MYC-Mediated Ribosomal Gene Expression Sensitizes Enzalutamide-resistant Prostate Cancer Cells to EP300/CREBBP Inhibitors.

Patients with advanced prostate cancer are frequently treated with the antiandrogen enzalutamide. However, resistance eventually develops in virtually all patients, and various mechanisms have been associated with this process. The histone acetyltransferases EP300 and CREBBP are involved in regulation of cellular events in advanced prostate cancer. This study investigated the role of EP300/CREBBP inhibitors in enzalutamide-resistant prostate cancer. EP300/CREBBP inhibitors led to the same inhibition of androgen receptor activity in enzalutamide-resistant and -sensitive cells. However, enzalutamide-resistant cells were more sensitive to these inhibitors in viability assays. As indicated by the RNA-sequencing-based pathway analysis, genes related to the ribosome and MYC activity were significantly altered upon EP300/CREBBP inhibitor treatment. EP300/CREBBP inhibitors led to the down-regulation of ribosomal proteins RPL36 and RPL29. High-level ribosomal proteins amplifications and MYC amplifications were observed in castration-resistant prostate cancer samples of the publicly available Stand Up to Cancer data set. An inhibitor of RNA polymerase I-mediated transcription was used to evaluate the functional implications of these findings. The enzalutamide-resistant cell lines were more sensitive to this treatment. In addition, the migration rate of enzalutamide-resistant cells was strongly inhibited by this treatment. Taken together, the current data show that EP300/CREBBP inhibitors affect the MYC/ribosomal protein axis in enzalutamide-resistant cells and may have promising therapeutic implications.

Novel Insights into the PKCß-dependent Regulation of the Oxidoreductase p66Shc.

Dysfunctional mitochondria contribute to the development of many diseases and pathological conditions through the excessive production of reactive oxygen species (ROS), and, where studied, ablation of p66Shc (p66) was beneficial. p66 translocates to the mitochondria and oxidizes cytochrome c to yield H2O2, which in turn initiates cell death. PKCß-mediated phosphorylation of serine 36 in p66 has been implicated as a key regulatory step preceding mitochondrial translocation, ROS production, and cell death, and PKCß thus may provide a target for therapeutic intervention. We performed a reassessment of PKCß regulation of the oxidoreductase activity of p66. Although our experiments did not substantiate Ser36 phosphorylation by PKCß, they instead provided evidence for Ser139 and Ser213 as PKCß phosphorylation sites regulating the pro-oxidant and pro-apoptotic function of p66. Mutation of another predicted PKCß phosphorylation site also located in the phosphotyrosine binding domain, threonine 206, had no phenotype. Intriguingly, p66 with Thr206 and Ser213 mutated to glutamic acid showed a gain-of-function phenotype with significantly increased ROS production and cell death induction. Taken together, these data argue for a complex mechanism of PKCß-dependent regulation of p66 activation involving Ser139 and a motif surrounding Ser213.

HUS1 as a Potential Therapeutic Target in Urothelial Cancer.

Platinum-based chemotherapy is the standard of care with concern to first-line systemic therapy for metastatic disease in urothelial cancer (UC). Resistance to chemotherapy despite an initial response is linked with the ability to remove platinum-based DNA adducts and to repair chemotherapy-induced DNA lesions by various DNA repair proteins. The Rad9-Rad1-HUS1 complex that is loaded onto DNA at sites of damage is involved in checkpoint activation as well as DNA repair. Here, we addressed for the first time the potential influence of HUS1 expression in urothelial carcinogenesis (using two human basal urothelial cancer cell lines UM-UC-3 and HT1197) and its role as a potential therapeutic target for predicting responses to platinum-based chemotherapy. Specific inhibition of HUS1 expression in both cell lines was achieved by specific siRNA and validated by Western blot. In order to define the possible importance of HUS1 in the regulation of cellular proliferation, parental and resistant cells were treated with increasing concentrations of either control or HUS1 siRNA. HUS1 protein expression was observed in both human basal urothelial cancer cell lines UM-UC-3 and HT1197. In cisplatin-sensitive cells, knock-down of HUS1 inhibited cellular proliferation in the presence of cisplatin. On the contrary, knock-down of HUS1 in resistant cells did not result in a re-sensitization to cisplatin. Finally, RNAseq data from the Cancer Genome Atlas provided evidence that HUS1 expression is a significant prognostic factor for poor survival in UC patients. In summary, HUS1 may acts as an oncogene in UC and might be a key determinant of the cellular response to cisplatin-based chemotherapy.

p300 is upregulated by docetaxel and is a target in chemoresistant prostate cancer.

Administration of the microtubule inhibitor docetaxel is a common treatment for metastatic castration-resistant prostate cancer (mCRPC) and results in prolonged patient overall survival. Usually, after a short period of time chemotherapy resistance emerges and there is urgent need to find new therapeutic targets to overcome therapy resistance. The lysine-acetyltransferase p300 has been correlated to prostate cancer (PCa) progression. Here, we aimed to clarify a possible function of p300 in chemotherapy resistance and verify p300 as a target in chemoresistant PCa. Immunohistochemistry staining of tissue samples revealed significantly higher p300 protein expression in patients who received docetaxel as a neoadjuvant therapy compared to control patients. Elevated p300 expression was confirmed by analysis of publicly available patient data, where significantly higher p300 mRNA expression was found in tissue of mCRPC tumors of docetaxel-treated patients. Consistently, docetaxel-resistant PCa cells showed increased p300 protein expression compared to docetaxel-sensitive counterparts. Docetaxel treatment of PCa cells for 72 h resulted in elevated p300 expression. shRNA-mediated p300 knockdown did not alter colony formation efficiency in docetaxel-sensitive cells, but significantly reduced clonogenic potential of docetaxel-resistant cells. Downregulation of p300 in docetaxel-resistant cells also impaired cell migration and invasion. Taken together, we showed that p300 is upregulated by docetaxel, and our findings suggest that p300 is a possible co-target in treatment of chemoresistant PCa.

The oxidoreductase p66Shc acts as tumor suppressor in BRAFV600E-transformed cells.

Metabolic reprogramming, as exemplified by the shift from oxidative phosphorylation to glycolysis, is a common feature of transformed cells. In many tumors, altered metabolism is also reflected in increased reactive oxygen species (ROS) levels, which contribute to proliferation and survival signaling. However, despite high ROS levels, cancer cells can be efficiently killed by further increasing ROS production. We have shown previously that both wild-type and oncogenic CRAF and BRAF prevent excessive mitochondrial ROS production. Subsequently, it has been demonstrated that raising ROS levels in BRAFV600E-transformed melanoma cells by inhibiting BRAF or MEK rendered them susceptible to cell death induction. To understand how oncogenic BRAF affects mitochondrial ROS production in melanoma, we studied the mitochondrial ROS-producing oxidoreductase p66Shc, which is frequently overexpressed in tumors. Using NIH 3T3 BRAFV600E fibroblasts and the melanoma cell lines A375 and M238 carrying the same BRAF mutation, we show that under treatment with the ROS-inducing agent phenethyl isothiocyanate (PEITC), oncogenic BRAF renders cells refractory to p66ShcS36 phosphorylation, which is essential for p66Shc activation and mitochondrial ROS production. Consistent with this, the activation of JNK1/2, which phosphorylate S36, was blunted, while other mitogen-activated protein kinases were not affected. Inhibition of JNK1/2 efficiently prevented ROS production, while BRAF and MEK inhibitors increased ROS levels. Vemurafenib-resistant M238R melanoma cells were impaired in S36 phosphorylation and ROS production following PEITC treatment. Moreover, they failed to increase ROS levels after MEK/BRAF inhibition. Finally, shRNA-mediated knockdown of p66Shc led to increased growth of BRAFV600E-transformed NIH 3T3 cells in soft agar assay. Taken together, these data suggest that phosphorylation-activated p66Shc functions as a tumor suppressor in melanoma cells.

Increased Expression of miR-23a Mediates a Loss of Expression in the RAF Kinase Inhibitor Protein RKIP.

RAF kinase inhibitor protein (RKIP) is a seminal regulator of intracellular signaling and exhibits both antimetastatic and antitumorigenic properties. Decreased expression of RKIP has been described in several human malignancies, including acute myelogenous leukemia (AML). As the mechanisms leading to RKIP loss in AML are still unclear, we aimed to analyze the potential involvement of miRNAs within this study. miRNA microarray and qPCR data of more than 400 AML patient specimens revealed correlation between decreased expression of RKIP and increased expression of miR-23a, a member of the miR-23a/27a/24-2 cluster. In functional experiments, overexpression of miR-23a decreased RKIP mRNA and protein expression, whereas miR-23a inhibition caused the opposite effect. By using an RKIP 3'-untranslated region luciferase reporter construct with and without mutation or deletion of the putative miR-23a-binding site, we could show that RKIP modulation by miR-23a is mediated via direct binding to this region. Importantly, miR-23a overexpression induced a significant increase of proliferation in hematopoietic cells. Simultaneous transfection of an RKIP expression construct lacking the miR-23a-binding sites reversed this phenotype, indicating that this effect is truly mediated via downregulation of RKIP. Finally, by analyzing more than 4,300 primary patient specimens via database retrieval from The Cancer Genome Atlas, we could highlight the importance of the miR-23a/RKIP axis in a broad range of human cancer entities. In conclusion, we have identified miR-23a as a negative regulator of RKIP expression in AML and have provided data that suggest the importance of our observation beyond this tumor entity. Cancer Res; 76(12); 3644-54. ©2016 AACR.