Tissue Array Research Articles

Integrated analysis of disulfidptosis-related immune genes signature to boost the efficacy of prognostic prediction in gastric cancer

BackgroundGastric cancer (GC) remains a malignant tumor with high morbidity and mortality, accounting for approximately 1,080,000 diagnosed cases and 770,000 deaths worldwide annually. Disulfidptosis, characterized by the stress-induced abnormal accumulation of disulfide, is a recently identified form of programmed cell death. Substantial studies have demonstrated the significant influence of immune clearance on tumor progression. Therefore, we aimed to explore the intrinsic correlations between disulfidptosis and immune-related genes (IRGs) in GC, as well as the potential value of disulfidptosis-related immune genes (DRIGs) as biomarkers.MethodsThis study incorporated the single-cell RNA sequencing (scRNA-seq) dataset GSE183904 and transcriptome RNA sequencing of GC from the TCGA database. Disulfidptosis-related genes (DRGs) and IRGs were derived from the representative literature on both cell disulfidptosis and immunity. The expression and distribution of DRGs were investigated at the single-cell level in different GC cell types. Pearson correlation analysis was used to identify the IRGs closely related to disulfidptosis. The prognostic signature of DRIGs was established using Cox and LASSO analyses. We then analyzed and evaluated the differences in long-term prognosis, Gene Set Enrichment Analysis (GSEA), immune infiltration, mutation profile, CD274 expression, and response to chemotherapeutic drugs between the two groups. A tissue array containing 63 paired GC specimens was used to verify the expression of 4 DRIGs and disulfidptosis regulator SLC7A11 through immunohistochemistry staining.ResultsThe scRNA-seq analysis found that SLC7A11, SLC3A2, RPN1 and NCKAP1 were enriched in specific cell types and closely related to immune infiltration. Four DIRGs (GLA, HIF-1α, VPS35 and CDC37) were successfully identified to establish a signature to potently predict the survival time of GC patients. Patients with high risk scores generally experienced worse prognoses and exhibited greater resistant to classical chemotherapy drugs. Furthermore, the expression of GLA, HIF-1α, VPS35, CDC37 and SLC7A11 were elevated in GC tissues. A high expression of GLA, HIF-1α, VPS35 or CDC37 was associated with more advanced clinical stage of GC and increased SLC7A11 expression.ConclusionCurrent study first highlights the potential value of DRIGs as biomarkers in GC. We successfully constructed a robust model incorporating four DRIGs to accurately predict the survival time and clinicopathological characteristics of GC patients.

Abstract 1527: Exploring the role of tertiary lymphoid structure in the tumor microenvironment of cholangiocarcinoma

Abstract Cholangiocarcinoma is the second most common hepatobiliary malignant tumor besides liver cancer, grows from bile duct epithelial cells. However, surgery is the only hope for most patients with cholangiocarcinoma, but the effectiveness of treatment is not outstanding. Even though other methods of treatment, such as chemotherapy and radiation therapy, have limited effects. The immunotherapy and precision treatment have become the latest expectations of patients. Here, we analyzed the TCGA and GEO database to classify the cold and hot tumor using hierarchical clustering. The MCP counter results demonstrated that two genes, MS4A1 and CD79A, could identify the hot and cold tumor subgroups after TCGA data training and similar results show in GEO database. We also performed the tissue array for 105 cholangiocarcinoma tissue samples and found patients with high expression levels of MS4A1 or CD79A have better disease free survival (DFS) and progression free survival (PFS) (p value =0.0131 and 0.0017). In addition, we tested the tertiary lymphoid structures (TLS) signatures in tissue array sections, which is associated with the present of MS4A1 and CD79A. The results shows that patients who received the immunotherapy with TLS signatures have longer overall survival (OS) and PFS (p value =0.017 and 0.006). Collectively, B cell lineages or TLS could be an ideal biomarker to lead the clinical treatment and wish can broader applications in other diseases. Citation Format: Shin-Yi Chung, Ming-Huang Chen, Yi-Chen Yeh, Yu-Chan Chang, Yu-Cha Wang. Exploring the role of tertiary lymphoid structure in the tumor microenvironment of cholangiocarcinoma [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 1527.

Abstract 6070: Identification of a molecular glue degrader that engages a cancer-specific E3 ligase

Abstract To date, most successful molecular glues have leveraged ubiquitously expressed E3 ligases, most notably Cereblon in the case of the thalidomide derivatives. However, a number of specific E3 ligases are highly over-expressed in certain tumors. Compounds that engage such tumor-specific E3 ligases might provide a unique opportunity to develop molecular glues with higher therapeutic indexes. One such tumor-specific E3 ligase is FBXO5, a member of the F-box family of E3 ligases. Expression analysis suggests this E3 ligase is highly expressed in a wide array of tumor tissues when compared to the corresponding normal tissue, and that within a given tumor type, higher FBXO5 expression correlates with advanced tumor stage and worse disease-free survival. Here, we describe a small molecule (MW~400 Daltons) that uses FBXO5 to function as a molecular glue degrader. This compound, CL-200, exerts cytotoxicity in the nanomolar range across a number of diverse tumor types. The cytotoxicity of CL-200 is abrogated when FBXO5 is knockdown using siRNA. We have further determined that CL-200 induces the FBXO5-dependent proteasomal degradation of at least two critical neosubstrates. One identified neosubstrate is the p150 isoform of ADAR1, an enzyme that catalyzes the posttranscriptional conversion of adenosine to inosine in double-stranded RNA (dsRNA). Evidence suggests that ADAR1p150 is the isoform that confers oncogenic and immune modulating effects in many tumors. Another neosubstrate targeted by CL-200 is c-myc, a critical transcription factor linked to a wide array of tumors. Of note, both p150 ADAR1 or c-myc have been viewed as difficult-to-drug if not undruggable targets. As such, these data suggests that CL-200 functions as a novel molecular glue degrader that engages the cancer-specific E3 ligase FBXO5 to degrade both p150 ADAR1 and c-myc. Structural alteration of CL-200 suggest that chemical modifications can be made that retain the engagement with FBXO5 while altering the neosubstrate profile. Together, these results demonstrate the possibility of leveraging cancer-specific E3 ligases to develop novel molecular glues. Citation Format: Yuan Liu, Mads Larsen, Bo Lin, Irene A. Cardenal, Jason R. Kennerdell, Toren Finkel, Bill B. Chen. Identification of a molecular glue degrader that engages a cancer-specific E3 ligase [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 6070.

Abstract 1044: Low-cost, high-sensitivity InSituPlex® mIF assays on the Parhelia Omni-Stainer™

Abstract Background: Multiplex immunofluorescence (mIF) is an important tool for extracting complex phenotypic information from normal or diseased tissues, and provides accurate and predictive insights on the tissue microenvironment faster. For patient samples in translational and clinical research, high sensitivity, specificity, and reproducibility are critical; however, throughput and cost considerations are also important in designing clinical studies. Enabling an easy-to-use high-performance assay such as InSituPlex® on robust and affordable auto-stainers like the Parhelia Omni-Stainer™ can allow researchers to gain a deeper understanding of the tissue micro-environment through high-quality multiplexed biomarker detection in a fraction of the time and cost. Methods: We performed Ultivue InSituPlex® assays with OmniVUE™ panels using the Parhelia Omni-Stainer™, a low-cost, high-throughput tissue auto-stainer with reduced requirements for reagents with a minimal dead volume. We compared the whole-slide staining performance across replicates using human tonsil and tumor tissue arrays across multiple independent runs over multiple days. The stained slides were scanned using the Zeiss AxioScan.Z1 high-throughput imaging system, and image batches were processed using Ultivue’s UltiStacker.AI and UltiAnalyzer.AI software. Results: Tighter temperature control of the slide in the Omni-Stainer supports optimal InSituPlex® signal amplification. Intra-run and inter-day analysis on serial sections of human tonsil and tumor tissue arrays demonstrated a high degree of consistency in InSituPlex® assay performance, specifically with marker-specific signal intensities and cell densities. The Omni-Stainer facilitates the use of low reagent volumes at each step of the assay due to its efficient reagent use, thus lowering the cost of reagents per slide. Conclusions: This study demonstrated a fast, simple and robust workflow to perform high-performance InSituPlex® mIF assays on the low-cost, high-throughput Parhelia Omni-Stainer™. Ultivue’s configurable OmniVUE™ mIF panels can be run on the Omni-Stainer with reduced associated reagent costs per slide. This workflow can support larger clinical and translational studies in a more cost-effective fashion, leading to in-depth analysis of tissue microenvironment through highly precise multiplexed biomarker detection. Citation Format: Kevin Hwang, Lauren Duro, Alex Veith, Nelson Wedin, Nikolay Samusik, Je H. Lee. Low-cost, high-sensitivity InSituPlex® mIF assays on the Parhelia Omni-Stainer™ [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 1044.

Abstract 390: Direct interaction of Pin1 with HIF-prolyl hydroxylase 2: Implications for breast cancer cell growth and survival

Abstract Prolyl hydroxylase domain 2 (PHD2) is the main hypoxia-inducible factor (HIF)-prolyl hydroxylase. PHD2 in normoxia hydroxylates specific proline residues in HIF-1α and HIF-2α, which facilitates their ubiquitination and proteasomal degradation. Although the activity of PHD2 is reduced in hypoxia, significant levels of residual activity of this monooxygenase are still detected under hypoxic conditions. However, its role under hypoxia is poorly understood. Peptidyl-prolyl isomerase (Pin1) binds to target proteins containing phosphorylated serine or threonine residues followed by proline (pS/T-P). As PHD2 harbors several pS/T-P motifs, it may be a potential substrate of Pin1. We found for the first time interaction between Pin1 and PHD2 in human breast cancer MDA-MB-231 cells in normoxic and hypoxic conditions. Additionally, the breast cancer tissue array showed elevated expression of PHD2 as well as Pin1 in tumors compared to adjacent normal tissues. LC-MS/MS spectrometry identified three amino acid residues (S125, T168, and S174) of PHD2 undergoing phosphorylation. Among these, serine 125 was found to be the principal site required for Pin1 binding. As a novel binding partner of Pin1, oncogenic PHD2 can be explored as a therapeutic target for the treatment of breast cancer. Citation Format: Yanymee Nimesia Guillen Quispe, Su-Jung Kim, Soma Saeidi, Gyo-Jin Choi, Chaithanya Chelakkot, Tianchi Zhou, Sang-Beom Bang, Tae-Won Kim, Young-Kee Shin, Young-Joon Surh. Direct interaction of Pin1 with HIF-prolyl hydroxylase 2: Implications for breast cancer cell growth and survival [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 390.

Abstract 2765: Novel WNT-RIP1-GDF15 pathway promotes migration and invasion of colorectal cancer cells

Abstract Growth-differentiation factor 15 (GDF15) is a member of the transforming growth factor β (TGF-β) family protein, also known as NSAID-activated gene 1 (NAG-1) or macrophage inhibitory cytokine 1 (MIC-1), and is a major cause of cancer cachexia, cardiovascular disease, and various inflammatory diseases. Roles of GDF15 as major inflammatory response factor also have been demonstrated in animal models, and it has also showed various malignant functions such as regulating metabolism, promoting metastasis by inducing EMT in cancer. Here, we revealed a novel potential role for RIP1 in colorectal cancer (CRC) in enhancing metastasis by controlling WNT-RIP1-GDF15 signaling. We showed that WNT3A treatment increased the expression of GDF15 and GFRAL. Immunohistochemical analysis of human CRC tissue arrays consisting of primary cancer and metastatic cancer also confirmed that the expression of GDF15 and GFRAL was increased in metastatic cancer. Human XL cytokine assay, western blot, and ELISA results confirmed that GDF15 expression was reduced in cell lines in which RIP1 was knocked down. Reduction of GDF15 expression and secretion by RIP1 knockdown showed suppression of EMT induction resulting to decrease of migration and invasion of CRC cells. Taken together, these results suggest a novel EMT-enhancing role for RIP1 in the WNT pathway and suggest a novel WNT-RIP1-GDF15 pathway contributing to CRC malignancy by promoting EMT. Citation Format: A-Ram Kang, Do Hoon Kim, Jong Kuk Park. Novel WNT-RIP1-GDF15 pathway promotes migration and invasion of colorectal cancer cells [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 2765.

Abstract 212: Repression of NKX2-1 promote epithelial-mesenchymal transition and tumor neutrophil recruitment in lung cancer progression

Abstract Background: Malignant transformation is associated with tumor aggressiveness and poor patient outcome. This cellular alteration is due to either activation of oncogenes or the silencing of tumor suppressor genes. NKX2-1 was identified as a lineage-specific gene downregulated in poorly differentiated lung adenocarcinoma (LUAD). However, the molecular mechanism and functional role of infiltrated immune cells in NKX2-1-negative tumors are not clearly understood. Methods: Clinical relevance of NKX2-1 expression and disease status were evaluated by immunohistochemical analysis of LUAD tissue arrays and the overall survival analysis of TCGA databases. In vitro and in vivo experiments were applied to confirm the role and mechanisms of NKX2-1 actions in transformed cells. The secretion of chemokines was evaluated by using chemokine array. Flow cytometry and single-cell RNA sequencing analysis of mouse tumor tissue were used to demonstrate the infiltration of neutrophils in mouse models. Results: Low expression of NKX2-1 was observed in high-grade LUAD tumors. LUAD patients who express a pattern of NKX2-1high/TGF-βlow had better survival outcomes than those with NKX2-1low/TGF-βhigh. Mechanistically, TGF-β induced the downregulation of NKX2-1 which led to enhanced epithelial-mesenchymal transition (EMT), cell motility, and CXC chemokine expression. The high release of these chemoattractants promoted the recruitment of neutrophils. Meanwhile, NKX2-1-dependent suppression of CXC chemokines gene expression may be via repressive histone methylation at the promoter region of CXC chemokines in lung cancer cells. In the in vivo syngeneic LUAD models, exacerbated neutrophil infiltration was noted. Single-cell RNA sequencing and flow cytometry both revealed that neutrophils were recruited via CXCLs/CXCR2 activation in LUAD tumors with NKX2-1 knockdown. Conclusion: Our data showed that NKX2-1 represses tumor growth, metastasis, and neutrophil recruitment via the CXCLs/CXCR2-dependent mechanisms. Notably, targeting CXCR2 in NKX2-1-negative tumors may improve LUAD patient outcomes. Citation Format: Shih-Hwa Chiou, Ping-Hsing Tsai, Mong-Lien Wang. Repression of NKX2-1 promote epithelial-mesenchymal transition and tumor neutrophil recruitment in lung cancer progression [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 212.

Abstract 3268: A novel signaling axis of APE1-redox-sensitive SOX9 activation underlying reflux-condition-associated chemoresistance in esophageal adenocarcinoma

Abstract Chronic gastroesophageal reflux disease (GERD), characterized by the reflux of acidic bile salts (ABS) into the esophagus, stands as the primary risk factor for esophageal adenocarcinoma (EAC). EAC, known for its aggressive nature and limited response to therapy, exhibits a disheartening 5-year survival rate of less than 20%. Overcoming drug resistance remains a pressing challenge in achieving optimal therapeutic outcomes for EAC patients. Previous studies from our team have unveiled that exposure to ABS leads to the overexpression of Apurinic/apyrimidinic endonuclease (APE1), which orchestrates various oncogenic pathways in EAC cells. In this study, we delve into the influence of ABS in driving APE1-redox-mediated activation of the SOX9 pathway, thus contributing to the chemoresistance observed in EAC. To identify the enrichment of the SOX9 pathway and the heightened downstream target genes in EAC, we analyzed RNA sequencing data and public databases. Cell models, patient tissue arrays, pL2-IL1β transgenic mice, and de-identified xenografts derived from EAC patients (PDXs) were employed to investigate the signaling axis of APE1-redox-SOX9 in oxaliplatin resistance of EAC. Our analysis of public databases unveiled a significant enrichment of the SOX9 pathway in EAC patients. RNA sequencing data from EAC cells exposed to ABS, with or without APE1-knockdown, suggested APE1's involvement in the GERD-activated SOX9 signaling pathway. Immunohistochemistry staining of human patient tissue arrays, along with immunofluorescent staining of L2-IL1ß transgenic mice, confirmed the co-overexpression of APE1, SOX9, and ALDH1A1 in EAC. Furthermore, APE1 silencing led to the downregulation of SOX9 and exhibited synergistic effects with oxaliplatin in chemo-resistant EAC cells. Mechanistically, we uncovered that APE1's redox function is crucial for AKT-GSK3ß-mediated SOX9 stability. Treatment with the APE1-redox-specific inhibitor, APX2009, sensitized patient-derived xenograft (PDX) tumors to oxaliplatin by repressing SOX9-regulated chemoresistance genes. This study sheds light on a novel signaling axis involving APE1-redox-AKT-GSK3ß-SOX9 in Barrett’s carcinogenesis. The pharmacological inhibition of APE1-redox function presents itself as a promising therapeutic approach to target the previously intractable SOX9 signals, potentially overcoming chemoresistance in EAC. Citation Format: Heng Lu, Lei Chen, Farah Ballout, Dunfa Peng, Zheng Chen, Jianwen Que, Steven Chen, Oliver Gene McDonald, Alexander Zaika, Wael El-Rifai. A novel signaling axis of APE1-redox-sensitive SOX9 activation underlying reflux-condition-associated chemoresistance in esophageal adenocarcinoma [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 3268.

Abstract 859: Development of non-smoking lung cancers by indoor carcinogenic aerosols through epigenetic reprogramming of lung stem cells: Bioinformatics and artificial intelligence analysis

Abstract Several scientific studies discovered the toxic effects of indoor aerosols on different organs’ adult stem cells of the human body. The most importantly, the indoor toxic aerosols comprised of numerous carcinogens namely, sodium dodecyl sulfate, phthalic acids from floor cleaners, formaldehyde, acetaldehyde, benzene, toluene, ethyl benzene, xylenes, from air fresheners, Hydramethylnon, Pyrethrins, imiprothrin, cypermethrin, tetramethrin, prallethrin and permethrin from ant and roach Killer. However, there was a lack of analysis revealing the mechanism of toxic effects and epigenetic reprogramming of human lung stem cells and development of non-smoking lung cancers. In this study, we analyzed the toxic effects of these chemicals and the mechanism of epigenetic reprograming on lung epithelial cells using Comparative Toxico-genomics Database (CTD), database of Environmental Health Perspectives (EHP), National Institute of Environmental Health Sciences (NIEHS), Google search browser with generative artificial intelligence (AI) and other biological informatics methods (Reactome, Cytoscape PSICQUIC services, and ChEMBL database). We observed from CTD analysis that the chemical gene interactions as risk factors from indoor aerosols showed transcriptionally highly expressed of 31 common genes, by phthalic acids, sodium dodecyl sulfate, from the total of 1347 genes, 107 common genes by formaldehyde, acetaldehyde from the total of 4193 genes and all other toxic ingredients of indoor aerosols. The epigenetic mechanism of histone modifications (H3K4me3, H3K9me3, H3K27me3, H3K27ac, H3K36me3, H3K4me1, Hsa-mir-1301 of lung epithelial, and miRNAs have been revealed by NIH Roadmap Epigenomics Mapping Consortium and the lung tissue arrays were analyzed with AI and matplotlib program. The heterogenic adult stem cell reprogramming is regulated by epigenetic bivalent histone modifications at transcript starts sequences (TSS) of target genes and our current study revealed that CD44, CD80, ALDH2, ALDH3A1, ALDH3B2 are epigenetically reprogrammed with close interaction of other genes by these toxic ingredients of indoor aerosols. Therefore, our results are significant for the carcinogenic effects of indoor aerosols causing for development of lung cancer epigenetically, which may give a clue for the prevention and treatment of nonsmoking lung cancer. Citation Format: Juan Anderson, Mariah Delgado, Malcolm Lovett, Maya Saunders, Geovannie Lake, Samuel Darko, Rose M. Stiffin, Ayivi Huisso, Marilyn Sherman, Latoya Appleton, Amalya Mihnea, Swarnava Das, Mohammad M. Algahtani, Ravi Vadapalli, Biswarup Basu, Arunima Biswas, Noor Neha, Madhumita Das, Marco A. Ruiz, Mayur Doke, Jayanta K. Das. Development of non-smoking lung cancers by indoor carcinogenic aerosols through epigenetic reprogramming of lung stem cells: Bioinformatics and artificial intelligence analysis [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 859.

Abstract 7046: Proteogenomic and immunologic profiles of head and neck cancer patients of African ancestry

Abstract Blacks/African Americans (BAAs) with head and neck squamous cell carcinoma (HNSCC) have worse survival than Whites. However, the complex molecular biomarkers and signatures of HNSCC among BAAs and molecular distinction associated with HNSCC disparities are largely unknown. Here we show the results of a multi-omics analysis of 500 patients (48 BAAs and 452 Whites). Comparative analyses between the racial groups were performed at the level of genomics, pathways, molecular subtype, tumor immune infiltration, protein alterations and prognosis. We found that overall survival in BAAs with HPV-unrelated HNSCC is significantly shorter than in White patients. Ancestry-related differential mutation spectrum, DNA methylation, gene and protein expression pattern and immune infiltration landscape were identified. Particularly, FAT1 mutation is significantly associated with its gene expression in BAA HNSCC and poor overall survival in this group. In BAA HNSCC, SALL3 gene expression is positively associated with its gene CNVs, while RTP4 gene expression is inversely correlated with its methylation. High EGFR_pY1068 and RAD50 levels correlate with poor prognosis in BAAs with HNSCC. The immune infiltration enrichment scores of Th17 cells, activated dendritic cells, monocytes and neutrophils in BAA HNSCC were significantly lower than those in White HNSCC. By combining multi-omics analysis and data validation using in-house-made tumor tissue arrays, this study comprehensively elucidates the genetic landscape, immune response, and tumor biology in BAA and White HNSCC patients, providing a molecular basis for future research in identifying appropriate screening modalities and therapeutic interventions leading to improved outcomes in BAAs with HNSCC. Citation Format: Fan Yang, Fanghui Chen, Chloe Shay, Nabil F. Saba, Yong Teng. Proteogenomic and immunologic profiles of head and neck cancer patients of African ancestry [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 7046.

Suppression of ITPKB degradation by Trim25 confers TMZ resistance in glioblastoma through ROS homeostasis

Temozolomide (TMZ) represents a standard-of-care chemotherapeutic agent in glioblastoma (GBM). However, the development of drug resistance constitutes a significant hurdle in the treatment of malignant glioma. Although specific innovative approaches, such as immunotherapy, have shown favorable clinical outcomes, the inherent invasiveness of most gliomas continues to make them challenging to treat. Consequently, there is an urgent need to identify effective therapeutic targets for gliomas to overcome chemoresistance and facilitate drug development. This investigation used mass spectrometry to examine the proteomic profiles of six pairs of GBM patients who underwent standard-of-care treatment and surgery for both primary and recurrent tumors. A total of 648 proteins exhibiting significant differential expression were identified. Gene Set Enrichment Analysis (GSEA) unveiled notable alterations in pathways related to METABOLISM_OF_LIPIDS and BIOLOGICAL_OXIDATIONS between the primary and recurrent groups. Validation through glioma tissue arrays and the Xiangya cohort confirmed substantial upregulation of inositol 1,4,5-triphosphate (IP3) kinase B (ITPKB) in the recurrence group, correlating with poor survival in glioma patients. In TMZ-resistant cells, the depletion of ITPKB led to an increase in reactive oxygen species (ROS) related to NADPH oxidase (NOX) activity and restored cell sensitivity to TMZ. Mechanistically, the decreased phosphorylation of the E3 ligase Trim25 at the S100 position in recurrent GBM samples accounted for the weakened ITPKB ubiquitination. This, in turn, elevated ITPKB stability and impaired ROS production. Furthermore, ITPKB depletion or the ITPKB inhibitor GNF362 effectively overcome TMZ chemoresistance in a glioma xenograft mouse model. These findings reveal a novel mechanism underlying TMZ resistance and propose ITPKB as a promising therapeutic target for TMZ-resistant GBM.

A multi-dimensional approach to unravel the intricacies of lactylation related signature for prognostic and therapeutic insight in colorectal cancer

BackgroundLactylation, a novel contributor to post-translational protein modifications, exhibits dysregulation across various tumors. Nevertheless, its intricate involvement in colorectal carcinoma, particularly for non-histone lactylation and its intersection with metabolism and immune evasion, remains enigmatic.MethodsEmploying immunohistochemistry on tissue microarray with clinical information and immunofluorescence on colorectal cell lines, we investigated the presence of global lactylation and its association with development and progression in colorectal cancer as well as its functional location. Leveraging the AUCell algorithm alongside correlation analysis in single-cell RNA sequencing data, as well as cox-regression and lasso-regression analysis in TCGA dataset and confirmed in GEO dataset, we identified a 23-gene signature predicting colorectal cancer prognosis. Subsequently, we analyzed the associations between the lactylation related gene risk and clinical characteristics, mutation landscapes, biological functions, immune cell infiltration, immunotherapy responses, and drug sensitivity. Core genes were further explored for deep biological insights through bioinformatics and in vitro experiments.ResultsOur study innovatively reveals a significant elevation of global lactylation in colorectal cancer, particularly in malignant tumors, confirming it as an independent prognostic factor for CRC. Through a comprehensive analysis integrating tumor tissue arrays, TCGA dataset, GEO dataset, combining in silico investigations and in vitro experiments, we identified a 23-gene Lactylation-Related Gene risk model capable of predicting the prognosis of colorectal cancer patients. Noteworthy variations were observed in clinical characteristics, biological functions, immune cell infiltration, immune checkpoint expression, immunotherapy responses and drug sensitivity among distinct risk groups.ConclusionsThe Lactylation-Related Gene risk model exhibits significant potential for improving the management of colorectal cancer patients and enhancing therapeutic outcomes, particularly at the intersection of metabolism and immune evasion. This finding underscores the clinical relevance of global lactylation in CRC and lays the groundwork for mechanism investigation and targeted therapeutic strategies given the high lactate concentration in CRC.

ATP6AP1 as a potential prognostic biomarker in CRC by comprehensive analysis and verification

The role of ATP6AP1 in colorectal cancer (CRC) remains elusive despite its observed upregulation in pan-cancer. Therefore, the current study aimed to assess the clinical significance of ATP6AP1 and its relationship with the immune infiltration in CRC. Transcriptome data of CRC were obtained from The Cancer Genome Atlas (TCGA) database and analyzed using the combination of R packages and tumor-related databases, including TIMER2, TISIDB, cBioPortal, and MethSurv. The tissue arrays and immunohistochemical staining were performed to verify the expression and clinical characteristics of ATP6AP1. The results revealed that ATP6AP1 expression was significantly elevated in CRC and associated with poor clinicopathological characteristics and prognosis. Furthermore, the analysis demonstrated ATP6AP1 expression was correlated with the infiltration of immune cells and cancer-associated fibroblasts in the microenvironment of CRC. Moreover, ATP6AP1 was found to be linked to various immune checkpoints and chemokines, with enrichment of cytoplasmic vesicle lumen, endopeptidase regulator activity, and endopeptidase inhibitor activity observed in the high ATP6AP1 expressional group. In conclusion, the findings of this study suggest that ATP6AP1 upregulation may serve as a biomarker for poor diagnosis in CRC and offer a potential target for immunotherapy in CRC.

Increasing Sufu gene dosage reveals its unorthodox role in promoting polydactyly and medulloblastoma tumorigenesis.

Suppressor of fused (SUFU) is widely regarded as a key negative regulator of the sonic hedgehog (SHH) morphogenic pathway and a known tumor suppressor of medulloblastoma (MB). However, we report here that SUFU expression was markedly increased in 75% of specimens compiled in a tissue array comprising 49 unstratified MBs. The SUFU and GLI1 expression levels in this MB array showed strong positive correlation, which was also identified in a large public data set containing 736 MBs. We further report that increasing Sufu gene dosage in mice caused preaxial polydactyly, which was associated with the expansion of the Gli3 domain in the anterior limb bud and heightened Shh signaling responses during embryonic development. Increasing Sufu gene dosage also led to accelerated cerebellar development and, when combined with ablation of the Shh receptor encoded by Patched1 (Ptch1), promoted MB tumorigenesis. These data reveal multifaceted roles of SUFU in promoting MB tumorigenesis by enhancing SHH signaling. This revelation clarifies potentially counterintuitive clinical observation of high SUFU expression in MBs and may pave way for novel strategies to reduce or reverse MB progression.

TGFβR1 inhibition drives hepatocellular carcinoma proliferation through induction of toll-like-receptor signalling.

Transforming growth factor (TGF)-β and toll-like receptors (TLRs) have been shown to independently modulate the proliferation of hepatocellular carcinoma (HCC). Since a direct cross-talk between these two signalling pathways in HCC has not been clearly described before, we aimed here to explore the possibility of such interaction. A human HCC tissue array (n = 20 vs. four control samples), human HCC samples (n = 10) and steatohepatitis-driven murine HCC samples (control, NASH and HCC; n = 6/group) were immunostained for TGFβR1, pSMAD2, TRAF6, IRAK1 and PCNA. The results were confirmed by immunoblotting. Effects of constant activation of the SMAD pathway by constitutive expression of ALK5 or knockdown of mediators of TLR signalling, IRAK1 and MyD88, on HCC proliferation, were investigated in the HCC cell line (HUH-7) after treatment with TGFβ1 cytokine or TGFβR1 kinase inhibitor (LY2157299) using PCNA and MTS assay. TGFβR1 expression is decreased in human and murine HCC and associated with downregulated pSMAD2, but increased IRAK1, TRAF6 and PCNA staining. TGFβR1 kinase inhibition abolished the cytostatic effects of TGFβ1 and led to the induction of IRAK1, pIRAK1 and elevated mRNA levels of TLR-9. Overexpression of ALK5 and knockdown of MyD88 or IRAK1 augmented the cytostatic effects of TGFβ1 on HUH-7. In another epithelial HCC cell line, that is, HepG2, TGFβR1 kinase inhibitor similarly elevated cellular proliferation. There is a balance between the canonical SMAD-driven tumour-suppressing arm and the non-canonical tumour-promoting arm of TGFβ signalling. Disruption of this balance, by inhibition of the canonical pathway, induces HCC proliferation through TLR signalling.

PARD3 drives tumorigenesis through activating Sonic Hedgehog signalling in tumour-initiating cells in liver cancer

BackgroundPar-3 Family Cell Polarity Regulator (PARD3) is a cellular protein essential for asymmetric cell division and polarized growth. This study aimed to study the role of PARD3 in hepatic tumorigenesis.MethodsThe essential role of PARD3 in mediating hepatic tumorigenesis was assessed in diet-induced spontaneous liver tumour and syngeneic tumour models. The mechanism of PARD3 was delineated by bulk and single-cell RNA sequencing. The clinical significance of PARD3 was identified by tissue array analysis.ResultsPARD3 was overexpressed in tumour tissues and PARD3 overexpression was positively correlated with high tumour stage as well as the poor prognosis in patients. In models of spontaneous liver cancer induced by choline-deficient, amino acid-defined (CDAA) and methionine-choline-deficient (MCD) diets, upregulation of PARD3 was induced specifically at the tumorigenesis stage rather than other early stages of liver disease progression. Site-directed knockout of PARD3 using an adeno-associated virus 8 (AAV8)-delivered CRISPR/Cas9 single-guide RNA (sgRNA) plasmid blocked hepatic tumorigenesis, while PARD3 overexpression accelerated liver tumour progression. In particular, single-cell sequencing analysis suggested that PARD3 was enriched in primitive tumour cells and its overexpression enhanced tumour-initiating cell (TICs). Overexpression of PARD3 maintained the self-renewal ability of the CD133+ TIC population within hepatocellular carcinoma (HCC) cells and promoted the in vitro and in vivo tumorigenicity of CD133+ TICs. Transcriptome analysis revealed that Sonic Hedgehog (SHH) signalling was activated in PARD3-overexpressing CD133+ TICs. Mechanistically, PARD3 interacted with aPKC to further activate SHH signalling and downstream stemness-related genes. Suppression of SHH signalling and aPKC expression attenuated the in vitro and in vivo tumorigenicity of PARD3-overexpressing CD133+ TICs. Tissue array analysis revealed that PARD3 expression was positively associated with the phosphorylation of aPKC, SOX2 and Gli1 and that the combination of these markers could be used to stratify HCC patients into two clusters with different clinicopathological characteristics and overall survival prognoses. The natural compound berberine was selected as a potent suppressor of PARD3 expression and could be used as a preventive agent for liver cancer that completely blocks diet-induced hepatic tumorigenesis in a PARD3-dependent manner.ConclusionThis study revealed PARD3 as a potential preventive target of liver tumorigenesis via TIC regulation.

Dynamic phenotypic reprogramming and chemoresistance induced by lung fibroblasts in small cell lung cancer.

Small cell lung cancer (SCLC) is heterogenous in phenotype and microenvironment. Dynamic phenotypic reprogramming, leading to heterogeneity, is prevalent in SCLC, while the mechanisms remain incompletely understood. Cancer-associated fibroblasts (CAFs) possess comprehensive roles in cancer progression, while their function in phenotypic reprogramming of SCLC remain elusive. Here, we obtained transcriptome data of SCLC tissues from publicly available databases, subsequently estimated abundance of CAFs. We found CAF-abundant SCLC exhibited non-neuroendocrine (Non-NE) characteristics. Supporting this, the positive correlation of expression level of α-SMA, the CAF marker, and expression level of REST, protein typically expressed in Non-NE type SCLC, was identified in SCLC tissue arrays. Moreover, we revealed that fibroblasts inhibited NE markers expression and cell proliferation of SCLC cells in the co-culture system comprising lung fibroblasts and SCLC cells, indicating a phenotypic reprogramming from NE to Non-NE. During this process, fibroblast-derived IL-6 activated the JAK2/STAT3 signaling, upregulated c-MYC expression, and subsequently activated the NOTCH pathway, driving phenotypic reprogramming. Moreover, CAF-enriched SCLC exhibited increased immune cell infiltration, elevated expression of immune activation-related signatures, and checkpoint molecules. Our data also highlighted the chemoresistance induced by fibroblasts in SCLC cells, which was effectively reversed by JAK inhibitor. In conclusion, fibroblasts induced phenotypic reprogramming of SCLC cells from NE to Non-NE, likely contributes to inflamed immune microenvironment and chemoresistance. These findings provide novel insights into the clinical implications of CAFs in SCLC.

RAB31 drives extracellular vesicle fusion and cancer‐associated fibroblast formation leading to oxaliplatin resistance in colorectal cancer

AbstractEpithelial‐mesenchymal transition (EMT) is associated with tumorigenesis and drug resistance. The Rab superfamily of small G‐proteins plays a role in regulating cell cytoskeleton and vesicle transport. However, it is not yet clear how the Rab family contributes to cancer progression by participating in EMT. By analysing various in silico datasets, we identified a statistically significant increase in RAB31 expression in the oxaliplatin‐resistant group compared to that in the parental or other chemotherapy drug groups. Our findings highlight RAB31's powerful effect on colorectal cancer cell lines when compared with other family members. In a study that analysed multiple online meta‐databases, RAB31 RNA levels were continually detected in colorectal tissue arrays. Additionally, RAB31 protein levels were correlated with various clinical parameters in clinical databases and were associated with negative prognoses for patients. RAB31 expression levels in all three probes were calculated using a computer algorithm and were found to be positively correlated with EMT scores. The expression of the epithelial‐type marker CDH1 was suppressed in RAB31 overexpression models, whereas the expression of the mesenchymal‐type markers SNAI1 and SNAI2 increased. Notably, RAB31‐induced EMT and drug resistance are dependent on extracellular vesicle (EV) secretion. Interactome analysis confirmed that RAB31/AGR2 axis‐mediated exocytosis was responsible for maintaining colorectal cell resistance to oxaliplatin. Our study concluded that RAB31 alters the sensitivity of oxaliplatin, a supplementary chemotherapy approach, and is an independent prognostic factor that can be used in the treatment of colorectal cancer.

Novel LIPA-Targeted Therapy for Treating Ovarian Cancer.

Ovarian cancer (OCa) is the most lethal form of gynecologic cancer, and the tumor heterogeneities at the molecular, cellular, and tissue levels fuel tumor resistance to standard therapies and pose a substantial clinical challenge. Here, we tested the hypothesis that the heightened basal endoplasmic reticulum stress (ERS) observed in OCa represents an exploitable vulnerability and may overcome tumor heterogeneity. Our recent studies identified LIPA as a novel target to induce ERS in cancer cells using the small molecule ERX-41. However, the role of LIPA and theutility of ERX-41 to treat OCa remain unknown. Expression analysis using the TNMplot web tool, TCGA data sets, and immunohistochemistry analysis using a tumor tissue array showed that LIPA is highly expressed in OCa tissues, compared to normal tissues. ERX-41 treatment significantly reduced the cell viability and colony formation ability and promoted the apoptosis of OCa cells. Mechanistic studies revealed a robust and consistent induction of ERS markers, including CHOP, elF2α, PERK, and ATF4, upon ERX-41 treatment. In xenograft and PDX studies, ERX-41 treatment resulted in a significant reduction in tumor growth. Collectively, our results suggest that ERX-41 is a novel therapeutic agent that targets the LIPA with a unique mechanism of ERS induction, which could be exploited to treat heterogeneity in OCa.

Treatment development targeting Hippo pathway for scirrhous type gastric cancer.

398 Background: Treatment outcome of gastric cancer (GC) has been improved by minimally invasive surgery, and development of new drugs including immune checkpoint inhibitors. However, survival for patients with scirrhous type (SGC, Type4) gastric cancer is dismal. Methods: We collected miRNAs form exosomes released from cancer associated fibroblasts which is developed from resected gastric cancer tissue. Pathway enrichment analysis (PEA) of top miRNAs showed that they are significantly associated with Hippo signaling pathway (Hippo pathway). The molecules relating Hippo pathway was evaluated by Western blotting in cell line, and IHC of tissue array. Results: PEA using top miRNAs showed the significant relationship to Hippo pathway. Yap is a final effector in this pathway, and we found that YAP is not expressed in MKN45 cells (diffuse type), which is supposed to be degraded in cytoplasm. On the contrary, MKN74 cells express both YAP and pYAP. IHC showed that YAP translocation to nucleus is significantly associated with CTGF expression. CTGF low expression is significantly associated with worse prognosis especially in diffuse type GC (Log rank p = 0.022). This trend is similar in the analysis of patients with SGC. Conclusions: The role of Hippo pathway is different between intestinal type and diffuse type of GC. YAP activator could be effective for the treatment of SGC, and we are focusing on the treatment development.