Stem-like State Research Articles

Aspirin Rescues Wnt-Driven Stem-like Phenotype in Human Intestinal Organoids and Increases the Wnt Antagonist Dickkopf-1.

Background & AimsAspirin reduces colorectal cancer (CRC) incidence and mortality. Understanding the biology responsible for this protective effect is key to developing biomarker-led approaches for rational clinical use. Wnt signaling drives CRC development from initiation to progression through regulation of epithelial-mesenchymal transition (EMT) and cancer stem cell populations. Here, we investigated whether aspirin can rescue these proinvasive phenotypes associated with CRC progression in Wnt-driven human and mouse intestinal organoids.MethodsWe evaluated aspirin-mediated effects on phenotype and stem cell markers in intestinal organoids derived from mouse (ApcMin/+ and Apcflox/flox) and human familial adenomatous polyposis patients. CRC cell lines (HCT116 and Colo205) were used to study effects on motility, invasion, Wnt signaling, and EMT.ResultsAspirin rescues the Wnt-driven cystic organoid phenotype by promoting budding in mouse and human Apc deficient organoids, which is paralleled by decreased stem cell marker expression. Aspirin-mediated Wnt inhibition in ApcMin/+ mice is associated with EMT inhibition and decreased cell migration, invasion, and motility in CRC cell lines. Chemical Wnt activation induces EMT and stem-like alterations in CRC cells, which are rescued by aspirin. Aspirin increases expression of the Wnt antagonist Dickkopf-1 in CRC cells and organoids derived from familial adenomatous polyposis patients, which contributes to EMT and cancer stem cell inhibition.ConclusionsWe provide evidence of phenotypic biomarkers of response to aspirin with an increased epithelial and reduced stem-like state mediated by an increase in Dickkopf-1. This highlights a novel mechanism of aspirin-mediated Wnt inhibition and potential phenotypic and molecular biomarkers for trials.

The Transcription Factor TCF1 in T Cell Differentiation and Aging.

The transcription factor T cell factor 1 (TCF1), a pioneer transcription factor as well as a downstream effector of WNT/β-catenin signaling, is indispensable for T cell development in the thymus. Recent studies have highlighted the additional critical role of TCF1 in peripheral T cell responses to acute and chronic infections as well as cancer. Here, we review the regulatory functions of TCF1 in the differentiation of T follicular helper cells, memory T cells and recently described stem-like exhausted T cells, where TCF1 promotes less differentiated stem-like cell states by controlling common gene-regulatory networks. These studies also provide insights into the mechanisms of defective T cell responses in older individuals. We discuss alterations in TCF1 expression and related regulatory networks with age and their consequences for T cell responses to infections and vaccination. The increasing understanding of the pathways regulating TCF1 expression and function in aged T cells holds the promise of enabling the design of therapeutic interventions aiming at improving T cell responses in older individuals.

Hypoxia induces an endometrial cancer stem-like cell phenotype via HIF-dependent demethylation of SOX2 mRNA

Endometrial cancer stem cells (ECSCs) are stem-like cells endowed with self-renewal and differentiation abilities, and these cells are essential for cancer progression in endometrial cancer (EC). As hallmarks of the tumour microenvironment (TME), hypoxia and hypoxia-inducing factors (HIFs) give rise to the dysregulation of tumour stemness genes, such as SOX2. Against this backdrop, we investigated the regulatory mechanisms regulated by HIFs and SOX2 in ECSCs during EC development. Here, ECSCs isolated from EC cell lines and tissues were found to express stemness genes (CD133 and aldehyde dehydrogenase, ALDH1) following the induction of their ECSC expansion. Notably, m6A methylation of RNA and HIF-1α/2α-dependent AlkB homologue 5 (ALKBH5) participate in the regulation of HIFs and SOX2 in EC, as confirmed by the observations that mRNA levels of m6A demethylases and ALKBH5 significantly increase under hypoxic conditions in ECSCs. Moreover, hypoxia and high ALKBH5 levels restore the stem-like state of differentiated ECSCs and increase the ECSC-like phenotype, whereas the knockdown of HIFs or ALKBH5 significantly reduces their tumour initiation capacity. In addition, our findings validate the role of ALKBH5 in promoting SOX2 transcription via mRNA demethylation, thereby maintaining the stem-like state and tumorigenicity potential of ECSCs. In conclusion, these observations demonstrate a critical role for m6A methylation-mediated regulation of the HIF-ALKBH5-SOX2 axis during ECSC expansion in hypoxic TMEs.

Decoding the Role of CD271 in Melanoma.

The evolution of melanoma, the most aggressive type of skin cancer, is triggered by driver mutations that are acquired in the coding regions of particularly BRAF (rat fibrosarcoma serine/threonine kinase, isoform B) or NRAS (neuroblastoma-type ras sarcoma virus) in melanocytes. Although driver mutations strongly determine tumor progression, additional factors are likely required and prerequisite for melanoma formation. Melanocytes are formed during vertebrate development in a well-controlled differentiation process of multipotent neural crest stem cells (NCSCs). However, mechanisms determining the properties of melanocytes and melanoma cells are still not well understood. The nerve growth factor receptor CD271 is likewise expressed in melanocytes, melanoma cells and NCSCs and programs the maintenance of a stem-like and migratory phenotype via a comprehensive network of associated genes. Moreover, CD271 regulates phenotype switching, a process that enables the rapid and reversible conversion of proliferative into invasive or non-stem-like states into stem-like states by yet largely unknown mechanisms. Here, we summarize current findings about CD271-associated mechanisms in melanoma cells and illustrate the role of CD271 for melanoma cell migration and metastasis, phenotype-switching, resistance to therapeutic interventions, and the maintenance of an NCSC-like state.

ERK-Mediated Loss of miR-199a-3p and Induction of EGR1 Act as a "Toggle Switch" of GBM Cell Dedifferentiation into NANOG- and OCT4-Positive Cells.

There is great interest in understanding how the cancer stem cell population may be maintained in solid tumors. Here, we show that tumor cells exhibiting stem-like properties and expression of pluripotency markers NANOG and OCT4 can arise from original differentiated tumor cells freshly isolated from human glioblastomas (GBM) and that have never known any serum culture conditions. Induction of EGR1 by EGFR/ERK signaling promoted cell conversion from a less aggressive, more differentiated cellular state to a self-renewing and strongly tumorigenic state, expressing NANOG and OCT4. Expression of these pluripotency markers occurred before the cells re-entered the cell cycle, demonstrating their capacity to change and dedifferentiate without any cell divisions. In differentiated GBM cells, ERK-mediated repression of miR-199a-3p induced EGR1 protein expression and triggered dedifferentiation. Overall, this signaling pathway constitutes an ERK-mediated "toggle switch" that promotes pluripotency marker expression and stem-like features in GBM cells. SIGNIFICANCE: This study defines an ERK-mediated molecular mechanism of dedifferentiation of GBM cells into a stem-like state, expressing markers of pluripotency.See related commentary by Koncar and Agnihotri, p. 3195.

Anarchy or Respect the Hierarchy? The Complexity of Glioblastoma.

Glioma stem-like cells (GSC) in glioblastoma (GBM) structure tumor cells into a hierarchical organization and are postulated to be recalcitrant to conventional treatments, resulting in fatal relapse of the disease. A better understanding of these cells would be essential for meaningful and lasting treatments. In this issue of Cancer Research, Virolle and colleagues report a fascinating phenotype whereby the extracellular signal-regulated kinase (ERK) pathway regulates a mechanism of dedifferentiation of GBM cells into a stem-like state expressing markers of pluripotency through an EGFR-ERK-EGR1-dependent axis. This aptly termed "toggle switch" may contribute to maintenance of GSCs, promote intratumor heterogeneity, and potentially provide innovative treatment options.See related article by Almairac et al., p. 3236.

Stimulation of the Gαq/phospholipase Cβ1 signaling pathway returns differentiated cells to a stem-like state.

Phospholipase Cβ1 is activated by Gαq to generate calcium signals in response to hormones and neurotransmitters. Besides carrying out this plasma membrane function, PLCβ1 has a cytosolic population that helps to drive the differentiation of PC12 cells by inhibiting a nuclease that promotes RNA-induced silencing (C3PO). Here, we show that down-regulating PLCβ1 or reducing its cytosolic population by activating Gαq to localize it to the plasma membrane returns differentiated PC12 and SK-N-SH cells to an undifferentiated state. In this state, PC12 cells have a spherical morphology, resume proliferation, and express the stem cell transcription factors nanog and Oct4. Similar changes are seen when C3PO is down-regulated. This return to a stem-like state is accompanied by shifts in multiple miR populations. Surprisingly, de-differentiation can be induced by extended stimulation of Gαq where cells return to a spherical morphology and levels of specific miRs return to their undifferentiated values. In complementary studies, we followed the real-time hydrolysis of a fluorescent-tagged miR in cells where PLCβ1 or C3PO were down-regulated in PC12 cells and find substantial differences in miR processing in the undifferentiated and differentiated states. Taken together, our studies suggest that PLCβ1, through its ability to regulate C3PO and endogenous miR populations, mediates the differentiation of two types of cultured neuronal cells.

An integrative ENCODE resource for cancer genomics

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

Superior breast cancer metastasis risk stratification using an epithelial-mesenchymal-amoeboid transition gene signature

BackgroundCancer cells are known to display varying degrees of metastatic propensity, but the molecular basis underlying such heterogeneity remains unclear. Our aims in this study were to (i) elucidate prognostic subtypes in primary tumors based on an epithelial-to-mesenchymal-to-amoeboid transition (EMAT) continuum that captures the heterogeneity of metastatic propensity and (ii) to more comprehensively define biologically informed subtypes predictive of breast cancer metastasis and survival in lymph node-negative (LNN) patients.MethodsWe constructed a novel metastasis biology-based gene signature (EMAT) derived exclusively from cancer cells induced to undergo either epithelial-to-mesenchymal transition (EMT) or mesenchymal-to-amoeboid transition (MAT) to gauge their metastatic potential. Genome-wide gene expression data obtained from 913 primary tumors of lymph node-negative breast cancer (LNNBC) patients were analyzed. EMAT gene signature-based prognostic stratification of patients was performed to identify biologically relevant subtypes associated with distinct metastatic propensity.ResultsDelineated EMAT subtypes display a biologic range from less stem-like to more stem-like cell states and from less invasive to more invasive modes of cancer progression. Consideration of EMAT subtypes in combination with standard clinical parameters significantly improved survival prediction. EMAT subtypes outperformed prognosis accuracy of receptor or PAM50-based BC intrinsic subtypes even after adjusting for treatment variables in 3 independent, LNNBC cohorts including a treatment-naïve patient cohort.ConclusionsEMAT classification is a biologically informed method that provides prognostic information beyond that which can be provided by traditional cancer staging or PAM50 molecular subtype status and may improve metastasis risk assessment in early stage, LNNBC patients, who may otherwise be perceived to be at low metastasis risk.

Loss of TET2 Affects Proliferation and Drug Sensitivity through Altered Dynamics of Cell-State Transitions.

A persistent puzzle in cancer biology is how mutations, which neither alter growth signaling pathways nor directly interfere with drug mechanism, can still recur and persist in tumors. One example is the mutation of the DNA demethylase tet methylcytosine dioxygenase 2 (TET2) in acute myeloid leukemias (AMLs) that frequently persists from diagnosis through remission and relapse, but whose fitness advantage in chemotherapy is unclear. Here, we use isogenic human AML cell lines to show that TET2 loss of function alters the dynamics of transitions between differentiated and stem-like states. A conceptual mathematical model and experimental validation suggest that these altered cell-state dynamics can benefit the cell population by slowing population decay during drug treatment and lowering the number of survivor cells needed to re-establish the initial population. These studies shed light on the functional and phenotypic effects of a TET2 mutation in AML and illustrate how a single gene mutation can alter a cells' phenotypic plasticity. A record of this paper's transparent peer review process is included in the Supplemental Information.

Visualizing Human Colorectal Cancer Intratumor Heterogeneity with Phylogeography.

Phylogeography combines ancestry with location and can be translated to intratumor heterogeneity (ITH) to visualize how tumors spread. ITH is common in human tumors, with many genetic and phenotypic differences between regions. The roles of ITH in progression are uncertain because many subclones lack discernable driver mutations. ITH can be visualized by mapping mutations onto microscopic sections, where subclones are directly associated with phenotypes, especially the deeper areas with the more invasive cells that confer worst clinical outcomes. Instead of a stepwise hierarchy where subclones segregate by phenotype with later branching subclones in more invasive areas, multiple subclones share superficial and invasive phenotype and are jigsaw arrayed in vertical columns. Phylogeography shows that both early and late subclones extend from the surface to the invasive front, suggesting that founder cells start with phenotypic plasticity and essentially all the drivers necessary to rapidly grow into large invasive tumors.

Abstract A36: Organoid models for deciphering roles of the evolving landscape of epigenetic heterogeneity during aging in cancer development

Abstract Background: Tumor initiation and development occur not necessarily by genetic changes alone, but in the context of the epigenome and cell microenvironment. Growing evidence indicate that cancer risk factors, such as aging and inflammation, may primarily modulate the epigenome, causing predisposition to cancer development by driver mutations. Thus, there is a need to study and model epigenetic changes preceding and associating with cancer development. We have developed ex vivo models for investigating the evolution and roles of DNA methylation changes during normal cell divisions and how these epigenetic changes play critical roles in early stages of tumor development. Results: We showed that spontaneous epigenetic changes, specifically promoter-DNA hypermethylation, have a very important role in tumor initiation by oncogenic driver mutations. We addressed the precursor role of aging-like spontaneous promoter-DNA hypermethylation in initiating tumorigenesis. Using mouse colon-derived organoids, we show promoter hypermethylation spontaneously arises in cells mimicking the human “aging-like” phenotype. The silenced genes activate the Wnt pathway, causing a stemlike state and differentiation defects. These changes render aged organoids profoundly more sensitive than young ones to transformation by BrafV600E producing the typical human proximal BRAFV600E-driven colon adenocarcinomas characterized by extensive, abnormal gene promoter CGI methylation or the methylator phenotype (CIMP). Conversely, CRISPR-mediated simultaneous inactivation of a panel of the silenced genes markedly sensitized to BrafV600E-induced transformation. We showed that “spontaneous” promoter hypermethylation, arising in an “aging-like” scenario, sufficiently affects genes with roles in negative regulation of Wnt pathway. Inactivation of these genes leads to increased stem cell state and defects in differentiation. Conclusion: Our studies tightly link aging-like epigenetic abnormalities to intestinal cell fate changes and predisposition to oncogene-driven colon tumorigenesis. The organoid-based model opens up an opportunity to study mechanisms underlying epigenetic heterogeneity arising during normal cellular processes and their role in cancer predisposition. Citation Format: Yong Tao, Byunghak Kang, Daniel Petkovich, Steve Baylin, Hariharan Easwaran. Organoid models for deciphering roles of the evolving landscape of epigenetic heterogeneity during aging in cancer development [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A36.

Hybrid Stem Cell States: Insights Into the Relationship Between Mammary Development and Breast Cancer Using Single-Cell Transcriptomics.

Similarities between stem cells and cancer cells have implicated mammary stem cells in breast carcinogenesis. Recent evidence suggests that normal breast stem cells exist in multiple phenotypic states: epithelial, mesenchymal, and hybrid epithelial/mesenchymal (E/M). Hybrid E/M cells in particular have been implicated in breast cancer metastasis and poor prognosis. Mounting evidence also suggests that stem cell phenotypes change throughout the life course, for example, through embryonic development and pregnancy. The goal of this study was to use single cell RNA-sequencing to quantify cell state distributions of the normal mammary (NM) gland throughout developmental stages and when perturbed into a stem-like state in vitro using conditional reprogramming (CR). Using machine learning based dataset alignment, we integrate multiple mammary gland single cell RNA-seq datasets from human and mouse, along with bulk RNA-seq data from breast tumors in the Cancer Genome Atlas (TCGA), to interrogate hybrid stem cell states in the normal mammary gland and cancer. CR of human mammary cells induces an expanded stem cell state, characterized by increased expression of embryonic stem cell associated genes. Alignment to a mouse single-cell transcriptome atlas spanning mammary gland development from in utero to adulthood revealed that NM cells align to adult mouse cells and CR cells align across the pseudotime trajectory with a stem-like population aligning to the embryonic mouse cells. Three hybrid populations emerge after CR that are rare in NM: KRT18+/KRT14+ (hybrid luminal/basal), EPCAM+/VIM+ (hybrid E/M), and a quadruple positive population, expressing all four markers. Pseudotime analysis and alignment to the mouse developmental trajectory revealed that E/M hybrids are the most developmentally immature. Analyses of single cell mouse mammary RNA-seq throughout pregnancy show that during gestation, there is an enrichment of hybrid E/M cells, suggesting that these cells play an important role in mammary morphogenesis during lactation. Finally, pseudotime analysis and alignment of TCGA breast cancer expression data revealed that breast cancer subtypes express distinct developmental signatures, with basal tumors representing the most “developmentally immature” phenotype. These results highlight phenotypic plasticity of normal mammary stem cells and provide insight into the relationship between hybrid cell populations, stemness, and cancer.

Cell Reversal From a Differentiated to a Stem-Like State at Cancer Initiation.

Even if the Somatic Mutation Theory of carcinogenesis explains many of the relevant experimental results in tumor origin and development, there are frequent events that are not justified, or are even contradictory to this widely accepted theory. A Cell Reversal Theory is presented, putting forward the hypothesis that cancer is originated by reversal of a differentiated cell into a non-differentiated stem-like state, by a change of its intrinsic epigenetic state, following a perturbation on the cell and/or its microenvironment. In the current proposal a cluster of cancer stem cells can be established, without the strict control mechanisms of a normal stem cell niche, and initiate a tumor. It is proposed that a reversal to a pluripotent state is at tumor origin and not tumor progress that prompts cell dedifferentiation. The uncontrolled proliferation of cancer stem cells causes a microenvironment disorganization, resulting in stressful conditions, like hypoxia and nutrient deprivation, which induces the genetic instability characteristic of a tumor; thus, in most cases, mutations are a consequence and not the direct cause of a tumor. It is also proposed that metastases result from dedifferentiation signaling dispersion instead of cell migration. However, conceivably, once the microenvironment is normalized, the stem cell-like state can differentiate back to a mature cell state and loose its oncogenic capacity. Therefore, this can be a reversible condition, suggesting important therapeutic opportunities.

Epigenetic plasticity potentiates a rapid cyclical shift to and from an aggressive cancer phenotype.

Highly tumorigenic, drug-resistant cancer stem-like cells drive cancer progression. These aggressive cells can arise repeatedly from bulk tumor cells independently of mutational events, suggesting an epigenetic mechanism. To test this possibility, we studied bladder cancer cells as they cyclically shifted to and from a cancer stem-like phenotype, and we discovered that these two states exhibit distinct DNA methylation and chromatin accessibility. Most differential chromatin accessibility was independent of methylation and affected the expression of driver genes such as E2F3, a cell cycle regulator associated with aggressive bladder cancer. Cancer stem-like cells exhibited increased E2F3 promoter accessibility and increased E2F3 expression that drove cell migration, invasiveness and drug resistance. Epigenetic interference using a DNA methylation inhibitor blocked the transition to a cancer stem-like state and reduced E2F3 expression. Our findings indicate that epigenetic plasticity plays a key role in the transition to and from an aggressive, drug-resistant phenotype.

Abstract GS2-02: Acquired activating mutations in RTKs confer endocrine resistance in ER+ metastatic breast cancer through ER-reprogramming, MAPK signaling, and an induced stem-like cell state

While recent studies have begun characterizing the metastatic breast cancer (MBC) genomics, our understanding of mechanisms of acquired endocrine-resistance, their induced cell-state, and their altered drug-response profile, remains lacking. We collected biopsies from patients with MBC with detailed clinicopathologic features. To date we profiled 520 exomes, and 291 transcriptomes, with 126 patients having multiple biopsy exomes. Curated endocrine-relapse set include 60 patients with pre-treatment and post-relapse exomes. Characterization of candidate mechanisms of resistance (MOR) included 909 RNA-seq profiles of T47D cells with introduced MOR under various drugs. In the acquired endocrine resistance cohort, as expected, we found frequent ESR1 acquired mutations (13 pt, 22%). Additionally, we identified acquired activating SNVs and amplifications in oncogenic receptor tyrosine kinases (RTKs) in 18/60 (30%), including EGF family - HER2 (n=7), ERRB3 (R525Q), and EGFR (S116F), and FGF family - FGFR1 (n=5), FGFR2 (n=4), and FGF3 amplicon (n=4). RNA-seq of T47D cells overexpressing HER2 activating mutations revealed a distinct cell-state (HER2-ACT). Similarly, FGFR activation revealed a district FGFR-ACT state. The transcriptional signatures of these HER2 and FGFR states were remarkably similar (odd-ratio= 91, p= 2.64E-94). To characterize the cell-state common to HER and FGFR, we defined RTK-ACT with 358 overlapping marker genes (see table). Canonical (estradiol) ER signaling is slightly elevated in RTK-ACT, however this state is strongly associated with growth-factor driven ER signaling, suggesting reprogramming of ER from AF2, to AF1 signaling. Consistent with this, RTK-ACT had significantly higher MAPK activation. Additionally, RTK-ACT Induced stronger similarity to Basal-state, enrichment in motility/migration, mesenchymal, and stem-like features, with top genes including CDH3, MBP7, and S100, ETV, DUSP, SPRY families (see table). To study RTK-driven state in-vivo, we analyzed RNA-seq from our MBC biopsies, and compared tumors with activating RTK mutations (n=38) with WT (n=118), and inferred activated RTK in-vivo (RTK.ACT.iv). Our in-vitro and in-vivo states show significant overlap (OR=4.71, p=9.42E-20). Furthermore, characterization of RTK.ACT.iv, recapitulated all the cell-state features observed in RTK.ACT - including higher growth-factors ER signaling, MAPK, and basal-like state (see table). We further studied the viability and transcription of these RTKs under various drugs (12 treatments), including fulvestrant, palbociclib, and specific tyrosine kinase inhibitors (TKIs) - Neratinib (pan-HER inhibitor) and FIIN-3 (FGFR-i). We found that HER2-ACT and FGFR-ACT signatures remain robust when treated with fulvestrant, palbociclib, and their combinations, as compared to TKIs suppression (see table). in-line with our viability results - suggesting intrinsic resistance to CDK4/6i and sensitivity to TKIs. This study demonstrated that activating RTKs constitute of prevalent modality of acquired resistance to endocrine therapies, inducing a distinct state with clinical implications - suggesting the potential benefit of combination therapies with specific TKIs over CDK4/6i. The common MAPK activity and our preliminary results - suggests the potential of convergence-node targeting strategy with added MEK or SHP2 inhibition. Citation Format: Ofir Cohen, Pingping Mao, Utthara Nayar, Jorge E Buendia-Bue, Dewey Kim, Esha Jain, Karla Helvie, Daniel Abravanel, Kailey J Kowalski, Christian Kapstad, Samuel Freeman, Victor Adalsteinsson, Seth A Wander, Adrienne G Waks, Gad Getz, Aviv Regev, Eric Winer P Winer, Nancy U Nancy U. Lin, Nikhil Wagle. Acquired activating mutations in RTKs confer endocrine resistance in ER+ metastatic breast cancer through ER-reprogramming, MAPK signaling, and an induced stem-like cell state [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr GS2-02.

Dual antivascular function of human fibulin-3 variant, a potential new drug discovery strategy for glioblastoma.

The ECM protein EFEMP1 (fibulin‐3) is associated with all types of solid tumor through its cell context‐dependent dual function. A variant of fibulin‐3 was engineered by truncation and mutation to alleviate its oncogenic function, specifically the proinvasive role in glioblastoma multiforme (GBM) cells at stem‐like state. ZR30 is an in vitro synthesized 39‐kDa protein of human fibulin‐3 variant. It has a therapeutic effect in intracranial xenograft models of human GBM, through suppression of epidermal growth factor receptor/AKT and NOTCH1/AKT signaling in GBM cells and extracellular MMP2 activation. Glioblastoma multiforme is highly vascular, with leaky blood vessels formed by tumor cells expressing endothelial cell markers, including CD31. Here we studied GBM intracranial xenografts, 2 weeks after intratumoral injection of ZR30 or PBS, by CD31 immunohistochemistry. We found a 70% reduction of blood vessel density in ZR30‐treated xenografts compared with that of PBS‐treated ones. Matrigel plug assays showed the effect of ZR30 on suppressing angiogenesis. We further studied the effect of ZR30 on genes involved in endothelial transdifferentiation (ETD), in 7 primary cultures derived from 3 GBMs under different culture conditions. Two GBM cultures formed mesh structures with upregulation of ETD genes shortly after culture in Matrigel Matrix, and ZR30 suppressed both. ZR30 also downregulated ETD genes in two GBM cultures with high expression of these genes. In conclusion, multifaceted tumor suppression effects of human fibulin‐3 variant include both suppression of angiogenesis and vasculogenic mimicry in GBM.

Transcompp: understanding phenotypic plasticity by estimating Markov transition rates for cell state transitions.

Gradual population-level changes in tissues can be driven by stochastic plasticity, meaning rare stochastic transitions of single-cell phenotype. Quantifying the rates of these stochastic transitions requires time-intensive experiments, and analysis is generally confounded by simultaneous bidirectional transitions and asymmetric proliferation kinetics. To quantify cellular plasticity, we developed Transcompp (Transition Rate ANalysis of Single Cells to Observe and Measure Phenotypic Plasticity), a Markov modeling algorithm that uses optimization and resampling to compute best-fit rates and statistical intervals for stochastic cell-state transitions. We applied Transcompp to time-series datasets in which purified subpopulations of stem-like or non-stem cancer cells were exposed to various cell culture environments, and allowed to re-equilibrate spontaneously over time. Results revealed that commonly used cell culture reagents hydrocortisone and cholera toxin shifted the cell population equilibrium toward stem-like or non-stem states, respectively, in the basal-like breast cancer cell line MCF10CA1a. In addition, applying Transcompp to patient-derived cells showed that transition rates computed from short-term experiments could predict long-term trajectories and equilibrium convergence of the cultured cell population. Freely available for download at http://github.com/nsuhasj/Transcompp. Supplementary data are available at Bioinformatics online.

Acquisition of stem associated-features on metastatic osteosarcoma cells and their functional effects on mesenchymal stem cells

BackgroundOsteosarcoma (OS) is the most frequent malignant bone tumor, affecting predominantly children and young adults. Metastases are a major clinical challenge in OS. In this context, 20% of OS patients are diagnosed with metastatic OS, but near 80% of all OS patients could present non-detectable micrometastases at the moment of diagnosis. MethodsOsteogenic differentiation; doxorubicin exclusion assay; fluorescence microscopy; RT-qPCR; proteomic analysis. ResultsOur results suggest that metastatic OS cells possess a diminished osteoblastic differentiation potential with a gain of metastatic traits like the capacity to modify intracellular localization of chemodrugs and higher levels of expression of stemness-related genes. On the opposite hand, non-metastatic OS cells possess bone-associated traits like higher osteoblastic differentiation and also an osteoblastic-inducer secretome. OS cells also differ in the nature of their interaction with mesenchymal stem cells (MSCs), with opposites impacts on MSCs phenotype and behavior. ConclusionsAll this suggests that a major trait acquired by metastatic cells is a switch into a stem-like state that could favor its survival in the pulmonary niche, opening new possibilities for personalized chemotherapeutic schemes. General significanceOur work provides new insights regarding differences among metastatic and non-metastatic OS cells, with particular emphasis on differentiation potential, multidrug resistance and interaction with MSCs.

Radiation induces an inflammatory response that results in STAT3-dependent changes in cellular plasticity and radioresistance of breast cancer stem-like cells

Purpose: Pro-inflammatory cytokines within the tumor microenvironment, such as IL-6, contribute to the maintenance of stem cells and promote their survival following treatment. The IL-6/STAT3 pathway is a key regulator of genes involved in cancer progression. Activation of STAT3 promotes expansion of cancer stem cells in triple negative breast cancer. Radiation has also been shown to expand cancer stem cell populations and can induce stemness in nonstem cells. However, the role of IL-6/STAT3 in radiation-induced changes in cellular plasticity is unclear.Materials and methods: Expression and secretion of IL-6 from triple-negative breast cancer cell lines SUM159PT and MDA-MB-231 were determined after radiation treatment by real-time PCR and ELISA. Activation of STAT3 after radiation was determined by western blotting. Changes in cellular plasticity induced by radiation were determined by examining ALDEFLUOR activity, gene expression analysis of aldehyde dehydrogenase isoforms and mammosphere forming assays with and without the addition of STAT3 inhibitors. To determine the effect of radiation on nonstem cell populations, experiments were also carried out in ALDEFLUOR sorted cells.Results: Radiation induced an inflammatory response in both cell lines that resulted in activation of STAT3. Additionally, radiation induced a stem-like state as evidenced by an increased activity and expression of the ALDH isoforms ALDH1A1 and ALDH1A3, and increased self-renewal capabilities. Radiation increased ALDH activity and self-renewal in non-stem cell (ALDH−) populations, suggesting radiation-induced cellular reprograming. However, inhibition of STAT3 blocked the radiation-induced stem-like state in both ALDEFLUOR positive and negative populations, and enhanced radiosensitivity.Conclusions: Radiation-induced changes in cellular plasticity are STAT3 dependent and may be a potential target to reduce radioresistance in TNBC and improve treatment outcome.