Phenotypic heterogeneity driven by plasticity of the intermediate EMT state governs disease progression and metastasis in breast cancer.

The epithelial-to-mesenchymal transition (EMT) is frequently co-opted by cancer cells to enhance migratory and invasive cell traits. It is a key contributor to heterogeneity, chemoresistance, and metastasis in many carcinoma types, where the intermediate EMT state plays a critical tumor-initiating role. We isolate multiple distinct single-cell clones from the SUM149PT human breast cell line spanning the EMT spectrum having diverse migratory, tumor-initiating, and metastatic qualities, including three unique intermediates. Using a multiomics approach, we identify CBFß as a key regulator of metastatic ability in the intermediate state. To quantify epithelial-mesenchymal heterogeneity within tumors, we develop an advanced multiplexed immunostaining approach using SUM149-derived orthotopic tumors and find that the EMT state and epithelial-mesenchymal heterogeneity are predictive of overall survival in a cohort of stage III breast cancer. Our model reveals previously unidentified insights into the complex EMT spectrum and its regulatory networks, as well as the contributions of epithelial-mesenchymal plasticity (EMP) in tumor heterogeneity in breast cancer.

Quantifying epithelial-mesenchymal heterogeneity and EMT scoring in tumor samples via tyramide signal amplification (TSA).

Tumor heterogeneity presents an ongoing challenge to disease progression and treatment in many solid tumor types. Understanding the roots of intra-tumoral heterogeneity and how it may relate to the high incidence of metastasis is critical in overcoming disease relapse and chemoresistance. The epithelial-to-mesenchymal transition is a dynamic cellular program that is co-opted by cancer cells to enhance, among others, migratory and invasive cell traits. It is a key contributor to heterogeneity, chemo-resistance, and metastasis in many carcinoma-types, with the intermediate or hybrid EMT state playing a critical role due to its increased tumor-initiating potential. A critical component in utilizing this knowledge in patient treatment is to first detect and score the impact of EMT in a patient sample. Here, we provide a detailed protocol to detect EMT states and quantify the resulting epithelial-mesenchymal heterogeneity within tumors using a novel multiplexed immunostaining approach and analysis method. This protocol and concept can easily be adapted using custom panels of markers to explore other sources of tumoral heterogeneity in addition to EMT.

Distinct cytosine modification profiles define epithelial-to-mesenchymal cell-state transitions.

Background: Epithelial-to-mesenchymal transition (EMT) is an early step in the invasion-metastasis cascade, involving progression through intermediate cell states. Due to challenges with isolating intermediate cell states, genome-wide cytosine modifications that define transition are not completely understood. Methods: The authors measured multiple DNA cytosine modification marks and chromatin accessibility across clonal populations residing in specific EMT states. Results: Clones exhibiting more intermediate EMT phenotypes demonstrated increased 5-hydroxymethylcytosine and decreased 5-methylcytosine. Open chromatin regions containing increased 5-hydroxymethylcytosine CpG loci were enriched in EMT transcription factor motifs and were associated with Rho GTPases. Conclusion: The results indicate the importance of both distinct and shared epigenetic profiles associated with EMT processes that may be targeted to prevent EMT progression.

Quantifying the Epithelial-to-Mesenchymal Transition (EMT) from Bench to Bedside.

The epithelial-to-mesenchymal transition (EMT) and its reversal, the mesenchymal-to-epithelial transition (MET) are critical components of the metastatic cascade in breast cancer and many other solid tumor types. Recent work has uncovered the presence of a variety of states encompassed within the EMT spectrum, each of which may play unique roles or work collectively to impact tumor progression. However, defining EMT status is not routinely carried out to determine patient prognosis or dictate therapeutic decision-making in the clinic. Identifying and quantifying the presence of various EMT states within a tumor is a critical first step to scoring patient tumors to aid in determining prognosis. Here, we review the major strides taken towards translating our understanding of EMT biology from bench to bedside. We review previously used approaches including basic immunofluorescence staining, flow cytometry, single-cell sequencing, and multiplexed tumor mapping. Future studies will benefit from the consideration of multiple methods and combinations of markers in designing a diagnostic tool for detecting and measuring EMT in patient tumors.

Limiting Self-Renewal of the Basal Compartment by PKA Activation Induces Differentiation and Alters the Evolution of Mammary Tumors.

Differentiation therapy utilizes our understanding of the hierarchy of cellular systems to pharmacologically induce a shift toward terminal commitment. While this approach has been a paradigm in treating certain hematological malignancies, efforts to translate this success to solid tumors have met with limited success. Mammary-specific activation of PKA in mouse models leads to aberrant differentiation and diminished self-renewing potential of the basal compartment, which harbors mammary repopulating cells. PKA activation results in tumors that are more benign, exhibiting reduced metastatic propensity, loss of tumor-initiating potential, and increased sensitivity to chemotherapy. Analysis of tumor histopathology revealed features of overt differentiation with papillary characteristics. Longitudinal single-cell profiling at the hyperplasia and tumor stages uncovered an altered path of tumor evolution whereby PKA curtails the emergence of aggressive subpopulations. Acting through the repression of SOX4, PKA activation promotes tumor differentiation and represents a possible adjuvant to chemotherapy for certain breast cancers.

Loss of heterozygosity of essential genes represents a widespread class of potential cancer vulnerabilities.

Alterations in non-driver genes represent an emerging class of potential therapeutic targets in cancer. Hundreds to thousands of non-driver genes undergo loss of heterozygosity (LOH) events per tumor, generating discrete differences between tumor and normal cells. Here we interrogate LOH of polymorphisms in essential genes as a novel class of therapeutic targets. We hypothesized that monoallelic inactivation of the allele retained in tumors can selectively kill cancer cells but not somatic cells, which retain both alleles. We identified 5664 variants in 1278 essential genes that undergo LOH in cancer and evaluated the potential for each to be targeted using allele-specific gene-editing, RNAi, or small-molecule approaches. We further show that allele-specific inactivation of either of two essential genes (PRIM1 and EXOSC8) reduces growth of cells harboring that allele, while cells harboring the non-targeted allele remain intact. We conclude that LOH of essential genes represents a rich class of non-driver cancer vulnerabilities.

Targeting Cancer Gene Dependencies with Anthrax-Mediated Delivery of Peptide Nucleic Acids.

Antisense oligonucleotide therapies are important cancer treatments, which can suppress genes in cancer cells that are critical for cell survival. SF3B1 has recently emerged as a promising gene target that encodes a key splicing factor in the SF3B protein complex. Over 10% of cancers have lost one or more copies of the SF3B1 gene, rendering these cancers vulnerable after further suppression. SF3B1 is just one example of a CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) gene, but over 120 additional candidate CYCLOPS genes are known. Antisense oligonucleotide therapies for cancer offer the promise of effective suppression for CYCLOPS genes, but developing these treatments is difficult due to their limited permeability into cells and poor cytosolic stability. Here, we develop an effective approach to suppress CYCLOPS genes by delivering antisense peptide nucleic acids (PNAs) into the cytosol of cancer cells. We achieve efficient cytosolic PNA delivery with the two main nontoxic components of the anthrax toxin: protective antigen (PA) and the 263-residue N-terminal domain of lethal factor (LFN). Sortase-mediated ligation readily enables the conjugation of PNAs to the C terminus of the LFN protein. LFN and PA work together in concert to translocate PNAs into the cytosol of mammalian cells. Antisense SF3B1 PNAs delivered with the LFN/PA system suppress the SF3B1 gene and decrease cell viability, particularly of cancer cells with partial copy-number loss of SF3B1. Moreover, antisense SF3B1 PNAs delivered with a HER2-binding PA variant selectively target cancer cells that overexpress the HER2 cell receptor, demonstrating receptor-specific targeting of cancer cells. Taken together, our efforts illustrate how PA-mediated delivery of PNAs provides an effective and general approach for delivering antisense PNA therapeutics and for targeting gene dependencies in cancer.

Copy-number and gene dependency analysis reveals partial copy loss of wild-type SF3B1 as a novel cancer vulnerability.

Genomic instability is a hallmark of human cancer, and results in widespread somatic copy number alterations. We used a genome-scale shRNA viability screen in human cancer cell lines to systematically identify genes that are essential in the context of particular copy-number alterations (copy-number associated gene dependencies). The most enriched class of copy-number associated gene dependencies was CYCLOPS (Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS) genes, and spliceosome components were the most prevalent. One of these, the pre-mRNA splicing factor SF3B1, is also frequently mutated in cancer. We validated SF3B1 as a CYCLOPS gene and found that human cancer cells harboring partial SF3B1 copy-loss lack a reservoir of SF3b complex that protects cells with normal SF3B1 copy number from cell death upon partial SF3B1 suppression. These data provide a catalog of copy-number associated gene dependencies and identify partial copy-loss of wild-type SF3B1 as a novel, non-driver cancer gene dependency.

Exhaled breath condensate pH does not discriminate asymptomatic gastroesophageal reflux or the response to lansoprazole treatment in children with poorly controlled asthma.

BACKGROUND: Although exhaled breath condensate (EBC) pH has been identified as an "emerging" biomarker of interest for asthma clinical trials, the clinical determinants of EBC pH remain poorly understood. Other studies have associated acid reflux-induced respiratory symptoms, for example, cough, with transient acidification of EBC. OBJECTIVE: We sought to determine the clinical and physiologic correlates of EBC acidification in a highly characterized sample of children with poorly controlled asthma. We hypothesized that (1) children with asymptomatic gastroesophageal reflux determined by 24-hour esophageal pH monitoring would have a lower EBC pH than children without gastroesophageal reflux, (2) treatment with lansoprazole would alter EBC pH in those children, and (3) EBC acidification would be associated with increased asthma symptoms, poorer asthma control and quality of life, and increased formation of breath nitrogen oxides (NOx). METHODS: A total of 110 children, age range 6 to 17 years, with poor asthma control and esophageal pH data enrolled in the Study of Acid Reflux in Children with Asthma (NCT00442013) were included. Children submitted EBC samples for pH and NOx measurement at randomization and at study weeks 8, 16, and 24. RESULTS: Serial EBC pH measurements failed to distinguish asymptomatic gastroesophageal reflux and was not associated with breath NOx formation. EBC pH also did not discriminate asthma characteristics such as medication and health care utilization, pulmonary function, and asthma control and quality of life both at baseline and across the study period. CONCLUSION: Despite the relative ease of EBC collection, EBC pH as a biomarker does not provide useful information of children with asthma who were enrolled in asthma clinical trials.

Residential proximity to a major roadway is associated with features of asthma control in children.

BACKGROUND: While several studies suggest that traffic-related air pollutants are detrimental for respiratory health, few studies have examined relationships between residential proximity to a major roadway and asthma control in children. Furthermore, a major limitation of existing research is reliance on self-reported outcomes. We therefore determined the spatial relationship between the distance from a major roadway and clinical, physiologic and inflammatory features of asthma in a highly characterized sample of asthmatic children 6-17 years of age across a wide range of severities. We hypothesized that a closer residential proximity to a major roadway would be associated with increased respiratory symptoms, altered pulmonary function and a greater magnitude of airway and systemic inflammation. METHODOLOGY/PRINCIPAL FINDINGS: 224 children 6-17 years with confirmed asthma completed questionnaires and underwent spirometry, plethysmography, exhaled nitric oxide determination, exhaled breath condensate collection and venipuncture. Residential distance from a major roadway was determined by mapping the geographic coordinates of the residential address in Geographic Information System software. The distance between the home address and the nearest major roadway was calculated according to the shortest distance between the two points (i.e., "as the crow flies"). Asthmatic children living in closer proximity to a major roadway had an increased frequency of wheezing associated with increased medication requirements and more hospitalizations even after controlling for potential confounders. These children also had increased airway resistance, increased airway inflammation reflected by a lower breath condensate pH, and higher plasma EGF concentrations. CONCLUSIONS/SIGNIFICANCE: These findings suggest that closer residential proximity to a major roadway is associated with poorer asthma control in school-age children. Assessment of residential proximity to major roadways may be useful in the clinical evaluation of asthma in children.

Glutathione oxidation is associated with airway macrophage functional impairment in children with severe asthma.

Airway cellular dysfunction is a differentiating feature of severe asthma in children that may be related to an imbalance of the antioxidant, glutathione (GSH). We hypothesized that oxidation of GSH to glutathione disulfide (GSSG) in the epithelial lining fluid (ELF) of children with severe asthma would contribute to altered airway macrophage (AM) GSH homeostasis and AM cellular dysfunction. Bronchoalveolar lavage (BAL) was performed in 64 asthmatic children (severe asthma, n = 43). GSH, GSSG, markers of lipid peroxidation and DNA oxidation, and IL-8 were quantified in the BAL supernatant. GSH, GSSG, activities of histone deacetylase (HDAC) and histone acetyltransferase, apoptosis, and phagocytosis were assessed in isolated AMs. Children with severe asthma had increased GSSG, lipid peroxidation, byproducts of DNA oxidation, and inflammation in the ELF. This imbalance of GSH homeostasis was also noted intracellularly within the AMs and was associated with decreased HDAC activities, increased apoptosis, and impaired phagocytosis. In vitro GSH supplementation inhibited apoptosis and rescued phagocytosis in children with severe asthma. Severe asthma in children is characterized by altered airway and intracellular AM GSH homeostasis that translates to impaired AM function. Interventions to restore airway GSH homeostasis may be warranted in children with severe asthma.