ER stress and distinct outputs of the IRE1α RNase control proliferation and senescence in response to oncogenic Ras
Abstract: Oncogenic Ras causes proliferation followed by premature senescence in primary cells, an initial barrier to tumor development. The role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in regulating these two cellular outcomes is poorly understood. During ER stress, the inositol requiring enzyme 1α (IRE1α) endoribonuclease (RNase), a key mediator of the UPR, cleaves Xbp1 mRNA to generate a potent transcription factor adaptive toward ER stress. However, IRE1α also promotes cleavage and degradation of ER-localized mRNAs essential for cell death. Here, we show that oncogenic HRas induces ER stress and activation of IRE1α. Reduction of ER stress or Xbp1 splicing using pharmacological, genetic, and RNAi approaches demonstrates that this adaptive response is critical for HRas-induced proliferation. Paradoxically, reduced ER stress or Xbp1 splicing promotes growth arrest and premature senescence through hyperactivation of the IRE1α RNase. Microarray analysis of IRE1α- and XBP1-depleted cells, validation using RNA cleavage assays, and 5′ RACE identified the prooncogenic basic helix–loop–helix transcription factor ID1 as an IRE1α RNase target. Further, we demonstrate that Id1 degradation by IRE1α is essential for HRas-induced premature senescence. Together, our studies point to IRE1α as an important node for posttranscriptional regulation of the early Ras phenotype that is dependent on both oncogenic signaling as well as stress signals imparted by the tumor microenvironment and could be an important mechanism driving escape from Ras-induced senescence.
CLIC4 is incorporated into extracellular vesicles of murine breast cancer cells and may influence metastatic burden
Abstract: Chloride intracellular channel 4 (CLIC4) is an evolutionarily conserved, 29kD, dimorphic protein that contributes to TGF-β signaling by preventing the de-phosphorylation of phospho-SMAD2/3 upon nuclear translocation. In several cancer types, CLIC4 is excluded from the nucleus and downregulated in the cytoplasm of the tumor cells as the tumor progresses, suggesting that CLIC4 acts as tumor suppressor. In a parallel sequence, CLIC4 becomes upregulated in the stromal compartment, where it enhances tumor growth and invasion. Recent reports have suggested that CLIC4 is detectable in the serum of cancer patients and incorporated into extracellular vesicles, and has potential as a biomarker. We hope to gain a better understanding of the role that CLIC4 plays in the tumor stromal and epithelial compartments as well as their respective release of extracellular vesicles. Using in-vitro and in-vivo assays, we have conducted experiments using the FVB mouse MMTV-c-MYC 6DT1 breast cancer model. By CRISPR/ Cas9 system, CLIC4 was deleted from wild type 6DT1 cells. Following clonal selection, the loss of the CLIC4 protein at both the cellular and released vesicle level was validated. Both functional assays on CLIC4 deleted clones and evaluation of their extra-cellular vesicles were undertaken in order to further understand their tumorigenic and metastatic capabilities. In-vitro, CLIC4 was not necessary for vesicle biogenesis and its deletion did not have a significant effect on cellular proliferation. In vivo, selected clones were orthotopically injected into the 4th mammary fat pad of wild type FVB mice. Compared to wild type 6DT1 clones, CLIC4 deleted clones formed primary tumors that had greater mass but a fewer number of lung metastasis. Future studies are designed to isolate vesicles circulating in tumor bearing hosts to determine their stromal or epithelial origin and to provide a better understanding of the role that CLIC4 may play in tumor growth, creating a metastatic niche and as a potential serological biomarker.