FoxA1/2-dependent epigenomic reprogramming drives lineage switching in lung adenocarcinoma.

The ability of cancer cells to alter their identity is essential for tumor survival and progression. Loss of the pulmonary lineage specifier NKX2-1 within KRAS-driven lung adenocarcinoma (LUAD) enhances tumor progression and results in a pulmonary-to-gastric lineage switch that is dependent upon the activity of pioneer factors FoxA1 and FoxA2; however, the underlying mechanism remains largely unknown. Here, we show that FoxA1/2 reprogram the epigenetic landscape of NKX2-1-negative LUAD to facilitate a gastric identity. After Nkx2-1 deletion, FoxA1/2 mediate demethylation of gastric-defining genes through recruitment of TET3, an enzyme that induces DNA demethylation. H3K27ac ChIP-seq and HiChIP show that FoxA1/2 also control the activity of regulatory elements and their 3D interactions at gastric loci. Furthermore, oncogenic KRAS is required for the FoxA1/2-dependent epigenetic reprogramming. This work demonstrates the role of FoxA1/2 in rewiring the methylation and histone landscape and cis-regulatory dynamics of NKX2-1-negative LUAD to drive cancer cell lineage switching.

FoxA1 and FoxA2 control growth and cellular identity in NKX2-1-positive lung adenocarcinoma.

Changes in cellular identity (also known as histologic transformation or lineage plasticity) can drive malignant progression and resistance to therapy in many cancers, including lung adenocarcinoma (LUAD). The lineage-specifying transcription factors FoxA1 and FoxA2 (FoxA1/2) control identity in NKX2-1/TTF1-negative LUAD. However, their role in NKX2-1-positive LUAD has not been systematically investigated. We find that Foxa1/2 knockout severely impairs tumorigenesis in KRAS-driven genetically engineered mouse models and human cell lines. Loss of FoxA1/2 leads to the collapse of a dual-identity state, marked by co-expression of pulmonary and gastrointestinal transcriptional programs, which has been implicated in LUAD progression. Mechanistically, FoxA1/2 loss leads to aberrant NKX2-1 activity and genomic localization, which in turn actively inhibits tumorigenesis and drives alternative cellular identity programs that are associated with non-proliferative states. This work demonstrates that FoxA1/2 expression is a lineage-specific vulnerability in NKX2-1-positive LUAD and identifies mechanisms of response and resistance to targeting FoxA1/2 in this disease.

An NKX2-1/ERK/WNT feedback loop modulates gastric identity and response to targeted therapy in lung adenocarcinoma.

Cancer cells undergo lineage switching during natural progression and in response to therapy. NKX2-1 loss in human and murine lung adenocarcinoma leads to invasive mucinous adenocarcinoma (IMA), a lung cancer subtype that exhibits gastric differentiation and harbors a distinct spectrum of driver oncogenes. In murine BRAFV600E-driven lung adenocarcinoma, NKX2-1 is required for early tumorigenesis, but dispensable for established tumor growth. NKX2-1-deficient, BRAFV600E-driven tumors resemble human IMA and exhibit a distinct response to BRAF/MEK inhibitors. Whereas BRAF/MEK inhibitors drive NKX2-1-positive tumor cells into quiescence, NKX2-1-negative cells fail to exit the cell cycle after the same therapy. BRAF/MEK inhibitors induce cell identity switching in NKX2-1-negative lung tumors within the gastric lineage, which is driven in part by WNT signaling and FoxA1/2. These data elucidate a complex, reciprocal relationship between lineage specifiers and oncogenic signaling pathways in the regulation of lung adenocarcinoma identity that is likely to impact lineage-specific therapeutic strategies.

When cells become cancerous they grow uncontrollably and spread into surrounding healthy tissue. As the cancer progresses different genes are switched on and off which can cause tumor cells to change their identity and transition into other types of cell. How closely tumor cells resemble the healthy tissue they came from can influence how well the cancer responds to treatment. Many lung cancers have an identity similar to normal lung cells. However, some turn off a gene that codes for a protein called NKX2-1, which leads to a type of cancer called invasive mucinous adenocarcinoma (or IMA for short). Cells from this type of cancer develop an identity similar to mucous cells that line the surface of the stomach. But it was unclear how IMA tumor cells that developed from a mutation in the BRAF gene are affected by this loss in NKX2-1, and how transitioning to a different cell type impacts their response to treatment. To investigate this, Zewdu et al. studied lung cells from patients with IMA tumors driven by a mutation in BRAF and cells from mice that have been genetically engineered to have a similar form of cancer. This revealed that the NKX2-1 protein is needed to initiate the formation of cancer cells but is not required for the growth of already established BRAF-driven tumors. Further experiments showed that removing the gene for NKX2-1 made these cancer cells less responsive to drugs known as BRAF/MEK inhibitors that are commonly used to treat cancer. These drugs caused the IMA cancer cells to change their identity and become another type of stomach cell. This identity change was found to depend on two signaling pathways which cells use to communicate. This study provides some explanation of how IMA lung cancers that lack the gene for NKX2-1 resist treatment with BRAF/MEK inhibitors. It also shows new relationships between key genes in these cancers and systems for cell communication. These findings could lead to better therapies for lung cancer, particularly for patients whose tumor cells are deficient in NKX2-1 and therefore require specialized treatment.

Multiplexed approaches correlating mitochondrial health to cell health using microcapillary cytometry.

Mitochondria are critical cellular organelles that play a fundamental role in cellular metabolism and oxidative stress and are well known to trigger multiple cell death pathways. The study of sequence of mitochondrial events as it relates to apoptotic/cell death events can provide critical insights into mechanism of cellular homeostasis, stress and death. Availability of rapid and simplified cytometric testing methods for evaluating mitochondrial changes, apoptosis and cell death in parallel can greatly enhance our understanding of mechanism of compound action. In this study, we investigated a series of compounds to evaluate apoptotic/cell death effects in context of mitochondrial changes using plate-based assays on Guava® easyCyte systems. Studies utilized multiplexed assays for mitochondrial membrane potential changes and apoptosis/cell death markers and allowed for easy identification of hit compounds. Dose and time response studies with Niclosamide, an anti-helmintic drug and comparison of effects with Gambogic acid and celastrol demonstrated early and significant mitochondrial impacts for niclosamide and gambogic acid. No apoptotic or cell death impacts were observed in parallel at low doses/short times of incubation for niclosamide, while increased time with niclosamide caused increase in mitochondrial, apoptotic and cell death response. The method demonstrates great power in being able to distinguish between potency of compounds and conditions in modulating mitochondrial/apoptotic changes. The simplicity of the assays described coupled with the ease of use of plate based microcapillary cytometry can provide researchers valuable tools to obtain a more comprehensive insight into how compounds modulate mitochondria and its relationship with subsequent apoptosis/cell death pathways.

Physiotherapy extended-role practitioner for individuals with hip and knee arthritis: patient perspectives of a rural/urban partnership.

PURPOSE: To explore the perspectives of people with hip and knee arthritis regarding a physiotherapy extended-role practitioner (ERP) model of care in a rural setting. METHOD: Using semi-structured interviews, a qualitative descriptive case study was undertaken with 13 participants from a rural family practice located in the province of Ontario, Canada, who had all been assessed by an ERP. Transcribed interviews were analyzed for emergent themes. RESULTS: Three main themes were identified: (1) timely access to care, (2) distance as a factor in seeking care, and (3) perceptions of the ERP model of care. CONCLUSIONS: Participants reported many positive experiences with the physiotherapy ERP rural model. Processes related to minimizing travel required to access care are important for those in rural communities. An ERP model of care offers competent care that includes musculoskeletal diagnosis as well as time for educating patients and addressing questions.

Objectif : Explorer les perspectives des personnes qui ont de l'arthrite dans la hanche et le genou en ce qui concerne un modèle de soins basé sur le rôle élargi du professionnel de la physiothérapie (REP) en contexte rural. Méthode   : Nous avons entrepris une étude de cas descriptive qualitative basée sur des entrevues structurées menées auprès de 13 participants d'un cabinet de médecine familiale en milieu rural dans la province d'Ontario, au Canada, qui avaient tous été évalués par un professionnel à rôle élargi. Nous avons analysé les entrevues transcrites pour en dégager des thèmes émergents. Résultats : Nous avons dégagé trois grands thèmes : (1) l'accès rapide aux soins; (2) la distance comme facteur de la recherche de soins; (3) les perceptions du modèle de soins basé sur le rôle élargi du professionnel. Conclusions : Des participants ont signalé de nombreuses expériences positives du modèle rural REP en physiothérapie. Les moyens de réduire au minimum les déplacements pour avoir accès aux soins sont importants pour les membres des communautés rurales. Un modèle de soins basé sur le rôle élargi du professionnel offre des soins compétents qui incluent la capacité de poser un diagnostic de l'appareil locomoteur et du temps pour l'éducation et pour répondre aux questions.

Simplified evaluation of apoptosis using the Muse cell analyzer.

The degree of apoptosis in a cell population is an important parameter of cell health and is characterized by distinct morphological changes. Current methods of accurate detection and measurement of cellular apoptosis require expensive and complicated instrument platforms and expertise. The Muse Cell Analyzer is a unique instrument that enables multidimensional cell health analysis on a single platform. In this study, we used the Muse Cell Analyzer for apoptosis studies using the Muse Annexin V & Dead Cell Assay. The assay is based on the detection of phosphatidylserine (PS) on the surface of apoptotic cells. The results obtained from Muse Cell Analyzer were compared with traditional methods for apoptosis analysis. Our results indicate that Muse Annexin V & Dead Cell Assay and software module enabled the acquisition of accurate and highly precise measurements of cellular apoptosis. The assay is versatile and works with both suspension and adherent cell lines and multiple treatment conditions.