Increased Ki-67 Expression Research Articles

Prognostic prediction for HER2-low breast cancer patients using a novel machine learning model

BackgroundsTo develop a machine learning (ML) model for predicting the prognosis of breast cancer (BC) patients with low human epidermal growth factor receptor 2 (HER2) expression, and to investigate the association between clinicopathological characteristics and outcomes in HER2-low BC (HLBC) patients.MethodsA retrospective analysis was conducted on data from 998 female HLBC patients treated at the Breast Center of the Fourth Hospital of Hebei Medical University (Hebei, China) between January 1, 2017, and December 31, 2020. To address class imbalance, the synthetic minority over-sampling technique was applied. Feature selection was performed using the least absolute shrinkage and selection operator, followed by construction of the prediction model using the random forest algorithm. Model performance, including specificity and accuracy, was assessed using the receiver operating characteristic (ROC) curve and confusion matrix, comparing it against other ML models. Additionally, the log-rank test was employed to examine the relationship between selected features and patient outcomes in HLBC.ResultsThe random survival forest model demonstrated superior accuracy and specificity in predicting survival outcomes for HLBC patients. Compared with other ML models, it achieved more precise predictions of Disease-Free Survival (DFS) at 1, 2, and 3 years, with the area under the ROC curve (AUC) in the test and training cohorts measured at 0.726 and 0.819, 0.712 and 0.776, and 0.685 and 0.774, respectively. The analysis further identified a strong correlation between poor prognosis in HLBC patients and factors such as axillary lymph node dissection, family history, elevated topoisomerase (TOPO)-2 expression, advanced clinical stage, negative progesterone receptor status, P53 mutation, and increased Ki67 expression, observed across both cohorts.ConclusionsA novel ML model was developed for accurate prognosis prediction in HLBC patients, offering valuable insights into prognostic risk factors. This model equips clinicians with enhanced data to guide treatment decisions, ultimately contributing to improved patient outcomes.

Adipose stem cell exosomes, stimulated by pro-inflammatory factors, enhance immune evasion in triple-negative breast cancer by modulating the HDAC6/STAT3/PD-L1 pathway through the transporter UCHL1

BackgroundTriple-negative breast cancer (TNBC) is characterized by high invasiveness and metastasis potential. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is strongly associated with breast cancer progression, although the underlying mechanisms are largely unknown.MethodsThe gene expression profiles of TNBC samples were downloaded from the TCGA database, and ubiquitination enzymes related to immune regulation were screened. UCHL1 expression in the TNBC tissues and in adipose-derived mesenchymal stem cells (ADSCs) stimulated in vitro with pro-inflammatory cytokines were analyzed. Exosomes were isolated from these stimulated ADSCs and transfected with scrambled (si-NC) or UCHL1-specific (si-UCHL1) siRNA constructs. TNBC cells were treated with the ADSCs-derived exosomes (ADSCs-Exos) and then co-cultured with macrophages or T cells. Finally, the tumorigenic potential of the ADSCs-Exos was evaluated by injecting the exosomes into mice bearing TNBC xenografts.ResultsUCHL1 was highly expressed in TNBC tissues and the stimulated ADSCs. The exosomes derived from stimulated ADSCs increased the viability and migration capacity of TNBC cells in vitro, and significantly increased Ki-67 expression through UCHL1. Furthermore, ADSCs-Exos induced M2 polarization of THP-1 monocytes by upregulating CD206 and Arg-1, and downregulating TNF-α and iNOS, and also decreased the proportion of CD3+CD8+ T cells. Mechanistically, UCHL1 regulated the STAT3 and PD-L1 signaling pathways through HDAC6. Exosomes derived from the control and cytokine-stimulated ADSCs also promoted tumor growth in vivo, and increased the expression of UCHL1, CD206, HDAC6, STAT3, and PD-L1. However, UCHL1 knockdown reversed the pro-tumorigenic effects of the ADSCs-derived exosomes in vivo and in vitro.ConclusionPro-inflammatory factors (IFN-γ + TNF-α) stimulating ADSCs-Exos enhance immune evasion in triple-negative breast cancer by regulating the HDAC6/STAT3/PD-L1 pathway via UCHL1 transporter. Thus, UCHL1 inhibition may enhance the response of TNBC to immunotherapy.Graphical

High Ki-67 Expression Predicting a Risk Factor for the Progression of Disease within 24 Months and Microenvironment in Follicular Lymphoma

Most follicular lymphomas (FLs) demonstrate an indolent clinical course with favorable outcomes; however, a fraction of patients experiences progression of disease within 24 months (POD24) and has adverse outcomes. This study aimed to determine the predictive risk factors for POD24 in patients with FL, and the characteristics of the microenvironment in FL with POD24. By multivariate analysis, we revealed that increased Ki-67 expression was associated with POD24 events in patients with FL (hazard ratio [HR]: 6.29, 95% confidence interval [CI]: 1.96–20.22, p = 0.0020). Additionally, patients with FL with POD24 demonstrated immune cell reduction by immunohistochemistry analysis. Our results help better understand the therapeutic strategies for FL with POD24.

Abstract We131: Systems-Enabled Drug Repurposing to Regulate Cardiomyocyte Proliferation

Introduction: The inability of the adult heart to regenerate results in prevalent morbidity and mortality related to myocardial infarction and heart failure. This lack of cardiac regeneration is driven by the loss of cardiomyocytes (CMs), with limited supporting endogenous CM renewal. While many individual regulators of CM proliferation have been implicated, the field lacks strategies to identify druggable proliferative signaling pathways and prioritize candidate therapeutics. Our goal is to predict and experimentally validate FDA-approved drugs and their respective mechanisms that regulate CM proliferation. Methods: A logic-based differential equation model of CM proliferation signaling was integrated with drug-target interactions from a publicly available database (DrugBank) to simulate the effect of drugs on CM proliferation phenotypes (Binucleation, Cytokinesis, DNA Replication, Mitosis, and Polyploid). A virtual screen with knockdown combinations of regulators identified signaling mechanisms responsible for mediating the effect of drugs, and literature validation was performed against published experimental data investigating the drugs or the drug targets in CMs. Neonatal rat CMs were treated with the YAP-activating drug TT10 or drugs predicted to increase CM proliferation. Proliferation was measured by high-content microscopy and DNA replication and mitosis positive cells were identified from image analysis (CellProfiler) to discern CM proliferation from endoreplication. Results: We identified 16 unique drug-target interactions predicted to regulate CM proliferation phenotypes. Four drugs were predicted to increase CM phenotypic proliferation outputs. Experimental validation confirmed lithium, a GSK3beta-antagonist, induces cell cycle activity in neonatal rat CMs as evidenced by increased Ki67 expression in cardiac Troponin T positive cells. The model predicts cyclin D is critical in mediating this response and subsequent experiments measured changes in cyclin D expression in response to lithium treatment. Retrospective analysis of clinical data (TriNetX and AERSMine) indicates more adverse events of congenital cardiac disorders were reported in patients exposed to lithium. Conclusion: We identified lithium to promote DNA replication in CMs by upregulating cyclin D. This study advances the current knowledge of CM proliferation regulators by providing insight into new pathways targeted with existing drugs that can ultimately impact cardiac regeneration.

Prognostic and clinicopathological value of Ki-67 in patients with oesophageal squamous cell carcinoma: a systematic review and meta-analysis

ObjectivesThe relationship between Ki-67 expression and the prognosis of patients with oesophageal squamous cell carcinoma (ESCC) has been extensively studied. However, their findings were inconsistent. Consequently, the present meta-analysis was...

Osteoarthritis as a Systemic Disease Promoted Prostate Cancer In Vivo and In Vitro.

Osteoarthritis (OA) is increasing worldwide, and previous work found that OA increases systemic cartilage oligomeric matrix protein (COMP), which has also been implicated in prostate cancer (PCa). As such, we sought to investigate whether OA augments PCa progression. Cellular proliferation and migration of RM1 murine PCa cells treated with interleukin (IL)-1α, COMP, IL-1α + COMP, or conditioned media from cartilage explants treated with IL-1α (representing OA media) and with inhibitors of COMP were assessed. A validated murine model was used for tumor growth and marker expression analysis. Both proliferation and migration were greater in PCa cells treated with OA media compared to controls (p < 0.001), which was not seen with direct application of the stimulants. Migration and proliferation were not negatively affected when OA media was mixed with downstream and COMP inhibitors compared to controls (p > 0.05 for all). Mice with OA developed tumors 100% of the time, whereas mice without OA only 83.4% (p = 0.478). Tumor weight correlated with OA severity (Pearson correlation = 0.813, p = 0.002). Moreover, tumors from mice with OA demonstrated increased Ki-67 expression compared to controls (mean 24.56% vs. 6.91%, p = 0.004) but no difference in CD31, PSMA, or COMP expression (p > 0.05). OA appears to promote prostate cancer in vitro and in vivo.

O07 Lower doses of retinol facilitate cell proliferation and epidermal thickening in photoaged skin

Abstract Introduction and aims Chronic exposure to solar ultraviolet radiation leads to cutaneous hypertrophic photoageing which clinically manifests as wrinkles, skin laxity and hyperpigmentation. This photoaged phenotype is underscored by a remodelled cutaneous microenvironment including epidermal thinning and rete ridge flattening. We have previously shown that cosmeceutical formulations of 0.3% and 1% retinol can effectively induce epidermal thickening and keratinocyte proliferation. However, these concentrations require careful usage to limit potential irritancy issues, highlighting a need to explore the benefits of lower dose retinol for daily skincare. Therefore, we compare the efficacy of lower doses of retinol in facilitating epidermal thickness and proliferation in photoaged skin, as this currently remains unknown. Methods Using the in vivo Manchester Patch Test Assay, test substances (vehicle and retinol at concentrations of 0.025%, 0.05%, 0.1% and 1%) were applied under occlusion to extensor forearms of healthy but photoaged volunteers (three men, three women; age range 66–86 years) for 12 days. Resultant skin biopsies were processed for histological assessment of epidermal thickness and keratinocyte proliferation. Results Epidermal thickness was significantly increased with 0.025% (P &amp;lt; 0.05), 0.05%, 0.1% and 1% (all P &amp;lt; 0.01) retinol treatment, compared with baseline or vehicle. Immunofluorescence staining of the proliferation marker Ki67 identified elevated keratinocyte proliferation following retinol treatment (P &amp;lt; 0.05 for 0.025%, 0.05% and 1%) compared with baseline. Although Ki67 expression in the 0.1% retinol treatment was elevated, significance was not achieved compared with baseline. However, compared with the vehicle, all retinol concentrations significantly increased Ki67 expression (P &amp;lt; 0.05). Clinically, 50% of volunteers showed an erythemal irritancy response to 1% retinol only. Conclusions We established that lower doses of retinol are capable of inducing keratinocyte proliferation and epidermal thickening without irritancy issues. Therefore, long-term effective treatment of photoaged skin with low-dose retinol products may be the preferred choice for individuals where retinol sensitivity has previously been an issue.

Abstract PO1-13-12: Mechanistic Analysis and Potential Therapeutic Implications in Breast Cancer with Fibrotic Focus

Abstract A fibrotic focus (FF) is a scar-like region that forms in the center of carcinoma due to excessive tumor stroma formation. In our earlier studies, we found FF as an independent poor prognostic factor for breast cancers. However, the underlying mechanisms of how the presence of FF contributed to a poor prognosis are still unknown. To improve our understanding of FF in breast cancer, we have investigated genes associated with FF and their impacts on breast cancer prognosis. Analysis of data from The Cancer Genome Atlas Program (TCGA) indicated that Bone Morphogenetic Protein 8A (BMP8A) was significantly upregulated in breast cancers with FF. To further validate this finding, immunohistochemistry (IHC) on BMP8A was performed on a local cohort of breast cancers (N= 900). Of these samples, 147 (16.3%) were found to be BMP8A positive, and there was a significant association between the presence of FF and high expression of BMP8A (p-value=0.026). We also found that high expression of BMP8A was positively correlated with apocrine phenotype, necrosis, increased Ki67 expression, and triple-negative breast cancer subtype; and most of these are poor prognostic factors for breast cancer. Kaplan-Meier survival estimate analysis revealed that high expression of BMP8A was significantly associated with poor overall survival (p-value=0.035). Gene ontology (GO) analysis showed that "Extracellular matrix organization" was the prominent biological process associated with BMP8A. KEGG pathway analysis suggested that BMP8A was significantly related to "Protein digestion and absorption", "ECM-receptor interaction", and "Focal adhesion" pathways, further validating its correlation with stromal FF. In cellular studies using MCF7 and MDA-MB-231 cell lines, overexpression of BMP8A was found to enhance breast cancer cell proliferation, invasion, and migration. Similar tumor-promoting phenotypes were exhibited when stimulating the cell lines with recombinant human BMP8A. In conclusion, our findings suggested that BMP8A was associated with FF in breast cancer, contributing at least partly to the aggressive features of these cancers. Our findings also provided new insights into the precision treatment and diagnosis of breast cancer. Further exploration of BMP8A as a therapeutic target and prognostic factor may contribute to improved management of breast cancer patients. Citation Format: Canbin Fang, Julia Y. S. Tsang, Gary M.K. Tse. Mechanistic Analysis and Potential Therapeutic Implications in Breast Cancer with Fibrotic Focus [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 PO1-13-12.

The effects of dark deprivation and chronic alcohol intoxication on the liver of rats

BACKGROUND: Nighttime exposure to artificial lighting promotes epiphyseal melatonin deficiency and disrupts the circadian cycle of most body functions in mammals. Several studies have associated these factors with the development of many liver pathologies. A considerable lack of melatonin, along with a mismatch in the daily cycle of vital processes, increases the sensitivity of the liver to alcohol-induced damage and the severity of alcoholic illness.&#x0D; AIM: This study aimed to identify the combined effect of constant lighting and chronic alcohol intoxication (CAI) on the liver structure of both sexes of rats.&#x0D; MATERIALS AND METHODS: TThe study was conducted on 120 male and 80 female outbred Wistar rats aged 6 months. The trial lasted for 3 weeks. Four groups of rats of each sex were formed (control group, CAI group, dark deprivation group, and group exposed to both factors). Preparations stained with hematoxylin and eosin were evaluated. Sudan III staining was used to assess the severity of fatty degeneration, and the expression of Ki-67 and p53 as markers of proliferation and apoptosis, respectively, was measured immunohistochemically.&#x0D; RESULTS: Chronic alcohol intoxication and dark deprivation cause similar morphological changes in the livers of male and female rats, resulting in fatty degeneration, necrosis, and increased hepatocyte apoptotic activity. The combined effect of these factors causes more dramatic changes in the structure of the liver of males, resulting in the development of cirrhotic changes in 13.3% of them and toxic hepatitis in 20%, whereas only 5% of females show signs of alcoholic hepatitis. Increased Ki-67 expression in male hepatocytes is found only when CAI and dark deprivation are combined. However, in females, an increase in Ki-67 expression is observed both alone and when these factors are combined.&#x0D; CONCLUSION: The findings allow us to conclude that female rats adapt more successfully to the independent and combined effects of CAI and dark deprivation than male rats.

Abstract A047: Tumor specific γδ T cells expand and respond to PD-1 blockade

Abstract With the breakthrough of checkpoint blockade, immunotherapies targeting PD-1/PD-L1 have had remarkable success in the clinic and are now widely used to treat a variety of malignancies. While the majority of research on T cell exhaustion and PD-1 blockade has been focused on conventional αβ T cells, the contribution of innate-like T cells such as γδ T cells to PD-1 blockade is not clear. γδ T cells are generally not restricted to conventional MHC-peptide molecules and can recognize a diverse range of ligands including phosphoantigens presented on butyrophilins and MHC class I-like family members MR1, and CD1 isoforms. Using flow cytometry, we evaluated the γδ T cell responses in tumor biopsies and peripheral blood from six MCC patients treated with pembrolizumab. Furthermore, bulk and single cell γδ TCR sequencing was used to measure diversity and clonal expansion in the blood upon PD-1 blockade. TCRs were cloned and expressed in Jurkat cells to screen for reactivity against tumor cells lines. Finally, we used CRISPR-Cas9 to knockout known γδ T cell ligands to identify potential tumor antigen recognized by γδ T cells. We identified a MCC patient who experienced a complete response to pembrolizumab treatment and had a ten-fold expansion of γδ T cells in their tumor biopsies. Tumor-infiltrating γδ T cells expressed high levels of inhibitory receptors PD-1 and TIGIT. Furthermore, γδ T cells characterized in peripheral blood were predominantly Vδ1 cells and had increased Ki-67 expression upon pembrolizumab treatment. From TCR sequencing of peripheral blood, we observed the emergence of a dominant γδ T cell clonotype that was also found in the tumor. Upon TCR gene transfer into Jurkat 76 cells, we identified a γδ TCR that was able to recognize multiple Merkel cancer cell lines. CRISPR knockout of B2M in tumor cells still retained γδ TCR reactivity, suggesting that γδ TCR recognition is independent of B2M expression. Together, these results show innate-like T cells such as γδ T cells also have the capacity to respond to checkpoint blockade. As mutations in antigen presentation can be a resistance mechanism to PD-1 blockade, our study provides the rationale to utilize γδ T cells for treating cancer variants that have escaped the immune system and warrants further investigation of the interplay between γδ T cells and checkpoint blockade. Citation Format: Scott C. Lien, Dalam Ly, S.Y. Cindy Yang, Ben X. Wang, Michael St. Paul, Ramy Gadalla, Babak Noamani, Sarah Boross-Harmer, Trevor J. Pugh, Anna Spreafico, Naoto Hirano, Albiruni R.A. Razak, Pamela S. Ohashi. Tumor specific γδ T cells expand and respond to PD-1 blockade [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A047.

The NK Cell Receptor NKp46 Acts As an Essential Regulator of ILC1 Proliferation, Effector Function and Anti-AML Activity

Innate lymphoid cells (ILCs) are tissue-resident innate immune cells that respond rapidly to infection and secrete inflammatory mediators. Group 1 includes ILC1s and NK cells. Both cell types produce IFN-γ and require T-BET for their function; however, at least in mice, ILC1s do not express EOMES while NK cells do. The NKp46 receptor, encoded by the Ncr1 gene, plays an important role in NK cells. Non-NK immune cells, including ILC1s also express NKp46. However, the role of NKp46 in these non-NK immune cells remains largely unknown. We previously reported that NKp46 deficiency reduces both the percentages and numbers of ILC1s in mice but not the proportions of NK cells, ILC2s, and ILC3s (Wang et al., Plos Biology, 2018), suggesting that NKp46 may play a unique role in ILC1s. However, the precise mechanism by which NKp46 regulates ILC1s is yet to be determined. Using RNA-seq, we identified 35 upregulated genes and 66 downregulated genes in mutant ILC1s ( Ncr1 gfp/gfp) compared to wild-type ( Ncr1 +/+)ILC1s ( p &amp;lt; 0.05). Among the genes that were differentially expressed, we noted decreased expression of the cytokine receptors Il-18r1, Il-7rα, and Il-2rα in Ncr1 gfp/gfpILC1s compared to Ncr1 +/+ILC1s. Using gene set enrichment analysis of RNA-seq data from freshly sorted ILC1s from the livers of Ncr1 +/+and Ncr1 gfp/gfpmice, we identified the top 10 pathways associated with downregulated genes. Notably, NKp46 deletion significantly affected NF-κB. We validated this finding by treating ILC1s with NKp46 antibody and found that the phosphorylation of NF-κB p65 subunit was increased. The downregulation of IL-2Rα and IL-7Rα on Ncr1 gfp/gfpILC1s led us to investigate whether NKp46 also regulates ILC1 proliferation, survival, or persistence. We observed comparable levels of apoptosis in ILC1s freshly isolated from Ncr1 +/+and Ncr1 gfp/gfpmice. Thus, lack of NKp46 does not affect ILC1 apoptosis. However, expression of the proliferation marker Ki67 was significantly downregulated in freshly isolated Ncr1 gfp/gfpILC1s compared to Ncr1 +/+ILC1s. We also intravenously ( i.v.) co-transferred equal numbers of FACS-sorted Ncr1 +/+ and Ncr1 gfp/gfpILC1s labeled with CellTrace™ Violet dye (to distinguish donor cells from host cells) into immunodeficient recipient Rag2 −/− Il2γc −/− mice. After one week, the population of Ncr1 gfp/gfpILC1s was less than those of Ncr1 +/+ILC1s in the recipient Rag2 −/− Il2γc −/− mice ( p = 0.0002, n = 5), indicating that ILC1 proliferation requires NKp46 in vivo. To investigate the functional relevance ofNKp46, we i.v. injected C1498 AML cells into Ncr1 +/+or Ncr1 gfp/gfpmice. The Ncr1 gfp/gfpmice exhibited significantly more tumor growth than the Ncr1 +/+mice. Animal survival was significantly shorter in the Ncr1 gfp/gfpmice compared to Ncr1 +/+mice ( p = 0.0023, n &amp;gt; 9). Injection of additional Ncr1 +/+ILC1s into Ncr1 +/+mice increased survival compared to animals without additional Ncr1 +/+ILC1 administration ( p = 0.0183, n = 6). Further, this injection managed to rescue the dramatic decrease in the number of ILC1s observed in the Ncr1 gfp/gfpmice. This increased survival was in stark contrast to the Ncr1 gfp/gfpmice without Ncr1 +/+ILC1 injection, or those who received Ncr1 gfp/gfpILC1 injection. We also investigated the role of NKp46 in human ILC1s. Human ILC1s isolated from peripheral blood mononuclear cells (PBMCs) were treated with a human NKp46 antibody, increasing Ki67 expression. Next, we sorted NKp46 - and NKp46 + ILC1s from human PBMCs and co-cultured them with MOLM13 AML cells for 24 h and found the human NKp46 - ILC1s were significantly less cytotoxic compared to NKp46 + ILC1s. In conclusion, NKp46 is indispensable for ILC1 expression of IL-2Rα. Deficiency of NKp46 in mice reduces IL-2Rα expression on ILC1s by downregulating NF-κB signaling, thus impairing ILC1 proliferation. In a mouse model of AML, we see the functional consequences of cellular impairment as NKp46 deficiency eliminates the ability to control tumor growth and reduces survival. These changes can be reversed by injection of sufficient numbers of NKp46 + ILC1s into mice deficient for the receptor. Human NKp46 + ILC1s also exhibit stronger cytokine production and cytotoxicity than their NKp46 -counterparts, suggesting that NKp46 plays a similar role in both humans and mice. Our findings suggest a novel strategy for activating NKp46 signaling to promote ILC1 proliferation and anti-tumor activity.

BIOM-23. IDENTIFICATION OF NOVEL BIOMARKERS FOR BRAIN TUMOR USING PRECISION MEDICINE APPROACHES: ORC6 PLAYS A CRITICAL ROLE IN GLIOBLASTOMA THERAPEUTICS

Abstract INTRODUCTION Brain tumors remain challenging to treat despite diagnostic advancements. Current approaches involving surgery, radiotherapy, and chemotherapy tend to fail due to drug resistance. Our team identified potential biomarkers associated with aggressive brain tumors, including ORC6 and GPC4. ORC6, implicated in DNA replication and overexpressed in many cancers, presents a promising therapeutic target. In this study, we leverage advanced bioinformatics and experimental validation to further elucidate ORC6's potential for cancer treatment. Material and methods: This study uses R programming and packages to identify biomarkers from genomic databases like TCGA, CGGA, and GLASS. Protein expression is also assessed via tissue microarray and immunohistochemistry. Changes in the ORC6 gene are studied using GO, KEGG, and gene set enrichment analysis techniques. The tumor microenvironment is predicted using CIBERSORT. RESULTS are presented with 3D CLARITY images, and a deep learning algorithm is introduced for potential clinical application optimization. RESULTS Utilizing bioinformatics analyses, we identified ORC6 and GPC4 as potential biomarkers impacting tumor growth. This study focuses on ORC6, which demonstrated significantly increased expression in IDH-wildtype astrocytoma versus normal brain tissue. This heightened ORC6 expression correlated with tumor grading, poor prognosis, and increased KI67 expression in all glioma subgroups. ATAC sequencing and gene set enrichment analysis revealed E2F factors as potential ORC6 regulators and showed that increased ORC6 expression affected cell proliferation signaling pathways. Single-cell sequencing data further indicated ORC6 expression correlation with tumor cells, proliferative stem cells, and M0, M1, and M2 macrophages. Additionally, high ORC6 expression was observed in several brain tumor cell lines (U87MG, U118MG, LN229, GBM8401) that could be down-regulated using siRNA, paving the way for more complex gene editing and functional modeling experiments. CONCLUSION This study employs sophisticated bioinformatics and experimental validation techniques to explore the potential of targeting ORC6 for cancer therapy. Keywords: ORC6, Glioblastoma, TCGA, CGGA, GLASS, biomarker

Cortactin contributes to the tumorigenesis of gastric cancer by activating ERK/MMP pathway

Gastric cancer is a malignant tumor with high mortality and high incidence. This study aims to explore the function and molecular mechanism of Cortactin on gastric cancer progression in vitro and in vivo. A bioinformatics analysis from TCGA displayed that Cortactin was highly expressed in gastric cancer samples, and patients with a high Cortactin level had a worse survival rate. Subsequently, we investigated the specific mechanism of action of A in gastric cancer by collecting patient samples for immunohistochemistry, WB, qRT-PCR, cell transfection, cell invasion and metastasis, and constructing tumor xenografts in nude mice. Overexpression of Cortactin inhibited apoptosis and enhanced cellular proliferation and mobility in AGS cells, while those activities were reversed by the knockdown of MMP2 or MMP9. Conversely, the deletion of Cortactin induced apoptosis and suppressed cell growth and metastasis in SGC7901 cells, whereas those behaviors were inhibited by overexpression of MMP2 or MMP9. Additionally, the ERK pathway was activated by Cortactin upregulation. In vivo studies presented that overexpression of Cortactin promoted tumor growth, increased Ki67 expression, and reduced caspase 3 expression, which was reversed by ERK inhibitor treatment. In conclusion, Cortactin acted as an oncogene in gastric cancer and exerted its function by ERK/MMP2/MMP9 signaling pathway.

Ki-67 Expression as a Prognostic Marker: A Comparative Immunohistochemical Analysis of Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma.

Introduction Oral dysplasia is a frequent precancerous condition that may lead to oral cancer. The histopathologic abnormalities exhibited in a chronic, progressive, and premalignant condition of the oral mucosa are referred to as oral epithelial dysplasia (OED). It might show up as erythroplakia, leukoplakia, or leukoerythroplakia. OED is a premalignancy histologic marker that predicts a higher likelihood of squamous cell carcinoma development. Aims and objectives The aim of this study is to identify an association between Ki-67 protein expression and histological grading of OED and oral squamous cell carcinoma (OSCC) and to compare the expression of Ki-67 in different grades of OED and OSCC with the prognosis. Materials and methods The current retrospective research is focused on evaluating epithelial dysplasia and analyzing the function of Ki-67 as a prognostic marker after receiving institutional ethical approval. Group I - normal oral mucosa (NOM), Group II - OED, and Group III - OSCC were included in the study. For statistical analysis, SPSS Statistics version 21.0 (IBM Corp. Released 2021. IBM SPSS Statistics for Windows, Version 28.0. Armonk, NY: IBM Corp) was utilized. The Cox regression model was employed to look at interactions between various prognostic variables. At p<0.05, differences were deemed statistically significant. Results Ki-67 expression was confined to the basal layers in the normal oral epithelium and in the basal, suprabasal, and spinous layers in OED.Ki-67 positive cells were mostly found on the perimeter of well, moderate, and poorly differentiated OSCC tumor nests with Ki-67 positive cells scattered throughout OSCC.According to statistical analysis, there is a substantial difference in expression between OEDand NOM, OSCC and NOM, and OED and OSCC. Conclusion Our study showed that there is a progressive increase in Ki-67 expression across various grades of OED, and the highest expression was noted in OSCC. Early identification and prompt treatment will help in improving the quality of life of such patients.

Inhibitory Receptor TIGIT is a critical regulator of Natural Killer Cell Activation and Survival Following Strong Stimulation

Abstract TIGIT has emerged as a critical regulator of phenotype and function in mouse and human NK cells with key studies showing TIGIT upregulation suppresses NK anti-tumor responses. However, the role of TIGIT in promoting NK survival has received less attention. We hypothesized that TIGIT exerts a dual role on NK cells balancing decreased activation with enhanced survival. Our results showed sorted human NK TIGIT+ and TIGIT-cell subsets upregulate TIGIT to near 100%, post IL-2 cytokine stimulation, suggesting TIGIT’s importance in NK survival following cytokine stimulation. Sorted human TIGIT-NK cells also showed significantly greater apoptosis compared to TIGIT+ NK cells. NK cells from TIGIT KO mice showed greater proliferation and expansion in vitro and in vivo with higher numbers of NK cells and increased Ki67 expression following acute cytokine stimulation. NK cells from TIGIT KO mice also display significantly lower viability and fold expansion (0.2±0.2 vs 2.0±0.7) when compared to C57BL/6 (WT) aged-matched mice. In vivo, there was a greater number of NK cells in the spleen and lung of WT mice following IL-15 exposure, peaking on day 4 but remaining elevated on day 8. NK cells from TIGIT KO mice also showed significantly higher apoptosis (66 ± 6% vs. 20 ± 6%, P&amp;lt;0.001 in vitro and 25±4% vs. 12±2% P&amp;lt;0.01 in vivo). Similar results were observed in reconstituting NK cells of mice from TIGIT KO donors post bone marrow transplant (BMT). Overall, our results indicate that TIGIT plays a critical role in regulating NK cell responses following cytokine stimulation, suggesting that TIGIT blocking strategies may lead to both greater acute expansion and anti-tumor efficacy, but subsequently greater depletion of NK cells long-term following activation.

What Is Known about Breast Cancer in Young Women?

Breast cancer (BC) is the second leading cause of cancer-related death in women under the age of 40 years worldwide. In addition, the incidence of breast cancer in young women (BCYW) has been rising. Young women are not the focus of screening programs and BC in younger women tends to be diagnosed in more advanced stages. Such patients have worse clinical outcomes and treatment complications compared to older patients. BCYW has been associated with distinct tumour biology that confers a worse prognosis, including poor tumour differentiation, increased Ki-67 expression, and more hormone-receptor negative tumours compared to women >50 years of age. Pathogenic variants in cancer predisposition genes such as BRCA1/2 are more common in early-onset BC compared to late-onset BC. Despite all these differences, BCYW remains poorly understood with a gap in research regarding the risk factors, diagnosis, prognosis, and treatment. Age-specific clinical characteristics or outcomes data for young women are lacking, and most of the standard treatments used in this subpopulation currently are derived from older patients. More age-specific clinical data and treatment options are required. In this review, we discuss the epidemiology, clinicopathologic characteristics, outcomes, treatments, and special considerations of breast cancer in young women. We also underline future directions and highlight areas that require more attention in future studies.

Abstract P2-03-19: The efficacy of neoadjuvant endocrine therapy during the waiting period for surgery in postmenopausal hormone receptor positive breast cancer

Abstract Background: Although neoadjuvant endocrine therapy has been used to improve breast conservation rate, its prognostic relevance is unknown. The search for a valid prognostic factor equivalent to pCR in neoadjuvant chemotherapy is a current challenge in neoadjuvant endocrine therapy. In this study, we investigated the efficacy of short term neoadjuvant endocrine therapy utilizing the waiting period for surgery and the prognostic factor including Preoperative Endocrine Prognostic Index (PEPI) score. Patients and Methods: A total of 269 postmenopausal women with hormone receptor-positive, HER2-negative breast cancer was treated with endocrine therapy with non-steroidal aromatase inhibitor during the waiting period for surgery between October 2012 and November 2021. Of the entire 269 patients, 92 and 177 patients had anastrozole and letrozole, respectively. The primary endpoint was change in tumor size by ultrasound and Ki67 before and after short-term endocrine therapy. The secondary endpoint was prognosis of patients divided by PEPI score which was calculated using tumor size, lymph node metastasis, Ki67, and ER Allred score. This study was approved by the institutional review board of Teikyo University. Results: Median age was 68 years old (range, 41-89). ER and PgR was positive in 266 (98%) and 232 (86%) of the entire 269 patients, respectively. Median tumor size was 1.65 cm (range, 0.4-7.5). Seventeen (6.3%) pts were clinically node-positive. Patients with histological grade I tumor were 190 (70.6%). The median duration of endocrine therapy was 39 days (range, 2-88). Average pretreatment Ki67 expression was 10% (range, 0-90). Tumor diameter was significantly decreased to 1.43cm (range,0.45-5.83) after short-term endocrine therapy (p=0.01). The Ki67 expression was significantly decreased to 3.0% (range, 0-85) after endocrine therapy (p&amp;lt; 0.01) and only five patients (1.9%) showed marked increase in Ki-67 expression. PEPI score 0, 1-3 and ≥ 4 was found in 83 (30.9%),147 (54.7%) and 39 patients (14.5%), respectively. After the median observation period of 928 days, patients with PEPI score ≥ 4 showed worse disease-free survival (Figure) compared with patients with PEPI score 0 and 1-3 (p=0.06). In terms of mortality, patients with PEPI score ≥ 4 had worse overall survival than patients with PEPI score 0 and 1-3 (p=0.07). Conclusions: These results suggested that neoadjuvant endocrine therapy during the waiting period for surgery might be effective in reducing the size and Ki67 expression level and PEPI score might be useful in predicting the prognosis of postmenopausal hormone receptor positive breast cancer patients. Citation Format: Yuka Maeda, Saki Naruse, Yuka Isono, Ayana Sato, Miki Yamada, Akiko Matsumoto, Tatsuhiko Ikeda, Hiromitsu Jinno. The efficacy of neoadjuvant endocrine therapy during the waiting period for surgery in postmenopausal hormone receptor positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-03-19.

Chlamydia trachomatis infection co-operatively enhances HPV E6-E7 oncogenes mediated tumorigenesis and immunosuppression.

Chlamydia trachomatis and human papilloma virus (HPV) are the two most common sexually transmitted infections among women. HPV infection can increase the risk of cervical cancer and infertility while C. trachomatis induces pelvic inflammatory disease. Here, we elucidate the molecular conundrum of the co-infection of HPV and C. trachomatis infection and their outcome with respect to cervical cancer. HPV infection was mimicked by overexpression of HPV 16 E6-E7 or using human cervical cell lines SiHa and C33a (with and without HPV 16 respectively). HPV transfected co-infection increased cell proliferation and resistance to H202 and TNFα-induced cell death compared to individual infections. These changes are brought by alteration in the cell cycle proteins (CDK2, CDK6 and Bcl2) and thus increasing the stemness of the epithelial cells as observed by increased colony forming units and CD133 expression. The co-infection also induces change in the mRNA levels of cells which are involved in mesenchymal phenotype. C. trachomatis in presence of E6-E7 overexpression caused cervical epithelial neoplasm in mice with increased Ki67 expression and decreased P53 levels. Stem cell marker, CD133 expression also increased in the cervical tissues of both infected and co-infected group of mice. The cells obtained from the cervix were able to grow continuously in ex vivo cultures. All these results indicate the co-existence of the C. trachomatis and HPV 16 might increase the risk of cervical cancer.

Effect of preterm birth in rats on proliferation and hyperplasia of cardiomyocytes

Aim. To identify the effect of preterm birth on proliferation and hyperplasia of cardiomyocytes in the early postnatal period of ontogenesis in rats.Materials and methods. Preterm birth (on day 21 and 21.5 of gestation) in Wistar rats was induced by subcutaneous administration of mifepristone. Immunohistochemistry was used to identify and calculate the number of Ki67-positive and Mklp2-positive cardiomyocytes in the left ventricle of preterm and full-term rats on days 1, 2, 3, 4, 5, and 6 of postnatal ontogenesis. Statistical analysis of morphometric parameters was performed using the Shapiro – Wilk test and Mann – Whitney test with the Bonferroni correction.Results. We revealed an increase in the number of Ki67-positive cardiomyocytes in the left ventricle of the rats: on day 1 of postnatal ontogenesis (in the rats born on day 21 of gestation) and on days 3–5 of postnatal ontogenesis (in the rats born on day 21.5 of gestation). Preterm birth in rats did not result in a change in the number of Mklp2-positive cardiomyocytes in the left ventricular wall.Conclusion. A change in the pattern of Ki67 expression by cardiomyocytes in the rats born 12 or 24 hours before full term was demonstrated in the early postnatal period of ontogenesis. An isolated increase in Ki67 expression without a change in Mklp2 expression by cardiomyocytes in the left ventricular wall of preterm rats indicates acceleration of cardiomyocyte hypertrophy. Shorter duration of prenatal development is associated with more pronounced morphological and functional rearrangements in the rat myocardium.

Fluorinated triphenylphosphonium analogs improve cell selectivity and invivo detection of mito-metformin.

Triphenylphosphonium (TPP+) conjugated compounds selectively target cancercells by exploiting their hyperpolarized mitochondrial membrane potential. Todate, studies have focused on modifying either the linker or the cargo of TPP+-conjugated compounds. Here, we investigated the biological effects ofdirect modification to TPP+ to improve the efficacy and detection of mito-metformin (MMe), a TPP+-conjugated probe we have shown to have promising preclinical efficacy against solid cancer cells. We designed, synthesized, and tested trifluoromethyl and methoxy MMe analogs (pCF3-MMe, mCF3-MMe, and pMeO-MMe) against multiple distinct human cancer cells. pCF3-MMe showed enhanced selectivity toward cancer cells compared to MMe, while retaining thesame signaling mechanism. Importantly, pCF3-MMe allowed quantitative monitoring of cellular accumulation via 19F-NMR invitro and invivo. Furthermore, adding trifluoromethyl groups to TPP+ reduced toxicity invivo while retaining anti-tumor efficacy, opening an avenue to de-risk these next-generation TPP+-conjugated compounds.