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...

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.

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.

Uterine carcinosarcoma is an aggressive variant of endometrial carcinoma characterized by unusual histologic features including discrete malignant epithelial and mesenchymal components (carcinoma and sarcoma). Recent studies have confirmed a monoclonal origin, and comprehensive genomic characterizations have identified mutations such asTp53andPten. However, the biological origins and specific combination of driver events underpinning uterine carcinosarcoma have remained mysterious. Here, we explored the role of the tumor suppressorFbxw7in endometrial cancer through defined genetic model systems. Inactivation ofFbxw7andPtenresulted in the formation of precancerous lesions (endometrioid intraepithelial neoplasia) and well-differentiated endometrioid adenocarcinomas. Surprisingly, all adenocarcinomas eventually developed into definitive uterine carcinosarcomas with carcinomatous and sarcomatous elements including heterologous differentiation, yielding a faithful genetically engineered model of this cancer type. Genomic analysis showed that most tumors spontaneously acquiredTrp53mutations, pointing to a triad of pathways (p53, PI3K, and Fbxw7) as the critical combination underpinning uterine carcinosarcoma, and to Fbxw7 as a key driver of this enigmatic endometrial cancer type. Lineage tracing provided formal genetic proof that the uterine carcinosarcoma cell of origin is an endometrial epithelial cell that subsequently undergoes a prominent epithelial–mesenchymal transition underlying the attainment of a highly invasive phenotype specifically driven by Fbxw7.

Tumor invasion can be induced by changes in gene expression that alter cell phenotype. The transcription factor ΔNp63α promotes basal-like breast cancer (BLBC) migration by inducing the expression of the mesenchymal genes Slug and Axl, which confers cells with a hybrid epithelial/mesenchymal state. However, the extent of the ΔNp63α regulated genes that support invasive behavior is not known. Here, using gene expression analysis, ChIP-seq, and functional testing, we find that ΔNp63α promotes BLBC motility by inducing the expression of the atypical cadherin FAT2, the vesicular binding protein SNCA, the carbonic anhydrase CA12, the lipid binding protein CPNE8 and the kinase NEK1, along with Slug and Axl. Notably, lung squamous cell carcinoma migration also required ΔNp63α dependent FAT2 and Slug expression, demonstrating that ΔNp63α promotes migration in multiple tumor types by inducing mesenchymal and non-mesenchymal genes. ΔNp63α activation of FAT2 and Slug influenced E-cadherin localization to cell-cell contacts, which can restrict spontaneous cell movement. Moreover, live-imaging of spheroids in organotypic culture demonstrated that ΔNp63α, FAT2 and Slug were essential for the extension of cellular protrusions that initiate collective invasion. Importantly, ΔNp63α is co-expressed with FAT2 and Slug in patient tumors and the elevated expression of ΔNp63α, FAT2 and Slug correlated with poor patient outcome. Together, these results reveal how ΔNp63α promotes cell migration by directly inducing the expression of a cohort of genes with distinct cellular functions and suggest that FAT2 is a new regulator of collective invasion that may influence patient outcome.