Kathryn E. King

ΔNp63α functions as both a positive and a negative transcriptional regulator and blocks in vitro differentiation of murine keratinocytes

ΔNp63 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, ΔNp63α protein expression is abundant in basal cells and decreases with differentiation. To explore the biological consequences of ΔNp63α overexpression in relation to squamous carcinogenesis, we evaluated its effect on normal squamous differentiation and p53 transactivation function in keratinocytes. Forced overexpression of ΔNp63α in primary murine keratinocytes in vitro inhibits morphological differentiation induced by elevated extracellular [Ca2+], abrogates Ca2+-induced growth arrest, and blocks expression of maturation-specific proteins keratin 10 and filaggrin. This suggests that ΔNp63 overexpression in squamous carcinomas may serve to maintain the basal cell phenotype and promote cell survival. ΔNp63α 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, ΔNp63α enhances transactivation of these reporter constructs by 2.2–12-fold over control. Maximal ΔNp63α-induced transactivation requires intact p53 responsive elements, but is independent of cellular p53 status. This positive transcriptional function of ΔNp63α appears to be cell-type specific, as it is not observed in primary dermal fibroblasts or Saos-2 cells. These findings support ΔNp63α as a master regulator of keratinocyte differentiation, and suggest a novel function of this protein in the maintenance of epithelial homeostasis.

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. Clin Cancer Res; 19(18); 4911–6. ©2013 AACR.

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.

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.

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.

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

ABSTRACT 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.

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 β‐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.

Abstract 768: A role for c-Rel in ΔNp63α/v-RASHA-driven carcinogenesis?.

The p63 isoform ΔNp63α is expressed in basal keratinocytes and overexpressed in human squamous cell carcinomas, but the mechanisms whereby this p53 homologue contributes to cancer pathogenesis have yet to be elucidated fully. In mimicking the overexpression of ΔNp63α observed in squamous cell carcinomas using transient adenoviral transduction of primary murine kerationcytes, we showed that overexpression of ΔNp63α inhibits Ca 2+ -mediated growth arrest and biochemical differentiation. We previously reported that this block in growth arrest is mediated via the NF-κB subunit, c-Rel, which accumulates in a phosphorylated form in the nucleus of ΔNp63α-overexpressing cells and physically associates in a phosphorylation-dependent manner with ΔNp63α. Consistent with the observed effects on growth regulation, ΔNp63α:c-Rel complexes bind to a p63 binding site on the cdk inhibitor p21 WAF1 . To explore the biological impact of long-term ΔNp63α overexpression in primary murine keratinocytes, lentiviruses were developed. Consistent with our transient adenoviral vector studies, keratinocytes expressing lenti-ΔNp63α have enhanced proliferation rates over 15 days in culture relative to lenti-GFP controls. Using a nude mouse grafting model that allows distinction between normal, benign, and malignant growths, we previously reported that lenti-GFP keratinocytes expressing oncogenic v-ras Ha form well differentiated papillomas at the graft site, while keratinocytes expressing lenti-ΔNp63α in combination with v-ras Ha form undifferentiated carcinomas. To expand this model system to study c-Rel we first confirmed that lentivirus-driven ΔNp63α overexpression results in sustained nuclear accumulation of the NF-kB subunit c-Rel, consistent with our observations using the adenovirally transduced cultures. In the lenti-ΔNp63α expressing cultures, enhancement of c-Rel is observed beginning at day 5 post-infection and is maintained through the latest time point tested, 14 days. Lentiviral c-Rel shRNAs have been developed to assess the contribution of altered nuclear c-Rel expression to ΔNp63α-mediated growth regulation in vitro and in vivo and have been successfully used to knockdown c-Rel to the latest time point tested, 28 days. Taken together, these data support a role for ΔNp63α in facilitating keratinocyte transformation and provide a model system for elucidating the contribution of NFkB/c-rel to ΔNp63α /v-ras Ha -driven carcinogenesis. Citation Format: Kathryn E. King, Roshini M. Ponnamperuma, Sa Ra Park, Steven Jay, Linan Ha, Wendy C. Weinberg. A role for c-Rel in ΔNp63α/v-RAS HA -driven carcinogenesis?. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 768. doi:10.1158/1538-7445.AM2013-768

PDA Cell Substrate Task Force: Emerging Technologies for Virus Detection Technical Document--A Progress Report.

CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1–3, 2010 Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA)

Abstract 3900: Nuclear c-REL displaces p73 in partnering with DNp63 to suppress p21 expression and promote proliferation in head and neck cancer

The tumor suppressor p53 is known as the ‘guardian of the genome’ owing to its ability to integrate many signals that control cell life or death. But the functions of the p53 tumor suppressor are commonly lost in cancer. The p53 family members p63 and p73 also contribute to tumor-suppressor function, because they share many common transcriptional targets with p53 and their activities contribute to the p53-dependent apoptosis and cell cycling of cancer cells. In p53 mutant tumors, TAp73 or TAp63 isoforms may compensate for p53 loss and respond to cell stress by upregulating genes involved in growth arrest and apoptosis. In contrast, DNp63 and DNp73 counteract the activities of TA isoforms, either by competing for common DNA binding elements or by blocking the TAp63 and TAp73 isoforms through direct protein-protein interactions. The mechanism regulating TA and DN crosstalk and balance remains uncertain. We recently reported that p53 and NF-KappaB family members DNp63 and c-Rel can interact to repress p21Cip1 and growth arrest in normal murine keratinocytes. Here we show that c-Rel, p63, and p73 exhibit aberrant expression and nuclear localization in a subset of head and neck squamous cell carcinoma (HNSCC). Co-immunoprecipitation, electromobility shift assay (EMSA), and chromatin immunoprecipitation (ChIP) studies revealed that TNFalpha-induced or overexpressed nuclear c-Rel interact with DNp63, displace TAp73 from the nucleus, and from p63 promoter regulatory motifs of cell cycle and apoptotic genes p21, NOXA and PUMA. TNFalpha-induced or overexpressed c-Rel repressed, while c-Rel siRNA knockdown enhanced their expression and inhibited cell proliferation and survival. We conclude that aberrant and TNFalpha-mediated activation and interaction of c-Rel/DNp63 can displace TAp73 and block its key function in growth arrest and apoptosis. Our findings support a novel mechanism whereby an inflammatory cytokine, such as TNF-alpha, can promote c-Rel nuclear translocation, interaction with DNp63, and displacement of TAp73, to inhibit expression of key cell cycle inhibitor and apoptotic genes, which are downregulated in HNSCC and other cancers. This represents a novel form of cross-talk between NF-kappaB and TP53 family members and pathways that govern the switch between survival and death signals in HNSCC and other malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3900.