Signaling Pathway In Cancer Cells Research Articles

Levistilide a Induces Ferroptosis by Activating the Nrf2/HO-1 Signaling Pathway in Breast Cancer Cells.

IntroductionBreast cancer (BC) is the leading female malignancy, with one million new cases diagnosed worldwide per year. However, the current treatment options for BC patients have difficulty achieving satisfactory efficacy. Ferroptosis is a new mode of regulated cell death that plays a key role in the inhibition of tumorigenesis. Levistilide A (LA), as an active compound extracted from Chuanxiong Rhizoma, might prevent the development of tumors by regulating the critical cellular processes of ferroptosis.MethodsIn this study, the underlying mechanisms of LA on ferroptosis in BC were explored in vitro. The effect of LA on the viability and mitochondrial function of BC cells was determined. Moreover, the effect of LA on the expression levels of key molecules involved in ferroptosis and the nuclear factor erythroid-2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway was evaluated.ResultsLA significantly reduced cell viability and damaged the mitochondrial structure and function of BC cells in a dose-dependent manner. Furthermore, LA treatment markedly enhanced reactive oxygen species (ROS)-induced ferroptosis by activating the Nrf2/HO-1 signaling pathway.ConclusionThese findings suggest that LA may be a potential lead compound for breast cancer therapy by inducing ferroptosis in tumor cells.

SEC61G Promotes Cervical Cancer Proliferation by Activating MAPK Signaling Pathway.

Objective The abnormal expression of SEC61G plays an important role in the development of various tumors. This study explored the effects of SEC61G on MAPK signaling pathway and proliferation of cervical cancer (CC) cells. Methods shRNA was used to inhibit the expression of SEC61G and EdU to observe its effect on the proliferation of CC cell SiHa. The effect of SEC61G on invasion was evaluated by Transwell assay. TCGA database was used to analyze the influence of high or low SEC61G expression level on the overall survival of CC patients. Western blot was used to detect the expressions of SEC61G, p-RAF1, Raf1, p-MEK1/2, MEK1/2, and p-ERK1/2 in cells. SiHa cells overexpressing SEC61G (SiHa-SEC61G) and control group (SiHa-mock) were subcutaneously implanted in nude mice. The tumor growth curve was measured at the specified time points between SiHa-SEC61G and SiHa-mock. The inhibitory effect of gefitinib on SEC61G was further evaluated. Results In patients with CC, high SEC61G expression predicted poor prognosis. Silencing SEC61G inhibited proliferation and invasion of CC cells in vitro. Overexpression of SEC61G can promote the proliferation and invasion of CC cells in vitro. Meanwhile, overexpression of SEC61G promoted the proliferation of CC xenografts. Knocking down SEC61G can inhibit MAPK signaling pathway. Gefitinib can inhibit CC proliferation and tumor growth by SEC61G. Conclusion SEC61G is highly expressed in CC and has poor prognosis. Inhibition of SEC61G expression can effectively inhibit the growth and proliferation of human CC cells. The mechanism may be related to the inhibition of MAPK signaling pathway.

Gui Shao Tea Extracts Inhibit Gastric Cancer Growth in Vitro and in Vivo and Prolong Survival in Nude Mice.

Gui Shao Tea (GST), a long-aged tea with a Chinese herbal aroma, can treat many stubborn and malignant diseases, according to traditional Chinese medicine. This research aimed to discover and define GST, study the anti-gastric cancer effects of GST extracts and preliminarily elucidate the mechanism of action in the PI3K/Akt signaling pathway and the gut microbiota. GST was analyzed by GC/MS and HPLC. Cell proliferation, the cell cycle and apoptosis were evaluated by a CCK8 assay and flow cytometry. The effects of GST extracts on tumor inhibition and survival time were explored by a gastric cancer xenograft model in nude mice. The PI3K/Akt signaling pathway was assessed by western blotting and immunohistochemistry. Gut microbiota detection and fecal microbiota transplantation were performed to examine whether the tumor inhibition observed in mice was related to gut microbiota changes. The ingredients in GST, mostly terpenes and their derivatives, were novel and more concentrated than those in tea made from the branches and leaves of the same plant species, Camellia sinensis, picked and produced the same year, while the levels of polyphenols and alkaloids were significantly reduced. In BGC-823, MGC-803, and SGC-7901 gastric cancer cells, GST extracts significantly inhibited proliferation (p = 0.037), induced G0/G1 arrest (p < 0.001) and promoted early apoptosis (p = 0.041). In mice, gastric tumor growth was significantly inhibited in both the high-dose (HTF) and middle-dose (MTF) GST-fed groups. The inhibition rate in the HTF group was 33.77% on Day 14 (p = 0.042), and that in the MTF group was 55.21% on Day 14 (p = 0.002) and 61.6% on Day 28 (p = 0.008). The survival time of MTF group mice was significantly prolonged by 22.2% (p = 0.013). GST extracts inhibited the PI3K/AKT signaling pathway in gastric cancer cells (p = 0.016) and tissues (p = 0.029), downregulated the protein p-Rb and further downregulated E2F1, thereby affecting the cell cycle and proliferation. GST extracts altered the gut microbiota in mice, but these alterations alone were insufficient to inhibit gastric cancer growth. We confirmed the anti-gastric cancer effects of GST extracts, which might provide new approaches and methods for research and development of gastric cancer drugs.

Herpesvirus entry mediator regulates the transduction of Tregs via STAT5/Foxp3 signaling pathway in ovarian cancer cells.

The ratio of regulatory T cells (Treg) in peripheral blood of cancer patients has a closely correlation to the occurrence and development of ovarian cancer. In this study, our aim to explore the expression of herpesvirus entry mediator (HVEM) in ovarian cancer and its correlation with Tregs. The expression of HVEM in peripheral blood of ovarian cancer patients was detected by ELISA, and the ratio of CD4+ CD25 + Foxp3 positive Tregs cells was detected by flow cytometry. Ovarian cancer cell lines with high- and low-HVEM expression were constructed. CD4+ cells were co-cultured with ovarian cancer (OC) cells, and the expressions of IL-2 and TGF-β1 in the supernatant of cells were detected by ELISA, and western blot was used to detect the expressions of STAT5, p-STAT5, and Foxp3. The results indicated that the number of Treg cells in the peripheral blood of OC patients increased, and the expression of HVEM increased, the two have a certain correlation. At the same time, the overexpression of HVEM promoted the expression of cytokines IL-2 and TGF- β1, promoted the activation of STAT5 and the expression of Foxp3, leading to an increase in the positive rate of Treg, while the HVEM gene silence group was just the opposite. Our results showed that the expression of HVEM in OC cells has a positive regulation effect on Tregs through the STAT5/Foxp3 signaling pathway. To provide experimental basis and related mechanism for the clinical treatment of ovarian cancer.

Mesenchymal Stem/Stromal Cells May Decrease Success of Cancer Treatment by Inducing Resistance to Chemotherapy in Cancer Cells.

Simple SummaryTumours consist of different cell types and an extracellular matrix, all of which together form a complex microenvironment. The tumour microenvironment plays a critical role in various aspects of tumour development and progression. Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells that have a tri-lineage differentiation capacity and are one of the key stromal cells in the tumour microenvironment. Following the interaction with cancer cells, they are transformed from naïve MSCs to tumour-associated MSCs, which substantially affect tumour growth and progression as well as the development of chemoresistance in cancer cells. The aim of this review article is to provide an overview of studies that have investigated how MSCs affect the susceptibility of cancer cells to chemotherapeutics. Their results show that MSCs protect cancer cells from chemotherapeutics by influencing several signalling pathways. This knowledge is crucial for the development of new treatment approaches that will lead to improved treatment outcomes.The tumour microenvironment, which is comprised of various cell types and the extracellular matrix, substantially impacts tumour initiation, progression, and metastasis. Mesenchymal stem/stromal cells (MSCs) are one of the key stromal cells in the tumour microenvironment, and their interaction with cancer cells results in the transformation of naïve MSCs to tumour-associated MSCs. The latter has an important impact on tumour growth and progression. Recently, it has been shown that they can also contribute to the development of chemoresistance in cancer cells. This review provides an overview of 42 studies published between 1 January 2001 and 1 January 2022 that examined the effect of MSCs on the susceptibility of cancer cells to chemotherapeutics. The studies showed that MSCs affect various signalling pathways in cancer cells, leading to protection against chemotherapy-induced damage. Promising results emerged from the use of inhibitors of various signalling pathways that are affected in cancer cells due to interactions with MSCs in the tumour microenvironment. These studies present a good starting point for the investigation of novel treatment approaches and demonstrate the importance of targeting the stroma in the tumour microenvironment to improve treatment outcomes.

GLUT3 Promotes Epithelial-Mesenchymal Transition via TGF-β/JNK/ATF2 Signaling Pathway in Colorectal Cancer Cells.

Glucose transporter (GLUT) 3, a member of the GLUTs family, is involved in cellular glucose utilization and the first step in glycolysis. GLUT3 is highly expressed in colorectal cancer (CRC) and it leads to poor prognosis to CRC patient outcome. However, the molecular mechanisms of GLUT3 on the epithelial–mesenchymal transition (EMT) process in metastatic CRC is not yet clear. Here, we identified that activation of the c-Jun N-terminal kinase (JNK)/activating transcription factor-2 (ATF2) signaling pathway by transforming growth factor-β (TGF-β) promotes GLUT3-induced EMT in CRC cells. The regulation of GLUT3 expression was significantly associated with EMT-related markers such as E-cadherin, α- smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), vimentin and zinc finger E-box binding homeobox 1 (ZEB1). We also found that GLUT3 accelerated the invasive ability of CRC cells. Mechanistically, TGF-β induced the expression of GLUT3 through the phosphorylation of JNK/ATF2, one of the SMAD-independent pathways. TGF-β induced the expression of GLUT3 by increasing the phosphorylation of JNK, the nuclear translocation of the ATF2 transcription factor, and the binding of ATF2 to the promoter region of GLUT3, which increased EMT in CRC cells. Collectively, our results provide a new comprehensive mechanism that GLUT3 promotes EMT process through the TGF-β/JNK/ATF2 signaling pathway, which could be a potential target for the treatment of metastatic CRC.

Tumor microbiome metabolism: A game changer in cancer development and therapy.

Accumulating recent evidence indicates that the human microbiome plays essential roles in pathophysiological states, including cancer. The tumor microbiome, an emerging concept that has not yet been clearly defined, has been proven to influence both cancer development and therapy through complex mechanisms. Small molecule metabolites produced by the tumor microbiome through unique biosynthetic pathways can easily diffuse into tissues and penetrate cell membranes through transporters or free diffusion, thus remodeling the signaling pathways of cancer and immune cells by interacting with biomacromolecules. Targeting tumor microbiome metabolism could offer a novel perspective for not only understanding cancer progression but also developing new strategies for the treatment of multiple cancer types. Here, we summarize recent advances regarding the role the tumor microbiome plays as a game changer in cancer biology. Specifically, the metabolites produced by the tumor microbiome and their potential effects on the cancer development therapy are discussed to understand the importance of the microbial metabolism in the tumor microenvironment. Finally, new anticancer therapeutic strategies that target tumor microbiome metabolism are reviewed and proposed to provide new insights in clinical applications.

Celastrol attenuates amphiregulin expression by inhibiting MAPK signaling pathway in triple-negative breast cancer cells

BackgroundAmphiregulin (AREG) plays an essential role in the metastasis and angiogenesis of various cancers. PurposeThe objective of this study was to investigate the pharmacological effects of celastrol on AREG expression in triple-negative breast cancer (TNBC) cells. MethodsWe analyzed the prognostic value of AREG, EGF, TGFA, and HB-EGF mRNA expression in TNBC patients. Cell proliferation was analyzed by MTT assay and colony-forming assay. Levels of protein and gene expression were analyzed by ELISA, Western blotting, confocal microscopy, and real-time PCR, respectively. The expression of angiogenic proteins was analyzed by using the Human angiogenesis array kit. Cell invasiveness was analyzed by Boyden chamber assay. ResultsOur results showed that abnormal AREG induction remarkably decreased the relapse-free survival (RFS) rate of TNBC patients, whereas epidermal growth factor (EGF), transforming growth factor-alpha (TGFA), or heparin-binding EGF-like growth factor (HBEGF) demonstrated no effect on RFS rate. In addition, the growth of TNBC cells was prevented by drugs targeting EGFR such as neratinib or lapatinib. On the contrary, cell growth and invasion were enhanced by AREG treatment. Interestingly, celastrol reduced AREG expression and the growth of TNBC cells. Furthermore, we found that levels of AREG expression were reduced by a potent inhibitor of MEK1/2, binimetinib (BIN). Our results showed that celastrol significantly decreased the levels of ERK phosphorylation in TNBC cells. Finally, we observed that celastrol also suppressed the AREG-induced EGFR signaling pathway. Taken together, these results suggest that celastrol can downregulate AREG expression by inhibiting the MEK/ERK signaling pathway. In addition, the AREG-induced EGFR signaling pathway was inhibited by celastrol. ConclusionCelastrol can be an effective drug for treating TNBC by inhibiting AREG/EGFR signaling pathways.

Forced Overexpression of Signal Transducer and Activator of Transcription 3 (STAT3) Activates Yes-Associated Protein (YAP) Expression and Increases the Invasion and Proliferation Abilities of Small Cell Lung Cancer (SCLC) Cells.

Background: We sought to investigate the interaction between signal transducer and activator of transcription 3 (STAT3) and the Yes-associated protein (YAP) signaling pathway in human small cell lung cancer (SCLC) cells. Methods: The STAT3-overexpressing SCLC cell lines H146 and H446 were established by plasmid DNA transfection for in vitro and in vivo experiments. Results: Overexpression of STAT3 increased YAP protein expression in H146 and H446 cells. STAT3 overexpression significantly increased YAP mRNA expression and the mRNA expression of the YAP signaling downstream genes CTGF and CYR61 in H146 and H446 cells (p < 0.05). We showed that STAT3 overexpression promoted EMT (epithelial–mesenchymal transition) with increased matrix metalloproteinase (MMP)-2 and MMP9 expression. Transwell assays showed that STAT3 overexpression increased the invasion ability of H146 and H446 cells. In addition, STAT3-overexpressing H146 cells grew significantly more rapidly than control H146 cells in the xenograft mouse model (p < 0.05). Immunohistochemistry (IHC) staining and Western blotting (WB) showed that STAT3-overexpressing H146 tumors had increased p-STAT3 and YAP staining and protein expression compared with control tumors. Increased EMT was also observed in STAT3-overexpressed xenograft tumors. Conclusions: The results of our study suggest that the overexpression of STAT3 promotes SCLC EMT, invasion, and proliferation through the activation of the YAP signaling pathway.

Pharmacological Properties of 4', 5, 7-Trihydroxyflavone (Apigenin) and Its Impact on Cell Signaling Pathways.

Plant bioactive compounds, particularly apigenin, have therapeutic potential and functional activities that aid in the prevention of infectious diseases in many mammalian bodies and promote tumor growth inhibition. Apigenin is a flavonoid with low toxicities and numerous bioactive properties due to which it has been considered as a traditional medicine for decades. Apigenin shows synergistic effects in combined treatment with sorafenib in the HepG2 human cell line (HCC) in less time and statistically reduces the viability of tumor cells, migration, gene expression and apoptosis. The combination of anti-cancerous drugs with apigenin has shown health promoting potential against various cancers. It can prevent cell mobility, maintain the cell cycle and stimulate the immune system. Apigenin also suppresses mTOR activity and raises the UVB-induced phagocytosis and reduces the cancerous cell proliferation and growth. It also has a high safety threshold, and active (anti-cancer) doses can be gained by consuming a vegetable and apigenin rich diet. Apigenin also boosted autophagosome formation, decreased cell proliferation and activated autophagy by preventing the activity of the PI3K pathway, specifically in HepG2 cells. This paper provides an updated overview of apigenin’s beneficial anti-inflammatory, antibacterial, antiviral, and anticancer effects, making it a step in the right direction for therapeutics. This study also critically analyzed the effect of apigenin on cancer cell signaling pathways including the PI3K/AKT/MTOR, JAK/STAT, NF-κB and ERK/MAPK pathways.

Investigation of the Effect of Lactobacillus Brevis Bacteria on the Expression of Rel A, IKB, and Casp3 Genes in HT29 Colon Cancer Cells

Aims Studies have shown that probiotic bacteria inhibit the onset and progression of carcinogenesis through different pathways. Our objective in this study was to determine the effect of probiotic bacteria on the expression of growth-related genes Rel A, IKB, and Casp3 in HT29 colon cancer cells Methods &amp; Materials In this study, the Lactobacillus brevis probiotic bacteria were first cultured, and after the supply of media conditioning, they were treated on HT29 cancer cells. The bacterium’s cytotoxic effect (bacterial T cells) was investigated using a microculture tetrazolium test (MTT). DNA was extracted from the treated cells, and a DNA Ladder assay was performed. Also, the 4′,6-diamidino-2-phenylindole (DAPI) test was performed to show cell apoptosis. After ribonucleic acid (RNA) extraction and complementary DNA (cDNA) preparation to determine the mechanism of the effect of this bacterium on cellular signaling, the expression of growth-related genes Rel A, IKB, and Cas3 was measured using a real-time polymerase chain reaction (PCR) method. Findings The microculture tetrazolium (MTT) test showed that L. b bacteria inhibit HT29 cells’ proliferation, induce apoptosis in these cells, and inhibit Rel A gene proliferation by increasing IKB gene expression. Also, 4′,6-diamidino-2-phenylindole (DAPI), and DNA ladder assay following the treatment of HT29 cells regarding the mentioned bacteria showed qualitative changes in cell apoptosis. In addition, real-time polymerase chain reaction (PCR) results showed that L. b increased Casp3 gene expression in HT29 colon cancer cells (P=0.038). Conclusion Our findings indicate that L. b stimulates the apoptotic cell signaling pathway in HT29 colon cancer cells. It can be used as a new treatment strategy or therapy for colon cancer treatment.

Tumor necrosis factor-α-inducing protein of Helicobacter pylori promotes epithelial-mesenchymal transition and cancer stem-like cells properties via activation of Wnt/β-catenin signaling pathway in gastric cancer cells.

Tumor necrosis factor-α-inducing protein (Tipα) is a newly identified toxin that promotes the inflammation and carcinogenesis caused by Helicobacter pylori. However, its mechanism of pathogenesis is still unclear. To investigate the carcinogenic mechanisms of Tipα, SGC7901 cells and SGC7901-derived cancer stem-like cells (CSCs) were stimulated by recombinant Tipα with or without Wnt/β-catenin signaling inhibitor XAV939. qRT-PCR and Western blotting were employed to detect expression of epithelial-mesenchymal transition (EMT), CSCs markers and downstream target genes of this signaling pathway. The cell migration ability was measured by wound healing assay and transwell assay. Our results indicated that Tipα promoted CSC properties of SGC7901 spheroids, including increased expression of CSC specific surface markers CD44, Oct4 and Nanog and an increased capacity for self-renewal. Tipα activated Wnt/β-catenin signaling in both SGC7901 cells or CSCs. Furthermore, Tipα induced the EMT and increased the expressions of downstream target genes of this signaling, including c-myc, cyclin D1 and CD44. However, XAV939 pretreatment inhibited Tipα-induced EMT and CSC properties in SGC7901 cells or CSCs. These results suggest that Tipα promotes EMT and CSC-like properties in gastric cancer cells through activation of Wnt/β-catenin signaling pathway, thereby accelerating the progression of gastric cancer.

Estrogenic activity of freeze-dried silkworm extracts through the activation of estrogen receptors in MCF-7 cells

Silkworm is an insect that feeds on mulberry leaves only, and silkworm powder has been reported to have antioxidant, hypoglycemic, and anticholesterol activities. In this study, we measured the content of 1-deoxynojirimycin (1-DNJ) in 24 different extracts from silkworm cocoons and also investigated the estrogenic activities of these extracts and their effects on the activation of intracellular signaling pathways in MCF-7 breast cancer cells. Among the 24 silkworm extracts, relatively high 1-DNJ content and estrogenic activity were shown by Extract 11 (30E3H20-1), which was prepared by a single extraction of silkworm powder with 20 times the volume of 30% ethanol against each powder weight for 3 h. The estrogen receptor activity of this extract was confirmed based on its promoting effects on the phosphorylation of the estrogen receptor α (ERα) and mRNA expression of the ESR1 and ESR2 genes. In addition, treatment with Extract 11 (30E3H20-1) increased the phosphorylation of AKT, p38, and JNK, which are downstream proteins of ERα. Based on our findings, a silkworm extract could be developed as a natural estrogen supplement in the future.

FSCN1 Promotes Glycolysis and Epithelial-Mesenchymal Transition in Prostate Cancer through a YAP/TAZ Signaling Pathway.

Objective The aim of the study is to investigate the role and possible mechanism of fascin-1 (FSCN1) in the invasion, migration, glycolysis, and epithelial-mesenchymal transition (EMT) of prostate cancer. Methods Real-time quantitative polymerase chain reaction (qRT-PCR) was utilized to determine the mRNA expression level of FSCN1 in prostate cancer tissues and prostate cancer cells PC-3 and DU145. The transwell and the scratch test were applied to detect the invasion and migration abilities of cells, respectively. A metabolic assay was used for measuring the glucose consumption, lactate production, and the extracellular acidification rate (ECAR) in cells; western blot was used for checking FSCN1, EMT, and yes-associated protein/transcriptional co-activators with the PDZ-binding motif (YAP/TAZ) signaling pathway-related protein expression level in cells or tissues. Results FSCN1 was significantly highly expressed in prostate cancer tissues and cells. On the one hand, interference with the expression of FSCN1 could inhibit the invasion, migration, EMT, and glycolysis of prostate cancer cells. On the other hand, overexpression of FSCN1 promoted the invasion, migration, EMT, and glycolysis of prostate cancer cells. Besides, further mechanistic studies revealed that FSCN1 could activate the YAP/TAZ signaling pathway in prostate cancer cells. Conclusion FSCN1 promotes invasion, migration, EMT, and glycolysis in prostate cancer cells by activating the YAP/TAZ signaling pathway. FSCN1 may be used as a biomarker for the diagnosis or treatment in prostate cancer.

Transcription factor p8 regulates autophagy in response to disulfiram via PI3K/mTOR/p70S6K signaling pathway in pancreatic cancer cells.

Disulfiram (DSF), which is an inhibitor of aldehyde dehydrogenase (ALDH) and approved by the FDA for the treatment of alcoholism previously, has been repurposed for use as a cancer treatment because of its potent effect in preclinical studies. In this study, we found that disulfiram forms potent complexes with copper (DSF/Cu) inhibited cell proliferation, induced apoptosis in human pancreatic cancer cells, which was detected by flow cytometry and western blotting. Meanwhile, autophagy and autophagic flux also clearly observed by transmission electron microscopy, confocal microscopy and flow cytometry. Our results also showed that DSF/Cu induced transcription factor p8 upregulation and PI3K/mTOR signaling pathway activation detected by real-time PCR and western blotting. Additionally, suppression of p8 inactivated the mTOR signaling pathway and autophagic flux maintained. Furthermore, mechanism study indicated that autophagy induced by DSF/Cu was regulated by p8 and wasrelated to PI3K/mTOR/p70S6K signaling pathway in pancreatic cancer cells. Our findings provide insights into the role of p8 in regulatingautophagy induced by DSF/Cu effectsin pancreatic cancer cells.

Stem Cell Models for Cancer Therapy.

Metastatic progression of female breast and colon cancer represents a major cause of mortality in women. Spontaneous/acquired resistance to conventional and targeted chemo-endocrine therapy is associated with the emergence of drug-resistant tumor-initiating cancer stem cell populations. The cancer-initiating premalignant stem cells exhibit activation of select cancer cell signaling pathways and undergo epithelial–mesenchymal transition, leading to the evolution of a metastatic phenotype. The development of reliable cancer stem cell models provides valuable experimental approaches to identify novel testable therapeutic alternatives for therapy-resistant cancer. Drug-resistant stem cell models for molecular subtypes of clinical breast cancer and for genetically predisposed colon cancer are developed by selecting epithelial cells that survive in the presence of cytostatic concentrations of relevant therapeutic agents. These putative stem cells are characterized by the expression status of select cellular and molecular stem cell markers. The stem cell models are utilized as experimental approaches to examine the stem-cell-targeted growth inhibitory efficacy of naturally occurring dietary phytochemicals. The present review provides a systematic discussion on (i) conceptual and experimental aspects relevant to the chemo-endocrine therapy of breast and colon cancer, (ii) molecular/cellular aspects of cancer stem cells and (iii) potential stem-cell-targeting lead compounds as testable alternatives against the progression of therapy-resistant breast and colon cancer.

Emerging Role of CREB in Epithelial to Mesenchymal Plasticity of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive solid malignancy with a high rate of metastasis and therapeutic resistance as its major hallmarks. Although a defining mutational event in pancreatic cancer initiation is the presence of oncogenic KRAS, more advanced PDAC lesions accumulate additional genomic alterations, including loss of tumor suppressor gene TP53. Co-occurrence of mutant KRAS and TP53 in PDAC promotes hyperactivation of cancer cell signaling pathways driving epithelial to mesenchymal plasticity (EMP). The cellular process of EMP influences the biological behavior of cancer cells by increasing their migratory and invasive properties, thus promoting metastasis. Our previous work has demonstrated that oncogenic KRAS-mediated activation of cyclic AMP response element-binding protein 1 (CREB) is one of the critical drivers of PDAC aggressiveness. The therapeutic approach of targeting this key transcription factor attenuates tumor burden in genetically engineered mouse models (GEMMs) of this disease. Herein, we discuss the significant role of CREB in perpetuating disease aggressiveness and therapeutic resistance through the EMP process. Furthermore, this review updates the therapeutic implications of targeting CREB, highlighting the challenges and emerging approaches in PDAC.

Alpiniakatsumadai Hayata induces growth inhibition and autophagy‑related apoptosis by regulating the AMPK and Akt/mTOR/p70S6K signaling pathways in cancer cells.

Alpinia katsumadai Hayata (AKH), a widely used traditional Chinese medicine, exerts various biological functions, including anti-inflammatory, antioxidant, anti-microbial and anti-asthmatic effects. However, studies on its anticancer activity and associated mechanisms are limited. The present study investigated the effects of ethanol extract from AKH on the viability of various human cancer and normal liver LX-2 cells using Cell Counting Kit-8 assay. Apoptosis was detected by Hoechst 33342/PI staining and Annexin-V-FITC/PI double staining. Autophagy was examined by Ad-GFP-LC3B transfection. The association between AKH-induced autophagy and apoptosis was investigated by pre-treatment of the cells with the autophagy inhibitors, 3-methyladenine (3MA) and bafilomycin A1 (Baf-A1), followed by treatment with AKH. The expression levels of cleaved poly(ADP-ribose) polymerase (PARP), caspase-8, caspase-3, caspase-9, phosphorylated (p-)AMP-activated protein kinase (AMPK), Akt, mTOR and p70S6K were examined using western blot analysis. The in vivo antitumor activity of AKH was investigated in nude mice bearing A549 lung cancer xenografts. The components of AKH were detected by liquid chromatography mass spectrometry-ion trap-time-of-flight mass spectrometry. The results revealed that AKH significantly inhibited the proliferation of various cancer cells with the half maximal inhibitory concentration (IC50) values of 203–284 µg/ml; however, its inhibitory effect was much less prominent against normal liver LX-2 cells with an IC50 value of 395 µg/ml. AKH markedly induced apoptosis and autophagy, and upregulated the protein expression of cleaved-caspase-3, caspase-8, caspase-9 and cleaved PARP in a concentration-dependent manner. Of note, the autophagy inhibitors (3MA and Baf-A1) significantly attenuated its pro-apoptotic effects on human pancreatic cancer Panc-28 and lung cancer A549 cells. Furthermore, AKH significantly increased the levels of p-AMPK, and decreased those of p-Akt, p-mTOR and p-p70S6K in Panc-28 and A549 cells. AKH markedly inhibited the growth of A549 tumor xenografts in vivo. In addition, a total of nine compounds were detected from AKH. The present study demonstrates that AKH markedly inhibits the growth and induces autophagy-related apoptosis in cancer cells by regulating the AMPK and Akt/mTOR/p70S6K signaling pathways. AKH and/or its active fractions may thus have potential to be developed as novel anticancer agents for clinical use.

Abstract 106: Diclofenac hybrids with oleanolic acid oximes modulate key signaling pathways in pancreatic cancer cells

Abstract Pancreatic cancer is characterized by constitutive activation of mitogen-activated protein kinases (MAPKs) like JNK and p38 determining its malignant phenotypes. Overexpression of NF-κB and COX-2 involved in inflammation contributes to increased cell growth and invasiveness. The latter might be related to the activated AKT pathway. Nonsteroidal anti-inflammatory drugs were suggested for pancreas cancers prophylaxis. Naturally occurring oleanolic acid (OA) was shown to possess anti-inflammatory and anti-tumorigenic activities. Coupling its analogs with NSAIDs may enhance this effect. Our study aimed to evaluate the effect of two oleanolic acid oximes derivatives conjugated with diclofenac (DCL), 4c (3-diclofenacoxyiminoolean-12-en-28-oic acid benzyl ester) and 4d (3-diclofenacoxyiminoolean-12-en-28-oic acid morpholide) on inflammation-related pathways, MAPK and NF-κB, cell cycle arrest and ROS formation in PSN-1 pancreatic cancer cells. PSN-1 cells were incubated with DCL, 4c or 4d at the concentration of 30µM. Activation of NF-κB was assessed by measuring binding of its p50 and p65 subunits to DNA using ELISA method. Their nuclear concentrations were determined by Western blot. RT-PCR was used to evaluate NF-κB subunits and COX-2 mRNA levels. Bead-based immunoassay determined the cytosolic levels of p38, JNK, CREB, AKT, P70S6K and STAT3 proteins. Cell cycle distribution and ROS formation were assessed using flow cytometry. Conjugate 4d significantly reduced nuclear level of p65 subunit and its mRNA expression, although compound 4c had more significant impact on COX-2 expression. Both derivatives significantly diminished p65 DNA binding, but only 4d had significant effect on p50. Both compounds reduced ROS production, but neither of them affected the cell cycle arrest. We observed decreased cytosolic AKT level along with increased levels of unphosphorylated JNK and p38 kinases which may have an antagonistic effect on proliferation by reducing activity of inflammatory mediators acting as tumor promoters. AKT decrease after treatment with 4d derivative might be the cause of increased unphosphorylated CREB level. Accumulation of p38 and JNK in cytosol could be a result of decreased ROS production and/or cause increased CREB protein level. Additionally, increased STAT3 cytosolic level suggests inhibition of mTORC1 pathway that explains reduced p70S6 kinase. Overall, these results suggest that OAO-DCL morpholide conjugate may affect both NF-κB and MAPK signaling pathways in pancreatic cancer cells. Further studies are required to explain detailed mechanism of these modifications. Funding: Studies were supported by Polish National Science Centre grant 2016/21/B/NZ7/01758. Acknowledgments: Authors thank Dr. Gary Gorbsky, Chair of Cell Cycle &amp; Cancer Biology Program in Oklahoma Medical Research Foundation for supporting financing Maria Narozna conference participation. Citation Format: Maria Narozna, Violetta Krajka-Kuzniak, Robert Kleszcz, Wanda Baer-Dubowska. Diclofenac hybrids with oleanolic acid oximes modulate key signaling pathways in pancreatic 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 106.

Abstract 2437: Identification of MDMX/MDM2 metastasis signaling pathways in breast cancer cells with mutant p53

Abstract Human breast cancers often overexpress MDM family proteins MDM2 and MDMX(MDM4). MDM2 is an E3 ubiquitin ligase and MDMX is an E4 that promotes ubiquitination of MDM2 targets (however this is an understudied area). The Cancer Genome Atlas (TCGA) shows high MDMX/MDM2 expression in all breast cancer subtypes. We identified that MDMX depletion reduces circulating tumor cells (CTCs) and metastasis of breast cancers with mtp53. Our ongoing objective is to determine the mechanisms of how MDMX promotes CTCs and metastasis, and the potential MDMX regulated ubiquitinated targets. In TNBC primary tumors, knockdown of mdmx correlates with reduced expression of the G protein coupled receptor, CXCR4. We used mtp53 expressing T47D ER+, and MDA-MB-468, and MDA-MB-231 metastatic TNBC cells and their derived CTC lines, and shRNA-mediated knockdown of MDM2 and MDMX derived lines, to address the potential MDMX targets. We assessed metastatic properties of migration and invasion changed by the knockdown of MDM2/MDMX in parental and CTC cell lines using multi-faceted cell biological and biochemical approaches. Depletion of MDM2 in T47D and MDA-MB-231 cells increased the level of MDMX, demonstrating a critical function of MDM2 for regulating MDMX. MDMX depletion surprisingly did not change MDM2 levels, but did reduce the migration of both MDA-MB-231 cells and their derived CTC lines. Interestingly, the parental MDA-MB-231 cells migrated more rapidly in scratch assays, compared to both their derived CTCs and MDMX-depleted CTCs, suggesting migration to be critical for intravasation, but not for latter cancer cell survival. Experiments are in progress to address if loss of MDMX perturbs CXCL12 ligand communication with the CXCR4 receptor in parental and CTC breast cancer lines. Preliminary data suggests activated MDMX expression correlates with the activation of these metastasis signal transduction pathways. The ubiquitination targets of MDMX/MDM2 in the parental and CTC cell lines are being defined by affinity purification in the presence, and absence, of MDMX/MDM2, and add back of purified MDMX/MDM2 to validate targets. As expected, purified MDMX increases MDM2 E3 ubiquitin ligase activity, and on its own MDMX is inert. Stable isotope labeling in cell culture suggested protein targets involved in DNA replication and repair pathways. This preliminary data informed our interest in studying DNA damage signaling by pharmacologically inhibiting the DNA repair protein PARP. We examined how MDMX/MDM2 pathways respond to DNA damage caused by PARP inhibition. Our preliminary data affirms regulation of the MDMX/MDM2 stress-response circuit by replication stress response pathways, and reveals the requirement of MDMX/MDM2 for their function in the studied cell lines. Disrupting MDMX function has potential to be beneficial for inhibition of breast cancer progression and metastasis. Supported by BCRF-20-011. Citation Format: Rusia Lee, Viola Ellison, Gu Xiao, Dominique Forbes, Pam Leybengrub, Alexandra Kern, Falande Alexandre, George Annor, Jill Bargonetti. Identification of MDMX/MDM2 metastasis signaling pathways in breast cancer cells with mutant p53 [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 2437.