Contact-Inhibited ERK Signaling is Determined by Cellular-Resolution Western Blotting.

Extracellular signal-regulated kinase (ERK) signaling is essential to regulated cell behaviors, including cell proliferation, differentiation, and apoptosis. The influence of cell-cell contacts on ERK signaling is central to epithelial cells, yet few studies have sought to understand the same in cancer cells, particularly with single-cell resolution. To acquire both phenotypic (cell-contact state) and proteomic profile (ERK phosphorylation) on the same HeLa cells, we prepend high-content, whole-cell imaging prior to endpoint cellular-resolution western blot analyses for hundreds of cancer cells cultured on chip. By indexing the phosphorylation level of ERK in each cell or cell-contact cluster to the imaged cell-contact state, we compare ERK signaling between isolated and in-contact cells. We observe attenuated (∼2×) ERK signaling in HeLa cells which are in contact versus isolated. Attenuation is sustained when the HeLa cells are challenged with hyperosmotic stress. The contact-dependent differential ERK-phosphorylation corresponds to the differential EGFR distribution on cell surfaces, suggesting the involvement of EGFRs in contact-inhibited ERK signaling. Our findings show the impact of cell-cell contacts on ERK activation with isolated and in-contact cells, hence providing a new tool into control and scrutiny of cell-cell interactions.

ASK1 suppresses NK cell-mediated intravascular tumor cell clearance in lung metastasis.

Tumor metastasis is the leading cause of death worldwide and involves an extremely complex process composed of multiple steps. Our previous study demonstrated that apoptosis signal-regulating kinase 1 (ASK1) deficiency in mice attenuates tumor metastasis in an experimental lung metastasis model. However, the steps of tumor metastasis regulated by ASK1 remain unclear. Here, we showed that ASK1 deficiency in mice promotes natural killer (NK) cell-mediated intravascular tumor cell clearance in the initial hours of metastasis. In response to tumor inoculation, ASK1 deficiency upregulated immune response-related genes, including interferon-gamma (IFNγ). We also revealed that NK cells are required for these anti-metastatic phenotypes. ASK1 deficiency augmented cytokine production chemoattractive to NK cells possibly through induction of the ligand for NKG2D, a key activating receptor of NK cells, leading to further recruitment of NK cells into the lung. These results indicate that ASK1 negatively regulates NK cell-dependent anti-tumor immunity and that ASK1-targeted therapy can provide a new tool for cancer immunotherapy to overcome tumor metastasis.

ASK family and cancer.

Cancer is a major problem in public health and is one of the leading causes of mortality worldwide. Many types of cancer cells exhibit aberrant cellular signal transduction in response to stress, which often leads to oncogenesis. Mitogen-activated protein kinase (MAPK) signal cascades are one of the important intracellular stress signaling pathways closely related to cancer. The key molecules in MAPK signal cascades that respond to various types of stressors are apoptosis signal-regulating kinase (ASK) family members; ASK1, ASK2 and ASK3. ASK family members are activated by a wide variety of stressors, and they regulate various cellular responses, such as cell proliferation, inflammation and apoptosis. In this review, we will discuss both the oncogenic and anti-oncogenic roles of the ASK family members in various contexts of cancer development with deeper insights into the involvement of ASK family members in cancer pathology.

16S rRNA methyltransferase KsgA contributes to oxidative stress resistance and virulence in Staphylococcus aureus.

We previously reported that the rRNA methyltransferases RsmI and RsmH, which are responsible for cytidine dimethylation at position 1402 of 16S rRNA in the decoding center of the ribosome, contribute to Staphylococcus aureus virulence. Here we evaluated other 16S rRNA methyltransferases, including KsgA (RsmA), RsmB/F, RsmC, RsmD, RsmE, and RsmG. Knockout of KsgA, which methylates two adjacent adenosines at positions 1518 and 1519 of 16S rRNA in the intersubunit bridge of the ribosome, attenuated the S. aureus killing ability against silkworms. The ksgA knockout strain was sensitive to oxidative stress and had a lower survival rate in murine macrophages than the parent strain. The ksgA knockout strain exhibited decreased translational fidelity in oxidative stress conditions. Administration of N-acetyl-l-cysteine, a free-radical scavenger, restored the killing ability of the ksgA knockout strain against silkworms. These findings suggest that the methyl-modifications of 16S rRNA by KsgA contribute to maintain ribosome function under oxidative conditions and thus to S. aureus virulence.