MiR-205-driven downregulation of cholesterol biosynthesis through SQLE-inhibition identifies therapeutic vulnerability in aggressive prostate cancer.

Prostate cancer (PCa) shows strong dependence on the androgen receptor (AR) pathway. Here, we show that squalene epoxidase (SQLE), an enzyme of the cholesterol biosynthesis pathway, is overexpressed in advanced PCa and its expression correlates with poor survival. SQLE expression is controlled by micro-RNA 205 (miR-205), which is significantly downregulated in advanced PCa. Restoration of miR-205 expression or competitive inhibition of SQLE led to inhibition of de novo cholesterol biosynthesis. Furthermore, SQLE was essential for proliferation of AR-positive PCa cell lines, including abiraterone or enzalutamide resistant derivatives, and blocked transactivation of the AR pathway. Inhibition of SQLE with the FDA approved antifungal drug terbinafine also efficiently blocked orthotopic tumour growth in mice. Finally, terbinafine reduced levels of prostate specific antigen (PSA) in three out of four late-stage PCa patients. These results highlight SQLE as a therapeutic target for the treatment of advanced PCa.

CIP2A regulates MYC translation (via its 5'UTR) in colorectal cancer.

BACKGROUND: Deregulated expression of MYC is a driver of colorectal carcinogenesis, suggesting that decreasing MYC expression may have significant therapeutic value. CIP2A is an oncogenic factor that regulates MYC expression. CIP2A is overexpressed in colorectal cancer (CRC), and its expression levels are an independent marker for long-term outcome of CRC. Previous studies suggested that CIP2A controls MYC protein expression on a post-transcriptional level. METHODS: To determine the mechanism by which CIP2A regulates MYC in CRC, we dissected MYC translation and stability dependent on CIP2A in CRC cell lines. RESULTS: Knockdown of CIP2A reduced MYC protein levels without influencing MYC stability in CRC cell lines. Interfering with proteasomal degradation of MYC by usage of FBXW7-deficient cells or treatment with the proteasome inhibitor MG132 did not rescue the effect of CIP2A depletion on MYC protein levels. Whereas CIP2A knockdown had marginal influence on global protein synthesis, we could demonstrate that, by using different reporter constructs and cells expressing MYC mRNA with or without flanking UTR, CIP2A regulates MYC translation. This interaction is mainly conducted by the MYC 5'UTR. CONCLUSIONS: Thus, instead of targeting MYC protein stability as reported for other tissue types before, CIP2A specifically regulates MYC mRNA translation in CRC but has only slight effects on global mRNA translation. In conclusion, we propose as novel mechanism that CIP2A regulates MYC on a translational level rather than affecting MYC protein stability in CRC.

OmoMYC blunts promoter invasion by oncogenic MYC to inhibit gene expression characteristic of MYC-dependent tumors.

MYC genes have both essential roles during normal development and exert oncogenic functions during tumorigenesis. Expression of a dominant-negative allele of MYC, termed OmoMYC, can induce rapid tumor regression in mouse models with little toxicity for normal tissues. How OmoMYC discriminates between physiological and oncogenic functions of MYC is unclear. We have solved the crystal structure of OmoMYC and show that it forms a stable homodimer and as such recognizes DNA in the same manner as the MYC/MAX heterodimer. OmoMYC attenuates both MYC-dependent activation and repression by competing with MYC/MAX for binding to chromatin, effectively lowering MYC/MAX occupancy at its cognate binding sites. OmoMYC causes the largest decreases in promoter occupancy and changes in expression on genes that are invaded by oncogenic MYC levels. A signature of OmoMYC-regulated genes defines subgroups with high MYC levels in multiple tumor entities and identifies novel targets for the eradication of MYC-driven tumors.

Cystathionase mediates senescence evasion in melanocytes and melanoma cells.

The development of malignant melanoma is a highly complex process, which is still poorly understood. A majority of human melanomas are found to express a few oncogenic proteins, such as mutant RAS and BRAF variants. However, these oncogenes are also found in nevi, and it is now a well-accepted fact that their expression alone leads to senescence. This renders the understanding of senescence escape mechanisms an important point to understand tumor development. Here, we approached the question of senescence evasion by expressing the transcription factor v-myc myelocytomatosis viral oncogene homolog (c-MYC), which is known to act synergistically with many oncogenes, in melanocytes. We observed that MYC drives the evasion of reactive-oxygen stress-induced melanocyte senescence, caused by activated receptor tyrosine kinase signaling. Conversely, MIZ1, the growth suppressing interaction partner of MYC, is involved in mediating melanocyte senescence. Both, MYC overexpression and Miz1 knockdown led to a strong reduction of endogenous reactive-oxygen species (ROS), DNA damage and senescence. We identified the cystathionase (CTH) gene product as mediator of the ROS-related MYC and MIZ1 effects. Blocking CTH enzymatic activity in MYC-overexpressing and Miz1 knockdown cells increased intracellular stress and senescence. Importantly, pharmacological inhibition of CTH in human melanoma cells also reconstituted senescence in the majority of cell lines, and CTH knockdown reduced tumorigenic effects such as proliferation, H2O2 resistance and soft agar growth. Thus, we identified CTH as new MYC target gene with an important function in senescence evasion.

Multiple myeloma is affected by multiple and heterogeneous somatic mutations in adhesion- and receptor tyrosine kinase signaling molecules.

Multiple myeloma (MM) is a largely incurable plasma cell malignancy with a poorly understood and heterogeneous clinical course. To identify potential, functionally relevant somatic mutations in MM, we performed whole-exome sequencing of five primary MM, corresponding germline DNA and six MM cell lines, and developed a bioinformatics strategy that also integrated published mutational data of 38 MM patients. Our analysis confirms that identical, recurrent mutations of single genes are infrequent in MM, but highlights that mutations cluster in important cellular pathways. Specifically, we show enrichment of mutations in adhesion molecules of MM cells, emphasizing the important role for the interaction of the MM cells with their microenvironment. We describe an increased rate of mutations in receptor tyrosine kinases (RTKs) and associated signaling effectors, for example, in EGFR, ERBB3, KRAS and MAP2K2, pointing to a role of aberrant RTK signaling in the development or progression of MM. The diversity of mutations affecting different nodes of a particular signaling network appears to be an intrinsic feature of individual MM samples, and the elucidation of intra- as well as interindividual redundancy in mutations that affect survival pathways will help to better tailor targeted therapeutic strategies to the specific needs of the MM patient.

Effect of different collegiate sports on cortical bone in the tibia.

OBJECTIVES: The purpose of this study was to determine the effect of sports participation on cortical bone in the tibia. METHODS: 53 female collegiate athletes (25 cross-country, 16 soccer, and 12 volleyball) and 20 inactive controls had the left distal 20% tibia scanned by pQCT. Cortical volumetric BMD (vBMD) was measured within the cortical shell at the anterior, posterior, medial, and lateral regions and standard deviations were calculated. RESULTS: Total vBMD was greater in the control group (1161±5 mg/mm(3)) than each of the sports (p<0.05). Soccer players (1147±5 mg/mm(3)) had greater vBMD than volleyball players (1136±7 mg/mm(3)) (p<0.05), but similar to cross-country runners (1145±5 mg/mm(3)). Cortical thickness was greatest in soccer players (4.1±0.1 mm), while cross-country and control subjects (3.8±0.1 mm) had greater thickness than volleyball players (3.4±0.1 mm)(p<0.05). Periosteal circumference was greater in volleyball players (71±1.4 mm) than soccer, cross-country, and control subjects (68±0.9, 69±0.8, and 66±1 mm, respectively; all, p<0.05). vBMD variation within the cortical shell was greater among control subjects (70±6 mg/cm(3)) than each of the athlete groups, with soccer players having lower variation than cross country runners (within-in person SD 36±6 mg/cm(3) and 54±5 mg/cm(3) respectively; p<0.05). CONCLUSION: These results indicate bone geometry and distribution within the cortical shell of the tibia varies depending upon sporting activities of young women.

Ubiquitination of Myc: proteasomal degradation and beyond.

The level of Myc proteins is a critical determinant of cellular proliferation and apoptosis. Ubiquitination of Myc plays a key role in controlling protein levels by stimulating proteasomal degradation of the protein. Some experiments suggest that ubiquitination may also regulate Myc function in addition to turnover. This review attempts to summarize current knowledge about this field.

Intrathecal neuropeptide Y reduces behavioral and molecular markers of inflammatory or neuropathic pain.

Our previous work indicates that the intrathecal administration of neuropeptide Y (NPY) acts at its cognate receptors to reduce behavioral signs of nociception in several models of inflammatory pain, including the formalin test. The present study extends these findings to a rat model of peripheral neuropathic pain, and then evaluates the hypothesis that NPY inhibits inflammation- and nerve injury-induced activation of spinal nociceptive transmission. Here we show that NPY dose-dependently reduced behavioral signs of mechanical and cold hypersensitivity in the spared nerve injury (SNI) model. Intrathecal administration of either a Y1 (BIBO3304) or a Y2 (BIIE0246) receptor antagonist dose-dependently reversed the anti-allodynic actions of NPY. To monitor the effects of NPY on the stimulus-induced activation of spinal nociresponsive neurons, we quantified protein expression of the immediate-early gene c-fos in lamina I-VI of the L4-L5 dorsal horn, with special attention to the mediolateral pattern of Fos immunohistochemical staining after SNI. Either tactile stimulation of the hindpaw ipsilateral to nerve injury, or intraplantar injection of noxious formalin, increased the number of Fos-like immunoreactive profiles. Tactile stimulation evoked a mediolateral pattern of Fos expression corresponding to the innervation territory of the uninjured (sural) nerve. We found that intrathecal NPY reduced both formalin- and SNI-induced Fos expression. NPY inhibition of SNI-induced Fos expression was localized to the sural (uninjured) innervation territory, and could be blocked by intrathecal BIBO3304 and BIIE0246. We conclude that NPY acts at spinal Y1 and Y2 receptors to reduce spinal neuron activity and behavioral signs of inflammatory or neuropathic pain.

Promoting social capital for the elderly.

The new gold standard has evolved from aging in place to aging in community. Having social capital by being an active member of a community has a positive effect and can decrease vulnerability to health risk. Federal and state monies have been utilized to support community activities for the elderly including the first meal programs and moving to community-based assisted living programs. While staying in the community is the ideal, a community can be created by progressive leadership in a traditional retirement community including a skilled nursing home. Retirement communities based on the six dimensions of wellness can create a nurturing environment. Nurse leaders can meet the challenge of creating strong environments for the elderly.

Mechanisms of transcriptional repression by Myc.

Myc proteins are nuclear proteins that exert their biological functions at least in part through the transcriptional regulation of large sets of target genes. Recent microarray analyses show that several percent of all genes may be directly regulated by Myc. A large body of data shows that Myc proteins both positively and negatively affect transcription. The basic mechanism underlying Myc's activation of transcription is well understood, but the mechanisms through which Myc negatively regulates or represses transcription are far less understood. In this chapter, we will review our current knowledge about this less-well-understood topic.

Inhibition of retinoic acid receptor signaling by Ski in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease with multiple different cytogenetic and molecular aberrations contributing to leukemic transformation. We compared gene expression profiles of 4608 genes using cDNA-arrays from 20 AML patients (nine with -7/del7q and 11 with normal karyotype) with 23 CD34+ preparations from healthy bone marrow donors. SKI, a nuclear oncogene, was highly up regulated. In a second set of 183 AML patients analyzed with real-time PCR, the highest expression level of SKI in AML with -7/del7q could be confirmed. As previously described, Ski associates with the retinoic acid receptor (RAR) complex and can repress transcription. We wanted to investigate the interference of Ski with RARalpha signaling in AML. Ski was co-immunoprecipitated and colocalized with RARalpha. We also found that overexpression of wild-type Ski inhibited the prodifferentiating effects of retinoic acid in U937 leukemia cells. Mutant Ski, lacking the N-CoR binding, was no more capable of repressing RARalpha signaling. The inhibition by wild-type Ski could partially be reverted by the histone deacetylase blocking agent valproic acid. In conclusion, Ski seems to be involved in the blocking of differentiation in AML via inhibition of RARalpha signaling.

Expression profiling of Wilms tumors reveals new candidate genes for different clinical parameters.

Wilms tumor is the most frequent renal neoplasm in children, but our understanding of its genetic basis is still limited. We performed cDNA microarray experiments using 63 primary Wilms tumors with the aim of detecting new candidate genes associated with malignancy grade and tumor progression. All tumors had received preoperative chemotherapy as mandated by the SIOP protocol, which sets this study apart from related approaches in the Unites States that are based on untreated samples. The stratification of expression data according to clinical criteria allowed a rather clear distinction between different subsets of Wilms tumors. Clear-cut differences in expression patterns were discovered between relapse-free as opposed to relapsed tumors and tumors with intermediate risk as opposed to high risk histology. Several differentially expressed genes, e.g.TRIM22, CENPF, MYCN, CTGF, RARRES3 and EZH2, were associated with Wilms tumor progression. For a subset of differentially expressed genes, microarray data were confirmed by real-time RT-PCR on the original set of tumors. Interestingly, we found the retinoic acid pathway to be deregulated at different levels in advanced tumors suggesting that treatment of these tumors with retinoic acid may represent a promising novel therapeutic approach.

Regulation of cyclin D2 gene expression by the Myc/Max/Mad network: Myc-dependent TRRAP recruitment and histone acetylation at the cyclin D2 promoter.

Myc oncoproteins promote cell cycle progression in part through the transcriptional up-regulation of the cyclin D2 gene. We now show that Myc is bound to the cyclin D2 promoter in vivo. Binding of Myc induces cyclin D2 expression and histone acetylation at a single nucleosome in a MycBoxII/TRRAP-dependent manner. Down-regulation of cyclin D2 mRNA expression in differentiating HL60 cells is preceded by a switch of promoter occupancy from Myc/Max to Mad/Max complexes, loss of TRRAP binding, increased HDAC1 binding, and histone deacetylation. Thus, recruitment of TRRAP and regulation of histone acetylation are critical for transcriptional activation by Myc.

Expression of P27(KIP1) is prognostic and independent of MYCN amplification in human neuroblastoma.

Amplification of the MYCN gene is significantly associated with an unfavorable prognosis and rapid progression in human neuroblastoma tumors. One potential mechanism by which MYCN may cause these effects is by deregulating cell proliferation. Tissue culture experiments support a model in which MYC genes stimulate cell cycle progression by antagonizing the function of the cell cycle inhibitor p27(kip1). In culture, activation of MYC induces both sequestration of p27(kip1) by cyclin D complexes and its subsequent proteolytic degradation. We have tested whether this model applies to human neuroblastoma in a retrospective study of 100 primary tumor biopsy samples from neuroblastoma patients with a documented follow-up. Consistent with this hypothesis, MYCN-amplified tumors express high levels of both cyclin A and proliferating cell nuclear antigen, 2 marker proteins of cell proliferation. Further, expression levels of p27(kip1) are of prognostic significance in human neuroblastoma patients. Similar to tissue culture systems, p27(kip1) is sequestered by cyclin D complexes in a subset of human neuroblastoma samples. Surprisingly, however, expression levels of p27(kip1) are prognostic independent of MYCN amplification, and tumors that have an amplified MYCN gene do not express elevated levels of D-type cyclins or contain significantly lower levels of p27(kip1). Our data do not support a model in which regulation of p27(kip1) function is an important mechanism by which amplified MYCN deregulates cell proliferation in neuroblastoma.

Repression of p15INK4b expression by Myc through association with Miz-1.

Deregulated expression of c-myc can induce cell proliferation in established cell lines and in primary mouse embryonic fibroblasts (MEFs), through a combination of both transcriptional activation and repression by Myc. Here we show that a Myc-associated transcription factor, Miz-1, arrests cells in G1 phase and inhibits cyclin D-associated kinase activity. Miz-1 upregulates expression of the cyclin-dependent kinases (CDK) inhibitor p15INK4b by binding to the initiator element of the p15INK4b promoter. Myc and Max form a complex with Miz-1 at the p15 initiator and inhibit transcriptional activation by Miz-1. Expression of Myc in primary cells inhibits the accumulation of p15INK4b that is associated with cellular senescence; conversely, deletion of c-myc in an established cell line activates p15INK4b expression. Alleles of c-myc that are unable to bind to Miz-1 fail to inhibit accumulation of p15INK4b messenger RNA in primary cells and are, as a consequence, deficient in immortalization.

TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b.

Transforming growth factor-beta (TGFbeta) is a cytokine that arrests epithelial cell division by switching off the proto-oncogene c-myc and rapidly switching on cyclin-dependent kinase (CDK) inhibitors such as p15INK4b. Gene responses to TGFbeta involve Smad transcription factors that are directly activated by the TGFbeta receptor. Why downregulation of c-myc expression by TGFbeta is required for rapid activation of p15INK4b has remained unknown. Here we provide evidence that TGFbeta signalling prevents recruitment of Myc to the p15INK4b transcriptional initiator by Myc-interacting zinc-finger protein 1 (Miz-1). This relieves repression and enables transcriptional activation by a TGFbeta-induced Smad protein complex that recognizes an upstream p15INK4b promoter region and contacts Miz-1. Thus, two separate TGFbeta-dependent inputs - Smad-mediated transactivation and relief of repression by Myc - keep tight control over p15INK4b activation.

c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disease presenting with isolated thrombocytopenia in infancy and developing into a pancytopenia in later childhood. Thrombopoietin (TPO) is the main regulator of thrombocytopoiesis and has also been demonstrated to be an important factor in early hematopoiesis. We analyzed 9 patients with CAMT for defects in TPO production and reactivity. We found high levels of TPO in the sera of all patients. However, platelets and hematopoietic progenitor cells of patients with CAMT did not show any reactivity to TPO, as measured by testing TPO-synergism to adenosine diphosphate in platelet activation or by megakaryocyte colony assays. Flow cytometric analysis revealed absent surface expression of the TPO receptor c-Mpl in 3 of 3 patients. Sequence analysis of the c-mpl gene revealed point mutations in 8 of 8 patients: We found frameshift or nonsense mutations that are predicted to result in a complete loss of c-Mpl function in 5 patients. Heterozygous or homozygous missense mutations predicted to lead to amino acid exchanges in the extracellular domain of the receptor were found in 3 other patients. The type of mutations correlated with the clinical course of the disease. We propose a defective c-Mpl expression due to c-mpl mutations as the cause for thrombocytopenia and progression into pancytopenia seen in patients with CAMT.

Chondrocyte death precedes structural damage in blunt impact trauma.

Joint impact trauma has been shown to cause fissures, fibrillation, and other structural damage of the cartilage or subchondral bone. Previous studies used impact energies sufficient to fracture the underlying bone. Under these circumstances, the initial influence of impact trauma on cellular components and cartilage structure is unknown. The goal of this study was to determine whether an impact trauma first causes cellular or structural damage to a cartilage layer. Such damage might be the starting point of degenerative changes found in osteoarthrosis. Porcine patellas (n = 12) were subjected to standardized low-impact loading of three magnitudes with a spherical impactor attached to a drop tower device (0.06, 0.1, and 0.2 J). India ink staining and scanning electron microscopic analysis were used for analysis and showed no evidence of gross structural disruption. Chondrocyte viability assessed with thiazole blue staining and propidium iodide counterstaining was reduced significantly in the tangential and middle zones with increasing impact energy. These results indicate that chondrocyte death may precede excessive structural damage reported in earlier studies and might be a crucial factor in posttraumatic osteoarthrosis.