Epithelial-mesenchymal Transition Gene Expression Research Articles

Pirfenidone Reduces Epithelial-Mesenchymal Transition and Spheroid Formation in Breast Carcinoma through Targeting Cancer-Associated Fibroblasts (CAFs).

Simple SummaryCancer-associated fibroblasts (CAFs) stimulate phenotypic transformation and acquisition of stemness in carcinoma cells. Targeting CAF-derived cytokines may suppress initiation of these events. This study aimed to show the inhibitory effects of pirfenidone on phenotypic transformation and stemness of cancer cells. To this end, we leverage the use of a 3D microfluidic device to analyze carcinoma progression phenotypes. We found that pirfenidone decreased tumor spheroid formation and epithelial–mesenchymal transition (EMT) the inhibition of cytokine production by CAFs. In the microfluidic model, we demonstrate that pirfenidone significantly inhibits the migration of carcinoma cells and CAFs. This study highlights the potential application of pirfenidone in suppressing invasion and potentially metastasis in breast cancer which can be further investigated in vivo.The aim of this study was to assess the effects of pirfenidone (PFD) on promoting epithelial–mesenchymal-transition (EMT) and stemness features in breast carcinoma cells through targeting cancer-associated-fibroblasts (CAFs). Using The Cancer Genome Atlas (TCGA) database, we analyzed the association between stromal index, EMT, and stemness-related genes across 1084 breast cancer patients, identifying positive correlation between YAP1, EMT, and stemness genes in samples with a high-stromal index. We monitored carcinoma cell invasion and spheroid formation co-cultured with CAFs in a 3D microfluidic device, followed by exposing carcinoma cells, spheroids, and CAFs with PFD. We depicted a positive association between the high-stromal index and the expression of EMT and stemness genes. High YAP1 expression in samples correlated with more advanced EMT status and stromal index. Additionally, we found that CAFs promoted spheroid formation and induced the expression of YAP1, VIM, and CD44 in spheroids. Treatment with PFD reduced carcinoma cell migration and decreased the expression of these genes at the protein level. The cytokine profiling showed significant depletion of various EMT- and stemness-regulated cytokines, particularly IL8, CCL17, and TNF-beta. These data highlight the potential application of PFD on inhibiting EMT and stemness in carcinoma cells through the targeting of critical cytokines.

Efficacy, Safety, and Biomarker Analysis of Combined PD-L1 (Atezolizumab) and VEGF (Bevacizumab) Blockade in Advanced Malignant Peritoneal Mesothelioma.

Malignant peritoneal mesothelioma (MPeM) is a rare but aggressive malignancy with limited treatment options. VEGF inhibition enhances efficacy of immune-checkpoint inhibitors by reworking the immunosuppressive tumor milieu. Efficacy and safety of combined PD-L1 (atezolizumab) and VEGF (bevacizumab) blockade (AtezoBev) was assessed in 20 patients with advanced and unresectable MPeM with progression or intolerance to prior platinum-pemetrexed chemotherapy. The primary endpoint of confirmed objective response rate per RECISTv1.1 by independent radiology review was 40% [8/20; 95% confidence interval (CI), 19.1-64.0] with median response duration of 12.8 months. Six (75%) responses lasted for >10 months. Progression-free and overall survival at one year were 61% (95% CI, 35-80) and 85% (95% CI, 60-95), respectively. Responses occurred notwithstanding low tumor mutation burden and PD-L1 expression status. Baseline epithelial-mesenchymal transition gene expression correlated with therapeutic resistance/response (r = 0.80; P = 0.0010). AtezoBev showed promising and durable efficacy in patients with advanced MPeM with an acceptable safety profile, and these results address a grave unmet need for this orphan disease. SIGNIFICANCE: Efficacy of atezolizumab and bevacizumab vis-à-vis response rates and survival in advanced peritoneal mesothelioma previously treated with chemotherapy surpassed outcomes expected with conventional therapies. Biomarker analyses uncovered epithelial-mesenchymal transition phenotype as an important resistance mechanism and showcase the value and feasibility of performing translationally driven clinical trials in rare tumors.See related commentary by Aldea et al., p. 2674.This article is highlighted in the In This Issue feature, p. 2659.

BOO induces fibrosis and EMT in urothelial cells which can be recapitulated in vitro through elevated storage and voiding pressure cycles.

To determine the unique contributions from elevated voiding and storage pressures in the development of fibrosis and the epithelial-to-mesenchymal transition (EMT) in urothelial cells, and how progressive BOO pressure cycling is an important mechanical cue leading to these pathological changes. Urothelial cells isolated from control, SHAM, 2 (acute)- or 6 (chronic)-week BOO rats treated with an inflammasome inhibitor or no drug. Total RNA was isolated and RT-PCR was conducted with custom primers for pro-fibrotic and EMT genes. In separate experiments, a rat urothelial cell line was exposed to cyclic pressure regimes characteristic of acute and chronic BOO in the presence or absence of an inflammasome inhibitor. Following exposure, RT-PCR was conducted, collagen content was determined and intracellular caspase-1 activity was measured. Urothelial cells isolated from acute and chronic BOO rat models demonstrated expression of pro-fibrotic and EMT genes. Similarly, MYP3 rat urothelial cells subjected to pressure cycling regimes that reflect intravesical pressures in the acute or chronic BOO bladder also demonstrated increased expression of pro-fibrotic and EMT genes, along with elevated soluble collagen. Treatment with inflammasome inhibitors reduced expression of pro-fibrotic genes in the rat model and pressure cycling model but had a limited effect on EMT. These results indicate that acute and chronic BOO pressure cycling are essential in the initiation and progression of fibrosis in the bladder via the NLRP3 inflammasome, but also provide new evidence that there is also an alternative NLRP3-independent pathway leading to EMT and fibrosis.

ZEB2 regulates endocrine therapy sensitivity and metastasis in luminal a breast cancer cells through a non-canonical mechanism.

The transcription factors ZEB1 and ZEB2 mediate epithelial-to-mesenchymal transition (EMT) and metastatic progression in numerous malignancies including breast cancer. ZEB1 and ZEB2 drive EMT through transcriptional repression of cell-cell junction proteins and members of the tumor suppressive miR200 family. However, in estrogen receptor positive (ER +) breast cancer, the role of ZEB2 as an independent driver of metastasis has not been fully investigated. In the current study, we induced exogenous expression of ZEB2 in ER + MCF-7 and ZR-75-1 breast cancer cell lines and examined EMT gene expression and metastasis using dose-response qRT-PCR, transwell migration assays, proliferation assays with immunofluorescence of Ki-67 staining. We used RNA sequencing to identify pathways and genes affected by ZEB2 overexpression. Finally, we treated ZEB2-overexpressing cells with 17β-estradiol (E2) or ICI 182,780 to evaluate how ZEB2 affects estrogen response. Contrary to expectation, we found that ZEB2 did not increase canonical epithelial nor decrease mesenchymal gene expressions. Furthermore, ZEB2 overexpression did not promote a mesenchymal cell morphology. However, ZEB1 and ZEB2 protein expression induced significant migration of MCF-7 and ZR-75-1 breast cancer cells in vitro and MCF-7 xenograft metastasis in vivo. Transcriptomic (RNA sequencing) pathway analysis revealed alterations in estrogen signaling regulators and pathways, suggesting a role for ZEB2 in endocrine sensitivity in luminal A breast cancer. Expression of ZEB2 was negatively correlated with estrogen receptor complex genes in luminal A patient tumors. Furthermore, treatment with 17β-estradiol (E2) or the estrogen receptor antagonist ICI 182,780 had no effect on growth of ZEB2-overexpressing cells. ZEB2 is a multi-functional regulator of drug sensitivity, cell migration, and metastasis in ER + breast cancer and functions through non-canonical mechanisms.

Abstract 2447: TLR4 expression as a determinant of EMT and stress response gene expression in UV exposed human keratinocytes

Abstract Toll-like receptor 4 (TLR4) is gaining attention for its role in several cancer-associated signaling pathways, including the regulation of inflammation, DNA repair and epithelial-mesenchymal transition (EMT). There is evidence that TLR4 contributes to ultraviolet (UV) radiation-induced stress signaling in skin cells, including keratinocytes. Blockade of TLR4 activity using the pharmacological antagonist resatorvid (TAK-242) inhibits not only acute UV-induced signaling in vitro and in vivo, but blocks UV-induced skin carcinogenesis in mouse models. In order to genetically explore the role of TLR4 in keratinocyte responses to UV-induced stress, we recently utilized CRISPR/Cas9 techniques to delete TLR4 from the genome of human HaCaT keratinocyte cells in culture. First, using differential gene expression analysis (employing the EMTRT2 Profiler PCR array technology) it was observed that TLR4 KO status is associated with pronounced downregulation of EMT-related gene expression (MMP9, VIM, FN1, SNAI1, TWIST1) confirmed by independent RT-qPCR analysis. TLR4 was also a determinant of UV-induced inflammatory enzyme expression including PTGS2. Phenotypically, TLR4 KO cells displayed an attenuation of Matrigel transwell migration. TLR4 KO cells demonstrated increased sensitivity to UV-induced apoptosis as detected by flow cytometry of annexin V/propidium iodide stained cells, and by immunodetection of increased PARP cleavage. Moreover, NanoString nCounter expression analysis of murine skin chronically exposed to tumorigenic UV indicated that topical resatorvid treatment was associated with MMP9 downregulation. Future studies will employ this novel TLR4 KO keratinocyte cell line as a mechanistic tool to dissect the role of TLR4 in inflammatory signaling and EMT response in the context of skin photodamage and carcinogenesis. Supported by NIH grants ES029579, R03CA230830, P01CA229112. Citation Format: Viktoria Kirschnerova, Prajakta Vaishampayan, Maria Khawam, Clara Curiel-Lewandrowski, Georg T. Wondrak, Sally E. Dickinson. TLR4 expression as a determinant of EMT and stress response gene expression in UV exposed human keratinocytes [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 2447.

Abstract 2667: RAS-induced transformation of mammary epithelial cells relies upon a ZEB1-dependent cellular reprogramming through a paracrine process

Abstract Despite an enhanced interest, the origin of cancer stem cells (CSCs) remains equivocal. A strong body of evidence suggests that, in some adult carcinomas, CSC may originate from normal mature differentiated cells through a dedifferentiation process and the acquisition of stem-like properties. At the crux of this concept is the epithelial-mesenchymal transition (EMT), an embryonic transdifferentiation process that can be abnormally reactivated over the course of tumor development as a result of oncogenic or microenvironmental cues. High expression of EMT genes and stemness features are hallmarks of claudin-low (CL) breast cancers, a rare subtype of breast malignancies, generally considered as the most primitive breast cancers. We have recently reported that this molecular subtype of breast cancers exhibits a significant diversity, comprising three main subgroups (CL1, CL2 and CL3), that emerge from unique evolutionary processes. Genetic and epigenetic analyses support the hypothesis that CL1 tumors generate from the transformation of a normal mammary stem cells, whereas CL2 and CL3 arise from the reactivation of an EMT process over the course of tumor progression. Interestingly, CL tumors share the frequent activation of the RAS-MAPK pathway, suggesting a potent role for the RAS pathway in the development of CL tumors. Based on these findings, we wondered whether RAS pathway activation could induce the reactivation of EMT-TFs allowing the emergence of CSCs displaying CL2 and CL3 characteristics. By expressing an oncogenic form of RAS in differentiated epithelial cells (HME), we observed a phenotypic switch towards a CD24-/low/CD44+ phenotype, associated with CSC properties, and suggesting an acquisition of cellular plasticity after RAS activation. By using CRISPR/Cas9 knock-down experiments we have shown i) the emergence of CD24-/low/CD44+ cells, ii) the acquisition of transformation and stemness features, and iii) that the cellular plasticity acquired after RAS activation in differentiated HME strongly rely on ZEB1. Single-cell RNA sequencing analyses after RAS activation in HME-RAS cells suggested two different outcomes: senescence or EMT. Treatment with a senolytic drug resulted in reduction of RAS-induced senescent cells associated with a decrease in the emergence of CD24-/low/CD44+ cells and ZEB1 expression. Finally, a co-culture reporter system and a depletive approach with cytokine-neutralizing antibodies highlighted a paracrine effect of IL-6 and IL-1α, produced by senescent RAS-expressing cells on their neighboring differentiated cells, leading to the acquisition of ZEB1-dependent cellular plasticity. Hence, we unveiled a new paracrine process, in which cytokines secreted by cells undergoing oncogene-induced senescence drive ZEB1 expression, thus promoting cellular plasticity correlated with the emergence of CSCs. Citation Format: ALAIN PUISIEUX, Hadrien De Blander, Laurie Tonon, Frédérique Fauvet, Roxane Pommier, Christelle Lamblot, Rahma Benhassoun, Benjamin Gibert, Maria Ouzounova, Anne-Pierre Morel. RAS-induced transformation of mammary epithelial cells relies upon a ZEB1-dependent cellular reprogramming through a paracrine process [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 2667.

Tadalafil Reverses the Effect of Three-Dimensional Cell Culture System on Stem Cell Features in A549 and SK-MES-1.

Background: Non-small cell lung cancer is the most common type of lung cancer and is a frequent cause of death. In our research, A549 and SK-MES-1 were used to assess the effect of three-dimensional (3D) culture compared to that of two-dimensional (2D) monolayers in cell proliferation, migration, and invasion, response to chemotherapy, as well as the expression of epithelial to mesenchymal transition (EMT) and cancer stem cell (CSC)-related markers. As tadalafil is a phosphodiesterase type 5 (PDE5) inhibitor with the potential to target CSC maintenance in multiple cancer cell lines, we assessed its function in 3D culture and detected the downstream pathway genes. Methods: We compared the viability and proliferation capacity of A549 and SK-MES-1 cells in 2D and 3D culture by cell counting kit (CCK)-8, foci formation, and Live/Dead double stain (Operetta CLS High content screening). Migration, invasion, and other functions were also assessed. To elucidate the underlying mechanisms, the expression of EMT and CSC markers was analyzed by quantitative real-time PCR (qPCR) and Western blot. Results: A549 and SK-MES-1 cells formed spheroids heterogeneous in shape and size. In our 3D spheroid systems, cells went through EMT, and were also capacitated with higher stemness and chemoresistance. Combination use of tadalafil and cisplatin showed higher chemotherapy efficiency in the 3D model, compared to that of the 2D cell culture. Conclusion: Our research aims at the notable differences between these two cellular systems in terms of cell functions, EMT, and stemness-associated gene expression and chemo-response. We showed that a commonly used drug, tadalafil, showed more pronounced inhibitory effects when cells were cultured in the 3D model. Since the 3D culture system could imitate the in vivo behavior of cancer cells within the tumor, we advocate that this system is superior to traditional 2D culture and should be used in the investigation of new therapeutic compounds.

Crosstalk of the IκB Kinase with Spliced X-Box Binding Protein 1 Couples Inflammation with Glucose Metabolic Reprogramming in Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) plays a critical role in airway injury, repair, and structural remodeling. IκB kinase (IKK)-NFκB signaling regulates late EMT-associated gene expression. However, IKK-mediated mesenchymal transition occurs earlier than NFκB/RelA subunit-dependent EMT gene expression, leading us to investigate the hypothesis that IKK plays an independent mechanism in transforming growth factor-β (TGFβ)-induced EMT. Time-resolved dissection of early proteome and phosphoproteome changes in response to TGFβ and a specific IKK inhibitor, BMS-345541, revealed that IKK regulates cascades of 23 signaling pathways essential in EMT, including TGFβ signaling, p38 mitogen associate protein kinase (MAPK), Toll receptor signaling, and integrin pathways. We identified early IKK-dependent phosphorylation of core regulatory proteins in essential EMT signaling cassettes, including ATF2, JUN, NFKB1/p105, and others. Interestingly, we found that IKKβ directly complexes with and phosphorylates the spliced X-box-binding protein 1 (XBP1s). XBP1s is an arm of the unfolded protein response (UPR) that activates the hexosamine biosynthetic pathway (HBP), a pathway that mediates protein N-glycosylation and survival from ER stress-induced apoptosis in EMT. We found that inhibition of IKK activity abolishes the phosphorylation of XBP1-T48, blocks XBP1s nuclear translocation, and inhibits the activation of HBP. Our study elucidates a previously unrecognized IKKβ-XBP1s-HBP crosstalk pathway that couples inflammation and glucose metabolic reprogramming in ETM. Because XBP1-HBP controls N-glycosylation of the extracellular matrix (ECM) in EMT, this novel IKKβ-XBP1-HBP pathway may contain therapeutic targets whose inhibition could prevent ECM remodeling in lung fibrosis or other airway remodeling diseases.

Histone Methyltransferase SETD1A Induces Epithelial-Mesenchymal Transition to Promote Invasion and Metastasis Through Epigenetic Reprogramming of Snail in Gastric Cancer.

Aberrant epigenetic modification induces oncogene expression and promotes cancer development. The histone lysine methyltransferase SETD1A, which specifically methylates histone 3 lysine 4 (H3K4), is involved in tumor growth and metastasis, and its ectopic expression has been detected in aggressive malignancies. Our previous study reported that SETD1A promotes gastric cancer (GC) proliferation and tumorigenesis. However, the function and molecular mechanisms of SETD1A in GC metastasis remain to be elucidated. In this study, we found that overexpression of SETD1A promoted GC migration and invasion, whereas knockdown of SETD1A suppressed GC migration and invasion in vitro. Moreover, knockdown of SETD1A suppressed GC epithelial-mesenchymal transition (EMT) by increasing the expression of epithelial marker E-cadherin and decreasing the expression of mesenchymal markers, including N-cadherin, Fibronectin, Vimentin, and α-smooth muscle actin (α-SMA). Mechanistically, knockdown of SETD1A reduced the EMT key transcriptional factor snail expression. SETD1A was recruited to the promoter of snail, where SETD1A could methylate H3K4. However, knockdown of SETD1A decreased the methylation of H3K4 on the snail promoter. Furthermore, SETD1A could be a coactivator of snail to induce EMT gene expression. Rescue of snail restored SETD1A knockdown-induced GC migration and invasion inhibition. In addition, knockdown of SETD1A suppressed GC metastasis in vivo. In summary, our data revealed that SETD1A mediated the EMT process and induced metastasis through epigenetic reprogramming of snail.

Characterization of the tumor immune microenvironment in clear cell renal cell carcinoma (ccRCC): Prognostic value and therapeutic implications of an M0-macrophage enriched subtype.

4572 Background: Metastatic clear cell renal cell carcinoma (ccRCC) has a 5-year survival of 12%, but the number of approved immune checkpoint blockade (ICB) agents is growing, necessitating the need to better identify responders. The composition and role of the tumor immune microenvironment (TIME) has yet to be comprehensively characterized in ccRCC. Here, we leveraged a genomic data driven approach to characterize TIME subtypes in ccRCC. Methods: Whole transcriptome data from patients with local and metastatic disease in the Cancer Genome Atlas KIRC (TCGA-KIRC) project was utilized (n = 537). CIBERSORT was used for immune cell deconvolution, and unsupervised hierarchical clustering divided the cohort based on similar immune profiles. Progression free (PFS) and overall (OS) survival of each cluster was analyzed, and Gene Set Enrichment analysis was performed among clusters. The tumor immune dysfunction and exclusion (TIDE) tool, which uses a genomic signature validated on immunotherapy treated melanoma patients to model tumor immune evasion, was then used to predict response to ICB in the TCGA-KIRC clusters. Results: There was a distinct M0hi cluster identified which demonstrated a higher proportion of patients with stage III/IV disease, decreased PFS and OS (Table). Additionally, the M0hi cluster was characterized by lower PD-L1 expression (ANOVA, p = 0.0045) and an enrichment of epithelial to mesenchymal transition (EMT) hallmark genes [Enrichment Score = 0.64, p = 0.001]. The M0hi cluster also showed a higher degree of T-Cell Exclusion (ANOVA, p = 2.2x10-16), predominance of Cancer Associated Fibroblasts (CAFs; ANOVA, p = 2.2x10-16) and Myeloid Derived Suppressor Cells (MDSCs; ANOVA, p = 4.1x10-10). The M0hi cluster had the lowest predicted response to immunotherapy using the TIDE tool (Table). Conclusions: Comprehensive characterization of the TCGA-KIRC cohort led to identification of a distinct cluster of ccRCC defined molecularly by decreased PD-L1 and increased EMT gene expression and cellularly by enrichment of M0 macrophages, CAFs, MDSCs, and an exclusion of T Cells. Patients within this cluster exhibited aggressive disease and poor predicted response to ICB. These findings warrant further validation to identify appropriate therapeutic approaches for this ccRCC subgroup.[Table: see text]

Molecular Orchestration of Epithelial Mesenchymal Transition During Trophoblast and Placental Development

In Eutherian mammals, placenta is a highly specialized extra-embryonic organ that provides the feto-maternal interface and modulates the uterine environment conducive to successful pregnancy. Placenta originates from trophectoderm cells of the blastocyst and is comprised of various lineages of trophoblast cells, endothelial and smooth muscle cells. Since trophectoderm cells are epithelial in nature, it is obvious that epithelial mesenchymal transition (EMT) is a hallmark of placentation. In this report, we aimed to identify the molecular signature that regulates EMT during trophoblast and placental development in mice. On E7.5 onwards the trophoblast stem cells located in ectoplacental cone rapidly divides, differentiate leading to formation of placenta proper. Functional EMT transcriptome in mouse implantation sites on E7.5 and E9.5 were analyzed using a RT2 profiler real time PCR array scanning 84 EMT related genes. Validation of the array was performed using real time q-PCR and western blot analyses. Array result showed that there was an overall reduction of EMT gene expression in the implantation site as gestation progressed from E7.5 to E9.5 albeit the levels of EMT gene expression were substantial on both days. The differentially expressed EMT genes were further assessed during a) placental development using mouse placental samples from various days of gestation, b) induction of either EMT or differentiation in trophoblast stem (TS) cells using western blot analysis. The genes thus identified include transcription factors (Esr1, Snai2, Stat3, Foxc2), mesenchymal markers (Vim, Fn1), extracellular matrix and cell adhesion genes (Col1a2, Col3a1, Itga5, Vcan), migration and motility genes (Msn, Fn1, Pdgfrb). Extent of EMT gene expression was highest in placenta from E12.5 of mouse gestation. In addition, ectopic administration of EMT inducer led to the induction of EMT in TS cells as evidenced by EMT gene expression profile. Furthermore, differentiation of TS cells to other trophoblast lineages led to decline in EMT gene expression. Our data thus provides new insights into the importance of EMT during trophoblast stem cell differentiation and placental morphogenesis.

542 BRAFV600E-inhibition drives EMT gene expression enhancing invasiveness and metastasis in a bioluminescent murine model of BRAFV600E/NRASQ61K melanoma

BRAF inhibitor (BRAFi) resistance compromises long term survivorship of malignant melanoma patients. Mutant NRASQ61K with constitutive activation of PI3K/AKT/mTOR signaling is a major mediator of BRAFi resistance, and BRAFi therapy can accelerate pre-existing RAS-mutant malignancies including NRAS-mutant leukemias in melanoma patients. Here, employing NanoString transcriptomic analysis of isogenic (A375-BRAFV600E versus A375-BRAFV600E/NRASQ61K) malignant melanoma cells we demonstrate that BRAFi treatment selectively targets BRAFV600E/NRASQ61K cells with induction of Epithelial to Mesenchymal Transition (EMT) gene expression, paradoxically promoting invasiveness and metastasis in vivo. Cancer progression nCounterTM pathway analysis identified ‘EMT’ and ‘Proliferative Control’ gene expression networks specific to BRAFV600E/NRASQ61K status. Strikingly, in contrast to BRAFi (vemurafenib, VEM)-induced antiproliferative and antiinvasive effects in BRAFV600E cells, VEM enhanced proliferation and invasiveness of BRAFV600E/NRASQ61K cells. RT2Profiler PCR array analysis confirmed VEM-upregulation of genes promoting EMT and proliferation [AKT1, MMP3, PDGFRB, RAC1, SPARC, ZEB1, ZEB2 (≤ 350-fold; p<0.05)] detectable only in BRAFV600E/NRASQ61K cells, while causing the expected downregulation of EMT-driver genes [CDH2, FN1, FOXC2, IGFBP4, MMP9, VIM, WNT5A (≤ 40-fold; p<0.05)] only in the BRAFV600E isogenic variant. Phenotypic transwell migration assays confirmed the seemingly opposing effects of VEM treatment on melanoma cell invasiveness [achieving blockade (BRAFV600E) or enhancement (BRAFV600E/NRASQ61K)]. In a bioluminescent SCID mouse metastasis model using A375-luc isogenic variants, VEM treatment (50 mg/kg; p.o., q.d.) enhanced lung tumor burden imposed by BRAFV600E/NRASQ61K cells, while blocking metastasis of BRAFV600E cells. Our data provide preclinical evidence that identifies a BRAFi-driven upregulation of EMT-related gene expression potentially enhancing invasiveness and metastasis in human BRAFV600E/NRASQ61K melanoma.

Localized EMT reprograms glial progenitors to promote spinal cord repair.

Anti-regenerative scarring obstructs spinal cord repair in mammals and presents a major hurdle for regenerative medicine. In contrast, adult zebrafish possess specialized glial cells that spontaneously repair spinal cord injuries by forming a pro-regenerative bridge across the severed tissue. To identify the mechanisms that regulate differential regenerative capacity between mammals and zebrafish, we first defined the molecular identity of zebrafish bridging glia and then performed cross-species comparisons with mammalian glia. Our transcriptomics show that pro-regenerative zebrafish glia activate an epithelial-to-mesenchymal transition (EMT) gene program and that EMT gene expression is a major factor distinguishing mammalian and zebrafish glia. Functionally, we found that localized niches of glial progenitors undergo EMT after spinal cord injury in zebrafish and, using large-scale CRISPR-Cas9 mutagenesis, we identified the gene regulatory network that activates EMT and drives functional regeneration. Thus, non-regenerative mammalian glia lack an essential EMT-driving gene regulatory network that reprograms pro-regenerative zebrafish glia after injury.

Abstract IA020: Chromatin regulation of the endometrium: What are our models telling us?

Abstract ARID1A and PI3-Kinase (PI3K) pathway alterations are common in neoplasms originating from the uterine endometrium. ARID1A is a SWI/SNF chromatin-remodeling complex subunit. ARID1A loss in the mouse endometrial epithelium leads vaginal bleeding when combined with PI3K pathway activation. Sorted mutant endometrial epithelial cells display gene expression and promoter chromatin signatures associated with epithelial-to-mesenchymal transition (EMT), inflammation and estrogen signaling in vivo. ARID1A loss leads to the expression of EMT genes, suggesting ARID1A normally maintains endometrial epithelial cell identity by repressing mesenchymal cell fates. In contrast to its repressive roles, ARID1A is required for the activation estrogen responsive genes, including the progesterone receptor (PGR). Estrogen responsive genes are marked by bivalent chromatin domains containing both histone H3 lysine 4 trimethylation (H3K4me3) and lysine 27 trimethylation (H3K27me3). High H3K27me3 correlates with low PGR expression, suggesting Polycomb Repressive Complex 2 (PRC2)-mediated H3K27me3 normally silences PGR. In support of this, pharmacological inhibition of PRC2 partially rescues PGR activation is estrogen treated, ARID1A-deficient cells. Given the known antagonistic relationship between SWI/SNF and PRC2 complexes, we propose that SWI/SNF normally opposes PRC2 mediated silencing of the PGR promoter in response to estrogen, leading to PGR expression. In the ARID1A mutant endometrium, PGR promoter silencing by PRC2 is maintained. In the absence of PGR, progesterone cannot oppose the actions estrogen in the endometrium, leading to unopposed estrogen signaling. Our data demonstrate ARID1A has both activating and repressive roles in the endometrium, and ARID1A loss contributes to multiple aspects of disease pathogenesis. Citation Format: Mike R. Wilson, Jake J. Reske, Ronald L. Chandler. Chromatin regulation of the endometrium: What are our models telling us? [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr IA020.

Platelets induce free and phospholipid-esterified 12-hydroxyeicosatetraenoic acid generation in colon cancer cells by delivering 12-lipoxygenase

Platelets promote tumor metastasis by inducing promalignant phenotypes in cancer cells and directly contributing to cancer-related thrombotic complications. Platelet-derived extracellular vesicles (EVs) can promote epithelial-mesenchymal transition (EMT) in cancer cells, which confers high-grade malignancy. 12S-hydroxyeicosatetraenoic acid (12-HETE) generated by platelet-type 12-lipoxygenase (12-LOX) is considered a key modulator of cancer metastasis through unknown mechanisms. In platelets, 12-HETE can be esterified into plasma membrane phospholipids (PLs), which drive thrombosis. Using cocultures of human platelets and human colon adenocarcinoma cells (line HT29) and LC-MS/MS, we investigated the impact of platelets on cancer cell biosynthesis of 12S-HETE and its esterification into PLs and whether platelet ability to transfer its molecular cargo might play a role. To this aim, we performed coculture experiments with CFSE[5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester]-loaded platelets. HT29 cells did not generate 12S-HETE or express 12-LOX. However, they acquired the capacity to produce 12S-HETE mainly esterified in plasmalogen phospholipid forms following the uptake of platelet-derived medium-sized EVs (mEVs) expressing 12-LOX. 12-LOX was detected in plasma mEV of patients with adenomas/adenocarcinomas, implying their potential to deliver the protein to cancer cells in vivo. In cancer cells exposed to platelets, endogenous but not exogenous 12S-HETE contributed to changes in EMT gene expression, mitigated by three structurally unrelated 12-LOX inhibitors. In conclusion, we showed that platelets induce the generation of primarily esterified 12-HETE in colon cancer cells following mEV-mediated delivery of 12-LOX. The modification of cancer cell phospholipids by 12-HETE may functionally impact cancer cell biology and represent a novel target for anticancer agent development.

GAPDH and PUM1: Optimal Housekeeping Genes for Quantitative Polymerase Chain Reaction-Based Analysis of Cancer Stem Cells and Epithelial-Mesenchymal Transition Gene Expression in Rectal Tumors.

BackgroundThe overwhelming majority of published articles have taken colon and rectal cancer as a single group, i.e., colorectal cancer, when normalizing gene expression data with housekeeping genes (HKG) in quantitative polymerase chain reaction (qPCR) experiments though there are published reports that suggest the differential expression pattern of genes between the colon and rectal cancer groups and hence the current experiment was attempted to find out the optimal set of housekeeping genes from the list of common HKG for rectal tumor gene expression analysis.MethodsThe expression of five potential housekeeping genes GAPDH, RPNI, PUM1, B2M, and PMM1 was analyzed through qPCR and Bestkeeper software (http://www.wzw.tum.de/gene-quantification/bestkeeper.html) in 20 stage II-IV rectal cancer samples to check for uniformity in their expression pattern. Cancer stem cell (CSC) marker ALDH1 and epithelial-mesenchymal transition marker (EMT) markers E cadherin, vimentin, Twist, and SNAI2 expression were evaluated in conjunction with the two optimal reference genes in 10 rectal cancers as part of validation.ResultsThe standard deviation of the cycle threshold value of GAPDH was found the lowest at 0.65 followed by RPN1 at 0.88, PUM1 at 0.94, PMM1 at 0.94, and B2M at 1.21 when analyzed with BestKeeper software. Using GAPDH and PUM1 as the reference gene for the validation phase, rectal cancer patients with stage III/IV showed a 4.79-fold change (P=0.006) in ALDH1 expression, and an 11.76-fold change in Twist expression (P=0.003) with respect to stage II rectal tumor when normalized with GAPDH and PUM1.ConclusionGAPDH and PUM1 can be used as an optimal set of housekeeping genes for gene expression-related experiments in rectal tumors. ALDH1 and Twist were found significantly overexpressed in stage III/IV rectal tumors in comparison to stage II rectal cancer. Genes associated with cancer stem cells and EMT markers could be optimally analyzed by normalizing them with GAPDH and PUM1 as housekeeping genes.

Increased Expressions of Matrix Metalloproteinases (MMPs) in Prostate Cancer Tissues of Men with Type 2 Diabetes.

Type 2 diabetes (T2D) is associated with worse prognosis of prostate cancer (PCa). The molecular mechanisms behind this association are still not fully understood. The aim of this study was to identify key factors, which contribute to the more aggressive PCa phenotype in patients with concurrent T2D. Therefore, we investigated benign and PCa tissue of PCa patients with and without diabetes using real time qPCR. Compared to patients without diabetes, patients with T2D showed a decreased E-cadherin/N-cadherin (CDH1/CDH2) ratio in prostate tissue, indicating a switch of epithelial-mesenchymal transition (EMT), which is a pivotal process in carcinogenesis. In addition, the gene expression levels of matrix metalloproteinases (MMPs) and CC chemokine ligands (CCLs) were higher in prostate samples of T2D patients. Next, prostate adenocarcinoma PC3 cells were treated with increasing glucose concentrations to replicate hyperglycemia in vitro. In these cells, high glucose induced expressions of MMPs and CCLs, which showed significant positive associations with the proliferation marker proliferating cell nuclear antigen (PCNA). These results indicate that in prostate tissue of men with T2D, hyperglycemia may induce EMT, increase MMP and CCL gene expressions, which in turn activate invasion and inflammatory processes accelerating the progression of PCa.

Abstract PR03: Nongenomic BAP1 aberrancy drives highly aggressive cutaneous melanoma phenotype

Abstract The purpose of this study was to determine the role of BAP1 levels in cutaneous melanoma (CM). BAP1 is a tumor suppressor in which loss of heterozygosity (LOH) from mutation and copy number alteration is well described in germline and somatic cancers. Although BAP1 genomic alterations in CM are extremely rare (2% of 665 samples from 5 datasets), marked variability in BAP1 expression is observed in CM. We show that low nuclear BAP1 levels portend a significantly worse clinical outcome in stage III CM (n=37, log rank p ≤0.01 for both overall survival and progression-free survival). Gene Set Enrichment Analysis (GSEA) revealed low BAP1 expression to be most highly ranked with an increased epithelial–mesenchymal transition (EMT) gene expression profile in CM tumors (n=379, FDR q = 1.34E-26) and cell lines (n=53, FDR q = 2.86E-116). We identify the expression of ZEB1, a master regulator of EMT, to be significantly associated with low BAP1 expression in CM tumors and cell lines (p= 1.5E-04 and 3.3E-05, respectively). Analysis of the BAP1 promoter indicates three canonical ZEB1 binding sites. Functional experiments show ZEB1 to bind to the BAP1 promoter, and luciferase activity assays indicate that ZEB1 acts as a transcriptional suppressor of BAP1 expression with differential utilization of the promoter binding sites. Targeted reduction of endogenous ZEB1 caused increased BAP1 levels, while targeted reduction of BAP1 did not modulate ZEB1 levels, consistent with ZEB1 having a suppressive effect on BAP1. Phenotypically, targeted reduction of BAP1 in CM cells resulted in a switch from a more differentiated, melanocytic state, to a less differentiated, more migratory and invasive phenotype. Extinguishing melanocyte-specific BAP1 in mice with a BRAF V600E mutant genetic background resulted in the emergence of primary melanoma tumors, with a marked EMT gene expression profile, and resultant metastases. Given the phenotypic changes associated with BAP1 levels in our mouse and human studies, we then tested the effect of modulating BAP1 on BRAF targeted therapy. Exogenous expression of BAP1 sensitized BRAF inhibitor (vemurafenib)-resistant melanoma cells, while the targeted reduction of BAP1 desensitized BRAF inhibitor-sensitive melanoma cells. BRAF mutant/BAP1 loss mice failed to exhibit a marked response to vemurafenib treatment compared to control mice. These data implicate regulation of BAP1 to be a major mechanism that characterizes a highly malignant and treatment-resistant subset of tumors. Our study indicates that nongenomic reduction in BAP1 through ZEB1 transcriptional modulation may be a key factor in aggressive CM. This abstract is also being presented as Poster A30. Citation Format: Haifeng Zhu, Junna Oba, Xiaoxing Yu, Caitlin A. Creasy, Marie-Andrée Forget, Fernando Carapeto, Cara L. Haymaker, Chang-Jiun Wu, Tatiana V. Karpinets, Wei-Lien Wang, Michael T. Tetzlaff, Alexander J. Lazar, Gordon B. Mills, Amanda R. Moore, Yu Chen, Jianhua Zhang, Jeffrey E. Gershenwald, Jennifer A. Wargo, Chantale Bernatchez, Patrick Hwu, P. Andrew Futreal, Scott E. Woodman. Nongenomic BAP1 aberrancy drives highly aggressive cutaneous melanoma phenotype [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR03.

Fibroblast Growth Factor 2 Augments Transforming Growth Factor Beta 1 Induced Epithelial-mesenchymal Transition in Lung Cell Culture Model.

Impaired lung epithelial cell regeneration following injury may contribute to the development of pulmonary fibrosis. Epithelial-mesenchymal transition (EMT) is a critical event in embryonic development, wound healing following injury, and even cancer progression. Previous studies have shown that the combination of transforming growth factor beta-1 (TGFβ1) and fibroblast growth factor 2 (FGF2) induces EMT during cancer metastasis. However, this synergy remains to be elucidated in inducing EMT associated with wound healing after injury. We set out this study to determine the effect of fibroblast growth factor 2 (FGF2) on TGFβ1-induced EMT in the human lung epithelium. BEAS-2B and A549 cells were treated with TGFβ1, FGF2, or both. EMT phenotype was investigated morphologically and by measuring mRNA expression levels; using quantitative real-time PCR. E-cadherin expression was assayed by western blot and immunofluorescence staining. Cell migration was confirmed using a wound-healing assay. TGFβ1 induced a morphological change and a significant increase in cell migration of BEAS-2B cells. TGFβ1 significantly reduced E-cadherin (CDH1) mRNA expression and markedly induced expression of N-cadherin (CDH2), tenascin C (TNC), fibronectin (FN), actin alpha 2 (ACTA2), and collagen I (COL1A1). While FGF2 alone did not significantly alter EMT gene expression, it enhanced TGFβ1-induced suppression of CDH1 and upregulation of ACTA2, but not TNC, FN, and CDH2. FGF2 significantly inhibited TGFβ1-induced COL1A1 expression. Furthermore, FGF2 maintained TGFβ1-induced morphologic changes and increased the migration of TGFβ1-treated cells. This study suggests a synergistic effect between TGFβ1 and FGF2 in inducing EMT in lung epithelial cells, which may play an important role in wound healing and tissue repair after injury.

Epigenetic Silencing of LMX1A Contributes to Cancer Progression in Lung Cancer Cells.

Epigenetic modification is considered a major mechanism of the inactivation of tumor suppressor genes that finally contributes to carcinogenesis. LIM homeobox transcription factor 1α (LMX1A) is one of the LIM-homeobox-containing genes that is a critical regulator of growth and differentiation. Recently, LMX1A was shown to be hypermethylated and functioned as a tumor suppressor in cervical cancer, ovarian cancer, and gastric cancer. However, its role in lung cancer has not yet been clarified. In this study, we used public databases, methylation-specific PCR (MSP), reverse transcription PCR (RT-PCR), and bisulfite genomic sequencing to show that LMX1A was downregulated or silenced due to promoter hypermethylation in lung cancers. Treatment of lung cancer cells with the demethylating agent 5-aza-2’-deoxycytidine restored LMX1A expression. In the lung cancer cell lines H23 and H1299, overexpression of LMX1A did not affect cell proliferation but suppressed colony formation and invasion. These suppressive effects were reversed after inhibition of LMX1A expression in an inducible expression system in H23 cells. The quantitative RT-PCR (qRT-PCR) data showed that LMX1A could modulate epithelial mesenchymal transition (EMT) through E-cadherin (CDH1) and fibronectin (FN1). NanoString gene expression analysis revealed that all aberrantly expressed genes were associated with processes related to cancer progression, including angiogenesis, extracellular matrix (ECM) remodeling, EMT, cancer metastasis, and hypoxia-related gene expression. Taken together, these data demonstrated that LMX1A is inactivated through promoter hypermethylation and functions as a tumor suppressor. Furthermore, LMX1A inhibits non-small cell lung cancer (NSCLC) cell invasion partly through modulation of EMT, angiogenesis, and ECM remodeling.