A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A/BAF250a is associated with AKT phosphorylation

BMC Cancer

Mark S Carey⁴  David G Huntsman²  Gordon B Mills¹  Blake Gilks³  Michael S Anglesio⁷  Fan Zhang¹  Yiling Lu¹  Katherine Stemke-Hale¹  Sarah Finlayson⁴  Steve E Kalloger⁷  Mollianne McGahren¹  Zhenlin Ju⁵  Yemin Wang⁷  Samuel Leung³  Bryan T Hennessy⁶  Kimberly C Wiegand⁷ 

[1]Department of Systems Biology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA

[2]Hereditary Cancer Program, British Columbia Cancer Agency, Vancouver, BC, Canada

[3]Genetic Pathology Evaluation Centre, Vancouver General Hospital, Vancouver, BC, Canada

[4]Division of Gynecologic Oncology, University of British Columbia, Vancouver, BC, Canada

[5]Department of Bioinformatics and Computational Biology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA

[6]Department of Medical Oncology, Beaumont Hospital, Dublin, Ireland

[7]Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada

关键词: Phosphorylation;    AKT;    PIK3CA mutation;    ARID1A/BAF250a;    Proteomics;    Ovarian cancer;   

Others  :  859037
DOI  :  10.1186/1471-2407-14-120

 received in 2013-11-08, accepted in 2014-02-12,  发布年份 2014

【 摘 要 】

Background

Ovarian cancer is now recognized as a number of distinct diseases primarily defined by histological subtype. Both clear cell ovarian carcinomas (CCC) and ovarian endometrioid carcinomas (EC) may arise from endometriosis and frequently harbor mutations in the ARID1A tumor suppressor gene. We studied the influence of histological subtype on protein expression with reverse phase protein array (RPPA) and assessed proteomic changes associated with ARID1A mutation/BAF250a expression in EC and CCC.

Methods

Immunohistochemistry (IHC) for BAF250a expression was performed on 127 chemotherapy-naive ovarian carcinomas (33 CCC, 29 EC, and 65 high-grade serous ovarian carcinomas (HGSC)). Whole tumor lysates were prepared from frozen banked tumor samples and profiled by RPPA using 116 antibodies. ARID1A mutations were identified by exome sequencing, and PIK3CA mutations were characterized by MALDI-TOF mass spectrometry. SAM (Significance Analysis of Microarrays) was performed to determine differential protein expression by histological subtype and ARID1A mutation status. Multivariate logistic regression was used to assess the impact of ARID1A mutation status/BAF250a expression on AKT phosphorylation (pAKT). PIK3CA mutation type and PTEN expression were included in the model. BAF250a knockdown was performed in 3 clear cell lines using siRNA to ARID1A.

Results

Marked differences in protein expression were observed that are driven by histotype. Compared to HGSC, SAM identified over 50 proteins that are differentially expressed in CCC and EC. These included PI3K/AKT pathway proteins, those regulating the cell cycle, apoptosis, transcription, and other signaling pathways including steroid hormone signaling. Multivariate models showed that tumors with loss of BAF250a expression showed significantly higher levels of AKT-Thr³⁰⁸ and AKT-Ser⁴⁷³ phosphorylation (p < 0.05). In 31 CCC cases, pAKT was similarly significantly increased in tumors with BAF250a loss on IHC. Knockdown of BAF250a by siRNA in three CCC cell lines wild type for ARID1A showed no increase in either pAKT-Thr³⁰⁸ or pAKT-S⁴⁷³ suggesting that pAKT in tumor tissues is indirectly regulated by BAF250a expression.

Conclusions

Proteomic assessment of CCC and EC demonstrates remarkable differences in protein expression that are dependent on histotype, thereby further characterizing these cancers. AKT phosphorylation is associated with ARID1A/BAF250a deficient tumors, however in ovarian cancers the mechanism remains to be elucidated.

【 授权许可】

   
2014 Wiegand et al.; licensee BioMed Central Ltd.

【 图 表 】

【 参考文献 】

• [1]Kobel M, Kalloger SE, Boyd N, McKinney S, Mehl E, Palmer C, Leung S, Bowen NJ, Ionescu DN, Rajput A, Prentice LM, Miller D, Santos J, Swenerton K, Gilks CB, Huntsman D: Ovarian carcinoma subtypes are different diseases: implications for biomarker studies. PLoS Med 2008, 5(12):e232.
• [2]Thigpen T, du Bois A, McAlpine J, DiSaia P, Fujiwara K, Hoskins W, Kristensen G, Mannel R, Markman M, Pfisterer J, Quinn M, Reed N, Swart AM, Berek J, Colombo N, Freyer G, Gallardo D, Plante M, Poveda A, Rubinstein L, Bacon M, Kitchener H, Stuart GC, Gynecologic Cancer InterGroup: First-line therapy in ovarian cancer trials. Int J Gynecol Cancer 2011, 21(4):756-762.
• [3]Kobel M, Kalloger SE, Huntsman DG, Santos JL, Swenerton KD, Seidman JD, Gilks CB, Cheryl Brown Ovarian Cancer Outcomes Unit of the British Columbia Cancer Agency VBC: Differences in tumor type in low-stage versus high-stage ovarian carcinomas. Int J Gynecol Pathol 2010, 29(3):203-211.
• [4]Crum CP, Drapkin R, Miron A, Ince TA, Muto M, Kindelberger DW, Lee Y: The distal fallopian tube: a new model for pelvic serous carcinogenesis. Curr Opin Obstet Gynecol 2007, 19(1):3-9.
• [5]Kuhn E, Kurman RJ, Vang R, Sehdev AS, Han G, Soslow R, Wang TL: Shih IeM: TP53 mutations in serous tubal intraepithelial carcinoma and concurrent pelvic high-grade serous carcinoma–evidence supporting the clonal relationship of the two lesions. J Pathol 2012, 226(3):421-426.
• [6]LaGrenade A, Silverberg SG: Ovarian tumors associated with atypical endometriosis. Hum Pathol 1988, 19(9):1080-1084.
• [7]Ogawa S, Kaku T, Amada S, Kobayashi H, Hirakawa T, Ariyoshi K, Kamura T, Nakano H: Ovarian endometriosis associated with ovarian carcinoma: a clinicopathological and immunohistochemical study. Gynecol Oncol 2000, 77(2):298-304.
• [8]Vigano P, Somigliana E, Chiodo I, Abbiati A, Vercellini P: Molecular mechanisms and biological plausibility underlying the malignant transformation of endometriosis: a critical analysis. Hum Reprod Update 2006, 12(1):77-89.
• [9]Munksgaard PS, Blaakaer J: The association between endometriosis and gynecological cancers and breast cancer: a review of epidemiological data. Gynecol Oncol 2011, 123(1):157-163.
• [10]Pearce CL, Templeman C, Rossing MA, Lee A, Near AM, Webb PM, Nagle CM, Doherty JA, Cushing-Haugen KL, Wicklund KG, Chang-Claude J, Hein R, Lurie G, Wilkens LR, Carney ME, Goodman MT, Moysich K, Kjaer SK, Hogdall E, Jensen A, Goode EL, Fridley BL, Larson MC, Schildkraut JM, Palmieri RT, Cramer DW, Terry KL, Vitonis AF, Titus LJ, Ziogas A, et al.: Association between endometriosis and risk of histological subtypes of ovarian cancer: a pooled analysis of case–control studies. Lancet Oncol 2012, 13(4):385-394.
• [11]Anglesio MS, Carey MS, Kobel M, Mackay H, Huntsman DG: Clear cell carcinoma of the ovary: a report from the first ovarian clear cell symposium, june 24th, 2010. Gynecol Oncol 2011, 121(2):407-415.
• [12]Wiegand KC, Shah SP, Al-Agha OM, Zhao Y, Tse K, Zeng T, Senz J, McConechy MK, Anglesio MS, Kalloger SE, Yang W, Heravi-Moussavi A, Giuliany R, Chow C, Fee J, Zayed A, Prentice L, Melnyk N, Turashvili G, Delaney AD, Madore J, Yip S, McPherson AW, Ha G, Bell L, Fereday S, Tam A, Galletta L, Tonin PN, Provencher D, et al.: ARID1A mutations in endometriosis-associated ovarian carcinomas. N Engl J Med 2010, 363(16):1532-1543.
• [13]Jones S, Wang TL, Shih I, Mao TL, Nakayama K, Roden R, Glas R, Slamon D, Diaz LA Jr, Vogelstein B, Kinzler KW, Velculescu VE, Papadopoulos N: Frequent mutations of chromatin remodeling gene ARID1A in ovarian clear cell carcinoma. Sci (New York, NY) 2010, 330(6001):228-231.
• [14]Guan B, Wang TL, Shih Ie M: ARID1A, a factor that promotes formation of SWI/SNF-mediated chromatin remodeling, is a tumor suppressor in gynecologic cancers. Cancer Res 2011, 71(21):6718-6727.
• [15]Lowery WJ, Schildkraut JM, Akushevich L, Bentley R, Marks JR, Huntsman D, Berchuck A: Loss of ARID1A-associated protein expression is a frequent event in clear cell and endometrioid ovarian cancers. Int J Gynecol Cancer 2012, 22(1):9-14.
• [16]Kuo KT, Mao TL, Jones S, Veras E, Ayhan A, Wang TL, Glas R, Slamon D, Velculescu VE, Kuman RJ, Shih IeM: Frequent activating mutations of PIK3CA in ovarian clear cell carcinoma. Am J Pathol 2009, 174(5):1597-1601.
• [17]Campbell IG, Russell SE, Choong DY, Montgomery KG, Ciavarella ML, Hooi CS, Cristiano BE, Pearson RB, Phillips WA: Mutation of the PIK3CA gene in ovarian and breast cancer. Cancer Res 2004, 64(21):7678-7681.
• [18]Gonzalez-Angulo AM, Ferrer-Lozano J, Stemke-Hale K, Sahin A, Liu S, Barrera JA, Burgues O, Lluch AM, Chen H, Hortobagyi GN, Mills GB, Meric-Bernstam F: PI3K pathway mutations and PTEN levels in primary and metastatic breast cancer. Mol Cancer Ther 2011, 10(6):1093-1101.
• [19]McConechy MK, Ding J, Cheang MC, Wiegand KC, Senz J, Tone AA, Yang W, Prentice LM, Tse K, Zeng T, McDonald H, Schmidt AP, Mutch DG, McAlpine JN, Hirst M, Shah SP, Lee CH, Goodfellow PJ, Gilks CB, Huntsman DG: Use of mutation profiles to refine the classification of endometrial carcinomas. J Pathol 2012, 228(1):20-30.
• [20]Liang H, Cheung LW, Li J, Ju Z, Yu S, Stemke-Hale K, Dogruluk T, Lu Y, Liu X, Gu C, Guo W, Scherer SE, Carter H, Westin SN, Dyer MD, Verhaak RG, Zhang F, Karchin R, Liu CG, Lu KH, Broaddus RR, Scott KL, Hennessy BT, Mills GB: Whole-exome sequencing combined with functional genomics reveals novel candidate driver cancer genes in endometrial cancer. Genome Res 2012, 22(11):2120-2129.
• [21]Hennessy BT, Smith DL, Ram PT, Lu Y, Mills GB: Exploiting the PI3K/AKT pathway for cancer drug discovery. Nat Rev Drug Discov 2005, 4(12):988-1004.
• [22]Tibes R, Qiu Y, Lu Y, Hennessy B, Andreeff M, Mills GB, Kornblau SM: Reverse phase protein array: validation of a novel proteomic technology and utility for analysis of primary leukemia specimens and hematopoietic stem cells. Mol Cancer Ther 2006, 5(10):2512-2521.
• [23]Anderson CL, Wang Y, Rustandi RR: Applications of imaged capillary isoelectric focussing technique in development of biopharmaceutical glycoprotein-based products. Electrophoresis 2012, 33(11):1538-1544.
• [24]Stemke-Hale K, Gonzalez-Angulo AM, Lluch A, Neve RM, Kuo WL, Davies M, Carey M, Hu Z, Guan Y, Sahin A, Symmans WF, Pusztai L, Nolden LK, Horlings H, Berns K, Hung MC, van de Vijver MJ, Valero V, Gray JW, Bernards R, Mills GB, Hennessy BT: An integrative genomic and proteomic analysis of PIK3CA, PTEN, and AKT mutations in breast cancer. Cancer Res 2008, 68(15):6084-6091.
• [25]Tusher VG, Tibshirani R, Chu G: Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci USA 2001, 98(9):5116-5121.
• [26]Ho CM, Lin MC, Huang SH, Huang CJ, Lai HC, Chien TY, Chang SF: PTEN promoter methylation and LOH of 10q22-23 locus in PTEN expression of ovarian clear cell adenocarcinomas. Gynecol Oncol 2009, 112(2):307-313.
• [27]Iacovides DC, Johnson AB, Wang N, Boddapatti S, Korkola J, Gray JW: Identification and quantification of AKT isoforms and phosphoforms in breast cancer using a novel nanofluidic immunoassay. Mol Cell Proteomics 2013, 12(11):3210-3220.
• [28]Kamel D, Brady B, Tabchy A, Mills GB, Hennessy B: Proteomic classification of breast cancer. Curr Drug Targets 2012, 13(12):1495-1509.
• [29]Yang JY, Yoshihara K, Tanaka K, Hatae M, Masuzaki H, Itamochi H, Takano M, Ushijima K, Tanyi JL, Coukos G, Lu Y, Mills GB, Verhaak RG: Predicting time to ovarian carcinoma recurrence using protein markers. J Clin Invest 2013, 123(9):3740-3750.
• [30]Carey MS, Agarwal R, Gilks B, Swenerton K, Kalloger S, Santos J, Ju Z, Lu Y, Zhang F, Coombes KR, Miller D, Huntsman D, Mills GB, Hennessy BT: Functional proteomic analysis of advanced serous ovarian cancer using reverse phase protein array: TGF-beta pathway signaling indicates response to primary chemotherapy. Clin Cancer Res 2010, 16(10):2852-2860.
• [31]Cheung LW, Hennessy BT, Li J, Yu S, Myers AP, Djordjevic B, Lu Y, Stemke-Hale K, Dyer MD, Zhang F, Ju Z, Cantley LC, Scherer SE, Liang H, Lu KH, Broaddus RR, Mills GB: High frequency of PIK3R1 and PIK3R2 mutations in endometrial cancer elucidates a novel mechanism for regulation of PTEN protein stability. Cancer Discov 2011, 1(2):170-185.
• [32]Cizkova M, Susini A, Vacher S, Cizeron-Clairac G, Andrieu C, Driouch K, Fourme E, Lidereau R, Bieche I: PIK3CA mutation impact on survival in breast cancer patients and in ERalpha, PR and ERBB2-based subgroups. Breast Cancer Res 2012, 14(1):R28. BioMed Central Full Text
• [33]Vasudevan KM, Barbie DA, Davies MA, Rabinovsky R, McNear CJ, Kim JJ, Hennessy BT, Tseng H, Pochanard P, Kim SY, Dunn IF, Schinzel AC, Sandy P, Hoersch S, Sheng Q, Gupta PB, Boehm JS, Reiling JH, Silver S, Lu Y, Stemke-Hale K, Dutta B, Joy C, Sahin AA, Gonzalez-Angulo AM, Lluch A, Rameh LE, Jacks T, Root DE, Lander ES, Lluch A, et al.: AKT-independent signaling downstream of oncogenic PIK3CA mutations in human cancer. Cancer Cell 2009, 16(1):21-32.
• [34]Fadare O, Gwin K, Desouki MM, Crispens MA, Jones HW 3rd, Khabele D, Liang SX, Zheng W, Mohammed K, Hecht JL, Parkash V: The clinicopathologic significance of BAF250a (AIRD1A) expression in clear cell carcinomas of the endometrium. Mol Pathol 2013, 26(8):1101-1110.
• [35]Balbas-Martinez C, Rodriguez-Pinilla M, Casanova A, Dominguez O, Pisano DG, Gomez G, Lloreta J, Lorente JA, Malats N, Real FX: ARID1A alterations are associated with FGFR3-wild type, poor prognosis, urothelial bladder tumors. PLoS One 2013, 8(5):e62483.
• [36]Agarwal R, Carey M, Hennessy B, Mills GB: PI3K pathway-directed therapeutic strategies in cancer. Curr Opin Investig Drugs 2010, 11:615-628.
• [37]Mackay HJ, Brady MF, Oza AM, Reuss A, Pujade-Lauraine E, Swart AM, Siddiqui N, Colombo N, Bookman MA, Pfisterer J, du Bois A, Gynecologic Cancer InterGroup: Prognostic relevance of uncommon ovarian histology in women with stage III/IV epithelial ovarian cancer. Int J Gynecol Cancer 2010, 20(6):945-952.
• [38]Takakura S, Takano M, Takahashi F, Saito T, Aoki D, Inaba N, Noda K, Sugiyama T, Ochiai K, Japanese Gynecologic Oncology G: Randomized phase II trial of paclitaxel plus carboplatin therapy versus irinotecan plus cisplatin therapy as first-line chemotherapy for clear cell adenocarcinoma of the ovary: a JGOG study. Int J Gynecol Cancer 2010, 20(2):240-247.