The tumor microenvironment (TME) is comprised of non-malignant cells that interact with each other and with cancer cells, critically impacting cancer biology. The TME is complex, and understanding it ...requires simplifying approaches. Here we provide an experimental-mathematical approach to decompose the TME into small circuits of interacting cell types. We find, using female breast cancer single-cell-RNA-sequencing data, a hierarchical network of interactions, with cancer-associated fibroblasts (CAFs) at the top secreting factors primarily to tumor-associated macrophages (TAMs). This network is composed of repeating circuit motifs. We isolate the strongest two-cell circuit motif by culturing fibroblasts and macrophages in-vitro, and analyze their dynamics and transcriptomes. This isolated circuit recapitulates the hierarchy of in-vivo interactions, and enables testing the effect of ligand-receptor interactions on cell dynamics and function, as we demonstrate by identifying a mediator of CAF-TAM interactions - RARRES2, and its receptor CMKLR1. Thus, the complexity of the TME may be simplified by identifying small circuits, facilitating the development of strategies to modulate the TME.
The role of mitochondria dynamics and its molecular regulators remains largely unknown during naïve-to-primed pluripotent cell interconversion. Here we report that mitochondrial MTCH2 is a regulator ...of mitochondrial fusion, essential for the naïve-to-primed interconversion of murine embryonic stem cells (ESCs). During this interconversion, wild-type ESCs elongate their mitochondria and slightly alter their glutamine utilization. In contrast, MTCH2
ESCs fail to elongate their mitochondria and to alter their metabolism, maintaining high levels of histone acetylation and expression of naïve pluripotency markers. Importantly, enforced mitochondria elongation by the pro-fusion protein Mitofusin (MFN) 2 or by a dominant negative form of the pro-fission protein dynamin-related protein (DRP) 1 is sufficient to drive the exit from naïve pluripotency of both MTCH2
and wild-type ESCs. Taken together, our data indicate that mitochondria elongation, governed by MTCH2, plays a critical role and constitutes an early driving force in the naïve-to-primed pluripotency interconversion of murine ESCs.
The tumor microenvironment (TME) presents cells with challenges such as variable pH, hypoxia, and free radicals, triggering stress responses that affect cancer progression. In this study, we examine ...the stress response landscape in four carcinomas—breast, pancreas, ovary, and prostate—across five pathways: heat shock, oxidative stress, hypoxia, DNA damage, and unfolded protein stress. Using a combination of experimental and computational methods, we create an atlas of stress responses across various types of carcinomas. We find that stress responses vary within the TME and are especially active near cancer cells. Focusing on the non-immune stroma we find, across tumor types, that NRF2 and the oxidative stress response are distinctly activated in immune-regulatory cancer-associated fibroblasts and in a unique subset of cancer-associated pericytes. Our study thus provides an interactome of stress responses in cancer, offering ways to intersect survival pathways within the tumor, and advance cancer therapy.
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•A network of stress responses is heterogeneously activated in the tumor microenvironment•Stress responses are highly activated near cancer cells•The oxidative stress response is active in cancer-associated fibroblast and pericyte subsets•NRF2 plays a role in stromal heterogeneity
Lior et al. examine the network of stress responses activated in the tumor microenvironment across carcinomas. They find that stress responses are heterogeneously activated in the microenvironment and highly activated near cancer cells. They identify specific subsets of cancer-associated fibroblasts and pericytes showing distinct activation of the oxidative stress response.
Cancer cells recruit and rewire normal fibroblasts in their microenvironment to become protumorigenic cancer-associated fibroblasts (CAF). These CAFs are genomically stable, yet their transcriptional ...programs are distinct from those of their normal counterparts. Transcriptional regulation plays a major role in this reprogramming, but the extent to which epigenetic modifications of DNA also contribute to the rewiring of CAF transcription is not clear. Here we address this question by dissecting the epigenetic landscape of breast CAFs. Applying tagmentation-based whole-genome bisulfite sequencing in a mouse model of breast cancer, we found that fibroblasts undergo massive DNA methylation changes as they transition into CAFs. Transcriptional and epigenetic analyses revealed RUNX1 as a potential mediator of this process and identified a RUNX1-dependent stromal gene signature. Coculture and mouse models showed that both RUNX1 and its stromal signature are induced as normal fibroblasts transition into CAFs. In breast cancer patients, RUNX1 was upregulated in CAFs, and expression of the RUNX1 signature was associated with poor disease outcome, highlighting the relevance of these findings to human disease. This work presents a comprehensive genome-wide map of DNA methylation in CAFs and reveals a previously unknown facet of the dynamic plasticity of the stroma.
The first genome-wide map of DNA methylation in breast cancer-associated fibroblasts unravels a previously unknown facet of the dynamic plasticity of the stroma, with far-reaching therapeutic implications.
Tumors are supported by cancer-associated fibroblasts (CAFs). CAFs are heterogeneous and carry out distinct cancer-associated functions. Understanding the full repertoire of CAFs and their dynamic ...changes as tumors evolve could improve the precision of cancer treatment. Here we comprehensively analyze CAFs using index and transcriptional single-cell sorting at several time points along breast tumor progression in mice, uncovering distinct subpopulations. Notably, the transcriptional programs of these subpopulations change over time and in metastases, transitioning from an immunoregulatory program to wound-healing and antigen-presentation programs, indicating that CAFs and their functions are dynamic. Two main CAF subpopulations are also found in human breast tumors, where their ratio is associated with disease outcome across subtypes and is particularly correlated with BRCA mutations in triple-negative breast cancer. These findings indicate that the repertoire of CAF changes over time in breast cancer progression, with direct clinical implications.
The accumulation of myeloid cells, particularly tumor‐associated macrophages (TAMs), characterizes the tumor microenvironment (TME) of many solid cancers, including breast cancer. Compared to healthy ...tissue‐resident macrophages, TAMs acquire distinct transcriptomes and tumor‐promoting functions by largely unknown mechanisms. Here, we hypothesize the involvement of TME signaling and subsequent epigenetic reprogramming of TAMs. Using the 4T1 mouse model of triple‐negative breast cancer, we demonstrate that the presence of cancer cells significantly alters the DNA methylation landscape of macrophages and, to a lesser extent, bone marrow‐derived monocytes (BMDMs). TAM methylomes, dissected into BMDM‐originating and TAM‐specific epigenetic programs, implicated transcription factors (TFs) and signaling pathways involved in TAM reprogramming, correlated with cancer‐specific gene expression patterns. Utilizing published single‐cell gene expression data, we linked microenvironmentally‐derived signals to the cancer‐specific DNA methylation landscape of TAMs. These integrative analyses highlighted the role of altered cytokine production in the TME (eg, TGF‐β, IFN‐γ and CSF1) on the induction of specific TFs (eg, FOSL2, STAT1 and RUNX3) responsible for the epigenetic reprogramming of TAMs. DNA methylation deconvolution identified a TAM‐specific signature associated with the identified signaling pathways and TFs, corresponding with severe tumor grade and poor prognosis of breast cancer patients. Similarly, immunosuppressive TAM functions were identified, such as induction of the immune inhibitory receptor‐ligand PD‐L1 by DNA hypomethylation of Cd274. Collectively, these results provide strong evidence that the epigenetic landscapes of macrophages and monocytes are perturbed by the presence of breast cancer, pointing to molecular mechanisms of TAM reprogramming, impacting patient outcomes.
What's new?
In many solid cancers, tumor‐associated macrophages (TAMs) congregate in the microenvironment around the tumor. These TAMs have distinct gene expression profiles and help the tumor survive and grow. Here, the authors investigated the differences between TAMs from a mouse model of triple‐negative breast cancer and mammary gland macrophages as well as monocytes from healthy mice. They show for the first time that cancer cells alter DNA methylation patterns in macrophages. These alterations, they found, affected transcription factors and signaling pathways associated with breast cancer progression and prognosis.
The
gene is mutated in approximately 30% of all breast cancer cases. Adipocytes and preadipocytes, which constitute a substantial fraction of the stroma of normal mammary tissue and breast tumors, ...undergo transcriptional, metabolic, and phenotypic reprogramming during breast cancer development and play an important role in tumor progression. We report here that p53 loss in breast cancer cells facilitates the reprogramming of preadipocytes, inducing them to acquire a unique transcriptional and metabolic program that combines impaired adipocytic differentiation with augmented cytokine expression. This, in turn, promotes the establishment of an inflammatory tumor microenvironment, including increased abundance of Ly6C+ and Ly6G+ myeloid cells and elevated expression of the immune checkpoint ligand PD-L1. We also describe a potential gain-of-function effect of common p53 missense mutations on the inflammatory reprogramming of preadipocytes. Altogether, our study implicates p53 deregulation in breast cancer cells as a driver of tumor-supportive adipose tissue reprogramming, expanding the network of non-cell autonomous mechanisms whereby p53 dysfunction may promote cancer. Further elucidation of the interplay between p53 and adipocytes within the tumor microenvironment may suggest effective therapeutic targets for the treatment of breast cancer patients.
Abstract
Cancer associated fibroblasts (CAFs) are prevalent in carcinomas. CAFs are also heterogeneous and perform various tumor-promoting tasks. Understanding whether distinct CAF-subsets exert ...specific functions, and how the composition of CAFs changes as tumors evolve could improve the accuracy of cancer treatment. Here, we analyzed thousands of CAFs by single-cell RNA-sequencing and index-sorting at several timepoints along breast tumor progression in mice, revealing distinct CAF-subsets. We discovered that the transcriptional programs of these subsets change over time, shifting from an immune-regulatory program at earlier timepoints to wound-healing and antigen-presenting programs at later timepoints, indicating that the composition and functions of CAFs are dynamic. We also found the two main CAF subsets in human breast tumors, wherein their ratio was associated with disease outcome. This association was particularly correlated with BRCA mutations in triple-negative breast cancer. Our findings indicate that the diverse composition of CAFs in breast cancer changes over time as tumors progress, and that these changes are linked to disease outcome.
Citation Format: Gil Friedman, Oshrat Levi-Galibov, Eyal David, Chamutal Bornstein, Amir Giladi, Maya Dadiani, Avi Mayo, Coral Halperin, Meirav Pevsner-Fischer, Hagar Lavon, Shimrit Mayer, Reinat Nevo, Yaniv Stein, Nora Balint-Lahat, Iris Barshack, H. Raza Ali, Carlos Caldas, Einav Nili Gal-Yam, Uri Alon, Ido Amit, Ruth Scherz-Shouval. Dynamic changes in the compositions of cancer associated-fibroblasts correlate with clinical outcome in breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB009.
Abstract
Cancer cells recruit and rewire normal cells in their microenvironment to support and protect them by creating a pro-tumorigenic tumor microenvironment (TME). We lack an overarching view of ...how, despite being genomically stable, stromal cells in the tumor microenvironment are heterogeneously reprogrammed across time and space to promote the evolution of aggressive disease. Recent work by us and others has shown that fibroblasts in the tumor microenvironment are transcriptionally rewired to become protumorigenic cancer associated fibroblasts (CAFs). Transcriptional regulation plays a major role in this reprogramming, but the extent to which epigenetic modifications of DNA also contribute to the rewiring of CAF transcription is not clear. Here we address this question by dissecting the epigenetic landscape of breast CAFs. We applied a sensitive method of whole genome bisulfide sequencing on a model of triple-negative breast cancer in mice to evaluate the methylome profile of CAFs compared to normal mammary fibroblasts (NMFs). we found that fibroblasts undergo massive DNA methylation changes as they transition into CAFs in mice. These changes inversely correlated with transcriptional changes between CAFs and NMFs. We characterized potential regulators of this process, and tested their expression in CAFs in human breast cancer patients, to confirm relevance of our findings to human disease. Our findings suggest that epigenetic alterations contribute to the transcriptional rewiring of fibroblasts to CAFs. This work presents a comprehensive map of DNA-methylation in CAFs, and reveals a previously unknown facet of the dynamic plasticity of the stroma.
Citation Format: Coral Halperin, Joschka Hey, Dieter Weichenhan, Yaniv Stein, Shimrit Mayer, Pavlo Lustik, Christoph Plass, Ruth Scherz-Shouval. Global DNA methylation analysis of cancer-associated fibroblasts reveals extensive epigenetic rewiring linked with RUNX1 upregulation in breast cancer stroma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5998.
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