STUB1 Enhances Bortezomib Sensitivity In Multiple Myeloma Via Repressing TFEB-mediated Autophagy

Background and objectives:Multiple myeloma(MM),the second most common hematological malignancy,is characterized by the aberrant proliferation of plasma cells within the bone marrow and excessive secretion of monoclonal immunoglobulin.Although bortezomib has become the cornerstone for MM treatment due to the high effectiveness,most patients will eventually experience drug resistance to bortezomib and disease recurrence.Up till now,the effects of STUB1 on MM tumorigenesis and its role in drug resistance still remain indistinct.The objective of this study is to explore the correlation between STUB1 expression level and clinical characteristics and survival outcomes of MM patients.Furthermore,we investigated the biological functions of STUB1 in MM and its underlying molecular mechanisms in regulating bortezomib sensitivity.Materials and Methods:1.We retrospectively analyzed the clinical characteristics and survival status of newly-diagnosed MM patients who then received bortezomib-based therapeutic regimen.Kaplan-Meier survival curves and Cox regression model were used to determine independent prognostic factors.2.Bioinformatic databases were utilized to analyze and predict the biological features and functions of targeted molecule,as well as its potential interacting substrates.3.Immunohistochemistry was conducted to demonstrate the protein expression level in the bone marrow tissue.4.ELISA assay was utilized to calculate the concentration of STUB1 in the bone marrow supernatant of prospectively enrolled MM patients and heathy control.5.By magnetic beads sorting system,CD138+ plasma cells were separated and enriched from bone marrow mononuclear cells of MM patients and healthy donors.6.Bortezomib-resistant cell lines U266-BR and H929-BR were established by long-term bortezomib induction of U266 and H929 parental cell lines.7.MM cell lines with stable overexpression or knockdown of targeted genes were constructed by means of lentivirus transfection and then were filtered out by puromycin or blasticidin.8.RT-PCR was employed to determine m RNA expression level of targeted molecules.Western Blot was conducted to determine the protein expression of targeted molecules.9.CCK-8(Cell counting kit-8)test was performed to determine cell viability in vitro and calculate the 50% inhibitory concentration(IC50).10.Soft agar clone-formation test was performed to demonstrate long-term cell proliferation status and drug resistance in vitro.11.Flow cytometry was used to measure cellular apoptosis(Annexin V-PE/7-AAD staining)and cell-cycle distribution(Propidium iodide staining).12.Transmission electron microscope was employed to visualized and quantified the autophagy-related membrane structures including autophagosomes,autolysomes and phagophores.13.Immunofluorescence co-localization,co-immunoprecipitation(CO-IP)and western blot were used to manifest the binding and interaction between molecules.14.Through transfection of GFP-m Cherry RFP-LC3 B adenovirus,the autophagic flux was observed and assessed with confocal laser scanning microscopy.15.Immunofluorescence,nucleoplasmic separation technique and western blot were used to determine the subcellular localization and nucleus-cytoplasm translocation of certain molecule.16.Xenograft tumor model was established through subcutaneous injection of human MM cell lines in the BALB/c female nude mice to evaluate the tumor growth in vivo.Bortezomib were administered through the tail vein.Hematoxylin-Eosin staining(HE staining)was used to exhibit the basic pathological features of xenograft tumor tissue.IHC was conducted to reveal the protein expression level in the xenograft tumor tissue.Fluorescence TUNEL assay was performed to detect the cell apoptosis within the tumor tissue.Results:1.In multiple myeloma,STUB1 expression was negatively correlated with TFEB expression.Patients with high expression of STUB1 in bone marrow tissue had a better prognosis than those with low expression.High STUB1 expression had close association with lower tumor burden,lower disease staging,deeper optimal remission during treatment,enhanced bortezomib sensitivity,lower frequency of high-risk abnormal cytogenetic karyotypes.The low concentration of STUB1 in the bone marrow microenvironment was related to the occurrence of bortezomib resistance.High STUB1 expression was an independent favorable prognostic factor(HR=0.343,95%CI0.121-0.975).2.STUB1 is mainly localized in the cytoplasm of myeloma cells.Overexpression of STUB1 repressed cell growth,proliferation,clone formation,and allowed more cells to enter the G0/G1 phase.Knockdown of STUB1 facilitated cell growth,proliferation,clone formation,and enabled more cells to enter the synthesis or mitosis phase.While,overexpression or knockdown alone did not influence cellular apoptosis.3.Overexpression of STUB1 greatly enhanced bortezomib-induced apoptosis by suppressing protective autophagy,while reversing drug resistance in bortezomib-resistant MM cells.Knockdown of STUB1 alleviated bortezomib-induced apoptosis and strengthened resistance to bortezomib by activating protective autophagy.4.Overexpression of STUB1 resulted in downregulation of TFEB,thus downregulating the expression of autophagy-related proteins ATG5,Beclin1,and LC3 B.Suppression of biological formation of autophagosomes or autolysosomes and blockade of autophagic flux significantly boosted the sensitivity of myeloma to bortezomib and overcame bortezomib resistance in drug-resistant cells,as evidenced by the increased apoptotic rate,decreased clone numbers and IC50 value.Knockdown of STUB1 increased TFEB expression.TFEB shuttled from cytoplasm into the nucleus.TFEB nulear translocation initiated the transcription and translation of autophagy-related genes,as evidenced by the elevation of ATG5,Beclin1,and LC3 B both in the m RNA and protein expression level.Activation of autophagic flux contributed to the biological formation of autophagosomes or autolysosomes.TFEB-mediated protective autophagy conferred bortezomib resistance on MM cells,which was manifested by decreased apoptosis,increased clone formation and IC50 value.5.Immunofluorescence co-localization test and CO-IP test corroborated the binding,combination and interaction between STUB1 and TFEB.Overexpression of STUB1 promoted TFEB retention in the cytoplasm,thus facilitating ubiquitination of TFEB and degradation through ubiquitin-proteasome pathway.6.In myeloma xenograft mice models,overexpression of STUB1 significantly suppressed tumor growth,aggravated bortezomib-induced apoptosis,thus augmenting the anti-myeloma effects of bortezomib and overcoming drug resistance via inhibiting protective autophagy.While,knockdown of STUB1 strengthened tumor growth and attenuated bortezomib-induced apoptosis by activating autophagy.Conclusion:STUB1 functioned as a tumor suppressor gene in MM,inhibiting cell growth and proliferation both in vitro and in vivo.STUB1 repressed prosurvival autophagy to enhance bortezomib sensitivity in MM through ubiquitination and degradation of TFEB.The STUB1-TFEB-autophagy signaling axis might serve as a potential target for overcoming bortezomib resistance,providing novel therapeutic strategy for relapsed and refractory MM.