The mutational landscape of the adult healthy parous and nulliparous human breast.

The accumulation of somatic mutations in healthy human tissues has been extensively characterized, but the mutational landscape of the healthy breast is still poorly understood. Our analysis of whole-genome sequencing shows that in line with other healthy organs, the healthy breast during the reproduction years accumulates mutations with age, with the rate of accumulation in the epithelium of 15.24 ± 5 mutations/year. Both epithelial and stromal compartments contain mutations in breast-specific driver genes, indicative of subsequent positive selection. Parity- and age-associated differences are evident in the mammary epithelium, partly explaining the observed difference in breast cancer risk amongst women of different childbearing age. Parity is associated with an age-dependent increase in the clone size of mutated epithelial cells, suggesting that older first-time mothers have a higher probability of accumulating oncogenic events in the epithelium compared to younger mothers or nulliparous women. In conclusion, we describe the reference genome of the healthy female human breast during reproductive years and provide evidence of how parity affects the genomic landscape of the mammary gland.

Molecular profiling of advanced breast cancer tumors is beneficial in assisting clinical treatment plans.

We used data obtained by Caris Life Sciences, to evaluate the benefits of tailoring treatments for a breast carcinoma cohort by using tumor molecular profiles to inform decisions. Data for 92 breast cancer patients from the commercial Caris Molecular Intelligence database was retrospectively divided into two groups, so that the first always followed treatment recommendations, whereas in the second group all patients received at least one drug after profiling that was predicted to lack benefit. The biomarker and drug associations were based on tests including fluorescent in situ hybridization and DNA sequencing, although immunohistochemistry was the main test used. Patients whose drugs matched those recommended according to their tumor profile had an average overall survival of 667 days, compared to 510 days for patients that did not (P=0.0316). In the matched treatment group, 26% of patients were deceased by the last time of monitoring, whereas this was 41% in the unmatched group (P=0.1257). We therefore confirm the ability of tumor molecular profiling to improve survival of breast cancer patients. Immunohistochemistry biomarkers for the androgen, estrogen and progesterone receptors were found to be prognostic for survival.

The benefit of tumor molecular profiling on predicting treatments for colorectal adenocarcinomas.

We evaluated the benefit of tailoring treatments for a colorectal adenocarcinoma cancer cohort according to tumor molecular profiles, by analyzing data collected on patient responses to treatments that were guided by a tumor profiling technology from Caris Life Sciences. DNA sequencing and immunohistochemistry were the main tests that predictions were based upon, but also fragment analysis, and in situ hybridization. The status of the IHC biomarker for the thymidylate synthase receptor was a good indicator for future survival. Data collected for the clinical treatments of 95 colorectal adenocarcinoma patients was retrospectively divided into two groups: the first group was given drugs that always matched recommended treatments as suggested by the tumor molecular profiling service; the second group received at least one drug after profiling that was predicted to lack benefit. In the matched treatment group, 19% of patients were deceased at the end of monitoring compared to 49% in the unmatched group, indicating a benefit in mortality by tumor molecular profiling colorectal adenocarcinoma patients.

Correction: Assessing tumor molecular profiling to guide treatments for patients with advanced female genital tract malignancy.

[This corrects the article DOI: 10.18632/oncotarget.23675.].

Correction: The benefit of tumor molecular profiling on predicting treatments for colorectal adenocarcinomas.

[This corrects the article DOI: 10.18632/oncotarget.24257.].

Correction: Does molecular profiling of tumors using the Caris molecular intelligence platform improve outcomes for cancer patients?

[This corrects the article DOI: 10.18632/oncotarget.24258.].

Assessing tumor molecular profiling to guide treatments for patients with advanced female genital tract malignancy.

Tumor molecular profiling has enabled selection of targeted therapies in a host of solid tumors. Here we used a retrospective clinical cohort, to evaluate the benefit of tailoring treatments for female genital tract malignancy, using tumor molecular profiles. Clinical outcome data for 112 patients was retrospectively separated into two groups. These either followed a matched treatment plan that incorporated at least one drug recommended according to their tumor profile and none that were expected to have no benefit (64 patients), or was unmatched with suggested treatments and received at least one drug that was anticipated to lack benefit for that tumor (48 patients). In the group of patients whose drugs matched those recommended by molecular profiling of their tumor, their overall survival was 593 days on average, compared to 449 days for patients that did not; removing drugs predicted to have no benefit from treatment regimens received after profiling increased survival by 144 days on average (P = 0.0265). In the matched treatment group, 30% of patients had died by the last time of monitoring, whereas this was 40% in the unmatched group (P = 0.2778). The IHC biomarker for the progesterone receptor was demonstrated to be prognostic for survival.

The Typhoid Toxin Promotes Host Survival and the Establishment of a Persistent Asymptomatic Infection.

Bacterial genotoxins, produced by several Gram-negative bacteria, induce DNA damage in the target cells. While the responses induced in the host cells have been extensively studied in vitro, the role of these effectors during the course of infection remains poorly characterized. To address this issue, we assessed the effects of the Salmonella enterica genotoxin, known as typhoid toxin, in in vivo models of murine infection. Immunocompetent mice were infected with isogenic S. enterica, serovar Typhimurium (S. Typhimurium) strains, encoding either a functional or an inactive typhoid toxin. The presence of the genotoxic subunit was detected 10 days post-infection in the liver of infected mice. Unexpectedly, its expression promoted the survival of the host, and was associated with a significant reduction of severe enteritis in the early phases of infection. Immunohistochemical and transcriptomic analysis confirmed the toxin-mediated suppression of the intestinal inflammatory response. The presence of a functional typhoid toxin further induced an increased frequency of asymptomatic carriers. Our data indicate that the typhoid toxin DNA damaging activity increases host survival and favours long-term colonization, highlighting a complex cross-talk between infection, DNA damage response and host immune response. These findings may contribute to understand why such effectors have been evolutionary conserved and horizontally transferred among Gram-negative bacteria.

Local inhibition of elastase reduces EMILIN1 cleavage reactivating lymphatic vessel function in a mouse lymphoedema model.

Lymphatic vasculature critically depends on the connections of lymphatic endothelial cells with the extracellular matrix (ECM), which are mediated by anchoring filaments (AFs). The ECM protein EMILIN1 is a component of AFs and is involved in the regulation of lymphatic vessel functions: accordingly, Emilin1(-/-) mice display lymphatic vascular morphological alterations, leading to functional defects such as mild lymphoedema, lymph leakage and compromised lymph drainage. In the present study, using a mouse post-surgical tail lymphoedema model, we show that the acute phase of acquired lymphoedema correlates with EMILIN1 degradation due to neutrophil elastase (NE) released by infiltrating neutrophils. As a consequence, the intercellular junctions of lymphatic endothelial cells are weakened and drainage to regional lymph nodes is severely affected. The local administration of sivelestat, a specific NE inhibitor, prevents EMILIN1 degradation and reduces lymphoedema, restoring a normal lymphatic functionality. The finding that, in human secondary lymphoedema samples, we also detected cleaved EMILIN1 with the typical bands of an NE-dependent pattern of fragmentation establishes a rationale for a powerful strategy that targets NE inhibition. In conclusion, the attempts to block EMILIN1 degradation locally represent the basis for a novel 'ECM' pharmacological approach to assessing new lymphoedema treatments.

Neutrophil elastase-dependent cleavage compromises the tumor suppressor role of EMILIN1.

Proteolysis of the extracellular matrix (ECM) is a key event in tumor growth and progression. The breakdown of ECM can lead to the generation of bioactive fragments that promote cell growth and spread. EMILIN1, a multidomain glycoprotein expressed in several tissues, exerts a crucial regulatory function through the engagement of α4/α9 integrins. Unlike the majority of ECM molecules that elicit a proliferative program, the signals emitting from EMILIN1 engaged by α4/α9ß1 integrins are antiproliferative. In this study, aimed to demonstrate if the suppressor role of EMILIN1 was related to its structural integrity, we tested the possibility that EMILIN1 could be specifically cleaved. Among the proteolytic enzymes released in the tumor microenvironment we showed that neutrophil elastase cleaved EMILIN1 in three/four major fragments. The consequence of this proteolytic process was the impairment of its anti-proliferative role. Accordingly, EMILIN1 was digested in sarcomas and ovarian cancers. Sarcoma specimens were infiltrated by neutrophils (PMNs) and stained positively for elastase. The present findings highlight the peculiar activity of PMN elastase in disabling EMILIN1 suppressor function.

Evolutionarily conserved heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins functionally interact with human and Drosophila TAR DNA-binding protein 43 (TDP-43).

Human TDP-43 represents the main component of neuronal inclusions found in patients with neurodegenerative diseases, especially frontotemporal lobar degeneration and amyotrophic lateral sclerosis. In vitro and in vivo studies have shown that the TAR DNA-binding protein 43 (TDP-43) Drosophila ortholog (TBPH) can biochemically and functionally overlap the properties of the human factor. The recent direct implication of the human heterogeneous nuclear ribonucleoproteins (hnRNPs) A2B1 and A1, known TDP-43 partners, in the pathogenesis of multisystem proteinopathy and amyotrophic lateral sclerosis supports the hypothesis that the physical and functional interplay between TDP-43 and hnRNP A/B orthologs might play a crucial role in the pathogenesis of neurodegenerative diseases. To test this hypothesis and further validate the fly system as a useful model to study this type of diseases, we have now characterized human TDP-43 and Drosophila TBPH similarity in terms of protein-protein interaction pathways. In this work we show that TDP-43 and TBPH share the ability to associate in vitro with Hrp38/Hrb98DE/CG9983, the fruit fly ortholog of the human hnRNP A1/A2 factors. Interestingly, the protein regions of TDP-43 and Hrp38 responsible for reciprocal interactions are conserved through evolution. Functionally, experiments in HeLa cells demonstrate that TDP-43 is necessary for the inhibitory activity of Hrp38 on splicing. Finally, Drosophila in vivo studies show that Hrp38 deficiency produces locomotive defects and life span shortening in TDP-43 with and without animals. These results suggest that hnRNP protein levels can play a modulatory role on TDP-43 functions.

EMILIN1/α9ß1 integrin interaction is crucial in lymphatic valve formation and maintenance.

Lymphatic vasculature plays a crucial role in the maintenance of tissue interstitial fluid balance. The role of functional collecting lymphatic vessels in lymph transport has been recently highlighted in pathologies leading to lymphedema, for which treatments are currently unavailable. Intraluminal valves are of paramount importance in this process. However, valve formation and maturation have not been entirely elucidated yet, in particular, the role played by the extracellular matrix (ECM). We hypothesized that EMILIN1, an ECM multidomain glycoprotein, regulates lymphatic valve formation and maintenance. Using a mouse knockout model, we show that in the absence of EMILIN1, mice exhibit defects in lymphatic valve structure and in lymph flow. By applying morphometric in vitro and in vivo functional assays, we conclude that this impaired phenotype depends on the lack of α9ß1 integrin engagement, the specific lymphatic endothelial cell receptor for EMILIN1, and the ensuing derangement of cell proliferation and migration. Our data demonstrate a fundamental role for EMILIN1-integrin α9 interaction in lymphatic vasculature, especially in lymphatic valve formation and maintenance, and underline the importance of this ECM component in displaying a regulatory function in proliferation and acting as a "guiding" molecule in migration of lymphatic endothelial cells.

An EMILIN1-negative microenvironment promotes tumor cell proliferation and lymph node invasion.

The evidence that EMILIN1 (Elastic Microfibril Interface Located proteIN) deficiency in Emilin1(-/-) mice caused dermal and epidermal hyperproliferation and an abnormal lymphatic phenotype prompted us to hypothesize the involvement of this extracellular matrix component in tumor development and in lymphatic metastasis. Using the 12-dimethylbenz(α)anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) two-stage model of skin carcinogenesis, we found that Emilin1(-/-) mice presented an accelerated formation, a higher incidence, and the development of a larger number of tumors compared with their wild-type littermates. EMILIN1-negative tumors showed more Ki67-positive proliferating cells and higher levels of pErk1/2. In these tumors, PTEN expression was lower. Emilin1(-/-) mice displayed enhanced lymphangiogenesis both in the tumor and in the sentinel lymph nodes. Accordingly, tumor growth and lymph node metastasis of transplanted syngenic tumors were also increased in Emilin1(-/-) mice. In vitro transmigration assays through lymphatic endothelial cells showed that EMILIN1 deficiency greatly facilitated tumor cell trafficking. Overall, these data established that EMILIN1 exerts a protective role in tumor growth, in tumor lymphatic vessel formation, as well as in metastatic spread to lymph nodes and reinforced the importance of its presence in the microenvironment to determine the tumor phenotype.

EMILIN1-α4/α9 integrin interaction inhibits dermal fibroblast and keratinocyte proliferation.

EMILIN1 promotes α4ß1 integrin-dependent cell adhesion and migration and reduces pro-transforming growth factor-ß processing. A knockout mouse model was used to unravel EMILIN1 functions in skin where the protein was abundantly expressed in the dermal stroma and where EMILIN1-positive fibrils reached the basal keratinocyte layer. Loss of EMILIN1 caused dermal and epidermal hyperproliferation and accelerated wound closure. We identified the direct engagement of EMILIN1 to α4ß1 and α9ß1 integrins as the mechanism underlying the homeostatic role exerted by EMILIN1. The lack of EMILIN1-α4/α9 integrin interaction was accompanied by activation of PI3K/Akt and Erk1/2 pathways as a result of the reduction of PTEN. The down-regulation of PTEN empowered Erk1/2 phosphorylation that in turn inhibited Smad2 signaling by phosphorylation of residues Ser245/250/255. These results highlight the important regulatory role of an extracellular matrix component in skin proliferation. In addition, EMILIN1 is identified as a novel ligand for keratinocyte α9ß1 integrin, suggesting prospective roles for this receptor-ligand pair in skin homeostasis.