Jennifer A Pietenpol

Publications of Jennifer A Pietenpol

• Identification of human triple-negative breast cancer subtypes and preclinical models for selection of targeted therapies.

  Authors: Brian D Lehmann, Joshua A Bauer, Xi Chen, Melinda E Sanders, A Bapsi Chakravarthy, Yu Shyr, Jennifer A Pietenpol

  The Journal of clinical investigation. 06/2011; 121(7):2750-67.

  Triple-negative breast cancer (TNBC) is a highly diverse group of cancers, and subtyping is necessary to better identify molecular-based therapies. In this study, we analyzed gene expression (GE)Triple-negative breast cancer (TNBC) is a highly diverse group of cancers, and subtyping is necessary to better identify molecular-based therapies. In this study, we analyzed gene expression (GE) profiles from 21 breast cancer data sets and identified 587 TNBC cases. Cluster analysis identified 6 TNBC subtypes displaying unique GE and ontologies, including 2 basal-like (BL1 and BL2), an immunomodulatory (IM), a mesenchymal (M), a mesenchymal stem-like (MSL), and a luminal androgen receptor (LAR) subtype. Further, GE analysis allowed us to identify TNBC cell line models representative of these subtypes. Predicted "driver" signaling pathways were pharmacologically targeted in these cell line models as proof of concept that analysis of distinct GE signatures can inform therapy selection. BL1 and BL2 subtypes had higher expression of cell cycle and DNA damage response genes, and representative cell lines preferentially responded to cisplatin. M and MSL subtypes were enriched in GE for epithelial-mesenchymal transition, and growth factor pathways and cell models responded to NVP-BEZ235 (a PI3K/mTOR inhibitor) and dasatinib (an abl/src inhibitor). The LAR subtype includes patients with decreased relapse-free survival and was characterized by androgen receptor (AR) signaling. LAR cell lines were uniquely sensitive to bicalutamide (an AR antagonist). These data may be useful in biomarker selection, drug discovery, and clinical trial design that will enable alignment of TNBC patients to appropriate targeted therapies.

• Differential regulation of the p73 cistrome by mammalian target of rapamycin reveals transcriptional programs of mesenchymal differentiation and tumorigenesis.

  Authors: Jennifer M Rosenbluth, Deborah J Mays, Aixiang Jiang, Yu Shyr, Jennifer A Pietenpol

  Proceedings of the National Academy of Sciences of the United States of America. 02/2011; 108(5):2076-81.

  The transcription factor p73 plays critical roles during development and tumorigenesis. It exhibits sequence identity and structural homology with p53, and can engage p53-like tumor-suppressiveThe transcription factor p73 plays critical roles during development and tumorigenesis. It exhibits sequence identity and structural homology with p53, and can engage p53-like tumor-suppressive programs. However, different pathways regulate p53 and p73, and p73 is not mutated in human tumors. Therefore, p73 represents a therapeutic target, and there is a critical need to understand genes and noncoding RNAs regulated by p73 and how they change during treatment regimens. Here, we define the p73 genomic binding profile and demonstrate its modulation by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) and inducer of p73. Rapamycin selectively increased p73 occupancy at a subset of its binding sites. In addition, multiple determinants of p73 binding, activity, and function were evident, and were modulated by mTOR. We generated an mTOR-p73 signature that is enriched for p73 target genes and miRNAs that are involved in mesenchymal differentiation and tumorigenesis, can classify rhabdomyosarcomas by clinical subtype, and can predict patient outcome.

• Preoperative concurrent paclitaxel-radiation in locally advanced breast cancer: pathologic response correlates with five-year overall survival.

  Authors: Sylvia Adams, A Bapsi Chakravarthy, Martin Donach, Darcy Spicer, Stella Lymberis, Baljit Singh, Joshua A Bauer, Tsivia Hochman, Judith D Goldberg, Franco Muggia, Robert J Schneider, Jennifer A Pietenpol, Silvia C Formenti

  Breast cancer research and treatment. 09/2010; 124(3):723-32.

  We have previously demonstrated high pathologic response rates after neoadjuvant concurrent chemoradiation in patients with locally advanced breast cancer (LABC). We now report disease-free survivalWe have previously demonstrated high pathologic response rates after neoadjuvant concurrent chemoradiation in patients with locally advanced breast cancer (LABC). We now report disease-free survival (DFS) and overall survival (OS) in the context of pathologic response. 105 LABC patients (White 46%, Non-White 54%) were treated with paclitaxel (30 mg/m² intravenously twice a week) for 10-12 weeks. Daily radiotherapy was delivered to breast, axillary, and supraclavicular lymph nodes during weeks 2-7 of paclitaxel treatment, at 1.8 Gy per fraction to a total dose of 45 Gy with a tumor boost of 14 Gy at 2 Gy/fraction. Pathological complete response (pCR) was defined as the absence of invasive cancer in breast and lymph nodes and pathological partial response (pPR) as the persistence of <10 microscopic foci of invasive carcinoma in breast or lymph nodes. Pathologic response (pCR and pPR) after neoadjuvant chemoradiation was achieved in 36/105 patients (34%) and was associated with significantly better DFS and OS. Pathological responders had a lower risk of recurrence or death (HR = 0.35, P = 0.01) and a longer OS (HR = 4.27, P = 0.01) compared with non-responders. Median DFS and OS were 57 and 84 months for non-responders, respectively, and have not yet been reached for responders. Importantly, pathologic response was achieved in 54% of patients with HR negative tumors (26/48). In conclusion, pathologic response to concurrent paclitaxel-radiation translated into superior DFS and OS. Half of the patients with HR negative tumors achieved a pathologic response.

• Identification of markers of taxane sensitivity using proteomic and genomic analyses of breast tumors from patients receiving neoadjuvant paclitaxel and radiation.

  Authors: Joshua A Bauer, A Bapsi Chakravarthy, Jennifer M Rosenbluth, Deming Mi, Erin H Seeley, Nara De Matos Granja-Ingram, Maria G Olivares, Mark C Kelley, Ingrid A Mayer, Ingrid M Meszoely, Julie A Means-Powell, Kimberly N Johnson, Chiaojung Jillian Tsai, Gregory D Ayers, Melinda E Sanders, Robert J Schneider, Silvia C Formenti, Richard M Caprioli, Jennifer A Pietenpol

  Clinical cancer research : an official journal of the American Association for Cancer Research. 01/2010; 16(2):681-90.

  To identify molecular markers of pathologic response to neoadjuvant paclitaxel/radiation treatment, protein and gene expression profiling were done on pretreatment biopsies. Patients with high-risk,To identify molecular markers of pathologic response to neoadjuvant paclitaxel/radiation treatment, protein and gene expression profiling were done on pretreatment biopsies. Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel followed by concurrent paclitaxel/radiation. Tumor tissue from pretreatment biopsies was obtained from 19 of the 38 patients enrolled in the study. Protein and gene expression profiling were done on serial sections of the biopsies from patients that achieved a pathologic complete response (pCR) and compared to those with residual disease, non-pCR (NR). Proteomic and validation immunohistochemical analyses revealed that alpha-defensins (DEFA) were overexpressed in tumors from patients with a pCR. Gene expression analysis revealed that MAP2, a microtubule-associated protein, had significantly higher levels of expression in patients achieving a pCR. Elevation of MAP2 in breast cancer cell lines led to increased paclitaxel sensitivity. Furthermore, expression of genes that are associated with the basal-like, triple-negative phenotype were enriched in tumors from patients with a pCR. Analysis of a larger panel of tumors from patients receiving presurgical taxane-based treatment showed that DEFA and MAP2 expression as well as histologic features of inflammation were all statistically associated with response to therapy at the time of surgery. We show the utility of molecular profiling of pretreatment biopsies to discover markers of response. Our results suggest the potential use of immune signaling molecules such as DEFA as well as MAP2, a microtubule-associated protein, as tumor markers that associate with response to neoadjuvant taxane-based therapy.

• ISG20L1 is a p53 family target gene that modulates genotoxic stress-induced autophagy.

  Authors: Kathryn G Eby, Jennifer M Rosenbluth, Deborah J Mays, Clayton B Marshall, Christopher E Barton, Seema Sinha, Kimberly N Johnson, Luojia Tang, Jennifer A Pietenpol

  Molecular cancer. 01/2010; 9:95.

  Autophagy is characterized by the sequestration of cytoplasm and organelles into multimembrane vesicles and subsequent degradation by the cell's lysosomal system. It is linked to many physiologicalAutophagy is characterized by the sequestration of cytoplasm and organelles into multimembrane vesicles and subsequent degradation by the cell's lysosomal system. It is linked to many physiological functions in human cells including stress response, protein degradation, organelle turnover, caspase-independent cell death and tumor suppression. Malignant transformation is frequently associated with deregulation of autophagy and several tumor suppressors can modulate autophagic processes. The tumor suppressor p53 can induce autophagy after metabolic or genotoxic stress through transcriptionally-dependent and -independent mechanisms. In this study we expand on the former mechanism by functionally characterizing a p53 family target gene, ISG20L1 under conditions of genotoxic stress. We identified a p53 target gene, ISG20L1, and show that transcription of the gene can be regulated by all three p53 family members (p53, p63, and p73). We generated an antibody to ISG20L1 and found that it localizes to the nucleolar and perinucleolar regions of the nucleus and its protein levels increase in a p53- and p73-dependent manner after various forms of genotoxic stress. When ectopically expressed in epithelial cancer-derived cell lines, ISG20L1 expression decreased clonogenic survival without a concomitant elevation in apoptosis and this effect was partially rescued in cells that were ATG5 deficient. Knockdown of ISG20L1 did not alter 5-FU induced apoptosis as assessed by PARP and caspase-3 cleavage, sub-G1 content, and DNA laddering. Thus, we investigated the role of ISG20L1 in autophagy, a process commonly associated with type II cell death, and found that ISG20L1 knockdown decreased levels of autophagic vacuoles and LC3-II after genotoxic stress as assessed by electron microscopy, biochemical, and immunohistochemical measurements of LC3-II. Our identification of ISG20L1 as a p53 family target and discovery that modulation of this target can regulate autophagic processes further strengthens the connection between p53 signaling and autophagy. Given the keen interest in targeting autophagy as an anticancer therapeutic approach in tumor cells that are defective in apoptosis, investigation of genes and signaling pathways involved in cell death associated with autophagy is critical.

• RNA interference (RNAi) screening approach identifies agents that enhance paclitaxel activity in breast cancer cells.

  Authors: Joshua A Bauer, Fei Ye, Clayton B Marshall, Brian D Lehmann, Christopher S Pendleton, Yu Shyr, Carlos L Arteaga, Jennifer A Pietenpol

  Breast cancer research : BCR. 01/2010; 12(3):R41.

  Paclitaxel is a widely used drug in the treatment of patients with locally advanced and metastatic breast cancer. However, only a small portion of patients have a complete response toPaclitaxel is a widely used drug in the treatment of patients with locally advanced and metastatic breast cancer. However, only a small portion of patients have a complete response to paclitaxel-based chemotherapy, and many patients are resistant. Strategies that increase sensitivity and limit resistance to paclitaxel would be of clinical use, especially for patients with triple-negative breast cancer (TNBC). We generated a gene set from overlay of the druggable genome and a collection of genomically deregulated gene transcripts in breast cancer. We used loss-of-function RNA interference (RNAi) to identify gene products in this set that, when targeted, increase paclitaxel sensitivity. Pharmacological agents that targeted the top scoring hits/genes from our RNAi screens were used in combination with paclitaxel, and the effects on the growth of various breast cancer cell lines were determined. RNAi screens performed herein were validated by identification of genes in pathways that, when previously targeted, enhanced paclitaxel sensitivity in the pre-clinical and clinical settings. When chemical inhibitors, CCT007093 and mithramycin, against two top hits in our screen, PPMID and SP1, respectively, were used in combination with paclitaxel, we observed synergistic growth inhibition in both 2D and 3D breast cancer cell cultures. The transforming growth factor beta (TGFbeta) receptor inhibitor, LY2109761, that targets the signaling pathway of another top scoring hit, TGFbeta1, was synergistic with paclitaxel when used in combination on select breast cancer cell lines grown in 3D culture. We also determined the relative paclitaxel sensitivity of 22 TNBC cell lines and identified 18 drug-sensitive and four drug-resistant cell lines. Of significance, we found that both CCT007093 and mithramycin, when used in combination with paclitaxel, resulted in synergistic inhibition of the four paclitaxel-resistant TNBC cell lines. RNAi screening can identify druggable targets and novel drug combinations that can sensitize breast cancer cells to paclitaxel. This genomic-based approach can be applied to a multitude of tumor-derived cell lines and drug treatments to generate requisite pre-clinical data for new drug combination therapies to pursue in clinical investigations.

• Evaluation of p63 and p73 antibodies for cross-reactivity.

  Authors: Jennifer M Rosenbluth, Kimberly Johnson, Luojia Tang, Tracy Triplett, Jennifer A Pietenpol

  Cell cycle (Georgetown, Tex.). 11/2009; 8(22):3702-6.

  The tumor suppressor p53 is commonly mutated in human cancers. However, two homologs of p53, p63 and p73, are frequently overexpressed in tumors and are associated with tumor subtypes, clinicalThe tumor suppressor p53 is commonly mutated in human cancers. However, two homologs of p53, p63 and p73, are frequently overexpressed in tumors and are associated with tumor subtypes, clinical outcomes, and responses to therapy. There are many isoforms of p53, p63 and p73 (the p53 family). Proper detection of and discrimination between the members of this tumor suppressor family in human tissues is of critical importance to cancer research and clinical care. In this study, we assessed the specificity of several commercially available and newly generated p73 antibodies, focusing on antibodies that distinguish between the TA p73 and DeltaNp73 isoforms by western analysis, immunohistochemistry, and immunofluorescence. In addition, we found that the pan-p63 and pan-p73 antibodies tested cross-react with p73 and p63 respectively. The results of this study have important implications for analysis of p63 and p73 expression and co-expression in human tumors, and for potential use of these reagents in molecular diagnostics and therapeutic decision-making.

• DeltaNp63 antagonizes p53 to regulate mesoderm induction in Xenopus laevis.

  Authors: Christopher E Barton, Emilios Tahinci, Christopher E Barbieri, Kimberly N Johnson, Alison J Hanson, Kristin K Jernigan, Tony W Chen, Ethan Lee, Jennifer A Pietenpol

  Developmental biology. 04/2009;

  p63, a homolog of the tumor suppressor p53, is critical for the development and maintenance of complex epithelia. The developmentally regulated p63 isoform, DeltaNp63, can act as a transcriptionalp63, a homolog of the tumor suppressor p53, is critical for the development and maintenance of complex epithelia. The developmentally regulated p63 isoform, DeltaNp63, can act as a transcriptional repressor, but the link between the transcriptional functions of p63 and its biological roles is unclear. Based on our initial finding that the mesoderm-inducing factor activin A is suppressed by DeltaNp63 in human keratinocytes, we investigated the role of DeltaNp63 in regulating mesoderm induction during early Xenopus laevis development. We find that down-regulation of DeltaNp63 by morpholino injection in the early Xenopus embryo potentiates mesoderm formation whereas ectopic expression of DeltaNp63 inhibits mesoderm formation. Furthermore, we show that mesodermal induction after down-regulation of DeltaNp63 is dependent on p53. We propose that a key function for p63 in defining a squamous epithelial phenotype is to actively suppress mesodermal cell fates during early development. Collectively, we show that there is a distinct requirement for different p53 family members during the development of both mesodermal and ectodermal tissues. These findings have implications for the role of p63 and p53 in both development and tumorigenesis of human epithelia.

• The jury is in: p73 is a tumor suppressor after all.

  Authors: Jennifer M Rosenbluth, Jennifer A Pietenpol

  Genes & development. 11/2008; 22(19):2591-5.

  While p53 has been extensively characterized as a tumor suppressor, it has been more difficult to determine whether p63 and/or p73 play a similar role. Every system in which these family members haveWhile p53 has been extensively characterized as a tumor suppressor, it has been more difficult to determine whether p63 and/or p73 play a similar role. Every system in which these family members have been studied, from cells to animal models to human tissues, seems to create more questions than answers. Tomasini and colleagues (2677-2691) demonstrate that one isoform of p73 is responsible for preventing tumor formation in vivo, providing critical validation of an isoform-based model of p73 function.

• A gene signature-based approach identifies mTOR as a regulator of p73.

  Authors: Jennifer M Rosenbluth, Deborah J Mays, Maria F Pino, Luo Jia Tang, Jennifer A Pietenpol

  Molecular and cellular biology. 09/2008;

  Although genomic technologies have advanced the characterization of gene regulatory networks downstream of transcription factors, the identification of pathways upstream of these transcriptionAlthough genomic technologies have advanced the characterization of gene regulatory networks downstream of transcription factors, the identification of pathways upstream of these transcription factors has been more challenging. In this study we present a gene signature-based approach for connecting signaling pathways to transcription factors, as exemplified by p73. We generated a p73 gene signature by integrating whole-genome chromatin immunoprecipitation and expression profiling. The p73 signature was linked to corresponding signatures produced by drug candidates, using the in silico Connectivity Map resource, to identify drugs that would induce p73 activity. Of the pharmaceutical agents identified, there was enrichment for direct or indirect inhibitors of mammalian Target of Rapamycin (mTOR) signaling. Treatment of both primary cells and cancer cell lines with rapamycin, metformin, and pyrvinium resulted in an increase in p73, as did RNAi-mediated knockdown of mTOR. Further, a subset of genes associated with insulin response or autophagy exhibited mTOR-mediated, p73-dependent expression. Thus, downstream gene signatures can be used to identify upstream regulators of transcription factor activity, and doing so, we identified a new link between mTOR, p73, and p73-regulated genes associated with autophagy and metabolic pathways.

• Identification of novel Smad2 and Smad3 associated proteins in response to TGF-beta1.

  Authors: Kimberly A Brown, Amy-Joan L Ham, Cara N Clark, Nahum Meller, Brian K Law, Anna Chytil, Nikki Cheng, Jennifer A Pietenpol, Harold L Moses

  Journal of cellular biochemistry. 09/2008;

  Transforming growth factor-beta 1 (TGF-beta1) is an important growth inhibitor of epithelial cells and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute toTransforming growth factor-beta 1 (TGF-beta1) is an important growth inhibitor of epithelial cells and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. TGF-beta1 signals through the TGF-beta type I and type II receptors, and activates the Smad pathway via phosphorylation of Smad2 and Smad3. Since little is known about the selective activation of Smad2 versus Smad3, we set out to identify novel Smad2 and Smad3 interacting proteins in epithelial cells. A non-transformed human cell line was transduced with Myc-His(6)-Smad2 or Myc-His(6)-Smad3-expressing retrovirus and was treated with TGF-beta1. Myc-His(6)-Smad2 or Myc-His(6)-Smad3 was purified by tandem affinity purification, eluates were subject to SDS-PAGE and Colloidal Blue staining, and select protein bands were digested with trypsin. The resulting tryptic peptides were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) and the SEQUEST algorithm was employed to identify proteins in the bands. A number of proteins that are known to interact with Smad2 or Smad3 were detected in the eluates. In addition, a number of putative novel Smad2 and Smad3 associated proteins were identified that have functions in cell proliferation, apoptosis, actin cytoskeleton regulation, cell motility, transcription, and Ras or insulin signaling. Specifically, the interaction between Smad2/3 and the Cdc42 guanine nucleotide exchange factor, Zizimin1, was validated by co-immunoprecipitation. The discovery of these novel Smad2 and/or Smad3 associated proteins may reveal how Smad2 and Smad3 are regulated and/or uncover new functions of Smad2 and Smad3 in TGF-beta1 signaling. J. Cell. Biochem. (c) 2008 Wiley-Liss, Inc.

• Prostate cancer serum biomarker discovery through proteomic analysis of alpha-2 macroglobulin protein complexes.

  Authors: Earle F Burgess, Amy-Joan L Ham, David L Tabb, Dean Billheimer, Bruce J Roth, Sam S Chang, Michael S Cookson, Timothy J Hinton, Kristin L Cheek, Salisha Hill, Jennifer A Pietenpol

  Proteomics. Clinical applications. 07/2008; 2(9):1223.

  Alpha-2 macroglobulin (A2M) functions as a universal protease inhibitor in serum and is capable of binding various cytokines and growth factors. In this study, we investigated if immunoaffinityAlpha-2 macroglobulin (A2M) functions as a universal protease inhibitor in serum and is capable of binding various cytokines and growth factors. In this study, we investigated if immunoaffinity enrichment and proteomic analysis of A2M protein complexes from human serum could improve detection of biologically relevant and novel candidate protein biomarkers in prostate cancer. Serum samples from six patients with androgen-independent, metastatic prostate cancer and six control patients without malignancy were analyzed by immunoaffinity enrichment of A2M protein complexes and MS identification of associated proteins. Known A2M substrates were reproducibly identified from patient serum in both cohorts, as well as proteins previously undetected in human serum. One example is heat shock protein 90 alpha (HSP90α), which was identified only in the serum of cancer patients in this study. Using an ELISA, the presence of HSP90α in human serum was validated on expanded test cohorts and found to exist in higher median serum concentrations in prostate cancer (n = 18) relative to control (n = 13) patients (median concentrations 50.7 versus 27.6 ng/mL, respectively, p = 0.001). Our results demonstrate the technical feasibility of this approach and support the analysis of A2M protein complexes for proteomic-based serum biomarker discovery.

• A tale of two proteins: differential roles and regulation of Smad2 and Smad3 in TGF-beta signaling.

  Authors: Kimberly A Brown, Jennifer A Pietenpol, Harold L Moses

  Journal of cellular biochemistry. 06/2007; 101(1):9-33.

  Transforming growth factor-beta (TGF-beta) is an important growth inhibitor of epithelial cells, and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute toTransforming growth factor-beta (TGF-beta) is an important growth inhibitor of epithelial cells, and insensitivity to this cytokine results in uncontrolled cell proliferation and can contribute to tumorigenesis. Smad2 and Smad3 are direct mediators of TGF-beta signaling, however little is known about the selective activation of Smad2 versus Smad3. The Smad2 and Smad3 knockout mouse phenotypes and studies comparing Smad2 and Smad3 activation of TGF-beta target genes, suggest that Smad2 and Smad3 have distinct roles in TGF-beta signaling. The observation that TGF-beta inhibits proliferation of Smad3-null mammary gland epithelial cells, whereas Smad3 deficient fibroblasts are only partially growth inhibited, suggests that Smad3 has a different role in epithelial cells and fibroblasts. Herein, the current understanding of Smad2 and Smad3-mediated TGF-beta signaling and their relative roles are discussed, in addition to potential mechanisms for the selective activation of Smad2 versus Smad3. Since alterations in the TGF-beta signaling pathway play an important role in promoting tumorigenesis and cancer progression, methods for therapeutic targeting of the TGF-beta signaling pathway are being pursued. Determining how Smad2 or Smad3 differentially regulate the TGF-beta response may translate into developing more effective strategies for cancer therapy.

• Epidermal growth factor receptor plays a significant role in hepatocyte growth factor mediated biological responses in mammary epithelial cells.

  Authors: Alyssa R Bonine-Summers, Mary E Aakre, Kimberly A Brown, Carlos L Arteaga, Jennifer A Pietenpol, Harold L Moses, Nikki Cheng

  Cancer biology & therapy. 05/2007; 6(4):561-70.

  Breast cancers often have deregulated hepatocyte growth factor (HGF) and c-Met signaling that results in increased tumor growth and invasion. Elucidating the mechanism responsible for HGF/c-MetBreast cancers often have deregulated hepatocyte growth factor (HGF) and c-Met signaling that results in increased tumor growth and invasion. Elucidating the mechanism responsible for HGF/c-Met action in breast cancer progression has been difficult as c-Met communicates with a number of secondary receptors that can lead to various pathological outcomes. Understanding how these secondary receptors facilitate HGF/c-Met cellular responses will aid in the development of better therapeutic treatment options for breast cancer patients with elevated HGF signaling. In the present study it was shown that the epidermal growth factor receptor (EGFR) plays a significant role in HGF/c-Met mediated biological activities indicative of advanced tumor pathology, including enhanced proliferation and invasion. The clinically relevant EGFR inhibitor gefitinib was used to determine the role of EGFR in HGF-induced proliferation and motility in several mammary carcinoma cells including PyVmT, MDA-MB-231 and 4T1. Our analyses indicated that EGFR inhibition significantly blocked HGF activation of c-Met and EGFR and that inhibition of these pathways mitigated HGF induced proliferation and motility. The data indicate that this inhibition was not through a direct effect of gefitinib on c-Met, but that EGFR is necessary for c-Met activation in the assays performed. These results provide a novel mechanism of action for EGFR as a mediator of HGF signaling thereby linking EGFR to the oncogenic potential of c-Met in mammary carcinomas cells.

• Transcriptional programs regulated by p63 in normal epithelium and tumors.

  Authors: Carmen A Perez, Jennifer A Pietenpol

  Cell cycle (Georgetown, Tex.). 03/2007; 6(3):246-54.

  The transcription factor p63 belongs to a family of regulatory proteins that bind DNA in a sequence-specific manner, close to a target gene, to activate or repress its transcription. These proteinsThe transcription factor p63 belongs to a family of regulatory proteins that bind DNA in a sequence-specific manner, close to a target gene, to activate or repress its transcription. These proteins display a striking conservation of functional domains, but are differentially involved in regulating cellular processes. Nearly a decade after the discovery of p63, its critical role for proper epithelial development, maintenance and tumorigenesis is widely recognized. Several important cellular endpoints have been linked to p63 regulation in epithelium, including differentiation, cell fate specification, proliferation, survival, senescence, apoptosis, cell-cell and cell-matrix interaction programs. Although a large number of genes have been associated with p63-dependent regulation, only a few of these have been validated as direct p63 transcriptional targets. The challenge still remains to define the p63 transcriptome in epithelial cells, and to dissect the cellular mechanisms used to modulate the expression of this transcriptional profile. In this article we will review current knowledge regarding the basic mechanisms used by p63 to regulate gene expression. Also, we will discuss specific p63-regulated biological endpoints in epithelial cells and the specific genes that have been linked to p63 regulation in these particular contexts.

• Loss of p63 leads to increased cell migration and up-regulation of genes involved in invasion and metastasis.

  Authors: Christopher E Barbieri, Luo Jia Tang, Kimberly A Brown, Jennifer A Pietenpol

  Cancer research. 09/2006; 66(15):7589-97.

  p63, a homologue of the tumor suppressor p53, is critical for the development and maintenance of squamous epithelia. p63 is specifically expressed in the basal layers of stratified epithelial tissuesp63, a homologue of the tumor suppressor p53, is critical for the development and maintenance of squamous epithelia. p63 is specifically expressed in the basal layers of stratified epithelial tissues and is considered a specific marker for cells of this type. The role of p63 in tumorigenesis remains poorly defined. Numerous studies have highlighted the oncogenic potential of the predominant p63 isoform DeltaNp63alpha; however, data suggest that other p63 proteins can act as tumor suppressors or alter the metastatic potential of tumors. DeltaNp63alpha can act as a transcriptional repressor, but the link between the transcriptional functions of p63 and its biological role is still unclear. In this study, we used a loss-of-function approach to investigate the transcriptional programs controlled by p63. Disruption of p63 in squamous cell lines resulted in down-regulation of transcripts specifically expressed in squamous tissues and a significant alteration of keratinocyte differentiation. Interestingly, we found that disruption of p63 led to up-regulation of markers of nonepithelial tissues (mesenchyme and neural tissue) in both primary and immortalized squamous cells. Many of these up-regulated genes are associated with increased capacity for invasion and metastasis in tumors. Furthermore, loss of p63 expression was accompanied by a shift toward mesenchymal morphology and an increase in motility in primary keratinocytes and squamous cell lines. We conclude that loss of endogenous p63 expression results in up-regulation of genes associated with invasion and metastasis, and predisposes to a loss of epithelial and acquisition of mesenchymal characteristics. These findings have implications for the role of p63 in both development and tumorigenesis.

• The DNA binding activity of p53 displays reaction-diffusion kinetics.

  Authors: Peter Hinow, Carl E Rogers, Christopher E Barbieri, Jennifer A Pietenpol, Anne K Kenworthy, Emmanuele DiBenedetto

  Biophysical journal. 08/2006; 91(1):330-42.

  The tumor suppressor protein p53 plays a key role in maintaining the genomic stability of mammalian cells and preventing malignant transformation. In this study, we investigated the intracellularThe tumor suppressor protein p53 plays a key role in maintaining the genomic stability of mammalian cells and preventing malignant transformation. In this study, we investigated the intracellular diffusion of a p53-GFP fusion protein using confocal fluorescence recovery after photobleaching. We show that the diffusion of p53-GFP within the nucleus is well described by a mathematical model for diffusion of particles that bind temporarily to a spatially homogeneous immobile structure with binding and release rates k1 and k2, respectively. The diffusion constant of p53-GFP was estimated to be Dp53-GFP=15.4 microm2 s-1, significantly slower than that of GFP alone, DGFP=41.6 microm2 s-1. The reaction rates of the binding and unbinding of p53-GFP were estimated as k1=0.3 s-1 and k2=0.4 s-1, respectively, values suggestive of nonspecific binding. Consistent with this finding, the diffusional mobilities of tumor-derived sequence-specific DNA binding mutants of p53 were indistinguishable from that of the wild-type protein. These data are consistent with a model in which, under steady-state conditions, p53 is latent and continuously scans DNA, requiring activation for sequence-specific DNA binding.

• p63 and epithelial biology.

  Authors: Christopher E Barbieri, Jennifer A Pietenpol

  Experimental cell research. 05/2006; 312(6):695-706.

  The transcription factor p63 is a homologue of the tumor suppressor p53. Unlike p53, which is dispensable for normal development, p63 is critical for the development of stratified epithelial tissuesThe transcription factor p63 is a homologue of the tumor suppressor p53. Unlike p53, which is dispensable for normal development, p63 is critical for the development of stratified epithelial tissues such as epidermis, breast, and prostate. p63 encodes multiple protein isoforms with both transactivating and transcriptional repressor activities that can regulate a wide spectrum of target genes. p63 is also implicated in tumor formation and progression in stratified epithelia, with evidence for both tumor suppressive and oncogenic properties. This review will examine current data and hypotheses regarding the role of p63 in the development, maintenance, and tumorigenesis of stratified epithelium.

• Neoadjuvant concurrent paclitaxel and radiation in stage II/III breast cancer.

  Authors: A Bapsi Chakravarthy, Mark C Kelley, Bernadette McLaren, Cristina I Truica, Dean Billheimer, Ingrid A Mayer, Ana M Grau, David H Johnson, Jean F Simpson, R Daniel Beauchamp, Catherine Jones, Jennifer A Pietenpol

  Clinical cancer research : an official journal of the American Association for Cancer Research. 04/2006; 12(5):1570-6.

  PURPOSE: The aim of this study was to determine the safety and pathologic response rates following neoadjuvant paclitaxel and radiation in patients with stage II/III breast cancer and to evaluate thePURPOSE: The aim of this study was to determine the safety and pathologic response rates following neoadjuvant paclitaxel and radiation in patients with stage II/III breast cancer and to evaluate the use of sequential biopsies to allow an in vivo assessment of biological markers as potential predictive markers of response to this regimen. PATIENTS AND METHODS: Patients with high-risk, operable breast cancer were treated with three cycles of paclitaxel 175 mg/m2 every 3 weeks, followed by twice-weekly paclitaxel 30 mg/m2 and concurrent radiation. Core biopsies were obtained at baseline and 24 to 72 hours after the first cycle of paclitaxel. After completing neoadjuvant treatment, patients underwent definitive surgery. The primary end point was pathologic complete response, which is defined as the absence of any invasive cancer at surgery. Potential markers of therapeutic response were evaluated including markers of proliferation, apoptosis, p21, HER2, estrogen receptor, and progesterone receptor status. RESULTS: Of the 38 patients enrolled, 13 (34%) had a pathologic complete response. There was no significant difference in baseline Ki-67 between responders (35%) and nonresponders (28%; P = 0.45). There was also no significant change in Ki-67 following paclitaxel administration. Despite this lack of immunohistologic change in proliferative activity, baseline mitotic index was higher for patients with pathologic complete response over nonresponders (27 versus 10, P = 0.003). Moreover, the increase in mitotic index following paclitaxel administration was associated with pathologic complete response. CONCLUSIONS: Neoadjuvant paclitaxel/radiation is effective and well tolerated. Tumor proliferation at baseline and response to chemotherapy as measured by mitotic activity may serve as an important indicator of pathologic response to neoadjuvant paclitaxel/radiation.

• Chromatin immunoprecipitation-based screen to identify functional genomic binding sites for sequence-specific transactivators.

  Authors: Jamie M Hearnes, Deborah J Mays, Kristy L Schavolt, Luojia Tang, Xin Jiang, Jennifer A Pietenpol

  Molecular and cellular biology. 12/2005; 25(22):10148-58.

  In various human diseases, altered gene expression patterns are often the result of deregulated gene-specific transcription factor activity. To further understand disease on a molecular basis, theIn various human diseases, altered gene expression patterns are often the result of deregulated gene-specific transcription factor activity. To further understand disease on a molecular basis, the comprehensive analysis of transcription factor signaling networks is required. We developed an experimental approach, combining chromatin immunoprecipitation (ChIP) with a yeast-based assay, to screen the genome for transcription factor binding sites that link to transcriptionally regulated target genes. We used the tumor suppressor p53 to demonstrate the effectiveness of the method. Using primary and immortalized, nontransformed cultures of human mammary epithelial cells, we isolated over 100 genomic DNA fragments that contain novel p53 binding sites. This approach led to the identification and validation of novel p53 target genes involved in diverse signaling pathways, including growth factor signaling, protein kinase/phosphatase signaling, and RNA binding. Our results yield a more complete understanding of p53-regulated signaling pathways, and this approach could be applied to any number of transcription factors to further elucidate complex transcriptional networks.