Study On The Role And Mechanism Of SHP2 And Obese Tumor Microenvironment Derived OLCarn In Breast Cancer

Breast cancer,the primary threat to women’s health globally,garners scientific and clinical attention due to its high incidence and metastatic potential.Despite the continuous progress in treatment methods,TNBC remains a challenging issue due to the absence of clear therapeutic targets.Meanwhile,as lifestyles change,the rate of obesity continues to increase annually,and its correlation with the development of breast cancer is receiving more attention.This study aims to investigate the pathogenesis and metastasis mechanisms of breast cancer from the perspective of cell death and obesity-induced metabolic remodeling.Pyroptosis is a type of programmed cell death that differs from ferroptosis and apoptosis in terms of cell morphology,biological changes,and molecular mechanisms.It is primarily characterized by the release of inflammatory mediators,cellular contents,and changes in intracellular and extracellular osmotic pressure.Inducing pyroptosis in tumor cells may be an effective strategy for cancer treatment since it can inhibit tumor progression.Although studies have shown that non-receptor protein tyrosine phosphatase SHP2 can regulate the growth,differentiation,and apoptosis of tumor and immune cells,it is still unclear whether SHP2 can participate in tumor cell pyroptosis and the related mechanism.To clarify the role of SHP2 in MDA-MB-231 cells,we assessed cell viability and proliferation using CCK8 and Ki-67 immunofluorescence assays,respectively,after SHP2 knockdown.The results showed that the reduction of SHP2 significantly inhibited cell proliferation.Subsequent Transwell assays also demonstrated that SHP2knockdown significantly attenuated cell migration and invasion.To determine whether SHP2 mediates TNBC pyroptosis,we observed the cell morphology by transmission electron microscopy.The findings indicate that SHP2 knockdown resulted in cell membrane damage and the appearance of vacuolar structures in HCC1806 and MDA-MB-231 cells,consistent with the characteristics of pyroptosis.Furthermore,the knockdown of SHP2 resulted in an increase in the proportion of PI~+cells.The levels of LDH,IL-1β,and IL-18 in cell supernatants and the activation of GSDMD and Caspase-1 were also upregulated,with SHP2 knockdown.Additionally,the upregulation of LDH,IL-1β,and IL-18 contents due to SHP2 knockdown was reversed upon the addition of NSA,a GSDMD inhibitor.The results indicate that SHP2 regulates TNBC pyroptosis via GSDMD.To uncover the potential mechanism of SHP2’s regulation of cellular pyroptosis,we utilized online databases to find that JNK is correlated with SHP2.Molecular docking also revealed a strong affinity between SHP2 and JNK.Subsequent Co-IP and immunofluorescence assays confirmed that SHP2 and JNK directly bind and co-localize cytoplasm.The phosphorylation level of JNK and NF-κB,was upregulated with the knockdown of SHP2.To confirm that SHP2 regulates pyroptosis via JNK,we used the JNK inhibitor SP600125 after knocking down SHP2.The results showed that SP600125 abolished the increase in LDH,IL-1β,and IL-18 levels in the cell supernatant caused by SHP2knockdown.It also reversed the proportion of PI~+cells,as well as the activation of JNK and NF-κB,and significantly inhibited the cleavege of GSDMD and Caspase-1.It was found that knockdown of SHP2 reduced the size and weight of tumor tissues and affected JNK,NF-κB phosphorylation,and pyroptosis-related biomarkers in TNBC xenografts.In summary,inhibiting SHP2 hinders TNBC progression by activating the JNK/NF-κB signaling pathway,which regulates pyroptosis.The relationship between diet-induced obesity and breast cancer development is well-established.However,the exact mechanism by which obesity contributes to breast cancer progression remains unclear.In recent decades,the obesity rate among the Chinese population has been rapidly increasing.It is projected that by 2035,18%of Chinese adults will be obese.Therefore,it is imperative to conduct an in-depth exploration of the pathological mechanisms linking obesity to the onset and progression of breast cancer.Previous studies have shown that obesity-induced changes in adipokines,chronic inflammation,oxidative stress,and remodeling of the tumor microenvironment can promote breast cancer invasion and metastasis.However,these studies have focused on changes in biomacromolecules,and few studies have confirmed whether obesity-related endogenous small molecule metabolites remodeling are involved in breast cancer progression.Based on the above background,we investigated whether serum from obese mice can promote EMT and metastasis of breast cancer cells.Through metabolomics,we confirmed that the small molecule metabolite OLCarn in serum is a key factor in promoting EMT of breast cancer.We prepared xenograft tumor and lung metastasis models of breast cancer based on a diet-induced obese mouse model to monitor tumor growth and metastasis.In mice with obesity,breast cancer growth and metastasis were accelerated,and lung metastasis was more pronounced.Immunofluorescence and Western blotting analyses revealed significant changes in the EMT markers in obese breast cancer tissues,indicating a higher degree of EMT and a stronger metastatic potential.To investigate the link between obesity and EMT in breast cancer,E0771cells were incubated with serum from obese mice for 72 hours.The results showed that the cells underwent EMT,cytoskeleton remodeling,enhanced migration and invasion,and increased lung metastasis.Subsequently,metabolomics was used to screen for differential metabolites,and it was discovered that only OLCarn was able to dose-dependently induce the EMT program in breast cancer cells and promote metastasis.Secondly,RNA-seq was used to identify the key factor KIFC1 that OLCarn regulates in inducing EMT in breast cancer cells.To investigate the molecular mechanism of OLCarn in regulating EMT in breast cancer cells,RNA-seq was conducted on HCC1806 cells treated with OLCarn.The KEGG and GO enrichment analyses revealed that differentially expressed genes were primarily enriched in signaling pathways such as cell adhesion and tight junctions,which are closely associated with EMT.Among the upregulated genes,KIFC1 can enhance the survival and cell polarity of tumor cells by stabilizing multiple centrosomes,inducing cytoskeletal remodeling,and promoting EMT and metastasis.Therefore,it is speculated that OLCarn can drive EMT in breast cancer through KIFC1.OLCarn upregulates the protein and m RNA levels of KIFC1 in breast cancer cells in a dose-dependent manner.Obese serum also promotes the expression of KIFC1 in E0771 cells.After knocking down or pharmacologically blocking KIFC1,the effects of OLCarn or obese serum on promoting KIFC1 expression,driving EMT programs,inducing cytoskeleton remodeling,and enhancing cell migration and invasion ability are reversed.Knocking down KIFC1 also eliminates the role of OLCarn in promoting lung metastasis.These results indicate that OLCarn can drive EMT of breast cancer through KIFC1.Finally,it was found that OLCarn directly binds with ADCY10 to activate the c AMP signaling pathway,promoting KIFC1 transcription through TCF-4,leading to EMT in breast cancer.To identify proteins that may bind to OLCarn,a target fishing technique based on the biotin-streptavidin system was utilized.Combined with RNA-seq results,it was ultimately determined that ADCY10 is the direct target of OLCarn.ADCY10 primarily mediates c AMP generation and is involved in various biological processes,including cell proliferation,differentiation,transcription,and translation.Additionally,the expression level of ADCY10 is not associated with overall survival in breast cancer patients,but high expression of ADCY10 is linked to shorter survival in patients with metastasis.Therefore,it is hypothesized that OLCarn binds directly to ADCY10,activating c AMP-dependent TCF-4 signaling and promoting the expression of KIFC1.Pulldown and immunofluorescence experiments demonstrate OLCarn’s ability to bind to ADCY10.CETSA and DARTS also confirm OLCarn’s improved stability through binding to ADCY10.Molecular docking analysis confirms Arg176 as the binding site for OLCarn on ADCY10.The mutated Arg176 ADCY10 protein is unable to bind to OLCarn.Co-incubation with OLCarn does not enhance heating stability or anti-enzymolysis ability.However,OLCarn does not affect the content of ADCY10 protein.Therefore,we speculate that OLCarn directly activates c AMP signaling by binding to ADCY10.The levels of c AMP in tumor cells incubated with OLCarn increased in a dose-dependent manner.Additionally,obese serum also increased the c AMP content in E0771 cells.These findings suggest that OLCarn activates ADCY10-dependent c AMP signaling.When ADCY10 was knocked down or pharmacologically blocked,the effects of OLCarn or obese serum on promoting an increase in c AMP content,KIFC1-specific transcription factor TCF-4 and KIFC1expression,driving the EMT program,inducing cytoskeleton remodeling,enhancing cell migration,and invasion ability are all reversed.Knocking down ADCY10 also eliminated the role of OLCarn in promoting lung metastasis.The results indicate that OLCarn binds directly to the Arg176 site of ADCY10,promoting EMT and metastasis of breast cancer through the c AMP/TCF-4/KIFC1 signaling pathway.In conclusion,the serum metabolic profile remodeling in obese mice leads to the abnormal accumulation of OLCarn in the tumor microenvironment,regulating EMT and metastasis of breast cancer cells by targeting the ADCY10/TCF-4/KIFC1signal cascade.Based on the aforementioned results,it was determined that SHP2 regulates cell pyroptosis through the JNK/NF-κB signaling pathway,providing a potential new method for targeted TNBC treatment.The study also revealed the significant role of obese tumor microenvironment derived OLCarn in driving breast cancer metastasis and its related signaling pathways,which provides a theoretical basis for the diagnosis and treatment of breast cancer.