Role of notch signaling in breast cancer progression

Breast cancer is the most common cancer and the most frequent cause of cancer related mortality among women worldwide. In recent years, major strides in breast cancer research have propelled advances in diagnostic and therapeutic tools that, in turn, have led to improvements in breast cancer survival rates. However, several critical stages of breast cancer remain poorly understood, including ductal carcinoma in situ (DCIS) as well as cancer recurrence after therapy. Here, using genetically engineered mouse models that faithfully recapitulate these processes as they occur in women, we demonstrate that Notch signaling plays a role in both the formation of DCIS and in the recurrence of invasive breast cancers following HER2/neu targeted therapy. First to investigate the role of Notch signaling in early stages of breast cancer progression as well as mammary epithelial cell fate determination, we generated transgenic mice in which Notch signaling is activated in the mammary epithelium upon doxycycline administration. We found that Notch signaling promotes the expansion of mature luminal epithelial cells resulting in ductal lesions that closely resemble micropapillary DCIS in humans. Next, using this model to generate an expression signature that classifies human breast cancers according to their predicted levels of Notch activity, we demonstrated that Notch signaling is associated with an elevated risk of recurrence in women with breast cancer. To determine whether Notch signaling plays a functional role in breast cancer recurrence, we used genetic and pharmacological approaches to activate or inhibit Notch signaling in a mouse model of HER2/neu targeted therapy. These experiments revealed that upregulation of endogenous Notch signaling following HER2/neu inhibition promotes the survival and recurrence of dormant residual tumor cells. Together, our results identify new roles for Notch signaling in regulating distinct stages of breast cancer progression and suggest that inhibition of Notch signaling may be effective in treating DCIS and limiting breast cancer recurrence

Expansion sequencing: Spatially precise in situ transcriptomics in intact biological systems

Methods for highly multiplexed RNA imaging are limited in spatial resolution and thus in their ability to localize transcripts to nanoscale and subcellular compartments. We adapt expansion microscopy, which physically expands biological specimens, for long-read untargeted and targeted in situ RNA sequencing. We applied untargeted expansion sequencing (ExSeq) to the mouse brain, which yielded the readout of thousands of genes, including splice variants. Targeted ExSeq yielded nanoscale-resolution maps of RNAs throughout dendrites and spines in the neurons of the mouse hippocampus, revealing patterns across multiple cell types, layer-specific cell types across the mouse visual cortex, and the organization and position-dependent states of tumor and immune cells in a human metastatic breast cancer biopsy. Thus, ExSeq enables highly multiplexed mapping of RNAs from nanoscale to system scale

Actomyosin purse strings: renewable resources that make morphogenesis robust and resilient

Dorsal closure in Drosophila is a model system for cell sheet morphogenesis and wound healing. During closure two sheets of lateral epidermis move dorsally to close over the amnioserosa and form a continuous epidermis. Forces from the amnioserosa and actomyosin-rich, supracellular purse strings at the leading edges of these lateral epidermal sheets drive closure. Purse strings generate the largest force for closure and occur during development and wound healing throughout phylogeny. We use laser microsurgery to remove some or all of the purse strings from developing embryos. Free edges produced by surgery undergo characteristic responses as follows. Intact cells in the free edges, which previously had no purse string, recoil away from the incision and rapidly assemble new, secondary purse strings. Next, recoil slows, then pauses at a turning point. Following a brief delay, closure resumes and is powered to completion by the secondary purse strings. We confirm that the assembly of the secondary purse strings requires RhoA. We show that α-actinin alternates with nonmuscle myosin II along purse strings and requires nonmuscle myosin II for its localization. Together our data demonstrate that purse strings are renewable resources that contribute to the robust and resilient nature of closure