Scaffold protein Scribble is a potent modulator of Sonic Hedgehog signaling

Abstract

<b>Abstract ID 28302</b> <b>Poster Board 39</b> Sonic Hedgehog (SHH) signaling plays a crucial role in cerebellum development by controlling the proliferation of granule neuron progenitors (GNP). Its aberrant expression results in SHH-Medulloblastoma (MB), which is among the most prevalent pediatric brain tumors in children younger than 3 years old. Though SHH-MB is genetically understood, current therapies are ineffective. There is an unmet need to identify new targetable regulators of SHH signaling to improve outcomes. We previously reported that the ABC transporter ABCC4 was expressed in MB and modulated the intensity of SHH pathway activity¹, and have recently identified Scribble (SCRIB) as a protein that binds to the PDZ motif of ABCC4. SCRIB is a conserved scaffold protein that has known roles in cell polarity and coordinates several signaling pathways². A recent study discussed SCRIB’s association with neurodevelopmental disorders³. However, it is unknown whether SCRIB regulates the SHH pathway. In mouse cerebellum, we found that Scrib is highly expressed at postnatal day 1-7 (when SHH signaling is most active) and specifically expressed in proliferating GNPs. We hypothesized that SCRIB might regulate SHH signaling by either affecting ABCC4 or acting independently. To test if SCRIB is required for SHH signaling, <i>Scrib</i> knockdown by siRNAs was performed in NIH-3T3 cells, a model of the SHH pathway. SHH signaling was initiated by either SHH ligand or Smoothened (Smo) agonist SAG. The activation was assessed by Gli1 expression, a target gene of SHH signaling. Our results showed that suppression of <i>Scrib</i> remarkably diminished both mRNA (p&lt;0.05) and protein level (p&lt;0.01) of Gli1, demonstrating that SCRIB is a positive regulator of SHH signaling. We then used CRISPR-Cas9 to develop NIH-3T3 Scrib knockout (KO) clones that affirmed <i>Scrib</i> absence impaired SHH signaling. Because ABCC4 regulates SHH signaling by affecting cAMP transport, we excluded the possibility that SCRIB modulates the pathway by affecting ABCC4’s expression, localization and function. We then investigated alternative molecular mechanisms by which SCRIB acts independently of ABCC4. The expression and localization of key elements in SHH signaling were evaluated in ScribKO clones, including Smo and Gli2. There was minimal effect on the upstream SHH activator Smo, but subcellular fractionation revealed that Scrib’s absence dramatically reduced the SHH-induced nuclear expression of Gli2 (p&lt;0.0001), a dominant downstream activator. Cycloheximide turnover studies suggested that SCRIB is required for the stability of GLI2 during SHH signaling. To understand the influence of SCRIB on the aberrant SHH signaling that drives some SHH-MBs, <i>Scrib</i> siRNAs were applied to mouse fibroblast cell lines lacking either <i>Ptch1</i> or <i>Sufu</i>. Loss of Scrib restrained the constitutive SHH pathway activation produced by <i>Ptch1</i> or <i>Sufu</i> absence as shown by the dramatic reduction in Gli1(p&lt;0.01). Moreover, <i>SCRIB</i> knockdown in human SHH-MB cell lines, DAOY and UW228, significantly decreased the proliferation of these tumor cells (p&lt;0.02), highlighting that SCRIB suppression is a viable strategy to reduce SHH-MB tumor growth. Overall, our data demonstrates that SCIRB is a new potent modulator of SHH signaling with the potential as a novel target in SHH-MB therapy. This work is supported by NIH and ALSAC. <b>Ref:</b> 1. Wijaya, J. <i>et&nbsp;al.</i><i>Cancer Res.</i>2020 2. Pires, H. R. &amp; Boxem, M. <i>J. Mol. Biol.</i>2018 3. Ezan, J. <i>et&nbsp;al.</i><i>Sci. Rep.</i>2021.