The Molecular Mechanisms Of GPCPD1-Regulated Mitophagy In Triple-Negative Breast Cancer

BackgroundGlobal cancer data showed that as of 2021,breast cancer has been the most common type of malignant tumor in women.And in recent years,the risk and mortality of breast cancer in women have gradually increased,which seriously reduced women’s quality of life and threatened women’s life and health.Breast cancer is a disease with a high degree of morphological and genetic heterogeneity,among them,triple negative breast cancer(TNBC)is a special molecular subtype of breast cancer,which is mainly manifested as negative in estrogen receptor(ER),progesterone receptor(PR),human epidermal growth factor receptor-2(HER-2).Studies have found that the clinical characteristics in TNBC are with young age of onset,high degree of malignancy,and early recurrence and metastasis.Because TNBC patients are lack of the expression of the above receptors,they cannot benefit from endocrine therapy and anti-HER2 targeted therapy,which increases the difficulty of subsequent treatment.At present,chemotherapy is the main adjuvant treatment for TNBC patients,but a considerable number of patients will develop chemotherapy resistance,which is more likely to occur distant metastasis and poor prognosis.Chemotherapy resistance and progressive metastasis of tumors is a complex biological process involving multiple stages,and more and more studies have shown that the biological behavior of tumors not only depends on the tumor cells themselves,but also plays an important role in tumor cell chemotherapy resistance and metastasis.Solid tumor of TNBC could cause the formation of the hypoxic environment due to the rapid growth rate.Under hypoxic conditions,tumor cells activate a series of downstream related signaling molecules through hypoxia-inducible factors(HIFs),coordinate and control the cellular response,adapt tumor cells to the hypoxic environment,and increase their own aggressiveness and treatment tolerance.The latest research showed that hypoxia can induce the occurrence of mitophagy,and cells selectively remove excess or damaged mitochondria through autophagy mechanisms to maintain the stability of mitochondrial quantity and quality.Mitophagy is currently a hot spot in the field of biomedical research.At present,the study of mitophagy induced by the tumor hypoxic microenvironment is still in its infancy,and its role in breast cancer,especially in TNBC,is still unknown.In order to study the effect and mechanism of hypoxia-induced mitophagy in TNBC progression and chemotherapy resistance,we used the high-throughput sequencing technology and mt-Keima evaluation system and select the GPCPD1 as the important regulator in mitophagy.We then elucidate its effects on progression and chemotherapy resistance in TNBC,and analyze its clinical application value by using tissue samples.Part Ⅰ Screening and expression analysis of GPCPD1Objective1.To screen genes related to hypoxia-associated mitophagy regulated by HIF1A in TNBC;2.To detect the GPCPD1 expression levels in breast cancer cell lines,and GPCPD1 expression levels at different time points induced by hypoxia and CCCP in TNBC cells.MethodsThe RNA-seq,ChIP-seq and mt-Keima evaluation system were used to screen GPCPD1,which is closely related to hypoxia-induced mitophagy in TNBC.qRT-PCR was used to detect the expression of GPCPD1 in breast cancer cell lines.And then qRT-PCR and western blot were used to detect the expression level of hypoxia or CCCP-induced GPCPD1 in TNBC cells.ResultsWe found that GPCPD1 can regulate mitophagy in hypoxia-induced TNBC,and the expression of GPCPD1 in TNBC cell lines is significantly higher than that in other cell lines.In addition,hypoxia and CCCP can promote GPCPD1 expression in TNBC cell lines.Conclusions1.GPCPD1 is the target gene which can regulate mitophagy in hypoxia-induced TNBC;2.The expression level of GPCPD1 was significantly higher in TNBC cell lines than that in other breast cancer cell lines;3.CCCP and hypoxia can upregulate the expression of GPCPDlin TNBC.Part Ⅱ GPCPD1 promotes the function of progression and metastasis of triple-negative breast cancer through mitophagyObjective1.To explore the effects of knockdown or overexpression of GPCPD1 on TNBC cell proliferation,migration and invasion,and chemotherapy resistance;2.To explore the regulation of mitophagy in TNBC by knockdown or overexpression of GPCPD1;3.To explore the ability to promote the proliferation,migration,and invasion of TNBC cells through mitophagy with knockdown or overexpression of GPCPD1;4.To explore the effects of knockout or overexpression of GPCPD1 on TNBC growth,metastasis,and chemotherapy resistance in vivo.MethodsFunctional experiments in vitro such as MTT and EdU were used to verify the effect of GPCPD1 on the proliferation capacity of TNBC cells.Transwell experiments were used to verify the effect of GPCPD1 on TNBC cell migration and invasion.The effect of GPCPD1 on mitophagy in TNBC was verified by RNA-Seq,ROS,JC-1,OCR,mPTP,ATP,mt-Keima,immunofluorescence and electron microscopy.Furthermore,the ability of GPCPD1 to regulate TNBC proliferation and progression metastasis through mitophagy was verified by using mitophagy promoters or inhibitors.Finally,we constructed a subcutaneous tumor xenograft model of TNBC and a nude mouse model of lung metastasis with TNBC cells,and the then the progression and chemotherapy resistance with GPCPD1 knockdown or overexpression were explored in vivo.ResultsIn vitro functional experiments,we found that with GPCPD1 knockdown can significantly supress the proliferation,migration,invasion and chemotherapy resistance in TNBC cells under normoxia or hypoxia conditions.In addition,overexpression of GPCPD1 can promote the phenotypes mentioned above in TNBC cells.The subcutaneous tumorigenic model and tail vein injection lung metastasis model of nude mice showed that overexpression of GPCPD1 could promote the progressive metastasis and chemotherapy resistance of TNBC cells in vivo.At the same time,we found that under hypoxic conditions,knockdown GPCPD1 can inhibit biological function in TNBC,which could be rescued by using mitophagy promoter;In addition,overexpression of GPCPD1 can promote biological function in TNBC,which could be diminished by using mitophagy inhibitor,suggesting that GPCPD1 could regulate the proliferation and progression of TNBC through mitophagy.Conclusions1.GPCPD1 promotes the proliferation,migration,invasion,and chemotherapy resistance of TNBC cells;2.TNBC cells with high expression of GPCPD1 have the stronger tumorigenic ability,lung metastasis ability and resistance to chemotherapy drugs;3.GPCPD1 promotes mitophagy in TNBC cells under hypoxic conditions;4.GPCPD1 promotes the proliferation,migration invasion and chemotherapy resistance of TNBC cells through mitophagy under hypoxic conditions.Part Ⅲ The molecular mechanism of GPCPD1 promoting the progression and metastasis through mitophagyObjective1.To explore the effect of HIF1A on the transcriptional regulation of GPCPD1,and the modification effect of methylation and acetylation on this process;2.To explore the mechanism of GPCPD1 nucleoplasmic shuttle and localization to mitochondria;3.To explore the GPCPD1’s mitochondrial downstream target VDAC 1;4.To explore the interaction and molecular mechanism between GPCPD1 and VDAC1;5.To elucidate the molecular mechanism by which GPCPD1 affects mitophagy in TNBC cells by regulating VADC1-PRKN.MethodsThe luciferase experiment and ChIP experiment were used to explore the regulatory effect of HIF1A on GPCPD1.The western blot experiment was used to detect the effect of GPCPD1 on the downstream target of mitophagy.Then bioinformatics prediction and Co-IP experiment were used to verify the interaction between GPCPD1 and LYPLA1,and molecular mechanism of LYPL A1 to regulate GPCPD1’s mitochondrial localization through depalmitoylation was explored.Furthermore,mass spectrometry and co-IP assay were used to verified the interaction between VDAC1 and GPCPD1.Then domain truncation experiments and Co-IP experiments were used to detect the effect of GPCPD1 on the formation of VDAC1 monomers and polymers.And immunofluorescence and western blot experiments were used to verify the VDAC1 monomers effect on PRKN recruitment and distribution.Finally,we explored whether GPCPD1 regulated mitophagy in TNBC through VDAC1-PRKN by MTT,clonal formation and Transwell experiments.ResultsExperiments have found that HIF1A can positively regulate the transcription of GPCPD1,while methylation and acetylation can also positively modify this process.At the same time,it was found that GPCPD1 was more localized to the cytoplasm under hypoxic conditions,and could bind to LYPLA1,which regulated the localization of GPCPD1 to mitochondria through depalmitoylation.At the same time,we found VDAC1 was the target protein of GPCPD1 in mitochondria,and and VDAC1 could interact with GPCPD1 and mainly bound to the CBM20 domain of GPCPD1.GPCPD1 promotes the formation of VDAC1 monomers,recruiting more PRKN proteins to distribute in mitochondria,and promotes mitopahgy.Later,it was found that GPCPD1 regulated the progression of TNBC through VDAC1-PRKN.Conclusions1.HIF1A can positively regulate GPCPD1,which is modified by methylation and acetylation;2.LYPLA1 can bind to GPCPD1 and regulate GPCPD1 localization to mitochondria through depalmitoylation;3.VDAC1 is the downstream direct target of GPCPD1;4.GPCPD1 can promote the formation of VDAC1 monomer,recruit more PRKN to target to mitochondria,and promote the occurrence of mitophagy;5.GPCPD1 promotes the progression of TNBC cells through VDAC1-PRKN.Part Ⅳ Clinical significance analysis of GPCPD1 in TNBCObjective1.To explore the relationship between HIF1A and GPCPD1 at the tissue level;2.To analyze the relationship between GPCPD1 expression and clinicopathological features such as ER,PR and HER2 receptor status,lymph node metastasis,distant metastasis,and patient prognosis.MethodsThe expression of GPCPD1 or/and HIF1A in TNBC tissues and normal tissues was detected by qRT-PCR and immunohistochemistry experiments.Finally,statistical methods were used to analyze the correlation between the expression levels of GPCPD1 and HIF1A and the clinicopathological characteristics and prognosis of TNBC patients.ResultsIt was found that GPCPD1 was highly expressed in breast cancer tissues,and GPCPD1 and HIF1A were positively correlated at the histological level.Further clinicopathological features and prognosis analysis showed that GPCPD1 was closely related to histological grade and distant metastasis in TNBC patients,and the high expression of GPCPD1 and HIF1A suggested a poor prognosis in TNBC patients.Conclusions1.The expression level of GPCPD1 in TNBC tissues was significantly higher than that in normal breast tissues.2.The expression levels of HIF1A and GPCPD1 were positively correlated.And high expression of GPCPD1 was closely related to the clinicopathological characteristics and prognosis of patients with triple-negative breast cancer.