Phenotypic Changes In Cancer Cells Research Articles

Abstract 3180: Suppression of the CPEB3 ribozyme modulates the progression of glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most aggressive primary malignant brain tumor in adults, with a poor prognosis that highlights a dire clinical need for innovative therapeutic interventions. Despite significant advances in diagnoses and multimodality therapies, the overall prognosis for patients with GBM remains poor, with a median survival time of 15-18 months. Therefore, there is an unmet medical need to develop alternative treatment strategies to improve clinical outcomes. Dysregulation of post-transcriptional control and translational machinery have been implicated in malignant tumor development. Cytoplasmic polyadenylation element binding proteins (CPEB1-CPEB4) are RNA-binding proteins that regulate poly(A) tail elongation of target mRNAs and subsequently contribute to phenotypic changes in cancer cells. Notably, a self-cleaving ribozyme was identified in the CPEB3 gene, but its role in cancer is wholly unexplored. Considering the role of CPEB3 as a tumor suppressor gene and the promotion of cancer progression through the downregulation of CPEB3, our hypothesis is that the CPEB3 ribozyme regulates CPEB3 expression, and its activity contributes to the progression of tumors. Using antisense oligonucleotides (ASOs) as an approach, we demonstrated that inhibition of CPEB3 ribozyme resulted in an increase of CPEB3 mRNA and protein expression. Blocking the CPEB3 ribozyme led to a significant reduction in cell proliferation, migration, and invasion in GBM cell lines. Gene set enrichment analysis (GSEA) revealed the downregulation of epithelial-mesenchymal transition (EMT), angiogenesis, and hypoxia gene sets in GBM cells treated with ASO compared to Ctrl-ASO. We further measured VEGFA mRNA and protein expression and found that ASO-treated GBM cells secreted significantly less VEGF in conditioned media. Inhibition of the CPEB3 ribozyme also mitigated the EMT process in GBM cells. Subsequently, ASO strategies were applied to patient-derived glioma stem cells (GSCs), representing a clinically relevant model for pre-clinical therapeutic intervention. We found that treatment of CPEB3 ribozyme ASO up-regulated CPEB3 mRNA and inhibited cell proliferation in GSCs. Furthermore, the combination of ASO and temozolomide chemotherapy exhibited a more pronounced decrease in GSCs proliferation compared to individual treatment alone. Collectively, this study highlights the significance of the CPEB3 ribozyme in GBM and explores therapeutic approaches focused on targeting CPEB3 in cancer. Citation Format: Claire Chen, Eric Wang, Lily Tong, Mehran Nikan, Daniela A. Bota, Claudia Benavente, Andrej Luptak. Suppression of the CPEB3 ribozyme modulates the progression of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3180.

Microenvironmental stiffness induces metabolic reprogramming in glioblastoma

The mechanical properties of solid tumors influence tumor cell phenotype and the ability to invade surrounding tissues. Using bioengineered scaffolds to provide a matrix microenvironment for patient-derived glioblastoma (GBM) spheroids, this study demonstrates that a soft, brain-like matrix induces GBM cells to shift to a glycolysis-weighted metabolic state, which supports invasive behavior. We first show that orthotopic murine GBM tumors are stiffer than peritumoral brain tissues, but tumor stiffness is heterogeneous where tumor edges are softer than the tumor core. We then developed 3D scaffolds with μ-compressive moduli resembling either stiffer tumor core or softer peritumoral brain tissue. We demonstrate that the softer matrix microenvironment induces a shift in GBM cell metabolism toward glycolysis, which manifests in lower proliferation rate and increased migration activities. Finally, we show that these mechanical cues are transduced from the matrix via CD44 and integrin receptors to induce metabolic and phenotypic changes in cancer cells.

Conventional Cancer Therapies Can Accelerate Malignant Potential of Cancer Cells by Activating Cancer-Associated Fibroblasts in Esophageal Cancer Models.

Esophageal cancer is one of the most aggressive tumors, and the outcome remains poor. One contributing factor is the presence of tumors that are less responsive or have increased malignancy when treated with conventional chemotherapy, radiotherapy, or a combination of these. Cancer-associated fibroblasts (CAFs) play an important role in the tumor microenvironment. Focusing on conventional cancer therapies, we investigated how CAFs acquire therapeutic resistance and how they affect tumor malignancy. In this study, low-dose chemotherapy or radiotherapy-induced normal fibroblasts showed enhanced activation of CAFs markers, fibroblast activation protein, and α-smooth muscle actin, indicating the acquisition of malignancy in fibroblasts. Furthermore, CAFs activated by radiotherapy induce phenotypic changes in cancer cells, increasing their proliferation, migration, and invasion abilities. In in vivo peritoneal dissemination models, the total number of tumor nodules in the abdominal cavity was significantly increased in the co-inoculation group of cancer cells and resistant fibroblasts compared to that in the co-inoculation group of cancer cells and normal fibroblasts. In conclusion, we demonstrated that conventional cancer therapy causes anti-therapeutic effects via the activation of fibroblasts, resulting in CAFs. It is important to select or combine modalities of esophageal cancer treatment, recognizing that inappropriate radiotherapy and chemotherapy can lead to resistance in CAF-rich tumors.

Role of Oncogenic Pathways on the Cancer Immunosuppressive Microenvironment and Its Clinical Implications in Hepatocellular Carcinoma.

Simple SummaryHepatocellular carcinoma is known to become resistant to treatments easily by mutations in genes involved in the key cellular pathways targeted by current molecular targeted agents (MTAs). However, the immune checkpoint inhibitor (ICI) is a promising modality for cancer treatment, in which the cancer cells are made recognizable by the immune system. Blockade of the PD1/PDL1 proteins, which help cancers evade the immune system, is currently being tested in clinical trials in combination with MTAs. In this review, several cellular signaling pathways that can alter the immune processes within the tumor and can subsequently affect the patient’s response to ICIs are detailed. This review may help scientists and clinicians to better understand the molecular factors that can influence ICI-based therapy and will help in identifying suitable cases for this type of treatment.The tumor immune microenvironment, including hepatocellular carcinoma (HCC), is complex, consisting of crosstalk among tumor components such as the cancer cells, stromal cells and immune cells. It is conceivable that phenotypic changes in cancer cells by genetic and epigenetic alterations affect the cancer–stroma interaction and anti-cancer immunity through the expression of immune checkpoint molecules, growth factors, cytokines, chemokines and metabolites that may act on the immune system in tumors. Therefore, predicting the outcome of ICI therapy requires a thorough understanding of the oncogenic signaling pathways in cancer and how they affect tumor immune evasion. In this review, we have detailed how oncogenic signaling pathways can play a role in altering the condition of the cellular components of the tumor immune microenvironment such as tumor-associated macrophages, regulatory T cells and myeloid-derived suppressor cells. The RAS/MAPK, PI3K/Akt, Wnt/β-catenin and JAK/STAT pathways have all been implicated in anti-tumor immunity. We also found that factors that reflect the immune microenvironment of the tumor, including the status of oncogenic pathways such as the volume of tumor-infiltrating T cells, expression of the immune checkpoint protein PD-1 and its ligand PD-L1, and activation of the Wnt/β-catenin signaling pathway, predict a response to ICI therapy in HCC cases.

Active elimination of intestinal cells drives oncogenic growth in organoids.

SummaryCompetitive cell interactions play a crucial role in quality control during development and homeostasis. Here, we show that cancer cells use such interactions to actively eliminate wild-type intestine cells in enteroid monolayers and organoids. This apoptosis-dependent process boosts proliferation of intestinal cancer cells. The remaining wild-type population activates markers of primitive epithelia and transits to a fetal-like state. Prevention of this cell-state transition avoids elimination of wild-type cells and, importantly, limits the proliferation of cancer cells. Jun N-terminal kinase (JNK) signaling is activated in competing cells and is required for cell-state change and elimination of wild-type cells. Thus, cell competition drives growth of cancer cells by active out-competition of wild-type cells through forced cell death and cell-state change in a JNK-dependent manner.

Development of a luciferase/luciferin cell proliferation (XenoLuc) assay for real-time measurements of Gfp-Luc2-modified cells in a co-culture system

BackgroundIn vitro modelling of cancer cells is becoming more complex due to prevailing evidence of intimate interactions between cancer cells and their surrounding stroma. A co-culture system which consists of more than one cell type is physiologically more relevant and thus, could serve as a useful model for various biological studies. An assay that specifically detects the phenotypic changes of cancer cells in a multi-cellular system is lacking for nasopharyngeal carcinoma (NPC).ResultsHere, we describe a luciferase/luciferin (XenoLuc) assay that could specifically measure changes in the proliferation of cancer cells in the co-culture system using two modified NPC patient-derived tumour xenograft (PDTXs) cells: Xeno284-gfp-luc2 and XenoB110-gfp-luc2. Through this assay, we are able to show that the growth of NPC xenograft cells in both two-dimensional (2D) and three-dimensional (3D) models was enhanced when co-cultured with normal human dermal fibroblasts (NHDFs). In addition, potential applications of this assay in in vitro drug or inhibitor screening experiments are also illustrated.ConclusionsXenoLuc assay is specific, sensitive, rapid and cost-effective for measuring the growth of luciferase-expressing cells in a co- or multiple-culture system. This assay may also be adapted for tumour microenvironment studies as well as drug screening experiments in more complex 3D co-culture systems.

Onco-Multi-OMICS Approach: A New Frontier in Cancer Research.

The acquisition of cancer hallmarks requires molecular alterations at multiple levels including genome, epigenome, transcriptome, proteome, and metabolome. In the past decade, numerous attempts have been made to untangle the molecular mechanisms of carcinogenesis involving single OMICS approaches such as scanning the genome for cancer-specific mutations and identifying altered epigenetic-landscapes within cancer cells or by exploring the differential expression of mRNA and protein through transcriptomics and proteomics techniques, respectively. While these single-level OMICS approaches have contributed towards the identification of cancer-specific mutations, epigenetic alterations, and molecular subtyping of tumors based on gene/protein-expression, they lack the resolving-power to establish the casual relationship between molecular signatures and the phenotypic manifestation of cancer hallmarks. In contrast, the multi-OMICS approaches involving the interrogation of the cancer cells/tissues in multiple dimensions have the potential to uncover the intricate molecular mechanism underlying different phenotypic manifestations of cancer hallmarks such as metastasis and angiogenesis. Moreover, multi-OMICS approaches can be used to dissect the cellular response to chemo- or immunotherapy as well as discover molecular candidates with diagnostic/prognostic value. In this review, we focused on the applications of different multi-OMICS approaches in the field of cancer research and discussed how these approaches are shaping the field of personalized oncomedicine. We have highlighted pioneering studies from “The Cancer Genome Atlas (TCGA)” consortium encompassing integrated OMICS analysis of over 11,000 tumors from 33 most prevalent forms of cancer. Accumulation of huge cancer-specific multi-OMICS data in repositories like TCGA provides a unique opportunity for the systems biology approach to tackle the complexity of cancer cells through the unification of experimental data and computational/mathematical models. In future, systems biology based approach is likely to predict the phenotypic changes of cancer cells upon chemo-/immunotherapy treatment. This review is sought to encourage investigators to bring these different approaches together for interrogating cancer at molecular, cellular, and systems levels.

Prostate Cancer Cells are Reprogrammed to Normal via Ultrasound-Induced Mechanotransduction

Introduction: Prostate cancer is the most incident cancer in men. Since the majority of prostate cancer patients are elderly men, often not suitable for invasive procedures, there is a need for minimally invasive therapies (e.g., focused ultrasound). We tested the hypothesis that focused ultrasound (FUS) drastically reduces the metastatic potential of chemically-treated cancer cells via overproduction of heat-shock protein 70 (HSP70), death receptor Fas and its ligand FasL. Materials and Methods: Suspensions of cultured DU145 and PC3 prostate cancer cells were placed in 0.2ml tubes. The cells were left untreated or exposed to FUS alone, 4% ethanol, or FUS+4% ethanol. The FUS signal was generated by a 1.1 MHz transducer, with acoustic power ranging from 4.1 to 20 W. Fas and FasL expression was measured by flow cytometry, and HSP 70 protein levels were determined by Western blot analysis at 2, 24, and 72 hours post-treatment. To confirm that cancer cells lose aggressiveness, we measured the number of cancer cells adhered to TNF-α-activated endothelium as well as tested the ability of cells to form multi-cellular spheroids. Results and Discussion: Prostate cancer cells significantly increased their expression of Fas, FasL, and HSP 70 immediately after being exposed to FUS+4% ethanol and continued to produce these molecules at a significantly higher amount than untreated, ethanol, or FUS alone alone-treated cells. Static adhesion assay showed that cells in the combined treatment were less likely to attach to endothelium. Cells exposed to both ethanol and FUS were unable to form three-dimensional tumor spheroids. We showed that FUS induces Hsp70 overproduction in prostate cancer cells and promotes cell apoptosis via an increase in expression of Fas and its ligand FasL. Both these factors lead to phenotypic changes in cancer cells that reduce their aggressiveness.

Dynamic Microenvironment Induces Phenotypic Plasticity of Esophageal Cancer Cells Under Flow.

Cancer microenvironment is a remarkably heterogeneous composition of cellular and non-cellular components, regulated by both external and intrinsic physical and chemical stimuli. Physical alterations driven by increased proliferation of neoplastic cells and angiogenesis in the cancer microenvironment result in the exposure of the cancer cells to elevated levels of flow-based shear stress. We developed a dynamic microfluidic cell culture platform utilizing eshopagael cancer cells as model cells to investigate the phenotypic changes of cancer cells upon exposure to fluid shear stress. We report the epithelial to hybrid epithelial/mesenchymal transition as a result of decreasing E-Cadherin and increasing N-Cadherin and vimentin expressions, higher clonogenicity and ALDH positive expression of cancer cells cultured in a dynamic microfluidic chip under laminar flow compared to the static culture condition. We also sought regulation of chemotherapeutics in cancer microenvironment towards phenotypic control of cancer cells. Such in vitro microfluidic system could potentially be used to monitor how the interstitial fluid dynamics affect cancer microenvironment and plasticity on a simple, highly controllable and inexpensive bioengineered platform.

Fc Gamma Rll (CD32b) Expression Affects Anti-CD20 Monoclonal Antibody Cellular Responses and Is Down Regulated in Rituximab Resistant Pre-Clinical Models

Rituximab's primary mechanisms-of-action include: antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CMC), induction of apoptosis and anti-proliferation. The acquirement of phenotypic changes in cancer cells or host immune cells over time may affect rituximab responsiveness and underscores the complexity of the potential mechanisms-of-resistance to anti-CD20 monoclonal antibodies (mAbs). Laboratory studies had demonstrated the importance of FcγRIIa and FcγRIII activating receptors in the biological activity of rituximab and other monoclonal antibodies (mAbs) in cancer medicine. Recently, investigators had focused their efforts in the study of FcγRIIb function expressed primarily on normal and malignant B-cells and its role in inflammatory conditions or hematological malignancies. FcγRIIb is an inhibitory low affinity Fcg receptor capable to induce potent inhibitory (apoptotic) signals via immune receptor tyrosine-based inhibition motif (ITIM). Previously, we generated several rituximab-resistance lymphoma cell lines (RRCL) and demonstrated a global down-regulation of CD20. On the other hand, forced expression of CD20 in RRCL did not restore rituximab sensitivity suggesting the existence of additional mechanisms of resistance to mAbs targeting CD20 (Tsai et al Clin Cancer Res. 2012;18:1039-50). To this end, we evaluated the expression and significance of FcγRIIb (CD32b) in RRCL. FcγRIIb gene and protein expression levels were determined in a panel of rituximab sensitive (RSCL) or RRCL by quantitative polymerase chain reaction (qPCR) and western blotting respectively. Subsequently, we silenced FcγRIIb in RSCL using siRNA interference and evaluated changes in the capacity of several mAbs targeting CD20 (rituximab, ofatumumab, and obinotuzumab) of inducing CMC or ADCC in vitro. RRCL were found to have almost a complete loss in FcgRIIb mRNA and protein levels when compared to RSCL. In addition, silencing of FcγRIIb in RSCL decreased the capacity of rituximab (P=0.04 [ADCC] and P=0.15 [CMC]), ofatumumab (P=0.02 [ADCC] and P =0.10 [CMC]), and obinituzumab (P=0.02 [ADCC] and P<0.01 [CMC]) in inducing effective ADCC and CMC when compared to scramble transfected cells. Together our data suggests that the chronic exposure of lymphoma cells to rituximab results in the decreased expression of FcγRIIb. In addition, FcγRIIb low affinity inhibitory receptors appear to contribute to rituximab anti-tumor activity in vitro. Additional in vivo experiments are ongoing to further define the role of FcγRII in the anti-tumor activity of mAbs targeting CD20 and how it is down regulated in the context of acquired resistance to rituximab-based therapies. (Research, in part, supported by The Eugene and Connie Corasanti Lymphoma Research Fund) DisclosuresNo relevant conflicts of interest to declare.

Abstract 5074: Activation of TGF β-IL6 axis mediates chemoresistance in non-small cell lung cancer by increasing epithelial-mesenchymal transition signaling

Abstract The tumor microenvironment is composed of different types of stromal cells that represent a key component of tumor progression, while it has been speculated that stromal components are functionally organized to promote cancer cell survival. A specific subset of stromal cells, termed cancer-associated fibroblasts (CAFs), secretes factors that promote tumorigenesis. We previously reported that CAFs increase the malignant potential of non-small-cell lung cancer (NSCLC) via the TGF β-IL6 axis. The purpose of this study was to clarify the role of the TGF β-IL6 axis in communication between CAFs and NSCLC cells for chemoresistance. Methods We used standard NSCLC cell lines (A549, NCI-H358), NSCLC cells obtained using a cancer tissue-originated spheroid (CTOS) method, lung normal fibroblasts (LNFs), and CAFs obtained from primary cultures of specimens from NSCLC patients to evaluate phenotypic changes in the presence of human IL-6, TGF-β1, and conditioned medium (CM) from the cells. Immunohistochemistry was also utilized to examine the distribution of fibroblasts in 75 tumor specimens obtained from patients with NSCLC after undergoing chemoradiotherapy. Results Expression levels of myofibroblast markers were elevated in CAFs as compared to LNFs, while CM from fibroblasts induced epithelial mesenchymal transition (EMT) in NSCLC cells. CM also enhanced the expression of stem cell markers such as CD44, and increased CD133 mRNA levels in cancer cells and resistance to cisplatin in lung cancer cells as well as CTOS produced cells, indicating that the CM from fibroblasts was biologically active. Furthermore, the concentration of IL-6 was higher in CM from CAFs as compared to that from LNFs, and CM-induced phenotypic changes of cancer cells were inhibited by addition of the IL-6 receptor neutralizing antibody. Cancer cells incubated with IL-6 and TGF-β1 showed significantly increased EMT changes and acquisition of cancer stemness as compared with those treated with TGF-β1 alone, suggesting that IL-6 enhances TGF-β signaling. Cisplatin treatment also increased TGF-β expression in cancer cells, and TGF-β1 activated LNFs and CAFs to increase IL-6 production. Immunohistochemistry was used to examine EMT status, fibroblast distribution, and IL-6 expression in tumor specimens obtained from patients with NSCLC after undergoing chemotherapy. EMT changes of cancer cells were correlated with a diffuse distribution of SMA-stained fibroblasts and stromal IL-6 expression. Conclusion IL-6 from CAFs enhanced epithelial cell EMT and acquisition of cancer stemness by enhancing TGF-β signaling. IL-6 and TGF-β may play contributing roles in maintenance of the paracrine loop between these two cytokines as part of the communication between CAFs and NSCLC cells, resulting in chemoresistance. Citation Format: Yasushi Shintani, Soichiro Funaki, Toru Kimura, Ayako Fujiwara, Tomohiro Kawamura, Toshiya Bessho, Masayoshi Inoue, Masato Minami, Meinoshin Okumura. Activation of TGF β-IL6 axis mediates chemoresistance in non-small cell lung cancer by increasing epithelial-mesenchymal transition signaling. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5074. doi:10.1158/1538-7445.AM2015-5074

Novel insights into the regulation of cyclooxygenase-2 expression by platelet-cancer cell cross-talk.

Platelets are activated by the interaction with cancer cells and release enhanced levels of lipid mediators [such as thromboxane (TX)A2 and prostaglandin (PG)E2, generated from arachidonic acid (AA) by the activity of cyclooxygenase (COX)-1], granule content, including ADP and growth factors, chemokines, proteases and Wnt proteins. Moreover, activated platelets shed different vesicles, such as microparticles (MPs) and exosomes (rich in genetic material such as mRNAs and miRNAs). These platelet-derived products induce several phenotypic changes in cancer cells which confer high metastatic capacity. A central event involves an aberrant expression of COX-2 which influences cell-cycle progression and contribute to the acquisition of a cell migratory phenotype through the induction of epithelial mesenchymal transition genes and down-regulation of E-cadherin expression. The identification of novel molecular determinants involved in the cross-talk between platelets and cancer cells has led to identify novel targets for anti-cancer drug development.

Profilin-1 overexpression in MDA-MB-231 breast cancer cells is associated with alterations in proteomics biomarkers of cell proliferation, survival, and motility as revealed by global proteomics analyses.

Despite early screening programs and new therapeutic strategies, metastatic breast cancer is still the leading cause of cancer death in women in industrialized countries and regions. There is a need for novel biomarkers of susceptibility, progression, and therapeutic response. Global analyses or systems science approaches with omics technologies offer concrete ways forward in biomarker discovery for breast cancer. Previous studies have shown that expression of profilin-1 (PFN1), a ubiquitously expressed actin-binding protein, is downregulated in invasive and metastatic breast cancer. It has also been reported that PFN1 overexpression can suppress tumorigenic ability and motility/invasiveness of breast cancer cells. To obtain insights into the underlying molecular mechanisms of how elevating PFN1 level induces these phenotypic changes in breast cancer cells, we investigated the alteration in global protein expression profiles of breast cancer cells upon stable overexpression of PFN1 by a combination of three different proteome analysis methods (2-DE, iTRAQ, label-free). Using MDA-MB-231 as a model breast cancer cell line, we provide evidence that PFN1 overexpression is associated with alterations in the expression of proteins that have been functionally linked to cell proliferation (FKPB1A, HDGF, MIF, PRDX1, TXNRD1, LGALS1, STMN1, LASP1, S100A11, S100A6), survival (HSPE1, HSPB1, HSPD1, HSPA5 and PPIA, YWHAZ, CFL1, NME1) and motility (CFL1, CORO1B, PFN2, PLS3, FLNA, FLNB, NME2, ARHGDIB). In view of the pleotropic effects of PFN1 overexpression in breast cancer cells as suggested by these new findings, we propose that PFN1-induced phenotypic changes in cancer cells involve multiple mechanisms. Our data reported here might also offer innovative strategies for identification and validation of novel therapeutic targets and companion diagnostics for persons with, or susceptibility to, breast cancer.

Abstract 1055: Syntenin positively regulates TGF-β1- and oncogenic Ras-mediated EMT-like phenotypic changes in cancer cells

Abstract Epithelial to mesenchymal transition (EMT) is biological process in polarized epithelial cells, which occurs in various physiological and pathological conditions. In carcinoma progression, EMT has emerged as a critical player in regulating cancer cell invasive phenotype. Syntenin, a tandem PDZ-domain containing scaffold protein, functions as a positive regulator of cancer cell progression in several human cancers. In present study, we investigated a role of syntenin in transforming growth factor (TGF)-β1- and Ras-V12-mediated EMT-like phenotypic changes in cancer cells. Knockdown of syntenin suppressed TGF-β1- and Ras-V12-induced morphological and cell biological alterations in A549, Ep/Ras, and BEAS-2B cells that are consistent with EMT; however, overexpression of syntenin stimulated TGF-β1- and Ras-V12-induced EMT, implying that syntenin is necessary for TGF-β1- and Ras-V12- induced EMT. We found that syntenin is associated with RSK (p90 ribosomal S6 kinase) via PDZ binding motif and syntenin at Ser6 residue may directly be phosphorylated by RSK. Mutation of syntenin Ser6 to Ala suppresses TGF-β1- and oncogenic Ras-mediated EMT in a dominant negative manner by abolishing the interaction of endogenous syntenin with RSK. Strikingly, expression of syntenin-S6E, a phosphomimetic mutant of syntenin at Ser6, into A549, Ep/Ras, or BEAS-2B cells induced EMT-like phenotypic changes, characterized by down-regulation of E-cadherin and up-regulation of vimentin, Snail and Slug, suggesting that Ser6 phosphorylation of syntenin by RSK is necessary and sufficient for the induction of EMT mediated by TGF-β1. In addition, we sought to determine whether Ser6 phosphorylation of syntenin would modulate in vivo metastasis in a mouse model. A549 cells expressing syntenin-S6E dramatically increased lung metastases. Based on these data, we conclude that phosphorylation of syntenin at Ser6 by RSK is necessary and sufficient for the induction of EMT and metastasis in certain cancer cells. Citation Format: Okhwa Kim, Cheol Hwangbo, Nara Tae, Suhyun Lee, Jeong-Hyung Lee. Syntenin positively regulates TGF-β1- and oncogenic Ras-mediated EMT-like phenotypic changes in cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1055. doi:10.1158/1538-7445.AM2014-1055

Pulmonary Fibroblasts Induce Epithelial Mesenchymal Transition and Some Characteristics of Stem Cells in Non-Small Cell Lung Cancer

Fibroblasts are key components of the tumor microenvironment. The purpose of this study was to clarify the role of fibroblasts in tumor progression in non-small cell lung cancer (NSCLC). Fibroblasts isolated from surgical exploration were co-cultured with human lung adenocarcinoma cell lines. We defined fibroblasts obtained from tumors as cancer associated fibroblasts (CAFs) and those from normal lung tissue as lung normal fibroblasts (LNFs). Expression levels of myofibroblast markers were higher in CAFs than LNFs within 5 passages in the absence of continuing interaction with carcinoma cells. Thus, we used at least 2 pairs of these CAFs and LNFs in the following experiments; conditioned medium (CM) from fibroblast-induced epithelial mesenchymal transition and acquisition of cancer stem cell-like qualities in lung cancer cells (A549 and NCI-H358), indicating that CM from fibroblasts was biologically active. Furthermore, the concentration of the transforming growth factor (TGF)-β1 was higher in CM from CAFs as compared with that from LNFs, and phenotypic changes of cancer cells by CM from CAFs were greater than those induced by CM from LNFs. These CAF-induced changes were inhibited by addition of the TGF-β inhibitor SB431542. Subcutaneous co-injection of lung cancer cells and CAFs in mice enhanced tumor growth when compared with cancer cells alone, which was attenuated by administration of SB431542. Fibroblasts were associated with increased malignant potential and the acquisition of stem cell-like properties in NSCLC tumors. Targeting CAFs as a therapeutic strategy against cancer is an intriguing concept that would benefit from further study.

The EMT regulator slug and lung carcinogenesis

Lung cancer is the leading cause of cancer death worldwide. Cancer metastasis and resistance to treatment (including radiotherapy, chemotherapy and targeted therapy) are two major causes for the poor survival of lung cancer patients. Epithelial-mesenchymal transition (EMT) is involved in cancer cell invasion, resistance to apoptosis and stem cell features. The process of EMT is controlled by a group of transcriptional factors, zinc finger proteins and basic helix-loop-helix factors. Signaling pathways activated by intrinsic or extrinsic stimuli converge on these transcriptional factors and regulated the phenotypic changes of cancer cells. These EMT regulators may play an important role in cancer progression. In lung cancer, Slug is the most thoroughly investigated EMT regulator. The expression of Slug is associated with lung cancer invasion and resistance to target therapy. In this review, we focus on the current understanding of the role of Slug in the carcinogenesis and progression of lung cancer.

Abstract 2914: A role for glycolysis in the c-Met/HGF signaling axis

Abstract Mitochondrial respiration is the most efficient form of energy production in the cell; however, in the absence of oxygen, such as within a solid tumor mass, cells rely on glycolysis as the main source of energy. Under hypoxic conditions the end product of glycolysis, pyruvate, is reduced to lactate by lactate dehydrogenase (LDH), which is then exported out of the cell. This reduction is essential to regenerate NAD+ consumed by the earlier steps of glycolysis. Recent evidence suggests that hepatocyte growth factor (HGF), the only known ligand for the c-Met receptor, causes an increase in glycolysis. This led our laboratory to hypothesize that glycolysis may be required for HGF-induced phenotypic changes in cancer cells. HGF signaling through the c-Met receptor induces an epithelial-mesenchymal transition (EMT), including loss of cell-cell adhesions through down-regulation of E-cadherin expression, as well as increased motility and cell scattering characteristic of the initiating steps leading to cancer cell invasion and metastasis. Using DU145 prostate cancer cells, we have found that inhibitors of glycolysis (2-DG) and lactate production via LDH activity (oxamate), were able to inhibit HGF-induced cell scattering. Using a wound healing assay, we observed that oxamate was able to prevent HGF-induced cell migration, suggesting a role for glycolysis in cell motility. Additionally, lactic acid was able to rescue cell scattering and wound healing blocked by oxamate, but not by 2-DG. Lactic acid measurements showed that oxamate prevented an increase in glycolysis stimulated by HGF. Measurement of ATP levels also suggested that 2-DG was able to block cell motility due to a reduction in ATP, while oxamate appeared to work through a yet undetermined method. Western blot analysis indicated that oxamate caused a decrease in HGF-induced phospho-Met and downstream phospho-Akt levels. Using H1993 lung cancer cells with constitutively active c-Met, we were able to determine that oxamate deactivated c-Met, while not affecting total c-Met levels. The ability of oxamate to reduce phosphorylation levels appeared to be Met specific based on its inability to block phosphorylation of other activated receptors. Oxamate not only inhibits LDH, but also aspartate aminotransferase (AAT), an enzyme involved in the malate-aspartate shuttle. Our data suggest, however, that the ability of oxamate to inhibit HGF-induced signaling and cell scattering is not through its ability to inhibit AAT. Taken together, our data suggest the c-Met/HGF signaling axis is dependent on glycolysis and perhaps lactic acid levels. Our long-term goals are to elucidate the specific mechanism for this dependence as a means of defining novel targets for preventing c-Met-mediated cancer cell invasion and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2914. doi:10.1158/1538-7445.AM2011-2914

Practical strategies for suppressing hypoxia-inducible factor activity in cancer therapy

The utility of anti-angiogenic strategies for cancer control is strongly compromised by hypoxia-driven phenotypic changes in cancer cells, which make cancer cells more invasive and more prone to give rise to metastases. A key mediator of this phenotypic shift is the transcription factor hypoxia-inducible factor-1 (HIF-1), which acts directly and indirectly to promote the epidermal-mesenchymal transition, boost cancer invasiveness, increase production of angiogenic factors, and induce chemoresistance. In some cancers, HIF-1 activity is constitutively elevated even in aerobic environments, making the cancer harder to treat and control. Practical strategies for suppressing HIF-1 activation may include the following: inhibiting NF-kappaB activation with salicylic acid and/or silibinin, which should decrease transcription of the HIF-1alpha gene; suppressing translation of HIF-1alpha mRNA with drugs that inhibit mTOR or topoisomerase I; supporting the effective activity of prolyl hydroxylases - which promote proteasomal degradation of HIF-1alpha under aerobic conditions - with antioxidant measures, alpha-ketoglutarate, and possibly dichloroacetate; promoting the O(2)-independent proteasomal degradation of HIF-1alpha with agents that inhibit the chaperone protein Hsp90; and blocking HIF-1 binding to its DNA response elements with anthracyclines. The utility of various combinations of these strategies should be tested in cancer cell cultures and rodent xenograft models; initial efforts in this regard have yielded encouraging results. Comprehensive strategies for suppressing HIF-1 activity can be expected to complement the efficacy of cancer chemotherapy and of effective anti-angiogenic regimens.

Computational methods for analysis of cellular functions and pathways collectively targeted by differentially expressed microRNA

This report presents computational methods of analysis of cellular processes, functions, and pathways affected by differentially expressed microRNA, a statistical basis of the gene enrichment analysis method, a modification of enrichment analysis method accounting for combinatorial targeting of Gene Ontology categories by multiple miRNAs and examples of the global functional profiling of predicted targets of differentially expressed miRNAs in cancer. We have also summarized an application of Ingenuity Pathway Analysis tools for in depth analysis of microRNA target sets that may be useful for the biological interpretation of microRNA profiling data. To illustrate the utility of these methods, we report the main results of our recent computational analysis of five published datasets of aberrantly expressed microRNAs in five human cancers (pancreatic cancer, breast cancer, colon cancer, lung cancer, and lymphoma). Using a combinatorial target prediction algorithm and statistical enrichment analysis, we have determined Gene Ontology categories as well as biological functions, disease categories, toxicological categories, and signaling pathways that are: targeted by multiple microRNAs; statistically significantly enriched with target genes; and known to be affected in specific cancers. Our recent computational analysis of predicted targets of co-expressed miRNAs in five human cancers suggests that co-expressed miRNAs provide systemic compensatory response to the abnormal phenotypic changes in cancer cells by targeting a broad range of functional categories and signaling pathways reportedly affected in a particular cancer.

Computational analysis of biological functions and pathways collectively targeted by co-expressed microRNAs in cancer

BackgroundMultiple recent studies have found aberrant expression profiles of microRNAome in human cancers. While several target genes have been experimentally identified for some microRNAs in various tumors, the global pattern of cellular functions and pathways affected by co-expressed microRNAs in cancer remains elusive. The goal of this study was to develop a computational approach to global analysis of the major biological processes and signaling pathways that are most likely to be affected collectively by co-expressed microRNAs in cancer cells.ResultsWe report results of computational analysis of five datasets of aberrantly expressed microRNAs in five human cancers published by the authors (pancreatic cancer) and others (breast cancer, colon cancer, lung cancer and lymphoma). Using the combinatorial target prediction algorithm miRgate and a two-step data reduction procedure we have determined Gene Ontology categories as well as biological functions, disease categories, toxicological categories and signaling pathways that are: targeted by multiple microRNAs; statistically significantly enriched with target genes; and known to be affected in specific cancers.ConclusionOur global analysis of predicted miRNA targets suggests that co-expressed miRNAs collectively provide systemic compensatory response to the abnormal phenotypic changes in cancer cells by targeting a broad range of functional categories and signaling pathways known to be affected in a particular cancer. Such systems biology based approach provides new avenues for biological interpretation of miRNA profiling data and generation of experimentally testable hypotheses regarding collective regulatory functions of miRNA in cancer.