Acquired platinum resistance involves epithelial to mesenchymal transition through ubiquitin ligase FBXO32 dysregulation.

To identify the molecules involved in epithelial to mesenchymal transition (EMT) in urothelial carcinoma (UC) after acquisition of platinum resistance, here we examined the changes in global gene expression before and after platinum treatment. Four invasive UC cell lines, T24, 5637, and their corresponding sublines T24PR and 5637PR with acquired platinum resistance, were assessed by microarray, and the ubiquitin E3 ligase FBXO32 was newly identified as a negative regulator of EMT in UC tumors after acquisition of platinum resistance. In vitro and in vivo studies showed an intimate relationship between FBXO32 expression and EMT, demonstrating that FBXO32 dysregulation in T24PR cells results in elevated expression of the mesenchymal molecules SNAIL and vimentin and decreased expression of the epithelial molecule E-cadherin. The association between FBXO32 expression and EMT was further validated using clinical samples. Knockdown of MyoD expression, a specific target of FBXO32 polyubiquitination, revealed upregulation of E-cadherin expression and downregulation of SNAIL and vimentin expression in T24PR cells. Comparative genomic hybridization array analysis demonstrated loss of heterozygosity at 8q24.13 in T24PR cells, which harbors FBXO32. Our findings suggest the importance of the association between EMT and ubiquitin-proteasome regulation when tumors develop acquired platinum resistance.

[1]  J. Witjes,et al.  EAU guidelines on muscle-invasive and metastatic bladder cancer: summary of the 2013 guidelines. , 2014, European urology.

[2]  P. Wade,et al.  Aberrant Expression of the Transcription Factors Snail and Slug Alters the Response to Genotoxic Stress , 2004, Molecular and Cellular Biology.

[3]  E. Kikuchi,et al.  Acquired platinum resistance enhances tumour angiogenesis through angiotensin II type 1 receptor in bladder cancer , 2011, British Journal of Cancer.

[4]  Y. Okada,et al.  Imaging , Diagnosis , Prognosis Expression of Snail in Upper Urinary Tract Urothelial Carcinoma : Prognostic Significance and Implications for Tumor Invasion , 2010 .

[5]  K. O'Byrne,et al.  Generation and Characterisation of Cisplatin-Resistant Non-Small Cell Lung Cancer Cell Lines Displaying a Stem-Like Signature , 2013, PloS one.

[6]  N. Offner,et al.  The initiation factor eIF3‐f is a major target for Atrogin1/MAFbx function in skeletal muscle atrophy , 2008, The EMBO journal.

[7]  Qiang Yu,et al.  Pharmacologic disruption of Polycomb-repressive complex 2-mediated gene repression selectively induces apoptosis in cancer cells. , 2007, Genes & development.

[8]  A. Russell,et al.  The role and regulation of MAFbx/atrogin-1 and MuRF1 in skeletal muscle atrophy , 2011, Pflügers Archiv - European Journal of Physiology.

[9]  I. Fabregat,et al.  Snail blocks the cell cycle and confers resistance to cell death. , 2004, Genes & development.

[10]  W. Lam,et al.  Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cells. , 2013, Cancer research.

[11]  M. Quinn,et al.  Cisplatin treatment of primary and metastatic epithelial ovarian carcinomas generates residual cells with mesenchymal stem cell‐like profile , 2011, Journal of cellular biochemistry.

[12]  Huamin Wang,et al.  Epithelial to mesenchymal transition contributes to drug resistance in pancreatic cancer. , 2009, Cancer research.

[13]  N. Offner,et al.  The initiation factor eIF 3f is a major target for Atrogin 1 / MAFbx function in skeletal muscle atrophy , 2013 .

[14]  P. Atadja,et al.  Superior Efficacy of a Combined Epigenetic Therapy against Human Mantle Cell Lymphoma Cells , 2012, Clinical Cancer Research.

[15]  D J Glass,et al.  Identification of Ubiquitin Ligases Required for Skeletal Muscle Atrophy , 2001, Science.

[16]  C. Dinney,et al.  Role of epithelial-to-mesenchymal transition (EMT) in drug sensitivity and metastasis in bladder cancer , 2009, Cancer and Metastasis Reviews.

[17]  Xifeng Wu,et al.  Sensitivity to Epidermal Growth Factor Receptor Inhibitor Requires E-Cadherin Expression in Urothelial Carcinoma Cells , 2008, Clinical Cancer Research.

[18]  G. Gallick,et al.  Development and Characterization of Gemcitabine-Resistant Pancreatic Tumor Cells , 2007, Annals of Surgical Oncology.

[19]  J. Thiery.  Epithelial–mesenchymal transitions in tumour progression , 2002, Nature Reviews Cancer.

[20]  G. Berx,et al.  Regulatory networks defining EMT during cancer initiation and progression , 2013, Nature Reviews Cancer.

[21]  J. Izbicki,et al.  Notch signaling activated by replication stress-induced expression of midkine drives epithelial-mesenchymal transition and chemoresistance in pancreatic cancer. , 2011, Cancer research.

[22]  M. Ohmura,et al.  CD44 variant regulates redox status in cancer cells by stabilizing the xCT subunit of system xc(-) and thereby promotes tumor growth. , 2011, Cancer cell.

[23]  Paulo A. S. Nuin,et al.  EMT transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer , 2012, BMC Cancer.

[24]  S. Sze,et al.  Identification of atrogin-1-targeted proteins during the myostatin-induced skeletal muscle wasting. , 2012, American journal of physiology. Cell physiology.

[25]  F. Portillo,et al.  SNAI1 is required for tumor growth and lymph node metastasis of human breast carcinoma MDA-MB-231 cells. , 2007, Cancer research.

[26]  Jun-ling Liu,et al.  Activation of notch‐1 enhances epithelial–mesenchymal transition in gefitinib‐acquired resistant lung cancer cells , 2012, Journal of cellular biochemistry.

[27]  M. Loda,et al.  Identification and Prognostic Significance of an Epithelial-Mesenchymal Transition Expression Profile in Human Bladder Tumors , 2007, Clinical Cancer Research.

[28]  S. Leibovitch,et al.  Identification of essential sequences for cellular localization in the muscle‐specific ubiquitin E3 ligase MAFbx/Atrogin 1 , 2012, FEBS letters.

[29]  Marco Sandri,et al.  Foxo Transcription Factors Induce the Atrophy-Related Ubiquitin Ligase Atrogin-1 and Cause Skeletal Muscle Atrophy , 2004, Cell.

[30]  S. Lippard,et al.  Structure, Recognition, and Processing of Cisplatin-DNA Adducts. , 1999, Chemical reviews.

[31]  Sergio Ricci,et al.  Long-term survival results of a randomized trial comparing gemcitabine plus cisplatin, with methotrexate, vinblastine, doxorubicin, plus cisplatin in patients with bladder cancer. , 2006, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[32]  S. Higashiyama,et al.  Cisplatin influences acquisition of resistance to molecular‐targeted agents through epithelial–mesenchymal transition‐like changes , 2013, Cancer science.

[33]  angesichts der Corona-Pandemie,et al.  UPDATE , 1973, The Lancet.

[34]  L. Ellis,et al.  Chronic Oxaliplatin Resistance Induces Epithelial-to-Mesenchymal Transition in Colorectal Cancer Cell Lines , 2006, Clinical Cancer Research.

[35]  S. Ricci,et al.  Long-Term Survival Results of a Randomized Trial Comparing Gemcitabine Plus Cisplatin, With Methotrexate, Vinblastine, Doxorubicin, Plus Cisplatin in Patients With Bladder Cancer , 2005 .

[36]  W. Guo,et al.  Aberrant methylation and decreased expression of the TGF‐β/Smad target gene FBXO32 in esophageal squamous cell carcinoma , 2014, Cancer.

[37]  Eric P Hoffman,et al.  Slug Is a Novel Downstream Target of MyoD , 2002, The Journal of Biological Chemistry.

[38]  F. Spinella,et al.  Acquisition of Chemoresistance and EMT Phenotype Is Linked with Activation of the Endothelin A Receptor Pathway in Ovarian Carcinoma Cells , 2011, Clinical Cancer Research.

[39]  A. Goldberg,et al.  Atrogin-1, a muscle-specific F-box protein highly expressed during muscle atrophy , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Héctor Peinado,et al.  Snail, Zeb and bHLH factors in tumour progression: an alliance against the epithelial phenotype? , 2007, Nature Reviews Cancer.

[41]  V. Sirri,et al.  Degradation of MyoD Mediated by the SCF (MAFbx) Ubiquitin Ligase* , 2005, Journal of Biological Chemistry.

[42]  Zhiwei Wang,et al.  Acquisition of epithelial-mesenchymal transition phenotype of gemcitabine-resistant pancreatic cancer cells is linked with activation of the notch signaling pathway. , 2009, Cancer research.

[43]  Daniel E. Deatherage,et al.  Promoter hypermethylation of FBXO32, a novel TGF-β/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer , 2010, Laboratory Investigation.

[44]  Da-Zhi Wang,et al.  Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex. , 2004, The Journal of clinical investigation.

[45]  Huidong Shi,et al.  Combined epigenetic therapy with the histone methyltransferase EZH2 inhibitor 3-deazaneplanocin A and the histone deacetylase inhibitor panobinostat against human AML cells. , 2009, Blood.

[46]  M. Ohmura,et al.  Reduced methylation of PFKFB3 in cancer cells shunts glucose towards the pentose phosphate pathway , 2014, Nature Communications.

[47]  M. Nieto,et al.  The Snail genes as inducers of cell movement and survival: implications in development and cancer , 2005, Development.

[48]  Richard Sylvester,et al.  European guidelines on upper tract urothelial carcinomas: 2013 update. , 2013, European urology.