Abstract 2386: Molecular-cytogenetic analysis of the maternal embryonic leucine-zipper kinase (MELK) oncogene in cancer

Abstract

Abstract Introduction: The MELK gene is located on chromosome 9p13.2 and encodes a serine/threonine kinase that is involved in cell cycle regulation and apoptosis. The MELK protein is abundantly expressed in various tumors including breast cancer, and is associated with poor patient survival. MELK is an attractive molecular target due to its critical roles in cancer stem cell maintenance. However, the function and mechanism of MELK overexpression remain elusive. Gene amplification/copy number gain is one of the potential mechanisms underlying MELK overexpression. In this pilot study, we used fluorescence in situ hybridization (FISH) to detect MELK gene amplification and analyzed other MELK gene copy number alterations (CNA) in endometrial, ovarian, and breast cancer cell lines. Methods: To date, 3 endometrial, 1 ovarian and 13 breast cancer cell lines of different subtypes were screened for MELK CNA. Of these, six breast cancer cell lines (BT20, MCF7, MDAMB231, SKBR3, BT549, and T47D) had known status of MELK: all carried high levels of protein expression by Western Blot. Cell harvest and metaphase slides were prepared according to standard protocols. MELK FISH probe (BAC RP11-450B8) directly labeled with SpectrumGreen was developed and validated in our laboratory. The chromosome 9 centromere enumeration probe (CEP9) labeled with SpectrumOrange (Abbott Molecular) was used to distinguish gene amplification from gene polysomy (gene and chromosome copy number gain ≥ 3). Mean copies of each signal per cell and copy number ratios per cell were calculated. Ratio of MELK to CEP9 ≥ 2.0 was a cut off point for MELK amplification. Results: Across all cell lines, the ratios of MELK to CEP9 ranged between 0.5 to 1.7, showing no amplification. However, 10 cell lines (59%) displayed 3-8 copies of MELK due to polysomy for chromosome 9, 4 (24%) harbored both gene polysomy and structural alterations (duplications and translocations); only two cell lines exhibited normal MELK and CEP9 copies and one presented with heterozygous deletion of MELK. Interestingly, all 6 MELK- overexpressed cell lines showed either gene polysomy or complex chromosomal alterations or both. Conclusions: Our pilot study suggests that amplification of MELK gene does not occur or is a rare event in human cancer cells in vitro. However, recurrent MELK structural alterations (duplications and translocations) and gene polysomy can cause elevated protein expression in breast cancer cell lines. MELK FISH study in primary tumors from breast cancer patients will be presented. Supported by: American Cancer Society Citation Format: Ashley Hardeman, Tatyana A. Grushko, Maria J. Gomez, Mariann Coyle, Yusuke Nakamura, Olufunmilayo I. Olopade. Molecular-cytogenetic analysis of the maternal embryonic leucine-zipper kinase (MELK) oncogene in cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2386. doi:10.1158/1538-7445.AM2014-2386