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...

Fungal polyketides with the resorcylic acid lactone (RAL) scaffold are of interest for growth stimulation, the treatment of cancer, and neurodegenerative diseases. The RAL radicicol is a nanomolar inhibitor of the chaperone Hsp90, whose repression leads to a combinatorial blockade of cancer-causing pathways. Clustered genes for radicicol biosynthesis were identified and functionally characterized from the endophytic fungus Chaetomium chiversii, and compared to recently described RAL biosynthetic gene clusters. Radicicol production is abolished upon targeted inactivation of a putative cluster-specific regulator, or either of the two polyketide synthases that are predicted to collectively synthesize the radicicol polyketide core. Genomic evidence supports the existence of flavin-dependent halogenases in fungi: inactivation of such a putative halogenase from the C. chiversii radicicol locus yields dechloro-radicicol (monocillin I). Inactivation of a cytochrome P450 epoxidase furnishes pochonin D, a deepoxy-dihydro radicicol analog.

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.

Purpose-CatalyCEST MRI compares the detection of an enzyme-responsive CEST agent with the detection of an unresponsive "control" CEST agent that accounts for other conditions that influence CEST. The purpose of this study was to investigate the feasibility of in vivo catalyCEST MRI.Methods-CEST agents that were responsive and unresponsive to the activity of urokinase Plasminogen Activator (uPA) were shown to have negligible interaction with each other. A CEST-FISP MRI protocol was used to acquire MR CEST spectroscopic images with a Capan-2 pancreatic tumor model after intravenous injection of the CEST agents. A function of (super)-Lorentzian line shapes was fit to CEST spectra of a region-of-interest that represented the tumor.Results-The CEST effects from each agent showed the same initial uptake into tumor tissues, indicating that both agents had the same pharmacokinetic transport rates. Starting five minutes after injection, CEST from the enzyme-responsive agent disappeared more quickly than CEST from the unresponsive agent, indicating that the enzyme responsive agent was being catalyzed by uPA while both agents also experienced net pharmacokinetic washout from the tumor.Conclusion-CatalyCEST MRI demonstrates that dynamic tracking of enzyme-responsive and unresponsive CEST agents during the same in vivo MRI study is feasible.

Genes that are normally biased towards expression in the testis are often induced in tumor cells. These gametogenic genes, known as cancer-testis antigens (CTAs), have been extenstively investigated as targets for immunotherapy. However, despite their frequent detection, the degree to which CTAs support neoplastic invasion is poorly understood. Here, we find that the CTA genes SPANX-A/C/D and CTAG2 are coordinately induced in breast cancer cells and regulate distinct features of invasive behavior. Our functional analysis revealed that CTAG2 interacts with Pericentrin at the centrosome and is necessary for directional migration. Conversely, SPANX-A/C/D interacts with Lamin A/C at the inner nuclear membrane and is required for the formation of actin-rich cellular protrusions that reorganize the extracellular matrix. Importantly, SPANX-A/C/D was required for breast cancer cells to spontaneously metastasize to the lung, demonstrating that CTA reactivation can be critical for invasion dependent phenotypes in vivo. Moreover, elevated SPANX-A/C/D expression in breast cancer patient tumors correlated with poor outcome. Together, our results suggest that distinct CTAs promote tumor progression by regulating complementary cellular functions that are integrated together to induce invasive behavior.