induced the invasion of non-trailblazer cells, thus revealing a new type of commensal relationship among naturally existing tumor subpopulations. Together, these results demonstrate how the epigenetic alteration of the signaling circuitry in a subpopulation of tumor cells can promote collective invasion through cellautonomous and non-cell-autonomous mechanisms. Results A distinct subpopulation of trailblazer cells has enhanced invasive ability.To begin defining the molecular traits that confer tumor cells with invasive ability, we analyzed spheroid invasion in an organotypic culture system that reconstitutes key features of collective invasion that are conserved in vivo (9, 13). Normal mammary epithelial cells form duct-like spheroids in this system (Supplemental Figure 1A; supplemental material available online with this article; doi:10.1172/JCI77767DS1), indicating that our model was testing for unique traits of tumor cells that promote cell-autonomous invasion, potentially during the transition from ductal carcinoma in situ (DCIS) to invasive breast cancer (13). To begin defining traits that promote collective invasion, we determined the percentage of invasive trailblazer spheroids that were formed in 7 different breast cancer cell lines that represent key known features of intertumor molecular diversity. Invasive spheroids were detected in 3 of the 7 cell lines evaluated, with the percentage of invasive spheroids ranging between 8% and 75% of the total population ( Figure 1, A and B). None of the cell lines contained a 100% pure population of invasive spheroids ( Figure 1B). The 3 cell lines that contained invasive spheroids were derived from patients with TNBC (no detectable estrogen receptor [ER], progesterone receptor, or human epidermal growth factor receptor 2 [HER2] expression) ( Figure 1A). TNBC accounts for 10% to 20% of diagnosed breast cancers and has a relatively worse outcome compared with that of ER + breast cancer (21). Importantly, the strand-like organization of the collectively invading cells observed in organotypic culture was also detected in primary breast tumors ( Figure 1C). Thus, our results indicate that there can be a distinct subpopulation within a community of tumor cells that has an enhanced capacity to lead collective invasion. We refer to this intrinsically invasive subpopulation as trailblazer cells to distinguish them from other types of leader cells, such as KRT14-expressing breast cancer cells, that are unable to invade under these conditions. The noninvasive subpopulation, which may require additional extrinsic factors to invade, is referred to as "opportunist" cells herein.Immunofluorescence analysis and time-lapse imaging showed that the leader trailblazer cells formed long cellular protrusions (LCPs) into the ECM before invading away from the main mass of cells (Figure 1, D and E, and Supplemental Video 1), similar to previous reports (9, 10). Additional trailblazer cells could then migrate into the space within the ECM created by the first invading cell, indicating tha...
The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.
Cell identity signals influence the invasive capability of tumor cells, as demonstrated by the selection for programs of epithelial-to-mesenchymal transition (EMT) during malignant progression. Breast cancer cells retain canonical epithelial traits and invade collectively as cohesive groups of cells, but the signaling pathways critical to their invasive capabilities are still incompletely understood. Here we report that the transcription factor ΔNp63α drives the migration of basal-like breast cancer (BLBC) cells by inducing a hybrid mesenchymal/epithelial state. Through a combination of expression analysis and functional testing across multiple BLBC cell populations, we determined that ΔNp63α induces migration by elevating the expression of the EMT program components Slug and Axl. Interestingly, ΔNp63α also increased the expression of miR205, which can silence ZEB1/2 to prevent the loss of epithelial character caused by EMT induction. In clinical specimens, co-expression of various elements of the ΔNp63α pathway confirmed its implication in motility signaling in BLBC. We observed that activation of the ΔNp63α pathway occurred during the transition from noninvasive ductal carcinoma in situ to invasive breast cancer. Notably, in an orthotopic tumor model, Slug expression was sufficient to induce collective invasion of E-cadherin expressing BLBC cells. Together, our results illustrate how ΔNp63α can drive breast cancer cell invasion by selectively engaging pro-migratory components of the EMT program while, in parallel, still promoting the retention of epithelial character.
Influenza A virus is a human pathogen whose genome is comprised of eight viral RNA segments that replicate in the nucleus. Two viral mRNAs are alternatively spliced. The unspliced M1 mRNA is translated into the matrix M1 protein while the ion channel M2 protein is generated after alternative splicing. These proteins are critical mediators of viral trafficking and budding. We show that influenza virus utilizes nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies, and exports the spliced M2 mRNA from the nucleus. Since nuclear speckles are storage sites for splicing factors, which leave these sites to splice cellular pre-mRNAs at transcribing genes, we reveal a functional subversion of nuclear speckles to promote viral gene expression.
Influenza viruses antagonize key immune defense mechanisms via the virulence factor NS1 protein. A major NS1 strategy is to block nuclear export of host mRNAs including those encoding immune factors1–3; however, the direct cellular target of NS1 and the mechanism of host mRNA export inhibition are not known. Here, we identify the target of NS1 as the mRNA export receptor NXF1•NXT1, which is the principal receptor to mediate docking and translocation of mRNAs through the nuclear pore complex by interacting with nucleoporins4,5. We determined the crystal structure of NS1 in complex with NXF1•NXT1 at 3.8 Å resolution. The structure reveals that NS1 prevents binding of NXF1•NXT1 to nucleoporins, thereby inhibiting mRNA export through the nuclear pore complex into the cytoplasm for translation. We demonstrate that a mutant influenza virus deficient in binding NXF1•NXT1 does not block host mRNA export and is attenuated. This attenuation is marked by the release of mRNAs encoding immune factors from the nucleus. Together, our study uncovers the molecular basis of a major nuclear function of influenza NS1 protein that causes potent blockage of host gene expression and contributes to inhibition of host immunity.
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