In Vitro Intracellular Trafficking of Virulence Antigen during Infection by Yersinia pestis

Yersinia pestis, the causative agent of plague, encodes several essential virulence factors on a 70 kb plasmid, including the Yersinia outer proteins (Yops) and a multifunctional virulence antigen (V). V is uniquely able to inhibit the host immune response; aid in the expression, secretion, and injection of the cytotoxic Yops via a type III secretion system (T3SS)-dependent mechanism; be secreted extracellularly; and enter the host cell by a T3SS-independent mechanism, where its activity is unknown. To elucidate the intracellular trafficking and target(s) of V, time-course experiments were performed with macrophages (MΦs) infected with Y. pestis or Y. pseudotuberculosis at intervals from 5 min to 6 h. The trafficking pattern was discerned from results of parallel microscopy, immunoblotting, and flow cytometry experiments. The MΦs were incubated with fluorescent or gold conjugated primary or secondary anti-V (antibodies [Abs]) in conjunction with organelle-associated Abs or dyes. The samples were observed for co-localization by immuno-fluorescence and electron microscopy. For fractionation studies, uninfected and infected MΦs were lysed and subjected to density gradient centrifugation coupled with immunoblotting with Abs to V or to organelles. Samples were also analyzed by flow cytometry after lysis and dual-staining with anti-V and anti-organelle Abs. Our findings indicate a co-localization of V with (1) endosomal proteins between 10–45 min of infection, (2) lysosomal protein(s) between 1–2 h of infection, (3) mitochondrial proteins between 2.5–3 h infection, and (4) Golgi protein(s) between 4–6 h of infection. Further studies are being performed to determine the specific intracellular interactions and role in pathogenesis of intracellularly localized V

Classification of TP53 mutations and HPV predict survival in advanced larynx cancer

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134126/1/lary25915-sup-0001-suppinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134126/2/lary25915_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134126/3/lary25915.pd

Chemotherapy alone for organ preservation in advanced laryngeal cancer

Background. For patients with advanced laryngeal cancer, a trial was designed to determine if chemotherapy alone, in patients achieving a complete histologic complete response after a single neoadjuvant cycle, was an effective treatment with less morbidity than concurrent chemoradiotherapy. Methods. Thirty-two patients with advanced laryngeal or hypopharyngeal cancer received 1 cycle of induction chemotherapy, and subsequent treatment was decided based on response. Results. A histologic complete response was achieved in 4 patients and were treated with chemotherapy alone. All 4 patients' cancer relapsed in the neck and required surgery and postoperative radiotherapy (RT). Twenty-five patients were treated with concomitant chemoradiation. Three patients were treated with surgery. Overall survival and disease-specific survival at 3 years were 68% and 78%, respectively. Conclusion. Chemotherapy alone is not feasible for long-term control of regional disease in patients with advanced laryngeal cancer even when they achieve a histologic complete response at the primary site. © 2009 Wiley Periodicals, Inc. Head Neck, 2010Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/77508/1/21285_ftp.pd

Expression of Six1 in luminal breast cancers predicts poor prognosis and promotes increases in tumor initiating cells by activation of extracellular signal-regulated kinase and transforming growth factor-beta signaling pathways

Abstract Introduction Mammary-specific overexpression of Six1 in mice induces tumors that resemble human breast cancer, some having undergone epithelial to mesenchymal transition (EMT) and exhibiting stem/progenitor cell features. Six1 overexpression in human breast cancer cells promotes EMT and metastatic dissemination. We hypothesized that Six1 plays a role in the tumor initiating cell (TIC) population specifically in certain subtypes of breast cancer, and that by understanding its mechanism of action, we could potentially develop new means to target TICs. Methods We examined gene expression datasets to determine the breast cancer subtypes with Six1 overexpression, and then examined its expression in the CD24low/CD44+ putative TIC population in human luminal breast cancers xenografted through mice and in luminal breast cancer cell lines. Six1 overexpression, or knockdown, was performed in different systems to examine how Six1 levels affect TIC characteristics, using gene expression and flow cytometric analysis, tumorsphere assays, and in vivo TIC assays in immunocompromised and immune-competent mice. We examined the molecular pathways by which Six1 influences TICs using genetic/inhibitor approaches in vitro and in vivo. Finally, we examined the expression of Six1 and phosphorylated extracellular signal-regulated kinase (p-ERK) in human breast cancers. Results High levels of Six1 are associated with adverse outcomes in luminal breast cancers, particularly the luminal B subtype. Six1 levels are enriched in the CD24low/CD44+ TIC population in human luminal breast cancers xenografted through mice, and in tumorsphere cultures in MCF7 and T47D luminal breast cancer cells. When overexpressed in MCF7 cells, Six1expands the TIC population through activation of transforming growth factor-beta (TGF-β) and mitogen activated protein kinase (MEK)/ERK signaling. Inhibition of ERK signaling in MCF7-Six1 cells with MEK1/2 inhibitors, U0126 and AZD6244, restores the TIC population of luminal breast cancer cells back to that observed in control cells. Administration of AZD6244 dramatically inhibits tumor formation efficiency and metastasis in cells that express high levels of Six1 ectopically or endogenously. Finally, we demonstrate that Six1 significantly correlates with phosphorylated ERK in human breast cancers. Conclusions Six1 plays an important role in the TIC population in luminal breast cancers and induces a TIC phenotype by enhancing both TGF-β and ERK signaling. MEK1/2 kinase inhibitors are potential candidates for targeting TICs in breast tumors

Biomarkers in advanced larynx cancer

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/102058/1/lary24245.pd

Mutational profiles of persistent/recurrent laryngeal squamous cell carcinoma

BackgroundWe sought to describe targeted DNA sequencing data of persistent/recurrent laryngeal squamous cell carcinoma (LSCC) and to compare gene‐specific alteration frequencies with that of primary, untreated LSCC specimens from The Cancer Genome Atlas (TCGA).MethodsThe tumors of 21 patients with persistent/recurrent LSCC were subjected to targeted DNA sequencing using the Ion AmpliSeq Comprehensive Cancer Panel. Gene‐specific alteration frequencies were compared (Chi‐Square test) to primary, untreated LSCC sequencing data from TCGA using the cBioPortal platform.ResultsPersistent/recurrent LSCC was characterized by a high rate of inactivating alterations in TP53 (38.1%) and CDKN2A (33%), amplification events of CCND1 (19.1%), and ERBB2 (14.3%), and NOTCH1 (19.1%) mutations. Comparison of primary vs persistent/recurrent LSCC revealed significant differences in alteration frequencies of eight critical genes: BAP1, CDKN2A, DCUN1D1, MSH2, MTOR, PIK3CA, TET2, and TP53.ConclusionsOur results provide preliminary support for a distinct mutational profile of persistent/recurrent LSCC that requires validation in larger cohorts.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147873/1/hed25444.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147873/2/hed25444_am.pd

Weekly chemotherapy with radiation versus high‐dose cisplatin with radiation as organ preservation for patients with HPV‐positive and HPV‐negative locally advanced squamous cell carcinoma of the oropharynx

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106948/1/hed23339.pd

Hsp90 orchestrates transcriptional regulation by Hsf1 and cell wall remodelling by MAPK signalling during thermal adaptation in a pathogenic yeast

Acknowledgments We thank Rebecca Shapiro for creating CaLC1819, CaLC1855 and CaLC1875, Gillian Milne for help with EM, Aaron Mitchell for generously providing the transposon insertion mutant library, Jesus Pla for generously providing the hog1 hst7 mutant, and Cathy Collins for technical assistance.Peer reviewedPublisher PD

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin