Crosstalk between ERK and MRTF-A signaling regulates TGFß1-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is a physiological process that is essential during embryogenesis and wound healing and also contributes to pathologies including fibrosis and cancer. EMT is characterized by marked gene expression changes, loss of cell-cell contacts, remodeling of the cytoskeleton, and acquisition of enhanced motility. In the late stages of EMT, cells can exhibit myofibroblast-like properties with enhanced expression of the mesenchymal protein marker α-smooth muscle actin and contractile activity. Transforming growth factor (TGF)-ß1 is a well-known inducer of EMT and it activates a plethora of signaling cascades including extracellular signal-regulated kinase (ERK). Previous reports have demonstrated a role for ERK signaling in the early stages of EMT, but the molecular impacts of ERK signaling on the late stages of EMT are still unknown. Here, we found that inhibition of the phosphorylation of ERK enhances focal adhesions, stress fiber formation, cell contractility, and gene expression changes associated with TGFß1-induced EMT in mammary epithelial cells. These effects are mediated in part by the phosphorylation state and subcellular localization of myocardin-related transcription factor-A. These findings indicate that the intricate crosstalk between signaling cascades plays an important role in regulating the progression of EMT and suggests new approaches to control EMT processes.

Chromosomal instability can favor macrophage-mediated immune response and induce a broad, vaccination-like anti-tumor IgG response.

Chromosomal instability (CIN), a state in which cells undergo mitotic aberrations that generate chromosome copy number variations, generates aneuploidy and is thought to drive cancer evolution. Although associated with poor prognosis and reduced immune response, CIN generates aneuploidy-induced stresses that could be exploited for immunotherapies. In such contexts, macrophages and the CD47-SIRPα checkpoint are understudied. Here, CIN is induced pharmacologically induced in poorly immunogenic B16F10 mouse melanoma cells, generating persistent micronuclei and diverse aneuploidy while skewing macrophages towards an anti-cancer M1-like phenotype, based on RNA-sequencing profiling, surface marker expression and short-term antitumor studies. These results further translate to in vivo efficacy: Mice bearing CIN-afflicted tumors with wild-type CD47 levels survive only slightly longer relative to chromosomally stable controls, but long-term survival is maximized when combining macrophage-stimulating anti-tumor IgG opsonization and some form of disruption of the CD47-SIRPα checkpoint. Survivors make multi-epitope, de novo anti-cancer IgG that promote macrophage-mediated phagocytosis of CD47 knockout B16F10 cells and suppress tumoroids in vitro and growth of tumors in vivo . CIN does not greatly affect the level of the IgG response compared to previous studies but does significantly increase survival. These results highlight an unexpected therapeutic benefit from CIN when paired with maximal macrophage anti-cancer activity: an anti-cancer vaccination-like antibody response that can lead to more durable cures and further potentiate cell-mediated acquired immunity.

Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG.

Solid tumors generally exhibit chromosome copy number variation, which is typically caused by chromosomal instability (CIN) in mitosis. The resulting aneuploidy can drive evolution and associates with poor prognosis in various cancer types as well as poor response to T-cell checkpoint blockade in melanoma. Macrophages and the SIRPα-CD47 checkpoint are understudied in such contexts. Here, CIN is induced in poorly immunogenic B16F10 mouse melanoma cells using spindle assembly checkpoint MPS1 inhibitors that generate persistent micronuclei and diverse aneuploidy while skewing macrophages toward a tumoricidal 'M1-like' phenotype based on markers and short-term anti-tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels succumb similar to controls, but long-term survival is maximized by SIRPα blockade on adoptively transferred myeloid cells plus anti-tumor monoclonal IgG. Such cells are the initiating effector cells, and survivors make de novo anti-cancer IgG that not only promote phagocytosis of CD47-null cells but also suppress tumor growth. CIN does not affect the IgG response, but pairing CIN with maximal macrophage anti-cancer activity increases durable cures that possess a vaccination-like response against recurrence.

Differences in cell shape, motility, and growth reflect chromosomal number variations that can be visualized with live-cell ChReporters.

Chromosome numbers often change dynamically in tumors and cultured cells, which complicates therapy as well as understanding genotype-mechanotype relationships. Here we use a live-cell "ChReporter" method to identify cells with a single chromosomal loss in efforts to better understand differences in cell shape, motility, and growth. We focus on a standard cancer line and first show clonal populations that retain the ChReporter exhibit large differences in cell and nuclear morphology as well as motility. Phenotype metrics follow simple rules, including migratory persistence scaling with speed, and cytoskeletal differences are evident from drug responses, imaging, and single-cell RNA sequencing. However, mechanotype-genotype relationships between fluorescent ChReporter-positive clones proved complex and motivated comparisons of clones that differ only in loss or retention of a Chromosome-5 ChReporter. When lost, fluorescence-null cells show low expression of Chromosome-5 genes, including a key tumor suppressor APC that regulates microtubules and proliferation. Colonies are compact, nuclei are rounded, and cells proliferate more, with drug results implicating APC, and patient survival data indicating an association in multiple tumor-types. Visual identification of genotype with ChReporters can thus help clarify mechanotype and mechano-evolution.