A Study Of The Role And Molecular Mechanism Of IASPP In Maintaining Er Ca²⁺ Homeostasis In Tumor Cells

As an important intracellular second messenger,Calcium ion(Ca²⁺)regulates a series of cellular biological processes.More and more evidences indicate that Ca²⁺homeostasis disorder is closely related to the occurrence and progression of cancer.Therefore,in-depth exploration of the molecular mechanisms regulating Ca²⁺homeostasis not only improve the understanding of tumor pathological processes,but also provide a basis for the development of new cancer treatment strategies.To accurately regulate Ca²⁺-related biological functions,cells form a Ca²⁺regulatory system composed of multiple Ca²⁺pumps and Ca²⁺channel proteins,which maintain intracellular Ca²⁺homeostasis and regulate the activation of Ca²⁺signals by regulating Ca²⁺in and out of cells and organelles.Endoplasmic reticulum（ER）is the largest intracellular Ca²⁺reservoir and multiple stress factors can cause Ca²⁺outflow from ER,resulting in increased local Ca²⁺concentration in cytoplasm and activation of Ca²⁺signaling pathway.As a newly discovered ER Ca²⁺channel protein,Transmembrane and Coiled-Coil Domains 1（TMCO1）can prevent ER Ca²⁺overfilling by inducing Ca²⁺outflow to maintain ER Ca²⁺homeostasis.TMCO1 gene mutation is closely related to glaucoma,craniofacial deformity and bone abnormality.However,whether TMCO1 plays a role in tumors or the molecular regulation mechanism of TMCO1 has not been reported.Oncogene inhibitor of Apoptosis Stimulating Protein of p53（iASPP）is highly expressed in a variety of tumors and is closely related to drug resistance and metastasis.Initial studies have shown that iASPP can bind p53 in the nucleus and inhibit the transcriptional activity of p53 and cancer cell apoptosis in a p53-dependent manner.Previous studies in our lab found that iASPP mainly locates in the cytoplasm and stabilizes and activates the activity of Nuclear Factor Erythroid-2 Related Factor 2（Nrf2）,an important antioxidant transcription factor,in a p53-independent manner,thereby promoting tumor growth and resisting oxidative stress and killing effects caused by 5-Fluorouracil（5-FU）.Therefore,iASPP has a variety of tumor-promoting activities depending on different cell localization.However,whether iASPP is involved in Ca²⁺homeostasis has not been investigated.To further understand the cancer-promoting function of iASPP,RNA-seq results of overexpressed iASPP were firstly analyzed and found that the expression level of iASPP was correlated with multiple tumor-related signaling pathways such as Ca²⁺signaling pathway.The following Ca²⁺flow analysis showed that iASPP could regulate the ER Ca²⁺storage.Overexpression of iASPP led to a decrease in ER Ca²⁺storage,while iASPP knockdown led to an increase.Studies on the distribution of iASPP in tumor cells showed that although iASPP was mainly located in the cytosol,part of iASPP protein could be aggregated in the perinuclear area.Co-location analysis of endoplasmic reticulum markers and isolation of cell subfractions showed that iASPP could be located on the endoplasmic reticulum.However,iASPP does not have a transmembrane domain,suggesting that iASPP may regulate ER Ca²⁺through other ER Ca²⁺pumps or Ca²⁺channel proteins.Indeed,overexpression or knockdown of iASPP can lead to significant changes in the expression level of ER localized Ca²⁺channel protein TMCO1,and the regulatory activity of iASPP on ER Ca²⁺storage is mainly dependent on TMCO1,rather than its known target molecules p53 and Nrf2.On this basis,our study further explored that the molecular mechanism of iASPP regulating calcium homeostasis by TMCO1.The results showed that iASPP inhibited TMCO1 ubiquitin-proteasome pathway degradation to improve the protein stability of TMCO1,but did not significantly regulate the m RNA level of TMCO1 and found the molecular mechanism of E3 ligase Gp78 binding and promoting TMCO1 ubiquitination degradation.90-188 fragment of TMCO1 binds to the 301-400 fragment of Gp78,resulting in Gp78-mediated polyubiquitination of K48 at K186 of TMCO1,and thereby promoting protein degradation of TMCO1,protecting the function of TMCO1-mediated E1R Ca²⁺release,preventing ER Ca²⁺overabundance,and maintaining ER Ca²⁺homeostasis of tumor cells.On the basis of revealing the mechanism of Ca²⁺homeostasis,our study further explored the role of this mechanism in tumor growth and drug resistance.Firstly,it was found that iASPP and TMCO1 protein levels were highly expressed in the tissues of colorectal cancer patients,and they were positively correlated.Knockdown of iASPP or TMCO1 significantly inhibited the growth of tumor cells in vitro and in nude mice.In addition,Thapsigargin（TG）/histamine/staurosporine can obviously improve ER Ca²⁺release and cytoplasm Ca²⁺level,promote caspase3/7 activation and apoptosis in iASPP/TMCO1 knockdown cancer cells.Further mechanistic studies revealed that this process relies on cytoplasmic Ca²⁺signaling to induce calpain activation.Subcutaneous tumor bearing assay in nude mice in vivo further confirmed the mechanism of inhibiting iASPP-mediated TMCO1 dependence,which can effectively promote staurosporine-induced apoptosis and tumor inhibition effect.In conclusion,our study confirmed the important function and mechanism of iASPP in maintaining ER Ca²⁺homeostasis through TMCO1,thus promoting tumor growth and resistance to Staurosporine,providing new ideas and clues for drug development of targeted regulation of Ca²⁺signal for cancer cell.