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

Plasticity of Lgr5-Negative Cancer Cells Drives Metastasis in Colorectal Cancer

Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5- and formed distant metastases in which Lgr5+ CSCs appeared. This p

Zooming in on Cellular Plasticity: Unveiling Cancer Cells Secrets and gaining Therapeutic Insights using Intravital Microscopy

Cancer is a broad term encompassing various disease. One common characteristic among all types of cancer is the exploitation of physiological processes to their advantage. This exploitation typically involves acquisition of genetic mutations, often in conjunction with alterations in the immediate environment. The ability of cancer to manipulate the body's own mechanisms, possess significant challenges to battle the disease. Therefore, it is crucial to possess in-depth knowledge about different cancer cells, their behavior, and their interaction with adjacent "healthy" cells. This understanding plays a pivotal role in optimizing cancer treatments in the long run. This dissertation utilizes intravital microscopy to gain fundamental insights into the mechanisms exploited. Hereby we focused not only on mechanisms cancer hijack from healthy tissue but also how this can influence cancer treatment. The first chapter provides a comprehensive summary of recent advancements in the field of tumor cell dynamics and plasticity, were we particularly have focused on migration behavior and spreading of cancer to other organs (Chapter 1). Migration is a process frequently exploited by cancer cells. In healthy tissue, migration is important for wound healing in the skin. Therefore, we examined migration behavior of keratinocytes in a "healthy", cancer-free skin. Our intravital microscopy observations revealed dynamic movement of keratinocytes, much more than previously thought, which likely facilitates wound healing, even in the presence of obstacles like hair roots (Chapter 2). Next, we explored epithelial-to-mesenchymal transition (EMT), an embryonic process also frequently employed by tumor cells. We assess and discuss our current understanding of EMT, reviewing historical studies in light of recent insights, such as hybrid E/M states (Chapter 3). Additionally, we developed various tumor models enabling visualization of different hybrid E/M states in the same cell over time. Through this, we demonstrated that EMT is a spectrum of distinct cellular states (Chapter 4) challenging the previous held belief of it being binary process. We also discovered that cells in an early hybrid E/M state exhibit the highest potential for metastasis and resistance to chemotherapy (Chapter 5). In the final chapters, we investigated influences of environmental factors on the behavior and dynamics of tumor cells, examining both microenvironmental and systemic influences. We demonstrated that CD8+ T-cell activity, particularly release of INFγ over long distances within the tumor, can be influence tumor cell behavior (Chapter 6). Furthermore, we explored the impact of the menstrual cycle on the behavior of breast tumor cells and revealed that tumor cells mimic behavior of healthy breast tissue. Additionally, we discovered that treatment is more effective when chemotherapy is synchronized with the hormonal cycle (Chapter 7). The diversity of the different chapters underscores the significance considering multiple factors to gain better understand of cancer and consequently enhance treatment outcomes. While we have still not fully grasped the its entire complexity, this dissertation demonstrates that tumor cells themselves, their cellular state, their immediate environment, and the entire organism can all play a role in the success of therapies. To improve treatments, it is important to expand this knowledge in the future

Epithelial-to-Mesenchymal Transition in the Light of Plasticity and Hybrid E/M States

Epithelial-to-mesenchymal transition (EMT) is a cellular program which leads to cells losing epithelial features, including cell polarity, cell–cell adhesion and attachment to the basement membrane, while gaining mesenchymal characteristics, such as invasive properties and stemness. This program is involved in embryogenesis, wound healing and cancer progression. Over the years, the role of EMT in cancer progression has been heavily debated, and the requirement of this process in metastasis even has been disputed. In this review, we discuss previous discrepancies in the light of recent findings on EMT, plasticity and hybrid E/M states. Moreover, we highlight various tumor microenvironmental cues and cell intrinsic signaling pathways that induce and sustain EMT programs, plasticity and hybrid E/M states. Lastly, we discuss how recent findings on plasticity, especially on those that enable cells to switch between hybrid E/M states, have changed our understanding on the role of EMT in cancer metastasis, stemness and therapy resistance

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.</p

Distinct contributions of partial and full EMT to breast cancer malignancy

Epithelial-mesenchymal transition (EMT) is a transient, reversible process of cell de-differentiation where cancer cells transit between various stages of an EMT continuum, including epithelial, partial EMT, and mesenchymal cell states. We have employed Tamoxifen-inducible dual recombinase lineage tracing systems combined with live imaging and 5-cell RNA sequencing to track cancer cells undergoing partial or full EMT in the MMTV-PyMT mouse model of metastatic breast cancer. In primary tumors, cancer cells infrequently undergo EMT and mostly transition between epithelial and partial EMT states but rarely reach full EMT. Cells undergoing partial EMT contribute to lung metastasis and chemoresistance, whereas full EMT cells mostly retain a mesenchymal phenotype and fail to colonize the lungs. However, full EMT cancer cells are enriched in recurrent tumors upon chemotherapy. Hence, cancer cells in various stages of the EMT continuum differentially contribute to hallmarks of breast cancer malignancy, such as tumor invasion, metastasis, and chemoresistance.ISSN:1534-5807ISSN:1878-155

Distinct contributions of partial and full EMT to breast cancer malignancy

Epithelial-mesenchymal transition (EMT) is a transient, reversible process of cell de-differentiation where cancer cells transit between various stages of an EMT continuum, including epithelial, partial EMT, and mesenchymal cell states. We have employed Tamoxifen-inducible dual recombinase lineage tracing systems combined with live imaging and 5-cell RNA sequencing to track cancer cells undergoing partial or full EMT in the MMTV-PyMT mouse model of metastatic breast cancer. In primary tumors, cancer cells infrequently undergo EMT and mostly transition between epithelial and partial EMT states but rarely reach full EMT. Cells undergoing partial EMT contribute to lung metastasis and chemoresistance, whereas full EMT cells mostly retain a mesenchymal phenotype and fail to colonize the lungs. However, full EMT cancer cells are enriched in recurrent tumors upon chemotherapy. Hence, cancer cells in various stages of the EMT continuum differentially contribute to hallmarks of breast cancer malignancy, such as tumor invasion, metastasis, and chemoresistance

Dynamic Visualization of TGF-β/SMAD3 Transcriptional Responses in Single Living Cells

Transforming growth factor-β (TGF-β) signaling is tightly controlled in duration and intensity during embryonic development and in the adult to maintain tissue homeostasis. To visualize the TGF-β/SMAD3 signaling kinetics, we developed a dynamic TGF-β/SMAD3 transcriptional fluorescent reporter using multimerized SMAD3/4 binding elements driving the expression of a quickly folded and highly unstable GFP protein. We demonstrate the specificity and sensitivity of this reporter and its wide application to monitor dynamic TGF-β/SMAD3 transcriptional responses in both 2D and 3D systems in vitro, as well as in vivo, using live-cell and intravital imaging. Using this reporter in B16F10 cells, we observed single cell heterogeneity in response to TGF-β challenge, which can be categorized into early, late, and non-responders. Because of its broad application potential, this reporter allows for new discoveries into how TGF-β/SMAD3-dependent transcriptional dynamics are affected during multistep and reversible biological processes