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-κB, 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 pre-clinical and clinical investigation.
Inflammation; cytokines; signal pathways; targeted therapies; head and neck cancer
Cancer stem cells (CSC) and genes have been linked to cancer development and therapeutic resistance, but the signaling mechanisms regulating CSC genes and phenotype are incompletely understood. CK2 has emerged as a key signal serine/threonine kinase that modulates diverse signal cascades regulating cell fate and growth. We previously showed that CK2 is often aberrantly expressed and activated in head and neck squamous cell carcinomas (HNSCC), concomitantly with mutant (mt) tumor suppressor TP53, and inactivation of its family member, TAp73. Unexpectedly, we observed that classical stem cell genes Nanog, Sox2, and Oct4, are overexpressed in HNSCC with inactivated TAp73 and mtTP53. However, the potential relationship between CK2, TAp73 inactivation, and CSC phenotype is unknown. We reveal that inhibition of CK2 by pharmacologic inhibitors or siRNA inhibits the expression of CSC genes and side population (SP), while enhancing TAp73 mRNA and protein expression. Conversely, CK2 inhibitor attenuation of CSC protein expression and the SP by was abrogated by TAp73 siRNA. Bioinformatic analysis uncovered a single predicted CK2 threonine phosphorylation site (T27) within the N-terminal transactivation domain of TAp73. Nuclear CK2 and TAp73 interaction, confirmed by co-immunoprecipitation, was attenuated by CK2 inhibitor, or a T27A point-mutation of this predicted CK2 threonine phospho-acceptor site of TAp73. Further, T27A mutation attenuated phosphorylation, while enhancing TAp73 function in repressing CSC gene expression and SP cells. A new CK2 inhibitor, CX-4945, inhibited CSC related SP cells, clonogenic survival, and spheroid formation. Our study unveils a novel regulatory mechanism whereby aberrant CK2 signaling inhibits TAp73 to promote the expression of CSC genes and phenotype.
CK2, Casein Kinase 2; CSC, Cancer Stem Cells; DMAT, 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole; HNSCC, Head and neck squamous cell carcinoma; HEKA, Human epidermal keratinocytes; HOK, Human oral keratinocytes; mt, Mutant; SP, Side population; TAp73, Transactivating p73; TP53, Transforming Protein p53; UM-SCC, University of Michigan Squamous Cell Carcinoma; wt, Wild-type
The Inhibitor-κB Kinase-Nuclear Factor-κB (IKK-NF-κB) and Epidermal Growth Factor Receptor-Activator Protein-1 (EGFR-AP-1) 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 enhancing 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 amongst different cell lines, but overexpression of both IKKs induced the strongest NF-κB activation. Conversely, siRNA knockdown of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα, and alternative p52 and RELB subunits. Knockdown 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 TGFα, EGFR, and AP1 transcription factors cJun, JunB, and Fra1. Knockdown 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 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.
IKKα; IKKβ; NF-κB; AP1; head and neck cancers
Inflammation has significant roles in all phases of tumor development, including initiation, progression and metastasis. Interleukin-10 (IL-10) is a well-known immuno-modulatory cytokine with an anti-inflammatory activity. Lack of IL-10 allows induction of pro-inflammatory cytokines and hinders anti-tumor immunity, thereby favoring tumor growth. The IL-10 network is among the most important paths linking cancer and inflammation. The simple node-and-edge network representation is useful, but limited, hampering the understanding of the mechanistic details of signaling pathways. Structural networks complete the missing parts, and provide details. The IL-10 structural network may shed light on the mechanisms through which disease-related mutations work and the pathogenesis of malignancies.
Using PRISM (a PRotein Interactions by Structural Matching tool), we constructed the structural network of IL-10, which includes its first and second degree protein neighbor interactions. We predicted the structures of complexes involved in these interactions, thereby enriching the available structural data. In order to reveal the significance of the interactions, we exploited mutations identified in cancer patients, mapping them onto key proteins of this network. We analyzed the effect of these mutations on the interactions, and demonstrated a relation between these and inflammation and cancer. Our results suggest that mutations that disrupt the interactions of IL-10 with its receptors (IL-10RA and IL-10RB) and α2-macroglobulin (A2M) may enhance inflammation and modulate anti-tumor immunity. Likewise, mutations that weaken the A2M-APP (amyloid precursor protein) association may increase the proliferative effect of APP through preventing β-amyloid degradation by the A2M receptor, and mutations that abolish the A2M-Kallikrein-13 (KLK13) interaction may lead to cell proliferation and metastasis through the destructive effect of KLK13 on the extracellular matrix.
Prediction of protein-protein interactions through structural matching can enrich the available cellular pathways. In addition, the structural data of protein complexes suggest how oncogenic mutations influence the interactions and explain their potential impact on IL-10 signaling in cancer and inflammation.
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.
TAK1; SMAD7; TGF-β; NF-κB; celastrol; head and neck cancer
In head and neck squamous cell carcinoma (HNSCC), mutations of p53 usually coexist with aberrant activation of NF-kappaB (NF-κB), other transcription factors and microRNAs, which promote tumor pathogenesis. However, how these factors and microRNAs interact to globally modulate gene expression and mediate oncogenesis is not fully understood. We devised a novel bioinformatics method to uncover interactive relationships between transcription factors or microRNAs and genes. This approach is based on matrix decomposition modeling under the joint constraints of sparseness and regulator-target connectivity, and able to integrate gene expression profiling and binding data of regulators. We employed this method to infer the gene regulatory networks in HNSCC. We found that the majority of the predicted p53 targets overlapped with those for NF-κB, suggesting that the two transcription factors exert a concerted modulation on regulatory programs in tumor cells. We further investigated the interrelationships of p53 and NF-κB with five additional transcription factors, AP1, CEBPB, EGR1, SP1 and STAT3, and microRNAs mir21 and mir34ac. The resulting gene networks indicate that interactions among NF-κB, p53, and the two miRNAs likely regulate progression of HNSCC. We experimentally validated our findings by determining expression of the predicted NF-κB and p53 target genes by siRNA knock down, and by examining p53 binding activity on promoters of predicted target genes in the tumor cell lines. Our results elucidating the cross-regulations among NF-κB, p53, and microRNAs provide insights into the complex regulatory mechanisms underlying HNSCC, and shows an efficient approach to inferring gene regulatory programs in biological complex systems.
Heat shock protein 90 (HSP90) is a chaperone protein that stabilizes proteins involved in oncogenic and therapeutic resistance pathways of epithelial cancers, including head and neck squamous cell carcinomas (HNSCCs). Here, we characterized the molecular, cellular, and preclinical activity of HSP90 inhibitor SNX5422/2112 in HNSCC overexpressing HSP90. SNX2112 inhibited proliferation, induced G2/M block, and enhanced cytotoxicity, chemosensitivity, and radiosensitivity between 25 and 250 nM in vitro. SNX2112 showed combinatorial activity with paclitaxel in wild-type (wt) TP53-deficient and cisplatin in mutant (mt) TP53 HNSCC lines. SNX2112 decreased expression or phosphorylation of epidermal growth factor receptor (EGFR), c-MET, v-akt murine thymoma viral oncogene homolog 1 (AKT), extracellular signal-regulated kinases (ERK) 1 and 2, inhibitor κB kinase, and signal transducer and transcription factor 3 (STAT3), corresponding downstream nuclear factor κB, activator protein-1, and STAT3 reporter genes, and target oncogenes and angiogenic cytokines. Furthermore, SNX2112 enhanced re-expression of TP53 and targets p21WAF1 and PUMA, while TP53 inhibitor Pifithrin or siRNA attenuated the antiproliferative activity of SNX2112 in wtTP53 HNSCC in vitro. Prodrug SNX5422 similarly down-modulated key signal targets, enhanced TP53 expression and apoptosis, and inhibited proliferation, angiogenesis, and tumorigenesis in a wtTP53-deficient HNSCC xenograft model. Thus, HSP90 inhibitor SNX5422/2112 broadly modulates multiple key nodes within the dysregulated signaling network, with corresponding effects upon the malignant phenotype. Our data support investigation of SNX5422/2112 in combination with paclitaxel, cisplatin, and radiotherapy in HNSCC with different TP53 status.
The molecular mechanisms that contribute to the initiation and progression of head and neck squamous cell carcinoma (HNSCC) have not been completely delineated. Our observations indicate that defects in the TGF-β and PI3K/Akt signaling pathways are common in human HNSCCs. Conditional activation of the PI3K/Akt pathway due to Pten deletion in the mouse head and neck epithelia gives rise to hyperproliferation, but only a few lesions progress to HNSCC. However, Pten-deficient mice developed full-penetrance HNSCC in combination with type I TGF-β receptor (Tgfbr1) deletion. Molecular analysis revealed enhanced cell proliferation, decreased apoptosis, and increased expression of CCND1 in the basal layer of the head and neck epithelia, as well as in the tumors of Tgfbr1/Pten double conditional knockout (2cKO) mice. Furthermore, neoplastic transformation involves senescence evasion and is associated with an increased number of putative cancer stem cells (CSCs). In addition, the NF-κB pathway activation, myeloid derived suppressor cell (MDSC) infiltration, angiogenesis, and immune suppression in the tumor microenvironment, all of which are characteristic of human HNSCCs, contribute significantly to head and neck carcinogenesis in 2cKO mice. These tumors display pathology and multiple molecular alterations resembling human HNSCCs. This suggests that the Tgfbr1/Pten 2cKO mouse model is suitable for preclinical intervention, and that it has significant implications in the development of diagnostic cancer biomarkers and effective strategies for prevention and treatment of HNSCCs.
TGF-β; PI3K/Akt; Head and Neck Squamous Cell Carcinoma (HNSCC); Conditional Knockout; Cancer Mouse Model
Constitutive activation of nuclear factor κB (NF-κB) is associated with poor prognosis. Irinotecan demonstrates single-agent activity in head and neck cancer but activates NF-κB, promoting cell survival and resistance. Bortezomib is a proteasome inhibitor that inactivates NF-κB.
Patients and Methods
We performed a randomized phase II trial of bortezomib on days 1, 4, 8, and 11 and irinotecan on days 1 and 8 of each 21-day cycle or single-agent bortezomib on days 1, 4, 8, and 11 on a 21-day cycle. The addition of irinotecan to bortezomib was allowed in patients who progressed on bortezomib alone.
The response rate of bortezomib and irinotecan was 13%. One patient had a partial response to bortezomib alone (response rate 3%). No responses were seen in patients with addition of irinotecan at time of progression on bortezomib.
The bortezomib-based regimens evaluated in this study have minimal activity in recurrent or metastatic head and neck cancer.
head and neck cancer; chemotherapy; bortezomib; irinotecan; phase II
Xue et al demonstrate response and increased survival but development of acquired resistance to proteasome and Inhibitor-kappaB Kinase inhibitors targeting NF-kappa B activation in adenocarcinomas of Kras-activated, p53 deficient mice
Inflammation-induced activation of proto-oncogenic NF-κB/REL and dysfunction of tumor suppressor TP53/p63/p73 family transcription factors are key events in cancer progression. How inflammatory signaling coordinates dysregulation of these two transcription factor families during oncogenesis remains incompletely understood. Here, we observed that oncoprotein c-REL and tumor suppressor TAp73 are co-expressed and complex with ΔNp63α in the nucleus of a subset of head and neck squamous cell carcinoma (HNSCC) cell lines with mutant (mt)TP53. TNF-α a pro-inflammatory cytokine, promoted nuclear translocation and c-REL/ΔNp63α interaction and dissociation of TAp73 from nuclear ΔNp63α to the cytoplasm, while c-REL siRNA depletion attenuated this effect. Overexpression of c-REL or a c-REL κB-site DNA binding mutant enhanced protein interaction withΔNp63α and TAp73 dissociation, implicating c-REL/ΔNp63α-specific interactions in these effects. We discovered TNF-α- or genetic alteration of c-REL expression inversely modulatesΔNp63α/TAp73 interactions on distinct p63 DNA binding sites, including those for key growth arrest and apoptotic genes p21WAF1, NOXA, and PUMA. Functionally, c-REL repressed these genes and the anti-proliferative effects of TNF-α or TAp73. Conversely, c-REL siRNA depletion enhanced TAp73 promoter interaction, and expression of genes mediating growth arrest and apoptosis. Similar to TNF-α treated HNSCC lines, human HNSCC tumors and hyperplastic squamous epithelia of transgenic mice overexpressing ΔNp63α that exhibit inflammation, also demonstrate increased nuclear c-REL/ΔNp63α and cytoplasmic TAp73 localization. These findings unveil a novel and reversible dynamic mechanism whereby pro-inflammatory cytokine TNF-α-induced c-REL/ΔNp63α interactions inactivate tumor suppressor TAp73 function, promoting TNF-α resistance and cell survival in cancers with mtTP53.
c-REL; ΔNp63; TAp73; TNF-α; squamous cell carcinoma
Strong epidemiologic evidence links smoking and cancer. An increased understanding of the molecular biology of tobacco-related cancers could advance progress toward improving smoking cessation and patient management. Knowledge gaps between tobacco addiction, tumorigenesis, and cancer brought an interdisciplinary group of investigators together to discuss “The Biology of Nicotine and Tobacco: Bench to Bedside.” Presentations on the signaling pathways and pathogenesis in tobacco-related cancers, mouse models of addiction, imaging and regulation of nicotinic receptors, the genetic basis for tobacco carcinogenesis and development of lung cancer, and molecular mechanisms of carcinogenesis were heard. Importantly, new opportunities to use molecular biology to identify and abrogate tobacco-mediated carcinogenesis and to identify high-risk individuals were recognized.
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.
Gene expression; inflammation; ΔNp63; NF-κB; head and neck cancer
A phase I clinical trial and molecular correlative studies were performed to evaluate preclinical evidence for combinatorial activity of proteasome inhibitor bortezomib, epidermal growth factor receptor (EGFR) inhibitor cetuximab, and radiation therapy.
Patients with radiotherapy-naïve stage IV or recurrent squamous cell carcinoma of the head and neck (SCCHN) were studied. Escalating doses of bortezomib (0.7, 1.0 and 1.3 mg/m2) were given intravenously twice weekly on days 1, 4, 8, 11, every 21 days, with weekly cetuximab beginning 1 week prior and concurrently with intensity modulated radiotherapy (IMRT), delivered in 2Gy fractions to 70-74 Gy. Molecular effects were examined in serial serum and SCCHN tumor specimens, and the cell line UMSCC-1.
Seven patients were accrued before the study was terminated when 5/6 previously untreated patients with favorable prognosis oropharyngeal SCCHN progressed within 1 year (PFS =4.8 months; 95% CI, 2.6-6.9). Three patients each received bortezomib 0.7 or 1.0 mg/m2, without dose-limiting toxicities; 1 patient treated at 1.3 mg/m2 was taken off study due to recurring cetuximab infusion reaction and progressive disease. Expected grade 3 toxicities included radiation mucositis (n=4), dermatitis (n=1), and rash (n=1). SCCHN-related cytokines increased in serial serum specimens of patients developing progressive disease (P=0.029). Bortezomib antagonized cetuximab- and radiation-induced cytotoxicity, degradation of EGFR, and enhanced prosurvival signal pathway activation in SCCHN tumor biopsies and UMSCC-1.
Combining bortezomib with cetuximab and radiation therapy demonstrated unexpected early progression, evidence for EGFR stabilization, increased prosurvival signaling and SCCHN cytokine expression, warranting avoidance of this combination.
bortezomib; cetuximab; epidermal growth factor receptor; radiotherapy; head and neck cancer
The epidermal growth factor receptor (EGFR) has been targeted for inhibition using tyrosine kinase inhibitors and monoclonal antibodies, with improvement in outcome in subsets of patients with head and neck, lung, and colorectal carcinomas. We have previously found that EGFR stability plays a key role in cell survival after chemotherapy and radiotherapy. Heat shock protein 90 (HSP90) is known to stabilize mutant EGFR and ErbB2, but its role in cancers with wild-type (WT) WT-EGFR is unclear. In this report, we demonstrate that fully mature, membrane-bound WT-EGFR interacts with HSP90 independent of ErbB2. Further, the HSP90 inhibitors geldanamycin (GA) and AT13387 cause a decrease in WT-EGFR in cultured head and neck cancer cells. This decrease results from a significantly reduced half-life of WT-EGFR. WT-EGFR was also lost in head and neck xenograft specimens after treatment with AT13387 under conditions that inhibited tumor growth and prolonged survival of the mice. Our findings demonstrate that WT-EGFR is a client protein of HSP90 and that their interaction is critical for maintaining both the stability of the receptor as well as the growth of EGFR-dependent cancers. Furthermore, these findings support the search for specific agents that disrupt HSP90's ability to act as an EGFR chaperone.
Importance of the field
Recent advances in understanding the oncogenesis of head and neck squamous cell carcinomas (HNSCC) have revealed multiple dysregulated signaling pathways. One frequently altered axis is the EGFR/PI3K/Akt/mTOR pathway. This pathway plays a central role in numerous cellular processes including metabolism, cell growth, apoptosis, survival and differentiation, which ultimately contributes to HNSCC progression.
What the reader will gain
This article reviews the current understanding of EGFR/PI3K/Akt/mTOR signaling in HNSCC, including the impact of both genetic and epigenetic alterations. This review further highlights the potential of targeting this signaling cascade as a promising therapeutic approach in the treatment of HNSCC.
Areas covered in this review
Books, journals, databases and websites have been searched to provide a current review on the subject.
Take home message
Genetic alterations of several nodes within this pathway, including both genetic and epigenetic changes, leading to either oncogene activation or inactivation of tumor suppressors, have frequently been implicated in HNSCC. Consequently, drugs that target the central nodes of this pathway have become attractive for molecular oriented cancer therapies. Numerous preclinical and clinical studies are being performed in HNSCC, however, more studies are still needed to better understand the biology of this pathway.
Akt; EGFR; Head and neck squamous cell carcinoma; mTOR; PI3K; targeted therapy
Epidermal growth factor receptor (EGFR) overexpression in head and neck squamous cell carcinoma (HNSCC) stimulates tumor cell proliferation, inhibits apoptosis, and increases chemotherapy and radiation resistance. We examined the toxicity, safety and the effects on EGFR signaling in tumor biopsies from patients with locally advanced HNSCC treated with the EGFR signaling inhibitor gefitinib (GEF) combined with weekly intravenous paclitaxel (PAC) and radiation therapy (RT).
Methods and Materials
A pilot phase I dose-escalation study. Eligibility included stage III-IVB HNSCC, age ≥18 years, no prior RT or chemotherapy, adequate organ function and informed consent. Endpoints included determination of maximum tolerated dose (MTD) and analysis of treatment effect on EGFR signaling, tumor cell proliferation and apoptosis in biopsies.
Ten patients were treated. The MTD of this combination was GEF 250 mg/d with PAC 36 mg/m2 I.V. weekly × 6 with concurrent RT. Grade 3/4 toxicities included prolonged (>8 weeks) stomatitis (7 patients), infection (1), and interstitial pneumonitis (1). There were five complete responses (CR) and two partial responses (PR). Of 7 patients undergoing serial biopsies, only one demonstrated a reduction in phosphorylated-EGFR, decreased downstream signaling and reduced cellular proliferation after initiating GEF.
GEF inhibition of EGFR was observed in only one of seven tumors studied. The addition of GEF to PAC and RT did not appear to improve the response of locally advanced HNSCC compared to our prior experience with PAC and RT alone. This treatment appeared to delay recovery from stomatitis.
Epidermal growth factor receptor; head and neck cancer; gefitinib; paclitaxel; radiation
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.
YAP; p53; ΔNp63; p73; Apoptosis; 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.
Protein kinase CK2, a protein serine/threonine kinase, plays a global role in activities related to cell growth, cell death and cell survival. CK2 has a large number of potential substrates localized in diverse locations in the cell including, e.g., NF-κB as an important downstream target of the kinase. In addition to its involvement in cell growth and proliferation it is also a potent suppressor of apoptosis, raising its key importance in cancer cell phenotype. CK2 interacts with diverse pathways which illustrates the breadth of its impact on the cellular machinery of both cell growth and cell death giving it the status of a “master regulator” in the cell. With respect to cancer, CK2 has been found to be dysregulated in all cancers examined demonstrating increased protein expression levels and nuclear localization in cancer cells compared with their normal counterparts. We originally proposed CK2 as a potentially important target for cancer therapy. Given the ubiquitous and essential for cell survival nature of the kinase, an important consideration would be to target it specifically in cancer cells while sparing normal cells. Towards that end, our design of a tenascin based sub-50 nm (i.e., less than 50 nm size) nanocapsule in which an anti-CK2 therapeutic agent can be packaged is highly promising because this formulation can specifically deliver the cargo intracellularly to the cancer cells in vivo. Thus, appropriate strategies to target CK2 especially by molecular approaches may lead to a highly feasible and effective approach to eradication of a given cancer.
Protein kinase CK2; casein kinase 2; signaling; cancer; NF-κB; nanocapsule; nanoparticle; tenfibgen; tenascin; antisense; siRNA
To investigate the expression of CK2 subunits, and CK2 effects on NF-κB 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.
Protein expression of CK2 subunits was investigated by Western blot and immunohistochemistry. CK2 subunits were knocked down by siRNA, and NF-κB activation was examined using DNA binding, Western blot, and luciferase reporter assays. Gene expression was measured by quantitative RT-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 (ODN) encapsulated in sub-50 nm tenfibgen nanocapsules.
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, while CK2α is critical to promote proliferation, anti-apoptosis and cisplatin resistance in vitro. Further, in vivo delivery of anti-CK2α/α′ ODN 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.
Our study reveals a novel role of CK2 in co-regulating NF-κB activation, and TP53/p63 expression, and downstream gene expression. Downregulation of CK2 in HNSCC models in vitro and in vivo demonstrates antitumor effects as well as sensitization to cisplatin.
CK2; nanocapsules; NF-κB; p53; head and neck cancer
Dyskeratosis congenita (DC) is a heterogeneous inherited bone marrow failure syndrome, characterized by abnormally short telomeres and mutations in telomere biology genes. The spectrum of telomere biology disorders is growing and the clinical management of these patients is complex. A DC-specific workshop was held at the NIH on September 19, 2008; participants included physicians, patients with DC, their family members, and representatives from other support groups. Data from the UK’s DC Registry and the NCI’s DC cohort were described. Updates on the function of the known DC genes were presented. Clinical aspects discussed included androgen therapy, stem cell transplant, cancer risk, and cancer screening. Families with DC met for the first time and formed a family support group (http://www.dcoutreach.com/). Ongoing, open collaboration between the clinical, scientific, and family communities is required for continued improvement in our understanding of DC and the clinical consequences of telomeric defects.
dyskeratosis congenita; bone marrow failure; telomere
To report the long-term outcomes and toxicity of a regimen of infusional paclitaxel delivered concurrently with radiotherapy in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN).
Between 1995 and 1999, 35 patients with non-metastatic stage III or IV SCCHN were treated with 3 cycles of paclitaxel as a 120-hour continuous infusion beginning days 1, 21, and 42 concurrent with radiation. The initial 16 patients received 105 mg/m2/cycle, and the subsequent 19 patients received 120 mg/m2/cycle. External beam radiation was delivered to 70.2–72 Gy in 5 fractions per week. Patients were followed to evaluate disease outcomes and the late toxicity of this regimen.
Median follow-up for all patients is 56.5 months. The median survival was 56.5 months and the median time to local recurrence was not reached. Fifteen patients (43%) developed hypothyroidism. Eleven of the 33 patients who underwent percutaneous endoscopic gastrostomy tube (PEG) placement were PEG-dependent until death or last follow-up. Five patients (14%) required a tracheostomy until death, while 3 patients (9%) suffered from severe esophageal stricture. All evaluated long term survivors exhibited salivary hypofunction. Fibrosis in the radiation field occurred in 24 patients (69%).
Concurrent chemo-radiation therapy with a 120-hour infusion of paclitaxel provides long-term local control and survival in patients with SCCHN.Xerostomia, hypothyroidism, esophageal and pharyngeal complications, and subcutaneous fibrosis were common long-term toxicities; however, the vast majority of toxicities were grade 1 or 2.
paclitaxel; radiation; toxicity; head and neck
Although constitutively activated NF-κB, attenuated 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 TNFα-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 IκBα 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 demonstrate 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.
TGFβ; NF-κB; TP53; head and neck cancer
Aberrant nuclear activation and phosphorylation of the canonical NF-κB subunit RELA/p65 at Serine-536 by Inhibitor Kappa B 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, gene transactivation, and as a potential target for therapy.
Phospho and total p65 protein expression and localization was determined in HNSCC tissue array and in cell lines. The effects of PKA inhibitor H-89 on cell proliferation and cell cycle and of H-89 and PKA specific siRNA knockdown on NF-κB activation and downstream gene expression were examined.
Nuclear 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, TNF-α significantly increased while H-89 inhibited constitutive and TNF-α induced nuclear p65-Ser276 phosphorylation, and significantly suppressed NF-κB and target gene IL-8 reporter activity. Knock down of PKA by siRNA 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.
NF-κB RELA Ser276 phosphorylation by PKA promotes the malignant phenotype and holds potential as a therapeutic target in HNSCC.
NF-κB p65 Serine276; phosphorylation; PKA; H-89; head and neck cancer