TGF-β plays a dual role in epithelial malignancies, including head and neck squamous cell carcinoma (HNSCC). Attenuation of canonical TGF-β signaling enhances de novo tumor development, while TGF-β overexpression and signaling paradoxically promotes malignant progression. We recently observed that TGF-β-induced growth arrest response is attenuated, in association with aberrant activation of Nuclear Factor-κB (NF-κB), a transcription factor which promotes malignant progression in HNSCC. However, what role cross-talk between components of the TGF-β and NF-κB pathways plays in altered activation of these pathways has not been established. Here, we show TGF-β receptor II and TGF-β-activated kinase 1 (TAK1) are predominantly expressed in a subset of HNSCC tumors with nuclear activation of NF-κB family member RELA (p65). Further, TGF-β1 treatment induced sequential phosphorylation of TAK1, IKK, IκBα, and RELA in human HNSCC lines. TAK1 enhances TGF-β-induced NF-κB activation, as TAK1 siRNA knock-down decreased TGF-β1-induced phosphorylation of IKK, IκB, and RELA, degradation of IκBα, nuclear translocation, and DNA binding of RELA, and NF–κB-induced reporter and target gene transcription. Functionally, TAK1 siRNA inhibited cell proliferation, migration and invasion. Celastrol, a TAK1 inhibitor and anti-inflammatory used in traditional Chinese medicine, also decreased TGF-β1-induced phosphorylation of TAK1 and RELA, suppressed basal, TGF-β1- and TNFα-induced NF-κB reporter gene activity, and cell proliferation, while increasing sub-G0 DNA fragmentation and Annexin V markers of apoptosis. Furthermore, TGF-β and RELA activation promoted SMAD7 expression. In turn, SMAD7 preferentially suppressed TGF-β-induced SMAD and NF-κB reporters when compared with constitutive or TNF-α-induced NF-κB reporter gene activation. Thus, cross-talk by TGF-β via TAK1 and NF-κB promotes the malignant phenotype of HNSCC. Moreover, NF-κB may contribute to the downstream attenuation of canonical TGF-β signaling through increased SMAD7 expression. Celastrol highlights the therapeutic potential of agents targeting TAK1 as a key node in this pro-oncogenic TGF-β-NF-κB signal pathway.

The mitochondrial serine hydroxymethyltransferase SHMT2, which catalyzes the rate-limiting step in serine catabolism, drives cancer cell proliferation, but how this role is regulated is undefined. Here, we report that the sirtuin SIRT5 desuccinylates SHMT2 to increase its activity and drive serine catabolism in tumor cells. SIRT5 interaction directly mediated desuccinylation of lysine 280 on SHMT2, which was crucial for activating its enzymatic activity. Conversely, hypersuccinylation of SHMT2 at lysine 280 was sufficient to inhibit its enzymatic activity and downregulate tumor cell growth in vitro and in vivo. Notably, SIRT5 inactivation led to SHMT2 enzymatic downregulation and to abrogated cell growth under metabolic stress. Our results reveal that SHMT2 desuccinylation is a pivotal signal in cancer cells to adapt serine metabolic processes for rapid growth, and they highlight SIRT5 as a candidate target for suppressing serine catabolism as a strategy to block tumor growth.Significance: These findings reveal a novel mechanism for controlling cancer cell proliferation by blocking serine catabolism, as a general strategy to impede tumor growth. Cancer Res; 78(2); 372-86.Ó2017 AACR.

Purpose: Head and neck squamous cell carcinomas have been reported to overexpress hypoxia-inducible factor (HIF)-1␣, a transcription factor that promotes expression of angiogenesis factors and resistance to programmed and therapy-induced cell death. 2-Methoxyestradiol (2ME2) is a natural compound with HIF-1␣ inhibitory activity that is currently being evaluated in phase 1 and 2 clinical trials for advanced solid tumors and multiple myeloma. To our knowledge, this is the first study to evaluate the effects of 2ME2 in head and neck squamous cell carcinoma.Experimental Design: In the present study, we investigated the effects of 2ME2 alone and in combination with paclitaxel, an active agent in recurrent or advanced head and neck squamous cell carcinoma.Results: 2ME2 exhibited antiproliferative and cytotoxic effects in a panel of five head and neck squamous cell carcinoma cell lines in the 0.5 to 10 mol/L range, including induction of G 2 -M blockade, caspase-3/7 activation, and apoptosis at 48 hours. 2ME2 resulted in decreased nuclear HIF-1␣-binding activity and affected the expression of downstream genes, such as bid, a proapoptotic bcl-2 family member, and vascular endothelial growth factor, a proangiogenic cytokine. The up-regulation of Bid (57.5% at 12 hours, P < 0.0006) and inhibition of vascular endothelial growth factor secretion (57.7% at 24 hours, P < 0.015; and 50.3% at 48 hours, P < 0.0006) could be partially attributed to the effects on HIF-1␣, because HIF-1␣ small interfering RNAs produced similar effects. Finally, in vivo, in a xenograft model of head and neck squamous cell carcinoma using UM-SCC-11A cells, 2ME2 exhibited antitumor and antiangiogenic activity, as measured by CD31 immunostaining.Conclusions: These results provide support for the use of 2ME2 in combination with paclitaxel for the treatment of recurrent or advanced head and neck squamous cell carcinoma.

Purpose: To determine if gene signatures differentially expressed in head and neck squamous cell carcinomas (HNSCC) are related to alterations in transcription factors nuclear factor-nB (NF-nB) andTP53previouslyassociatedwithdecreasedcelldeath,responsetotherapy,andworseprognosis. Experimental Design: Unique gene signatures expressed by HNSCC lines were identified by cDNA microarray, principal components, and cluster analyses and validated by quantitative reverse transcription-PCR (RT-PCR) and in situ hybridization. Bioinformatic analysis of the promoters and ontogeny of these clustered genes was done. Expression of proteins encoded by genes of a putative NF-nB signature, NF-nB p65, and TP53 were examined in HNSCC tissue specimens by immunostaining. Predicted promoter binding and modulation of expression of candidate NF-nB genes and cell survival were evaluated by p65 chromatin immunoprecipitation (ChIP) and small interfering RNA (siRNA) knockdown. Results: Two groups of HNSCC exhibiting distinct gene signatures were identified: cluster A enriched for histone genes, with a higher prevalence of TP53 promoter binding motifs; and cluster B enriched for injury response genes with NF-nB regulatory motifs. Coexpression of cluster B proteins was observed with strong NF-nB phospho-p65 and weak TP53 staining, and NF-nB phospho-p65 was inversely associated with TP53 (P = 0.02). Promoter binding of the NF-nB signature genes was confirmed by p65 ChIP, and down-modulation of their expression and cell death were induced by p65 siRNA. Conclusion: NF-nB promotes expression of a novel NF-nB^related gene signature and cell survival in HNSCC that weakly express TP53, a subset previously associated with inactivated wild-typeTP53, greater resistance to chemoradiotherapy, and worse prognosis.

Summary Identification of key regulators of lipid metabolism and thermogenic functions has important therapeutic implications for the current obesity and diabetes epidemic. Here we show that Grb10, a newly identified direct substrate of mechanistic/mammalian target of rapamycin (mTOR), is expressed highly in brown adipose tissue, and its expression in white adipose tissue is markedly induced by cold exposure. In adipocytes, mTOR-mediated phosphorylation at Ser501/503 switches the binding preference of Grb10 from the insulin receptor to raptor, leading to the dissociation of raptor from mTOR and down-regulation of mTOR complex 1 (mTORC1) signaling. Fat-specific disruption of Grb10 increased mTORC1 signaling in adipose tissues, suppressed lipolysis, and reduced thermogenic function. The effects of Grb10 deficiency on lipolysis and thermogenesis were diminished by rapamycin administration in vivo. Our study has uncovered a novel feedback mechanism regulating mTORC1 signaling in adipose tissues and identified Grb10 as a key regulator of adiposity, thermogenesis, and energy expenditure.

Highly efficient capture and enrichment is always the key for rapid analysis of airborne pathogens. Herein we report a simple microfluidic device which is capable of fast and efficient airborne bacteria capture and enrichment. The device was validated with Escherichia coli (E. coli) and Mycobacterium smegmatis. The results showed that the efficiency can reach close to 100% in 9 min. Compared with the traditional sediment method, there is also great improvement with capture limit. In addition, various flow rate and channel lengths have been investigated to obtain the optimized condition. The high capture and enrichment might be due to the chaotic vortex flow created in the microfluidic channel by the staggered herringbone mixer (SHM) structure, which is also confirmed with flow dynamic mimicking. The device is fabricated from polydimethylsiloxane (PDMS), simple, cheap, and disposable, perfect for field application, especially in developing countries with very limited modern instruments.

In inducing photoperiodic conditions, plants produce a signal dubbed “florigen” in leaves. Florigen moves through the phloem to the shoot apical meristem (SAM) where it induces flowering. In Arabidopsis, the FLOWERING LOCUS T (FT) protein acts as a component of this phloem-mobile signal. However whether the transportable FT mRNA also contributes to systemic florigen signalling remains to be elucidated. Using non-conventional approaches that exploit virus-induced RNA silencing and meristem exclusion of virus infection, we demonstrated that the Arabidopsis FT mRNA, independent of the FT protein, can move into the SAM. Viral ectopic expression of a non-translatable FT mRNA promoted earlier flowering in the short-day (SD) Nicotiana tabacum Maryland Mammoth tobacco in SD. These data suggest a possible role for FT mRNA in systemic floral signalling, and also demonstrate that cis-transportation of cellular mRNA into SAM and meristem exclusion of pathogenic RNAs are two mechanistically distinct processes.

Purpose: To determine the nature and potential pharmacologic reversibility of deficient TP53 expression and function in head and neck squamous cell carcinomas (HNSCC) with wild-type TP53, previously associated with decreased sensitivity to cisplatin therapy. Experimental Design: TP53 genotype, mRNA and protein expression, TP53-induced p21 expression, and TP53 DNA^binding and reporter gene function were determined in a panel of nine previously characterized HNSCC cell lines from the University of Michigan squamous cell carcinoma (UM-SCC) series. The genotoxic drug doxorubicin and the anti-inflammatory and antimalarial drug quinacrine, previously identified as inducers of TP53, were used to examine the nature and potential reversibility of deficient TP53 expression and function. The specific role of inducibleTP53 on function and cellular proliferation was confirmed using selectiveTP53 inhibitor pifithrin-a or short hairpin RNA knockdown. The capability of quinacrine to sensitize HNSCC to the cytotoxic effects of cisplatin was assessed. Results: UM-SCC cell lines with wild-type TP53 genotype underexpressed TP53 mRNA and protein when compared with normal human keratinocytes or UM-SCC with mutant TP53. Although doxorubicin failed to induce TP53 expression or functional activity, quinacrine induced TP53 mRNA and protein expression, increased TP53 reporter activity and p21 protein expression, and induced growth inhibition in these wild-type TP53 cell lines. Quinacrineinduced TP53 reporter activity and growth suppression were attenuated by pifithrin-a and TP53 short hairpin RNA knockdown. Furthermore, quinacrine sensitized UM-SCC to cisplatin in vitro. Conclusions: Deficient TP53 mRNA and protein expression underlies decreased function in a subset of HNSCC with wild-type TP53 and can be restored together with cisplatin sensitization by quinacrine. TP53, a tumor suppressor gene important in regulating cellcycle arrest, apoptosis, and therapeutic sensitivity, represents one of the most common targets for alterations underlying the development of cancer, including head and neck squamous cell carcinomas (HNSCC; refs. 1 -5). Mutation of TP53 occurs in f40% to 50% of HNSCC, resulting in altered TP53 expression and function (3 -5). In HNSCC that retain wild-type TP53 genotype, approximately one half expressed detectable TP53 protein, whereas approximately one half exhibited deficient expression by immunohistochemistry (6). In the latter subset of HNSCC that retain wildtype TP53 genotype, the alterations resulting in a defect in TP53 expression or function have been shown to include protein inactivation after infection with human papilloma virus (HPV), defects of p16INK4a and other components of the DNA damage response pathway, or unknown mechanisms. As a consequence, the differing nature and effects of these multiple alterations on TP53 expression and function likely contributed to the frequent discordance of results between studies using immunohistochemistry, genotyping, or other methods to define the role of TP53 in...