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Mutant p53(s) are widely considered as oncogenes and promote several gain-of-function oncogenic activities. p53 mutations correlate with higher rates of metastasis and poor survival, therefore, it is paramount to inhibit mutant p53 protein either directly or indirectly. Although some compounds have been developed, none of them have achieved a desirable level of specificity. Some of these compounds only targeted specific mutations. In search of less-toxic compounds, we tested plant-derived compounds on mutant p53 triple-negative breast cancer cell lines. Here, we show that the compounds tested reduced the protein levels of one of the more frequent oncogenic p53 mutants (R249S, hot spot mutation), and its important targets that promote invasion and metastasis, including GMPS and IMPDH1. All compounds tested perturbed the invasion potential of the breast cancer cell line. These compounds downregulated several nucleotide metabolism genes (NMGs) which are essential for cell cycle progression. We observed S-phase arrest correlating to reduced cell proliferation and increased replication stress. Moreover, we also show a reduction of key ETS transcription family members including ETS2, ETS1, ETV1, and ETV4, which are involved in invasion and metastasis. We propose that these compounds may inhibit invasion by interfering with multiple pathways. Our findings exemplify that these tested compounds could inhibit invasion and cell growth in TNBC in a nucleotide-dependent manner.

Targeted inhibition of proteins modulating epigenetic changes is an increasingly important priority in cancer therapeutics, and many small molecule inhibitors are currently being developed. In the case of neuroblastoma (NB), a paediatric solid tumour with a paucity of intragenic mutations, epigenetic deregulation may be especially important. In this study we validate the histone methyltransferase G9a/EHMT2 as being associated with indicators of poor prognosis in NB. Immunological analysis of G9a protein shows it to be more highly expressed in NB cell-lines with MYCN amplification, which is a primary determinant of dismal outcome in NB patients. Furthermore, G9a protein in primary tumours is expressed at higher levels in poorly differentiated/undifferentiated NB, and correlates with high EZH2 expression, a known co-operative oncoprotein in NB. Our functional analyses demonstrate that siRNA-mediated G9a depletion inhibits cell growth in all NB cell lines, but, strikingly, only triggers apoptosis in NB cells with MYCN amplification, suggesting a synthetic lethal relationship between G9a and MYCN. This pattern of sensitivity is also evident when using small molecule inhibitors of G9a, UNC0638 and UNC0642. The increased efficacy of G9a inhibition in the presence of MYCN-overexpression is also demonstrated in the SHEP-21N isogenic model with tet-regulatable MYCN. Finally, using RNA sequencing, we identify several potential tumour suppressor genes that are reactivated by G9a inhibition in NB, including the CLU, FLCN, AMHR2 and AKR1C1-3. Together, our study underlines the under-appreciated role of G9a in NB, especially in MYCN-amplified tumours.

Neuroblastoma is one of the commonest and deadliest solid tumours of childhood, and is thought to result from disrupted differentiation of the developing sympathoadrenergic lineage of the neural crest. Neuroblastoma exhibits intra-and intertumoural heterogeneity, with high risk tumours characterised by poor differentiation, which can be attributable to MYCN-mediated repression of genes involved in neuronal differentiation. MYCN is known to co-operate with oncogenic signalling pathways such as Alk, Akt and MEK/ERK signalling, and, together with c-MYC has been shown to be activated by Wnt signalling in various tissues. However, our previous work demonstrated that Wnt3a/Rspo2 treatment of some neuroblastoma cell lines can, paradoxically, decrease c-MYC and MYCN proteins. This prompted us to define the neuroblastoma-specific Wnt3a/Rspo2-driven transcriptome using RNA sequencing, and characterise the accompanying changes in cell biology.Here we report the identification of ninety Wnt target genes, and show that Wnt signalling is upstream of numerous transcription factors and signalling pathways in neuroblastoma. Using live-cell imaging, we show that Wnt signalling can drive differentiation of SK-N-BE(2)-C and SH-SY5Y cell-lines, but, conversely, proliferation of SK-N-AS cells. We show that cell-lines that differentiate show induction of pro-differentiation BMP4 and EPAS1 proteins, which is not apparent in the SK-N-AS cells. In contrast, SK-N-AS cells show increased CCND1, phosphorylated RB and E2F1 in response to Wnt3a/Rspo2, consistent with their proliferative response, and these proteins are not increased in differentiating lines. By meta-analysis of the expression of our 90 genes in primary tumour gene expression databases, we demonstrate discrete expression patterns of our Wnt genes in patient cohorts with different prognosis. Furthermore our analysis reveals interconnectivity within subsets of our Wnt genes, with one subset comprised of novel putative drivers of neuronal differentiation repressed by MYCN. Assessment of β-catenin immunohistochemistry shows high levels of β-catenin in tumours with better differentiation, further supporting a role for canonical Wnt signalling in neuroblastoma differentiation.

p53(R172H/+) mice inherit a p53 mutation found in Li-Fraumeni syndrome and develop metastatic tumors at much higher frequency than p53(+/-) mice. To explore the mutant p53 metastatic phenotype, we used expression arrays to compare primary osteosarcomas from p53(R172H/+) mice with metastasis to osteosarcomas from p53(+/-) mice lacking metastasis. For this study, 213 genes were differentially expressed with a P value

Drug resistance is the major obstacle to successful cancer therapy. Our study focuses on resistance to Aurora kinase inhibitors tested as anti-cancer drugs in clinical trials. We have used 2D electrophoresis in the pH ranges of 4-7 and 6-11 followed by protein identification using MALDI-TOF/TOF to compare the protein composition of HCT116 colon cancer cells either sensitive to CYC116 and ZM447439 inhibitors or resistant toward these drugs. The analysis also included p53(+/+) and p53(-/-) phenotypes of HCT116 cells. Our findings demonstrate that platelet-activating factor acetylhydrolase and GTP-binding nuclear protein Ran contribute to the development of resistance to ZM447439 only where resistance is related to p53. On the other hand, serine hydroxymethyltransferase was found to promote the tumor growth in cells resistant to CYC116 without the influence of p53. Computer modeling of interaction networks highlighted a direct link of the p53-independent mechanism of resistance to CYC116 with autophagy. Importantly, serine hydroxymethyltransferase, serpin B5, and calretinin represent the target proteins that may help overcome resistance in combination therapies. In addition, serpin B5 and calretinin appear to be candidate biomarkers that may be accessible in patients for monitoring of cancer therapy with ease.

Aurora kinases are a recently discovered family of kinases (A, BC) consisting of highly conserved serine\threonine protein kinases found to be involved in multiple mitotic events: regulation of spindle assembly checkpoint pathway, function of centrosomes and cytoskeleton, and cytokinesis. Aberrant expression of Aurora kinases may lead to cancer. For this reason the Aurora kinases are potential targets in the treatment of cancer. In this review we discuss the biology of these kinases: structure, function, regulation and association with cancer.A literature search.Many of the multiple functions of mitosis are mediated by the Aurora kinases. Their aberrant expression can lead to the deregulation of cell division and cancer. For this reason, the Aurora kinases are currently one of the most interesting targets for cancer therapy. Some Aurora kinase inhibitors in the clinic have proven effectively on a wide range of tumor types. The clinical data are very encouraging and promising for development of novel class of structurally different Aurora kinase inhibitors. Hopefully the Aurora kinases will be potentially useful in drug targeted cancer treatment.

Mutant p53 (mtp53) is an oncogene that drives cancer cell proliferation. We found that mtp53 associates with the promoters of numerous nucleotide metabolism genes (NMG). Mtp53 knockdown reduced NMG expression and substantially depleted nucleotide pools. Decreased GTP levels correlated with reduced activation of GTP dependent proteins (GTPases) and reduced invasiveness. Addition of exogenous GTP restored the invasiveness of mtp53 knockdown cells, suggesting that mtp53 promotes invasion by increasing GTP. Additionally, mtp53 creates a dependency on the nucleotide salvage pathway enzyme deoxycytidine kinase (dCK) for the maintenance of a proper balance in dNTP pools required for proliferation. These data indicate that mtp53 harboring cells have acquired a synthetic sick or lethal phenotype relationship with the nucleotide salvage pathway. Finally, elevated expression of NMG correlates with mutant p53 status and poor prognosis in breast cancer patients. Thus, mtp53's control of nucleotide biosynthesis has both a driving and sustaining role in cancer development. Citation Format: Luis A. Martinez, Madhusudhan Kollareddy. Regulation of nucleotide metabolism by mutant p53 contributes to its gain-of-function activities. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2108. doi:10.1158/1538-7445.AM2015-2108

Carboplatin, cisplatin and oxaliplatin are structurally similar anticancer drugs and their mechanism of action is permanent coordination to DNA and interfering with DNA repair. However, these drugs have different anticancer activities in human tumors, adverse effects and dose limiting toxicities. Therefore, we performed complex proteomic profiling in order to characterize cellular effects of all three platinum based anticancer drugs. To identify the molecular basis of different pharmacological properties, expressional characteristics of cells exposed to vincristine and daunorubicin were used in parallel as prototypes of drugs with different mechanism of action. T-lymphoblastic leukemia CCRF-CEM cells were treated with 5x IC50 concentrations of individual drugs for time equivalent to half-time to induction apoptosis. Untreated (control) cells were cultured with heavy amino acid labeled media in order to enable relative quantification of peptide expression using SILAC technology, LC-MS/MS and LC-MALDI-TOF/TOF. Results were processed by Proteinscape software equipped by Mascot and Phenyx analytical algorithms. We identified 646 ± 133 proteins by ESI and 312 ± 85 proteins by MALDI in average of all fifteen replicates. Identified results were further processed by in-house written script to remove outlier peptides and to normalize individual runs. All replicates were summed, and statistic analysis was done. To see up- and down- regulated proteins, the hierarchical clustering and PCA analysis was performed. Hierarchical clustering revealed that cisplatin and carboplatin shares the same cluster what is conclusive with literature. Surprisingly, the cluster of daunorubicin and vincristine was close to cisplatin and carboplatin and the oxaliplatin was in the separated cluster from rest of four drugs. PCA analysis revealed 30 proteins with contribution higher than 1 %. The highest contribution (7.5 %) to drug clustering have proteins involved in DNA remodeling. These proteins could be used as specific biomarkers of therapy by platinum based compounds. Citation Format: Petr Dzubak, Tomas Ozdian, Dusan Holub, Gabriela Rylova, Jiri Rehulka, Madhusudhan kollareddy, Daniela Mlcuchova, Marian Hajduch. Proteomic profiling of platinum based anticancer drugs. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4652. doi:10.1158/1538-7445.AM2013-4652