The RING finger family of proteins possess ubiquitin ligase activity and play pivotal roles in protein degradation and receptor-mediated endocytosis. In this study, we examined whether the breast cancer-associated gene 2 (BCA2), a novel RING domain protein, has E3 ubiquitin ligase activity and investigated its expression status in breast tumors. The fulllength BCA2 gene was cloned from the human breast cancer cell line MDA-MB-468. It encodes an open reading frame of 304 amino acids and contains a RING-H2 domain. BCA2 maps to chromosome 1q21.1, a region known to harbor cytogenetic aberrations in breast cancers. We found that the BCA2 protein has an intrinsic autoubiquitination activity, the hallmark of E3 ligases, whereas mutant RING protein is not autoubiquitinated. This indicates that the BCA2 ubiquitin ligase activity is dependent on the RING-H2 domain. Using tissue microarrays and immunohistochemistry, we found strong to intermediate BCA2 staining in 56% of 945 invasive breast cancers cases, which was significantly correlated with positive estrogen receptor status [odds ratio (OR), 1.51; P = 0.004], negative lymph node status (OR, 0.73; P = 0.02), and an increase in disease-free survival for regional recurrence (OR, 0.45; P = 0.03). Overexpression of BCA2 increased proliferation and small interfering RNA inhibited growth of T47D human breast cancer cells and NIH3T3 mouse cells. The autoubiquitination activity of BCA2 indicates that it is a novel RING-type E3 ligase. Its association with clinical measures and its effects on cell growth indicate that BCA2 may be important for the ubiquitin modification of proteins crucial to breast carcinogenesis and growth. (Cancer Res 2005; 65(22): 10401-12)

Accumulating evidence suggests that ubiquitination plays a role in cancer by changing the function of key cellular proteins. Previously, we isolated BCA2 gene from a library enriched for breast tumor mRNAs. The BCA2 protein is a RING-type E3 ubiquitin ligase and is overexpressed in human breast tumors. In order to deduce the biochemical and biological function of BCA2, we searched for BCA2-binding partners using human breast and fetal brain cDNA libraries and BacterioMatch two-hybrid system. We identified 62 interacting partners, the majority of which were found to encode ubiquitin precursor proteins including ubiquitin C and ubiquitin A-52. Using several deletion and point mutants, we found that the BCA2 zinc finger (BZF) domain at the NH 2 terminus specifically binds ubiquitin and ubiquitinated proteins. The autoubiquitination activity of BCA2, RING-H2 mutant, BZF mutant, and various lysine mutants of BCA2 were investigated. Our results indicate that the BCA2 protein is strongly ubiquitinated and no ubiquitination is detected with the BCA2 RING-H2 mutant, indicating that the RING domain is essential for autoubiquitination. Mutation of the K26 and K32 lysines in the BZF domain also abrogated autoubiquitination activity. Interestingly, mutation of the K232 and K260 lysines in and near the RING domain resulted in an increase in autoubiquitination activity. Additionally, in cellular migration assays, BCA2 mutants showed altered cell motility compared with wild-type BCA2. On the basis of these findings, we propose that BCA2 might be an important factor regulating breast cancer cell migration/metastasis. We put forward a novel model for BCA2 E3 ligase -mediated cell regulation. (Mol Cancer Res 2008;6(9):1385 -96)

Insulin-like growth factor binding protein 7 (IGFBP7) has been shown to be a tumor suppressor in a variety of cancers. We previously have shown that IGFBP7 expression is inversely correlated with disease progression and poor outcome in breast cancer. Overexpression of IGFBP7 in MDA-MB-468, a triple-negative breast cancer (TNBC) cell line, resulted in inhibition of growth and migration. Xenografted tumors bearing ectopic IGFBP7 expression were significantly growth-impaired compared to IGFBP7-negative controls, which suggested that IGFBP7 treatment could inhibit breast cancer cell growth. To confirm this notion, 14 human patient primary breast tumors were analyzed by qRTPCR for IGFBP7 expression. The TNBC tumors expressed the lowest levels of IGFBP7 expression, which also correlated with higher tumorigenicity in mice. Furthermore, when breast cancer cell lines were treated with IGFBP7, only the TNBC cell lines were growth inhibited. Treatment of NOD/SCID mice harboring xenografts of TNBC cells with IGFBP7 systemically every 3-4 days inhibited tumorigenesis, with associated anti-angiogenic effects, together with increased apoptosis. Upon examining the mechanism of IGFBP7-mediated growth inhibition in TNBC cells, we found that cells not only were arrested in G1 phase of the cell cycle but also underwent senescence as a result of treatment with IGFBP7. Interestingly, IGFBP7 treatment was also associated with strong activation of the stress-associated p38 MAPK pathway, together with upregulation of p53 and the cyclin-dependent protein kinase (CDK) inhibitor, p21(cip1). Prolonged treatment of cells with IGFBP7 resulted in increased cell death, marked by an increase in apoptotic cells and associated cleaved PARP. This is the first study showing that exogenous IGFBP7 inhibits TNBC cell growth both in vitro and in vivo. Taken together, these results suggest IGFBP7 treatment might have therapeutic potential for TNBC.

Defects in ubiquitin E3 ligases are implicated in the pathogenesis of several human diseases, including cancer, because of their central role in the control of diverse signaling pathways. RING E3 ligases promote the ubiquitination of proteins that are essential to a variety of cellular events. Identification of which ubiquitin ligases specifically affect distinct cellular processes is essential to the development of targeted therapeutics for these diseases. Here we discuss two novel RING E3 ligases, BCA2 and RNF11, that are closely linked to human breast cancer. BCA2 E3 ligase is coregulated with estrogen receptor and plays a role in the regulation of epidermal growth factor receptor (EGF-R) trafficking. RNF11 is a small RING E3 ligase that affects transforming growth factorbeta and EGF-R signaling and is overexpressed in invasive breast cancers. These two proteins demonstrate the complexity of RING E3 ligase interactions in breast cancer and are potential targets for therapeutic interventions.

Müllerian adenosarcoma harbors low malignant potential, except in cases with myometrial invasion or sarcomatous overgrowth. The presence of a high-grade stromal component has been proposed as an important pathologic predictor of outcome. We hypothesized that high-grade adenosarcoma has distinct clinical and molecular features, distinct from low-grade adenosarcoma. We analyzed the clinicopathologic features and follow-up of 9 high-grade adenosarcomas and a control group of 9 low-grade adenosarcomas. Comprehensive genomic analysis of the high-grade group was performed targeting exons of 409 oncogenes and tumor suppressor genes. In 1 case, the high-grade and low-grade components were separately sequenced. High-grade and low-grade adenosarcomas were comparable in patient age, myometrial invasion, and stage at presentation. Sarcomatous overgrowth was observed in 2/9 (22%) low-grade and 8/9 (89%) high-grade adenosarcomas. Six of 9 (67%) patients with high-grade adenosarcoma developed rapid recurrence; 1 died of her disease. Conversely, no low-grade tumors recurred or metastasized. Sequencing of high-grade adenosarcomas revealed frequent TP53 pathway alterations, identified in 7/9 (78%) cases. p53 expression by immunohistochemistry highly correlated with mutation status. Copy number variations occurred at a mean of 28.8 per tumor; most frequently involved genes included CDK4, MDM2, GNAS, SGK1, and DICER1. High-grade adenosarcoma is an aggressive neoplasm with propensity for short-interval recurrence and metastasis. The proportion of copy number alterations is similar to that reported for adenosarcoma with sarcomatous overgrowth. However, the high frequency of TP53 abnormalities is a novel finding, indicating that high-grade adenosarcoma is a distinct subset with driver TP53 pathway alterations. p53 immunohistochemistry can be used to confirm the presence of a high-grade component. Given its aggressive potential, the presence of any high-grade component in an adenosarcoma should be reported, even in the absence of sarcomatous overgrowth.

The pattern-based classification system for HPV-related endocervical adenocarcinoma, which classifies tumors based on the destructiveness of stromal invasion, is predictive of the risk of nodal metastases and adverse outcome. Previous studies have demonstrated clinically important molecular alterations in endocervical adenocarcinoma, including KRAS and PIK3CA mutations; however, correlation between the molecular landscape and pathological variables including pattern of invasion has not been thoroughly explored. In this study, 20 endocervical adenocarcinomas were classified using the pattern-based classification system and were subjected to targeted sequencing using the Ion AmpliSeq Cancer Hotspot Panel v2 (ThermoFisher Scientific, Waltham, MA, USA) that surveys hotspot regions of 50 oncogenes and tumor suppressor genes. Single-nucleotide polymorphisms were correlated with clinical and pathologic variables including pattern of invasion. Five (25%), six (30%), and nine (45%) cases were classified as patterns A, B, and C respectively. Lymph node metastases, advanced stage at presentation and mortality from disease were exclusively seen in destructively invasive tumors (patterns B or C). Prevalent mutations in the cohort involved PIK3CA (30%), KRAS (30%), MET (15%), and RB1 (10%). Most (94%) relevant genomic alterations were present in destructively invasive tumors with PIK3CA, KRAS, and RB1 mutations seen exclusively in pattern B or C subgroups. KRAS mutations correlated with advanced stage at presentation (FIGO stage II or higher). Our findings indicate that the pattern of stromal invasion correlates with genomic abnormalities detected by next-generation sequencing, suggesting that tumors without destructive growth (pattern A) are biologically distinct from those with destructive invasion (patterns B and C), and that pattern B endocervical adenocarcinoma is more closely related to its pattern C counterpart. The pattern-based classification may be used as a triage tool when considering molecular testing for prognostic or therapeutic purposes.

Previously, we have shown that insulin-like growth factor binding protein-7 (IGFBP-7) expression is inversely correlated with disease progression in breast cancer and is associated with poor outcome. To further investigate the role of IGFBP-7 in the growth and metastatic behavior of breast cancer, primary breast tumors and metastatic tumors derived from the same patients were analyzed for IGFBP-7 expression. Immunohistochemical analysis revealed that IGFBP-7 is downregulated in half of the human metastatic breast tumors tested. IGFBP-7 has been linked to suppression of oncogenic pathways and can directly restore cellular senescence in melanomas, leading to their regression. It is possible that breast tumors with metastatic potential have escaped from IGFBP-7-induced suppression by its down-regulation. Twenty-two human primary breast tumor specimens were transplanted into human-bone NOD/SCID mice. One of the two triple negative primary breast tumors was serially xenotransplanted more than five times. Each serial transplant resulted in increased tumor take and rate of growth. Expression of IGFBP-7 was downregulated upon each serial implantation. To investigate the role of IGFBP-7 in breast tumor suppression, IGFBP-7 was overexpressed in the triple negative MDA-MB-468 human breast cancer line by stable transfection of a pSec-tag2-IGFBP-7 vector. The parental MDA-MB-468 breast cancer cells expressed extremely low levels of endogenous IGFBP-7. The production of IGFBP-7 protein by the MDA-MB-468 cells stably transfected with IGFBP-7 was confirmed by immunoblotting with anti-IGFBP-7 antibody. Ectopic overexpression of IGFBP-7 significantly reduced the growth of the IGFBP-7 transfected MDA-MB-468 cells compared to the parental MDA-MB-468 cells. We also assessed the role of IGFBP-7 on cell migration, a key determinant of malignant progression and metastasis. When parental MDA-MB-468 cells were treated with various amounts of conditioned medium derived from the IGFBP-7 overexpressing cell line, a significant difference in cell migration rate was observed between untreated and treated cells. IGFBP-7 strongly suppressed the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK-1/2, suggesting that IGFBP-7 mediates its anti-proliferative effects through negative feedback signaling. Levels of phospho-ERK-1/2 were higher in the parental MDA-MB-468 than in IGFBP-7-expressing cells derived from it. When injected subcutaneously into NOD/SCID mice, the increased expression of IGFBP-7 in the MDA-MB-468 transfected cells reduced the rate of tumor growth in comparison to the parental MDA-MB-468 controls. These results suggest that the growth of breast cancer could be prevented by the forced expression of IGFBP-7 protein.

Background Liquid biopsy is promising for early detection, monitoring of response and recurrence of cancer. The blood-brain barrier (BBB) limits the shedding of biomarker, such as cell-free DNA (cfDNA), into the blood, and their detection by conventional assays. Transcranial MR-guided focused ultrasound (MRgFUS) can safely and transiently open the BBB, providing an opportunity for less-invasive access to brain pathology. We hypothesized MRgFUS can enrich the signal of circulating brain-derived biomarkers to aid in liquid biopsy. Methods Nine patients were treated in a prospective single-arm, open-label trial to investigate serial MRgFUS and adjuvant temozolomide combination in patients with glioblastoma (NCT03616860). Blood samples were collected as an exploratory measure within the hours before and after sonication, with control samples from non-brain tumor patients undergoing BBB opening alone (NCT03739905). Results Brain regions averaging 7.8±6.0 cm 3 (range 0.8–23.1 cm 3) were successful treated within 111±39 minutes without any serious adverse events. We found MRgFUS acutely enhanced plasma cfDNA (2.6±1.2 fold, p<0.01, Wilcoxon signed-rank test), neuron-derived extracellular vesicles (3.2±1.9 fold, p<0.01), and brain specific protein S100b (1.4±0.2 fold, p<0.01). Further comparison of the cfDNA methylation profiles suggests a signature that is disease and post-BBB opening specific, in keeping with our hypothesis. We also found cfDNA mutant copies of isocitrate dehydrogenase 1 (IDH1) increased, although this was in only one patient known to harbour the tumor mutation. Conclusions This first-in-human proof-of concept study shows MRgFUS enriches the signal of circulating brain-derived biomarkers, demonstrating the potential of the technology to support liquid biopsy for the brain.