Fat1 suppresses the CSCs properties through CaMKII-mediated activation of the IFN pathway

doi:10.21203/rs.3.rs-3335299/v1

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Fat1 suppresses the CSCs properties through CaMKII-mediated activation of the IFN pathway

https://doi.org/10.21203/rs.3.rs-3335299/v1

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FAT atypical cadherin 1 (Fat1), which encodes an atypical cadherin-coding protein, exhibits a high mutation rate and is commonly regarded as a tumor-suppressor gene in head and neck squamous cell carcinoma (HNSCC). Nonetheless, the potential regulatory mechanisms by which Fat1 influences the progression of HNSCC remain an unresolved enigma. In this context, we reported FAT1 was down-regulated in tumor tissues/cells when compared to the normal tissues/cells and correlated with the clinicopathological features and prognosis of HNSCC. Knockdown of FAT1 enhanced the CSCs properties and decreased the apoptosis rate of tumor cells. Mechanically, FAT1 knockdown increased the phosphorylation levels of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), subsequently resulting in a diminished interaction between phosphorylated STAT1 and interferon regulatory factor 9 (IRF9), which inactivated the interferons pathways and facilitated the malignant phenotype of HNSCC cells. Overexpression of STAT1 and IRF9 alleviated the malignant behavior caused by FAT1 inhibition. In summary, our study unveils the role of FAT1 in suppressing the CSCs properties of HNSCC via the CaMKII /STAT1/IRF9 pathway and targeting FAT1 might be a promising treatment for HNSCC.

Biological sciences/Cancer/Head and neck cancer

Biological sciences/Cancer/Cancer stem cells

FAT1

CSCs

STAT1

IRF9

HNSCC

The emergence of cancer is an intricate, multifaceted process intricately linked to both environmental and genetic factors[1]. Mutations in oncogenes and tumor suppressor genes are prevalent across a spectrum of cancers[2–4]. Gain-of-function mutations in oncogenes or loss-of-function mutations in suppressor genes can precipitate abnormal cell proliferation and trigger tumorigenesis[5–8]. FAT atypical cadherin 1 (Fat1) exhibited a mutation rate of approximately 22.79% in head and neck squamous cell carcinoma (HNSCC), second only to Tp53 [9–11]. As a member of the cadherin-protein family, FAT1 is implicated in a variety of biological processes including cell proliferation, intercellular communication, and flat cell polarity [11]. Previous investigations demonstrate that FAT1 deletion strengthens a hybrid epithelial-to-mesenchymal transition (EMT) state, leading to the occurrence and metastasis of human squamous cell carcinoma[12]. Nonetheless, the connection between FAT1 and the progression of HNSCC, along with the potential underlying regulatory mechanisms, necessitates further elucidation.

Interferons (IFNs) are widely known for the role in antitumor therapy, and usually classified into three types, including type I (IFNα, IFNβ), type II (IFNγ), and type III (IFNλ)[13–15]. Among most cell types, cGAS-STING phosphorylates IRF3 after sensing pathogenic DNA, leading to the secretion of type I and type III IFNs [16, 17]. Typically, type I and type III IFNs bind to membrane surface receptors, such as IFNAR1/2 (type I IFNs) and IFNLR1(type III IFNs), induce the sequential activation of JAKs, TYKs, and STATs to form the transcriptional complex ISGF3 composed of P-STAT1/P-STAT2/IRF9 [16, 18]. IFNs exert anti-cancer function by inhibiting the vitality of tumor cells and augmenting the cytotoxic activity of immune cells[19]. Gozgit et al find the enhanced type I interferon signaling caused by PARP7 inhibition simultaneously inhibit cell proliferation and activate the immune response results in cancer regression[20]. However, the relationship between FAT1 and IFNs has not been reported yet, and whether type I interferon signaling pathway is involved in FAT1-driven tumor regression still requires clarification.

In this study, we found the down-regulation of FAT1 was closely connected with the clinicopathological features of HNSCC. Suppressing FAT1 promoted the phosphorylation of CaMKII and restrained the formation of P-STAT1/IRF9 complex, resulting in the deactivation of interferon pathway and enhancement of malignant phenotype of HNSCC. Our study revealed the role of FAT1 as a tumor suppressor and underscored its potential as a promising therapeutic target for HNSCC.

Downregulated FAT1 predicts poor prognosis of HNSCC

Based on the Cancer Genome Atlas Project (TCGA) (https://www.cancer.gov/ccg/) database, we found that Fat1 was one of the most frequently mutated genes in HNSSC, second only to Tp53, with a mutation rate of 22.79% (Fig. 1A). The analysis results acquired from the online tool SangerBox (https://www.SangerBox.com/) indicated that the expression of FAT1 was significantly decreased following mutation in HNSCC (Fig. 1B). Besides, FAT1 was negatively correlated with distant metastasis and cervical lymph node metastasis but not to the stage, grade, age, and gender. (Fig. 1G, H and Fig.S1A-D). Subsequently, we detected the expression of FAT1 in the clinical specimens of HNSCC and found that the expression of FAT1 was significantly lower in the tumor tissues than that in the normal epithelial tissues (Fig. 1C-F). Furthermore, we examined the expression of FAT1 in the normal epithelial cell line HIOEC, and the HNSCC lines HN4, SCC9, SCC25, and CAL27, the results showed that FAT1 was highly expressed in the normal epithelial cell lines when compared with the squamous carcinoma cell lines. Among the four HNSCC cell lines, FAT1 remained relatively higher level in CAL27 and SCC25 (Fig. 1I). In conclusion, FAT1 was down-regulated upon mutation in HNSCC, and its expression was negatively correlated with the poor prognosis of HNSCC, implying its tumor-suppressive role in HNSCC.

Knockdown of FAT1 enhances the CSCs properties.

As downregulated FAT1 predicted a poor prognosis of HNSCC, we wondered whether FAT1 could regulate the malignant behaviors of tumor cells. Through the results acquired from SangerBox, we found FAT1 expression was negatively correlated with the CSCs properties of HNSCC (Fig. 2A). Considering the expression of FAT1 in SCC25 and CAL27 cells was higher among the four HNSCC cells lines (Fig. 1F), we used three different siRNAs to interfere with the FAT1 expression in CAL27 and SCC25 and chose the most potent sequence for the following experiments (Fig. S2H). As shown, the expression of the CSCs-related markers, such as CD133, CD44, ALDH1A1, OCT4, NANOG and BMI1, were increased in both protein and RNA levels after FAT1 knockdown (Fig. 2B and Fig. S2A). Besides, inhibition of FAT1 enhanced the sphere formation and colony formation abilities of tumor cells (Fig. 2C, E), and the CSCs-related markers were robustly expressed in the formed spheres (Fig. 2D). Limit dilution assay is commonly used to detect tumor initiating frequency of CSCs [21]. Here, we found FAT1 knockdown markedly increased the stemness of tumor cells (Fig. 2F), and the proliferative capacity was also augmented (Fig. S2C, D). In addition, the migration and invasion abilities of cancer cells were enhanced after FAT1 knockdown (Fig. S2E, F). Tumor cells with stronger CSCs properties are resistant to apoptosis [22, 23]. The Western blot and RT-qPCR results implied that Bcl-2 was upregulated, while Bax and Caspase3 were downregulated in the FAT1 knockdown cells (Fig. 2G and Fig. S2G). Moreover, the FAT1 knockdown group had fewer dead cells, indicating FAT1 inhibition enhanced the anti-apoptosis ability of tumor cells (Fig. 2H, I). In a nutshell, these results demonstrated FAT1 knockdown enhanced the CSCs properties of HNSCC.

FAT1 regulates type Ⅰ interferon pathway independent of IFNs secretion

Previous results showed that FAT1 deletion enhanced the CSCs properties of HNSCC. To further explore the underlying mechanisms by which FAT1 regulates the CSCs phenotype, RNA-sequencing of the SiFAT1 group and the normal group were performed. The Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene ontology (GO) analysis showed that FAT1 knockdown markedly influenced the signal transduction that closed related to type Ⅰ interferon signaling pathway (Fig. 3A, B). Growing evidence demonstrates interferon signaling pathway can inhibit tumor growth in a direct manner [1–3]. Stimulation by exogenous IFNs leads to the activation of interferon pathway [24, 25]. Therefore, exogenous IFN-α was used to treat HNSCC cells in our research. Western blot, RT-qPCR and immunofluorescence results showed that the expression of CSCs-related markers decreased obviously in CAL27 and SCC25 cells (Fig. 3C, D and Fig. S3A). Besides, the self-renewal and proliferation abilities of the treated group were dampened (Fig. 3E-G). Correspondingly, Bcl-2 was downregulated, Bax and Caspase3 were upregulated in cells exposed to the IFN-α (Fig. 3H and Fig. S3B). The dead cells also increased robustly in the IFN-α treated group (Fig. 4I, J and Fig. S3C). The above results demonstrated that the activation of the interferon pathway did inhibit the CSCs properties of HNSCC. Therefore, we hypothesized that knockdown FAT1 might inactivate the type I interferon signaling pathway by blocking the secretion of type I IFNs. However, we found FAT1 inhibition had a slight effect on the secretion of IFN-α and IFN-β (Figure. 3K). And IRF3, a critical protein for type I IFNs production, was nearly unchanged (Figure. 3L), suggesting that FAT1 regulated the interferon pathway through a non-classical manner independent of IFNs secretion. All in all, the above results indicated that FAT1 regulated the interferon pathway through a noncanonical pathway independent of IFNs secretion, thereby controlling the CSCs properties of HNSCC.

FAT1 activates type Ⅰ interferon pathway through CaMKII dephosphorylation

RNA-sequencing results showed that 58 genes downregulated after FAT1 knockdown (Fig. S4A). Among the top 20 differentially expressed genes (DEGs), STAT1 and IRF9, which are closely related to the type I interferon signaling pathway, were both downregulated (Fig. 4A). Next, we selected 8 DEGs from the top 20 for Protein-Protein Interaction Networks (PPI) analysis, and found STAT1 was closely related to IRF9 (Fig. S4B). In accordance with the RNA-sequencing results, western blotting analysis showed that inhibition of FAT1 remarkably reduced the expression of IRF9 and the phosphorylation of STAT1. However, the other component of the ISGF3 complex, P-STAT2, showed no significant changes (Fig. 4B and Fig. S4C). Next, equal amounts of magnetic beads were used to conjugate the IRF9 antibody, upon which IRF9 was used to pull down P-STAT1 to ensure consistent levels of IRF9 in different groups, and we found FAT1 knockdown directly inhibited the formation of P-STAT1/IRF9 complex (Fig. 4C and Fig. S4D). Besides, observed by confocal microscopy, we found the co-localized dots of P-STAT1 and IRF9 were significantly reduced (Fig. 4D and Fig. S4E). Previous studies have shown that CaMKII is the kinase that act downstream of FAT1 [4]. Therefore, we explored whether FAT1 could regulate the activity of CaMKII. And found FAT1 knockdown did result in the phosphorylation of CaMKII. Moreover, we found that activation of CaMKII significantly inhibited the phosphorylation of STAT1, whereas had a slight effect on IRF9 expression. CaMKII inhibitor KN93 decreased the level of CaMKII phosphorylation, and promoted the level of P-STAT1 (Fig. 4E). The co-immunoprecipitation (CO-IP) and fluorescence colocalization results validated that P-CaMKII could bind to and inhibit formation of P-STAT1/IRF9 complex by dephosphorylation of STAT1 (Figure. 4F, G). Taken together, these results demonstrated that the knockdown of FAT1 blocked the formation of the P-STAT1/IRF9 complex by phosphorylating CaMKII.

Overexpression of STAT1 and IRF9 alleviates FAT1 knockdown-driven CSCs properties.

In order to explore whether STAT1 and IRF9 participated in the FAT1-mediated phenotypic transition of HNSCC we constructed five groups, including the empty carrier group, Si FAT1 group (using siRNA to interfere with the expression of FAT1), Lv-STAT1 (STAT1 overexpression) and Si FAT1 group, Lv-IRF9 (IRF9 overexpression) and Si FAT1 group, Lv-STAT1 + Lv-IRF9 (STAT1 was co-transfected with IRF9) and Si FAT1 group. The results showed that overexpression of either STAT1 or IRF9 alleviated the enhanced CSCs phenotype caused by the knockdown of FAT1 to a certain extent, whereas simultaneously overexpression of STAT1 and IRF9 maximally blocked the effect caused by FAT1 knockdown (Fig. 5A-E and Fig. S5A, B). Consistent with these, overexpression of STAT1 and IRF9 inhibited the enhanced anti-apoptotic ability caused by FAT1 knockdown. Besides, Lv-STAT1 and Si FAT1 group, Lv-IRF9 and Si FAT1 group, Lv-STAT1 + Lv-IRF9 and Si FAT1 group had lowly expressed Bcl-2 and highly expressed Bax and Caspase3 expression when compared to the Si FAT1 group (Fig. 5F-I and Fig. S5C). Taken together, we identified that STAT1/IRF9 axis participated in FAT1-mediated phenotypic transition of HNSCC, overexpression of STAT1 and IRF9 alleviates FAT1 knockdown-driven CSCs properties.

FAT1 knockdown promotes tumor growth in vivo

To investigate the impact of FAT1 on the growth of HNSCC, we constructed a subcutaneous tumor model in nude mice. Five groups of cells were prepared in vitro, including the control and Sh FAT1 (cells were transfected with the lentivirus-Si FAT1#3) group, Lv-STAT1 and Sh FAT1 group, Lv-IRF9 and Sh FAT1 group, Lv-STAT1 + Lv-IRF9 and Sh FAT1 group. In terms of tumor weight and size, the Sh FAT1 group was the highest among all groups, whereas the Lv-STAT1 + Lv-IRF9 and Sh FAT1 groups were the smallest, and there was no significant difference among the remaining three groups (Fig. 6A-C). The results of IHC suggested that the increased expression of CSCs-related proteins or decreased expression of apoptosis-related proteins caused by FAT1 knockdown could be eliminated by overexpression of STAT1/IRF9 (Fig. 6D and E). The results of Tunel assay demonstrated that overexpression of STAT1/IRF9 counteracted the enhanced anti-apoptosis ability caused by FAT1 inhibition (Fig. 6F). Collectively, inhibition of FAT1 promoted tumor growth in vivo, whereas overexpression of STAT1/IRF9 inhibited the tumor-promoting effect induced by FAT1 knockdown.

In this study, we proved FAT1 suppressed the malignant phenotype of HNSCC through activating the type I interferon signaling pathway. Specifically, we identified FAT1 could phosphorylate CaMKII and further impede the formation of P-STAT1/IRF9 complex, thereby exerting its cancer suppressive role (Fig. 7). Based on our findings, we posit that Fat1 holds significant promise as a viable target for the treatment of HNSCC.

Recently, Fat1 mutations have been identified in various cancer types, with its mutation rate in HNSCC second only to that of Tp53[9, 10, 26]. In the present study, we found that FAT1 was lowly expressed in HNSCC tissues/cells, and patients with higher FAT1 expression had better prognosis, suggesting that FAT1 might play a tumor-suppressor role in HNSCC, which was consist with previous studies [12, 27–30]. The CSCs theory proposes that tumor consist of a heterogeneous population of cells, and the hierarchy of cells within tumor is maintained by a small group of cells called CSCs, which have continuous self-renewal ability and stronger resistant to chemoradiotherapy [31–34]. Recently, Li et al reported that loss of FAT1 activated Hippo signaling pathway, which was closely related to the pluripotency of CSCs [35]. Zhai et al uncovered that FAT1 inhibited the stemness and ABCC3-related cisplatin resistance of ESCC cells via Wnt/β-catenin signaling pathway [36]. Hence, we wondered whether FAT1 exerted its cancer-suppressive activity by the modulation of CSCs properties of HNSCC. We interfered with the expression of FAT1 in CAL27 and SCC25 cells using siRNAs and demonstrated that inhibition of FAT1 endowed HNSCC cells with heightened CSCs properties and a diminished apoptosis rate.

By means of transcriptomic sequencing, we found that knockdown of FAT1 affected the type I interferon signaling pathway, which has been recognized to act as a tumor suppressor [37, 38]. Here, we noticed treatment with IFN-α resulted in decreased CSCs properties and increased apoptosis rate of tumor cells, indicating that the activation of interferon pathway did suppress the malignant phenotype of tumors. Zhang et al observed IFN-α strengthened the therapeutic efficacy of EGFR-targeted therapies by augmenting RIG-I, and the m6A demethylase ALKBH5 could negatively regulate RIG-I expression and IFN-α production through the IKKε/TBK1/IRF3 pathway in HNSCC[39, 40]. However, scant efforts have been directed towards investigating the interplay between FAT1 and interferon pathway. STAT1 and IRF9 serve as pivotal transcription factors within the type I interferon signaling pathway, and the formation of P-STAT1/IRF9 complex represents the activation of the interferon signaling pathway [41]. The transcriptomic sequencing results revealed that the expression of STAT1 and IRF9 were significantly upregulated after FAT1 knockdown, signifying the engagement of interferon pathway. However, we found that IRF3, which triggers the secretion of type I IFNs, remained largely unaffected upon FAT1 knockdown [17], and the secretion of type I IFNs was only slightly higher than that in the control group. As such, we hypothesized that FAT1 did not activate the STAT1/IRF9 pathway through the IRF3-engaged pattern. Pastushenko et al reported that FAT1 regulated the phosphorylation of CaMKII, which then directly or indirectly phosphorylated YES and SRC [12]. Here, we found FAT1 directly regulated the type I interferon pathway by inhibiting the phosphorylation of CaMKII, which subsequently phosphorylated of STAT1 and promoted the formation of the P-STAT1/IRF9 complex. Using KN93, an inhibitor of CaMKII phosphorylation, blocking the effect of FAT1 on the type I interferon pathway. Additionally, the overexpression of STAT1 and IRF9 rescued the inactivation of the type I interferon pathway following FAT1 knockdown.

In the present study, we uncovered a previously unreported mechanism through which FAT1 exerts its tumor-suppressor role by impeding type I interferon pathway. This novel insight enriched the role of FAT1 in the field of HNSCC. However, our focus was confined to the role of FAT1 on the malignant attributes of tumor cells, omitting an investigation into the underlying causes of of Fat1 mutation and its contribution to the transformation of normal cells into malignant tumor cells. In addition, the type I interferon pathway is intricately intertwined with immunity, yet our exploration did not extend to investigating the influence of FAT1 on tumor immunity.

In summary, our findings indicate that FAT1 knockdown could prevent the activation of the type I interferon pathway through noncanonical CaMKII-dependent pathway. As a result, directing therapeutic attention towards FAT1 holds significant promise as a potentially effective strategy for addressing HNSCC.

Cell lines and culture

The human HNSCC cell lines, HN4、SCC9、SCC25, and CAL27 were purchased from the Chinese Academy of Science (Shanghai, China). The human immortalized oral epithelial cell (HIOEC) was gifted from Professor Chengzhang Li, Wuhan University. Both SCC9 and SCC25 were cultured in DMEM/F12, and HN4 and CAL27 were cultivated in DMEM/high glucose. HIOEC was cultured in KGM™ Gold Keratinocyte Cell Basal Medium (Lonza, Switzerland) with associated growth factors. All cells were maintained in a medium with 10% fetal bovine serum (Gibco, USA) at humidified 37 ℃ with 5% CO2 incubators. All cells have been tested and confirmed to be free of mycoplasma and chlamydia contamination.

Specimen collection

The HNSCC tumor tissues and normal oral mucosa specimens were collected from the Hospital of Stomatology, Wuhan University. Our experiment was approved by the Ethics Committee of School and Hospital of Stomatology, Wuhan University (2022A05). Written informed consent was obtained from all patients.

Co-Immunoprecipitation (CO-IP)

CAL27 and SCC25 cells transfected with Si FAT1 or control siRNAs were lysed for 0.5 hour on the ice after transfection in the presence of KN93. Following lysate centrifugation, the supernatants of samples were mixed well with primary antibody/lgG at 4℃ overnight. The next day, the mixture was mixed with protein magnetic A/G beads (Bimake, B23202, China) at room temperature for 1 hour, heated at 95℃ for 10 minutes after adding 5×SDS-PAGE buffer, and then analyzed by western blotting.

Sphere formation assay and colony formation assay

For the sphere formation assay, tumor cells were digested with pancreatic enzymes to form a single-cell suspension. They were then cultured in ultra-low attachment plates at s density of 10,000 cells/well in serum-free DMEM/F12 accompanied with 2% B27 (Thermo Fisher Scientific, USA), human recombinant EGF (20ng/ml, PeproTech, Bubendorf, Switzerland), and human recombinant basic fibroblast growth factor (10ng/mL, PeproTech, Bubendorf, Switzerland). Took a picture to record the shape and size of spheres after 10 ~ 14 days later and then counted the number of spheres per well.

Similar to the Sphere formation assay, the isolated cells were plated in a 12-well plate with full medium, fixed with 4% paraformaldehyde, and stained with crystal violet. Lastly, took a photo and counted the number of colonies with more than 50 cells.

In vitro limiting dilution assay

Initially, a single-cell suspension was obtained. Subsequently, four doses (1000, 100, 10, and 1) of CAL27 and SCC25 cells were plated in ultra-low attachment 96-well plates. The frequency of CSCs was determined by the Extreme Limiting Dilution Analysis (ELDA) web tool (http://bioinf.wehi.edu.au/software/elda/). Specific experimental steps could be followed according to the protocol provided in Bioprotocol [21].

Mouse xenografts

Five-week-old female BALB/c nude mice were purchased from the Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China), and were housed under standard laboratory conditions. The xenograft studies were approved by the Ethical Committee on Animal Experiments of the Animal Care Committee of Wuhan University (S0792203066). These female BALB/c nude mice were randomly divided into five groups after approximately 1×10⁶ CAL27 cells transfected with lentivirus injections. Harvesting tumors upon reaching the end point of the trial.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 8 software. Comparisons between groups were analyzed using the two-tailed unpaired Student’s t-test (two groups) and one-way ANOVA tests followed by Dunnett’s multiple comparison tests (more than two groups). Results were expressed as means ± SD and considered statistically significant when p < 0.05.

Acknowledgements

We thank the assistance of the Department of Oral and Maxillofacial-Head and Neck Oncology, School & Hospital of Stomatology, Wuhan University. Thank you Tianhao Luo for accompanying me through this complicated road.

Authors’ contributions

Study concept and design: Jingjing Wang and Yang Chen; Experiments: Jingjing Wang and Yang Chen, Data analysis: Yunqing Sun, Hanzhe Liu, Ruixue Du and Xuewen Wang. Manuscript drafting: Zhe Shao. Critical revisions of the study design and the manuscript: Ke Liu and Zhengjun Shang.

All the authors went through and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (Grant number: 82273306)

Availability of data and materials

All data generated or analyzed in this study are included in this published article and its supplementary information files.

Ethics approval and consent to participate

This study was approved by the Ethics Committee of School and Hospital of Stomatology, Wuhan University (2022A05). Animal experiments were approved by the the Ethical Committee on Animal Experiments of the Animal Care Committee of Wuhan University (S0792203066).

Competing interests

All authors declare that they have no competing interests.

Author details

¹State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Wuhan University.

²Department of Oral and Maxillofacial-Head and Neck Oncology, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, China.

³Department of Oral and Maxillofacial Surgery, School & Hospital of Stomatology, Wuhan University, Wuhan 430079, China.

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There is NO conflict of interest to disclose.

Supplementary.docx
Figures8.png
Supplement Figure. 1 (A) The biometric analysis results acquired from SangerBox showed the relationship between Fat1 and tumor stage.(B) The biometric analysis results acquired from SangerBox showed the relationship between Fat1 and tumor grade. (C) The biometric analysis results acquired from SangerBox showed the relationship between Fat1 and the age of patients with HNSC. (D) The biometric analysis results acquired from SangerBox showed the relationship between Fat1 and the gender of patients with HNSCC.
Figures9.png
Supplement Figure. 2 (A) RT-qPCR results of CSC-related markers in CAL27 and SCC25 cells after FAT1 knockdown. GAPDH was used as a control. Student’s t test. (B) CCK8 assay was used to test cell viability. (C) EdU assay was used to test cell viability. Scale bars, 50 μm. (D, F) Matrigel invasion assay was used to examine the invasion ability of HNSCC cells with FAT1 knockdown. Student’s t test. Scale bar, 50 μm. (E) Wound healing assay was used to determine the migration ability of HNSCC cells with FAT1 knockdown. Student’s t test. Scale bars, 100 μm. (G) RT-qPCR results of Bcl-2, Bax and Caspase3 in CAL27 and SCC25 cells after FAT1 knockdown. GAPDH was used as a control. Student’s t test. (H) Western blot results showed the FAT1 knockdown efficiency of three siRNAs chains in CAL27 and SCC25 cells. Independent experiments (in vitro) performed in triplicate. Data are presented as mean±SD. *, P < 0.05, **, P < 0.01, ***, P< 0.001, ****, P < 0.0001.
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Supplement Figure. 3 (A) RT-qPCR results of CSC-related markers in CAL27 and SCC25 cells following exposure to the IFN-α for up to 24h at 100ng/ml concentration. GAPDH was used as a control. Student’s t test. (B) Cell immunofluorescence of Bcl-2 and Caspase3 expression in cells exposed to the IFN-α for up to 24h at 100ng/ml concentration. Scale bars, 50 μm. (C) Live/dead viability assay was conducted to examine apoptosis following exposure to the IFN-α for up to 24h at 100ng/ml concentration. Scale bars, 100 μm. Student’s t test.`Independent experiments (in vitro) performed in triplicate. Data are presented as mean±SD. *, P < 0.05, **, P < 0.01, ***, P< 0.001, ****, P < 0.0001.
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Supplement Figure. 4 (A) RNA-sequencing results suggested that 58 mRNA significantly downregulated after FAT1 knockdown (B) Protein-Protein Interaction Networks (PPI) which displayed STAT1 was closely linked to IRF9. (C) Western blot results of STAT2 and P-STAT2 after FAT1 knockdown. (D)HNSCC cells transfected with FAT1 or control siRNAs were lysed for 0.5 hours on the ice. Lysates were processed for immunoprecipitation (IP) with a control IgG (IgG) or anti-IRF9 antibody. Blots were probed to detect P-STAT1 co-precipitated with IRF9 in the IgG group. (E) HNSCC cells transfected with FAT1 or control siRNAs were lysed for 0.5 hours on the ice after being treated with KN93 at 10μM/ml concentration for 24h.Lysates were processed for immunoprecipitation (IP) with a control IgG or anti-IRF9 antibody. Blots were probed to detect P-STAT1 co-precipitated with IRF9 in the IgG group. Independent experiments (in vitro) performed in triplicate. Data are presented as mean±SD. *, P < 0.05, **, P < 0.01, ***, P< 0.001, ****, P < 0.0001.
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Supplement Figure. 5 (A) Five groups were described as Figure 5A, the expressions of CD133 and ALDH1A1 in CAL27 cells were determined by cell immunofluorescence. Scale bars, 100 μm. One-way ANOVA. (B) Colony formation assay was performed to examine the colony formation ability of CAL27 cells. Scale bars, 100 μm. One-way ANOVA. (C) The expressions of Bcl-2 and Caspase3 were determined by cell immunofluorescence in CAL27 cells. Scale bars, 100 μm. One-way ANOVA. Independent experiments (in vitro) performed in triplicate. Data are presented as mean±SD. *, P < 0.05, **, P < 0.01, ***, P< 0.001, ****, P < 0.0001.

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Fat1 suppresses the CSCs properties through CaMKII-mediated activation of the IFN pathway

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Abstract

Figures

Introduction

Results

Downregulated FAT1 predicts poor prognosis of HNSCC

FAT1 regulates type Ⅰ interferon pathway independent of IFNs secretion

FAT1 activates type Ⅰ interferon pathway through CaMKII dephosphorylation

FAT1 knockdown promotes tumor growth in vivo

Discussion

Materials and Methods

Cell lines and culture

Specimen collection

Co-Immunoprecipitation (CO-IP)

Sphere formation assay and colony formation assay

In vitro limiting dilution assay

Mouse xenografts

Statistical analysis

Declarations

References

Additional Declarations

Supplementary Files

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