor (VEGF) protein expression. ResultsThe study established that the tumor inhibition rate of AD combined with DOX reached 79.51%, which was significantly higher than that of AD (25.14%) or DOX (49.48%) alone. Additionally, the Q-value characterizing the synergy between AD and DOX was 1.72, demonstrating a strong synergistic effect. Furthermore, compared to AD or DOX administration alone, the combined administration group significantly decreased the alpha-fetoprotein (AFP) level in the serum, increased the tumor necrosis area, increased the Bax/Bcl-2, Cyt-c, caspase-9, Fas, Fasl, caspase-8, and caspase-3 protein expression, and significantly increased the CD31 and Ki67 protein expression in tumor tissue. Compared to AD or DOX alone, AD combined with DOX treatment had a synergistic effect on H22 cells (combination index values < 0.9), which inhibited cell viability, reduced mitochondrial membrane potential (MMP), induced apoptosis, promoted MMP loss, and increased ROS generation, cleaved caspase-3/caspase-3 levels, and caspase-3 activity. Moreover, combined administration showed a more pronounced inhibition of cell viability, migration, invasion, tube formation, and VEGF protein expression in HUVECs. ConclusionsAD enhances the anti-tumor effect of DOX by promoting apoptosis and inhibiting angiogenesis and cell proliferation. The findings of this study lay experimental foundations for the clinical combination of AD and DOX.

A network pharmacology approach and experimental validation to investigate the anticancer mechanism and potential active targets of ethanol extract of Wei-Tong-Xin against colorectal cancer through induction of apoptosis via PI3K/AKT signaling pathway

Ethnopharmacological relevanceWei-Tong-Xin (WTX), derives from the Chinese herbal decoction (CHD) of Wan-Ying-Yuan in ancient China, has been shown to be effective therapeutic herbal decoction for treating gastrointestinal diseases. Present studies have demonstrated that WTX had potential to alleviate the symptoms of gastrointestinal inflammation, gastric ulcer and improve gastric motility. Aim of the studyThe study primarily focused on exploring the therapeutic effect and possible pharmacological mechanism of WTX on colorectal cancer (CRC) based on network pharmacology, in vitro and in vivo experiments. Materials and methodsFirstly, colorectal cancer and WTX associated with targets were searched from GeneCards database and TCM Systems Pharmacology Database and Analysis Platform (TCMSP) respectively. The protein–protein interaction (PPI) network also was constructed to screening key targets. In addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to predict the underlying biological function and mechanism involving in the anti-colorectal cancer effect of WTX. Next, CCK-8, colony formation and transwell assays were performed to verify the influence of proliferation and metastasizing ability of HCT116 cells after treated with WTX. Cell cycle, apoptosis and reactive oxygen species (ROS) were analysis by flow cytometry. Hoechst 33258 staining was conducted to observe nuclear morphology changes. Protein expression of apoptosis and PI3K/AKT signaling as well as mRNA expression of ferroptosis and apoptosis were determined by Western Blotting and RT-qPCR. The effects of WTX and LY294002 combination on the PI3K/Akt/mTOR signaling pathway were measured by Western Blotting. Finally, the xenograft tumor mouse model was established by subcutaneous injection of CT26 cells to measure tumors volume and weight. Hematoxylin and eosin (HE) staining and immunohistochemical analysis were used to observe the pathological changes and the protein expression in tumor tissues. ResultsThere were 286 potential treatment targets from 130 bioactive compounds in WTX, 1349 CRC-related targets were identified. Eleven core targets (TP53, AKT1, STAT3, JUN, TNF, HSP90AA1, IL-6, MAPK3, CASP3, EGFR, MYC) were found by PPI network analysis constructed of 142 common targets. The results of KEGG enrichment displayed PI3K/AKT signaling pathway as core pathway. After the treatment of WTX, the inhibitory of viability, metastases and cell cycle arrest at G2/M phase were observed in HCT116 cells. Moreover, WTX induced an increase in the expression of apoptosis proteins (Bak, cytochrome c, cleaved caspase-9/caspase-9 and cleaved caspase-3/caspase-3) and the levels of ROS and MDA, a decrease in the expression of PI3K/AKT signaling related proteins (PI3K, p-PI3K, p-AKT/AKT and p-mTOR/mTOR) and the level of SOD. WTX treatment significantly reduced the tumor weight, increased cleaved caspase-3 positive area and decreased that of ki67 in xenograft mouse model. ConclusionThrough a network pharmacology approach and in vitro experiments, we predicted and verified the effect of WTX on colorectal cancer cells mainly depended on the regulation of intrinsic apoptosis via PI3K/AKT signaling pathway, and further animal experiments proved that WTX has a good anti-colon cancer effect in vivo.

Overexpression of Copines-1 is associated with clinicopathological parameters and poor outcome in gastric cancer.

Copines-1 (CPNE1) is a soluble membrane-binding protein that includes two tandem C2 domains at the N-terminus and a C terminal A domain. Importantly, it is associated with the prognosis of various tumors, but there are only a few studies regarding the role of CPNE1 in gastric cancer (GC). This study aimed to explore the clinicopathological significance and prognostic potential of CPNE1 expression in GC. Data from the TIMER2.0 and UALCAN were analyzed to assess CPNE1 mRNA levels in GC. The prognostic role of CPNE1 mRNA was examined via the Kaplan-Meier plotter. CPNE1 protein expression in tumor tissues was analyzed via immunohistochemistry of clinical samples from 99 GC patients. The relationship of CPNE1 expression with clinicopathological parameters and overall survival (OS) was evaluated using Cox proportional hazards regression models and Kaplan-Meier survival curves. Copines-1 mRNA levels were higher in GC tissues than in adjacent normal tissue (ANT) (p< 0.05). Further, high CPNE1 mRNA expression indicated poor OS (p= 9.4 e-10) and was significantly associated with first progression (FP) (p= 1.6 e-06) and post-progression survival (PPS) (p= 1.5 e-12). In addition, CPNE1 protein expression was higher in GC tissues than in ANT (p< 0.0001). Moreover, CPNE1 high expression was significantly related to advanced tumor-node-metastasis (TNM) stage (p= 0.004), lymph node metastasis (p= 0.003), and vascular invasion (p= 0.001). Kaplan-Meier analysis showed that GC patients with high expression CPNE1 group had worse OS than low expression group (p= 0.003). Univariate analysis showed that age (hazard ratio [HR]=1.992; 95% confidence interval [CI], 1.009-3.934; p= 0.047), advanced TNM stage (HR=4.941; 95% CI, 2.052-11.897; p= 0.000), tumor invasion (HR=3.472; 95% CI, 1.349-8.937; p= 0.010), lymph node metastasis (HR=8.846; 95% CI, 2.708-28.897; p= 0.000), vascular invasion (HR=3.237; 95% CI, 1.521-6.891; p= 0.002), nervous invasion (HR=2.324; 95% CI, 1.205-4.479; p= 0.012), and CPNE1 expression (HR=3.464; 95% CI, 1.440-8.334; p= 0.006) were correlated with OS. In the multivariate analysis, age (HR=2.514; 95% CI, 1.264-4.999; p= 0.009), lymph node metastasis (HR=8.441; 95% CI, 2.553-27.906; p< 0.05), and CPNE1 expression (HR=2.549; 95% CI, 1.051-6.186; p= 0.039) were significant prognostic predictors for GC. Copines-1 overexpression in GC is significantly associated with poor prognosis. Thus, CPNE1 levels may serve as a prognostic biomarker in GC patients.

Upregulated Apelin Signaling in Pancreatic Cancer Activates Oncogenic Signaling Pathways to Promote Tumor Development.

Despite decades of effort in understanding pancreatic ductal adenocarcinoma (PDAC), there is still a lack of innovative targeted therapies for this devastating disease. Herein, we report the expression of apelin and its receptor, APJ, in human pancreatic adenocarcinoma and its protumoral function. Apelin and APJ protein expression in tumor tissues from patients with PDAC and their spatiotemporal pattern of expression in engineered mouse models of PDAC were investigated by immunohistochemistry. Apelin signaling function in tumor cells was characterized in pancreatic tumor cell lines by Western blot as well as proliferation, migration assays and in murine orthotopic xenograft experiments. In premalignant lesions, apelin was expressed in epithelial lesions whereas APJ was found in isolated cells tightly attached to premalignant lesions. However, in the invasive stage, apelin and APJ were co-expressed by tumor cells. In human tumor cells, apelin induced a long-lasting activation of PI3K/Akt, upregulated β-catenin and the oncogenes c-myc and cyclin D1 and promoted proliferation, migration and glucose uptake. Apelin receptor blockades reduced cancer cell proliferation along with a reduction in pancreatic tumor burden. These findings identify the apelin signaling pathway as a new actor for PDAC development and a novel therapeutic target for this incurable disease.

Aberrant TRPC1 expression reflects stromal cervical invasion, lymphovascular invasion, elevated FIGO stage, and poor survival in resectable endometrial carcinoma patients.

BackgroundTransient receptor potential channel 1 (TRPC1) promotes tumor growth and metastasis in endometrial carcinoma (EC) cell lines, whereas its clinical role in EC management remains unclear. Therefore, this study aimed to investigate the association of TRPC1 protein expression with the clinical features and survival of EC patients, then was further validated by TRPC1 mRNA measurement and data from The Human Protein Atlas.MethodsTRPC1 protein expression in tumor tissues and normal endometria of 176 resectable EC patients was determined using immunohistochemistry. Besides, TRPC1 mRNA expression of partial patients (n = 80) was detected using RT‐qPCR. Additionally, survival data from The Human Protein Atlas (derived from The Cancer Genome Atlas [TCGA]) was analyzed.ResultsTRPC1 protein expression was up‐regulated in tumor tissue compared with normal endometrium (p < 0.001). Up‐regulated TRPC1 protein expression was associated with stromal cervical invasion (p = 0.044), lymphovascular invasion (p = 0.032), and increased federation of gynecology and obstetrics (FIGO) stage (p = 0.005). Tumor TRPC1 protein high was linked with shortened accumulating disease‐free survival (DFS) (p = 0.009) and overall survival (OS) (p = 0.026), which were also confirmed by multivariate Cox's regression analysis (both p < 0.050). Further, TRPC1 mRNA validation disclosed that TRPC1 mRNA high was related to shortened accumulating DFS (p = 0.038) and exhibited a correlating trend with declined OS (lacked statistical significance) (p = 0.162). Meanwhile, survival analysis on the data from The Human Protein Atlas (derived from TCGA) also exhibited that TRPC1 mRNA high was correlated with reduced accumulating OS (p < 0.001).ConclusionOur findings support TRPC1 as a prognostic biomarker in resectable EC patients.

NMT1 Enhances the Stemness of NSCLC Cells by Activating the PI3K/AKT Pathway

Introduction: Abundant studies have disclosed that proteins can function as pivotal tumor promoters or suppressors in cancers’ progression. This work was planned to investigate the regulatory function of N-myristoyltransferase-1 (NMT1) on non-small cell lung cancer (NSCLC) and the underlying molecular mechanisms. Methods: The self-renewal abilities were assessed through a spheroid-formation assay. The tumorigenic abilities were examined through nude mice in vivo assay. The proteins’ expression was measured through Western blot. The NMT1 protein expression in tumor tissues was measured through an IHC assay. The cell migration and invasion was confirmed through a transwell assay. The IC50 was verified through a CCK-8 assay. The NMT1 mRNA expression in NSCLC tissues was detected through RT-qPCR. Results: It was demonstrated that NMT1 exhibited higher expression in spheroid cells. Additionally, NMT1 facilitated the stemness in NSCLC. It was also found that NMT1 accelerated NSCLC tumor metastasis and the resistance to cisplatin. Moreover, NMT1 activated the PI3K/AKT pathway to facilitate stemness in NSCLC. NMT1 was also higher in tumor tissues of NSCLC patients and resulted in a poor survival rate. Conclusion: NMT1 enhanced the stemness of NSCLC cells by activating the PI3K/AKT pathway. This discovery suggested that NMT1 may be a valid therapeutic biomarker for NSCLC.

Abstract 714: Weight changes between one year before and following breast cancer diagnosis in relation to mTOR protein expression in tumor tissues in the Women’s Circle of Health Study

Abstract Background: Obesity is an important prognostic factor for breast cancer. Aberrant activation of the mechanistic Target of Rapamycin (mTOR) pathway is associated with obesity, cross-sectionally, as well as with breast cancer survival. This study aimed to examine the association of weight change between one year before and following cancer diagnosis with a panel of mTOR protein markers in breast tumors. The study hypothesis was that greater weight gain compared to less or no weight gain would be associated with higher mTOR protein expression in breast tumors. Methods: Study participants were 698 newly-diagnosed breast cancer cases (545 Black women and 153 White women) from the Women’s Circle of Health Study (WCHS). Surgical tissue microarrays were stained for mTOR, phosphorylated mTOR, phosphorylated AKT, and phosphorylated P70S6K using immunohistochemistry. In-home interviews were conducted approximately 10 months after breast cancer diagnosis, and anthropometric measurement was taken by trained staff. Participants reported body weight one year before the diagnosis, excluding the time of pregnancy or nursing. Linear regression analyses were conducted to estimate percent differences in protein expression across quartiles of weight change, adjusting for age, race, BMI one year before diagnosis, menopausal status, history of diabetes, and molecular tumor subtype. Results: Study participants had a mean age of 52.7 (SD = 10.8) years and BMI one year before diagnosis of 30.7 (SD = 6.9) kg/m2. The proportions of participants who had loss ≥5 lbs, loss &amp;lt;5 lbs, no change to gain &amp;lt;5 lbs, and gain ≥5 lbs in weight were 33.7%, 16.2%, 16.2%, and 33.7%, respectively. The fourth vs. first quartile of the weight change was associated with a higher protein expression of mTOR (35.3%, 95% CI = 3.8%, 76.5%), and there was a dose-response pattern (P-trend = 0.0273). No associations were observed between weight change and phosphorylated protein expression. Conclusion: Short-term weight gain before breast cancer diagnosis and following diagnosis may play a role in breast tumor mTOR protein expression. Our findings warrant validation in a study with a prospective measurement of weight gain before breast cancer surgery. Citation Format: Mmadili N. Ilozumba, Angela R. Omilian, Song Yao, Weizhou Zhang, Susmita Datta, Warren Davis, Chi-Chen Hong, Elisa V. Bandera, Thaer Khoury, Christine B. Ambrosone, Ting-Yuan David Cheng. Weight changes between one year before and following breast cancer diagnosis in relation to mTOR protein expression in tumor tissues in the Women’s Circle of Health Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 714.

Clinical and prognostic significance of expression of phosphoglycerate mutase family member 5 and Parkin in advanced colorectal cancer.

BACKGROUNDDrugs targeting mitochondria can induce mitophagy and restrain proliferation in colorectal cancer (CRC) cells. Phosphoglycerate mutase family member 5 (PGAM5) activates serine/threonine PTEN-induced putative kinase 1/Parkin pathway-mediated mitophagy. However, there are few studies on the clinical and prognostic significance of expression of PGAM5 protein and mitophagy-related protein Parkin in patients.AIMTo assess the clinical significance of PGAM5 and Parkin proteins, as biomarkers for diagnosis and prognosis of CRC, by studying their expression in advanced CRC tissues and their association with clinicopathological parameters.METHODSThe expression of PGAM5 and Parkin in CRC tissues from 100 patients was determined by immunohistochemistry. Each case was evaluated by using a combined scoring method based on signal intensity staining (scored 0-3) and the proportion of positively stained cancer cells (scored 0-4). The final staining score was calculated as the intensity score multiplied by the proportion score. Specimens were categorized as either high or low expression according to the Youden index, and the association between the expression of PGAM5 or Parkin and clinicopathological factors was ascertained. Additionally, we employed western blot to measure PGAM5 and Parkin protein expression in six matched pairs of CRC and adjacent non-tumor tissues.RESULTSImmunohistochemical and western blot findings showed that both PGAM5 and Parkin protein expression in tumor tissues was significantly higher than that in the adjacent tissues: PGAM5 and Parkin were mainly expressed in the cytoplasm of colonic epithelial cells. PGAM5 and Parkin protein levels were significantly positively correlated in advanced CRC tissues. Moreover, reduced Parkin protein expression was an independent prognostic factor for overall survival and progression-free survival in CRC patients as evinced by multivariate analysis.CONCLUSIONThe expression of PGAM5 protein and mitophagy-related protein Parkin has diagnostic significance for CRC and may become new biomarkers. Parkin may be a potential marker for the survival of CRC patients.

The Anti-Proliferative and Apoptotic Effects of Rutaecarpine on Human Esophageal Squamous Cell Carcinoma Cell Line CE81T/VGH In Vitro and In Vivo.

The overall five-year survival rate for patients with esophageal cancer is low (15 to 25%) because of the poor prognosis at earlier stages. Rutaecarpine (RTP) is a bioalkaloid found in the traditional Chinese herb Evodia rutaecarpa and has been shown to exhibit anti-proliferative effect on tumor cells. However, the mechanisms by which RTP confer these effects and its importance in esophageal squamous cell carcinoma treatment remain unclear. Thus, in the present study, we first incubated human esophageal squamous cell carcinoma cell line, CE81T/VGH, with RTP to evaluate RTP’s effects on tumor cell growth and apoptosis. We also performed a xenograft study to confirm the in vitro findings. Furthermore, we determined the expression of p53, Bax, bcl-2, caspase-3, caspase-9, and PCNA in CE81T/VGH cells or the tumor tissues to investigate the possible mechanisms. All the effects of TRP were compared with that of cisplatin. The results showed that RTP significantly inhibits CE81T/VGH cell growth, promotes arrest of cells in the G2/M phase, and induces apoptosis. Consistently, the in vivo study showed that tumor size, tumor weight, and proliferating cell nuclear antigen protein expression in tumor tissue are significantly reduced in the high-dose RTP treatment group. Furthermore, the in vitro and in vivo studies showed that RTP increases the expression of p53 and Bax proteins, while inhibiting the expression of Bcl-2 in cancer cells. In addition, RTP significantly increases the expression of cleaved caspase-9 and cleaved caspase-3 proteins in tumor tissues in mice. These results suggest that RTP may trigger the apoptosis and inhibit growth in CE81T/VGH cells by the mechanisms associated with the regulation of the expression of p53, Bax, Bcl-2, as well as caspase-9 and caspase-3.

Swimming attenuates tumor growth in CT-26 tumor-bearing mice and suppresses angiogenesis by mediating the HIF-1α/VEGFA pathway.

Low physical activity correlates with increased cancer risk in various cancer types, including colorectal cancer (CRC). However, the ways in which swimming can benefit CRC remain largely unknown. In this study, mice bearing tumors derived from CT-26 cells were randomly divided into the control and swimming groups. Mice in the swimming group were subjected to physical training (swimming) for 3 weeks. Compared with the control group, swimming clearly attenuated tumor volume and tumor weight in CT-26 tumor-bearing mice. RNA sequencing (RNA-seq) identified 715 upregulated and 629 downregulated transcripts (including VEGFA) in tumor tissues of mice in the swimming group. KEGG pathway analysis based on differentially expressed transcripts identified multiple enriched signaling pathways, including angiogenesis, hypoxia, and vascular endothelial growth factor (VEGF) pathways. Consistently, IHC analysis revealed that swimming significantly downregulated CD31, HIF-1α, VEGFA, and VEGFR2 protein expression in tumor tissues. In conclusion, swimming significantly attenuates tumor growth in CT-26 tumor-bearing mice by inhibiting tumor angiogenesis via the suppression of the HIF-1α/VEGFA pathway.