<p>  </p> <p>Non-small cell lung cancer (NSCLC), the most common type of lung cancer, is essentially the leading cause of cancer related deaths in United States. Only 24% of NSCLC patients survive 5-years post diagnosis, largely attributed to the lack of efficient treatment strategies at the metastatic stage. Thus, understanding the biological mechanisms that promote NSCLC metastasis is critical for the development of effective cancer-specific therapeutic agents. The development of cancer metastasis is greatly driven and influenced by intercellular communication. Key mediators of cell-to-cell communication are extracellular vesicles (EVs). For the past years, the study of EVs released by tumor cells have gained attention, given their impact in modulating the tumor immunity, supporting tumorigenesis, and contributing to the development of metastasis. However, the mechanisms though which tumor EVs contribute to tumor development are still understudied. In this study, we isolated and characterized small EVs, also referred as exosomes, from NSCLC cell lines (H358, Calu6, H460, SKMES-1) and investigated their release, uptake, and impact in non-tumorigenic lung epithelial cells recipient cells (BEAS-2B and HBEC). Our results demonstrated that EVs from NSCLC can induce migration and invasion of non-tumorigenic epithelial cells, and impair epithelial barrier permeability, suggesting their role in supporting tumorigenesis and metastasis. Furthermore, we assessed the immunomodulatory effects of NSCLC EVs on anti-tumor immune cells, particularly T cells. Our findings revealed a suppressive effect of EVs derived from mutant KRASG12C NSCLC (H358) on T-cell proliferation, suggesting their contribution to immune evasion mechanisms in mutant KRAS tumors. To dissect the underlying mechanisms, we employed a dual approach utilizing genetic manipulation (shRNA knockdown) and a small molecule inhibitor (ARS-1620) targeting the oncogenic KRASG12C. Our data demonstrated that targeting KRASG12C impaired the EV-driven cancer phenotypes, highlighting the pivotal role of KRAS oncogenic signaling in tumorigenesis and immune suppression mediated by EVs. Overall, our study sheds light on the crucial involvement of tumor derived EVs in NSCLC progression, both in terms of promoting cellular migration and invasion, as well as dampening anti-tumor immune responses. By elucidating the mechanisms underlying EV-driven tumorigenesis and highlighting the therapeutic potential of targeting KRAS signaling, our findings pave the way for the development of novel and effective therapeutic agents for NSCLC.</p>

In wetland soils, hydrology is considered to be one of the primary factors shaping wetland function and microbial community structure, but plant-soil interactions are also important mechanisms affecting microbial nutrient transformations. The research presented here considered the interactive effect to describe how hydrology and the presence of plants alter the soil profile, the development of the bacterial community, and their associated functions. To achieve this goal, plots were established in three hydrologically-distinct regimes (Wet, Intermediate, and Dry) within a non-tidal freshwater wetland along the James River (Charles City County, Virginia). Inside each main plot, ten subplots were cleared of all aboveground plant material; five plots were left to re-grow (“Vegetated” reference), while the remaining five were weeded each week to maintain bare soil (“Clipped” treatment subplots). Manipulations were started at the beginning of the growing season, and sampling continued until the following winter. Every eight weeks, soil cores (30 cm) were collected and analyzed for a variety of soil properties (e.g., pH, OM, C:N, redox, vegetation and root biomass), microbial community structure (16S-rDNA-based T-RFLP),bacterial abundance (Acridine Orange Direct Count), and soil function (Extracellular Enzyme Activity (EEA)). A mixed-effects repeated measures analysis of variance (ANOVA) was used to better understand how each variable responded within each hydrological regime and treatment. Principal component analysis (PCA) and Partial Mantel tests were used to elucidate how saturation and vegetation influence the microbial community structure and soil enzyme function. Bacterial community properties and soil functions followed differences in soil saturation and associated physicochemical parameters (i.e., pH and redox). Correlations with wetland vegetation were primarily related to seasonal changes in plant community composition and biomass, and differences between experimental treatments were small. Evidence suggests the present plant species and the amount of above- and belowground biomass plays a more selective role shaping bacterial communities and soil function. Due to the short-term of this study and tight soil correlations, it is difficult to determine if observed differences are a product of the plant community or soil saturation, but it is clear that each is important. Based on the literature, plant effects were smaller in this wetland than might be expected. This experiment took place in a recently exposed lake basin, so plant-soil-microbe interaction may not be well established. As the wetland matures, relative importance of vegetation is expected to increase and impact bacterial composition and function. Collectively, these results demonstrate that wetlands are not a product of one separate variable, but result from various factors interlinked to shape microbial communities and soil functions.

Acquisition of invasiveness through extracellular matrix is a crucial characteristic of transition to malignancy in the breast. It was previously shown that Polo-like kinase 1 (PLK-1), a mitotic kinase and genome stability regulator, is involved in acquisition of invasiveness in a breast cell model (HMT-3522 cell line) of pre-invasive to invasive transition. This and other data led to the suggestion that a new class of genes called GISEM for Genome Instability and Extracellular Matrix Invasiveness may exist. Previous lab data show that XRCC3 is found downregulated in progression from preinvasive to invasive phenotype. This led to the hypothesis that XRCC3 may be a negative regulator of invasion. To support this hypothesis, overexpression of XRCC3 in the invasive T4-2 cells downregulated invasion, but also growth. In order to verify the role of XRCC3 in invasiveness, and determine whether it is independent from any effects on growth, we tested the effect of downregulating XRCC3 on the invasiveness of T4-2 cells. Short-term downregulation of XRCC3 using siRNAs produced a significant increase in invasiveness, suggesting a role for XRCC3 as a negative regulator of invasion. During the invasion assay time course, XRCC3 downregulation had no effect on growth or apoptosis supporting the idea that this is a direct effect on invasion and not an artifact of the assay. XRCC3 is one amongst the five members of the RAD51 paralog family, consisting of accessory proteins or RAD51 cofactors (namely RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3) which interact with each other to form complexes (BCDX2, BC, DX2 and CX3) that collaboratively assist RAD51 in homologous recombinational repair (HRR) of DNA double-strand breaks. To see if these interactions are important in terms of invasion, as they have been demonstrated for DNA repair, we studied the effect of XRCC3 downregulation on the levels of RAD51 paralogs. We found lowered levels of RAD51C, but not RAD51B or RAD51D, when XRCC3 was downregulated. Since XRCC3 forms the CX3 complex with RAD51C, we downregulated RAD51C using siRNAs in T4-2 cells and found this to significantly increase invasiveness. Consistent with previous findings by other groups, downregulating RAD51C also lead to decreased levels of XRCC3 in invasive T4-2 cells. These results suggest that the XRCC3-RAD51C interaction is important for invasion as well as the previously studied DNA repair function. In delineating the mechanism by which XRCC3 acts as a negative regulator of invasion, we further questioned if XRCC3 alters secreted factors that are important for the invasiveness of T4-2 cells and tested the effects of conditioned medium (CM) from XRCC3 altered T4-2 cells on parental T4-2 cells’ ability to invade. Results show a significant increase in invading T4-2 cells when suspended in CM from XRCC3 siRNA transfected T4-2 cells, suggesting a direct effect of XRCC3 siRNAs on the ability of T4-2 CM to induce invasiveness in T4-2 cells. Furthermore, we investigated the effects of XRCC3 inhibition on cell surface integrins and focal adhesion kinase (FAK). Indirect immunofluorescence results show increased formation of focal adhesions containing two phosphorylated FAK residues- autophosphorylated FAK-Y397 and FAK-Y861 (previously implicated in increased migration and invasion of tumor cells) in XRCC3 siRNA transfected T4-2 cells. Overall, these results support a new role of XRCC3 in invasion, in addition to its previously reported role in DNA repair. These findings imply that loss of XRCC3 function in cancer progression would upregulate invasion as well as downregulate DNA repair and genome stability. Therefore, stabilization of XRCC3 function could provide a promising therapeutic against breast cancer progression. The dual role of XRCC3 in invasion and DNA repair also renders it an attractive candidate risk biomarker of breast pre-cancer to invasive cancer progression.