Pattatheyil Arun

Current and potential inflammation targeted therapies in head and neck cancer.

Inflammation often exists in the tumor microenvironment and is induced by inflammatory mediators (cytokines, chemokines, and growth factors) produced by the tumor, stroma, and infiltrating cells. These factors modulate tissue remodeling and angiogenesis and actively promote tumor cell survival and chemoresistance through autocrine and paracrine mechanisms. Head and neck squamous cell carcinomas (HNSCCs) are highly inflammatory and aggressive in nature, and they express a number of cytokines and growth factors involved in inflammation. These cytokines and growth factors activate important signal transduction pathways, including NF-kappaB, JAK/STAT, and PI3K/Akt/mTOR, which regulate the expression of genes controlling growth, survival, and chemosensitivity. This review provides an update on recent advances in the understanding of the mechanisms driving cancer-related inflammation in HNSCC and on molecular targeted therapies under preclinical and clinical investigation.

NF-κB in Carcinoma Therapy and Prevention

Background: NF-κB includes a family of signal-activated transcription factors that normally regulate responses to injury and infection but which are aberrantly activated in many carcinomas. Objective: To review the activation and role of NF-κB in pathogenesis and as a target for treatment and prevention in carcinoma. Methods: Evidence from experimental, epidemiological, preclinical studies and clinical trials cited in the literature are reviewed. Results/conclusion: Cumulative evidence implicates NF-κB in cell survival, inflammation, angiogenesis, spread and therapeutic resistance during tumor development, progression and metastasis of carcinomas. Non-specific natural and synthetic agents that inhibit NF-κB have demonstrated activity and safety in prevention or therapy. NF-κB-activating kinases and the proteasome are under investigation for targeted prevention and therapy of carcinoma.

Bortezomib-Induced Apoptosis with Limited Clinical Response Is Accompanied by Inhibition of Canonical but not Alternative Nuclear Factor-κB Subunits in Head and Neck Cancer

Purpose: Nuclear factor-κB (NF-κB)/REL transcription factors promote cancer cell survival and progression. The canonical (NF-κB1/RELA or cREL) and alternate (NF-κB2/RELB) pathways require the proteasome for cytoplasmic-nuclear translocation, prompting the investigation of bortezomib for cancer therapy. However, limited clinical activity of bortezomib has been observed in many epithelial malignancies, suggesting this could result from incomplete inhibition of NF-κB/RELs or other prosurvival signal pathways. Experimental Design: To examine these possibilities, matched biopsies from 24 h posttreatment were obtained from accessible tumors of patients who received low-dose bortezomib (0.6 mg/m2) before reirradiation in a phase I trial for recurrent head and neck squamous cell carcinoma (HNSCC). Effects of bortezomib on apoptosis and proliferation by TUNEL and Ki67 staining were compared with nuclear staining for all five NF-κB subunits, phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), and phosphorylated signal transducers and activators of transcription 3 (STAT3) in tumor biopsies, and by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTP) and DNA binding assay for the five NF-κB subunits in HNSCC cell lines. Results: HNSCC showed increased nuclear staining for all five NF-κB subunits, phosphorylated ERK1/2, and phosphorylated STAT3. Bortezomib treatment significantly enhanced apoptosis with inhibition of nuclear RELA in three of four tumors, but other NF-κB subunits, ERK1/2, and STAT3 were variably or not affected, and tumor progression was observed within 3 months. In HNSCC cell lines, 10−8 mol/L bortezomib inhibited cell density while inhibiting tumor necrosis factor-α–induced and partially inhibiting basal activation of NF-κB1/RELA, but not NF-κB2/RELB. Conclusions: Although low-dose bortezomib inhibits activation of subunits of the canonical pathway, it does not block nuclear activation of the noncanonical NF-κB or other prosurvival signal pathways, which may contribute to the heterogeneous responses observed in HNSCC.

CK2 Modulation of NF-κB, TP53, and the Malignant Phenotype in Head and Neck Cancer by Anti-CK2 Oligonucleotides In vitro or In vivo via Sub-50-nm Nanocapsules

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

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

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)

Aberrant IKKα and IKKβ cooperatively activate NF-κB and induce EGFR/AP1 signaling to promote survival and migration of head and neck cancer

The inhibitor-κB kinase-nuclear factor-κB (IKK-NF-κB) and epidermal growth factor receptor-activator protein-1 (EGFR-AP1) pathways are often co-activated and promote malignant behavior, but the underlying basis for this relationship is unclear. Resistance to inhibitors of IKKβ or EGFR is observed in head and neck squamous cell carcinomas (HNSCC). Here, we reveal that both IKKα and β contribute to nuclear activation of canonical and alternate NF-κB/REL family transcription factors, and overexpression of signal components that enhance co-activation of the EGFR-AP1 pathway. We observed that IKKα and IKKβ exhibit increased protein expression, nuclear localization, and phosphorylation in HNSCC tissues and cell lines. Individually, IKK activity varied among different cell lines, but overexpression of both IKKs induced the strongest NF-κB activation. Conversely, siRNA knock down of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα and alternative p52 and RELB subunits. Knock down of both IKKs more effectively inhibited NF-κB activation, broadly modulated gene expression and suppressed cell proliferation and migration. Global expression profiling revealed that NF-κB, cytokine, inflammatory response and growth factor signaling are among the top pathways and networks regulated by IKKs. Importantly, IKKα and IKKβ together promoted the expression and activity of transforming growth factor α, EGFR and AP1 transcription factors cJun, JunB and Fra1. Knock down of AP1 subunits individually decreased 8/15 (53%) of IKK-targeted genes sampled and similarly inhibited cell proliferation and migration. Mutations of NF-κB and AP1-binding sites abolished or decreased IKK-induced interleukin-8 (IL-8) promoter activity. Compounds such as wedelactone with dual IKK inhibitory activity and geldanomycins that block IKKα/β and EGFR pathways were more active than IKKβ-specific inhibitors in suppressing NF-κB activation and proliferation and inducing cell death. We conclude that IKKα and IKKβ cooperatively activate NF-κB and EGFR/AP1 networks of signaling pathways and contribute to the malignant phenotype and the intrinsic or acquired therapeutic resistance of HNSCC.

Attenuated Transforming Growth Factor β Signaling Promotes Nuclear Factor-κB Activation in Head and Neck Cancer

Although constitutively activated nuclear factor-kappaB (NF-kappaB), attenuated transforming growth factor beta (TGFbeta) 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-kappaB-related genes, reduced expression of TGFbeta receptor (TbetaR) subunits and downstream targets, and TP53 genotype in head and neck squamous cell carcinoma (HNSCC). In response to recombinant TGFbeta1, both growth inhibition and TGFbeta target gene modulation were attenuated or absent in a panel of human HNSCC lines. However, in HNSCC cells that retained residual TGFbeta signaling, TGFbeta1 inhibited both constitutive and tumor necrosis factor alpha-stimulated NF-kappaB activity. Furthermore, HNSCC lines overexpressing mutant (mt) TP53 and human tumor specimens with positive TP53 nuclear staining exhibited reduced TbetaRII and knocking down mtTP53 induced TbetaRII, increasing TGFbeta downstream gene expression while inhibiting proinflammatory NF-kappaB target gene expression. Transfection of ectopic TbetaRII directly restored TGFbeta signaling while inhibiting inhibitor kappaBalpha degradation and suppressing serine-536 phosphorylation of NF-kappaB p65 and NF-kappaB transcriptional activation, linking these alterations. Finally, experiments with TbetaRII conditional knockout mice show that abrogation of TGFbeta signaling promotes the sustained induction of NF-kappaB and its proinflammatory target genes during HNSCC tumorigenesis and progression. Together, these findings elucidate a regulatory framework in which attenuated TGFbeta signaling promotes NF-kappaB activation and squamous epithelial malignancy in the setting of altered TP53 status.

Attenuated Transforming Growth Factor Signaling Promotes Nuclear Factor- B Activation in Head and Neck Cancer

Although constitutively activated nuclear factor-kappaB (NF-kappaB), attenuated transforming growth factor beta (TGFbeta) 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-kappaB-related genes, reduced expression of TGFbeta receptor (TbetaR) subunits and downstream targets, and TP53 genotype in head and neck squamous cell carcinoma (HNSCC). In response to recombinant TGFbeta1, both growth inhibition and TGFbeta target gene modulation were attenuated or absent in a panel of human HNSCC lines. However, in HNSCC cells that retained residual TGFbeta signaling, TGFbeta1 inhibited both constitutive and tumor necrosis factor alpha-stimulated NF-kappaB activity. Furthermore, HNSCC lines overexpressing mutant (mt) TP53 and human tumor specimens with positive TP53 nuclear staining exhibited reduced TbetaRII and knocking down mtTP53 induced TbetaRII, increasing TGFbeta downstream gene expression while inhibiting proinflammatory NF-kappaB target gene expression. Transfection of ectopic TbetaRII directly restored TGFbeta signaling while inhibiting inhibitor kappaBalpha degradation and suppressing serine-536 phosphorylation of NF-kappaB p65 and NF-kappaB transcriptional activation, linking these alterations. Finally, experiments with TbetaRII conditional knockout mice show that abrogation of TGFbeta signaling promotes the sustained induction of NF-kappaB and its proinflammatory target genes during HNSCC tumorigenesis and progression. Together, these findings elucidate a regulatory framework in which attenuated TGFbeta signaling promotes NF-kappaB activation and squamous epithelial malignancy in the setting of altered TP53 status.

Abstract B103: HSP90 inhibition targets the dysregulated signal and transcription factor network and malignant phenotype of head and neck squamous cell carcinoma.

Purpose: Activation of multiple oncogenic signaling pathways and transcription factors, concurrent with defective tumor suppressor TP53, have been implicated in pathogenesis and therapeutic resistance of epithelial cancers. Many proteins upon which these cancers depend are stabilized by the chaperone, heat shock protein 90 (HSP90). Here we characterized the effects of HSP90 inhibitor, PF-04928473, against the dysregulated network and malignant phenotype in head and neck squamous cell carcinoma (HNSCC). Experimental Design: HSP90 expression was analyzed in HNSCC tissue array and cell lines. Expression and function of oncogenic molecules and cytokines were analyzed by Western blot, reporter gene, and multiplex Luminex assays. Drug effects on proliferation, cell cycle, and radiosensitivity were evaluated by MTT, flow cytometry, and clonogenic assays. Anti-tumor effects and modulation of immunohistochemical markers were evaluated in a TP53-deficient xenograft model resistant to targeted therapies. Results: HNSCC tissues and lines overexpressed HSP90. PF-04928473 inhibited dysregulated EGFR, c-MET, AKT, ERK1/2, IKK signal molecules, AP-1, STAT3, and NF-B reporter genes, and expression of target oncogenes and angiogenic cytokines. PF-04928473 induced re-expression of TP53 and targets p21 and PUMA in TP53-deficient HNSCC. Corresponding to effects on these molecules implicated in HNSCC pathogenesis, PF-04928473 inhibited proliferation, induced G2/M block, apoptosis, and enhanced radiosensitivity in vitro. PF-04928473 similarly modulated key targets, inducing apoptosis, and inhibiting proliferation, angiogenesis and xenograft tumorigenesis in vivo. Conclusion: HSP90 antagonist PF-04928473 modulated the dysregulated signal and transcriptional network, inhibited the malignant phenotype, and radiosensitized HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B103.

Abstract LB-289: Synthetic Hsp90 inhibitor, PF-04928473 and prodrug PF-04929113, demonstrates potent antitumor activity in head and neck cancer by inhibiting multiple proliferative and prosurvival signaling pathways

Purpose: Heat shock protein 90 (Hsp90) is a molecular chaperone that stabilizes client substrates involved in cell proliferation, survival, and apoptosis. Cancer cells display increased dependency on Hsp90 and client oncoproteins, and Hsp90 has been identified as a druggable target. This study investigated the biological activity of a synthetic small molecule inhibitor of Hsp90, PF-04928473 and the orally available pro-drug PF-04929113 in head and neck squamous cell carcinoma (HNSCC). Experimental Design: Hsp90 expression was examined in nine human HNSCC cell lines (UMSCC). In vitro, the effect of PF-04928473 on cell proliferation, death and cell cycle were examined by MTT assay and flow cytometry. The effects on signaling molecules and cytokine production were determined using Western blot, reporter gene, and Luminex assays. In vitro combination with radiation was examined using the clonogenic survival assay. The effect of pro-drug PF-04929113 on UMSCC-11A tumor growth and Hsp90 client proteins were examined in the murine xenograft model and by immunohistochemistry (IHC). Results: UMSCC cell lines over-expressed Hsp90 mRNA and protein when compared with normal keratinocytes. In vitro, PF-04928473 potently inhibited UMSCC cell growth, induced cell death and modulated cell cycle, with an IC50 range of 32-73nmol/L. Combining radiation and PF-04928473 increased radiation sensitivity by a dose modifying effect of 1.4. PF-04928473 significantly modulated signaling molecules, including down-regulation of c-Met receptor, IKK, IKK, and BCLXL, as well as up-regulation of cleaved PARP, pro-apoptotic p53, PUMA, and p21 protein expression. The drug inhibited AKT, ERK, and STAT3 phosphorylation, suppressed NF- B, AP-1, BCLXL, and IL8 reporter activities, and blocked IL8, IL6, and VEGF production. In vivo, pro-drug PF-04929113 20mg/kg daily for 3-7 weeks significantly inhibited tumor growth. After treatment, tumor specimens showed increased IHC staining for p53, PUMA, and TUNEL, and decreased staining for c-Met, SRC, p-STAT3, p-AKT, p-ERK, IKK, RELB, Ki67 and CD31. Conclusion: Synthetic Hsp90 inhibitor PF-04928473 effectively modulated proliferation, cell survival, and inflammatory and angiogeneic cytokine production in HNSCC in vitro. Pro-drug PF-04929113 inhibits tumor growth in vivo by promoting apoptosis through inhibition of several key signaling pathways. [Supported by NIDCD project ZIA-DC-000073] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-289.