Unlocking the Role of Age-Related Changes to Fibroblasts in Pancreatic Cancer.

Pancreatic cancer prevalence increases with age, and disease prognosis is poorer in older individuals. The increased prevalence is driven, undoubtedly, by the multistep accumulation of oncogenic mutations in cancer cells with age. However, fibroblasts are major constituents and key players in pancreatic cancer, and they too undergo age-related changes that may contribute to disease severity. In this issue of Cancer Research, Zabransky and colleagues set out to dissect the effect of age-related changes in pancreatic fibroblasts on pancreatic ductal adenocarcinoma growth and metastasis. They discovered that aged fibroblasts secrete GDF-15, which in turn activates AKT signaling and accelerates tumor progression. These findings provide a mechanistic role for aged fibroblasts in pancreatic cancer, underpinning the importance of normal physiologic processes in tumor progression. See related article by Zabransky et al., p. 1221.

Loss of EIF4G2 mediates aggressiveness in distinct human endometrial cancer subpopulations with poor survival outcome in patients.

The non-canonical translation initiation factor EIF4G2 plays essential roles in cellular stress responses via translation of selective mRNA cohorts. Currently there is limited and conflicting information regarding its involvement in cancer development and progression. Here we assessed its role in endometrial cancer (EC), in a cohort of 280 EC patients across different types, grades, and stages, and found that low EIF4G2 expression highly correlated with poor overall- and recurrence-free survival in Grade 2 EC patients, monitored over a period of up to 12 years. To establish a causative connection between low EIF4G2 expression and cancer progression, we stably knocked-down EIF4G2 in two human EC cell lines in parallel. EIF4G2 depletion resulted in increased resistance to conventional therapies and increased the prevalence of molecular markers for aggressive cell subsets, altering their transcriptional and proteomic landscapes. Prominent among the proteins with decreased abundance were Kinesin-1 motor proteins, KIF5B and KLC1, 2, 3. Multiplexed imaging of the EC patient tumor cohort showed a correlation between decreased expression of the kinesin proteins, and poor survival in patients with tumors of certain grades and stages. These findings reveal potential novel biomarkers for Grade 2 EC with ramifications for patient stratification and therapeutic interventions.

p53 deficient breast cancer cells reprogram preadipocytes toward tumor-protective immunomodulatory cells.

The TP53 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.

The tumor microenvironment shows a hierarchy of cell-cell interactions dominated by fibroblasts.

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.

Breast Cancer-Secreted Factors Promote Lung Metastasis by Signaling Systemically to Induce a Fibrotic Premetastatic Niche.

Metastatic cancer is largely incurable and is the main cause of cancer-related deaths. The metastatic microenvironment facilitates formation of metastases. Cancer-associated fibroblasts (CAF) are crucial players in generating a hospitable metastatic niche by mediating an inflammatory microenvironment. Fibroblasts also play a central role in modifying the architecture and stiffness of the extracellular matrix (ECM). Resolving the early changes in the metastatic niche could help identify approaches to inhibit metastatic progression. Here, we demonstrate in mouse models of spontaneous breast cancer pulmonary metastasis that fibrotic changes and rewiring of lung fibroblasts occurred at premetastatic stages, suggesting systemic influence by the primary tumor. Activin A (ActA), a TGFß superfamily member, was secreted from breast tumors and its levels in the blood were highly elevated in tumor-bearing mice. ActA upregulated the expression of profibrotic factors in lung fibroblasts, leading to enhanced collagen deposition in the lung premetastatic niche. ActA signaling was functionally important for lung metastasis, as genetic targeting of ActA in breast cancer cells significantly attenuated lung metastasis and improved survival. Moreover, high levels of ActA in human patients with breast cancer were associated with lung metastatic relapse and poor survival. This study uncovers a novel mechanism by which breast cancer cells systemically rewire the stromal microenvironment in the metastatic niche to facilitate pulmonary metastasis. SIGNIFICANCE: ActA mediates cross-talk between breast cancer cells and cancer-associated fibroblasts in the lung metastatic niche that enhances fibrosis and metastasis, implicating ActA as a potential therapeutic target to inhibit metastatic relapse.

Keeping IPMNs in Check: A Novel Role for the Transcription Factor NKX6-2 in Preserving an Indolent Cell Identity in Pancreatic Cystic Lesions.

SUMMARY: In this issue of Cancer Discovery, Sans and colleagues identify the transcription factor NKX6-2 as a principal element in maintaining the low-grade gastric cell phenotype of intraductal papillary mucinous neoplasms (IPMN) in the pancreas. Their discoveries in patient cohorts and dissection in animal models provide a novel molecular understanding underpinning IPMN differentiation, with implications for risk stratification and therapeutic intervention in pancreatic cancer. See related article by Sans et al., p. 1844 (7).

Insights into the co-evolution of epithelial cells and fibroblasts in the esophageal tumor microenvironment.

Cancer-associated fibroblasts (CAFs) are recruited and rewired by cancer cells to become protumorigenic. The molecular mechanisms underlying this crosstalk in esophageal cancer are completely unknown. Chen et al. discover that premalignant epithelial cells of the esophagus rewire normal resident fibroblasts into CAFs through the downregulation of ANXA1-FRP2 signaling.

From gatekeepers to providers: regulation of immune functions by cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) are major protumorigenic components of the tumor microenvironment in solid cancers. CAFs are heterogeneous, consisting of multiple subsets that display diverse functions. Recently, CAFs have emerged as major promoters of immune evasion. CAFs favor T cell exclusion and exhaustion, promote recruitment of myeloid-derived suppressor cells, and induce protumoral phenotypic shifts in macrophages and neutrophils. With the growing appreciation of CAF heterogeneity came the understanding that different CAF subpopulations may be driving distinct immune-regulatory effects, interacting with different cell types, and perhaps even driving opposing effects on malignancy. In this review we discuss the current understanding of CAF-immune interactions, their effect on tumor progression and therapeutic response, and the possibility of exploiting CAF-immune interactions as potential targets for cancer therapy.

Second Virtual International Symposium on Cellular and Organismal Stress Responses, September 8-9, 2022.

The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8-9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI.

DNA methylation landscape of tumor-associated macrophages reveals pathways, transcription factors and prognostic value relevant to triple-negative breast cancer patients.

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-ß, 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.

BRCA mutational status shapes the stromal microenvironment of pancreatic cancer linking clusterin expression in cancer associated fibroblasts with HSF1 signaling.

Tumors initiate by mutations in cancer cells, and progress through interactions of the cancer cells with non-malignant cells of the tumor microenvironment. Major players in the tumor microenvironment are cancer-associated fibroblasts (CAFs), which support tumor malignancy, and comprise up to 90% of the tumor mass in pancreatic cancer. CAFs are transcriptionally rewired by cancer cells. Whether this rewiring is differentially affected by different mutations in cancer cells is largely unknown. Here we address this question by dissecting the stromal landscape of BRCA-mutated and BRCA Wild-type pancreatic ductal adenocarcinoma. We comprehensively analyze pancreatic cancer samples from 42 patients, revealing different CAF subtype compositions in germline BRCA-mutated vs. BRCA Wild-type tumors. In particular, we detect an increase in a subset of immune-regulatory clusterin-positive CAFs in BRCA-mutated tumors. Using cancer organoids and mouse models we show that this process is mediated through activation of heat-shock factor 1, the transcriptional regulator of clusterin. Our findings unravel a dimension of stromal heterogeneity influenced by germline mutations in cancer cells, with direct implications for clinical research.

Global DNA Methylation Analysis of Cancer-Associated Fibroblasts Reveals Extensive Epigenetic Rewiring Linked with RUNX1 Upregulation in Breast Cancer Stroma.

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. SIGNIFICANCE: 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.

Cancer-associated fibroblasts in the single-cell era.

Cancer-associated fibroblasts (CAFs) are central players in the microenvironment of solid tumors, affecting cancer progression and metastasis. CAFs have diverse phenotypes, origins and functions and consist of distinct subpopulations. Recent progress in single-cell RNA-sequencing technologies has enabled detailed characterization of the complexity and heterogeneity of CAF subpopulations in multiple tumor types. In this Review, we discuss the current understanding of CAF subsets and functions as elucidated by single-cell technologies, their functional plasticity, and their emergent shared and organ-specific features that could potentially be harnessed to design better therapeutic strategies for cancer.

T Cells Promote Metastasis by Regulating Extracellular Matrix Remodeling following Chemotherapy.

Metastasis is the main cause of cancer-related mortality. Despite intense efforts to understand the mechanisms underlying the metastatic process, treatment of metastatic cancer is still challenging. Here we describe a chemotherapy-induced, host-mediated mechanism that promotes remodeling of the extracellular matrix (ECM), ultimately facilitating cancer cell seeding and metastasis. Paclitaxel (PTX) chemotherapy enhanced rapid ECM remodeling and mechanostructural changes in the lungs of tumor-free mice, and the protein expression and activity of the ECM remodeling enzyme lysyl oxidase (LOX) increased in response to PTX. A chimeric mouse model harboring genetic LOX depletion revealed chemotherapy-induced ECM remodeling was mediated by CD8+ T cells expressing LOX. Consistently, adoptive transfer of CD8+ T cells, but not CD4+ T cells or B cells, from PTX-treated mice to naïve immunodeprived mice induced pulmonary ECM remodeling. Lastly, in a clinically relevant metastatic breast carcinoma model, LOX inhibition counteracted the metastasis-promoting, ECM-related effects of PTX. This study highlights the role of immune cells in regulating ECM and metastasis following chemotherapy, suggesting that inhibiting chemotherapy-induced ECM remodeling represents a potential therapeutic strategy for metastatic cancer. SIGNIFICANCE: Chemotherapy induces prometastatic pulmonary ECM remodeling by upregulating LOX in T cells, which can be targeted with LOX inhibitors to suppress metastasis.See related commentary by Kolonin and Woodward, p. 197.

Impact of TP53 Genomic Alterations in Large B-Cell Lymphoma Treated With CD19-Chimeric Antigen Receptor T-Cell Therapy.

PURPOSE: Tumor-intrinsic features may render large B-cell lymphoma (LBCL) insensitive to CD19-directed chimeric antigen receptor T cells (CAR-T). We hypothesized that TP53 genomic alterations are detrimental to response outcomes in LBCL treated with CD19-CAR-T. MATERIALS AND METHODS: Patients with LBCL treated with CD19-CAR-T were included. Targeted next-generation sequencing was performed on pre-CAR-T tumor samples in a subset of patients. Response and survival rates by histologic, cytogenetic, and molecular features were assessed. Within a cohort of newly diagnosed LBCL with genomic and transcriptomic profiling, we studied interactions between cellular pathways and TP53 status. RESULTS: We included 153 adults with relapsed or refractory LBCL treated with CD19-CAR-T (axicabtagene ciloleucel [50%], tisagenlecleucel [32%], and lisocabtagene maraleucel [18%]). Outcomes echoed pivotal trials: complete response (CR) rate 54%, median overall survival (OS) 21.1 months (95% CI, 14.8 to not reached), and progression-free survival 6 months (3.4 to 9.7). Histologic and cytogenetic LBCL features were not predictive of CR. In a subset of 82 patients with next-generation sequencing profiling, CR and OS rates were comparable with the unsequenced cohort. TP53 alterations (mutations and/or copy number alterations) were common (37%) and associated with inferior CR and OS rates in univariable and multivariable regression models; the 1-year OS in TP53-altered LBCL was 44% (95% CI, 29 to 67) versus 76% (65 to 89) in wild-type (P = .012). Transcriptomic profiling from a separate cohort of patients with newly diagnosed lymphoma (n = 562) demonstrated that TP53 alterations are associated with dysregulation of pathways related to CAR-T-cell cytotoxicity, including interferon and death receptor signaling pathway and reduced CD8 T-cell tumor infiltration. CONCLUSION: TP53 is a potent tumor-intrinsic biomarker that can inform risk stratification and clinical trial design in patients with LBCL treated with CD19-CAR-T. The role of TP53 should be further validated in independent cohorts.

Multiplexed profiling facilitates robust m6A quantification at site, gene and sample resolution.

N6-methyladenosine (m6A) is the most prevalent modification of messenger RNA in mammals. To interrogate its functions and dynamics, there is a critical need to quantify m6A at three levels: site, gene and sample. Current approaches address these needs in a limited manner. Here we develop m6A-seq2, relying on multiplexed m6A-immunoprecipitation of barcoded and pooled samples. m6A-seq2 allows a big increase in throughput while reducing technical variability, requirements of input material and cost. m6A-seq2 is furthermore uniquely capable of providing sample-level relative quantitations of m6A, serving as an orthogonal alternative to mass spectrometry-based approaches. Finally, we develop a computational approach for gene-level quantitation of m6A. We demonstrate that using this metric, roughly 30% of the variability in RNA half life in mouse embryonic stem cells can be explained, establishing m6A as a main driver of RNA stability. m6A-seq2 thus provides an experimental and analytic framework for dissecting m6A-mediated regulation at three different levels.

Evolution of fibroblasts in the lung metastatic microenvironment is driven by stage-specific transcriptional plasticity.

Mortality from breast cancer is almost exclusively a result of tumor metastasis, and lungs are one of the main metastatic sites. Cancer-associated fibroblasts are prominent players in the microenvironment of breast cancer. However, their role in the metastatic niche is largely unknown. In this study, we profiled the transcriptional co-evolution of lung fibroblasts isolated from transgenic mice at defined stage-specific time points of metastases formation. Employing multiple knowledge-based platforms of data analysis provided powerful insights on functional and temporal regulation of the transcriptome of fibroblasts. We demonstrate that fibroblasts in lung metastases are transcriptionally dynamic and plastic, and reveal stage-specific gene signatures that imply functional tasks, including extracellular matrix remodeling, stress response, and shaping the inflammatory microenvironment. Furthermore, we identified Myc as a central regulator of fibroblast rewiring and found that stromal upregulation of Myc transcriptional networks is associated with disease progression in human breast cancer.

First Virtual International Congress on Cellular and Organismal Stress Responses, November 5-6, 2020.

Members of the Cell Stress Society International (CSSI), Patricija van Oosten-Hawle (University of Leeds, UK), Mehdi Mollapour (SUNY Upstate Medical University, USA), Andrew Truman (University of North Carolina at Charlotte, USA) organized a new virtual meeting format which took place on November 5-6, 2020. The goal of this congress was to provide an international platform for scientists to exchange data and ideas among the Cell Stress and Chaperones community during the Covid-19 pandemic. Here we will highlight the summary of the meeting and acknowledge those who were honored by the CSSI.

Cancer-Associated Fibroblasts Promote Aggressive Gastric Cancer Phenotypes via Heat Shock Factor 1-Mediated Secretion of Extracellular Vesicles.

Gastric cancer is the third most lethal cancer worldwide, and evaluation of the genomic status of gastric cancer cells has not translated into effective prognostic or therapeutic strategies. We therefore hypothesize that outcomes may depend on the tumor microenvironment (TME), in particular, cancer-associated fibroblasts (CAF). However, very little is known about the role of CAFs in gastric cancer. To address this, we mapped the transcriptional landscape of human gastric cancer stroma by microdissection and RNA sequencing of CAFs from patients with gastric cancer. A stromal gene signature was associated with poor disease outcome, and the transcription factor heat shock factor 1 (HSF1) regulated the signature. HSF1 upregulated inhibin subunit beta A and thrombospondin 2, which were secreted in CAF-derived extracellular vesicles to the TME to promote cancer. Together, our work provides the first transcriptional map of human gastric cancer stroma and highlights HSF1 and its transcriptional targets as potential diagnostic and therapeutic targets in the genomically stable tumor microenvironment. SIGNIFICANCE: This study shows how HSF1 regulates a stromal transcriptional program associated with aggressive gastric cancer and identifies multiple proteins within this program as candidates for therapeutic intervention. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/7/1639/F1.large.jpg.