Higher mRNA Expression Levels Research Articles

The Transcription Factor SOX18 Inhibitor Small Molecule 4 Is a Potential Treatment of Cancer-Induced Lymphatic Metastasis and Lymphangiosarcoma.

Malignant tumors release growth factors, promoting lymphangiogenesis in primary tumors and draining sentinel lymph nodes, ultimately facilitating lymph node metastasis. As a malignant lymphatic tumor entity, lymphangiosarcomas are characterized by low survival rates and limited treatment options. The transcription factor SOX18 plays a crucial role in both lymphatic endothelial cell differentiation and cancer-induced lymphangiogenesis. In this invitro study, we investigated the potential therapeutic effect of a small molecule called Sm4, which inhibits SOX18, on lymphatic endothelial and lymphangiosarcoma cells invitro. Human dermal lymphatic endothelial cells (HDLECs), lymphangiosarcoma cells (MO-LAS), and other endothelial cell lines were cultured. We found that Sox18 exhibited high mRNA expression levels in both HDLEC and MO-LAS. Sm4 treatment decreased the Sox18 expression level at the mRNA and protein levels in both HDLEC and MO-LAS significantly, a phenomenon confirmed through immunofluorescence images. Additionally, Sm4 treatment suppressed the expression of key lymphatic phenotype markers (Prox1, Flt4, and Lyve1) and hindered migration in both HDLEC and MO-LAS, all while maintaining cell viability. These findings suggest that targeting SOX18 with Sm4 may hold potential as a therapeutic strategy for lymphangiosarcoma and cancer-induced lymphatic metastasis. Further invitro studies are warranted to investigate the mechanisms and conduct dose-response analyses to evaluate Sm4's potential as a targeted therapy for lymphangiosarcoma and cancer-induced lymphangiogenesis in the future.

Prognostic and diagnostic value of SPINK mRNAs expression in head and neck squamous cell carcinoma based on genome-wide analysis

Aim: Head and neck squamous cell carcinoma (HNSC) is a major contributor to the global cancer burden. The serine protease inhibitor Kazal-type (SPINK) gene family has been linked to various cancers. This study explores the prognostic value of SPINK genes in predicting overall survival (OS) in HNSC patients. Methods: We analyzed RNA sequencing and clinical data from 504 cancer and 44 non-cancer samples from the TCGA database. Differential expression and functional enrichment analyses gene ontology and Kyoto encyclopedia of genes and genomes (GO and KEGG) were performed using clusterProfiler. Protein-protein interaction (PPI) networks were built with STRING and visualized. Immune infiltration was evaluated using single-sample Gene Set Enrichment Analysis (ssGSEA). Survival analysis utilized Kaplan-Meier curves and Cox regression models. Results: Our results showed that SPINK5, SPINK7, SPINK8, SPINK9, and SPINK14 were significantly overexpressed in normal tissues compared to carcinoma tissues, whereas SPINK1, SPINK4, and SPINK6 showed higher expression in carcinoma tissues. Correlation analysis revealed significant relationships among SPINK family members. GO and KEGG analyses highlighted their involvement in processes such as negative regulation of peptidase activity and serine-type endopeptidase inhibitor activity. PPI network analysis indicated close interactions between several SPINK proteins and other relevant proteins. Immune infiltration analysis showed that NK cells and Th2 cells were negatively correlated with SPINK genes, while mast cells and neutrophils were positively correlated. Survival analysis revealed that high mRNA expression levels of SPINK1, SPINK5, and SPINK6 were significantly associated with OS in HNSC patients. Receiver operating characteristic (ROC) curve analysis indicated that these genes have diagnostic value. We developed a nomogram model that combines tumor stage and SPINK gene expression providing a predictive tool for patient prognosis. Conclusions: This study elucidates the multifaceted roles of the SPINK gene family in HNSC. These findings offer valuable insights into their potential as diagnostic biomarkers and therapeutic targets.

Multi-omics uncovers the potential functions of transcription factor Dp-1 in human digestive cancers

Previous studies have reported the critical effects of TFDP1 on the occurrence and progression of multiple cancers. Nevertheless, these studies were confined to a single cancer type rather than human digestive cancers. Therefore, using various integrated databases available, we conducted a multi-omics analysis of the potential roles of TFDP1 in digestive cancers. Firstly, high TFDP1 mRNA expression levels were observed in all seven digestive cancers, and were closely related to survival differences, pathological features, as well as immune and molecular subtypes. Epigenetic alterations of TFDP1 were evident in the four digestive cancers, and TFDP1 promoter methylation was negatively correlated with TFDP1 mRNA expression. Subsequently, using MuTarget, we identified several mutant genes accountable for TFDP1 overexpression. In addition, function enrichment analysis unveiled that TFDP1 could influence the cell cycle process and the pathways of the cell cycle and FoxO signaling. Intriguingly, our single-cell analysis demonstrated high expression of TFDP1 in blood and immune cells. We also found the statistically significant correlation of TFDP1 expression with six infiltration cell types, four immunosuppressive cell types, and various drugs. Finally, the TIDE results revealed that TFDP1 had a higher predictive ability of TFDP1 than four classical biomarkers and TFDP1 expression was associated with various types of cohorts. Taking the CRISPR screen cohorts, we observed the powerful effect of TFDP1-knockout on MC38 colon cancer cells. These findings could help elucidate the potential functions of TFDP1 and facilitate the development of TFDP1-related diagnostic and therapeutic strategies for different digestive cancers.

Cell-autonomous IL6ST activation suppresses prostate cancer development via STAT3/ARF/p53-driven senescence and confers an immune-active tumor microenvironment

BackgroundProstate cancer ranks as the second most frequently diagnosed cancer in men worldwide. Recent research highlights the crucial roles IL6ST-mediated signaling pathways play in the development and progression of various cancers, particularly through hyperactivated STAT3 signaling. However, the molecular programs mediated by IL6ST/STAT3 in prostate cancer are poorly understood.MethodsTo investigate the role of IL6ST signaling, we constitutively activated IL6ST signaling in the prostate epithelium of a Pten-deficient prostate cancer mouse model in vivo and examined IL6ST expression in large cohorts of prostate cancer patients. We complemented these data with in-depth transcriptomic and multiplex histopathological analyses.ResultsGenetic cell-autonomous activation of the IL6ST receptor in prostate epithelial cells triggers active STAT3 signaling and significantly reduces tumor growth in vivo. Mechanistically, genetic activation of IL6ST signaling mediates senescence via the STAT3/ARF/p53 axis and recruitment of cytotoxic T-cells, ultimately impeding tumor progression. In prostate cancer patients, high IL6ST mRNA expression levels correlate with better recurrence-free survival, increased senescence signals and a transition from an immune-cold to an immune-hot tumor.ConclusionsOur findings demonstrate a context-dependent role of IL6ST/STAT3 in carcinogenesis and a tumor-suppressive function in prostate cancer development by inducing senescence and immune cell attraction. We challenge the prevailing concept of blocking IL6ST/STAT3 signaling as a functional prostate cancer treatment and instead propose cell-autonomous IL6ST activation as a novel therapeutic strategy.

Overexpression of TPD52L2 in HNSCC: prognostic significance and correlation with immune infiltrates.

Evidence has been presented that the tumor protein D52 (TPD52) family plays a critical role in tumor development and progression. As a member of the TPD52 family, the changes in TPD52L2 gene status are instrumental in kinds of cancer development. However, its effects on patient prognosis and immune infiltration in Head and Neck Squamous Carcinoma (HNSCC) are still poorly understood. The Tumor Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and c-BioPortal database was used to explore the expression pattern, prognostic value, and variation of gene status in HNSCC. The LinkedOmics database was used to obtain the co-expression genes of TPD52L2 and identify the diagnostic value of TPD52L2 in HNSCC. The correlations between TPD52L2 expression and six main types of immune cell infiltrations and immune signatures were explored using Tumor Immune Estimation Resource (TIMER). The correlation between TPD52L2 expression and immune checkpoint genes (ICGs) was analyzed by TCGA database. Immunohistochemistry (IHC) was performed to validate the expression of three ICGs (PDL1, PDL2, EGFR) and TPD52L2 using 5 paired HNSCC and normal head and neck tissues. Polymerase Chain Reaction (PCR) and Western Blot (WB) of HNSCC and normal head and neck cell lines were performed to verify the high level of TPD52L2 mRNA and protein expression. protein expression of TPD52L2 in pan-cancer was also validated using UALCAN. TPD52L2 was overexpressed in tumor tissues, and it predicted worse survival status in HNSCC. ROC analysis suggested that TPD52L2 had a diagnostic value. Multivariate Cox analysis identified TPD52L2 as an independent negative prognostic marker of overall survival. Functional network analysis suggested that TPD52L2 was associated with immune-related signaling pathways, cell migration pathways, and cancer-related pathways. High expression of TPD52L2 was associated with a more mutant frequency of TP53. Notably, we found that the expression of TPD52L2 was closely negatively correlated with the infiltration levels of 15 types of immune cells and positively correlated with several immune markers. PCR, WB experiments, and UALCAN database verified the high level of TPD52L2 mRNA and protein expression. TPD52L2 is upregulated in HNSCC, which is an independent factor for adverse prognosis prediction. It probably plays a role in the negative regulation of immune cell infiltration. TPD52L2 might be a promising prognostic biomarker and therapeutic target in HNSCC.

Changes in the molecular nodes of the Notch and NRF2 pathways in cervical cancer tissues from the precursor stages to invasive carcinoma.

Cancer is a multifactorial disease characterized by the loss of control in the expression of genes known as cancer driver genes. Cancer driver genes trigger uncontrolled cell replication, which leads to the development of malignant tumors. A cluster of signal transduction pathways that contain cancer driver genes involved in cellular processes, such as cell proliferation, differentiation, apoptosis and dysregulated organ growth, are associated with cancer initiation and progression. In the present study, three signal transduction pathways involved in cervical cancer (CC) development were analyzed: The Hippo pathway (FAT atypical cadherin, yes-associated protein 1, SMAD4 and TEA domain family member 2), the Notch pathway [cellular-MYC, cAMP response element-binding binding protein (CREBBP), E1A-associated cellular p300 transcriptional co-activator protein and F-Box and WD repeat domain containing 7] and the nuclear factor erythroid 2-related factor 2 (NRF2) pathway [NRF2, kelch-like ECH-associated protein 1 (KEAP1), AKT and PIK3-catalytic subunit α]. Tumor samples from patients diagnosed with various stages of CC, including cervical intraepithelial neoplasia (CIN) 1, CIN 2, CIN 3, in situ CC and invasive CC, were analyzed. The mRNA expression levels were analyzed using reverse transcription-quantitative PCR assays, whereas protein expression levels were assessed through immunohistochemical tissue microarrays. High mRNA expression levels of c-MYC and AKT and low expression levels of NRF2 and KEAP1 were associated with a decreased survival time of patients with CC. Additionally, increased expression levels of c-MYC were detected in the invasive CC stage. At the protein level, increased NRF2 expression levels were observed in all five stages of CC samples compared with those in the cancer-free control samples. AKT1 was found to be dysregulated in the CIN 1 and CIN 2 stages, PI3K in the in situ and invasive stages, and CREBBP in the CIN 3 and in situ stages. In summary, the present study demonstrated significant changes in proteins of the Notch and NRF2 pathways in CC. NRF2 was overexpressed in all cervical cancer stages (cervical intraepithelial neoplasia, in situ CC and invasive CC). The present study makes an important contribution to the possible biomarker proteins to be analyzed for the presence of premalignant and malignant lesions in the cervix.

High Glycolytic Activity Signature Reveals CCNB2 as a Key Therapeutic Target in Triple-Negative Breast Cancer.

Aerobic glycolysis and the cell cycle are well-established tumor hallmarks. Understanding their relationship could help to unravel the pathogenic mechanisms of breast cancer (BC) and suggest potential new strategies for treatment. Glycolysis-related genes (GRGs) were downloaded from the Reactome database and screened using univariate Cox analysis. The consensus clustering method was employed to identify a glycolytic activity signature (GAS) using the Gene Expression Omnibus (GEO) dataset. A nomogram risk prediction model was constructed using coefficients from univariate Cox analysis. Immune cell infiltration was evaluated using single-sample gene set enrichment analysis (ssGSEA) and the ESTIMATE algorithm. Gene co-expression modules were created using weighted correlation network analysis (WGCNA) to identify hub genes. Gene expression in three BC cell lines was quantified using Quantitative Reverse Transcriptase Polymera (qRT-PCR). Single-cell RNA sequencing (scRNA-seq) data was used to examine the relationship between GAS and hub genes. The sensitivity of different groups to cell cycle-related clinical drugs was also examined. BC with high GAS (HGAS) showed high tumor grade and recurrence rate. HGAS was a prognostic indicator of worse overall survival (OS) in BC patients. HGAS BC showed more abundant immune cells and significantly higher expression of immunomodulators compared to BC with low GAS (LGAS). HGAS BC also showed enhanced cell cycle pathway, with high mRNA and protein expression levels of Cyclin B2 (CCNB2), a key component of the cell cycle pathway. Importantly, scRNA-seq analysis revealed that elevated CCNB2 expression was positively correlated with HGAS in triple-negative BC (TNBC). This was validated in clinical samples from TNBC patients. High expression of CCNB2 was found in three BC cell lines, and was also an indicator of poor prognosis. HGAS BC showed high sensitivity to several cell cycle-related clinical drugs, with 9 of these also showing activity in BC with high CCNB2 expression. HGAS was associated with enhanced cell cycle pathway and immune activity in BC. These results suggest that CCNB2 is a potential key therapeutic target in BC patients.

Activating Transcription Factor 5; A Potential Prognostic Molecular Biomarker for Cervical Cancer (CC)

Background: Prognostic biomarkers with high sensitivity and specificity that accurately predict disease prognosis are crucial for influencing cancer patients' treatment and disease progression choices. This research attempts to determine ATF5's cervical cancer prognostic value and find a theoretical basis for cervical cancer treatment. Method: This study began with a survival analysis, then univariate and multivariate analyses using transcriptomics, methylation, and TCGA ATF5 clinical data. Second, ATF5's role in cervical cancer development was explored using GO, KEGG, and TIMER databases. We assessed ATF5's cervical cancer treatment potential using CMap, a drug development tool. Results: After conducting several analyses, we discovered that high levels of ATF5 mRNA expression and low levels of methylation were beneficial for patients with cervical cancer prognosis. An important finding is that ATF5 may promote apoptosis in cervical carcinoma cells. In addition, we assessed the therapeutic potential of four small molecules against cervical cancer. ATF 5 function as a tumor suppressor gene in cervical cancer has been demonstrated for the first time using numerous databases and bioinformatics techniques from multiple dimensions, which is a novel aspect of this research. Moreover, the finding that ATF5 is a biomarker that can contribute to the apoptosis of cervical cancer cells and predict a favorable prognosis for the disease was an additional innovation. In the future, ATF5 can be used as a therapeutic target and to develop anti-cancer medications.

DNA methylation and mRNA expression of ZNF577 as biomarkers for the detection and prognosis of lung adenocarcinoma.

Despite advances in science and technology, lung cancer remains a major public health issue. The discovery of early diagnostic and prognostic markers is still needed to reduce the mortality rate of lung cancer, which is the highest among all cancer types. Aberrations in the DNA methylation system have an important role in human cancer and are promising for the development of early diagnostic and prognostic markers. The present study focused on zinc finger protein (ZNF)577, whose encoding gene was indicated to exhibit promoter hypermethylation together with 9 other genes in lung adenocarcinoma (LADC) in a previous study by our group. ZN577 is a member of the ZNG family and its functional role has so far remained elusive. LADC tissue samples surgically resected at Tokushima University Hospital (Tokushima, Japan) between April 1999 and November 2013 were collected. A total of 73 tumors and 27 paired tumor-adjacent normal tissues were examined for DNA methylation and mRNA expression of ZNF577. A total of 149 LADC tissue samples were collected and evaluated by immunohistochemistry (IHC) for the tissue expression of ZNF577. High methylation (n=27, P<0.0001) and low mRNA expression levels (n=27, P<0.031) of ZNF577 were identified in LADC tissues, and it was demonstrated that methylation levels were inversely correlated with mRNA expression levels (P=0.0116, ρ=-0.2515). Among the LADC tissues, lepidic-patterned samples had lower methylation levels of ZNF577 than other pathological types. In addition, mRNA expression levels of ZNF577 were significantly higher in females, non-smokers and stage I samples. Overall survival [P<0.0001; area under curve (AUC)=0.8658] and disease-free survival (DFS; P<0.0004; AUC=0.7232) rates were significantly higher in the ZNF577 high mRNA expression group than in the ZNF577 low mRNA expression group. Among the 149 LADC samples examined by IHC, 105 were negative and 44 were positive for the tissue expression of ZNF577. Cox regression analysis showed poorer DFS (hazard ratio: 3.917; P=0.023) in patients with lower expression of ZNF577. In conclusion, higher methylation levels of ZNF577 were observed in LADC tissues than in normal lung tissue and low mRNA expression of ZNF577 was associated with unfavorable prognosis.

Potentially functional variants of ERRFI1 in hypoxia-related genes predict survival of non-small cell lung cancer patients.

Hypoxia is often involved in tumor microenvironment, and the hypoxia-induced signaling pathways play a key role in aggressive cancer phenotypes, including angiogenesis, immune evasion, and therapy resistance. However, it is unknown what role genetic variants in the hypoxia-related genes play in survival of patients with non-small cell lung cancer (NSCLC). We evaluated the associations between 16,092 single-nucleotide polymorphisms (SNPs) in 182 hypoxia-related genes and survival outcomes of NSCLC patients. Data from the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial were used as the discovery dataset, and the Harvard Lung Cancer Susceptibility (HLCS) Study served as the replication dataset. We also performed additional linkage disequilibrium analysis and a stepwise multivariable Cox proportional hazards regression analysis in the PLCO dataset. An independent SNP, ERRFI1 rs28624 A > C, was identified with an adjusted hazards ratio (HR) of 1.31 (95% CI = 1.14-1.51, p = 0.0001) for overall survival (OS). In further analyses, unfavorable genotypes AC and CC, compared with the AA genotype, were associated a worse OS (HR = 1.20, 95% CI = 1.03-1.39, p = 0.014) and disease-specific survival (HR = 1.21, 95% CI = 1.04-1.42, p = 0.016). Further expression quantitative trait loci analysis indicated that ERRFI1 rs28624C genotypes were significantly associated with higher ERRFI1 mRNA expression levels in the whole blood. Additional analysis showed that high ERRFI1 mRNA expression levels were associated with a worse OS in patients with lung adenocarcinoma. Our findings suggest that genetic variants in the hypoxia-related gene ERRFI1 may modulate NSCLC survival, potentially through their effect on the gene expression.

The combined inhibition of SLC1A3 and glutaminase in osimertinib-resistant EGFR mutant cells

Background: We investigated the unknown mechanisms of osimertinib-resistant EGFR-mutant lung cancer. Methods: An osimertinib-resistant cell line (PC-9/OsmR2) was established through continuous exposure to osimertinib using an EGFR exon 19 deletion (19Del) lung adenocarcinoma cell line (PC-9). EGFR 19Del (M1), L858R/T790M/C797S (M6), and L858R/C797S (M8) expression vectors were introduced into Ba/F3 cells. A second osimertinib-resistant line (M1/OsmR) was established through continuous exposure to osimertinib using M1 cells. Results: SLC1A3 had the highest mRNA expression level in PC-9/OsmR2 compared to PC-9 cells by microarray analysis and SLC1A3 was increased by flow cytometry. In PC-9/OsmR2 cells, osimertinib sensitivity was significantly increased in combination with siSLC1A3. Because SLC1A3 functions in glutamic acid transport, osimertinib with a glutaminase inhibitor (CB-839) or an SLC1A3 inhibitor (TFB-TBOA) increased the sensitivity. Also, CB-839 plus TFB-TBOA without osimertinib resulted in greater susceptibility than did CB-839 or TFB-TBOA plus osimertinib. Comprehensive metabolome analysis showed that the M1/OsmR cells had significantly more glutamine and glutamic acid than M1 cells. CB-839 plus osimertinib exerted a synergistic effect on M6 cells and an additive effect on M8 cells. Conclusion: Targeting glutaminase and glutamic acid may overcome the osimertinib-resistant EGFR-mutant lung cancer.

KRAS-mutant non-small cell lung cancer (NSCLC) therapy based on tepotinib and omeprazole combination

BackgroundKRAS-mutant non-small cell lung cancer (NSCLC) shows a relatively low response rate to chemotherapy, immunotherapy and KRAS-G12C selective inhibitors, leading to short median progression-free survival, and overall survival. The MET receptor tyrosine kinase (c-MET), the cognate receptor of hepatocyte growth factor (HGF), was reported to be overexpressed in KRAS-mutant lung cancer cells leading to tumor-growth in anchorage-independent conditions.MethodsCell viability assay and synergy analysis were carried out in native, sotorasib and trametinib-resistant KRAS-mutant NSCLC cell lines. Colony formation assays and Western blot analysis were also performed. RNA isolation from tumors of KRAS-mutant NSCLC patients was performed and KRAS and MET mRNA expression was determined by real-time RT-qPCR. In vivo studies were conducted in NSCLC (NCI-H358) cell-derived tumor xenograft model.ResultsOur research has shown promising activity of omeprazole, a V-ATPase-driven proton pump inhibitor with potential anti-cancer properties, in combination with the MET inhibitor tepotinib in KRAS-mutant G12C and non-G12C NSCLC cell lines, as well as in G12C inhibitor (AMG510, sotorasib) and MEK inhibitor (trametinib)-resistant cell lines. Moreover, in a xenograft mouse model, combination of omeprazole plus tepotinib caused tumor growth regression. We observed that the combination of these two drugs downregulates phosphorylation of the glycolytic enzyme enolase 1 (ENO1) and the low-density lipoprotein receptor-related protein (LRP) 5/6 in the H358 KRAS G12C cell line, but not in the H358 sotorasib resistant, indicating that the effect of the combination could be independent of ENO1. In addition, we examined the probability of recurrence-free survival and overall survival in 40 early lung adenocarcinoma patients with KRAS G12C mutation stratified by KRAS and MET mRNA levels. Significant differences were observed in recurrence-free survival according to high levels of KRAS mRNA expression. Hazard ratio (HR) of recurrence-free survival was 7.291 (p = 0.014) for high levels of KRAS mRNA expression and 3.742 (p = 0.052) for high MET mRNA expression.ConclusionsWe posit that the combination of the V-ATPase inhibitor omeprazole plus tepotinib warrants further assessment in KRAS-mutant G12C and non G12C cell lines, including those resistant to the covalent KRAS G12C inhibitors.

Abstract PO2-03-07: Immune Landscape in PD-L1-positive and TROP2-positive Triple-Negative Breast Cancer

Abstract Background: Triple-negative breast cancer (TNBC) tends to be more aggressive, and more likely to recur than other subtypes of breast cancer, and patients with metastatic TNBC have a poor prognosis. The recent approvals and data of pembrolizumab (anti-PD-1 inhibitor) in combination with chemotherapy in advanced-metastatic and early high-risk TNBC, and sacituzumab govitecan (an ADC that targets the tumor cell surface antigen TROP2 coupled with the irinotecan metabolite SN-38) in metastatic TNBC, has shed light on some of the immune and cancer signaling associated with TNBC. Although exciting progress has been made in the treatment of TNBC, there are still important gaps to fill to understand the biology of TNBC and identify additional therapies. Materials and Methods: We identified a total of 1500 patients whose tumors were negative for the expression of ER and HER2 receptors by gene array. PD-L1 and TROP2 expression data were available for 802 early TNBC patients, of whom 657 were treated with adjuvant chemotherapy. A comprehensive identification and analysis of immune-related genes (n= 1169) was conducted. Mann-Whitney test comparing the gene expression for all immune related-genes was evaluated in patients expressing PD-L1 and TROP2 at low and high expression levels. Immune gene modules resembling key immune gene signaling were analyzed in all subgroups identified. Fold changes and Mann-Whitney P values were calculated in each subgroup. Results: Patients were stratified based on high and low expression levels of PD-L1 and TROP-2 mRNA. PD-L1 highly expressing (PD-L1+) tumors showed a marked difference in immune-related genes as compared to PD-L1 low-level tumors (PD-L1-) with PD-L1+ tumors demonstrating a significantly higher proportion of correlated immune genes (CXCL9/10/11, IFN-γ, CCL5 and STAT1). TROP2 highly and low expressing tumors (TROP2+ and TROP2-, respectively) showed a relatively limited difference in terms of immune-related gene expression with key immune-related genes having statistical differential expression in the two subgroups. TROP2+ tumors showed a high presence of several Mucin family gene members (MUC1, -5AC, -4). In addition, CLDN 4, VTCN1, and MET were among the key immune genes significantly expressed in TROP2+ tumors (P = 5.73E-20; P = 6.2E-13, P = 2.1E-11). Overall, PD-L1+ tumors and TROP2+ tumors showed an immune suppressive genomic landscape but were potentially regulated by different signaling. Of note, tumors with very high expression of PD-L1 showed an inverse correlation with VTCN1 gene expression. Conclusions: This preliminary analysis confirmed that only a limited number of early TNBC highly expressed PD-L1 and that a different immune suppressive landscape for PD-L1± and TROP2± tumors exists. In addition, our data suggest that early TNBC cases lacking PD-L1 might escape immune surveillance by virtue of the upregulation of key alternate signaling. Ongoing efforts investigating the expression of such markers on stroma/epithelial tumor components will help to clarify their biological, clinical, and potential therapeutic role in TNBC. Citation Format: Balazs Gyorffy, Libero Santarpia. Immune Landscape in PD-L1-positive and TROP2-positive Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-03-07.

Abstract PO1-24-08: Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer

Abstract Background: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) overexpression. This aggressive form of breast cancer carries a higher risk of recurrence compared to other subtypes, presenting a significant clinical challenge. Although chemotherapy is commonly used as the primary treatment for a substantial number of TNBC patients, it is often accompanied by drug resistance and frequent tumor recurrence. Urgent efforts are needed to identify novel molecular targets specific to TNBC and develop effective therapies to combat this aggressive disease. The ultimate objective is to discover treatments capable of overcoming drug resistance and improving the overall survival of patients with TNBC. Methods: Immunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. LIVE/DEAD Cell Imaging was to detect live and dead cells. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our newly developed anti-HER3 monoclonal antibody (mAb) 4A7 could potentiate the antitumor activity of paclitaxel in vivo. Results: The chemo-resistant subline HCC1806-TR was obtained by subjecting TNBC HCC1806 cells to prolonged treatment with paclitaxel. In comparison to the parental cell line HCC1806-P, HCC1806-TR exhibited enhanced HER3 expression and activation. It is noteworthy that approximately half of the tested TNBC specimens and cell lines displayed elevated expression of HER3. Analyzing TCGA databases revealed that TNBC patients with high levels of HER3 mRNA expression in their tumors had significantly poorer overall survival (OS) and relapse-free survival (RFS) compared to those with low HER3 expression. Specifically knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Blockade of HER3 signaling with our mAb (4A7) in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo. Conclusions: Our data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of paclitaxel in TNBC. Keywords: HER3, Anbibody, Chemotherapy, Triple Negative Breast Cancer Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Ann Thor, Bolin Liu. Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-08.

The high FKBP1A expression in WBCs as a potential screening biomarker for pancreatic cancer

Given the limitation of current routine approaches for pancreatic cancer screening and detection, the mortality rate of pancreatic cancer cases is still critical. The development of blood-based molecular biomarkers for pancreatic cancer screening and early detection which provide less-invasive, high-sensitivity, and cost-effective, is urgently needed. The goal of this study is to identify and validate the potential molecular biomarkers in white blood cells (WBCs) of pancreatic cancer patients. Gene expression profiles of pancreatic cancer patients from NCBI GEO database were analyzed by CU-DREAM. Then, mRNA expression levels of three candidate genes were determined by quantitative RT-PCR in WBCs of pancreatic cancer patients (N = 27) and healthy controls (N = 51). ROC analysis was performed to assess the performance of each candidate gene. A total of 29 upregulated genes were identified and three selected genes were performed gene expression analysis. Our results revealed high mRNA expression levels in WBCs of pancreatic cancer patients in all selected genes, including FKBP1A (p < 0.0001), PLD1 (p < 0.0001), and PSMA4 (p = 0.0002). Among candidate genes, FKBP1A mRNA expression level was remarkably increased in the pancreatic cancer samples and also in the early stage (p < 0.0001). Moreover, FKBP1A showed the greatest performance to discriminate patients with pancreatic cancer from healthy individuals than other genes with the 88.9% sensitivity, 84.3% specificity, and 90.1% accuracy. Our findings demonstrated that the alteration of FKBP1A gene in WBCs serves as a novel valuable biomarker for patients with pancreatic cancer. Detection of FKBP1A mRNA expression level in circulating WBCs, providing high-sensitive, less-invasive, and cost-effective, is simple and feasible for routine clinical setting that can be applied for pancreatic cancer screening and early detection.

Abstract 633: Integrin αVβ3 activation by thyroid hormones triggers JAK/STAT oncogenic pathways that promote T cell lymphoma dissemination

Abstract T cell lymphomas (TCL) are a heterogeneous group of lymphoproliferative disorders with poor prognosis. Aberrant activation of JAK/STAT pathway is associated with lymphoma dissemination in TCL patients. Although the FDA approved the use of inhibitors to target this pathway (Ruxolitinib), significant toxic effects have been reported and their use is limited. Thus making an excellent opportunity to study biological factors that regulate the activation of these pathways. In this sense, we recently showed that thyroid hormones (THs) acting through integrin αVβ3 induced TCL proliferation, which is reduced in vitro and in vivo in the presence of integrin αVβ3 pharmacological inhibitor. Our aim is to evaluate the impact of THs on JAK/STAT pathway activation and their implications on anti-lymphoma therapy. For the in vitro assays we used human and murine TCL cell lines. We used EL4 cells and c57bl/6 syngeneic mice for in vivo analysis. Proteomic profiles were evaluated by LC-MS-MS analysis. The GSE58445 database was used for in silico analysis. We found that THs induced STAT1, 3 and 5 phosphorylation in TCL cells and that the integrin αVβ3 inhibitor, Cilengitide (Cile) blunted these effects (p&amp;lt;0.05). Interestingly, we found that high mRNA expression levels of integrin β3 significantly correlate with worse overall survival in a cohort of TCL patients (p&amp;lt;0.05). Additionally, Ruxolitinib (Ruxo) diminished THs-induced STATs phosphorylation (p&amp;lt;0.05). As Ruxo reported toxic effects we aim to study alternative therapies such as Bexarotene (Bex), a synthetic retinoid used for cutaneous LCT treatment. We found that Bex significantly reduced cell viability in vitro of all TCL subtypes analyzed and combination with Cile resulted in improved anti-lymphoma activity (p&amp;lt;0.01). Also, Ruxo significantly decreased TCL cell viability but to a lesser extent than Bex+Cile (p&amp;lt;0.05). We previously found that BexT4+Cile (T4+, thyroxine supplementation) significantly decreases the in vivo growth of TCL tumors. To understand the mechanisms under this effects we analyzed tumor proteomics profiles and metalloprotease activity after 12 days of treatment. We found that BexT4+Cile regulates proteomic profiles related not only to lymphoma progression (JAK/STAT and PI3K-Akt signaling) but also pathways that impact the tumor microenvironment (TNF and angiogenic factors secretion and Th1/Th2 T cell differentiation). Regarding metalloprotease activity, we found that BexT+Cile inhibited MMP2 and MMP9 activity (p&amp;lt;0.05). Finally, we evaluated the dissemination of TCL in an in vivo model (by vain tail) and found that BexT4+Cile reduced EL4 cell implantation into the liver and kidneys (p&amp;lt;0.05). These results described the mechanisms by which THs induce the activation of JAK/STAT oncogenic pathway and showed how the inhibition of integrin αvβ3 in combination with bexarotene has therapeutic potential for TCL. Citation Format: María M. Debernardi, María V. Revuelta, Helena Sterle, Gonzalo Gonzalez, Ingrid L M Souza, Alejandro Correa, Leandro Cerchietti, Graciela Cremaschi, Florencia Cayrol. Integrin αVβ3 activation by thyroid hormones triggers JAK/STAT oncogenic pathways that promote T cell lymphoma dissemination [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 633.

Growth, biofilm formation, and motility of Listeria monocytogenes strains isolated from food and clinical samples located in Shanghai (China)

Listeria monocytogenes is a common foodborne pathogen that frequently causes global outbreaks. In this study, the growth characteristics, biofilm formation ability, motility ability and whole genome of 26 L. monocytogenes strains isolated from food and clinical samples in Shanghai (China) from 2020 to 2022 were analyzed. There are significant differences among isolates in terms of growth, biofilm formation, motility, and gene expression. Compared with other sequence type (ST) types, ST1930 type exhibited a significantly higher maximum growth rate, the ST8 type demonstrated a stronger biofilm formation ability, and the ST121 type displayed greater motility ability. Furthermore, ST121 exhibited significantly high mRNA expression levels compared with other ST types in virulence genes mpl, fbpA and fbpB, the quorum sensing gene luxS, starvation response regulation gene relA, and biofilm adhesion related gene bapL. Whole-genome sequencing (WGS) analyses indicated the isolates of lineage I were mostly derived from clinical, and the isolates of lineage II were mostly derived from food. The motility ability, along with the expression of genes associated with motility (motA and motB), exhibited a significantly higher level in lineage II compared with lineage I. The isolates from food exhibited significantly higher motility ability compared with isolates from clinical. By integrating growth, biofilm formation, motility phenotype with molecular and genotyping information, it is possible to enhance comprehension of the association between genes associated with these characteristics in L. monocytogenes.

Evaluation of the lncRNA-miRNA-mRNA ceRNA network in lungs of miR-147 -/- mice.

Background: Previous studies have documented important roles for microRNA-147 (miR-147) in inflammation, radiation-induced injury, cancer, and a range of other diseases. Murine lungs exhibit high levels of miRNA, mRNA, and lncRNA expression. However, very little research to date has focused on the lncRNA-miRNA-mRNA competing endogenous RNA (ceRNA) networks associated with miR-147, and the regulation of lncRNAs and miRNAs in this setting remains poorly understood. Methods: After establishing a miR-147-/- model mouse, samples of lung tissue were harvested for RNA-sequencing, and differentially expressed lncRNAs, miRNAs, and mRNAs were identified. The miRNA targets of these lncRNAs and the identified miRNAs were first overlapped to facilitate the prediction of target mRNAs, with analyses then examining the overlap between these targets and mRNAs that were differentially expressed. Then, these target mRNAs were subjected to pathway enrichment analyses. These results were ultimately used to establish a miR-147-related ceRNA network. Results: Relative to wild-type mice, the lungs of miR-147-/- mice exhibited 91, 43, and 71 significantly upregulated lncRNAs, miRNAs, and mRNAs, respectively, together with 114, 31, and 156 that were significantly downregulated. The lncRNA-miRNA-mRNA network established based on these results led to the identification of Kcnh6 as a differentially expressed hub gene candidate and enabled the identification of a range of regulatory relationships. KEGG pathway enrichment showed that the mRNA targets of differentially expressed lncRNAs and miRNAs in the mice were associated with tumor-related signaling, endometrial cancer, bladder cancer, and ErbB signaling. Conclusion: These results suggest that the identified ceRNA network in miR-147-/- mice shapes tumor-associated signaling activity, with miR-147 potentially regulating various lncRNAs and miRNAs through Kcnh6, ultimately influencing tumorigenesis. Future studies of the lncRNA, miRNA, and mRNA regulatory targets shown to be associated with miR-147 in the present study may ultimately lead to the identification of novel clinically relevant targets through which miR-147 shapes the pathogenesis of cancer and other diseases.

Biomarker cystatin B expression correlates with pathogenesis in cervical cancer.

Cervical cancer (CC) is one of the most common gynecologic malignancies worldwide. Although rapid improvements have been made regarding its prevention and treatment, little is known about disease pathogenesis and the clinical relevance of reliable biomarkers. The present study evaluated the expression of cystatin B (CSTB) as a potential biomarker of CC. Tissue microarray analysis and immunohistochemical staining were performed to detect CSTB expression, while CSTB mRNA and protein expression levels of freshly isolated CC tissue were measured by quantitative real-time PCR and western blot, respectively. Bioinformatics were used to analyze the CSTB co-expression network and functional enrichments. We observed high CSTB mRNA and protein expression levels in CC tissues, which was confirmed by tissue microarray in a comparison with paired adjacent non-cancerous cervical tissue samples. CSTB gene enrichments and associations with co-expressed genes were also observed. Further analysis showed that elevated CSTB expression was associated with pathological progress in CC. Our data demonstrate that CSTB has the potential to be used as a tissue biomarker with clinical value in patients with CC, which may aid the development of intervention strategies.

Isolation and characterization of rabbit limbal niche cells

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs.Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRβ) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRβ and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRβ in rabbit-LNCs P4.In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRβ+).