Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug 1;21(15):3480-91.
doi: 10.1158/1078-0432.CCR-14-3286. Epub 2015 Apr 15.

An Integrated Molecular Analysis of Lung Adenocarcinomas Identifies Potential Therapeutic Targets among TTF1-Negative Tumors, Including DNA Repair Proteins and Nrf2

Robert J G Cardnell  1 , Carmen Behrens  1 , Lixia Diao  2 , YouHong Fan  1 , Ximing Tang  3 , Pan Tong  2 , John D Minna  4 , Gordon B Mills  5 , John V Heymach  1 , Ignacio I Wistuba  3 , Jing Wang  2 , Lauren A Byers  6
Affiliations

An Integrated Molecular Analysis of Lung Adenocarcinomas Identifies Potential Therapeutic Targets among TTF1-Negative Tumors, Including DNA Repair Proteins and Nrf2

Robert J G Cardnell et al. Clin Cancer Res. .

Abstract

Purpose: Thyroid transcription factor-1 (TTF1) immunohistochemistry (IHC) is used clinically to differentiate primary lung adenocarcinomas (LUAD) from squamous lung cancers and metastatic adenocarcinomas from other primary sites. However, a subset of LUAD (15%-20%) does not express TTF1, and TTF1-negative patients have worse clinical outcomes. As there are no established targeted agents with activity in TTF1-negative LUAD, we performed an integrated molecular analysis to identify potential therapeutic targets.

Experimental design: Using two clinical LUAD cohorts (274 tumors), one from our institution (PROSPECT) and The Cancer Genome Atlas, we interrogated proteomic profiles (by reverse phase protein array, RPPA), gene expression, and mutational data. Drug response data from 74 cell lines were used to validate potential therapeutic agents.

Results: Strong correlations were observed between TTF1 IHC and TTF1 measurements by RPPA (Rho = 0.57, P < 0.001) and gene expression (NKX2-1, Rho = 0.61, P < 0.001). Established driver mutations (e.g., BRAF and EGFR) were associated with high TTF1 expression. In contrast, TTF1-negative LUAD had a higher frequency of inactivating KEAP1 mutations (P = 0.001). Proteomic profiling identified increased expression of DNA repair proteins (e.g., Chk1 and the DNA repair score) and suppressed PI3k/mTOR signaling among TTF1-negative tumors, with differences in total proteins confirmed at the mRNA level. Cell line analysis showed drugs targeting DNA repair to be more active in TTF1-low cell lines.

Conclusions: Combined genomic and proteomic analyses demonstrated infrequent alteration of validated lung cancer targets (including the absence of BRAF mutations in TTF1-negative LUAD), but identified novel potential targets for TTF1-negative LUAD, including KEAP1/Nrf2 and DNA repair pathways.

PubMed Disclaimer

Conflict of interest statement

Conflicts of interest: The other authors report no relevant conflicts of interest.

Figures

Figure 1
Figure 1. Robust quantification of TTF1/NKX2-1 in clinical samples
TTF1 as measured by IHC (the gold standard) and by RPPA correlate well (A) in the PROSPECT cohort (n=94). NKX2-1 mRNA expression and TTF1 IHC scores also correlate well (B) across a larger PROSPECT cohort (n=152) for which both gene expression and IHC data were available. Representative images of 0, 1+, 2+ and 3+ TTF1 IHC stating correlate well to IHC, RPPA and mRNA scores (C). By way of cross validation, NKX2-1 gene expression and TTF1 RPPA also correlated well (D, n=94).
Figure 2
Figure 2. TTF1-negative defined by RPPA is associated with worse clinical outcome
Density plot analysis of both cohorts reveals are bimodal distribution of TTF1 expression by RPPA (A) categorizing LUAD biopsy samples into new TTF1-negative and –positive groups. TTF1-negative disease is observed across stages I-III (B) and all smoking statuses (C). As previously described TTF1-negative disease is associated with worse overall survival (D).
Figure 3
Figure 3. Association between TTF1 and mutation status
Comparison of TTF1 protein levels between wild type and mutant samples reveals a distinct association between TTF-1 positive disease and mutations (A) in EGFR (TCGA and PROSPECT), PIK3CA (TCGA and PROSPECT, and BRAF (TCGA). Particularly notable is the absence of TTF-1 negative samples in the BRAF mutant group. TCGA TTF1-negative samples show an enrichment of KEAP1 mutations (B & C, p=0.001). KEAP1 negatively regulates NRF2; as predicted analysis of Nrf2 reveals higher expression in TTF1-negative LUAD (D, p=0.001).
Figure 4
Figure 4. Protein signature of TTF-1 negative LUAD
Unsupervised hierarchal clustering of protein expression (RPPA) in two clinical cohorts of LUAD (A). Comparison of protein expression between bimodally distributed TTF1-negative and positive LUAD identifies 25 proteins common to both cohorts (B & D, p<0.05). Validation of the 18 total proteins identified using mRNA expression analysis (p<0.05) identifies a five component signature commonly upregulated in TTF1-negative disease across both cohorts and platforms (D). TTF1-negative LUAD also have a higher DNA repair score (20) identifying agents that target DNA repair as potential therapeutic target for these patients (C)
Figure 5
Figure 5. Therapeutic targets in TTF1-low LUAD cell lines
Comparison of the top and bottom thirds of cell lines by NKX2-1 expression (A) to IC50 values from drug sensitivity databases identify TTF1-low LUAD cell lines as being more sensitive to agents that impair DNA repair (B & C). Potential mechanisms for the higher expression of thymidylate synthase and DNA repair proteins via E2F1 in TTF1-negative LUAD and elevated PI3K/MAPK signaling in TTF1-positive LUAD (D) combining genetic and protein observations. Proteins/genes in red are higher/more active in TTF1-negative, those in black in TTF-positive LUAD. Copy number loss of SOX2 or gain of CDK2NA can both lead to increased E2F1.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bingle CD. Thyroid transcription factor-1. Int J Biochem Cell Biol. 1997;29:1471–3. - PubMed
    1. Yamaguchi T, Hosono Y, Yanagisawa K, Takahashi T. NKX2-1/TTF-1: an enigmatic oncogene that functions as a double-edged sword for cancer cell survival and progression. Cancer Cell. 2013;23:718–23. - PubMed
    1. Tang X, Kadara H, Behrens C, Liu DD, Xiao Y, Rice D, et al. Abnormalities of the TITF-1 lineage-specific oncogene in NSCLC: implications in lung cancer pathogenesis and prognosis. Clin Cancer Res. 2011;17:2434–43. - PMC - PubMed
    1. Solis LM, Behrens C, Raso MG, Lin HY, Kadara H, Yuan P, et al. Histologic patterns and molecular characteristics of lung adenocarcinoma associated with clinical outcome. Cancer. 2012;118:2889–99. - PMC - PubMed
    1. Takeuchi T, Tomida S, Yatabe Y, Kosaka T, Osada H, Yanagisawa K, et al. Expression profile-defined classification of lung adenocarcinoma shows close relationship with underlying major genetic changes and clinicopathologic behaviors. J Clin Oncol. 2006;24:1679–88. - PubMed

Publication types

MeSH terms