NCAPD2 Promotes Colorectal Cancer Growth By Inhibiting Autophagy Through The Ca²⁺/CaMKK2/AMPK/mTOR Pathway And PARP-1/SIRT1 Axis

The incidence rate and mortality rate of colorectal cancer ranks third and second in global cancer respectively.The pathogenesis is very complex,and the cause of the disease is not yet fully clear.Therefore,more in-depth study on the molecular mechanism of colorectal cancer development is needed to provide the basis for further improving the molecular etiology of colorectal cancer and also provide theoretical guidance for clinical treatment of colorectal cancer.NCAPD2 is a non-SMC subunit of condensin complex I.Previous studies have shown that NCAPD2 plays an important role in mitotic and meiotic chromatin condensation.However,its role in tumor development and specific molecular mechanisms need further investigation.From our results,we find that NCAPD2 is highly expressed in colorectal cancer tissues through transcriptome analysis of clinical samples.Previous experimental studies also confirmed that NCAPD2 can activate m TORC1,promote the growth of colorectal cancer cells,inhibit autophagy and block autophagy flow.In this study,the colorectal cell line,clinical tissue samples and model mice are used as experimental materials to explore the molecular mechanism of NCAPD2 inhibiting autophagy in colorectal cancer cells and promoting the occurrence and development of colorectal cancer through immunohistochemistry,Western blot and functional assays.It will provide a theoretical basis and experimental foundation for the development of drugs targeting NCAPD2.First,we find that the expression of NCAPD2 is significantly higher in different stages of colorectal cancer than that in normal tissues by using online database analysis;at the same time,Western blot detection with clinical tissue samples find that NCAPD2 is highly expressed in colorectal cancer tissues compared with adjacent tissues,which is consistent with our previous experimental results.To further explore the mechanism of NCAPD2 in colorectal cancer,we examine a series of molecules upstream of m TORC1 and find that NCAPD2 is able to affect AMPK phosphorylation.It has been reported that there are two signaling pathways regulating autophagy through AMPK:LKB1/AMPK and Ca²⁺/Ca MKK2/AMPK.We find that NCAPD2 only regulates the Ca²⁺/Ca MKK2/AMPK signaling pathway in colorectal cancer,combined with the use of the Ca²⁺-ATPase inhibitor Thapsigargin（TG）and the calcium chelator BAPTA-AM to activate and inhibit calcium flow,respectively.The experimental results show that NCAPD2 inhibits autophagy in colorectal cancer cells through the Ca²⁺/Ca MKK2/AMPK/m TORC1 signaling pathway.Through literature review,NCAPD2 is found to be able to interact with PARP-1,and our immunoprecipitation experiment results have confirmed this result.According to the literature,PARP-1 is the major intracellular NAD⁺consumer,the deacetylase activity of SIRT1 is dependent on NAD⁺levels,while SIRT1 can affect the acetylation levels of autophagy-related genes,which then regulates autophagy.Therefore,we designed an experiment to detect whether NCAPD2 is involved in the regulation of autophagy and the growth of colorectal cancer cells.The results of Western blot and other experiments show that NCAPD2 enhances the activity of PARP-1,depletes NAD⁺,decreases NAD⁺levels,reduces SIRT1 activity and enhances the acetylation of ATG5.Pharmacological inhibition assays show that NCAPD2-overexpressed cells followed by treatment with PJ34,an inhibitor of PARP-1 activity,the levels of SIRT1 and ATG5 are increased by compared with those in cells NCAPD2-overexpressed only.Similar results are obtained by using PARP-1si RNA treatment in SW480 cells.These results suggest that NCAPD2 promotes the acetylation of ATG5 through the PARP-1/SIRT1 axis,which in turn downregulates ATG5levels to inhibit autophagy.The results of functional experiments show that NCAPD2 plays a role in promoting cancer in colorectal cancer,and NCAPD2 promotes the colonization and cell migration of colorectal cancer cells through the Ca²⁺/Ca MKK2/AMPK/m TORC1 and PARP-1/SIRT1axes.Further,subcutaneous xenograft tumor model experiments show that overexpression of NCAPD2 promotes transplant tumor growth,the results of Western blot and immunohistochemical detection are consistent with those of in vitro experiments.In addition,we use NCAPD2^[+/-]knockout mice to induce spontaneous colorectal cancer mouse model by AOM/DSS treatment.From the results,although the body weights of both NCAPD2^[+/-]mice and wild-type mice are all decreased after DSS treatment,the body weights of wild-type mice are decreased more significantly;and the colorectal length of NCAPD2^[+/-]mice are longer and the number of colorectal tumors are less than those of wild-type mice,which indicates that NCAPD2^[+/-]mice are less sensitive to DSS-induced inflammatory response than that of wild-type mice.Western blot and immunohistochemistry results are all opposite to those of the NCAPD2-overexpressed HCT116 cells subcutaneous graft tumors.All the results of these in vivo experiments are consistent with those of in vitro experiments in cell level.In conclusion,this study finds that NCAPD2 inhibits autophagy and promotes the development of colorectal cancer through Ca²⁺/Ca MKK2/AMPK/m TORC1 pathway and PARP-1/SIRT1 axis,indicating that NCAPD2 is a cancer-promoting factor in colorectal cancer,and may become a potential target for the prevention or treatment of colorectal cancer.