Decrease In Cellular Proliferation Research Articles

FAP Serves as a Prognostic Biomarker in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) poses significant challenges with poor survival rates and limited therapeutic strategies. Our study, using The Cancer Genome Atlas (TCGA) data, assesses cancer-associated fibroblast (CAF) gene signatures’ clinical relevance. In our analysis across TCGA tumor types, differential gene expression analysis revealed that fibroblast activation protein (FAP) is upregulated in tumor tissues and associated with poorer survival rates in HNSCC. Furthermore, mechanistic studies employing gene-silencing techniques substantiated that FAP knockout led to a significant decrease in cellular proliferation, invasion, and migration in HNSCC cell lines. Through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, we established that high FAP expression correlates with vital biological processes such as extracellular matrix organization, angiogenesis, and cellular motility. Importantly, FAP was found to regulate these processes by promoting the expression of key proteins involved in epithelial–mesenchymal transition-related pathways. Additionally, our analysis revealed a significant correlation between FAP expression and the expression profiles of immune checkpoint molecules, underscoring its potential role in immune modulation. Collectively, our findings illuminate FAP’s pivotal role in HNSCC pathogenesis and its potential as a prognostic biomarker and therapeutic target. This research lays the groundwork for understanding the multifaceted roles and regulatory mechanisms of CAFs in HNSCC, thereby offering valuable perspectives for the development of targeted therapeutic strategies aimed at improving patient outcomes.

Prenatal aripiprazole induces alterations of rat placenta: a histological, immunohistochemical and ultrastructural study.

Antipsychotic drugs (APDs) are used to treat many psychiatric illnesses as schizophrenia. Typical antipsychotic drugs (TAPDs) are being used; however, they have many side effects. Atypical antipsychotic drugs (AAPDs) are newer medications with known fewer side effects. Aripiprazole (ARI) is an AAPD, recommended by healthcare providers, even during pregnancy. It can cross the placental barrier and enter fetal circulation, so it might be possible that ARI can adversely impair normal placental development and growth, if it is given prenatally. ARI was applied orally to pregnant female rats in two doses (3& 6mg/kg body weight). On gestation day 20, the mothers were sacrificed, and the placentas were removed and processed for general histological and electron microscopic evaluations. Immunohistochemistry was done using anti-PCNA (proliferating cell nuclear antigen), anti-Bax (for apoptosis) and anti-vascular endothelial growth factor alpha (VEGFA). Morphological evaluation revealed degenerative changes in the placenta as dark nuclei, vacuolization, and cyst formation. Ultra-structurally, there was degeneration of cellular components including organelles and nuclei. These changes were found in different cells of the basal and labyrinth zones and were dose dependent. Immunohistochemistry revealed upregulation of Bax and VEGFA and downregulation of PCNA. Prenatal administration of the AAPD, ARI to pregnant female rats resulted in histological changes in the placenta. Additionally, there was a decrease in cellular proliferation and increase in apoptosis, and vascular impairment. This indicates placental atrophy and dysgenesis and might suggest possible teratogenic effects to ARI, which needs further evaluation.

Effect of Ivospemin and Difluoromethylornithine Polyamine Inhibitors on Viability of Myeloma Cell Lines

Abstract Introduction: Multiple myeloma (MM) is caused by heterogeneous genomic alterations that lead to activation of the MYC oncogenic pathway (Larrayoz et al., 2023). Moreover, MYC activity plays a role in immune escape by MM cells and alteration of the bone marrow microenvironment as the disease progresses, which in turn affects response to immunotherapy. Downstream targets and pathways of MYC, such as polyamine (PA) pathway, are an important avenue for therapeutic anticancer interventions for targeting MYC that is generally considered to be “undruggable”. In the present study, we therefore evaluated the effect of two polyamine inhibitors, ivospemin and difluoromethylornithine (DFMO), on growth and viability of myeloma cell lines. SBP-101, an analogue of the naturally occurring PA, spermine, is a polyamine metabolic inhibitor (PMI) that reduces native PA pools by inhibiting key synthetic enzymes and induction of catabolic enzymes. DFMO is an irreversible inhibitor of the ornithine decarboxylase and polyamine synthesis. Methods: Four myeloma cell lines: NCI-H929, RPMI-8226, ARP-1, and MM.1S, were exposed to varying concentrations of two PA inhibitors, SBP-101 and DFMO, based on the data available from clinical trials conducted for other malignancies. We analyzed the effects of these treatments on cell proliferation, cell viability, and the induction of apoptosis. The analyses were done separately for each compound as well as their combination. To assess cytotoxicity and cell proliferation, we used the Incucyte® SX5 Live-Cell Analysis System. Additionally, the detection of apoptosis was performed using Annexin V and propidium iodide flow cytometric assay. Results: Exposure of the MM cell lines to Ivospemin at increasing concentrations ranging from 1µM to 10 µM showed a significant decrease in cellular proliferation in all four MM cell lines. The mean percentage of cytotoxicity observed at 10 µM concentrations of Ivospemin in NCI-H929, RPMI-8226, ARP-1, and MM.1S cell lines were 69.17, 20.75, 83.92, and 48.71 respectively (Fig. 1a). Similarly, when we exposed the MM cell lines to DFMO at increasing concentrations ranging from 1 µM to 100 µM, we also saw a significant decrease in cellular proliferation in all four cell lines, with the most pronounced effect observed in RPMI-8226. The percentage of cells killed at DFMO 10 µM in NCI-H929, RPMI-8226, ARP-1, and MM.1S cell lines were 32.67, 63.61, 28.24, and 16.06 respectively. Moreover, when we combined Ivospemin at 10 µM and DFMO at 100 µM, we observed a complete abolition of cell growth in all the cell lines (Fig. 1b). Additionally, the treatment of the myeloma cell lines with either Ivospemin, DFMO alone, or in combination, showed an induction of apoptosis, as revealed by flow cytometric analysis (data not shown). These findings indicate that both Ivospemin and DFMO have significant anti-proliferative effects on MM cell lines, and their combination leads to even more potent growth inhibition and induction of apoptosis. Conclusion: The polyamine inhibitors,Ivospemin and DFMO, exhibited a marked anti-proliferative effect in the four human MM cell lines studied when used alone or in combination. These results confirm the anti-neoplastic potential of SBP-101 and DFMO and offer a compelling rationale for its clinical development as a potentially promising treatment option for multiple myeloma. Fig. 1. Effect of varying concentration of Ivospemin (SBP-101) treatment on myeloma cell lines (a). The data is represented as mean ± SEM of three replicates. A combination of both Ivospemin (SBP-101) and DFMO improved the cytotoxicity as compared to when used alone (b).

MiR‐30a‐3p inhibits the proliferation of laryngeal cancer cells by targeting DNMT3a through regulating DNA methylation of PTEN

Abstract Objectives The study aims to examine how miR-30a-3p impacts the growth of laryngeal cancer by exploring its underlying mechanism. Our hypothesis suggests that the regulation of DNMT3a through PTEN by miR-30a-3p plays a significant role in the proliferation of laryngeal cancer. Methods To predict the role of miR-30a-3p in laryngeal cancer and its binding site to DNA methyltransferase 3a (DNMT3a), we utilized data from TCGA, GEO, and starBase. We employed Western blot and qRT-PCR to measure the expression levels of miR-30a-3p, DNMT3a, and PTEN. The interaction between miR-30a-3p and DNMT3a was evaluated using a Luciferase reporter assay. Cell proliferation and invasive abilities were assessed through the CCK-8 kit, EdU staining, and transwell assays. Results Analysis of TCGA data revealed that the expression of miR-30a-3p could impact the survival of patients with head and neck cancer. In Hep-2 cells, we observed down-regulated miR-30a-3p and up-regulated DNMT3a, with a negative correlation between the two. Furthermore, we discovered that miR-30a-3p directly targeted DNMT3a and suppressed its expression in Hep-2 cells, resulting in a decrease in cellular proliferation and invasive capabilities. Additionally, overexpression of miR-30a-3p in Hep-2 cells activated PTEN by reducing DNMT3a expression. Conclusions Our findings indicate that miR-30a-3p, acting as a tumor suppressor gene, plays a regulatory role in the growth and progression of laryngeal cancer through its interaction with DNMT3a and PTEN.

MiR-199a-5p Decreases Esophageal Cancer Cell Proliferation Partially through Repression of Jun-B.

MicroRNA (miR)-199a-5p has been shown to function as a tumor suppressor in some malignancies but its role in esophageal cancer is poorly understood. To further explore its role in esophageal cancer, we sought to investigate the interaction between miR-199a-5p and Jun-B, an important component of the AP1 transcription factor, which contains a potential binding site for miR-199a-5p in its mRNA. We found that levels of miR-199a-5p are reduced in both human esophageal cancer specimens and in multiple esophageal cancer cell lines compared to esophageal epithelial cells. Jun-B expression is correspondingly elevated in these tumor specimens and in several cell lines compared to esophageal epithelial cells. Jun-B mRNA expression and stability, as well as protein expression, are markedly decreased following miR-199a-5p overexpression. A direct interaction between miR-199a-5p and Jun-B mRNA was confirmed by a biotinylated RNA-pull down assay and luciferase reporter constructs. Either forced expression of miR-199a-5p or Jun-B silencing led to a significant decrease in cellular proliferation as well as in AP-1 promoter activity. Our results provide evidence that miR-199a-5p functions as a tumor suppressor in esophageal cancer cells by regulating cellular proliferation, partially through repression of Jun B.

IRAK2 Downregulation in Triple-Negative Breast Cancer Cells Decreases Cellular Growth In Vitro and Delays Tumour Progression in Murine Models

Breast cancer stem cells (BCSCs) are responsible for tumour recurrence and therapy resistance. We have established primary BCSC cultures from human tumours of triple-negative breast cancer (TNBC), a subgroup of breast cancer likely driven by BCSCs. Primary BCSCs produce xenografts that phenocopy the tumours of origin, making them an ideal model for studying breast cancer treatment options. In the TNBC cell line MDA-MB-468, we previously screened kinases whose depletion elicited a differentiation response, among which IRAK2 was identified. Because primary BCSCs are enriched in IRAK2, we wondered whether IRAK2 downregulation might affect cellular growth. IRAK2 was downregulated in primary BCSCs and MDA-MB-468 by lentiviral delivery of shRNA, causing a decrease in cellular proliferation and sphere-forming capacity. When orthotopically transplanted into immunocompromised mice, IRAK2 knockdown cells produced smaller xenografts than control cells. At the molecular level, IRAK2 downregulation reduced NF-κB and ERK phosphorylation, IL-6 and cyclin D1 expression, ERN1 signalling and autophagy in a cell line-dependent way. Overall, IRAK2 downregulation decreased cellular aggressive growth and pathways often exploited by cancer cells to endure stress; therefore, IRAK2 may be considered an interesting target to compromise TNBC progression.

Abstract 1812: Therapeutic targeting and development of IDH inhibitors for the treatment of chondrosarcoma

Abstract Chondrosarcomas are the second most frequently occurring type of bone malignancy, and account for approximately 25% of all bone sarcomas. They are often highly aggressive neoplasms that rapidly progress and eventually recur and give distant metastases. They are largely considered to be resistant to conventional chemotherapy and radiotherapy. Recurrent somatic mutations in the isocitrate dehydrogenase I (IDH1) genes have been identified in a spectrum of human malignancies, including chondrosarcoma. Mutations in these genes lead to impaired ability of IDH1 to catalyze the conversion of isocitrate to alpha ketoglutarate and to gain of a neomorphic enzymatic activity which results in production of oncometabolite 2-hydroxyglutarate (2HG). Our preliminary data indicated that compared to samples that were wild type, IDH1 mutant samples showed a significant increase in intra-tumoral 2HG levels. In the present study, we hypothesized that 2-HG accumulation induces DNA and histone hypermethylation and altered gene expression, ultimately resulting in a block in cellular differentiation by competitively inhibiting α-ketoglutarate-dependent dioxygenases involved in histone and DNA demethylation. Therefore, we proposed that inhibition of IDH should mediate its antitumor effects through the induction of differentiation. To test this hypothesis, we utilized human chondrosarcoma cell lines including IDH wild type (CH2879) and IDH1 mutant (JJ012) and the IDH1 inhibitor FT6535. Results from our in vitro cell viability assay and histone methylation showed that selective inhibition of IDH1 resulted in modest inhibition of cell proliferation and suppression of histone methylation in IDH1 mutant chondrosarcoma cells. Next we elected to test the IDH inhibitor in combination with differentiating agents including retinoic acid. Cell viability assays showed that when compared to either agent alone, the combination of retinoic acid and FT6535 resulted in a significant decrease in cellular proliferation. Western blot analysis showed induction of COL1A2 and SOX9 (differentiation markers) with suppression of histone methylation H3K27me3 or H3K9me3, Taken together, our data strongly suggests that combination treatment with the IDH1 inhibitor FT6535 and retinoic acid represents a novel approach to suppress cell growth by epigenetic modulation to induce differentiation and merits further evaluation as a potential treatment modality in chondrosarcoma. Citation Format: Tahir N. Sheikh, Chao Lu, Gary K. Schwartz. Therapeutic targeting and development of IDH inhibitors for the treatment of chondrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1812.

Abstract 1486: MicroRNA (miR)199a-5p targets JUN-B to decrease cell proliferation in esophageal cancer cells

Abstract Objectives: MiR-199a-5p has been shown to be downregulated in multiple malignancies and function as a tumor suppressor. We have previously demonstrated that miR-199a-5p is markedly downregulated in esophageal cancer cell lines compared to esophageal epithelial cells. In several miR-target sequence analysis programs, miR-199a-5p is predicted to bind JUN-B mRNA with high affinity. JUN-B is an important component of the AP1 transcription factor. The objective of this study was to determine expression of JUN-B in esophageal cancer cells as well as to investigate the interaction between miR-199a-5p and JUNB in these cells and to characterize the functional implications of this interaction. Methods: Studies were performed in human esophageal epithelial (hESO) cells and in TE7, SK-GT-4 and OE21 human esophageal cancer cells. Expression of miR-199a-5p and JUNB mRNA in these cell lines was measured by real-time PCR. JUNB protein expression levels were examined by Western blot. Function of miR-199a-5p was tested through its overexpression and silencing. Cellular proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Levels of miR-199a-5p in TE7, SK-GT-4, and OE21 esophageal cancer cells are markedly reduced compared to hESO cells. JUN-B mRNA and protein expression are correspondingly elevated in these cells compared to hESO cells. JUN-B mRNA and protein expression levels were decreased following miR-199a-5p overexpression in TE7, SK-GT-4 and OE21 cells. In reciprocal experiments, silencing miR-199a-5p in hESO cells resulted in increased JUN-B protein and mRNA levels. Forced expression of miR-199a-5p in TE7, SK-GT-4 and OE21 cells led to a marked decrease in cellular proliferation. Conclusions: MiR-199a-5p expression is significantly reduced in TE7, SK-GT-4, and OE21 esophageal cancer cells relative to hESO cells. Forced expression of miR-199a-5p results in significantly decreased JUN-B mRNA and protein expression in these cells, leading to decreased cellular proliferation. These results add our understanding of the roles by which miR-199a-5p functions as a tumor suppressor in esophageal cancer cells. Further studies will investigate the association of miR-199a-5p with JUN-B mRNA using biotinylated RNA pull-down and luciferase reporter assays, in addition to determining the effect of Jun-B downregulation on AP1 promoter activity. Citation Format: Pornima Phatak, Mohan E. Tulapurkar, James M. Donahue. MicroRNA (miR)199a-5p targets JUN-B to decrease cell proliferation in esophageal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1486.

Ethacrynic acid and cinnamic acid combination exhibits selective anticancer effects on K562 chronic myeloid leukemia cells.

Despite the recent advances in chemotherapy, the outcomes and the success of these treatments still remain insufficient. Novel combination treatments and treatment strategies need to be developed in order to achieve more effective treatment. This study was designed to investigate the combined effect of ethacrynic acid and cinnamic acid on cancer cell lines. The anti-proliferative effect of ethacrynic acid and cinnamic acid was investigated by MTT cell viability assay in three different cancer cell lines. Combination indexes were calculated using CompuSyn software. Apoptosis was assessed by flow cytometric Annexin V-FITC/PI double-staining. The effect of the inhibitors on cell cycle distribution was measured by propidium iodide staining. The combination treatment of ethacrynic acid and cinnamic acid decreased cell proliferation significantly, by 63%, 75% and 70% for K562, HepG2 and TFK-1 cells, respectively. A 5.5-fold increase in the apoptotic cell population was observed after combination treatment of K562 cells. The population of apoptotic cells increased by 9.3 and 0.4% in HepG2 and TFK-1 cells, respectively. Furthermore, cell cycle analysis shows significant cell cycle arrest in S and G2/M phase for K562 cells and non-significant accumulation in G0/G1 phase for TFK-1 and HepG2 cells. Although there is a need for further investigation, our results suggest that the inhibitors used in this study cause a decrease in cellular proliferation, induce apoptosis and cause cell cycle arrest.

Truncated WT1 Protein Isoform Expression Is Increased in MCF-7 Cells with Long-Term Estrogen Depletion.

Background The wt1 gene codes for a transcription factor that presents several protein isoforms with diverse biological properties, capable of positively and negatively regulating genes involved in proliferation, differentiation, and apoptosis. WT1 protein is overexpressed in more than 90% of breast cancer; however, its role during tumor progression is still unknown. Methodology. In this work, we analyzed the expression of WT1 isoforms in several breast cancer cells with different tumor marker statuses and an in vitro assay using MCF-7 cells cultured with long-term estrogen depletion (MCF-7 LTED cells) with the finality to mimic the process of switching from hormone-dependent to hormone-independent. Moreover, growth kinetics, sensitivity to tamoxifen, and relative expression analysis of ER and Her2/neu were performed. Results Initially, the expression of 52-54 kDa protein isoform of WT1 in the breast cancer cell line ER (+) was detected by western blot and was absent in ER (-), and the 36-38 kDa protein isoform of WT1 was detected in all cell lines analyzed. The analysis of alternative splicing by RT-PCR shows that the 17AA (+)/KTS (-) isoform of WT1 was the most frequent in the four cell lines analyzed. In vitro, the MCF-7 cells in the estrogen depletion assay show an increase in the expression of the 52-54 kDa isoform of WT1 in the first 48 hours, and this was maintained until week 13, and later, this expression was decreased, and the 36-38 kDa isoform of WT1 did not show change during the first 48 hours but from week 1 showed an increase of expression, and this remained until week 27. Growth kinetic analysis showed that MCF-7 LTED cells presented a 1.4-fold decrease in cellular proliferation compared to MCF-7 cells cultured under normal conditions. In addition, MCF-7 LTED cells showed a decrease in sensitivity to the antiproliferative effect of tamoxifen (p ≤ 0.05). Samples collected until week 57 analyzed by qRT-PCR showed an increase in the relative expression of the Her2/neu and ER. Conclusions Modulation of protein isoforms showed differential expression of WT1 isoforms dependent on estrogen receptor. The absence of 52-54 kDa and the presence of the 36-38 kDa protein isoform of WT1 were detected in ER-negative breast cancer cell lines classified as advanced stage cells. Long-term estrogen depletion assay in MCF-7 cells increased the expression of the 36-38 kDa isoform and reduced the 52-54 kDa isoform, and these cells show an increase in the expression of tumor markers of ER and Her2/neu. MCF-7 LTED cells showed low proliferation and insensitivity to tamoxifen compared to MCF-7 cells in normal conditions. These results support the theory about the relationship of the 36-38 kDa isoform of WT1 and the absence of ER function in advanced breast cancer.

Eukaryotic initiation factor-2, gamma subunit, suppresses proliferation and regulates the cell cycle via the MAPK/ERK signaling pathway in acute myeloid leukemia.

The expression of eukaryotic translation initiation factor-2 subunit 3 (EIF2S3) in patients with non-small cell lung and colorectal cancer is lower than that in healthy individuals. However, the functions of EIF2S3 remain unclear, and its study in leukemia has not been reported. The article aims to explore the role of EIF2S3 in AML (acute myeloid leukemia) and its underlying mechanism. Reverse transcription-quantitative PCR was performed to evaluate the expression levels of EIF2S3, and its association with patient prognosis was determined. Inducible HEL-EIF2S3 and HL-60-EIF2S3 cell lines were established by retrovirus infection. Cellular proliferation and the cell cycle were analyzed using Cell Counting Kit-8 and flow cytometric analyses. Tumorigenic ability was evaluated using xenograft nude mouse model. Gene expression profiles were analyzed in HL-60-EIF2S3 cells by next-generation sequencing, and WB analysis was performed to detect the expression of related proteins. The expression of EIF2S3 in patients with AML was lower than that experiencing CR (P = 0.02). Furthermore, EIF2S3 overexpression inhibited cellular proliferation, halted G0/1 to S phase cell cycle progression, and inhibited tumorigenicity (P = 0.015). 479 differentially expressed genes were identified between HL60-EIF2S3 DOX (-) and HL60-EIF2S3 DOX ( +) cells via NGS and several of them involved in MAPK/ERK signaling pathway. The phosphorylation levels of ERK decreased when EIF2S3 was overexpressed (P < 0.050). EIF2S3 overexpression may result in a decrease in cellular proliferation, cell cycle arrest, and tumorigenic inhibition via the MAPK/ERK signaling pathway in AML cells.

Downregulation of high mobility group box 1 enhances the radiosensitivity of non-small cell lung cancer by acting as a crucial target of microRNA-107.

High mobility group box 1 (HMGB1) has been reported to regulate the sensitivity of several types of cancer cell to chemoradiotherapy. The present study aimed to investigate the changes in HMGB1 expression after radiotherapy, as well as its regulatory role in the radiosensitivity of non-small cell lung cancer (NSCLC) cells. The expression levels of HMGB1 in the serum of 73 patients with NSCLC were analyzed by ELISA. HMGB1 mRNA and microRNA (miR)-107 expression in NSCLC cells were assessed using reverse transcription-quantitative PCR. Receiver operating characteristic analysis was used to evaluate the diagnostic value of HMGB1. Cell counting kit-8, Transwell invasion and clonogenic assays were used to determine cellular viability, invasiveness and colony formation ability, respectively. Following radiotherapy, the levels of HMGB1 were significantly decreased in the serum of patients with NSCLC, and lower serum levels had relatively high diagnostic accuracy in radiosensitive patients. Furthermore, HMGB1-knockdown retarded cellular proliferation and invasion with or without irradiation, and enhanced NSCLC cell radiosensitivity. Furthermore, knocking down miR-107 reversed the decreases in cellular proliferation and invasiveness both with and without irradiation, and reduced the survival fractions induced by sh-HMGB1. HMGB1-knockdown leads to radiosensitivity that may result from suppression of the Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. Collectively, decreased expression of HMGB1 was found to be a putative diagnostic predictor of radiosensitivity in patients with NSCLC. HMGB1-knockdown inhibited the proliferation and enhanced the radiosensitivity of NSCLC cells, which may be regulated via miR-107 by mediating the TLR4/NF-κB signaling pathway. Thus, HMGB1 may be a potential regulator of radioresistance in NSCLC, and the HMGB1/miR-107 axis may represent a promising therapeutic target.

Discovery of Novel CCR5 Ligands as Anticolorectal Cancer Agents by Sequential Virtual Screening.

C-C chemokine receptor type 5 (CCR5) is a member of the G protein-coupled receptor. CCR5 and its interaction with chemokine ligands have been crucial for understanding and tackling human immunodeficiency virus (HIV)-1 entry into target cells. In recent years, the change in CCR5 expression has been related to the progression of different cancer types. Patients treated with the CCR5 ligand, maraviroc (MVC), showed a deceleration in tumor development especially for metastatic colorectal cancer. Based on the crystal structure of CCR5, we herein describe a multistage virtual screening protocol including pharmacophore screening, molecular docking, and protein–ligand interaction fingerprint (PLIF) postdocking filtration for discovery of novel CCR5 ligands. The applied virtual screening protocol led to the identification of four hits with binding modes showing access to the major and minor pockets of the MVC binding site. Compounds 2–4 showed a decrease in cellular proliferation upon testing on the metastatic colorectal cancer cell line, SW620, displaying 12, 16, and 4 times higher potency compared to MVC, respectively. Compound 3 induced apoptosis by arresting cells in the G0/G1 phase of the cell cycle similar to MVC. Further in vitro assays showed compound 3 drastically decreasing the CCR5 expression and cellular migration 48 h post treatment, indicating its ability to inhibit metastatic activity in SW620 cells. The discovered hits represent potential leads for the development of novel classes of anticolorectal cancer agents targeting CCR5.

Identification of nephronectin as a new target for IGF1 action.

The growth hormone (GH)-insulin-like growth factor-1 (IGF1) endocrine axis has a key role in normal growth and development. Laron syndrome (LS) is a type of dwarfism that results from mutation of the GH receptor, leading to congenital IGF1 deficiency. Epidemiological studies have shown that LS patients are protected from cancer. Genome-wide profiling led to the identification of a series of metabolic genes whose differential expression in LS might be linked to cancer protection. Nephronectin (NPNT) is an intracellular and secreted extracellular matrix protein with important roles in kidney development. NPNT was identified as the top-downregulated gene in LS-derived cells in comparison with ethnic-, age- and gender-matched controls (p-value=0.0148; fold-change=-3.12 versus controls). NPNT has not been previously linked to the IGF1 signaling pathway. The present study was aimed at evaluating the hypothesis that NPNT is a new target for IGF1 action and that decreased expression of NPNT in LS is correlated with cancer protection. Basal and IGF1-stimulated NPNT expression were assessed in LS lymphoblastoid cells as well as in human breast and prostate cancer cells. NPNT silencing experiments were conducted using siRNA methodology. We provide evidence that IGF1 stimulates NPNT expression in LS-derived lymphoblastoids and various cancer cell lines. In addition, we demonstrate that NPNT silencing results in diminished activation of the AKT and ERK1/2 pathways, with ensuing decreases in cellular proliferation. Our data identified the NPNT gene as a target for IGF1 action. The clinical implications of the functional and physical interactions between NPNT and the IGF1 pathway merit further investigation.

Merkel Cell Polyomavirus Large T Antigen Unique Domain Regulates Its Own Protein Stability and Cell Growth.

Merkel cell polyomavirus (MCV) is the only known human oncogenic virus in the polyomaviridae family and the etiological agent of most Merkel cell carcinomas (MCC). MCC is an aggressive and highly metastatic skin cancer with a propensity for recurrence and poor prognosis. Large tumor antigen (LT), is an essential oncoprotein for MCV transcription, viral replication, and cancer cell proliferation. MCV LT is a short-lived protein that encodes a unique domain: MCV LT unique regions (MURs). These domains consist of phosphorylation sites that interact with multiple E3 ligases, thus limiting LT expression and consequently, viral replication. In this study, we show that MURs are necessary for regulating LT stability via multiple E3 ligase interactions, resulting in cell growth arrest. While expression of wild-type MCV LT induced a decrease in cellular proliferation, deletion of the MUR domains resulted in increased LT stability and cell proliferation. Conversely, addition of MURs to SV40 LT propagated E3 ligase interactions, which in turn, reduced SV40 LT stability and decreased cell growth activity. Our results demonstrate that compared to other human polyomaviruses (HPyVs), MCV LT has evolved to acquire the MUR domains that are essential for MCV LT autoregulation, potentially leading to viral latency and MCC.

Abstract 5714: Targeting serine metabolism impairs tumor growth in preclinical models of Ewing sarcoma

Abstract Despite a growing body of knowledge about the genomic landscape of Ewing sarcoma (ES), translation of basic discoveries into targeted therapies and significant clinical gains has remained elusive. Recent insights have revealed that the oncogenic transcription factor EWS-FLI1 can impact ES cellular metabolism, raising the possibility of targeting metabolic dependencies as a novel therapeutic strategy. Here, we show that 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in de novo serine synthesis, is highly expressed in human ES cells and that EWS-FLI1 regulates PHGDH expression and de novo serine synthesis. PHGDH knockdown resulted in decreased ES cell proliferation, especially under serine limitation, and significantly inhibited xenograft tumorigenesis in preclinical orthotopic models of ES. Additionally, the PHGDH inhibitor NCT-503 caused a dose-dependent decrease in cellular proliferation. Moreover, we report a novel drug combination in which nicotinamide phosphoribosyltransferase (NAMPT) inhibition, which blocks production of the PHGDH substrate NAD+, synergized with NCT-503 to abolish ES cellular proliferation and tumor growth. Furthermore, we show that serine deprivation inhibited ES cell proliferation and tumorigenesis, indicating that ES cells depend on exogenous serine in addition to de novo serine synthesis. Our findings suggest that serine metabolism is critical for ES tumorigenesis, and that targeting serine metabolism should be further investigated as a potential therapeutic strategy for ES. Citation Format: Sameer Issaq, Arnulfo Mendoza, Tracy Rosales, Christine Heske, Craig Thomas, Ralph DeBerardinis, Lee Helman. Targeting serine metabolism impairs tumor growth in preclinical models of Ewing sarcoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5714.

EWS-FLI1-regulated Serine Synthesis and Exogenous Serine are Necessary for Ewing Sarcoma Cellular Proliferation and Tumor Growth.

Despite a growing body of knowledge about the genomic landscape of Ewing sarcoma, translation of basic discoveries into targeted therapies and significant clinical gains has remained elusive. Recent insights have revealed that the oncogenic transcription factor EWS-FLI1 can impact Ewing sarcoma cellular metabolism, regulating expression of 3-phosphoglycerate dehydrogenase (PHGDH), the first enzyme in de novo serine synthesis. Here, we have examined the importance of serine metabolism in Ewing sarcoma tumorigenesis and evaluated the therapeutic potential of targeting serine metabolism in preclinical models of Ewing sarcoma. We show that PHGDH knockdown resulted in decreased Ewing sarcoma cell proliferation, especially under serine limitation, and significantly inhibited xenograft tumorigenesis in preclinical orthotopic models of Ewing sarcoma. In addition, the PHGDH inhibitor NCT-503 caused a dose-dependent decrease in cellular proliferation. Moreover, we report a novel drug combination in which nicotinamide phosphoribosyltransferase (NAMPT) inhibition, which blocks production of the PHGDH substrate NAD+, synergized with NCT-503 to abolish Ewing sarcoma cell proliferation and tumor growth. Furthermore, we show that serine deprivation inhibited Ewing sarcoma cell proliferation and tumorigenesis, indicating that Ewing sarcoma cells depend on exogenous serine in addition to de novo serine synthesis. Our findings suggest that serine metabolism is critical for Ewing sarcoma tumorigenesis, and that targeting metabolic dependencies should be further investigated as a potential therapeutic strategy for Ewing sarcoma. In addition, the combination strategy presented herein may have broader clinical applications in other PHGDH-overexpressing cancers as well.

The impact of photobiomodulation therapy on the biology and behavior of head and neck squamous cell carcinomas cell lines

Photobiomodulation therapy (PBMT) is an emerging therapeutic modality designed to prevent and treat chemotherapy-driven oral mucositis (OM). However, the response of tumor cells to the effects of PBMT remains poorly understood. Our study explores the effects of PBMT in head and neck squamous cell carcinoma (HNSCC) based on cellular proliferation, migration, and survival of tumor cells and its population of cancer stem cells (CSC). We explored the behavior of two HNSCC cell lines (HN6 and HN13) under two distinct conditions, a physiological growing condition (10% FBS), and under stress growing condition (2% FBS) prior to irradiation using diode laser (InGaAlP; MM Optics, São Carlos, SP, Brazil). Diode laser (660 nm) was applied with a power of 100 mW delivering a total energy per point of 0.24 J. MTT and wound healing test (scratch assay) were performed to evaluate, respectively, proliferation and migration of tumor cells. Clonogenic and spheres formation assays were also performed to evaluate the survival and percentage of CSC upon irradiation. Overall, we observed that PBMT does not exacerbate the behavior of HNSCC. We could only observe a decrease in cellular proliferation of one cell line (HN6) when cultured under nutritional stress conditions (p < .05). There were no significant differences between the control and the PBMT groups regarding cell migration, survival and the percentage of CSC. Collectively, our results suggest that in vitro administration of PBMT to HNSCC does not modify the behavior of tumor cells.

Abstract 708: Targeting fatty acid synthase induces apoptosis and senescence

Abstract Fatty acid synthase (FASN) is a novel therapeutic target for cancer as it has increased expression in many different types of cancers. There has been significant understanding that cancer cells can hijack and modify tightly regulated metabolic pathway for its own survival. FASN is the enzyme that synthesizes palmitate from malonyl-CoA for energy storage, structure and biomolecules. FASN expression is low to undetectable in normal cells with the exception of liver and adipose tissue. In this study, we investigated the role of FASN in breast cancer cells. We observed that in highly expressing FASN breast cancer cells, TVB-3166, FASN inhibitor, results in a decrease in cellular proliferation and an increase in cellular death. FASN inhibition increased reactive oxygen species (ROS) and altered NADPH/NADP+ ratio. Increase in ROS and NADPH have been linked to the intrinsic apoptosis pathway. Additionally, there is an increase in expression of the BH3-only proteins Bim, Puma and Noxa, leading to the permeabilization of the mitochondria and the release of cytochrome c. Furthermore, after prolong exposure to TVB-3166 a number of cells enter into a senescent state. This was observed the in detection of β-galactosidase and confirmed by the expression of senescent markers including ARF, INK4A, and CIP. Additionally, when the cells were treated with a CDK4/6 inhibitor in combination with the FASN inhibitor TVB-3166, an increase in stress induced premature senescence was observed. Our data demonstrates that inhibition of FASN induces intrinsic apoptosis while it stimulates cellular senescence to the apoptotic resistant cells. Therefore, the ability for TVB-3166 to both eradicate and overturn cell growth makes it potential therapeutic modality. Citation Format: Travis Van der Steen, George Kemble, Ruth Lupu. Targeting fatty acid synthase induces apoptosis and senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 708.

Abstract 658: Antiproliferative effects of type II diabetes agents in Fanconi derived oral squamous carcinoma

Abstract Fanconi anemia is a rare disease characterized by bone marrow failure requiring transplantation at a young age. Patients with Fanconi Anemia are at risk for the development of squamous cell carcinomas of the skin and mucosal surfaces at risks several hundred fold higher than control populations and these malignancies can pursue an aggressive, often fatal, course. Our group has been interested in repurposing Type 2 diabetes agents for oral cancer prevention and treatment. Presently, we examined the effects of the thiazolidinedione drug, pioglitazone, and an experimental biguanide, N1-hexyl-N5-benzyl-biguanide (HBB), on the proliferation of Fanconi-derived oral carcinoma cells. We incubated MOP FA1,a, VU-1131, and VU-1365 b, and C squamous cell carcinoma cell lines with vehicle controls or the thiazolidinedione pioglitazone or biguanide (HBB). We found dose-dependent decreases in cellular proliferation in all three Fanconi-derived cell lines at physiologically relevant concentrations (10 - 50 µM) after 1 to 3 days in several experiments. The effects were not due to simple cytotoxicity as judged by trypan blue staining. In other non-squamous cell lines of cervical and oral cavity origins we have previously discovered similar effects. These agents affect PPAR gamma associated pathways (pioglitazone) or mTOR and mitochondrial glucose metaboliscm pathways (HBB). Since Fanconi anemia patients are highly susceptible to DNA damaging effects of cytotoxic chemotherapy, these classes of agents might be attractive in Fanconi Anemia oral cancer treatment or prevention. Metformin, another biguanide, is currently postulated to delay bone marrow failure in the Fanconi population and is currently in use in an early phase clinical trial. Our results may, therefore, be attratciveattractive for oral cancer prevention and treatment in this population, as they are well tolerated. Further examination of molecular mechanisms will likely generate putative pharmacodynamics markers for future clinical trials. Citation Format: Kim Miller, Beverly Wuertz, David Potter, Frank G. Ondrey. Antiproliferative effects of type II diabetes agents in Fanconi derived oral squamous carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 658.