Bradford Hall

Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts

Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin-null mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

Loss of TGF-β signaling and PTEN promotes head and neck squamous cell carcinoma through cellular senescence evasion and cancer-related inflammation.

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 transforming growth factor-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. In addition, the nuclear factor-κB pathway activation, myeloid-derived suppressor cell infiltration, angiogenesis and immune suppression in the tumor microenvironment, all of which are characteristics 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.

Overview: Generation of Gene Knockout Mice

The technique of gene targeting allows for the introduction of engineered genetic mutations into a mouse at a determined genomic locus. The process of generating mouse models with targeted mutations was developed through both the discovery of homologous recombination and the isolation of murine embryonic stem cells (ES cells). Homologous recombination is a DNA repair mechanism that is employed in gene targeting to insert a designed mutation into the homologous genetic locus. Targeted homologous recombination can be performed in murine ES cells through electroporation of a targeting construct. These ES cells are totipotent and, when injected into a mouse blastocyst, they can differentiate into all cell types of a chimeric mouse. A chimeric mouse harboring cells derived from the targeted ES cell clone can then generate a whole mouse containing the desired targeted mutation. The initial step for the generation of a mouse with a targeted mutation is the construction of an efficient targeting vector that will be introduced into the ES cells. Curr. Protoc. Cell Biol. 44:19.12.1‐19.12.17.

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.

Control of the development of CD8[alpha][alpha]+ intestinal intraepithelial lymphocytes by TGF-[beta]

The molecular mechanisms directing the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IEL) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IEL. Mice with either a TGF-β1 null mutation or a T cell-specific deletion of the TGF-β receptor I lacked TCRαβ+CD8αα+ IEL, whereas transgenic mice that over-expressed TGF-β1 had an increased population of TCRαβ+CD8αα+ IEL. Defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4-CD8-TCRαβ+CD5+) was observed in the absence of TGF-β. In addition, we showed that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. These data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IEL and the expression of CD8 in T cells.The molecular mechanisms that direct the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-β1 or T cell–specific deletion of TGF-β receptor I lacked TCRαβ+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-β1 had a larger population of TCRαβ+CD8αα+ IELs. We observed defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4−CD8−TCRαβ+CD5+) in the absence of TGF-β. In addition, we found that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IELs and the expression of CD8α in T cells.

Chemopreventive and Chemotherapeutic Actions of mTOR Inhibitor in Genetically Defined Head and Neck Squamous Cell Carcinoma Mouse Model

Purpose: To assess the efficacy of rapamycin treatment in chemoprevention and chemotherapy of tumorigenesis in a genetically defined mouse model of head and neck squamous cell carcinoma (HNSCC). Experimental design: Knockdown of Tgfbr1 and/or Pten using siRNA-mediated RNA interference was carried out in human HNSCC cell lines to analyze molecular changes in the mTOR pathway. Tgfbr1flox/flox; Ptenflox/flox; K14-CreERtam mice were treated with oral gavage of tamoxifen for the conditional deletion of Tgfbr1 and Pten in oral mucosa, resulting in HNSCC. Tgfbr1 and Pten conditonal deletion (2cKO) mice were treated with rapamycin before or after the onset of HNSCC, and the efficacy of this treatment was assessed by determining tumor burden, longevity, and molecular analysis of the mTOR pathway. Molecular changes observed in human HNSCC cell lines and 2cKO mice were compared to identify key alterations in the mTOR pathway. Results: Knockdown of Tgfbr1 and/or Pten in human HNSCC cell lines resulted in activation of mTOR activity complex 1 and increased levels of survivin. Furthermore, we observed similar changes in HNSCC of the 2cKO mouse. In the human HNSCC tissue array, a loss of Tgfbr1 expression correlated with increased survivin levels. Chemopreventive rapamycin treatment significantly delayed the onset of the HNSCC tumors and prolonged survival in 2cKO mice. In addition, we also found that rapamycin had a therapeutic effect on squamous cell carcinomas in these mice. In 2cKO HNSCC tongue tumors, rapamycin treatment induced apoptosis, inhibited cell proliferation and phosphorylation of Akt and S6, and decreased survivin expression. Conclusions: These findings indicate that tumorigenesis in 2cKO HNSCC is associated with activation of the Akt/mTOR/survivin pathway, and inhibition of this pathway by rapamycin treatment successfully ameliorates the onset and progression of tumorigenesis. Clin Cancer Res; 18(19); 5304–13. ©2012 AACR.

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.

Conditional overexpression of TGF-β1 disrupts mouse salivary gland development and function

Transforming growth factor-β (TGF-β) signaling is known to affect salivary gland physiology by influencing branching morphogenesis, regulating ECM deposition, and controlling immune homeostasis. To study the role of TGF-β1 in the salivary gland, we created a transgenic mouse (β1glo) that conditionally overexpresses active TGF-β1 upon genomic recombination by Cre recombinase. β1glo mice were bred with an MMTV (mouse mammary tumor virus)-Cre (MC) transgenic line that expresses the Cre recombinase predominantly in the secretory cells of both the mammary and salivary glands. Although most of the double positive (β1glo/MC) pups die either in utero or just after birth, clear defects in salivary gland morphogenesis such as reduced branching and increased mesenchyme could be seen. Those β1glo/MC mice that survived into adulthood, however, had hyposalivation due to salivary gland fibrosis and acinar atrophy. Increased TGF-β signaling was observed in the salivary gland with elevated phosphorylation of Smad2 and concomitant increase in ECM deposition. In particular, aberrant TGF-β1 overexpression caused salivary gland hypofunction in this mouse model because of the replacement of normal glandular parenchyma with interstitial fibrous tissue. These results further implicate TGF-β in pathological cases of salivary gland inflammation and fibrosis that occur with chronic infections in the glands or with the autoimmune disease, Sjögrens syndrome, or with radiation therapy given to head-and-neck cancer patients.

Control of the development of CD8αα + intestinal intraepithelial lymphocytes by TGF-β

The molecular mechanisms directing the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IEL) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IEL. Mice with either a TGF-β1 null mutation or a T cell-specific deletion of the TGF-β receptor I lacked TCRαβ+CD8αα+ IEL, whereas transgenic mice that over-expressed TGF-β1 had an increased population of TCRαβ+CD8αα+ IEL. Defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4-CD8-TCRαβ+CD5+) was observed in the absence of TGF-β. In addition, we showed that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. These data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IEL and the expression of CD8 in T cells.The molecular mechanisms that direct the development of TCRαβ+CD8αα+ intestinal intraepithelial lymphocytes (IELs) are not thoroughly understood. Here we show that transforming growth factor-β (TGF-β) controls the development of TCRαβ+CD8αα+ IELs. Mice with either a null mutation in the gene encoding TGF-β1 or T cell–specific deletion of TGF-β receptor I lacked TCRαβ+CD8αα+ IELs, whereas mice with transgenic overexpression of TGF-β1 had a larger population of TCRαβ+CD8αα+ IELs. We observed defective development of the TCRαβ+CD8αα+ IEL thymic precursors (CD4−CD8−TCRαβ+CD5+) in the absence of TGF-β. In addition, we found that TGF-β signaling induced CD8α expression in TCRαβ+CD8αα+ IEL thymic precursors and induced and maintained CD8α expression in peripheral populations of T cells. Our data demonstrate a previously unrecognized role for TGF-β in the development of TCRαβ+CD8αα+ IELs and the expression of CD8α in T cells.

Modulating Innate and Adaptive Immunity by (R)-Roscovitine: Potential Therapeutic Opportunity in Cystic Fibrosis

(R)-Roscovitine, a pharmacological inhibitor of kinases, is currently in phase II clinical trial as a drug candidate for the treatment of cancers, Cushings disease and rheumatoid arthritis. We here review the data that support the investigation of (R)-roscovitine as a potential therapeutic agent for the treatment of cystic fibrosis (CF). (R)-Roscovitine displays four independent properties that may favorably combine against CF: (1) it partially protects F508del-CFTR from proteolytic degradation and favors its trafficking to the plasma membrane; (2) by increasing membrane targeting of the TRPC6 ion channel, it rescues acidification in phagolysosomes of CF alveolar macrophages (which show abnormally high pH) and consequently restores their bactericidal activity; (3) its effects on neutrophils (induction of apoptosis), eosinophils (inhibition of degranulation/induction of apoptosis) and lymphocytes (modification of the Th17/Treg balance in favor of the differentiation of anti-inflammatory lymphocytes and reduced production of various interleukins, notably IL-17A) contribute to the resolution of inflammation and restoration of innate immunity, and (4) roscovitine displays analgesic properties in animal pain models. The fact that (R)-roscovitine has undergone extensive preclinical safety/pharmacology studies, and phase I and II clinical trials in cancer patients, encourages its repurposing as a CF drug candidate.