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Translation and transcription are frequently dysregulated in cancer. These two processes are generally regulated by distinct sets of factors. The CBFB gene, which encodes a transcription factor, has recently emerged as a highly mutated driver in a variety of human cancers including breast cancer. Here we report a noncanonical role of CBFB in translation regulation. RNA immunoprecipitation followed by deep sequencing (RIP-seq) reveals that cytoplasmic CBFB binds to hundreds of transcripts and regulates their translation. CBFB binds to mRNAs via hnRNPK and enhances translation through eIF4B, a general translation initiation factor. Interestingly, the RUNX1 mRNA, which encodes the transcriptional partner of CBFB, is bound and translationally regulated by CBFB. Furthermore, nuclear CBFB/RUNX1 complex transcriptionally represses the oncogenic NOTCH signaling pathway in breast cancer. Thus, our data reveal an unexpected function of CBFB in translation regulation and propose that breast cancer cells evade translation and transcription surveillance simultaneously through downregulating CBFB., CBFB is highly mutated in breast cancers and is known to interact with RUNX proteins to regulate transcription. Here, the authors describe a non-canonical role of CBFB in translation regulation in which it binds to mRNAs through hnRNPK, facilitating translation by eIF4B.

Body weight is controlled by the activation of signal transduction pathways in both the brain and peripheral tissues. Interestingly, although many hypothalamic neuropeptides and receptors have been implicated in the regulation of body weight, the transcriptional and posttranscriptional mechanisms through which these genes are expressed in response to changes in energy balance remain unclear. Our laboratory studies a mouse in which targeted deletion of the neuronal basic helix-loop-helix (bHLH) transcription factor, nescient helix-loop-helix 2 protein (Nhlh2), results in adult-onset obesity. The aim of this work was to use the phenotype of the Nhlh2 knockout mouse and the expression pattern of Nhlh2 to identify genes that are regulated by this transcription factor. In this article, we show that Nhlh2 is expressed throughout the adult hypothalamus. Using dual-label in situ hybridization, we demonstrate that, in the arcuate nucleus of the adult hypothalamus (ARC), Nhlh2 expression can be found in rostral proopiomelanocortin (POMC) neurons, whereas in the paraventricular nucleus (PVN), Nhlh2 is expressed in TRH neurons. In addition, we find that hypothalamic POMC-derived alphaMSH in the ARC and TRH in the PVN are regulated posttranscriptionally via Nhlh2-mediated control of prohormone convertase I and II mRNA levels. This is the first report in which regulation of body weight is linked to the action of a neuronal bHLH transcription factor on prohormone convertase mRNA levels. Furthermore, this work supports a direct role for transcriptional control of neuropeptide processing enzymes in the etiology of adult-onset obesity.

Chloride intracellular channel 4 (CLIC4) is an evolutionarily conserved, 29kD, dimorphic protein that contributes to TGF-β signaling by preventing the de-phosphorylation of phospho-SMAD2/3 upon nuclear translocation. In several cancer types, CLIC4 is excluded from the nucleus and downregulated in the cytoplasm of the tumor cells as the tumor progresses, suggesting that CLIC4 acts as tumor suppressor. In a parallel sequence, CLIC4 becomes upregulated in the stromal compartment, where it enhances tumor growth and invasion. Recent reports have suggested that CLIC4 is detectable in the serum of cancer patients and incorporated into extracellular vesicles, and has potential as a biomarker. We hope to gain a better understanding of the role that CLIC4 plays in the tumor stromal and epithelial compartments as well as their respective release of extracellular vesicles. Using in-vitro and in-vivo assays, we have conducted experiments using the FVB mouse MMTV-c-MYC 6DT1 breast cancer model. By CRISPR/ Cas9 system, CLIC4 was deleted from wild type 6DT1 cells. Following clonal selection, the loss of the CLIC4 protein at both the cellular and released vesicle level was validated. Both functional assays on CLIC4 deleted clones and evaluation of their extra-cellular vesicles were undertaken in order to further understand their tumorigenic and metastatic capabilities. In-vitro, CLIC4 was not necessary for vesicle biogenesis and its deletion did not have a significant effect on cellular proliferation. In vivo, selected clones were orthotopically injected into the 4th mammary fat pad of wild type FVB mice. Compared to wild type 6DT1 clones, CLIC4 deleted clones formed primary tumors that had greater mass but a fewer number of lung metastasis. Future studies are designed to isolate vesicles circulating in tumor bearing hosts to determine their stromal or epithelial origin and to provide a better understanding of the role that CLIC4 may play in tumor growth, creating a metastatic niche and as a potential serological biomarker. Citation Format: Alayna B. Craig-Lucas, Vanesa C. Sanchez, Abigail Read, Ji Lou, Anjali Shukla, Stuart H. Yuspa. CLIC4 is incorporated into extracellular vesicles of murine breast cancer cells and may influence metastatic burden [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1042. doi:10.1158/1538-7445.AM2017-1042

Regulation of energy balance by leptin involves regulation of several neuropeptides, including thyrotropin-releasing hormone (TRH). Synthesized from a larger inactive precursor, its maturation requires proteolytic cleavage by prohormone convertases 1 and 2 (PC1 and PC2). Since this maturation in response to leptin requires prohormone processing, we hypothesized that leptin might regulate hypothalamic PC1 and PC2 expression, ultimately leading to coordinated processing of prohormones into mature peptides. Using hypothalamic neurons, we found that leptin stimulated PC1 and PC2 mRNA and protein expression and also increased PC1 and PC2 promoter activities in transfected 293T cells. Starvation of rats, leading to low serum leptin levels, decreased PC1 and PC2 gene and protein expression in the paraventricular nucleus (PVN) of the hypothalamus. Exogenous administration of leptin to fasted animals restored PC1 levels in the median eminence (ME) and the PVN to approximately the level found in fed control animals. Consistent with this regulation of PCs in the PVN, concentrations of TRH in the PVN and ME were substantially reduced in the fasted animals relative to the fed animals, and leptin reversed this decrease. Further analysis showed that proteolytic cleavage of pro-thyrotropin-releasing hormone (proTRH) at known PC cleavage sites was reduced by fasting and increased in animals given leptin. Combined, these findings suggest that leptin-dependent stimulation of hypothalamic TRH expression involves both activation of trh transcription and stimulation of PC1 and PC2 expression, which lead to enhanced processing of proTRH into mature TRH.

The nucleoside reverse transcriptase inhibitor AZT induces multiple manifestations of genotoxicity, among which aneuploidy, observed as micronuclei containing whole chromosomes, has been reported (Borojerdi et al., Mut Res, 2009). We have seen a loss of β-tubulin polymerization in about 23% of normal human mammary epithelial cells (NHMECs) exposed for 24 hr to 200 μM AZT, and demonstrated down-regulation of hsa-miR-770-5p, a microRNA known to target STMN1, in the same AZT-exposed cells. STMN1 prevents microtubule polymerization, resulting in destabilization of the mitotic spindle and chromosome misalignment. We therefore hypothesized that, if AZT exposure downregulates hsa-miR-770-5p, there should be increased expression of STMN1, which would result in loss of microtubule polymerization leading to aneuploidy. In this study we exposed cells of the human breast line MCF-10A to 100 μM AZT continuously for 1 to 3 passages (P). By Western Blot we showed that total STMN1 was 3.5-fold increased at P1, in AZT-exposed cells, compared to the unexposed controls. Using the same STMN1 antiserum, by immunohistochemistry (IHC), we observed that some cells stained heavily for STMN1, while others had virtually no staining, and that the overall STMN1 fluorescence was increased in AZT-exposed cells, compared to unexposed controls. When tubulin polymerization was evaluated by IHC, using β-tubulin antiserum, 10.1% of AZT-exposed cells showed a loss of tubulin polymerization, while only 0.7% of unexposed cells showed the same effect (p=0.004). The data suggest that AZT induced aneuploidy may be mediated by inhibition of hsa-miR-770-5p, upregulation of STMN1, impaired polymerization of β-tubulin, aberrant mitotic spindle formation and consequent chromosome misalignment resulting in aneuploidy. If confirmed, this will constitute a novel mechanism of AZT-induced genotoxicity that may or may not be related to AZT-DNA incorporation. Ongoing experiments will explore these relationships further by localizing STMN1 and β-tubulin in specific cells, and examining the same cultures for spindle aberrations at mitosis. Citation Format: Andrea V. Rivera, Vanesa C. Sanchez, Miriam C. Poirier, Ofelia A. Olivero. Zidovudine (AZT)-induced aneuploidy, mediated by Stathmin 1 (STMN1), involves a loss of polymerized β-tubulin. [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 3603. doi:10.1158/1538-7445.AM2013-3603