FASN Inhibits Anoikis In Osteosarcoma Cells By Regulating The "NDRG1/autophagy" Axis

Background and Purpose:Osteosarcoma,the most common malignant bone tumor,predominantly affects teenagers and middle-aged individuals around 50 years of age.In the early stages,osteosarcoma often presents with no apparent symptoms,resulting in approximately30% of patients having pulmonary micrometastases at the time of diagnosis.Subsequently,about 50% of patients will develop metastatic bone tumors,and the prognosis for those with distant metastases remains poor.Despite existing treatment options,high recurrence rates and drug resistance pose significant challenges in managing osteosarcoma,leading to distant metastasis in some cases.Therefore,elucidating the key drivers of osteosarcoma metastasis and understanding the specific underlying mechanisms is crucial to improving patient outcomes.In our previous study,we observed elevated expression of fatty acid synthase(FASN)in osteosarcoma and demonstrated that knocking down FASN in vitro induced apoptosis in osteosarcoma cell lines,specifically triggering anoikis,a critical step in the metastatic process.FASN was found to be upregulated during osteosarcoma anoikis,concomitant with increased anoikis resistance(AR)of osteosarcoma cells.However,the precise mechanism by which FASN mediates AR in osteosarcoma cells remains unclear.Further experimental findings suggest that FASN may mediate AR in osteosarcoma cells by maintaining heightened autophagy levels.Thus,the aim of this study is to provide further insights into the potential regulatory mechanisms by which FASN mediates AR in osteosarcoma cells and promotes distant metastasis,offering new perspectives for comprehending the intricate molecular regulatory processes involved in osteosarcoma progression and identifying novel therapeutic targets for future development,based on sound theoretical foundations.Methods:1.Investigation of FASN’s role in promoting anoikis resistance in osteosarcoma cells through autophagy induction: AR-OS cell lines were constructed and FASN was knocked down using lentiviral vector(Lv-sh RNA).Stable cell lines were generated,and Western Blot and LC3 double-label fluorescence experiments were performed to detect FASN and autophagy levels in these cells and their parental OS cell lines following standard protocols.Small molecule compounds chloroquine and rapamycin were used to modulate autophagy in AR-OS cells with FASN knockdown or osteoblasts overexpressing FASN,and autophagy levels were evaluated using Western Blot and flow cytometry for apoptosis.The potential mediation of autophagy in FASN-induced changes in AR ability was verified.2.FASN induces autophagy and AR in osteosarcoma cells by inhibiting the expression of NDRG1: i TRAQ proteomics was employed to identify downstream gene(s)affected by FASN after changing its expression.Western Blot,real-time quantitative PCR,and dual luciferase assays were conducted to investigate the mechanism of NDRG1 changes in response to FASN alteration.NDRG1 was replenished in AR-OS cell lines with stable FASN knockdown,and Western Blot,LC3 double-label fluorescence experiments,and flow cytometry were performed to assess autophagy and apoptosis changes,thus confirming the role of FASN in regulating autophagy and AR in OS cells through NDRG1.3.Study of the molecular biological mechanism underlying FASN-mediated transcriptional inhibition of NDRG1: Based on the correlation between autophagy and anoikis in the transcription factor database,transcription factors of NDRG1 were screened and further investigated using Western Blot,co-immunoprecipitation,realtime quantitative PCR,and dual luciferase assays to understand the alteration pattern of these transcription factors and their regulatory mechanism upon changing FASN.The function of the regulatory protein SYVN1 was verified through flow cytometry,tumor sphere formation experiments,and a lung metastasis experiment in an orthotopic tumor formation model in nude mice.Results:1.Knockdown of FASN led to an increase in autophagy,as indicated by the LC3double-labeled fluorescence experiment,and a decrease in autophagy when compared to the negative control group in AR-OS cells.Flow cytometry detection of AR-OS cells treated with the autophagy inhibitor chloroquine showed a significant increase in the proportion of apoptotic cells(p<0.05),while addition of the autophagy agonist rapamycin to stable AR-OS cells with knocked down FASN resulted in a decrease in apoptotic cells(p<0.01).Western Blot analysis revealed that chloroquine treatment increased the level of apoptotic marker protein in AR-OS,whereas the level of apoptotic marker protein decreased when rapamycin was added to stable AR-OS cells with knockdown of FASN.These findings suggest that overexpression of FASN in osteoblasts promotes autophagy in a manner similar to rapamycin,and this effect is reversed by chloroquine treatment.2.Proteomics analysis of AR-143 B cells with knocked down FASN revealed 173 differentially expressed proteins compared to the control group,including 11autophagy-related proteins.Among these,NDRG1,an autophagy inhibitory protein,showed a significant increase in protein expression with the smallest degree of value dispersion.Further testing demonstrated that knockdown of FASN in AR-OS cells led to increased NDRG1 promoter activity(p<0.01),transcript levels(p<0.001),and protein expression.Subsequent knockdown of NDRG1 in AR-OS cells with stable knockdown of FASN reversed the levels of autophagy and apoptosis.3.To investigate the potential intermediary transcription factors through which FASN affects NDRG1 expression,a transcription factor library TF-BIND and UCSC annotated anoikis-related genes and autophagy-related molecule library HADb were screened,and HIF-1α was found to be the intersecting factor.Knockdown of FASN did not significantly change the transcript level of HIF-1α(p=0.81),but resulted in a significant decrease in protein level.Overexpression of HIF1 A after knockdown of FASN inhibited the increase of NDRG1 protein level.Degradation and ubiquitination experiments revealed that the ubiquitination level of HIF-1α increased upon knockdown of FASN.Further online predictive analysis identified 43 intermediate E3 ligases that may be involved in the ubiquitination modification of HIF-1α by FASN.Co-immunoprecipitation experiments showed that only SYVN1 and FASN could enrich each other,and ubiquitination experiments revealed that overexpression of SYVN1 increased the level of ubiquitination modification of HIF-1α.Moreover,as the level of FASN increased,the ubiquitination level of HIF-1α decreased and the level of protein binding to SYVN1 increased,suggesting a possible competition between FASN and HIF-1α for binding to SYVN1,resulting in mutual stabilization through reduction of ubiquitination level.Further verification demonstrated that overexpression of SYVN1 promoted AR-143 B cell anoikis and inhibited lung metastasis in an osteosarcoma orthotopic model.Conclusion1.FASN promotes anoikis resistance in osteosarcoma cells through mediating autophagy;2.FASN promotes autophagy and anoikis resistance in osteosarcoma cells by inhibiting NDRG1 transcription3.FASN inhibits NDRG1 transcription by competitively binding SYVN1 and reducing HIF-1α ubiquitination,and SYVN1 inhibits anoikis resistance and distant metastasis in osteosarcoma.