Supplementary Figure 1 from ΔNp63 Versatilely Regulates a Broad NF-κB Gene Program and Promotes Squamous Epithelial Proliferation, Migration, and Inflammation

Supplementary Methods, References, Legends for Figures 1-5 from ΔNp63 Versatilely Regulates a Broad NF-κB Gene Program and Promotes Squamous Epithelial Proliferation, Migration, and Inflammation

Supplementary Figure 2 from ΔNp63 Versatilely Regulates a Broad NF-κB Gene Program and Promotes Squamous Epithelial Proliferation, Migration, and Inflammation

Supplementary Data from Nuclear NF-κB p65 Phosphorylation at Serine 276 by Protein Kinase A Contributes to the Malignant Phenotype of Head and Neck Cancer

Supplementary Tables 1-4 from ΔNp63 Versatilely Regulates a Broad NF-κB Gene Program and Promotes Squamous Epithelial Proliferation, Migration, and Inflammation

Abstract Although constitutively activated nuclear factor-κB (NF-κB), attenuated transforming growth factor β (TGFβ) 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-κB–related genes, reduced expression of TGFβ receptor (TβR) subunits and downstream targets, and TP53 genotype in head and neck squamous cell carcinoma (HNSCC). In response to recombinant TGFβ1, both growth inhibition and TGFβ target gene modulation were attenuated or absent in a panel of human HNSCC lines. However, in HNSCC cells that retained residual TGFβ signaling, TGFβ1 inhibited both constitutive and tumor necrosis factor α–stimulated NF-κB activity. Furthermore, HNSCC lines overexpressing mutant (mt) TP53 and human tumor specimens with positive TP53 nuclear staining exhibited reduced TβRII and knocking down mtTP53 induced TβRII, increasing TGFβ downstream gene expression while inhibiting proinflammatory NF-κB target gene expression. Transfection of ectopic TβRII directly restored TGFβ signaling while inhibiting inhibitor κBα degradation and suppressing serine-536 phosphorylation of NF-κB p65 and NF-κB transcriptional activation, linking these alterations. Finally, experiments with TβRII conditional knockout mice show that abrogation of TGFβ signaling promotes the sustained induction of NF-κB and its proinflammatory target genes during HNSCC tumorigenesis and progression. Together, these findings elucidate a regulatory framework in which attenuated TGFβ signaling promotes NF-κB activation and squamous epithelial malignancy in the setting of altered TP53 status. [Cancer Res 2009;69(8):3415–24]

Supplementary Methods, Figures 1-6 from Attenuated Transforming Growth Factor β Signaling Promotes Nuclear Factor-κB Activation in Head and Neck Cancer

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.

Head and neck squamous cell carcinoma (HNSCC) and many other epithelial malignancies exhibit increased proliferation, invasion, and inflammation, concomitant with aberrant nuclear activation of TP53 and NF-kB family members DNp63, cRel, and RelA. However, the mechanisms of cross-talk by which these transcription factors coordinate gene expression and the malignant phenotype remain elusive. In this study, we showed that DNp63 regulates a cohort of genes involved in cell growth, survival, adhesion, and inflammation, which substantially overlaps with the NF-kB transcriptome. DNp63 with cRel and/or RelA are recruited to form novel binding complexes on p63 or NF-kB/Rel sites of multitarget gene promoters. Overexpressed DNp63or TNF-a–induced NF-kB and inflammatory cytokine interleukin-8 (IL-8) reporter activation depended on RelA/cRel regulatory binding sites. Depletion of RelA or DNp63 by small interfering RNA (siRNA) significantly inhibited NF-kB–specific, or TNF-a–induced IL-8 reporter activation. DNp63 siRNA significantly inhibited proliferation, survival, and migration by HNSCC cells in vitro. Consistent with these data, an increase in nuclear DNp63, accompanied by increased proliferation (Ki-67) and adhesion (b4 integrin) markers, and induced inflammatory cell infiltration was observed throughout HNSCC specimens, when compared with the basilar pattern of protein expression and minimal inflammation seen in nonmalignant mucosa. Furthermore, overexpression of DNp63a in squamous epithelial cells in transgenic mice leads to increased suprabasilar cRel, Ki-67, and cytokine expression, together with epidermal hyperplasia and diffuse inflammation, similar to HNSCC. Our study reveals DNp63 as a master transcription factor that, in coordination with NF-kB/Rels, orchestrates a broad gene program promoting epidermal hyperplasia, inflammation, and the malignant phenotype of HNSCC. Cancer Res; 71(10); 3688–700. 2011 AACR.

AbstractHead and neck squamous cell carcinoma (HNSCC) and many other epithelial malignancies exhibit increased proliferation, invasion, and inflammation, concomitant with aberrant nuclear activation of TP53 and NF-κB family members ΔNp63, cRel, and RelA. However, the mechanisms of cross-talk by which these transcription factors coordinate gene expression and the malignant phenotype remain elusive. In this study, we showed that ΔNp63 regulates a cohort of genes involved in cell growth, survival, adhesion, and inflammation, which substantially overlaps with the NF-κB transcriptome. ΔNp63 with cRel and/or RelA are recruited to form novel binding complexes on p63 or NF-κB/Rel sites of multitarget gene promoters. Overexpressed ΔNp63- or TNF-α–induced NF-κB and inflammatory cytokine interleukin-8 (IL-8) reporter activation depended on RelA/cRel regulatory binding sites. Depletion of RelA or ΔNp63 by small interfering RNA (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 these data, an increase in nuclear ΔNp63, accompanied by increased proliferation (Ki-67) and adhesion (β4 integrin) markers, and induced inflammatory cell infiltration was observed throughout HNSCC specimens, when compared with the basilar pattern of protein expression and minimal inflammation seen in nonmalignant mucosa. Furthermore, overexpression of ΔNp63α in squamous epithelial cells in transgenic mice leads to increased suprabasilar cRel, 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. Cancer Res; 71(10); 3688–700. ©2011 AACR.

Overexpression of the Yes-associated protein (YAP), and TP53 family members ΔNp63 and p73, have been independently detected in subsets of head and neck squamous cell carcinomas (HNSCCs). YAP may serve as a nuclear cofactor with ΔNp63 and p73, but the functional role of YAP and their potential relationship in HNSCCs are unknown. In this study, we show 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 with decreased expression of ΔNp63 and p73. In another subset, YAP expression is decreased but detectable in the nucleus in association with lower AKT and YAP phosphorylation, and with increased ΔNp63 and p73 expression. Inhibiting AKT decreased serine-127 phosphorylation and enhanced nuclear translocation of YAP. ΔNp63 bound to the YAP promoter and suppressed its expression. 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/or ΔNp63 are potential targets for enhancing YAP-mediated apoptosis and chemosensitivity in HNSCCs.

AbstractPurpose: 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. Clin Cancer Res; 16(8); 2295–307. ©2010 AACR.

Abstract 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 G1-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 p21CIP1/WAF1, while suppressing proliferative marker Ki67. Conclusions: NF-κB p65 (Ser276) phosphorylation by PKA promotes the malignant phenotype and holds potential as a therapeutic target in HNSCC. (Clin Cancer Res 2009;15(19):5974–84)

Although constitutively activated nuclear factor-kappa beta (NF-kappa B), attenuated transforming growth factor beta (TGF beta) 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-kappa B-related genes, reduced expression of TGF beta receptor (T beta R) subunits and downstream targets, and TP53 genotype in head and neck squamous cell carcinoma (HNSCC). In response to recombinant TGF beta 1, both growth inhibition and TGIF( target gene modulation were attenuated or absent in a panel of human HNSCC lines. However, in HNSCC cells that retained residual TGF beta signaling, TGF beta 1 inhibited both constitutive and tumor necrosis factor alpha-stimulated NF-kappa B activity. Furthermore, HNSCC lines overexpressing mutant (mt) TP53 and human tumor specimens with positive TP53 nuclear staining exhibited reduced T beta RII and knocking down mtTP53 induced T beta RII, increasing TGF beta downstream gene expression while inhibiting proinflammatory NF-kappa B target gene expression. Transfection of ectopic T beta RII directly restored TGF beta signaling while inhibiting inhibitor kappa B alpha degradation and suppressing serine-536 phosphorylation of NF-kappa B p65 and NF-kappa B transcriptional activation, linking these alterations. Finally, experiments with T beta RII conditional knockout mice show that abrogation of TGF beta signaling promotes the sustained induction of NF-kappa B and its proinflammatory target genes during HNSCC tumorigenesis and progression. Together, these findings elucidate a regulatory framework in which attenuated TGF beta signaling promotes NF-kappa B activation and squamous epithelial malignancy in the setting of altered TP53 status. [Cancer Res 2009;69(8):3415-24]

Abstract We previously reported that protein kinase CK2 contributes to aberrant NF-kappaB activation, but the molecular mechanisms and antitumor effects of targeting CK2 have not been thoroughly investigated in head and neck squamous cell carcinoma (HNSCC). In the present study, we showed elevated CK2alpha, alpha' and beta mRNA and protein expression in a panel of HNSCC lines and tissues. siRNA specifically knocked down CK2 subunits and differentially inhibited IKKbeta mediated IkappaBalpha degradation, p65 synthesis and phosphorylation at serine 536 and 529, p65 and p50 binding, and NF-kappaB reporter activities. CK2alpha knockdown also upregulated two genes promoting apoptosis, TP53 and TAp63, which together with inhibition of NF-kappaB downregulated genes promoting growth, survival and adhesion, such as CyclinD1, BCL-XL, ITGA3, and ITGB4. Knockdown of CK2alpha' or beta also exhibited differential effects on gene expression. Consequently, CK2alpha siRNA most significantly inhibited proliferation, cell cycle, and migration, while inducing apoptosis and cisplatin sensitivity in vitro. Furthermore, delivery of antisense targeting CK2alpha and alpha' using tenfibgen (fibrinogen fragment of tenascin) coated nanocapsules significantly suppressed tumor growth in HNSCC xenograft models. Consistent with CK2 inhibition, antisense-treated tumor tissue specimens showed decreased expression of CK2alpha and alpha', NF-kappaB p65, Cyclin D1, Bcl-XL and Bcl2 proteins, decreased NF-kappaB p65 phosphorylation at serine 536 and 529, as well as increased TP53 and p63 by immunohistochemistry. Our study revealed the novel role of CK2 subunits in co-regulating NF-kappaB and TP53/p63 expression and signaling, as well as downstream gene expression, which result in anti-tumor effects in vitro and in vivo. Targeting the CK2 subunits may be an effective antitumor strategy with the ability to enhance the efficacy of cisplatin chemotherapy. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C156.

Aberrant activation of NF-\#954;B is prevalent in head and neck squamous cell carcinoma (HNSCC), mainly through nuclear translocation and phosphorylation of p65 at Ser 536 by the IKK\#945;/\#946;/\#947; complex. However, the role of other kinases in NF-\#954;B p65 phosphorylation in HNSCC has not been delineated. This study underscores the importance of an alternative site of p65 phosphorylation at Ser 276 by Protein Kinase A (PKA) that contributes to NF-\#954;B gene transactivation and the malignant phenotype. Immunohistochemistry and Western blot were performed on human tumor tissue arrays and HNSCC cell lines to detect presence and localization of phosphorylated and total p65. PKA inhibitor, H-89 was used to study effects of PKA inhibition on cell proliferation by MTT assay and cell cycle by flow cytometry. The effects of H-89 and PKA siRNA knockdown on NF-\#954;B activation and downstream gene expression were studied using luciferase reporter assays and by real time RT-PCR. Nuclear localized p65 is phosphorylated at Ser 276 in HNSCC and adjacent mucosal margins, but not in the normal mucosa. In HNSCC cell lines, TNF-\#945; significantly increased nuclear phosphorylated p65 at Ser 276. PKA inhibitor H-89, inhibited constitutive and TNF induced p65 Ser 276 phosphorylation, and significantly suppressed NF-\#954;B and its dependent IL-8 reporter gene activities. Knock down of PKA by siRNA also inhibited NF-\#954;B, IL-8 and Bcl-xL reporter activities. In addition, H-89 suppressed cell proliferation, induced cell death and blocked the cell cycle in G1/S phase. Consistent with its biological effects, H-89 modulated expression of NF-\#954;B related genes, such as cyclin D1, Bcl2, Bcl-xL, Cox2, IL-8, and VEGF, as well as induced p21, which controls cell cycle, and suppressed Ki67, which is a biomarker for proliferation. We have , for the first time, shown that a PKA mediated nuclear phosphorylation of p65 at Ser 276 is observed in HNSCC and premalignant lesions, in contrast to cytoplasmic localization in normal mucosa. HNSCC cell lines showed similar constitutive and TNF-\#945;-inducible nuclear activation. This site specific phosphorylation has been found to be important for NF-\#954;B transactivation and has been shown to regulate expression of genes controlling various components of the cancer phenotype such as cell cycle, apoptosis, inflammation, angiogenesis and proliferation. The study highlights the potential of kinases other than IKK to activate the NF-\#954;B signaling cascade and highlights the fact that inhibition of these kinases or their effects on site specific phosphorylation may need to be addressed in future prevention and treatment strategies. The aberrant activation of p65(Ser 276) at the tumor margins and in dysplastic epithelium may hold promise as a biomarker to predict the behavior of premalignant lesions of head and neck and to assess efficacy of chemopreventive interventions. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2758.

Tumor progression of oral and other head and neck squamous cell carcinomas (HNSCCs) from dysplasia to metastasis involves a series of pathologic phenotypic changes considered to be the hallmarks of cancer, and these have been associated with a number of genetic, epigenetic, and molecular alterations (Fig. 10.1). Pathologic phenotypic changes precede metastasis and include increased cell proliferation, survival, and horizontal spread, which require certain molecular changes that together with later events contribute to the metastatic phenotype. These steps commonly include altered expression of molecules regulating the cell cycle and death (e.g., p53), growth factor response (epidermal growth factor receptor, EGFR), protein synthesis and metabolism (mammalian targets of rapamycin, mTOR), and cell immortality (telomerase). The subsequent steps of invasion and metastasis involve penetration and breakdown of the extracellular matrix (ECM) comprising the basement membrane and interstitial connective tissue; formation and invasion of a new stroma of host inflammatory and mesenchymal cells; neoangiogenesis and lymphangiogenesis; and distant spread via these lymphatics and blood vessels to secondary regional and distant sites. The molecular events accompanying the metastatic stage commonly include loss of expression or function of tumor suppressor genes or increased expression or function of oncogenes including numerous growth factors, cytokines, cell adhesion molecules and proteases, signal kinases, and nuclear transcription factors. Nuclear transcription factors appear to play a central role in malignant transformation and metastasis, since direct overexpression, mutation or activation of these molecules, or components of various upstream signaling pathways that modulate their function (Chaps. 8, 9, 11, 13) result in altered regulation of expression of diverse gene programs that produce the phenotypic changes characteristic of cancer and metastasis (Chaps. 4, 5, 12-14).