Multiciliated cells (MCCs) in the brain include the ependymal cells and choroid plexus (CP) epithelial cells. The CP secretes cerebrospinal fluid that circulates within the ventricular system, driven by ependymal cilia movement. However, the mechanisms and functional significance of multiciliogenesis in the CP remain unknown. Deregulated oncogenic signals cause CP carcinoma (CPC), a rare but aggressive pediatric brain cancer. Here we show that aberrant NOTCH and Sonic Hedgehog signaling in mice drive tumors that resemble CPC in humans. NOTCH-driven CP tumors were monociliated, whereas disruption of the NOTCH complex restored multiciliation and decreased tumor growth. NOTCH suppressed multiciliation in tumor cells by inhibiting the expression of GEMC1 and MCIDAS, early regulators of multiciliogenesis. Consistently, GEMC1-MCIDAS function is essential for multiciliation in the CP, and is critical for correcting multiciliation defect in tumor cells by a NOTCH inhibitor. Disturbances to the GEMC1 program are commonly observed in human CPCs characterized by solitary cilia. Consistently, CPC driven by deletion of Trp53 and Rb1 in mice exhibits a cilia deficit consequent to loss of Gemc1-Mcidas expression. Taken together, these findings reveal a GEMC1-MCIDAS multiciliation program in the CP critical for inhibiting tumorigenesis, and it may have therapeutic implications for the treatment of CPC.

Multiciliated cells (MCCs) in the brain reside in the ependyma and the choroid plexus (CP) epithelia. The CP secretes cerebrospinal fluid that circulates within the ventricular system, driven by ependymal cilia movement. Tumors of the CP are rare primary brain neoplasms mostly found in children. CP tumors exist in three forms: CP papilloma (CPP), atypical CPP, and CP carcinoma (CPC). Though CPP and atypical CPP are generally benign and can be resolved by surgery, CPC is a particularly aggressive and little understood cancer with a poor survival rate and a tendency for recurrence and metastasis. In contrast to MCCs in the CP epithelia, CPCs in humans are characterized by solitary cilia, frequent TP53 mutations, and disturbances to multiciliogenesis program directed by the GMNC-MCIDAS transcriptional network. GMNC and MCIDAS are early transcriptional regulators of MCC fate differentiation in diverse tissues. Consistently, components of the GMNC-MCIDAS transcriptional program are expressed during CP development and required for multiciliation in the CP, while CPC driven by deletion of Trp53 and Rb1 in mice exhibits multiciliation defects consequent to deficiencies in the GMNC-MCIDAS program. Previous studies revealed that abnormal NOTCH pathway activation leads to CPP. Here we show that combined defects in NOTCH and Sonic Hedgehog signaling in mice generates tumors that are similar to CPC in humans. NOTCH-driven CP tumors are monociliated, and disruption of the NOTCH complex restores multiciliation and decreases tumor growth. NOTCH suppresses multiciliation in tumor cells by inhibiting the expression of GMNC and MCIDAS, while Gmnc-Mcidas overexpression rescues multiciliation defects and suppresses tumor cell proliferation. Taken together, these findings indicate that reactivation of the GMNC-MCIDAS multiciliogenesis program is critical for inhibiting tumorigenesis in the CP, and it may have therapeutic implications for the treatment of CPC.

Choroid plexus (CP) tumors are rare primary brain neoplasms found most commonly in children and are thought to arise from CP epithelial cells. Sox2 is a transcription factor that not only plays a role in development in the ventricular zone, CP, and roof plate, but also contributes to cancer stemness, tumorigenesis, and drug resistance. Gene expression studies demonstrate aberrant Sox2 expression in human CP tumors, suggesting a role in tumor development. A subset of CP tumors exhibit abnormal NOTCH pathway activity. Using animal models, we previously show that sustained NOTCH activity leads to CP tumors. Immunofluorescence, RT-qPCR, and RNA scope assays have revealed increased Sox2 levels in NOTCH-driven CP tumors compared to wild type CP in mice. To investigate the role of Sox2 in CP tumors, we eliminated Sox2 expression in NOTCH-driven CP tumors. Loss of Sox2 almost completely blocked NOTCH-driven CP tumor growth in these mice, supporting a role for Sox2 in these tumors. Ciliation regulation is one proposed functional pathway for tumorigenesis in CP tumors. Using immunofluorescence assays for cilia (ARL13b) and aquaporin transport protein 1 (AQP1) in combination with super resolution microscopy, we observe a stark contrast between wild type CP epithelial cells which are multiciliated and homogeneously express AQP1, indicative of normal epithelial differentiation, compared to NOTCH-driven CP tumors consisting of mono-ciliated cells with loss of AQP1 expression. In Sox2-deficient NOTCH-driven CP tumors, we observe tumor cells remain mono-ciliated and AQP1-negative, indicating that Sox2 loss does not affect the ciliation machinery. Together this warrants further study into the mechanisms of Sox2 functions in CP tumors. By unraveling the role of Sox2 in CP tumors, we may better understand their origin and biology to ultimately design improved treatment options.

Tumors of the choroid plexus (CP) are rare primary brain neoplasms mostly found in children. CP tumors exist in three forms: CP papilloma (CPP), atypical CPP, and CP carcinoma (CPC). Though CPP is more benign, CPC is a highly lethal and little understood cancer with poor survival rate and a tendency for recurrence and metastasis. CP tumors are thought to arise from CP epithelial cells that secrets cerebral spinal fluid and generate multiple cilia on their apical surface. Here we show that aberrant NOTCH and Sonic Hedgehog signaling in mice drive tumors that resemble CPC in humans. In contrast to CP epithelial cells with clusters of multiple cilia, NOTCH-driven CP tumors were monociliated, and disruption of the NOTCH complex restored multiciliation and decreased tumor growth. NOTCH suppressed multiciliation in tumor cells by inhibiting the expression of Geminin Coiled-Coil Domain Containing 1 (GEMC1), and multiciliate differentiation and DNA synthesis associated cell cycle protein (MCIDAS), early transcriptional regulators of multiciliated cell (MCC) differentiation. Consistently, Gemc1-Mcidas deficiency led to a lack of MCCs in the CP, and impaired the correction of the multiciliation defect in tumor cells by a NOTCH inhibitor. Disturbances to the GEMC1 program are commonly observed in human CPCs characterized by solitary cilia and frequent somatic TP53 mutations. Accordingly, CPC driven by deletion of tumor suppressors Trp53 and Rb1 in mice exhibits a cilia deficit consequent to loss of Gemc1-Mcidas expression. Taken together, these findings reveal that the GEMC1-MCIDAS multiciliogenesis program in the CP is critical for inhibiting tumorigenesis, whereas a defective multiciliation program promotes CPC and may represent a therapeutic avenue for this cancer.