Novel Regulator Of Epithelial-mesenchymal Transition Research Articles

Abstract A57: Characterization of novel regulators of epithelial-mesenchymal transition in human cancer cells

Abstract The epithelial-mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits by cancer cells of several cancer types. Recently, a few kinases, such as FYN and c-Met, were reported to participate in regulating EMT. However, the roles of the majority of >700 human kinases in EMT and cancer progression are still unknown. Here we carried out human kinase cDNA screens to identify novel regulators of EMT. Many unknown EMT regulators and a few known ones such as PI3K, PDGFRA, FYN and c-Met, were found to up-regulate mesenchymal marker vimentin. Some also increase expression of fibronectin and N-cadherin, as well as EMT transcriptional regulators, such as ZEB1/2, Snail and Slug. At the same time, the cells acquired mesenchymal phenotypes, such as increased migration/invasion activity, enhanced capability to survive and proliferate in growth factor deprivation condition or under drug treatments. Interestingly, FACS analysis also showed a substantial increase in CD44hiCD24low population in kinase-transducted mammary gland epithelial cells. The increase of CD44hi population could be attributed to increase of a CD44 mesenchymal variant — the standard form CD44s and decrease of the epithelial variant CD44 v8-10. And this alternation was associated with down-regulation of ESRP1 and ESRP2, two splicing factors that regulate CD44 splicing and EMT. Furthermore, the mammary gland epithelial cells undergoing EMT showed increase capability of forming mammosphere and differentiating into other lineage, such as osteoblast and adipocyte. Importantly, some of these novel EMT regulators are associated with tumor progression and stemness of human cancers. Further study of these novel EMT regulators promises to provide new mechanisms of cancer progression, and may also lead to development of new prognostic markers and therapeutics for metastasis. Citation Format: Linna Li, Nanping Ai, Mohit Hulsurkar, Wenliang Li. Characterization of novel regulators of epithelial-mesenchymal transition in human cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A57.

Abstract IA6: Epithelial-mesenchymal plasticity in breast cancer metastasis

Abstract Increasing evidence suggests that the normal genetic programs underlying various developmental processes can often be usurped in pathological conditions such as cancer metastasis. Epithelial—mesenchymal transition (EMT) is a complex process that is associated with dramatic changes in cell adhesion, polarity, and migratory properties, and is intimately associated both with cell fate transitions during embryonic development and with the acquisition of invasive properties during cancer metastasis. Several transcription factors, including the Snail, Zeb and Twist families, have been identified as major drivers of EMT by repressing the transcription of E-cadherin. However, our knowledge about the regulatory network and pleiotropic effects of epithelial-mesenchymal plasticity remains largely incomplete, and the importance of reverse process, mesenchymal-epithelial transition (MET), is poorly understood. In our study of the early stage of breast cancer metastasis, we found that the transcription factor Elf5, a key regulator of alveologenesis in the mammary gland, regulates EMT in both mammary gland development and metastasis. This specific role for Elf5 in suppressing EMT and metastasis was uncovered via analyses of Elf5 conditional knockout animals, various in vitro and in vivo models of EMT and metastasis, an MMTV-neu transgenic model of mammary gland tumor progression, and clinical breast cancer samples. Furthermore, we demonstrate that this role of Elf5 is mediated by the direct transcriptional repression of Snail2/Slug, a master regulator of mammary stem cells and a known inducer of EMT. These findings establish a broad molecular function of Elf5 that extends from being a key cell lineage regulator during normal mammary gland development to serving as a suppressor of EMT and metastasis in breast cancer. Focusing on non-coding RNAs as potential novel regulators of EMT, we identified the miR-200 family miRNAs as suppressors of EMT and tumor invasion that function via direct targeting of Zeb1 and Zeb2. Contrary to the expectation that miR-200s should inhibit metastasis, we found that miR-200 overexpression is in fact associated with poor metastasis-free survival of breast cancer patients and functionally promotes metastatic colonization in mouse models. Through an integrated genomic/proteomic analysis, we identified the Sec23a secretory pathway as a prominent functional target of miR-200s with an active role in suppressing metastatic colonization. Secretome analysis further identified IGFBP4 and TINAGL1 as important Sec23a-dependent secreted proteins with metastasis suppressive functions. Thus, the dichotomous functions of miR-200s in inhibiting early steps of invasion while promoting late step of metastatic colonization is achieved through targeting of both tumor cell-intrinsic and —extrinsic regulatory pathways. Taken together, these findings support dynamic and pleiotropic roles of epithelial-mesenchymal plasticity in early and late stages of metastatic progression: while EMT is necessary for initial invasion, MET may be required for efficient colonization. Citation Format: Yibin Kang. Epithelial-mesenchymal plasticity in breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA6.

Abstract P5-04-08: Identifying and characterizing human kinases for novel regulators of epithelial-mesenchymal transition in breast cancer cells

Abstract The epithelial-mesenchymal transition (EMT) confers mesenchymal properties on epithelial cells and has been closely associated with the acquisition of aggressive traits by breast cancer cells. Recently, a few kinases, such as FYN and c-Met, were reported to participate in regulating EMT. However, the roles of the majority of ∼700 human kinases in EMT and breast cancer progression are still unknown. Here we carried out human kinase cDNA screens to identify novel regulators of EMT. Many unknown EMT regulators and a few known ones such as PI3K, PDGFRA, FYN and c-Met, were found to up-regulate mesenchymal marker vimentin. Some also increase fibronectin and N-cadherin expression in kinase-transducted human mammary gland epithelial cells (HMLER). At the same time, these HMLER cells acquired mesenchymal phenotypes, such as increased in vitro migration activity, enhanced capability to survive and proliferate in growth factor deprivation condition or under drug treatments. RT-PCR results also exhibited increased expression of some EMT transcriptional regulators, such as ZEB1, Snail and Slug, which suggested our selected kinases may regulate EMT through these transcription factors. Interestingly, FACS analysis also showed a substantial increase in CD44hiCD24− population in these kinase-transducted HMLER cells. The increase of CD44hiCD24− mesenchymal phenotype could be attributed to increase of standard form CD44 (CD44s) and decrease of the variant form CD44 (CD44v8-10). And this alternation was associated with down-regulation of ESRP1 and ESRP2, two splicing factors that regulate CD44 splicing and EMT. Therefore, these selected kinases may be considered as novel regulators of EMT, and may aid the development of new prognostic markers or drug targets for breast cancer progression. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-04-08.

Downregulation of miR-153 contributes to epithelial-mesenchymal transition and tumor metastasis in human epithelial cancer

The epithelial-mesenchymal transition (EMT) is a crucial step in epithelial cancer invasion and metastasis. The aims of this study were to investigate and validate unidentified micro RNAs (miRNAs) that regulate EMT and to reveal their clinical relevance in epithelial cancer patients. By applying miRNA array screening in a natural epithelial-mesenchymal phenotype cell line pair and in a transforming growth factor β-induced EMT cell model, we found miR-153 was markedly downregulated in the cells that underwent an EMT. A close association was confirmed between inhibition of miR-153 and the EMT phenotype, as well as the invasive ability of epithelial cancer cells. Ectopic expression of miR-153 in mesenchymal-like cells resulted in an epithelial morphology change with decreased cellular invasive ability. On the contrary, transfection of a miR-153 inhibitor in epithelial-like cells led to a mesenchymal phenotype change. In vivo ectopic expression of miR-153 significantly inhibited tumor cell metastasis formation. Data from the dual-luciferase reporter gene assay showed, for the first time, that SNAI1 and ZEB2 were direct targets of miR-153. Inverse correlations were also observed between miR-153 and SNA1 and ZEB2 levels in oral cancer patients' samples. Furthermore, low expression level of miR-153 was found to be significantly related to metastasis and poor prognosis in oral cancer patients. These data demonstrate that miR-153 is a novel regulator of EMT by targeting SNAI1 and ZEB2 and indicate its potential therapeutic value for reducing cancer metastasis.

Let-7d functions as novel regulator of epithelial-mesenchymal transition and chemoresistant property in oral cancer

Oral squamous cell carcinoma (OSCC) is a prevalent cancer worldwide. Let-7 family has been shown to function as a tumor suppressor through regulating multiple oncogenic signaling. Recent study reported that combined underexpression of miR-205 and let-7d showed negative correlation with the survival prognosis of head and neck cancer patients. However, the let-7d-involved mechanism in regulating OSCC is still unclear. In this study, we first demonstrated that let-7d expression was significantly decreased while Twist and Snail expression was increased in OSCC cancer cell lines and primary cultures as compared to normal human oral keratinocyte cells. To further investigate the role of let-7d in OSCC, we applied the SPONGE method to knock down let-7d in OECM-1 and two primary OSCC cell types. The results showed that knockdown of let-7d promote epithelial-mesenchymal transition (EMT) traits and migratory/invasive capabilities in OSCC cells. Furthermore, down-expression of let-7d significantly activated Twist and Snail expressions and chemo-resistant abilities of OSCC cells. Notably, overexpression of let-7d effectively reversed the EMT phenotype, blocked migratory/invasive abilities, and further increased the chemosensitivity in oral cancer tumor initiating ALDH1+ cells. In sum, these results show that let-7d negatively modulates EMT expression and also plays a role in regulating chemo-resistant ability in oral cancer.

Abstract 967: N-Myc interactor, a novel regulator of EMT

Abstract Dissemination of tumor cells and subsequent colonization at distant sites is the most lethal event in cancer progression. The epithelial-mesenchymal transition (EMT) has recently been recognized as a foremost phenomenon underlying such events. Because of the importance of EMT, it is vital to understand what triggers it, as well as the mechanisms by which it is regulated. We have shown that N-myc interactor (NMI) plays a role in restricting malignant activities of cancerous cell lines in vivo and in vitro. This study describes a novel function of NMI in reducing the mesenchymal phenotype of breast cancer cell lines. Constitutive expression of NMI in the mesenchymal cell line MDA-MB-435, showed remarkable morphologic transition towards an epithelial appearance, accompanied by reduction or loss of several mesenchymal markers such as TWIST, MMP2, SNAI2 and ZEB1 and gain of epithelial markers such as E-cadherin and Keratin 18. Conversely, silencing NMI from immortalized breast epithelial line MCF10A, renders the cells more mesenchymal in appearance. Studies using a variety of additional cell lines consistently revealed that silencing of NMI expression coincided with increased expression of both ZEB1 and SNAI2, while NMI overexpression brought about decreased expression of ZEB1 and SNAI2. Assessment of NMI in breast cancer specimens revealed a significant loss of NMI transcript with increasing grade of breast cancer. Thus, overall, our data strongly suggests that loss of NMI may be a key event allowing increased expression of EMT-inducing transcription factors that drive EMT-dependent malignant progression. Acknowledgement: We acknowledge grant support from the NCI: 1R01CA140472 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 967. doi:10.1158/1538-7445.AM2011-967

Protein Kinase D1 Suppresses Epithelial-to-Mesenchymal Transition through Phosphorylation of Snail

Cancer cells undergo epithelial-mesenchymal transition (EMT) as a program of increased invasion and metastasis during cancer progression. Here, we report that a novel regulator of EMT in cancer cells is protein kinase D1 (PKD1), which is downregulated in advanced prostate, breast, and gastric cancers. Ectopic reexpression of PKD1 in metastatic prostate cancer cells reversibly suppressed expression of mesenchyme-specific genes and increased epithelial markers such as E-cadherin, whereas small interfering RNA-mediated knockdown of PKD1 increased expression of mesenchyme markers. Further, PKD1 inhibited tumor growth and metastasis in a tumor xenograft model. PKD1 phosphorylates Ser(11) (S11) on transcription factor Snail, a master EMT regulator and repressor of E-cadherin expression, triggering nuclear export of Snail via 14-3-3σ binding. Snail S11 mutation causes acquisition of mesenchymal traits and expression of stem cell markers. Together, our results suggest that PKD1 functions as a tumor and metastasis suppressor, at least partly by regulating Snail-mediated EMT, and that loss of PKD1 may contribute to acquisition of an aggressive malignant phenotype.