Head and neck squamous cell carcinoma (HNSCC) and many epithelial malignancies exhibit increased proliferation, invasion and inflammation, concomitant with aberrant nuclear activation of TP53 and NF-κB family members ΔNp63, c-REL and RELA. However, the mechanisms of crosstalk by which these transcription factors coordinate gene expression and the malignant phenotype remain elusive. Here we demonstrate thatΔNp63 regulates a cohort of genes involved in cell growth, survival, adhesion and inflammation, which substantially overlaps with the NF-κB transcriptome. ΔNp63 with c-REL and/or RELA are recruited to form novel binding complexes on p63 or NF-κB/REL sites of multiple target gene promoters. Overexpressed ΔNp63- or TNF-α-induced NF-κB and inflammatory cytokine IL-8 reporter activation depended upon RELA/c-REL regulatory binding sites. Depletion of RELA or ΔNp63 by siRNA significantly inhibited NF-κB-specific, or TNF-α-induced IL-8 reporter activation. ΔNp63 siRNA significantly inhibited proliferation, survival, and migration by HNSCC cells in vitro. Consistent with the above, an increase in nuclear ΔNp63 accompanied by increased proliferation (Ki67), and adhesion (β4 integrin) markers, and induced inflammatory cell infiltration was observed throughout HNSCC specimens, when compared to the basilar pattern of protein expression and minimal inflammation seen in non-malignant mucosa. Further, overexpression of ΔNp63α in squamous epithelia in transgenic mice leads to increased suprabasilar c-REL, Ki-67, and cytokine expression, together with epidermal hyperplasia and diffuse inflammation, similar to HNSCC. Our study reveals ΔNp63 as a master transcription factor that in coordination with NF-κB/RELs, orchestrates a broad gene program promoting epidermal hyperplasia, inflammation, and the malignant phenotype of HNSCC.
This is an in-depth review of the history of quinacrine as well as its pharmacokinetic properties and established record of safety as an FDA-approved drug. The potential uses of quinacrine as an anti-cancer agent are discussed with particular attention to its actions on nuclear proteins, the arachidonic acid pathway, and multi-drug resistance, as well as its actions on signaling proteins in the cytoplasm. In particular, quinacrine's role on the NF-κB, p53, and AKT pathways are summarized.
Purpose: The aim of this study is to investigate the expression of CK2 subunits and CK2 effects on NF-κB-mediated and TP53-mediated signal activation and gene expression, the malignant phenotype, and chemosensitivity in head and neck squamous cell carcinoma (HNSCC) in vitro and in vivo.Experimental Design: Protein expression of CK2 subunits was investigated by Western blot and immunohistochemistry. CK2 subunits were knocked down by small interfering RNA, and NF-κB activation was examined using DNA binding, Western blot, and luciferase reporter assays. Gene expression was measured by quantitative reverse transcription-PCR. Cell growth, survival, motility, and sensitivity to cisplatin were measured by MTT, flow cytometry, and migration assays. In vivo targeting of CK2α/α′ in HNSCC xenograft models was achieved using anti-CK2α/α′ oligodeoxynucleotide encapsulated in sub-50-nm tenfibgen nanocapsules.Results: CK2 subunit proteins were overexpressed in HNSCC lines and tissues. Knockdown of CK2 subunits differentially inhibited IκBα degradation, NF-κB nuclear localization, phosphorylation, DNA binding, and reporter activity. CK2 subunits modulated gene expression and the malignant phenotype involved in cell cycle and migration, whereas CK2α is critical to promote proliferation, antiapoptosis, and cisplatin resistance in vitro. Furthermore, in vivo delivery of anti-CK2α/α′ oligodeoxynucleotide nanocapsules significantly suppressed tumor growth in HNSCC xenograft models, in association with modulation of CK2 and NF-κB regulated molecules, TP53 family proteins, and induction of apoptosis.Conclusions: Our study reveals a novel role of CK2 in coregulating NF-κB activation, TP53/p63 expression, and downstream gene expression. Downregulation of CK2 in HNSCC models in vitro and in vivo shows antitumor effects as well as sensitization to cisplatin.
Over-expression of Yes-associated protein (YAP), and TP53 family members ΔNp63 and p73 with which YAP may serve as a nuclear co-factor, have been independently detected in subsets of head and neck squamous cell carcinomas (HNSCC). Their potential relationship and functional role of YAP in HNSCC are unknown. Here we reveal that in a subset of HNSCC lines and tumors, YAP expression is increased but localized in the cytoplasm in association with increased AKT and YAP phosphorylation, and decreased expression of ΔNp63 and p73. Conversely, YAP expression is decreased but detectable in the nucleus in association with lower AKT and YAP phosphorylation, and increased ΔNp63 and p73 expression, in another subset. Inhibiting AKT decreased Serine-127 phosphorylation and enhanced nuclear translocation of YAP. ΔNp63 repressed YAP expression and bound its promoter. Transfection of a YAP-Serine-127-Alanine phosphoacceptor-site mutant or ΔNp63 knockdown significantly increased nuclear YAP and cell death. Conversely, YAP knockdown enhanced cell proliferation, survival, migration, and cisplatin chemoresistance. Thus, YAP function as a tumor suppressor may alternatively be dysregulated by AKT phosphorylation at Serine-127 and cytoplasmic sequestration, or by transcriptional repression by ΔNp63, in different subsets of HNSCC. AKT and/orΔNp63 are potential targets for enhancing YAP-mediated apoptosis and chemosensitivity in HNSCC.
Purpose: Aberrant nuclear activation and phosphorylation of the canonical NF-κB subunit RELA/p65 at Serine-536 by inhibitor κB kinase is prevalent in head and neck squamous cell carcinoma (HNSCC), but the role of other kinases in NF-κB activation has not been well defined. Here, we investigated the prevalence and function of p65-Ser276 phosphorylation by protein kinase A (PKA) in the malignant phenotype and gene transactivation, and studied p65-Ser276 as a potential target for therapy. Experimental Design: Phospho and total p65 protein expression and localization were determined in HNSCC tissue array and in cell lines. The effects of the PKA inhibitor H-89 on NF-κB activation, downstream gene expression, cell proliferation and cell cycle were examined. Knockdown of PKA by specific siRNA confirmed the specificity. Results: NF-κB p65 phosphorylated at Ser276 was prevalent in HNSCC and adjacent dysplastic mucosa, but localized to the cytoplasm in normal mucosa. In HNSCC lines, tumor necrosis factor-α (TNF-α) significantly increased, whereas H-89 inhibited constitutive and TNF-α-induced nuclear p65 (Ser276) phosphorylation, and significantly suppressed NF-κB and target gene IL-8 reporter activity. Knockdown of PKA by small interfering RNA inhibited NF-κB, IL-8, and BCL-XL reporter gene activities. H-89 suppressed cell proliferation, induced cell death, and blocked the cell cycle in G 1 -S phase. Consistent with its biological effects, H-89 down-modulated expression of NF-κB-related genes Cyclin D1, BCL2, BCL-XL, COX2, IL-8, and VEGF, as well as induced cell cycle inhibitor p21 CIP1/WAF1, while suppressing proliferative marker Ki67. NF-κB is a signal transcription factor that has emerged as an important modulator of altered gene expression programs and malignant phenotype in the development and progression of cancer. We reported previously that NF-κB is aberrantly activated and promotes altered expression of multiple genes important in the malignant phenotype of human and murine head and neck squamous cell carcinoma (HNSCC; ref. 1, 2). The canonical mechanism for NF-κB nuclear translocation and activation involves the phosphorylation and subsequent degradation of its cytoplasmic inhibitor κB (IκB) by a trimeric inhibitor-κ B kinase α/β/γ complex in response to a wide range of stimuli such as oncogenic viruses, carcinogens, growth factors, and cytokines. Degradation of IκB results in the liberation of the p65-p50 heterodimer for its translocation to the nucleus where it transactivates target genes (3). The classical pathway of activation of NF-κB by physical dissociation of p65-p50 subunits from IκBα and subsequent nuclear translocation has been well studied (4, 5), but the differential dynamics of NF-κB transactivation following the phosphorylation of p65 at various sites is increasingly being appreciated. There is evidence that inhibitor-κ B kinase and various other kinases induce phosphorylation of p65 at Ser 536, a posttranslational modification critical for the transactivation of NF-κB in response to var...
Although constitutively activated nuclear factor-KB (NF-KB), attenuated transforming growth factor B (TGFB) signaling, and TP53 mutations frequently occur in human cancers, how these pathways interact and together contribute to malignancy remains uncertain. Here, we found an association between overexpression of NF-KB-related genes, reduced expression of TGFB receptor (TBR) subunits and downstream targets, and TP53 genotype in head and neck squamous cell carcinoma (HNSCC). In response to recombinant TGFB1, both growth inhibition and TGFB target gene modulation were attenuated or absent in a panel of human HNSCC lines. However, in HNSCC cells that retained residual TGFB signaling, TGFB1 inhibited both constitutive and tumor necrosis factor A-stimulated NF-KB activity. Furthermore, HNSCC lines overexpressing mutant (mt) TP53 and human tumor specimens with positive TP53 nuclear staining exhibited reduced TBRII and knocking down mtTP53 induced TbRII, increasing TGFB downstream gene expression while inhibiting proinflammatory NF-KB target gene expression. Transfection of ectopic TBRII directly restored TGFB signaling while inhibiting inhibitor KBA degradation and suppressing serine-536 phosphorylation of NF-KB p65 and NF-KB transcriptional activation, linking these alterations. Finally, experiments with TbRII conditional knockout mice show that abrogation of TGFB signaling promotes the sustained induction of NF-KB and its proinflammatory target genes during HNSCC tumorigenesis and progression. Together, these findings elucidate a regulatory framework in which attenuated TGFB signaling promotes NF-KB activation and squamous epithelial malignancy in the setting of altered TP53 status. [Cancer Res 2009;69(8):3415-24]
Study design Retrospective analysis. Objective To assess the impact of mean arterial blood pressure (MAP) during surgical intervention for spinal cord injury (SCI) on motor recovery. Setting Level-one Trauma Hospital and Acute Rehabilitation Hospital in San Jose, CA, USA. Methods Twenty-five individuals with traumatic SCI who received surgical and acute rehabilitation care at a level-one trauma center were included in this study. The Surgical Information System captured intraoperative MAPs on a minute-byminute basis and exposure was quantified at sequential thresholds from 50 to 104 mmHg. Change in International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) motor score was calculated based on physiatry evaluations at the earliest postoperative time and at discharge from acute rehabilitation. Linear regression models were used to estimate the rate of recovery across the entire MAP range. Results An exploratory analysis revealed that increased time within an intraoperative MAP range (70-94 mmHg) was associated with ISNCSCI motor score improvement. A significant regression equation was found for the MAP range 70-94 mmHg (F[1, 23] = 4.65, r 2 = 0.168, p = 0.042). ISNCSCI motor scores increased 0.036 for each minute of exposure to the MAP range 70-94 mmHg during the operative procedure; this represents a significant correlation between intraoperative time with MAP 70-94 and subsequent motor recovery. Blood pressure exposures above or below this range did not display a positive association with motor recovery. Conclusions Hypertension as well as hypotension during surgery may impact the trajectory of recovery in individuals with SCI, and there may be a direct relationship between intraoperative MAP and motor recovery.
In the general population, females experience depression at significantly higher rates than males. Individuals with traumatic brain injury (TBI) are at substantially greater risk for depression compared to the overall population. Treatment of, and recovery from, TBI can be hindered by depression; comorbid TBI and depression can lead to adverse outcomes and negatively affect multiple aspects of individuals’ lives. Gender differences in depression following TBI are not well understood, and relevant empirical findings have been mixed. Utilizing the Patient Health Questionnaire-9 (PHQ-9) 1 year after TBI, we examined whether women would experience more severe depressive symptoms, and would endorse higher levels of depression within each category of depression severity, than would men. Interestingly, and contrary to our hypothesis, men and women reported mild depression at equal rates; PHQ-9 total scores were slightly lower in women than in men. Men and women did not differ significantly in any PHQ-9 depression severity category. Item analyses, yielded significant gender differences on the following items: greater concentration difficulties (cognitive problems) in men and more sleep disturbances (psychosomatic issues) in women per uncorrected two-sample Z-test for proportions analyses; however, these results were not significant after the family-wise Bonferroni correction. Our results indicate that, in contrast to the general population, mild depression in persons with moderate to severe TBI may not be gender-specific. These findings underscore the need for early identification, active screening, and depression treatment equally for men and women to improve emotional well-being, promote recovery, and enhance quality of life following TBI.
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