Epithelial-mesenchymal Phenotype Research Articles

PO-012 Pre-clinical insight into how platelet count affects the activity of HDACi resminostat in combination with the multi-kinase inhibitor sorafenib in HCC

Introduction In a recent phase I/II clinical trial in hepatocellular carcinoma (HCC), (NCT02400788), the combination of HDAC-inhibitor resminostat (YHI1001) with a multi-kinase inhibitor sorafenib was clinically beneficial compared to sorafenib mono-treatment in patients with platelet counts ≥1.5 × 10 5 /µl, but not below that threshold. In HCC, platelets have been linked to bad prognosis, tumour growth and metastasis as well as to resistance against the standard treatment with sorafenib. This begs the question how platelets affect HCC cells and modulate their drug response. Material and methods Several HCC cell lines were used to compare phenotypical features, susceptibility to platelets and drug-mediated effects. To determine effects on cancer cell features, in vitro cell growth and transwell invasion assays were performed. Furthermore, the underlying mechanism of a platelet-modulated drug response on the molecular level was explored. Results and discussions In HCC cells, the anti-proliferative potency of sorafenib was counteracted by platelet factors and the mesenchymal phenotype. However, resminostat alone and in combination with sorafenib effectively triggered an anti-proliferative response independently of platelets or the mesenchymal phenotype. Therefore, resminostat determined the anti-proliferative response of the drug combination. Moreover, recent reports highlight HCC cell subpopulations which express cancer stem cell genes and harbour clonogenic growth and cell invasive capacities as critical for metastasis. Intriguingly, we found that platelets induced the cell-invasive capacity in HCC cells with detectable levels of several cancer stem cell markers and which featured a mixed epithelial-mesenchymal phenotype. Importantly, only the combination of resminostat with sorafenib, but not the mono-treatments, significantly reversed the platelet-induced cell invasion. Conclusion Our pre-clinical data provide evidence on how platelets mediate pro-tumorigenic effects and modulate the therapeutic response to the resminostat/sorafenib drug combination. Platelet factors negatively modulated the drug response to sorafenib. This was overcome by the anti-proliferative activities of resminostat. Importantly, providing an explanation for the clinical benefit of the combination therapy, the platelet-induced invasive capacity was reversed only by the combination of resminostat with sorafenib, but not the mono-treatments.

Combination of two anti-tubulin agents, eribulin and paclitaxel, enhances anti-tumor effects on triple-negative breast cancer through mesenchymal-epithelial transition.

Improved prognosis for triple-negative breast cancer (TNBC) has currently plateaued and the development of novel therapeutic strategies is required. Therefore, we aimed to explore the anti-tumor effect of eribulin and paclitaxel combination therapy for TNBC. The effect of eribulin and paclitaxel in combination was tested, with both concurrent and sequential administration, using four TNBC cell lines (MDA-MB-231, Hs578T, MDA-MB-157, and Mx-1) in vitro and in an MDA-MB-231 BALB/c-nu/nu mouse xenograft model. The expression of epithelial-mesenchymal phenotypic markers was analyzed by western blotting and immunohistochemical analyses. Simultaneous administration of eribulin and paclitaxel resulted in a synergistic anti-tumor effect with MDA-MB-231 and Hs578T cells, but not MDA-MB-157 and Mx-1 cells, in vitro. Moreover, pre-treatment with one drug significantly enhanced sensitivity to the subsequently administrated second drug in MDA-MB-231 and Hs578T cells. Eribulin increased E-cadherin expression and decreased the expression of mesenchymal markers in MDA-MB-231 and Hs578T cells. In contrast, paclitaxel increased the expression of mesenchymal markers. When epithelial-mesenchymal transition was induced by TGF-β1, eribulin sensitivity was enhanced. In contrast, a TGF-β receptor kinase inhibitor decreased eribulin sensitivity. In MDA-MB-231 tumor-bearing mice, concurrent administration of low doses of eribulin and paclitaxel significantly inhibited tumor growth compared to that with either monotherapy. Moreover, single administration of eribulin before the initiation of paclitaxel treatment decreased vimentin expression and reduced the average tumor volume in a mouse xenograft model. Eribulin and paclitaxel show synergistic anti-tumor effect by altering the epithelial-mesenchymal phenotype. This combination therapy could represent a novel therapeutic strategy for TNBC.

The correlation between the clinical characteristics of HCC patients with different phenotype circulating tumor cells in the peripheral blood

Objective To study the correlation between the clinical characteristics of HCC patients with different phenotype circulating tumor cells in the peripheral blood. Methods 77 patients with HCC and 21 patients with benign liver diseases treated at Zhujiang Hospital of Southern Medical University from April 2015 to December 2016 were retrospectively studied. The CanPatrol™ System was adoptedto detect epithelial-mesenchymal phenotype CTCs in the peripheral blood. This study aimed to clarify whether the epithelial-mesenchymal phenotype CTCs were specifically originated from the tumorand to investigate the correlation between CTCs and the clinical features of the patients. Results Using epithelial and mesenchymal markers, the CTCs were divided into three subtypes: epithelial, mesenchymal and mixed CTCs. No epithelial-mesenchymal phenotype CTCs were detected in patients with benign liver diseases. The positivity rates of epithelial, mixed and mesenchymal CTCs in the peripheral blood of 77 patients with HCC were 37.7%(29/77), 74.0%(57/77) and 50.6%(39/77), respectively. The positivity rates of epithelial-mesenchymal phenotype CTCs in patients with HCC were significantly higher than the group of patients with benign liver diseases (P<0.05). The positivity rates of epithelial CTCs were significantly correlated with AFP, number of tumor and portal vein tumor thrombus (P<0.05). The positivity rates of mixed CTCs were significantly correlated with portal vein tumor thrombus, tumor differentiation and BCLC stage (P<0.05). The positivity rates of mesenchymal CTCs were significantly correlated with tumor size, portal vein tumor thrombus, tumor differentiation and BCLC stage (P<0.05). Conclusions The positivity rates of epithelial, mixed and mesenchymal CTCs were closely related to different aspects of clinical characteristics of patients with HCC. These CTCs may serve as indicatorsto evaluatethe progress of HCC. Key words: Epithelial-mesenchymal phenotype; Hepatocellular carcinoma; Circulating tumor cells; Clinical characteristics

Abstract 2374: Mixed-Lineage Kinase 3 signals through the FRA1-MMP1 axis to drive triple-negative breast cancer invasion and transendothelial migration

Abstract Mixed-lineage kinase 3 (MLK3), a mitogen-activated kinase kinase kinase (MAP3K), contributes to metastasis of triple-negative breast cancer (TNBC), in part through its regulation of paxillin phosphorylation and focal adhesion turnover. However the signaling mechanisms through which MLK3 promotes invasion and metastasis are not fully understood. We found that in non-metastatic, estrogen receptor positive (ER+) breast cancer cells, induced MLK3 expression robustly upregulates the oncogenic transcription factor, FOS-related antigen-1 (FRA-1), as well as matrix metalloproteinases (MMPs), MMP-1 and MMP-9. MLK3-induced MMP-1 induction is abrogated by FRA-1 silencing, demonstrating that MLK3 signals through FRA-1 to control MMP-1. In complementary experiments performed in metastatic TNBC models, high levels of FRA-1 are significantly reduced upon gene silencing of MLK3 or by MLK3 disruption using the CRISPR/Cas9n approach. Furthermore, both MMP-1 and MMP-9 are decreased upon ablation of MLK3 or MLK inhibitor treatment. Reduced endothelial permeability and transendothelial migration is observed in experiments with MLK3-depleted TNBC cells compared with parental counterparts. In addition, MLK inhibitor treatment or MLK3 depletion renders TNBC cells defective in Matrigel invasion. Furthermore, circulating tumor cells (CTCs) derived from a TNBC-bearing mouse which show an intermediate epithelial-mesenchymal phenotype display increased levels of MLK3, FRA-1, and MMP-1 compared with parental cells, supporting a role for the MLK3-FRA-1-MMP-1 signaling axis in tumor cell invasion and vascular intravasation. Consistent with the role of MMP-1 in multiple steps of metastasis, high levels of MMP-1 in breast cancer patient tumor samples are associated with increased distant metastases and poorer overall survival, particularly in TNBC. Our results demonstrating the requirement for MLK3 in controlling the FRA-1/MMP-1 axis suggest that MLK3 is a potentially effective therapeutic target for TNBC. Citation Format: Chotirat Rattanasinchai, Brandon Llewellyn, Susan E. Conrad, Kathleen A. Gallo. Mixed-Lineage Kinase 3 signals through the FRA1-MMP1 axis to drive triple-negative breast cancer invasion and transendothelial migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2374. doi:10.1158/1538-7445.AM2017-2374

Abstract 3053: Stability and stemness of the hybrid epithelial-mesenchymal phenotype

Abstract Transitions between epithelial and mesenchymal phenotypes – EMT and MET – are hallmarks of cellular plasticity during embryonic development and cancer metastasis. During these transitions, cells can also adopt a hybrid epithelial/mesenchymal (hybrid E/M) phenotype enabling them to migrate collectively as observed during gastrulation, wound healing, and clusters of Circulating Tumor Cells (CTCs). The hybrid E/M phenotype has largely been tacitly assumed to be 'metastable', i.e. transient state. Here, we integrate mathematical modeling with in vitro experiments to identify certain 'phenotypic stability factors' (PSFs) - GRHL2, OVOL2 and ΔNP63α that can stabilize a hybrid E/M phenotype. We show that H1975 (NSCLC cell line) cells can display a hybrid E/M phenotype stably and migrate collectively, a behavior that is impaired by knockdown of GRHL2 or OVOL2. Further, our computational model predicts that these PSFs can also associate hybrid E/M phenotype with high tumor-initiating potential, a prediction strengthened by the observation that the higher levels of one or more of these PSFs may predict poor patient outcome. Overall, our results suggest that a hybrid E/M phenotype need not be 'metastable', and bolster the notion that a hybrid E/M phenotype, but not necessarily full EMT, associates with aggressive tumor progression. Citation Format: Mohit Kumar Jolly, Dongya Jia, Satyendra C. Tripathi, Steve Mooney, Muge Celiktas, Samir M. Hanash, Sendurai A. Mani, Kenneth J. Pienta, Eshel Ben-Jacob, Herbert Levine. Stability and stemness of the hybrid epithelial-mesenchymal phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3053. doi:10.1158/1538-7445.AM2017-3053

Pediatric Mesenchymal Hamartomas of the Liver can Show Both Foregut and Hindgut Phenotype.

Cystic epithelium in mesenchymal hamartoma (MH) is typically biliary type. Mucinous differentiation of the epithelium and increased hepatocellular component may pose a diagnostic challenge. We studied MH in 7 children (6 M, 1 F; age 4 months to 8 years, median 1 year). Resected tumors varied from 3.0 to 17.0 cm. All tumors showed biliary epithelium in the cystic component with strong and diffuse reactivity for CK7 and CK19. Expression of CK20 and CDX2 was additionally seen in 2 tumors, rare/focal in 1, and diffuse with mucinous differentiation in the other. Strong and continuous nuclear reactivity for Sox9 was seen in the cyst epithelium in all 7 tumors including focal staining in mucinous areas. It was also positive in the hepatocellular component. CD56 and vimentin were variably positive in the cystic epithelium of all cases. Alpha-fetoprotein showed patchy weak staining in the hepatocellular component and was negative in the cystic epithelium. Hepar-1 showed focal staining in the cystic epithelium in 4/7 cases and diffuse in the hepatocellular component. Both patients showing CK20 and CDX2 expression had abnormal chromosomal analysis: one with balanced translocation between chromosomes 2 and 19 and other with loss of chromosome Y. Among others, one showed 46,XY,inv(9)(p11q12), one did not grow cells, and the remaining 3 had normal karyotype. Six patients underwent resection and one had liver transplantation. All were alive and well with a median follow-up of 1.5 years. In conclusion, mucinous epithelium can be seen in MH. Expression of Sox9 in the cyst epithelium and hepatocytes suggests a common origin for these components. Expression of vimentin indicates an overlapping epithelial-mesenchymal phenotype. Hepatic MH can show divergent epithelial differentiation including both foregut and hindgut phenotype, which may provide insights into the pathogenesis of this rare neoplasm but does not seem to affect the outcome in this limited series.

Silencing of GP73 inhibits invasion and metastasis via suppression of epithelial-mesenchymal transition in hepatocellular carcinoma.

Epithelial-mesenchymal transition (EMT) is associated with invasion and metastasis of cancer cells. Golgi protein 73 (GP73) is a serum biomarker for hepatocellular carcinoma (HCC) and our previous study demonstrated that the expression of GP73 correlated with aggressive behavior and EMT molecules in HCC. However, its role in metastatic mechanism of HCC is not clear. The aim of this study was to investigate the effect of GP73 on invasion and migration, and underlying mechanism of GP73 involved in EMT of HCC. The expression of GP73 was downregulated by small interfering RNA (siRNA). The metastatic and invasive abilities were analyzed using scratch assay and Transwell assay. Changes in EMT-related molecules were evaluated by western blot and qRT‑PCR analyses, and epithelial-mesenchymal phenotype changes were also observed. Expression of GP73 was upregulated in the more metastatic HCC cell lines. Knockdown of GP73 by siRNA resulted in a significant decrease in migratory and invasive abilities in both MHCC97H and Bel-7404 cell lines. Importantly, EMT-related markers and morphological phenotypes significantly changed following by the inhibition of GP73. Silencing GP73 contributed to the reduction of invasion and metastasis via suppressing EMT in HCC. GP73 may serve as a novel molecular target against EMT in HCC metastasis therapy.

Epithelial-mesenchymal transition and nuclear β-catenin induced by conditional intestinal disruption of Cdh1 with Apc is E-cadherin EC1 domain dependent.

Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to β-catenin. Here, we evaluate whether E-cadherin binding can inhibit β-catenin when there is loss of Adenomatous polyposis coli (APC) from the β-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear β-catenin localization, suggesting that E-cadherin inhibits β-catenin. Combination with an intestinal stem cell Cre (Lgr5CreERT2) resulted in ApcΔ/Δ recombination and adenoma, but intact Cdh1fl/fl alleles. Cultured ApcΔ/ΔCdh1fl/fl adenoma cells infected with adenovirus-Cre induced Cdh1fl/fl recombination (Cdh1Δ/Δ), disruption of organoid morphology, nuclear β-catenin localization, and cells with an epithelial-mesenchymal phenotype. Complementation with adenovirus expressing wild-type Cdh1 (Cdh1-WT) rescued adhesion and β-catenin membrane localization, yet an EC1 specific double mutant defective in homophilic adhesion (Cdh1-MutW2A, S78W) did not. These data suggest that E-cadherin inhibits β-catenin in the context of disruption of the APC-destruction complex, and that this function is also EC1 domain dependent. Both binding functions of E-cadherin may be required for its tumour suppressor activity.

PD-L1 expression is associated with epithelial-to-mesenchymal transition in adenocarcinoma of the lung

PD-1/PD-L1-targeted immunotherapy has emerged as a promising therapeutic strategy for pulmonary adenocarcinoma (pADC). Epithelial-to-mesenchymal transition (EMT) is involved in the progression and immune evasion of cancers. Therefore, we investigated the association between PD-L1 expression and EMT phenotype in pADC. Immunohistochemistry for E-cadherin (epithelial marker), ZEB1, SNAIL, SLUG, vimentin (mesenchymal markers), PD-L1, CD8, and PD-1 was performed on 477 cases of pADC. Cases were classified into epithelial, mesenchymal, epithelial-mesenchymal, and unspecified types based on immunohistochemical results. PD-L1 expression was scored as 0 in 14.0% (n=67), 1 in 26.4% (n=126), 2 in 51.2% (n=244), and 3 in 8.4% (n=40). PD-L1 score was positively correlated with SNAIL and vimentin H scores (P<.001, both). After dichotomizing patients into PD-L1-negative and PD-L1-positive groups, PD-L1 positivity was significantly higher in patients with mesenchymal (71.2%; 84/118) and epithelial-mesenchymal (62.7%; 84/134) phenotypes compared with those with epithelial (50.6%; 44/87) and unspecified (50.0%; 35/70) phenotypes (P=.005). The significant association between PD-L1 expression and EMT phenotype was maintained in EGFR-mutated pADCs. Moreover, cases with EMT phenotype (ie, mesenchymal and epithelial-mesenchymal) were infiltrated by higher numbers of CD8+ and PD-1+ cells than those with epithelial and unspecified phenotypes in EGFR-mutated pADCs (P=.043 for CD8+ cells and P<.001 for PD-l+ cells). Particularly, cases with EMT phenotype and PD-L1 expression showed the greatest amount of CD8+ and PD-1+ cells in EGFR-mutated cases (P=.043 for CD8+ cells and P=.005 for PD-1+ cells). This study demonstrates that EMT phenotype is related to PD-L1 overexpression in pADC cells and patients with EMT-phenotype pADC may benefit from PD-1/PD-L1-blocking immunotherapy.

Musashi-2 (MSI2) supports TGF-β signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis

Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-β receptor 1 (TGFβR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFβRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFβR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.

Tumor Budding: The Name is EMT. Partial EMT.

Tumor budding is a histological phenomenon encountered in various cancers, whereby individual malignant cells and/or small clusters of malignant cells are seen in the tumor stroma. Postulated to be mirror epithelial-mesenchymal transition, tumor budding has been associated with poor cancer outcomes. However, the vast heterogeneity in its exact definition, methodology of assessment, and patient stratification need to be resolved before it can be routinely used as a standardized prognostic feature. Here, we discuss the heterogeneity in defining and assessing tumor budding, its clinical significance across multiple cancer types, and its prospective implementation in clinical practice. Next, we review the emerging evidence about partial, rather than complete, epithelial-mesenchymal phenotype at the tumor bud level, and its connection with tumor proliferation, quiescence, and stemness. Finally, based on recent literature, indicating a co-expression of epithelial and mesenchymal markers in many tumor buds, we posit tumor budding to be a manifestation of this hybrid epithelial/mesenchymal phenotype displaying collective cell migration.

Conversion to stem-cell state in response to microenvironmental cues is regulated by balance between epithelial and mesenchymal features in lung cancer cells

Cancer cells within a tumor are functionally heterogeneous and specific subpopulations, defined as cancer initiating cells (CICs), are endowed with higher tumor forming potential. The CIC state, however, is not hierarchically stable and conversion of non-CICs to CICs under microenvironment signals might represent a determinant of tumor aggressiveness. How plasticity is regulated at the cellular level is however poorly understood. To identify determinants of plasticity in lung cancer we exposed eight different cell lines to TGFβ1 to induce EMT and stimulate modulation of CD133+ CICs. We show that response to TGFβ1 treatment is heterogeneous with some cells readily switching to stem cell state (1.5–2 fold CICs increase) and others being unresponsive to stimulation. This response is unrelated to original CICs content or extent of EMT engagement but is tightly dependent on balance between epithelial and mesenchymal features as measured by the ratio of expression of CDH1 (E-cadherin) to SNAI2. Epigenetic modulation of this balance can restore sensitivity of unresponsive models to microenvironmental stimuli, including those elicited by cancer-associated fibroblasts both in vitro and in vivo. In particular, tumors with increased prevalence of cells with features of partial EMT (hybrid epithelial/mesenchymal phenotype) are endowed with the highest plasticity and specific patterns of expression of SNAI2 and CDH1 markers identify a subset of tumors with worse prognosis. In conclusion, here we describe a connection between a hybrid epithelial/mesenchymal phenotype and conversion to stem-cell state in response to external stimuli. These findings have implications for current endeavors to identify tumors with increased plasticity.

Splicing of a non-coding antisense transcript controlsLEF1gene expression

In this report we have analyzed the role of antisense transcription in the control of LEF1 transcription factor expression. A natural antisense transcript (NAT) is transcribed from a promoter present in the first intron of LEF1 gene and undergoes splicing in mesenchymal cells. Although this locus is silent in epithelial cells, and neither NAT transcript nor LEF1 mRNA are expressed, in cell lines with an intermediate epithelial-mesenchymal phenotype presenting low LEF1 expression, the NAT is synthesized and remains unprocessed. Contrarily to the spliced NAT, this unspliced NAT down-regulates the main LEF1 promoter activity and attenuates LEF1 mRNA transcription. Unspliced LEF1 NAT interacts with LEF1 promoter and facilitates PRC2 binding to the LEF1 promoter and trimethylation of lysine 27 in histone 3. Expression of the spliced form of LEF1 NAT in trans prevents the action of unspliced NAT by competing for interaction with the promoter. Thus, these results indicate that LEF1 gene expression is attenuated by an antisense non-coding RNA and that this NAT function is regulated by the balance between its spliced and unspliced forms.

Short-term expansion of breast circulating cancer cells predicts response to anti-cancer therapy.

Circulating tumor cells (CTCs) are considered as surrogate markers for prognosticating and evaluating patient treatment responses. Here, 226 blood samples from 92 patients with breast cancer, including patients with newly diagnosed or metastatic refractory cancer, and 16 blood samples from healthy subjects were cultured in laser-ablated microwells. Clusters containing an increasing number of cytokeratin-positive (CK+) cells appeared after 2 weeks, while most blood cells disappeared with time. Cultures were heterogeneous and exhibited two distinct sub-populations of cells: 'Small' (≤ 25 μm; high nuclear/cytoplasmic ratio; CD45-) cells, comprising CTCs, and 'Large' (> 25 μm; low nuclear/cytoplasmic ratio; CD68+ or CD56+) cells, corresponding to macrophage and natural killer-like cells. The Small cell fraction also showed copy number increases in six target genes (FGFR1, Myc, CCND1, HER2, TOP2A and ZNF217) associated with breast cancer. These expanded CTCs exhibited different proportions of epithelial-mesenchymal phenotypes and were transferable for further expansion as spheroids in serum-free suspension or 3D cultures. Cluster formation was affected by the presence and duration of systemic therapy, and its persistence may reflect therapeutic resistance. This novel and advanced method estimates CTC clonal heterogeneity and can predict, within a relatively short time frame, patient responses to therapy.

Abstract 1133: Partial EMT, hybrid epithelial-mesenchymal phenotype and epithelial plasticity: role of (miR-200/ZEB) loop

Abstract Epithelial to Mesenchymal Transition (EMT) and Mesenchymal to Epithelial Transition (MET) are critical for embryonic development and tissue repair. Aberrantly regulated transitions are also implicated in cancer metastasis. The core regulatory genetic network for these transitions appears to allow for the existence of a hybrid epithelial/mesenchymal phenotype (E/M). Such hybrid cells are seen during wound healing and branching morphogenesis, and have mixed epithelial and mesenchymal characteristics, which enable them to migrate collectively. The core regulatory network for the cell fate determination between these three phenotypes is composed of two highly interconnected mutual-inhibition feedback circuits - miR-34/SNAIL and miR-200/ZEB. Here we applied a novel theoretical framework to model the microRNA-based regulation to study this core regulatory unit. More specifically, we looked into the functions of the two loops in isolation, and subsequently of the combined unit when the two loops are integrated into the full regulatory circuit. We found that miR-200/ZEB forms a tristable circuit that acts as a ternary decision making switch, driven by miR-34/SNAIL that is a monostable loop which acts as noise-buffering integrator of internal and external signals that induce or inhibit EMT/MET. Our results are corroborated by recent experiments indicating the (miR-200/ZEB) to be the commitment point and decision making loop (eg. Cells don't revert to epithelial phenotype on removal of EMT-inducing signal, if ZEB expression has been turned on.) .We associate the three stable states of the miR-200/ZEB circuit to different phenotypes - (1,0) - (high miR-200)/(low ZEB) - epithelial, (0,1) - (low miR-200)/(high ZEB) - mesenchymal, and (1/2,1/2) - (medium miR-200)/(medium ZEB) - hybrid. Thus, we propose that the hybrid epithelial - mesenchymal phenotype has concurrent expression of both miR-200 and ZEB. Our hypothesis is consistent with recent experimental studies (e.g. ZEB expression measurements in collectively migrating cells), and also explains the lack of hybrid phenotype observed during MET in colonization and induced pluripotent stem cells (iPSCs). We also present testable predictions of dynamic gene expression during complete and partial transitions (EMT/MET). Citation Format: Mohit Kumar Jolly, Mingyang Lu, Herbert Levine, Jose N. Onuchic, Eshel Ben-Jacob. Partial EMT, hybrid epithelial-mesenchymal phenotype and epithelial plasticity: role of (miR-200/ZEB) loop. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1133. doi:10.1158/1538-7445.AM2014-1133

Role of non-coding RNAs in pancreatic cancer: the bane of the microworld.

Our understanding of the mechanisms underlying the development of pancreatic cancer has been greatly advanced. However, the molecular events involved in the initiation and development of pancreatic cancer remain inscrutable. None of the present medical technologies have been proven to be effective in significantly improving early detection or reducing the mortality/morbidity of this disease. Thus, a better understanding of the molecular basis of pancreatic cancer is required for the identification of more effective diagnostic markers and therapeutic targets. Non-coding RNAs (ncRNAs), generally including microRNAs and long non-coding RNAs, have recently been found to be deregulated in many human cancers, which provides new opportunities for identifying both functional drivers and specific biomarkers of pancreatic cancer. In this article, we review the existing literature in the field documenting the significance of aberrantly expressed and functional ncRNAs in human pancreatic cancer, and discuss how oncogenic ncRNAs may be involved in the genetic and epigenetic networks regulating functional pathways that are deregulated in this malignancy, particularly of the ncRNAs' role in drug resistance and epithelial-mesenchymal transition biological phenotype, with the aim of analyzing the feasibility of clinical application of ncRNAs in the diagnosis and treatment of pancreatic cancer.

MiR-300 inhibits epithelial to mesenchymal transition and metastasis by targeting Twist in human epithelial cancer.

BackgroundEpithelial-to-mesenchymal transition (EMT) is a key step of the progression of tumor cell metastasis. Recent work has demonstrated some miRNAs play critical roles in EMT. In this study, we focused on the roles of miR-300 in regulating EMT.MethodsThe expression levels of miR-300 were examined in epithelial carcinoma cells that underwent an EMT using quantitative reverse transcription-PCR. The role of miR-300 in EMT was investigated by transfection of the miR-300 mimic or inhibitor in natural epithelial-mesenchymal phenotype cell line pairs and in transforming growth factor (TGF) beta-induced EMT cell models. A luciferase reporter assay and a rescue experiment were conducted to confirm the target gene of miR-300. The efficacy of miR-300 against tumor invasion and metastasis was evaluated both in vitro and in vivo. Correlation analysis between miR-300 expression and the expression levels of its target gene, as well as tumor metastasis was performed in specimens from patients with head and neck squamous cell carcinoma (HNSCC).ResultsMiR-300 was found down-regulated in the HNSCC cells and breast cancer cells that underwent EMT. Ectopic expression of miR-300 effectively blocked TGF-beta-induced EMT and reversed the phenotype of EMT in HN-12 and MDA-MB-231 cells, but inhibition of miR-300 in the epithelial phenotype cells, HN-4 and MCF-7 cells, could induce EMT. The luciferase reporter assay and the rescue assay results showed that miR-300 directly targets the 3′UTR of Twist. Enforced miR-300 expression suppressed cell invasion in vitro and experimental metastasis in vivo. Clinically, miR-300 expression was found inversely correlated with Twist expression and reduced miR-300 was associated with metastasis in patient specimens.ConclusionsDown-regulation of miR-300 is required for EMT initiation and maintenance. MiR-300 may negatively regulate EMT by direct targeting Twist and therefore inhibit cancer cell invasion and metastasis, which implicates miR-300 as an attractive candidate for cancer therapy.

Fibronectin overexpression is associated with latent membrane protein 1 expression and has independent prognostic value for nasopharyngeal carcinoma

Despite recent improvements in the diagnosis and treatment, the final outcomes in patients with nasopharyngeal carcinomas (NPC) still remain suboptimal. Through data mining from published transcriptomic database with further bioinformatic validation, fibronectin (FN1) was identified as a differentially upregulated gene in NPC tissues, which implicates the transition from epithelial to mesenchymal phenotype (EMT) and promotes metastasis. Given the roles of fibronectin in risk stratification and in the frontline therapeutics of common carcinomas, such as renal cell cancer, we explored fibronectin immunoexpression status and its associations with clinicopathological variables and survival in a well-defined cohort of NPC patients. Fibronectin immunohistochemistry was retrospectively performed and analyzed using H-score for 124 biopsy specimens from NPC patients who received standard treatment without distant metastasis at initial diagnosis. Those cases with H-score higher than the median value were regarded as fibronectin overexpression. The findings were correlated with clinicopathological variables, EBV latent membrane protein 1 (LMP1) expression, disease-specific survival (DSS), distant metastasis-free survival (DMFS), and local recurrence-free survival (LRFS). Fibronectin overexpression was significantly associated with American Joint Committee on Cancer (AJCC) stages III-IV (p = 0.019) and LMP1 expression (p = 0.004), and univariately predictive of adverse outcomes for DSS, DMFS, and LRFS (all p < 0.0001). In the multivariate comparison, fibronectin overexpression still remained prognostically independent to portend worse DSS (p < 0.01, hazard ratio = 5.958), DMFS (p < 0.01, hazard ratio = 5.728), and LRFS (p < 0.01, hazard ratio = 5.411) together with a vanced AJCC stages III-IV. Fibronectin is upregulated in a subset of NPCs, and its increased immunoexpression significantly correlated with advanced features, justifying the potentiality of fibronectin as a theragnostic biomaker of NPC.

Abstract 321: Sunitinib alternated with everolimus in renal cell carcinoma: a sequential combination strategy for antitumor efficacy in mice clinical trial.

Abstract Background: Sunitinib (VEGFR/PDGFR inhibitor) and everolimus (mTOR inhibitor) are approved for advanced renal cell carcinoma (RCC), as first-line and second-line therapy, respectively. No data have been reported on programmed sequential strategies combining alternative use of sunitinib and everolimus prior to progression. Such strategy is expected to delay the emergence of first-line acquired resistance and improve tumor control. The aim of our study was to assess the effects of three sequences administration of sunitinib and everolimus. Methods: Nude mice (n=5/group) were engrafted with human RCC cells (CAKI-1) then randomized to vehicle, everolimus, sunitinib, sunitinib alternated with everolimus every week (S1), every two weeks (S2), and every three weeks (S3). Median time to tumor progression (mTTP) was determined in each group. Angiogenesis and necrosis were evaluated using CD31 and HE staining. Inhibition of mTOR pathway was evaluated using phospho-S6 (pS6) and e-cadherin/vimentin expressions were analyzed by immunofluorescence staining. Results: The S1, S2 and S3 regimens increased mTTP by 30 to 80% relative to sunitinib or everolimus alone. The vascular surface areas (vessels size and number) in tumors were reduced in all treated groups relative to control, especially S1 and S2. All three sequential therapies induced larger necrosis areas in tumors as compared to control. In each group, tumor response seemed associated with a decrease in pS6 expression and changes in epithelial-mesenchymal phenotype with a decrease in vimentin and an increase in e-cadherin expression Conclusion: Sunitinib and everolimus programmed sequential regimens prior to progression yielded longer mTTP than sunitinib and everolimus monotherapies. The sequential combinations of these two agents in a RCC mouse clinical trial, increased antiangiogenic effects, leading potentially to tumor necrosis, especially with the S1 and S2 regimens. mTTP for each group and effects of sequential treatments in tumors angiogenesis and necrosis Treatment groups mTTP (in days) Vascular Surface Area per field (% vs control) Number of vessels per field % of necrosis surface per field Control 24 100 22 23 Sunitinib 36 30 5 47 Everolimus 50 28 7 27 S1 64 15 5 37 S2 59 20 4 51 S3 59 42 5 37 Citation Format: Célia Dos Santos, Annemilaï Tijeras-Raballand, Maria Serova, Chantal Dreyer, Khemaies Slimane, Armand de Gramont, Sandrine Faivre, Eric Raymond. Sunitinib alternated with everolimus in renal cell carcinoma: a sequential combination strategy for antitumor efficacy in mice clinical trial. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 321. doi:10.1158/1538-7445.AM2013-321

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.