Citations

Abstract Lung cancer is the most lethal malignancy around the world. In Hong Kong, chronic tobacco use is prevalent in male but <20% of female patients smoke. Epidermal growth factor receptor (EGFR) mutations affect 30% and 67% of male and female lung adenocarcinomas, respectively. Treatment responses to tyrosine kinase inhibitors (TKI) are not uniform and all patients eventually develop drug resistance. Mutation screening of immediate pre-treatment tumors is the most ideal for assessing drug targets but in practice, excision specimens of primary or metastatic sites are more efficient in terms of tumor quantity, timeliness and patient acceptance. However, information on the applicability of this approach and correlation with the clinical outcome is limited. For this purpose, we compared the whole exome mutation profiles of 39 excised EGFR mutant cancers treated with first line TKI with the clinical response type, including 15 stage I/II and 24 stage III/IV tumors. They included 16 non-responders (NR) defined clinically as those with stable or progressive disease, and 23 responders (R) defined by partial or complete tumor shrinkage. Considering only the non-synonymous SNV and INDEL mutations of coding regions of known actionable targets and genes listed in both the CGC and COSMIC cancer databases, the tumors harbored 54 mutated genes including 26 recurrently mutated and 28 genes involving only 1 tumor. Excluding EGFR, TP53 was the most commonly mutated gene occurring in 25/39 (64.1%) cases. Most known resistant genes involved in the EGFR and bypass signaling network showed mutations only in the NR group including recurrent mutations of PTEN (3/16), PIK3CA (2/16) and NF1 (2/16), and single case mutations of AKT1, ALK, RAF1 and KDR. Two EGFR network candidates showed mutations in both the NR and R groups, including HGF (2/16 NR, 1/23 R) and ROS1 (1 case in either group). Four post-treatment (post-TKI) tumors of acquired resistance were also analyzed, all of which showed EGFR T790M while no pre-treatment NR or R tumor harbored this mutation. Notably, mutations in the β-catenin pathway were prominent in the NR and post-TKI tumors, including APC (2/16 NR), CTNNB1 (1/16 NR, 1/4 post-TKI) and c-MYC (1/16 NR, 1/4 post-TKI) while they were not detected in the R tumors. Also, nonsense mutations of ARID1A was observed in 2/16 NR but none of the R tumors. In summary, our findings revealed candidate TKI resistant mutations involving the EGFR and bypass signaling networks in pre-treatment excision specimens particularly in non-responding patients. The EGFR T790M was not detectable in pre-treatment samples but was prevalent in post-TKI treated cancers. While this study is limited by its small cohort size, the findings indicate deep sequencing analysis of pre-treatment excision specimens of EGFR-mutant lung adenocarcinomas is warranted for detection of resistant mutations and predicting treatment response. Note: This abstract was not presented at the meeting. Citation Format: Xu-yuan Gao, Hang Xu, James CM Ho, Oscar SH Chan, Feng Xu, Junwen Wang, Victor HF Lee, Vicky PC Tin, Zhijie Xiao, Siqi Wang, Judy WP Yam, Maria P. Wong. Whole-exome sequencing reveals tyrosine kinase-resistant mutations in pretreatment EGFR-mutant lung adenocarcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-274. doi:10.1158/1538-7445.AM2017-LB-274

Tumor-initiating cells (TIC) are dynamic cancer cell subsets that display enhanced tumor functions and resilience to treatment but the mechanism of TIC induction or maintenance in lung cancer is not fully understood. In this study, we show the calcium pathway transcription factor NFATc2 is a novel regulator of lung TIC phenotypes, including tumorspheres, cell motility, tumorigenesis, as well as in vitro and in vivo responses to chemotherapy and targeted therapy. In human lung cancers, high NFATc2 expression predicted poor tumor differentiation, adverse recurrence-free and cancer-specific overall survivals. Mechanistic investigations identified NFATc2 response elements in the 3’ enhancer region of SOX2, and NFATc2/SOX2 coupling upregulates ALDH1A1 by binding to its 5’ enhancer. Through this axis, oxidative stress induced by cancer drug treatment is attenuated, leading to increased resistance in a mutation-independent manner. Targeting this axis provides a novel approach for the long-term treatment of lung cancer through TIC elimination.

Abstract We have previously reported that the protease activity of uPA delays tumor progression in a syngeneic model of breast cancer, and that the inhibitory effects are due to the protease activity of uPA (Merchan et. al, JNCI, 2006: 7; 98(11)). We also reported that changes in the uPA/PAI-1 balance favoring the proteolytic activity of uPA are associated with delayed tumor growth and reduction in lung metastases in vivo (Jing, et al, AACR 2009 meeting, #1124). The aim of this study was to extend the above findings in other in vivo syngeneic cancer models and to characterize the changes in gene expression mediated by uPA overexpression. We generated stably uPA overexpressing murine renal cancer (RENCA) and murine colon cancer (MC-38) cell lines using lentiviral vectors carrying m-uPA and selecting high uPA expressing clones for in vivo studies. uPA protein expression and in vitro growth was similar between the uPA overexpressing and control cancer cells. Similar with our findings in the 4T1 model, mice harboring RENCA tumors overexpressing uPA had reduced tumor growth than controls. Conversely, mice carrying uPA-overexpressing MC-38 tumors had significantly enhanced tumor growth compared to controls. To understand the causes for the opposite in vivo effects of uPA overexpressing RENCA and MC-38 tumors, we first compared uPA's proteolytic activity vs. protein concentration between both cell lines. To our surprise, the uPA proteolytic activity of (uPA overexpressing) MC-38 conditioned medium (CM) was significantly lower (> 15-fold) than that of stable RENCA cell lines even though uPA protein levels were comparable. To assess if the differences in uPA proteolytic activity between MC38 and RENCA transfectants was due to differential endogenous PAI-1 expression, we measured PAI-1 levels in MC38 and RENCA transfectants CM (ELISA). We found that PAI-1 expression in MC 38 was significantly higher (30-fold) than RENCA cells. The above data strongly suggest that in MC-38 tumors, uPA overexpression promoted tumor growth in part due to high endogenous PAI-1 levels. On the other hand, in the RENCA model, uPA overexpression (and low PAI-1 expression) was associated with significantly enhanced proteolytic activity and delayed tumor growth. Gene expression arrays of early murine mammary tumors overexpressing active uPA, showed significant tumor down regulation of several members of the MMP family, which may explain the in vivo antitumor effects. The above data suggest that the pro-or anti tumorigenic/metastatic effects of uPA overexpression depend on its proteolytic activity, and indicate that the status of endogenous tumor PAI-1 may regulate uPA's effects on tumor progression and metastases. The above data may help reconcile contradictory experimental and clinical findings on the role of proteases and protease inhibitors on tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 516.