Integral membrane protein 2A inhibits cell growth in human breast cancer via enhancing autophagy induction

BackgroundBreast cancer is a life-threatening disease in females and the leading cause of mortality among the female population, presenting huge challenges for prognosis and treatment. ITM2A is a member of the BRICHOS superfamily, which are thought to have a chaperone function. ITM2A has been identified to related to ovarian cancer progress recently. However, the biological role of ITM2A in breast cancer remains largely unclear.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR), western blotting assay and immunohistochemistry staining were used to analyzed the expression level of ITM2A. The patient overall survival versus ITM2A expression level was evaluated by Kaplan-Meier analysis. MTT assay, EdU incorporation assay and colony formation assay were used to evaluated the role of ITM2A on breast cancer cell proliferation. Autophagy was explored through autophagic flux detection using a confocal microscope and autophagic vacuoles investigation under a transmission electron microscopy (TEM). In vitro kinase assay was used to investigated the phosphorylation modification of ITM2A by HUNK.ResultsOur data showed that the expression of integral membrane protein 2A (ITM2A) was significantly down-regulated in human breast cancer tissues and cell lines. Kaplan-Meier analysis indicated that patients presenting with reduced ITM2A expression exhibited poor overall survival, and expression significantly correlated with age, progesterone receptor status, TNM classification and tumor stage. ITM2A overexpression significantly inhibited the proliferation of breast cancer cells. By studying several autophagic markers and events in human breast cancer SKBR-3 cells, we further demonstrated that ITM2A is a novel positive regulator of autophagy through an mTOR-dependent manner. Moreover, we found that ITM2A was phosphorylated at T35 by HUNK, a serine/threonine kinase significantly correlated with human breast cancer overall survival and HER2-induced mammary tumorigenesis.ConclusionOur study provided evidence that ITM2A functions as a novel prognostic marker and represents a potential therapeutic target.

[1]  M. Hall,et al.  mTORC2 Promotes Tumorigenesis via Lipid Synthesis. , 2017, Cancer cell.

[2]  J. Merregaert,et al.  In vitro studies on Itm2a reveal its involvement in early stages of the chondrogenic differentiation pathway , 2004, Biology of the cell.

[3]  Marc Liesa,et al.  Pancreatic cancers require autophagy for tumor growth. , 2011, Genes & development.

[4]  H. Gardner,et al.  Protein kinase expression during murine mammary development. , 2000, Developmental biology.

[5]  T. Noda,et al.  Rheb localized on the Golgi membrane activates lysosome-localized mTORC1 at the Golgi–lysosome contact site , 2018, Journal of Cell Science.

[6]  A. Linge,et al.  SLC3A2/CD98hc, autophagy and tumor radioresistance: a link confirmed , 2019, Autophagy.

[7]  A. Regev,et al.  Acquired HER2 mutations in ER+ metastatic breast cancer confer resistance to estrogen receptor–directed therapies , 2018, Nature Genetics.

[8]  E. White.  Deconvoluting the context-dependent role for autophagy in cancer , 2012, Nature Reviews Cancer.

[9]  Hilde van der Togt,et al.  Publisher's Note , 2003, J. Netw. Comput. Appl..

[10]  K. Guan,et al.  Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy , 2013, Autophagy.

[11]  C. Parise,et al.  Breast Cancer Survival Defined by the ER/PR/HER2 Subtypes and a Surrogate Classification according to Tumor Grade and Immunohistochemical Biomarkers , 2014, Journal of cancer epidemiology.

[12]  B. Viollet,et al.  AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1 , 2011, Nature Cell Biology.

[13]  G. Lianos,et al.  Breast cancer in young women: an overview , 2017, Updates in Surgery.

[14]  D. Hailey,et al.  Autophagy termination and lysosome reformation regulated by mTOR , 2010, Nature.

[15]  Y. Eishi,et al.  Autophagy-deficient mice develop multiple liver tumors. , 2011, Genes & development.

[16]  E. Yeh,et al.  Regulation of cell survival by HUNK mediates breast cancer resistance to HER2 inhibitors , 2014, Breast Cancer Research and Treatment.

[17]  D. Green,et al.  Autophagy-Independent Functions of the Autophagy Machinery , 2019, Cell.

[18]  A. Jemal,et al.  Cancer statistics, 2018 , 2018, CA: a cancer journal for clinicians.

[19]  Gordon B. Mills,et al.  The energy sensing LKB1–AMPK pathway regulates p27kip1 phosphorylation mediating the decision to enter autophagy or apoptosis , 2007, Nature Cell Biology.

[20]  W. Liu,et al.  ITM2A Expands Evidence for Genetic and Environmental Interaction in Graves Disease Pathogenesis , 2017, The Journal of clinical endocrinology and metabolism.

[21]  Yi Zhang,et al.  Prognostic significance of PLIN1 expression in human breast cancer , 2016, Oncotarget.

[22]  Seamus J. Martin,et al.  Autophagy in malignant transformation and cancer progression , 2015, The EMBO journal.

[23]  B. Persson,et al.  BRICHOS - a superfamily of multidomain proteins with diverse functions , 2009, BMC Research Notes.

[24]  N. Harbeck,et al.  Breast cancer , 2017, The Lancet.

[25]  Maria Chikina,et al.  Causal network perturbations for instance-specific analysis of single cell and disease samples , 2019, Bioinformatics.

[26]  K. Zaret,et al.  Itm2a Is a Pax3 Target Gene, Expressed at Sites of Skeletal Muscle Formation In Vivo , 2013, PloS one.

[27]  S. Wesselborg,et al.  Role of AMPK-mTOR-Ulk1/2 in the Regulation of Autophagy: Cross Talk, Shortcuts, and Feedbacks , 2011, Molecular and Cellular Biology.

[28]  Loss of ITM2A, a novel tumor suppressor of ovarian cancer through G2/M cell cycle arrest, is a poor prognostic factor of epithelial ovarian cancer. , 2016, Gynecologic oncology.

[29]  M. Wang,et al.  Identification of glycerol-3-phosphate dehydrogenase 1 as a tumour suppressor in human breast cancer , 2017, Oncotarget.

[30]  H. Iwase,et al.  [Breast cancer]. , 2006, Nihon rinsho. Japanese journal of clinical medicine.

[31]  Rebecca L. Siegel Mph,et al.  Cancer statistics, 2018 , 2018 .

[32]  S. Pai,et al.  Itm2a, a Target Gene of GATA-3, Plays a Minimal Role in Regulating the Development and Function of T Cells , 2014, PloS one.

[33]  M. Campone,et al.  bc-GenExMiner: an easy-to-use online platform for gene prognostic analyses in breast cancer , 2012, Breast Cancer Research and Treatment.

[34]  Masaaki Komatsu,et al.  Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice , 2005, The Journal of cell biology.

[35]  L. Chodosh,et al.  Hunk negatively regulates c-myc to promote Akt-mediated cell survival and mammary tumorigenesis induced by loss of Pten , 2013, Proceedings of the National Academy of Sciences.

[36]  P. Soares,et al.  The mTOR Signalling Pathway in Human Cancer , 2012, International journal of molecular sciences.

[37]  P. Fasching,et al.  NAB-Paclitaxel Improves Disease-Free Survival in Early Breast Cancer: GBG 69-GeparSepto. , 2019, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[38]  E. van Marck,et al.  Isolation of Markers for Chondro-osteogenic Differentiation Using cDNA Library Subtraction , 1996, The Journal of Biological Chemistry.

[39]  A. Kimmelman,et al.  The dynamic nature of autophagy in cancer. , 2011, Genes & development.

[40]  I. Jang,et al.  The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase , 2015, Autophagy.

[41]  K. Nagata,et al.  Itm2a Expression in the Developing Mouse First Lower Molar, and the Subcellular Localization of Itm2a in Mouse Dental Epithelial Cells , 2014, PloS one.

[42]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[43]  Jun Dai,et al.  Pygo2 functions as a prognostic factor for glioma due to its up-regulation of H3K4me3 and promotion of MLL1/MLL2 complex recruitment , 2016, Scientific Reports.

[44]  David M Sabatini,et al.  Defining the role of mTOR in cancer. , 2007, Cancer cell.

[45]  Thomas W. Yang,et al.  Hunk is required for HER2/neu-induced mammary tumorigenesis. , 2011, The Journal of clinical investigation.

[46]  E. White,et al.  Autophagy and Tumor Metabolism. , 2017, Cell metabolism.