Multiple Cell Signaling Pathways Research Articles

Abstract P5-08-06: MDX-124, a novel annexin-A1 antibody, shows anti-tumor efficacy in several preclinical models of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases and is a particularly aggressive form of the disease, with poorer prognosis compared to other breast cancer subtypes. Advances in TNBC treatment have been made with the introduction of checkpoint inhibitors, PARP inhibitors and antibody drug conjugates. However, these therapies still have several limitations including low patient response rates, treatment resistance or relapse, and considerable side effects. Therefore, a significant unmet clinical need remains to develop novel targeted agents to treat TNBC. Annexin-A1 (ANXA1) is a phospholipid binding protein secreted in response to several physiological stimuli where it activates formyl peptide receptors (FPR1/2) triggering multiple oncogenic cell signaling pathways. Overexpression of ANXA1 by TNBC cells has been shown to influence several cancer-related processes including cell growth, cell cycle progression, angiogenesis, migration and invasion. In addition, ANXA1 has been shown to have immunomodulatory effects on T-cells, macrophages and dendritic cells. High ANXA1 expression in TNBC patients correlates with both poor overall survival and progression-free survival, indicating ANXA1 is a therapeutic target in TNBC. MDX-124 is a novel humanized antibody targeting ANXA1, and we have previously demonstrated its significant anti-proliferative activity. Here we present additional data showing the efficacy of MDX-124 in preclinical models of TNBC. Methods: The impact of MDX-124 on cell cycle progression was evaluated by measuring changes in DNA content using flow cytometry and analyzed with FlowJo™ software.The modulation of key oncogenic cell signalling pathways by MDX-124 was assessed using human phospho-kinase and XL Oncology Proteome Profiler™ antibody arrays. The effect of MDX-124 in combination therapy with chemotherapy was studied in-vitro using MTT cell proliferation assays, whilst synergy with anti-PD-1 therapy was evaluated using the EMT6 syngeneic mouse model of TNBC. Results: MDX-124 treatment dramatically decreased the proportion of HCC1806 TNBC cells in S-phase by 29.1% with a concomitant increase in G1 of 33.5% versus untreated cells. This occurred in a dose-dependent manner and is consistent with an MDX-124 mediated increase in cell cycle arrest.Treatment of HCC1806 TNBC cells with MDX-124 altered the expression of key oncogenic proteins and the phosphorylation of several kinases that regulate cell signaling pathways involved in proliferation, survival and migration. Notably, MDX-124 substantially reduced ERK and AKT phosphorylation by 84% and 72% respectively versus untreated control cells. In the HCC1806 TNBC cell line, proliferation was significantly reduced after 72 h treatment with MDX-124 and chemotherapy versus control.In the EMT6 syngeneic mouse model of TNBC, the murine analogue of MDX-124 (MDX-001) potentiated mean tumor growth inhibition of single agent anti-PD-1 treatment by 15%. Additionally, 30% of treated mice showed tumor regression in the MDX-001 combination therapy group versus 10% in the single agent anti-PD-1 group. This suggest that anti-ANXA1 antibody therapy acts synergistically with anti-PD-1 immunotherapy.Conclusion: MDX-124 binds to secreted and extracellular ANXA1 disrupting interactions with FPR1/2. This results in altered expression levels of several key cancer-related proteins preventing the activation of oncogenic signaling pathways that promote cancer progression. MDX-124 has demonstrated anti-cancer activity in several TNBC cell line and mouse models, as both a single agent and in combination with other drugs, including anti-PD-1 immunotherapy. Medannex plan to initiate a First-In-Human study in Q4 2021 to evaluate MDX-124 in solid malignancies, including TNBC. Citation Format: Fiona C Dempsey, Hussein Al-Ali, Scott J Crichton, Charlene Fabian, Chris Pepper, Christopher N Parris. MDX-124, a novel annexin-A1 antibody, shows anti-tumor efficacy in several preclinical models of triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-06.

Dimethyl Fumarate Combined With Vemurafenib Enhances Anti-Melanoma Efficacy via Inhibiting the Hippo/YAP, NRF2-ARE, and AKT/mTOR/ERK Pathways in A375 Melanoma Cells.

Melanoma is a deadly form of skin cancer with high rates of resistance to traditional chemotherapy and radiotherapy. BRAF inhibitors (BRAFi) can achieve initial efficacy when used to treat melanoma patients, but drug resistance and relapse are common, emphasizing the need for new therapeutic strategies. Herein, we reported that combination of dimethyl fumarate (DMF) and vemurafenib (Vem) inhibited melanoma cell proliferation more significantly and induced more cell death than single agent did both in vitro and in vivo. DMF/Vem treatment induced cell death through inhibiting the expression and transcriptional activity of NRF2 thereby resulting in more reactive oxygen species (ROS) and via inhibiting the expression of YAP, a key downstream effector of Hippo pathway. DMF/Vem treatment also reduced phosphorylation of AKT, 4EBP1, P70S6K and ERK in AKT/mTOR/ERK signaling pathways. RNA-seq analysis revealed that DMF/Vem treatment specifically suppressed 4561 genes which belong to dozens of cell signaling pathways. These results indicated that DMF/Vem treatment manifested an enhanced antitumor efficacy through inhibiting multiple cell signaling pathways, and thus would be a novel promising therapeutic approach targeted for melanoma.

Shp2 in uterine stromal cells critically regulates on time embryo implantation and stromal decidualization by multiple pathways during early pregnancy.

Approximately 75% of failed pregnancies are considered to be due to embryo implantation failure or defects. Nevertheless, the explicit signaling mechanisms governing this process have not yet been elucidated. Here, we found that conditional deletion of the Shp2 gene in mouse uterine stromal cells deferred embryo implantation and inhibited the decidualization of stromal cells, which led to embryonic developmental delay and to the death of numerous embryos mid-gestation, ultimately reducing female fertility. The absence of Shp2 in stromal cells increased the proliferation of endometrial epithelial cells, thereby disturbing endometrial epithelial remodeling. However, Shp2 deletion impaired the proliferation and polyploidization of stromal cells, which are distinct characteristics of decidualization. In human endometrial stromal cells (hESCs), Shp2 expression gradually increased during the decidualization process. Knockout of Shp2 blocked the decidual differentiation of hESCs, while Shp2 overexpression had the opposite effect. Shp2 knockout inhibited the proliferation of hESCs during decidualization. Whole gene expression profiling analysis of hESCs during the decidualization process showed that Shp2 deficiency disrupted many signaling transduction pathways and gene expression. Analyses of hESCs and mouse uterine tissues confirmed that the signaling pathways extracellular regulated protein kinases (ERK), protein kinase B (AKT), signal transducer and activator of transcription 3 (STAT3) and their downstream transcription factors CCAAT/enhancer binding protein β (C/EBPβ) and Forkhead box transcription factor O1 (FOXO-1) were involved in the Shp2 regulation of decidualization. In summary, these results demonstrate that Shp2 plays a crucial role in stromal decidualization by mediating and coordinating multiple signaling pathways in uterine stromal cells. Our discovery possibly provides a novel key regulator of embryo implantation and novel therapeutic target for pregnancy failure.

ORAI1 Limits SARS-CoV-2 Infection by Regulating Tonic Type I IFN Signaling.

ORAI1 and stromal interaction molecule 1 (STIM1) are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of the type I IFN (IFN-I) response. To examine their potential role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated ORAI1 and STIM1 knockout human HEK293-angiotensin-converting enzyme 2 cells and checked their responses. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection as a result of enhanced IFN-I response. On the contrary, ORAI1 deletion induced high susceptibility to SARS-CoV-2 infection. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic IFN-I signaling. Transcriptome analysis showed downregulation of multiple antiviral signaling pathways in ORAI1 knockout cells, likely because of reduced expression of the Ca2+-dependent transcription factors of the AP-1 family and MEF2C Accordingly, modulation of homeostatic Ca2+ concentration by pretreatment with ORAI1 blocker or agonist could influence baseline IFNB expression and resistance to SARS-CoV-2 infection in a human lung epithelial cell line. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the tonic IFN-I levels, which determine host resistance to SARS-CoV-2 infection.

Cancer Stem Cells: Innovative Approach for Testable Alternatives against Therapy Resistant Cancer

Chemo-resistant cancer stem cells represent a sub-population of cancer initiating phenotype in primary cancer. These cells evolve in to a metastatic phenotype via activation of multiple cell signaling pathways for cancer cell survival and epithelial-mesenchymal transition. Reliable cancer stem cell models represent a valuable experimental approach for drug discovery platform to identify efficacious testable alternatives against therapy-resistant cancer. Present commentary provides a systematic discussion of relevant conceptual and technical aspects of cancer stem cell biology and its significance for therapeutic alternatives.

Roles of mTOR in thoracic aortopathy understood by complex intracellular signaling interactions.

Thoracic aortopathy–aneurysm, dissection, and rupture–is increasingly responsible for significant morbidity and mortality. Advances in medical genetics and imaging have improved diagnosis and thus enabled earlier prophylactic surgical intervention in many cases. There remains a pressing need, however, to understand better the underlying molecular and cellular mechanisms with the hope of finding robust pharmacotherapies. Diverse studies in patients and mouse models of aortopathy have revealed critical changes in multiple smooth muscle cell signaling pathways that associate with disease, yet integrating information across studies and models has remained challenging. We present a new quantitative network model that includes many of the key smooth muscle cell signaling pathways and validate the model using a detailed data set that focuses on hyperactivation of the mechanistic target of rapamycin (mTOR) pathway and its inhibition using rapamycin. We show that the model can be parameterized to capture the primary experimental findings both qualitatively and quantitatively. We further show that simulating a population of cells by varying receptor reaction weights leads to distinct proteomic clusters within the population, and that these clusters emerge due to a bistable switch driven by positive feedback in the PI3K/AKT/mTOR signaling pathway.

Minor Cannabinoids: Biosynthesis, Molecular Pharmacology and Potential Therapeutic Uses.

The medicinal use of Cannabis sativa L. can be traced back thousands of years to ancient China and Egypt. While marijuana has recently shown promise in managing chronic pain and nausea, scientific investigation of cannabis has been restricted due its classification as a schedule 1 controlled substance. A major breakthrough in understanding the pharmacology of cannabis came with the isolation and characterization of the phytocannabinoids trans-Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). This was followed by the cloning of the cannabinoid CB1 and CB2 receptors in the 1990s and the subsequent discovery of the endocannabinoid system. In addition to the major phytocannabinoids, Δ9-THC and CBD, cannabis produces over 120 other cannabinoids that are referred to as minor and/or rare cannabinoids. These cannabinoids are produced in smaller amounts in the plant and are derived along with Δ9-THC and CBD from the parent cannabinoid cannabigerolic acid (CBGA). While our current knowledge of minor cannabinoid pharmacology is incomplete, studies demonstrate that they act as agonists and antagonists at multiple targets including CB1 and CB2 receptors, transient receptor potential (TRP) channels, peroxisome proliferator-activated receptors (PPARs), serotonin 5-HT1a receptors and others. The resulting activation of multiple cell signaling pathways, combined with their putative synergistic activity, provides a mechanistic basis for their therapeutic actions. Initial clinical reports suggest that these cannabinoids may have potential benefits in the treatment of neuropathic pain, neurodegenerative diseases, epilepsy, cancer and skin disorders. This review focuses on the molecular pharmacology of the minor cannabinoids and highlights some important therapeutic uses of the compounds.

Dual Hyaluronic Acid and Folic Acid Targeting pH-Sensitive Multifunctional 2DG@DCA@MgO-Nano-Core-Shell-Radiosensitizer for Breast Cancer Therapy.

Simple SummaryIn this study, we have developed CD44 and folate receptor-targeting multi-functional dual drug-loaded nanoparticles. This comprises hyaluronic acid (HA) and folic acid (FA) conjugated to 2-deoxy glucose (2DG) and a shell linked to a dichloroacetate (DCA) and magnesium oxide (MgO) core (2DG@DCA@MgO; DDM) to enhance the localized chemo-radiotherapy for effective breast cancer (BC) treatment. The physicochemical properties of nanoparticles including stability, selectivity, responsive release to pH, cellular uptake, and anticancer efficacy were comprehensively examined. Mechanistically, we identified multiple component signal pathways as important regulators of BC metabolism and mediators for the inhibitory effects exerted by DDM. Nanoparticles exhibited sustained DDM release properties in bio-relevant media, which was responsive to acidic pH providing edibility to the control of drug release from nanoparticles. DDM-loaded and HA–FA-functionalized nanoparticles exhibited increased selectivity and uptake by BC cells. Cell-based assays indicated that the functionalized DDM significantly suppressed cancer cell growth and boosted radiotherapy (RT) efficacy via inducing cell cycle arrest, enhancing apoptosis, and modulating glycolytic and OXPHOS pathways. Accordingly, the inhibition of glycolysis/OXPHOS by DDM and RT treatment may result in cancer metabolic reprogramming via a novel PI3K/AKT/mTOR/P53NF-κB/VEGF pathway in BC cells. Therefore, the dual targeting of glycolysis/OXPHOS pathways is suggested as a promising antitumor strategy.Globally, breast cancer (BC) poses a serious public health risk. The disease exhibits a complex heterogeneous etiology and is associated with a glycolytic and oxidative phosphorylation (OXPHOS) metabolic reprogramming phenotype, which fuels proliferation and progression. Due to the late manifestation of symptoms, rigorous treatment regimens are required following diagnosis. Existing treatments are limited by a lack of specificity, systemic toxicity, temporary remission, and radio-resistance in BC. In this study, we have developed CD44 and folate receptor-targeting multi-functional dual drug-loaded nanoparticles. This composed of hyaluronic acid (HA) and folic acid (FA) conjugated to a 2-deoxy glucose (2DG) shell linked to a layer of dichloroacetate (DCA) and a magnesium oxide (MgO) core (2DG@DCA@MgO; DDM) to enhance the localized chemo-radiotherapy for effective BC treatment. The physicochemical properties of nanoparticles including stability, selectivity, responsive release to pH, cellular uptake, and anticancer efficacy were thoroughly examined. Mechanistically, we identified multiple component signaling pathways as important regulators of BC metabolism and mediators for the inhibitory effects elicited by DDM. Nanoparticles exhibited sustained DDM release properties in a bio-relevant media, which was responsive to the acidic pH enabling eligibility to the control of drug release from nanoparticles. DDM-loaded and HA–FA-functionalized nanoparticles exhibited increased selectivity and uptake by BC cells. Cell-based assays revealed that the functionalized DDM significantly suppressed cancer cell growth and improved radiotherapy (RT) through inducing cell cycle arrest, enhancing apoptosis, and modulating glycolytic and OXPHOS pathways. By highlighting DDM mechanisms as an antitumor and radio-sensitizing reagent, our data suggest that glycolytic and OXPHOS pathway modulation occurs via the PI3K/AKT/mTOR/NF-κB/VEGFlow and P53high signaling pathway. In conclusion, the multi-functionalized DDM opposed tumor-associated metabolic reprogramming via multiple signaling pathways in BC cells as a promising targeted metabolic approach.

Potential of baicalein in the prevention and treatment of cancer: A scientometric analyses based review

• Baicalein, a pleiotropic agent, isolated from the roots of Scutelleria baicaleinsis . • Scientometric analysis reveals historical and recent trends in baicalein’s research. • Studies have reported the immense potential of baicalein against various cancers. • It modulates PI3K/AKT/mTOR, Wnt/β-catenin, MAPK, NF-κB, Notch, ROS, and Shh signalling. • Further studies are warranted to establish baicalein as a promising anti-cancer agent. Cancer is one of the deadliest diseases worldwide which kills approximately 10 million people annually. Although, substantial improvements have been made in the treatment modalities of this disease, radio-resistance, chemo-resistance, and adverse side-effects limit their potential. It is now well-established that natural products provide a less toxic and efficacious alternative in developing drugs for the treatment of cancer. Hence, we investigated the potential of baicalein, a natural compound isolated from the roots of Scutelleria baicaleinsis, which targets wide spectrum of molecular alterations involved in the progression of cancer . At the molecular level, this compound has shown to exhibit anti-inflammatory and anti-cancerous properties via modulation of multiple cell signaling pathways and transcription factors, including PI3K/Akt/mTOR, Wnt/β-catenin, MAPK, NF-κB, Smad4, Notch 1/Hes, Nrf2/Keap 1, AMPK, Src/Id1, ROS signaling, miR 183/ezrin, and Shh signaling pathways. Overall, this review provides a strong evidence for the potential of baicalein as a therapeutic agent for the treatment of different malignancies.

HAGLROS promotes cell proliferation and angiogenesis and inhibits apoptosis by activating multiple signaling pathways in LSCC cells.

HAGLROS is a long noncoding RNA involving in the development of a variety of cancers, but its mechanism of action in laryngeal squamous cell carcinomas (LSCC) is still unclear. We aim to unveil the effect and mechanism of HAGLROS on LSCC. The expression of HAGLROS in LSCC patients' tissues, serum, and LSCC cell lines was quantified by quantitative real-time PCR. AMC-HN-8 and SNU-46 cells were transfected with the overexpression plasmid of HAGLROS and shHAGLROS, and the functional assay (colony formation assays, flow cytometry, and tube formation) was performed. Western blot was used to determine the expressions of vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), P27 and cleaved caspase-3, as well as phosphorylated-c-Jun-N-terminal kinase (p-JNK), JNK, phosphorylated-extracellular signal-regulated kinase 1/2 (p-Erk1/2), Erk1/2, phosphorylated-protein kinase B (p-AKT) and AKT. HAGLROS was highly expressed in LSCC tissues and cells, and it was correlated to lymph node, tumor depth, and clinical stage of LSCC patients. The proliferation ability of LSCC cells was higher than that of HuLa-PC cells. Meanwhile, HAGLROS overexpression promoted the abilities of proliferation and angiogenesis and reduced apoptosis, whereas silencing of HAGLROS exerted the opposite effects in LSCC cell lines. Moreover, overexpressed HAGLROS upregulated the expressions of VEGF and PCNA yet downregulated the expressions of P27 and cleaved caspase-3 by activating Erk1/2 and AKT or JNK signaling pathways in different LSCC cell lines. Overexpressed HAGLROS promoted the proliferation and angiogenesis yet inhibited apoptosis of LSCC cells by activating Erk1/2 and AKT or JNK signaling pathways.

Antitumoral Activities of Curcumin and Recent Advances to ImProve Its Oral Bioavailability.

Curcumin, a main bioactive component of the Curcuma longa L. rhizome, is a phenolic compound that exerts a wide range of beneficial effects, acting as an antimicrobial, antioxidant, anti-inflammatory and anticancer agent. This review summarizes recent data on curcumin’s ability to interfere with the multiple cell signaling pathways involved in cell cycle regulation, apoptosis and the migration of several cancer cell types. However, although curcumin displays anticancer potential, its clinical application is limited by its low absorption, rapid metabolism and poor bioavailability. To overcome these limitations, several curcumin-based derivatives/analogues and different drug delivery approaches have been developed. Here, we also report the anticancer mechanisms and pharmacokinetic characteristics of some derivatives/analogues and the delivery systems used. These strategies, although encouraging, require additional in vivo studies to support curcumin clinical applications.

Proteome-Wide Characterizations of N6-Methyl-Adenosine Triphosphate- and N6-Furfuryl-Adenosine Triphosphate-Binding Capabilities of Kinases.

Kinases catalyze the transfer of the γ-phosphate group from adenosine triphosphate (ATP) to their protein and small-molecule substrates, and this phosphorylation is a crucial element of multiple cell signaling pathways. Herein, we employed isotope-coded ATP acyl-phosphate probes, in conjunction with a multiple-reaction monitoring (MRM)-based targeted proteomic method for proteome-wide identifications of endogenous kinases that can bind to two N6-modified ATP derivatives, N6-methyl-ATP (N6-Me-ATP), and N6-furfuryl-ATP (a.k.a. kinetin triphosphate, KTP). We found that, among the ∼300 quantified kinases, 27 and 18 are candidate kinases that can bind to KTP and N6-Me-ATP, respectively. Additionally, GSK3α and GSK3β are among the kinases that can bind to both ATP analogues. Moreover, the in vitro biochemical assay showed that GSK3β could employ N6-Me-ATP but not KTP as the phosphate group donor to phosphorylate its substrate peptide. Molecular modeling studies provided insights into the differences between N6-Me-ATP and KTP in enabling the GSK3β-mediated phosphorylation. Together, our chemoproteomic approach led to the identification of endogenous kinases that can potentially be targeted by the two ATP analogues. The approach should be generally applicable for assessing endogenous kinases targeted by other ATP and purine analogues.

Ouabain Suppresses Cell Migration and Invasion in Human Gastric Cancer AGS Cells Through the Inhibition of MMP Signaling Pathways.

Ouabain has been shown to induce human cancer cell death via apoptosis. Still, its anti-metastatic effect on cell migration and invasion of human gastric cancer cells has not been addressed. Cell proliferation and viability were measured by the MTT assay and flow cytometry, respectively. Cell motitlity was analysed by wound healing assay. Cell migration and invasion were analysed by the transwell system. Protein expression was assayed by western blotting. Ouabain decreased AGS cell proliferation, cell viability, and motility. In addition, ouabain inhibited AGS cell migration and invasion. Furthermore, ouabain decreased matrix metalloproteinase-2 (MMP-2) activity at 48 h. Ouabain reduced the levels of proteins associated with PI3K/AKT and p38/MAPK pathways. In addition, ouabain decreased the expressions of N-cadherin, tissue inhibitor of metalloproteinases-1 (TIMP-1), urokinase-type plasminogen activator (c-uPA), and MMP-2 at 48 h. Ouabain suppresses cell metastasis through multiple signaling pathways in AGS cells.

Galectin‑3 blockade suppresses the growth of cetuximab‑resistant human oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a cancer associated with high mortality (accounting for 3.1/100,000 deaths per year in Brazil in 2013) and a high frequency of amplification in the expression of the epidermal growth factor receptor (EGFR). Treatment with the EGFR inhibitor cetuximab leads to drug resistance in patients with OSCC due to unknown mechanisms. Galectin-3 (Gal-3) is a β-galactoside binding lectin that regulates multiple signaling pathways in cells. The present study aimed to investigate the effect of Gal-3 in cetuximab-resistant (cet-R) OSCC. The OSCC HSC3 cell line was selected to establish a mouse xenograft model, which was treated with cetuximab to induce resistance. Subsequently, a Gal-3 inhibitor was used to treat cet-R tumors, and the tumor volume was monitored. The expression of Gal-3, phosphorylated (p)-ERK1/2 and p-Akt was assessed using immunohistochemistry. The combined effect of cetuximab and the Gal-3 inhibitor on HSC3 tumor xenografts was also investigated. HSC3 cells were cultured in vitro to investigate the regulatory effects of Gal-3 on ERK1/2 and Akt via western blotting. In addition, the effects of the Gal-3 inhibitor on the proliferation, colony formation, invasion and apoptosis of HSC3 cells were investigated by performing Cell Counting Kit-8, colony formation, Transwell and apoptosis assays, respectively. In cet-R OSCC tumors, increased expression of Gal-3, p-ERK1/2 and p-Akt was observed. Further research demonstrated that Gal-3 regulated the expression of both ERK1/2 and Akt in HSC3 cells by promoting phosphorylation. Moreover, the Gal-3 inhibitor decreased the proliferation and invasion, but increased the apoptosis of cet-R HSC3 cells. In addition, the Gal-3 inhibitor suppressed the growth of cet-R tumors. Collectively, the results indicated that the Gal-3 inhibitor and cetuximab displayed a synergistic inhibitory effect on OSCC tumors. In summary, the present study demonstrated that Gal-3 may serve an important role in cet-R OSCC. The combination of cetuximab and the Gal-3 inhibitor may display a synergistic antitumor effect, thereby inhibiting the development of cetuximab resistance in OSCC.

Abstract 2371: MicroRNA-99b-5p targets the mTOR/AR axis and induced autophagic cell death in prostate cancer cells

Abstract MicroRNAs are the small non-coding regulatory RNA molecules involved in a wide array of cellular and molecular processes. MicroRNAs negatively regulate gene expression via base-pairing with complementary sequences within mRNA molecules. In cancer, the dysregulation of miRNAs expression is associated with cancer development or suppression through the regulation of key gene expression followed by modulation of multiple cell signaling pathways. In the present study, we investigated the role of microRNA-99b-5p (miR-99b) on prostate cancer (PCa) cell survival, cell migration, and apoptosis. Overexpression of miR-99b in PCa cells inhibited cell proliferation, cell colony-forming capability, spheroid formation, and induced cell apoptosis. On the contrary, inactivation of miR-99b by miR-99b inhibitor promotes PCa cell proliferation, cell colony-forming capability, and cell migration. Mechanistically, miR-99b binds with the 3'UTR region of the mammalian target of the rapamycin (mTOR) gene and controls the expression of mTOR. Our preliminary data also suggest that miR-99b also inhibits androgen receptor (AR) activity in LNCaP cells and induced cell apoptosis. Further by inhibition of mTOR, miR-99b induced autophagy in PCa cells and autophagic cell death. Expression of miR-99b and inactivation of mTOR by rapamycin increased the sensitivity of PCa cells to anti-PCa drug docetaxel (DTX). Collectively the data suggest that miR-99b functions as a tumor suppressor in PCa cells, and therefore, miR-99b may be considered as novel biomarkers and therapeutic targets for PCa management in PCa patients. Citation Format: Suryakant Niture, Lucas Tricoli, Kala Hayes, Deepak Kumar. MicroRNA-99b-5p targets the mTOR/AR axis and induced autophagic cell death in prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2371.

Targeting the Epigenetic and Metabolic Vulnerabilities of Ezh2-Deficiency-Induced Myeloid Neoplasms

Myeloproliferative neoplasms (MPNs) are clonal, progressive hematopoietic malignancies characterized by alterations of multiple signaling and epigenetic pathways in hematopoietic stem cells (HSCs). The identification of driver oncogenic mutations in JAK2, CALR or MPL has transformed our knowledge of MPNs; however, patients with non-mutated JAK2, CALR and MPL (so-called ‘triple-negative‘) have the highest incidence of leukemic transformation and the lowest overall survival (Rumi et al., 2014 Blood 124:1062-1069), indicating that other factors may contribute to MPNs. The molecular processes controlling MPN progression from chronic to acute leukemic transformation remain unknown. This is a major impediment for developing target-based therapeutics to selectively eliminate the mutant stem cells to prevent disease progression and/or relapse. Therefore, the challenge is twofold: 1) to identify and characterize the genetic complexity of MPN pathophysiology, and 2) to understand and control the progression of MPNs from chronic to life-threatening stages.We have developed a new mouse model of MPNs by hematopoietic-selective (Mx1-cre) activation of oncogenic Ras (NRasG12D+/-) and inactivation of Ezh2, the enzymatic subunit of the Polycomb Repressive Complex 2 (PRC2), which mimics concurrent mutations in human MPNs. While NRasG12D+/- alone led to an indolent myeloproliferative disorder, Ezh2 loss markedly accelerated disease progression to life-threatening stages, resulting in thrombocytosis, neutrophilia, splenomegaly, primary myelofibrosis, leukemic transformation and mortality. Strikingly, while loss of Ezh1, a homolog of Ezh2, had no effect on normal hematopoiesis, concurrent inactivation of Ezh1 and Ezh2 with NRasG12D+/- completely abolished MPN development and myelofibrosis in vivo . These results establish distinct functions of Ezh1 and Ezh2 in myeloid neoplasms, and identify a selective epigenetic vulnerability for MPNs induced by Ezh2-deficiency. Follow-up studies of Ezh1/2-mediated chromatin (by ChIP-seq) and transcriptomic (by RNA-seq) changes in normal HSCs and leukemia-initiating cells (LICs) revealed non-overlapping gene targets and cellular pathways required for the distinct cellular phenotypes.Reprogrammed cellular metabolism underlies the progression of various human cancers including leukemia. We noted by RNA-seq transcriptomic profiling that Bcat1, the first and rate-limiting enzyme catalyzing the transamination of branched-chain amino acids (BCAAs), was aberrantly activated in Ezh2-deficient leukemia-initiating cells (LICs). By ChIP-seq analysis, we observed that the Bcat1 promoter is highly enriched with tri-methylated H3-Lys27 (H3K27me3), suggesting that it is epigenetically silenced by Ezh2-PRC2 in normal HSCs. Furthermore, genetic or pharmacologic inhibition of BCAT1 activity selectively abrogated the clonogenic activity of Ezh2-deficient LICs in vitro and the leukemogenic potential in transplanted recipients in vivo, highlighting a metabolic dependency for Ezh2-mutant myeloid neoplasms. Follow-up targeted metabolomic analysis of Ezh2-deficiency-induced metabolic changes established the critical role of Bcat1 and the associated BCAA metabolism in the progression of myeloid neoplasms and leukemia.Collectively, our results not only elucidate the mechanisms by which Ezh1 is specifically required for Ezh2-deficient LICs, but also validate an important epigenetic vulnerability for MPN patients carrying loss-of-function Ezh2 mutations. Moreover, in-depth analysis of BCAA metabolism in myeloid neoplasms identifies a new metabolic dependency for Ezh2-deficient LICs. Because normal hematopoietic stem cells are unaffected by loss of Ezh1 or Bcat1, the selective vulnerabilities raise the possibility of leveraging Ezh1 or BCAA metabolism as targeted therapies to specifically eradicate Ezh2-mutant LICs. Hence, our study promises to provide critical insights into developing new and generalizable therapeutic strategies to selectively eliminate leukemia-initiating cells harboring alterations of epigenetic pathways. DisclosuresDeberardinis:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Gene 33/Mig6/ERRFI1, an Adapter Protein with Complex Functions in Cell Biology and Human Diseases.

Gene 33 (also named Mig6, RALT, and ERRFI1) is an adapter/scaffold protein with a calculated molecular weight of about 50 kD. It contains multiple domains known to mediate protein–protein interaction, suggesting that it has the potential to interact with many cellular partners and have multiple cellular functions. The research over the last two decades has confirmed that it indeed regulates multiple cell signaling pathways and is involved in many pathophysiological processes. Gene 33 has long been viewed as an exclusively cytosolic protein. However, recent evidence suggests that it also has nuclear and chromatin-associated functions. These new findings highlight a significantly broader functional spectrum of this protein. In this review, we will discuss the function and regulation of Gene 33, as well as its association with human pathophysiological conditions in light of the recent research progress on this protein.

Anticancer activities, structure-activity relationship, and mechanism of action of 12-, 14-, and 16-membered macrolactones.

Cancer remains one of the major causes of death worldwide despite the encouraging breakthroughs in the discovery of novel chemotherapeutic agents in recent years. The development of new effective anticancer candidates still represents a challenging endeavor due to the severe anticancer demands and the emergence of drug-resistant, especially multidrug-resistant, cancers. Macrolactones could regulate multiple signaling pathways in cancer cells and demonstrated potential anticancer effects, including inhibition of proliferation, metastasis, and angiogenic activity. Accordingly, macrolactones possess excellent efficacy against both drug-sensitive and drug-resistant cancer cells, and the rational design of macrolactones may provide valuable therapeutic interventions for cancers. The purpose of this review is as follows: (1) outline the recent advances made in the development of 12-, 14-, and 16-membered macrolactones with anticancer potential; (2) summarize the structure-activity relationship; and (3) discuss their anticancer mechanisms.

From Stem Cells to Populations-Using hiPSC, Next-Generation Sequencing, and GWAS to Explore the Genetic and Molecular Mechanisms of Congenital Heart Defects.

Congenital heart defects (CHD) are developmental malformations affecting the heart and the great vessels. Early heart development requires temporally regulated crosstalk between multiple cell types, signaling pathways, and mechanical forces of early blood flow. While both genetic and environmental factors have been recognized to be involved, identifying causal genes in non-syndromic CHD has been difficult. While variants following Mendelian inheritance have been identified by linkage analysis in a few families with multiple affected members, the inheritance pattern in most familial cases is complex, with reduced penetrance and variable expressivity. Furthermore, most non-syndromic CHD are sporadic. Improved sequencing technologies and large biobank collections have enabled genome-wide association studies (GWAS) in non-syndromic CHD. The ability to generate human to create human induced pluripotent stem cells (hiPSC) and further differentiate them to organotypic cells enables further exploration of genotype–phenotype correlations in patient-derived cells. Here we review how these technologies can be used in unraveling the genetics and molecular mechanisms of heart development.

Nkx2-2 expressing taste cells in endoderm-derived taste papillae are committed to the type III lineage

In mammals, multiple cell-signaling pathways and transcription factors regulate development of the embryonic taste system and turnover of taste cells in the adult stage. Using single-cell RNA-Seq of mouse taste cells, we found that the homeobox-containing transcription factor Nkx2-2, a target of the Sonic Hedgehog pathway and a key regulator of the development and regeneration of multiple cell types in the body, is highly expressed in type III taste cells but not in type II or taste stem cells. Using in situ hybridization and immunostaining, we confirmed that Nkx2-2 is expressed specifically in type III taste cells in the endoderm-derived circumvallate and foliate taste papillae but not in the ectoderm-derived fungiform papillae. Lineage tracing revealed that Nkx2-2-expressing cells differentiate into type III, but not type II or type I cells in circumvallate and foliate papillae. Neonatal Nkx2-2-knockout mice did not express key type III taste cell marker genes, while the expression of type II and type I taste cell marker genes were unaffected in these mice. Our findings indicate that Nkx2-2-expressing cells are committed to the type III lineage and that Nkx2-2 may be critical for the development of type III taste cells in the posterior tongue, thus illustrating a key difference in the mechanism of type III cell lineage specification between ectoderm- and endoderm-derived taste fields.