Paired primary tumor and lymph node metastases (n = 44; Table S1) for methylation (n = 44) and gene expression analysis (n = 36) were collected at the time of primary surgery and banked at MSKCC. All samples were independently reviewed by a breastcancer pathologist and microdissected to obtain .70% tumor cell content. DNA and RNA were isolated using DNeasy Blood and Tissue and RNeasy Miniprep kits, respectively (QIAGEN). Methylation and gene expression profiling using Illumina Infinium 450 K methylation chip and Affymetrix GeneChip Human Genome U133 2.0 chip, respectively, were performed by the Figure 2. Chromosome characterization of subtype-specific methylation change inmetastasis. A. Chromosome view of smoothed averaged paired differential methylation between metastasis and primary tumors shown for luminal A (purple), luminal B (orange), basal-like (red) and Her2-enriched (green) subtypes along human chromosome 2. CpG islands (CGI) are shown in grey below. B. Methylation profile of a 20 Mb region is shown. Location of RNAseq transcripts for+and – strands is shown above. CGIs, lamin B1-associated domains (LAD), and peaks for H3K4- trimethylated (H3K4me3), H3K4-monomethylated (H3K4me1) and H3K9-acetylated chromatin marks from human mammary endothelial cells (HMEC) are shown (from http://www.genome.ucsc.edu/cgi-bin/hgTables). C. Volcano plots of differentially methylated sites between metastasis and primaries by subtype-specific ANOVA. b-value difference is shown on the x-axis, -log10 of FDR-corrected p-value is on the y-axis. b-values of top three loci from luminal A, luminal B and basal primaries and metastases are shown in figure S1.
Breastcancer is the most frequently diagnosed cancer and the leading cause ofcancer death among females worldwide, with an estimated 1.7 million cases and 521,900 deaths in 2012, It alone accounts for 25% of all cancer cases and 15% of all cancer deaths among females. The incidence rates and mortality rates are stable or decreasing in more developed countries but increasing in less developed countries. The lack of better adjuvant therapy is still the main cause of death in patients with recurrence and metastasisofbreastcancer. Nowadays, the tumor, node, and metastasis (TNM) staging system ofthe American Joint Committee on Can- cer (AJCC) has been broadly recognized, and the plasma cancer antigen 15–3 level has conven- tionally become a simple and clinically useful method for routine surveillance, diagnosis, and the evaluation of prognosis, but the worldwide recognized system or marker for preoperatively predicting the prognosis of patients with breastcancer is uncertain. And with the application of neoadjuvant chemotherapy and endocrine therapy, postoperative clinicopathological parameters often change, which influences the judgment of real prognosis. Therefore, a biologi- cal characteristics that can predict recurrence and metastasis is very important for patients with operable breastcancer, maybe it can develop the treatment plans ofbreastcancer.
mRNA remained low compared to the other cell lines. Re-expression of claudin 1 protein on the surface of treated HCC1569 and MDA‑MB‑453 cells was confirmed by flow cytometry. Similar to what was observed by qRT-PCR analysis, decitabine caused an increase in claudin 1 signal in both cell lines (Figure 3D, empty arrows) and 5 µM azacitidine was again the most effective treatment in HCC1569 as it caused an increase inthe proportion of claudin 1-positive cells compared to the untreated controls (Figure 3D, filled arrow). To confirm that the re-expression of claudin 1 could be ascribed to demethylation of its promoter CpG island, we analyzed claudin 1 methylation by MSP inthe cells treated with 1µM decitabine, as this particular treatment had consistently caused re- expression in all the cell lines tested. MSP analysis shows that the MDA‑MB‑453 has the strongest methylationofthe claudin 1 promoter among these cell lines, and although decitabine was effective, there was considerable residual methylation after treatment (Figure 3E). Conversely, inthe less methylated HCC1569 and BT‑474 cells treatment results in a less pronounced upregulation but in a higher overall expression of claudin 1. Thus, themethylation status ofthe claudin 1 promoter correlates well with both basal expression and response to treatment, which is consistent with epigenetic regulation. As a control, we treated the EMF19 cell line, which is not methylated at the claudin 1 CpG island according to both the microarray and MSP analysis. As expected, neither drug had significant effect on claudin 1 expression in EMF19 cells (Figure 3C, E).
Brain metastasisofbreastcancer is an important clinical problem, with few therapeutic options and a poor prognosis. Recent data have implicated mixed lineage kinase 3 (MLK3) in controlling thein vitro migratory capacity ofbreastcancer cells, as well as themetastasisof MDA-MB-231 breastcancer cells from the mammary fat pad to distant lymph nodes in a mouse xenograft model. We therefore set out to test whether MLK3 plays a role in brain metastasisofbreastcancer cells. To address this question, we used a novel, brain penetrant, MLK3 inhibitor, URMC099. URMC099 efficiently inhibited the migration ofbreastcancer cells in an in vitro cell monolayer wounding assay, and an in vitro transwell migration assay, but had no effect on in vitro cell growth. We also tested the effect of URMC099 on tumor formation in a mouse xenograft model ofbreastcancer brain metastasis. This analysis showed that URMC099 had no effect on the either the frequency or size ofbreastcancer brain metastases. We conclude that pharmacologic inhibition of MLK3 by URMC099 can reduce thein vitro migratory capacity ofbreastcancer cells, but that it has no effect on either the frequency or size ofbreastcancer brain metastases, in a mouse xenograft model.
recent report showing that increased levels of ICAM-1 inbreast tumors are associated with a more aggressive phenotype , and by studies highlighting the importance of vascular cell adhesion molecules inthe establishment ofbreastcancer cells at the secondary site . Other genes encoding adhesion molecules Figure 3. AngII transcriptionally regulates a panel of connected genes. (A). Gene networks differentially regulated by AngII. Up- and down- regulated genes related to angiotensinogen (AGT) are indicated in red and green, respectively. Filled lines indicate direct interactions, filled and dashed arrows indicate direct and indirect regulations, respectively. Note two groups of connected genes centered around MAPK1 and MMP2/9, respectively. (B). RT-PCR analysis of MMP9, MMP2 and MMP3 mRNA expression in MDA-MB-231 cells treated for 24 hrs with increasing doses of AngII as indicated, or LPS (Lipopolysaccharide, 100 ng/ml) as a positive control. GAPDH amplification was used as internal control. Shown is one out of 3 to 5 independent experiments performed in duplicate. (C). Quantification (Image J software) of PCR amplification of MMP9, MMP2 and MMP3 relative to GAPDH and normalized to expression levels in cells treated with 1 nM AngII. (D). Gelatin-based zymography analysis of MMP9 activity in conditioned medium of cells treated as in B. Shown is one representative out of 3 independent experiments (Upper panel). Quantification (ImageJ software) of results normalized to the quantity of proteins in cell lysate and expressed relative to control (lower panel). (E). FACS analysis of ICAM-1 expression at the plasma membrane of MDA-MB-231 cells treated with AngII (100 nM) or vehicle for 24 hrs. Results are means +/2 SEM of 3 independent experiments and expressed as fold-increase ofthe control. **p,0.01.
Axillary staging of patients with early-stage breastcancer is essential inthe treatment planning. Currently such staging is intraoperatively performed, but there is a tendency to seek a preoperative and less invasive technique to detect lymph node metastasis. Ultrasonography is widely utilized for this purpose, many times in association with fine-needle aspiration biopsy or core needle biopsy. However, the sonographic criteria for determining malignancy in axillary lymph nodes do not present significant predictive values, producing discrepant results in studies evaluating the sensitivity and specificity of this method. The present study was aimed at reviewing the literature approaching the utilization of ultrasonography inthe axillary staging as well as the main morphological features of metastatic lymph nodes.
ABSTRACT – Background: Breastcancer is the most common malignant neoplasm inthe female population. However, stomach is a rare site for metastasis, and can show up many years after initial diagnosis and treatment ofthe primary tumor. Aim: Analyze a case series of this tumor and propose measures that can diagnose it with more precocity. Methods: Were analyzed 12 patients with secondary gastric tumors. Immunohistochemistry has demonstrated that primary tumor was breastcancer. We retrieved information of age, histological type, interval between diagnosis ofthe primary breastcancer and its metastases, immunohistochemistry results, treatment and survival. Results: The mean age was 71.3 years (ranging 40-86). Ten cases had already been underwent mastectomy inthe moment ofthe diagnosis of gastric metastasis. Two patients had diagnosis of both primary and secondary tumors concomitantly. At average, diagnosis of gastric metastasis was seven years after diagnosis of primary breastcancer (ranging 0-13). Besides, nine cases had also metastases in other organs, being bones the most affected ones. Immunohistochemistry ofthe metastases has shown positivity for CK7 antibody in 83.34%, estrogen receptor in 91.67%, progesterone receptor in 66.67% and AE1AE3 antibody in 75%, considering all 12 cases. Moreover, CK20 was absent significantly (66.67%). The positivity of BRST2 marker did not present statistical significance (41.67%). Eight cases were treated with chemotherapy associated or not with hormonal blockade. Surgical treatment of gastric metastasis was performed in four cases: three of them with total gastrectomy and one with distal gastrectomy. Follow-up has shown a mean survival of 14.58 months after diagnosis ofmetastasis, with only two patients still alive. Conclusion: Patients with a history ofbreastcancer presenting endoscopic diagnosis of gastric cancer it is necessary to consider the possibility of gastric metastasisofbreastcancer. The confirmation is by immunohistochemistry and gastrectomy should be oriented inthe absence of other secondary involvement and control ofthe primary lesion.
The metastatic pattern of male breastcancer follows that of female breastcancerin that the bones, lungs, and liver are the most common sites. Splenic metastasisofbreastcancer, as shown in this case, is rare inthe literature, and the few cases reported have all been in women. Metastases to the spleen are fairly uncom- mon, can be single or multiple, and often occur inthe context of multi-organ metastatic carcinoma, usually without clinical sig- nificance, splenectomy being palliative in symptomatic patients.
Thebreast tumor was classified as clinical stage IV, with metastasis to the lungs (lymphangitis carcinomatosa identified on CT) and bones, and surgery for thebreast lesion was therefore not indicated. Chemotherapy followed by endocrine therapy was the treatment strategy elected. After a year, thecancer was re- staged. A CT scan ofthe upper abdomen showed parenchymal nodules suggestive of secondary implants inthe spleen, which were also seen on ultrasound (Figure 2A). On the basis of an ultra- sound-guided percutaneous biopsy, the patient was diagnosed with splenic metastasisofbreast carcinoma (Figure 2B), and a new chemotherapy regimen was started exclusively for the splenic pro- gression.
Identification and characterization of crucial gene target(s) that will allow focused therapeutics development remains a challenge. We have interrogated the putative therapeutic targets associated with the transcription factor Grainy head-like 2 (GRHL2), a critical epithelial regulatory factor. We demonstrate the possibility to define the molecular functions of critical genes in terms of their personalized expression profiles, allowing appropriate functional conclusions to be derived. A novel methodology, relative expression analysis with gene-set pairs (RXA-GSP), is designed to explore the potential clinical utility ofcancer-biology discovery. Observing that Grhl2-overexpression leads to increased metastatic potential in vitro, we established a model assuming Grhl2-induced or -inhibited genes confer poor or favorable prognosis respectively for cancermetastasis. Training on public gene expression profiles of 995 breastcancer patients, this method prioritized one gene-set pair (GRHL2, CDH2, FN1, CITED2, MKI67 versus CTNNB1 and CTNNA3) from all 2717 possible gene-set pairs (GSPs). The identified GSP significantly dichotomized 295 independent patients for metastasis-free survival (log-rank tested p = 0.002; severe empirical p = 0.035). It also showed evidence of clinical prognostication in another independent 388 patients collected from three studies (log-rank tested p = 3.3e–6). This GSP is independent of most traditional prognostic indicators, and is only significantly associated with the histological grade ofbreastcancer (p = 0.0017), a GRHL2-associated clinical character (p = 6.8e–6, Spearman correlation), suggesting that this GSP is reflective of GRHL2-mediated events. Furthermore, a literature review indicates the therapeutic potential ofthe identified genes. This research demonstrates a novel strategy to integrate both biological experiments and clinical gene expression profiles for extracting and elucidating the genomic impact of a novel factor, GRHL2, and its associated gene-sets on thebreastcancer prognosis. Importantly, the RXA-GSP method helps to individualize breastcancer treatment. It also has the potential to contribute considerably to basic biological investigation, clinical tools, and potential therapeutic targets.
Tumor spreading occurs mainly by two pathways: through blood vessels and by lymphatic vessels, but the last is preferred by breast tumor cells. Some proteins are involved in cell adhesion and proteolysis, causing metastasis, such as ADAMs, a family of multi-domain and multi-functional proteins that contribute in these processes. ADAM9, a member of this family, has been increased in a large number of human carcinomas, including, breastcancer. In this context, the aim of this study was to evaluate the role of ADAM9 in tumor spread ing via blood and lymphatic systems, inthe search for new targets and focusing the development of new therapeutical tools. Therefore, MDA-MB-231 breast tumor cells were silenced for ADAM9 and tested with respect to their adhesive and invasive activity against blood and lymphatic endothelium. Our results showed that ADAM9 silencing in MDA-MB-231 breastcancer cells inhibited the invasion of this cells in matrigel (71.51 ± 8.02%) when compared to control cells, without affecting cell adhesion, proliferation, migration, and gene expression ofthe ADAM10, ADAM12, ADAM-17, cMyc, MMP9, VEGF-A, VEGF-C, Osteopontin and Collagen XVII, however, there was a decrease inthe expression ofthe ADAM15 and increased expression of MMP2 when compared to controls. Furthermore, ADAM9 silencing did not affect the adhesion under flow to these vascular endothelial cells (HMEC-1 and HUVEC) and lymphatic (HMVEC-dLyNeo-Der). However, there was a decrease inthe rate of trans-endothelial migration through the monolayer endothelial cells (HUVEC, HMEC-1 and HMVEC-dLyNeo-Der) by approximately 50%, 40% and 32%, respectively. In conclusion, ADAM9 showed to be essential in invasion and extravasation of MDA-MB-231 breastcancer cells through the blood and lymphatic vessels in vitro.
Figure 2. Loss of Rb expression promotes metastatic behavior in human breastcancer cell lines. (A) Phase contrast images and quantification of mammosphere-forming potency of MCF7ras and T47D cells with Rb knockdown. Arrows indicate protrusions formed by invading cells and cell clusters. Scale bar, 50 mm; unequal variance Student’s t-test, * p,0.05, ** p,0.01, *** p,0.001. (B) Morphology of primary tumors and lymphovascular (LV) or mammary fat pad (MFP) invasion. MCF7ras cells stably expressing shRNA against Rb or scrambled sequence were injected into the MFP of NOD/SCID mice. Primary tumors and other tissues were harvested after 8 weeks and H&E stained. Bar graph depicts primary tumor weight in each group expressed as mean 6 SD where n is the number of animals in each group. Arrows indicate sites of lymphovascular invasion in Rb knockdown experiment. Scale bar, 50 mm (left panel) and 25 mm (middle panel). (C) Fluorescent and H&E stained images of metastatic tumor growth in lungs. Metastases containing EGFP-expressing Rb knockdown or control cells originated from orthotopic site. Images from n number of animals were quantified using ImageJ software (NIH). Data are presented as mean 6 SD. Scale bar, 5 mm or 100 mm; equal variance Student’s t-test, ** p,0.01, *** p,0.001. (D) Scheme showing isolation of circulating cancer cells (CCC) from mice bearing primary tumors. CCC were co-separated with the mononuclear fraction of blood and adhered to Poly-L-lysine-coated plates. (E) Fluorescent images and quantification of isolated EGFP- expressing circulating cancer cells and cell clusters. Scale bar, 200 mm; unequal variance Student’s t-test, * p,0.05, ** p,0.01. (F) Western blot analysis of cell lysates from in vitro cultures of MCF7ras cells expressing Rb or control shRNA. b-tubulin staining was used as a loading control. (G) Western blot analysis of lysates from primary tumors. Total Akt was used as a loading control.
Histopathological examination ofthe tumor revealed an undifferentiated carcinoma (T3N0Mx). The lack of dysplasia and atypia ofthe intestinal epithelium and the normal glands surrounding the malignant cells favored diagnosis of a metastatic lesion. Immunohistochemistry (IHC) performed on the specimen was positive for cytokeratin 7 (CK7) and CAM 5.2 (Figure 2) and negative for ER, PgR, and HER2 receptors, cytokeratin-20 (CK20), and gross cystic disease fluid protein-15 (GCDFP-15). Histologic slides revision was performed in other institutions with agreeing results: IHC was negative for mammaglobin and GCDFP-15. GATA3 expression is not performed in both institutions. Notwith- standing this, IHC along with morphological aspects ofthe tumor were consistent with breastcancer metastases. Based on these findings and the clinical history, we concluded this was a jejunal metastasis from breast carcinoma.
expression of CDKN2A and MTAP; however. the MTAP promoter was partially methylated. Bisogna et al.  also described a deletion of CDKN2A in this cell line but not a deletion of CDKN2B or INK4A genes, which are closely located on the chromosome. Perhaps this is not a case of co-deletion, and MTAP is not expressed in MCF-7 cells due to DNA methylation. T47-D cells show strong CDKN2A mRNA expression but no evidence of protein expression by Western blotting. Bisogna et al.  observed DNA methylationofthe CDKN2A promoter in this cell line, which could explain the absence of protein in our study. However, the presence of mRNA suggests post-transcriptional regulation, for example, via RNA interference. Kim et al.  analyzed a set of gastric cancer cell lines and found a correlation between mRNA down-reg- ulation and homozygous deletion of MTAP and CKN2A because 8 of 10 cell lines expressed both genes. However, the proteins were absent in two out of ten cell lines with a homozygous deletion. We observed no difference in mRNA MTAP expression in respect to their ER, PR and HER2 expressionamong the cell lines and these data partially constrast with the fact that in FFEE triple negative samples did express higher MTAp mRNA levels. The low number of cell lines considered can be at the basis of this discrepancy.
The purpose of this study was to develop quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the analysis of proteins involved inmetastasisofbreastcancer for diagnosis and determining disease prognosis, as well as to further our understand of metastatic mechanisms. We have previously demonstrated that the protein type XIV collagen may be specifically expressed in metastatic tissues by two dimensional LC-MS/MS. In this study, we developed quantitative LC-MS/MS methods for type XIV colla- gen. Type XIV collagen was quantified by analyzing 2 peptides generated by digesting type XIV collagen using stable isotope-labeled peptides. The individual concentrations were equivalent between 2 different peptides of type XIV collagen by evaluation of imprecise tran- sitions and using the best transition for the peptide concentration. The results indicated that type XIV collagen is highly expressed in metastatic tissues of patients with massive lymph node involvement compared to non-metastatic tissues. These findings were validated by quantitative real-time RT-PCR. Further studies on type XIV collagen are desired to verify its role as a prognostic factor and diagnosis marker for metastasis.
The function of Rrp1b and its exact role inmetastasis remain unclear at this time. Sipa1 was originally cloned as a mitogen-inducible protein  that was subsequently shown to be a negative regulator of Rap1 by serving as a GAP for Rap1 . Sipa1 has signiﬁcant effects on cellular adhesion , primarily related to its effects on Rap1, which has been implicated in maintaining the integrity of polarized epithelia  and intercellular adherens junctions , and potentially integrating signaling between cadherins and integrins . Rrp1b may therefore mediate tumor cell adhesion properties by altering intercellular and cell–ECM contacts in a Sipa1- dependent and Rap1-dependent manner. It should be noted, however, that the human polymorphism in RRP1B falls outside ofthe domain that directly interacts with the PDZ domain of Sipa1. Whether this polymorphism impacts the enzymatic function of Sipa1 or mediates metastatic potential through some other mechanism is unclear and currently under investigation. Similarly, it is unclear whether the amino acid substitution in human RRP1B directly affects function. Based on the mouse model, where increased expression confers protection against malignant progression, the variant leucine inthe human ortholog may phenocopy the mouse situation by activating some function of RRP1B. Further in vitro analysis will be required to clarify the different situations inthe two species and is currently under inves- tigation in our laboratory.
ening overt metastasis remains largely unknown, in large part due to the lack of appropriate animal models that closely reca- pitulate the process. We developed a novel mouse model to mimic the activation of indolent micrometastases to aggressive lesions. In this model, the single cell progeny clone number 6 (SCP6) single-cell derived clone ofthe MDA-MB-231 breastcancer cell line was known to have no basal bone metastatic ability after intracardiac injection into nude mice recipients . Interestingly, a small number of mice eventually devel- oped osteolytic bone lesions after more than 6 months of ap- parent bone metastasis free survival, suggesting that certain ge- netic/epigenetic changes occurs inthe DTCs that endowed them with bone metastatic ability. Careful gene expression pro- filing analysis and functional studies led to the identification and validation of vascular cell adhesion molecule 1 (VCAM1) as a crucial functional driver of conversion from indolent mi- crometastases to overt osteolytic bone metastasis . Mecha- nistically, we determined that tumor-derived soluble VCAM1 serves as a chemoattractant to recruit circulating monocytic precursors of osteoclasts. By interacting with its cognate recep- tor, α4β1 integrin, VCAM1 also promotes the adhesion of pre- osteoclasts to the surface of tumor cells, leading to cell fusion and differentiation of pre-osteoclasts and initiation of bone de- struction (Fig. 1).
fat pad xenograft, breastcancer development has shown a low success rate in most trials due to the technically limited occurrence of mammary glands in neonatal mice. Also, an intra-tibia inoculation ofbreastcancer is frequently applied as a cancer-bone metastasis model. In this model, cancer cells are inserted extra-capsulary through the tibial crest, epiphysis, and growth plate and then cells are sequentially injected into the bone marrow space. The intra-tibia model does not precisely relect the characteristics of bone-invasive OSCC 8,13 . Thus,
Recently, with the advent of high-throughput technolo- gies, gene expression profiling has enabled a more com- prehensive view ofthe molecular identity ofbreastcancer. Five major molecular and outcome related BC subtypes, known as PAM50 subtypes, were identified based on genome-wide expression analyses: Luminal-A, Luminal-B, HER-2, Normal-like and Basal-like [2, 6–8]. Breastcancer classification based on PAM50 subtypes and risk of recur- rence (ROR) score have shown to significantly contribute to prognostic assessment and to facilitate more precise therapeutic decisions . Other genomic tests, such as Mammaprint (Agendia, Huntington Beach, CA) and Oncotype DX (Genomic Health, Redwood City, CA) may also be used to provide prognostic and/or predictive infor- mation in early-stage breastcancer beyond the standard clinicopathological assessment and to determine the likeli- hood of benefit from adjuvant chemotherapy [5, 10]. Tai- loring treatment to individual tumor subtypes has the potential to greatly improve breastcancer management and survival [11, 12].
Previous studies have identified gene expression signatures that define breastcancermetastasis . Microarray and RNA-seq performed in this report provide a comprehensive view of changes in gene expression elicited by over-expression of WNT5A in metastatic breastcancer cells. We identified several potential targets of WNT5A that are associated with cell migration including chemokines, Cxcl1 and Il1a, ECM associated proteins, Mmp13 and Lamb3, as well as an inflammation responsive enzyme, Nos2. All of these genes were down-regulated in WNT5A expressing cells and have known roles in tumor progression. Cxcl1has an important role in promoting breastcancermetastasis and, recently, was shown to link metastasis to drug resistance . Polymorphisms in Il1a are associated with increased breastcancer risk . Many studies have implicated Mmps in promoting metastasis. Recently, a selective inhibitor of Mmp13 was shown to delay the onset of tumor associated osteolytic lesions in a model of bone metastasis; however, no effects on soft organ metastasis were observed . Lamb3 is the beta chain for laminin 5, which has been shown to promote migration ofbreastcancer cells . Increased inducible Nos2 was associated with poor survival in estrogen receptor-negative breastcancer patients . Down- regulation of any of these genes by WNT5A would be expected to contribute to inhibition of tumor progression. The list of differentially expressed genes provides information to guide future mechanistic studies aimed at determining how WNT5A affects tumor progression and metastasis.