Cell Proliferation-related Genes Research Articles

Enhanced cancer cell proliferation and aggressive phenotype counterbalance in breast cancer with high BRCA1 gene expression.

While comprehensive research exists on the mutation of the DNA repair gene BRCA1, limited information is available regarding the clinical significance of BRCA1 gene expression. Given that cancer cell proliferation is aggrevated by DNA repair, we hypothesized that high BRCA1 gene expression breast cancer (BC) might be linked with aggressive tumor biology and poor clinical outcomes. The cohorts: The Cancer Genome Atlas (TCGA, n = 1069), METABRIC (n = 1903), and SCAN-B (n = 3273) were utilzed to obtain data of 6245 BC patients. BC patients without BRCA1 mutation exhibited higher BRCA1 expression, which was associated with DNA repair functionality. However, no such correlation was observed with BRCA2 expression. The association of high BRCA1 expression with cancer cell proliferation was evidenced by significant enrichment of cell proliferation-related gene sets, higher histological grade, and proliferation score. Furthermore, increased levels of homologous recombination deficiency, intratumoral heterogeneity, and altered fractions were associated with high BRCA1 expression. Moreover, BC with high BRCA1 expression exhibited reduced infiltration of dendritic cells and CD8 T-cells, while showing increased infiltration of Th1 cells. Surprisingly, BRCA1 expression was not associated with the survival of BC irrespective of the subtypes. Conversely, BC with low BRCA1 expression enriched cancer aggravating pathway gene sets, such as Cancer Stem Cell-related signaling (NOTCH and HEDGEHOG), Angiogenesis, Epithelial-Mesenchymal Transition, Inflammatory Response, and TGF-beta signaling. Despite being linked to heightened proliferation of cancer cells and unassertive phenotype, BRCA1 expression did not show any association with survival in BC.

IGFBP7 promotes endothelial cell repair in the recovery phase of acute lung injury.

IGFBP7 has been found to play an important role in inflammatory diseases, such as acute lung injury (ALI). However, the role of IGFBP7 in different stages of inflammation remains unclear. Transcriptome sequencing was used to identify the regulatory genes of IGFBP7, and endothelial IGFBP7 expression was knocked down using Aplnr-Dre mice to evaluate the endothelial proliferation capacity. The expression of proliferation-related genes was detected by Western blotting and RT-PCR assays. In the present study, we found that knockdown of IGFBP7 in endothelial cells significantly decreases the expression of endothelial cell proliferation-related genes and cell number in the recovery phase but not in the acute phase of ALI. Mechanistically, using bulk-RNA sequencing and CO-IP, we found that IGFBP7 promotes phosphorylation of FOS and subsequently up-regulates YAP1 molecules, thereby promoting endothelial cell proliferation. This study indicated that IGFBP7 has diverse roles in different stages of ALI, which extends the understanding of IGFBP7 in different stages of ALI and suggests that IGFBP7 as a potential therapeutic target in ALI needs to take into account the period specificity of ALI.

T cell proliferation-related subtypes, prognosis model and characterization of tumor microenvironment in head and neck squamous cell carcinoma

BackgroundThirty-three synthetic driver genes of T-cell proliferation have recently been identified through genome-scale screening. However, the tumor microenvironment (TME) cell infiltration, prognosis, and response to immunotherapy mediated by multiple T cell proliferation-related genes (TRGs) in patients with head and neck squamous cell carcinoma (HNSC) remain unclear. MethodsThis study examined the genetic and transcriptional changes in 771 patients with HNSC by analyzing the TRGs from two independent datasets. Two different subtypes were analyzed to investigate their relationship with immune infiltrating cells in the TME and patient prognosis. The study also developed and validated a risk score to predict overall survival (OS). Furthermore, to enhance the clinical utility of the risk score, an accurate nomogram was constructed by combining the characteristics of this study. ResultsThe low-risk score observed in this study was associated with high levels of immune checkpoint expression and TME immune activation, indicating a favorable OS outcome. Additionally, various factors related to risk scores were depicted. ConclusionThrough comprehensive analysis of TRGs in HNSC, our study has revealed the characteristics of the TME and prognosis, providing a basis for further investigation into TRGs and the development of more effective immunotherapy and targeted therapy strategies.

Potential involvement of KANK1 haploinsufficiency in centrosome aberrations

KANK1 was found as a tumor suppressor gene based on frequent deletions in renal cell carcinoma and the inhibitory activity of tumor cell proliferation. Previously, we reported that knockdown of KANK1 induced centrosomal amplification, leading to abnormal cell division, through the hyperactivation of RhoA small GTPase. Here, we investigated the loss of KANK1 function by performing CRISPR/Cas9-based genome editing to knockout the gene. After several rounds of genome editing, however, there were no cell lines with complete loss of KANK1, and the less the wild-type KANK1 dosage, the greater the number of cells with abnormal numbers of centrosomes and rates of cell-doubling and apoptosis, suggesting the involvement of KANK1 haploinsufficiency in centrosome aberrations. The rescue of KANK1-knockdown cells with a KANK1-expressing plasmid restored the rates of cells exhibiting centrosomal amplification to the control level. RNA-sequencing analysis of the cells with reduced dosages of functional KANK1 revealed potential involvement of other cell proliferation-related genes, such as EGR1, MDGA2, and BMP3, which have been reported to show haploinsufficiency when they function. When EGR1 protein expression was reduced by siRNA technology, the number of cells exhibiting centrosomal amplification increased, along with the reduction of KANK1 protein expression, suggesting their functional relationship. Thus, KANK1 haploinsufficiency may contribute to centrosome aberrations through the network of haploinsufficiency-related genes.

Discovery of a T cell proliferation-associated regulator signature correlates with prognosis risk and immunotherapy response in bladder cancer.

The efficacy of immunotherapy is heavily influenced by T cell activity. This study aimed to examine how T cell proliferation regulators can predict the prognosis and response to immunotherapy in patients with bladder cancer (BCa). T cell proliferation-related subtypes were determined by employing the non-negative matrix factorization (NMF) algorithm that analyzed the expression patterns of T cell proliferation regulators. Subtypes were assessed for variations in prognosis, immune infiltration, and functional behaviors. Subsequently, a risk model related to T cell proliferation was created through Cox and Lasso regression analyses in the TCGA cohort and then confirmed in two GEO cohorts and an immunotherapy cohort. BCa patients were categorized into two subtypes (C1 and C2) according to the expression profiles of 31T cell proliferation-related genes (TRGs) with distinct prognoses and immune landscapes. The C2 subtype had a shorter overall survival (OS), with higher levels of M2 macrophage infiltration, and the activation of cancer-related pathways than the C1 subtype. Following this, thirteen prognosis-related genes that were involved in T cell proliferation were utilized to create the prognostic signature. The model's predictive accuracy was confirmed by analyzing both internal and external datasets. Individuals in the high-risk category experienced a poorer prognosis, increased immunosuppressive factors in the tumor microenvironment, and diminished responses to immunotherapy. Additionally, the immunotherapeutic prediction efficacy of the model was further confirmed by an immunotherapy cohort (anti-PD-L1 in the IMvigor210 cohort). Our study characterized two subtypes linked to T cell proliferation in BCa patients with distinct prognoses and tumor microenvironment (TME) patterns, providing new insights into the heterogeneity of T cell proliferation in BCa and its connection to the immune landscape. The signature has prospective clinical implications for predicting outcomes and may help physicians to select prospective responders who prioritize current immunotherapy.

Proteogenomic characterization of difficult-to-treat breast cancer with tumor cells enriched through laser microdissection

BackgroundBreast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer death among women globally. Despite advances, there is considerable variation in clinical outcomes for patients with non-luminal A tumors, classified as difficult-to-treat breast cancers (DTBC). This study aims to delineate the proteogenomic landscape of DTBC tumors compared to luminal A (LumA) tumors.MethodsWe retrospectively collected a total of 117 untreated primary breast tumor specimens, focusing on DTBC subtypes. Breast tumors were processed by laser microdissection (LMD) to enrich tumor cells. DNA, RNA, and protein were simultaneously extracted from each tumor preparation, followed by whole genome sequencing, paired-end RNA sequencing, global proteomics and phosphoproteomics. Differential feature analysis, pathway analysis and survival analysis were performed to better understand DTBC and investigate biomarkers.ResultsWe observed distinct variations in gene mutations, structural variations, and chromosomal alterations between DTBC and LumA breast tumors. DTBC tumors predominantly had more mutations in TP53, PLXNB3, Zinc finger genes, and fewer mutations in SDC2, CDH1, PIK3CA, SVIL, and PTEN. Notably, Cytoband 1q21, which contains numerous cell proliferation-related genes, was significantly amplified in the DTBC tumors. LMD successfully minimized stromal components and increased RNA–protein concordance, as evidenced by stromal score comparisons and proteomic analysis. Distinct DTBC and LumA-enriched clusters were observed by proteomic and phosphoproteomic clustering analysis, some with survival differences. Phosphoproteomics identified two distinct phosphoproteomic profiles for high relapse-risk and low relapse-risk basal-like tumors, involving several genes known to be associated with breast cancer oncogenesis and progression, including KIAA1522, DCK, FOXO3, MYO9B, ARID1A, EPRS, ZC3HAV1, and RBM14. Lastly, an integrated pathway analysis of multi-omics data highlighted a robust enrichment of proliferation pathways in DTBC tumors.ConclusionsThis study provides an integrated proteogenomic characterization of DTBC vs LumA with tumor cells enriched through laser microdissection. We identified many common features of DTBC tumors and the phosphopeptides that could serve as potential biomarkers for high/low relapse-risk basal-like BC and possibly guide treatment selections.

Abstract PO5-14-09: Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients?

Abstract Background: Intratumor heterogeneity (ITH) refers to the presence of distinct cancer cell populations with different clones within a single tumor mass. Tumors with high ITH are more prone to progression, exhibit resistance to therapies, and are associated with an unfavorable prognosis. Therefore, evaluation of ITH is anticipated to serve as a breakthrough biomarker for prognosis and treatment strategies. Several computational algorithms have been proposed to quantify ITH. However, with the exception of DEPTH2, all of these algorithms require both DNA and RNA-sequence data from the same tumor, which can be challenging. In this study, we investigated whether DEPTH2 which solely analyzes RNA-sequence data, accurately reflects the biological characteristics of ITH. Methods: We analyzed clinicopathological and gene expression data from a total of 6573 breast cancer patients from several large independent cohorts: METABRIC (n = 1903), GSE96058 (n = 3273), GSE21974 (n = 57), GSE28844 (n = 61), GSE87455 (n = 153), GSE180280 (n = 57), and The Cancer Genome Atlas (TCGA, n = 1069). Results: DEPTH2 score was high in Triple Negative Breast Cancer (TNBC) subtype (p < 0.001), but no consistent correlation was observed with lymph node or distal metastasis. Higher DEPTH2 score was associated with cell proliferation, as evidenced by higher Nottingham histological grade and Ki67 gene expression (both p < 0.001). In agreement, high DEPTH2 tumors enriched all cell proliferation-related Hallmark gene sets such as E2F targets, G2M checkpoint, MYC targets v1 and v2, and Mitotic Spindle (FDR < 0.25). Additionally, high DEPTH2 tumors exhibited reduced immune cell infiltrations, such as CD8, CD4, Th1, Th2, B cells, and macrophages, and increased infiltrations of stromal cells, such as adipocytes, fibroblasts, blood and lymphatic endothelial cells (all p < 0.05). In TCGA cohort, DEPTH2 score exhibited a similar trend to intratumoral heterogeneity, homologous recombination deficiency, fraction altered, silent and nonsilent mutations, as well as SNV and Indel neoantigens. However, the Pearson’s correlation coefficient was very weak (p < 0.05). DEPTH2 score decreased in the GSE21974 and GSE87455 cohorts after neoadjuvant chemotherapy (NAC), whereas no change was observed in the GSE28844 and GSE180280 cohorts. DEPTH2 high tumors showed a lower rate of pathologic complete response (pCR) after NAC in estrogen receptor (ER)+HER2- subtype across all cohorts, however, opposite pattern was observed in the HER2+ and TNBC subtypes in all cohorts. Lastly, high DEPTH2 score was associated with lower disease free and overall survival in METABRIC and GSE96058 (p < 0.05), but not in the TCGA cohort. Conclusions: Our findings suggest that DEPTH2 score may not completely capture the biological characteristics associated with intratumor heterogeneity. Citation Format: Kohei Chida, Rongrong Wu, Arya Mariam Roy, Takashi Ishikawa, Kazuaki Takabe. Does transcriptome based DEPTH2 score reflect intratumor heterogeneity in breast cancer patients? [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-09.

Abstract PO3-13-12: ER-positive/HER2-negative breast cancer in Adolescent & Young Adult (AYA) age group has a specific biological feature and worse outcome compared to other generations

Abstract Background: Adolescents & Young Adults (AYA) is commonly defined as patients younger than 40 years old. Breast cancer patients in AYA has been reported to have poor outcome compared to other generation, however, the clinicopathological and biological features of AYA breast cancer remains controversial partly because many previous studies did not consider the difference in subtypes by age. We aim to clarify the features of AYA compared with the other age group in ER-positive/HER2-negative breast cancer, assuming the effects of age-related hormonal status. Material and Methods: Each analyzed cohort were divided into four age groups; AYA (15-39yo), perimenopausal (40-55yo), menopausal (55-65yo), and old (65+yo). Clinicopathological and biological features were analyzed using gene set variation analysis and the xCell algorithm using mRNA expression profiles of large independent public databases (METABRIC; n = 1,903, GSE96058; n = 3,273). Results: We found that both clinical and pathological T-category of AJCC staging were larger in AYA and Old compared from the other groups. Pathological N-category as well as pathological Stage were higher in AYA. Nottingham histological grade 3 was significantly higher in the AYA compared with the other older groups. Among the subtypes, AYA breast cancer was significantly associated with higher rate of triple-negative breast cancer compared to the others, as expected. Considering the whole cohort, ER and PgR positivity trended to be lower, but HER2-positivity trended to be higher in AYA. Within the ER-positive/HER2-negative subtype, PgR positivity was higher in the AYA and Perimenopausal groups compared to others. AYA had poorer disease-specific survival than other groups (p = 0.010 and p = 0.002, respectively), as well as overall survival, especially compared to perimenopausal group. The survival difference was more pronounced in ER-positive/HER2-negative breast cancer patients. In biological analysis in ER-positive/HER2-negative subtype, we found that estrogen response late signaling level decreased as the age group gets older. AYA breast cancer had significant enrichment in cell proliferation-related gene sets (G2M checkpoint, E2F targets, and MYC target v1), other pro-cancerous gene sets (MTORC1, unfolded protein response, and PI3K/ACT/MTOR signaling), consistently in two cohorts (all p ≦ 0.028), when compared to the other age groups. Interestingly, the features were observed even in the small tumor size (T1/T2) subgroup. Furthermore, AYA breast cancer showed significantly high BRCAness (all p≦0.002) and DNA repair (all p≦0.023) compared to other age groups. The infiltration fraction of several immune cells: CD8+ T cells, M1 macrophages, regulatory T cells, helper type 2 T cells, were significantly higher, and M2 macrophages were significantly lower in AYA, consistently in two cohorts. Conclusion: ER-positive/HER2-negative breast cancer in AYA was highly proliferative with high immune cell infiltrations compared to other generations. This may be related to the difference in outcomes in each age group. Considering these unique features may enable us to fine tune treatment strategies for AYA. Citation Format: Masanori Oshi, Akimitsu Yamada, Mahato Sasamoto, Kei Kawashima, Yoshie Fujiwara, Shoko Adachi, Shinya Yamamoto, Kazutaka Narui, Takashi Ishikawa, Itaru Endo, Kazuaki Takabe. ER-positive/HER2-negative breast cancer in Adolescent & Young Adult (AYA) age group has a specific biological feature and worse outcome compared to other generations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-12.

Abstract PO3-15-10: Enhanced Cancer Cell Proliferation and Aggressive Phenotype Counterbalance in Breast Cancer with High BRCA1 Gene Expression

Abstract Introduction: It is estimated that 5–10% of all breast cancer cases have a hereditary component with BRCA1 mutation being one of the most frequently observed gene mutations in breast cancer patients. BRCA1 plays a crucial role in DNA repair, and its mutation has been extensively studied. However, the clinical significance of BRCA1 gene expression remains largely unexplored. Given that heightened DNA repair mechanisms can potentially increase cancer cell proliferation, we hypothesized that breast cancer with high BRCA1 gene expression might be associated with aggressive tumor biology and worse survival. Methods: The clinical, pathological and gene expression levels of a total of 6,245 breast cancer patients were analyzed from three large independent cohorts: METABRIC (n = 1,903), GSE96058 (n = 3,273), and The Cancer Genome Atlas (TCGA, n = 1,069). The data was retrieved through cBioPortal. The xCell algorithm was used to correlate the BRCA1 gene expression with the stromal and immune cell fractions infiltrating the tumor microenvironment (TME). Results: BRCA1 gene expression was higher in breast cancer patients without BRCA1 mutation, and it correlated with the DNA repair activity (p = 0.026 and p < 0.01 respectively), however, it did not correlate with BRCA2 gene expression (p = 0.38). Breast cancer with high BRCA1 gene expression was associated with cancer cell proliferation as evidenced by higher Nottingham histological grade (p < 0.001), Ki67 expression (p < 0.001), significant enrichment of cell proliferation-related gene sets such as E2F Targets, G2M Checkpoint, MYC Targets V1 and V2, Mitotic Spindle (all FDR < 0.25), and significantly less infiltration of stromal cells such as adipocytes, fibroblasts, microvascular and lymphatic endothelial cells, and pericytes (all p < 0.05). High BRCA1 gene expression was associated with higher homologous recombination deficiency, intratumor genomic heterogeneity, and fraction altered (all p < 0.001); however, there was no correlation observed with mutation rates or neoantigens. It was observed that breast cancer with high BRCA1 gene expression had less infiltration of CD8 T cells, dendritic cells, regulatory T cells, and B cells and had higher infiltration of Th1 cells (all p < 0.05). To our surprise, there was no significant difference in overall survival regardless of the subtypes (except for ER+/HER2- subtype in METABRIC cohort, p = 0.03). Breast cancer with low BRCA1 gene expression enriched cancer aggravating pathway gene sets such as Cancer Stem Cell-related signaling pathways (NOTCH and HEDGEHOG), Angiogenesis, Epithelial Mesenchymal Transition, Inflammatory Response, and TGF-beta signaling (all FDR < 0.25). Conclusion: Contrary to our initial hypothesis, we did not find any significant association between BRCA1 gene expression and survival outcomes in breast cancer patients. Based on the results of our study, we speculate that the interplay between enhanced cancer cell proliferation and aggressive tumor characteristics may counterbalance the effect of high BRCA1 expression in breast cancer. Citation Format: Kohei Chida, Masanori Oshi, Arya Mariam Roy, Itaru Endo, Kazuaki Takabe. Enhanced Cancer Cell Proliferation and Aggressive Phenotype Counterbalance in Breast Cancer with High BRCA1 Gene Expression [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-15-10.

Abstract PO3-25-03: Can intratumoral microRNA-125a levels be a prognostic biomarker of a ER-positive breast cancer patients？

Abstract Background: MicroRNA-125a (miR-125a) has been shown to function as tumor suppressive miRNA by pre-clinical in vitro studies. However, to the best of our knowledge, there has been no study that investigated the clinical relevance of miR-125a in breast cancer patients. We hypothesized that miR-125a expression in breast cancer associate with its aggressiveness and can be a prognostic biomarker. Material and Methods: Non-tumorigenic epithelial cell line, MCF-10A and breast cancer cell lines, MCF-7, T47D, and MDA-MB231, were used to analyze the expression levels of miR-125a as well as cell viability. The data of 2042 patients were extracted from publicly available databases, The Molecular Taxonomy of Breast Cancer International Consortium (METARBRIC), The Cancer Genome Atlas (TCGA), and GSE57897. These databases included clinicopathological and survival data as well as transcriptome data. The survival analysis, gene set enrichment analysis (GSEA) were conducted comparing miR-125a high expressing and low expressing tumors, divided by the median cutoff. The association between the miR-125a expression and tumor immune microenvironment was assessed by xCell. Results: The expression levels of miR-125a were suppressed in breast cancer cell lines, MCF-7, T47D, and MDA-MB231compared with MCF-10A. This was concordant with human samples. The expression levels of miR-125a were lower in tumors compared with normal breast tissues in both TCGA and GSE57897 cohorts. These results were in agreement with the notion that it is a tumor suppressive miRNA (p< 0.001 and p< 0.001, respectively). ER-positive/HER2-negative (ER+/HER2-) showed the highest miR-125a expression among the subtypes in TCGA (p< 0.001). MiR-125a expression was not associated with cancer staging in any of the subtypes in neither TCGA nor METABRIC cohorts. To our surprise, high miR-125a tumors demonstrated worse disease free survival (DFS), disease specific survival (DSS), and overall survival (OS) compared with low expressing in ER+/HER2- breast cancer patients (p=0.008, p=0.005, and p=0.037) which was not the case for the other subtypes in METABRIC cohort. Interestingly, miR-125a expression was associated with Nottingham histological grade only in ER+/HER2- among the subtypes (p< 0.001). Also, high miR-125a tumors significantly demonstrated higher expression of MKI67, one of the most commonly used parameters for cell proliferation, correlated with miR-125a expression in ER+/HER2- (p=0.02), but not in other subtypes. Furthermore, high miR-125a tumors enriched four out of five cell proliferation-related gene sets in Hallmark collection, such as E2F Targets, G2M Checkpoint, Mitotic Spindle, and MYC Targets V2 in ER+/HER2- subtype, but not in TNBC. This was also the case in immune-related gene sets; interferon-alpha response and interferon-gamma response that enriched to high miR-125a tumors in ER+/HER2-, but not in TNBC. Anti-tumor immune cells, CD8 cells, T-helper type 1 cells, and M1 macrophages, were all elevated in high miR-125a tumors in ER+/HER2- subtype (all p< 0.03), but none in TNBC. Also, pro-cancerous immune cell such as T-helper type 2 cells were similarly highly infiltrated with high miR-125a tumors only in ER+/HER2- subtype (p=0.026). Given its association with the survival outcome, we cannot help but speculate that miR-125a expression is parallel with highly proliferative ER+/HER2- breast cancer, but its tumor suppressive effect is not enough to improve survival outcome. Conclusion: High miR-125a levels in tumors were associated with aggressive biology and worse survival, thus it can be a candidate for a prognostic biomarker in ER+/HER2- patients. Citation Format: Yoshihisa Tokumaru, Masanori Oshi, Mai Okawa, Saki Ando, Yoshimi Niwa, Ryutaro Mori, Kazuaki Takabe, Manabu Futamura. Can intratumoral microRNA-125a levels be a prognostic biomarker of a ER-positive breast cancer patients？ [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-03.

Cytoplasmic tail of transmembrane Dscam controls antibacterial responses by regulating cell proliferation-related genes in hemocytes of Chinese mitten crab (Eriocheir sinensis)

In arthropods, the involvement of Dscam (Down syndrome cell adhesion molecule) in innate immunity has been extensively demonstrated. Its cytoplasmic tail contains multiple conserved functional sites, which indicates its involvement in different intracellular signaling pathways. In this study, we focused on the role of the cytoplasmic tail of Dscam in the Chinese mitten crab (Eriocheir sinensis) immune defense. In the group with cytoplasmic tail knockdown (the site was located on constant exons 37 and 38), 3885 differentially expressed genes (DEGs) were identified. The DEGs were enriched in small molecule binding, protein-containing complex binding, and immunity-related pathways. The expression of selected genes were validated using quantitative real-time reverse transcription PCR. We identified key Cell cycle, Janus kinase (JAK)-signal transducer, activator of transcription (STAT) and mitogen-activated protein kinase (MAPK) signaling pathway genes, the results indicated that the cytoplasmic tail of Dscam controls antibacterial responses by regulating cell proliferation-related genes in hemocytes.

Moringa oleifera Leaf Powder – Silver Nanoparticles (MOLP-AgNPs) efficiently inhibit metastasis and proliferative signaling in HT-29 human colorectal cancer cells

Colorectal cancer is the second most common cancer which caused large number of deaths worldwide. It has been widely known that its undergoing therapies were related to cell metastasis and drug resistance. In line with the endeavor to reduce the side effects of those therapies, bio-fabricated silver nanoparticles (AgNPs), which are synthesized using plant extracts, are massively explored due to their potential as anticancer agents. Specific analytical techniques were used to characterize the AgNPs, such as UV–visible spectrophotometry (UV–Vis), Particle sizer (PSA), Zeta potential, FT-IR and TEM. HT-29 cells were treated at different single-dose concentrations and combined treatment using Doxorubicin as a well-established cancer drug. Based on the MTT assay result, Moringa oleifera Leaf Powder – Silver Nanoparticles (MOLP-AgNPs) induced significant dose-dependent results according to decreased HT-29 cell viability and proliferation. MOLP-AgNPs reduced cell cycle and proliferation-related genes, such as Ki-67, Wnt, β-catenin, and Cyclin D1 in a dose-dependent manner. Furthermore, MOLP-AgNPs also reduced the expression of metastasis-related genes, such as TGF-β and Snail, known as metastasis inducers. These findings suggest that MOLP-AgNPs could be considered a potential strategy for colon cancer treatment.

High Ki67 Gene Expression Is Associated With Aggressive Phenotype in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) with high Ki67 protein expression, the most commonly used cell proliferation marker, is associated with an aggressive biologic phenotype; however, conventional immunostaining is hampered by variability in institutional protocol, specific antibody probe, and by assessor subjectivity. To this end, we hypothesized that Ki67 gene (MKi67) expression would identify highly proliferative HCC, and clarify its association with oncologic outcome, tumor progression, and immune cell population in the tumor microenvironment (TME). Furthermore, we sought to identify the cell-cycle gene expression profile that confers this aggressive phenotype. A total of 473 HCC patients with clinicopathological data associated with transcriptome were selected for this study: 358 patients from The Cancer Genome Atlas (TCGA) as the testing cohort, and 115 from GSE76427 as the validation cohort. Each cohort was divided into a highly proliferative group (MKi67-high) and the low MKi67 group (MKi67-low) by the median of Ki67 gene (MKi67) expression levels. MKi67-high HCC patients had worse disease-free survival (DFS), disease-specific survival (DSS), and overall survival (OS) independent of histological grade in the TCGA cohort. MKi67 expression correlated with histological grade and tumor size. MKi67 expression increased throughout the HCC carcinomatous sequence from normal liver, cirrhotic liver, early HCC, and advanced HCC. MKi67-high HCC was associated with higher intratumor heterogeneity, homologous recombination deficiency, and altered fraction as well as intratumoral infiltration of T helper type 1 (Th1) and Th2 cells, but lower interferon-gamma response and M2 macrophage infiltration. Cell proliferation-related gene sets in the Hallmark collection (E2F targets, G2M checkpoint, Myc target v1 and mitotic spindle), MTORC1 signaling, DNA repair, PI3K MTOR signaling, and unfolded protein response were all enriched in the MKi67-high HCC (false discovery rate (FDR) < 0.25). High MKi67 gene expression identified highly proliferative HCC with aggressive biology involving classical pathways in cell cycle regulation and DNA repair, as well as poor overall oncologic outcomes. This suggests potential for personalized treatment strategies, but validation and refinement of these observations require further research to elucidate the underlying mechanisms and validate therapeutic targeting of these pathways in MKi67-high HCC tumors.

Spinster Homologue 2 Expression Correlates With Improved Patient Survival in Hepatocellular Carcinoma Despite Association With Lymph-Angiogenesis.

Spinster homologue 2 (SPNS2) is a transporter of sphingosine-1-phosphate (S1P), a bioactive lipid linked to cancer progression. We studied the link between SPNS2 gene expression, tumor aggressiveness, and outcomes in patients with hepatocellular carcinoma (HCC). Gene expression in patients with HCC was analyzed from the Cancer Genome Atlas (TCGA) (n = 350) and GSE76427 (n = 115) as a validation cohort, as well as liver tissue cohort GSE6764 (n = 75). High-SPNS2 HCC was significantly associated with high level of lymph-angiogenesis-related factors. SPNS2 expression was significantly higher in normal liver and early HCC versus advanced HCC (P < 0.02). High SPNS2 levels enriched immune response-related gene sets; inflammatory, interferon (IFN)-α, IFN-γ responses, and tumor necrosis factor (TNF)-α, interleukin (IL)-6/Janus kinase/signal transducer and activator of transcription (JAK/STAT3) signaling, complement and allograft rejection, but did not significantly infiltrate specific immune cells nor cytolytic activity score. High-SPNS2 HCC enriched tumor aggravating pathway gene sets such as KRAS (Kirsten rat sarcoma virus) signaling, but inversely correlated with Nottingham histological grade, MKI67 (marker of proliferation Ki-67) expression, and cell proliferation-related gene sets. Further, high-SPNS2 HCC had significantly high infiltration of stromal cells, showing that low-SPNS2 HCC is highly proliferative. Finally, high-SPNS2 HCC was associated with better disease-free, disease-specific, and overall survival (P = 0.031, 0.046, and 0.040, respectively). Although SPNS2 expression correlated with lymph-angiogenesis and other cancer-promoting pathways, it also enriched immune response. SPNS2 levels were higher in normal liver compared to HCC, and inversely correlated with cancer cell proliferation and better survival. SPNS2 expression may be beneficial in HCC patients despite detrimental in-vitro effects.

Enhanced epithelial-mesenchymal transition signatures are linked with adverse tumor microenvironment, angiogenesis and worse survival in gastric cancer.

Epithelial-mesenchymal transition (EMT) is a crucial mechanism that facilitates cancer cell metastasis. Despite its importance, the clinical significance of EMT in gastric cancer (GC) patients has yet to be clearly demonstrated. For gauging the extent of EMT in GC, we employed gene set variation analysis to score 807 patient samples from two large cohorts: TCGA and GSE84437. In both cohorts, EMT high GC showed a significant association with worse overall survival (hazard ratio (HR) = 1.74, p = 0.011 and HR = 2.01, p < 0.001, respectively). This association was stronger when considering the EMT signature score compared to the individual expressions of EMT-related genes (CDH1, CDH2, VIM, and FN1). While the EMT signature level did not differ among various cancers, high EMT signature specifically correlated with survival in GC alone. Mucinous and diffuse histological types exhibited higher EMT levels compared to others (p < 0.001), and the EMT signature level was correlated with tumor depth and AJCC stage (all p < 0.001). Interestingly, the EMT score was an independent factor for overall and disease-specific survival (multivariate; p = 0.006 and 0.032, respectively). EMT high GC displayed a lower fraction of Th1 cells and a higher fraction of dendritic cells, M1 macrophages and several stromal cells. EMT high GC exhibited an inverse correlation with cell proliferation-related gene sets. While they significantly enriched multiple pro-cancerous gene sets, such as TGF-β signaling, hypoxia, and angiogenesis. The presence of EMT signature in a bulk tumor was linked to TGF-β signaling, hypoxia, and angiogenesis, and was also associated with poorer survival outcomes in GC patients.

Abstract 2569: VEGFA gene expression is associated with cell proliferation, less immune cell infiltration, and worse survival, but better response to chemotherapy and immunotherapy

Abstract Introduction: Vascular endothelial growth factor-A (VEGFA) is a well-known factor that induces angiogenesis, one of the hallmarks of cancer, which generates new blood vessels in the tumor microenvironment (TME) and provides conduit for cancer to metastasize. Interestingly, an anti-VEGFA drug (Avastin) failed to show benefit in clinical trials for breast cancer (BC). In this study we investigated the clinical relevance of VEGFA gene expression in BC. Methods: A total of 7336 BC patients from eight independent cohorts, TCGA (n= 1073), METABRIC (n= 1903), SCAN-B (GSE96058, n= 3069), GSE20194 (n=278), GSE34138 (n=178), GSE163882 (n=222), GSE25066 (n=508), and ISPY2 (GSE173839, n= 105) were analyzed. These cohorts were divided into high and low VEGFA gene expression groups using the median. Results: High VEGFA expression in BC was associated with worse overall survival in SCAN-B cohort which was validated in METABRIC cohort with overall, disease-specific, and disease-free survival (all p&amp;lt;0.02). VEGFA expression was higher in Triple Negative BC (TNBC) but was lower in BC with lymph node metastasis. High VEGFA BC was associated with significantly high intratumoral heterogeneity, homologous recombination defect, silent and non-silent mutations, and SNV neoantigens in TCGA, and was also associated with higher cell proliferation, evidenced by higher Nottingham histological grade, higher Ki67 gene expression, and enrichment of all the Hallmark cell proliferation-related gene sets: Mitotic spindle, G2M Checkpoint, E2F Targets, MYC Targets v1 and v2, and mTorC1 signaling, consistently, in SCAN-B, METABRIC, and TCGA cohorts. Reflecting its enhanced growth, high VEGFA expression was associated with less infiltration of fibroblasts and adipocytes (all p&amp;lt;0.05). Surprisingly, VEGFA expression did not enrich angiogenesis gene set in any of the cohorts, nor was related with infiltrations of blood or lymphatic vascular endothelial cells, besides pericytes. But high VEGFA BC was associated with significantly less infiltrations of anti-cancer immune cells; CD8, CD4, and dendritic cells were all significantly less, and T helper type 1 cells and regulatory T cells were significantly higher consistently in SCAN-B, METABRIC, and TCGA cohorts. VEGFA expression was significantly higher in BC that achieved pathological complete response after Anthracycline and Taxane based neoadjuvant therapy in both ER+/HER2- and TNBC subtypes in the GSE25066 cohort, and after immunotherapy in ER+/HER2- subtype, but not TNBC, in the ISPY2 clinical trial cohort. This was not validated in GSE20194, GSE34138, GSE163882 cohorts. Conclusion: Our research indicates that high VEGFA BC is associated with high cell proliferation, less immune cell infiltration, and worse survival, but better response to Anthracycline and Taxane based chemotherapy, and immunotherapy. Citation Format: Pia Sharma, Kohei Chida, Kenichi Hakamada, Kazuaki Takabe. VEGFA gene expression is associated with cell proliferation, less immune cell infiltration, and worse survival, but better response to chemotherapy and immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2569.

Abstract 2520: CD133 expression is associated with less DNA repair, better response to chemotherapy, and survival in ER-positive/ER-negative breast cancer

Abstract Introduction: Cancer stem cells (CSC) constitute only 0.1-1% of all the cells in a bulk tumor, yet they play a crucial role in treatment resistance and relapses. Its role in breast cancer (BC) has been studied but a consensus remains elusive. We focused on gene expression of a CSC marker CD133 and studied its clinical significance in ER-positive/HER2-negative (ER+/HER2-) BC, the most prevalent subtype. We utilized multiple independent large patient cohorts of gene expression and clinical variables for transcriptome analysis. Methods: A total of 2969 patients from The Cancer Genome Atlas (TCGA, n=1065) and the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC, n=1904) BC cohorts, as well as BC patients who underwent neoadjuvant chemotherapy (NAC; GSE25066, GSE20194, GSE32646 cohorts) were analyzed. Results: By analyzing SCP1039 and SCP1106 single-cell sequencing cohorts, we found that epithelial cells were the predominant source of CD133 gene expression in the tumor microenvironment. High expression of CD133 in ER+/HER2- BC correlated with elevated gene expressions of the other CSC markers; CD24, NOTCH1, DLL1, and ALDH1A1 (all p&amp;lt;0.001), and significantly enriched WNT/β-Catenin, Hedgehog, and Notch signaling (all FDR&amp;lt;0.25), which are all known pathways related with CSC in TCGA and validated in METABRIC cohort. Consistent with CSC phenotype, CD133-high BC was negatively enriched with Hallmark cell proliferation-related gene sets, such as E2F Targets, G2M Checkpoint, MYC Targets V1, Ki67 gene expression and proliferation score (FDR&amp;lt;0.25 and p&amp;lt;0.001, respectively) in both cohorts. CD133-high BC was negatively associated with DNA repair genes, such as BRCA1, E2F7 in both cohorts (all p&amp;lt;0.001). In agreement, CD133-high BC was associated with less homologous recombination deficiency, lower silent and non-silent mutation rates. Surprisingly, CD133-high BC exhibited a proinflammatory microenvironment, as represented by higher cytolytic activity in the METABRIC and higher lymphocyte infiltrations, TGF-beta response, and TCR richness in TCGA (both p&amp;lt;0.001), but less Th1 cells, M1 macrophages and dendritic cell infiltrations consistently in both cohorts. It also enriched signaling pathways related to inflammation and immune responses such as inflammatory response, allograft rejection, TNF-alpha, IL-6, TGF-beta signaling, and complement signaling (all FDR&amp;lt;0.25). CD133-high tumors had better pathological complete response after NAC in GSE25066 cohort and improved Disease-Free Survival and Overall Survival in both TCGA and METABRIC cohorts (all p&amp;lt;0.05). Conclusion: CD133-high ER+/HER2- BC is associated with CSC phenotype, reduced cell proliferation, and impaired DNA repair, but enhanced inflammation, potentially explaining the better response to NAC and favorable patient prognosis. Citation Format: Takumi Sato, Masanori Oshi, Jing Lee, Arya M. Roy, Kohei Chida, Itaru Endo, Masato Obayashi, Kazuaki Takabe. CD133 expression is associated with less DNA repair, better response to chemotherapy, and survival in ER-positive/ER-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2520.

Abstract 2568: High infiltration of neurons was associated with lower cell proliferation and less immune response but not with response to treatments or survival in estrogen receptor-positive breast cancer

Abstract Introduction: Psychological stress aggravates cancer cell biology which leads to cancer progression and metastasis through its influence on the sympathetic nervous system. Recently, infiltration of a local anesthetic lidocaine around a tumor prior to its removal was reported to lead to better survival in early-stage breast cancer (BC). Therefore, we hypothesized that neuron infiltration within the bulk tumor of BC could be associated with cancer aggressiveness and worse survival. Methods: A total of 5755 BC patients from five independent cohorts, including METABRIC (n=1903), SCAN-B (n=3273), GSE25066 (n=508), and ISPY2 (GSE173839; n=105), which have both clinical and transcriptomic data, were included in the study. The fraction of neuron infiltration was calculated using the xCell algorithm. Results: High neuron infiltration in BC was not associated with survival in either the METABRIC or SCAN-B cohorts. BC with high neuron infiltration had high expressions of neurotransmitter receptors: adrenergic receptor α1B and 2A, dopamine receptor D2, muscarinic acetylcholine receptor M3 and M4, and TAC1, which encodes TACR1, the receptor for substance P, but had lower expression of adrenergic receptor β2 and cholinergic acetylcholine receptor 1 and 10. Further, levels of neuron infiltration were highest in estrogen receptor-positive/HER2-negative (ER+/HER2-) among the subtypes and lower in tumors with lymph node metastasis, but was not associated with tumor size. Neuron infiltration was associated with higher AJCC staging in METABRIC, which was not validated in SCAN-B cohort. High neuron infiltration in BC was associated with reduced homologous recombination defects, mutation rate, and SNV neoantigen, and with less cell proliferation, evidenced by lower Nottingham histological grade and Ki67 gene expression, as well as less enrichment of Hallmark cell proliferation-related gene sets: E2F targets, G2M checkpoint, and Mitotic spindle. Despite its association with less cell proliferation, BC with high neuron infiltration was not associated with pathological complete response (pCR) in the GSE25066 cohort. High neuron infiltration in BC was associated with higher infiltration of lymph endovascular cells as well as less immune cell infiltration; CD8, CD4 memory, T-helper 1 and 2, dendritic cells, M1 and M2 macrophages, regulatory T cells, and B cells, lower cytolytic activity, and immune response; interferon-alpha and interferon-gamma and IL2 stat signaling. Despite this association, BC with high neuron infiltration was not associated with pCR after immunotherapy in the ISPY2 clinical trial cohort. Conclusion: BC with high infiltration of neurons was associated with lower cell proliferation and less immune response but not with response to systemic therapies or patient survival in ER+/HER2- subtype. Citation Format: Kohei Chida, Masanori Oshi, Arya Mariam Roy, Jun Arima, Pia Sharma, Itaru Endo, Kenichi Hakamada, Kazuaki Takabe. High infiltration of neurons was associated with lower cell proliferation and less immune response but not with response to treatments or survival in estrogen receptor-positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2568.

Abstract 2570: Gastric cancer with high neuron infiltration was associated with enhanced epithelial-to-mesenchymal transition, angiogenesis and poor clinical outcomes

Abstract Introduction: Neurons within the tumor microenvironment of gastric cancer (GC), as identified through immunohistochemistry, have been reported to be associated with worse overall survival (OS) of GC patients. However, histology-based analyses have limitations in quantifying nerve cells and investigating the underlying biology. Thus, we aim to study the clinical significance of intratumoral nerve cells in GC using computational deconvolution of transcriptomes from bulk tumors. Methods: Total of 807 GC patients from two independent cohorts (TCGA; n = 375, GSE84437; n = 432) were included, encompassing both clinical and transcriptomic profiles. The neuron infiltration fraction was determined using the xCell algorithm. Results: GC with high neuron infiltration was associated with high expressions of neurotransmitter receptors; beta-adrenaline, dopamine, muscarinic acetylcholine, and neurokinin receptors in TCGA cohort (all p &amp;lt; 0.02). It was associated with reduced mutation rate and increased intratumoral heterogeneity (all p ≦ 0.002), as well as less immune response; interferon-alpha and interferon-gamma, and with low infiltration of immune cells; CD4 memory, T-helper 1, M1 and M2 macrophages, and NK cells (all p ≦ 0.02). Further, neuron highly infiltrated GC enriched Epithelial-to-Mesenchymal Transition and Angiogenesis gene sets and had higher lymphatic and microvessel endothelial cells and pericyte infiltrations (all p ≦ 0.001). Conversely, GC with high neuron infiltration showed an inverse relationship with cell proliferation-related gene sets; E2F Targets, G2M Checkpoint, and MYC Targets v1 and v2, and had lower Ki67 gene expression (FDR &amp;lt; 0.25 and p &amp;lt; 0.001, respectively). High neuron infiltration in GC was associated with worse patient OS in TCGA cohort, which was validated in GSE84437 cohort (both p &amp;lt; 0.05). It was an independent prognostic factor of OS in both univariate and multivariate Cox-regression analysis in the TCGA cohort (hazard ratio [HR]: 1.46; 95% confidence interval [CI]: 1.01 to 2.12; p = 0.045 and [HR]:1.54; 95% CI: 1.05 to 2.26; p = 0.026). Conclusion: Our research indicates that high infiltration of neurons in GC correlates with high expressions of neurotransmitter receptors, high intratumoral heterogeneity, less immune response and immune cell infiltrations, and less cell proliferation. It enriched EMT and angiogenesis which may explain its association with worse patient survival. Citation Format: Kohei Chida, Masanori Oshi, Arya Mariam Roy, Jun Arima, Pia Sharma, Gabriella Kim Mann, Itaru Endo, Kenichi Hakamada, Kazuaki Takabe. Gastric cancer with high neuron infiltration was associated with enhanced epithelial-to-mesenchymal transition, angiogenesis and poor clinical outcomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2570.

Abstract 2549: Activity-regulated cytoskeleton-associated protein (ARC) gene expression is associated with high infiltration of stromal cells and immune cells, but with less cancer cell proliferation and better overall survival in ER-positive/HER2-negative breast cancers

Abstract Introduction: Recently peritumoral lidocaine infiltration prior to removal was reported to be associated with better survival in early-stage breast cancer (BC). This led us to hypothesize that innervation to the tumor affects its biology and patient survival. Activity-regulated cytoskeleton-associated protein (ARC) gene expression is known to be regulated by neuronal activity. Therefore, we studied the clinical relevance of ARC gene expression as a surrogate of neuronal activity in BC. Methods: Sweden Cancerome Analysis Network-Breast (SCAN-B (GSE96058), n=3273) cohort was analyzed, and the results were validated using The Cancer Genome Atlas (TCGA, n=1069). Results: ER+/HER2- and Luminal A type cancers expressed significantly higher ARC compared to the other subtypes in both cohorts (p&amp;lt;0.005). In the tumor microenvironment, the stromal cells (fibroblasts, endothelial cells and adipocytes) were all found to be significantly infiltrated in high ARC BC (p&amp;lt;0.01). Multiple immune cells were significantly infiltrated in high ARC BC, including CD8, CD4 memory cells, helper type II T cells, regulatory T cells, M1 and M2 macrophages, dendritic cells and B cells (all p&amp;lt;0.03 in both cohorts). In terms of cancer characteristics, there was no difference in silent or nonsilent mutations, single nucleotide variant or indel neoantigens between tumors with low or high ARC expression. However, high ARC BC was significantly associated with less homologous recombination deficiency, intratumor heterogeneity and fraction altered mutation rate compared to low ARC BC in TCGA (p&amp;lt;0.001). High ARC expression was significantly associated with smaller tumor size (p&amp;lt;0.001) and without lymph node metastasis in the SCAN-B cohort (p&amp;lt;0.02), and less Stage IV disease in the TCGA cohort (p&amp;lt;0.02); however, these results were not validated by the other. High ARC BC was significantly associated with lower Nottingham histologic grade and lower MKi67 expression in both cohorts (p&amp;lt;0.001). Cell proliferation-related gene sets in the Hallmark collection (E2F targets, G2M checkpoint, and MTORC1 signaling) were significantly less enriched to high ARC BC consistently in both cohorts. Overall survival (OS) was significantly better in high ARC BC in the ER+/HER2- subtype consistently in both cohorts (p&amp;lt;0.01) and when including all subtypes in the SCAN-B cohort (p&amp;lt;0.001); however this was not validated in TCGA. No significant difference in OS was found between low and high ARC gene expression in triple negative BC in either cohort. Conclusion: ARC gene expression as a surrogate of neuronal activity in BC was associated with high infiltration of stromal cells and immune cells but with less cancer cell proliferation and better overall survival, particularly in the ER+/HER2- subtype. Citation Format: Gabrielle Yee, Rongrong Wu, Takashi Ishikawa, Kazuaki Takabe. Activity-regulated cytoskeleton-associated protein (ARC) gene expression is associated with high infiltration of stromal cells and immune cells, but with less cancer cell proliferation and better overall survival in ER-positive/HER2-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2549.