Epithelial-mesenchymal Transition In Breast Cancer Research Articles

A Feedback Loop Comprising EGF/TGFα Sustains TFCP2-Mediated Breast Cancer Progression.

Stemness and epithelial-mesenchymal transition (EMT) are two fundamental characteristics of metastasis that are controlled by diverse regulatory factors, including transcription factors. Compared with other subtypes of breast cancer, basal-type or triple-negative breast cancer (TNBC) has high frequencies of tumor relapse. However, the role of alpha-globin transcription factor CP2 (TFCP2) has not been reported as an oncogenic driver in those breast cancers. Here, we show that TFCP2 is a potent factor essential for EMT, stemness, and metastasis in breast cancer. TFCP2 directly bound promoters of EGF and TGFα to regulate their expression and stimulate autocrine signaling via EGFR. These findings indicate that TFCP2 is a new antimetastatic target and reveal a novel regulatory mechanism in which a positive feedback loop comprising EGF/TGFα and AKT can control malignant breast cancer progression. SIGNIFICANCE: TFCP2 is a new antimetastatic target that controls TNBC progression via a positive feedback loop between EGF/TGFα and the AKT signaling axis.

A combinatorially regulated RNA splicing signature predicts breast cancer EMT states and patient survival.

During breast cancer metastasis, the developmental process epithelial–mesenchymal transition (EMT) is abnormally activated. Transcriptional regulatory networks controlling EMT are well-studied; however, alternative RNA splicing also plays a critical regulatory role during this process. A comprehensive understanding of alternative splicing (AS) and the RNA binding proteins (RBPs) that regulate it during EMT and their impact on breast cancer remains largely unknown. In this study, we annotated AS in the breast cancer TCGA data set and identified an AS signature that is capable of distinguishing epithelial and mesenchymal states of the tumors. This AS signature contains 25 AS events, among which nine showed increased exon inclusion and 16 showed exon skipping during EMT. This AS signature accurately assigns the EMT status of cells in the CCLE data set and robustly predicts patient survival. We further developed an effective computational method using bipartite networks to identify RBP-AS networks during EMT. This network analysis revealed the complexity of RBP regulation and nominated previously unknown RBPs that regulate EMT-associated AS events. This study highlights the importance of global AS regulation during EMT in cancer progression and paves the way for further investigation into RNA regulation in EMT and metastasis.

Role of E2F3 and shugoshin I in epithelial-to-mesenchymal transition and cell invasion in breast cancer.

e13100 Background: Triple-negative breast cancer (TNBC) is the most aggressive and poorly prognostic breast cancer subtype, yet there are currently no biological therapies against this subtype. Our laboratory is finding the sources of novel biological targets in TNBC by studying the E2F transcription factors, which are essential for cellular proliferation and maintenance of genomic stability. While the deregulated Rb/E2F pathway signals the epithelial-to-mesenchymal transition (EMT), the underlining mechanism of how E2Fs drive EMT in TNBC remains unknown. We recently published that the E2F transcriptional activators (E2Fs) are overexpressed in the vast majority of TNBC and that their overexpression upregulates mitotic kinases such as TTK, which we have shown to induce EMT and invasion in TNBC cells. We also demonstrated that the E2Fs maintain genomic integrity in part through Shugoshin I (SGO1), which normally controls chromosome cohesion; however, the role of SGO1 in EMT in breast cancer is unknown. Our hypothesis is that E2F3 and SGO1 are highly expressed in TNBC and that their overexpression modulates EMT genes, thus promoting cell invasion. Methods: To test our hypothesis, we conducted siRNA transfection to knockdown E2F3 and SGO1 in MDA-MB-231 and Hs578t, which are TNBC cells. After 48 hours, we evaluated mRNA levels of EMT-related genes after E2F3 or SGO1 depletion using RT-PCR analysis. We also evaluated the effects of SGO1 depletion in protein localization by immunofluorescence. Furthermore, we evaluated the invasive behavior of MDA-MB-231 and Hs578t cells after SGO1 depletion using a Boyden Chamber Assay. Results: Our results demonstrate that E2F3 and SGO1 depletion decrease MMP3 mRNA levels. Moreover, E2F3 and SGO1 depletion restore E-cadherin expression and localization. Furthermore, E2F3 and SGO1 depletion significantly reduce cell invasion in MDA-MB-231 and Hs578t cells. Conclusions: Our results suggest that SGO1 is a promising drug target for breast cancer metastasis since EMT and invasion are essential early steps in breast cancer metastasis and E2F3 is presently undruggable.

Regulation of cell growth and migration by miR-96 and miR-183 in a breast cancer model of epithelial-mesenchymal transition

Breast cancer is the most commonly diagnosed malignancy in women, and has the second highest mortality rate. Over 90% of all cancer-related deaths are due to metastasis, which is the spread of malignant cells from the primary tumor to a secondary site in the body. It is hypothesized that one cause of metastasis involves epithelial-mesenchymal transition (EMT). When epithelial cells undergo EMT and transition into mesenchymal cells, they display increased levels of cell proliferation and invasion, resulting in a more aggressive phenotype. While many factors regulate EMT, microRNAs have been implicated in driving this process. MicroRNAs are short noncoding RNAs that suppress protein production, therefore loss of microRNAs may promote the overexpression of specific target proteins important for EMT. The goal of this study was to investigate the role of miR-96 and miR-183 in EMT in breast cancer. Both miR-96 and miR-183 were found to be downregulated in post-EMT breast cancer cells. When microRNA mimics were transfected into these cells, there was a significant decrease in cell viability and migration, and a shift from a mesenchymal to an epithelial morphology (mesenchymal-epithelial transition or MET). These MET-related changes may be facilitated in part by the regulation of ZEB1 and vimentin, as both of these proteins were downregulated when miR-96 and miR-183 were overexpressed in post-EMT cells. These findings indicate that the loss of miR-96 and miR-183 may help facilitate EMT and contribute to the maintenance of a mesenchymal phenotype. Understanding the role of microRNAs in regulating EMT is significant in order to not only further elucidate the pathways that facilitate metastasis, but also identify potential therapeutic options for preventing or reversing this process.

WHSC1 promotes wnt/β-catenin signaling in a FoxM1-dependent manner facilitating proliferation, invasion and epithelial-mesenchymal transition in breast cancer

Objectives: Wolf-Hirschhorn syndrome candidate gene-1 (WHSC1) is highly expressed in various malignant tumors. We investigated the correlation and regulatory pathway of WHSC1 in the progression of breast cancer (BC).Methods: The expression and distribution of WHSC1 in the BC tissues and cell lines were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemical staining. Spearman correlation analysis demonstrated the correlation between WHSC1 high expression level and the clinical characteristics of BC patients. The effects of WHSC1 on the proliferation, apoptosis, migration and invasion of BC cells were analyzed by cell transfection, MTT, colony formation, scratch assay, and transwell. Furthermore, the expression of Forkhead box M1 (FoxM1) and the location of β-catenin were detected by qRT-PCR and western blot.Results: Firstly, WHSC1 expression was up-regulated in BC tissues and cell lines. The high expression of WHSC1 in BC is associated with the tumor size (p = 0.027), metastasis (p = 0.018) and pathological stages (p = 0.025) of the BC patients. The knockdown of WHSC1 inhibited the growth, proliferation migration, invasion and EMT of BC cell lines. Furthermore, WHSC1 could promote the expression of FoxM1 in BC cells and tissues. WHSC1 enhanced the expression of FoxM1, and promoted the nuclear localization of β-catenin, and thus activated the downstream genes expression of Wnt/β-catenin signaling pathway to regulate the development of BC.Conclusion: In summary, our study elucidates the correlation and specific regulatory mechanism between WHSC1 and the progression of BC, thus implying that WHSC1 may function as molecular diagnosis, prognosis and molecular targeted therapy of BC.

UTMD inhibit EMT of breast cancer through the ROS/miR-200c/ZEB1 axis

As a potential drug/gene delivery system, the ultrasound-targeted microbubble destruction (UTMD) system can be used as a vehicle as well as increasing the permeability of biological barriers to enhance the effect of tumor treatment. However, the effect of UTMD in the tumor EMT process is unknown. In this study, we aimed to investigate the potential and mechanism of UTMD induced oxidative stress in inhibiting EMT of breast cancer. Human breast MDA231 cells were treated with microbubble (MB), ultrasound (US) and UTMD, respectively. The generation of oxidative stress, the levels of miR-200c, ZEB1 and vimentin, and the numbers of migratory cells were evaluated quantitatively and qualitatively by the measurement of intracellular reactive oxygen species (ROS), qRT-PCR, western blot assay, and transwell assay. Then, to evaluate the role of UTMD-induced oxidative stress and miR-200c in the epithelial-mesenchymal transition (EMT) inhibition, the ROS scavenger N-acetyl-L-cysteine (NAC) and miR-200c inhibitor were used before UTMD treatment. We found that UTMD induced oxidative stress, upregulated the expression of miR-200c, downregulated the expression of ZEB1 and vimentin and suppressed the MDA231 cell migration. The addition of NAC and miR-200c inhibitor had an opposite impact on the expression of miR-200c and ZEB1, thus hindered the effects of UTMD on MDA231 cells EMT. In conclusion, UTMD can inhibit the EMT characteristics of MDA231 cells. The mechanism may be related to the regulation of the miR-200c/ZEB1 axis through the generation of ROS induced by UTMD, which may provide a new strategy to prevent the tumor cells EMT under UTMD treatment.

EMT related circular RNA expression profiles identify circSCYL2 as a novel molecule in breast tumor metastasis.

Substantial evidence indicates that circular RNAs (circRNAs) play vital roles in several diseases, especially in cancer development. However, the functions of circRNAs in breast cancer metastasis remain to be investigated. This study aimed to identify the key circRNAs involved in epithelial mesenchymal transition (EMT) of breast cancer and evaluated their molecular function and roles in pathways that may be associated with tumor metastasis. An EMT model was constructed by treating breast cancer cells MCF-7 and MDA-MB-231 with transforming growth factor-β1. High-throughput RNA sequencing was used to identify the differentially expressed circRNAs in EMT and blank groups of two cells, and reverse transcription-quantitative PCR was used to validate the expression of circSCYL2 in human breast cancer tissues and cells. The effects of circSCYL2 on breast cancer cells were explored by transfecting with plasmids and the biological roles were assessed using transwell assays. EMT groups of breast cancer cells exhibited the characteristics of mesenchymal cells. Furthermore, the present study found that 7 circRNAs were significantly upregulated in both the MCF-7 EMT and MDA-MB-231 EMT groups, while 16 circRNAs were significantly downregulated. The current study identified that circSCYL2 was downregulated in breast cancer tissues and cell lines, and that circSCYL2 overexpression inhibited cell migration and invasion. This study provides expression profiles of circRNAs in EMT groups of breast cancer cells. circSCYL2, which is downregulated in breast cancer tissues and cells, may play an important role in breast cancer EMT progression.

RUNX1 and RUNX2 transcription factors function in opposing roles to regulate breast cancer stem cells.

Breast cancer stem cells (BCSCs) are competent to initiate tumor formation and growth and refractory to conventional therapies. Consequently BCSCs are implicated in tumor recurrence. Many signaling cascades associated with BCSCs are critical for epithelial-to-mesenchymal transition (EMT). We developed a model system to mechanistically examine BCSCs in basal-like breast cancer using MCF10AT1 FACS sorted for CD24 (negative/low in BCSCs) and CD44 (positive/high in BCSCs). Ingenuity Pathway Analysis comparing RNA-seq on the CD24-/low versus CD24+/high MCF10AT1 indicates that the top activated upstream regulators include TWIST1, TGFβ1, OCT4, and other factors known to be increased in BCSCs and during EMT. The top inhibited upstream regulators include ESR1, TP63, and FAS. Consistent with our results, many genes previously demonstrated to be regulated by RUNX factors are altered in BCSCs. The RUNX2 interaction network is the top significant pathway altered between CD24-/low and CD24+/high MCF10AT1. RUNX1 is higher in expression at the RNA level than RUNX2. RUNX3 is not expressed. While, human-specific quantitative polymerase chain reaction primers demonstrate that RUNX1 and CDH1 decrease in human MCF10CA1a cells that have grown tumors within the murine mammary fat pad microenvironment, RUNX2 and VIM increase. Treatment with an inhibitor of RUNX binding to CBFβ for 5 days followed by a 7-day recovery period results in EMT suggesting that loss of RUNX1, rather than increase in RUNX2, is a driver of EMT in early stage breast cancer. Increased understanding of RUNX regulation on BCSCs and EMT will provide novel insight into therapeutic strategies to prevent recurrence.

MicroRNA-548m Suppresses Cell Migration and Invasion by Targeting Aryl Hydrocarbon Receptor in Breast Cancer Cells.

Breast cancer is the most commonly diagnosed cancer among women and one of the leading causes of cancer mortality worldwide, in which the most severe form happens when it metastasizes to other regions of the body. Metastasis is responsible for most treatment failures in advanced breast cancer. Epithelial–mesenchymal transition (EMT) plays a significant role in promoting metastatic processes in breast cancer. MicroRNAs (miRNAs) are highly conserved endogenous short noncoding RNAs that play a role in regulating a broad range of biological processes, including cancer initiation and development, by functioning as tumor promoters or tumor suppressors. Expression of miR-548m has been found in various types of cancers, but the biological function and molecular mechanisms of miR-548m in cancers have not been fully studied. Here we demonstrated the role of miR-548m in modulating EMT in the breast cancer cell lines MDA-MB-231 and MCF-7. Expression data for primary breast cancer obtained from NCBI GEO data sets showed that miR-548m expression was downregulated in breast cancer patients compared with healthy group. We hypothesize that miR-548m acts as a tumor suppressor in breast cancer. Overexpression of miR-548m in both cell lines increased E-cadherin expression and decreased the EMT-associated transcription factors SNAI1, SNAI2, ZEB1, and ZEB2, as well as MMP9 expression. Consequently, migration and invasion capabilities of both MDA-MB-231 and MCF-7 cells were significantly inhibited in miR-548m-overexpressing cells. Analysis of 1,059 putative target genes of miR-548m revealed common pathways involving both tight junction and the mTOR signaling pathway, which has potential impacts on cell migration and invasion. Furthermore, this study identified aryl hydrocarbon receptor (AHR) as a direct target of miR-548m in breast cancer cells. Taken together, our findings suggest a novel function of miR-548m in reversing the EMT of breast cancer by reducing their migratory and invasive potentials, at least in part via targeting AHR expression.

MiR-526b suppresses cell proliferation, cell invasion and epithelial-mesenchymal transition in breast cancer by targeting Twist1.

The aberrant expression of microRNAs (miRNAs) acts as crucial regulators in the tumorigenesis of breast cancer (BC). The aim of the study is to investigate the functional effects of miR-526b expression in breast cancer progression. The expression level of miR-526b in breast cancer tissues and cell lines was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Cell proliferation, migration, and invasion capacity was detected by CCK-8 cell proliferation, colony formation, and transwell invasion assays after up-regulating or down-regulating miR-526b expression in breast cancer cells. Bioinformatics analysis and Dual-Luciferase reporter gene assays were used to demonstrate that Twist1 was a target of miR-526b. Western blot analysis was also performed. We showed that miR-526b expression was significantly downregulated in breast cancer tissues compared to adjacent normal tissues. Lower miR-526b expression was associated with lymph node metastasis in breast cancer patients. Function assays showed that upregulation of miR-526b expression suppressed cell proliferation, cell colony formation, and cell invasion ability in breast cancer. Furthermore, the upregulation of miR-526b suppressed EMT makers Vimentin expression but increased the E-cadherin expression. Mechanically, we showed that miR-526b inhibited cell EMT process by targeting Twist1 expression. Thus, our evidence indicated that miR-526b may serve as a potential target of breast cancer treatment.

Abstract P6-07-06: Shugoshin I signals epithelial-mesenchymal transition and promotes cell invasion in breast cancer

Abstract Despite being the most aggressive and poorly prognostic breast cancer subtype, there are currently no biological therapies against triple-negative (TN, or ER-PR-Her2-) breast cancers. Our laboratory is finding the sources of novel biological targets in TNBC by studying the E2F transcription factors, which are essential for cellular proliferation and maintenance of genomic stability. We recently published that the E2F transcriptional activators are overexpressed in the vast majority of TNBC and can dictate the high percentage of mitotic cells in mammary tumors. We also demonstrated that that the E2Fs control multiple mitotic regulators and maintain genomic integrity through Shugoshin I (SGO1) and BubR1. SGO1 is essential for chromosome segregation by preventing premature dissociation of the cohesion complex and sister chromatids after prophase. Until recently, SGO1 research was restricted to embryogenesis; however, recent studies have shown a role for SGO1 in tumorigenesis and epithelial-mesenchymal transition (EMT), but none have been done in breast cancer. Thus, we investigated SGO1 expression and genetic alteration in open databases such as KM-Plotter and cBioPortal and found that SGO1 is highly expressed in triple-negative breast cancer subtype and that its expression correlates with poor prognostic factors. Therefore, we decided to investigate the role of SGO1 in breast cancer EMT and cell invasion. Our hypothesis is that SGO1 modulates EMT genes, thus promoting cell invasion. To test our hypothesis, we conducted siRNA transfection to knockdown SGO1 in MDA-MB-231 and Hs578t, which are TNBC cells. After 48 hours, we evaluated mRNA levels of EMT-related genes after SGO1 depletion using RT-PCR analysis. We also evaluated the effects of SGO1 depletion in protein localization by immunofluorescence analysis. Furthermore, we evaluated the invasive behavior of MDA-MB-231 and Hs578t cells after SGO1 depletion using a Boyden Chamber Assay. Our results demonstrate that SGO1 depletion signals EMT by decreasing N-cadherin and increasing E-cadherin mRNA levels. SGO1 depletion also restores protein expression of E-cadherin in MDA-MB-231 cells. Strikingly, SGO1 depletion significantly reduces invasion in MDA-MB-231 and Hs578t cells. Our results suggest that SGO1 is a promising drug target for breast cancer metastasis since EMT and invasion are essential early steps in breast cancer metastasis. Currently, we are evaluating SGO1 expression in tissue microarrays derived from breast cancer patients (including Luminal and TNBC subtypes), in order to determine the percentage of those patients expressing Sgo1, as well as to establish important correlations with surrogate markers of prognosis (EMT, proliferation, mitosis). Our results may help establish new druggable targets in order to prevent and reduce metastasis in TNBC patients. Citation Format: Shirley Jusino, Harold I. Saavedra. Shugoshin I signals epithelial-mesenchymal transition and promotes cell invasion in breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-07-06.

Long Noncoding RNA RP11-334E6.12 Promotes the Proliferation, Migration and Invasion of Breast Cancer Cells Through the EMT Pathway by Activating the STAT3 Cascade.

BackgroundRP11-334E6.12 is a dysregulated long noncoding RNA (lncRNA) that has never been studied in breast cancer. The biological function and potential mechanism of RNA RP11-334E6.12 in tumorigenesis are still unknown.MethodsWe scanned the Cancer Genome Atlas (TCGA) database and identified RP11-334E6.12 as one of the most dysregulated lncRNAs. The level of RP11-334E6.12 was assessed in breast cancer (BC) tissue samples and BC cell lines. The survival and RP11-334E6.12 expression of patients were analysed. The biological influence of RP11-334E6.12 on BC cell lines was studied using proliferation, Transwell migration, and invasion assays.ResultsRP11-334E6.12 was upregulated in both the TCGA database and our own database. Moreover, survival analyses indicated that RP11-334E6.12 was related to poor overall survival. Moreover, RP11-334E6.12 promoted the proliferation, migration and invasion of BC cells. RP11-334E6.12 promotes the epithelial mesenchymal transition of BC by activating the STAT3 pathway.ConclusionTaken together, our results demonstrate that RP11-334E6.12 is associated with the progression of breast cancer. Our findings indicate that long noncoding RNA RP11-334E6.12 promotes the proliferation, migration and invasion of breast cancer cells by activating the STAT3 pathway.

H19/let‑7/Lin28 ceRNA network mediates autophagy inhibiting epithelial‑mesenchymal transition in breast cancer.

Long non‑coding RNA (lncRNA) H19 and Lin28protein have been shown to participate in various pathophysiological processes, including cellular proliferation, autophagy and epithelial‑mesenchymal transition (EMT). A number of studies have investigated lncRNAs, microRNAs and mRNAs, and their roles in the initiation and progression of cancer, in doing so identifying competitive endogenous RNA (ceRNA) networks, including the H19/let‑7/Lin28 network. However, whether the H19/let‑7/Lin28 ceRNA network is involved in autophagy and EMT in breast cancer (BC) remains unclear. The present study demonstrated that the H19/let‑7/Lin28 loop was required for the downregulation of autophagy in BCcells via western blot analysis, reverse transcription‑quantitative PCR and autophagy flux monitoring. Using wound healing, migration and invasion assays, and morphological assays, the H19/let‑7/Lin28 loop was revealed to promote EMT in BCcells. Moreover, the H19/let‑7/Lin28 network was found to contribute to autophagy by inhibiting EMT in BCcells. To the best of our knowledge, the present study is the first to suggest the important roles of the H19/let‑7/Lin28 ceRNA network in BC autophagy and EMT, thus providing insight for the use of these molecules as prognostic biomarkers and therapeutic targets in BC metastasis.

Effect of aloin on some gene biomarkers for epithelial-mesenchymal transition in breast cancer in vitro compared to an anthracycline analog (Doxorubicin)

Effect of aloin on some gene biomarkers for epithelial-mesenchymal transition in breast cancer in vitro compared to an anthracycline analog (Doxorubicin)

WSZG inhibits BMSC-induced EMT and bone metastasis in breast cancer by regulating TGF-β1/Smads signaling

Bone metastasis of breast cancer causes severe skeletal-related events and poor prognosis. Wensheng Zhuanggu Formula (WSZG), a traditional Chinese prescription, is used to adjunctively treat breast cancer bone metastases in clinical practice. This study was undertaken to investigate the antibone-metastatic activities and mechanisms of WSZG extract by evaluating the effect of this formula on the cross-talk between bone marrow-derived mesenchymal stem cells (BMSCs) and breast cancer cells in triggering epithelial-mesenchymal transition (EMT) in vivo and in vitro. The results demonstrated that BMSCs might enhance the invasive and metastatic potentials of breast cancer cells as a consequence of EMT induction through direct cell-to-cell contact. WSZG treatment remarkably suppressed motility, invasion, EMT-related gene, and protein markers in BMSC-conditioned breast cancer cells and ameliorated bone metastases and damages in nude mice following co-injection of BMSCs and MDA-MB-231BO breast cancer cells. Further investigation showed that the transforming growth factor-β1 (TGF-β1)/Smads pathway was an important mechanism enabling BMSCs to induce EMT occurrence of breast cancer cells. WSZG treatment reversed BMSC-induced EMT by downregulating TGF-β1/Smads signaling. Thus, WSZG extracts may be regarded as a potential antibone-metastatic agent for breast cancer therapy.

Rhizoma Pleionis inhibits of breast cancer epithelial-mesenchymal transition by VEGF-A related angiogenesis

Objective To investigate the Rhizoma Pleionis mediating vascular endothelial growth factor A (VEGF-A)-related angiogenesis in breast cancer cells to inhibit epithelial-mesenchymal transition (EMT) in breast cancer. Methods Firstly, the proliferation activity of MDA-MB-231 and 4T1 of breast cancer cells was detected by methyl thiazolyl tetrazolium (MTT) method at different concentrations (0.2 g/ml, 0.4 g/ml, 0.6 g/ml, 0.8 g/ml, 1.0 g/ml); secondly, the specific concentration of Rhizoma Pleionis was selected and tested by Transwell test for its effect on breast cancer cell metastasis and invasion. The formation of human umbilical vein endothelial cells (HUVEC) capillary wall was measured to detect the effect of Rhizoma Pleionis on cell angiogenesis. The EMT-related protein and angiogenesis-related molecules were detected by Western blot. Results MTT results showed that Rhizoma Pleionis of 0.8 g/ml and 1.0 g/ml can inhibit the MDA-MB-231 and 4T1 cells proliferation in a dose-dependent manner, with statistically significant difference (P<0.05). Rhizoma Pleionis can inhibit the invasion of breast cancer cells and inhibit the formation of blood vessel walls in HUVECs. Western blot confirmed that the expression level of EMT-related proteins: E-cadherin was significantly increased and the expression level of N-cadherin, Vimentin and Snail was significantly decreased after treatment with Rhizoma Pleionis (P<0.05); and the level of the related molecule hypoxia inducible factor-1α (HIF-1α), VEGF-A and vascular endothelial growth factor receptor-2 (VEGFR-2) was significantly reduced (P<0.05). Conclusions Rhizoma Pleionis inhibits the epithelial-mesenchymal transition of breast cancer by influencing the angiogenesis of VEGF-A, and inhibits the invasion and metastasis of breast cancer. Key words: CREMASTRA VARIABILIS; Vascular endothelial growth factor A; Breast neoplasms; Tumor cells, cultured; Epithelial-mesenchymal transition

32P - Delineating the mechanisms of alpha 1-3 fucosyltransferase FUT11 in ovarian cancer

BackgroundOvarian cancer is the most lethal gynecological cancer and the biological mechanisms remains unclear. The prevalence of abnormal fucosylation has been reported to be closely related to cancer progression and prognosis. It was reported that only FUT3 and/or FUT6 in the α 1-3/4 fucosyltransferase subfamily were required for TGF-β-mediated epithelial-to-mesenchymal transition (EMT) in colorectal cancer. However, it was also reported that the α 1-6 fucosyltransferase FUT8 facilitated TGF-β binding to receptor and stimulated the EMT in breast cancer. As an α 1-3 fucosyltransferase distinct from the α 1-3/4 and 1-6 fucosyltransferase subfamily, the role of FUT11 in ovarian cancer development is less understood. MethodsFUT11 was identified as a novel oncogene related with the EMT process in ovarian cancer by bioinformatic analysis and real-time PCR. The transwell chamber assays were used to examine the changes of the invasion and migration ability after FUT11 knocked-down by siRNA treatment in ovarian cancer cells SK-OV-3 and HEY. Western-blot was usd to determine the changes of EMT genes by FUT11 depletion in response to TGF-β 1. Furthermore, expression and location of R-Smads and Co-Smad were analyzed by western blot and immunofluorescence assay, to evaluate the impact of FUT11 depletion on the TGF-β/Smad pathway. ResultsFUT11 was highly expressed in ovarian cancer, and increased FUT11 expression was significantly correlated with poor survival of the ovarian patients. FUT11 depletion impaired the invasion and migration of the ovarian cancer cell lines, through regulation of the TGF-β 1-induced EMT process. These inhibitory effect was due to downregulation of the expression of SMAD2 and SMAD3, as well as prevention of their translocation from cytoplasm to nucleus. Although expression of SMAD4 were slightly affected, their translocation from cytoplasm to nucleus were inhibited by FUT11 depletion. ConclusionsFUT11 promotes invasion and migration of ovarian cancer through regulation of the TGF-β 1-induced EMT process. FUT11 affects the response to TGF-β 1 by regulation of the expression level of R-Smads and Co-Smad, as well as their translocation from cytoplasm to nucleus. Legal entity responsible for the studyBeijing Obstetrics and Gynecology Hospital, Capital Medical University. FundingThe National Natural Science Foundation of China (81502353 & 81672838), Beijing Municipal Science & Technology Commission (No. Z181100001718193), and Beijing Obstetrics and Gynecology Hospital, Capital Medical University (FCYY201713). DisclosureAll authors have declared no conflicts of interest.

Abstract 1881: Role of E2F activators and mitotic regulators controlled by E2Fs in EMT, invasion, and metastasis of breast cancer

Abstract The E2 Promoter Binding Factors (E2Fs) are a group of eight transcription factors that are highly deregulated in breast cancer and control several cellular processes, including proliferation and centrosome duplication. Broadly, E2Fs can be divided into transcriptional activators and repressors based on their role in gene transcription. In addition to their traditional cell cycle functions, it is known that deregulated Rb/E2F pathway signals the epithelial-to-mesenchymal transition (EMT); yet the underlining mechanism of how E2Fs drive EMT in breast cancer remains unknown. Published data from our laboratory demonstrates that overexpression of activating E2Fs leads to increased expression of mitotic regulators such as BUBR1, MPS1 (TTK), and SGO1, among others. Furthermore, published data from our laboratory shows that TTK promotes mesenchymal signaling through several mechanisms in breast cancer. Hence, we are investigating to what extent deregulation of E2F activators promotes EMT, invasion, and metastasis through the remaining mitotic regulators. Our hypothesis is that the overexpression of E2F activators contributes to tumorigenesis by promoting EMT through the induction of the transcription of specific mitotic regulators, thus leading to increased invasion and metastasis. To test our hypothesis, E2Fs (1, 2 and 3a) were knockdown individually or in combination of two or three E2F activators simultaneously using specific siRNAs sequences in MDA-MB-231 cells. After 48 hours, western blot analysis was performed to confirm the knockdown and to evaluate the effects of knockdown of these E2Fs on protein levels of mitotic regulators and EMT markers. Real-time PCR was performed to evaluate EMT mRNA expression levels. Additionally, a wound healing assay was conducted to evaluate cell migration, as an indicator of metastasis. All experiments were performed in triplicates and a non-parametric Mann Whitney test was used to evaluate statistical significance. Our results show that knockdown of E2F1 and the combination of all activating E2Fs decreased the expression of mitotic regulators and EMT proteins. Likewise, knockdown of E2Fs decreased the expression of certain EMT genes (but not all) and knockdown of E2F1 decreased cell migration. These results suggest that E2F1 has a prominent role in breast cancer EMT and cell migration. Currently, we are exploring the effects of knockdown of E2F-controlled mitotic regulators in breast cancer EMT and cell migration. Citation Format: Shirley Jusino, Melanie J. Hidalgo-Vargas, Jaya Padmanabhan, Srikumar P. Chellapppan, Harold I. Saavedra. Role of E2F activators and mitotic regulators controlled by E2Fs in EMT, invasion, and metastasis of breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1881.

Abstract 1882: Molecular context dictates the effects of eribulin on key EMT regulators: Snail and Slug

Abstract Microtubule targeting agents (MTAs) are a mainstay in the treatment of breast cancer and a growing body of evidence demonstrates that they have non-mitotic effects that contribute to their anticancer actions. Even after decades of clinical use, there is much to be learned about the mechanisms of action these drugs, and differences among them, for optimal utility. MTAs rapidly alter microtubule dynamics, often within minutes, leading to significant changes in oncogenic cellular signaling. We evaluated the early effects of eribulin on key oncogenic signaling pathways following a 2 h incubation using clinically relevant concentrations and compared the effects to those initiated by other MTAs. These studies led to the identification of novel mechanisms by which MTAs disrupt oncogenic signaling and contribute to the reversal of epithelial to mesenchymal transition (EMT), including unanticipated differences among drugs. The TGF-β-mediated expression of the Snail transcription factor is a key driver pathway of EMT in breast cancer. The effects of eribulin and other MTAs on TGF-β-induced, Smad-dependent expression of Snail were evaluated. Our results show that eribulin and vinorelbine inhibit the ability of TGF-β to promote the transcriptional induction of Snail by impeding the nuclear transport of Smad2/3 proteins in 4 triple-negative breast cancer cell lines. The effects of microtubule destabilizers contrasted with those of paclitaxel, which had no effect on the expression of Snail. This study begins to explain how microtubule disruption might contribute to the eribulin-mediated EMT reversal. Slug is another member of the Snail family of transcription factors that plays a central role in breast cancer EMT. Although Snail and Slug are often grouped together due to functional similarities, it is now understood that they regulate non-overlapping sets of genes. In contrast to our findings with Snail, eribulin and vinorelbine, but not paclitaxel or ixabepilone, induced Slug over-expression in a subset of breast cancer cell lines. We have identified molecular contexts where eribulin and vinorelbine induced Slug expression, resulting in the identification of a potential biomarker for response to microtubule destabilization. This work highlights the multifaceted nature of MTA-mediated effects on EMT-associated signaling pathways in breast cancer cells. These studies are supported by Eisai Inc. Citation Format: Roma Kaul, April L. Risinger, Susan L. Mooberry. Molecular context dictates the effects of eribulin on key EMT regulators: Snail and Slug [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1882.

Abstract 2602: AKT1-mediated activation of HSF1 by phosphorylation and an association with metastasis-free survival

Abstract Breast cancer is the most common cancer affecting women worldwide. The overwhelming majority of deaths from breast cancer are caused by metastasis. The transcription factor heat shock factor (HSF1), which classically regulates the heat shock response, has been linked to tumor progression and metastasis. Our previous data indicate HSF1 is activated in approximately 50% of patient breast tumors, independent of subtype and is associated with promoting epithelial-to-mesenchymal transition (EMT). Our laboratory has shown that AKT1 phosphorylates Ser326 of HSF1, leading to HSF1 activation in breast cancer cells and EMT. However, it remains unclear what phosphorylation sites are most critical for HSF1 transcriptional activity and whether AKT1 also regulates other HSF1 sites. To answer this question, HSF1 was subjected to an in vitro kinase assay. We observed Ser326 phosphorylation via immunoblotting. These phosphorylated HSF1 proteins were further subjected to mass spectrometry wherein we identified three novel sites of AKT1-mediated phosphorylation in Thr142, Ser230, and Thr527. All of these sites, along with Ser326, have previously been shown to promote HSF1 transcriptional activity. Interestingly, incubation with a pan-AKT inhibitor also suppressed the heat shock response by HSF1. However, when HSF1 protein was incubated with AKT2 or AKT3, there was no phosphorylation at Ser326. Furthermore, transcriptional activity of HSF1 and expression of Hsp70, a known HSF1 target gene, were increased by AKT1 but not by AKT2 or AKT3. Thus, we have further identified that AKT1 phosphorylates several activating residues on the HSF1 protein whereas AKT2 and AKT3 do not phosphorylate HSF1 to promote its activation. The importance of promoting HSF1 transcriptional activity by AKT1 cannot be understated as the PI3K pathway has been found to be genetically activated in ~77% of breast cancer. Additionally, it has recently been reported that this pathway is activated in almost 40% of all human tumors. However, whether HSF1 activity has any relation to EMT or metastasis in these other tumor types is unknown. To address this, we developed a gene expression signature for HSF1. We found this gene signature was strongly associated with metastasis-free survival in a broad range of solid tumors including breast cancer, lung cancer, ovarian cancer, melanoma, pancreatic cancer, and prostate cancer. In summary, we established a more definitive mechanism by which AKT1 phosphorylates and activates the HSF1 protein by phosphorylation of several activating sites. Furthermore, due to the seemingly ubiquitous nature of PI3K/AKT signaling across human tumors, we found that HSF1 is also potentially activated in these tumor types and has a strong association with patient outcomes, in particular metastasis. Citation Format: Wen-Cheng Lu, Haimanti Ray, Ramsey Omari, Sha Cao, Richard Carpenter. AKT1-mediated activation of HSF1 by phosphorylation and an association with metastasis-free survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2602.