Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

The reprogramming of tumor stroma by HSF1 is a potent enabler of malignancy.

Stromal cells within the tumor microenvironment are essential for tumor progression and metastasis. Surprisingly little is known about the factors that drive the transcriptional reprogramming of stromal cells within tumors. We report that the transcriptional regulator heat shock factor 1 (HSF1) is frequently activated in cancer-associated fibroblasts (CAFs), where it is a potent enabler of malignancy. HSF1 drives a transcriptional program in CAFs that complements, yet is completely different from, the program it drives in adjacent cancer cells. This CAF program is uniquely structured to support malignancy in a non-cell-autonomous way. Two central stromal signaling molecules-TGF-ß and SDF1-play a critical role. In early-stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. Thus, tumors co-opt the ancient survival functions of HSF1 to orchestrate malignancy in both cell-autonomous and non-cell-autonomous ways, with far-reaching therapeutic implications.

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.

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.

Expanding the Organismal Proteostasis Network: Linking Systemic Stress Signaling with the Innate Immune Response.

Stress response pathways regulate proteostasis and mitigate macromolecular damage to promote long-term cellular health. Intercellular signaling is an essential layer of systemic proteostasis in an organism and is facilitated via transcellular signaling molecules that orchestrate the activation of stress responses across tissues and organs. Accumulating evidence indicates that components of the immune response act as signaling factors that regulate the cell-non-autonomous proteostasis network. Here, we review emergent advances in our understanding of cell-non-autonomous regulators of proteostasis networks in multicellular settings, from the model organism, Caenorhabditis elegans, to humans. We further discuss how innate immune responses can be players of the organismal proteostasis network and discuss how both are linked in cancer.

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.

The Role of HSF1 and the Chaperone Network in the Tumor Microenvironment.

Tumors are stressful environments. As tumors evolve from single mutated cancer cells into invasive malignancies they must overcome various constraints and barriers imposed by a hostile microenvironment. To achieve this, cancer cells recruit and rewire cells in their microenvironment to become pro-tumorigenic. We propose that chaperones are vital players in this process, and that activation of stress responses helps tumors adapt and evolve into aggressive malignancies, by enabling phenotypic plasticity in the tumor microenvironment (TME). In this chapter we will review evidence supporting non-cancer-cell-autonomous activity of chaperones in human patients and mouse models of cancer, discuss the mechanisms by which this non-cell-autonomous activity is mediated and provide an evolutionary perspective on the basis of this phenomenon.

Fitness trade-offs restrict the evolution of resistance to amphotericin B.

The evolution of drug resistance in microbial pathogens provides a paradigm for investigating evolutionary dynamics with important consequences for human health. Candida albicans, the leading fungal pathogen of humans, rapidly evolves resistance to two major antifungal classes, the triazoles and echinocandins. In contrast, resistance to the third major antifungal used in the clinic, amphotericin B (AmB), remains extremely rare despite 50 years of use as monotherapy. We sought to understand this long-standing evolutionary puzzle. We used whole genome sequencing of rare AmB-resistant clinical isolates as well as laboratory-evolved strains to identify and investigate mutations that confer AmB resistance in vitro. Resistance to AmB came at a great cost. Mutations that conferred resistance simultaneously created diverse stresses that required high levels of the molecular chaperone Hsp90 for survival, even in the absence of AmB. This requirement stemmed from severe internal stresses caused by the mutations, which drastically diminished tolerance to external stresses from the host. AmB-resistant mutants were hypersensitive to oxidative stress, febrile temperatures, and killing by neutrophils and also had defects in filamentation and tissue invasion. These strains were avirulent in a mouse infection model. Thus, the costs of evolving resistance to AmB limit the emergence of this phenotype in the clinic. Our work provides a vivid example of the ways in which conflicting selective pressures shape evolutionary trajectories and illustrates another mechanism by which the Hsp90 buffer potentiates the emergence of new phenotypes. Developing antibiotics that deliberately create such evolutionary constraints might offer a strategy for limiting the rapid emergence of drug resistance.

Regulation of autophagy by ROS: physiology and pathology.

Reactive oxygen species (ROS) are small and highly reactive molecules that can oxidize proteins, lipids and DNA. When tightly controlled, ROS serve as signaling molecules by modulating the activity of the oxidized targets. Accumulating data point to an essential role for ROS in the activation of autophagy. Be the outcome of autophagy survival or death and the initiation conditions starvation, pathogens or death receptors, ROS are invariably involved. The nature of this involvement, however, remains unclear. Moreover, although connections between ROS and autophagy are observed in diverse pathological conditions, the mode of activation of autophagy and its potential protective role remain incompletely understood. Notably, recent advances in the field of redox regulation of autophagy focus on the role of mitochondria as a source of ROS and on mitophagy as a means for clearance of ROS.

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.

Multi-parametric atlas of the pre-metastatic liver for prediction of metastatic outcome in early-stage pancreatic cancer.

Metastasis occurs frequently after resection of pancreatic cancer (PaC). In this study, we hypothesized that multi-parametric analysis of pre-metastatic liver biopsies would classify patients according to their metastatic risk, timing and organ site. Liver biopsies obtained during pancreatectomy from 49 patients with localized PaC and 19 control patients with non-cancerous pancreatic lesions were analyzed, combining metabolomic, tissue and single-cell transcriptomics and multiplex imaging approaches. Patients were followed prospectively (median 3 years) and classified into four recurrence groups; early (<6 months after resection) or late (>6 months after resection) liver metastasis (LiM); extrahepatic metastasis (EHM); and disease-free survivors (no evidence of disease (NED)). Overall, PaC livers exhibited signs of augmented inflammation compared to controls. Enrichment of neutrophil extracellular traps (NETs), Ki-67 upregulation and decreased liver creatine significantly distinguished those with future metastasis from NED. Patients with future LiM were characterized by scant T cell lobular infiltration, less steatosis and higher levels of citrullinated H3 compared to patients who developed EHM, who had overexpression of interferon target genes (MX1 and NR1D1) and an increase of CD11B+ natural killer (NK) cells. Upregulation of sortilin-1 and prominent NETs, together with the lack of T cells and a reduction in CD11B+ NK cells, differentiated patients with early-onset LiM from those with late-onset LiM. Liver profiles of NED closely resembled those of controls. Using the above parameters, a machine-learning-based model was developed that successfully predicted the metastatic outcome at the time of surgery with 78% accuracy. Therefore, multi-parametric profiling of liver biopsies at the time of PaC diagnosis may determine metastatic risk and organotropism and guide clinical stratification for optimal treatment selection.

p53-dependent regulation of autophagy protein LC3 supports cancer cell survival under prolonged starvation.

The p53 tumor suppressor is mutated in a high percentage of human tumors. However, many other tumors retain wild-type (wt) p53 expression, raising the intriguing possibility that they actually benefit from it. Recent studies imply a role for p53 in regulation of autophagy, a catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles, particularly under conditions of nutrient deprivation. Here, we show that, in many cell types, p53 confers increased survival in the face of chronic starvation. We implicate regulation of autophagy in this effect. In HCT116 human colorectal cancer cells exposed to prolonged nutrient deprivation, the endogenous wt p53 posttranscriptionally down-regulates LC3, a pivotal component of the autophagic machinery. This enables reduced, yet sustainable autophagic flux. Loss of p53 impairs autophagic flux and causes excessive LC3 accumulation upon starvation, culminating in apoptosis. Thus, p53 increases cell fitness by maintaining better autophagic homeostasis, adjusting the rate of autophagy to changing circumstances. We propose that some cancer cells retain wt p53 to benefit from the resultant increased fitness under limited nutrient supply.

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.

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.

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.