Mesenchymal-to-epithelial Research Articles

The bacterial metabolite, lithocholic acid, has antineoplastic effects in pancreatic adenocarcinoma

Lithocholic acid (LCA) is a secondary bile acid. LCA enters the circulation after bacterial synthesis in the gastrointestinal tract, reaches distantly located cancer cells, and influences their behavior. LCA was considered carcinogenic, but recent studies demonstrated that LCA has antitumor effects. We assessed the possible role of LCA in pancreatic adenocarcinoma. At the serum reference concentration, LCA induced a multi-pronged antineoplastic program in pancreatic adenocarcinoma cells. LCA inhibited cancer cell proliferation and induced mesenchymal-to-epithelial (MET) transition that reduced cell invasion capacity. LCA induced oxidative/nitrosative stress by decreasing the expression of nuclear factor, erythroid 2-like 2 (NRF2) and inducing inducible nitric oxide synthase (iNOS). The oxidative/nitrosative stress increased protein nitration and lipid peroxidation. Suppression of oxidative stress by glutathione (GSH) or pegylated catalase (pegCAT) blunted LCA-induced MET. Antioxidant genes were overexpressed in pancreatic adenocarcinoma and decreased antioxidant levels correlated with better survival of pancreatic adenocarcinoma patients. Furthermore, LCA treatment decreased the proportions of cancer stem cells. Finally, LCA induced total and ATP-linked mitochondrial oxidation and fatty acid oxidation. LCA exerted effects through the farnesoid X receptor (FXR), vitamin D receptor (VDR), and constitutive androstane receptor (CAR). LCA did not interfere with cytostatic agents used in the chemotherapy of pancreatic adenocarcinoma. Taken together, LCA is a non-toxic compound and has antineoplastic effects in pancreatic adenocarcinoma.

Alternative splicing downstream of EMT enhances phenotypic plasticity and malignant behavior in colon cancer.

Phenotypic plasticity allows carcinoma cells to transiently acquire the quasi-mesenchymal features necessary to detach from the primary mass and proceed along the invasion-metastasis cascade. A broad spectrum of epigenetic mechanisms is likely to cause the epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) transitions necessary to allow local dissemination and distant metastasis. Here, we report on the role played by alternative splicing (AS) in eliciting phenotypic plasticity in epithelial malignancies with focus on colon cancer. By taking advantage of the coexistence of subpopulations of fully epithelial (EpCAMhi) and quasi-mesenchymal and highly metastatic (EpCAMlo) cells in conventional human cancer cell lines, we here show that the differential expression of ESRP1 and other RNA-binding proteins (RBPs) downstream of the EMT master regulator ZEB1 alters the AS pattern of a broad spectrum of targets including CD44 and NUMB, thus resulting in the generation of specific isoforms functionally associated with increased invasion and metastasis. Additional functional and clinical validation studies indicate that both the newly identified RBPs and the CD44s and NUMB2/4 splicing isoforms promote local invasion and distant metastasis and are associated with poor survival in colon cancer. The systematic elucidation of the spectrum of EMT-related RBPs and AS targets in epithelial cancers, apart from the insights in the mechanisms underlying phenotypic plasticity, will lead to the identification of novel and tumor-specific therapeutic targets.

Epithelial to mesenchymal transition (EMT) of feto-maternal reproductive tissues generates inflammation: a detrimental factor for preterm birth

Human pregnancy is a delicate and complex process where multiorgan interactions between two independent systems, the mother, and her fetus, maintain pregnancy. Intercellular interactions that can define homeostasis at the various cellular level between the two systems allow uninterrupted fetal growth and development until delivery. Interactions are needed for tissue remodeling during pregnancy at both fetal and maternal tissue layers. One of the mechanisms that help tissue remodeling is via cellular transitions where epithelial cells undergo a cyclic transition from epithelial to mesenchymal (EMT) and back from mesenchymal to epithelial (MET). Two major pregnancy-associated tissue systems that use EMT, and MET are the fetal membrane (amniochorion) amnion epithelial layer and cervical epithelial cells and will be reviewed here. EMT is often associated with localized inflammation, and it is a well-balanced process to facilitate tissue remodeling. Cyclic transition processes are important because a terminal state or the static state of EMT can cause accumulation of proinflammatory mesenchymal cells in the matrix regions of these tissues and increase localized inflammation that can cause tissue damage. Interactions that determine homeostasis are often controlled by both endocrine and paracrine mediators. Pregnancy maintenance hormone progesterone and its receptors are critical for maintaining the balance between EMT and MET. Increased intrauterine oxidative stress at term can force a static (terminal) EMT and increase inflammation that are physiologic processes that destabilize homeostasis that maintain pregnancy to promote labor and delivery of the fetus. However, conditions that can produce an untimely increase in EMT and inflammation can be pathologic. These tissue damages are often associated with adverse pregnancy complications such as preterm prelabor rupture of the membranes (pPROM) and spontaneous preterm birth (PTB). Therefore, an understanding of the biomolecular processes that maintain cyclic EMT-MET is critical to reducing the risk of pPROM and PTB. Extracellular vesicles (exosomes of 40-160 nm) that can carry various cargo are involved in cellular transitions as paracrine mediators. Exosomes can carry a variety of biomolecules as cargo. Studies specifically using exosomes from cells undergone EMT can carry a pro-inflammatory cargo and in a paracrine fashion can modify the neighboring tissue environment to cause enhancement of uterine inflammation.

Emergence of Resistance to MTI-101 Selects for a MET Genotype and Phenotype in EGFR Driven PC-9 and PTEN Deleted H446 Lung Cancer Cell Lines.

Simple SummaryMTI-101 is a first-in-class novel cyclic peptide shown to have anti-tumor activity in both multiple myeloma and castrate-resistant prostate in vivo cancer models. These data suggest the potential for a broad spectrum of anti-cancer activity for this class of compounds. To further delineate determinants of sensitivity and resistance that were not dependent on oncogenic drivers, two isogenic drug-resistant cell lines were generated with chronic exposure to MTI-101 in a non-small cell lung cancer (NSCLC) PC-9 (EGFR driven) and a small cell lung cancer (SCLC) H446 (PTEN deleted and c-MYC amplified). Our data indicate that the chronic exposure of MTI-101 selects for a stable mesenchymal-to-epithelial (MET) genotype and phenotype in both PC-9 and H446 lung cancer cell lines.MTI-101 is a first-in-class cyclic peptide that kills cells via calcium overload in a caspase-independent manner. Understanding biomarkers of response is critical for positioning a novel therapeutic toward clinical development. Isogenic MTI-101-acquired drug-resistant lung cancer cell line systems (PC-9 and H446) coupled with differential RNA-SEQ analysis indicated that downregulated genes were enriched in the hallmark gene set for epithelial-to-mesenchymal transition (EMT) in both MTI-101-acquired resistant cell lines. The RNA-SEQ results were consistent with changes in the phenotype, including a decreased invasion in Matrigel and expression changes in EMT markers (E-cadherin, vimentin and Twist) at the protein level. Furthermore, in the EGFR-driven PC-9 cell line, selection for resistance towards MTI-101 resulted in collateral sensitivity toward EGFR inhibitors. MTI-101 treatment showed synergistic activity with the standard of care agents erlotinib, osimertinib and cisplatin when used in combination in PC-9 and H446 cells, respectively. Finally, in vivo data indicate that MTI-101 treatment selects for increased E-cadherin and decreased vimentin in H446, along with a decreased incident of bone metastasis in the PC-9 in vivo model. Together, these data indicate that chronic MTI-101 treatment can lead to a change in cell state that could potentially be leveraged therapeutically to reduce metastatic disease.

Smart Material‐Integrated Systems for Isolation and Profiling of Rare Cancer Cells and Emboli

This review presents a broad aspect of smart material‐integrated systems for isolating and profiling rare circulating tumor cells (CTCs) and circulating tumor microemboli (CTM) to provide physiological, biological, and mechanistic insights into cancer research. In particular, CTCs/CTM have emanated as essential pieces of evidence that can reveal clonal evolution, tumor heterogeneity, and disease progression within the metastatic cascade. Morphologies, cellular compositions, and rarity of CTCs/CTM make them difficult to track and isolate for profiling distinct molecular characteristics in the case of metastatic potential. Accordingly, with the advanced‐characterization techniques, examining the aspects of the specific surface markers of CTCs/CTM, epithelial‐to‐mesenchymal (EMT), mesenchymal‐to‐epithelial (MET) transitions, and timing of tumor cell dissemination would assist us to understand cancer biology and metastatic characteristics. Existing clinical and research methods for the enrichment and isolation of these sporadic cells depend on mainly conventional methods with low‐yield and expensive features. Owing to their specialized functions and analytical performances, smart material‐based technologies hold an enormous impact not only on cell detection, but also on cell isolation for downstream analyses. Herein, the main reasons for cell isolation are discussed and the recent developments in CTCs/CTM approaches for identifying further methods and future perspectives are elaborated on.

Epithelial-to-Mesenchymal Transition Is Not a Major Modulating Factor in the Cytotoxic Response to Natural Products in Cancer Cell Lines.

Numerous natural products exhibit antiproliferative activity against cancer cells by modulating various biological pathways. In this study, we investigated the potential use of eight natural compounds (apigenin, curcumin, epigallocatechin gallate, fisetin, forskolin, procyanidin B2, resveratrol, urolithin A) and two repurposed agents (fulvestrant and metformin) as chemotherapy enhancers and mesenchymal-to-epithelial (MET) inducers of cancer cells. Screening of these compounds in various colon, breast, and pancreatic cancer cell lines revealed anti-cancer activity for all compounds, with curcumin being the most effective among these in all cell lines. Although some of the natural products were able to induce MET in some cancer cell lines, the MET induction was not related to increased synergy with either 5-FU, irinotecan, gemcitabine, or gefitinib. When synergy was observed, for example with curcumin and irinotecan, this was unrelated to MET induction, as assessed by changes in E-cadherin and vimentin expression. Our results show that MET induction is compound and cell line specific, and that MET is not necessarily related to enhanced chemosensitivity.

Glucocorticoid receptor-targeted liposomal delivery system for delivering small molecule ESC8 and anti-miR-Hsp90 gene construct to combat colon cancer

High mortality rate in colon cancer patients is often attributed to late diagnosis. To overcome the conventional chemotherapy associated challenges, chemotherapeutic drugs (single or combination) or genetic drugs are often delivered using ligand-modified delivery systems that selectively target over expressed receptors or particular receptors that act abnormally in cancer cells. In the current investigation, first we assessed anti-colon cancer effect of a cationic estrogenic molecule, ESC8 which was earlier shown to act against estrogen receptor (ER) ± breast cancer cells. We found that against both colon and breast cancer cells the anticancer activity is intervened by AMPK-mTOR pathway and at the same time it acts as anti-angiogenic agent. It also showed enhancement of mesenchymal-to-epithelial (MET) transition as well as reduction of cyclin D in both cells. Earlier we demonstrated the use of glucocorticoid receptor (GR) targeted cationic liposomal delivery system carrying anti-Hsp90 plasmid and ESC8 to act as potent anti-skin cancer therapeutics. As ESC8 demonstrated anti-colon cancer effect in vitro, in here, we used the same GR-targeted liposomal formulation but carrying a more fusogenic cationic lipid D1 and used against colon tumor orthotopic model in mice. We show that GR targeted formulation (D1XE-Hsp90) exhibited efficient cellular uptake, transfection and selective cytotoxicity in colon cancer cells, tumor-targeted bio-distribution and enhanced survivability, reduced tumor size in orthotopic colon tumor-bearing mice. The tumor sections exhibited reduced tumor proliferation as well as neo-vascularization, thus supporting the holistic antitumor effect of the D1XE-Hsp90 formulation. Over all our results establish the GR-targeted D1XE-Hsp90 formulation as potent anti-colon cancer therapeutics.

Quick, Coordinated and Authentic Reprogramming of Ribosome Biogenesis during iPSC Reprogramming.

Induction of pluripotent stem cells (iPSC) by OCT4 (octamer-binding transcription factor 4), SOX2 (SR box 2), KLF4 (Krüppel-Like Factor 4), and MYC (cellular Myelocytomatosis, c-MYC or MYC) (collectively OSKM) is revolutionary, but very inefficient, slow, and stochastic. It is unknown as to what underlies the potency aspect of the multi-step, multi-pathway, and inefficient iPSC reprogramming. Mesenchymal-to-epithelial (MET) transition is known as the earliest pathway reprogrammed. Using the recently established concepts of reprogramome and reprogramming legitimacy, the author first demonstrated that ribosome biogenesis (RB) is globally enriched in terms of human embryonic stem cells in comparison with fibroblasts, the popular starting cells of pluripotency reprogramming. It is then shown that the RB network was reprogrammed quickly in a coordinated fashion. Human iPSCs also demonstrated a more robust ribosome biogenesis. The quick and global reprogramming of ribosome biogenesis was also observed in an independent fibroblast line from a different donor. This study additionally demonstrated that MET did not initiate substantially at the time of proper RB reprogramming. This quick, coordinated and authentic RB reprogramming to the more robust pluripotent state by the OSKM reprogramming factors dramatically contrasts the overall low efficiency and long latency of iPSC reprogramming, and aligns well with the potency aspect of the inefficient OSKM reprogramming.

Novel pathways of inflammation in human fetal membranes associated with preterm birth and preterm pre-labor rupture of the membranes

Spontaneous preterm birth (PTB) and preterm pre-labor rupture of the membranes (pPROM) are major pregnancy complications. Although PTB and pPROM have common etiologies, they arise from distinct pathophysiologic pathways. Inflammation is a common underlying mechanism in both conditions. Balanced inflammation is required for fetoplacental growth; however, overwhelming inflammation (physiologic at term and pathologic at preterm) can lead to term and preterm parturition. A lack of effective strategies to control inflammation and reduce the risk of PTB and pPROM suggests that there are several modes of the generation of inflammation which may be dependent on the type of uterine tissue. The avascular fetal membrane (amniochorion), which provides structure, support, and protection to the intrauterine cavity, is one of the key contributors of inflammation. Localized membrane inflammation helps tissue remodeling during pregnancy. Two unique mechanisms that generate balanced inflammation are the progressive development of senescence (aging) and cyclic cellular transitions: epithelial to mesenchymal (EMT) and mesenchymal to epithelial (MET). The intrauterine build-up of oxidative stress at term or in response to risk factors (preterm) can accelerate senescence and promote a terminal state of EMT, resulting in the accumulation of inflammation. Inflammation degrades the matrix and destabilizes membrane function. Inflammatory mediators from damaged membranes are propagated via extracellular vesicles (EV) to maternal uterine tissues and transition quiescent maternal uterine tissues into an active state of labor. Membrane inflammation and its propagation are fetal signals that may promote parturition. This review summarizes the mechanisms of fetal membrane cellular senescence, transitions, and the generation of inflammation that contributes to term and preterm parturitions.

CCAAT/enhancer binding protein delta (C/EBPδ) demonstrates a dichotomous role in tumour initiation and promotion of epithelial carcinoma

BackgroundCCAAT/enhancer binding protein delta (C/EBPδ,CEBPD), a gene part of the highly conserved basic-leucine zipper (b-ZIP) domain of transcriptional factors, is downregulated in 65% of high grade serous carcinomas of the ovary (HGSC). Overexpression of C/EBPδ in different tumours, such as glioblastoma and breast cancer either promotes tumour progression or inhibits growth and has low expression in normal tissue until activated by cytotoxic stressors. MethodsHigher overall expression of C/EBPδ in the luteal phase of the menstrual cycle prompted us to investigate the role of C/EBPδ in carcinogenesis. In vitro experiments were conducted in fallopian tube cell samples and cancer cell lines to investigate the role of C/EBPδ in proliferation, migration, and the epithelial to mesenchymal transition. FindingsExpression of C/EBPδ induced premature cellular arrest and decreased soft agar colony formation. Loss of C/EBPδ in epithelial cancer cell lines did not have significant effects on proliferation, yet overexpression demonstrated downregulation of growth, similar to normal fallopian tube cells. C/EBPδ promoted a partial mesenchymal to epithelial (MET) phenotype by upregulating E-cadherin and downregulating Vimentin and N-cadherin in FTE cells and increased migratory activity, which suggests a regulatory role in the epithelial-mesenchymal plasticity of these cells. InterpretationOur findings suggest that C/EBPδ regulates the phenotype of normal fallopian tube cells by acting on downstream regulatory factors that are implicated in the development of ovarian serous carcinogenesis. FundThis study was funded by the CDMRP Ovarian Cancer program (W81WH-0701-0371, W81XWH-18-1-0072), the Princess Margaret Cancer Centre Foundation, Foundation for Women's Cancer – The Belinda-Sue/Mary-Jane Walker Fund, Colleen's Dream Foundation and Sylvester Comprehensive Cancer Center.

SCD5 restored expression favors differentiation and epithelial-mesenchymal reversion in advanced melanoma.

Our previous data supported a role for the Stearoyl-CoA desaturase (SCD5) in protection against malignancy, whereby it appears to functionally modify tumor stroma impairing tumor spread. SCD5 is significantly expressed in primary melanoma, but becomes barely detectable at tumor advanced stages. Looking for the regulatory mechanisms underlying SCD5 reduced expression during melanoma progression, we demonstrated a significantly lower stability of SCD5 protein as well as the direct targeting of SCD5 mRNA by the oncogenic miR-221&222 in metastatic cell lines. Moreover, our results indicated the existence of a negative feedback loop between SCD5 and miR-221&222, in good agreement with their opposite functions. Also, we showed how SCD5 re-expression and the direct supplementation of its main product oleic acid (OA) can drive advanced melanoma cell lines toward differentiation and reversion of the epithelial-mesenchymal (EMT)-like process, eventually inducing a less malignant phenotype. Indeed, SCD5 re-established the sensitivity to all-trans retinoic acid in A375M metastatic melanoma, associated with increased levels of Tyrosinase, melanin production and reduced proliferation. As evidenced by the correct modulation of some key transcription factors, SCD5 managed by favoring a partial mesenchymal-to-epithelial (MET) transition in in vitro studies. Interestingly, a more complete MET, including E-cadherin re-expression correctly localized at cell membranes, was obtained in in vivo xenograft models, thus indicating the requirement of direct contacts between tumor cells and the surrounding microenvironment as well as the presence of some essential factors for SCD5 complete function.

Oxygen-dependent regulation of tumor growth and metastasis in human breast cancer xenografts.

BackgroundTumor hypoxia is relevant for tumor growth, metabolism, resistance to chemotherapy and metastasis. We have previously shown that hyperoxia, using hyperbaric oxygen treatment (HBOT), attenuates tumor growth and shifts the phenotype from mesenchymal to epithelial (MET) in the DMBA-induced mammary tumor model. This study describes the effect of HBOT on tumor growth, angiogenesis, chemotherapy efficacy and metastasis in a triple negative MDA-MB-231 breast cancer model, and evaluates tumor growth using a triple positive BT-474 breast cancer model.Materials and methods5 x 105 cancer cells were injected s.c. in the groin area of NOD/SCID female mice. The BT-474 group was supplied with Progesterone and Estradiol pellets 2-days prior to tumor cell injection. Mice were divided into controls (1 bar, pO2 = 0.2 bar) or HBOT (2.5 bar, pO2 = 2.5 bar, 90 min, every third day until termination of the experiments). Treatment effects were determined by assessment of tumor growth, proliferation (Ki67-staining), angiogenesis (CD31-staining), metastasis (immunostaining), EMT markers (western blot), stromal components collagen type I, Itgb1 and FSP1 (immunostaining) and chemotherapeutic efficacy (5FU).ResultsHBOT significantly suppressed tumor growth in both the triple positive and negative tumors, and both MDA-MB-231 and BT-474 showed a decrease in proliferation after HBOT. No differences were found in angiogenesis or 5FU efficacy between HBOT and controls. Nevertheless, HBOT significantly reduced both numbers and total area of the metastastatic lesions, as well as reduced expression of N-cadherin, Axl and collagen type I measured in the MDA-MB-231 model. No change in stromal Itgb1 and FSP1 was found in either tumor model.ConclusionDespite the fact that behavior and prognosis of the triple positive and negative subtypes of cancer are different, the HBOT had a similar suppressive effect on tumor growth, indicating that they share a common oxygen dependent anti-tumor mechanism. Furthermore, HBOT significantly reduced the number and area of metastatic lesions in the triple negative model as well as a significant reduction in the EMT markers N-cadherin, Axl and density of collagen type I.

Complex Determinants of Epithelial: Mesenchymal Phenotypic Plasticity in Ovarian Cancer

Unlike most epithelial malignancies which metastasize hematogenously, metastasis of epithelial ovarian cancer (EOC) occurs primarily via transcoelomic dissemination, characterized by exfoliation of cells from the primary tumor, avoidance of detachment-induced cell death (anoikis), movement throughout the peritoneal cavity as individual cells and multi-cellular aggregates (MCAs), adhesion to and disruption of the mesothelial lining of the peritoneum, and submesothelial matrix anchoring and proliferation to generate widely disseminated metastases. This exceptional microenvironment is highly permissive for phenotypic plasticity, enabling mesenchymal-to-epithelial (MET) and epithelial-to-mesenchymal (EMT) transitions. In this review, we summarize current knowledge on EOC heterogeneity in an EMT context, outline major regulators of EMT in ovarian cancer, address controversies in EMT and EOC chemoresistance, and highlight computational modeling approaches toward understanding EMT/MET in EOC.

Deep-proteome mapping of WM-266-4 human metastatic melanoma cells: From oncogenic addiction to druggable targets.

Cutaneous melanoma is a malignant tumor of skin melanocytes that are pigment-producing cells located in the basal layer (stratum basale) of epidermis. Accumulation of genetic mutations within their oncogenes or tumor-suppressor genes compels melanocytes to aberrant proliferation and spread to distant organs of the body, thereby resulting in severe and/or lethal malignancy. Metastatic melanoma’s heavy mutational load, molecular heterogeneity and resistance to therapy necessitate the development of novel biomarkers and drug-based protocols that target key proteins involved in perpetuation of the disease. To this direction, we have herein employed a nano liquid chromatography-tandem mass spectrometry (nLC-MS/MS) proteomics technology to profile the deep-proteome landscape of WM-266-4 human metastatic melanoma cells. Our advanced melanoma-specific catalogue proved to contain 6,681 unique proteins, which likely constitute the hitherto largest single cell-line-derived proteomic collection of the disease. Through engagement of UNIPROT, DAVID, KEGG, PANTHER, INTACT, CYTOSCAPE, dbEMT and GAD bioinformatics resources, WM-266-4 melanoma proteins were categorized according to their sub-cellular compartmentalization, function and tumorigenicity, and successfully reassembled in molecular networks and interactomes. The obtained data dictate the presence of plastically inter-converted sub-populations of non-cancer and cancer stem cells, and also indicate the oncoproteomic resemblance of melanoma to glioma and lung cancer. Intriguingly, WM-266-4 cells seem to be subjected to both epithelial-to-mesenchymal (EMT) and mesenchymal-to-epithelial (MET) programs, with 1433G and ADT3 proteins being identified in the EMT/MET molecular interface. Oncogenic addiction of WM-266-4 cells to autocrine/paracrine signaling of IL17-, DLL3-, FGF(2/13)- and OSTP-dependent sub-routines suggests their critical contribution to the metastatic melanoma chemotherapeutic refractoriness. Interestingly, the 1433G family member that is shared between the BRAF- and EMT/MET-specific interactomes likely emerges as a novel and promising druggable target for the malignancy. Derailed proliferation and metastatic capacity of WM-266-4 cells could also derive from their metabolic addiction to pathways associated with glutamate/ammonia, propanoate and sulfur homeostasis, whose successful targeting may prove beneficial for advanced melanoma-affected patients.

Eribulin sensitizes oral squamous cell carcinoma cells to cetuximab via induction of mesenchymal-to-epithelial transition

Inhibition of epidermal growth factor receptor (EGFR) signalling has emerged as a new treatment strategy for oral squamous cell carcinoma (OSCC). Previously, we found that loss of EGFR expression in OSCC was associated with epithelial-mesenchymal transition (EMT), and may have functional implications with regard to resistance to cetuximab, a monoclonal anti-EGFR antibody. Eribulin (a microtubule inhibitor) reportedly renders breast cancer less aggressive, and less likely to metastasise, by triggering mesenchymal-to-epithelial (MET) transition. In the present study we evaluated whether eribulin-induced MET was associated with re-sensitization of resistant OSCC cell lines to cetuximab. In vitro antiproliferative activities were determined in three human OSCC lines (OSC-20, OSC-19 and HOC313) treated with eribulin. These three human OSCC represented different EMT/MET states. Interestingly, HOC313 cells (mesenchymal phenotype) were highly sensitive to eribulin in comparison with other cell lines, and significantly enhanced the anti-proliferative effect of cetuximab in response to the drug. Eribulin also underwent a MET-associated gene switch that resulted in morphological changes and high EGFR expression in HOC313 cells, and abrogated a TGF-β-induced EMT gene expression signature. Eribulin-dependent sensitization of OSCC to cetuximab is likely due to induction of MET. Combination therapies based on eribulin and cetuximab have potential as a novel treatment regimen in OSCC.

Highlights of This Issue

The oncogenic epithelial-to-mesenchymal transition (EMT) contributes to tumor progression in several ways, including resistance to anoikis, resulting in metastatic potential. Here, an interesting mechanism is described for EMT to confer anoikis resistance. Through the up-regulation of glutamate dehydrogenase, EMT enhances mitochondrial metabolism, while simultaneously reducing reactive oxygen species (ROS), thereby protecting against anoikis. These effects are reversed by the master programmer of the epithelial phenotype, Grainyhead-like 2 (GRHL2). These results demonstrate that EMT and mesenchymal-to-epithelial (MET) regulate anoikis through metabolic pathways that are amenable to pharmacologic intervention.This study conclusively demonstrates the central role played by the chemokine receptor CX3CR1 in the seeding and colonization of the skeleton by breast cancer cells and also reports the synthesis, functional characterization and pre-clinical validation of a new small-molecule inhibitor of CX3CR1. Interfering with CX3CR1 activation dramatically impairs both spreading and growth of existing metastases in animal models. As emerging evidence emphasizes the impact that the cross-seeding of existing metastases exerts on clinical progression, blocking the seeding and initial colonization of new lesions by inhibiting CX3CR1 could prevent, decelerate or even stabilize metastatic breast cancer.Here, Zhang and colleagues, from the Duke Cancer Institute, have developed a novel circulating tumor cell (CTC) detection platform, modified from the original Janssen Cellsearch assay, to detect c-MET amplified tumor cells from the blood. The investigators developed and validated their CTC assay in appropriate healthy volunteers and spiked cell line controls. Importantly, the study also identified patients with metastatic gastric, renal, colorectal, and bladder cancer with detectable MET-amplified CTCs. This novel CTC capture approach serves as a predictive biomarker for future studies involving c-MET pathway inhibitors in patients with advanced solid tumors.Protein Tyrosine Kinase 6 (PTK6) promotes epithelial differentiation in the gastrointestinal tract and skin, while membrane-associated active PTK6 promotes oncogenic signaling in breast and prostate tumors. Mathur and colleagues report that PTK6 maintains the epithelial phenotype of colon cancer cells in a kinase-independent manner and PTK6 expression decreases during colon cancer progression. However, kinase active PTK6 is also able to promote oncogenic signaling in these same cells. Understanding the distinct context-dependent adaptor and kinase functions of PTK6 is critical for determining its potential as a therapeutic target.

Oncogenic ALK regulates EMT in non-small cell lung carcinoma through repression of the epithelial splicing regulatory protein 1.

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy.

Abstract 4726: Gene expression signatures of isolated circulating tumor cells from metastatic breast cancer patients reveal presence of breast cancer stem cells with EMT or MET phenotypes

Abstract Breast cancer stem cells are responsible for tumor recurrence, metastasis and drug resistance. One of the important steps in metastasis is entry of tumor cells into the blood circulation. These circulating tumor cells (CTCs) may serve as markers of cancer progression and as a “liquid biopsy” to provide information on tumor biology at single cell resolution. Current methods of CTC capture are not fully inclusive to isolate all phenotypic variations of cancer stem cells (CSCs) among the CTCs. Using newly developed microfluidic methods to isolate CTCs from blood samples of metastatic breast cancer patients, we assessed gene expression profiles in these cells. The gene expression signatures of isolated CTCs were determined using a highly sensitive microfluidic-based 96-plex RT-qPCR method. Analyzed multiplex RT-qPCR data revealed distinct gene expression patterns among CTCs isolated from different patients. Cancer stem cell markers such as ALDH1a1, ALDH1a2 and ALDH1a3 as well as CD44 and CD24 genes were differentially expressed in isolated CTCs. In addition, markers of the epithelial to mesenchymal (EMT) and mesenchymal to epithelial (MET) phenotypes such as Vimentin, EpCAM, HER2, CDH1, CDH2, TGFb1 and cytokeratins were also expressed variably in the CTC samples. Furthermore, MMP9, TIMP1, CD90, CD14, SPARC, BRAF, KRAS, PI3K and ERCC1 genes which play a role in cancer initiation, promotion, metastasis and drug resistance were also differentially expressed in the isolated CTCs. Overall, these studies further our understanding into the heterogeneity of CTCs and CSCs as well as providing a potential tool for real-time monitoring of cancer patients on clinical trials. Citation Format: Shamileh Fouladdel, Hyeun Joong Yoon, Eric Lin, Tae Hyun Kim, Yadwinder S. Deol, Tahra K. Luther, Shawn G. Clouthier, Hui Jiang, Monika L. Burness, Sunitha Nagrath, Ebrahim Azizi, Max S. Wicha. Gene expression signatures of isolated circulating tumor cells from metastatic breast cancer patients reveal presence of breast cancer stem cells with EMT or MET phenotypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4726. doi:10.1158/1538-7445.AM2015-4726

Abstract 4834: Single cell multiplex gene expression analysis to unravel heterogeneity of PDX samples established from tumors of breast cancer patients with different ethnicity

Abstract Intra-tumor heterogeneity represents one of the main challenges in cancer therapeutics. This heterogeneity is generated at the genetic level primarily by genomic alterations and clonal selection and at the epigenetic level by tumor cell differentiation. In addition, inter-tumor heterogeneities may also be related to differences in tumor biology in different ethnic populations. To explore the cell heterogeneity in breast cancer, we studied patient derived xenograft (PDX) models generated from African (Ghana), African-American, and Caucasian breast cancer patients. Gene expression of a selected panel of cancer associated genes was determined in single cells using C1 and BioMark HD platforms. Analyzed multiplex RT-qPCR data revealed significant heterogeneity in mRNA expression within and between studied PDX samples at single cell resolution. We observed a significantly different mRNA expression profile within each ethnic group of breast cancer PDX samples. In addition, distinct gene expression signatures were observed between the PDX samples of different ethnic origin. ALDH1a1, ALDH1a3, CD44, CD24, and CD133 were among the CSC markers that were differentially expressed in different PDX single cell samples. Vimentin, EpCAM, HER2, CDH1, CDH2, TGFb1, cytokeratins, GATA3 and MKI67 genes related to the epithelial to mesenchymal (EMT) and mesenchymal to epithelial (MET) phenotypes also demonstrated significantly variable expression in the studied PDX samples. YAP1, TM4SF1, TSPAN6, AMOTL2, STAP2 and ANXA3 genes were differentially expressed in the PDX samples. These candidate biomarkers represent a collection of genes that may warrant further investigation for their potential involvement in cancer initiation, promotion and progression to metastasis, and drug resistance. These data emphasize the importance of studying tumors at single cell resolution to unravel gene expression variation. Upon validation in clinical tumor specimens, this approach could help to identify cellular subpopulations within tumors and possibly identify novel targets for cancer therapy. Citation Format: Ebrahim Azizi, Evelyn M. Jiagge, Shamileh Fouladdel, Shukmei Wong, Michele L. Dziubinski, Mary Sehl, Anahita Kyani, Jun Li, Hui Jiang, Tahra K. Luther, Shawn G. Clouthier, Sean P. McDermott, John Carpten, Lisa A. Newman, Sofia D. Merajver, Max S. Wicha. Single cell multiplex gene expression analysis to unravel heterogeneity of PDX samples established from tumors of breast cancer patients with different ethnicity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4834. doi:10.1158/1538-7445.AM2015-4834

Abstract LB-185: Novel activity of selective inhibitors of nuclear export in epithelial-to-mesenchymal transition models

Abstract Epithelial-to-mesenchymal transition (EMT) that confers tumors an aggressive phenotype is a fine tuned process regulated by a number of molecules that are strategically distributed in the nuclear and cytosolic compartments of cancer cells. Major EMT regulating proteins and transcription factors (TFs), such as wnt/βcatenin, notch, TGF-β, Twist and Snail are recognized to undergo nuclear-cytosolic shuttling using specialized transporters; karyopherins. The export of most of the EMT regulating TFs is mediated exclusively by Exportin1/XPO1/CRM1 through nuclear exclusion sequence (NES) recognition. These observations indicate that nuclear transport may play a critical role in the development of EMT. However, to date there have been no studies evaluating the impact of nuclear export inhibition on EMT. We have earlier developed Specific Inhibitors of Nuclear Export (SINEs) that block XPO1 leading to nuclear retention of various tumor suppressor proteins (TSPs) and TFs. This is the first report showing that XPO1 inhibition by SINEs induce global re-organization of proteins leading to reversal of EMT in Robert Weinberg's snail transduced immortalized human mammary epithelial (HMLE-Snail) cells. Snail is a NES carrying transcription factor that has been documented to regulate EMT from either the nucleus or cytosol. Exposure of HMLE-Snail cells to SINEs to clinically relevant concentration of 0-200 nM [i.e. KPT-185, its inactive analog KPT-TRANS, and clinical agent Selinexor (KPT-330)] resulted in reversal of cellular morphology from mesenchymal to epithelial (MET) that was concurrent with growth inhibition and apoptosis. KPT-185 treatment at 150 nM (IC50) concentration suppressed spheroid forming ability of HMLE-snail cells. Molecular analysis of KPT-185 treated HMLE-snail cells and corresponding spheroids showed suppression of key EMT markers including snail, Twist, vimentin and EpCAM. Co-immunoprecipitation studies verified the disruption of the pre-dominantly cytosolic snail-XPO1 interaction upon SINE treatment. In order to understand the mechanism of EMT reversal, RNA from quadruplet SINE treated HMLE-Snail cells were subjected to mRNA microarrays and systems biology analysis. KPT-185 treatment resulted in differential expression of 720 genes (fold change p<0.001). The statistically significant differentially expressed genes were subjected to pathway analysis (upstream and downstream regulator analysis and similarity analysis to known EMT gene networks), which showed downregulation in Snail, HDAC1 and Glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE) networks. Conversely, the activated networks included apoptosis and p73 signaling. The network genes were validated at the expression level using, RT-PCR and western blotting. The anti-tumor activity of SINEs against HMLE-snail derived spheroids grown subcutaneously in mice is currently being evaluated. In conclusion, this is the first study demonstrating the role of XPO1 in EMT. Selinexor is currently being evaluated in 3 phase 1 studies in patients with advanced solid tumor and hematological malignancies. Therefore, in view of our results, XPO1 inhibition through SINEs can be a new form of therapy for highly aggressive and metastatic tumors that harbor EMT. Citation Format: Asfar S. Azmi, Sharon Shacham, Michael Kauffman, Dilara McCauley, Amro Aboukameel, Ramzi M. Mohammad. Novel activity of selective inhibitors of nuclear export in epithelial-to-mesenchymal transition models. [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 LB-185. doi:10.1158/1538-7445.AM2014-LB-185