Reversal Of Epithelial-mesenchymal Transition Research Articles

Abstract 1596: Induction of mesenchymal-to-epithelial transition through pan-MEK inhibition in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) presents a clinical challenge due to the aggressive nature of the disease and a lack of targeted therapies. Constitutive activation of the MAPK/extracellular signal-regulated kinases (MEK) pathways has been linked to chemoresistance and metastatic progression through distinct mechanisms, including activation of epithelial-to-mesenchymal transition (EMT). Here we proposed to investigate dual inhibition of MEK1/2 and MEK5 as a more efficacious method for intervention to target mesenchymal and highly metastatic breast cancer cells than MEK1/2 or MEK5 alone through the use of a novel pan-MEK inhibitor SC-151. Interestingly, TNBC cells demonstrated a change in cell morphology indicative of mesenchymal-to-epithelial transition (MET) and exhibited a significant decrease in migration potential following pan-MEK inhibition. Additionally, immuno-compromised mice inoculated with MDA-MB-231 cells and treated with SC-151 demonstrated decreased tumor volumes compared to vehicle-treated animals. To parse the roles of MEK1/2 and MEK5 in EMT and tumorigenesis, we used the CRISPR/Cas9 approach to knock out ERK5 expression in the TNBC cell line MDA-MB-231. Similar to biological changes induced by pan-MEK inhibition, loss of ERK5 promoted epithelial characteristics in TNBC cells at the morphological and molecular level and impaired tumor formation in vivo. Treatment of ERK5-ko cells with SC-151 further enhanced these effects in vitro, suggesting that MEK1/2 and MEK5 play distinct roles in maintaining the mesenchymal phenotype. Further analysis revealed that constitutive activation of MEK5 abrogated the effects of SC-151 on the reversal of EMT, highlighting the requirement for MEK5 inhibition in MET induction. Taken together, these findings show that while the MEK5-ERK5 pathway may be sufficient in EMT regulation, MEK1/2 signaling further sustains the mesenchymal state of TNBC cells. Thus, dual MEK inhibition exerts optimal effects in the reversal of EMT. These data present a novel compound and viable therapeutic strategy to target both MEK1/2 and MEK5 in phenotypically mesenchymal and clinically aggressive breast cancer cells, warranting further investigation into mechanisms by which MEK1/2 and MEK5 individually modulate the EMT axis. Additionally, as MEK inhibition has been shown to sensitize resistant cancer cells to targeted therapies, synergistic and sensitizing effects of SC-151 combined with inhibitors of alternative signaling pathways as well as kinases upstream of MEK will be examined. Citation Format: Van T. Hoang, Steven Elliott, Elizabeth C. Martin, Lyndsay V. Rhodes, Hope E. Burks, Margarite Matossian, Suravi Chakrabarty, Darlene Monlish, Theresa B. Phamduy, Lowry Curley, Muralidharan Anbalagan, Brian G. Rowan, Doug Chrisey, Jane E. Cavanaugh, Patrick T. Flaherty, Bridgette M. Collins-Burow, Matthew E. Burow. Induction of mesenchymal-to-epithelial transition through pan-MEK inhibition in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1596.

Abstract 1680: Targeting subcellular localization of Kaiso to limit dissemination through MAPK signaling

Abstract Breast cancer is the second most commonly diagnosed cancer among American women. The American Cancer Society has estimated over 230,000 new cases of breast cancer, and over 40,000 deaths for the year 2015. Unfortunately, metastasis is the major contributor for the significant number of these deaths. The Epithelial-Mesenchymal Transition (EMT) is promoted by the Master Regulator known as Kaiso, a transcriptional repressor that is a member of the BTB/POZ family of zinc finger transcription factors. We have previously demonstrated that Epidermal Growth Factor Receptor (EGFR) signaling shuttles Kaiso into the nucleus, although, its mechanism is still not understood. EGFR signaling activates multiple downstream signaling cascades including the MAP Kinase cascade, which has a significant influence on proliferation, cell adhesion, gene expression, and cell survival. Mesenchymal cells demonstrate high levels of MAPK activity and disruption or inhibition of the cascade has proven to be a method for decreasing tumor growth. Therefore we hypothesize that subcellular localization and expression of Kaiso is modulated by EGFR induced MAPK activity. Using the TCGA Breast cancer dataset, we observed that Kaiso was altered in about 11% of patients, compared to 15% of E-cad expression and 13% of MAPK3 of 959 patients/cases of Breast cancers. MAPK3 (eRK1); MAPK6, MAPK7, MAPK9 AND MAPK14 (p38) seems to have trends in mutual exclusivity or co-occurrence with Kaiso (ZBTB33). To determine if MAPK3 is responsible for increased Kaiso expression, MCF-7 cells, which express low levels of Kaiso in the cytoplasm, were treated with 10ng/ml of EGF in the presence or absence of MAPK inhibitor, 20μM PD98093. EGF stimulation caused robust increase in Kaiso protein expression, while PD98093 inhibited EGF induced expression. Similarly, in Kaiso overexpressing, MDA-MB-231, blocking EGFR kinase activity with 500nM PD153035 resulted in an opposite effect as EGF stimulation in MCF-7 cells. Furthermore, we observed a significant decrease in p-ERK expression, re-expression of E-cadherin, and low EGFR expression levels in sh-Kaiso MDA-MB-231 cells compared to sh-Scr cells. The decreased p-ERK expression in sh-Kaiso MDA-MB-231 cells was also associated with decreased expression of mesenchymal markers ZEB1, ZEB2, Twist and SNAIL at both the mRNA and protein levels compared to the scrambled control. Similarly, we observed significant decrease in the somatic cell reprogramming factors Klf4, Oct4, Nanog expression in sh-Kaiso MDA-MB-231 as well, which coincide with a reversal of EMT. To our knowledge, this is the first study to investigate the molecular mechanism and/or functional consequences of increased Kaiso expression in breast cancer, and highlights the multiple roles that Kaiso plays in promoting aggressive disease. Citation Format: Shweta Tripathi, Balasubramanyam Karanam, Bo Ma, Alan Wells, Clayton Yates. Targeting subcellular localization of Kaiso to limit dissemination through MAPK signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1680.

Abstract 344: Antimitotic effect and complex of nonmitotic effect on tumor biology of eribulin mesilate in soft tissue sarcoma model

Abstract Background and objectives: Eribulin mesilate (Eribulin), a first-in-class halichondrin B-based microtubule dynamics inhibitor, has been shown to have novel mechanism of actions (reversion of epithelial-mesenchymal transition of tumor cell and promotion of vascular remodeling) beyond its established antimitotic activity in breast cancer models, leading to decrease of tumor metastatic potency. Recently, eribulin showed trends towards greater overall survival compared to Dacarbazine in subjects with leiomyosarcoma and liposarcoma patients in phase 3 trial. The objective of this study was to investigate the effect of eribulin on tumor cell proliferation of soft tissue sarcoma (STS). Furthermore, the effects of eribulin on morphology and gene expression of differentiation markers against STS and tumor blood perfusion in a leiomyosarcoma xenograft model were examined. Methods: Anti-proliferation activity of eribulin was examined in 6 human STS cell lines. Anti-tumor activity of eribulin was examined in xenografts of A-673 (Ewing's sarcoma), SK-LMS-1 (leiomyosarcoma), SW 872 (liposarcoma), and HT-1080 (fibrosarcoma) at several doses. To analyze the effect of eribulin on in vitro morphological change, 3 human STS cell lines were treated with eribulin at 1, 3, and 10 nmol/L for 7 days. Gene expression change was analyzed by microarray and differentiation marker genes in 2 cell lines were confirmed by qPCR analysis. For tumor blood perfusion analysis, eribulin was administered at doses of 0.38, 0.75, and 1.5 mg/kg one shot in SK-LMS-1 xenografts, and tumor blood perfusion was calculated as Hoechst-positive areas in tumor cryosections 5 days after the administration. Results: Eribulin showed anti-proliferation activity in vitro against all 6 cell lines in a dose dependent manner with 50% inhibitory concentration values of around 1 nmol/L. Eribulin showed significant antitumor activity against 4 xenografts in a dose dependent manner. Eribulin treatment for 7 days induced apparent morphological change in 3 cell lines. Of these, expression level of smooth muscle differentiation marker gene CNN1 was 7.1-fold upregulated in SK-UT-1 and adipocyte differentiation marker genes MYLK, C/EBPβ, and KIF23 were 1.9-, 1.4-, and 1.3-fold upregulated in SW 872, respectively, by 10 nmol/L treatment compared to vehicle control. In addition, eribulin significantly enhanced tumor blood perfusion compared to vehicle control 5 days after the administration in SK-LMS-1 xenografts at all doses.Conclusions: Eribulin showed anti-proliferation activity against all of STS cell lines we tested in vitro and in vivo. Furthermore, eribulin might cause differentiation of STS cells and remodeling of tumor vasculature to enhance tumor blood perfusion. These data supported that eribulin showed mitotic effect and non-mitotic effect, which may decrease tumor metastatic potency in STS, as well in breast cancer. Citation Format: Satoshi Kawano, Makoto Asano, Yusuke Adachi, Junji Matsui. Antimitotic effect and complex of nonmitotic effect on tumor biology of eribulin mesilate in soft tissue sarcoma model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 344.

Abstract 684: Targeting an unconventional kinase-invasion axis in breast cancer metastasis

Abstract Triple negative breast cancer (TNBC) is associated with poor survival, metastatic recurrence, increased mortality, and has few viable therapeutic options. Given the high mortality associated with metastasis of this disease, discovery of therapeutics targeting the metastasis axis is imperative. Kinases have been demonstrated to have an essential role in regulation of epithelial-mesenchymal transition (EMT), a process involved in the initiation of cancer metastasis. Elucidating specific signaling pathways that promote EMT has the potential to provide novel targeting strategies in the treatment of TNBC. Here we demonstrate a partially non-selective polo-like kinase 1 (PLK1) inhibitor, GSK346294, is capable of EMT reversal in phenotypically mesenchymal TNBC cell lines, as evidenced by enhanced E-cadherin expression and loss of cellular migration potential. Structurally analogous but highly specific PLK inhibitors did not exhibit similar EMT changes, which led us to investigate off-target kinases inhibited by GSK346294. Follow-up kinase profiling studies revealed members of the NEver-in-mitosis-related Kinase family (NEK5 and NEK9) as additional kinases inhibited by GSK346294, leading us to investigate their individual roles in the progression of EMT. NEK5 overexpression in a non-invasive breast cancer cell line increased both migration and invasion in trans-well assays. Additionally, enhanced NEK5 expression altered the TNBC gene expression profile, enhancing expression of genes associated with growth factor signaling (MET, EGFR) and the mesenchymal gene signature (SLUG, VIM). To date, no studies exist which delineate the regulatory roles of NEK5 and NEK9 in EMT or cellular invasion/motility. Further investigation into the function of NEK5 and/or NEK9 in the metastatic progression may provide novel therapeutic targets for the treatment of TNBC, the implication of which could impact the treatment and management of neoplastic disease and metastasis beyond breast cancer. Citation Format: Margarite D. Matossian, Steven Elliott, Van T. Hoang, Hope E. Burks, Theresa B. Phamudy, Doug Chrisey, William J. Zuercher, David H. Drewry, Carrow Wells, Bridgette Collins-Burow, Matthew E. Burow. Targeting an unconventional kinase-invasion axis in breast cancer metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 684.

Abstract 1613: A novel live imaging system for inflammation-induced epithelial-mesenchymal transition in colorectal cancers

Abstract Background: Epithelial-mesenchymal transition (EMT) is a biological process, by which epithelial cancer cells acquire the mesenchymal phenotype with malignant properties for invasion and metastasis, leading to poor prognosis. However, as EMT is a reversible process and depends on tumor microenvironment, the precise role of EMT in cancer progression remains unclear. Inflammatory microenvironment has been shown to be responsible for the development and progression of colorectal cancers. To evaluate the implication of EMT in the inflammation-mediated colorectal cancer progression, a live imaging system for EMT is needed on the in vitro and in vivo experiments. In this study, we generated the EMT-detectable colorectal cancer cells by introducing mesenchymal cell marker promoter-driven fluorescence protein expression vector, and investigated whether inflammation-induced EMT is detectable in vitro. Methods: To generate the EMT-detectable colorectal cancer cells HCT116-VIM635, human colorectal cancer cell line HCT116 was stably transfected with vimentin promoter-driven red fluorescence protein TurboFP635 expression vector. EMT was induced in HCT116-VIM635 cells by treatment with inflammatory cytokines, IL-1β (1 ng/ml) and TNF-α (20 ng/ml), or by co-culture with mouse macrophage cell line RAW264.7 in the presence of lipopolysaccharide (LPS) (200 ng/ml). The time-lapse live imaging was observed by confocal laser scanning microscope. Migration and invasion properties were examined by transwell chamber assays. The fluorescence intensity was measured by microplate reader and flow cytometric analysis. The expression of EMT-related markers was assessed by Western blot analysis. Results: Administration of IL-1β or TNF-α induced the TurboFP635 expression in consistent with morphological change like mesenchymal phenotype in HCT116-VIM635 cells. Removal of these inflammatory cytokines attenuated the TurboFP635 expression and morphological change in HCT116-VIM635 cells, suggesting the detection of reverse EMT process in these cells. Inflammatory cytokines also induced the migration and invasion properties and the expression of EMT-related markers in HCT116-VIM635 cells. Moreover, co-culture with RAW264.7 cells stimulated with LPS also induced the TurboFP635 expression and morphological change as well as inflammatory cytokines in HCT116-VIM635 cells. Conclusions: Our results suggest that this unique live imaging system for EMT has a great potential to detect reversible EMT process during inflammation-induced cancer progression. This system would be useful strategy to assess the role of EMT during inflammation-mediated cancer progression in vivo. Citation Format: Takeshi Ieda, Hiroshi Tazawa, Satoru Kikuchi, Shinji Kuroda, Toshiaki Ohara, Kazuhiro Noma, Hiroyuki Kishimoto, Takeshi Nagasaka, Masahiko Nishizaki, Shunsuke Kagawa, Toshiyoshi Fujiwara. A novel live imaging system for inflammation-induced epithelial-mesenchymal transition in colorectal cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1613.

TIM-3 promotes the metastasis of esophageal squamous cell carcinoma by targeting epithelial-mesenchymal transition via the Akt/GSK-3β/Snail signaling pathway.

T-cell immunoglobulin and mucin domain-con-taining protein-3(TIM-3), a negative regulator of antitumor immune response, has been demonstrated to be involved in the onset and progression of several types of malignancies. The present study aimed to determine whether and how TIM‑3 plays such a role in esophageal squamous cell carcinoma(ESCC). TIM-3 expression was analyzed by immunohistochemistry and real‑time fluorescence quantitative PCR(qRT‑PCR) in ESCC and matched adjacent normal tissues. Functional experimentsinvitro were performed to elucidate the effect of TIM‑3 knockdown on the proliferation, apoptosis, migration, invasion and epithelial-mesenchymal transition(EMT) in Eca109 and TE‑1 cell lines. Our data revealed that TIM‑3 expression was significantly elevated at both the mRNA and protein levels in ESCC tissues compared with the levels in the matched adjacent normal tissues (both P<0.001). TIM‑3 expression was significantly associated with lymph node metastasis (P=0.008), tumor‑node‑metastasis(TNM) stage (P=0.042) and depth of tumor invasion (P=0.042). In addition, we observed a strong correlation between high TIM‑3 expression and a worse overall survival of ESCC patients (P=0.001). Functional study demonstrated that TIM‑3 knockdown markedly inhibited proliferation, migration and invasion of ESCC cell lines without affecting apoptosis. In addition, TIM‑3 depletion was associated with downregulation of matrix metalloproteinase(MMP)-9 and upregulation of tissue inhibitor of metalloproteinase(TIMP)-1, and with reversion of EMT, as reflected by higher levels of the epithelial marker E‑cadherin and lower levels of the mesenchymal markers N‑cadherin and vimentin. Further study found that TIM‑3 depletion suppressed the signaling pathway involving p‑Akt, p‑GSK‑3β and Snail. Taken together, these results suggest that TIM‑3 is a novel therapeutic target and prognostic biomarker for ESCC and promotes metastasis of ESCC by inducing EMT via, at least partially, the Akt/GSK-3β/Snail signaling pathway.

γ-Tocotrienol reversal of epithelial-to-mesenchymal transition in human breast cancer cells is associated with inhibition of canonical Wnt signalling.

Frizzled-7 (FZD7) receptor-dependent activation of the canonical Wnt/β-catenin pathway plays a crucial role in epithelial-to-mesenchymal transition (EMT) and breast cancer metastasis. FZD7 and its co-receptor, low-density lipoprotein receptor-related protein 6 (LRP6), are highly expressed in MDA-MB-231 and T-47D breast cancer cells, and endogenous ligands for FZD7 include Wnt3a and Wnt5a/b. γ-Tocotrienol, a natural isoform of vitamin E, inhibits human breast cancer cell proliferation and EMT. Here, studies have been conducted to investigate the role of the canonical Wnt pathway in mediating inhibitory effects of γ-tocotrienol on EMT in human breast cancer cells. MDA-MB-231, T-47D and MCF-10A cells were maintained in serum-free defined media containing selected doses of γ-tocotrienol. Cell viability was determined using the MTT colorimetric assay, Western blot analysis was used to measure protein expression and the wound-healing assay was employed to study cell mobility and migration. Immunohistochemical fluorescence staining visualized expression and localization of EMT cell markers. γ-Tocotrienol was found to induce dose-responsive inhibition of MDA-MB-231 and T-47D cell growth at doses that had no effect on immortalized normal MCF-10A mammary epithelial cells. These growth inhibitory effects were associated with suppression in canonical Wnt signalling, reversal of EMT and significant reduction in breast cancer cell motility. γ-Tocotrienol suppression of metastatic breast cancer cell proliferation and EMT was associated with suppression of the canonical Wnt/β-catenin signalling pathway.

Giant obscurins regulate the PI3K cascade in breast epithelial cells via direct binding to the PI3K/p85 regulatory subunit.

Obscurins are a family of giant cytoskeletal proteins, originally identified in striated muscles where they have structural and regulatory roles. We recently showed that obscurins are abundantly expressed in normal breast epithelial cells where they play tumor and metastasis suppressing roles, but are nearly lost from advanced stage breast cancer biopsies. Consistent with this, loss of giant obscurins from breast epithelial cells results in enhanced survival and growth, epithelial to mesenchymal transition (EMT), and increased cell migration and invasion in vitro and in vivo. In the current study, we demonstrate that loss of giant obscurins from breast epithelial cells is associated with significantly increased phosphorylation and subsequent activation of the PI3K signaling cascade, including activation of AKT, a key regulator of tumorigenesis and metastasis. Pharmacological and molecular inhibition of the PI3K pathway in obscurin-depleted breast epithelial cells results in reversal of EMT, (re)formation of cell-cell junctions, diminished mammosphere formation, and decreased cell migration and invasion. Co-immunoprecipitation, pull-down, and surface plasmon resonance assays revealed that obscurins are in a complex with the PI3K/p85 regulatory subunit, and that their association is direct and mediated by the obscurin-PH domain and the PI3K/p85-SH3 domain with a KD of ∼50 nM. We therefore postulate that giant obscurins act upstream of the PI3K cascade in normal breast epithelial cells, regulating its activation through binding to the PI3K/p85 regulatory subunit.

Epithelial-to-Mesenchymal Transition in Paclitaxel-Resistant Ovarian Cancer Cells Is Downregulated by Luteolin.

Ovarian cancer (OVCA) is the deadliest of all gynecological cancers which is attributed to late presentation, persistence, and development of chemoresistance. The objectives were to evaluate the association between OVCA paclitaxel-resistance and epithelial-to-mesenchymal transition (EMT) and to determine the capability of luteolin to chemosensitize OVCA cells. X10 and X22 cells were 11.8-25.3-fold and 7.8-8.6-fold resistant to paclitaxel than 1AP cells. X10 and X22 cells exhibited a mesenchymal phenotype, while 1AP has an epithelial characteristics. Furthermore, the expression of the epithelial marker E-cadherin was downregulated, while mesenchymal markers Vimentin and N-cadherin were upregulated in X10 and X22 cells when compared to 1AP cells. Transcription factors Snail, Slug, and Twist1 were upregulated in X10 cells, while Twist1 was highly expressed in X22 cells. Luteolin treatment caused cytotoxicity being most potent to X10 OVCA cells. Treatment of non-cytotoxic dose of luteolin at 15.625 μM chemosensitized X10 and X22 OVCA cells to paclitaxel as evidenced by reduced ED50 values from 11.8 to 0.2 μM and 8.6 to 3.6 μM for X10 and X22 cells, respectively. Moreover, luteolin treatment led to a more epithelial phenotype of X10 and X22 cells and modification of EMT markers indicating reversal of EMT. The mechanism involved is through reduction of phosphorylation of FAK and ERK leading to reduced nuclear translocation of p65. Our results highlight the significance of EMT in OVCA resistance to paclitaxel and warrant the investigation of luteolin as a potential therapeutic agent in chemoresistant OVCA. J. Cell. Physiol. 232: 391-401, 2017. © 2016 Wiley Periodicals, Inc.

Inositol induces mesenchymal-epithelial reversion in breast cancer cells through cytoskeleton rearrangement

Inositol displays multi-targeted effects on many biochemical pathways involved in epithelial-mesenchymal transition (EMT). As Akt activation is inhibited by inositol, we investigated if such effect could hamper EMT in MDA-MB-231 breast cancer cells. In cancer cells treated with pharmacological doses of inositol E-cadherin was increased, β-catenin was redistributed behind cell membrane, and metalloproteinase-9 was significantly reduced, while motility and invading capacity were severely inhibited. Those changes were associated with a significant down-regulation of PI3K/Akt activity, leading to a decrease in downstream signaling effectors: NF-kB, COX-2, and SNAI1. Inositol-mediated inhibition of PS1 leads to lowered Notch 1 release, thus contributing in decreasing SNAI1 levels. Overall, these data indicated that inositol inhibits the principal molecular pathway supporting EMT. Similar results were obtained in ZR-75, a highly metastatic breast cancer line. These findings are coupled with significant changes on cytoskeleton. Inositol slowed-down vimentin expression in cells placed behind the wound-healing edge and stabilized cortical F-actin. Moreover, lamellipodia and filopodia, two specific membrane extensions enabling cell migration and invasiveness, were no longer detectable after inositol addiction. Additionally, fascin and cofilin, two mandatory required components for F-actin assembling within cell protrusions, were highly reduced. These data suggest that inositol may induce an EMT reversion in breast cancer cells, suppressing motility and invasiveness through cytoskeleton modifications.

Thrombomodulin reduces tumorigenic and metastatic potential of lung cancer cells by up-regulation of E-cadherin and down-regulation of N-cadherin expression

Thrombomodulin (TM) is an endothelial cell membrane protein and plays critical roles in anti-thrombosis, anti-inflammation, vascular endothelial protection, and is traditionally regarded as a “vascular protection god”. In recent years, although TM has been reported to be down-regulated in a variety of malignant tumors including lung cancer, the role and mechanism of TM in lung cancer are enigmatic. In this study, we found that induction of TM overexpression by cholesterol-reducing drug atorvastatin significantly diminished the tumorigenic capability of the lung cancer cells. Moreover, we demonstrated that TM overexpression caused G0/G1 phase arrest and markedly reduced the colony forming capability of the cells. Furthermore, overexpression of TM inhibited cell migration and invasion. Consistently, depletion of TM promoted cell growth, reduced the cell population at the G0/G1 phase, and enhanced cell migratory ability. Mechanistic study revealed that TM up-regulated E-cadherin but down-regulated N-cadherin expression, resulting in reversal of epithelial-mesenchymal transition (EMT) in the lung cancer cells. Moreover, silencing TM expression led to decreased E-cadherin and increased N-cadherin. Taken together, our study suggests that TM functions as a tumor suppressive protein, providing a conceptual framework for inducing TM overexpression as a sensible strategy and approach for novel anti-lung cancer drug discovery.

Ferulic acid exerts antitumor activity and inhibits metastasis in breast cancer cells by regulating epithelial to mesenchymal transition.

Metastasis, which frequently occurs in breast cancer, is the major cause of mortality; therefore, new treatment strategies are urgently needed. Ferulic acid, isolated from Ferulafoetida, a perennial herb, has shown antineoplastic activity in various types of cancers, such as colon and lung cancer, and central nervous system tumors. However, its potential role in suppressing breast cancer metastasis has not been fully understood. In the present study, we evaluated the antitumor activity of ferulic acid in breast cancer cell line-based invitro and invivo models. We first showed that ferulic acid treatment resulted in decreased viability, increased apoptosis and suppression of metastatic potential in breast cancer cell line MDA-MB-231. Furthermore, it was demonstrated that the antitumor activity of ferulic acid and its role in suppressing metastasis were regulated by the reversal of epithelial-mesenchymal transition (EMT). Consistent with our findings invitro, the antitumor potential of ferulic acid was also verified in an MDA-MB-231 xenograft mouse model where significantly decreased tumor volume, weight and increased apoptosis were observed. Taken together, these results indicate that ferulic acid may be used as an effective therapeutic agent against breast cancer.

Autophagy links β-catenin and Smad signaling to promote epithelial-mesenchymal transition via upregulation of integrin linked kinase

TGF-β1 induces epithelial-mesenchymal transition (EMT) and autophagy in a variety of cells. However, the role of autophagy in TGF-β1-induced EMT has not been clearly elucidated and the underlying mechanisms are unclear. In the present study, we found that TGF-β1 induced both autophagy and EMT in mouse tubular epithelial C1.1 cells. Inhibition of autophagy by 3-methyladenine or siRNA knockdown of Beclin 1 reduced TGF-β1-induced increase of vimentin and decreased E-cadherin expression. In contrast, rapamycin-associated enhancement of TGF-β1-induced autophagy increased EMT of C1.1 cells. Serum rescue inhibited autophagy followed by reversal of EMT. Blocking of autophagosome-lysosomal but not proteosomal degradation reduced the decrease of E-cadherin, demonstrating a role for autophagy in degradation of E-cadherin during EMT. Autophagy promoted the activation of Src and Src-associated phosphorylation of β-catenin at Y-654 leading to pY654-β-catenin/p-Smad2 complex formation. Chromatin immunoprecipitation assay demonstrated binding by the pY654-β-catenin/p-Smad2 complex to ILK promoter thus increasing ILK expression. Taken together, our results demonstrate that TGF-β1-induced autophagy links β-catenin and Smad signaling to promote EMT in C1.1 cells through a novel pY654-β-catenin/p-Smad2/ILK pathway. The pathway delineated links disruption of E-cadherin/β-catenin-mediated cell–cell contact to induction of EMT via upregulation of ILK.

Bit1—a potential positive regulator of epithelial–mesenchymal transition in lens epithelial cells

Posterior capsule opacification (PCO) is a common postoperative complication of cataract surgery. Epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is an important initial step of PCO pathogenesis. We have previously shown that Bit1 expresses in rat LECs. In this study, we aim to investigate the role of Bit1 in the EMT of human LECs. The expression of Bit1 was firstly detected in human PCO-attached LECs and human lens cell line SRA01/04 by real-time PCR and immunofluorescence staining. The proliferation and migration of Bit1 knockdown SRA01/04 cells were evaluated by cell counting, wound-healing assay, and transwell migration assay. The EMT of LECs was triggered by TGF-β2, and then the effect of Bit1 on EMT with a key biomarker of α-smooth muscle actin (α-SMA) was analyzed by siRNA knockdown assay, and the reversal of EMT was validated by real-time PCR and western blot. Bit1 was obviously augmented in LECs derived from PCO tissues and Bit1 expressed with high levels in the cytoplasm of cultured SRA01/04 cells. Cell proliferation, invasion, and migration, as well as α-SMA expression, were significantly decreased in Bit1 knockdown SRA01/04 cells. While TGF-β2 elevated Bit1 and α-SMA expression levels in a dose-dependent manner, with peak levels at 10ng/ml of TGF-β2 treatment, suppression of Bit1 in SRA01/04 cells reversed the EMT process. TGF-β2 induced elevation of α-SMA expression level, as well as migration, and invasion abilities were all suppressed by Bit1 deficiency. These findings reveal that Bit1 promotes TGF-β2 induced α-SMA expression and acts as an positive regulator of EMT. Suppressing Bit1 inhibits the proliferation, migration, and EMT of LECs. Bit1 may be a potential novel therapeutic target for the prevention and treatment of PCO.

Abstract IA29: Identifying epithelial morphogenesis inhibitors in neural crest development and cancer

Abstract The epithelial to mesenchymal transition (EMT) is a highly conserved morphogenesis program that is essential for re-shaping and mobilizing epithelial cells during gastrulation, neural crest development and tissue regeneration. In cancer cells, EMT induction promotes acquisition of invasive cellular morphologies, stem cell-like properties and pro-survival mechanisms, which contribute to disease progression, therapy resistance and decreased overall survival. The identification of compounds that block or reverse EMT therefore represents an important therapeutic strategy to prevent cancer invasion and eradicate disseminated tumor cells. Unfortunately, current EMT inhibitors have shown limited clinical benefit, in part due to an incomplete understanding of the molecular mechanisms controlling EMT in development and cancer as well as a lack of screening platforms that recapitulate the complex physiological environment of EMT in the living animal. To overcome these obstacles, we have established a zebrafish Snail1-GFP lineage reporter strain to label dorsal neural tube progenitor cells before they undergo EMT to become neural crest, which allows us to visualize epithelial morphogenesis independent of later cell migration events. Thousands of Snail1-GFP embryos can be easily generated to perform whole animal-based screens with small molecule libraries to identify compounds that inhibit EMT in vivo. Our initial screening results using previously characterized EMT inhibitors showed that many compounds inhibited neural crest migration after EMT had occurred, but only one compound, an AXL receptor tyrosine kinase inhibitor called TP-0903, inhibited both epithelial morphogenesis and neural crest migration. TP-0903 significantly decreased twist1a expression, a canonical EMT transcription factor and blocked down-regulation of epithelial Cadherins. RNA-Seq analysis and chemical rescue experiments revealed that TP-0903 acts by inducing retinoic acid (RA) biosynthesis and triggering a RA-mediated transcriptional program. TP-0903 treatment of a number of human cell lines and mouse xenograph tumors showed that TP-0903 also inhibits EMT programs and survival in human cancer cells. These studies demonstrate the value and feasibility of using zebrafish neural crest development to identify effective EMT compounds in vivo. As such, we have identified TP-0903 as a new potential therapeutic for inhibiting EMT in cancer, and our findings support the hypothesis that RA-induced inhibition of EMT contributes to its current success in treating minimal residual disease in humans. Citation Format: Laura Jimenez, Clifford Whatcott, Jindong Wang, Steven Warner, Monique Morrison, David Bearss, Rodney A. Stewart. Identifying epithelial morphogenesis inhibitors in neural crest development and cancer. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr IA29.

Topoisomerase IIα mediates TCF-dependent epithelial-mesenchymal transition in colon cancer.

Aberrant T-cell factor (TCF) transcription is implicated in the majority of colorectal cancers (CRCs). TCF transcription induces epithelial–mesenchymal transition (EMT), promoting a tumor-initiating cell (TIC) phenotype characterized by increased proliferation, multidrug resistance (MDR), invasion and metastasis. The data presented herein characterize topoisomerase IIα (TopoIIα) as a required component of TCF transcription promoting EMT. Using chromatin immunoprecipitation (ChIP) and protein co-immunoprecipitation (co-IP) studies, we show that TopoIIα forms protein–protein interactions with β-catentin and TCF4 and interacts with Wnt response elements (WREs) and promoters of direct target genes of TCF transcription, including: MYC, vimentin, AXIN2 and LEF1. Moreover, both TopoIIα and TCF4 ChIP with the N-cadherin promoter, which is a new discovery indicating that TCF transcription may directly regulate N-cadherin expression. TopoIIα N-terminal ATP-competitive inhibitors, exemplified by the marine alkaloid neoamphimedine (neo), block TCF activity in vitro and in vivo. Neo effectively inhibits TopoIIα and TCF4 from binding WREs/promoter sites, whereas protein–protein interactions remain intact. Neo inhibition of TopoIIα-dependent TCF transcription also correlates with significant antitumor effects in vitro and in vivo, including the reversion of EMT, the loss of TIC-mediated clonogenic colony formation, and the loss of cell motility and invasion. Interestingly, non-ATP-competitive inhibitors of TopoIIα, etoposide and merbarone, were ineffective at preventing TopoIIα-dependent TCF transcription. Thus, we propose that TopoIIα participation in TCF transcription may convey a mechanism of MDR to conventional TopoIIα inhibitors. However, our results indicate that TopoIIα N-terminal ATP-binding sites remain conserved and available for drug targeting. This article defines a new strategy for targeted inhibition of TCF transcription that may lead to effective therapies for the treatment of CRC and potentially other Wnt-dependent cancers.

Drug resistance originating from a TGF-β/FGF-2-driven epithelial-to-mesenchymal transition and its reversion in human lung adenocarcinoma cell lines harboring an EGFR mutation.

Epithelial-to-mesenchymal transition (EMT) is a malignant cancer phenotype characterized by augmented invasion and metastasis, chemoresistance, and escape from host-immunity. This study sought to identify efficient methods for inducing EMT reversion, to evaluate alterations in chemosensitivity and immune-protectiveness, and to elucidate the underlying mechanisms. In this study, the human lung adenocarcinoma cell lines PC-9 and HCC-827, harboring an EGFR mutation, were treated with TGF-β and FGF-2 to induce EMT. The phenotypic alterations were evaluated by RT-PCR, fluorescent immunohistochemistry, cell-mobility, and flow cytometry. Chemosensitivity to gefitinib and cisplatin was evaluated using an MTT assay and apoptosis. Immune-protectiveness was evaluated by PD-L1 expression. A combination of TGF-β and FGF-2 efficiently induced EMT in both cell lines: through Smad3 pathway in PC-9, and through Smad3, MEK/Erk, and mTOR pathways in HCC-827. The mTOR inhibitor PP242, metformin, and DMSO reverted EMT to different extent and through different pathways, depending on the cell lines. EMT induction reduced the sensitivity to gefitinib in both cell lines and to cisplatin in HCC-827, and it increased PD-L1 expression in both cell lines. EMT reversion using each of the 3 agents partly restored chemosensitivity and suppressed PD-L1 expression. Thus, chemoresistance and increased PD-L1 expression caused by EMT can be successfully reverted by EMT-reverting agents.

Low doses of PEG-coated gold nanoparticles sensitize solid tumors to cold plasma by blocking the PI3K/AKT-driven signaling axis to suppress cellular transformation by inhibiting growth and EMT

Metastasis, the primary cause of tumor cell transformation, is often activated during cancer invasion and progression and is associated with poor therapeutic outcomes. The effects of combined treatments that included PEG-coated gold nanoparticles (GNP) and cold plasma on epithelial–mesenchymal transition (EMT) and the maintenance of cancer stem cells (CSC) have not been described so far. Here, we report that co-treatment with GNP and cold plasma inhibited proliferation in cancer cells by abolishing the activation of the PI3K/AKT signaling axis. In addition, co-treatment reversed EMT in solid tumor cells by reducing the secretion of a number of proteins, resulting in the upregulation of epithelial markers such as E-cadherin along with down-regulation of N-Cadherin, Slug and Zeb-1. The inhibition of the PI3K/AKT pathway and the reversal of EMT by co-treatment prevented tumor cells growth in solid tumors. Furthermore, we show that GNP and plasma also suppresses tumor growth by decreasing mesenchymal markers in tumor xenograft mice models. Importantly, co-treatment resulted in a substantial decrease in sphere formation and the self-renewal capacity of glioma-like stem cells. Together, these results indicate a direct link between a decrease of EMT and an increase in cell death in solid tumors following co-treatment with cold plasma and GNP.

Abstract P2-05-05: Expression profiling of in vivo DCIS progression models identified BCL9 as a molecular driver of invasive progression

Abstract Introduction: There are an estimated 60,000 new cases of ductal carcinoma in situ (DCIS) each year. At present, it is not clear why some DCIS remain non-invasive for decades while others become invasive. A lack of understanding in DCIS pathobiology has led to overtreatment of more than half of DCIS patients. To identify factors that promote DCIS invasion, we have profiled the temporal molecular changes during DCIS transition to invasive ductal carcinoma (IDC) using two in vivo models, MIND (mouse-intraductal) and DCIS/IDC tandem lesions. These studies led to the identification of B cell lymphoma-9 as a potential molecular driver of early invasion. BCL9 is a newly found co-activator of Wnt-stimulated β-catenin-mediated transcription. BCL9 has been shown to promote progression of multiple myeloma and colon carcinoma. However its role in breast cancer progression had not been recognized. Methods: Microarray and RNA sequencing were utilized to characterize the sequential and temporal changes in mRNA expression during DCIS invasive transition. BCL9 shRNA knockdown was performed to assess the role of BCL9 in in vivo invasion, EMT and canonical Wnt signaling. Immunofluorescence of 28 patient DCIS samples was used to assess a correlation between the expression of BCL9 and biomarkers of high risk DCIS. TCGA data was analyzed to assess the status of BCL9 gene alterations in 959 human breast cancers. Results: Analysis of BCL9, by RNA and protein showed BCL9 up-regulation to be associated with DCIS transition to IDC. Analysis of patient DCIS revealed a significant correlation between high nuclear BCL9 and pathologic characteristics associated with DCIS recurrence: ER and PR negative, high nuclear grade, and high HER2. In vivo silencing of BCL9 resulted in the inhibition of DCIS invasion and reversal of epithelial-mesenchymal transition (EMT). Analysis of the TCGA data showed BCL9 gene to be altered in 26% of breast cancers. This is a significant alteration when compared to ERBB2 (19%) and ESR1 (8%). A significantly higher proportion of basal like invasive breast cancers showed BCL9 amplification. Conclusion: BCL9 is a molecular driver of DCIS invasive progression and may predispose to the development of basal like invasive breast cancers. As such, BCL9 has the potential to serve as a biomarker of high risk DCIS and as a therapeutic target for prevention of IDC. Citation Format: Behbod F, Elsarraj H, Hong Y, Valdez K, Chien J, Godwin A, Fields T. Expression profiling of in vivo DCIS progression models identified BCL9 as a molecular driver of invasive progression. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-05-05.

The histone variant H2A.X is a regulator of the epithelial-mesenchymal transition.

The epithelial–mesenchymal transition (EMT), considered essential for metastatic cancer, has been a focus of much research, but important questions remain. Here, we show that silencing or removing H2A.X, a histone H2A variant involved in cellular DNA repair and robust growth, induces mesenchymal-like characteristics including activation of EMT transcription factors, Slug and ZEB1, in HCT116 human colon cancer cells. Ectopic H2A.X re-expression partially reverses these changes, as does silencing Slug and ZEB1. In an experimental metastasis model, the HCT116 parental and H2A.X-null cells exhibit a similar metastatic behaviour, but the cells with re-expressed H2A.X are substantially more metastatic. We surmise that H2A.X re-expression leads to partial EMT reversal and increases robustness in the HCT116 cells, permitting them to both form tumours and to metastasize. In a human adenocarcinoma panel, H2A.X levels correlate inversely with Slug and ZEB1 levels. Together, these results point to H2A.X as a regulator of EMT.