Abstract 2305: Centriolin depletion inhibits cellular proliferation of rhabdomyosarcoma cells
Authors: Takeda Yoshinori , Cole Ariel , Boyle Caitlin , Gromley Adam 
Published: June 2022

Abstract
Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, accounts for approximately five percent of all childhood cancers. It develops from rhabdomyoblasts and can arise in a variety of anatomic locations. Recent findings have assisted the prediction of disease prognosis and treatment regimens based on genetic and cellular differences among the major types of RMS. One potential target is a cellular appendage called the primary cilium.
Primary cilia are thought to play a critical role in the regulation of myogenesis, a process deregulated in RMS. During myogenesis, Gli protein accumulates at the primary cilia in myoblasts and induces proliferation. The centrosome protein, centriolin (CNTRL), is known to be a key structural component of primary cilia. Therefore, we hypothesized that disruption of CNTRL, using CRISPR/Cas9, may affect Gli protein function and, therefore, the proliferation of these cells.
Two cell lines were used: A204, a rhabdoid cell line, and CCL-136, an embryonal RMS cell line. When CNTRL was depleted and analyzed 2 days post-transfection, a significant reduction in cell viability was observed in CCL-136 compared to control, whereas no significant increase in cell death in A204 was observed. Gli1 expression decreased in both cells lines when CNTRL was eliminated, while cell cycle progression was also reduced, as confirmed by immunofluorescence staining of Ki67. Unexpectedly, primary cilia formation, indicated by Arl13B expression, increased in A204 with CNTRL loss, with no perceptible change in CCL-136 cells.
To assess differentiation potential, A204 and CCL-136 were cultured in differentiation media using 2% horse serum. Separately, cells were cultured in 0% FBS (serum) to test differentiation potential under starvation conditions. As expected, both cell lines expressed robust levels of MyoG, a muscle differentiation marker, with 2% horse serum. However, loss of CNTRL leads to a perceptible decrease in MyoG in the CCL-136, and a slight decrease in A204. Additionally, wound healing assays were conducted and showed CCL-136 had inherently greater ability to migrate 24 hours post-wound compared to A204., However, CCL-136 showed diminished migration under stressed conditions. Interestingly, cell migration was significantly diminished in A204 under starvation conditions when CNTRL was depleted (p&lt;0.05).
We show that CNTRL loss inhibits the cell cycle in two different forms of RMS. This arrest occurs with a corresponding reduction in Gli1 expression. This confirms previous studies that suggest Gli1 is a critical factor in maintaining the proliferative state. In addition, our results show that in cells normally producing primary cilia, like A204, CNTRL loss increases the number of ciliated cells. Taken together, our data support the possibility of inhibiting cellular proliferation of RMS cells through manipulation of CNTRL and suggest that CNTRL may be a viable future treatment target.
Citation Format: Yoshinori Takeda, Ariel Cole, Caitlin Boyle, Adam Gromley. Centriolin depletion inhibits cellular proliferation of rhabdomyosarcoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2305.