Abstract
Functional reactivation of p53 pathway, although arduous, can potentially provide a broad-based strategy for cancer therapy owing to frequent p53 inactivation in human cancer. Using a phosphoprotein-screening array, we found that Benzyl Isothiocynate, (BITC) increases p53 phosphorylation in breast cancer cells and reveal an important role of ERK and PRAS40/MDM2 in BITC-mediated p53 activation. We show that BITC rescues and activates p53-signaling network and inhibits growth of p53-mutant cells. Mechanistically, BITC induces p73 expression in p53-mutant cells, disrupts the interaction of p73 and mutant-p53, thereby releasing p73 from sequestration and allowing it to be transcriptionally active. Furthermore, BITC-induced p53 and p73 axes converge on tumor-suppressor LKB1 which is transcriptionally upregulated by p53 and p73 in p53-wild-type and p53-mutant cells respectively; and in a feed-forward mechanism, LKB1 tethers with p53 and p73 to get recruited to p53-responsive promoters. Analyses of BITC-treated xenografts using LKB1-null cells corroborate in vitro mechanistic findings and establish LKB1 as the key node whereby BITC potentiates as well as rescues p53-pathway in p53-wild-type as well as p53-mutant cells. These data provide first in vitro and in vivo evidence of the integral role of previously unrecognized crosstalk between BITC, p53/LKB1 and p73/LKB1 axes in breast tumor growth-inhibition.
Highlights
With approximately 50 to 55% human cancers exhibiting loss of wild-type p53 activity, tumor suppressor p53 is the most commonly silenced or mutated gene in cancer[1,2]
Our results show that Benzyl Isothiocyanate (BITC) treatment effectively inhibits the growth of breast cancer cells in vitro and in vivo but our phosphoprotein array studies led us to novel discoveries that extend beyond the popular notion of inhibiting oncogenic signals to achieve tumor-inhibition
extracellular-signal-regulated kinase (ERK) is popularly known for its pro-proliferation role but this study illustrates that BITC despite effectively inhibiting breast tumor growth, increases ERK-phosphorylation
Summary
With approximately 50 to 55% human cancers exhibiting loss of wild-type p53 activity, tumor suppressor p53 is the most commonly silenced or mutated gene in cancer[1,2]. BITC suppresses proliferation and induces apoptosis in multiple cancer-types[14,15], including breast cancer[16,17] but molecular understanding of BITC-mediated signaling-networks is still emerging. Modulation of phosphorylation-status of key proteins including kinases, oncogenes and tumor-suppressors is an important regulatory mechanism with functional consequences; in the present study we utilize phosphorylation-array to gain insight into the intricacies of BITC-induced signaling pathways and their impact on p53-signaling network. We designed this study to examine the role of tumor-suppressors p53 and p73 and the underlying molecular mechanisms how BITC-mediated activation of p53/p73 leads to growth-inhibition of breast cancer cells. Our study uncovers that BITC concertedly modulates tumor-suppressors- p53, p73 and LKB1 and activates p53-signaling networks in p53-wild-type breast cancer and functionally restores p53-signaling in p53-mutant breast cancer. Our study suggests that BITC could be a useful strategy to potentiate p53-signaling in p53-wild-type cells as well as rescue p53-signaling in p53-mutant cells offering a broad-based strategy that can be useful for multiple cancer types
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