Late-stage breast cancer metastasis is driven by dysregulated TGF-β signaling, but the underlying molecular mechanisms have not been fully elucidated. We attempted to recapitulate tumor and metastatic microenvironments via the use of biomechanically compliant or rigid 3D organotypic cultures and combined them with global microRNA (miR) profiling analyses to identify miRs that were upregulated in metastatic breast cancer cells by TGF-β. Here we establish miR-181a as a TGF-β-regulated "metastamir" that enhanced the metastatic potential of breast cancers by promoting epithelial-mesenchymal transition, migratory, and invasive phenotypes. Mechanistically, inactivation of miR-181a elevated the expression of the proapoptotic molecule Bim, which sensitized metastatic cells to anoikis. Along these lines, miR-181a expression was essential in driving pulmonary micrometastatic outgrowth and enhancing the lethality of late-stage mammary tumors in mice. Finally, miR-181a expression was dramatically and selectively upregulated in metastatic breast tumors, particularly triple-negative breast cancers, and was highly predictive for decreased overall survival in human breast cancer patients. Collectively, our findings strongly implicate miR-181a as a predictive biomarker for breast cancer metastasis and patient survival, and consequently, as a potential therapeutic target in metastatic breast cancer.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is an enduring public health problem globally, particularly in sub-Saharan Africa. Several studies have suggested a role for host genetic susceptibility in increased risk for TB but results across studies have been equivocal. As part of a household contact study of Mtb infection and disease in Kampala, Uganda, we have taken a unique approach to the study of genetic susceptibility to TB, by studying three phenotypes. First, we analyzed culture confirmed TB disease compared to latent Mtb infection (LTBI) or lack of Mtb infection. Second, we analyzed resistance to Mtb infection in the face of continuous exposure, defined by a persistently negative tuberculin skin test (PTST-); this outcome was contrasted to LTBI. Third, we analyzed an intermediate phenotype, tumor necrosis factor-alpha (TNFα) expression in response to soluble Mtb ligands enriched with molecules secreted from Mtb (culture filtrate). We conducted a full microsatellite genome scan, using genotypes generated by the Center for Medical Genetics at Marshfield. Multipoint model-free linkage analysis was conducted using an extension of the Haseman-Elston regression model that includes half sibling pairs, and HIV status was included as a covariate in the model. The analysis included 803 individuals from 193 pedigrees, comprising 258 full sibling pairs and 175 half sibling pairs. Suggestive linkage (p<10−3) was observed on chromosomes 2q21-2q24 and 5p13-5q22 for PTST-, and on chromosome 7p22-7p21 for TB; these findings for PTST- are novel and the chromosome 7 region contains the IL6 gene. In addition, we replicated recent linkage findings on chromosome 20q13 for TB (p = 0.002). We also observed linkage at the nominal α = 0.05 threshold to a number of promising candidate genes, SLC11A1 (PTST- p = 0.02), IL-1 complex (TB p = 0.01), IL12BR2 (TNFα p = 0.006), IL12A (TB p = 0.02) and IFNGR2 (TNFα p = 0.002). These results confirm not only that genetic factors influence the interaction between humans and Mtb but more importantly that they differ according to the outcome of that interaction: exposure but no infection, infection without progression to disease, or progression of infection to disease. Many of the genetic factors for each of these stages are part of the innate immune system.

Circulating exosomes provide a promising approach to assess novel and dynamic biomarkers in human disease, due to their stability, accessibility and representation of molecules from source cells. However, this potential has been stymied by lack of approaches for molecular profiling of individual exosomes, which have a diameter of 30–150 nm. Here we report a rapid analysis approach to evaluate heterogeneous surface protein expression in single circulating exosomes from human blood. Our studies show a differential CD47 expression in blood-derived individual circulating exosomes that is correlated with breast cancer status, demonstrating a great potential of individual exosome profiles in biomarker discovery. The sensitive and high throughput platform of single exosome analysis can also be applied to characterizing exosomes derived from other patient fluids.

PD-L1 expression in the tumor immune microenvironment is recognized as both a prognostic and predictive biomarker in patients with cutaneous melanoma, a finding closely related to its adaptive (IFN-γ-mediated) mechanism of expression. Approximately 35% of cutaneous melanomas express PD-L1, however, the expression patterns, levels, and prevalence in rarer melanoma subtypes are not well described. We performed immunohistochemistry for PD-L1 and CD8 on 200 formalin-fixed paraffin-embedded specimens from patients with acral (n = 16), mucosal (n = 36), uveal (n = 103), and chronic sun-damaged (CSD) (n = 45) melanomas (24 lentigo maligna, 13 ‘mixed’ desmoplastic, and 8 ‘pure’ desmoplastic melanomas). CD8+ tumor-infiltrating lymphocyte (TIL) densities were characterized as mild, moderate, or severe, and their geographic association with PD-L1 expression was evaluated. Discrete lymphoid aggregates, the presence of a spindle cell morphology, and the relationship of these features with PD-L1 expression were assessed. PD-L1 expression was observed in 31% of acral melanomas, 44% of mucosal melanomas, 10% of uveal melanomas, and 62% of CSD melanomas (P<0.0001). Compared to our previously characterized cohort of cutaneous melanomas, the proportion of PD-L1(+) tumors was lower in uveal (P = 0.0002) and higher in CSD (P = 0.0073) melanomas, while PD-L1 expression in the acral and mucosal subtypes was on par. PD-L1 expression in all subtypes correlated with a moderate–severe grade of CD8+ TIL (all, P<0.003), supporting an adaptive mechanism of expression induced during the host antitumor response. The tumor microenvironments observed in CSD melanomas segregated by whether they were the pure desmoplastic subtype, which showed lower levels of PD-L1 expression when compared to other CSD melanomas (P = 0.047). The presence of lymphoid aggregates was not associated with the level of PD-L1 expression, while PD-L1(+) cases with spindle cell morphology demonstrated higher levels of PD-L1 than those with a nested phenotype (P<0.0001). Our findings may underpin the reported clinical response rates for anti-PD-1 monotherapy, which vary by subtype.

Genome-wide platforms for high-throughput profiling of circulating miRNA (oligoarray or miR-Seq) offer enormous promise for agnostic discovery of circulating miRNA biomarkers as a pathway for development in breast cancer detection. By harmonizing data from 15 previous reports, we found widespread inconsistencies across prior studies. Whether this arises from differences in study design, such as sample source or profiling platform, is unclear. As a reproducibility experiment, we generated a genome-wide plasma miRNA dataset using the Illumina oligoarray and compared this to a publically available dataset generated using an identical sample size, substrate and profiling platform. Samples from 20 breast cancer patients, 20 mammography-screened controls, as well as 20 breast cancer patients after surgical resection and 10 female lung or colorectal cancer patients were included. After filtering for miRNAs derived from blood cells, and for low abundance miRNAs (non-detectable in over 10% of samples), a set of 522 plasma miRNAs remained, of which 46 were found to be differentially expressed between breast cancer patients and healthy controls (p<0.05), of which only 3 normalized to baseline levels in post-resection cases and were unique to breast cancer vs. lung or colorectal cancer (miR-708*, miR-92b* and miR-568, none previously reported). We were unable to demonstrate reproducibility by various measures between the two datasets. This finding, along with widespread inconsistencies across prior studies, highlight the need for better understanding of factors influencing circulating miRNA levels as prerequisites to progress in this area of translational research.

The FERM domain containing protein Kindlin-3 has been recognized as a major regulator of integrin function in hematopoietic cells, but its role in neoplasia is totally unknown. We have examined the relationship between Kindlin-3 and breast cancer in mouse models and human tissues. Human breast tumors showed a ∼7-fold elevation in Kindlin-3 mRNA compared with nonneoplastic tissue by quantitative polymerase chain reaction. Kindlin-3 overexpression in a breast cancer cell line increased primary tumor growth and lung metastasis by 2.5- and 3-fold, respectively, when implanted into mice compared with cells expressing vector alone. Mechanistically, the Kindlin-3-overexpressing cells displayed a 2.2-fold increase in vascular endothelial growth factor (VEGF) secretion and enhanced β1 integrin activation. Increased VEGF secretion resulted from enhanced production of Twist, a transcription factor that promotes tumor angiogenesis. Knockdown of Twist diminished VEGF production, and knockdown of β1 integrins diminished Twist and VEGF production by Kindlin-3-overexpressing cells, while nontargeting small interfering RNA had no effect on expression of these gene products. Thus, Kindlin-3 influences breast cancer progression by influencing the crosstalk between β1 integrins and Twist to increase VEGF production. This signaling cascade enhances breast cancer cell invasion and tumor angiogenesis and metastasis.

BACKGROUND: Short duration and poor quality of sleep have been associated with increased risks of obesity, cardiovascular disease, diabetes mellitus, and total mortality. However, few studies have investigated their associations with risk of colorectal neoplasia. METHODS: In a screening colonoscopy-based case-control study, the Pittsburg Sleep Quality Index (PSQI) was administered to 1240 study participants before colonoscopy. RESULTS: Three hundred thirty-eight (27.3%) of the participants were diagnosed with incident colorectal adenomas. Although there was no appreciable difference in the overall PSQI score between cases and adenoma-free controls (5.32 vs 5.11; P ¼ .37), the authors found a statistically significant association of colorectal adenoma with the PSQI component 3, which corresponds to sleep duration (P ¼ .02). Cases were more likely to average less than 6 hours of sleep per night (28.9% vs 22.1% in controls, P ¼ .01). In multivariate regression analysis adjusted for age, gender, race, smoking, family history of colorectal cancer, and waist-to-hip ratio, individuals averaging less than 6 hours per night had an almost 50% increase in risk of colorectal adenomas (OR ¼ 1.47; CI ¼ 1.05-2.06, P for trend ¼ .02) as compared with individuals sleeping at least 7 hours per night. Cases were also more likely to report being diagnosed with sleep apnea (9.8% vs 6.5%, P ¼ .05) and more likely to have worked alternate shifts (54.0% vs 46.1%, P ¼ .01), although these differences were not significant in multivariate models. CONCLUSIONS: Shorter duration of sleep significantly increases risk of colorectal adenomas. The authors' results suggest sleep duration as a novel risk factor for colorectal neoplasia.

Background It is postulated that high serum levels of insulin and insulin growth factor 1 (IGF-1) mediate obesity-associated carcinogenesis. The relationship of insulin, IGF-1 and IGF binding proteins (IGFBP) with Barrett’s oesophagus (BO) has not been well examined. Methods Serum levels of insulin and IGFBPs in patients with BO were compared with two separate control groups: subjects with gastro-oesophageal reflux disease (GORD) and screening colonoscopy controls. Fasting insulin, IGF-1 and IGFBPs were assayed in the serum of BO cases (n = 135), GORD (n = 135) and screening colonoscopy (n = 932) controls recruited prospectively at two academic hospitals. Logistic regression was used to estimate the risk of BO. Results Patients in the highest tertile of serum insulin levels had an increased risk of BO compared with colonoscopy controls (adjusted OR 2.02, 95% CI 1.15 to 3.54) but not compared with GORD controls (adjusted OR 1.55, 95% CI 0.76 to 3.15). Serum IGF-1 levels in the highest tertile were associated with an increased risk of BO (adjusted OR 4.05, 95% CI 2.01 to 8.17) compared with the screening colonoscopy control group but were not significantly different from the GORD control group (adjusted OR 0.57, 95% CI 0.27 to 1.17). IGFBP-1 levels in the highest tertile were inversely associated with a risk of BO in comparison with the screening colonoscopy controls (adjusted OR 0.11, 95% CI 0.05 to 0.24) but were not significantly different from the GORD control group (adjusted OR 1.04, 95% CI 0.49 to 2.16). IGFBP-3 levels in the highest tertile were inversely associated with the risk of BO compared with the GORD controls (OR 0.36, 95% CI 0.16 to 0.81) and also when compared with the colonoscopy controls (OR 0.40, 95% CI 0.20 to 0.79). Conclusions These results provide support for the hypothesis that the insulin/IGF signalling pathways have a role in the development of BO.