Mast cells impair melanoma cell homing and metastasis by inhibiting HMGA1 secretion.

Metastatic disease is the major cause of death from cancer. From the primary tumour, cells remotely prepare the environment of the future metastatic sites by secreted factors and extracellular vesicles. During this process, known as pre-metastatic niche formation, immune cells play a crucial role. Mast cells are haematopoietic bone marrow-derived innate immune cells whose function in lung immune response to invading tumours remains to be defined. We found reduced melanoma lung metastasis in mast cell-deficient mouse models (Wsh and MCTP5-Cre-RDTR), supporting a pro-metastatic role for mast cells in vivo. However, due to evidence pointing to their antitumorigenic role, we studied the impact of mast cells in melanoma cell function in vitro. Surprisingly, in vitro co-culture of bone-marrow-derived mast cells with melanoma cells showed that they have an intrinsic anti-metastatic activity. Mass spectrometry analysis of melanoma-mast cell co-cultures secretome showed that HMGA1 secretion by melanoma cells was significantly impaired. Consistently, HMGA1 knockdown in B16-F10 cells reduced their metastatic capacity in vivo. Importantly, analysis of HMGA1 expression in human melanoma tumours showed that metastatic tumours with high HMGA1 expression are associated with reduced overall and disease-free survival. Moreover, we show that HMGA1 is reduced in the nuclei and enriched in the cytoplasm of melanoma metastatic lesions when compared to primary tumours. These data suggest that high HMGA1 expression and secretion from melanoma cells promote metastatic behaviour. Targeting HMGA1 expression intrinsically or extrinsically by mast cells actions reduce melanoma metastasis. Our results pave the way to the use of HMGA1 as anti-metastatic target in melanoma as previously suggested in other cancer types.

Inactivation of EMILIN-1 by Proteolysis and Secretion in Small Extracellular Vesicles Favors Melanoma Progression and Metastasis.

Several studies have demonstrated that melanoma-derived extracellular vesicles (EVs) are involved in lymph node metastasis; however, the molecular mechanisms involved are not completely defined. Here, we found that EMILIN-1 is proteolyzed and secreted in small EVs (sEVs) as a novel mechanism to reduce its intracellular levels favoring metastasis in mouse melanoma lymph node metastatic cells. Interestingly, we observed that EMILIN-1 has intrinsic tumor and metastasis suppressive-like properties reducing effective migration, cell viability, primary tumor growth, and metastasis. Overall, our analysis suggests that the inactivation of EMILIN-1 by proteolysis and secretion in sEVs reduce its intrinsic tumor suppressive activities in melanoma favoring tumor progression and metastasis.

The influence of secreted factors and extracellular vesicles in ovarian cancer metastasis.

Ovarian cancer cells mainly metastasise within the peritoneal cavity, the lethal consequence of tumour progression in this cancer type. Classically, changes in tumour cells, such as epithelial to mesenchymal transition, involve the down-regulatinon of E-cadherin, activation of extracellular proteases and integrin-mediated adhesion. However, our current understanding of ovarian tumour progression suggests the implication of both intrinsic and extrinsic factors. It has been proposed that ovarian cancer metastases are a consequence of the crosstalk between cancer cells and the tumour microenvironment by soluble factors and extracellular vesicles. Characterisation of the alterations in both the tumour cells and the surrounding microenvironment has emerged as a new research field to understand ovarian cancer metastasis. In this mini review, we will summarise the most recent findings, focusing our attention on the role of secreted factors and extracellular vesicles in ovarian cancer metastasis.

A role for the transducer of the Hippo pathway, TAZ, in the development of aggressive types of endometrial cancer.

Although TAZ, the final effector of the Hippo pathway that modulates epithelial to mesenchymal transition and stemness, has been implicated in the development of different types of cancer, its role in endometrial cancer has not yet been studied. Thus, we evaluated the expression of TAZ in different types of endometrial cancer by immunohistochemistry. TAZ expression was detected in 76% of undifferentiated endometrial carcinomas, 54% of endometrial carcinosarcomas, 46% of endometrial serous carcinomas, 36% of grade 3 endometrioid carcinomas, and 18% of grade 1-2 endometrioid carcinomas, with statistically significant differences. We analyzed the WWTR1 gene that encodes TAZ by FISH and MassARRAY spectrometry, ruling out gene amplification and differential promoter methylation as the main mechanisms that modulate TAZ expression in endometrial tumors. However, we did detect a significant association between Scribble hypoexpression and delocalization with TAZ expression. Moreover, we demonstrated that TAZ promoted invasiveness, and it favored cell motility and tumor growth, in endometrial cancer cell lines. In addition, TAZ expression was associated with the transition from an epithelial to mesenchymal phenotype, both in vitro and in human tumors. Together, these data reveal a previously unknown role for TAZ and the Hippo pathway in the progression of aggressive subtypes of endometrial cancer.

Extracellular Vesicle DNA Extraction and Sequencing in Ancient Serum Samples From Patients With Breast Cancer.

BACKGROUND/AIM: Extracellular vesicle DNA (EV-DNA) has emerged as a novel biomarker for tumor mutation detection using liquid biopsies, exhibiting biological advantages compared to cell-free DNA (cfDNA). This study assessed the feasibility of EV-DNA and cfDNA extraction and sequencing in old serum samples of patients with breast cancer (BC). PATIENTS AND METHODS: A total of 28 serum samples of 27 patients with corresponding clinical information were collected between 1983 and 1991. EV-DNA was extracted using Exo-GAG kit (Nasabiotech) and cfDNA using QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen), respectively. Subsequently, 10 matched samples (EV-DNA n=5, cfDNA n=5) of five patients were subjected to sequencing using the Oncomine™ Breast cfDNA Research Assay v2 (Thermo Fisher Scientific). RESULTS: Samples were collected on median 1.9 years after primary diagnosis [interquartile range (IQR)=0.2-7.2]. Median follow-up was 9.5 years (IQR=5.2-14.2). Median age of serum samples was 36.1 years (IQR=34.5-37.3). EV-DNA and cfDNA were extracted from 100% (28/28) of the included samples. Both, DNA quantity and concentration were comparable between EV-DNA and cfDNA. Sequencing was successfully performed in 100% (10/10) of the included samples. Two matched analyses yielded equivalent results in EV-DNA and cfDNA (no mutations, n=1; PIK3CA mutation, n=1), whilst in two analyses, PIK3CA mutation was only found in cfDNA, and in one analysis, a TP53 mutation was only found in EV-DNA. CONCLUSION: EV-DNA extraction and sequencing in old serum samples of patients with BC is feasible and has the potential to address clinically relevant questions in longitudinal studies.

Characterization of plasma circulating small extracellular vesicles in patients with metastatic solid tumors and newly diagnosed brain metastasis.

Nearly 40% of the advanced cancer patients will present brain metastases during the course of their disease, with a 2-year life expectancy of less than 10%. Immune system impairment, including the modulation of both STAT3 and PD-L1, is one of the hallmarks of brain metastases. Liquid biopsy could offer several advantages in brain metastases management, such as the possibility of noninvasive dynamic monitoring. Extracellular vesicles (EVs) have been recently proposed as novel biomarkers especially useful in liquid biopsy due to their secretion in biofluids and their role in cell communication during tumor progression. The main aim of this work was to characterize the size and protein cargo of plasma circulating EVs in patients with solid tumors and their correlation with newly diagnosed brain metastases, in addition to their association with other relevant clinical variables. We analyzed circulating EVs in the plasma of 123 patients: 42 patients with brain metastases, 50 without brain metastases and 31 healthy controls. Patients with newly diagnosed brain metastases had a lower number of circulating EVs in the plasma and a higher protein concentration in small EVs (sEVs) compared to patients without brain metastases and healthy controls. Interestingly, melanoma patients with brain metastases presented decreased STAT3 activation and increased PD-L1 levels in circulating sEVs compared to patients without central nervous system metastases. Decreased STAT3 activation and increased PD-L1 in plasma circulating sEVs identify melanoma patients with brain metastasis.

TUNAR lncRNA Encodes a Microprotein that Regulates Neural Differentiation and Neurite Formation by Modulating Calcium Dynamics.

Long noncoding RNAs (lncRNAs) are regulatory molecules which have been traditionally considered as "non-coding". Strikingly, recent evidence has demonstrated that many non-coding regions, including lncRNAs, do in fact contain small-open reading frames that code for small proteins that have been called microproteins. Only a few of them have been characterized so far, but they display key functions in a wide variety of cellular processes. Here, we show that TUNAR lncRNA encodes an evolutionarily conserved microprotein expressed in the nervous system that we have named pTUNAR. pTUNAR deficiency in mouse embryonic stem cells improves their differentiation potential towards neural lineage both in vitro and in vivo. Conversely, pTUNAR overexpression impairs neuronal differentiation by reduced neurite formation in different model systems. At the subcellular level, pTUNAR is a transmembrane protein that localizes in the endoplasmic reticulum and interacts with the calcium transporter SERCA2. pTUNAR overexpression reduces cytoplasmatic calcium, consistent with a possible role of pTUNAR as an activator of SERCA2. Altogether, our results suggest that our newly discovered microprotein has an important role in neural differentiation and neurite formation through the regulation of intracellular calcium. From a more general point of view, our results provide a proof of concept of the role of lncRNAs-encoded microproteins in neural differentiation.

Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism.

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR+ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.

Functional characterization of E- and P-cadherin in invasive breast cancer cells.

BACKGROUND: Alterations in the cadherin-catenin adhesion complexes are involved in tumor initiation, progression and metastasis. However, the functional implication of distinct cadherin types in breast cancer biology is still poorly understood. METHODS: To compare the functional role of E-cadherin and P-cadherin in invasive breast cancer, we stably transfected these molecules into the MDA-MB-231 cell line, and investigated their effects on motility, invasion and gene expression regulation. RESULTS: Expression of either E- and P-cadherin significantly increased cell aggregation and induced a switch from fibroblastic to epithelial morphology. Although expression of these cadherins did not completely reverse the mesenchymal phenotype of MDA-MB-231 cells, both E- and P-cadherin decreased fibroblast-like migration and invasion through extracellular matrix in a similar way. Moreover, microarray gene expression analysis of MDA-MB-231 cells after expression of E- and P-cadherins revealed that these molecules can activate signaling pathways leading to significant changes in gene expression. Although the expression patterns induced by E- and P-cadherin showed more similarities than differences, 40 genes were differentially modified by the expression of either cadherin type. CONCLUSION: E- and P-cadherin have similar functional consequences on the phenotype and invasive behavior of MDA-MB-231 cells. Moreover, we demonstrate for the first time that these cadherins can induce both common and specific gene expression programs on invasive breast cancer cells. Importantly, these identified genes are potential targets for future studies on the functional consequences of altered cadherin expression in human breast cancer.

"New" molecular taxonomy in breast cancer.

Advances in the analysis of expression profiles, using genomic techniques, have revealed the high heterogeneity present in breast cancers. These approaches have served to identify different breast cancer subgroups with specific molecular characteristics that could sub-classify these tumours as carcinomas expressing hormone receptors, denominated Luminal subtype, and tumours with negative expression of hormone receptors, the Basal and HER2+ phenotypes. Therefore, during recent years, identification of markers characteristic of each subtype has been the focus of many research groups. All of these breast tumour subtypes probably have specific clinical and morphological features; however, this hypothesis needs to be confirmed by analysing more homogenous series. Although this "new" classification has limitations, it could be useful in the clinical practice, allowing not only a more accurate prognosis in breast cancer patients but also a selective treatment for each predefined subtype.