Loss of primary cilia promotes mitochondria-dependent apoptosis in thyroid cancer

www.nature.com/scientificreports OPEN Loss of primary cilia promotes mitochondria‑dependent apoptosis in thyroid cancer Junguee Lee1*, Ki Cheol Park2, Hae Joung Sul1, Hyun Jung Hong3, Kun‑Ho Kim4, Jukka Kero5 & Minho Shong6* The primary cilium is well-preserved in human differentiated thyroid cancers such as papillary and follicular carcinoma. Specific thyroid cancers such as Hürthle cell carcinoma, oncocytic variant of papillary thyroid carcinoma (PTC), and PTC with Hashimoto’s thyroiditis show reduced biogenesis of primary cilia; these cancers are often associated the abnormalities in mitochondrial function. Here, we examined the association between primary cilia and the mitochondria-dependent apoptosis pathway. Tg-Cre;Ift88flox/flox mice (in which thyroid follicles lacked primary cilia) showed irregularly dilated follicles and increased apoptosis of thyrocytes. Defective ciliogenesis caused by deleting the IFT88 and KIF3A genes from thyroid cancer cell lines increased VDAC1 oligomerization following VDAC1 overexpression, thereby facilitating upregulation of mitochondria-dependent apoptosis. Furthermore, VDAC1 localized with the basal bodies of primary cilia in thyroid cancer cells. These results demonstrate that loss-of-function of primary cilia results in apoptogenic stimuli, which are responsible for mitochondrial-dependent apoptotic cell death in differentiated thyroid cancers. Therefore, regulating primary ciliogenesis might be a therapeutic approach to targeting differentiated thyroid cancers. The primary cilium is a non-motile, microtubule-based sensory organelle that receives mechanical and chemical stimuli from the environment and transduces external signals into the c ell1. The tips of primary cilia, which are present in the apical membrane of thyroid follicular cells (thyrocytes), face into the follicular lumen2. The primary cilia of murine thyroid follicular cells play a role in maintaining globular follicle structures by acting on cell polarity3. Loss-of-function (LOF) of primary cilia in murine thyroid follicles results in abnormal and irregular follicles that eventually develop into papillary and solid proliferative nodules3. The primary cilium is well-preserved in human differentiated thyroid cancers, including papillary and follicular carcinoma, and their frequency and length appear similar to those of normal thyroid f ollicles2. Interestingly, the frequency of ciliated thyroid cancer cells is markedly lower in Hürthle cell carcinoma, oncocytic variant of papillary carcinoma (PTCov), and PTC with Hashimoto’s thyroiditis (PTC-HT), which are usually associated with mitochondrial d ysfunction2. However, we do not know whether ciliogenesis is linked with mitochondrial function in thyroid cancer cells. Mitochondria are crucial regulators of cell death through a process called the mitochondria-dependent (intrinsic) pathway of apoptosis. Typically, mitochondrial outer membrane permeabilization (MOMP) is responsible for mediating the intrinsic apoptotic pathway. The voltage-dependent anion channel (VDAC), a component of MOMP, participates in mitochondria-dependent apoptosis by promoting cytochrome c release4,5. VDAC oligomerization, followed by VDAC overexpression, may represent a common mechanism by which various apoptogens act through different initiating c ascades6. Moreover, VDAC function extends beyond the mitochondria, and VDACs localize to the basal body of the primary cilium, where VDAC1 and VDAC3 negatively regulate ciliogenesis7. Recent reports show that dysfunction of primary cilia increases apoptotic cell death in 1 Department of Pathology, Daejeon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Seoul 06591, Republic of Korea. 2Clinical Research Institute, Daejeon St. Mary’s Hospital, College of Medicine, The Catholic University of Korea, Daejeon 34943, Republic of Korea. 3Research Center for Endocrine and Metabolic Diseases, Chungnam National University School of Medicine, Daejeon 35015, Republic of Korea. 4Department of Nuclear Medicine, Chungnam National University Hospital and College of Medicine, Daejeon 35015, Republic of Korea. 5Research Centre for Integrative Physiology and Pharmacology, Institute of Biomedicine, University of Turku, Kiinamyllynkatu 10, 20520 Turku, Finland. 6Department of Internal Medicine, Chungnam National University School of Medicine, 266 Munhwaro, Daejeon 35015, Republic of Korea. *email: junguee@ catholic.ac.kr; Scientific Reports | (2021) 11:4181 | https://doi.org/10.1038/s41598-021-83418-3 1 Vol.:(0123456789) www.nature.com/scientificreports/ Scientific Reports | Vol:.(1234567890) (2021) 11:4181 | https://doi.org/10.1038/s41598-021-83418-3 2 www.nature.com/scientificreports/ ◂Figure 1.  Loss of function of Ift88-mediated resulted in ciliary loss and increased apoptosis. (A) Immunofluorescence images showing primary cilia in thyroid of Tg-Cre;Ift88+/+ and Tg-Cre;Ift88flox/flox mice. Primary cilia were confirmed by staining with anti-acetylated α-tubulin (Ac-α-Tub, green) and anti-γtubulin (γ-Tub, red) antibodies. Primary cilia are indicated by arrows. Scale bar, 10 μm. (B) The thyroid of Tg-Cre;Ift88flox/flox mice (aged 14 weeks) shows irregular dilated follicles with a flat epithelium and luminal colloid depletion. Scale bar, 10 μm. (C) Tg-Cre;Ift88flox/flox mice (aged 14 weeks) showed irregularly dilated thyroid follicles comprising shrunken eosinophilic cells with compact nuclei (H&E). Scale bar, 10 μm. The number of TUNEL-positive follicular cells per follicle in the wild-type control and Tg-Cre;Ift88flox/flox mice was 25 ± 20% and 70 ± 18%, respectively (P < 0.0001). BCL-2-positive follicular cells were rarely observed in the irregularly dilated thyroid follicles of Tg-Cre;Ift88flox/flox mice. ***P < 0.001. (D) In 35 week-old Tg-Cre;Ift88flox/flox mice, the dilated follicles developed into solid proliferative thyroid nodules. Scale bar, 10 μm. glioblastoma, or induce neuron apoptosis in m ice8,9. However, the relationship between primary cilia and cell death via activation of the mitochondrial apoptotic pathway is unclear. Here we established a mouse model with thyrocyte-specific loss of primary cilia (Tg-Cre;Ift88flox/flox) and human thyroid cancer cell lines with ciliary loss by silencing the KIF3A or IFT88 gene. To identify the role of ciliogenesis with respect to the viability of normal thyrocytes and thyroid cancer cells, we examined apoptotic cell death in murine thyroid follicular cells and human thyroid cancer cells devoid of primary cilia. We found that mice lacking primary cilia in thyroid follicular cells showed upregulated apoptotic cell death, resulting in altered follicular structure, and that inhibiting ciliogenesis in thyroid cancer cell lines resulted in VDAC1 oligomerization following VDAC1 overexpression, leading ultimately to apoptosis. Additionally, we demonstrate that VDAC1 is localized to the primary cilia of thyroid follicular cells. Tak (...truncated)