Tumor Adjacent Normal Tissues Research Articles

RIG-I Promotes Tumorigenesis and Confers Radioresistance of Esophageal Squamous Cell Carcinoma by Regulating DUSP6.

We investigated the expression and biological function of retinoic acid inducible gene I (RIG-I) in esophageal squamous cell carcinoma (ESCC). Materials and methods: An immunohistochemical analysis was performed on 86 pairs of tumor tissue and adjacent normal tissue samples of patients with ESCC. We generated RIG-I-overexpressing ESCC cell lines KYSE70 and KYSE450, and RIG-I- knockdown cell lines KYSE150 and KYSE510. Cell viability, migration and invasion, radioresistance, DNA damage, and cell cycle were evaluated using CCK-8, wound-healing and transwell assay, colony formation, immunofluorescence, and flow cytometry and Western blotting, respectively. RNA sequencing was performed to determine the differential gene expression between controls and RIG-I knockdown. Tumor growth and radioresistance were assessed in nude mice using xenograft models. RIG-I expression was higher in ESCC tissues compared with that in matched non-tumor tissues. RIG-I overexpressing cells had a higher proliferation rate than RIG-I knockdown cells. Moreover, the knockdown of RIG-I slowed migration and invasion rates, whereas the overexpression of RIG-I accelerated migration and invasion rates. RIG-I overexpression induced radioresistance and G2/M phase arrest and reduced DNA damage after exposure to ionizing radiations compared with controls; however, it silenced the RIG-I enhanced radiosensitivity and DNA damage, and reduced the G2/M phase arrest. RNA sequencing revealed that the downstream genes DUSP6 and RIG-I had the same biological function; silencing DUSP6 can reduce the radioresistance caused by the overexpression of RIG-I. RIG-I knockdown depleted tumor growth in vivo, and radiation exposure effectively delayed the growth of xenograft tumors compared with the control group. RIG-I enhances the progression and radioresistance of ESCC; therefore, it may be a new potential target for ESCC-targeted therapy.

Abstract P1-05-18: Orphan non-coding RNAs for early detection of breast cancer with liquid biopsy

Abstract Background: Early detection of breast cancer is crucial for optimal patient outcomes but cannot always be accomplished based on symptoms or screening mammography. Biomarker-based screening could aid early detection of breast cancer by improving sensitivity and specificity. Exai Bio has developed a novel liquid biopsy technology that detects and analyzes small non-coding RNAs that are cancer specific, termed orphan non-coding RNAs (oncRNAs). Previous work in patients with diagnosed breast cancer demonstrated that changes in oncRNAs in serum reflected treatment response and event-free survival. In this study, we developed an assay that measures oncRNAs in serum to detect breast cancer across the range of tumor stages and sizes. Methods: Previously, a library of ~260,000 oncRNAs from 32 different cancers was compiled based on smRNA sequences found in tumor tissues and largely absent in tumor-adjacent normal tissues from The Cancer Genome Atlas (TCGA). To refine this library for applications in serum, we sequenced smRNA in 31 control serum samples. These smRNA sequences were filtered from the larger library, reducing its size to 250,332 oncRNAs. The diagnostic performance of these oncRNAs was then assessed in an independent cohort of archived serum samples from 96 female patients with clinically diagnosed, untreated breast cancer and 95 age- and sex-matched individuals with no known history of cancer. We sequenced smRNAs at an average depth of 17.7 million 50-bp single-end reads per sample. Of the 250,332 oncRNAs in our library, 171,981 (68.7%) were detected in our independent study cohort. An ensemble of logistic regression models was trained with 5-fold cross-validation, using only those oncRNAs yielding an odds ratio >1 and observed in >6% of samples within each training set. Results: The cohort of 96 breast cancer patients and 95 matched controls had mean ages of 59.4 and 56.3 years, respectively. Area under the receiver operating characteristic curve (AUC) for detecting breast cancer was 0.94 (95% CI, 0.85–0.96). Sensitivities for detecting breast cancer at 95% specificity ranged from 0.75 to 0.87 among the four breast cancer stages, including a sensitivity of 0.81 for tumor stage I (Table 1); and from 0.67 to 0.87 among the four main TNM T categories (Table 2). Sensitivities at 95% specificity were relatively high for small tumors, at 0.75 (95% CI, 0.40–0.97) for T1b (>5mm to ≤10mm; n = 9) and 0.80 (0.68–0.94) for T1c (>10mm to ≤20mm; n = 37). Conclusions We have demonstrated the potential value of an oncRNA-based liquid biopsy assay by showing that oncRNAs can be used to detect breast cancer in serum samples with high sensitivity, and that detection requires fewer reads than are needed with other platforms. Moreover, we found that this oncRNA-based assay performed well in detecting early-stage breast cancer and small tumors. This suggests that an oncRNA-based liquid biopsy assay may be beneficial for early detection of breast cancer. Table 1. Model sensitivity by tumor stage. For the indicated numbers of cases (N), sensitivity and Pearson-Clopper 95% confidence intervals are reported for tumor detection by the oncRNA-based model at 95% specificity by tumor stage, as defined by the AJCC 7th Edition breast cancer staging system. Table 2. Model sensitivity by tumor size. For the indicated numbers of cases (N), sensitivity and Pearson-Clopper 95% confidence intervals are reported for tumor detection by the oncRNA-based model at 95% specificity by TNM T category, as defined by the AJCC 7th Edition breast cancer staging system. Citation Format: Taylor B. Cavazos, Jeffrey Wang, Oluwadamilare I. Afolabi, Alice Huang, Dung Ngoc Lam, Seda Kilinc, Jieyang Wang, Lisa Fish, Xuan Zhao, Andy Pohl, Helen Li, Kimberly H. Chau, Patrick A. Arensdorf, Fereydoun Hormozdiari, Hani Goodarzi, Babak Alipanahi. Orphan non-coding RNAs for early detection of breast cancer with liquid biopsy [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 P1-05-18.

Abstract P2-20-06: Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK and T Cell–Mediated Cytotoxicity against Breast Cancer Cells

Abstract One of the main factors contributing to breast cancer (BC) initiation and metastasis is immune suppression by tumor cells. Immune checkpoint blockade has recently been shown to overcome tumor-induced immune suppression, but a significant proportion of patients do not respond, implying that more effective cancer immunotherapies are required. B7-H3 belongs to B7 family of immune checkpoint proteins that is overexpressed in various human malignancies. Here, we hypothesize that blocking B7-H3 using monoclonal antibodies (mAbs) enhances immune cell proliferation and function. To test our hypothesis, B7-H3 expression was determined using RNA-seq data from primary and metastatic breast tumors, and adjacent normal tissues, from the TCGA and METABRIC databases. To assess B7-H3 protein expression in BC, we performed immunohistochemistry (IHC) on frozen primary tumor tissues (n=50) and adjacent normal tissues (n=23) from TNBC patients. In addition, to investigate the immunomodulatory effect of B7-H3 in BC, we performed IHC for T-cell markers in subsets of patient tissues with high and low levels of B7-H3 expression. Next, we assessed B7-H3 expression in over 13 BC cell lines, including TNBC and ER+, PR+, and HER2+ cell lines, as well as TNBC PDX-derived cells. Moreover, to determine the effect of B7-H3 knockdown on NK- and T-cell activity, we co-cultured control and B7H3–KD BC cells in the presence and absence of NK and T cells and measured the induction of apoptosis in BC cells through IncuCyte live-cell imaging system. The effect of a novel B7H3-blocking mAb (clone T-1A5, isotype IgG1) on NK-cell and T-cell-mediated cytotoxicity in BC cell lines was examined using live-cell imaging. In the TCGA and METABRIC databases, B7-H3 was found to be significantly overexpressed (P< 0.0001) in tumor tissues than in adjacent normal tissues of BC patients. A survival analysis by the log-rank test indicated that patients with B7-H3high tumors had significantly lower progression-free (P=0.01) and relapse-free (P=0.0026) survival than patients with B7-H3low tumors. Moreover, B7-H3 is significantly upregulated in all the BC subtypes including basal, luminal A, luminal B and Her2-enriched with highest expression in basal type BC. Relative mRNA quantification and flow cytometry analysis demonstrated strong B7-H3 expression in most of the BC cell lines including TNBC and ER+, PR+, and HER2+ cell lines, as well as TNBC PDX-derived cells. Furthermore, IHC analysis revealed that compared to the matched normal tissue, B7-H3 expression was substantially higher in tumor tissue (N=16, P< 0.001). Also, patients with high B7-H3 expression had significantly lower numbers of CD3+, CD4+, and CD8+ T-cell, compared to patients with low B7-H3 expression (P< 0.001), indicating immunosuppressive role of B7-H3. Furthermore, NK cell and T cell mediated killing was significantly higher in B7H3-KD BC cell lines compared to control cells. We observed a significant increase in the killing of BC cells by NK cells and T cells in the presence of anti-B7H3 mAb T-1A5 in a concentration dependent manner (P < 0.001), suggesting that anti-B7H3 antibodies suppress the immunomodulatory function of B7-H3 and enhance NK and T cell–mediated ADCC in BC. To determine the antibody-dependent cell-mediated cytotoxicity, we developed a human-mouse chimera of T-1A5 (chT-1A5) and tested in combination with NK cells. Interestingly, we found a concentration and time dependent increase in ADCC in BC cells in the presence of chT-1A5 antibody. Our data suggests that B7-H3 is overexpressed in primary BC and inhibits immune-cell infiltration. Moreover, blocking the immunomodulatory functions of B7-H3 using anti-B7H3 antibody T-1A5 enhances NK and T cell–mediated killing of BC cells. Citation Format: Vivek Anand, Anudishi Tyagi, Venkata Lokesh Battula. Anti-B7-H3 Antibody (T-1A5) Blocks Immunomodulatory Function of B7-H3 and Enhances NK and T Cell–Mediated Cytotoxicity against Breast Cancer 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 P2-20-06.

TPM2 attenuates progression of prostate cancer by blocking PDLIM7-mediated nuclear translocation of YAP1

BackgroundProstate cancer (PCa) is a common malignant tumor of the genitourinary system. Clinical intervention in advanced PCa remains challenging. Tropomyosins 2 (TPM2) are actin-binding proteins and have been found as a biomarker candidate for certain cancers. However, no studies have explored the role of TPM2 in PCa and its regulatory mechanism.MethodsTPM2 expression was assessed in Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) PCa patient dataset. The effect of TPM2 on PCa progression was assessed in vitro and in vivo by quantifying proliferation, migration, invasion and tumor growth assays, and the mechanism of TPM2 in PCa progression was gradually revealed by Western blotting, immunoprecipitation, and immunofluorescence staining arrays.ResultsTPM2 was found to be severely downregulated in tumor tissues of PCa patients compared with tumor-adjacent normal tissues. In vitro experiments revealed that TPM2 overexpression inhibited PCa cell proliferation, invasion and androgen-independent proliferation. Moreover, TPM2 overexpression inhibited the growth of subcutaneous xenograft tumors in vivo. Mechanistically, this effect was noted to be dependent on PDZ-binding motif of TPM2. TPM2 competed with YAP1 for binding to PDLIM7 through the PDZ-binding motif. The binding of TPM2 to PDLIM7 subsequently inhibited the nuclear transport function of PDLIM7 for YAP1. YAP1 sequestered in the cytoplasm phosphorylated at S127, resulting in its inactivation or degradation which in turn inhibited the expression of YAP1 downstream target genes.ConclusionsThis study investigated the role of TPM2, PDLIM7, and YAP1 in PCa progression and castration resistance. TPM2 attenuates progression of PCa by blocking PDLIM7-mediated nuclear translocation of YAP1. Accordingly, targeting the expression or functional modulation of TPM2, PDLIM7, or YAP1 has the potential to be an effective therapeutic approach to reduce PCa proliferation and prevent the progression of castration-resistant prostate cancer (CRPC).

HNRNPA2B1 as a potential therapeutic target for thymic epithelial tumor recurrence: An integrative network analysis

Thymic epithelial tumors (TETs) are rare malignant tumors, and the molecular mechanisms of both primary and recurrent TETs are poorly understood. Here we established comprehensive proteomic signatures of 15 tumors (5 recurrent and 10 non-recurrent) and 15 pair wised tumor adjacent normal tissues. We then proposed an integrative network approach for studying the proteomics data by constructing protein-protein interaction networks based on differentially expressed proteins and a machine learning-based score, followed by network modular analysis, functional enrichment annotation and shortest path inference analysis. Network modular analysis revealed that primary and recurrent TETs shared certain common molecular mechanisms, including a spliceosome module consisting of RNA splicing and RNA processing, but the recurrent TET was specifically related to the ribosome pathway. Applying the shortest path inference to the collected seed gene module identified that the ribonucleoprotein hnRNPA2B1 probably serves as a potential target for recurrent TET therapy. The drug repositioning combined molecular dynamics simulations suggested that the compound ergotamine could potentially act as a repurposing drug to treat recurrent TETs by targeting hnRNPA2B1. Our study demonstrates the value of integrative network analysis to understand proteotype robustness and its relationships with genotype, and provides hits for further research on cancer therapeutics.

Tumor necrosis factor-α-inducible protein 8-like protein 3 (TIPE3): a novel prognostic factor in colorectal cancer

BackgroundTo explore the correlation of tumor necrosis factor-α-induced protein 8-like protein 3 (TIPE3) expressions in colorectal cancer (CRC) with tumor-immune infiltration and patient prognosis.MethodsFormalin-fixed paraffin-embedded tumor samples from CRC patients (n = 110) were used in this study. Immunohistochemistry staining of TIPE3 and three prognostic immune biomarkers (CD8, CD20, and CD66b) was conducted in the tumor tissues and adjacent normal tissues. A Cox regression analysis of univariate and multivariate variables was performed to assess the correlation between TIPE3 and patient prognosis.ResultWe found that TIPE3 was mainly expressed in the cytoplasm, with a small amount in the nucleus. The expression of TIPE3 in tumor tissues is significantly higher than in adjacent normal tissues, and it is significantly correlated with the survival rate of patients in tumor tissues (p = 0.0038) and adjacent normal tissues (p<0.0001). Patients with a high TIPE3 expression had a lower survival rate, while patients with a low TIPE3 expression had a higher survival rate. Univariate regression analysis showed that the TIPE3 expression in tumor tissues (p = 0.007), the TIPE3 expression in adjacent normal tissues (p<0.001), the number of CD8+ T cells in tumor tissues (p = 0.020), the number of CD20+ B cells in tumor tissues (p = 0.023), the number of CD20+ B cells in adjacent normal tissues (p = 0.023), the number of CD66b+ neutrophils in tumor tissues (p = 0.005), the number of CD66b+ neutrophils in adjacent normal tissues (p<0.001), lymphatic metastasis (p = 0.010), TNM stage (p = 0.013), and tumor grade (p = 0.027) were significantly correlated with overall survival (OS). These prognostic factors were then subjected to multivariate regression analysis, and the results showed that the expression of TIPE3, the number of CD8+ T cells, and the number of CD66b+ neutrophils were prognostic factors affecting the OS rate of CRC patients.ConclusionWe found that the TIPE3 protein is upregulated in CRC cancer tissues and is correlated with survival rate.

Vascular endothelial growth factor A is a potential prognostic biomarker and correlates with immune cell infiltration in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths among cancer patients. Vascular endothelial growth factor A (VEGFA) is involved in regulating biological processes, such as angiogenesis and vascular permeability, and is very closely related to the pathogenesis of various tumours, especially vascular-rich, solid tumours. Clinical data of patients with HCC and other tumours were analysed through public databases, such as the TCGA database, Gene Expression Omnibus database, Human Protein Atlas database, STRING, Tumour Immune Estimation Resource and Kaplan-Meier Plotter. The tumour tissues and adjacent normal tissues of patients with HCC from Hunan Provincial People's Hospital were collected to verify the expression of VEGFA by immunohistochemistry, immunofluorescence, Western blotting and qPCR. VEGFA expression is elevated in multiple tumour types and correlates with the prognosis of tumour patients. VEGFA is involved in regulating the tumour microenvironment and immune cell function in tumour development. Inhibition of VEGFA reduces proliferation, invasion, and migration and promotes apoptosis in HCC cells. VEGFA is a potential predictive biomarker for the diagnosis and prognosis of HCC.

Prognostic and predictive value of a lncRNA signature in patients with stage II colon cancer

The current staging method is inadequate to identify high-risk recurrence patients with stage II colon cancer (CC). Using a systematic and comprehensive-biomarker discovery and validation method, we aimed to construct a lncRNA-based signature to improve the prognostic prediction of stage II CC. We identified 1,377 differently expressed lncRNAs by analyzing 16 paired stage II CC tumor tissue and adjacent normal mucosal tissue from the TCGA dataset. Subsequently, using a univariable and step multivariable Cox regression model, we trained an 11-lncRNA signature in the training cohort (n = 141), which could divide patients into high-risk and low-risk groups (AUC at 3 years = 0.801, 95% CI: 0.724–0.877; AUC at 5 years = 0.801, 95% CI: 0.718–0.885). Significantly, patients in the high-risk group had poorer recurrence-free survival (RFS) compared with the low-risk group (log-rank test, P < 0.001 in the training cohort). This lncRNA-based signature was further confirmed in the validation cohort (P < 0.001). Multivariate Cox regression and stratified survival analyses showed that the prognostic value of this signature was independent of other clinicopathological risk factors (CEA, T stage, and chemotherapy). Time-dependent receiver operating characteristic (ROC) analysis demonstrated that this signature had better prognostic ability than any other clinical risk factors or single lncRNAs (all P < 0.05). A nomogram was constructed for clinical use, which integrated both the lncRNA-based signature and clinical risk factors (CEA and T stage) and performed well in the calibration plots. Altogether, our lncRNA-based signature was an independent prognostic factor and possessed a stronger predictive power compared with the currently used clinicopathological risk factors when predicting the recurrence of patients with stage II CC. Collectively, this lncRNA-based signature might facilitate individualized treatment decisions and postoperative counseling, ultimately contributing to improved survival.

Analysis of the expression and prognostic significance of DDK complex in Hepatocarcinoma

BackgroundHepatocellular carcinoma (HCC) remains one of the most common and lethal malignancies worldwide. Although DBF4-dependent kinase (DDK) complex composed of CDC7 kinase and its regulatory subunit DBF4 has been shown to be overexpressed in primary tumors and promotes tumor development, while its role and prognostic value in HCC remain largely unknown. In the present study, the expression of DBF4 and CDC7 and their relationship with clinical characteristics were comprehensively analyzed.MethodsThe mRNA expression profiles of HCC and the corresponding clinical data of HCC patients were downloaded from TCGA and GEO databases, respectively. The differences in DBF4 and CDC7 expression in tumor tissues and adjacent normal tissues were analyzed. HCC-derived tissue microarray (TMA) was used to evaluate and score the expression of CDC7 by immunohistochemistry (IHC) staining. The Kaplan–Meier method and the Cox regression method were used to analyze the relationship between overall survival and clinical characteristics of the patients. Gene set enrichment analysis (GSEA) was used to analyze the pathway enrichment of DBF4 and CDC7.ResultsDBF4 and CDC7 had similar expression patterns in HCC patients. Detailly, compared with adjacent tissues, both mRNA and protein of DBF4 and CDC7 were significantly higher in HCC, and their expression was positively correlated with AJCC_T stage, clinical stage and G stage (grade) of liver cancer patients, and higher DBF4 or CDC7 expression predicted a worse prognosis in HCC patients with shorter overall survival (OS), recurrence-free survival (RFS), disease-specific survival (DSS) and progress-free survival (PFS). Cox regression analysis suggested that both DBF4 and CDC7 were independent risk factors for the prognosis of HCC patients in TCGA dataset. GSEA suggested that both DBF4 and CDC7 were positively correlated with cell cycle and DNA replication. Finally, the prognostic value of CDC7 was furtherly confirmed by TMA-based IHC staining results.ConclusionsOur study showed that DDK complex was significantly increased in HCC. Both DBF4 and CDC7 may be potential diagnostic and prognostic markers for HCC, and high expression of DDK members predicts a worse prognosis in patients with HCC, which may be associated with high tumor cell proliferation rate.

Identifying Mitochondrial Transcription Factor A As a Potential Biomarker for the Carcinogenesis and Prognosis of Prostate Cancer.

Aims: Mitochondrial functional transformation contributes to the carcinogenesis of the prostate by meeting the metabolic needs of cancer cells. Mitochondrial transcription factor A (TFAM) is a pivotal regulator that maintains homeostasis of mitochondrial function. However, its role in prostate carcinogenesis has not been well elucidated. Materials and Methods: In the present study, we analyzed the expression of TFAM in normal prostate tissue and prostate cancer using public databases; a prostate-tissue chip was used to verify the results. The expression of TFAM in normal cells and in prostate cancer cells was determined by western blotting analysis. We knocked down TFAM in the prostate cancer cell line PC3 using a specific shRNA to explore the potential effects of TFAM in prostatic carcinogenesis. Results: We observed higher expression levels of TFAM in prostate cancer tissue than in normal prostate tissue and tumor adjacent normal tissues. A receiver operating characteristic curve was drawn that demonstrated the diagnostic efficacy of using TFAM expression for prostate cancer prognoses. Elevated levels of TFAM may indicate poorer overall survival in prostate cancer patients. Western blotting assays also showed that relative to the normal prostatic epithelial cell line RWPE-1, prostate cancer cell lines PC3 and DU145 expressed more TFAM protein. Furthermore, knockdown of TFAM inhibited the colony-formation capability of PC3 cells. Conclusion: Collectively, these results suggest that TFAM promotes carcinogenesis of the prostate, and may constitute a marker to be used in the diagnosis and prognosis of prostate cancer.

Dishevelled Segment Polarity Protein 3: A Novel Prognosis-related Marker in Pan-driver-gene-negative Lung Adenocarcinoma.

Purpose: In this study, we aimed to investigate the potential prognostic molecular marker in patients with "pan-driver-gene-negative" lung adenocarcinoma (PDGN-LUAD). LUAD patients who were negative for mutations in EGFR, KRAS, BRAF, HER2, MET, ALK, RET and ROS1 were identified as PDGN-LUAD. Methods: In the screening phase, we profiled the mRNA expression levels in 52 paired PDGN-LUAD tumor tissues and adjacent normal tissues using a genome-wide microarray, and the results revealed that the expression level of dishevelled segment polarity protein 3 (DVL3) was higher in PDGN-LUAD tumor tissues than in normal lung tissues. Then, we enrolled 626 PDGN-LUAD patients from three independent hospital centers and divided them into a training cohort, an internal validation cohort and two external validation cohorts. In the training cohort, tissue microarrays (TMAs) were used to confirm the protein expression level of DVL3. Statistical methods were applied to explore the prognostic role of DVL3. Results: The results indicated that the level of DVL3 could be used to classify patients with PDGN-LUAD in the training cohort into a high-risk group (high expression level of DVL3) and a low-risk group (low expression level of DVL3). In the training cohort, high-risk patients had shorter overall survival (OS) times (hazard ratio [HR] 2.27; 95% CI, 1.57-2.97; p<0.001) than low-risk patients. In the validation phase, the performance of DVL3 as a prognostic marker was successfully validated in the internal and external cohorts. Conclusions: In conclusion, DVL3 is an important prognostic indicator for PDGN-LUAD and may provide new insights into the treatment of PDGN-LUAD.

Study of LncRNA BANCR Expression in Tumor Tissues and Adjacent Normal Tissues in Gastric Cancer Patients.

Long non-coding RNAs (lncRNAs) have emerged as crucial regulators in various biological processes, including cancer development and progression. This study aimed to investigate the expression differences of the BRAF-activated non-coding RNA (BANCR) gene in GC tissues compared to adjacent normal tissues. The potential diagnostic significance of BANCR in GC was explored, with the aim of improving diagnostic and therapeutic approaches for this global health burden. Tissue samples from 100 gastric cancer (GC) patients were collected, and BANCR expression was analyzed using quantitative real-time PCR. Correlations between BANCR expression and clinicopathological features were assessed, and its biomarker potential was evaluated. In individuals diagnosed with GC, the expression of BANCR was notably elevated in tumor tissues compared to adjacent normal tissues (P < 0.0001). However, the analysis of gene expression data did not demonstrate any statistically significant correlation between elevated BANCR expression and clinicopathological features. According to the ROC analysis, BANCR demonstrated an AUC of 0.6733 (P < 0.0001), with a sensitivity of 73% and a specificity of 45%. However, further evaluation is required to determine its potential as a biomarker (CI 95% = 0.5992 to 0.7473). The observed upregulation of BANCR in GC tissues implies its potential involvement as an oncogenic lncRNA in GC patients. Furthermore, BANCR may serve as a promising biomarker for identification and treatment of GC.

Screening of VHL mutation in different types of kidney cancer in patients of West Bengal (India)

Introduction: The purpose of this study was to screen the patients of West Bengal (India) with different types of renal cancer to see the presence of von Hippel–Lindau (VHL) gene mutations. Materials and Methods: This prospective study included 25 patients with renal cancer operated on between December 2019 and January 2022. Tumor tissue and adjacent normal tissue samples were taken and subjected to genomic DNA isolation, polymerase chain reaction, DNA sequencing, and identification of polymorphism in renal cell carcinoma (RCC) patients by comparing with a database using “ENSEMBL” (genome browser) followed by the role of identified variants in disease-causing using different software. Results: After analysis, we identified six exonic and one intronic variant in the VHL gene. rs34661876 A&gt;G, AG genotype in intron 2–3 has increased the risk of RCC against the odd 6.729 times (P = 0.0041). It is present in 17 out of 25 case samples. rs1642742 G&gt;A, AA genotype in exon 3 has increased the risk of RCC against the odd 22.167 times (P = 0.0001). It is present in 14 out of 25 case samples. The effect of these Single nucleotide polymorphism SNPs/mutations on VHL function were predicted by various bioinformatics software and it was found that rs1399097617 C&gt;T, rs5030830 T&gt;C, and rs1553620326 G&gt;C are disease-causing. Conclusion: If any of the above-mentioned variants are detected in RCC patients, then they will be benefited from the agents that modulate the VHL-hypoxia-inducible factor pathway and will help in developing new strategies for the management of RCC.

Cisplatin or Doxorubicin Reduces Cell Viability via the PTPIVA3-JAK2-STAT3 Cascade in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for 80% of all liver cancers and is the 2nd leading cause of cancer-related death in Taiwan. Various factors, including rapid cell growth, a high recurrence rate and drug resistance, make HCC difficult to cure. Moreover, the survival rate of advanced HCC patients treated with systemic chemotherapy remains unsatisfactory. Hence, the identification of novel molecular targets and the underlying mechanisms of chemoresistance in HCC and the development more effective therapeutic regimens are desperately needed. An MTT assay was used to determine the cell viability after cisplatin or doxorubicin treatment. Western blotting, qRT‒PCR and immunohistochemistry were utilized to examine the protein tyrosine phosphatase IVA3 (PTP4A3) level and associated signaling pathways. ELISA was utilized to analyze the levels of the inflammatory cytokine IL-6 influenced by cisplatin, doxorubicin and PTP4A3 silencing. In this study, we found that PTP4A3 in the cisplatin/doxorubicin-resistant microarray was closely associated with the overall and recurrence-free survival rates of HCC patients. Cisplatin or doxorubicin significantly reduced cell viability and decreased PTP4A3 expression in hepatoma cells. IL-6 secretion increased with cisplatin or doxorubicin treatment and after PTP4A3 silencing. Furthermore, PTP4A3 was highly expressed in tumor tissues versus adjacent normal tissues from HCC patients. In addition, we evaluated the IL-6-associated signaling pathway involving STAT3 and JAK2, and the levels of p-STAT3, p-JAK2, STAT3 and JAK2 were obviously reduced with cisplatin or doxorubicin treatment in HCC cells using Western blotting and were also decreased after silencing PTP4A3. Collectively, we suggest that cisplatin or doxorubicin decreases HCC cell viability via downregulation of PTP4A3 expression through the IL-6R-JAK2-STAT3 cascade. Therefore, emerging evidence provides a deep understanding of the roles of PTP4A3 in HCC cisplatin/doxorubicin chemoresistance, which can be applied to develop early diagnosis strategies and reveal prognostic factors to establish novel targeted therapeutics to specifically treat HCC.

Methyltransferase 3A-mediated promoter methylation represses retinoic acid receptor responder 3 expression in basal-like breast cancer

Retinoic acid receptor responder 3 (RARRES3) has been characterized as a tumor suppressor in multiple types of cancer. This study aimed to examine the expression profile of RARRES3 across the PAM50 subtypes of breast cancer. The DNA methylation status of RARRES3 was checked in the basal-like subtype, and the underlying mechanisms of its dysregulation were explored. RNA-sequencing (seq) and methylation data from The Cancer Genome Atlas were used for in-silico analysis. Basal-like representative SUM149 and MDA-MB-468 cell lines were used for in vitro and in vivo studies. Compared to tumor-adjacent normal tissues, only the basal-like tumor tissues had significantly downregulated RARRES3 expression. The methylation level of four CpG sites in the promoter region showed a strong negative correlation with RARRES3 expression. The gene coding for DNA methyltransferase 3A (DNMT3A) had consistent positive correlations with the methylation of the CpG sites. Chromatin Immunoprecipitation-quantitative polymerase chain-reaction and bisulfite sequencing PCR showed that DNMT3A could bind to the promoter region of RARRES3 and promote methylation of the CpG sites within the region. DNMT3A knockdown significantly restored RARRES3 expression at the mRNA and protein level in the two cell lines. CCK-8, colony formation, and flow cytometric analysis showed that RARRES3 overexpression attenuated the growth-promoting effects of DNMT3A overexpression and also weakened the DNMT3A overexpression-induced activation of ERK1/2 and PI3K/AKT signaling. In summary, this study revealed that DNMT3A enhances promoter methylation of the RARRES3 gene and suppresses its transcription in basal-like breast cancer. The DNMT3A-RARRES3 signaling pathway might be a potential target for the treatment of this tumor subtype.

ENO1 contributes to 5-fluorouracil resistance in colorectal cancer cells via EMT pathway.

Chemoresistance is a major barrier in the treatment of colorectal cancer (CRC) and many other cancers. ENO1 has been associated with various biological characteristics of CRC. This study aimed to investigate the function of ENO1 in regulating 5-Fluorouracil (5-FU) resistance in CRC. ENO1 level in 120 pairs of tumor tissues and adjacent normal tissues was examined by immunohistochemistry, and the correlation between ENO1 expression and prognosis was explored by survival analysis. Its role and potential mechanisms in regulating 5-FU resistance in CRC were studied by Western blotting, MTT assay, colony formation assay and transwell invasion assay. Murine xenograft assay was implied to verify the results in vivo. Our study indicated that ENO1 was elevated in CRC tissues and was associated with poor patient prognosis. High levels of ENO1 expression were detected as a significant influencing factor for overall survival. Furthermore, ENO1 expression was found to have increased in drug-resistant cells (HCT116/5-FU and SW620/5-FU) constructed by increasing concentrations of 5-FU. Knockdown of ENO1 markedly increased the drug susceptibility and inhibited the proliferation and migration ability of HCT116/5-FU and SW620/5-FU cells. It was found that down-regulation of ENO1 inhibited the epithelial-mesenchymal transformation (EMT) signaling process. Finally, a murine xenograft assay verified that the depletion of ENO1 alleviated 5-FU resistance. This study identified that ENO1 regulated 5-FU resistance via the EMT pathway and may be a novel target in the prevention and treatment of 5-FUresistant CRC.

Antitumor activity of RUNX3: Upregulation of E-cadherin and downregulation of the epithelial-mesenchymal transition in clear-cell renal cell carcinoma.

RUNX3 is a transcription factor and tumor suppressor that is silenced or inactivated in diverse tumors. The effect of RUNX3 on the epithelial-mesenchymal transition in clear-cell renal cell carcinoma (CCRCC) remains unclear. We determined the expression of RUNX3 and E-cadherin in tumor tissues and adjacent normal tissues of 30 CCRCC patients; established cultured CCRCC cells with the overexpression of RUNX3; and examined the in vivo tumorigenic function of RUNX3 in a nude mouse xenograft model of CCRCC. RUNX3 and E-cadherin were downregulated in human CCRCC samples. Cell lines with RUNX3 overexpression had reduced cell proliferation, invasion, and migration, a prolonged cell cycle, increased apoptosis, and increased expression of E-cadherin. In the nude mouse xenograft model of CCRCC, tumors with the overexpression of RUNX3 had smaller volumes and weights and had increased expression of E-cadherin. In conclusion, RUNX3 overexpression increased the level of E-cadherin and inhibited the proliferation, invasion, and migration of CCRCC in vitro and in vivo. RUNX3 has potential use as a biomarker for prognostic monitoring of CCRCC and as a therapeutic target for the treatment of this cancer.

Molecular linking of HIPEC (Hyperthermic Intraperitoneal Chemotherapy) and T-regulatory cells (FOXp3) and Th-17 (RORɣ) in advanced epithelial ovarian cancer

Despite of multimodality treatment of Ovarian Cancer (OC), about two-thirds of patients have peritoneal recurrence with 5-year survival of 15–35%. HIPEC (Hyperthermic Intraperitoneal Chemotherapy) delivers cell-cycle non-specific chemotherapeutic agents along with the temperature of 41°C to 43 °C into the peritoneal cavity after achieving complete cytoreduction, improving overall survival around 50% and recurrence-free survival significantly. Cytoreductive surgery (CRS) and HIPEC may be Upfront OR Primary - when performed primarily , Interval - when performed after 3 to 6 six cycles of Neo-adjuvant chemotherapy(NACT) and S econdary- when performed for patients who have a recurrence after CRS. OC is associated frequently with tumor-infiltrating lymphocytes in OC microenvironment. Here, we sought to study the interaction of HIPEC on FOXp3 and RORɣ T cells in primary, interval and recurrent settings of HIPEC. We have seen the expression of FOXp3, RORɣ in tumor tissue and surrounding normal tissue of ovarian cancer patients. We also performed IHC of tumor tissue and qRT-PCR of cDNA, isolated from tumor tissue and adjacent normal tissue for the same expression and compared intra-group and among the groups.We studied the expression of FOXp3,RORɣ, Interleukin-10 (IL-10) and Interleukin-17 (IL-17) in peripheral blood mononuclear cells(PBMC) before and after HIPEC in one week and four weeks and compared the changes of expressions. We included 30 patients with FIGO stage III to IVA advanced Epithelial ovarian cancer patients, with minimum 10 patients in each subgroup i.e.,upfront, interval and secondary. The pilot study was conducted between June 2017 to February 2021 at the premier Institute, India after Ethical approval. Though there was highest expression of FOXp3 mRNA in recurrent group followed by upfront and interval. However, there was no difference in RORɣ T cell mRNA (by qRT-PCR) expression among three subgroups. We found the change in the expression of FOXp3 and RORɣ T cell in Peripheral blood mononuclear cells by flowcytometry, before and after HIPEC (around 1 week and 4 week after HIPEC) to be significant. In the interval group, there was decreased expression of pre-op Foxp3 compared to the upfront ( p = 0.0121) and control ( p = 0.0187). These findings suggest that neoadjuvant chemotherapy may reverse the immunosuppressive environment in ovarian cancers. In the upfront group, there was a transient increase of FOXp3 in one week post HIPEC ( p = 0.005) and then it decreased to below pre-op values in 4 weeks ( p = 0.022). There was no difference in pre-op RORγ and IL 17 among the three subgroups.However, RORγ was increased at four weeks post-HIPEC in the upfront group ( p = 0.009), interval group ( p = 0.032), and the recurrent group. ( p = 0.001). Overall these findings suggest that HIPEC can reverse the immunosuppressive mechanism in ovarian tumor microenvironment (TME),thereby improving survival In this study, we could come to a conclusion that there is a complex nature of interaction at a molecular and immunological level in epithelial ovarian cancer patients underwent HIPEC in different settings. There is an urgent need to explore bio-molecular markers to improve treatment outcomes of ovarian cancer patients. This basic research study exploring the role of FOXp3 Tregs and RORγ T-helper cells indicated their potential role as biomarkers in understanding ovarian cancer prognosis and the impact of therapeutic interventions like HIPEC in this study, on outcomes.

Pan-cancer analysis of DNA epigenetic modifications by hydrophilic interaction liquid chromatography-tandem mass spectrometry

Accumulating evidence in recent years indicates that DNA methylation (5-methyl-2′-deoxycytidine, 5-mdC) and hydroxymethylation (5-hydroxymethyl-2′-deoxycytidine, 5-hmdC) have been implicated in various biological processes, and the aberrations of these DNA cytosine modifications is tightly associated with cancer. N6-methyl-2′-deoxyadenosine (m6dA), as a newly discovered epigenetic modification in genome of mammals, has been demonstrated to play vital regulatory roles in tumorigenesis. However, the content information of m6dA in human tumor tissues is still limited and pan-cancer analysis of these DNA epigenetic modifications is lacked. Herein, we developed a sensitive and robust stable isotope-diluted hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for accurate quantification of m6dA, 5-mdC and 5-hmdC in genomic DNA from 82 pairs of human tumor tissues and matched tumor-adjacent normal tissues. The types of tumors included esophagus cancer, lung cancer, breast cancer, liver cancer, pancreatic cancer, gastric cancer, stromal tumor and colorectal cancer. Compared to the normal tissues, we revealed the level of m6dA was increased in tumor tissues of esophagus cancer, lung cancer and liver cancer, whereas the level of m6dA was diminished in tumor tissues of pancreatic cancer and gastric cancer; while the contents of 5-mdC and 5-hmdC exhibited significant decrease in tumor tissues of most types of cancer. It is worth noting that we revealed, for the first time, the content of genomic m6dA in pancreatic cancer, stromal tumor and colorectal cancer. The significant changes of these DNA epigenetic modifications indicate they may serve as indicators of cancers. In addition, this study will benefit for better understanding of the regulatory roles of these DNA epigenetic modifications in cancers.

ABO blood groups and expression of blood group antigens of epithelial ovarian cancer in Chinese women.

ABO blood groups has been associated with risk of several cancers; however, the results for an association with ovarian cancer are inconsistent and little is known about the expression of histo-blood group (ABH) antigens and ABO gene in ovarian tumor tissues. To assess the impact of genotype-derived ABO blood types on the risk of EOC, we conducted a case-control study in 1,870 EOC and 4,829 controls. Expression of A and B antigen in 70 pairs of ovarian tumor tissues and adjacent normal tissues were detected by immunohistochemistry. Gene expression and DNA methylation profiling was conducted in ovarian tumor tissues. We identified that blood group A was associated with increased risk for EOC compared to blood group O (OR=1.18, 95% CI=1.03-1.36, p=0.019). Increased frequency of aberrant expression of histo-blood group antigens was observed in patients with blood group A (76.5%) compared to patients with blood group O (21.1%) and B (5.0%) by immunohistochemistry (p < 0.001). ABO gene expression was down-regulated in ovarian tumor tissues compared with paired adjacent normal tissues (p=0.027). In addition, ABO gene expression was positively correlated with NFYB (r=0.38, p < 0.001) and inversely correlated with DNA methylation level of four CpG sites on ABO gene (cg11879188, r=- 0.3, p=0.002; cg22535403, r=- 0.30, p=0.002; cg13506600, r=- 0.22, p=0.025; cg07241568, r=- 0.21, p=0.049) in ovarian tumor tissues. We identified blood group A was associated with increased EOC risk in Chinese women and provided the clues of the possible molecular mechanisms of blood group A related to ovarian cancer risk.