MicroRNA-155 (miR-155) is frequently up-regulated in various types of human cancer; however, its role in cancer angiogenesis remains unknown. Here, we demonstrate the role of miR-155 in angiogenesis through targeting von Hippel-Lindau tumour suppressor (VHL) in breast cancer. Ectopic expression of miR-155 induced whereas knockdown of miR-155 inhibited HUVEC network formation, proliferation, invasion, and migration. Furthermore, mammary fat pad xenotransplantation of ectopically expressed miR-155 resulted in extensive angiogenesis, proliferation, tumour necrosis, and recruitment of pro-inflammatory cells such as tumour associated macrophages. Expression of VHL abrogated these miR-155 effects. Moreover, miR-155 expression inversely correlates with VHL expression level and is associated with late stage, lymph node metastasis, and poor prognosis as well as triple-negative tumour in breast cancer. These findings indicate that miR-155 plays a pivotal role in tumour angiogenesis by downregulation of VHL, and provide a basis for miR-155-expressing tumours to embody an aggressive malignant phenotype, and therefore, miR-155 is an important therapeutic target in breast cancer.

The AKT/PKB kinase is a key signaling component of one of the most frequently activated pathways in cancer and is a major target of cancer drug development. Most studies have focused on its activation by Receptor Tyrosine Kinase (RTK) mediated Phosphatidylinositol-3-OH kinase (PI3K) activation or loss of Phosphatase and Tensin homolog (PTEN). We have uncovered that growth factors binding to RTKs lead to activation of a non-receptor tyrosine kinase, Ack1 (also known as ACK or TNK2), which directly phosphorylates AKT at an evolutionarily conserved tyrosine 176 in the kinase domain. Tyr176-phosphorylated AKT localizes to the plasma membrane and promotes Thr308/Ser473-phosphorylation leading to AKT activation. Mice expressing activated Ack1 specifically in the prostate exhibit AKT Tyr176-phosphorylation and develop murine prostatic intraepithelial neoplasia (mPINs). Further, expression levels of Tyr176-phosphorylated-AKT and Tyr284-phosphorylated-Ack1 were positively correlated with the severity of disease progression, and inversely correlated with the survival of breast cancer patients. Thus, RTK/Ack1/AKT pathway provides a novel target for drug discovery.

NAD (nicotinamide adenine dinucleotide in its oxidized state) is an essential molecule for a variety of physiological processes. It is synthesized in distinct subcellular compartments by three different synthases (NMNAT-1, -2, and -3). We found that compartmentalized NAD synthesis by NMNATs integrates glucose metabolism and adipogenic transcription during adipocyte differentiation. Adipogenic signaling rapidly induces cytoplasmic NMNAT-2, which competes with nuclear NMNAT-1 for the common substrate, nicotinamide mononucleotide, leading to a precipitous reduction in nuclear NAD levels. This inhibits the catalytic activity of poly[adenosine diphosphate (ADP)-ribose] polymerase-1 (PARP-1), a NAD-dependent enzyme that represses adipogenic transcription by ADP-ribosylating the adipogenic transcription factor C/EBPβ. Reversal of PARP-1-mediated repression by NMNAT-2-mediated nuclear NAD depletion in response to adipogenic signals drives adipogenesis. Thus, compartmentalized NAD synthesis functions as an integrator of cellular metabolism and signal-dependent transcriptional programs.

PARP inhibitors (PARPi) prevent cancer cell growth by inducing synthetic lethality with DNA repair defects (e.g., in BRCA1/2 mutant cells). We have identified an alternative pathway for PARPi-mediated growth control in BRCA1/2-intact breast cancer cells involving rDNA transcription and ribosome biogenesis. PARP-1 binds to snoRNAs, which stimulate PARP-1 catalytic activity in the nucleolus independent of DNA damage. Activated PARP-1 ADP-ribosylates DDX21, an RNA helicase that localizes to nucleoli and promotes rDNA transcription when ADP-ribosylated. Treatment with PARPi or mutation of the ADP-ribosylation sites reduces DDX21 nucleolar localization, rDNA transcription, ribosome biogenesis, protein translation, and cell growth. The salient features of this pathway are evident in xenografts in mice and human breast cancer patient samples. Elevated levels of PARP-1 and nucleolar DDX21 are associated with cancer-related outcomes. Our studies provide a mechanistic rationale for efficacy of PARPi in cancer cells lacking defects in DNA repair whose growth is inhibited by PARPi.

Background Androgen receptor (AR) plays a critical role in the progression of both androgen-dependent and androgen-independent prostate cancer (AIPC). Ligand-independent activation of AR in AIPC or castration resistant prostate cancer (CRPC) is often associated with poor prognosis. Recently, tyrosine kinase Ack1 has been shown to regulate AR activity by phosphorylating it at tyrosine 267 and this event was shown to be critical for AIPC growth. However, whether a small molecule inhibitor that can mitigate Ack1 activation is sufficient to abrogate AR activity on AR regulated promoters in androgen-depleted environment is not known. Methods We have generated two key resources, antibodies that specifically recognize pTyr267-AR and synthesized a small molecule inhibitor of Ack1, 4-amino-5,6-biaryl-furo[2,3-d]pyrimidine (named here as AIM-100) to test whether AIM-100 modulates ligand-independent AR activity and inhibits prostate cell growth. Results Prostate tissue microarray analysis indicates that Ack1 Tyr284 phosphorylation correlates positively with disease progression and negatively with the survival of prostate cancer patients. Interestingly, neither pTyr267-AR expression nor its transcriptional activation was affected by anti-androgens in activated Ack1 expressing or EGF stimulated prostate cells. However, the Ack1 inhibitor, AIM-100, not only inhibited Ack1 activation but also able to suppress pTyr267-AR phosphorylation, binding of AR to PSA, NKX3.1, and TMPRSS2 promoters, and inhibit AR transcription activity. Conclusion Ack1 Tyr284 phosphorylation is prognostic of progression of prostate cancer and inhibitors of Ack1 activity could be novel therapeutic agents to treat AIPC.

ADP-ribosylation (ADPRylation) is a posttranslational modification of proteins discovered nearly six decades ago, but many important questions remain regarding its molecular functions and biological roles, as well as the activity of the ADP-ribose (ADPR) transferase enzymes (PARP family members) that catalyze it. Growing evidence indicates that PARP-mediated ADPRylation events are key regulators of the protein biosynthetic pathway, leading from rDNA transcription and ribosome biogenesis to mRNA synthesis, processing, and translation. In this review we describe the role of PARP proteins and ADPRylation in all facets of this pathway. PARP-1 and its enzymatic activity are key regulators of rDNA transcription, which is a critical step in ribosome biogenesis. An emerging role of PARPs in alternative splicing of mRNAs, as well as direct ADPRylation of mRNAs, highlight the role of PARP members in RNA processing. Furthermore, PARP activity, stimulated by cellular stresses, such as viral infections and ER stress, leads to the regulation of mRNA stability and protein synthesis through posttranscriptional mechanisms. Dysregulation of PARP activity in these processes can promote disease states. Collectively, these results highlight the importance of PARP family members and ADPRylation in gene regulation, mRNA processing, and protein abundance. Future studies in these areas will yield new insights into the fundamental mechanisms and a broader utility for PARP-targeted therapeutic agents.

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PAGE 6862:Lanes 5 and 6 of the U6 panel of Fig. 4C were missing in the original published figure. The revised figure shows these lanes. This correction does not affect the results or conclusions of this work.