MicroRNA-193b Enhances Tumor Progression via Down Regulation of Neurofibromin 1

<div><p>Despite improvements in therapeutic approaches for head and neck squamous cell carcinomas (HNSCC), clinical outcome has remained disappointing, with 5-year overall survival rates hovering around 40–50%, underscoring an urgent need to better understand the biological bases of this disease. We chose to address this challenge by studying the role of micro-RNAs (miRNAs) in HNSCC. MiR-193b was identified as an over-expressed miRNA from global miRNA profiling studies previously conducted in our lab, and confirmed in HNSCC cell lines. <em>In vitro</em> knockdown of miR-193b in FaDu cancer cells substantially reduced cell proliferation, migration and invasion, along with suppressed tumour formation <em>in vivo</em>. By integrating <em>in silico</em> prediction algorithms with <em>in vitro</em> experimental mRNA profilings, plus mRNA expression data of clinical specimens, neurofibromin 1 (NF1) was identified to be a target of miR-193b. Concordantly, miR-193b knockdown decreased NF1 transcript and protein levels significantly. Luciferase reporter assays confirmed the direct interaction of miR-193b with NF1. Moreover, p-ERK, a downstream target of NF1 was also suppressed after miR-193b knockdown. FaDu cells treated with a p-ERK inhibitor (U0126) phenocopied the reduced cell proliferation, migration and invasion observed with miR-193b knockdown. Finally, HNSCC patients whose tumours expressed high levels of miR-193b experienced a lower disease-free survival compared to patients with low miR-193b expression. Our findings identified miR-193b as a potentially novel prognostic marker in HNSCC that drives tumour progression <em>via</em> down-regulating NF1, in turn leading to activation of ERK, resulting in proliferation, migration, invasion, and tumour formation.</p> </div

Identification of mRNA targets of miR-193b.

<p>(A) qRT-PCR of 13 predicted miR-193b targets, 72 hours post transfection with LNA-193b (40 nM), fold change relative to LNA-scramble (40 nM). (B) Basal mRNA expression of PER2, DUSP1 and NF1 was assessed using qRT-PCR in all three HNSCC cell lines relative to NOE cells. (C) NF1 transcript expression in FaDu cells was measured 72 hours post transfection with LNA-193b (40 nM) or LNA-scramble (40 nM). (D) Western blotting of NF1 in FaDu cells was determined 72 hours post transfection, images (above), quantification (below). (E) Relative luciferase activity of FaDu cells after co-transfection with pMIR-NF1 UTR (100 ng) or pMIR-NF1 Mutant (100 ng) vectors, and LNA-193b (40 nM) or LNA-scramble (40 nM), 72 hours post transfection. (F) Immunoprecipitation of GST-RBD in FaDu cells post transfection with LNA 193b (40 nM) or LNA Scramble (40 nM); images (above), quantification (below). *P<0.05, **P<0.005, ***P<0.0005, P = ns (not significant).</p

miR-193b downregulation reduced cell proliferation, migration and invasion <i>in vitro,</i> and delayed tumor formation <i>in vivo</i>.

<p>(A) FaDu cells were transfected with 40 nM of LNA-scramble or LNA-193b. Cell viability was assessed in FaDu cells by the MTS assay 24–72 hours post transfection. (B) Clonogenic survival of FaDu cells was measured 10 to 12 days after re-seeding cells treated with LNA-scramble (40 nM) or LNA-193b (40 nM) for 72 hours. (C) Representative images (right) and quantification (left) depicting the reduced ability of FaDu cells to migrate after transfection with LNA-193b (40 nM) compared to LNA-scrambled (40 nM). (D) Representative images (right) and quantification (left) depicting the reduced ability of FaDu cells to invade after transfection with LNA-193b (40 nM) compared to LNA-scrambled (40 nM). (E) FaDu cells were transfected with LNA-193b (40 nM) or LNA-scramble (40 nM) and 48 hours later implanted intramuscularly (IM) into the left gastrocemius of SCID mice. Tumor plus leg diameter was measured two to three times a week (y-axis). Data presented as mean ± SE n = 5 (LNA-scrambled), n = 6 (LNA-193b). *P<0.05, **P<0.005, ***P<0.0005, P = ns (not significant).</p

MiR-193b and p-ERK expression in a primary HNSCC patient sample.

<p>(A) A representative image of miR-193b <i>in situ</i> hybridization of a primary HNSCC biopsy, arrows indicating tumor cells exhibiting cytoplasmic signal. (B) A representative image of immunohistochemical analysis of p-ERK expression in the same patient’s HNSCC biopsy sample; arrows indicating expression in both the tumor nucleus and the cytoplasm. (C) Kaplan-Meier plots of disease free survival for HNSCC patients dichotomized based on high (>median) <i>vs</i>. low (≤ median) miR-193b expression. P = ns (not significant).</p

Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies

Proteomic Analysis of Cancer-Associated Fibroblasts Reveals a Paracrine Role for MFAP5 in Human Oral Tongue Squamous Cell Carcinoma

Bidirectional communication between cells and their microenvironment is crucial for both normal tissue homeostasis and tumor growth. During the development of oral tongue squamous cell carcinoma (OTSCC), cancer-associated fibroblasts (CAFs) create a supporting niche by maintaining a bidirectional crosstalk with cancer cells, mediated by classically secreted factors and various nanometer-sized vesicles, termed as extracellular vesicles (EVs). To better understand the role of CAFs within the tumor stroma and elucidate the mechanism by which secreted proteins contribute to OTSCC progression, we isolated and characterized patient-derived CAFs from resected tumors with matched adjacent tissue fibroblasts (AFs). Our strategy employed shotgun proteomics to comprehensively characterize the proteomes of these matched fibroblast populations. Our goals were to identify CAF-secreted factors (EVs and soluble) that can functionally modulate OTSCC cells in vitro and to identify novel CAF-associated biomarkers. Comprehensive proteomic analysis identified 4247 proteins, the most detailed description of a pro-tumorigenic stroma to date. We demonstrated functional effects of CAF secretomes (EVs and conditioned media) on OTSCC cell growth and migration. Comparative proteomics identified novel proteins associated with a CAF-like state. Specifically, MFAP5, a protein component of extracellular microfibrils, was enriched in CAF secretomes. Using in vitro assays, we demonstrated that MFAP5 activated OTSCC cell growth and migration via activation of MAPK and AKT pathways. Using a tissue microarray of richly annotated primary human OTSCCs, we demonstrated an association of MFAP5 expression with patient survival. In summary, our proteomics data of patient-derived stromal fibroblasts provide a useful resource for future mechanistic and biomarker studies

The Mid-infrared Instrument for JWST and Its In-flight Performance

The Mid-Infrared Instrument (MIRI) extends the reach of the James Webb Space Telescope (JWST) to 28.5 μm. It provides subarcsecond-resolution imaging, high sensitivity coronagraphy, and spectroscopy at resolutions of λ/Δλ ∼ 100–3500, with the high-resolution mode employing an integral field unit to provide spatial data cubes. The resulting broad suite of capabilities will enable huge advances in studies over this wavelength range. This overview describes the history of acquiring this capability for JWST. It discusses the basic attributes of the instrument optics, the detector arrays, and the cryocooler that keeps everything at approximately 7 K. It gives a short description of the data pipeline and of the instrument performance demonstrated during JWST commissioning. The bottom line is that the telescope and MIRI are both operating to the standards set by pre-launch predictions, and all of the MIRI capabilities are operating at, or even a bit better than, the level that had been expected. The paper is also designed to act as a roadmap to more detailed papers on different aspects of MIRI.</p