Cell Proliferation-related Genes Research Articles

Abstract 404: A novel 4-gene score predict patient survival as well as pathologically complete (R0) resection in pancreatic cancer

Abstract BACKGROUND: Pancreatic cancer is one of the most aggressive cancers with a 5-year survival of less than 10%. Surgery is the only cure for pancreatic cancer but the majority of patients present with metastatic disease at the time of diagnosis. Systemic chemotherapy remains the primary treatment option. Given the insidious clinical presentation and poor treatment response, there is an urgent need to discover a predictive biomarker for appropriate patient selection. We hypothesized that the 4-gene score, which reflects tumor cell proliferation, is both a prognostic and predictive biomarker for pancreatic cancer. METHODS: The 4-gene score is derived from tumor expression of DOK4, HCCS, PGF, and SHCBP1 genes, which were identified based on differential mRNA expression analysis of a human breast cell line, its metastatic variant cells, and clinical outcome data of breast cancer patient cohorts, as previously reported. A total of 954 pancreatic cancer patients were analyzed for both discovery and validation of the 4-gene score from publicly available datasets to investigate the relationship between the score with clinical features such as metastasis, cancer aggressiveness, immune cell infiltration, patient survival, and resectability. RESULTS: We found that the 4-gene score correlation in pancreatic cancer was higher than in breast cancer cohorts, specifically in clinically aggressive parameters such as pathological grade and MKI67 expression. Also, the score in metastatic tumor cohorts was higher than in primary cancer cohorts (p < 0.001). These findings were mirrored in gene set analysis, in which the score high tumors enriched cell proliferation-related gene sets such as E2F targets, G2M checkpoint, MYC targets v1 and v2, mitotic spindle, and metastasis-related gene sets in three cohorts (TCGA, GSE62452, and GSE57495). A high score was associated with increased mutation rates in KRAS and CDKN2A genes (p = 0.006 and 0.044) in the TCGA cohort, as well as enhancement of KRAS, CDKN2A, p53, TGF-β, and E2F pathways in three cohorts. Furthermore, a high score tumor had less infiltration of CD8+ T cells but was associated with high cytolytic activity and IFN-γ scores, which reflect overall cancer immunity. The high score was correlated with the level of drug sensitivity area under the curve of Sorafenib (r = 0.648) in primary cancer, Paclitaxel (r = 0.624) and Doxorubicin (r = 0.575) in metastatic cancer. Finally, a high 4-gene score was significantly associated with worse clinical outcomes in three cohorts (p = 0.044, 0.048, and 0.022), a low rate of R0 resection (p = 0.013), and worse survival after R1/2 resection (p = 0.027). CONCLUSION: The 4-gene score identified poor survival in pancreatic cancer and has potential as a predictive biomarker for R0 resection and treatment response in metastatic pancreatic cancer. Evaluating the degree of the 4-gene score could be a valuable potential prognostic tool. Citation Format: Masanori Oshi, Lan Le, Yoshihisa Tokumaru, Ankit Patel, Ryusei Matsuyama, Itaru Endo, Li Yan, Matthew H.G. Katz, Kazuaki Takabe. A novel 4-gene score predict patient survival as well as pathologically complete (R0) resection in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 404.

Abstract 1966: Cell proliferation-related pathway scores (G2M Checkpoint, E2F Targets, MYC Targets V1 and V2, Mitotic Spindle, p53 pathway) as predictive and prognostic biomarkers in pancreatic cancer

Abstract Background: Given the poor survival and severe side effects of treatments for pancreatic cancer, we need prognostic and predictive biomarkers to guide management. We hypothesized that the cell proliferation-related pathway is associated with drug response and survival in pancreatic cancer. Methods: We studied six hallmark cell proliferation-related gene sets (G2M Checkpoint, E2F Targets, MYC Targets V1 and V2, Mitotic Spindle, p53 pathway) defined by MSigDB in gene set variant analysis (GSVA) was evaluated in 3 independent cohorts, TCGA-PDAC (n = 176), GSE57495 (n = 63), and GSE62452 (n = 69). Results: We found that although G2M and E2F correlated strongly with each other (spearman's r = 0.976), Mitotic and p53 pathway scores correlated highly with the other cell function gene sets. All pathways were significantly associated with high expression of MKI67 and with proliferation score (all p > 0.001), but none with pathologically complete resection (R0). Mitotic Spindle pathway alone associated with high cytolytic activity (p = 0.020). All pathways were significantly associated with high expression of KRAS and TP53 genes and with high rate of KRAS gene alteration except for MYC v1. The G2M, E2F, and p53 pathway were significantly associated with a high rate of TP53 gene alteration (p = 0.026, 0.011, and 0.07, respectively). Interestingly, different pathway correlated with AUC of different agents, such as Gemcitabine (Mitotic: r=0.706 [p = 0.01]), Paclitaxel (MYC v2: r = -0.636 [p < 0.05]), Apatinib (Mitotic: r = -0.556 [p = 0.03]), Palbociclib (E2F: r =0.675 [p < 0.01]), and Sorafenib (G2M: r = -0.593 [p = 0.03]), for pancreatic cancer. Among all six pathways, only G2M was significantly associated with worse patient survival consistently in all 3 cohorts. Conclusion: Each proliferation-related pathway was predictive of unique agent, and G2M score predicts survival of pancreatic cancer. Citation Format: Masanori Oshi, Lan Le, Yoshihisa Tokumaru, Li Yan, Ryusei Matsuyama, Itaru Endo, Kazuaki Takabe. Cell proliferation-related pathway scores (G2M Checkpoint, E2F Targets, MYC Targets V1 and V2, Mitotic Spindle, p53 pathway) as predictive and prognostic biomarkers in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1966.

Gene expression profiling of anaplastic thyroid carcinoma and coexisting differentiated thyroid carcinoma using the NanoString nCounter system.

e18049 Background: Anaplastic thyroid carcinoma (ATC) is one of the worst prognostic malignancies with a 1-year survival rate of 5-20%. ATC is thought to be originated from differentiated thyroid carcinoma (DTC) caused by undifferentiated transformation because ATCs frequently have preexisting or coexisting region of differentiated thyroid carcinoma (co-DTC), but the detailed mechanism is unclear. In this study, we analyzed mRNA expression among ATCs, DTCs and co-DTCs, and compared their molecular characteristics. Methods: Twelve patients with ATC and eight patients with DTC, who were diagnosed at Tsukuba University Hospital between 2009 to 2020, were included in this study. Of 12 ATCs, four cases had co-DTCs. Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples of ATCs, DTCs and co-DTCs. We analyzed mRNA expression profiles among three groups using nCounter PanCancer IO 360 Panel (NanoString Technologies) and nCounter Advanced Analysis (version 2.0.134). The p-values were adjusted using Benjamini-Hochberg method and adjusted p-value < 0.05 was considered as significant. Results: The 1-year survival rate of the 12 ATC cases in this study was 37.5%. Heatmap of pathway revealed that most ATCs showed up-regulation of almost all tumor-related pathways included in the panel except for autophagy while three of ATCs didn’t show immune-related pathways up-regulation, and one co-DTC sample showed similar behavior of pathways to ATCs. The top 10 up-regulated gene in co-DTCs compare to DTCs were BATF3, TDO2, PRF1, BIRC5, MKI67, ANLN, FCGR3A/B, PPARGC1B, C2 and PFKFB3, and gene set enrichment analysis revealed that the genes with differential expression were enriched in gene set of cell proliferation. The top 10 genes with differential expression in ATCs compare to co-DTCs were CXCL5, BRCA2, MMP1, CSCL6, CXCL1, DKK1 and CXCL8, for up-regulation and ID4, ARID1A and EPM2AIP1 for down-regulation. In ATCs, gene sets of cell proliferation, cytokine and chemokine signaling, and myeloid compartment were especially up-regulated. Immune cell profiling showed that ATC had abundant inflammatory cells, especially TILs, neutrophils and macrophages, while co-DTC showed intermediate characteristics between ATC and DTC. Of note, three case of ATCs had lower inflammatory cell scores than the other ATCs except for macrophages and neutrophils, and were clustered as “DTC-like”. Conclusions: These data revealed that co-DTCs showed up-regulation of cell proliferation related genes compare to DTCs, and some ATCs had different immune cell profiles. co-DTCs showed an intermediate immune cell profile between ATC and DTC. Biological profiling of ATCs, DTCs and co-DTCs may help to understand the developmental mechanism of ATCs. In this presentation, we will report the detailed results of mRNA expression analysis with discussion of the previous reports.

G2M checkpoint signaling among six-cell proliferation-related pathway as a biomarker for prediction and prognosis in pancreatic cancer.

e16264 Background: Given the poor survival and severe side effects of treatments for pancreatic cancer, prognostic and predictive biomarkers to guide management are in urgent need. We hypothesized that the cell proliferation-related pathway is associated with drug response and survival in pancreatic cancer. Methods: Six hallmark cell proliferation-related gene sets (G2M checkpoint, E2F targets, MYC targets V1 and V2, mitotic spindle, p53 pathway) defined by MSigDB in gene set variant analysis (GSVA) was evaluated in 3 independent cohorts, TCGA-PDAC ( n = 176), GSE57495 ( n = 63), and GSE62452 ( n = 69). Results: Although G2M and E2F correlated strongly with each other (Spearman’s r = 0.976), Mitotic and p53 pathway scores correlated highly with the other cell function gene sets. All pathways were significantly associated with high expression of MKI67 and with proliferation score (all p > 0.001), but none with pathologically complete resection (R0). Mitotic spindle pathway alone associated with high cytolytic activity ( p = 0.020). All pathways were significantly associated with high expression of KRAS and TP53 genes and with high rate of KRAS gene alteration except for MYC v1. G2M, E2F, and p53 pathways were significantly associated with high rate of TP53 gene alteration ( p = 0.026, 0.011, and 0.07, respectively). Interestingly, different pathway correlated with AUC of different agents, such as gemcitabine (mitotic: r=0.706 [ p = 0.01]), paclitaxel (MYC v2: r = -0.636 [ p < 0.05]), apatinib (mitotic: r = -0.556 [ p = 0.03]), palbociclib (E2F: r =0.675 [ p < 0.01]), and sorafenib (G2M: r = -0.593 [ p = 0.03]), for pancreatic cancer. Among all six pathways, only G2M was significantly associated with worse patient survival consistently in all 3 cohorts. Conclusions: Each proliferation-related pathway was predictive of the unique agent, and the G2M score predicts survival of pancreatic cancer.

A Novel Three-Gene Score as a Predictive Biomarker for Pathologically Complete Response after Neoadjuvant Chemotherapy in Triple-Negative Breast Cancer.

Simple SummaryNeoadjuvant chemotherapy is now a standard of care not only to decrease tumor size for breast conserving operation but also to assess drug response of an in situ cancer. Although the triple-negative subtype typically responds better compared to the other subtypes, a pathological complete response, which is a surrogate of survival, is achieved in less than half of the cases. For the most efficient patient selection, and avoiding unnecessary side effects and financial toxicity, an accurate predictive biomarker is urgently needed. We developed a novel three-gene score that associated with immune cell infiltration and pathological complete response not only in the training cohort but also in the validation triple-negative cohort. High-score TNBC was significantly associated with better survival in patients who received chemotherapy but not in patients who did not receive chemotherapy. Our score is a predictive and prognostic biomarker of response to neoadjuvant chemotherapy in triple-negative breast cancer patients.Although triple-negative breast cancer (TNBC) typically responds better to neoadjuvant chemotherapy (NAC) compared to the other subtypes, a pathological complete response (pCR) is achieved in less than half of the cases. We established a novel three-gene score using genes based on the E2F target gene set that identified pCR after NAC, which showed robust performance in both training and validation cohorts (total of n = 3862 breast cancer patients). We found that the three-gene score was elevated in TNBC compared to the other subtypes. A high score was associated with Nottingham histological grade 3 in TNBC. Across multiple cohorts, high-score TNBC enriched not only E2F targets but also G2M checkpoint and mitotic spindle, which are all cell proliferation-related gene sets. High-score TNBC was associated with homologous recombination deficiency, high mutation load, and high infiltration of Th1, Th2, and gamma-delta T cells. However, the score did not correlate with drug sensitivity for paclitaxel, 5-fluorouracil, cyclophosphamide, and doxorubicin in TNBC human cell lines. High-score TNBC was significantly associated with a high rate of pCR not only in the training cohort but also in the validation cohorts. High-score TNBC was significantly associated with better survival in patients who received chemotherapy but not in patients who did not receive chemotherapy. The three-gene score is associated with a high mutation rate, immune cell infiltration, and predicts response to NAC in TNBC.

High BRCA2 Gene Expression is Associated with Aggressive and Highly Proliferative Breast Cancer.

Mutations of BRCA genes are the most studied in breast cancer, but the clinical relevance of BRCA2 gene expression has been less well studied. Given that BRCA2 is a DNA repair gene, we hypothesized that high BRCA2 expression is associated with highly proliferative and aggressive biology in breast cancer. A total of 4342 breast cancer patients were analyzed from The Cancer Genome Atlas (TCGA, n = 1069) as the testing cohort and Gene Expression Omnibus (GEO) dataset GSE96058 (n = 3273) as a validation cohort. There was no relationship between BRCA2 mutation and BRCA2 gene expression. BRCA2 high expression breast cancer was associated with higher Nottingham grade (p < 0.001), with high proliferation (MKI-67, p < 0.001), and with higher intratumor heterogeneity, homologous recombination deficiency, mutation rate, fraction altered, and neoantigens (all p < 0.001). BRCA2 high expression was associated with E2F1, RB1, PALB2, and PARP, as well as cell proliferation-related gene sets, E2F targets, G2M checkpoints, and mitotic spindle, and with less ESR1 and ER response early and late gene sets. They were associated with significantly reduced number of stroma cells and with high infiltration of both favorable and unfavorable immune cells. BRCA2 high expression significantly correlated with response to olaparib, a PARP inhibitor, and inversely with cyclophosphamide in ER-positive/HER2-negative tumors, but not in TNBC. BRCA2 high gene expression was associated with highly proliferative and aggressive breast cancer that was highly immunogenic with better response to PARP inhibitors in ER-positive patients. BRCA2 gene expression may become a candidate marker for aggressive biology and to tailor specific treatment strategies in the future.

The haplolethality paradox of the wupA gene in Drosophila.

Haplolethals (HL) are regions of diploid genomes that in one dose are fatal for the organism. Their biological meaning is obscure because heterozygous loss-of-function mutations result in dominant lethality (DL) and, consequently, should be under strong negative selection. We report an in depth study of the HL associated to the gene wings up A (wupA). It encodes 13 transcripts (A-M) that yield 11 protein isoforms (A-K) of Troponin I (TnI). They are functionally diverse in their control of muscle contraction, cell polarity and cell proliferation. Isoform K transfers to the nucleus where it increases transcription of the cell proliferation related genes CDK2, CDK4, Rap and Rab5. The nuclear translocation of isoform K is prevented by the co-expression of A or B isoforms, which illustrates isoform interactions. The corresponding DL mutations are, either DNA rearrangements clustered towards the gene 3’ end, thus affecting the genomic organization of all transcripts, or CRISPR-induced mutations in one of the two ATG sites which eliminate a subset of wupA products. The joint elimination of isoforms C, F, G and H, however, do not cause DL phenotypes. Genetically driven expression of single isoforms rescue neither DL nor any of the mutants known in the gene, suggesting that normal function requires properly regulated expression of specific combinations, rather than single, TnI isoforms. We conclude that the wupA associated HL results from the combined haploinsufficiency of a large set of TnI isoforms. The qualitative and quantitative normal expression of which, requires the chromosomal integrity of the wupA genomic region. Since all fly TnI isoforms are encoded in the same gene, its HL condition becomes unavoidable. These wupA features are comparable to those of dpp, the only other HL studied to some extent, and reveal a scenario of strict dosage dependence with implications for gene expression regulation and splitting.

Ocoxin Increases the Antitumor Effect of BRAF Inhibition and Reduces Cancer Associated Fibroblast-Mediated Chemoresistance and Protumoral Activity in Metastatic Melanoma.

Whereas the prevalence of several cancer types is decreasing, skin malignancies are growing more common every year. Malignant melanoma is the most aggressive form of skin cancer with high metastatic capacity. In most cases, malignant melanoma shows acquired therapy resistance. We evaluated the ability of Ocoxin, a natural compound-based antioxidant and anti-inflammatory nutritional complement, to exert an antitumor effect in melanoma. To do so, the cytotoxicity of Ocoxin in a panel of BRAF-mutated murine and human melanoma cell lines was tested alone and in combination with BRAF inhibitor Vemurafenib. Our results revealed a potent cytotoxic effect of Ocoxin against melanoma cells and a synergic effect when combined with Vemurafenib, reducing viability and increasing apoptosis. Besides, Ocoxin interferes with the cell cycle, impairs the inherent and fibroblast-mediated melanoma cell migration, and reduces resistance to BRAF inhibition. Proteomic analysis revealed reduced tumor secretion of inflammatory factors Galectin-1, Osteopontin, CCL5, and CCL9 upon treatment with Ocoxin. Moreover, RNASeq showed that Ocoxin downregulated the cell cycle and proliferation-related genes. In vivo, Ocoxin reduced the number of lung metastasis of YUMM-1.7 melanoma cells. Therefore, Ocoxin arises as a good candidate for clinical trials analyzing the beneficial effects in patients suffering from this cutaneous malignancy.

Abstract PS4-35: The E2F cell cycle pathway score to predict treatment response among patients with breast cancer

Abstract Cell cycle progression is a critical component of cell proliferation, and continuous proliferation is one of the hallmarks of cancer. Components regulating the E2F pathway have been identified in nearly every human malignancy and many of them including E2F transcription factors themselves play major roles in cancer progression, metastasis and treatment response of breasts cancer. Their activity therefore is expected to reflect tumor aggressiveness and responsiveness to therapy. We scored 3,905 tumors of nine breast cancer cohorts for this activity based on their 200 gene expression for the Hallmark E2F targets gene set. As expected, tumors with a high score had increased expression of cell proliferation-related genes, including G2M checkpoint, MYC targets v1 and v2, MITOTIC spindle, MTORC1 signaling, UNFOLDED protein response, and DNA repair in both the TCGA and METABRIC cohorts (false discovery rate &amp;lt; 0.001 in both cohorts). A high score was significantly associated with greater MKI67 expression (p &amp;lt; 0.001 in both cohorts), histological grade (p &amp;lt; 0.001 in both cohorts), and AJCC pathological stage (p &amp;lt; 0.001 in both cohorts). And Indel and single nucleotide variation neoantigen loads were associated with a high E2F pathway score (p = 0.006 and 0.047 respectively). Furthermore, the E2F pathway score correlated positively with copy number alteration (Spearman r = 0.55, p &amp;lt; 0.001). Intra-tumoral genome heterogeneity and proliferation score were significantly associated with the E2F pathway score as well (p &amp;lt; 0.001). The high E2F pathway score group demonstrated significantly higher fractions of not only pro-cancerous regulatory T cells, helper T cell (Th2), but also anti-cancerous CD4 memory T cell, helper T cell (Th1), and M1 macrophage as well as B cells compared to the low score group in the TCGA cohort (p &amp;lt; 0.001). Similar trends were observed in the METABRIC cohort. Furthermore, metastatic tumors had higher E2F scores than the primary tumors from which they arose especially luminal and normal subtype (p = 0.006 and p &amp;lt; 0.001). Interestingly, metastases with a high E2F score were associated with significantly worse PFS in the whole cohort, as well as patient sub-groups with local recurrence and with liver metastasis (p = 0.018, p = 0.025 and p = 0.027). Additionally, the E2F pathway score was significantly decreased with good response to chemotherapy (p &amp;lt; 0.001) and demonstrated higher pCR rate after neoadjuvant therapies in the high E2F pathway score group of estrogen receptor (ER)-positive/HER2-negative cancer (p &amp;lt; 0.001). The E2F score was significantly associated with expression of cyclin-dependent kinase (CDK)-related genes, including CCNE1, CDKN2A, CDKN2D, CDK2, CDK4 and CDK6 (all p &amp;lt; 0.001), and immune checkpoint molecule-related genes including PD-L1/2, CTLA4, IDO1, LAG3 and TIGIT (p = 0.013, 0.014, &amp;lt; 0.001, &amp;lt; 0.001, &amp;lt; 0.001 and 0.002, respectively). These results were validated by other cohort. Finally, the E2F score was strongly correlated with sensitivity to CDK inhibition in ER+/HER2- breast cancer cell-lines (Spearman r = 0.90, p = 0.04). In conclusion, the E2F score is a marker of breast cancer aggressiveness and predicts responsiveness of ER+/HER2- patients to neoadjuvant chemotherapy and possibly to CDK and immune checkpoint inhibitors. Citation Format: Masanori Oshi, Hideo Takahashi, Yoshihisa Tokumaru, Ryusei Matsuyama, Itaru Endo, Kazuaki Takabe. The E2F cell cycle pathway score to predict treatment response among patients with breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS4-35.

Expression of Twist1, SIRT1, FGF2 and TGF-β3 genes and its regulatory effect on the proliferation of placenta, umbilical cord and dental pulp mesenchymal stem cells

To compare the mRNA level of cell proliferation-related genes Twist1, SIRT1, FGF2 and TGF-β3 in placenta mesenchymal stem cells (PA-MSCs), umbilical cord mensenchymals (UC-MSCs) and dental pulp mesenchymal stem cells (DP-MSCs). The morphology of various passages of PA-MSCs, UC-MSCs and DP-MSCs were observed by microscopy. Proliferation and promoting ability of the three cell lines were detected with the MTT method. Real-time PCR (RT-PCR) was used to determine the mRNA levels of Twist1, SIRT1, FGF2, TGF-β3. The morphology of UC-MSCs and DP-MSCs was different from that of PA-MSCs. Proliferation ability and promoting ability of the PA-MSCs was superior to that of UC-MSCs and DP-MSCs. In PA-MSCs, expression level of Twist1 and TGF-β3 was the highest and FGF2 was the lowest. SIRT1 was highly expressed in UC-MSCs. With the cell subcultured, different expression levels of Twist1, SIRT1, FGF2, TGF-β3 was observed in PA-MSCs, UC-MSCs and DP-MSCs. Up-regulated expression of the Twist1, SIRT1 and TGF-β3 genes can promote proliferation of PA-MSCs, UC-MSCs and DP-MSCs, whilst TGF-β3 may inhibit these. The regulatory effect of Twist1, SIRT1, FGF2 and TGF-β3 genes on PA-MSCs, UC-MSCs and DP-MSCs are different.

Effects of omega-7 palmitoleic acids on skeletal muscle differentiation in a hyperglycemic condition.

Maternal obesity and diabetes are known to be involved in fetal myogenesis, but the later stages of myogenesis are not well understood. In this study, we investigated the influence of a hyperglycemic environment on L6 skeletal myoblast differentiation and the function of omega-7 palmitoleic acids. Exposure to a high concentration of glucose (25 mM) in high-glucose culture medium (HG) increased the expression of myogenic genes (MyoD, Myogenin, MRF4, Myhc2x, and Myhc2a) and the synthesis of myosin. HG also activated the PI3K/AKT pathway revealed muscle cell differentiation. Furthermore, the levels of reactive oxygen species (ROS) and an inflammatory cytokine (Tnfaip3; tumor necrosis factor alpha-induced protein 3), which are crucial for the growth and differentiation of skeletal muscle, were increased by HG. Palmitoleic acids suppressed the expression levels of myogenic regulatory genes and increased the expression level of a cell proliferation-related gene (Pax3). Trans-palmitoleic acid and eicosapentaenoic acid (TPA and EPA) increased the phosphorylation level of MAPK/ERK1/2 and downregulated ROS generation and Tnfaip3 expression. In contrast, cis-palmitoleic acid inactivated MAPK/ERK1/2, leading to increased ROS generation. In conclusion, a hyperglycemic environment mediated by HG induced excessive muscle differentiation. Palmitoleic acids inhibited myoblast differentiation by downregulating muscle-specific genes. Moreover, trans-palmitoleic acids may have beneficial antioxidant and/or anti-inflammatory effects in cells.

A Novel Four-Gene Score to Predict Pathologically Complete (R0) Resection and Survival in Pancreatic Cancer.

Simple SummaryA biomarker to predict survival is a critical need in pancreatic cancer treatment. We hypothesized that a four-gene score, which was previously reported to reflect cell proliferation, can be used as a predictive biomarker for pancreatic cancer. A total of 954 patients were analyzed for both discovery and validation of the four-gene score from publicly available datasets for pancreatic cancer, in order to investigate the relationship between the score and clinical features of pancreatic cancer, such as metastasis, cancer aggressiveness, immune cell infiltration, patient survival, and resectability. We found that the score correlated with clinical aggressiveness in pancreatic cancer, and did so to a higher degree compared to breast cancer cohorts. We found that the four-gene score identified poor survival in pancreatic cancer, and has potential as a predictive biomarker of treatment response in metastatic pancreatic cancer, as well completion of a pathologically complete (R0) resection. Appropriately utilized, the four-gene score could be a valuable prognostic and predictive tool for pancreatic cancer in the future.Pathologically complete (R0) resection is essential for prolonged survival in pancreatic cancer. Survival depends not only on surgical technique, but also on cancer biology. A biomarker to predict survival is a critical need in pancreatic treatment. We hypothesized that this 4-gene score, which was reported to reflect cell proliferation, is a translatable predictive biomarker for pancreatic cancer. A total of 954 pancreatic cancer patients from multiple cohorts were analyzed and validated. Pancreatic cancer had the 10th highest median score of 32 cancers in The Cancer Genome Atlas (TCGA) cohort. The four-gene score significantly correlated with pathological grade and MKI67 expression. The high four-gene score enriched cell proliferation-related and cancer aggressiveness-related gene sets. The high score was associated with activation of KRAS, p53, transforming growth factor (TGF)-β, and E2F pathways, and with high alteration rate of KRAS and CDKN2A genes. The high score was also significantly associated with reduced CD8+ T cell infiltration of tumors, but with high levels of interferon-γ and cytolytic activity in tumors. The four-gene score correlated with the area under the curve of irinotecan and sorafenib in primary pancreatic cancer, and with paclitaxel and doxorubicin in metastatic pancreatic cancer. The high four-gene score was associated with significantly fewer R0 resections and worse survival. The novelty of the study is in the application of the four-gene score to pancreatic cancer, rather than the bioinformatics technique itself. Future analyses of inoperable lesions are expected to clarify the utility of our score as a predictive biomarker of systemic treatments.

Abstract 14257: Endothelial SOX17 Deficiency Induces Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is a disaster disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Hypothesis: Human genome-wide association studies identified that SOX17 locus variants are associated with PAH. SOX17 mutation is also found in patients with PAH. We hypothesis that endothelial SOX17 deficiency contributes to the pathogenesis of PAH. Methods: Mice with EndoSCL-CreERT mediated deletion of Sox17 ( Sox17 iCKO ) were generated. Sox17 iCKO and Sox17 f/f mice after tamoxifen injection were incubated with hypoxia (10% O 2 ) for 3 weeks to induced PAH. Hemodynamics and histological examination were measured to determine the PAH phenotypes and vascular remodeling. EC proliferation and apoptosis were assessed in SiRNA-mediated SOX17 knockdown in human lung microvascular endothelial cells (hLMVECs). The RNA-sequencing analysis was performed to understand the molecular mechanisms of SOX17 deficiency in ECs. Results: Sox17 iCKO mice exhibited exaggerative PAH evident by the increase of RVSP and RV hypertrophy after hypoxia treatment compared to Sox17 f/f WT mice. SOX17 knockdown in hLMVECs induced cell proliferation and reduced starvation-induced apoptosis. RNA-seq analysis and DAVID pathway analysis demonstrated that there was dysregulation of cell proliferation-related genes, which are enriched in the pathways related to cell cycle, cell division, and mitotic cell cycle. Transcriptional factor, target, and motif discovery analysis of the dysregulated gene set revealed the involvement of transcriptional factors FOXM1 and E2F1. siRNA mediated knockdown of E2F1 but not FOXM1 normalized SOX17 deficiency-induced hLMVECs proliferation and anti-apoptosis. Conclusions: Our study demonstrated that endothelial SOX17 deficiency exaggerates hypoxia-induced PAH. Loss of SOX17 promotes EC proliferation and anti-apoptosis via the upregulation of transcription factor E2F1.

Transcriptomic Analyses Reveal the Protective Immune Regulation of Conjugated Linoleic Acids in Sheep Ruminal Epithelial Cells.

The ruminal epithelium is continuously challenged by antigens released by the lysis of dead microbial cells within the rumen. However, the innate immune system of the ruminal epithelium can almost always actively respond to these challenges. The cross talk between the ruminal microbiota and innate immune cells in the ruminal epithelium has been suggested to play an important role in sustaining the balance of immune tolerance and inflammatory response in the rumen. We hypothesized that conjugated linoleic acid (CLA), a functional microbial metabolite in the rumen, may contribute to the immune regulation in rumen epithelial cells (RECs); therefore, we first established an immortal REC line and then investigated the regulatory effects of CLA on the immune responses in these RECs. The results showed that long-term REC cultures were successfully established via SV40T-induced immortalization. Transcriptome analysis showed that a 100 μM CLA mixture consisting of 50:50 cis-9, trans-11:trans-10, cis-12 CLA significantly downregulated the expression of the inflammatory response-related genes TNF-α, IL-6, CX3CL1, IRF1, ICAM1 and EDN1, and upregulated the expression of the cell proliferation-related genes FGF7, FGF21, EREG, AREG and HBEGF and the lipid metabolism-related genes PLIN2, CPT1A, ANGPTL4, ABHD5 and SREBF1 in the RECs upon LPS stimulation. Correspondingly, the GO terms regulation of cell adhesion, response to stimulus and cytokine production and KEGG pathways TNF and HIF-1 signaling, ECM-receptor interaction and cell adhesion molecules were identified for the significantly downregulated genes, while the GO terms epithelial cell proliferation and regulation of epithelial cell migration and the KEGG pathways PPAR, ErbB and adipocytokine signaling were identified for the RECs with significantly upregulated CLA-pretreated genes upon LPS stimulation. These findings revealed that CLA conferred protective immunity onto the RECs by inhibiting proinflammatory processes, promoting cell proliferation and regulating lipid metabolism related to the immune response.

ITPKC as a Prognostic and Predictive Biomarker of Neoadjuvant Chemotherapy for Triple Negative Breast Cancer.

Simple SummaryInositol 1,4,5-trisphosphate 3-kinase C (ITPKC) gene is a negative regulator of T cell activation and is a proven promoter of Kawasaki disease. Given the critical role of immune response in breast cancer, we aimed to determine the clinical relevance of ITPKC expression in breast cancer. ITPKC expression was highest in triple negative breast cancer, associated with its survival, and was its independent prognostic factor. Although high ITPKC expression was not associated with immune function nor with any immune cell fraction, low ITPKC triple negative breast cancer was significantly associated with activated cell proliferation, and they achieved a significantly rate in pathological complete response (disappearance of tumor) after neoadjuvant chemotherapy. To the best of our knowledge, this is the first report to demonstrate that ITPKC gene expression may be useful as a prognostic and predictive biomarker in triple negative breast cancer.Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with higher mortality than the others. Pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) is considered as a surrogate to predict survival. Inositol 1,4,5-trisphosphate 3-kinase C (ITPKC) is a negative regulator of T cell activation, and reduction in ITPKC function is known to promote Kawasaki disease. Given the role of tumor infiltrating lymphocytes in NAC and since TNBC has the most abundant immune cell infiltration in breast cancer, we hypothesized that the ITPKC expression level is associated with NAC response and prognosis in TNBC. The ITPKC gene was expressed in the mammary gland, but its expression was highest in breast cancer cells among other stromal cells in a bulk tumor. ITPKC expression was highest in TNBC, associated with its survival, and was its independent prognostic factor. Although high ITPKC was not associated with immune function nor with any immune cell fraction, low ITPKC significantly enriched cell proliferation-related gene sets in TNBC. TNBC with low ITPKC achieved a significantly higher pCR rate after NAC. To the best of our knowledge, this is the first report to demonstrate that ITPKC gene expression may be useful as a prognostic and predictive biomarker in TNBC.

Prevalence and clinical relevance of tumor-associated tissue eosinophilia (TATE) in breast cancer

BackgroundTumor-associated tissue eosinophilia (TATE) has been associated with outcomes in a variety of solid tumors; however, its role in breast cancer is not well defined. We hypothesized that tumor-associated tissue eosinophilia is associated with a high mutation and neoantigen load, and we assessed its correlation with cancer outcomes. MethodsThe Cancer Genome Atlas was analyzed for eosinophil signatures in breast cancer specimens. Descriptive analyses were performed, including the tumor-infiltrating cell composition using CIBERSORT, cytolytic activity score, and gene set enrichment analysis. Overall survival and disease-free survival were calculated using the Kaplan-Meier method. ResultsOut of 1069 cases analyzed, 40 (3.7%) had tissue eosinophils (the tumor-associated tissue eosinophilia group). Tumor-associated tissue eosinophilia was noted in 32.5% luminal, 5% HER2-positive, and 15% triple-negative breast cancer subtypes. The single nucleotide variant–neoantigen load was significantly higher in the tumor-associated tissue eosinophilia group (P = .005), with a higher nonsilent mutation rate (P = .01). The tumor-associated tissue eosinophilia group had lower cytolytic activity (P = .02) but had enriched MYC-targeted (P = .002), E2F-targeted (P = .04), deoxyribonucleic acid repair (P = .03), and unfolded protein response gene sets (P = .05). Tumor-associated tissue eosinophilia was associated with a trend toward improved disease-free survival (P = .06) but presented no differences in overall survival (P = .56). ConclusionTumor-associated tissue eosinophilia was noted in 3.7% of breast cancers and was associated with a higher single nucleotide variant–neoantigen load and nonsilent mutation rate, similar to that of tumor-infiltrating lymphocytes in the triple-negative subtype. However, a lower cytolytic activity score and enriched cell proliferation–related gene sets implicate different roles for tumor-associated tissue eosinophilia than for tumor-infiltrating lymphocytes.

Arginine infusion rescues ovarian follicular development in feed-restricted Hu sheep during the luteal phase

The aim of this study was to investigate the molecular mechanisms of arginine (Arg) on follicular development of acute feed-restricted ewes during the luteal phase. From day 6 of the estrous cycle, 24 multiparous Hu sheep were randomly assigned into three groups: control group (a maintenance diet; n = 6), feed restriction group (0.5 maintenance diet, saline infusion; n = 9) and Arg treatment group (0.5 maintenance diet, infusion with 155 μmol of Arg-HCl/kg body weight; n = 9). The intravenous administrations were performed three times per day from day 6 to day 15 of the estrous cycle. At the end of treatment, the hypothalamus and pituitary were collected, as well as the follicular fluid (FF) and granulose cells (GCs) in the ≥2.5 mm follicles. The transcription level of NPVF was significantly increased, and the expression level of GNRH was significantly decreased in the hypothalamus with feed restriction. In addition, feed restriction significantly decreased the number of ≥2.5 mm follicles in the ovaries. In the ≥2.5 mm follicles, feed restriction significantly increased estradiol (E2) level in FF and the expression levels of steroidogenesis related genes (STAR, 3BHSD and CYP19A1) in GCs, while significantly decreased the expressions of FSHR and cell proliferation related genes (YAP1, CCND1 and PCNA) in GCs. Moreover, the activities of glucose metabolism enzymes (PFKP and G6PDH) were significantly decreased in GCs of the ≥2.5 mm follicles with feed restriction. Interestingly, as a precursor of nitric oxide, Arg supplementation can rescue the effects of feed restriction on follicular development by enhancing glucose metabolism and cell proliferation of GCs, and alleviating the abnormal E2 secretion in the ≥2.5 mm follicles, accompanied with recovering the expressions of NPVF and GNRH in the hypothalamus. These findings will be helpful for understanding the role of nutrition and Arg in sheep follicular development.

Impact of low-intensity pulsed ultrasound on transcription and metabolite compositions in proliferation and functionalization of human adipose-derived mesenchymal stromal cells

To investigate the effect of low-intensity pulsed ultrasound (LIPUS) on the proliferation of human adipose-derived mesenchymal stromal cells (hASCs) and uncovered its stimulation mechanism. LIPUS at 30 mW/cm2 was applied for 5 min/day to promote the proliferation of hASCs. Flow cytometry was used to study the cell surface markers, cell cycle, and apoptosis of hASCs. The proliferation of hASCs was detected by cell counting kit-8, cell cycle assay, and RT-PCR. The expression of hASCs cytokines was determined by ELISA. The differences between transcriptional genes and metabolites were analyzed by transcript analysis and metabolomic profiling experiments. The number of cells increased after LIPUS stimulation, but there was no significant difference in cell surface markers. The results of flow cytometry, RT-PCR, and ELISA after LIPUS was administered showed that the G1 and S phases of the cell cycle were prolonged. The expression of cell proliferation related genes (CyclinD1 and c-myc) and the paracrine function related gene (SDF-1α) were up-regulated. The expression of cytokines was increased, while the apoptosis rate was decreased. The results of transcriptome experiments showed that there were significant differences in 27 genes;15 genes were up-regulated, while 12 genes were down-regulated. The results of metabolomics experiments showed significant differences in 30 metabolites; 7 metabolites were up-regulated, and 23 metabolites were down-regulated. LIPUS at 30 mW/cm2 intensity can promote the proliferation of hASCs cells in an undifferentiating state, and the stem-cell property of hASCs was maintained. CyclinD1 gene, c-myc gene, and various genes of transcription and products of metabolism play an essential role in cell proliferation. This study provides an important experimental and theoretical basis for the clinical application of LIPUS in promoting the proliferation of hASCs cells.

Intratumoral Adipocyte-High Breast Cancer Enrich for Metastatic and Inflammation-Related Pathways but Associated with Less Cancer Cell Proliferation.

Cancer-associated adipocytes are known to cause inflammation, leading to cancer progression and metastasis. The clinicopathological and transcriptomic data from 2256 patients with breast cancer were obtained based on three cohorts: The Cancer Genome Atlas (TCGA), GSE25066, and a study by Yau et al. For the current study, we defined the adipocyte, which is calculated by utilizing a computational algorithm, xCell, as “intratumoral adipocyte”. These intratumoral adipocytes appropriately reflected mature adipocytes in a bulk tumor. The amount of intratumoral adipocytes demonstrated no relationship with survival. Intratumoral adipocyte-high tumors significantly enriched for metastasis and inflammation-related gene sets and are associated with a favorable tumor immune microenvironment, especially in the ER+/HER2- subtype. On the other hand, intratumoral adipocyte-low tumors significantly enriched for cell cycle and cell proliferation-related gene sets. Correspondingly, intratumoral adipocyte-low tumors are associated with advanced pathological grades and inversely correlated with MKI67 expression. In conclusion, a high amount of intratumoral adipocytes in breast cancer was associated with inflammation, metastatic pathways, cancer stemness, and favorable tumor immune microenvironment. However, a low amount of adipocytes was associated with a highly proliferative tumor in ER-positive breast cancer. This cancer biology may explain the reason why patient survival did not differ by the amount of adipocytes.

The E2F Pathway Score as a Predictive Biomarker of Response to Neoadjuvant Therapy in ER+/HER2- Breast Cancer.

E2F transcription factors play critical roles in the cell cycle. Therefore, their activity is expected to reflect tumor aggressiveness and responsiveness to therapy. We scored 3905 tumors of nine breast cancer cohorts for this activity based on their gene expression for the Hallmark E2F targets gene set. As expected, tumors with a high score had an increased expression of cell proliferation-related genes. A high score was significantly associated with shorter patient survival, greater MKI67 expression, histological grade, stage, and genomic aberrations. Furthermore, metastatic tumors had higher E2F scores than the primary tumors from which they arose. Although tumors with a high score had greater infiltration by both pro- and anti-cancerous immune cells, they had an increased expression of immune checkpoint genes. Estrogen receptor (ER)-positive/human epidermal growth factor receptor 2 (HER2)-negative cancer with a high E2F score achieved a significantly higher pathological complete response (pCR) rate to neoadjuvant chemotherapy. The E2F score was significantly associated with the expression of cyclin-dependent kinase (CDK)-related genes and strongly correlated with sensitivity to CDK inhibition in cell lines. In conclusion, the E2F score is a marker of breast cancer aggressiveness and predicts the responsiveness of ER-positive/HER2-negative patients to neoadjuvant chemotherapy and possibly to CDK and immune checkpoint inhibitors.