Journal of Carcinogenesis BioMed Central Research Open Access Implications of tyrosine phosphoproteomics in cervical carcinogenesis Bernice L Robinson-Bennett*1, James DeFord2, Concepcion Diaz-Arrastia1, Lyuba Levine1, Hui-Qui Wang3, Edward V Hannigan1 and John Papaconstantinou2 Address: 1Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, Texas, USA, 2Department of Biochemistry and Molecular Biology, The University of Texas and Medical Branch, Galveston, Texas, USA and 3Department of Histology, The University of Texas Medical Branch, Galveston, Texas, USA Email: Bernice L Robinson-Bennett* - [email protected]; James DeFord - [email protected]; Concepcion Diaz- Arrastia - [email protected]; Lyuba Levine - [email protected]; Hui-Qui Wang - [email protected]; Edward V Hannigan - [email protected]; John Papaconstantinou - [email protected] * Corresponding author Published: 17 July 2008 Received: 9 February 2008 Accepted: 17 July 2008 Journal of Carcinogenesis 2008, 7:2 doi:10.1186/1477-3163-7-2 This article is available from: http://www.carcinogenesis.com/content/7/1/2 © 2008 Robinson-Bennett et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Worldwide cervical cancer remains a leading cause of mortality from gynecologic malignancies. The link between cervical cancer and persistent infection with HPV has been established. At a molecular level little is known about the transition from the precancerous state to invasive cancer. To elucidate this process, cervical biopsies from human specimens were obtained from precancerous state to stage III disease. Methods: Cervical biopsies were obtained from patients with a diagnosis of cervical cancer undergoing definitive surgery or staging operation. Biopsies were obtained from patients with precancerous lesions at the time of their excisional procedure. Control samples were obtained from patients undergoing hysterectomy for benign conditions such as fibroids. Samples were subjected to proteomic profiling using two dimensional gel electrophoresis with subsequent trypsin digestion followed by MALDI-TOF protein identification. Candidate proteins were then further studied using western blotting, immunoprecipitation and immunohistochemistry. Results: Annexin A1 and DNA-PKcs were found to be differentially expressed. Phosphorylated annexin A1 was up regulated in diseased states in comparison to control and its level was strongly detected in the serum of cervical cancer patients compared to controls. DNA-PKcs was noted to be hyperphosphorylated and fragmented in cancer when compared to controls. By immunohistochemistry annexin A1 was noted in the vascular environment in cancer and certain precancerous samples. Conclusion: This study suggests a probable role for protein tyrosine phosphorylation in cervical carcinogenesis. Annexin A1 and DNA-PK cs may have synergistic effects with HPV infection. Precancerous lesions that may progress to cervical cancer may be differentiated from lesions that will not base on similar immunohistochemical profile to invasive squamous cell carcinoma. Page 1 of 10 (page number not for citation purposes) Journal of Carcinogenesis 2008, 7:2 http://www.carcinogenesis.com/content/7/1/2 Introduction functions and binding capacity compared to the native Worldwide cervical cancer remains the second leading forms [12]. cause of cancer mortality in females [1]. The five year sur- vival for locally advanced cervical cancer is within the DNA-Protein Kinase catalytic subunit (DNA-PKcs), a mac- range of 18% to 34% [2,3]. This poor prognosis and lack romolecule found to be involved in the repair of double of treatment for late stage and recurrent disease probably stranded DNA breaks through activation of p53 was reflects the sparse understanding of the molecular patho- found to be expressed in cancer specimens in its tyrosine genesis of disease progression. The link between cervical phosphorylated and cleaved form [13]. In contrast, in cancer and the Human Papilloma Virus (HPV) has long normal specimens DNA-PKcs existed in its intact full been established. However, the multi-step progression in length non-phosphorylated form. cervical carcinogenesis from the precancerous lesions of high grade cervical intraepithelial neoplasia (high grade The aim of the study was to identify differential expression dysplasia) to invasive carcinoma is still to be elucidated. and modification of proteins that could suggest aberrant Unlike colon cancer which has a definite precancerous pathways which could serve as novel targets for develop- state and an elucidated model for carcinogenesis, cervical ing new therapies in the treatment of cervical cancer and carcinogenesis with a known precancerous lesion has not in monitoring disease recurrence or progression. yet been completely elucidated. Materials and methods In general, at a molecular level cancer is considered a state Antibodies of altered signaling. One of the most common mecha- Annexin A1 mouse monoclonal (Santa Cruz, CA, USA), nisms of activating and/or inactivating signaling pathways Calgranulin A8 rabbit polyclonal (Santa Cruz, CA, USA), is phosphorylation and de-phosphorylation at serine, DNA-PK catalytic subunit, multiple clones (USBiological, threonine and tyrosine residues. This modification con- MA, USA). Phospho-ERK44/42 (Cell Signaling, Beverly, trols a variety of cellular processes including cellular MA), Phospho-MEK (Cell Signaling, Beverly, MA, USA), growth, proliferation, cell cycle control, cytoskeletal MEK (Cell Signaling, Beverly, MA, USA), Phosphotyrosine mobility and receptor regulation [4]. Phosphorylation mouse monoclonal antibody (Santa Cruz, CA, USA), p53 leads to allosteric modifications that may result in suffi- rabbit polyclonal antibody (Santa Cruz, CA, USA), Goat cient conformational change to cause activation or inacti- Anti-mouse secondary HRP (Alpha Diagnostics, San vation of various proteins and associated altered Antonio, TX, USA), Anti-rabbit IgG HRP secondary (Alpha functioning. We hypothesized that identification of phos- Diagnostics, San Antonio, TX, USA). phoproteins associated with the various stages of cervical cancer may provide information on the mechanism of Buffers tumorigenesis and insight in the development of diagnos- Buffer M (100 mM HEPES-KOH, pH 7.4, 5 mM EGTA, 20 tic and therapeutic procedures. mM EDTA, 100 mM sodium orthovanadate, 0.5 M sodium fluoride, 10 mM sodium molybdate, 0.2 M β- The mitogen activated protein kinase (MAPK) pathways glycerophosphate); 2-D Lysis Buffer (8 M urea, 4% are known be deregulated in many human malignancies CHAPS); Rehydration Buffer (8 M Urea, 2% CHAPS, [5]. The best studied with regards to malignancy are the 0.002% bromophenol blue, 0.75% pH 4–7 pharmalyte); extracellular signal regulated protein kinases (ERK). ERKs Equilibration Buffer (50 mM Tris-HCL, pH 8.8, 6 M Urea, phosphorylate cytoplasmic targets or migrate to the 30% of 87% Glycerol, 2% SDS, 0.002% Bromophenol nucleus where they can activate transcription factors blue); Transfer Buffer (25 mM Tris, 200 mM Glycine, 20% involved in cellular proliferation. Aberrant signaling in Methanol); the MAPK/ERK has been described in prostate; breast and colon cancers in in vitro as well as in vivo model [6-8]. In TBS-T (10 mM Tris pH 7.4, 154 mM sodium chloride, cervical cancer, one study has described decreased activa- 0.1% Tween 20); Blocking Buffer (5% carnation nonfat tion of ERK1/2 in invasive cervical carcinoma [9]. dried milk or 5% BSA with phosphatase inhibitors for phosphoamino antibodies). 3× Sample Buffer (187.5 mN Annexin A1 is a calcium dependent phospholipid binding Tris-HCL, 6% SDS, 30% Glycerol, 150 mM DTT, 0.3% protein that has been linked to membrane trafficking bromophenol blue). through exocytosis and endocytosis [10]. Other studies have evaluated its role in the modulation of the MAPK/ Patients ERK [11]. Many members of the Annexin family are After obtaining IRB approval and written consents, known to undergo alternate splicing yielding a number of patients were recruited from the University of Texas Med- isoforms. The resultant variant forms may have different ical Branch after having a biopsy proven diagnosis of high grade dysplasia (pre-cancer) or frankly invasive cervical Page 2 of 10 (page number not for citation purposes) Journal of Carcinogenesis 2008, 7:2 http://www.carcinogenesis.com/content/7/1/2 cancer at different stages. A small biopsy was obtained μl of PBS. 100 μl of 3× sample buffer was added to immu- from these patients. The patients' ages ranged from 16–69 nopellets which were then boiled for 5 minutes. Mixtures years old; and the most advanced stage recruited was IIIB were centrifuged at 13,000 rpm for 2 minutes and the disease. Normal cervical tissue was obtained from women supernatant collected. 15 μl aliquots of the supernatant undergoing hysterectomy for benign diseases such as were then subjected to separation by SDS-PAGE on 5% or fibroids or endometriosis without a history of abnormal 4–20% Tris-Glycine precast BioRad gels. Pap smears. The study cohort consisted of normal (n = 10), precancerous (n = 10), stage I (n = 6), stage II (n = 6) Western blotting and stage III (n = 5). Tissue lysates and immunoprecipitates were prepared as described above