ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Triple negative breast cancer (TNBC) has poor survival, exhibits rapid metastases, lacks targeted therapies and reliable prognostic markers. Here, we examined metastasis promoting role of cancer testis antigen SPANXB1 in TNBC and its utility as a therapeutic target and prognostic biomarker. Expression pattern of SPANXB1 was determined using matched primary cancer, lymph node metastatic tissues and circulating small extracellular vesicles (sEVs). cDNA microarray analysis of TNBC cells stably integrated with a metastasis suppressor SH3GL2 identified SPANXB1 as a potential target gene. TNBC cells overexpressing SH3GL2 exhibited decreased levels of both SPANXB1 mRNA and protein. Silencing of SPANXB1 reduced migration, invasion and reactive oxygen species production of TNBC cells. SPANXB1 depletion augmented SH3GL2 expression and decreased RAC-1, FAK, A-Actinin and Vinculin expression. Phenotypic and molecular changes were reversed upon SPANXB1 re-expression. SPANXB1 overexpressing breast cancer cells with an enhanced SPANXB1:SH3GL2 ratio achieved pulmonary metastasis within 5 weeks, whereas controls cells failed to do so. Altered expression of SPANXB1 was detected in the sEVs of SPANXB1 transduced cells. Exclusive expression of SPANXB1 was traceable in circulating sEVs, which was associated with TNBC progression. SPANXB1 represents a novel and ideal therapeutic target for blocking TNBC metastases due to its unique expression pattern and may function as an EV based prognostic marker to improve TNBC survival. Uniquely restricted expression of SPANXB1 in TNBCs, makes it an ideal candidate for targeted therapeutics and prognostication.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Biomarkers, Tumor/metabolism , Liver Neoplasms/diagnosis , Lung Neoplasms/diagnosis , Nuclear Proteins/metabolism , Triple Negative Breast Neoplasms/pathology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Breast/pathology , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cell-Derived Microparticles/metabolism , Disease Progression , Female , Gene Expression Profiling , Gene Knockdown Techniques , Humans , Kaplan-Meier Estimate , Liver/pathology , Liver Neoplasms/blood , Liver Neoplasms/prevention & control , Liver Neoplasms/secondary , Lung/pathology , Lung Neoplasms/blood , Lung Neoplasms/prevention & control , Lung Neoplasms/secondary , Mice , Neoplasm Invasiveness/pathology , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/blood , Nuclear Proteins/genetics , Oligonucleotide Array Sequence Analysis , Prognosis , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Triple Negative Breast Neoplasms/blood , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/mortality , Xenograft Model Antitumor AssaysABSTRACT
Human papilloma virus-16 (HPV-16) associated oropharyngeal cancer (HPVOPC) is increasing alarmingly in the United States. We performed whole genome sequencing of a 44 year old, male HPVOPC subject diagnosed with moderately differentiated tonsillar carcinoma. We identified new somatic mutation in MUC16 (A.k.a. CA-125), MUC12, MUC4, MUC6, MUC2, SIRPA, HLA-DRB1, HLA-A and HLA-B molecules. Increased protein expression of MUC16, SIRPA and decreased expression of HLA-DRB1 was further demonstrated in this HPVOPC subject and an additional set of 15 HPVOPC cases. Copy number gain (3 copies) was also observed for MUC2, MUC4, MUC6 and SIRPA. Enhanced expression of MUC16, SIRPA and HPV-16-E7 protein was detectable in the circulating exosomes of numerous HPVOPC subjects. Treatment of non-tumorigenic mammary epithelial cells with exosomes derived from aggressive HPVOPC cells harboring MUC16, SIRPA and HPV-16-E7 proteins augmented invasion and induced epithelial to mesenchymal transition (EMT) accompanied by an increased expression ratio of the EMT markers Vimentin/E-cadherin. Exosome based screening of key HPVOPC associated molecules could be beneficial for early cancer diagnosis, monitoring and surveillance.
Subject(s)
Exosomes/metabolism , Mutation/genetics , Oropharyngeal Neoplasms/virology , Papillomaviridae/genetics , Adult , DNA Copy Number Variations/genetics , Epithelial-Mesenchymal Transition , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Genome, Human , Human papillomavirus 16/genetics , Humans , Male , Neoplasm Invasiveness , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Oropharyngeal Neoplasms/pathology , Reproducibility of ResultsABSTRACT
PURPOSE: The goal of this study was to understand the role of altered mitochondrial function in breast cancer progression and determine the potential of the molecular alteration signature in developing exosome-based biomarkers. EXPERIMENTAL DESIGN: This study was designed to characterize the critical components regulating mitochondrial function in breast tumorigenesis. Experiments were conducted to assess the potential of these molecules for exosome-based biomarker development. RESULTS: We observed a remarkable reduction in spontaneous metastases through the interplay in mitochondria by SH3GL2, vesicular endocytosis-associated protein and MFN2, an important regulator of mitochondrial fusion. Following its overexpression in breast cancer cells, SH3GL2 translocated to mitochondria and induced the production of superoxide and release of cytochrome C from mitochondria to the cytoplasm. These molecular changes were accompanied by decreased lung and liver metastases and primary tumor growth. SH3GL2 depletion reversed the above phenotypic and associated molecular changes in nontumorigenic and tumorigenic breast epithelial cells. Loss of SH3GL2 and MFN2 expression was evident in primary human breast cancer tissues and their positive lymph nodes, which was associated with disease progression. SH3GL2 and MFN2 expression was detected in sera exosomes of normal healthy women, but barely detectable in the majority of the women with breast cancer exhibiting SH3GL2 and MFN2 loss in their primary tumors. CONCLUSIONS: This study identified a new mitochondria reprogramming pathway influencing breast cancer progression through SH3GL2 and MFN2. These proteins were frequently lost in breast cancer, which was traceable in the circulating exosomes. Clin Cancer Res; 22(13); 3348-60. ©2016 AACR.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Breast Neoplasms/pathology , GTP Phosphohydrolases/metabolism , Mitochondrial Proteins/metabolism , Tumor Suppressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Apoptosis/genetics , Breast Neoplasms/diagnosis , Cell Line, Tumor , Cell Proliferation , Cytochromes c/metabolism , Disease Progression , Exosomes/metabolism , Female , GTP Phosphohydrolases/genetics , Humans , Liver Neoplasms/secondary , Lung Neoplasms/secondary , MCF-7 Cells , Mitochondria/metabolism , Mitochondrial Proteins/genetics , Superoxides/metabolism , Tumor Suppressor Proteins/geneticsABSTRACT
Little is known about the molecular pathways regulating poor differentiation and invasion of head and neck squamous cell carcinoma (HNSCC). In the present study, we aimed to determine the role of MDA-9/Syntenin, a metastasis associated molecule in HNSCC tumorigenesis. Elevated MDA-9/Syntenin expression was evident in 67% (54/81) primary HNSCC tumors (p=0.001-0.002) and 69% (9/13) pre-neoplastic tissues (p=0.02-0.03). MDA-9/Syntenin overexpression was associated with the stage (p=0.001), grade (p=0.001) and lymph node metastasis (p=0.0001). Silencing of MDA-9/Syntenin in 3 poorly differentiated HNSCC cell lines induced squamous epithelial cell differentiation, disrupted angiogenesis and reduced tumor growth in vitro and in vivo. We confirmed SPRR1B and VEGFR1 as the key molecular targets of MDA-9/Syntenin on influencing HNSCC differentiation and angiogenesis respectively. MDA-9/Syntenin disrupted SPRR1B expression interacting through its PDZ1 domain and altered VEGFR1 expression in vitro and in vivo. VEGFR1 co-localized with MDA-9/Syntenin in HNSCC cell lines and primary tumor. Downregulation of growth regulatory molecules CyclinD1, CDK4, STAT3, PI3K and CTNNB1 was also evident in the MDA-9/Syntenin depleted cells, which was reversed following over-expression of MDA-9/Syntenin in immortalized oral epithelial cells. Our results suggest that early induction of MDA-9/Syntenin expression influences HNSCC progression and should be further evaluated for potential biomarker development.