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1.
BMC Cancer ; 21(1): 1075, 2021 Oct 02.
Article in English | MEDLINE | ID: mdl-34600526

ABSTRACT

BACKGROUND: Monitoring circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), known as liquid biopsies, continue to be developed as diagnostic and prognostic markers for a wide variety of cancer indications, mainly due to their minimally invasive nature and ability to offer a wide range of phenotypic and genetic information. While liquid biopsies maintain significant promising benefits, there is still limited information regarding the kinetics of ctDNA and CTCs following radiation therapy which remains a vital treatment modality in head and neck cancers. This study aims to describe the kinetics of ctDNA and CTCs following radiation exposure in a preclinical rabbit model with VX2 induced buccal carcinoma. METHODS: Seven rabbits were inoculated with VX2 cells in the buccal mucosa and subjected to radiation. At selected time points, blood sampling was performed to monitor differing levels of ctDNA and CTC. Plasma ctDNA was measured with quantitative PCR for papillomavirus E6 while CTCs were quantified using an immunomagnetic nanoparticles within a microfluidic device. Comparisons of CTC detection with EpCAM compared to multiple surface markers (EGFR, HER2 and PSMA) was evaluated and correlated with the tumor size. RESULTS: Plasma ctDNA reflects the overall tumor burden within the animal model. Analysis of correlations between ctDNA with tumor and lymph node volumes showed a positive correlation (R = 0.452 and R = 0.433 [p < 0.05]), respectively. Over the course of treatment, ctDNA levels declined and quickly becomes undetectable following tumor eradication. While during the course of treatment, ctDNA levels were noted to rise particularly upon initiation of radiation following scheduled treatment breaks. Levels of CTCs were observed to increase 1 week following inoculation of tumor to the primary site. For CTC detection, the use of multiple surface markers showed a greater sensitivity when compared to detection using only EpCAM. Plasma CTC levels remained elevated following radiation therapy which may account for an increased shedding of CTCs following radiation. CONCLUSION: This study demonstrates the utility of ctDNA and CTCs detection in response to radiation treatment in a preclinical head and neck model, allowing for better understanding of liquid biopsy applications in both clinical practice and research development.


Subject(s)
Carcinoma, Squamous Cell/blood , Carcinoma, Squamous Cell/radiotherapy , Cell-Free Nucleic Acids/blood , Mouth Neoplasms/blood , Mouth Neoplasms/radiotherapy , Animals , Biomarkers, Tumor/blood , Carcinoma, Squamous Cell/chemically induced , Circulating Tumor DNA/blood , Cottontail rabbit papillomavirus , Epithelial Cell Adhesion Molecule/blood , Head and Neck Neoplasms/blood , Head and Neck Neoplasms/chemically induced , Head and Neck Neoplasms/radiotherapy , Immunomagnetic Separation/methods , Liquid Biopsy/methods , Male , Mouth Neoplasms/chemically induced , Mouth Neoplasms/virology , Nanoparticles , Neoplasm Transplantation , Open Reading Frames , Rabbits , Radiotherapy Dosage , Tumor Burden
2.
JCO Precis Oncol ; 2: 1-8, 2018 Nov.
Article in English | MEDLINE | ID: mdl-35135145

ABSTRACT

PURPOSE: To determine whether plasma human papillomavirus (HPV) DNA predates clinical recurrence and compare its accuracy with 3-month fluorodeoxyglucose positron emission tomography (FDG-PET) in locally advanced cervical cancer. METHODS: This prospective multicenter study accrued 23 women with stage IB to IVA cervical cancer planned for definitive chemoradiation therapy (CRT). Plasma HPV DNA was measured serially by digital polymerase chain reaction, and FDG-PET was performed at 3 months post-CRT. RESULTS: Of the 19 women with HPV+ cervical cancer included in this analysis, 32% were stage IB, 58% IIB, and 10% IIIB/IVA. Median follow-up was 24 months (range, 18 to 30 months). All patients had detectable plasma HPV DNA before treatment. Six patients had detectable plasma HPV DNA at the end of CRT, and three of them developed metastases at 3 months. Of the 13 patients with undetectable plasma HPV DNA at end of CRT, to date, only one has developed recurrence. Six of those 13 patients had a positive 3-month FDG-PET with no definite residual disease on subsequent imaging or clinical examination to date, and four of these six had undetectable plasma HPV DNA at 3 months. Patients with undetectable plasma HPV DNA at end of CRT had significantly higher 18-month progression-free survival than those with detectable plasma HPV DNA (92% v 50%; P = .02). The area under the receiver operating characteristic curve (accuracy) of 3-month plasma HPV DNA and 3-month FDG-PET imaging for predicting recurrence at 18 months were 77% and 60%, respectively (P = .008). CONCLUSION: Detectable plasma HPV DNA at end of CRT predates the clinical diagnosis of metastases and is associated with inferior progression-free survival. Moreover, 3-month plasma HPV DNA level is more accurate than 3-month FDG-PET imaging in detecting residual disease. The clinical utility of plasma HPV DNA detection for guiding adjuvant/salvage therapy should be evaluated in future studies.

3.
Sci Rep ; 7(1): 16723, 2017 12 01.
Article in English | MEDLINE | ID: mdl-29196748

ABSTRACT

In cancer patients, circulating tumour-derived DNA (ctDNA) levels imperfectly reflect disease burden apparent on medical imaging. Further evaluation of ctDNA levels over time is needed to better understand the correlation with tumour growth and therapeutic response. We describe ctDNA kinetics within an orthotopic, immunocompetent preclinical rabbit model of local-regionally advanced head and neck squamous cell carcinoma (HNSCC). Monitoring primary tumour and metastatic lymph node volume by computed tomography (CT), we observed a correlation between ctDNA levels and tumour burden. We found that ctDNA detection could precede evidence of tumour on CT. Sensitivity and specificity of ctDNA detection in this model was 90.2% (95% C.I.: 76.9-97.3%) and 85.7% (95% C.I.: 67.3-96.0%), respectively. Rapid tumour growth followed by auto-necrosis and tumour volume contraction produced a spike in ctDNA levels, suggesting that viable tumour cells may be required for sustained ctDNA release. Following surgical resection, both ctDNA and total plasma DNA were correlated with recurrent tumour volume. Our results reveal the complex kinetic behaviour of ctDNA and total plasma DNA upon tumour growth or surgery. This pre-clinical model could be useful for future studies focused on elucidating mechanisms of ctDNA release into the circulation from primary and metastatic sites.


Subject(s)
Cell-Free Nucleic Acids/blood , Circulating Tumor DNA/genetics , Head and Neck Neoplasms/pathology , Squamous Cell Carcinoma of Head and Neck/pathology , Animals , Head and Neck Neoplasms/diagnostic imaging , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/surgery , Kinetics , Male , Neoplasm Transplantation , Rabbits , Sensitivity and Specificity , Squamous Cell Carcinoma of Head and Neck/diagnostic imaging , Squamous Cell Carcinoma of Head and Neck/genetics , Squamous Cell Carcinoma of Head and Neck/surgery , Tomography Scanners, X-Ray Computed , Tumor Burden
4.
Sci Transl Med ; 7(317): 317ra197, 2015 Dec 09.
Article in English | MEDLINE | ID: mdl-26659571

ABSTRACT

Osteosarcoma (OS) is the most common primary bone cancer, which occurs primarily in children and adolescents, severely affecting survivors' quality of life. Despite its chemosensitivity and treatment advances, long-term survival rates for OS patients have stagnated over the last 20 years. Thus, it is necessary to develop new molecularly targeted therapies for this metastatic bone cancer. Mutations in TP53 and RB are linked to OS predisposition and to the evolution of spontaneous OS. We established receptor activator of nuclear factor κB ligand (RANKL) as a therapeutic target for suppression and prevention of OS. Combined conditional osteoblast-specific deletions of Rb, p53, and the protein kinase A (PKA) regulatory subunit Prkar1α genes in genetically engineered mouse models (GEMMs) generate aggressive osteosarcomas, characterized by PKA, RANKL, and osteoclast hyperactivity. Whole-body Rankl deletion completely abrogates tumorigenesis. Although osteoblastic Rank deletion has little effect, osteoclastic Rank deletion delays tumorigenesis and prolongs life span. The latter is associated with inactivation of osteoclastogenesis and up-regulation of the tumor suppressor phosphatase and tensin homolog (PTEN). Further, we use these GEMMs as preclinical platforms to show that RANKL blockade with RANK-Fc arrests tumor progression and improves survival and also inhibits lung metastasis. Moreover, preemptive administration of RANK-Fc completely prevents tumorigenesis in mice highly predisposed to this aggressive cancer. Denosumab, a fully human monoclonal antibody against RANKL, is currently used to treat patients with osteoporosis or bone metastases. Our studies provide a strong rationale to consider RANKL blockade for the treatment and prevention of aggressive RANKL-overexpressing OS in humans.


Subject(s)
Osteosarcoma/metabolism , Osteosarcoma/pathology , RANK Ligand/antagonists & inhibitors , Animals , Carcinogenesis/genetics , Carcinogenesis/pathology , Gene Deletion , Humans , Mice , Neoplasm Invasiveness , Organ Specificity , Osteoblasts/metabolism , Osteosarcoma/genetics , Osteosarcoma/therapy , PTEN Phosphohydrolase/metabolism , RANK Ligand/metabolism , Retinoblastoma Protein/genetics , Retinoblastoma Protein/metabolism , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
5.
Stem Cell Reports ; 5(1): 31-44, 2015 Jul 14.
Article in English | MEDLINE | ID: mdl-26095608

ABSTRACT

Systemic and local signals must be integrated by mammary stem and progenitor cells to regulate their cyclic growth and turnover in the adult gland. Here, we show RANK-positive luminal progenitors exhibiting WNT pathway activation are selectively expanded in the human breast during the progesterone-high menstrual phase. To investigate underlying mechanisms, we examined mouse models and found that loss of RANK prevents the proliferation of hormone receptor-negative luminal mammary progenitors and basal cells, an accompanying loss of WNT activation, and, hence, a suppression of lobuloalveologenesis. We also show that R-spondin1 is depleted in RANK-null progenitors, and that its exogenous administration rescues key aspects of RANK deficiency by reinstating a WNT response and mammary cell expansion. Our findings point to a novel role of RANK in dictating WNT responsiveness to mediate hormone-induced changes in the growth dynamics of adult mammary cells.


Subject(s)
Mammary Glands, Animal/metabolism , Receptor Activator of Nuclear Factor-kappa B/genetics , Stem Cells/cytology , Thrombospondins/genetics , Animals , Cell Proliferation/genetics , Female , Humans , Mammary Glands, Animal/growth & development , Mice , Receptor Activator of Nuclear Factor-kappa B/antagonists & inhibitors , Thrombospondins/biosynthesis , Wnt Signaling Pathway/genetics
6.
J Cell Mol Med ; 17(11): 1465-74, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24251770

ABSTRACT

Interleukin (IL)-12 is the key cytokine in the initiation of a Th1 response and has shown promise as an anti-cancer agent; however, clinical trials involving IL-12 have been unsuccessful due to toxic side-effects. To address this issue, lentiviral vectors were used to transduce tumour cell lines that were injected as an autologous tumour cell vaccine. The focus of the current study was to test the efficacy of this approach in a solid tumour model. SCCVII cells that were transduced to produce IL-12 at different concentrations were then isolated. Subcutaneous injection of parental SCCVII cells results in tumour development, while a mixture of IL-12-producing and non-producing cells results in tumour clearance. Interestingly, when comparing mice injected a mixture of SCCVII and either high IL-12-producing tumour cells or low IL-12-producing tumour cells, we observed that mixtures containing small amounts of high producing cells lead to tumour clearance, whereas mixtures containing large amounts of low producing cells fail to elicit protection, despite the production of equal amounts of total IL-12 in both mixtures. Furthermore, immunizing mice with IL-12-producing cells leads to the establishment of both local and systemic immunity against challenge with SCCVII. Using depletion antibodies, it was shown that both CD4(+) and CD8(+) cells are crucial for therapy. Lastly, we have established cell clones of other solid tumour cell lines (RM-1, LLC1 and moto1.1) that produce IL-12. Our results show that the delivery of IL-12 by cancer cells is an effective route for immune activation.


Subject(s)
Carcinoma, Squamous Cell/therapy , Head and Neck Neoplasms/therapy , Immunotherapy , Interleukin-12/physiology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Carcinoma, Squamous Cell/immunology , Cell Line, Tumor , Female , Head and Neck Neoplasms/immunology , Immunologic Memory , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Neoplasm Transplantation
7.
Trends Endocrinol Metab ; 23(6): 299-309, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22613704

ABSTRACT

Adult stem cells are recruited in response to specific physiological demands to regenerate, repair or maintain essential cellular components of tissues, while preserving self-renewal capacity. Signals that activate adult stem cells are not simply cell autonomous and stem cells are part of a larger dynamic framework, the stem cell 'niche', which integrates systemic and local cues to sustain stem cell functionality. The mammary stem cell niche responds readily to hormonal stimuli, generating pertinent signals that activate stem cells, culminating in stem cell expansion and tissue growth. We review here current knowledge of the mammary stem cell niche with attention to the potent stimulation rendered by ovarian hormones, relevant cellular and molecular players, and the implication of a deregulated niche, for breast cancer risk.


Subject(s)
Gonadal Steroid Hormones/physiology , Mammary Glands, Animal/growth & development , Mammary Glands, Human/growth & development , Stem Cell Niche/physiology , Stem Cells/physiology , Animals , Breast Neoplasms/epidemiology , Cell Differentiation/physiology , Female , Humans , Mammary Glands, Animal/cytology , Mammary Glands, Animal/physiology , Mammary Glands, Human/cytology , Mammary Glands, Human/physiology , Models, Animal , Risk Factors
8.
J Cell Sci ; 125(Pt 4): 943-55, 2012 Feb 15.
Article in English | MEDLINE | ID: mdl-22421365

ABSTRACT

RANKL (receptor activator of NF-κB ligand) is a crucial cytokine for regulating diverse biological systems such as innate immunity, bone homeostasis and mammary gland differentiation, operating through activation of its cognate receptor RANK. In these normal physiological processes, RANKL signals through paracrine and/or heterotypic mechanisms where its expression and function is tightly controlled. Numerous pathologies involve RANKL deregulation, such as bone loss, inflammatory diseases and cancer, and aberrant RANK expression has been reported in bone cancer. Here, we investigated the significance of RANK in tumor cells with a particular emphasis on homotypic signaling. We selected RANK-positive mouse osteosarcoma and RANK-negative preosteoblastic MC3T3-E1 cells and subjected them to loss- and gain-of-RANK function analyses. By examining a spectrum of tumorigenic properties, we demonstrate that RANK homotypic signaling has a negligible effect on cell proliferation, but promotes cell motility and anchorage-independent growth of osteosarcoma cells and preosteoblasts. By contrast, establishment of RANK signaling in non-tumorigenic mammary epithelial NMuMG cells promotes their proliferation and anchorage-independent growth, but not motility. Furthermore, RANK activation initiates multiple signaling pathways beyond its canonical target, NF-κB. Among these, biochemical inhibition reveals that Erk1/2 is dominant and crucial for the promotion of anchorage-independent survival and invasion of osteoblastic cells, as well as the proliferation of mammary epithelial cells. Thus, RANK signaling functionally contributes to key tumorigenic properties through a cell-autonomous homotypic mechanism. These data also identify the likely inherent differences between epithelial and mesenchymal cell responsiveness to RANK activation.


Subject(s)
Cell Movement , Cell Proliferation , Epithelial Cells/pathology , Osteosarcoma/pathology , Receptor Activator of Nuclear Factor-kappa B/metabolism , Signal Transduction , Animals , Autocrine Communication , Cell Line, Tumor , Cell Survival , Cell Transformation, Neoplastic/pathology , Contact Inhibition , Epithelial Cells/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Mammary Glands, Animal/pathology , Mesoderm/cytology , Mesoderm/metabolism , Mice , Neoplasm Invasiveness , Osteoblasts/cytology , Osteoblasts/metabolism , Osteosarcoma/metabolism , Phosphorylation , Proto-Oncogene Proteins c-akt/metabolism , RANK Ligand/metabolism
9.
J Clin Invest ; 120(9): 3310-25, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20697156

ABSTRACT

Some cancers have been stratified into subclasses based on their unique involvement of specific signaling pathways. The mapping of human cancer genomes is revealing a vast number of somatic alterations; however, the identification of clinically relevant molecular tumor subclasses and their respective driver genes presents challenges. This information is key to developing more targeted and personalized cancer therapies. Here, we generate a new mouse model of genomically unstable osteosarcoma (OSA) that phenocopies the human disease. Integrative oncogenomics pinpointed cAMP-dependent protein kinase type I, alpha regulatory subunit (Prkar1a) gene deletions at 11qE1 as a recurrent genetic trait for a molecularly distinct subclass of mouse OSA featuring RANKL overexpression. Using mouse genetics, we established that Prkar1a is a bone tumor suppressor gene capable of directing subclass development and driving RANKL overexpression during OSA tumorigenesis. Finally, we uncovered evidence for a PRKAR1A-low subset of human OSA with distinct clinical behavior. Thus, tumor subclasses develop in mice and can potentially provide information toward the molecular stratification of human cancers.


Subject(s)
Cyclic AMP-Dependent Protein Kinase RIalpha Subunit/genetics , Genes, Tumor Suppressor , Osteosarcoma/genetics , Animals , Disease Models, Animal , Gene Deletion , Mice , Mice, Transgenic , Phenotype
10.
J Clin Invest ; 120(8): 2731-44, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20628198

ABSTRACT

The cell death receptor Fas plays a role in the establishment of fulminant hepatitis, a major cause of drug-induced liver failure. Fas activation elicits extrinsic apoptotic and hepatoprotective signals; however, the mechanisms by which these signals are integrated during disease are unknown. Tissue inhibitor of metalloproteinases 3 (TIMP3) controls the critical sheddase a disintegrin and metalloproteinase 17 (ADAM17) and may dictate stress signaling. Using mice and cells lacking TIMP3, ADAM17, and ADAM17-regulated cell surface molecules, we have found that ADAM17-mediated ectodomain shedding of TNF receptors and EGF family ligands controls activation of multiple signaling cascades in Fas-induced hepatitis. We demonstrated that TNF signaling promoted hepatotoxicity, while excessive TNF receptor 1 (TNFR1) shedding in Timp3-/- mice was protective. Compound Timp3-/-Tnf-/- and Timp3-/-Tnfr1-/- knockout conferred complete resistance to Fas-induced toxicity. Loss of Timp3 enhanced metalloproteinase-dependent EGFR signaling due to increased release of the EGFR ligands TGF-alpha, amphiregulin, and HB-EGF, while depletion of shed amphiregulin resensitized Timp3-/- hepatocytes to apoptosis. Finally, adenoviral delivery of Adam17 prevented acetaminophen-induced liver failure in a clinically relevant model of Fas-dependent fulminant hepatitis. These findings demonstrate that TIMP3 and ADAM17 cooperatively dictate cytokine signaling during death receptor activation and indicate that regulated metalloproteinase activity integrates survival and death signals during acute hepatotoxic stress.


Subject(s)
ADAM Proteins/physiology , Apoptosis , Chemical and Drug Induced Liver Injury/pathology , ErbB Receptors/physiology , Hepatocytes/pathology , Liver Failure, Acute/pathology , Receptors, Tumor Necrosis Factor, Type I/physiology , Tissue Inhibitor of Metalloproteinase-3/physiology , ADAM17 Protein , Animals , Cells, Cultured , Cytoprotection , Extracellular Signal-Regulated MAP Kinases/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Male , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Phosphorylation , Protein Structure, Tertiary , Proto-Oncogene Proteins c-akt/physiology , Signal Transduction , fas Receptor/physiology
11.
Nature ; 465(7299): 803-7, 2010 Jun 10.
Article in English | MEDLINE | ID: mdl-20445538

ABSTRACT

Reproductive history is the strongest risk factor for breast cancer after age, genetics and breast density. Increased breast cancer risk is entwined with a greater number of ovarian hormone-dependent reproductive cycles, yet the basis for this predisposition is unknown. Mammary stem cells (MaSCs) are located within a specialized niche in the basal epithelial compartment that is under local and systemic regulation. The emerging role of MaSCs in cancer initiation warrants the study of ovarian hormones in MaSC homeostasis. Here we show that the MaSC pool increases 14-fold during maximal progesterone levels at the luteal dioestrus phase of the mouse. Stem-cell-enriched CD49fhi cells amplify at dioestrus, or with exogenous progesterone, demonstrating a key role for progesterone in propelling this expansion. In aged mice, CD49fhi cells display stasis upon cessation of the reproductive cycle. Progesterone drives a series of events where luminal cells probably provide Wnt4 and RANKL signals to basal cells which in turn respond by upregulating their cognate receptors, transcriptional targets and cell cycle markers. Our findings uncover a dynamic role for progesterone in activating adult MaSCs within the mammary stem cell niche during the reproductive cycle, where MaSCs are putative targets for cell transformation events leading to breast cancer.


Subject(s)
Aging/physiology , Mammary Glands, Animal/cytology , Progesterone/pharmacology , Stem Cells/cytology , Stem Cells/drug effects , Animals , Cell Count , Cell Division/drug effects , Cell Transformation, Neoplastic , Estrogens/pharmacology , Estrous Cycle/blood , Estrous Cycle/physiology , Female , Homeostasis/drug effects , Integrin alpha6/metabolism , Mice , Ovariectomy , Paracrine Communication/drug effects , Progesterone/blood , Progesterone/metabolism , RANK Ligand/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Stem Cell Niche/cytology , Stem Cell Niche/drug effects , Stem Cell Niche/metabolism , Stem Cells/metabolism , Wnt Proteins/metabolism , Wnt4 Protein
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