Delineating functional mechanisms of the p53/p63/p73 family of transcription factors through identification of protein-protein interactions using interface mimicry.

Members of the p53 family of transcription factors-p53, p63, and p73-share a high degree of homology; however, members can be activated in response to different stimuli, perform distinct (sometimes opposing) roles and are expressed in different tissues. The level of complexity is increased further by the transcription of multiple isoforms of each homolog, which may interact or interfere with each other and can impact cellular outcome. Proteins perform their functions through interacting with other proteins (and/or with nucleic acids). Therefore, identification of the interactors of a protein and how they interact in 3D is essential to fully comprehend their roles. By utilizing an in silico protein-protein interaction prediction method-HMI-PRED-we predicted interaction partners of p53 family members and modeled 3D structures of these protein interaction complexes. This method recovered experimentally known interactions while identifying many novel candidate partners. We analyzed the similarities and differences observed among the interaction partners to elucidate distinct functions of p53 family members and provide examples of how this information may yield mechanistic insight to explain their overlapping versus distinct/opposing outcomes in certain contexts. While some interaction partners are common to p53, p63, and p73, the majority are unique to each member. Nevertheless, most of the enriched pathways associated with these partners are common to all members, indicating that the members target the same biological pathways but through unique mediators. p63 and p73 have more common enriched pathways compared to p53, supporting their similar developmental roles in different tissues.

Intersection of the p63 and NF-κB pathways in epithelial homeostasis and disease.

Overexpression of ΔNp63α, a member of the p53/p63/p73 family of transcription factors, is a molecular attribute of human squamous cancers of the head and neck, lung and skin. The TP63 gene plays important roles in epidermal morphogenesis and homeostasis, regulating diverse biological processes including epidermal fate decisions and keratinocyte proliferation and survival. When overexpressed experimentally in primary mouse keratinocytes, ΔNp63α maintains a basal cell phenotype including the loss of normal calcium-mediated growth arrest, at least in part through the activation and enhanced nuclear accumulation of the c-rel subunit of NF-κB (Nuclear Factor-kappa B). Initially identified for its role in the immune system and hematopoietic cancers, c-Rel has increasingly been associated with solid tumors and other pathologies. ΔNp63α and c-Rel have been shown to be associated in the nuclei of ΔNp63α overexpressing human squamous carcinoma cells. Together, these transcription factors cooperate in the transcription of genes regulating intrinsic keratinocyte functions, as well as the elaboration of factors that influence the tumor microenvironment (TME). This review provides an overview of the roles of ΔNp63α and c-Rel in normal epidermal homeostasis and elaborates on how these pathways may intersect in pathological conditions such as cancer and the associated TME.

Molecular Mechanisms of p63-Mediated Squamous Cancer Pathogenesis.

The p63 gene is a member of the p53/p63/p73 family of transcription factors and plays a critical role in development and homeostasis of squamous epithelium. p63 is transcribed as multiple isoforms; ΔNp63α, the predominant p63 isoform in stratified squamous epithelium, is localized to the basal cells and is overexpressed in squamous cell cancers of multiple organ sites, including skin, head and neck, and lung. Further, p63 is considered a stem cell marker, and within the epidermis, ΔNp63α directs lineage commitment. ΔNp63α has been implicated in numerous processes of skin biology that impact normal epidermal homeostasis and can contribute to squamous cancer pathogenesis by supporting proliferation and survival with roles in blocking terminal differentiation, apoptosis, and senescence, and influencing adhesion and migration. ΔNp63α overexpression may also influence the tissue microenvironment through remodeling of the extracellular matrix and vasculature, as well as by enhancing cytokine and chemokine secretion to recruit pro-inflammatory infiltrate. This review focuses on the role of ΔNp63α in normal epidermal biology and how dysregulation can contribute to cutaneous squamous cancer development, drawing from knowledge also gained by squamous cancers from other organ sites that share p63 overexpression as a defining feature.

Dysregulated ΔNp63α negatively regulates the maspin promoter in keratinocytes via blocking endogenous p73 binding.

While overexpression of the p63 isoform, ΔNp63α, has been reported in squamous cell cancers, the contribution of p63 to cancer pathogenesis remains unclear. We previously demonstrated that overexpressed ΔNp63α aberrantly maintains proliferation of primary mouse keratinocytes under conditions that normally induce growth arrest and differentiation. To identify genes downstream of dysregulated ΔNp63α that may contribute to squamous cancer development and progression, we performed microarray analyses using primary mouse keratinocytes. Herein we report that elevated ΔNp63α differentially regulates genes involved in a variety of cellular functions. Of note, multiple protease inhibitor mRNAs were downregulated including: maspin (serpinB5); plasminogen activator inhibitor-2 (PAI-2; serpinB2); and tissue inhibitor of metalloproteinase-3 (TIMP-3). Correspondingly, secreted TIMP-3 and PAI-2 protein declined in the presence of dysregulated ΔNp63α, however secreted maspin remained stable. Intracellular maspin protein expression decreased in response to overexpressed ΔNp63α, as did PAI-2. In contrast, TIMP-3 protein was not detected intracellularly, supporting a solely extracellular function. Electrophoretic mobility shift assays (EMSAs) using a maspin promoter p53/p63 consensus sequence revealed endogenous transcription factor(s) binding to this sequence in keratinocytes that was disrupted by overexpressed ΔNp63α. This was confirmed by ChIP assays. This binding was interrupted by the addition of antibodies recognizing p73, but not p53 or p63, and significantly diminished in EMSA reactions from p73(-/-) keratinocytes, confirming p73 as a constituent. Physical association between p73/ΔNp63α was observed in control ß-gal overexpressing keratinocytes and was enhanced in the presence of overexpressed ΔNp63α These findings underscore the importance of properly balanced p53 homologs for tissue homeostasis.

Booster Breaks in the workplace: participants' perspectives on health-promoting work breaks.

Increasing sedentary work has been associated with greater cardiovascular and metabolic risk, as well as premature mortality. Interrupting the sedentary workday with health-promoting work breaks can counter these negative health effects. To examine the potential sustainability of work-break programs, we assessed the acceptance of these breaks among participants in a Booster Break program. We analyzed qualitative responses from 35 participants across five worksites where one 15-min physical activity break was taken each workday. Two worksites completed a 1-year intervention and three worksites completed a 6-month intervention. Responses to two open-ended questions about the acceptance and feasibility of Booster Breaks were obtained from a survey administered after the intervention. Three themes for benefits and two themes for barriers were identified. The benefit themes were (i) reduced stress and promoted enjoyment, (ii) increased health awareness and facilitated behavior change, and (iii) enhanced workplace social interaction. The barrier themes were the need for (iv) greater variety in Booster Break routines and (v) greater management support. This study provides empirical support for the acceptance and feasibility of Booster Breaks during the workday. Emphasizing the benefits and minimizing the barriers are strategies that can be used to implement Booster Breaks in other workplaces.

Oncogenic Ras and ΔNp63α cooperate to recruit immunosuppressive polymorphonuclear myeloid-derived suppressor cells in a mouse model of squamous cancer pathogenesis.

Introduction: Amplification of human chromosome 3q26-29, which encodes oncoprotein ΔNp63 among other isoforms of the p63 family, is a feature common to squamous cell carcinomas (SCCs) of multiple tissue origins. Along with overexpression of ΔNp63, activation of the protooncogene, RAS, whether by overexpression or oncogenic mutation, is frequently observed in many cancers. In this study, analysis of transcriptome data from The Cancer Genome Atlas (TCGA) demonstrated that expression of TP63 mRNA, particularly ΔNp63 isoforms, and HRAS are significantly elevated in advanced squamous cell carcinomas of the head and neck (HNSCCs), suggesting pathological significance. However, how co-overexpressed ΔNp63 and HRAS affect the immunosuppressive tumor microenvironment (TME) is incompletely understood. Methods: Here, we established and characterized an immune competent mouse model using primary keratinocytes with retroviral-mediated overexpression of ΔNp63α and constitutively activated HRAS (v-rasHa G12R) to evaluate the role of these oncogenes in the immune TME. Results: In this model, orthotopic grafting of wildtype syngeneic keratinocytes expressing both v-rasHa and elevated levels of ΔNp63α consistently yield carcinomas in syngeneic hosts, while cells expressing v-rasHa alone yield predominantly papillomas. We found that polymorphonuclear (PMN) myeloid cells, experimentally validated to be immunosuppressive and thus representing myeloid-derived suppressor cells (PMN-MDSCs), were significantly recruited into the TME of carcinomas arising early following orthotopic grafting of ΔNp63α/v-rasHa-expressing keratinocytes. ΔNp63α/v-rasHa-driven carcinomas expressed higher levels of chemokines implicated in recruitment of MDSCs compared to v-rasHa-initiated tumors, providing a heretofore undescribed link between ΔNp63α/HRAS-driven carcinomas and the development of an immunosuppressive TME. Conclusion: These results support the utilization of a genetic carcinogenesis model harboring specific genomic drivers of malignancy to study mechanisms underlying the development of local immunosuppression.

FDA Approval Summary: Margetuximab plus Chemotherapy for Advanced or Metastatic HER2-Positive Breast Cancer.

On December 16, 2020, the FDA granted regular approval to margetuximab-cmkb (MARGENZA), in combination with chemotherapy, for the treatment of adult patients with HER2-positive (HER2+) metastatic breast cancer who have received two or more prior anti-HER2 regimens, at least one of which was for metastatic disease. Approval was based on data from SOPHIA, a multicenter, randomized, open-label, active controlled study comparing margetuximab with trastuzumab, in combination with chemotherapy. The primary efficacy endpoint was progression-free survival (PFS) by blinded independent central review. SOPHIA demonstrated a 0.9-month difference in median PFS between the two treatment arms [5.8 vs. 4.9 months, respectively; stratified HR, 0.76 (95% confidence interval: 0.59-0.98; P = 0.0334)]. Overall survival (OS) was immature at the data cut-off date of September 10, 2019. Infusion-related reactions (IRR) are an important safety signal associated with margetuximab plus chemotherapy. In SOPHIA, 13% of patients treated with margetuximab plus chemotherapy reported IRRs, of which 1.5% were grade 3. The most commonly reported adverse drug reactions (>10%) with margetuximab in combination with chemotherapy were fatigue/asthenia, nausea, diarrhea, vomiting, constipation, headache, pyrexia, alopecia, abdominal pain, peripheral neuropathy, arthralgia/myalgia, cough, decreased appetite, dyspnea, IRR, palmar-plantar erythrodysesthesia, and extremity pain. Overall, the favorable risk-benefit profile for margetuximab when added to chemotherapy supported its approval for the intended indication.

The transcriptional regulatory function of p53 is essential for suppression of mouse skin carcinogenesis and can be dissociated from effects on TGF-beta-mediated growth regulation.

Transcriptional regulation by p53 is critical for p53-mediated tumour suppression; however, p53-mediated transactivation has been dissociated from p53-mediated biological processes including apoptosis, DNA repair, and differentiation. We compared the effects of a mutant allele, p53(QS - val135), containing a double mutation in the amino-terminus abrogating transactivation activity and a modification at amino acid 135 partially affecting DNA binding, to complete loss of p53. We applied in vitro endpoints correlated with epithelial tumourigenesis and an in vivo assay of tumour phenotype to assess whether loss of p53-mediated transcriptional regulation underlies the malignant phenotype of p53(-/-)/v-ras(Ha)-overexpressing keratinocytes. Transactivation deficiency of p53QS-val135 was confirmed by reporter gene assays in fibroblasts and differentiating keratinocytes. Ras oncogene-induced senescence was lost in both p53(QS - val135/QS - val135) and p53(-/-) keratinocytes. Similarly, p53(QS - val135/QS - val135), like p53(-/-), cooperated with v-ras(Ha) to enhance malignant conversion. The tumours arising in p53(QS - val135/QS - val135) keratinocytes displayed strong nuclear p53 expression; thus, the p53(QS - val135) allele was maintained and the deficient transactivation function of the expressed p53QS mutant protein was supported by absence of p21(waf1) in these tumours. The p53(QS - val135) allele did not confer a dominant-negative phenotype, as p53(+/QS - val135) keratinocytes senesced normally in response to v-ras(Ha) expression and formed benign tumours. While p53(-/-) keratinocytes displayed diminished response to TGF-beta, p53(QS - val135/QS - val135) and p53(+/+) keratinocytes responded equivalently, indicating that the requirement for p53 in maximizing TGF-beta-mediated growth regulation is independent of its transactivation domain and that the ability of keratinocytes to respond to TGF-beta is insufficient to suppress the malignant phenotype in this model. Furthermore, TGF-beta enhances p53QS-induced activation of a dual p53-TGF-beta responsive reporter in a keratinocyte cell line. These findings support an essential role for p53-mediated transcriptional regulation in suppressing malignancies arising from ras-induced skin tumours, consistent with previous findings in spontaneous carcinogenesis in other organs, and highlight the potential importance of senescence for tumour suppression in vivo.

Increasing Numbers of Staphylococcal Scalded Skin Syndrome Cases Caused by ST121 in Houston, Texas.

BACKGROUND: The molecular epidemiology of Staphylococcus aureus strains causing staphylococcal scalded skin syndrome (SSSS) in the United States has not been described. We analyzed patient and S. aureus isolate characteristics associated with SSSS in children at Texas Children's Hospital. METHODS: Patients with SSSS were identified by ICD9/10 codes and available S. aureus isolates were identified from an ongoing S. aureus surveillance study. Medical records were reviewed for 58 patients with available S. aureus isolates. Isolate analyses included PCR for agr group, pvl (lukSF-PV), tst, eta and etb, pulsed-field gel electrophoresis, multi-locus sequence typing and antimicrobial susceptibilities. RESULTS: Cases of SSSS increased from 2.3/10,000 admissions in 2008 to 52.6/10,000 admissions in 2017 (P < 0.0001). The 58 study cases (57 methicillin-susceptible S. aureus, 1 MRSA) with isolates were from 2013 to 2017. The majority (88%) of isolates was of clonal cluster (CC) 121, agr group IV, pvl, tst and carried eta and/or etb and 26% were clindamycin resistant. Twelve ST121 isolates had high level resistance to mupirocin. Patients were treated with standard supportive care plus systemic antibiotics [clindamycin alone or in combination with another antibiotic (n = 44)]. One patient had a recurrent SSSS and one patient was transferred to a burn unit on day 3. CONCLUSIONS: Cases of SSSS are increasing at our hospital. Most S. aureus strains isolated were of one CC, CC121 and carried eta and etb. Supportive care plus clindamycin was effective treatment. We speculate that CC121 was recently introduced to our region and is responsible for the increasing numbers of SSSS cases observed at Texas Children's Hospital.

Cardiotoxicity of ErbB2-targeted therapies and its impact on drug development, a spotlight on trastuzumab.

INTRODUCTION: Trastuzumab, a therapeutic monoclonal antibody directed against ErbB2, is often noted as a successful example of targeted therapy. Trastuzumab improved outcomes for many patients with ErbB2-positive breast and gastric cancers, however, cardiac side effects [e.g., left ventricular dysfunction and congestive heart failure (CHF)] were reported in the early phase clinical studies. This finding, subsequently corroborated by multiple clinical studies, raised concerns that the observed cardiotoxicity induced by trastuzumab might adversely impact the clinical development of other therapeutics targeting ErbB family members. Areas covered: In this review we summarize both basic research and clinical findings regarding trastuzumab-induced cardiotoxicity and assess if there has been an impact of trastuzumab-induced cardiotoxicity on the development of other agents targeting ErbB family members. Expert opinion: There are a number of scientific gaps that are critically important to address for the continued success of HER2-targeted agents. These include: 1) elucidating the molecular mechanisms contributing to cardiotoxicity; 2) developing relevant preclinical testing systems for predicting cardiotoxicity; 3) developing clinical strategies to identify patients at risk of cardiotoxicity; and 4) enhancing management of clinical symptoms of cardiotoxicity.

Advanced Virus Detection Technologies Interest Group (AVDTIG): Efforts on High Throughput Sequencing (HTS) for Virus Detection.

Several nucleic-acid based technologies have recently emerged with capabilities for broad virus detection. One of these, high throughput sequencing, has the potential for novel virus detection because this method does not depend upon prior viral sequence knowledge. However, the use of high throughput sequencing for testing biologicals poses greater challenges as compared to other newly introduced tests due to its technical complexities and big data bioinformatics. Thus, the Advanced Virus Detection Technologies Users Group was formed as a joint effort by regulatory and industry scientists to facilitate discussions and provide a forum for sharing data and experiences using advanced new virus detection technologies, with a focus on high throughput sequencing technologies. The group was initiated as a task force that was coordinated by the Parenteral Drug Association and subsequently became the Advanced Virus Detection Technologies Interest Group to continue efforts for using new technologies for detection of adventitious viruses with broader participation, including international government agencies, academia, and technology service providers.

deltaNp63alpha functions as both a positive and a negative transcriptional regulator and blocks in vitro differentiation of murine keratinocytes.

deltaNp63 is overexpressed in squamous carcinomas where it is associated with proliferation and is believed to enhance cell growth by blocking p53-mediated transactivation. In normal epithelium, deltaNp63alpha protein expression is abundant in basal cells and decreases with differentiation. To explore the biological consequences of deltaNp63alpha overexpression in relation to squamous carcinogenesis, we evaluated its effect on normal squamous differentiation and p53 transactivation function in keratinocytes. Forced overexpression of deltaNp63alpha in primary murine keratinocytes in vitro inhibits morphological differentiation induced by elevated extracellular [Ca(2+)], abrogates Ca(2)(+)-induced growth arrest, and blocks expression of maturation-specific proteins keratin 10 and filaggrin. This suggests that deltaNp63 overexpression in squamous carcinomas may serve to maintain the basal cell phenotype and promote cell survival. deltaNp63alpha blocks transactivation of p53 responsive reporter constructs mediated by endogenous or exogenous p53 at 17 h postinfection, as expected. However, at 41 h, when p53-mediated transactivation is diminished, deltaNp63alpha enhances transactivation of these reporter constructs by 2.2-12-fold over control. Maximal deltaNp63alpha-induced transactivation requires intact p53 responsive elements, but is independent of cellular p53 status. This positive transcriptional function of deltaNp63alpha appears to be cell-type specific, as it is not observed in primary dermal fibroblasts or Saos-2 cells. These findings support deltaNp63alpha as a master regulator of keratinocyte differentiation, and suggest a novel function of this protein in the maintenance of epithelial homeostasis.

Delineating Molecular Mechanisms of Squamous Tissue Homeostasis and Neoplasia: Focus on p63.

Mouse models have informed us that p63 is critical for normal epidermal development and homeostasis. The p53/p63/p73 family is expressed as multiple protein isoforms due to a combination of alternative promoter usage and C-terminal alternative splicing. These isoforms can mimic or interfere with one another, and their balance ultimately determines biological outcome in a context-dependent manner. While not frequently mutated, p63, and in particular the ΔNp63 subclass, is commonly overexpressed in human squamous cell cancers. In vitro keratinocytes and murine transgenic and transplantation models have been invaluable in elucidating the contribution of altered p63 levels to cancer development, and studies have identified the roles for ΔNp63 isoforms in keratinocyte survival and malignant progression, likely due in part to their transcriptional regulatory function. These findings can be extended to human cancers; for example, the novel recognition of NF κ B/c-Rel as a downstream effector of p63 has identified a role for NF κ B/c-Rel in human squamous cell cancers. These models will be critical in enhancing the understanding of the specific molecular mechanisms of cancer development and progression.

First FDA approval of dual anti-HER2 regimen: pertuzumab in combination with trastuzumab and docetaxel for HER2-positive metastatic breast cancer.

On June 8, 2012, the U.S. Food and Drug Administration (FDA) approved pertuzumab (Perjeta, Genentech) for use in combination with trastuzumab (Herceptin, Genentech) and docetaxel for the treatment of patients with HER2-positive metastatic breast cancer (MBC) who have not received prior anti-HER2 therapy or chemotherapy for metastatic disease. Approval was based on the results of a randomized, double-blind, placebo-controlled trial conducted in 808 patients with HER2-positive MBC. Patients were randomized (1:1) to receive pertuzumab (n = 402) or placebo (n = 406) in combination with trastuzumab and docetaxel. The primary endpoint was progression-free survival (PFS) and a key secondary endpoint was overall survival (OS). A statistically significant improvement in PFS (difference in medians of 6.1 months) was observed in patients receiving pertuzumab [HR, 0.62; 95% confidence interval (CI), 0.51-0.75; P < 0.0001]. A planned interim analysis suggested an improvement in OS (HR, 0.64; 95% CI, 0.47-0.88; P = 0.0053) but the HR and P value did not cross the stopping boundary. Common adverse reactions (>30%) observed in patients on the pertuzumab arm included diarrhea, alopecia, neutropenia, nausea, fatigue, rash, and peripheral neuropathy. No additive cardiac toxicity was observed. Significant manufacturing issues were identified during the review. On the basis of substantial evidence of efficacy for pertuzumab in MBC and the compelling public health need, FDA did not delay availability to patients pending final resolution of all manufacturing concerns. Therefore, FDA approved pertuzumab but limited its approval to lots not affected by manufacturing problems. The applicant agreed to multiple manufacturing and testing postmarketing commitments under third-party oversight to resolve manufacturing issues.

PDA/FDA Adventitious Agents and Novel Cell Substrates: Emerging Technologies and New Challenges, Nov. 3-4, 2011, Rockville, MD.

In response to the challenges of addressing potential safety concerns related to the use of novel cell substrates, the Parenteral Drug Association (PDA) and the U.S. Food and Drug Administration (FDA) co-sponsored a conference on November 3-4, 2011 in Rockville, Maryland, USA. The following were discussed: the current adventitious agent testing methods and the use of emerging nucleic acid-based technologies for broad detection of known and novel viruses; issues associated with manufacture of biologicals in novel cell substrates such as insect cell lines and plants, with specific focus on potential safety concerns related to endogenous retroviral elements; and raw material controls and strategies to mitigate risk of adventitious virus contamination during product manufacture.

TNF-α promotes c-REL/ΔNp63α interaction and TAp73 dissociation from key genes that mediate growth arrest and apoptosis in head and neck cancer.

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 coexpressed 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 proinflammatory cytokine, promoted c-REL nuclear translocation, c-REL/ΔNp63α interaction, and dissociation of TAp73 from ΔNp63α and the nucleus to the cytoplasm, whereas c-REL siRNA knockdown attenuated this effect. Overexpression of c-REL or a c-REL κB-site DNA-binding mutant enhanced protein interaction with ΔNp63α and TAp73 dissociation, implicating c-REL/ΔNp63α-specific interactions in these effects. We discovered that TNF-α or genetic alteration of c-REL expression inversely modulates ΔNp63α/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 antiproliferative 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 show increased nuclear c-REL/ΔNp63α and cytoplasmic TAp73 localization. These findings unveil a novel and reversible dynamic mechanism whereby proinflammatory cytokine TNF-α-induced c-REL/ΔNp63α interactions inactivate tumor suppressor TAp73 function, promoting TNF-α resistance and cell survival in cancers with mtTP53.

The p53 homologue DeltaNp63alpha interacts with the nuclear factor-kappaB pathway to modulate epithelial cell growth.

The p53 homologue DeltaNp63alpha is overexpressed and inhibits apoptosis in a subset of human squamous cell carcinomas (SCC). Here, we report that in normal keratinocytes overexpressing DeltaNp63alpha and in human squamous carcinoma cells, DeltaNp63alpha physically associates with phosphorylated, transcriptionally active nuclear c-Rel, a nuclear factor-kappaB family member, resulting in increased c-Rel nuclear accumulation. This accumulation and the associated enhanced proliferation driven by elevated DeltaNp63alpha are attenuated by c-Rel small interfering RNA or overexpression of mutant IkappaBalphaM, indicating that c-Rel-containing complex formation is critical to the ability of elevated DeltaNp63alpha to maintain proliferation in the presence of growth arresting signals. Consistent with a role in growth regulation, DeltaNp63alpha-c-Rel complexes bind a promoter motif and repress the cyclin-dependent kinase inhibitor p21WAF1 in both human squamous carcinoma cells and normal keratinocytes overexpressing DeltaNp63alpha. The relationship between DeltaNp63alpha and activated c-Rel is reflected in their strong nuclear staining in the proliferating compartment of primary head and neck SCC. This is the first report indicating that high levels of DeltaNp63alpha interact with activated c-Rel in keratinocytes and SCC, thereby promoting uncontrolled proliferation, a key alteration in the pathogenesis of cancers.

p63: defining roles in morphogenesis, homeostasis, and neoplasia of the epidermis.

p63 is a member of a gene family also including the p53 tumor suppressor and p73. In contrast to p53, p63 is rarely mutated in human cancers. Rather, gene amplification and dysregulated expression of p63 protein have been observed, particularly in squamous cell carcinomas. p63 is essential for development of stratified squamous epithelium, including the epidermis. The p63 gene is expressed as multiple protein isoforms with different functional capacities, and the balance of these isoforms, along with the presence or absence of the other family members, p53 and p73, can impact biological outcome. Both gene silencing and overexpression approaches have been utilized to elucidate the contributions of specific p63 isoforms to normal epidermal morphogenesis and tissue maintenance. While numerous studies have established the essential nature of p63 in the epidermis, the basis of this requirement, and the unique, as well as, overlapping functions of the individual isoforms, remain controversial. In this review, we summarize the current understanding of roles played by specific p63 isoforms within the context of epidermal morphogenesis and homeostasis of the established epidermis, and the potential impact of p63 dysregulation on cancer development.

Krüppel-like factor 4 (KLF4/GKLF) is a target of bone morphogenetic proteins and transforming growth factor beta 1 in the regulation of vascular smooth muscle cell phenotype.

Vascular smooth muscle cell (VSMC) differentiation and phenotypic modulation is characterized by changes in mRNA expression for smooth muscle (SM) marker contractile proteins such as alpha-SM actin and SM22 alpha. Transforming growth factor beta1 (TGF-beta 1) is a potent VSMC differentiation factor; however, it is not known if other TGF-beta-superfamily members, in particular the bone morphogenetic proteins (BMPs), modulate VSMC phenotype. Here we demonstrate that a large subset of TGF-beta-superfamily members and their type I receptors are differentially co-expressed as VSMC phenotype changes during fetal/neonatal development and that BMP2, -4, and -6 reciprocally regulate SM-marker mRNA and protein expression in vitro. BMP2 and BMP6 decrease expression of the SM markers alpha-SM actin, SM22alpha, and calponin in rat VSMCs, whereas BMP4 increases their expression. The effects of BMP-2, -4, and -6 on SM marker gene transcription are mediated through a consensus TGF-beta-controlling element, the TCE, which is common to regulatory regions of SM-marker genes. Moreover, co-treatment experiments revealed that BMP-2, -4, and -6 each inhibit TGF-beta 1-modulated increases in SM22alpha reporter gene activity. Regardless of whether they positively or negatively regulate SM marker expression, TGF-beta 1 and BMP-2, -4, and -6 all induced binding of the Krüppel-like transcription factor, GKLF/KLF4, to the TGF-beta control element. Induction of KLF4 was confirmed by immunocytochemistry and Western blotting, which revealed that a lower molecular weight KLF4 protein is induced after treatment with TGF-beta-superfamily members. Taken together, our results demonstrate that multiple members of the TGF-beta superfamily act in concert to modulate VSMC phenotype.

HOW GIBBERELLIN REGULATES PLANT GROWTH AND DEVELOPMENT: A Molecular Genetic Analysis of Gibberellin Signaling.

Gibberellins are hormones that control growth and a wide variety of other plant developmental processes. In recent years, significant progress has been made on the biochemistry of gibberellin biosynthesis and on the mechanisms by which gibberellin levels are regulated in plants. There have also been major advances in the understanding of gibberellin signaling, with several key genes being cloned. This review discusses our current understanding of gibberellin signaling, as seen from the perspective of molecular genetic analysis, and relates these observations to previous biochemical studies. In particular, we highlight an important conclusion of recent years: that GAI/RGA and orthologs play major roles in gibberellin signaling in diverse plant species, and that gibberellin probably stimulates growth by derepression of GAI/RGA.