Objective: To investigate the molecular basis of drug resistance in pancreatic cancer. Methods: The expression of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) levels in pancreatic cancer tissues and cell lines was analyzed. Clinical relevance between Nrf2 activation and drug resistance was demonstrated by measuring cell viability after Nrf2 and adenosine 5′-triphosphate-binding cassette, subfamily G member 2 (ABCG2) regulation by overexpression or knock-down of these genes. Activity of ABCG2 was measured by Hoechst 33342 staining. Results: Abnormally elevated Nrf2 protein levels were observed in pancreatic cancer tissues and cell lines relative to normal pancreatic tissues. Increasing Nrf2 protein levels either by overexpression of exogenous Nrf2 or by activating endogenous Nrf2 resulted in increased drug resistance. Conversely, a reduction in endogenous Nrf2 protein levels or inactivation of endogenous Nrf2 resulted in decreased drug resistance. These changes in drug resistance or sensitivity were also positively correlated to the expression levels of Nrf2 downstream genes. Similarly, the expression of ABCG2 was correlated with drug resistance. Conclusions: Because the intrinsic drug resistance of pancreatic cancers is, in part, due to abnormally elevated Nrf2 protein levels, further research on regulating Nrf2 activity may result in the development of novel pancreatic cancer therapies. Abbreviations: Nrf2 - nuclear factor (erythroid-derived 2)-like 2, ABCG2 - adenosine 5′-triphosphate-binding cassette, subfamily G member 2, MRP - multidrug resistance protein

Many types of mutations in tumor suppressor p53 are oncogenic through gain-of-function. Therefore, targeting mutant p53 (mtp53) is a promising therapeutic approach to fight against many types of cancers. We report here a small molecule compound YK-3-237 that reduces acetylation of mtp53 and exhibits anti-proliferative effects toward triple-negative breast cancer (TNBC) cells carrying mtp53. YK-3-237 activates SIRT1 enzyme activities in vitro and deacetylation of both mtp53 and wild type p53 (WTp53) in a SIRT1-dependent manner. Deacetylation of mtp53 resulted in depletion of mtp53 protein level and up-regulated the expression of WTp53-target genes, PUMA and NOXA. YK-3-237 also induces PARP-dependent apoptotic cell death and arrests the cell cycle at G2/M phase in mtp53 TNBC cells. Taken together, our data suggest that targeting acetylation of mtp53 is a potential target to treat human cancers.

Gemcitabine (GEM) is the front-line standard chemotherapy used for the treatment of pancreatic cancer; however, chemoresistance to GEM remains the major obstacle to the successful control of this disease. Both the expression levels and activity of aldehyde dehydrogenase 1A1 (ALDH1A1) are important features of tumor-initiating and/or cancer stem cell properties in multiple types of human cancer. As one of the intrinsic properties of cancer stem cells is drug resistance, in this study, we examined the correlation between the level and activity of endogenous ALDH1A1 and GEM resistance in the MIA PaCa-2 cell line that contains high expression levels and activity of ALDH1A1. We used small interfering RNAs (siRNAs) to deplete ALDH1A1 and investigate its potential role in conferring GEM resistance. The ALDH1A1 knockdown markedly reduced ALDH1A1 expression and activity and inhibited cell proliferation. Moreover, the combination of ALDH1A1-siRNA and GEM significantly decreased cell viability, increased apoptotic cell death and increased the accumulation of cells at the S-phase compared to the controls. Our data also demonstrated that ALDH1A1 expression and activity were significantly higher in the GEM-resistant MIA PaCa-2 cell line (MIA PaCa-2/GR), compared to the parental MIA PaCa-2 cell line (MIA PaCa-2/P). In the MIA PaCa-2/GR cells, the combination of ALDH1A1-siRNA and GEM also showed a significant decrease in cell viability and an increase in apoptotic cell death, emphasizing the importance of ALDH1A1 in both intrinsic and acquired GEM resistance. This potentially powerful combination treatment of ALDH1A1-siRNA and GEM warrants further investigation as an effective therapeutic regimen to overcome the resistance of pancreatic cancer to GEM.

Triple‐negative breast cancers (TNBCs) are known to be intrinsically resistant to inhibitors for epidermal growth factor receptor (EGFR). Until now, clinical trials for TNBCs using EGFR inhibitors (EGFRis) as single agents have yielded disappointing results. Here, we report that combinatorial treatment using EGFRis, such as gefitinib or erlotinib, with PI3K/AKT pathway inhibitors (PI3K/AKTis) demonstrated a synergistic, anti‐proliferative effect in cell lines of the basal‐like (BL) subtype, a subtype of TNBC. Western blot analysis revealed that the gefitinib/PI‐103 combination significantly reduced the level of both phospho‐AKT and phospho‐ERK in two susceptible BL subtype cell lines, SUM149PT and MDA‐MB‐468, whereas it had little or no effect on the level of phospho‐ERK in two non‐susceptible cell lines (HS578T and MDA‐MB‐231) of mesenchymal stem‐like (MSL) TNBC subtype. The gefitinib/PI‐103 combination also significantly induced caspase‐3/7‐mediated PARP cleavage and reduced two anti‐apoptotic proteins, XIAP and Bcl‐2 in the susceptible cell lines. In addition, the level of myeloid cell leukemia 1 (Mcl‐1) protein was markedly decreased by gefitinib/PI‐103 combination in the BL TNBC cells, but showed no significant change by this combination in MSL subtype cells. These results suggest that pharmacological inhibition of EGFR used in combination of PI3K/AKTis is a potential therapeutic approach to treat a subtype of TNBCs.

Free radicals generated by oxidative stress cause damage that can contribute to numerous chronic diseases. Mammalian cells respond to this damage by increased transcription of cytoprotective phase II genes, which are regulated by NRF2. Previously, it has been shown that NRF2 protein levels increase after oxidative stress because its negative regulator, KEAP1, loses its ability to bind NRF2 and cause its proteasome-mediated degradation during oxidative stress. Here, we show that CRIF1, a protein previously known as cell cycle regulator and transcription cofactor, is also able to negatively regulate NRF2 protein stability. However, in contrast to KEAP1, which regulates NRF2 stability only under normal reducing conditions, CRIF1 regulates NRF2 stability and its target gene expression under both reducing and oxidative stress conditions. Thus, CRIF1-NRF2 interactions and their consequences are redox-independent. In addition, we found that CRIF1, unlike KEAP1 (which only interacts with N-terminal region of NRF2), physically interacts with both N- and C-terminal regions of NRF2 and promotes NRF2 ubiquitination and subsequent proteasome-mediated NRF2 protein degradation.

We describe the potential benefit of PIK-75 in combination of gemcitabine to treat pancreatic cancer in a preclinical mouse model. The effect of PIK-75 on the level and activity of NRF2 was characterized using various assays including reporter gene, quantitative PCR, DNA-binding and western blot analyses. Additionally, the combinatorial effect of PIK-75 and gemcitabine was evaluated in human pancreatic cancer cell lines and a xenograft model. PIK-75 reduced NRF2 protein levels and activity to regulate its target gene expression through proteasome-mediated degradation of NRF2 in human pancreatic cancer cell lines. PIK-75 also reduced the gemcitabine-induced NRF2 levels and the expression of its downstream target MRP5. Co-treatment of PIK-75 augmented the antitumor effect of gemcitabine both in vitro and in vivo. Our present study provides a strong mechanistic rationale to evaluate NRF2 targeting agents in combination with gemcitabine to treat pancreatic cancers.