Upon transforming growth factor-␤ (TGF-␤) binding to its cognate receptor, Smad3 and Smad4 form heterodimers and transduce the TGF-␤ signal to the nucleus. In addition to the Smad pathway, another pathway involving a member of the mitogen-activated protein kinase kinase kinase family of kinases, TGF-␤-activated kinase-1 (TAK1), is required for TGF-␤ signaling. However, it is unknown how these pathways function together to synergistically amplify TGF-␤ signaling. Here we report that the transcription factor ATF-2 (also called CRE-BP1) is bound by a hetero-oligomer of Smad3 and Smad4 upon TGF-␤ stimulation. ATF-2 is one member of the ATF/CREB family that binds to the cAMP response element, and its activity is enhanced after phosphorylation by stress-activated protein kinases such as c-Jun N-terminal kinase and p38. The binding between ATF-2 and Smad3/4 is mediated via the MH1 region of the Smad proteins and the basic leucine zipper region of ATF-2. TGF-␤ signaling also induces the phosphorylation of ATF-2 via TAK1 and p38. Both of these actions are shown to be responsible for the synergistic stimulation of ATF-2 trans-activating capacity. These results indicate that ATF-2 plays a central role in TGF-␤ signaling by acting as a common nuclear target of both Smad and TAK1 pathways.

Mice lacking p63, a single gene that encodes a group of transcription factors that either contain (TA) or lack (⌬N) a transactivation domain, fail to develop stratified epithelia as well as epithelial appendages and limbs. ⌬Np63 isoforms are predominantly expressed during late embryonic and postnatal epidermal development, however, the function of these proteins remains elusive. Using an epidermal-specific inducible knockdown mouse model, we demonstrate that ⌬Np63 proteins are essential for maintaining basement membrane integrity and terminal differentiation of keratinocytes. Furthermore, we have identified two ⌬Np63␣ target genes that mediate these processes. We propose that ⌬Np63␣ initially induces expression of the extracellular matrix component Fras1, which is required for maintaining the integrity of the epidermal-dermal interface at the basement membrane. Subsequently, induction of I B kinase-␣ by ⌬Np63␣ initiates epidermal terminal differentiation resulting in the formation of the spinous layer. Our data provide insights into the role of ⌬Np63␣ in epidermal morphogenesis and homeostasis, and may contribute to our understanding of the pathogenic mechanisms underlying disorders caused by p63 mutations.

IkappaB kinase-alpha (IKK-alpha) exhibits protein-kinase-dependent and -independent functions. Its kinase activity is required for lymphoid organogenesis and mammary gland development, whereas a kinase-independent activity is required for epidermal keratinocyte differentiation. In addition to failed epidermal differentiation, IKK-alpha-deficient mice exhibit abnormal skeletal and craniofacial morphogenesis. As similar defects are not exhibited by mice that experience systemic inhibition of NF-kappaB, we postulated that the morphogenetic defects in IKK-alpha-deficient mice are not caused by reduced NF-kappaB activity but instead are due to failed epidermal differentiation that disrupts proper epidermal-mesodermal interactions. We tested this hypothesis by introducing an epidermal-specific Ikka (also known as Chuk) transgene into IKK-alpha-deficient mice. Mice lacking IKK-alpha in all cell types including bone and cartilage, but not in basal epidermal keratinocytes, exhibit normal epidermal differentiation and skeletal morphology. Thus, epidermal differentiation is required for proper morphogenesis of mesodermally derived skeletal elements. One way by which IKK-alpha controls skeletal and craniofacial morphogenesis is by repressing expression of fibroblast growth factor (FGF) family members, such as FGF8, whose expression is specifically elevated in the limb bud ectoderm of IKK-alpha-deficient mice.

Cell-cycle exit and differentiation of suprabasal epidermal keratinocytes require nuclear IB kinase ␣ (IKK␣), but not its protein kinase activity. IKK␣ also is a suppressor of squamous cell carcinoma (SCC), but its mode of action remains elusive. Postulating that IKK␣ may serve as a transcriptional regulator in keratinocytes, we searched for cell-cycle-related genes that could illuminate this function. IKK␣ was found to control several Myc antagonists, including Mad1, Mad2, and Ovol1, through the association with TGF␤-regulated Smad2/3 transcription factors and is required for Smad3 recruitment to at least one of these targets. Surprisingly, Smad2/3-dependent Mad1 induction and keratinocyte differentiation are independent of Smad4, the almost universal coregulator of canonical TGF␤ signaling. IKK␣ also is needed for nuclear accumulation of activated Smad2/3 in the epidermis, and Smad2/3 are required for epidermal differentiation. We suggest that a TGF␤-Smad2/3-IKK␣ axis is a critical Smad4-independent regulator of keratinocyte proliferation and differentiation.epidermis ͉ cornification ͉ terminal differentiation A critical mediator of NF-B activation (1), IB kinase (IKK) consists of two catalytic subunits, IKK␣ and IKK␤ (2-5), and a regulatory subunit, IKK␥/NEMO (6, 7). Despite structural similarity, IKK␣ and IKK␤ have nonredundant functions, with IKK␤ being the predominant IKK (1, 8) and IKK␣ being a critical regulator of keratinocyte differentiation (9, 10). Without IKK␣, epidermal keratinocytes exhibit enhanced proliferation and failure to differentiate. Consequently, Ikk␣ Ϫ/Ϫ mice are born enshrouded in a taut and thickened, nonstratified, epidermal sheet devoid of barrier function.The mammalian epidermis is a stratified squamous epithelium in which basal keratinocytes undergo asymmetric cell divisions, giving rise to nonproliferative progeny that embark on a differentiation program as they delaminate and move upward through the spinous and granular layers before generating the cornified layer, which provides the crucial barrier function (11, 12). Without IKK␣, this process is blocked, and basal keratinocytes fail to exit the cell cycle (9, 10, 13). Isolated Ikk␣ Ϫ/Ϫ keratinocytes proliferate uncontrollably and do not respond to differentiation-inducing signals such as high Ca 2ϩ (9, 13). The reexpression of IKK␣ in Ikk␣ Ϫ/Ϫ keratinocytes induces growth arrest and allows terminal differentiation, but this function depends neither on IKK␣'s protein kinase activity nor on NF-B. Instead, it requires nuclear accumulation of IKK␣ (14).Recently, IKK␣ was identified as a tumor suppressor in squamous cell carcinoma (SCC), a type of cancer derived from squamous epithelia of the skin, oral and nasal cavities, esophagus, and other sites (15). Decreased nuclear IKK␣ expression was found in about one third of oral SCCs, mainly those that exhibit poorly differentiated phenotype and poor prognosis (16). These results strongly suggest that loss of nuclear IKK␣ contributes to malignant conversion of keratinocytes to less dif...

Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell-redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid "on-target off-tumor" toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G-structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein-4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.

Transcription factor ATF-2 is a nuclear target of stress-activated protein kinases, such as p38, which are activated by various extracellular stresses, including UV light. Here, we show that ATF-2 plays a critical role in hypoxia-and high-cell-density-induced apoptosis and the development of mammary tumors. Compared to wild-type cells, Atf-2 ؊/؊ mouse embryonic fibroblasts (MEFs) were more resistant to hypoxia-and anisomycininduced apoptosis but remained equally susceptible to other stresses, including UV. Atf-2 ؊/؊ and Atf-2MEFs could not express a group of genes, such as Gadd45␣, whose overexpression can induce apoptosis, in response to hypoxia. Atf-2 ؊/؊ MEFs also had a higher saturation density than wild-type cells and expressed lower levels of Maspin, the breast cancer tumor suppressor, which is also known to enhance cellular sensitivity to apoptotic stimuli. Atf-2 ؊/؊ MEFs underwent a lower degree of apoptosis at high cell density than wild-type cells. Atf-2 ؉/؊ mice were highly prone to mammary tumors that expressed reduced levels of Gadd45␣ and Maspin. The ATF-2 mRNA levels in human breast cancers were lower than those in normal breast tissue. Thus, ATF-2 acts as a tumor susceptibility gene of mammary tumors, at least partly, by activating a group of target genes, including Maspin and Gadd45␣.

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