Abstract Cancer progression is a complex series of events thought to incorporate the reversible developmental process of epithelial-to-mesenchymal transition (EMT). In vitro , the microRNA-200 family ...maintains the epithelial phenotype by posttranscriptionally inhibiting the E-cadherin repressors, ZEB1 and ZEB2 . Here, we used in situ hybridization and immunohistochemistry to assess expression of miR-200 and EMT biomarkers in formalin-fixed paraffin-embedded human colorectal adenocarcinomas. In addition, laser capture microdissection and quantitative real-time polymerase chain reaction were employed to quantify levels of miR-200 in the normal epithelium, tumor core, invasive front, and stroma. We find that miR-200 is downregulated at the invasive front of colorectal adenocarcinomas that have destroyed and invaded beyond the basement membrane. However, regional lymph node metastases and vascular carcinoma deposits show strong expression of miR-200, suggesting this family of miRNAs is involved in the recapitulation of the primary tumor phenotype at metastatic sites. In contrast, adenomas and adenocarcinomas with intact basement membranes showed uniform miR-200 expression from the tumor core to the tumor-host interface. Taken together, these data support the involvement of EMT and mesenchymal-to-epithelial transition (MET) in the metastasis cascade and show that miR-200 is downregulated in the initial stages of stromal invasion but is restored at metastatic sites.
Epithelial to mesenchymal transition (EMT) facilitates tissue remodelling during embryonic development and is viewed as an essential early step in tumour metastasis. We found that all five members of ...the microRNA-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) and miR-205 were markedly downregulated in cells that had undergone EMT in response to transforming growth factor (TGF)-β or to ectopic expression of the protein tyrosine phosphatase Pez. Enforced expression of the miR-200 family alone was sufficient to prevent TGF-β-induced EMT. Together, these microRNAs cooperatively regulate expression of the E-cadherin transcriptional repressors ZEB1 (also known as δEF1) and SIP1 (also known as ZEB2), factors previously implicated in EMT and tumour metastasis. Inhibition of the microRNAs was sufficient to induce EMT in a process requiring upregulation of ZEB1 and/or SIP1. Conversely, ectopic expression of these microRNAs in mesenchymal cells initiated mesenchymal to epithelial transition (MET). Consistent with their role in regulating EMT, expression of these microRNAs was found to be lost in invasive breast cancer cell lines with mesenchymal phenotype. Expression of the miR-200 family was also lost in regions of metaplastic breast cancer specimens lacking E-cadherin. These data suggest that downregulation of the microRNAs may be an important step in tumour progression.
The microRNAs of the miR‐200 family maintain the central characteristics of epithelia and inhibit tumor cell motility and invasiveness. Using the Ago‐HITS‐CLIP technology for transcriptome‐wide ...identification of direct microRNA targets in living cells, along with extensive validation to verify the reliability of the approach, we have identified hundreds of miR‐200a and miR‐200b targets, providing insights into general features of miRNA target site selection. Gene ontology analysis revealed a predominant effect of miR‐200 targets in widespread coordinate control of actin cytoskeleton dynamics. Functional characterization of the miR‐200 targets indicates that they constitute subnetworks that underlie the ability of cancer cells to migrate and invade, including coordinate effects on Rho‐ROCK signaling, invadopodia formation, MMP activity, and focal adhesions. Thus, the miR‐200 family maintains the central characteristics of the epithelial phenotype by acting on numerous targets at multiple levels, encompassing both cytoskeletal effectors that control actin filament organization and dynamics, and upstream signals that locally regulate the cytoskeleton to maintain cell morphology and prevent cell migration.
Synopsis
miR‐200 microRNAs are involved in the maintenance of epithelial integrity. Direct, transcriptome‐wide target detection and validation identifies genes functionally grouped as regulators of Rho GTPase signaling, invadopodia formation, metalloprotease activity and cell adhesion, which together regulate cell motility, migration and cancer metastasis.
The global profile of miR‐200 targets in breast cancer cells reveals a network of cytoskeletal regulators
miR‐200 is found to control invadopodia, focal adhesions and Rho GTPase signaling.
Canonical seed‐3′UTR target site interactions are dominant, but target sites in coding regions and non‐canonical interactions are also detected.
Target genes identified in cell lines negatively correlate with miR‐200 across human breast cancer samples as well.
miR‐200 microRNAs are involved in the maintenance of epithelial integrity. Direct, transcriptome‐wide target detection and validation identifies genes functionally grouped as regulators of Rho GTPase signaling, invadopodia formation, metalloprotease activity and cell adhesion, which together regulate cell motility, migration and cancer metastasis.
Identifying tumor antigen-specific T cells from cancer patients has important implications for immunotherapy diagnostics and therapeutics. Here, we show that CD103
CD39
tumor-infiltrating CD8 T cells ...(CD8 TIL) are enriched for tumor-reactive cells both in primary and metastatic tumors. This CD8 TIL subset is found across six different malignancies and displays an exhausted tissue-resident memory phenotype. CD103
CD39
CD8 TILs have a distinct T-cell receptor (TCR) repertoire, with T-cell clones expanded in the tumor but present at low frequencies in the periphery. CD103
CD39
CD8 TILs also efficiently kill autologous tumor cells in a MHC-class I-dependent manner. Finally, higher frequencies of CD103
CD39
CD8 TILs in patients with head and neck cancer are associated with better overall survival. Our data thus describe an approach for detecting tumor-reactive CD8 TILs that will help define mechanisms of existing immunotherapy treatments, and may lead to future adoptive T-cell cancer therapies.
Members of the miR‐200 family are critical gatekeepers of the epithelial state, restraining expression of pro‐mesenchymal genes that drive epithelial–mesenchymal transition (EMT) and contribute to ...metastatic cancer progression. Here, we show that miR‐200c and another epithelial‐enriched miRNA, miR‐375, exert widespread control of alternative splicing in cancer cells by suppressing the RNA‐binding protein Quaking (QKI). During EMT, QKI‐5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI‐5 is both necessary and sufficient to direct EMT‐associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial‐derived cancer types. Importantly, several actin cytoskeleton‐associated genes are directly targeted by both QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR‐200/miR‐375/QKI axis that impacts cancer‐associated epithelial cell plasticity through widespread control of alternative splicing.
Synopsis
miR‐200 and miR‐375 regulate the level of the RNA‐binding protein, Quaking (QKI), which orchestrates widespread control of alternative splicing during epithelial‐mesenchymal transition (EMT), thereby affecting multiple facets of cancer‐associated epithelial cell plasticity.
miR‐200c and miR‐375 control EMT‐associated alternative splicing by suppressing QKI.
QKI‐5 influences cell plasticity, invasion and tumour growth by modulating alternative splicing.
Actin cytoskeleton‐associated genes are targeted both by QKI and miR‐200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT.
While miRNAs of the miR‐200 family are known to promote EMT and cancer progression via target mRNA repression, they exert a separate effect on epithelial cell plasticity by modulating alternative splicing signatures.
Although autophagy controls cell death and survival, underlying mechanisms are poorly understood, and it is unknown whether autophagy affects only whether or not cells die or also controls other ...aspects of programmed cell death. MAP3K7 is a tumor suppressor gene associated with poor disease-free survival in prostate cancer. Here, we report that Map3k7 deletion in mouse prostate cells sensitizes to cell death by TRAIL (TNF-related apoptosis-inducing ligand). Surprisingly, this death occurs primarily through necroptosis, not apoptosis, due to assembly of the necrosome in association with the autophagy machinery, mediated by p62/SQSTM1 recruitment of RIPK1. The mechanism of cell death switches to apoptosis if p62-dependent recruitment of the necrosome to the autophagy machinery is blocked. These data show that the autophagy machinery can control the mechanism of programmed cell death by serving as a scaffold rather than by degrading cargo.
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•MAP3K7 loss sensitizes cells to TRAIL-induced necroptotic cell death•The necrosome associates with autophagy machinery•Autophagy inhibition promotes or inhibits cell death depending on the blocked step•Autophagy machinery can serve as a scaffold to modulate mode of programmed cell death
Goodall et al. show that in the context of Map3k7 loss, the autophagy machinery has a scaffolding role in modulating the mode of cell death between necroptosis and apoptosis. p62/SQSTM1 recruits RIPK1 and mediates necrosome assembly in association with autophagic machinery for TRAIL-induced necroptosis. Blocking this recruitment results in apoptosis.
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