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Regulatory effects of TDP2 in non-small cell lung carcinoma (NSCLC)

Annie Chou

Appointment Period: 2015, Grant Year: [29]

Annie Chou

TDP2 (also known as TTRAP/EAPII) is both a DNA repair and a signal transduction protein, being implicated as an inhibitor of the TRAF/TNF receptor and NF-κB pathways and as a DNA damage repair enzyme that resolves protein-linked double-stranded breaks mediated by topoisomerase 2 through its phosphodiesterase (PDE) activity. Notably, TDP2 is expressed at higher levels in over 80% of non-small cell lung carcinoma (NSCLC) lines compared to immortalized bronchial epithelia. shRNA knockdown of TDP2 in NSCLC lines increases apoptosis and inhibits proliferation whereas reintroduction or de novo expression restores and accelerates growth, both in culture and in mouse xenographs. TDP2 expression in NSCLC lines has been positively correlated with ERK/MAPK signaling, marked by increased phosphorylation of Raf1, MEK and ERK. The opposite phenotype is observed in osteosarcoma (OS) cell lines, where TDP2 expression results in PDE-dependent inhibition of cell proliferation. This dual functionality of TDP2 suggests this protein may be a pivotal regulator governing cell proliferation vs. death. How TDP2 regulates these processes differently remains unknown; understanding how TDP2 accelerates NSCLC while inhibiting OS will be important in evaluating TDP2 as a chemotherapeutic target. Our research on the regulation and regulatory effects of TDP2 will evaluate its potential as a candidate for targeted therapies.

TDP2 has been observed to shift from a nuclear to a cytoplasmic localization during NSCLC progression, and this shift in localization might be key in regulating the effects of TDP2 on cell proliferation by spatially differentiating it as a signaling molecule in cytoplasmic growth pathways and a DNA repair enzyme in the nucleus. Through sequence analysis, I have identified and characterized a novel nuclear localization signal (NLS) in the N-terminus of TDP2. My evidence suggests this NLS is physiologically relevant and can contribute to the observed relocalization, since many NSCLC lines express a 43 kDa variant isoform in addition to the full-length 49 kDa isoform which, through antibody screening, was deemed to be an N-terminal truncation variant lacking the NLS.

My first goal will be to identify the Tdp2 isoforms present in NSCLC. I will characterize the 43 kDa isoform and its effects on proliferation in NSCLC lines. Our hypothesis is that the 43 kDa form arises from an alternative translation start site, since TDP2 has an ATG at codon 54 flanked by the 2nd strongest Kozak consensus, predicting a ~5.5 kDa shorter form lacking the NLS. We are currently using mass spectrometry (MS) analysis to identify peptides differentiating the two isoforms. We will also utilize RACE-PCR, northern analysis and in vitro transcription/translation assays to test whether the variant results from alternative splicing, alternative translational start site, or proteolytic cleavage.

Second, I will characterize the phenotype of cells expressing one or both isoforms and determine the effectiveness of TDP2 phosphodiesterase inhibition. The isoforms will be evaluated for PDE activity using in vitro assays, and their effects on proliferation and apoptosis will be characterized in immortalized Tdp2-/- MEFs (from Felipe Cortés’ lab) re-expressing Tdp2 isoforms and mutants using retroviral vectors. In addition, I will use the CRISPR/Cas9 system to modify the endogenous TDP2 gene in NSCLC, ovarian, and OS cell lines so that only either the long or short isoform, or neither, is expressed. We will evaluate the effectiveness of toxoflavins and deazaflavins, selective inhibitors of TDP2 PDE activity, on cell lines expressing both vs. individual isoforms.

PUBLICATIONS (resulting from this training)

Trainee recently appointed to grant: publications are still in progress.