Epidermal growth factor receptor (EGFR) inhibitors have demonstrated efficacy in squamous cell carcinoma of the head and neck (SCCHN). In addition to EGFR, other ErbB family members are expressed and activated in SCCHN. Afatinib is an ErbB family blocker that has been approved for treating patients with EGFR-mutated nonsmall cell lung cancer. We sought to determine the efficacy of afatinib in preclinical models and compare this to other EGFR-targeted agents. Afatinib efficacy was characterized in a panel of ten SCCHN cell lines and found to be most effective against cell lines amplified for EGFR. Afatinib had lower IC50 values than did gefitinib against the same panel. Two EGFR-amplified cell lines that are resistant to gefitinib are sensitive to afatinib. Cetuximab was not found to have a synergistic effect with afatinib either in vitro or in vivo. Both afatinib and cetuximab were effective in tumor xenograft model. Afatinib is an effective agent in SCCHN especially in models with EGFR amplification.

Maternal embryonic leucine-zipper kinase (MELK) regulates cell cycle progression and is highly expressed in many cancers. The molecular mechanism of MELK dysregulation has not been determined in aggressive forms of breast cancer, such as triple negative breast cancer (TNBC). To evaluate molecular markers of MELK aberrations in aggressive breast cancer, we assessed MELK gene amplification and expression in breast tumors. MELK mRNA expression is highly up-regulated in basal-like breast cancer (BLBC), the major molecular subtype of TNBC, compared to luminal or other subtypes of breast tumors. MELK copy number (CN) gains are significantly associated with BLBC, whereas no significant association of CpG site methylation or histone modifications with breast cancer subtypes was observed. Accordingly, the CN gains appear to contribute to an increase in MELK expression, with a significant correlation between mRNA expression and CN in breast tumors and cell lines. Furthermore, immunohistochemistry (IHC) assays revealed that both nuclear and cytoplasmic staining scores of MELK were significantly higher in invasive ductal carcinoma (IDC) tumors compared to ductal carcinoma in situ (DCIS) and normal breast tissues. Our data showed that upregulation of MELK in BLBC may be in part driven by CN gains, rather than epigenetic modifications, indicating a potential for overexpression and CN gains of MELK to be developed as a diagnostic and prognostic marker to identify patients who have more aggressive breast cancer.

Women of African ancestry have the highest mortality from triple-negative breast cancer (TNBC) of all racial groups. To understand the genomic basis of breast cancer in the populations, we previously conducted genome-wide association studies (GWAS) and identified single nucleotide polymorphisms (SNPs) associated with breast cancer in Black women. In this study, we investigated the functional significance of the top associated SNP rs13074711. We found the SNP served as an enhancer variant and regulated TNFSF10 (TRAIL) expression in TNBC cells, with a significant association between the SNP genotype and TNFSF10 expression in breast tumors. Mechanistically, rs13074711 modulated the binding activity of c-MYB at the motif and thereby controlled TNFSF10 expression. Interestingly, TNFSF10 expression in many cancers was consistently lower in African Americans (AA) compared to European Americans (EA). Furthermore, TNFSF10 expression in TNBC was significantly correlated with expression of antiviral immune genes and was regulated by type I interferons (IFNs). Accordingly, loss of TNFSF10 resulted in a profound decrease in apoptosis of TNBC cells in response to type I IFNs and poly (I:C), a synthetic analogue of double stranded virus. Lastly, in a syngeneic mouse model of breast cancer, TNFSF10-deficiency in breast tumors decreased tumor-infiltrated CD4+ and CD8+ T cell quantities. Collectively, our results suggested that TNFSF10 plays an important role in the regulation of antiviral immune responses in TNBC, and the expression is in part regulated by a genetic variant associated with breast cancer in Black women. Our results underscore the important contributions of genetic variants to immune defense mechanisms.

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