Yansong Bian

TGF-β and NF-κB signal pathway cross-talk is mediated through TAK1 and SMAD7 in a subset of head and neck cancers

Transforming growth factor-beta (TGF-β) has 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, whereas 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 knockdown decreased TGF-β1-induced phosphorylation of IKK, IκB and RELA, degradation of IκBα, RELA nuclear translocation and DNA binding, 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 compound used in traditional Chinese medicine, also decreased TGF-β1-induced phosphorylation of TAK1 and RELA, and suppressed basal, TGF-β1- and tumor necrosis factor-alpha (TNF-α)-induced NF-κB reporter gene activity. Celastrol also inhibited 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.

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

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-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.

Progressive Tumor Formation in Mice with Conditional Deletion of TGF-β Signaling in Head and Neck Epithelia Is Associated with Activation of the PI3k/Akt Pathway

The precise role of transforming growth factor (TGF)-beta signaling in head and neck squamous cell carcinoma (SCC) is not yet fully understood. Here, we report generation of an inducible head- and neck-specific knockout mouse model by crossing TGF-beta receptor I (Tgfbr1) floxed mice with K14-CreER(tam) mice. By applying tamoxifen to oral cavity of the mouse to induce Cre expression, we were able to conditionally delete Tgfbr1 in the mouse head and neck epithelia. On tumor induction with 7,12-dimethylbenz(a)anthracene (DMBA), 45% of Tgfbr1 conditional knockout (cKO) mice (n = 42) developed SCCs in the head and neck area starting from 16 weeks after treatment. However, no tumors were observed in the control littermates. A molecular analysis revealed an enhanced proliferation and loss of apoptosis in the basal layer of the head and neck epithelia of Tgfbr1 cKO mice 4 weeks after tamoxifen and DMBA treatment. The most notable finding of our study is that the phosphoinositide 3-kinase (PI3K)/Akt pathway was activated in SCCs that developed in the Tgfbr1 cKO mice on inactivation of TGF-beta signaling through Smad2/3 and DMBA treatment. These observations suggest that activation of Smad-independent pathways may contribute cooperatively with inactivation of Smad-dependent pathways to promote head and neck carcinogenesis in these mice. Our results revealed the critical role of the TGF-beta signaling pathway and its cross-talk with the PI3K/Akt pathway in suppressing head and neck carcinogenesis.

PI3K/mTOR Inhibitor PF-04691502 Antitumor Activity Is Enhanced with Induction of Wild-Type TP53 in Human Xenograft and Murine Knockout Models of Head and Neck Cancer

Purpose: Phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway activation is often associated with altered expression or mutations of PIK3CA, TP53/p73, PTEN, and TGF-β receptors (TGFBR) in head and neck squamous cell carcinomas (HNSCC). However, little is known about how these alterations affect response to PI3K/mTOR–targeted agents. Experimental Design: In this preclinical study, PI3K/Akt/mTOR signaling was characterized in nine HNSCC (UM-SCC) cell lines and human oral keratinocytes. We investigated the molecular and anticancer effects of dual PI3K/mTOR inhibitor PF-04691502(PF-502) in UM-SCC expressing PIK3CA with decreased wild-type TP53, mutant TP53−/+ mutantTGFBR2, and in HNSCC of a conditional Pten/Tgfbr1 double knockout mouse model displaying PI3K/Akt/mTOR activation. Results: UM-SCC showed increased PIK3CA expression and Akt/mTOR activation, and PF-502 inhibited PI3K/mTORC1/2 targets. In human HNSCC expressing PIK3CA and decreased wtTP53 and p73, PF-502 reciprocally enhanced TP53/p73 expression and growth inhibition, which was partially reversible by p53 inhibitor pifithrin-α. Most UM-SCC with wtTP53 exhibited a lower IC50 than those with mtTP53 status. PF-502 blocked growth in G0–G1 and increased apoptotic sub-G0 DNA. PF-502 suppressed tumorigenesis and showed combinatorial activity with radiation in a wild-type TP53 UM-SCC xenograft model. PF-502 also significantly delayed HNSCC tumorigenesis and prolonged survival of Pten/Tgfbr1-deficient mice. Significant inhibition of p-Akt, p-4EBP1, p-S6, and Ki67, as well as increased p53 and TUNEL were observed in tumor specimens. Conclusions: PI3K-mTOR inhibition can enhance TP53/p73 expression and significantly inhibit tumor growth alone or when combined with radiation in HNSCC with wild-type TP53. PIK3CA, TP53/p73, PTEN, and TGF-β alterations are potential modifiers of response and merit investigation in future clinical trials with PI3K-mTOR inhibitors. Clin Cancer Res; 19(14); 3808–19. ©2013 AACR.

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.

TGF-beta receptor I conditional knockout mice develop spontaneous squamous cell carcinoma

We generated a mouse model with a conditional deletion of TGF-β signaling in the neurons by crossing TGF-β receptor I (TβRI) floxed mice with neurofilament-H (NF-H) Cre mice. 35% of F1 conditional knockout (COKO) mice developed spontaneous squamous cell carcinomas (SCCs) in periorbital and/or perianal regions. Transplantation of these tumors into athymic nude mice resulted in 62% tumorigenicity. To determine whether evasion of the immune response plays any role in this tumorigenesis, we analyzed the expression levels of receptors for interleukin-13 (mIL-13R), a key negative regulator of tumor immunosurveillance, and found that 33% of COKO tumors expressed the IL-13Rα2 chain. Primary cultures of the SCCs expressing IL-13Rα2 were sensitive to the cytotoxic effect of IL-13R-directed cytotoxin treatment. This is the first demonstration that loss of TβRI can lead to spontaneous tumor formation. These mice can serve as a unique mouse model of SCC to evaluate the tumorigenicity and effect of anti-cancer therapeutics.

MEK Inhibitor PD-0325901 Overcomes Resistance to CK2 Inhibitor CX-4945 and Exhibits Anti-Tumor Activity in Head and Neck Cancer

The serine-threonine kinase CK2 exhibits genomic alterations and aberrant overexpression in human head and neck squamous cell carcinomas (HNSCC). Here, we investigated the effects of CK2 inhibitor CX-4945 in human HNSCC cell lines and xenograft models. The IC50s of CX-4945 for 9 UM-SCC cell lines measured by MTT assay ranged from 3.4-11.9 μM. CX-4945 induced cell cycle arrest and cell death measured by DNA flow cytometry, and inhibited prosurvival mediators phospho-AKT and p-S6 in UM-SCC1 and UM-SCC46 cells. CX-4945 decreased NF-κB and Bcl-XL reporter gene activities in both cell lines, but upregulated proapoptotic TP53 and p21 reporter activities, and induced phospho-ERK, AP-1, and IL-8 activity in UM-SCC1 cells. CX-4945 exhibited modest anti-tumor activity in UM-SCC1 xenografts. Tumor immunostaining revealed significant inhibition of PI3K-Akt-mTOR pathway and increased apoptosis marker TUNEL, but also induced p-ERK, c-JUN, JUNB, FOSL1 and proliferation (Ki67) markers, as a possible resistance mechanism. To overcome the drug resistance, we tested MEK inhibitor PD-0325901 (PD-901), which inhibited ERK-AP-1 activation alone and in combination with CX-4945. PD-901 alone displayed significant anti-tumor effects in vivo, and the combination of PD-901 and CX-4945 slightly enhanced anti-tumor activity when compared with PD-901 alone. Immunostaining of tumor specimens after treatment revealed inhibition of p-AKT S129 and p-AKT T308 by CX-4945, and inhibition of p-ERK T202/204 and AP-1 family member FOSL-1 by PD-901. Our study reveals a drug resistance mechanism mediated by the MEK-ERK-AP-1 pathway in HNSCC. MEK inhibitor PD-0325901 is active in HNSCC resistant to CX-4945, meriting further clinical investigation.

Targeting of interleukin-13 receptor α2 for treatment of head and neck squamous cell carcinoma induced by conditional deletion of TGF-β and PTEN signaling

BackgroundThe sixth leading class of cancer worldwide is head and neck cancer, which typically arise within the squamous epithelium of the oral mucosa. Human head and neck squamous cell carcinoma (HNSCC) is known to be difficult to treat and has only a 50% five-year survival rate. With HNSCC, novel therapeutics are needed along with a means of rapidly screening anti-cancer agents in vivo, such as mouse models.MethodsIn order to develop new animal models of cancer to test safety and efficacy of novel therapeutic agents for human HNSCC, tumors resembling clinical cases of human HNSCC were induced in the head and neck epithelium of a genetically engineered mouse model. This mouse model was generated by conditional deletion of two tumor suppressors, Transforming Growth Factor-β Receptor 1 (TGFβRI) and Phosphatase and Tensin homolog (PTEN), in the oral epithelium. We discovered that the tumors derived from these Tgfbr1/Pten double conditional knockout (2cKO) mice over-expressed IL-13Rα2, a high affinity receptor for IL-13 that can function as a tumor antigen. To demonstrate a proof-of-concept that targeted therapy against IL-13Rα2 expression would have any antitumor efficacy in this spontaneous tumor model, these mice were treated systemically with IL-13-PE, a recombinant immunotoxin consisting of IL-13 fused to the Pseudomonas exotoxin A.ResultsTgfbr1/Pten 2cKO mice when treated with IL-13-PE displayed significantly increased survival when compared to the untreated control mice. The untreated mice exhibited weight loss, particularly with the rapid onset of tongue tumors, but the treated mice gained weight while on IL-13-PE therapy and showed no clinical signs of toxicity due to the immunotoxin. Expression of IL-13Rα2 in tumors was significantly decreased with IL-13-PE treatment as compared to the controls and the number of myeloid-derived suppressor cells (MDSC) was also significantly reduced in the spleens of the IL-13-PE treated mice.ConclusionsOur study demonstrates that the Tgfbr1/Pten 2cKO mouse model of human HNSCC is a useful model for assessing antitumor activity of new cancer therapeutic agents, and that IL-13-PE has therapeutic potential to treat human head and neck cancer.

CK2 phosphorylates and inhibits TAp73 tumor suppressor function to promote expression of cancer stem cell genes and phenotype in 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.

Selective blockade of B7‐H3 enhances antitumour immune activity by reducing immature myeloid cells in head and neck squamous cell carcinoma

Immature myeloid cells including myeloid‐derived suppressor cells (MDSCs) and tumour‐associated macrophages (TAMs) promote tumour growth and metastasis by facilitating tumour transformation and angiogenesis, as well as by suppressing antitumour effector immune responses. Therefore, strategies designed to reduce MDSCs and TAMs accumulation and their activities are potentially valuable therapeutic goals. In this study, we show that negative immune checkpoint molecule B7‐H3 is significantly overexpressed in human head and neck squamous cell carcinoma (HNSCC) specimen as compared with normal oral mucosa. Using immunocompetent transgenic HNSCC models, we observed that targeting inhibition of B7‐H3 reduced tumour size. Flow cytometry analysis revealed that targeting inhibition of B7‐H3 increases antitumour immune response by decreasing immunosuppressive cells and promoting cytotoxic T cell activation in both tumour microenvironment and macroenvironment. Our study provides direct in vivo evidence for a rationale for B7‐H3 blockade as a future therapeutic strategy to treat patients with HNSCC.