Master Regulator Of Epithelial-mesenchymal Transition Research Articles

Synthetic Epigenetic Reprogramming of Mesenchymal to Epithelial States Using the CRISPR/dCas9 Platform in Triple Negative Breast Cancer.

Epithelial-mesenchymal transition (EMT) is a reversible transcriptional program invoked by cancer cells to drive cancer progression. Transcription factor ZEB1 is a master regulator of EMT, driving disease recurrence in poor-outcome triple negative breast cancers (TNBCs).Here, this work silences ZEB1 in TNBC models by CRISPR/dCas9-mediated epigenetic editing, resulting in highly-specific and nearly complete suppression of ZEB1 in vivo, accompanied by long-lasting tumor inhibition. Integrated "omic" changes promoted by dCas9 linked to the KRAB domain (dCas9-KRAB) enabled the discovery of a ZEB1-dependent-signature of 26 genes differentially-expressed and -methylated, including the reactivation and enhanced chromatin accessibility in cell adhesion loci, outlining epigenetic reprogramming toward a more epithelial state. In the ZEB1 locus transcriptional silencing is associated with induction of locally-spread heterochromatin, significant changes in DNA methylation at specific CpGs, gain of H3K9me3, and a near complete erasure of H3K4me3 in the ZEB1 promoter. Epigenetic shifts induced by ZEB1-silencing are enriched in a subset of human breast tumors, illuminating a clinically-relevant hybrid-like state. Thus, the synthetic epi-silencing of ZEB1 induces stable "lock-in" epigenetic reprogramming of mesenchymal tumors associated with a distinct and stable epigenetic landscape. This work outlines epigenome-engineering approaches for reversing EMT and customizable precision molecular oncology approaches for targeting poor outcome breast cancers.

Abstract P2-19-02: Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors

Abstract Background: Epithelial-mesenchymal transition (EMT) phenotype is a complex process and plays a central role in tumor progression, aggression, invasion, metastasis, and resistance to therapy. Role of androgen receptor (AR) and androgens in inducing EMT has been well established in experimental systems of prostate cancer. AR is widely expressed in all subtypes of breast cancer (BC) and is considered to have context dependent role based on the steroid hormone microenvironment. Previous studies have shown AR as the only sex steroid receptor detectable in BC metastases and in metastatic lesion. We investigated the role of androgens in driving the EMT phenotype and association of AR induced genes with EMT in BC tumors using public dataset. Methods: We treated AR positive breast cancer cell line MDA-MB-453 with the androgen, dihydrotestosterone (DHT) followed by AR antagonist bicalutamide (Bic) and examined the expression levels of AR induced genes by quantitative real time PCR (qPCR) and AR induced protein GCDFP-15 by western blot. AR driven genes (SEC14L2, C1orf116, FKBP5, UGT2B11, UGT2B28, KCNMA1, PIP and ABCC11) were identified by bioinformatic pipeline using a systematic bioinformatic approach from public microarray data sets derived from AR positive breast cancer cell lines (S Rajarajan et al, SABCS 2021). Mesenchymal phenotype of the cell lines under DHT/Bic was evaluated morphologically and expression levels of EMT markers (ZEB1, Slug, Vimentin) and E-cadherin was estimated by qPCR, western blot and immunofluorescence. Migratory potential of the cell line under treatment was evaluated by wound healing assay and proliferation was examined by the MTT assay. Association of AR induced genes and levels of enzymes involved in intracrinal testosterone metabolism (SRD5A1 & SRD5A3) with EMT in breast tumors was evaluated using transcript data from METABRIC database. EMT scores were derived from previous publications (Byers et al, 2013, Mak et al, 2016) and correlation was tested with AR induced genes. Results: The MDAMB-453 cell line was treated with 10nM DHT for 72hrs and this increased the expression of AR downstream protein GCDFP-15 by 31.47% (p=0.022) when compared to the control. Further, a significant increase in the expression levels of AR driven genes with a fold change >2 was observed in the DHT treated cells (p< 0.05). The expression of GCDP15 protein (p=0.028) and the AR downstream genes (p< 0.05) were significantly repressed upon treatment with bicalutamide. DHT treatment also showed a significant higher expression of EMT master regulator, SLUG (fold change>3, p=0.045) and a wound healing assay showed a 22% increase in migration (p=0.027). The expression of SLUG was significantly repressed (p=0.003) and the migratory ability of the DHT treated cells reduced upon treatment with bicalutamide (p=0.145). We also observed a significant loss of E-cadherin protein (by 23.65%; p=0.024) with DHT treatment and this was completely reversed upon treatment with bicalutamide (p=0.047). No significant change in proliferation was observed among the different treatment conditions. A significant positive correlation was observed between AR induced genes and the EMT scores (p< 0.05). Expression level of SRD5A3 was also positively correlated with EMT score (p=0.043) and AR induced genes (p< 0.05), except SEC14L2. Conclusion: Androgen receptor plays an important role in the biology of breast cancer and is considered a useful marker for prognosis. However, antiandrogen therapies haven’t seen the expected success in clinical trial settings. Our results support the controversial role played by AR in higher androgenic environment within breast cancer and warrant the consideration of the intratumoral levels of sex steroid hormones. Citation Format: Savitha Rajarajan, Snijesh VP, Madhumathy G Nair, Apoorva D, Chandrakala M, Vidya P Nimbalkar, Maalavika Pillai, Mohit Kumar Jolly, Jyothi S Prabhu. Androgen mediated signaling induces epithelial to mesenchymal transition phenotype in MDA-MB-453 breast cancer cells and human breast tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-19-02.

TGFβ1-Induced EMT in the MCF10A Mammary Epithelial Cell Line Model Is Executed Independently of SNAIL1 and ZEB1 but Relies on JUNB-Coordinated Transcriptional Regulation.

Epithelial-mesenchymal transition (EMT) fosters cancer cell invasion and metastasis, the main cause of cancer-related mortality. Growing evidence that SNAIL and ZEB transcription factors, typically portrayed as master regulators of EMT, may be dispensable for this process, led us to re-investigate its mechanistic underpinnings. For this, we used an unbiased computational approach that integrated time-resolved analyses of chromatin structure and differential gene expression, to predict transcriptional regulators of TGFβ1-inducible EMT in the MCF10A mammary epithelial cell line model. Bioinformatic analyses indicated comparatively minor contributions of SNAIL proteins and ZEB1 to TGFβ1-induced EMT, whereas the AP-1 subunit JUNB was anticipated to have a much larger impact. CRISPR/Cas9-mediated loss-of-function studies confirmed that TGFβ1-induced EMT proceeded independently of SNAIL proteins and ZEB1. In contrast, JUNB was necessary and sufficient for EMT in MCF10A cells, but not in A549 lung cancer cells, indicating cell-type-specificity of JUNB EMT-regulatory capacity. Nonetheless, the JUNB-dependence of EMT-associated transcriptional reprogramming in MCF10A cells allowed to define a gene expression signature which was regulated by TGFβ1 in diverse cellular backgrounds, showed positively correlated expression with TGFβ signaling in multiple cancer transcriptomes, and was predictive of patient survival in several cancer types. Altogether, our findings provide novel mechanistic insights into the context-dependent control of TGFβ1-driven EMT and thereby may lead to improved diagnostic and therapeutic options.

Epithelial-Mesenchymal Transition-Inducing Factors Involved in the Progression of Lung Cancers.

Although there have been advances in cancer therapy and surgical improvement, lung cancer has the lowest survival rate (19%) at all stages. This is because most patients are diagnosed with concurrent metastasis, which occurs due to numerous related reasons. Especially, lung cancer is one of the most common and malignant cancers in the world. Although there are advanced therapeutic strategies, lung cancer remains one of the main causes of cancer death. Recent work has proposed that epithelial-mesenchymal transition (EMT) is the main cause of metastasis in most cases of human cancers including lung cancer. EMT involves the conversion of epithelial cells, wherein the cells lose their epithelial abilities and become mesenchymal cells involved in embryonic development, such as gastrulation and neural crest formation. In addition, recent research has indicated that EMT contributes to altering the cancer cells into cancer stem cells (CSCs). Although EMT is important in the developmental stages, this process also activates lung cancer progression, including complicated and diverse signaling pathways. Despite the numerous investigations on signaling pathways involved in the progression of lung cancer, this malignancy is considered critical for treatment. EMT in lung cancer involves many transcription factors and inducers, for example, Snail, TWIST, and ZEB are the master regulators of EMT. EMT-related factors and signaling pathways are involved in the progression of lung cancer, proposing new approaches to lung cancer therapy. In the current review, we highlight the signaling pathways implicated in lung cancer and elucidate the correlation of these pathways, indicating new insights to treat lung cancer and other malignancies.

Abstract IA-005: Metabolic Stress in Pancreatic Cancer Progression and Therapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) cells depend on glutamine to meet their unique metabolic needs. The elevated consumption of glutamine leads to intratumoral nutrient depletion, causing metabolic stress that has the potential to impact tumor progression and therapy. We will show that nutrient stress caused by glutamine deprivation leads to the induction of epithelial-mesenchymal transition (EMT) in PDAC cells. Mechanistically, we demonstrate that glutamine deficiency regulates EMT through the upregulation of the EMT master regulator Slug, a process that is dependent on both MEK/ERK signaling and ATF4. We find that Slug is required in PDAC cells for glutamine deprivation-induced EMT, cell motility and nutrient stress survival. Importantly, we decipher that Slug is associated with nutrient stress in PDAC tumors and is required for metastasis. These results delineate a novel role for Slug in the nutrient stress response and provide insight into how nutrient depletion might influence PDAC progression. During the development of a pharmacological in vivo system aimed at mimicking glutamine stress, we have serendipitously discovered an approach to profoundly affect PDAC tumor growth and metastasis. We will outline the metabolic mechanisms underlying these effects and discuss additional targeting strategies for PDAC. Citation Format: Cosimo Commisso. Metabolic Stress in Pancreatic Cancer Progression and Therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr IA-005.

Transcriptional repression of E-cadherin in nickel-exposed lung epithelial cells mediated by loss of Sp1 binding at the promoter.

E-cadherin plays a central role in the stability of epithelial tissues by facilitating cell-cell adhesion. Loss of E-cadherin expression is a hallmark of epithelial-mesenchymal transition (EMT), a major event in the pathogenesis of several lung diseases. Our earlier studies showed that nickel, a ubiquitous environmental toxicant, induced EMT by persistently downregulating E-cadherin expression in human lung epithelial cells and that the EMT remained irreversible postexposure. However, the molecular basis of persistent E-cadherin downregulation by nickel exposure is not understood. Here, our studies show that the binding of transcription factor Sp1 to the promoter of E-cadherin encoding gene, CDH1, is essential for its expression. Nickel exposure caused a loss of Sp1 binding at the CDH1 promoter, resulting in its downregulation and EMT induction. Loss of Sp1 binding at the CDH1 promoter was associated with an increase in the binding of ZEB1 adjacent to the Sp1 binding site. ZEB1, an EMT master regulator persistently upregulated by nickel exposure, is a negative regulator of CDH1. CRISPR-Cas9-mediated knockout of ZEB1 restored Sp1 binding at the CDH1 promoter. Furthermore, ZEB1 knockout rescued E-cadherin expression and re-established the epithelial phenotype. Since EMT is associated with a number of nickel-exposure-associated chronic inflammatory lung diseases including asthma, fibrosis and cancer and metastasis, our findings provide new insights into the mechanisms associated with nickel pathogenesis.

Epigenetic Regulation and Post-Translational Modifications of SNAI1 in Cancer Metastasis.

SNAI1, a zinc finger transcription factor, not only acts as the master regulator of epithelial-mesenchymal transition (EMT) but also functions as a driver of cancer progression, including cell invasion, survival, immune regulation, stem cell properties, and metabolic regulation. The regulation of SNAI1 occurs at the transcriptional, translational, and predominant post-translational levels including phosphorylation, acetylation, and ubiquitination. Here, we discuss the regulation and role of SNAI1 in cancer metastasis, with a particular emphasis on epigenetic regulation and post-translational modifications. Understanding how signaling networks integrate with SNAI1 in cancer progression will shed new light on the mechanism of tumor metastasis and help develop novel therapeutic strategies against cancer metastasis.

Osteopontin Expression in Thyroid Cancer: Deciphering EMT-Related Molecular Mechanisms.

Thyroid cancer is the most common tumor arising from the endocrine system and generally presents good prognosis. However, its aggressive subtypes are related to therapeutic resistance and early metastasis. Epithelial–mesenchymal transition (EMT) and its reverse process, the mesenchymal–epithelial transition (MET), are key events mediating cancer progression, including in thyroid cancer. The matricellular protein osteopontin (OPN) has been reported as a master regulator of EMT in many tumor types. Although high OPN expression has been described and associated with important aspects of thyroid cancer progression, there is no clear evidence regarding OPN as a regulator of EMT in thyroid cancer. Thus, taking together the known roles of OPN in the modulation of EMT in cancer and the information reporting the expression of OPN in thyroid tumor progression, this review aims at summarizing and discussing data related to EMT in thyroid cancer and its putative relation to the roles of OPN in the development of thyroid cancer. These data provide new insights into the molecular mechanisms by which OPN could potentially modulate EMT in thyroid tumors, generating evidence for future studies that may contribute to new therapeutic, prognostic and/or diagnostic tools.

Evaluating the Epithelial-Mesenchymal Program in Human Breast Epithelial Cells Cultured in Soft Agar Using a Novel Macromolecule Extraction Protocol.

Simple SummaryAnchorage-independent soft agar colony formation assays have been widely used as an in vitro surrogate for in vivo tumour formation in xenograft studies, and has found much utility in studies such as cancer drug development. However, molecular characterisation of cells grown in soft agar has proven difficult and sometimes even impossible. We developed a set of new methods that allow DNA, RNA and proteins (including phosphoproteins) to be extracted from cells grown in soft agar, even without visible colony formation. We used these methods to demonstrate the role of the epithelial-mesenchymal program in the malignant transformation of a classical human mammary epithelial cell model.The ability to grow in anchorage-independent conditions is an important feature of malignant cells, and it is well-established that cellular phenotypes in adherent cultures can differ widely from phenotypes observed in xenografts and anchorage-independent conditions. The anchorage-independent soft-agar colony formation assay has been widely used as a bridge between adherent cell cultures and animal tumor studies, providing a reliable in vitro tool to predict the tumorigenicity of cancer cells. However, this functional assay is limited in its utility for molecular mechanistic studies, as currently there is no reliable method that allows the extraction of biological macromolecules from cells embedded in soft-agar matrices, especially in experimental conditions where no visible colonies form. We developed a set of new methods that enable the extraction of DNA, RNA and proteins directly from cells embedded in soft agar, allowing for a wide range of molecular signaling analysis. Using the new methods and human mammary epithelial cells (HMECs), we studied the role of epithelial-mesenchymal transition (EMT) in the ability of HMECs to form colonies in soft agar. We found that, when cultured in soft agar instead of in adherent cultures, immortalized non-malignant HME-hTERT cells upregulated the epithelial program, which was noted to be necessary for their survival in this anchorage-independent condition. Overexpression of SV40 small T antigen (ST) or the EMT master-regulator SNAI1 negates this requirement and significantly enhances colony formation in soft agar driven by mutant-RAS. Interestingly, we found that, similar to SNAI1, ST also promotes EMT changes in HMECs, providing further support for EMT as a prerequisite for the efficient anchorage-independent colony formation driven by mutant-RAS in our HMEC model.

MTA2 promotes the metastasis of esophageal squamous cell carcinoma via EIF4E-Twist feedback loop.

Metastasis‐associated protein 2 (MTA2) is frequently amplified in many types of cancers; however, the role and underlying molecular mechanism of MTA2 in esophageal squamous cell carcinoma (ESCC) remain unknown. Here, we reported that MTA2 is highly expressed in ESCC tissue and cells, and is closely related to the malignant characteristics and poor prognosis of patients with ESCC. Through in vitro and in vivo experiments, we demonstrated that MTA2 significantly promoted ESCC growth, metastasis, and epithelial‐mesenchymal transition (EMT) progression. This integrative analysis combined with expression microarray showed that MTA2 could interact with eukaryotic initiation factor 4E (EIF4E), which positively regulates the expression of Twist, known as a master regulator of EMT. Moreover, the results of chromatin immunoprecipitation revealed that MTA2 was recruited to the E‐cadherin promoter by Twist, which reduced the acetylation level of the promoter region and thus inhibited expression of E‐cadherin, and subsequently promoted the aggressive progression of ESCC. Collectively, our study provided novel evidence that MTA2 plays an aggressive role in ESCC metastasis by a novel EIF4E‐Twist positive feedback loop, which may provide a potential therapeutic target for the management of ESCC.

The miR-5694/AF9/Snail Axis Provides Metastatic Advantages and a Therapeutic Target in Basal-like Breast Cancer

Epigenetic deregulation, especially mutagenesis or the abnormal expression of epigenetic regulatory factors (ERFs), plays an important role in malignant tumorigenesis. To screen natural inhibitors of breast cancer metastasis, we adopted small interfering RNAs (siRNAs) to transiently knock down 591 ERF-coding genes in luminal breast cancer MCF-7 cells and found that depletion of AF9 significantly promoted MCF-7 cell invasion and migration. A mouse model of metastasis further confirmed the suppressive role of AF9 in breast cancer metastasis. RNA profiling revealed enrichment of AF9 targets genes in the epithelial-mesenchymal transition (EMT). Mechanistically, tandem mass spectrometry showed that AF9 interacts with Snail, which hampers Snail transcriptional activity in basal-like breast cancer (BLBC) cells. AF9 reconstitutes an activated state on the promoter of Snail, which is a master regulator of EMT, and derepresses genes by recruiting CBP or GCN5. Additionally, microRNA-5694 (miR-5694) targeted and degraded AF9 messenger RNA (mRNA) in BLBC cells, further enhancing cell invasion and migration. Notably, AF9 and miR-5694 expression in BLBC clinical samples correlated inversely. Hence, miR-5694 mediates downregulation of AF9 and provides metastatic advantages in BLBC. Restoring expression of the metastasis suppressor AF9 is a possible therapeutic strategy against metastatic breast cancer.

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.

Abstract B06: Candidate differentiation stall in epithelial mesenchymal transition in H3K27M diffuse midline glioma

Abstract The purpose of our study was to elucidate the molecular mechanisms by which the histone H3 K27M mutation drives tumorigenesis of pediatric gliomas. Though it has potential implications on the treatment of diffuse midline glioma as a disease driver, the timing, cell type of origin, and effect of the H3K27M mutation on early embryonic brain development are not fully characterized. Here, we performed differential expression analysis on a cohort of H3K27M and H3WT pediatric gliomas, revealing that genes in the epithelial-mesenchymal transition (EMT) pathway were significantly differentially expressed. SNAI1, the EMT master regulator, was significantly overexpressed in the H3K27M tumor cohort. Overall, pre-EMT genes were overexpressed in H3K27M tumors while post-EMT genes were underexpressed. We hypothesized that H3K27M may lead to gliomagenesis by stalling an EMT in early brain development and employed single-cell and bulk RNA sequencing data from cerebral organoids at multiple developmental timepoints to test this hypothesis. We observed that a long noncoding RNA (lncRNA) signature identified as transiently expressed in early brain development was preferentially expressed in H3K27M tumors. Cell type-specific lncRNA signatures had higher expression in H3K27M tumors for pre-EMT cell types, and higher expression in H3WT tumors for post-EMT cell types. Finally, t-SNE clustering of single-cell glioma RNA sequencing data with single-cell organoid data revealed transcriptional similarities between H3K27M and pre-EMT neural stem cells. In conclusion, we observed aberrant activity of the EMT in H3K27M gliomas. Our data suggest that the H3K27M mutation is associated with a pre-EMT cell phenotype, and that this mutation may cause EMT arrest or de-differentiation. Citation Format: Allison R. Cheney, Lauren M. Sanders, Lucas Seninge, Holly C. Beale, Ellen Towle Kephart, Jacob Pfeil, Katrina Learned, A. Geoffrey Lyle, Isabel Bjork, David Haussler, Sofie R. Salama, Olena M. Vaske. Candidate differentiation stall in epithelial mesenchymal transition in H3K27M diffuse midline glioma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B06.

Glutamine depletion regulates Slug to promote EMT and metastasis in pancreatic cancer

Tumor cells rely on glutamine to fulfill their metabolic demands and sustain proliferation. The elevated consumption of glutamine can lead to intratumoral nutrient depletion, causing metabolic stress that has the potential to impact tumor progression. Here, we show that nutrient stress caused by glutamine deprivation leads to the induction of epithelial-mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) cells. Mechanistically, we demonstrate that glutamine deficiency regulates EMT through the up-regulation of the EMT master regulator Slug, a process that is dependent on both MEK/ERK signaling and ATF4. We find that Slug is required in PDAC cells for glutamine deprivation-induced EMT, cell motility, and nutrient stress survival. Importantly, we decipher that Slug is associated with nutrient stress in PDAC tumors and is required for metastasis. These results delineate a novel role for Slug in the nutrient stress response and provide insight into how nutrient depletion might influence PDAC progression.

Aurora B induces epithelial-mesenchymal transition by stabilizing Snail1 to promote basal-like breast cancer metastasis.

Aurora B is a serine/threonine kinase that has been implicated in regulating cell proliferation in distinct cancers, including breast cancer. Here we show that Aurora B expression is elevated in basal-like breast cancer (BLBC) compared with other breast cancer subtypes. This high level of expression seems to correlate with poor metastasis-free survival and relapse-free survival in affected patients. Mechanistically, we show that elevated Aurora B expression in breast cancer cells activates AKT/GSK3β to stabilize Snail1 protein, a master regulator of epithelial-mesenchymal transition (EMT), leading to EMT induction in a kinase-dependent manner. Conversely, Aurora B knock down by short-hairpin RNAs (shRNAs) suppresses AKT/GSK3β/Snail1 signaling, reverses EMT and reduces breast cancer metastatic potential in vitro and in vivo. Finally, we identified a specific OCT4 phosphorylation site (T343) responsible for mediating Aurora B-induced AKT/GSK3β/Snail1 signaling and EMT that could be attenuated by Aurora B kinase inhibitor treatment. These findings support that Aurora B induces EMT to promote breast cancer metastasis via OCT4/AKT/GSK3β/Snail1 signaling. Pharmacologic Aurora B inhibition might be a potential effective treatment for breast cancer patients with metastatic disease.

Long Non-Coding RNA HCP5 Facilitates Cell Invasion And Epithelial-Mesenchymal Transition In Oral Squamous Cell Carcinoma By miR-140-5p/SOX4 Axis.

BackgroundOral squamous cell carcinoma (OSCC) is the predominant histological type of human oral cancer. In this study, we sought to investigate the functional role of lncRNA HCP5 in OSCC progression.MethodsThe HCP5 and miR-140-5p expression level was determined in 73 paired OSCC tissues and their adjacent normal tissues. Knockdown or overexpression of HCP5 was conducted to investigate the effects of HCP5 on malignant behaviors of OSCC cells. Then, bioinformatic prediction and dual-luciferase reporter assay were conducted to study the interaction between HCP5 and miR-140-5p in OSCC.ResultsOur results demonstrated that HCP5 expression was significantly increased in OSCC tissues and cell lines. High HCP5 level was associated with the aggressive clinicopathological characteristics and poor prognosis of OSCC patients. In vitro gain- and loss-of-function experiments showed that HCP5 overexpression promoted, whereas HCP5 knockdown inhibited the proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of OSCC cells. Mechanistically, we confirmed that HCP5 might serve as a competitive endogenous RNA (ceRNA) for miR-140-5p to alleviate the repression of its downstream target, SOX4, a master regulator of EMT. Furthermore, restoration of miR-140-5p expression diminished the oncogenic effects of HCP5 on OSCC cells.ConclusionOverall, the present study indicated that HCP5/miR-140-5p/SOX4 axis might be a ponderable and promising therapeutic target for OSCC.

Drug Design Targeting T-Cell Factor-Driven Epithelial-Mesenchymal Transition as a Therapeutic Strategy for Colorectal Cancer.

Metastasis is the cause of 90% of mortality in cancer patients. For metastatic colorectal cancer (mCRC), the standard-of-care drug therapies only palliate the symptoms but are ineffective, evidenced by a low survival rate of ∼11%. T-cell factor (TCF) transcription is a major driving force in CRC, and we have characterized it to be a master regulator of epithelial-mesenchymal transition (EMT). EMT transforms relatively benign epithelial tumor cells into quasi-mesenchymal or mesenchymal cells that possess cancer stem cell properties, promoting multidrug resistance and metastasis. We have identified topoisomerase IIα (TOP2A) as a DNA-binding factor required for TCF-transcription. Herein, we describe the design, synthesis, biological evaluation, and in vitro and in vivo pharmacokinetic analysis of TOP2A ATP-competitive inhibitors that prevent TCF-transcription and modulate or reverse EMT in mCRC. Unlike TOP2A poisons, ATP-competitive inhibitors do not damage DNA, potentially limiting adverse effects. This work demonstrates a new therapeutic strategy targeting TOP2A for the treatment of mCRC and potentially other types of cancers.

Immunohistochemical Analysis of Transcription Factors and Markers of Epithelial-Mesenchymal Transition (EMT) in Human Tumors.

Epithelial-mesenchymal transition (EMT) is a key multi-step process which enables cancer cells to detach from the epithelial primary tumor mass and allows them to metastasize to distant organs. We immunohistochemically analyzed the expression of the transcription factors (TWIST-1, SLUG, ZEB1, ZEB2) and components of the extracellular matrix (laminin-5, fibronectin) which influence the EMT. Primary human breast (MDA-MB-231), colon (HT29, HCT116), ovarian (SKOV3, OVCAR3) and head and neck squamous cell carcinoma cell lines (UTSCC2, UTSCC24A) grown as xenografts were immunohistochemically analyzed in vitro and in vivo. A high SLUG expression was observed in every cancer entity both in vitro and in vivo. ZEB1 and ZEB2 showed a high in vivo expression especially in SKOV3 and in in vitro grown MDA-MB-231 cells. SLUG expression showed the highest expression in all cancer entities investigated. Hence, it presumably represents the master regulator of EMT in these metastatic tumor entities.

Sphingosine 1-phosphate signaling induces SNAI2 expression to promote cell invasion in breast cancer cells.

Epithelial-mesenchymal transition (EMT) is a critical process implicated in the initial stage of cancer metastasis, which is the major cause of tumor recurrence and mortality. Although key transcription factors that regulate EMT, such as snail family transcriptional repressor 2 (SNAI2), are well characterized, the upstream signaling pathways controlling these transcriptional mediators are largely unknown, which limits therapeutic strategies. Sphingosine 1-phosphate (S1P) is a bioactive lipid mediator, generated by sphingosine kinases (SPHK1 and SPHK2), that mainly exerts its effects by binding to the following 5 GPCRs: S1P1 to S1P5. S1P signaling has been reported to regulate different aspects of cancer progression including cell proliferation, apoptosis, and migration; nevertheless, its role in cancer metastasis, specifically via EMT, is not established. Here we show that SPHK1 expression correlates significantly with EMT score in breast cancer cell lines, and with SNAI2 in patient-derived breast tumors. Cell-based assays demonstrate that S1P can rapidly up-regulate the expression of SNAI2 in breast cancer cells via the activation of cognate receptors S1P2 and S1P3. Knockdown studies suggest that S1P2 and S1P3 mediate this effect by activating myocardin-related transcription factor A (MRTF-A) and yes-associated protein (YAP), respectively. Michigan Cancer Foundation 7 cells stably overexpressing S1P2 or S1P3 exhibit a more invasive phenotype, when compared to control cells. Taken together, our findings suggest that S1P produced by SPHK1 induces SNAI2 expression via S1P2-YAP and S1P3-MRTF-A pathways, leading to enhanced cell invasion. Cumulatively, this study reveals a novel mechanism by which S1P activates parallel pathways that regulate the expression of SNAI2, a master regulator of EMT, and provides new insights into druggable therapeutic targets that may limit cancer metastasis. Wang, W., Hind, T., Lam, B. W. S., Herr, D. R. Sphingosine 1-phosphate signaling induces SNAI2 expression to promote cell invasion in breast cancer cells.

Abstract P1-07-01: Novel role of the tumor suppressor ductal epithelium associated ring chromosome 1 (DEAR1) in regulation of breast stem/progenitor cell properties

Abstract Introduction: Breast cancer is the most commonly diagnosed cancer and third-leading cause of cancer-related deaths in women in America. A quarter of lesions diagnosed annually are ductal carcinoma in situ (DCIS), one of the earliest pre-invasive forms of invasive ductal carcinoma (IDC). Without therapeutic intervention, 30-50% of DCIS cases can progress to IDC. Understanding the mechanisms regulating progression from DCIS to IDC would help identify biomarkers to stratify DCIS patients at higher risk of progression or recurrence. Cumulative literature suggests the earliest phase of dissemination from the primary tumor is driven by the epithelial-mesenchymal transition (EMT) program. DEAR1 is a tumor suppressor gene which is mutated, undergoes loss of heterozygosity in breast cancer, and is downregulated in DCIS lesions. DEAR1 regulates acinar morphogenesis and cell polarity, and is a negative regulator of TGF-beta-driven EMT by binding to and ubiquitinating SMAD3, thereby limiting the amount of SMAD3 available to activate an EMT signature. Overexpression of EMT master regulators, or exposure to TGF-beta in immortalized human mammary epithelial cells (HMECs), results in mammosphere formation and breast stem cell markers, thus linking the EMT process to acquisition of stem cell characteristics. Methods: Stable lentiviral shRNA knockdown, in vitro mammosphere assay, cytospin, immunofluorescence, immunoprecipitation, western blot analysis, ubiquitination and real-time quantitative PCR were performed. Results: DEAR1 knockdown in immortal HMECs resulted in a significant enhancement of primary mammosphere formation and growth compared to controls, suggesting that DEAR1 may regulate stem/progenitor cell properties; this effect was greater when cells were exposed to TGF-beta. To determine if DEAR1 regulates stem cell properties through regulation of SMAD3 levels, DEAR1-SMAD3 double knockdown clones were examined for mammosphere formation. Results indicated fewer mammospheres in double knockdown clones, but only in the presence of TGF-beta, suggesting that the mammosphere phenotype is partially dependent on the TGF-beta-SMAD3 pathway. The observation that loss of DEAR1 alone results in a stem/progenitor cell phenotype indicates that DEAR1 may regulate mammosphere formation independent of its role in regulating the TGF-beta pathway. HMEC mammospheres express higher levels of stem cell marker aldehyde dehydrogenase (ALDH1) and co-express luminal and basal cytokeratins, suggesting bipotential capacity. We also observed upregulation of SNAI2 and ZEB1, master EMT and stem cell regulators, in DEAR1 knockdown HMECs. DEAR1 interacted with SNAI2 by co-immunoprecipitation analysis and also promoted the polyubiquitination of SNAI2. Conclusions: Loss of the tumor suppressor/polarity regulator DEAR1 promotes stem cell properties in part through the DEAR1-TGF-beta-SMAD3 axis. We demonstrate a DEAR1-SNAI2 axis that may partially regulate stem/progenitor cell properties in HMECs through the polyubiquitination of SNAI2, a master regulator of EMT and stemness. These mechanisms governing acquisition of the stem cell properties may contribute to understanding how DCIS progress to IDC. Citation Format: Le U, Chen N, Balasenthil S, Killary AM. Novel role of the tumor suppressor ductal epithelium associated ring chromosome 1 (DEAR1) in regulation of breast stem/progenitor cell properties [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-07-01.