Signaling pathways regulating stem cell fate decisions

Division Of Basic Sciences - Nci

Investigators

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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, I have demonstrated that regulation of cAMP signaling is vital to the coordination of epithelial stem cell self-renewal and differentiation. Inactivation of Galphas or the cAMP-activated protein kinase A (PKA) induces BCC formation in the skin. Mechanistically, Galphas and PKA disruption promote the concomitant, cell-autonomous activation of Hedgehog GLI and Hippo YAP1, two pathways associated with stem cell maintenance and cancer formation. Cell fate regulation by Galphas, cAMP, and PKA have implications beyond skin progenitor cells. Galphas knockout in mouse neural progenitors results in medulloblastoma formation, and PKA activation promotes differentiation of tumor-initiating cells in breast cancer. My previous work revealed that regulation of cAMP and PKA by Galphas is essential for epithelial stem cell differentiation. However, the role of GPCR-coupled Galphai proteins in the skin was unclear. Utilizing a chemo-genetic approach, my laboratory explored the role of Galphai signaling in keratinocytes. GPCR-Galphai activation led to increased proliferation and decreased differentiation in mouse skin in vivo and human skin basal keratinocytes in 2D and 3D cultures, resulting in epidermal hyperplasia. Galphai signaling increased MAPK, JNK, and YAP1 activity. Remarkably, GPCR-Galphai effects in mouse skin were limited to interfollicular keratinocytes, while hair follicle morphology and Hedgehog signaling activation were unaffected. The fact that GPCR-Galphai activation did not phenocopy epidermal Galphas knockout indicated that Galphas inhibition of Hedgehog signaling is dominant over the described activation of this pathway by Galphai. Supporting this notion, we found that in skin keratinocytes, expression levels of Galphas were consistently higher compared to Galphai. Our results emphasize the importance of cAMP signaling in skin homeostasis and point towards overlapping and divergent roles of Galphas and Galphai in the modulation of epithelial cell fate. Our data showed 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 BCC and SCC 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 this inhibitor to understand the roles of TEADs in keratinocytes. We found that YAP1/TAZ-TEAD activity is necessary to maintain keratinocytes in a progenitor, undifferentiated state by regulating epithelial cell homeostasis at two independent levels: (1) controlling the expression of critical factors necessary for cell cycle entry and proliferation, including E2F1 and Cyclin D1; and (2), via a regulatory loop with KLF4, a master regulator of commitment to differentiation in keratinocytes. Blockage of YAP1/TAZ binding to TEAD resulted in KLF4 activation, and we demonstrated that KLF4 can interact and block the transcriptional activity of YAP1/TAZ-TEAD complexes. To validate our results in vivo, we developed a tetracycline-inducible TEADi transgenic mouse model. Blockage of TEAD activity in basal keratinocytes reduced proliferation and increased differentiation, leading to depletion of progenitor/stem cells and disruption of skin integrity. Our results exemplify how TEADi can dissect the consequences of TEAD blockage in more detail and serve as a resource to differentiate TEAD-dependent and independent effects. We next utilized TEADi in a BCC mouse model driven by the oncogenic Hedgehog GPCR, SMO. Our research unveiled that TEAD inhibition in BCC triggers rapid activation of differentiation programs in cell culture and mouse tumors in vivo. We found that the regulation of differentiation by TEAD was indirect and dependent on the repression of KLF4. YAP1/TAZ-TEAD regulated the expression of SNAI2, a known KLF4 repressor that could mediate the effects of TEAD in differentiation. Analysis of TEAD dependent and independent effects in BCC cells showed evidence that YAP1/TAZ but not TEAD regulate the activation of IFN-gamma, STAT, and NF-KB gene networks. Overall, our results indicated that repression of KLF4 by YAP1/TAZ-TEAD is essential for maintaining basal cell identity and blocking differentiation downstream of oncogenic Hedgehog-SMO activity.