Cancer Cell Plasticity Research Articles

Integrating Biological and Mathematical Models to Explain and Overcome Drug Resistance in Cancer, Part 2: from Theoretical Biology to Mathematical Models

Despite the promise of targeted cancer therapy (i.e., drugs targeted towards specific signaling pathways supposed to be essential to the survival and proliferation of cancer cells), unexpected treatment failures in the clinic are common. Tumor cell heterogeneity, which can result from the plasticity of individual cancer cells and/or evolutionary dynamics, has revealed the importance to study cancer at the level of the entire cell population. Here, we explore mathematical models that describe tumor dynamics under pressure of anticancer drugs by integrating cancer cell population heterogeneity and evolutionary behavior. We further explore mathematical modeling as a theoretical tool to analyze and predict the behavior of cancer cell populations as a whole, and not only of individual cells, which may reveal new clues to therapy failure and ways to overcome it. An evolutionary perspective that relies on the “atavistic theory of cancer” together with the so-called “cold genes”, and the involvement of “bet hedging” in tumors, has recently changed our vision of cell plasticity in cancer. These new perspectives provide a sound theoretical basis to the emergence of resistance in cancer cell populations and to its possible reversibility. Continuous mathematical models of the evolutionary dynamics of proliferative cell populations already exist, that take into account the heterogeneity of cancer cell populations, allowing to study the evolutionary potential of cancer cell populations and predict their behavior. Those models, in turn, can be used to probe population growth control by incorporating functions (in the mathematical model) that represent the action of drugs on the mechanisms driving proliferation, and furthermore to suggest new therapeutic strategies in the clinic of cancers. In this second part of our review, that can be taken independently of Part I, we focus on the level of cell populations, the only one amenable to completely take into account phenotype plasticity in its observable consequences on the evolution of proliferative diseases, and on heterogeneity, that makes sense only in the context of cell populations, together with the fundamental evolutionary potential of cancer cell populations (and not only of single cancer cells). These are modern views that come from the transposition from ecology of species to the biology of cancer (for further reading on evolution and cancer, the reader is referred to the recent comprehensive book on the subject Ujvari et al. 2017). Finally, we present a brief review of mathematical models, with their features and their use in cell population studies, to account for phenomena found in cancer, focusing on drug resistance. We contend that a good understanding of what mathematical models can do to tackle the question of drug resistance in cancer can shed light on the mechanisms of resistance and means to control them and help design principles for biological experiments to be performed at the lab and therapeutical strategies to be applied in the clinic of cancers.

Proteomic Analysis of the E3 Ubiquitin-Ligase Hakai Highlights a Role in Plasticity of the Cytoskeleton Dynamics and in the Proteasome System.

Carcinoma, the most common type of cancer, arises from epithelial cells. The transition from adenoma to carcinoma is associated with the loss of E-cadherin and, in consequence, the disruption of cell-cell contacts. E-cadherin is a tumor suppressor, and it is down-regulated during epithelial-to-mesenchymal transition (EMT); indeed, its loss is a predictor of poor prognosis. Hakai is an E3 ubiquitin-ligase protein that mediates E-cadherin ubiquitination, endocytosis and finally degradation, leading the alterations of cell-cell contacts. Although E-cadherin is the most established substrate for Hakai activity, other regulated molecular targets for Hakai may be involved in cancer cell plasticity during tumor progression. In this work we employed an iTRAQ approach to explore novel molecular pathways involved in Hakai-driven EMT during tumor progression. Our results show that Hakai may have an important influence on cytoskeleton-related proteins, extracellular exosome-associated proteins, RNA-related proteins and proteins involved in metabolism. Moreover, a profound decreased expression in several proteasome subunits during Hakai-driven EMT was highlighted. Since proteasome inhibitors are becoming increasingly used in cancer treatment, our findings suggest that the E3 ubiquitin-ligase, such as Hakai, may be a better target than proteasome for using novel specific inhibitors in tumor subtypes that follow EMT.

Epithelial-to-Mesenchymal Transition in the Female Reproductive Tract: From Normal Functioning to Disease Pathology

Epithelial-to-mesenchymal transition (EMT) is a physiological process that is vital throughout the human lifespan. In addition to contributing to the development of various tissues within the growing embryo, EMT is also responsible for wound healing and tissue regeneration later in adulthood. In this review, we highlight the importance of EMT in the development and normal functioning of the female reproductive organs (the ovaries and the uterus) and describe how dysregulation of EMT can lead to pathological conditions, such as endometriosis, adenomyosis, and carcinogenesis. We also summarize the current literature relating to EMT in the context of ovarian and endometrial carcinomas, with a particular focus on how molecular mechanisms and the tumor microenvironment can govern cancer cell plasticity, therapy resistance, and metastasis.

Abstract 4166: Role of CD44 as a regulator of adaptive plasticity in pancreatic cancer cells

Abstract Purpose: Patients with pancreatic ductal adenocarcinoma (PDAC) may show an initial objective response to chemotherapy that is usually of short duration. Our research goal is to understand the molecular mechanisms for this short duration response and to more fully define differences between chemosensitive and chemoresistant cells. Cancer cells that undergo an EMT and metastatic tumors show an increase in CD44s expression. In this study, we use CD44 Low and CD44 High isogenic cell models to identifying key molecular networks and potential targets that could benefit therapy. Experiments design: CFPAC1-CD44 Hi and Low cells were sorted by flowcytometry and single clone selection. Isogenic model systems were generated by overexpression CD44s in CF-Low cells. Western blot, MTT, migration and invasion assays had been used to characterize cell growth, invasiveness and gemcitabine sensitivity. Receptor Tyrosine Kinase arrays were used to identify the CD44 regulated molecular networks in these isogenic cell models treated with/without CD44 ligand hyaluronan acid (HA). Summary: CD44s high cells show an EMT phenotype (increase of vimentin and loss of E-cadherin) and increase invasion. Expression of CD44s in CD44 low cells was sufficient to drive EMT partially and drive invasion. By in vitro testing over expressing CD44s is not sufficient to decrease sensitivity to gemcitabine. By Receptor Tyrosine Kinase arrays, CD44 Hi clone cells show increase in IGF-1R phosphorylation. Preliminary data had shown that HA may stimulate ROR2, VEGFR1, and EphB2 phosphorylation in CD44 Hi clone cells. Conclusion: Our previous study suggested that CD44 could be a therapeutic target in Gemcitabine resistant PDAC cells. The current study will further identify the molecular targets that regulated by CD44 pathways. These newly identified targets could be hopefully used for the optimized multimodality therapy to sensitize the PDAC cells to therapy and to benefit the pancreatic cancer patients. Citation Format: Chen Chen, Shujie Zhao, Xiangru Zhao, James W. Freeman. Role of CD44 as a regulator of adaptive plasticity in pancreatic cancer cells [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 4166. doi:10.1158/1538-7445.AM2017-4166

Abstract 1975: FAM83B: A novel regulator of cell plasticity in breast cancer

Abstract Epithelial to mesenchymal transition (EMT) is an important process in both metastasis and cancer stem cell (CSC) enrichment. Recent studies suggest that instead of simply transitioning from one cell state to another, cancer cells exist along a continuum of epithelial and mesenchymal cell states. Identifying novel regulators of tumor cell plasticity will be critical to fully understand this continuum and to target metastasis and CSC phenotypes in the future. FAM83B is a novel oncogene identified by the Jackson Laboratory in a forward genetic screen for drivers of Human Mammary Epithelial Cell (HMEC) transformation. Previous work showed that FAM83B expression is elevated in triple negative breast cancers and that FAM83B also activates key cell signaling pathways (EGFR, MAPK, and PI3K) in breast cancers. Here, we implicate FAM83B as a novel regulator of epithelial-mesenchymal plasticity using a HMEC transformation model. Transformation of primary HMEC by expressing shp16, shp53, c-Myc, and RAS results in the generation of two distinct transformed cell populations. One population retains epithelial characteristics, while an emergent population spontaneously acquires a mesenchymal morphology and a CD24LOCD44HI (CSC) cell surface marker profile. Moreover, isolation of epithelial, CD24HICD44LO cells (non-CSC) and exposure to Oncostatin M (OSM), an EMT-inducing cytokine, can also generate cells with a mesenchymal/CSC phenotype. Interestingly, FAM83B expression was significantly elevated in the epithelial/non-CSC population compared to the mesenchymal/CSC population. Moreover, treatment of purified epithelial/non-CSC cells with OSM resulted in decreased FAM83B expression levels, indicating FAM83B expression is suppressed as the cells move through EMT. FAM83B expression also correlated with an epithelial phenotype in a panel of breast cancer cell lines. shRNA-mediated knock-down of FAM83B from epithelial/non-CSC resulted in increased spontaneous EMT, concomitant with elevated expression of the mesenchymal marker Vimentin and master EMT transcription factor Zeb1. Following treatment with OSM, sh83B cells maintain prolonged, elevated p-Stat3 and p-ERK signaling, known contributors to the mesenchymal/CSC phenotype. Conversely, OSM-induced EMT was blunted in epithelial/non-CSC expressing exogenous FAM83B. Thus, FAM83B expression is a determinant in maintaining an epithelial phenotype, while suppression of FAM83B is important during EMT. Future work will seek to define novel FAM83B-mediated signaling pathways important for regulating cellular plasticity. Citation Format: Courtney A. Bartel, Damian J. Junk, Mark W. Jackson. FAM83B: A novel regulator of cell plasticity in breast cancer [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 1975. doi:10.1158/1538-7445.AM2017-1975

Abstract 4483: TGF-β downregulates osteopontin isoforms and induce and epithelial plasticity in PC3 metastatic prostate cancer cells

Abstract Total osteopontin (OPN) has been described as a master regulator of epithelial-mesenchymal transition (EMT), including in prostate cancer (PCa). However, the contribution of each OPN splicing isoform (OPN-SI), named OPNa, OPNb, and OPNc in the epithelial plasticity dynamic process is currently unknown. Besides, the contribution of alternative splicing regulation in this scenario is poorly understood. Here we aimed to investigate the expression of OPN-SI and the associated splicing pattern regulation in response to metastatic prostate cancer (PCa) cells treatment with TGF-β . Further, we aimed to investigate the resulting cell phenotypic features. To achieve this, we treated PC3 cells with TGF-β 10ng/ml for 48 h as a model to induce epithelial plasticity in these cells. Total RNA was extracted and the corresponding cDNA was tested for transcriptional levels of OPN-SI, SR and HnRNP splicing regulators as well as classical (EMT) markers by using real time PCR. EMT markers and cell morphology have also been analyzed by immunofluoresce. PC3 cell phenotype has also been evaluated by using cell migration and viabilitity assays in response to β-lapachone cytotoxic reagent. PC3 cells treated with TGF-β displayed an intermediate EMT phenotype, as evidenced by a significant downregulation of both epithelial (E-cadherin, claudin-3 andcytokeratin 18) and mesenchymal markers (vimentin, N-cadherin, snailandslug). These cells presented a higher cytoplasmic E-cadherin expression, as well as a lower vimentin cytoplasmic staining. A major cortical distribution of actin filaments beneath the cell cortex has been observed. Further, a significant downregulation of OPNa and mainly, OPNb and OPNc isoforms, as well as most tested splicing regulators has been shown. TGF-β treatment also promoted a significant inhibition on cell migration as well as higher sensitivity to β-lapachone treatment, as compared to control cells. Altogether, our data evidence that in our in vitro experimental model, TGF-β promoted a an intermediate EMT phenotype, with a predominance of epithelial features, associated to downregulation of OPN-SI and most tested splicing regulators. We then hypothesize that TGF-β may also be an EMT-like inducer, in which metastatic PCa cells could acquire major epithelial features that facilitate cell anchorage and adhesion in distant tumor sites. OPN-SI dowregulation in this process could favour epithelial-like features, as apposed to OPN-SI overexpression during tumor progression towards metastic circulating cells typically displaying mesechymal phenotypes. Citation Format: Durval Marques, Rodrigo A. Peres, Paula P. de Freitas, Abigail C. Resende, Nataly dos Santos Melo, Waldemir Fernandes de Souza, José A. Morgado-Diaz, Etel Rodrigues Pereira Gimba. TGF-β downregulates osteopontin isoforms and induce and epithelial plasticity in PC3 metastatic prostate cancer cells [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 4483. doi:10.1158/1538-7445.AM2017-4483

Abstract LB-139: PRMT6-dependent CRAF/ERK signaling regulates cancer stem cell plasticity in liver cancer

Abstract Hepatocellular carcinoma (HCC), the major type of liver cancer, remains one of the most prevalent and deadliest cancer types in the world. Contemporary challenge in treating HCC has been the common therapy resistance and recurrence after therapy, all of which have been reported to be associated with stem-like behavior of cancer cells. Our group has previously identified a functional liver cancer stem cell (CSC) subset marked by the CD133 cell surface phenotype. Utilizing a PCR array encompassing diverse human chromatin modifiers, protein arginine methyltransferase 6 (PRMT6) was found to be differentially down-regulated in CD133+ liver CSCs of human HCC cells as well as CD133 enriched chemoresistant hepatospheres as compared to their counterparts. Clinically, reduced PRMT6 expression was detected in HCC specimens and correlated with a higher risk of metastasis. PRMT6 negatively regulated diverse in vitro cancer stem cell properties of HCC cells including self-renewal, therapy resistance, metastasis and expression of CSC and pluripotency markers. In addition, PRMT6 also suppressed in vivo tumor initiation and serial transplantation. Surprising, contrary to its usual localization in the nucleus as a chromatin modification enzyme mediating histone H3R2 methylation, we found PRMT6 to be predominantly expressed in the cytoplasm in normal liver and HCC cells. Through tandem affinity purification and subsequent mass spectrometry profiling, we identified CRAF, a serine/threonine-protein kinase, as a novel cytoplasmic protein partner of PRMT6. Binding of PRMT6 to CRAF inhibited its kinase activity through site-specific arginine methylation, resulting in inhibition of ERK-mediated CSC plasticity in HCC, demonstrated through in vivo / in vitro methylation assays, kinase assay and functional rescue experiments with the ERK inhibitor U0126. The link between PRMT6, ERK and cancer stemness was further substantiated in primary human normal liver and HCC organoids with or without PRMT6 modulated. Taken together, we found PRMT6 to be down-regulated in the liver CSC subset and to be functionally involved in regulating liver CSC plasticity via an unprecedented role in the cytoplasm through suppression of CRAF/ERK cascade. Citation Format: Lok Hei Chan, Lei Zhou, Kai Yu Ng, Tin Lok Wong, Stella Chai, Terence K Lee, Xin Yuan Guan, Yick Pang Ching, Chung Mau Lo, Kwan Man, Benedetta Artegiani, Hans Clevers, Helen H Yan, Suet Yi Leung, Stèphane Richard, Michael SY Huen, Stephanie Ma. PRMT6-dependent CRAF/ERK signaling regulates cancer stem cell plasticity in liver cancer [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 LB-139. doi:10.1158/1538-7445.AM2017-LB-139

Abstract 4915: Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in breast cancer cells

Abstract Background and Objective: The epidemiological and experimental studies suggest that alcohol consumption is associated with increased risk for breast cancer development and metastasis. However, the mechanisms of alcohol-induced breast cancer progression and metastasis remain unknown. In this study, we examined the roles of CCN5, a tumor suppressor gene in alcohol induced breast cancer cell migration/invasion, epithelial to mesenchymal transition (EMT) and stemness. Methods: Estrogen receptor-positive (ER+) human breast cancer cell line MCF-7 and immortalized normal human epithelial breast cell line MCF-12A were treated with ethanol at various concentrations in the presence and absence of human recombinant human CCN5 protein (hrCCN5) . Invasive/migratory ability of treated and untreated cells was measured by Boyden chamber assays. The mRNA and protein expression level of CCN5 was determined by real-time qRT-PCR and Western blot. EMT and stemness markers were evaluated by Western blot. Single-cell suspensions from pre-treated cells were re-suspended at a density of 500 cells/ml mammocult media in ultralow attachment dishes. Number as well as the size of the mammosphere in specified experimental set-up was monitored and recorded alternate day for 8-10 days. Results: The studies demonstrated that alcohol promotes significantly the invasive/migratory ability and EMT phenotypes of breast cancer cells through the suppression of CCN5. Interestingly, hrCCN5 protein treatment suppresses the effect of alcohol and reprograms MET (mesenchymal to epithelial transition). In addition, alcohol-induced mammosphere formation efficiency is also suppressed by hrCCN5 protein-treatment. Conclusions: Our findings suggest that the CCN5 signaling plays a preventive role in alcohol-induced reprogramming of tumor plasticity in breast cancer cells. Citation Format: Inamul Haque, Arvind Subramanian, Vijayalaxmi Gupta, Sandipto Sarkar, Snigdha Banerjee, Sushanta K. Banerjee. Alcohol-induced reprogramming of tumor plasticity is mediated via suppression of CCN5 signaling in breast cancer cells [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 4915. doi:10.1158/1538-7445.AM2017-4915

Correction: Epigenetic Switch between SOX2 and SOX9 Regulates Cancer Cell Plasticity.

Correction: Epigenetic Switch between SOX2 and SOX9 Regulates Cancer Cell Plasticity.

Phenotypic Plasticity and Cell Fate Decisions in Cancer: Insights from Dynamical Systems Theory.

Waddington’s epigenetic landscape, a famous metaphor in developmental biology, depicts how a stem cell progresses from an undifferentiated phenotype to a differentiated one. The concept of “landscape” in the context of dynamical systems theory represents a high-dimensional space, in which each cell phenotype is considered as an “attractor” that is determined by interactions between multiple molecular players, and is buffered against environmental fluctuations. In addition, biological noise is thought to play an important role during these cell-fate decisions and in fact controls transitions between different phenotypes. Here, we discuss the phenotypic transitions in cancer from a dynamical systems perspective and invoke the concept of “cancer attractors”—hidden stable states of the underlying regulatory network that are not occupied by normal cells. Phenotypic transitions in cancer occur at varying levels depending on the context. Using epithelial-to-mesenchymal transition (EMT), cancer stem-like properties, metabolic reprogramming and the emergence of therapy resistance as examples, we illustrate how phenotypic plasticity in cancer cells enables them to acquire hybrid phenotypes (such as hybrid epithelial/mesenchymal and hybrid metabolic phenotypes) that tend to be more aggressive and notoriously resilient to therapies such as chemotherapy and androgen-deprivation therapy. Furthermore, we highlight multiple factors that may give rise to phenotypic plasticity in cancer cells, such as (a) multi-stability or oscillatory behaviors governed by underlying regulatory networks involved in cell-fate decisions in cancer cells, and (b) network rewiring due to conformational dynamics of intrinsically disordered proteins (IDPs) that are highly enriched in cancer cells. We conclude by discussing why a therapeutic approach that promotes “recanalization”, i.e., the exit from “cancer attractors” and re-entry into “normal attractors”, is more likely to succeed rather than a conventional approach that targets individual molecules/pathways.

Epithelial-to-mesenchymal transition transcription factors in cancer-associated fibroblasts.

Beyond inducing epithelial‐to‐mesenchymal transcription (EMT), transcriptional factors of the Snail, ZEB and Twist families (EMT‐TFs) control global plasticity programmes affecting cell stemness and fate. Literature addressing the reactivation of these factors in adult tumour cells is very extensive, as they enable cancer cell plasticity and fuel both tumour initiation and metastatic spread. Incipient data reveal that EMT‐TFs are also expressed in fibroblasts, providing these with additional properties. Here, I will review recent reports on the expression of EMT‐TFs in cancer‐associated fibroblasts (CAFs). The new model suggests that EMT‐TFs can be envisioned as essential metastasis and chemoresistance‐promoting molecules, thereby enabling coordinated plasticity programmes in parenchyma and stroma–tumour compartments.

The lncRNA H19 mediates breast cancer cell plasticity during EMT and MET plasticity by differentially sponging miR-200b/c and let-7b.

Metastasis is a multistep process by which tumor cells disseminate from their primary site and form secondary tumors at a distant site. The pathophysiological course of metastasis is mediated by the dynamic plasticity of cancer cells, which enables them to shift between epithelial and mesenchymal phenotypes through a transcriptionally regulated program termed epithelial-to-mesenchymal transition (EMT) and its reverse process, mesenchymal-to-epithelial transition (MET). Using a mouse model of spontaneous metastatic breast cancer, we investigated the molecular mediators of metastatic competence within a heterogeneous primary tumor and how these cells then manipulated their epithelial-mesenchymal plasticity during the metastatic process. We isolated cells from the primary mammary tumor, the circulation, and metastatic lesions in the lung in TA2 mice and found that the long noncoding RNA (lncRNA) H19 mediated EMT and MET by differentially acting as a sponge for the microRNAs miR-200b/c and let-7b. We found that this ability enabled H19 to modulate the expression of the microRNA targets Git2 and Cyth3, respectively, which encode regulators of the RAS superfamily member adenosine 5'-diphosphate (ADP) ribosylation factor (ARF), a guanosine triphosphatase (GTPase) that promotes cell migration associated with EMT and disseminating tumor cells. Decreasing the abundance of H19 or manipulating that of members in its axis prevented metastasis from grafts in syngeneic mice. Abundance of H19, GIT2, and CYTH3 in patient samples further suggests that H19 might be exploited as a biomarker for metastatic cells within breast tumors and perhaps as a therapeutic target to prevent metastasis.

Dual Targeting of Mesenchymal and Amoeboid Motility Hinders Metastatic Behavior.

Commonly upregulated in human cancers, the scaffolding protein NEDD9/HEF1 is a known regulator of mesenchymal migration and cancer cell plasticity. However, the functional role of NEDD9 as a regulator of different migration/invasion modes in the context of breast cancer metastasis is currently unknown. Here, it is reported that NEDD9 is necessary for both mesenchymal and amoeboid individual cell migration/invasion in triple-negative breast cancer (TNBC). NEDD9 deficiency results in acquisition of the amoeboid morphology, but severely limits all types of cell motility. Mechanistically, NEDD9 promotes mesenchymal migration via VAV2-dependent Rac1 activation, and depletion of VAV2 impairs the ability of NEDD9 to activate Rac1. In addition, NEDD9 supports a mesenchymal phenotype through stimulating polymerization of actin via promoting CTTN phosphorylation in an AURKA-dependent manner. Interestingly, an increase in RhoA activity in NEDD9-depleted cells does not facilitate a switch to functional amoeboid motility, indicating a role of NEDD9 in the regulation of downstream RhoA signaling effectors. Simultaneous depletion of NEDD9 or inhibition of AURKA in combination with inhibition of the amoeboid driver ROCK results in an additional decrease in cancer cell migration/invasion. Finally, we confirmed that a dual targeting strategy is a viable and efficient therapeutic approach to hinder the metastasis of breast cancer in xenograft models, showcasing the important need for further clinical evaluation of this regimen to impede the spread of disease and improve patient survival.Implications: This study provides new insight into the therapeutic benefit of combining NEDD9 depletion with ROCK inhibition to reduce tumor cell dissemination and discovers a new regulatory role of NEDD9 in the modulation of VAV2-dependent activation of Rac1 and actin polymerization. Mol Cancer Res; 15(6); 670-82. ©2017 AACR.

Abstract TMEM-026: ZEB1–MEDIATED NNMT EXPRESSION ELICITS PHENOTYPIC AND METABOLIC PLASTICITY OF OVARIAN CANCER CELLS UNDER NUTRITIONAL STRESS

Abstract Glucose is considered the primary energy source for all cells, and some cancers are addicted to glucose. However, poorly vascularized regions of advanced human cancers, such as high grade serous ovarian cancers (HGSC), may have limited glucose access and suffer from nutritional stress. Here, we investigated the functional consequences of chronic glucose deprivation in serous ovarian cancer cell lines (OVCAR3, OVCAR4 and OAW28). Cells selected for resistance to glucose deprivation (glucose-independent sublines) demonstrated increased metabolic plasticity in utilization of additional energy sources compared to glucose-dependent precursor cells. Furthermore, some cells acquired mesenchymal gene expression and functional characteristics of EMT, such as increased migration efficiency; however classic EMT was not a prerequisite for glucose independence as majority of sublines retained their epithelial-like morphology. Instead, we identified the metabolic enzyme Nicotinamide N-methyltransferase (NNMT) to be commonly upregulated in all glucose-independent sublines. NNMT catalyzes methylation reactions that may alter histones and DNA methylation status. Also, NNMT expression is enriched in the mesenchymal subtype of HGSC and correlates with an invasive phenotype in vitro. We showed that NNMT overexpression in glucose-independent cells is mediated by genetic changes (genomic gain of NNMT), as well as transcriptional changes, such as induction of ZEB1. Ectopic expression of ZEB1 in ovarian cancer cell lines strongly induced NNMT, rendered cells more resistant to glucose deprivation and recapitulated many of the metabolic adaptations and mesenchymal gene expression observed in glucose-independent sublines. We further showed that ZEB1-driven glucose independence was mediated by NNMT because depletion of NNMT via shRNA or CRISPR/Cas9 reversed the metabolic plasticity of these cells and significantly impaired proliferation and de novo formation of glucose-independent colonies in the absence of glucose. In addition to its role in glucose independence, we found that NNMT was required for other ZEB1-induced phenotypes, such as increased migration. Our analysis also showed that increased NNMT expression correlated with worse overall and progression-free survival in patients. In line with these results, our tissue microarray data demonstrated that NNMT protein levels were elevated in metastatic and recurrent tumors compared to matched primary carcinomas, while normal ovary and fallopian tube tissue had no detectable NNMT expression. Our studies define a novel ZEB1/NNMT signaling axis, which elicits phenotypic and metabolic plasticity of ovarian cancer cells upon chronic glucose starvation. Understanding the causes of cancer cell plasticity is crucial for the development of therapeutic strategies to counter intratumoral heterogeneity, acquired drug resistance and recurrence in HGSC. Citation Format: Justyna Kanska, Barbie Taylor-Harding, Paul-Joseph Aspuria, Beth Y. Karlan, Simon Gayther and W. Ruprecht Wiedemeyer. ZEB1–MEDIATED NNMT EXPRESSION ELICITS PHENOTYPIC AND METABOLIC PLASTICITY OF OVARIAN CANCER CELLS UNDER NUTRITIONAL STRESS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-026.

MEDU-24. INSTRUMENTAL ROLE OF THE PHOTORECEPTOR PROGRAM IN GROUP 3 MEDULLOBLASTOMA THROUGH THE TRANSCRIPTION FACTOR NRL

Medulloblastoma (MB) is the most common malignant brain tumor of childhood arising in the cerebellum. Although multimodal treatments have significantly increased the survival, 20–30% of patients remain incurable. Most of them belong to a poorly characterized MB subgroup, called group3, which expresses a retina photoreceptor-specific differentiation program. Cancer cells often express such differentiation programs unrelated to their tissue of origin. They likely result from cancer cell plasticity but are usually not thought to contribute to cancer progression. Accordingly, the functional relevance of this aberrant differentiation program to MB remains unknown. By analyzing group3 enhancers (Enh) and super-enhancers (SE), we identified key interconnected transcription factors of this subgroup, Among those, two lineage restricted transcription factors, NRL and CRX, are known to play key roles during retina development and in particular for photoreceptor specification. We identified a list of genes harboring a co-occurrence of NRL and CRX binding sites within Enh/SE, and displaying higher expression in group3. Interestingly, photoreceptors genes are enriched among this list. This analysis showed that NRL and CRX are important players in group3 MB and control the photoreceptor differentiation program. By loss-of-function experiments in cell lines and PDXs, we showed that NRL and CRX are absolutely required for MB growth in vitro and in vivo upon orthotopic grafting. We then confirmed that NRL controls the expression of photoreceptor genes in group3 MB and established that it protects MB from apoptosis. By ChIP experiments we identified one of its key target genes in this process: the anti-apoptotic BCL-XL gene. We confirmed its key role downstream of NRL by rescue experiments. Importantly, we established the potential of anti-BCL therapies in MB. In conclusion, we showed that the photoreceptor program is instrumental to MB through NRL and CRX and that the anti-BCL therapies could represent an alternative therapeutic strategy.

Cancer Cell Plasticity: Rapid Reversal of Chemosensitivity and Expression of Stemness Markers in Lung and Breast Cancer Tumorspheres.

In cancer cells, the reversible nature of the stemness status in terms of chemoresistance has been poorly characterized. In this study, we have simulated one cycle of environmental conditions to study such reversibility by first generating floating tumorspheres (FTs) from lung and breast cancer cells by culturing them in serum-free media without the addition of any external mitogenic stimulation, and subsequently (after 2 weeks) re-incubating them back in serum-containing media to simulate routine culture conditions (RCCs). We found that cancer cells are extremely plastic: cells grown under RCCs become multidrug-resistant when grown as FTs, but upon re-incubation under RCCs quickly re-attach and lose the acquired resistance. These phenotypic changes are accompanied by concomitant changes in the expression of key proteins associated with multiple pathways important for chemoresistance, survival, and stemness maintenance. Therefore, our strategy provides an excellent experimental model to study environmental factors that modulate the plasticity of cancer cells. J. Cell. Physiol. 232: 2280-2286, 2017. © 2016 Wiley Periodicals, Inc.

Oncostatin M promotes cancer cell plasticity through cooperative STAT3-SMAD3 signaling

Increasing evidence supports the idea that cancer cell plasticity promotes metastasis and tumor recurrence, resulting in patient mortality. While it is clear that the tumor microenvironment (TME) contributes to cancer cell plasticity, the specific TME factors most actively controlling plasticity remain largely unknown. Here, we performed a screen to identify TME cytokines and growth factors that promote epithelial–mesenchymal plasticity, and acquisition of cancer stem cell (CSC) properties. Of 28 TME cytokines and growth factors tested, we identified Oncostatin M (OSM) as the most potent inducer of mesenchymal/CSC properties. OSM-induced plasticity was Signal Transducer and Activator of Transcription 3 (STAT3)-dependent, and also required a novel intersection with transforming growth factor-β (TGF-β)/SMAD signaling. OSM/STAT3 activation promoted SMAD3 nuclear accumulation, DNA binding and induced SMAD3-dependent transcriptional activity. Suppression of TGF-β receptor activity or ablation of SMAD3 or SMAD4, but not SMAD2, strongly suppressed OSM/STAT3-mediated plasticity. Moreover, removal of OSM or inhibition of STAT3 or SMAD3 resulted in a marked reversion to a non-invasive, epithelial phenotype. We propose that targeted blockade of the STAT3/SMAD3 axis in tumor cells may represent a novel therapeutic approach to prevent the plasticity required for metastatic progression and tumor recurrence.

Nigericin decreases the viability of multidrug-resistant cancer cells and lung tumorspheres and potentiates the effects of cardiac glycosides.

Multiple factors including tumor heterogeneity and intrinsic or acquired resistance have been associated with drug resistance in lung cancer. Increased stemness and the plasticity of cancer cells have been identified as important mechanisms of resistance; therefore, treatments targeting cancer cells independent of stemness phenotype would be much more effective in treating lung cancer. In this article, we have characterized the anticancer effects of the antibiotic Nigericin in cells displaying varying degrees of stemness and resistance to anticancer drugs, arising from (1) routine culture conditions, (2) prolonged periods of serum starvation. These cells are highly resistant to conventional anticancer drugs such as Paclitaxel, Hydroxyurea, Colchicine, Obatoclax, Wortmannin, and LY294002, and the multidrug-resistant phenotype of cells growing under prolonged periods of serum starvation is likely the result of extensive rewiring of signaling pathways, and (3) lung tumorspheres that are enriched for cancer stem-like cells. We found that Nigericin potently inhibited the viability of cells growing under routine culture conditions, prolonged periods of serum starvation, and lung tumorspheres. In addition, we found that Nigericin downregulated the expression of key proteins in the Wnt canonical signaling pathway such as LRP6, Wnt5a/b, and β-catenin, but promotes β-catenin translocation into the nucleus. The antitumor effects of Nigericin were potentiated by the Wnt activator HLY78 and by therapeutic levels of the US Food and Drug Administration-approved drug Digitoxin and its novel synthetic analog MonoD. We believe that Nigericin may be used in a co-therapy model in combination with other novel chemotherapeutic agents in order to achieve potent inhibition of cancers that display varying degrees of stemness, potentially leading to sustained anticancer effects.

Selective elimination of neuroblastoma cells by synergistic effect of Akt kinase inhibitor and tetrathiomolybdate.

Neuroblastoma is the most common extracranial solid tumour of infancy. Pathological activation of glucose consumption, glycolysis and glycolysis‐activating Akt kinase occur frequently in neuroblastoma cells, and these changes correlate with poor prognosis of patients. Therefore, several inhibitors of glucose utilization and the Akt kinase activity are in preclinical trials as potential anti‐cancer drugs. However, metabolic plasticity of cancer cells might undermine efficacy of this approach. In this work, we identified oxidative phosphorylation as compensatory mechanism preserving viability of neuroblastoma cells with inhibited glucose uptake/Akt kinase. It was oxidative phosphorylation that maintained intracellular level of ATP and proliferative capacity of these cells. The oxidative phosphorylation inhibitors (rotenone, tetrathiomolybdate) synergized with inhibitor of the Akt kinase/glucose uptake in down‐regulation of both viability of neuroblastoma cells and clonogenic potential of cells forming neuroblastoma spheroids. Interestingly, tetrathiomolybdate acted as highly specific inhibitor of oxygen consumption and activator of lactate production in neuroblastoma cells, but not in normal fibroblasts and neuronal cells. Moreover, the reducing effect of tetrathiomolybdate on cell viability and the level of ATP in the cells with inhibited Akt kinase/glucose uptake was also selective for neuroblastoma cells. Therefore, efficient elimination of neuroblastoma cells requires inhibition of both glucose uptake/Akt kinase and oxidative phosphorylation activities. The use of tetrathiomolybdate as a mitochondrial inhibitor contributes to selectivity of this combined treatment, preferentially targeting neuroblastoma cells.

Glucose deprivation elicits phenotypic plasticity via ZEB1-mediated expression of NNMT.

Glucose is considered the primary energy source for all cells, and some cancers are addicted to glucose. Here, we investigated the functional consequences of chronic glucose deprivation in serous ovarian cancer cells. We found that cells resistant to glucose starvation (glucose-restricted cells) demonstrated increased metabolic plasticity that was dependent on NNMT (Nicotinamide N-methyltransferase) expression. We further show that ZEB1 induced NNMT, rendered cells resistant to glucose deprivation and recapitulated metabolic adaptations and mesenchymal gene expression observed in glucose-restricted cells. NNMT depletion reversed metabolic plasticity in glucose-restricted cells and prevented de novo formation of glucose-restricted colonies. In addition to its role in glucose independence, we found that NNMT was required for other ZEB1-induced phenotypes, such as increased migration. NNMT protein levels were also elevated in metastatic and recurrent tumors compared to matched primary carcinomas, while normal ovary and fallopian tube tissue had no detectable NNMT expression. Our studies define a novel ZEB1/NNMT signaling axis, which elicits mesenchymal gene expression, as well as phenotypic and metabolic plasticity in ovarian cancer cells upon chronic glucose starvation. Understanding the causes of cancer cell plasticity is crucial for the development of therapeutic strategies to counter intratumoral heterogeneity, acquired drug resistance and recurrence in high-grade serous ovarian cancer (HGSC).