Cancer Stem Research Articles

Phosducin-like 3 is a novel prognostic and onco-immunological biomarker in glioma: A multi-omics analysis with experimental verification.

Malignant glioma is the most frequent primary tumor of the central nervous system. PDCL3 is a member of the phosducin-like protein family, and its imbalance has been shown to be associated with several human diseases. However, the underlying role of PDCL3 in human malignant cancers, especially in malignant gliomas, is unclear. In this study, we combined public database analysis and experimental verification to explore the differential expression, prognostic value and potential functions and mechanisms of PDCL3. The results revealed that PDCL3 is upregulated in multiple cancers and acts as a potential prognostic biomarker of glioma. Mechanistically, PDCL3 expression is associated with epigenetic modifications and genetic mutations. PDCL3 may directly interact with the chaperonin-containing TCP1 complex, regulating cell malignancy, cell communication and the extracellular matrix. More importantly, the association of PDCL3 with the infiltration of immune cells, immunomodulatory genes, immune checkpoints, cancer stemness and angiogenesis suggested that PDCL3 may regulate the glioma immune landscape. Furthermore, PDCL3 interference also decreased the proliferation, invasion and migration of glioma cells. In conclusion, PDCL3 is a novel oncogene and can be adopted as a biomarker with value in assisting clinical diagnosis, predicting patient outcomes and assessing the immune landscape of the tumor microenvironment in glioma.

Correlating mechanical and gene expression data on the single cell level to investigate metastatic phenotypes

Stiffness has been observed to decrease for many cancer cell types as their metastatic potential increases. Although cell mechanics and metastatic potential are related, the underlying molecular factors associated with these phenotypes remain unknown. Therefore, we have developed a workflow to measure the mechanical properties and gene expression of single cells that is used to generate large linked-datasets. The process combines atomic force microscopy to measure the mechanics of individual cells with multiplexed RT-qPCR gene expression analysis on the same single cells. Surprisingly, the genes that most strongly correlated with mechanical properties were not cytoskeletal, but rather were markers of extracellular matrix remodeling, epithelial-to-mesenchymal transition, cell adhesion, and cancer stemness. In addition, dimensionality reduction analysis showed that cell clustering was improved by combining mechanical and gene expression data types. The single cell genomechanics method demonstrates how single cell studies can identify molecular drivers that could affect the biophysical processes underpinning metastasis.

Pan-cancer investigation of C-to-U editing reveals its important role in cancer development and new targets for cancer treatment.

RNA editing is prevalent in the transcriptome and is important for multiple cellular processes. C-to-U RNA editing sites (RES) are relatively rare and understudied in humans, compared to A-to-I editing. However, the functional impact of C-to-U editing in human cancers also remains elusive. Here, we conducted the first comprehensive survey of pan-cancer C-to-U RESs. Surprisingly, we found that the same subset of RESs were associated with multiple features, including patient survival, cancer stemness, tumor mutation burden (TMB), and tumor-infiltrated immune cell compositions (ICC), suggesting an RES-mediated close relationship between these features. For example, editing sites for GALM or IFI6 that led to higher expression were linked to lower survival and more cancer stemness. Also, TMB was found to be lower in prostate cancer cases with ICC-associated RESs in CAVIN1 or VWA8 or higher in prostate cancer cases with thymoma. With experimental support, we also found RESs in CST3, TPI1, or TNC that are linked to immune checkpoint blockade by anti-PD1. We also confirmed through experiments that two C-to-U RESs in CSNK2B or RPS14 had different effects on colon cancer cells. Patients with CSNK2B editing, which increased the expression of the oncogene CLDN18, had a lower response to drugs. On the other hand, drugs worked better on people who had RPS14 editing, which greatly increased ribosome production. In summary, our study demonstrated the important roles of C-to-U RESs across cancers and shed light on personalized cancer therapy.

Abstract P2-21-12: Targeting ADAM8 dependent maintenance of the myCAF phenotype in the breast cancer tumor microenvironment

Abstract Background: Cancer associated fibroblasts (CAFs) regulate breast cancer growth, differentiation and metastasis. Targeting the signaling pathways between myofibroblast CAFs (myCAF) and cancer cells required for myCAF maintenance provides a mechanism to reverse engineer the tumor microenvironment to inhibit cancer growth and metastasis. Methods and Results: We co-cultured human triple negative breast cancer (MDA-MB-231) with human mesenchymal stem cells (MSC). After 72h, MSC mRNA expression of the myCAF markers, alpha-smooth muscle actin (α-SMA)/vimentin (Vim)/tenascin C (Ten-C) are all significantly increased. In parallel, MB-231 mRNA expression of stemness markers, SRY-Box Transcription factor 2 (Sox2)/Octamer-binding transcription factor 4 (Oct4)/Nanog, are also significantly increased. Migration assay and morphology imaging confirm the induction of the myCAF phenotype to promote MB-231 cancer stemness. shRNA lentivirus was used to knock down Sox2 gene expression in MB-231. We performed RNA-seq in MB-231 first comparing MB-231 vs. MB-231+MSC and then MB-231(-/-sox2)+MSC vs. MB-231+MSC. There are 104 overlapping genes in MB-231+MSC from the two analyses indicating upregulation with myCAF differentiation and cancer stemness. Using the Human Cancer Secretome Database, 9 genes are either membrane or secreted proteins. Using antibody blockade in co-culture, antibody depleted co-culture medium, and adding activated proteins to culture, A Disintegrin And Metalloprotease 8 (Adam8) gene was found to be required for myCAF maintenance. Using SELEX, we isolated 5 RNA aptamers targeting the human Adam8 soluble MP domain. Using in vitro quantification of Adam8 MP activity with human Adam8 fluorogenic peptide substrate and recombinant human Adam8 protein, Adam8 Apt-1 with IC50=33nM and Kd=29nM was selected. APt-1 does not inhibit human Adam 10 or 17 MP activity and remains extracellular. The core inhibitory domain of Apt-1 aptamer contains 23nt. Following induction of the myCAF phenotype and cancer stemness in MB-231+MSC coculture, addition of Apt-1 significantly decreased myCAF and cancer stemness markers. In murine NOD/scid xenograft model, 10^6 luciferase-MB-231 and 10^6 MSC were implanted in the R3 position at week 0. Apt1 was administered (10mg/kg via tail vein every 2 days) from week 3 to week 6. MB-231 volume (bioluminescence) was significantly decreased following Apt1. (Table) Conclusion: Adam8 is expressed in MB-231 breast cancer cells in the context of MSC/myCAF coculture and MB231 stemness and is required for MB-231 cancer growth. Aptamer ablation of Adam8 extracellular activity reverses MB-231 growth. MB-231 and MSC Murine Xenograft Model In murine NOD/scid xenograft model, 10^6 luciferase-MB-231 and 10^6 MSC were implanted in the R3 position at week 0. Apt1 was administered (10mg/kg via tail vein every 2 days) from week 3 to week 6. Citation Format: Zhiyong Mi, Paul C. Kuo. Targeting ADAM8 dependent maintenance of the myCAF phenotype in the breast cancer tumor microenvironment [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-21-12.

Abstract P6-11-02: Mitochondria-Nuclear Crosstalk Regulates the WNT Pathway in Triple-Negative Breast Cancer

Abstract Compared to hormone receptor (HR) positive breast cancer (BC), basal or triple-negative BC (TNBC) suffers a poor prognosis caused by a limited understanding of its driver signaling pathways. We used the transmitochondrial cybrid approach to understand the metabolic reprogramming and mitochondria-nuclear crosstalk in metastatic TNBC. In cybrids, mitochondria from cancer and benign cells are compared under a commonly defined nuclear background. Using cybrid-based discovery and its validation in cell lines, patient-derived xenografts, and clinical data, we have previously reported the activation of fatty acid oxidation (FAO) in metastatic TNBC. Recently, we developed a unique gene (DEG) signature by integrating gene expression data from cybrids with different omnibus databases such as TCGA and METABRIC. Our DEG could effectively distinguish the PAM50 subtypes of BC. Further analyses nominated negative regulation of the canonical WNT signaling pathway as one of the major pathways altered by mitochondrial character. The WNT pathway is known to regulate cell signaling, metabolism, epithelial-mesenchymal transition (EMT), and cancer stemness. Cybrids with benign mitochondria showed transcriptional activation of negative WNT regulators, including DKK1, SOST, DAB2, and CAV1. Citrate is a key metabolite from the tricarboxylic acid cycle (TCA cycle) and is an intermediate of many other metabolic pathways. Analysis of TNBC cybrids, cell lines, and BC tissues suggest that in TNBC, citrate levels are increased with the activation of CPT1A, the rate-limiting enzyme of FAO. We confirmed the reduction of the beta-catenin protein in TNBC cells after pharmacological and genetic inhibition of CPT1A and mitochondrial citrate transporter (SLC25A1). We further analyzed the gene set enrichment analysis (GSEA) using gene expression data developed from short and long-term CPT1A inhibited TNBC cells and observed that the FAO alters polycomb repressive complex 2 (PRC2) activity. PRC2 is a critical modulator of the H3K27 methylation pattern as well as pathways related to cancer stemness, including WNT/beta-catenin and Hedgehog (SHH) pathways. These results suggest that the mitochondrial FAO transcriptionally inhibits WNT antagonist genes through repressive histone modification and consequently activates the canonical WNT pathway and cancer stemness. Overall, our current observations provide new insights into the regulation WNT pathway by mitochondria-nuclear crosstalk in TNBC. Citation Format: Sukjin Yang, Jun Hyoung Park, Suna Kim, Meron Ghidey, Benny Kaipparettu. Mitochondria-Nuclear Crosstalk Regulates the WNT Pathway in Triple-Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-11-02.

Abstract P6-12-04: An Inducible Lineage Tracing System for the Identification of Tumor-Initiating Cells

Abstract Background: Metastasis and therapeutic resistance are a major clinical challenge, responsible for the vast majority of cancer deaths. A subpopulation of tumor cells known to have intrinsic resistance to standard therapies and contribute to metastasis function as “cancer stem”, or tumor-initiating, cells (TICs). Enriched populations of TICs are typically identified by markers such as aldehyde dehydrogenase activity, the cell surface marker combination of CD44+/CD24-, or fluorescent reporters for signaling pathways that regulate TIC function such as STAT3. Despite their utility, TIC markers and reporters have limitations. Marker expression can be unstable and there is no established method to lineage trace long-lived TICs or to follow them as they undergo cell state changes. Methods: To augment existing TIC reporters, we developed a two component TAM-inducible, Cre recombinase-dependent, STAT3 signaling-specific lentiviral LT system. The first component is a vector that labels cells with active STAT3 signaling (EGFP+), followed by a self-cleaving peptide and TAM-inducible Cre-recombinase (4M67-EGFP-P2A-CreERT2). The second component is a constitutively expressed dual-color switching Cre-dependent reporter vector (EFS-loxPdsRedloxP-mNeptune2). Addition of TAM drives color switching from dsRed to mNeptune2 via CreERT2 recombination in STAT3 signaling cells. Both lentiviral vectors were constructed using Gateway® Cloning. Sum159 cells were transduced with the LT system and reporter activity was validated both in vitro and in vivo using confocal microscopy and flow cytometry. Four LT cell populations (EGFP+/mNeptune2+, EGFP+/dsRed+, EGFP-/mNeptune2+, and EGFP-/dsRed+) were enriched using fluorescence activated cell sorting, then analyzed by single cell RNA sequencing (scRNA seq). Results: Our results confirm the STAT3 LT reporter identifies STAT3 signaling cells (EGFP+), which can be labelled (mNeptune2+) upon addition of TAM. We conducted a TAM dose response curve to identify the optimal TAM dose for complete and partial labeling of STAT3 signaling cells. scRNA seq uncovered gene expression patterns within the TIC compartment and revealed similarities and differences in gene expression between the TIC compartment and the remaining LT reporter populations. Conclusion: These data demonstrate our LT system is a powerful tool that can be applied to uncover the role of TICs in metastasis and therapeutic resistance, as well as identify genetic vulnerabilities to specifically target TICs. Citation Format: Eric P. Souto, Michael T. Lewis. An Inducible Lineage Tracing System for the Identification of Tumor-Initiating Cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-04.

Abstract P4-08-03: Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC

Abstract Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC Background and Premise: Triple-negative breast cancer (TNBC) is an aggressive, metastatic disease with high mortality. The standard-of-care neoadjuvant chemotherapy (NACT) in TNBC shows an initial response but followed by an increase in tumor relapse and distant metastases. Furthermore, metastatic TNBC (mTNBC) patients often develop resistance to NACT and targeted therapies. Immunotherapy with therapeutic antibodies such as anti-programmed death ligand-1 (PD-L1) has shown efficacy in treating a subset of TNBC patients, but currently only 20% of patients qualify for that treatment. Hence, there is an unmet need for developing a precision approach against novel actionable targets along with immunotherapy to treat mTNBC. Eukaryotic initiation factor 4A1 (eIF4A1) is an integral part of the translational machinery for many oncogenic mRNAs including survivin, c-MYC, Rho kinase1, and Cyclins D1 and D3, which all require the helicase activity of eIF4A1 for their translation. Our lab previously established that the eIF4A1 is a vulnerable target in breast cancer stem-like cells (BCSCs) and bulk tumor cells. We further demonstrated a key role for eIF4A1 in cancer stemness and drug resistance. Interestingly, we also observed a reduction in levels of PD-L1 when eIF4A1 is pharmacologically inhibited or genetically ablated. Many downstream effectors of eIF4A1 such as c-MYC, STAT1 and ARF6 (involved in recycling of PD-L1 back to the plasma membrane) are indeed upstream regulators of PD-L1 gene expression and cell surface expression of PD-L1. In this study, we examine human TNBC biospecimens for total eIF4A1 level, levels of STAT1, c-MYC, ARF6 and PD-L1. This will help establish the differential operation of the eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant primary and mTNBC. Approach: Human biospecimens were obtained from CHTN NIH repositories and now we have a collection of more than 100 TNBC tumor samples as well as some lymph node, lung and brain metastases and one sample of male breast primary tumor. These specimens are analyzed for role of eIF4A1 in translation of oncogenic mRNAs that contribute to the gene expression of immune checkpoint PD-L1 and other emerging immune checkpoint markers such as VISTA, TIGIT, and LAG3. To mimic the inclusion criteria for current PD-L1 monoclonal antibody treatment, TNBC tumors are examined for PD-L1 expression via immunohistochemistry after subjecting the samples to deglycosylation for effective detection and also by immunoblotting. Results: The levels of total eIF4A1 from 16 matched, therapy naive TNBC tissue were compared to adjacent normal breast tissue from the same patient via immunoblotting. Total eIF4A1 levels were found to be significantly elevated in TNBC compared to adjacent normal tissue. The downstream effectors of eIF4A1 such as STAT1 and ARF6 were also significantly elevated indicating a robust helicase activity of eIF4A1 in tumor samples. Importantly, PD-L1 levels was found to be elevated. Additionally, the total eIF4A1 level from tumor lysates from TNBC patients that had received a variety of NACT (i.e., patients with demonstrated drug resistance) were compared against normal untreated breast tissue from volunteers who underwent reduction mammoplasty by immunoblotting. The total eIF4A1 was found to be elevated in drug-resistant TNBC specimens compared to control, therapy-naïve TNBC samples. Conclusion: The eIF4A1-STAT1-PD-L1 axis is highly active in TNBC especially in drug-resistant situations. Currently, we are evaluating emerging immune checkpoints such as VISTA, TIGIT, and LAG3. Citation Format: Andrew Boring, Dharmindra Dulal, Dayanidhi Raman. Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-03.

Construction of a TTN Mutation-Based Prognostic Model for Evaluating Immune Microenvironment, Cancer Stemness, and Outcomes of Colorectal Cancer Patients.

Colorectal cancer (CRC) is one of the commonest cancers worldwide. As conventional biomarkers cannot clearly define the heterogeneity of CRC, it is essential to establish novel prognostic models. For the training set, data pertaining to mutations, gene expression profiles, and clinical parameters were obtained from the Cancer Genome Atlas. Consensus clustering analysis was used to identify the CRC immune subtypes. CIBERSORT was used to analyze the immune heterogeneity across different CRC subgroups. Least absolute shrinkage and selection operator regression was used to identify the genes for constructing the immune feature-based prognostic model and to determine their coefficients. A gene prognostic model was then constructed to predict patient outcomes; the model was then externally validated using data from the Gene Expression Omnibus. As a high-frequency somatic mutation, the titin (TTN) mutation has been identified as a risk factor for CRC. Our results demonstrated that TTN mutations have the potential to modulate the tumor microenvironment, converting it into the immunosuppressive type. In this study, we identified the immune subtypes of CRC. Based on the identified subtypes, 25 genes were selected for prognostic model construction; a prediction model was also constructed, and its prediction accuracy was tested using the validation dataset. The potential of the model in predicting immunotherapy responsiveness was then explored. TTN-mutant and TTN-wild-type CRC demonstrated different microenvironment features and prognosis. Our model provides a robust immune-related gene prognostic tool and a series of gene signatures for evaluating the immune features, cancer stemness, and prognosis of CRC.

Distinct roles of ADIPOR1 and ADIPOR2: A pan-cancer analysis.

AdipoR1 and AdipoR2 proteins, encoded by ADIPOR1 and ADIPOR2 genes respectively, are the receptors of adiponectin secrected by adipose tissue. Increasing studies have identified the vital role of adipose tissue in various diseases, including cancers. Hence, there is an urgent need to explore the roles of AdipoR1 and AdipoR2 in cancers. We conducted a comprehensive pan-cancer analysis for the roles of AdipoR1 and AdipoR2 via several public databases, including expression differences, prognostic value, and the correlations with tumor microenvironment, epigenetic modification, and drug sensitivity. Both ADIPOR1 and ADIPOR2 genes are dysregulated in most cancers, but their genomic alteration frequencies are low. In addition, they are also correlated with the prognosis of some cancers. Although they are not strongly correlated with tumor mutation burden (TMB) or microsatellite instability (MSI), ADIPOR1/2 genes display a significant association with cancer stemness, tumor immune microenvironment, immune checkpoint genes (especially CD274 and NRP1), and drug sensitivity. ADIPOR1 and ADIPOR2 play critical roles in diverse cancers, and it is a potential strategy to treat tumors through targeting ADIPOR1 and ADIPOR2.

Establishment of a novel 70K Mac-2 binding protein antibody through screening of fucosylation-related antibodies.

Mac-2 binding protein (Mac-2bp) is a serum glycoprotein that contains seven N-glycans, and Mac-2bp serum levels are increased in patients with several types of cancer and liver disease. Mac-2bp glycosylation isomer has been applied as a clinical biomarker of several diseases, including liver fibrosis. In the present study, we identified fucosylated Mac-2bp in the conditioned medium of cancer cells resistant to anticancer therapies using glycoproteomic analyses. Fucosylation is one of the most important types of glycosylation involved in carcinogenesis and cancer stemness. To establish a next-generation glycan antibody for fucosylated Mac-2bp, we used fucosylation-deficient HEK293T cells to prepare reference Mac-2bp antigens and performed antibody screening. Unexpectedly, the 19-8H mAb obtained with our screen recognized 70K Mac-2bp, which is C-terminus-truncated product, rather than specifically recognizing fucosylated Mac-2bp. We performed immunocytochemistry using our novel 19-8H mAb, which resulted in strong cell surface staining of anticancer drug-resistant cancer cells. Therefore, our novel 19-8H mAb represents a valuable tool for cancer biology research that can help elucidate the biological function of 70K Mac-2bp.

Nuclear Deformability of Breast Cells Analyzed from Patients with Malignant and Benign Breast Diseases.

Breast cancer is a heterogeneous and dynamic disease, in which cancer cells are highly responsive to alterations in the microenvironment. Today, conventional methods of detecting cancer give a rather static image of the condition of the disease, so dynamic properties such as invasiveness and metastasis are difficult to capture. In this study, conventional molecular-level evaluations of the patients with breast adenocarcinoma were combined with in vitro methods on micropatterned poly(methyl methacrylate) (PMMA) biomaterial surfaces that deform cells. A correlation between deformability of the nuclei and cancer stemness, invasiveness, and metastasis was sought. Clinical patient samples were from regions of the breast with different proximities to the tumor. Responses at the single-cell level toward the micropatterned surfaces were studied using CD44/24, epithelial cell adhesion marker (EpCAM), MUC1, and PCK. Results showed that molecular markers and shape descriptors can discriminate the cells from different proximities to the tumor center and from different patients. The cells with the most metastatic and invasive properties showed both the highest deformability and the highest level of metastatic markers. In conclusion, by using a combination of molecular markers together with nuclear deformation, it is possible to improve detection and separation of subpopulations in heterogenous breast cancer specimens at the single-cell level.

Comprehensive Analysis of KCNJ14 Potassium Channel as a Biomarker for Cancer Progression and Development.

Cancer is a global epidemic that has affected millions of lives. Discovering novel cancer targets is widely viewed as a key step in developing more effective therapies for cancer and other fatal illnesses. More recently, potassium (K+) channels have been studied as a potential biological target for the creation of cancer treatments. Potassium Inwardly Rectifying Channel Subfamily J Member 14 (KCNJ14) is one of the cancer genome's least investigated genes. This study conducted a comprehensive examination of the relationships between KCNJ14 gene expression analysis, survival, RNA modification, immunotherapy participation, and cancer stemness using several databases. KCNJ14 was shown to be dysregulated in a variety of cancers, including lung, intestinal, head and neck, oesophageal, and stomach. Additionally, KCNJ14 was shown to be linked to RNA and DNA stemness in 18 and 15 different tumour types, respectively. Moreover, KCNJ14 was discovered to be positively linked with immunological checkpoints and suppressor cells and to have a negative immunophenoscore (IPS). KCNJ14 was linked to tumour mutation burden (TMB), microsatellite instability (MSI), neoantigen (NEO), and programmed death ligand 1 (PD-L1); all four are potential targets for immunotherapies. In addition, a favourable relationship between genomic-instability markers such as heterozygosity (LOH), homologous recombination deficiency (HRD), and mutant-allele tumour heterogeneity (MATH) was demonstrated with KCNJ14. Based on these novel findings, KCNJ14 may be a useful independent prognostic biomarker for a range of cancers.

Molecular subtypes based on cuproptosis-related genes and tumor microenvironment infiltration characteristics in pancreatic adenocarcinoma

BackgroundMultiple molecular subtypes with distinct clinical outcomes in pancreatic adenocarcinoma (PAAD) have been identified in recent years. Cuproptosis is a new form of cell death that likely involved in tumor progression. However, the cuproptosis-related molecular subtypes as well as its mediated tumor microenvironment (TME) cell infiltration characteristics largely remain unclear.MethodsExpression profiles of 10 cuproptosis-related genes (CRGs) and their association with patient survival, TME, cancer stemness and drug resistance were studied in 33 cancer types using the TCGA pan-cancer data. Using 437 PAAD samples from five cohorts (TCGA-PAAD cohort and four GEO cohorts), we explored the molecular subtypes mediated by CRGs, along with the associated TME cell infiltration. Unsupervised methods were utilized to perform cuproptosis subtype clustering. The cuproptosis score was constructed using the COX regression model with least absolute shrinkage and selection operator regression (LASSO) algorithm to quantify the cuproptosis characteristics of a single tumor.ResultsThe expression of 10 CRGs varies in different cancer types with striking inter- and intra- cancer heterogeneity. We integrated the genomic profiling of the CRGs and identified three distinct cuproptosis subtypes, and found that multi-layer CRG alterations were correlated with patient prognosis and TME cell infiltration characteristics. In addition, a cuproptosis score signature was constructed to predict prognosis, and its clinical impacts were characterized in the TCGA-PAAD cohort. The cuproptosis signature was significantly associated with prognosis, tumor subtypes, CD8 T-cell infiltration, response to immune checkpoint inhibitors (ICIs) and chemotherapeutic drug sensitivity. Furthermore, the expression patterns of CRGs in pancreatic cancer cells and normal controls were validated, which was almost consistent with the results from the public database. The expression level and prognostic predictive capability of DLAT were verified in 97 PAAD patients from our patient cohort.ConclusionsThese findings may help understand the roles of CRGs in PAAD and the molecular characterization of cuproptosis subtypes. In addition, the cuproptosis score could serve as a promising biomarker for predicting prognosis and response to immunotherapy in PAAD patients.

The stromal tumor-infiltrating lymphocytes, cancer stemness, epithelial-mesenchymal transition, and B7-H4 expression in ovarian serous carcinoma

BackgroundB7-H4 is expressed in various types of cancers and its expression inversely correlates with the degree of tumor-infiltrating lymphocytes (TILs). Studies have shown the relationship between B7-H4, cancer stem cell (CSC) properties, and epithelial-mesenchymal transition (EMT) in various cancers. However, very few studies have investigated the relationship between B7-H4, TILs, cancer stemness, and EMT in epithelial ovarian cancer (EOC). The present study aimed to elucidate whether B7-H4 is involved in immune evasion and examine whether B7-H4 is associated with cancer stemness or EMT in ovarian serous carcinoma, the most common type of EOC. The clinical significance of B7-H4 was also investigated to evaluate its potential as a therapeutic target.MethodsA total of 145 patients included in this study. The degree of stromal TILs was evaluated using hematoxylin and eosin (H&E)-stained slides. Immunohistochemical analysis of B7-H4, CSC-related biomarkers (CD24, CD44s, CD133, and ALDH1), and EMT-related biomarkers (E-cadherin, N-cadherin, and vimentin) was performed using tissue microarray. qRT-PCR for VTCN1, CD24, CD44, PROM1, ALDH1, CDH1, CDH2, and VIM genes was performed on 38 frozen tissue samples. The mRNA expression levels were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) online analysis tool.ResultsB7-H4 protein expression positively correlated with the degree of stromal TILs. CD24, CD44s, and CD133 expression showed a positive correlation with B7-H4 expression at both the protein and mRNA levels, but ALDH1 correlated only at the protein level. E-cadherin expression was positively correlated with B7-H4 expression at both the protein and mRNA levels. N-cadherin and vimentin expression was inversely related to B7-H4 expression only at the mRNA level. B7-H4 positive patients were associated with higher tumor grade and lower overall survival rate than B7-H4 negative patients, especially in ovarian serous carcinoma with low stromal TILs.ConclusionsThe present study demonstrates that B7-H4 may not be involved in the immune evasion mechanism, but is involved in cancer stemness and mesenchymal-epithelial transition. In addition, B7-H4 may be a therapeutic target for the treatment of ovarian serous carcinoma, especially with low stromal TILs.

Abstract C054: Characterizing differences in the chromatin accessibility landscape by donor ancestry in human triple-negative breast cancer cell lines

Abstract African American (AA) women are at increased risk of developing and dying from Triple-Negative Breast Cancer (TNBC), an aggressive breast cancer subtype, compared to European American (EA) women in the United States. In addition to social determinants, further investigation into biologic factors that contribute to these disparities is needed to fully understand this multi-factorial problem. In particular, the epigenetics of racial/population diversity and its influence on breast cancer incidence and outcomes remain underexplored. Using ATAC-sequencing, we characterized differences in chromatin accessibility between EA-derived versus AA-derived breast cancer cell lines across a range of breast cancer subtypes. Interestingly, our principal component analysis of the top 50,000 peaks with the most variance revealed separation of chromatin profiles by genetic ancestry specifically in TNBC cell lines (N=10). Digital footprinting analysis of differentially open chromatin regions using TOBIAS revealed significant differences in transcription factor (TF) binding by ancestry (61 TFs with a differential binding score > 0.25 or < -0.25 and FDR < 0.01). AA TNBC cell lines exhibited increased binding of transcription factors associated with epithelial-to-mesenchymal transition (ZEB1, SNAI1, GRHL2), cancer stemness and chemotherapeutic resistance (TFAP2C, NRF1), and others such as KAISO, whose aberrant expression has been previously linked to a distinct biology and poor outcomes in AA breast and prostate cancer patients. Pathway analysis of genes located within promoters of differentially open chromatin regions reveals enrichment of the following KEGG pathways in AA TNBC cells: inflammatory mediator regulation of TRP channels (p-adj = 0.009), Hippo signaling (p-adj = 0.006), Wnt signaling (p-adj = 0.031), and Rap-1 signaling (p-adj = 0.046). Together, these data reveal a differential chromatin landscape associated with the aberrant activity of critical TFs and downstream gene expression changes that may contribute to worsened TNBC biology in women of African ancestry. Additionally, as many of these cell lines are used routinely in biomedical research, these findings also indicate that the ancestral origin of patient derived cell lines matters and may contribute to biologic variation in experimental data, suggesting that inclusion of diversely sourced cell lines should be considered in experimental design. Citation Format: Alexandra R. Harris, Vishal Koparde, Gatikrushna Panigrahi, Maeve Bailey-Whyte, Tiffany Dorsey, Stefan Ambs. Characterizing differences in the chromatin accessibility landscape by donor ancestry in human triple-negative breast cancer cell lines [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C054.

ZIC5 promotes aggressiveness and cancer stemness in cervical squamous cell carcinoma.

Cervical cancer is one of the major malignancies causing morbidity and mortality in women in developing countries. ZIC5 has been found to be associated with a variety of cancers, yet the expression and molecular function of ZIC5 in cervical squamous cell carcinoma (CESC) is unknown. We examined the expression of ZIC5 in tumors and normal tissues of CESC patients using immunohistochemistry, immunoblotting and fluorescent quantitative PCR, and used statistical methods to explore its relationship with clinical manifestations. Next, we constructed ZIC5 knockdown and overexpression CESC cell lines to observe the effect of ZIC5 on the proliferation and metastasis of CESC cells. Finally, we applied a nude mouse xenograft tumor model to observe the effect of ZIC5 on tumorigenesis in vivo. Our results showed that the expression of ZIC5 was higher in cancer tissues than in normal tissues. Prognostic analysis showed that ZIC5 expression level was an independent prognostic factor in CESC patients, and the results of Transwell, CCK-8 and wound healing assays confirmed that overexpression of ZIC5 could promote the proliferation and migration of CESC cells. A nude mouse xenograft tumor model showed that knockdown of ZIC5 inhibited tumor growth in vivo. Database, immunoblotting assay and in vitro sphere-forming assay confirmed that ZIC5 could promote the stemness of CESC cells. ZIC5 is a factor that indicates a poor prognosis of CESC patients and promotes stemness in CESC cells. ZIC5 may be a potential biomarker and therapeutic target for CESC patients.

Podoplanin promotes cell proliferation, survival, and migration of canine non-tonsillar squamous cell carcinoma.

Podoplanin (PDPN) is a prognostic factor and is involved in several mechanisms of tumor progression in human squamous cell carcinoma (SCC). Canine non-tonsillar SCC (NTSCC) is a common oral tumor in dogs and has a highly invasive characteristic. In this study, we investigated the function of PDPN in canine NTSCC. In canine NTSCC clinical samples, PDPN overexpression was observed in 80% of dogs with NTSCC, and PDPN expression was related to ki67 expression. In PDPN knocked-out canine NTSCC cells, cell proliferation, cancer stemness, and migration were suppressed. As the mechanism of PDPN-mediated cell proliferation, PDPN knocked-out induced apoptosis and G2/M cell cycle arrest in canine NTSCC cells. These findings suggest that PDPN promotes tumor malignancies and may be a novel biomarker and therapeutic target for canine NTSCC.

Mebendazole prevents distant organ metastases in part by decreasing ITGβ4 expression and cancer stemness

Breast cancer is the most diagnosed cancer among women. Approximately 15–20% of all breast cancers are highly invasive triple-negative breast cancer (TNBC) and lack estrogen, progesterone, and ERBB2 receptors. TNBC is challenging to treat due to its aggressive nature with far fewer targeted therapies than other breast cancer subtypes. Current treatments for patients with TNBC consist of cytotoxic chemotherapies, surgery, radiation, and in some instances PARP inhibitors and immunotherapy. To advance current therapeutics, we repurposed mebendazole (MBZ), an orally available FDA-approved anthelmintic that has shown preclinical efficacy for cancers. MBZ has low toxicity in humans and efficacy in multiple cancer models including breast cancer, glioblastoma multiforme, medulloblastoma, colon cancer, pancreatic and thyroid cancer. MBZ was well-tolerated in a phase I clinical trial of adults recently diagnosed with glioma. We determined that the half-maximal inhibitory concentration (IC50) of MBZ in four breast cancer cell lines is well within the range reported for other types of cancer. MBZ reduced TNBC cell proliferation, induced apoptosis, and caused G2/M cell cycle arrest. MBZ reduced the size of primary tumors and prevented lung and liver metastases. In addition, we uncovered a novel mechanism of action for MBZ. We found that MBZ reduces integrin β4 (ITGβ4) expression and cancer stem cell properties. ITGβ4 has previously been implicated in promoting “cancer stemness,” which may contribute to the efficacy of MBZ. Collectively, our results contribute to a growing body of evidence suggesting that MBZ should be considered as a therapeutic to slow tumor progression and prevent metastasis.

Succinylation and redox status in cancer cells.

Succinylation is a post-translational modification (PTM) event that associates metabolic reprogramming with various pathological disorders including cancers via transferring a succinyl group to a residue of the target protein in an enzymic or non-enzymic manner. With our incremental knowledge on the roles of PTM played in tumor initiation and progression, relatively little has been focused on succinylation and its clinical implications. By delineating the associations of succinylation with cancer hallmarks, we identify the, in general, promotive roles of succinylation in manifesting cancer hallmarks, and conceptualize two working modes of succinylation in driving oncogenic signaling, i.e., via altering the structure and charge of target proteins towards enhanced stability and activity. We also characterize succinylation as a reflection of cellular redox homeostatic status and metabolic state, and bring forth the possible use of hyper-succinylated genome for early cancer diagnosis or disease progression indication. In addition, we propose redox modulation tools such as cold atmospheric plasma as a promising intervention approach against tumor cells and cancer stemness via targeting the redox homeostatic environment cells established under a pathological condition such as hypoxia. Taken together, we emphasize the central role of succinylation in bridging the gap between cellular metabolism and redox status, and its clinical relevance as a mark for cancer diagnosis as well as a target in onco-therapeutics.

Potential roles of Cornichon Family AMPA Receptor Auxiliary Protein 4 (CNIH4) in head and neck squamous cell carcinoma.

It is of great clinical significance to discover novel biomarkers for neck squamous cell carcinoma (HNSCC) treatments. We discovered a potential cancer-related gene, Cornichon Family AMPA Receptor Auxiliary Protein 4 (CNIH4), that can be a biomarker for HNSCC. We access multiple open databases and analyzed bulk mRNA-sequencing, protein staining, and single-cell mRNA-sequencing data of HNSCC and investigated the diagnostic and prognostic value of CNIH4 in HNSCC. The potential association between CNIH4 and the immune microenvironment of HNSCC was also estimated. CNIH4 was significantly up-regulated in HNSCC compared with non-cancer tissues. Higher CNIH4 resulted in a shorter overall survival time and we further constructed a survival nomogram for clinical applications. 2012 and 421 genes were identified as positive and negative differentially expressed genes of CNIH4 in HNSCC respectively. These genes were mostly mapped to "Cell cycle", "DNA replicate", "Cytokine-cytokine receptor interaction" KEGG pathways. Functions associated with CNIH4 were "stemness", "cell cycle", and "DNA repair" in single-cell data. CNIH4 potentially affected immune cell infiltration levels and cancer immune therapy. CNIH4 is a potential diagnostic and prognostic biomarker associated with cancer stemness and immunity in HNSCC.