CRISPR-Cas9 for selective targeting of somatic mutations in pancreatic cancers.

Somatic mutations are desirable targets for selective elimination of cancer, yet most are found within noncoding regions. We have adapted the CRISPR-Cas9 gene editing tool as a novel, cancer-specific killing strategy by targeting the subset of somatic mutations that create protospacer adjacent motifs (PAMs), which have evolutionally allowed bacterial cells to distinguish between self and non-self DNA for Cas9-induced double strand breaks. Whole genome sequencing (WGS) of paired tumor minus normal (T-N) samples from three pancreatic cancer patients (Panc480, Panc504, and Panc1002) showed an average of 417 somatic PAMs per tumor produced from single base substitutions. Further analyses of 591 paired T-N samples from The International Cancer Genome Consortium found medians of ∼455 somatic PAMs per tumor in pancreatic, ∼2800 in lung, and ∼3200 in esophageal cancer cohorts. Finally, we demonstrated 69-99% selective cell death of three targeted pancreatic cancer cell lines using 4-9 sgRNAs designed using the somatic PAM discovery approach. We also showed no off-target activity from these tumor-specific sgRNAs in either the patient's normal cells or an irrelevant cancer using WGS. This study demonstrates the potential of CRISPR-Cas9 as a novel and selective anti-cancer strategy, and supports the genetic targeting of adult cancers.

Hsp90 chaperone facilitates E2F1/2-dependent gene transcription in human breast cancer cells.

The 90 kDa heat shock protein, Hsp90, is involved in the conformational stabilization and functional maturation of diverse cancer-promoting proteins. To date, more than 300 Hsp90 clients have identified, suggesting that Hsp90 plays a central role in deciding cancer cell fate. In this study, we present the nuclear functions of Hsp90 in regulating the E2F-dependent gene transcription. We show that the conformation specific Hsp90 inhibitor, 17AAG decreases the total cellular E2F levels more selectively in cancer cells than transformed cells. With the help of coimmunoprecipitation experiments, we show that Hsp90 interacts with E2F1 and E2F2 in cancer cells, whereas in transformed cells, only E2F1 interacts with Hsp90. Retention of E2F2 in the nucleus of cancer cells upon MG132 combination with 17AAG has suggested that Hsp90 is required for E2F2 stability and function. The HDAC6 inhibitor tubacin treatment did not interfere with E2F1/2 stability and nuclear accumulation. However, the HDAC3 inhibitor, RGFP966 treatment, decreased nuclear E2F1/2 and its target gene expression. The nuclear accumulation of E2F1 and E2F2 upon cell cycle inhibition correlated with decreased acetylated Hsp90. We expose the nuclear functions of Hsp90 in facilitating the cell cycle progression through stabilizing E2F1/2.

Hsp90 regulates HDAC3-dependent gene transcription while HDAC3 regulates the functions of Hsp90.

Deregulated DNA methylation and post-translational histone modifications are majorly associated with cancer progression. Histone modification regulates the gene expression patterns that contribute to the emergence of sporadic cancers. Histone deacetylases (HDACs) act as erasers of acetylation marks, and their functions are often deregulated in cancer. Since non-histone proteins can also act as substrates for HDACs, identifying their involvement in vital regulatory molecules contributing to cancer progression is essential. Hsp90 is a cancer chaperone that contributes to kinase evolution and, thus, cellular adaptations. Acetylated Hsp90 loses its chaperoning functions and client protein interactions. Robust cell proliferation is one of the hallmarks of cancer. However, Hsp90 involvement in cancer promoting gene transcription is less understood. Using human breast cancer cells, we demonstrate that nuclear Hsp90 functions are regulated by HDAC3, while Hsp90 regulates HDAC3 nuclear translocation. Pharmacological inhibition of Hsp90 decreased the HDAC3 nuclear translocation and increased the gene expression relevant to epithelial to mesenchymal transition. Further, inhibition of HDAC3 resulted in the nuclear accumulation of acetylated Hsp90. Additionally, Hsp90 inhibition affected the global histone acetylation and methylation patterns, whereas HDAC3 inhibition exhibited less impact. Our results display a novel regulatory mechanism mediated by Hsp90 and HDAC3 in tumor cells. Considering that Hsp90 and histone deacetylase inhibitors are emerging as novel anticancer agents, our findings may have clinical relevance.