Justin M. Cates

The receptor tyrosine kinase EphA2 promotes mammary adenocarcinoma tumorigenesis and metastatic progression in mice by amplifying ErbB2 signaling

Overexpression of the receptor tyrosine kinase EPH receptor A2 (EphA2) is commonly observed in aggressive breast cancer and correlates with a poor prognosis. However, while EphA2 has been reported to enhance tumorigenesis, proliferation, and MAPK activation in several model systems, other studies suggest that EphA2 activation diminishes these processes and inhibits the activity of MAPK upon ligand stimulation. In this study, we eliminated EphA2 expression in 2 transgenic mouse models of mammary carcinoma. EphA2 deficiency impaired tumor initiation and metastatic progression in mice overexpressing ErbB2 (also known as Neu) in the mammary epithelium (MMTV-Neu mice), but not in mice overexpressing the polyomavirus middle T antigen in mammary epithelium (MMTV-PyV-mT mice). Histologic and ex vivo analyses of MMTV-Neu mouse mammary epithelium indicated that EphA2 enhanced tumor proliferation and motility. Biochemical analyses revealed that EphA2 formed a complex with ErbB2 in human and murine breast carcinoma cells, resulting in enhanced activation of Ras-MAPK signaling and RhoA GTPase. Additionally, MMTV-Neu, but not MMTV-PyV-mT, tumors were sensitive to therapeutic inhibition of EphA2. These data suggest that EphA2 cooperates with ErbB2 to promote tumor progression in mice and may provide a novel therapeutic target for ErbB2-dependent tumors in humans. Moreover, EphA2 function in tumor progression appeared to depend on oncogene context, an important consideration for the application of therapies targeting EphA2.

The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair.

Cell-based therapies, using multipotent mesenchymal stem cells (MSCs) for organ regeneration, are being pursued for cardiac disease, orthopedic injuries and biomaterial fabrication. The molecular pathways that regulate MSC-mediated regeneration or enhance their therapeutic efficacy are, however, poorly understood. We compared MSCs isolated from MRL/MpJ mice, known to demonstrate enhanced regenerative capacity, to those from C57BL/6 (WT) mice. Compared with WT-MSCs, MRL-MSCs demonstrated increased proliferation, in vivo engraftment, experimental granulation tissue reconstitution, and tissue vascularity in a murine model of repair stimulation. The MRL-MSCs also reduced infarct size and improved function in a murine myocardial infarct model compared with WT-MSCs. Genomic and functional analysis indicated a downregulation of the canonical Wnt pathway in MRL-MSCs characterized by significant up-regulation of specific secreted frizzled-related proteins (sFRPs). Specific knockdown of sFRP2 by shRNA in MRL-MSCs decreased their proliferation and their engraftment in and the vascular density of MRL-MSC-generated experimental granulation tissue. These results led us to generate WT-MSCs overexpressing sFRP2 (sFRP2-MSCs) by retroviral transduction. sFRP2-MSCs maintained their ability for multilineage differentiation in vitro and, when implanted in vivo, recapitulated the MRL phenotype. Peri-infarct intramyocardial injection of sFRP2-MSCs resulted in enhanced engraftment, vascular density, reduced infarct size, and increased cardiac function after myocardial injury in mice. These findings implicate sFRP2 as a key molecule for the biogenesis of a superior regenerative phenotype in MSCs.

Low Levels of Tumor Necrosis Factor α Increase Tumor Growth by Inducing an Endothelial Phenotype of Monocytes Recruited to the Tumor Site

Microenvironmental cues instruct infiltrating tumor-associated myeloid cells to drive malignant progression. A subpopulation of tumor-associated myeloid cells coexpressing endothelial and myeloid markers, although rare in peripheral blood, are primarily associated with tumors where they enhance tumor growth and angiogenesis. These biphenotypic vascular leukocytes result from the endothelial differentiation of myeloid progenitors, a process regulated by tumor necrosis factor (TNF)alpha in vitro. An in vivo increase in tumor-derived TNFalpha expression promoted tumor growth and vascularity of mouse melanoma, lung cancer, and mammary tumors. Notably, tumor growth was accompanied by a significant increase in myeloid/endothelial biphenotypic populations. TNFalpha-associated tumor growth, vascularity, and generation of tumor vascular leukocytes in mouse melanoma tumors were dependent on intact host TNFalpha receptors. Importantly, TNFalpha-expressing tumors did not exhibit increased inflammation over control tumors, suggesting a unique action related to myeloid to endothelial differentiation. Our studies suggest that TNFalpha constitutes a tumor microenvironment signal that biases recruited monocytes toward a proangiogenic/provasculogenic myeloid/endothelial phenotype.

Targeted Next Generation Sequencing Identifies Markers of Response to PD-1 Blockade

Mutational load, by whole exome sequencing, can correlate with immunotherapy responses. Assessing melanoma mutational load of a fraction of the genome, by hybrid capture-based NGS, provided an accurate surrogate for WES determinations, and predicted response to anti-PD-1. Therapeutic antibodies blocking programmed death-1 and its ligand (PD-1/PD-L1) induce durable responses in a substantial fraction of melanoma patients. We sought to determine whether the number and/or type of mutations identified using a next-generation sequencing (NGS) panel available in the clinic was correlated with response to anti–PD-1 in melanoma. Using archival melanoma samples from anti–PD-1/PD-L1-treated patients, we performed hybrid capture–based NGS on 236–315 genes and T-cell receptor (TCR) sequencing on initial and validation cohorts from two centers. Patients who responded to anti–PD-1/PD-L1 had higher mutational loads in an initial cohort (median, 45.6 vs. 3.9 mutations/MB; P = 0.003) and a validation cohort (37.1 vs. 12.8 mutations/MB; P = 0.002) compared with nonresponders. Response rate, progression-free survival, and overall survival were superior in the high, compared with intermediate and low, mutation load groups. Melanomas with NF1 mutations harbored high mutational loads (median, 62.7 mutations/MB) and high response rates (74%), whereas BRAF/NRAS/NF1 wild-type melanomas had a lower mutational load. In these archival samples, TCR clonality did not predict response. Mutation numbers in the 315 genes in the NGS platform strongly correlated with those detected by whole-exome sequencing in The Cancer Genome Atlas samples, but was not associated with survival. In conclusion, mutational load, as determined by an NGS platform available in the clinic, effectively stratified patients by likelihood of response. This approach may provide a clinically feasible predictor of response to anti–PD-1/PD-L1. Cancer Immunol Res; 4(11); 959–67. ©2016 AACR.

Docetaxel/gemcitabine followed by gemcitabine and external beam radiotherapy in patients with pancreatic adenocarcinoma

BackgroundPancreatic cancer remains highly lethal. Previous attempts with neoadjuvant therapy in this disease have been inconclusive, but a potential for benefit exists. We conducted a phase II trial of dose-intense docetaxel and gemcitabine followed by twice-weekly gemcitabine and external beam radiotherapy in patients with pancreatic adenocarcinoma.MethodsPatients with stage I to III disease were eligible. Docetaxel 65 mg/m2 intravenously over 1 hour and gemcitabine 4000 mg/m2 given intravenously over 30 minutes were given on days 1, 15, and 29. On day 43, radiotherapy was begun at 50.4 Gy with gemcitabine 50 mg/m2 intravenously over 30 minutes twice weekly for 12 doses. After treatment, patients were considered for resection.ResultsTwenty-four assessable patients were recruited onto the trial. All but one patient completed a full 12 weeks of therapy. Grade 3 and 4 hematological and nonhematological toxicities were common but manageable, and neutropenic fever did not occur. No patient had local tumor progression. Twelve patients (50%) responded by Response Evaluation Criteria in Solid Tumors Group (RECIST) criteria, including one radiographic complete response. Seventeen patients underwent resection after therapy. Margin-negative resections were performed in 13 patients, including 9 patients whose disease was borderline or unresectable before treatment. A treatment effect was seen in all resection specimens. There have been no local recurrences of tumor, and several patients remain alive without evidence of disease.ConclusionsDocetaxel/gemcitabine followed by gemcitabine/radiotherapy is active in the treatment of pancreatic adenocarcinoma, with manageable toxicity. Tumor downstaging occurs in some patients to allow complete resection. Further investigation of this regimen is warranted.

Epithelial-Mesenchymal Transition Markers in Pancreatic Ductal Adenocarcinoma

Objectives: Expression of transcription factors that mediate epithelial-mesenchymal transition (EMT), such as Twist and Slug, is correlated with poor prognosis in many tumor types. Selected EMT markers were studied in a series of pancreatic ductal adenocarcinomas (PDAs) and benign pancreatic tissues to determine whether expression levels correlated with diagnosis, histologic grade, or patient outcome. Methods: Immunohistochemical stains for Twist, Slug, and N-cadherin were performed using a tissue microarray containing 68 PDAs and 38 samples of normal pancreas or chronic pancreatitis tissues. Results: Twist and Slug were identified in both the nucleus and cytoplasm of benign pancreatic ductal epithelium, chronic pancreatitis, and PDA. Compared with normal ductal epithelium, nuclear levels of Twist are decreased in PDA. None of the other EMT markers showed significant differences in staining indices among the diagnostic groups. There were no correlations between EMT marker expression and histologic grade. Epithelial-mesenchymal transition marker expression was not associated with N-cadherin expression, patient outcome, or duration of survival. Conclusions: Decreased expression of nuclear Twist is observed in malignant pancreatic epithelium. However, use of Twist as a diagnostic marker is precluded because decreased expression is also seen in chronic pancreatitis. None of the markers studied were predictive of patient outcome.

Loss of the Urothelial Differentiation Marker FOXA1 Is Associated with High Grade, Late Stage Bladder Cancer and Increased Tumor Proliferation

Approximately 50% of patients with muscle-invasive bladder cancer (MIBC) develop metastatic disease, which is almost invariably lethal. However, our understanding of pathways that drive aggressive behavior of MIBC is incomplete. Members of the FOXA subfamily of transcription factors are implicated in normal urogenital development and urologic malignancies. FOXA proteins are implicated in normal urothelial differentiation, but their role in bladder cancer is unknown. We examined FOXA expression in commonly used in vitro models of bladder cancer and in human bladder cancer specimens, and used a novel in vivo tissue recombination system to determine the functional significance of FOXA1 expression in bladder cancer. Logistic regression analysis showed decreased FOXA1 expression is associated with increasing tumor stage (p<0.001), and loss of FOXA1 is associated with high histologic grade (p<0.001). Also, we found that bladder urothelium that has undergone keratinizing squamous metaplasia, a precursor to the development of squamous cell carcinoma (SCC) exhibited loss of FOXA1 expression. Furthermore, 81% of cases of SCC of the bladder were negative for FOXA1 staining compared to only 40% of urothelial cell carcinomas. In addition, we showed that a subpopulation of FOXA1 negative urothelial tumor cells are highly proliferative. Knockdown of FOXA1 in RT4 bladder cancer cells resulted in increased expression of UPK1B, UPK2, UPK3A, and UPK3B, decreased E-cadherin expression and significantly increased cell proliferation, while overexpression of FOXA1 in T24 cells increased E-cadherin expression and significantly decreased cell growth and invasion. In vivo recombination of bladder cancer cells engineered to exhibit reduced FOXA1 expression with embryonic rat bladder mesenchyme and subsequent renal capsule engraftment resulted in enhanced tumor proliferation. These findings provide the first evidence linking loss of FOXA1 expression with histological subtypes of MIBC and urothelial cell proliferation, and suggest an important role for FOXA1 in the malignant phenotype of MIBC.

Bronchial Secretory Immunoglobulin A Deficiency Correlates With Airway Inflammation and Progression of Chronic Obstructive Pulmonary Disease

RATIONALE Although airway inflammation can persist for years after smoking cessation in patients with chronic obstructive pulmonary disease (COPD), the mechanisms of persistent inflammation are largely unknown. OBJECTIVES We investigated relationships between bronchial epithelial remodeling, polymeric immunoglobulin receptor (pIgR) expression, secretory IgA (SIgA), airway inflammation, and mural remodeling in COPD. METHODS Lung tissue specimens and bronchoalveolar lavage were obtained from lifetime nonsmokers and former smokers with or without COPD. Epithelial structural changes were quantified by morphometric analysis. Expression of pIgR was determined by immunostaining and real-time polymerase chain reaction. Immunohistochemistry was performed for IgA, CD4 and CD8 lymphocytes, and cytomegalovirus and Epstein-Barr virus antigens. Total IgA and SIgA were measured by ELISA and IgA transcytosis was studied using cultured human bronchial epithelial cells. MEASUREMENTS AND MAIN RESULTS Areas of bronchial mucosa covered by normal pseudostratified ciliated epithelium were characterized by pIgR expression with SIgA present on the mucosal surface. In contrast, areas of bronchial epithelial remodeling had reduced pIgR expression, localized SIgA deficiency, and increased CD4(+) and CD8(+) lymphocyte infiltration. In small airways (<2 mm), these changes were associated with presence of herpesvirus antigens, airway wall remodeling, and airflow limitation in patients with COPD. Patients with COPD had reduced SIgA in bronchoalveolar lavage. Air-liquid interface epithelial cell cultures revealed that complete epithelial differentiation was required for normal pIgR expression and IgA transcytosis. CONCLUSIONS Our findings indicate that epithelial structural abnormalities lead to localized SIgA deficiency in COPD airways. Impaired mucosal immunity may contribute to persistent airway inflammation and progressive airway remodeling in COPD.

Surgical treatment of hemangiomas of soft tissue.

Hemangioma is one of the most common soft tissue tumors comprising 7% of all benign tumors. The etiology is unclear. Many treatment modalities for the symptomatic deep subdermal or intramuscular hemangioma have been used, but surgical excision is the preferred treatment. During the past 20 years, 89 patients with soft tissue hemangiomas were treated by surgical excision at the authors’ institution. This study was done to define the clinical characteristics of pathologically proven hemangiomas and to evaluate the outcome of the operative procedures. Intralesional or marginal excision for symptomatic hemangiomas yields satisfactory results for pain relief, functional recovery, and avoidance of recurrence. According to the data a hemangioma of the soft tissues is a benign lesion in which more aggressive surgery (wide or radical excision) or other modalities such as radiation usually are not warranted.

A Novel Model of Urinary Tract Differentiation, Tissue Regeneration, and Disease: Reprogramming Human Prostate and Bladder Cells into Induced Pluripotent Stem Cells

Background Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation. Objective To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation. Design, setting, and participants Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs. Outcome measurements and statistical analysis The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used. Results and limitations Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies. Conclusions IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.