Triple negative breast cancer (TNBC) patients exhibit poor survival outcomes and lack effective targeted therapies. Using unbiased in vivo genome-wide CRISPR screening, we interrogated cancer vulnerabilities in TNBC and identified an interplay between oncogenic and tumor suppressor pathways. This study reveals tumor regulatory functions for essential components of the mTOR and Hippo pathways in TNBC. Using in vitro drug matrix synergy models and in vivo patient-derived xenografts, we further establish the therapeutic relevance of our findings and show that pharmacological inhibition of mTORC1/2 and oncoprotein YAP efficiently reduces tumorigenesis in TNBC. At the molecular level, we find that while verteporfin-induced YAP inhibition leads to apoptosis, torin1-mediated mTORC1/2 inhibition promotes macropinocytosis. Torin1-induced macropinocytosis further facilitates verteporfin uptake, thereby greatly enhancing its pro-apoptotic effects in cancer cells. Overall, our study underscores the power and robustness of in vivo CRISPR genome-wide screens in identifying clinically relevant and innovative therapeutic modalities in cancer.

Breast cancer consists of a range of tumor subtypes with different clinical characteristics, disease prognosis, and treatment-response. Luminal breast cancer has the best prognosis while basal-like breast cancer (BLBC) represents the worst subtype. Transforming growth factor-beta (TGFβ) plays a prominent role in stimulating the migration and invasion of malignant breast cancer cells contributing to tumor progression. In this study, we identified the Ephrin type-A receptor 4 (EPHA4) as a novel target of TGFβ in breast cancer. Moreover, we show that TGFβ induction of EPHA4 gene expression is specific to basal-like tumors and is required for TGFβ-mediated cell migration. We further addressed the mechanism and found EPHA4 to be required for TGFβ-mediated cell migration in breast cancer through TGFβ-induced short term and long term activation of RhoGTPases. Finally, our data revealed a strong association between high EPHA4 expression and advanced tumor stage, aggressive BLBC molecular subtype and poor prognosis. Importantly, we found significant co-expression of EPHA4 and the TGFβ receptor type-2 (TGFβR2) in breast cancer subtypes associated with increased tumor relapse and drug resistance. Together, this study highlight the important role of the TGFβ/EPHA4 signaling axis in mediating tumor aggressiveness and poor patient survival in human breast cancer.

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer–ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Although the cyclin-dependent kinases CDK4 and CDK6 play fundamental roles in cancer, the specific pathways and downstream targets by which they exert their tumorigenic effects remain elusive. In this study, we uncover distinct and novel functions for these kinases in regulating tumor formation and metastatic colonization in various solid tumors, including those of the breast, prostate, and pancreas. Combining in vivo CRISPR-based CDK4 and CDK6 gene editing with pharmacologic inhibition approaches in orthotopic transplantation and patient-derived xenograft preclinical models, we defined clear functions for CDK4 and CDK6 in facilitating tumor growth and progression in metastatic cancers. Transcriptomic profiling of CDK4/6 CRISPR knockouts in breast cancer revealed these two kinases to regulate cancer progression through distinct mechanisms. CDK4 regulated prometastatic inflammatory cytokine signaling, whereas CDK6 mainly controlled DNA replication and repair processes. Inhibition of CDK6 but not CDK4 resulted in defective DNA repair and increased DNA damage. Multiple CDK6 DNA replication/repair genes were not only associated with cancer subtype, grades, and poor clinical outcomes, but also facilitated primary tumor growth and metastasis in vivo. CRISPR-based genomic deletion of CDK6 efficiently blocked tumor formation and progression in preestablished cell- and patient-derived xenograft preclinical models of breast cancer, providing a potential novel targeted therapy for these deadly tumors. Significance: In-depth transcriptomic analysis identifies cyclin-dependent kinases CDK4 and CDK6 as regulators of metastasis through distinct signaling pathways and reveals the DNA replication/repair pathway as central in promoting these effects.

Basal-like triple-negative breast cancers (TNBCs) display poor prognosis, have a high risk of tumor recurrence, and exhibit high resistance to drug treatments. The TNBC aggressive features are largely due to the high proportion of cancer stem cells present within these tumors. In this study, we investigated the interplay and networking pathways occurring between TGFβ family ligands in regulating stemness in TNBCs. We found that TGFβ stimulation of TNBCs resulted in enhanced tumorsphere formation efficiency and an increased proportion of the highly tumorigenic CD44high/CD24low cancer stem cell population. Analysis of the TGFβ transcriptome in TNBC cells revealed bone morphogenetic protein4 (BMP4) as a main TGFβ-repressed target in these tumor cells. We further found that BMP4 opposed TGFβ effects on stemness and potently decreased cancer stem cell numbers, thereby acting as a differentiation factor in TNBC. At the molecular level, we found that TGFβ inhibition of BMP4 gene expression is mediated through the Smad pathway and cyclin D1. In addition, we also found BMP4 to act as a pro-differentiation factor in normal mammary epithelial cells and promote mammary acinar formation in 3D cell culture assays. Finally, and consistent with our in vitro results, in silico patient data analysis defined BMP4 as a potential valuable prognosis marker for TNBC patients.