Lymphotoxin-ß receptor-NIK signaling induces alternative RELB/NF-κB2 activation to promote metastatic gene expression and cell migration in head and neck cancer.

Head and neck squamous cell carcinomas (HNSCC) preferentially spread to regional cervical tissues and lymph nodes. Here, we hypothesized that lymphotoxin-ß (LTß), receptor LTßR, and NF-κB-inducing kinase (NIK), promote the aberrant activation of alternative NF-κB2/RELB pathway and genes, that enhance migration and invasion of HNSCC. Genomic and expression alterations of the alternative NF-kB pathway were examined in 279 HNSCC tumors from The Cancer Genome Atlas (TCGA) and a panel of HNSCC lines. LTßR is amplified or overexpressed in HNSCC of the larynx or oral cavity, while LTß, NIK, and RELB are overexpressed in cancers arising within lymphoid oropharyngeal and tonsillar sites. Similarly, subsets of HNSCC lines displayed overexpression of LTßR, NIK, and RELB proteins. Recombinant LTß, and siRNA depletion of endogenous LTßR and NIK, modulated expression of LTßR, NIK, and nuclear translocation of NF-κB2(p52)/RELB as well as functional NF-κB promoter reporter activity. Treatment with a NIK inhibitor (1,3[2H,4H]-Iso-Quinoline Dione) reduced the protein expression of NIK and NF-κB2(p52)/RELB, and blocked LTß induced nuclear translocation of RELB. NIK and RELB siRNA knockdown or NIK inhibitor slowed HNSCC migration or invation in vitro. LTß-induces expression of migration and metastasis related genes, including hepatocyte growth/scatter factor receptor MET. Knockdown of NIK or MET similarly inhibited the migration of HNSCC cell lines. This may help explain why HNSCC preferentially migrate to local lymph nodes, where LTß is expressed. Our findings show that LTß/LTßR promotes activation of the alternative NIK-NF-κB2/RELB pathway to enhance MET-mediated cell migration in HNSCC, which could be potential therapeutic targets in HNSCC.

Exquisite light sensitivity of Drosophila melanogaster cryptochrome.

Drosophila melanogaster shows exquisite light sensitivity for modulation of circadian functions in vivo, yet the activities of the Drosophila circadian photopigment cryptochrome (CRY) have only been observed at high light levels. We studied intensity/duration parameters for light pulse induced circadian phase shifts under dim light conditions in vivo. Flies show far greater light sensitivity than previously appreciated, and show a surprising sensitivity increase with pulse duration, implying a process of photic integration active up to at least 6 hours. The CRY target timeless (TIM) shows dim light dependent degradation in circadian pacemaker neurons that parallels phase shift amplitude, indicating that integration occurs at this step, with the strongest effect in a single identified pacemaker neuron. Our findings indicate that CRY compensates for limited light sensitivity in vivo by photon integration over extraordinarily long times, and point to select circadian pacemaker neurons as having important roles.

Targeting Notch1 and IKKα Enhanced NF-κB Activation in CD133+ Skin Cancer Stem Cells.

Cancer stem-like cells are hypothesized to be the major tumor-initiating cell population of human cutaneous squamous cell carcinoma (cSCC), but the landscape of molecular alterations underpinning their signaling and cellular phenotypes as drug targets remains undefined. In this study, we developed an experimental pipeline to isolate a highly enriched CD133+CD31-CD45-CD61-CD24- (CD133+) cell population from primary cSCC specimens by flow cytometry. The CD133+ cells show enhanced stem-like phenotypes, which were verified by spheroid and colony formation in vitro and tumor generation in vivo Gene expression profiling of CD133+/- cells was compared and validated, and differentially expressed gene signatures and top pathways were identified. CD133+ cells expressed a repertoire of stemness and cancer-related genes, including NOTCH and NOTCH1-mediated NF-κB pathway signaling. Other cancer-related genes from WNT, growth factor receptors, PI3K/mTOR, STAT pathways, and chromatin modifiers were also identified. Pharmacologic and genetic targeting of NOTCH1, IKKα, RELA, and RELB modulated NF-κB transactivation, the CD133+ population, and cellular and stemness phenotypes. Immunofluorescent staining confirmed colocalization of CD133+ and IKKα expression in SCC tumor specimens. Our functional, genetic, and pharmacologic studies uncovered a novel linkage between NOTCH1, IKKα, and NF-κB pathway activation in maintaining the CD133+ stem SCC phenotypes. Studies investigating markers of activation and modulators of NOTCH, IKK/NF-κB, and other pathways regulating these cancer stem gene signatures could further accelerate the development of effective therapeutic strategies to treat cSCC recurrence and metastasis. Mol Cancer Ther; 17(9); 2034-48. ©2018 AACR.

Genomic and Transcriptomic Characterization Links Cell Lines with Aggressive Head and Neck Cancers.

Cell lines are important tools for biological and preclinical investigation, and establishing their relationship to genomic alterations in tumors could accelerate functional and therapeutic discoveries. We conducted integrated analyses of genomic and transcriptomic profiles of 15 human papillomavirus (HPV)-negative and 11 HPV-positive head and neck squamous cell carcinoma (HNSCC) lines to compare with 279 tumors from The Cancer Genome Atlas (TCGA). We identified recurrent amplifications on chromosomes 3q22-29, 5p15, 11q13/22, and 8p11 that drive increased expression of more than 100 genes in cell lines and tumors. These alterations, together with loss or mutations of tumor suppressor genes, converge on important signaling pathways, recapitulating the genomic landscape of aggressive HNSCCs. Among these, concurrent 3q26.3 amplification and TP53 mutation in most HPV(-) cell lines reflect tumors with worse survival. Our findings elucidate and validate genomic alterations underpinning numerous discoveries made with HNSCC lines and provide valuable models for future studies.

SMAC Mimetic Birinapant plus Radiation Eradicates Human Head and Neck Cancers with Genomic Amplifications of Cell Death Genes FADD and BIRC2.

Comparison of tumors from The Cancer Genome Atlas (TCGA) reveals that head and neck squamous cell carcinomas (HNSCC) harbor the most frequent genomic amplifications of Fas-associated death domain (FADD), with or without Baculovirus inhibitor of apoptosis repeat containing BIRC2 (cIAP1), affecting about 30% of patients in association with worse prognosis. Here, we identified HNSCC cell lines harboring FADD/BIRC2 amplifications and overexpression by exome sequencing, RT-PCR, and Western blotting. In vitro, FADD or BIRC2 siRNA knockdown inhibited HNSCC displaying amplification and increased expression of these genes, supporting their functional importance in promoting proliferation. Birinapant, a novel SMAC mimetic, sensitized multiple HNSCC lines to cell death by agonists TNFα or TRAIL and inhibited cIAP1>XIAP>IAP2. Combination of birinapant and TNFα induced sub-G0 DNA fragmentation in sensitive lines and birinapant alone also induced significant G2-M cell-cycle arrest and cell death in UM-SCC-46 cells. Gene transfer and expression of FADD sensitized resistant UM-SCC-38 cells lacking FADD amplification to birinapant and TNFα, supporting a role for FADD in sensitization to IAP inhibitor and death ligands. HNSCC varied in mechanisms of cell death, as indicated by reversal by inhibitors or protein markers of caspase-dependent apoptosis and/or RIPK1/MLKL-mediated necroptosis. In vivo, birinapant inhibited tumor growth and enhanced radiation-induced TNFα, tumor responses, and host survival in UM-SCC-46 and -11B xenograft models displaying amplification and overexpression of FADD+/- BIRC2 These findings suggest that combination of SMAC mimetics such as birinapant plus radiation may be particularly active in HNSCC, which harbor frequent FADD/BIRC2 genomic alterations. Cancer Res; 76(18); 5442-54. ©2016 AACR.

MEK Inhibitor PD-0325901 Overcomes Resistance to PI3K/mTOR Inhibitor PF-5212384 and Potentiates Antitumor Effects in Human Head and Neck Squamous Cell Carcinoma.

PURPOSE: Head and neck squamous cell carcinomas exhibit variable sensitivity to inhibitors of the PI3K/mTOR pathway, an important target of genomic alterations in this cancer type. The mitogen-activated protein kinase kinase (MEK)/ERK/activator protein 1 (AP-1) and nuclear factor-κB (NF-κB) pathways are also frequently co-activated, but their roles in resistance mechanisms to PI3K/mTOR inhibitors and as therapeutic targets in head and neck squamous cell carcinoma (HNSCC) are not well defined. EXPERIMENTAL DESIGN: We determined the IC50s of dual PI3K/mTOR inhibitor PF-05212384 (PF-384) by XTT assays in 14 HNSCC lines with PI3K/Akt/mTOR cascade alterations. In two resistant models, we further characterized the molecular, cellular, and in vivo attributes and effects of combining PF-384 with MEK inhibitor PD-0325901 (PD-901). RESULTS: PF-384 IC50s varied between 0.75 and 133 nmol/L in 14 HNSCC lines with overexpression or mutations of PIK3CA, and sensitivity correlated with increased phospho-AKT(T308/S473). In resistant UMSCC-1 and -46 models, PF-384 increased G0-/G1-phase accumulation but weakly induced sub-G0 cell death. PF-384 inhibited direct targets of PI3K/mTOR, but incompletely attenuated co-activated ERK and UMSCC-1 xenograft growth in vivo. PD-901 strongly inhibited MEK/ERK targets, and the combination of PF-384 and PD-901 inhibited downstream NF-κB and AP-1 transactivation, and IL8 and VEGF production in vitro. PD-901 potently inhibited tumor growth alone and with PF384, enhanced antiproliferative, apoptotic, and anti-angiogenesis activity in vivo. CONCLUSIONS: PI3K/mTOR inhibitor PF-384 exhibits variable activity in a panel of HNSCC cell lines with differing PIK3CA expression and mutation status. MEK inhibitor PD-901 overcomes resistance and enhances antitumor effects observed with PF-384 in vivo.

PI3K/mTOR inhibitor PF-04691502 antitumor activity is enhanced with induction of wild-type TP53 in human xenograft and murine knockout models of head and neck cancer.

PURPOSE: Phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway activation is often associated with altered expression or mutations of PIK3CA, TP53/p73, PTEN, and TGF-ß receptors (TGFBR) in head and neck squamous cell carcinomas (HNSCC). However, little is known about how these alterations affect response to PI3K/mTOR-targeted agents. EXPERIMENTAL DESIGN: In this preclinical study, PI3K/Akt/mTOR signaling was characterized in nine HNSCC (UM-SCC) cell lines and human oral keratinocytes. We investigated the molecular and anticancer effects of dual PI3K/mTOR inhibitor PF-04691502(PF-502) in UM-SCC expressing PIK3CA with decreased wild-type TP53, mutant TP53-/+ mutantTGFBR2, and in HNSCC of a conditional Pten/Tgfbr1 double knockout mouse model displaying PI3K/Akt/mTOR activation. RESULTS: UM-SCC showed increased PIK3CA expression and Akt/mTOR activation, and PF-502 inhibited PI3K/mTORC1/2 targets. In human HNSCC expressing PIK3CA and decreased wtTP53 and p73, PF-502 reciprocally enhanced TP53/p73 expression and growth inhibition, which was partially reversible by p53 inhibitor pifithrin-α. Most UM-SCC with wtTP53 exhibited a lower IC50 than those with mtTP53 status. PF-502 blocked growth in G0-G1 and increased apoptotic sub-G0 DNA. PF-502 suppressed tumorigenesis and showed combinatorial activity with radiation in a wild-type TP53 UM-SCC xenograft model. PF-502 also significantly delayed HNSCC tumorigenesis and prolonged survival of Pten/Tgfbr1-deficient mice. Significant inhibition of p-Akt, p-4EBP1, p-S6, and Ki67, as well as increased p53 and TUNEL were observed in tumor specimens. CONCLUSIONS: PI3K-mTOR inhibition can enhance TP53/p73 expression and significantly inhibit tumor growth alone or when combined with radiation in HNSCC with wild-type TP53. PIK3CA, TP53/p73, PTEN, and TGF-ß alterations are potential modifiers of response and merit investigation in future clinical trials with PI3K-mTOR inhibitors.

Roles of dopamine in circadian rhythmicity and extreme light sensitivity of circadian entrainment.

Light has profound behavioral effects on almost all animals, and nocturnal animals show sensitivity to extremely low light levels [1-4]. Crepuscular, i.e., dawn/dusk-active animals such as Drosophila melanogaster are thought to show far less sensitivity to light [5-8]. Here we report that Drosophila respond to extremely low levels of monochromatic blue light. Light levels three to four orders of magnitude lower than previously believed impact circadian entrainment and the light-induced stimulation of locomotion known as positive behavioral masking. We use GAL4;UAS-mediated rescue of tyrosine hydroxylase (DTH) mutant (ple) flies to study the roles of dopamine in these processes. We present evidence for two roles of dopamine in circadian behaviors. First, rescue with either a wild-type DTH or a DTH mutant lacking neural expression leads to weak circadian rhythmicity, indicating a role for strictly regulated DTH and dopamine in robust circadian rhythmicity. Second, the DTH rescue strain deficient in neural dopamine selectively shows a defect in circadian entrainment to low light, whereas another response to light, positive masking, has normal light sensitivity. These findings imply separable pathways from light input to the behavioral outputs of masking versus circadian entrainment, with only the latter dependent on dopamine.