Signaling pathways regulating stem cell fate decisions

Division Of Basic Sciences - Nci

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Abstract

This project focuses on generating a mechanistic signaling framework for the regulation of epithelial cells by GPCRs, the heterotrimeric G proteins Galphas and Galphai, and their downstream pathways. Galphas- and Galphai-coupled GPCRs either stimulate (Galphas) or inhibit (Galphai) production of the intracellular second-messenger cyclic AMP (cAMP). Utilizing basal-skin keratinocytes as a stem cell model, we have demonstrated that regulation of cAMP and PKA by Galphas and Galphai is essential for epithelial stem cell differentiation. More recently, to study the receptors that might modulate epithelial stem cell fate upstream of heterotrimeric G-proteins, we performed RNA sequencing (RNAseq) analysis in primary human keratinocytes (HEK). We identified 53 GPCRs expressed at significant levels. Using a pooled interference RNA (siRNA) library, we found that knockdown of specific GPCRs that couple to Galphai leads to reduced proliferation in keratinocytes. We are currently elucidating the downstream signaling mechanisms responsible for the effect of these GPCRs in keratinocyte proliferation and migration. Since GPCRs are attractive targets for drug development, identifying the role of specific receptors in epithelial cell fate will allow us to identify potential GPCRs that can be targeted to regulate stem cell activity. Downstream of GPCR activity, we have found that Galphas and Galphai signaling in the skin are central regulators of YAP1 translocation to the nucleus and activation of downstream targets. YAP1 and its paralog TAZ (WWTR1) are co-transcriptional regulators downstream of the Hippo pathway essential for skin homeostasis and epithelial stem cell maintenance. YAP1 and TAZ are also implicated in skin cancer formation, and this axis has been listed as one of the top 10 signaling pathways altered in human cancer. Interaction with TEAD transcription factors is the primary way YAP1 and TAZ execute their regulatory and oncogenic functions. As such, efforts are underway to develop YAP1/TAZ-TEAD interaction inhibitors to treat hyperproliferative diseases. However, the lack of preclinical models to characterize the consequences of TEAD inhibition is a significant challenge in studying the efficacy of this approach. To circumvent some of the limitations to study TEAD inhibition in cells and tissues, our group developed TEADi. This genetically encoded fluorescently traceable dominant-negative protein blocks the nuclear interaction of TEAD with YAP1 and TAZ. TEADi rapidly inhibits TEAD transcription and concomitantly blocks both YAP1 and TAZ without altering the structural or cytoplasmic functions of these proteins. Ultimately, unveiling YAP1/TAZ-TEAD dependent and independent effects could provide additional clues to suppress YAP1/TAZ activity in tumors. After validating the usefulness of TEADi to study TEAD transcription specifically, we utilized TEADi in a mouse model of skin Basal Cell Carcinoma (BCC) to analyze the transcriptional and cell fate consequences of TEAD blockage in cancer. Both TEAD inhibitor and YAP1/TAZ knockdown reduced proliferation and increased differentiation of mouse BCC driven by oncogenic hedgehog-smoothened (SmoM2) activity. Although TEAD-transcriptional networks were essential to inactivate differentiation, this inactivation was indirect and potentially mediated through KLF4 repression by SNAI2. By comparing the transcriptional effects of TEAD inhibition with those caused by YAP1/TAZ depletion, we determined YAP1/TAZ TEAD independent effects in cancer cells that impact STAT3 and NF-KB. Overall, our results indicate that repression of KLF4 transcriptional networks by YAP1/TAZ TEAD is essential for maintaining basal cell identity and block differentiation in BCC downstream of oncogenic hedgehog-smoothened activity.