Abcg2 Overexpression Represents a Novel Mechanism for Acquired Resistance to the Multi-Kinase Inhibitor Danusertib in BCR-ABL-Positive Cells In Vitro

The success of Imatinib (IM) therapy in chronic myeloid leukemia (CML) is compromised by the development of IM resistance and by a limited IM effect on hematopoietic stem cells. Danusertib (formerly PHA-739358) is a potent pan-aurora and ABL kinase inhibitor with activity against known BCR-ABL mutations, including T315I. Here, the individual contribution of both signaling pathways to the therapeutic effect of Danusertib as well as mechanisms underlying the development of resistance and, as a consequence, strategies to overcome resistance to Danusertib were investigated. Starting at low concentrations, a dose-dependent inhibition of BCR-ABL activity was observed, whereas inhibition of aurora kinase activity required higher concentrations, pointing to a therapeutic window between the two effects. Interestingly, the emergence of resistant clones during Danusertib exposure in vitro occurred considerably less frequently than with comparable concentrations of IM. In addition, Danusertib-resistant clones had no mutations in BCR-ABL or aurora kinase domains and remained IM-sensitive. Overexpression of Abcg2 efflux transporter was identified and functionally validated as the predominant mechanism of acquired Danusertib resistance in vitro. Finally, the combined treatment with IM and Danusertib significantly reduced the emergence of drug resistance in vitro, raising hope that this drug combination may also achieve more durable disease control in vivo

Ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) is a potential tumour suppressor in prostate cancer and is frequently silenced by promoter methylation

<p>Abstract</p> <p>Background</p> <p>We have previously reported significant downregulation of ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) in prostate cancer (PCa) compared to the surrounding benign tissue. UCHL1 plays an important role in ubiquitin system and different cellular processes such as cell proliferation and differentiation. We now show that the underlying mechanism of UCHL1 downregulation in PCa is linked to its promoter hypermethylation. Furthermore, we present evidences that UCHL1 expression can affect the behavior of prostate cancer cells in different ways.</p> <p>Results</p> <p>Methylation specific PCR analysis results showed a highly methylated promoter region for UCHL1 in 90% (18/20) of tumor tissue compared to 15% (3/20) of normal tissues from PCa patients. Pyrosequencing results confirmed a mean methylation of 41.4% in PCa whereas only 8.6% in normal tissues. To conduct functional analysis of UCHL1 in PCa, UCHL1 is overexpressed in LNCaP cells whose UCHL1 expression is normally suppressed by promoter methylation and found that UCHL1 has the ability to decrease the rate of cell proliferation and suppresses anchorage-independent growth of these cells. In further analysis, we found evidence that exogenous expression of UCHL1 suppress LNCaP cells growth probably via p53-mediated inhibition of Akt/PKB phosphorylation and also via accumulation of p27kip1 a cyclin dependant kinase inhibitor of cell cycle regulating proteins. Notably, we also observed that exogenous expression of UCHL1 induced a senescent phenotype that was detected by using the SA-ß-gal assay and might be due to increased p14ARF, p53, p27kip1 and decreased MDM2.</p> <p>Conclusion</p> <p>From these results, we propose that UCHL1 downregulation via promoter hypermethylation plays an important role in various molecular aspects of PCa biology, such as morphological diversification and regulation of proliferation.</p

Identification of Clinically Relevant Protein Targets in Prostate Cancer with 2D-DIGE Coupled Mass Spectrometry and Systems Biology Network Platform

Prostate cancer (PCa) is the most common type of cancer found in men and among the leading causes of cancer death in the western world. In the present study, we compared the individual protein expression patterns from histologically characterized PCa and the surrounding benign tissue obtained by manual micro dissection using highly sensitive two-dimensional differential gel electrophoresis (2D-DIGE) coupled with mass spectrometry. Proteomic data revealed 118 protein spots to be differentially expressed in cancer (n = 24) compared to benign (n = 21) prostate tissue. These spots were analysed by MALDI-TOF-MS/MS and 79 different proteins were identified. Using principal component analysis we could clearly separate tumor and normal tissue and two distinct tumor groups based on the protein expression pattern. By using a systems biology approach, we could map many of these proteins both into major pathways involved in PCa progression as well as into a group of potential diagnostic and/or prognostic markers. Due to complexity of the highly interconnected shortest pathway network, the functional sub networks revealed some of the potential candidate biomarker proteins for further validation. By using a systems biology approach, our study revealed novel proteins and molecular networks with altered expression in PCa. Further functional validation of individual proteins is ongoing and might provide new insights in PCa progression potentially leading to the design of novel diagnostic and therapeutic strategies

A–D: Meso scale network models for apoptosis induction.

<p>(<b>A</b>) Distribution of the Pearson coefficient between individual protein expression and the mean component of factor analysis which represents the dominant co-regulation mechanism. (<b>B</b>) Shows that the proteins can be decomposed into two groups differing with respect to the impact of DANU. Most proteins show no different co-regulation behaviour if DANU is omitted from the data set, whereas three proteins show a significantly higher degree of co-regulation (increased r value) when DANU is omitted indicating a second mode of action of DANU. (<b>C</b>) Shows that with exception of two treatments the mean protein expression, represented by the value of the mean component of the factor analysis (y-axis) is correlated to the observed induction of apoptosis (x-axis) indicating a similar efficacy in apoptosis induction for most drugs. The exceptions indicate that protein expression is induced which does not contribute to apoptosis induction. (<b>D</b>) Depicts the meso scale set of pathways, which fits two the observations.</p

Schematic representation of meso network architecture and experimental design.

<p>(<b>A</b>) Exemplifies an abstract meso-scale network representing (abstract) pathways of drug action of drugs A and B on induction of proteins (red, yellow and orange bullets). Drug A uses two pathways (blue and black), whereas the blue pathway induces expression shifts only on a subset of the proteins (red, yellow), whereas the black pathway induces expression shifts in all proteins. Drug B acts only via one pathway which joins the black pathway of Drug A in an abstract node (represented by the green bullet). The mutation inhibits both pathways between the green and blue bullet resulting in an interference with drug induced expression shift for all proteins. The blue pathway from Drug A to the proteins, however, is not affected by the mutation. Hence the mutation may have a strong impact on the efficacy of drug B, whereas the profile of action of drug A is only altered by the mutation. (B) Imatinib sensitive and resistant cells were treated with four tyrosine kinase inhibitor and mesoscale network were reengineered based on specific proteome expression patterns.</p

A–D: Western blot analyses revealed posttranslational modification of eIF5A and up regulation of TGM2 after treatment with IM.

<p>(<b>A</b>) Enlarged regions from a coomassie stained 2D-PAGE from Ba/F3-p210 cells after treatment with IM or DMSO as a control. The arrows indicate two spots for eIF5A, one at pI of 5.2 and the other one at a pI of 6.1. The latter appeared after IM treatment. (<b>B</b>) 2D-WB validated the appearance of a second spot for eIF5A at a pI of 6.2 after IM treatment. (<b>C</b>) Enlarged regions from a coomassie stained 2D-PAGE from Ba/F3-p210 cells after treatment with IM or DMSO as a control. One spot for TGM2 (arrow) demonstrated an increased expression after IM treatment. (<b>D</b>) The increased expression of TGM2 after treatment with rising concentrations of IM could be validated in human Bcr-Abl K562 cells.</p

A–D: Meso scale networks Ba/F3-M351T.

<p>(<b>A</b>) Shows the protein expressions for wt and T351 cell type for all drugs. (<b>B</b>) Shows the sensitivity of protein expression with respect to the dominant activation mechanism, quantified by the mean component of factor analysis. (<b>C</b>) Shows, that surprisingly the overall level of protein expression induced by NILO increases, although the sensitivity decreases. (<b>D</b>) Shows the modifications which are induced by the analysis of the M351I mutation to the meso scale pathway network depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053668#pone-0053668-g005" target="_blank">Figure 5E</a>. Black block represents induction of the protein expression by the main pathway, whereas the red block is indicating an inhibition via the main pathway. Green block represents the unique effect of NILO on the overall protein expression level.</p