| Background and ObjectiveColorectal cancer(CRC)is the third most frequently diagnosed malignant tumor in the world and the second leading cause of cancer-related deaths[1].Metabolic regulation is essential for cancer cell proliferation,differentiation and metastasis[2].The expression and regulation of metabolic enzymes profoundly affect the occurrence and development of tumors.However,there is also evidence that metabolic enzymes can directly or indirectly affect the transcription and post-translational modification of oncogenes,thereby affecting cancer progression[3].Mitochondrial serine hydroxymethyl transferase 2(SHMT2)plays a key role in the metabolism of serine-glycine to drive the proliferation of cancer cells.However,the nonmetabolic function of SHMT2 in tumorigenesis is unknown.This research explores the important role of non-metabolic function of SHMT2 in the development of colorectal cancer.It provides a potential treatment strategy for the treatment of colorectal cancer.Methods1.To detect the distribution of SHMT2 in human CRC cellsFirst,Quantitative Real-time PCR(qRT-PCR)and Western Blot(WB)were used to detect the expression of SHMT2 in multiple human CRC cell lines.CRC cell lines with high expression of SHMT2 were selected.The cytoplasm,nucleus and mitochondrial protein were extracted from the mitochondrial and nucleus separation kits;The expression and distribution of SHMT2 in cytoplasm,nucleus and mitochondria were detected by WB and immunofluorescence(IF).2.To detect SHMT2 on human colorectal cancer cell line proliferation,invasion and migration regulation2.1 To detect SHMT2 on human rectal cancer cell proliferation,invasion and migration regulation in vitroFirst,siRNA was used to interfere with SHMT2 expression in CRC cell lines,and qRTPCR and WB were used to verify the success of the interference.Cell Counting Kit-8(CCK8)method was used to detect the proliferation of human CRC cells after interference with SHMT2 in vitro.And the above experiment was performed after adding serine,glycine and sodium formate.The clone formation experiment and 5-Ethynyl-2’-deoxyuridine(EdU)incorporation experiment were used to further determine the proliferation effect of SHMT2 on CRC cells.After Annexin 5/Propidium iodide(Annexin V/Propidium Iodide,Annexin V/PI)double-stained cells,flow cytometry was used to detect whether it induced apoptosis after interference with SHMT2.Scratch test and Transwell cell test were used to detect cell migration and invasion after interference with SHMT2.Meanwhile,SHMT2 was overexpressed in CRC cell lines with low expression of SHMT2,and the above in vitro experiments were conducted again to detect SHMT2 overexpression Effects on the proliferation and metastasis of CRC cells.2.2 To detect the effect of SHMT2 on the proliferation and metastasis of CRC cells in vivo In order to investigate whether interfering SHMT2 inhibited CRC cell lines growth in vivo,we established xenograft model in nude mice with CRC cell line with shSHMT2 and SHMT2 overexpression.After 4 days,measure the long and short diameter of the tumor every 3 days,and calculate the tumor volume according to the following formula:tumor volume(mm3)=0.52 × length × width2.After 4 weeks,the mice were sacrificed and the tumors were stripped,weighed and stored in liquid nitrogen.In order to test whether SHMT2 knockdown inhibits CRC metastasis in vivo,this study established an animal model of colorectal cancer metastasis model system,intrasplenic-nude mouse model system(ISMS).CRC cells were injected into the spleen,mice were sacrificed 6 weeks after injection,the liver was dissected and collected,and the number of nodules on the surface of the liver was counted.The number of nodules on the surface of the liver was counted to determine the difference in cell transfer ability.Also need to use HE and immunohistochemistry to further verify the results.3.To explore the changes of signal pathways related to the proliferation and metastasis of human CRC cells after SHMT2 knockdownAfter HCT116 cells with SHMT2 knockdown in vitro,the mRNAs were collected for mRNA-seq.Explore the gene expression of HCT116 cells before and after interfering with SHMT2,and summarize the differential signaling pathways.According to the differential signaling pathway,the genes with significant changes were selected to verify the mRNA changes of related genes through qRT-PCR.And the target genes mRNA and protein levels of the Wnt/β-catenin pathway were detected to determine the regulatory relationship between SHMT2 and Wnt/β-catenin pathway.4.To explore the regulation of SHMT2 on key signaling factors P-cateninBased on SHMT2 causes significant changes in the Wnt/β-catenin pathway,using Coimmunoprecipitation(Co-IP)to find signal transduction factors regulated by SHMT2.Using molecular biology methods to perform truncation mutations and site mutations on SHMT2,explore the regulatory effects of SHMT2 domains or key sites on β-catenin.Overexpression of SHMT2 mutants or β-catenin in CRC cells with shSHMT2.The CCK8 method,Transwell invasion and migration test were used to detect that SHMT2 affects the proliferation and metastasis of colorectal cancer cells by regulating β-catenin.5.To explore the upstream regulation mechanism of SHMT2By analyzing the DNA sequence of SHMT2,we searched for DNA sequences that could bind to transcription factors related to the signaling pathway regulated by SHMT2.Experiments such as Chromatin Immunoprecipitation-Quantitative PCR(ChIP-qPCR)verify the predicted transcription factor binding to SHMT2.Transcription factor-related inhibitors were given,and the expression of SHMT2 was detected by RT-qPCR and WB.Results1.SHMT2 is located in cytoplasm and nucleus of human CRC cells besides mitochondriaWe determined the subcellular localization of SHMT2 in CRC cells.The cytosolic,mitochondrial and nuclear fractions of the cells were prepared,and WB analysis showed SHMT2 was localized predominantly in the cytoplasm and nucleus besides mitochondrion in CRC cells.Similar distribution pattern was observed in IF staining assay.Collectively,these data indicated that SHMT2 is located in cytoplasm and nucleus of human CRC cells besides mitochondria.This also laid the foundation for exploring the non-metabolic function of SHMT2.2.SHMT2 regulates human CRC cells proliferation and apoptosis in vitroThe effect of SHMT2 on cells proliferation was investigated by CCK-8 assay.The results showed that SHMT2 knockdown led to a significantly proliferation inhibition in HCT116 and DLD-1 cells.Remarkably,this proliferation inhibition cannot be restored by the special addition of serine,glycine,and formate,suggesting that there might have non-enzymatic function of SHMT2 that is involved in proliferation inhibition of cells.Meanwhile,EdU incorporation assay and the colony assay further confirmed that SHMT2 knockdown suppressed CRC cells proliferation.Moreover,SHMT2 knockdown in HCT116 and DLD-1 cells significantly increased the AnnexinV/PI-positive cell numbers.Furthermore,the result showed that upregulation of Bax and cleaved caspase 3,and downregulation of Bcl2 in CRC cells after SHMT2 knockdown.Conversely,exogenous expression of SHMT2 led to proliferation promotion of HT-29 cells.These results suggest overexpression of SHMT2 is necessary for supporting CRC cells proliferation.3.SHMT2 promotes the migration and invasion of CRC cells in vitroTo explore whether SHMT2 would drive the migration and invasion,we conducted Wound-healing assay,Transwell and Matrigel invasion assays in CRC cells.The results showed that SHMT2 knockdown significantly weakened the migration and invasion of HCT116 and DLD-1 cells.Given the essential role of Epithelial-Mesenchymal Transition(EMT)in tumor metastasis,we investigated the expression of Vimentin and E-cadherin,two important markers of EMT.The immunofluorescence staining results showed that SHMT2 knockdown significantly downregulated Vimentin expression and upregulated E-cadherin expression in HCT116 and DLD-1 cells.Similar results were obtained in WB.We further evaluated the expression of a panel of EMT-associated transcription factors.SHMT2 knockdown significantly decreased the protein levels of EMT-associated transcription factors such as SNAIL、SLUG、Twist、ZEB1 and ZEB2 in HCT116 and DLD-1 cells.Meanwhile,the overexpression of SHMT2 obviously promoted the migratory and invasive property in HT-29 cells.In addition,the mRNA level of EMT-associated transcription factors decreased after SHMT2 knockdown,and increased after SHMT2 overexpression.SHMT2 overexpression in HT-29 cells clearly downregulated E-cadherin and upregulated Vimentin.Whereas SHMT2 overexpression in HT-29 cells resulted in upregulated EMT-associated transcription factors.Thus,our results show that SHMT2 induces EMT in CRC cells,through regulating EMT-associated transcription factors.4.SHMT2 depletion impairs Wnt/β-catenin pathwayWe applied mRNA sequence technology to explore the factors regulated by SHMT2.The results showed SHMT2 knockdown profoundly altered the expression of almost 200 genes in HCT116 cells.By Gene Ontology(GO)Consortium,SHMT2 knockdown clearly suppressed multiple signal pathways,including TGF-β pathway,alanine and glutamic acid metabolism,β-alanine metabolism.Furthermore,by using these significantly modified genes,the cell signaling networks analysis displayed Wnt/β-catenin signaling was critical in these pathways and closely connected with TGF-β and Notch pathways.To further investigate the effect of SHMT2 on Wnt/β-catenin signaling,we measured the mRNA level of β-catenin target genes which are important in CRC progression.Importantly,SHMT2 knockdown significantly decreased the level of these β-catenin targets such as CD44,MMP2,MMP9.WB further confirmed the results.Conversely,SHMT2 overexpression obviously enhanced the mRNA and protein levels of these Wnt/β-catenin targets.SHMT2 knockdown did not alter β-catenin mRNA level.Interestingly,we found protein level of β-catenin was significantly decreased after SHMT2 knockdown,and increased after SHMT2 overexpression.We further investigated the protein expression of β-catenin in nucleus and cytoplasm,respectively.SHMT2 knockdown remarkably suppressed protein level of βcatenin in both nucleus and cytoplasm.The gray-scale analysis was used to quantify and compare the changes of SHMT2 and β-catenin in cytoplasm and nucleus caused by SHMT2 knockdown.The results showed that the decrease of SHMT2 was more obvious in cytoplasm,while β-catenin decreased mainly occurred in nucleus.5.SHMT2 maintains β-catenin stability by inhibiting its ubiquitylationSince SHMT2 knockdown decreased protein level of β-catenin without mRNA level change.As ubiquitylation is a key step of β-catenin degradation which results in decrease ofβ-catenin protein level,we first examined the β-catenin ubiquitination level in CRC cells after SHMT2 deletion.We found that significant co-immunoprecipitation of β-catenin with ubiquitin after SHMT2 knockdown,suggesting that SHMT2 knockdown enhanced the ubiquitination of β-catenin in CRC cells.We detected the phosphorylation level of β-catenin markedly increased with SHMT2 knockdown in HCT116 and DLD-1 cells.To further detect the β-TrCP recognition for β-catenin after SHMT2 knockdown,Co-IP assay showed that the binding of β-TrCP with β-catenin in both HCT116 and DLD-1 cells.We interfered BRCC36 expression with specific siRNA in both HCT116 and DLD-1 cells.We found that the protein expression of β-catenin was reduced after BRCC36 knockdown.The above results confirm that SHMT2 inhibits β-catenin ubiquitination degradation through SHMT2BRISC deubiquitination regulatory complex.6.Lys 64 of SHMT2 is required for β-catenin interactionWe detected whether SHMT2 is co-localized with β-catenin in CRC cells by IF staining.The results indicated that SHMT2 partially co-localized with β-catenin both in HCT116 and DLD-1 cells.Then,we performed Co-IP experiments to confirm the interaction between endogenous SHMT2 and β-catenin.Based on the truncation mutation and point mutation of SHMT2,it is further proved that the K64 of SHMT2 mediates the interaction between SHMT2 and β-catenin.7.SHMT2 depends on β-catenin to regulate the proliferation and metastasis of colorectal cancer cells in vitroIn order to test whether the interaction between SHMT2 and β-catenin plays an important role in cell proliferation and migration regulated by SHMT2 in vitro,this study detected the re-expression of SHMT2WT restored the expression of β-catenin in CRC cells with SHMT knockdown,whereas re-expression of SHMT2 K64R did not.Meanwhile,the proliferation,migration and invasion of CRC cells re-expressed with SHMTWT or SHMT2K64R after SHMT2 knockdown.The re-expression of SHMT2WT restored the proliferation migration and invasion inhibition of CRC cells induced by SHMT knockdown,whereas re-expression of SHMT2 K64R did not.Moreover,we expressed β-catenin in CRC cells after SHMT2 knockdown and detected the cells proliferation.The results showed that overexpression of β-catenin significantly rescued CRC cells proliferation migration and invasion inhibition induced by SHMT2 knockdown.Taken together,these results suggest that SHMT2 regulates CRC cells proliferation,migration and invasion by modulating βcatenin.8.SHMT2 is reversely regulated by β-catenin in CRC cellsIn order to detect the upstream regulator of SHMT2,this study analyzed the DNA sequence of SHMT2 through PROMO software.The data showed two putative binding sites of TCF4 located on the exon of SHMT2 gene.We performed ChIP with a specific TCF4 antibody and RT-qPCR assay in CRC cells.Remarkably,TCF4 binding at the open reading frame(ORF)of SHMT2 gene in HCT116 and DLD-1 cells,respectively.β-catenin binds to TCF4 to regulate its target gene expression,this study used Wnt/β-catenin inhibitor iCRT 14 and XAV939 to inhibit Wnt/β-catenin signaling pathway.The mRNA and protein level of SHMT2 were decreased in HCT116 and DLD-1 cells.Taken together,these results demonstrate that SHMT2 is a target gene of Wnt/β-catenin pathway.9.SHMT2 promotes CRC cells growth and metastasis in vivoTo confirm the biological function of SHMT2 on tumor growth in vivo,this study constructed HCT 116 and DLD-1 cells with shSHMT2 and stable overexpression of SHMT2 in HT-29 cells.The above-mentioned cells were injected subcutaneously into nude mice to construct a nude mouse xenograft model,and the tumor volume and body weight of mice were detected every three days.SHMT2 knockdown significantly prevented tumor growth in HCT-116 and DLD-1 cell mice models.On the contrary,SHMT2 overexpression in HT29 cells obviously increased tumor volume compared with that in control.In addition,this study used the intrasplenic-nude mouse model system(ISMS)model with the above cells.We found that SHMT2 knockdown inhibited the formation of metastatic nodules in liver In contrast,more liver metastatic nodules were observed in mice with HT29-SHMT2 cells compared with control mice.Collectively,these data demonstrate that SHMT2 promotes growth and metastasis of CRC in vivo.10.SHMT2 and β-catenin are associated with CRC T stage,lymph node metastasis and poor prognosisIn this study,WB was used to detect the expression of SHMT2 in human CRC tissues.The results showed that SHMT2 was highly expressed in human CRC tissues.SHMT2 andβ-catenin expression were validated by using CRC tissue array which contained 85 human CRC tissues and corresponding normal adjacent tissues(NATs)by immunohistochemical(IHC)staining.We found that the expression of SHMT2 and β-catenin in CRC tissues were predominantly higher than that in NATs.The expression of SHMT2 in CRC tissues was associated with advanced T stage and lymph node metastasis(P=0.007).We then analyzed the potential correlation of SHMT2 and β-catenin based on IHC data.Kaplan-Meier survival analysis showed the expression of SHMT2 in CRC tissues were negatively correlated with survival rate of patients.The results showed that protein expression of SHMT2 is closely associated with that of β-catenin.Statistics found that among patients with higher SHMT2 expression,β-catenin expressed more and stronger in the nucleus.Furthermore,we examined the correlation of SHMT2 and β-catenin expression with the cancer progression and patient’ s survival.Notably,the patients with high SHMT2 and high β-catenin expression exhibited significantly poor survival rate of patients compared with low SHMT2 and low β-cateninConclusionSHMT2 plays a key role in the metabolism of serine-glycine to drive the proliferation of cancer cells.This study proves that SHMT2 is not only distributed in mitochondria,but also in the cytoplasm and nucleus.SHMT2 knockdown results in colorectal cancer cell proliferation inhibition,and serine,glycine,or formate supplements cannot restore the proliferation inhibition.After SHMT2 knocked down,the migration and invasion of colorectal cancer cells were inhibited.The molecular mechanism study found that SHMT2 interacts with β-catenin in the cytoplasm.This interaction inhibits the ubiquitination degradation of β-catenin and regulates the expression of its target genes,which leads to the proliferation and metastasis of colorectal cancer cells.It is worth noting that the 64-position lysine residue of SHMT2 mediates the interaction with β-catenin.In addition,β-actenin affects the transcription of SHMT2 by regulating the transcription factor TCF4;thereby increasing the expression of SHMT2 and forming a positive feedback loop of SHMT2/βcatenin.In addition,the experiment confirmed that SHMT2 promotes the proliferation and metastasis of colorectal cancer cells in vivo.Finally,the level of SHMT2 in human colorectal cancer tissues increased significantly,and was associated with increased levels of β-catenin,related to the progress of human colorectal cancer,and related to the poor prognosis of patients.All in all,our findings reveal the new non-metabolic enzyme function of SHMT2 in colorectal cancer,stabilize β-catenin,antagonize ubiquitination degradation and provide a potential therapeutic strategy for colorectal cancer treatment. |
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