Chemotherapy-induced intestinal epithelial damage directly promotes galectin-9-driven modulation of T cell behavior.

The intestine is vulnerable to chemotherapy-induced damage due to the high rate of intestinal epithelial cell (IEC) proliferation. We have developed a human intestinal organoid-based 3D model system to study the direct effect of chemotherapy-induced IEC damage on T cell behavior. Exposure of intestinal organoids to busulfan, fludarabine, and clofarabine induced damage-related responses affecting both the capacity to regenerate and transcriptional reprogramming. In ex vivo co-culture assays, prior intestinal organoid damage resulted in increased T cell activation, proliferation, and migration. We identified galectin-9 (Gal-9) as a key molecule released by damaged organoids. The use of anti-Gal-9 blocking antibodies or CRISPR/Cas9-mediated Gal-9 knock-out prevented intestinal organoid damage-induced T cell proliferation, interferon-gamma release, and migration. Increased levels of Gal-9 were found early after HSCT chemotherapeutic conditioning in the plasma of patients who later developed acute GVHD. Taken together, chemotherapy-induced intestinal damage can influence T cell behavior in a Gal-9-dependent manner which may provide novel strategies for therapeutic intervention.

Regulation of a progenitor gene program by SOX4 is essential for mammary tumor proliferation.

In breast cancer the transcription factor SOX4 has been shown to be associated with poor survival, increased tumor size and metastasis formation. This has mostly been attributed to the ability of SOX4 to regulate Epithelial-to-Mesenchymal-Transition (EMT). However, SOX4 regulates target gene transcription in a context-dependent manner that is determined by the cellular and epigenetic state. In this study we have investigated the loss of SOX4 in mammary tumor development utilizing organoids derived from a PyMT genetic mouse model of breast cancer. Using CRISPR/Cas9 to abrogate SOX4 expression, we found that SOX4 is required for inhibiting differentiation by regulating a subset of genes that are highly activated in fetal mammary stem cells (fMaSC). In this way, SOX4 re-activates an oncogenic transcriptional program that is regulated in many progenitor cell-types during embryonic development. SOX4-knockout organoids are characterized by the presence of more differentiated cells that exhibit luminal or basal gene expression patterns, but lower expression of cell cycle genes. In agreement, primary tumor growth and metastatic outgrowth in the lungs are impaired in SOX4KO tumors. Finally, SOX4KO tumors show a severe loss in competitive capacity to grow out compared to SOX4-proficient cells in primary tumors. Our study identifies a novel role for SOX4 in maintaining mammary tumors in an undifferentiated and proliferative state. Therapeutic manipulation of SOX4 function could provide a novel strategy for cancer differentiation therapy, which would promote differentiation and inhibit cycling of tumor cells.

C/EBPɑ is crucial determinant of epithelial maintenance by preventing epithelial-to-mesenchymal transition.

Extracellular signals such as TGF-ß can induce epithelial-to-mesenchymal transition (EMT) in cancers of epithelial origin, promoting molecular and phenotypical changes resulting in pro-metastatic characteristics. We identified C/EBPα as one of the most TGF-ß-mediated downregulated transcription factors in human mammary epithelial cells. C/EBPα expression prevents TGF-ß-driven EMT by inhibiting expression of known EMT factors. Depletion of C/EBPα is sufficient to induce mesenchymal-like morphology and molecular features, while cells that had undergone TGF-ß-induced EMT reverted to an epithelial-like state upon C/EBPα re-expression. In vivo, mice injected with C/EBPα-expressing breast tumor organoids display a dramatic reduction of metastatic lesions. Collectively, our results show that C/EBPα is required for maintaining epithelial homeostasis by repressing the expression of key mesenchymal markers, thereby preventing EMT-mediated tumorigenesis. These data suggest that C/EBPα is a master epithelial "gatekeeper" whose expression is required to prevent unwarranted mesenchymal transition, supporting an important role for EMT in mediating breast cancer metastasis.

Global transcriptional analysis identifies a novel role for SOX4 in tumor-induced angiogenesis.

The expression of the transcription factor SOX4 is increased in many human cancers, however, the pro-oncogenic capacity of SOX4 can vary greatly depending on the type of tumor. Both the contextual nature and the mechanisms underlying the pro-oncogenic SOX4 response remain unexplored. Here, we demonstrate that in mammary tumorigenesis, the SOX4 transcriptional network is dictated by the epigenome and is enriched for pro-angiogenic processes. We show that SOX4 directly regulates endothelin-1 (ET-1) expression and can thereby promote tumor-induced angiogenesis both in vitro and in vivo. Furthermore, in breast tumors, SOX4 expression correlates with blood vessel density and size, and predicts poor-prognosis in patients with breast cancer. Our data provide novel mechanistic insights into context-dependent SOX4 target gene selection, and uncover a novel pro-oncogenic role for this transcription factor in promoting tumor-induced angiogenesis. These findings establish a key role for SOX4 in promoting metastasis through exploiting diverse pro-tumorigenic pathways.

SOX4 can redirect TGF-ß-mediated SMAD3-transcriptional output in a context-dependent manner to promote tumorigenesis.

Expression of the transcription factor SOX4 is often elevated in human cancers, where it generally correlates with tumor-progression and poor-disease outcome. Reduction of SOX4 expression results in both diminished tumor-incidence and metastasis. In breast cancer, TGF-ß-mediated induction of SOX4 has been shown to contribute to epithelial-to-mesenchymal transition (EMT), which controls pro-metastatic events. Here, we identify SMAD3 as a novel, functionally relevant SOX4 interaction partner. Genome-wide analysis showed that SOX4 and SMAD3 co-occupy a large number of genomic loci in a cell-type specific manner. Moreover, SOX4 expression was required for TGF-ß-mediated induction of a subset of SMAD3/SOX4-co-bound genes regulating migration and extracellular matrix-associated processes, and correlating with poor-prognosis. These findings identify SOX4 as an important SMAD3 co-factor controlling transcription of pro-metastatic genes and context-dependent shaping of the cellular response to TGF-ß. Targeted disruption of the interaction between these factors may have the potential to disrupt pro-oncogenic TGF-ß signaling, thereby impairing tumorigenesis.