Cancer Cell Plasticity Research Articles

Abstract 5458: Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta

Abstract Transcriptional plasticity of cancer cells promotes tumor progression and treatment resistance via generation of phenotypically distinct cell states. We previously showed that Ewing sarcoma (EwS) cells exhibit inter- and intra-tumoral transcriptional heterogeneity and that they undergo state changes in response to cues from the tumor microenvironment (TME). More recently, we identified phenotypically distinct EwS tumor cell subpopulations in vivo that deposit pro-tumorigenic extracellular matrix (ECM) proteins, adopting similar functions to cancer-associated fibroblasts (CAFs). Here, we sought to identify the upstream regulators of these CAF-like EwS cells with the goal of defining molecular mechanisms that control EwS plasticity and creation of ECM-remodeling states. Integration of single cell proteogenomic sequencing (CITE-seq) with immunofluorescence and digital spatial profiling of cell line xenografts and patient tumors validated the cell-surface protein CD73 (NT5E) as a biomarker of ECM-secreting EwS cell subpopulations in vitro and in vivo. The transcriptomes of CD73+ CAF-like cells were compared to isogenic CD73- cells (n=9 cell lines) and highly significant enrichment of gene ontology terms related to TGF-beta signaling (p.adj = <4.4x10-14) was noted. EwS cells were previously reported to be non-responsive to TGF-beta due to profound transcriptional repression of the TGF-beta type 2 receptor (TGFBR2) by EWS::FLI1. In support of this, TGFBR2 expression was low in bulk measurements of EwS cells. However, TGFBR2 mRNA and surface protein expression were significantly increased in CD73+ subpopulations. Exposure of EwS cells to TGF-beta ligands (TGFB1 or TGFB2) strongly induced the CAF-like gene signature, including ECM genes BGN, COL1A1, SPARC, and TNC (p. adj < 8.5x10-5), and this was augmented in CD73+ cell populations. Transduction of EwS cells with dominant negative-TGFBR2 inhibited both basal and TGF-beta-induced expression of CAF-like signature genes and blocked SMAD2 phosphorylation. To test the functional significance of the TGF-beta response, unsorted EwS cells were plated in 2D or as tumor spheroids in collagen I-rich 3D cultures and exposed to TGFB1 or SB-505124, a TGF-beta inhibitor. TGFB1 treatment had no effect on cell viability or proliferation in 2D but increased invasion into collagen in 3D. Inhibition of TGF-beta reduced CAF-like gene expression in 2D cultures and diminished both viability and invasion in 3D. Finally, our studies revealed that NT5E (CD73) is a TGF-beta induced target in EwS cells and that CD73+ cells upregulate expression of TGFB2. Thus, an autocrine signaling circuit is created in which TGF-beta activity in CAF-like tumor cell subpopulations is sustained by CD73+ tumor cell-derived ligand. Together these studies demonstrate that TGF-beta is a key determinant of cell plasticity and ECM remodeling in EwS. Citation Format: Emma Wrenn, April A. Apfelbaum, Katherine Braun, Erin R. Rudzinski, Xuemei Deng, Aya Miyaki, Trisha Lipson, Nicolas M. Garcia, Virginia J. Hoglund, Elizabeth R. Lawlor. Plasticity of CAF-like states in Ewing sarcoma is mediated by TGF-beta [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5458.

Abstract 1530: Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice

Abstract The relevance of preclinical models has vastly improved with mice bearing a human immune system, especially in the context of immunotherapy. BRGSF (Balb/C Rag2−/−, IL2Rγ−/−, SIRPαNOD and Flt3−/−) is a highly immunodeficient mouse featuring reduced murine myeloid cells. BRGSF mice reconstituted with human cord blood CD34+ cells (BRGSF-HIS) develop functional lymphoid and myeloid compartments. This engraftment is stable for over a year (Labarthe et al., 2019) and mice do not develop GvHD. Myeloid compartment can be transiently boosted with exogenous human Flt3L injections. In contrary to other models overexpressing human cytokines to develop human myeloid cells, Flt3L-treated BRGSF-HIS mice do not show side effects. BRGSF-HIS mice are permissive to mouse and human cancer cell lines engraftment and represent a valuable preclinical model to study cancer development and test novel therapeutics. Effect of Flt3L-induced boost on myeloid compartment is seen in a non-small cell lung cancer (A549) tumor model. A single boost prior to tumor cell inoculation associates with increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, and reduced tumor-infiltrating NK cells in the tumor microenvironment (TME). Human triple negative cancer cell (TNBC) MDA-MB-231 and human pancreatic adenocarcinoma HPAFII cell lines are widely used in cancer research and drug development. We show that upon implantation in BRGSF-HIS mice, in vivo growth of human cell line-derived xenograft (CDX) is CD34+ donor independent and the TME composition varies according to cell line used. As seen in TNBC patients (Zheng et al., 2021); TME of MDA-MB-231 bearing BRGSF-HIS mice is enriched in myeloid cells, mostly CD206+/CD163+ M2 macrophages. CD163-expressing M2 macrophages represent the main tumor-associated macrophages, and are known to promote breast cancer initiation, angiogenesis, invasion, and metastasis by generating an immunosuppressive TME. Interestingly, TME composition evolves over time, with increased T cells frequency in later stage (tumor volume over 1000 mm3 compared to 500 mm3) depicting cancer cell plasticity. Conversely, TME of HPAFII bearing mice is mainly composed of T cells enabling T cells-based therapy. Treatment of BRGSF-HIS mice bearing HPAFII tumor cells with combotherapy based on CD3xTAA + CD28xTAA efficiently reduces tumor growth in vivo compared to vehicle-treated mice or mice injected with CD3xTAA alone. Induced systemic immunomodulation is observed in the TME where increased numbers of both CD4 and CD8 T cells are observed upon combotherapy. Remarkably, while no CD34+ donor effect is observed in CDX growth kinetic, response to treatment appears to be donor dependent with apparition of “weak” and “good” responders. This heterogeneity of response mimics what is observed in clinic and enables further investigations of treatment mode of action and immune escape mechanisms. Citation Format: Perrine Martin-Jeantet, Siham Hedir, Fabiane Sonego, Gaëlle Martin, Yacine Cherifi, Kader Thiam. Tumor cell line-derived xenograft subtype shapes tumor microenvironment composition in BRGSF-HIS mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1530.

Abstract 5981: Treatment of cholangiocarcinoma using humanized monoclonal antibodies targeting claudin-1

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly aggressive adenocarcinoma of the hepatobiliary system showing an alarming rise in incidence and mortality with unsatisfactory treatment options. Claudin-1 (CLDN1) is a transmembrane protein expressed in tight junctions, but exposed at the cell surface on cancer epithelial cells. Using highly specific monoclonal antibodies (mAbs) targeting the extracellular loop 1 of exposed CLDN1 with an excellent safety profile (Roehlen, Saviano et al. Science Transl Med 2022), we aimed to investigate the role of CLDN1 as therapeutic target for CCA. Methods: Integrative CCA patient CLDN1 expression analyses, spatial transcriptomics and mouse models were used to evaluate the role of CLDN1 as an oncogenic driver for CCA. Proof-of-concept studies were performed in cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse as well as patient CCA organoid models using humanized CLDN1 mAbs. Results: Integrative expression analyses of CLDN1 in CCA patient tissues revealed robust CLDN1 upregulation across several cohorts and patients with well-characterized driver mutations. scRNASeq and spatial transcriptomics of patient CCA showed that CLDN1 expression in cancer cells is associated with stemness, oncogenic signaling and EMT. Gain-of-function studies using an orthotopic HDTVi and syngeneic mouse model revealed a decrease in survival and an enhanced tumor growth, unraveling a functional role of CLDN1 as an oncogenic driver in CCA. Targeting exposed CLDN1 using highly CLDN1-specific mAb demonstrated a robust anti-tumoral effect across intra- and extra-hepatic CCA mouse models, with a significant inhibition of metastatic disease including models with medium or low CLDN1 expression. Functional studies in patient-derived CCA organoids demonstrated that CLDN1 mAb decreased cellular viability and altered cancer cell plasticity and fate. Mechanistically, CLDN1 mAb treatment suppressed gene expression of pathways mediating proliferation, stemness and EMT by inhibition of Notch1, SRC-FAK, and Hippo-YAP signaling. Conclusion: These results demonstrate that CLDN1 is a CCA driver and therapeutic target. The proof-of-concept studies in patient-derived models pave the way for the clinical development of CLDN1 mAbs to improve the outcomes of patients with advanced CCA. Citation Format: Zeina Nehme, Marion Muller, Emilie Crouchet, Frank Juehling, Julien Moehlin, Romain Désert, Jade Brochon, Fabio Del Zompo, Natascha Roehlen, Christine Thumann, Patrick Pessaux, Emanuele Felli, Aïna Venkatasamy, Patrice Marchand, Mihaela Alina Onea, Roberto Iacone, Markus Meyer, Alberto Toso, Nabeel Bardeesy, Lipika Goyal, Vikas Prakash Ranvir, Mirian Fernández-Vaquero, Mathias Heikenwälder, Tessa Ostyn, Tania Roskams, Patrice Laquerriere, Catherine Schuster, Laurent Mailly, Thomas F. Baumert. Treatment of cholangiocarcinoma using humanized monoclonal antibodies targeting claudin-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5981.

Abstract 6935: Non-genetic routes of lung cancer evolution

Abstract Cancer cell plasticity underlies the progression of several solid tumors. In lung adenocarcinoma, morphological changes characterizing the transition from indolent to aggressive disease exhibit profound epigenetic and transcriptional reprogramming1. However, the extent, features, and drivers of cancer cell plasticity in the progression of human lung adenocarcinoma is still largely unexplored. Here, we assembled and analyzed a meta-cohort of 119 human lung tumors analyzed by single-cell RNA-sequencing, for a total of ~730,000 cells. Cancer cell heterogeneity was the result of differential activation of 17 transcriptional programs associated with common cancer phenotypes, such as proliferation, cell migration, and response to hypoxia, as well as alveolar lineage identity (AT2). In particular, AT2-specific expression of MHC-II was down-regulated with the progression of the disease and markedly reduced in KEAP1 and/or STK11 mutated samples, providing a rationale for the association between these mutations and immunotherapy resistance. Co-activation of transcriptional programs clustered tumor cells in 4 major cell states, which emerged with the progression of the disease. To explore spatial distribution of different cancer cell states, we generated and analyzed multiple spatial transcriptomics and proteomics lung adenocarcinoma cohorts. To this purpose we developed a new algorithm called CellCharter2. CellCharter identified distinct cancer-enriched niches within individual samples, corresponding to the cancer cell states that we discovered in the scRNA-seq cohort and exhibited specific interactions with the tumor microenvironment (TME). In particular, we identified a cancer cell state characterized by response-to-hypoxia and migratory phenotypes, which spatially segregated from highly proliferative cancer cell niches and formed specific interactions with tumor associated neutrophils. This cell state was enriched in advanced primary samples, it uniquely characterized metastatic samples, and was an independent marker of poor prognosis across multiple independent datasets. Overall, our work uncovered cell plasticity trajectories associated with the progression of lung adenocarcinoma and shed new light into the tumor-TME interactions at their origin. 1 Tavernari et al., Cancer Discovery, 20212 Varrone et al, Nat. Genetics, (in press) Citation Format: Giovanni Ciriello, Daniele Tavernari, Marco Varrone. Non-genetic routes of lung cancer evolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6935.

Deciphering cellular and molecular mechanism of MUC13 mucin involved in cancer cell plasticity and drug resistance.

There has been a surge of interest in recent years in understanding the intricate mechanisms underlying cancer progression and treatment resistance. One molecule that has recently emerged in these mechanisms is MUC13 mucin, a transmembrane glycoprotein. Researchers have begun to unravel the molecular complexity of MUC13 and its impact on cancer biology. Studies have shown that MUC13 overexpression can disrupt normal cellular polarity, leading to the acquisition of malignant traits. Furthermore, MUC13 has been associated with increased cancer plasticity, allowing cells to undergo epithelial-mesenchymal transition (EMT) and metastasize. Notably, MUC13 has also been implicated in the development of chemoresistance, rendering cancer cells less responsive to traditional treatment options. Understanding the precise role of MUC13 in cellular plasticity, and chemoresistance could pave the way for the development of targeted therapies to combat cancer progression and enhance treatment efficacy.

Magnetically Stimulated Integrin Binding Alters Cell Polarity and Affects Epithelial-Mesenchymal Plasticity in Metastatic Cancer Cells.

Inorganic nanoparticles (NPs) have been widely recognized for their stability and biocompatibility, leading to their widespread use in biomedical applications. Our study introduces a novel approach that harnesses inorganic magnetic nanoparticles (MNPs) to stimulate apical-basal polarity and induce epithelial traits in cancer cells, targeting the hybrid epithelial/mesenchymal (E/M) state often linked to metastasis. We employed mesocrystalline iron oxide MNPs to apply an external magnetic field, disrupting normal cell polarity and simulating an artificial cellular environment. These led to noticeable changes in the cell shape and function, signaling a shift toward the hybrid E/M state. Our research suggests that apical-basal stimulation in cells through MNPs can effectively modulate key cellular markers associated with both epithelial and mesenchymal states without compromising the structural properties typical of mesenchymal cells. These insights advance our understanding of how cells respond to physical cues and pave the way for novel cancer treatment strategies. We anticipate that further research and validation will be instrumental in exploring the full potential of these findings in clinical applications, ensuring their safety and efficacy.

Abstract PR013: An agent-based modelling framework to study cell plasticity in non-small cell lung cancer

Abstract Recent breakthroughs in phylogenetic analysis of bulk tissue have allowed researchers to reconstruct the evolutionary histories of tumours. The hope is that knowledge of the order and timing of genetic mutations will allow us to characterise the properties of early-stage cancer cells, to better identify and target them. This is complicated by the phenomenon of tumour plasticity, the ability of cells to acquire the hallmarks of cancer via mechanisms other than heritable genetic mutation. In a tumour with only genetic heritability, changes in cell phenotype would occur only as a result of genetic mutations. By contrast, non-genetic heritability could lead to a disconnect between genotype and phenotype. Whilst both mechanisms are likely to impact on cancer development, we ask whether one has a significantly stronger influence than the other by simulating tumour growth under each regime and comparing the distribution of mutations obtained in either case to patient data. Here, we address this question in a real patient cohort by adapting a mathematical model of lung tumour evolution, which we use to test the validity of two scenarios. In a ‘low-plasticity’ scenario, changes to reproductive fitness occur when cells acquire mutations in the exome. Mutations may be ‘drivers’ (beneficial) or ‘passengers’ (deleterious or neutral). In a ‘high-plasticity’ scenario, mutations have no effect on cell fitness. Cells experience ‘driver-like’ or ‘passenger-like’ cell fitness changes on division, without leaving a genetic mark. The model describes three-dimensional growth of a tumour from a single cell and incorporates biologically-informed death patterns and local competition for space and resources. When the simulation has reached a realistic size, cells on the surface are sampled and sequenced to predict the relatedness of mutations present at detectable frequencies in each of several regions. The outputs are designed to allow comparison with those of the TRACERx cohort of 421 non-small- cell lung cancer patients (NSCLC), comprising multi-region whole-exome (WXS) and bulk RNA sequencing. In this ongoing work, we show results from a large cohort of simulations under both scenarios and predict corresponding patterns of genetic similarity. This allows us to use approximate Bayesian computation (ABC) to predict the mechanisms at play in the TRACERx cohort of 421 non-small-cell lung cancer patients (NSCLC). We present a novel ‘meta-inference’ approach, where evolutionary parameters are fit to each patient’s data using well-chosen summary statistics. Given the size of the TRACERx cohort, this enables the evaluation of each scenario by examining the plausibility and similarity of output parameters obtained across patients. We hope that this work will shed light on the role of heritability in lung cancer development and guide future research into therapeutic approaches. Citation Format: Helena M. K. Coggan, Carlos Martínez-Ruiz, James R. M. Black, Kristiana Grigoriadis, Nicholas McGranahan, Jasmin Fisher. An agent-based modelling framework to study cell plasticity in non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr PR013.

Abstract B056: Chronic hypoxia in TNBC breast cancer induces development of a novel population of CD24+/CD49b-high cells characterized by high expression of CST1

Abstract Triple-negative breast cancer (TNBC) is the subtype of breast cancer that has the worst clinical outcome due to the absence of specific targeting agents. TNBC is highly heterogeneous and within the tumor microenvironment, hypoxia emerges as a pivotal factor contributing to its aggressive biology. Solid tumors experience a fluctuating oxygen supply, leading to the formation of zones with prolonged oxygen deprivation. There have been few studies describing the response to chronic hypoxia [7-14 days] and this project investigates how chronic hypoxia contributes to tumour heterogeneity in a temporal manner. We performed scRNA-seq analysis on MDA-MB-231 cells subjected to a 14-day hypoxia treatment at 1% oxygen. We collected samples from both normoxia and hypoxia conditions on Days 1, 2, 7, and 14. A total of 40,000 cells were sequenced. There were 6 hypoxic cell clusters which evolved over the time course. Interestingly, chronic hypoxia induces various transient subpopulations. Cells in hypoxia Day 7 grew more slowly, before recovering growth at Day 14, marking a crucial adaptation point at Day 7 in chronic hypoxia. Our study suggests that chronic hypoxia cells conserve energy through G1 cell cycle arrest and Myc suppression as an adaptive mechanism to cope with chronic hypoxia. We also observed a higher expression of cancer stem cell markers [CD44, CXCR4 and KLF4] in hypoxia subpopulations relative to normoxia subpopulations, especially in the chronic hypoxia subpopulations. Notably, we identified a distinct CD24+/CD49b-high population, comprising the largest proportion of the hypoxic Day 7 samples which disappears by Day 14. This population is characterised by the upregulation of cystatin family genes- CST 1, 4 and 7 and may be pro-metastatic [upregulation of EMP1, ST3GAL6 and KRT19 genes] compared to earlier and later hypoxia phases. Interestingly, KRT19 is more abundant in peripheral blood of breast cancer patients with node metastasis, although its role in stemness of breast cancer is controversial. The heterogeneity we found here may explain this, with the existence of a transient population. Chronic hypoxia could perhaps have a role in reprogramming cancer stem cell-like cells into a pro-metastatic state. In summary, our study provides insights on how hypoxia induces protective translational reprogramming and drives plasticity in cancer cells. Understanding these processes may facilitate the development of targeted treatments to combat therapy resistance in TNBC. Citation Format: May Sin Ke, Badran Elshenawy, Anjali Arora, Helen Sheldon, Adrian Harris, Francesca Buffa. Chronic hypoxia in TNBC breast cancer induces development of a novel population of CD24+/CD49b-high cells characterized by high expression of CST1 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B056.

Abstract B022: Targeting epithelial-mesenchymal plasticity and CD73 to enhance responses of breast cancers to immune checkpoint blockade therapies

Abstract Immune checkpoint blockade (ICB) therapy has generated dramatic responses in certain types of human tumors however, the response of breast cancers has been largely limited. Epithelial-mesenchymal plasticity (EMP) enables carcinoma cells to metastasize, gain tumor-initiating ability and mount resistance to chemotherapies. In addition, we have demonstrated that this plasticity program also contributes to the establishment of an immunosuppressive tumor microenvironment and confers resistance to ICB therapy (Dongre and Weinberg 2019). By establishing novel, preclinical models of more-epithelial or more-mesenchymal breast tumors, we observed that epithelial tumors recruit CD8+ T-cells to their microenvironment and are sensitive to anti-CTLA4 ICB. In contrast, mesenchymal tumors recruit Tregs and M2-like macrophages and are resistant to the same treatment (Dongre et al., 2017). Strikingly, abrogation of CD73 from more-mesenchymal cells completely sensitizes otherwise refractory tumors to anti-CTLA4 ICB (Dongre et al., 2021). These findings demonstrated for the first time that more-mesenchymal breast cancer cells can be completely sensitized to anti-tumor immunity by interrupting certain cell-intrinsic signaling channels. However, the underlying mechanisms of such sensitization remain elusive. Additionally, the regulatory networks that control the expression of CD73 itself in more-mesenchymal but not epithelial breast cancer cells are not well defined. To determine the subset of T-cells that is functionally important in sensitizing mesenchymal tumors lacking CD73, we depleted either conventional CD4+ T-cells or CD8+ cytotoxic T-cells in combinatoin with ICB. While responding mice recruited both T-cells to their tumors, only CD4+ T-cells (and not CD8+ T-cells) were functionally important in driving sensitization. Along these lines, more-mesenchymal cancer cells lacking CD73 failed to respond to anti-CTLA4 ICB when propagated in genetically engineered mice lacking CD4+ T-cells relative to wild type controls. Importantly, depletion of CD4+ T-cells also prevented the recruitment of peripheral CD8+ T-cells to responding tumors, thereby driving resistance to ICB. In addition, we observed that human triple negative breast cancer cell lines expressed the highest amount of CD73 relative to hormone receptor-positive cell lines. Most importantly, activating EMP in human breast cancer cells by doxycycline-controlled expression of various transcription factors, induced the expression of CD73. Taken together, our work demonstrates that (i) the phenotypic plasticity of breast cancer cells can predict responses to ICB (ii) targeting CD73 can completely sensitize more-mesenchymal tumors to anti-CTLA4 ICB in a CD4+ T-cell dependent fashion and; (iii) altering the cellular plasticity of cancer cells can regulate CD73 expression. Given the highly refractory nature of more-mesenchymal cancer cells, our findings hold tremendous potential in aiding translational efforts to target cancer plasticity to enhance responses of breast cancers to ICB therapy. Citation Format: Anushka Dongre. Targeting epithelial-mesenchymal plasticity and CD73 to enhance responses of breast cancers to immune checkpoint blockade therapies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B022.

Abstract B015: KDM6A, lysine demethylase, loss affects epithelial-to-mesenchymal related gene expression and breast cancer growth

Abstract Epithelial-to-mesenchymal transition (EMT) or its reversal mesenchymal-to-epithelial transition (MET) are of critical importance throughout development and is implicated in wound healing, fibrotic disorders, and cancer metastasis. The ability of a cell to undergo EMT and MET, cellular plasticity, underlies tumor progression through multi-step metastasis and acquisition of chemotherapy resistance as alterations in the cellular and molecular level confer changes in motility, stemness properties, gene expression, and cellular dynamics. These changes facilitate invasion and dissemination of cancer cells. Epigenetic modifications leading to cancer cell plasticity are partially driven by histone modifying proteins including lysine (K)-specific demethylase 6A (KDM6A). KDM6A is a member of the COMPASS-like protein complex, and catalyzes the removal of methyl groups from H3K27me3, facilitating gene expression. KDM6A regulation of H3K27me3 has been associated with bivalent programs in gene expression, associates with oncogenic pathways, and contributes to low survival prognoses in breast cancer. There is critical need to elucidate the mechanisms of KDM6A suppression to understand its control on cellular plasticity. Previously we have shown that KDM6A is suppressed upon EMT. In this study, we sought to identify the regulatory mechanisms of KDM6A expression and how its activity is required to maintain epithelial cellular identity. To investigate the role of KDM6A in the context of EMT, KDM6A expression was compromised using CRISPR-Cas9 to determine the impact on H3K27me3, gene expression and cellular identity. Breast cancer cell lines were transduced with CRISPR-Cas9 and GFP via lentiviral transduction. KDM6A KO and control cell lines were generated via a CRISPR KO. We observed that the KDM6A suppression or inhibition profoundly impacts cellular identity, leading to gain of mesenchymal and stemness properties. Moreover, KDM6A was determined to be regulated through sub-cellular localization leading to partial sequestration of COMPASS-like protein complex outside the nucleus. These findings were validated via Western blot analysis of subcellular protein fractions of breast cancer cell lines and KDM6A KO cell lines and well as immunofluorescence and enzyme activity analysis. We conclude that KDM6A functions as a master regulator of epithelial cellular identity which is controlled by multi-factorial mechanisms including transcriptional suppression and altered protein localization. Citation Format: Charli Worth, Provas Das, Kelsey Johnson, Joseph Taube. KDM6A, lysine demethylase, loss affects epithelial-to-mesenchymal related gene expression and breast cancer growth [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B015.

Tumor-educated Gr1+CD11b+ cells drive breast cancer metastasis via OSM/IL-6/JAK-induced cancer cell plasticity.

Cancer cell plasticity contributes to therapy resistance and metastasis, which represent the main causes of cancer-related death, including in breast cancer. The tumor microenvironment drives cancer cell plasticity and metastasis, and unraveling the underlying cues may provide novel strategies for managing metastatic disease. Using breast cancer experimental models and transcriptomic analyses, we show that stem cell antigen-1 positive (SCA1+) murine breast cancer cells enriched during tumor progression and metastasis had higher in vitro cancer stem cell-like properties, enhanced in vivo metastatic ability, and generated tumors rich in Gr1hiLy6G+CD11b+ cells. In turn, tumor-educated Gr1+CD11b+ (Tu-Gr1+CD11b+) cells rapidly and transiently converted low metastatic SCA1- cells into highly metastatic SCA1+ cells via secreted oncostatin M (OSM) and IL-6. JAK inhibition prevented OSM/IL-6-induced SCA1+ population enrichment, while OSM/IL-6 depletion suppressed Tu-Gr1+CD11b+-induced SCA1+ population enrichment in vitro and metastasis in vivo. Moreover, chemotherapy-selected highly metastatic 4T1 cells maintained high SCA1+ positivity through autocrine IL-6 production, and in vitro JAK inhibition blunted SCA1 positivity and metastatic capacity. Importantly, Tu-Gr1+CD11b+ cells invoked a gene signature in tumor cells predicting shorter overall survival (OS), relapse-free survival (RFS), and lung metastasis in breast cancer patients. Collectively, our data identified OSM/IL-6/JAK as a clinically relevant paracrine/autocrine axis instigating breast cancer cell plasticity and triggering metastasis.

DPYSL5 is highly expressed in treatment-induced neuroendocrine prostate cancer and promotes lineage plasticity via EZH2/PRC2

Treatment-induced neuroendocrine prostate cancer (t-NEPC) is a lethal subtype of castration-resistant prostate cancer resistant to androgen receptor (AR) inhibitors. Our study unveils that AR suppresses the neuronal development protein dihydropyrimidinase-related protein 5 (DPYSL5), providing a mechanism for neuroendocrine transformation under androgen deprivation therapy. Our unique CRPC-NEPC cohort, comprising 135 patient tumor samples, including 55 t-NEPC patient samples, exhibits a high expression of DPYSL5 in t-NEPC patient tumors. DPYSL5 correlates with neuroendocrine-related markers and inversely with AR and PSA. DPYSL5 overexpression in prostate cancer cells induces a neuron-like phenotype, enhances invasion, proliferation, and upregulates stemness and neuroendocrine-related markers. Mechanistically, DPYSL5 promotes prostate cancer cell plasticity via EZH2-mediated PRC2 activation. Depletion of DPYSL5 decreases proliferation, induces G1 phase cell cycle arrest, reverses neuroendocrine phenotype, and upregulates luminal genes. In conclusion, DPYSL5 plays a critical role in regulating prostate cancer cell plasticity, and we propose the AR/DPYSL5/EZH2/PRC2 axis as a driver of t-NEPC progression.

Mutually exclusive teams-like patterns of gene regulation characterize phenotypic heterogeneity along the noradrenergic-mesenchymal axis in neuroblastoma

ABSTRACT Neuroblastoma is the most frequent extracranial pediatric tumor and leads to 15% of all cancer-related deaths in children. Tumor relapse and therapy resistance in neuroblastoma are driven by phenotypic plasticity and heterogeneity between noradrenergic (NOR) and mesenchymal (MES) cell states. Despite the importance of this phenotypic plasticity, the dynamics and molecular patterns associated with these bidirectional cell-state transitions remain relatively poorly understood. Here, we analyze multiple RNA-seq datasets at both bulk and single-cell resolution, to understand the association between NOR- and MES-specific factors. We observed that NOR-specific and MES-specific expression patterns are largely mutually exclusive, exhibiting a “teams-like” behavior among the genes involved, reminiscent of our earlier observations in lung cancer and melanoma. This antagonism between NOR and MES phenotypes was also associated with metabolic reprogramming and with immunotherapy targets PD-L1 and GD2 as well as with experimental perturbations driving the NOR-MES and/or MES-NOR transition. Further, these “teams-like” patterns were seen only among the NOR- and MES-specific genes, but not in housekeeping genes, possibly highlighting a hallmark of network topology enabling cancer cell plasticity.

The exostosin glycosyltransferase 1/STAT3 axis is a driver of breast cancer aggressiveness

The epithelial-mesenchymal transition (EMT) program is crucial for transforming carcinoma cells into a partially mesenchymal state, enhancing their chemoresistance, migration, and metastasis. This shift in cell state is tightly regulated by cellular mechanisms that are not yet fully characterized. One intriguing EMT aspect is the rewiring of the proteoglycan landscape, particularly the induction of heparan sulfate proteoglycan (HSPG) biosynthesis. This proteoglycan functions as a co-receptor that accelerates cancer-associated signaling pathways through its negatively-charged residues. However, the precise mechanisms through which EMT governs HSPG biosynthesis and its role in cancer cell plasticity remain elusive. Here, we identified exostosin glycosyltransferase 1 (EXT1), a central enzyme in HSPG biosynthesis, to be selectively upregulated in aggressive tumor subtypes and cancer cell lines, and to function as a key player in breast cancer aggressiveness. Notably, ectopic expression of EXT1 in epithelial cells is sufficient to induce HSPG levels and the expression of known mesenchymal markers, subsequently enhancing EMT features, including cell migration, invasion, and tumor formation. Additionally, EXT1 loss in MDA-MB-231 cells inhibits their aggressiveness-associated traits such as migration, chemoresistance, tumor formation, and metastasis. Our findings reveal that EXT1, through its role in HSPG biosynthesis, governs signal transducer and activator of transcription 3 (STAT3) signaling, a known regulator of cancer cell aggressiveness. Collectively, we present the EXT1/HSPG/STAT3 axis as a central regulator of cancer cell plasticity that directly links proteoglycan synthesis to oncogenic signaling pathways.

Mitochondrial Localized In Situ Self-Assembly Reprogramming Tumor Immune and Metabolic Microenvironment for Enhanced Cancer Therapy.

The inherent immune and metabolic tumor microenvironment (TME) of most solid tumors adversely affect the antitumor efficacy of various treatments, which is an urgent issue to be solved in clinical cancer therapy. In this study, a mitochondrial localized in situ self-assembly system is constructed to remodel the TME by improving immunogenicity and disrupting the metabolic plasticity of cancer cells. The peptide-based drug delivery system can be pre-assembled into nanomicelles in vitro and form functional nanofibers on mitochondria through a cascade-responsive process involving reductive release, targeted enrichment, and in situ self-assembly. The organelle-specific in situ self-assemblyeffectively switches the role of mitophagy from pro-survival to pro-death, which finally induces intense endoplasmic reticulum stress and atypical type II immunogenic cell death. Disintegration of the mitochondrial ultrastructure also impedes the metabolic plasticity of tumor cells, which greatly promotes the immunosuppresive TME remodeling into an immunostimulatory TME. Ultimately, the mitochondrial localized in situ self-assembly system effectively suppresses tumor metastases, and converts cold tumors into hot tumors with enhanced sensitivity to radiotherapy and immune checkpoint blockade therapy. This study offers a universal strategy for spatiotemporally controlling supramolecular self-assembly on sub-organelles to determine cancer cell fate and enhance cancer therapy.

Crosstalk between cancer cell plasticity and immune microenvironment in cholangiocarcinoma

Cholangiocarcinoma (CCA) is a highly aggressive tumor of the biliary tree characterized by an intense desmoplastic tumor microenvironment (TME). To date, treatment of CCA remains challenging; tumor resection is the only curative treatment with a high recurrence probability. Besides resection, therapeutic options have moved forward with the advent of immunotherapies, but these remain limited and low effective. Our knowledge about the cellular interplays in CCA is still fragmentary. An area is currently emerging regarding the potential role of cancer cell plasticity in the genesis of an immunosuppressive microenvironment. The cancer cells’ ability to acquire stemness properties and to disseminate through an epithelial-mesenchymal transition (EMT) shape a tumor immune microenvironment that supports cancer progression by attracting immunosuppressive cells including myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), M2 macrophages, and by increasing the expression of inhibitory immune checkpoints such as PD-1/PD-L-1. EMT-inducing transcription factors (EMT-TF) have recently emerged as regulators of tumor immunity by creating an immunosuppressive microenvironment. This review delves into the molecular mechanisms underlying the existing links between EMT/stemness and tumor immune microenvironment, as well as the last discoveries in CCA.

Targeting the key players of phenotypic plasticity in cancer cells by phytochemicals.

Plasticity of phenotypic traits refers to an organism's ability to change in response to environmental stimuli. As a result, the response may alter an organism's physiological state, morphology, behavior, and phenotype. Phenotypic plasticity in cancer cells describes the considerable ability of cancer cells to transform phenotypes through non-genetic molecular signaling activities that promote therapy evasion and tumor metastasis via amplifying cancer heterogeneity. As a result of metastable phenotypic state transitions, cancer cells can tolerate chemotherapy or develop transient adaptive resistance. Therefore, new findings have paved the road in identifying factors and agents that inhibit or suppress phenotypic plasticity. It has also investigated novel multitargeted agents that may promise new effective strategies in cancer treatment. Despite the efficiency of conventional chemotherapeutic agents, drug toxicity, development of resistance, and high-cost limit their use in cancer therapy. Recent research has shown that small molecules derived from natural sources are capable of suppressing cancer by focusing on the plasticity of phenotypic responses. This systematic, comprehensive, and critical review analyzes the current state of knowledge regarding the ability of phytocompounds to target phenotypic plasticity at both preclinical and clinical levels. Current challenges/pitfalls, limitations, and future perspectives are also discussed.

Blockade of tumor cell-intrinsic PD-L1 signaling enhances AURKA-targeted therapy in triple negative breast cancer.

Triple negative breast cancer (TNBC) accounts for 15-20% of all breast cancers and mainly affects pre-menopausal and minority women. Because of the lack of ER, PR or HER2 expression in TNBC, there are limited options for tailored therapies. While TNBCs respond initially to standard of care chemotherapy, tumor recurrence commonly occurs within 1 to 3 years post-chemotherapy and is associated with early organ metastasis and a high incidence of mortality. One of the major mechanisms responsible for drug resistance and emergence of organ metastasis is activation of epithelial to mesenchymal transition (EMT) reprogramming. EMT-mediated cancer cell plasticity also promotes the enrichment of cancer cells with a CD44high/CD24low and/or ALDHhigh cancer stem-like phenotype [cancer stem cells (CSCs)], characterized by an increased capacity for tumor self-renewal, intrinsic drug resistance, immune evasion and metastasis. In this study we demonstrate for the first time a positive feedback loop between AURKA and intra-tumoral PD-L1 oncogenic pathways in TNBC. Genetic targeting of intra-tumoral PD-L1 expression impairs the enrichment of ALDHhigh CSCs and enhances the therapeutic efficacy of AURKA-targeted therapy. Moreover, dual AURKA and PD-L1 pharmacological blockade resulted in the strongest inhibition of tumor growth and organ metastatic burden. Taken together, our findings provide a compelling preclinical rationale for the development of novel combinatorial therapeutic strategies aimed to inhibit cancer cell plasticity, immune evasion capacity and organ metastasis in patients with advanced TNBC.

Polymeric nanoformulations aimed at cancer metabolism reprogramming with high specificity to inhibit tumor growth.

Metabolic disorders of cancer cells create opportunities for metabolic interventions aimed at selectively eliminating cancer cells. Nevertheless, achieving this goal is challenging due to cellular plasticity and metabolic heterogeneity of cancer cells. This study presents a dual-drug-loaded, macrophage membrane-coated polymeric nanovesicle designed to reprogram cancer metabolism with high specificity through integrated extracellular and intracellular interventions. This nanoformulation can target cancer cells and largely reduce their glucose intake, while the fate of intracellular glucose internalized otherwise is redirected at the specially introduced oxidation reaction instead of inherent cancer glycolysis. Meanwhile, it inhibits cellular citrate intake, further reinforcing metabolic intervention. Furthermore, the nanoformulation causes not only H2O2 production, but also NADPH down-regulation, intensifying redox damage to cancer cells. Consequently, this nanoformulation displays highly selective toxicity to cancer cells and minimal harm to normal cells mainly due to metabolic vulnerability of the former. Once administered into tumor-bearing mice, this nanoformulation is found to induce the transformation of pro-tumor tumor associated macrophages into the tumor-suppressive phenotype and completely inhibit tumor growth with favourable biosafety.

Targeting cancer stem cell plasticity in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype with limited treatment options. Cancer stem cells (CSCs) are thought to play a crucial role in TNBC progression and resistance to therapy. CSCs are a small subpopulation of cells within tumors that possess self-renewal and differentiation capabilities and are responsible for tumor initiation, maintenance, and metastasis. CSCs exhibit plasticity, allowing them to switch between states and adapt to changing microenvironments. Targeting CSC plasticity has emerged as a promising strategy for TNBC treatment. This review summarizes recent advances in understanding the molecular mechanisms underlying CSC plasticity in TNBC and discusses potential therapeutic approaches targeting CSC plasticity.