Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms. The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover, phospho-Akt levels are increased in most clinical specimens obtained from EGFR-mutant non-small-cell lung cancer patients with acquired EGFR tyrosine kinase inhibitor resistance. Our findings provide a rationale for clinical trials testing Akt and EGFR inhibitor co-treatment in patients with elevated phospho-Akt levels to therapeutically combat the heterogeneity of EGFR tyrosine kinase inhibitor resistance mechanisms.

Highlights d Cholesterol biosynthesis is essential for breast cancer stem cell propagation d Expression of cholesterol synthesis proteins correlates with TNBC patient outcome d Inhibition of cholesterol biosynthesis reduces mammosphere formation

Triple-negative breast cancer (TNBC) is a heterogeneous subtype with varying disease outcomes. Tumor-infiltrating lymphocytes (TILs) are frequent in TNBC and have been shown to correlate with outcome, suggesting an immunogenic component in this subtype. However, other factors intrinsic to the cancer cells may also influence outcome. To identify proteins and molecular pathways associated with recurrence in TNBC, 34 formalin-fixed paraffin-embedded (FFPE) primary TNBC tumors were investigated by global proteomic profiling using mass spectrometry. Approximately, half of the patients were lymph node-negative and remained free of local or distant metastasis within 10 y follow-up, while the other half developed distant metastasis. Proteomic profiling identified >4,000 proteins, of which 63 exhibited altered expression in primary tumors of recurrence versus recurrence-free patients. Importantly, downregulation of proteins in the major histocompatibility complex (MHC) class I antigen presentation pathways were enriched, including TAP1, TAP2, CALR, HLA-A, ERAP1 and TAPBP, and were associated with significantly shorter recurrence-free and overall survival. In addition, proteins involved in cancer cell proliferation and growth, including GBP1, RAD23B, WARS and STAT1, also exhibited altered expression in primary tumors of recurrence versus recurrence-free patients. The association between the antigen-presentation pathway and outcome were validated in a second sample set of 10 primary TNBC tumors and corresponding metastases using proteomics and in a large public gene expression database of 249 TNBC and 580 basal-like breast cancer cases. Our study demonstrates that downregulation of antigen presentation is a key mechanism for TNBC cells to avoid immune surveillance, allowing continued growth and spread.

Prognostic and predictive biomarkers of disease and treatment outcome are needed to ensure optimal treatment of patients with triple-negative breast cancer (TNBC). In a mass spectrometry-based global proteomic study of 44 formalin-fixed, paraffin-embedded (FFPE) primary TNBC tumors and 10 corresponding metastases, we found that Cytochrome P450 reductase (CYPOR) expression correlated with patient outcome. The correlation between CYPOR expression and outcome was further evaluated in a Danish cohort of 113 TNBC patients using immunohistochemistry and publicly available gene expression data from two cohorts of TNBC and basal-like breast cancer patients, respectively (N = 249 and N = 580). A significant correlation between high CYPOR gene expression and shorter recurrence-free survival (RFS), but not overall survival, was found in the cohort of 249 TNBC patients (p = 0.018, HR = 1.77, 95% CI 1.1-2.85), and this correlation was recapitulated in a cohort of 580 basal-like breast cancer patients (p = 0.018, HR = 1.4, 95% CI 1.06-1.86). High CYPOR protein expression was also associated with shorter RFS in the cohort of 113 TNBC patients (p = 0.017, HR = 2.73, 95% CI 1.20-6.19), particularly those who were lymph node tumor-negative (p = 0.029, HR = 5.22). Multivariate Cox regression analysis identified CYPOR as an independent prognostic factor for shorter RFS in TNBC patients (p = 0.032, HR = 2.19, 95% CI 1.07-4.47). Together, these data suggest high expression of CYPOR as an independent prognostic biomarker of shorter RFS, which could be used to identify patients who should receive more extensive adjuvant treatment and more aggressive surveillance.

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Triple-negative breast cancer (TNBC) represents a major subtype of breast cancer (BC) and no biomarkers or targeted treatments are available. Retrospective proteomic analysis of formalin-fixed paraffin-embedded (FFPE) BC tissue has the potential to identify novel biomarkers and pathways associated with disease recurrence in TNBC, since sample data can be coupled with clinical data, including treatment and patient outcome. Primary TNBC tissue of patients who experienced distant metastasis or no recurrence within a 10 year follow-up period was retrieved to investigate whether proteomics using liquid chromatography tandem mass spectrometry (LC-MS/MS) can identify proteins and pathways associated with their metastatic potential. FFPE material on PEN-membrane slides were deparaffinized according to standard procedure with xylene/ethanol and stained with hematoxylin and eosin. Cancer cells were dissected by UV laser capture microdissection (LCM) and subjected to protein extraction in triethylammonium bicarbonate/acetonitrile buffer by heat-induced antigen retrieval. Extracted proteins were trypsinized and generated peptides were isobarically labelled with 6-plex tandem mass tags, combined 1:1 and subjected to hydrophilic interaction liquid chromatography LC-MS/MS. Preliminary experiments identified >500 unique proteins from BC FFPE tissue, verifying a robust setup. Cancer cells from primary TNBC FFPE tissues of 15 patients who experienced distant metastatic and 20 patients who did not have recurrence within ten years were isolated by UV-LCM and analyzed according to the above setup. The patients were stratified into four working groups based upon their menopausal status (pre or post) and their recurrence (recurrence or no-recurrence) outcome. The median aged varied from 42.2 to 63.4 years between the four groups with most tumors being diagnosed as T1 or T2 (TNM classification system). All tumors were grade 2 or higher at diagnosis, with no distant metastasis present prior to removal of the primary tumor. We identified 2564 high-confident proteins across the 35 patient samples with ≥2 unique peptides. Statistical analysis was done in RStudio and GproX and revealed distinct molecular profiles capable of separating metastatic from non-metastatic patients with a p-value ≤0.05. Furthermore three proteins were found to be differentially regulated between the two groups with a p-value <0.001 making these highly interesting targets in the prevention of metastatic lesions in TNBC. Research funded by the Danish Research Council, the A.P. Møller foundation and The Regional Strategically Research Council of Southern Denmark Citation Format: Martin H. Pedersen, Brian L. Hood, Thomas P. Conrads, Henrik J. Ditzel, Rikke Leth-Larsen. Quantitative proteomics of formalin-fixed paraffin-embedded, primary triple-negative breast cancer tissues of patients who experienced distant metastasis or no recurrence. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C10.