Hyperproliferation Of Epithelial Cells Research Articles

Abstract 3623: MIEN1 promoter ablation provides novel evidence for colorectal cancer genome editing-based therapeutics

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer associated mortalities worldwide. It starts with hyperproliferation of epithelial cells forming a polyp & is characterized by four stages, where stages 3 & 4 are associated with acquisition of metastasis. Chemotherapy majorly focuses on attenuating symptoms with no promising cure. Hence, studying the underlying mechanism of CRC progression & identifying novel therapeutic targets is critical for early diagnosis & treatment. The process of metastasis involves a complex interplay of signaling pathways, where various proteins play crucial roles. One such important protein that aids the process of migration is Migration & Invasion ENhancer 1 (MIEN1). In CRC, MIEN1 expression is predominantly upregulated in cancerous tissue in comparison to normal colorectal tissue, which is closely associated with invasive behavior. But the exact mechanism involved in the process of metastasis is yet unexplored. It is established that MIEN1 overexpression is a result of 17q12 chromosomal amplification, & such dysregulated expression is linked to the trans-regulation of its minimal promoter region. Therefore, we aim to investigate the effect of MIEN1 promoter ablation on CRC migration properties. CRISPR-Cas9 gene editing technology was used for deleting MIEN1 promoter region in the CRC cell line HT29. RNA seq & bioinformatics tools were employed to assess the transcriptomic consequences of genome-editing. Several DEGs discerned by RNA seq analysis were involved in different biological processes & molecular pathways such as cell adhesion, migration, invasion, & angiogenesis. Out of these the genes vital to CRC biogenesis were evaluated at both RNA & protein levels. Additionally, we analyzed the effect of MIEN1 knock-out on CRC metastatic potential using functional assays such as wound healing, Matrigel invasion, hanging drop cell - cell adhesion, & found that migration potential of HT29 cells was significantly reduced in absence of MIEN1 protein. We also successfully demonstrated that MIEN1 deletion disrupts the cytoskeletal rearrangement by affecting F-actin reorganization using phalloidin staining. Confocal staining of different proteins participating in actin cytoskeleton rearrangement such as paxillin, FAK, MIEN1 gave us an insight about the role of MIEN1 in mediating phosphorylation of FAK at different phosphorylation sites such as Tyr 397 & 925. Immunoblotting analysis of an array of proteins further confirmed the role of MIEN1 in actin cytoskeleton dynamics. Taken together, our results prove that MIEN1 is involved in different signaling pathways responsible for CRC migration & its deletion leads to perturbation of several biological processes especially the actin cytoskeleton rearrangement, involved in metastasis. Hence, targeting MIEN1 would be a potentially effective therapeutic strategy for CRC patients. Citation Format: Payal Ranade, Rucha Trivedi, Jamboor K. Vishwanatha. MIEN1 promoter ablation provides novel evidence for colorectal cancer genome editing-based therapeutics. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3623.

Loss of bone morphogenetic protein signaling in fibroblasts results in CXCL12-driven serrated polyp development

Mutations in Bone Morphogenetic Protein (BMP) Receptor (BMPR)1A and SMAD4 are detected in 50% of juvenile polyposis syndrome (JPS) patients, who develop stroma-rich hamartomatous polyps. The established role of stromal cells in regulating BMP activity in the intestine implies a role for stromal cells in polyp development. We used conditional Cre-LoxP mice to investigate how specific loss of BMPR1A in endothelial cells, fibroblasts, or myofibroblasts/smooth muscle cells affects intestinal homeostasis. Selective loss of BMPR1A in fibroblasts causes severe histological changes in the intestines with a significant increase in stromal cell content and epithelial cell hyperproliferation, leading to numerous serrated polyps. This phenotype suggests that crucial changes occur in the fibroblast secretome that influences polyp development. Analyses of publicly available RNA expression databases identified CXCL12 as a potential candidate. RNAscope in situ hybridization showed an evident increase of Cxcl12-expressing fibroblasts. In vitro, stimulation of fibroblasts with BMPs resulted in downregulation of CXCL12, while inhibition of the BMP pathway resulted in gradual upregulation of CXCL12 over time. Moreover, neutralization of CXCL12 in vivo in the fibroblast-specific BMPR1A KO mice resulted in a significant decrease in polyp formation. Finally, in CRC patient specimens, mRNA-expression data showed that patients with high GREMLIN1 and CXCL12 expression had a significantly poorer overall survival. Significantly higher GREMLIN1, NOGGIN, and CXCL12 expression were detected in the Consensus Molecular Subtype 4 (CMS4) colorectal cancers, which are thought to arise from serrated polyps. Taken together, these data imply that fibroblast-specific BMP signaling–CXCL12 interaction could have a role in the etiology of serrated polyp formation.

Preventative Effect of Astaxanthin Against Helicobacter pylori-Induced Proliferation of Gastric Epithelial Cells via the Suppression of Wnt/β-Catenin Signaling Pathway.

ObjectivesHelicobacter pylori (H. pylori) infection has been regarded as a major risk factor for gastric cancer. Various In vivo and in vitro studies have reported that H. pylori infection induces hyper-proliferation, a hallmark of carcinogenesis, in gastric epithelial cells by activating Wnt/β-catenin signaling pathway. Wnt/β-catenin signaling is associated with cell proliferation, which is believed to be linked to various types of human cancer when β-catenin is over-accumulated in the cytoplasm and translocated into the nucleus. Astaxanthin is a reddish carotenoid pigment that has powerful anti-oxidant, anti-inflammatory, and anti-microbial properties. We aim to investigate whether astaxanthin inhibits H. pylori-induced hyper-proliferation in gastric epithelial cells by suppressing Wnt/β-catenin signaling pathway. MethodsThe AGS cell line (a human gastric adenocarcinoma cell-line), H. pylori (NCTC 11,637 strain), and Astaxanthin were used. Cell viability was assessed by WST assay and Colony forming assay. Levels of proteins involved in Wnt/β-catenin signaling pathway were measured by Western blot assay. Real-time PCR assay was used to measure mRNA expression of oncogenes such as c-myc and cyclin E. ResultsH. pylori stimulation increased levels of proteins involved in Wnt/β-catenin signaling pathway. mRNA expression of oncogenes such as c-myc and cyclin E were also elevated by H. pylori stimulation. Astaxanthin suppressed cell growth in H. pylori–stimulated AGS cells. Levels of Wnt/β-catenin signaling pathway proteins were decreased by astaxanthin treatment in H. pylori-stimulated AGS cells. ConclusionsThis study suggested that AST may inhibit H. pylori-induced hyper-proliferation by suppressing Wnt/β-catenin signaling pathway. Funding SourcesBK21 FOUR.

Notch1 signaling in keratocytes maintains corneal transparency by suppressing VEGF expression.

SummaryThe cornea fends off chemicals, dirt, and infectious particles and provides most of the eye’s focusing power. Corneal transparency is of paramount importance to normal vision, yet how it is established and maintained remains unclear. Here, we ablated Notch1 in keratocytes using Twist2-Cre mice and found that Twist2-Cre; Notch1f/f mice developed stroma expansion and neovascularization, followed by hyperproliferation and metaplasia of corneal epithelial progenitor cells and plaque formation at central cornea, leading to loss of transparency. Development of these phenotypes does not involve bacteria-caused inflammation; instead, Notch1 deletion upregulates Vegfa and Vegfc via Hif1α in keratocytes. Vascular endothelial growth factor (VEGF) receptor inhibitor axitinib prevented development of these anomalies in Twist2-Cre; Notch1f/f mice, suggesting that VEGFs secreted by keratocytes promote not only neovascularization but also proliferation and metaplasia of epithelial progenitor cells at central cornea. This study uncovers a Notch1-Hif1α-VEGF pathway in keratocytes that maintains corneal transparency and represents a potential target for treatment of related corneal disorders.

A bio-functional polymer that prevents retinal scarring through modulation of NRF2 signalling pathway

One common cause of vision loss after retinal detachment surgery is the formation of proliferative and contractile fibrocellular membranes. This aberrant wound healing process is mediated by epithelial-mesenchymal transition (EMT) and hyper-proliferation of retinal pigment epithelial (RPE) cells. Current treatment relies primarily on surgical removal of these membranes. Here, we demonstrate that a bio-functional polymer by itself is able to prevent retinal scarring in an experimental rabbit model of proliferative vitreoretinopathy. This is mediated primarily via clathrin-dependent internalisation of polymeric micelles, downstream suppression of canonical EMT transcription factors, reduction of RPE cell hyper-proliferation and migration. Nuclear factor erythroid 2–related factor 2 signalling pathway was identified in a genome-wide transcriptomic profiling as a key sensor and effector. This study highlights the potential of using synthetic bio-functional polymer to modulate RPE cellular behaviour and offers a potential therapy for retinal scarring prevention.

Sleep deprivation induces corneal epithelial progenitor cell over-expansion through disruption of redox homeostasis in the tear film.

SummarySleep deficiency, a common public health problem, causes ocular discomfort and affects ocular surface health. However, the underlying mechanism remains unclear. Herein, we identified that short-term sleep deprivation (SD) resulted in hyperproliferation of corneal epithelial progenitor cells (CEPCs) in mice. The expression levels of p63 and Keratin 14, the biomarkers of CEPCs, were upregulated in the corneal epithelium after short-term SD. In addition, SD led to elevated levels of reactive oxygen species (ROS), and subsequent decrease in antioxidant capacity, in the tear film. Exogenous hydrogen peroxide (H2O2) could directly stimulate the proliferation of CEPCs in vivo and in vitro. Topical treatment of antioxidant L-glutathione preserved the over-proliferation of CEPCs and attenuated corneal epithelial defects in SD mice. Moreover, the activation of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway is essential to ROS-stimulated cell proliferation in CEPCs. However, long-term SD ultimately led to early manifestation of limbal stem cell deficiency.

Phycocyanin inhibits Helicobacter pylori-induced hyper-proliferation in AGS cells via activation of the ROS/MAPK signaling pathway.

BackgroundReactive oxygen species (ROS)-induced oxidative stress (OS) and hyper-proliferation of gastric epithelial cells (GECs) due to Helicobacter pylori (Hp) infection are important mechanisms that lead to gastric carcinoma. Phycocyanin is a marine functional food additive with antioxidant and anti-inflammatory properties.MethodsThe flow cytometry was used to detect the effect of 150 µM phycocyanin intervention on the cell cycle of human gastric adenocarcinoma cell line (AGS) infected with Hp. The transcriptomics of AGS cells intervened by 150 µM phycocyanin for 24 h and infected by Hp were detected. Differential gene expression analysis was performed using a cutoff at the normalized gene expression (log2) of 2 and a P-value of <0.05. Comparisons of the transcriptomes were made between the following groups: (I) AGS cells not infected with Hp and not using phycocyanin action and AGS cells infected with Hp only; (II) AGS cells not infected with Hp and not using phycocyanin action and AGS cells infected with phycocyanin action only; and (III) AGS cells infected with Hp only and phycocyanin treated and infected with Hp cells. c-myc and CyclinD1 was detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunoblotting. Phosphorylation and non-phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 Mitogen-activated protein kinase (p38MAPK) were detected by immunoblotting. Intracellular ROS was detected by immunofluorescence.ResultsPhycocyanin alleviates the Hp infection-induced increased cell viability and expression of cell cycle regulatory proteins c-myc and CyclinD1. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the differentially expressed genes in phycocyanin-treated Hp-infected AGS cells were most significantly enriched in the mitogen-activated protein kinase (MAPK) signaling pathway. Phycocyanin could inhibit the Hp infection-induced phosphorylation of JNK, ERK, and p38MAPK and reduce the level of cellular ROS.ConclusionsThis study suggests that phycocyanin can regulate the ROS/MAPK signaling pathway and reduce c-myc and CyclinD1 expression to inhibit the hyper-proliferation of AGS cells. Phycocyanin may serve as an inhibitor of malignant progression of Hp infection-induced gastric disease.

P008 Modulation of Wnt signaling via a Wnt mimetic improved epithelial healing without causing hyperplasia in a mouse colitis model

Abstract Background Disruption of the colon epithelial barrier is a characteristic of ulcerative colitis (UC), allowing luminal microbes to come into contact with intestinal immune cells resulting in inflammation. All approved UC treatments are anti-inflammatory drugs and because they do not directly heal the epithelial barrier, the intestinal immune system continues to be exposed to gut microbes, resulting in low remission rates. Physiological Wnt signaling is fundamental to intestine epithelial homeostasis and renewal. Modulation of Wnt signaling might present an opportunity to achieve histological remission, which is associated with an improved clinical course, in UC. Previously, in vivo overexpression or administration of exogenous R-Spondin (RSPO), which amplifies Wnt signaling by increasing the amount of Wnt receptors on the cell surface, was reported to show effects on the intestine epithelium and helped regenerate intestine epithelium in various injury models. However, RSPO was also reported, to induce hyperplasia in normal intestine epithelium. Wnt signaling may also be modulated with recently developed Wnt mimetics which mimic endogenous Wnt proteins and activate downstream β-catenin signaling upon engaging with receptors of Wnts. The objective of the current study was to compare the effect of these two approaches in an acute DSS model. Methods Utilizing a mouse acute DSS colitis model, we compared the epithelial healing effects of a Surrozen proprietary Wnt mimetic, R2M3-26, RSPO2, and combination treatments of R2M3-26 and RSPO2. Animals received 4% (wt/vol) dextran sulfate sodium (DSS) in drinking water for 7 days followed by 1% DSS in drinking water from day 8. Protein treatments began on day 4. Disease activity, body weight (BW), fecal score, and occult blood were measured daily. Animals were terminated on day 10 for colon and small intestine histology as well as a serum cytokine panel. Results DSS damage to the colon epithelium was visible by H&amp;E stain at day 4 and day 7 and continued to progress. We observed reduced mRNA expression of Wnt target genes and Wnt ligands in the colon, suggesting compromised Wnt signaling in the tissue. R2M3-26, RSPO2, and the combination treatments were efficacious in improving Disease Activity Index (DAI) and reducing serum cytokine levels. However, both RSPO treatments and combination treatments caused hyperproliferation of epithelium in the small intestine and the colon, consistent with what was reported in normal mice. Conclusion The Surrozen Wnt mimetic, R2M3-26, repaired the DSS damaged colon epithelium without causing hyperproliferation of epithelial cells in the intestine, making it a potential therapeutic platform for IBD.

GPR120 prevents colorectal adenocarcinoma progression by sustaining the mucosal barrier integrity

GPR120 (encoded by FFAR4 gene) is a receptor for long chain fatty acids, activated by ω-3 Polyunsaturated Fatty Acids (PUFAs), and expressed in many cell types. Its role in the context of colorectal cancer (CRC) is still puzzling with many controversial evidences. Here, we explored the involvement of epithelial GPR120 in the CRC development. Both in vitro and in vivo experiments were conducted to mimic the conditional deletion of the receptor from gut epithelium. Intestinal permeability and integrity of mucus layer were assessed by using Evans blue dye and immunofluorescence for MUC-2 protein, respectively. Microbiota composition, presence of lipid mediators and short chain fatty acids were analyzed in the stools of conditional GPR120 and wild type (WT) mice. Incidence and grade of tumors were evaluated in all groups of mice before and after colitis-associated cancer. Finally, GPR120 expression was analyzed in 9 human normal tissues, 9 adenomas, and 17 primary adenocarcinomas. Our work for the first time highlights the role of the receptor in the progression of colorectal cancer. We observed that the loss of epithelial GPR120 in the gut results into increased intestinal permeability, microbiota translocation and dysbiosis, which turns into hyperproliferation of epithelial cells, likely through the activation of β -catenin signaling. Therefore, the loss of GPR120 represents an early event of CRC, but avoid its progression as invasive cancer. these results demonstrate that the epithelial GPR120 receptor is essential to maintain the mucosal barrier integrity and to prevent CRC developing. Therefore, our data pave the way to GPR120 as an useful marker for the phenotypic characterization of CRC lesions and as new potential target for CRC prevention.

SALBUTAMOL AMELIORATES THE PHENOTYPE OF THE SKIN INFLAMMATORY DISEASE PSORIASIS ACCORDING TO SKIN SPHEROID MODELS

Psoriasis is a multifactorial chronic inflammatory disorder resulting by the interplay of genetics, the immune system and the environment. It is characterized by the hyperproliferation of epithelial cells, generating red, itchy psoriatic plaques which have no cure but have great negative impact in patients’ life. Although corticosteroids or vitamin D analogs might help recovery to some extent, there is yet no total cure for the disease. In this study, we sought to generate three-dimensional (3D) stress-related psoriatic skin spheroids with the screening of the potential efficacy of a β2-adrenergic receptor agonist, salbutamol. 3D Culture spheroids with human dermal fibroblasts (HDF), human epithelial keratinocytes (HEK) and human monocytic cell line (THP-1) were generated as a representative model of skin and the protocol of stress-related modelling was conducted. The efficacy of the drug salbutamol was evaluated by the changes in mRNA and protein expression levels of selected genes, as well as by several metabolic assays. We developed a method for culturing spherical organoid models of psoriasis in vitro. We tested the potential theurapetic effects of salbutamol on psoriasis spheroids. Spheroids treated with salbutamol indicated the effictiveness of the treatment. 3D spheroid system was found partially efficient for mimicking the physiological features of psoriasis in vitro. This present work may be a starting point for future investigation as it is the first to generate a stress-related psoriatic model and first to try a β2 agonist as a potential treatment option. Considering the effects and suitability of topical application of salbutamol, its efficacy should not be underestimated and should be investigated further for translating this knowledge into clinics.

Histomorphological Characteristics and Pathological Types of Hyperproliferation of Gastric Surface Epithelial Cells.

Objective To investigate the histomorphological characteristics and pathological types of hyperproliferation of gastric surface epithelial cells. Methods Hematoxylin and Eosin, Periodic acid–Schiff, and immunohistochemical staining were performed on biopsy specimens obtained from 723 patients with hyperproliferation of gastric surface epithelial cells and/or hyperplasia of gastric pits. Follow-up gastroscopic reexaminations were performed on 475 patients included. Improvement probability was analyzed using Kaplan-Meyer as well as Cox proportional hazards models. Results Seven different histomorphologies and clinicopathologies of hyperproliferation of gastric surface epithelial cells were identified: (1) common hyperplasia of gastric epithelial cells, which was characterized by focal glandular epithelial hyperplasia of gastric pits with chronic inflammation; (2) drug-induced hyperplasia of gastric epithelial cells, which was characterized by increased hyperplasia of gastric pits and cells arranged in a monolayer; (3) Helicobacter pylori (Hp) infection-induced hyperplasia of gastric epithelial cells, which was characterized by the disappearance of oval, spherical, and bounded membrane-enclosed mucus-containing granules in the cytoplasm and on the nucleus together with cytoplasmic swelling and vacuolation; (4) metaplastic hyperplasia of gastric epithelial cells, which was characterized by the coexistence of intestinal metaplastic cells with hyperplastic gastric epithelial cells; (5) atrophic hyperplasia of gastric epithelial cells, which was characterized by the mucosal atrophy accompanied with hyperplasia of gastric pits; (6) low-grade neoplasia of epithelial cells, which was characterized by the mild to moderate dysplasia of gastric epithelial cells; and (7) high-grade neoplasia of epithelial cells, which was characterized by the evident dysplasia of hyperplastic epithelial cells and losses of cell polarity. The different pathological types are associated with different improvement probabilities. Conclusions This study demonstrated the histomorphological characteristics and pathological types, which might guide clinicians to track malignant cell transformation, perform precise treatment, predict the clinical prognosis, and control the development of gastric cancer.

Abstract PS19-02: Gut pathogen, Bacteroides fragilis promotes breast cancer liver and lung metastasis

Abstract Background: The last decade established significant contributions of microbiome to many organ specific cancers. Existence of distinct breast microbiota has been recently established but their biological impact in breast cancer progression remains elusive. A few recent studies suggested the existence of distinct breast microbiota and a shift in microbial community composition in diseased breast compared to normal breast however, their functional impact and underlying mechanisms are unknown. Present study was designed to examine the contribution of pro-carcinogenic bacteria in breast cancer initiation, progression and metastasis. Utilizing extensive data mining and metagenomic analyses, we discovered the presence of toxin producing enterotoxigenic Bacteroides fragilis (ETBF) in malignant breast. ETBF is a pro-carcinogenic bacteria known for its potential to initiate and/or promote colon cancer and its pathogenicity has been attributed to its unique toxin B. fragilis toxin (BFT)’. Results: Using mammary intraductal model we discovered that ETBF can successfully colonize the breast confirmed by qPCR and Fluorescent in situ hybridization where it induces local inflammation, fibrosis and hyperproliferation of breast epithelial cells. Mice bearing gut ETBF infection exhibit significant circulating BFT confirmed by qPCR and ELISA and distinct morphological alterations in mammary gland as observed from whole breast mounting and histological evaluation. Gut colonization with ETBF rapidly induces hyperplasia in mammary glands with systemic and local breast inflammation validated by flow cytometry, immunohistochemistry and cytokine profiling. While no changes are observed in cell growth and clonogenicity upon BFT treatment, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells are observed. BFT leads to prominent cytoskeletal reorganization, and increase in migration, invasion and stemness potential of breast cancer cells. Our results indicate that breast cancer cells exposed to BFT ensue to exhibit increase tumor growth, form multifocal tumors and show a striking increase in tumor-initiating cells upon in vivo limiting dilution in immunocompromised mice exhibiting retention of ‘BFT memory’ from the initial exposure. Mechanistically, RNA-sequencing shows enrichment of βcatenin and Notch pathway in secondary tumors derived from BFT-exposed breast cancer cells. Inhibitors of βcatenin and Notch axis abrogates BFT-induced migration and invasion potential indicating the functional importance of this axis. Intriguingly, gut colonization with ETBF at a physiologically relevant level strongly induces growth and metastatic progression of 4T1 tumor cells implanted in mammary ducts monitored by whole animal bioluminescent imaging. In vivo and ex vivo analyses of tumors and distant organs reveal a significant induction of lung and liver metastasis of breast cancer by ETBF while gut colonization with non-toxigenic Bacteroides fragilis (NTBF) does not exhibit any tumor-augmenting impact. We mechanistically evaluate the oncogenic impact of alpha bug ETBF on breast cancer progression and its role in promoting liver and lung metastasis using multiple mice models and multiple techniques including multi-color flow cytometry, immunohistochemistry, quantitative PCR, multiplexed ELISA, ex vivo functional assays and western blotting. Conclusion: Collectively, these findings present the first evidence to show that gut colonization with Bacteroides fragilis rapidly induces inflammation, fibrosis and hyperplasia in the breast. In syngeneic breast cancer model, gut colonization with ETBF aggravates breast cancer progression and induces enhanced lung and liver metastasis via systemic immune modulation, cytokine synthesis and activation of pro-oncogenic pathways. Citation Format: Sheetal Parida, Sumit Siddharth, Guannan Wang, Himavanth Gatla, Shaoguang Wu, Brian Ladle, Kathleen Gabrielson, Cynthia L Sears, Dipali Sharma. Gut pathogen, Bacteroides fragilis promotes breast cancer liver and lung metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-02.

Serenoa repens and Urtica dioica Fixed Combination: In-Vitro Validation of a Therapy for Benign Prostatic Hyperplasia (BPH).

Benign prostatic hyperplasia (BPH) is an age-related chronic disorder, characterized by the hyperproliferation of prostatic epithelial and stromal cells, which drives prostate enlargement. Since BPH aetiology and progression have been associated with the persistence of an inflammatory stimulus, induced both by Nuclear Factor-kappa B (NF-κB) activation and reactive oxygen species (ROS) production, the inhibition of these pathways could result in a good tool for its clinical treatment. This study aimed to evaluate the antioxidant and anti-inflammatory activity of a combined formulation of Serenoa repens and Urtica dioica (SR/UD) in an in vitro human model of BPH. The results confirmed both the antioxidant and the anti-inflammatory effects of SR/UD. In fact, SR/UD simultaneously reduced ROS production, NF-κB translocation inside the nucleus, and, consequently, interleukin 6 (IL-6) and interleukin 8 (IL-8) production. Furthermore, the effect of SR/UD was also tested in a human androgen-independent prostate cell model, PC3. SR/UD did not show any significant antioxidant and anti-inflammatory effect, but was able to reduce NF-κB translocation. Taken together, these results suggested a promising role of SR/UD in BPH and BPH-linked disorder prevention.

Abstract A16: Understanding the principles of tissue repair that accelerate tumor initiation

Abstract Cancer development depends on the capacity of malignant cells to boost growth-promoting signals and restrain growth-inhibiting mechanisms, thus resulting in unbalanced homeostasis. Among growth-promoting mechanisms, sustained proliferation has been described as a fundamental trait of cancer cells. Hyperproliferative behavior is usually acquired in cancer by the constitutive activation of oncogenes such as H-Ras (G12V activating mutation). Previous work showed that H-Ras-G12V promotes epithelial cell hyperproliferation, a feature required but not sufficient to trigger cancer formation. Interestingly, injury has been linked to skin cancer emergence across several mutations. Yet it remains elusive how wound-triggered behaviors lead to cancer. Wound repair process increases cell proliferation, a key event to re-establish epithelial integrity. This observation suggests a possible cooperation between wound-induced and H-Ras-driven proliferation to reach the homeostatic breaking point that promotes cancer. To understand if enhanced proliferation during wound repair could contribute to cancer emergence, I tested the sufficiency of this behavior in promoting tumor in H-RasG12V-model in comparison to other hyperproliferative models, loss of function of the cell cycle inhibitor p21 (p21-/-) and loss of function of Tgf-beta Receptor I (Tgf-beta-RI-/-). Interestingly, in both Tgf-beta-RI-/- and p21-/- models, despite wound-increased cell proliferation, the tissue repairs normally without emergence of tumor. Surprisingly, in H-RasG12V-model wound repair does not increase significantly proliferation, yet tumors consistently develop in the wounded area after two weeks, in line with previous studies. These results indicate that wound-induced proliferation is insufficient to promote tumor initiation. To determine how differences in H-RasG12V cell numbers affect the wound repair outcome, I have activated H-RasG12V mutation in a mosaic fashion, conditions under which H-Ras cells outcompete wild-type neighboring cells via hyperproliferation during homeostatic conditions (Pineda and Greco, in revision). Unexpectedly, the repaired epithelium does not develop tumor after two weeks. Analyses of cellular behaviors show that wild-type cells display an hyperproliferative behaviors in response to the wound settings and they appear to outcompete the H-Ras cells, reversing the relationship established in homeostatic conditions. These data show that wound-induced proliferation seems to provide homeostatic protection against tumor formation. Wild-type cells force Hras-mutant cells to follow physiologic wound repair process suppressing their tumorigenic potential. In conclusion, my discoveries suggest that hyperproliferation is dispensable for tumor initiation. Furthermore, enhanced proliferation of the wild-type population through wounding may help eliminate oncogenic mutant cells that would otherwise integrate and expand into the tissue. Citation Format: Sara Gallini, Valentina Greco. Understanding the principles of tissue repair that accelerate tumor initiation [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A16.

Abstract P4-08-01: Tumor suppressor PLK2 may serve as a biomarker in triple negative breast cancer for combinational therapy of PLK1 inhibitors with the standard-of-care chemotherapy

Abstract Polo-like kinase (PLK) family members play important roles in cell cycle regulation. The founding member PLK1 is oncogenic and preclinically validated as a cancer therapeutic target. Surprisingly, PLK2 is frequently deleted in human breast cancer, preferentially in triple negative breast cancer (TNBC). Pilot studies using an RNAi screen and colony formation assays suggested that PLK2 might act as a tumor suppressor in breast cancer. This paradox stimulated us to investigate the functions of PLK1 and PLK2 in preclinical models and to understand their clinical implications. In preliminary experiments, we found that knockdown of PLK1 mitigated PLK2-deletion induced phenotypes including inhibiting colony formation, and rescued Plk2 null mammary epithelial cell phenotypes in vivo, including gland hyperbranching, epithelial cell hyperproliferation, and abnormal spindle orientation. Furthermore, we demonstrated that PLK2 interacted with PLK1 using a bimolecular fluorescence complementation assay and by co-immunoprecipitation, and using a proximity ligation assay this interaction was shown to occur in prometaphase. Due to the lack of targeted therapy, chemotherapy, such as carboplatin usually in combination with Taxol, is still one of the only systematic treatments for TNBC. Although PLK2 is frequently deleted in TNBC, there is currently no viable strategy to target this loss of function. The relationship between PLK2 and PLK1 suggests targeting PLK1 might provide a potential target to treat TNBC patients with loss of PLK2. In order to test this hypothesis, we used p53 null mammary epithelial cells as a sensitized background to generate a cohort of p53/Plk2 double null TNBC tumors. RNA-seq analysis showed that these tumors can be classified into three distinct molecular TNBC sublines: luminal-like, basal-like and claudin-low. Combinational treatment with the PLK1 inhibitor Volasertib and carboplatin showed a strong synergistic effect on tumor growth and led to regression of the Plk2/p53 null claudin-low tumors, but not control Plk2+/p53 null claudin-low tumors. The effect of combinational treatment of PLK1 inhibitor and carboplatin on the other two sublines as well as PLK2-low TNBC patient-derived xenograft (PDX) models is currently under investigation. We hope that these preclinical experiments will provide a rationale for the application PLK1 inhibitors in combination with chemotherapy in a subset of TNBC patients with deletion of PLK2. [Supported by Susan G. Komen Foundation grant SAC110031] Citation Format: Yang Gao, Elena Kabotyanski, Deanna Acosta, Elizabeth Villegas, Thomas F Westbrook, Charles M Perou, Jeffrey M Rosen. Tumor suppressor PLK2 may serve as a biomarker in triple negative breast cancer for combinational therapy of PLK1 inhibitors with the standard-of-care chemotherapy [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-08-01.

Inhibitory Effect of β-Carotene on Helicobacter pylori-Induced TRAF Expression and Hyper-Proliferation in Gastric Epithelial Cells.

Helicobacter pylori infection causes the hyper-proliferation of gastric epithelial cells that leads to the development of gastric cancer. Overexpression of tumor necrosis factor receptor associated factor (TRAF) is shown in gastric cancer cells. The dietary antioxidant β-carotene has been shown to counter hyper-proliferation in H. pylori-infected gastric epithelial cells. The present study was carried out to examine the β-carotene mechanism of action. We first showed that H. pylori infection decreases cellular IκBα levels while increasing cell viability, NADPH oxidase activity, reactive oxygen species production, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation, and TRAF1 and TRAF2 gene expression, as well as protein–protein interaction in gastric epithelial AGS cells. We then demonstrated that pretreatment of cells with β-carotene significantly attenuates these effects. Our findings support the proposal that β-carotene has anti-cancer activity by reducing NADPH oxidase-mediated production of ROS, NF-κB activation and NF-κB-regulated TRAF1 and TRAF2 gene expression, and hyper-proliferation in AGS cells. We suggest that the consumption of β-carotene-enriched foods could decrease the incidence of H. pylori-associated gastric disorders.

Apoptotic Potency of Bawang Dayak(Elutherine americana) Bulb Extract as Inhibitor of BPH through Computational Studies

Benign Prostatic Hyperplasia (BPH) is indicated by hyperproliferation of prostate epithelial and stromal cells. Bawang Dayak (Elutherine americana) is an Indonesian endemic plant which is used as herbal medicine for BPH. This study aimed to reveal the potency of Bawang Dayak bulbs extracts as an inhibitor agent of BPH cells. The research was conducted based on in silica approach. The 3D structure of active compound and protein target were studied and analyzed molecular interaction, calculated binding affinity, and pharmacophore, visualized biomolecules, and analyzed biological activity of each active compound. They are naphthalene, naphthoquinone, and anthraquinone. All active compounds have a higher binding affinity to COX-2. It has the highest binding affinity value around -8.7 Kcal/mol. Anthraquinone has an ability as anti-inflammatory and apoptosis agonist, which blocks the COX-2 active site, so, the Bcl- 2 would be inactivated and apoptosis can occur immediately.&#x0D; Keywords:

β-carotene Inhibits Expression of c-Myc and Cyclin E in Helicobacter pylori-infected Gastric Epithelial Cells.

BackgroundHelicobacter pylori infection is a major risk factor in the development of gastric cancer. H. pylori infection of gastric epithelial cells increases the levels of reactive oxygen species (ROS), activates oncogenes, and leads to β-catenin-mediated hyper-proliferation. β-Carotene reduces ROS levels, inhibits oxidant-mediated activation of inflammatory signaling and exhibits anticancer properties. The present study was carried out to determine if β-carotene inhibits H. pylori-induced cell proliferation and the expression of oncogenes c-myc and cyclin E by reducing the levels of β-catenin and phosphorylated glycogen synthase kinase 3β (p-GSK3β).MethodsGastric epithelial AGS cells were pre-treated with β-carotene (5 and 10 μM) for 2 hours prior to H. pylori infection and cultured for 6 hours (for determination of the levels of p-GSK3β, GSK3β, and β-catenin) and 24 hours (for determination of cell viability and protein levels of c-myc and cyclin E). Cell viability was determined by the MTT assay and protein levels were determined via western blot-based analysis.Resultsβ-Carotene inhibited H. pylori-induced increases in the percentage of viable cells, phosphorylated GSK3β (p-GSK3β), and the levels of β-catenin, c-myc and cyclin E.Conclusionsβ-Carotene inhibits H. pylori-induced hyper-proliferation of gastric epithelial cells by suppressing β-catenin signaling and oncogene expression.

Papillomavirus Immune Evasion Strategies Target the Infected Cell and the Local Immune System.

Persistent infection with human papillomavirus (HPV) initiates ~5% of all human cancers, and particularly cervical and oropharyngeal cancers. HPV vaccines prevent HPV infection, but do not eliminate existing HPV infections. Papillomaviruses induce hyperproliferation of epithelial cells. In this review we discuss how hyperproliferation renders epithelial cells less sensitive to immune attack, and impacts upon the efficiency of the local immune system. These observations have significance for the design of therapeutic HPV cancer immunotherapies.

Stimulation of Prostate Cells by the Senescence Phenotype of Epithelial and Stromal Cells: Implication for Benign Prostate Hyperplasia.

Hyperproliferation of prostate transition‐zone epithelial and stromal cells leads to benign prostate hyperplasia (BPH), a prevalent pathology in elderly men. Senescent cells in BPH tissue induce a senescence‐associated secretory phenotype (SASP) which, by generating inflamed microenvironment and reactive stroma, promotes leukocyte infiltration, cellular hyperproliferation, and nodular prostate growth. We examined human prostate epithelial (BPH‐1, PNT‐1α) and stromal (HPS‐19I) cells for SASP induction by ionizing radiation and assessed SASP's impacts on cell proliferation and on signal transducers that promote cellular growth, proliferation, and survival. Radiation‐induced DNA damage led to cellular senescence, evident from elevated expression of senescence‐associated β‐galactosidase and the cell‐cycle inhibitor p16/INK4a. Clinical BPH tissue showed p16 accumulation. SASP induced mRNA expression for inflammatory cytokines (IL‐1α, IL‐6, IL‐8, TNF‐α); chemokines (GM‐CSF, CXCL12); metalloproteases (MMP‐1, MMP‐3, MMP‐10); growth factor binding IGFBP‐3. Media from irradiated epithelial or stromal cells enhanced BPH‐1 proliferation. ERK1/2 and AKT, which enhance cell growth/survival and STAT5, which facilitates cell cycle progression and leukocyte recruitment to epithelial microenvironment, were activated by SASP components. The radiation‐induced cellular senescence model can be a platform for identification of individual SASP components and pathways that drive BPH etiology/progression in vivo and targeting them may form the basis for novel BPH therapy.