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La metástasis cerebral se ha descrito hasta en un 40% de los pacientes con tumores diseminados. Esta complicación se asocia con una supervivencia global de menos del 10% de los pacientes a los 2 años desde el diagnóstico y un deterioro significativo en la calidad de vida. La inducción de un estado de inmunosupresión es una de las señas de identidad en el desarrollo de las metástasis cerebrales. Durante este proceso se produce normalmente la modulación de la actividad de STAT3 y de la expresión de PD-L1 tanto en células tumorales como inmunes, entre otras moléculas. La identificación de nuevos biomarcadores de biopsia líquida para el diagnóstico de metástasis cerebrales ofrece diversas ventajas, como su fácil accesibilidad y la posibilidad de una monitorización dinámica no invasiva, y podría permitir una mejor comprensión de los mecanismos moleculares de la diseminación al sistema nervioso central (SNC), con impacto positivo en el pronóstico de los pacientes. Entre otros biomarcadores útiles en biopsia líquida, las vesículas extracelulares se han señalado como especialmente útiles debido a su secreción en fluidos corporales y a su papel en la comunicación intercelular implicada en procesos claves de la biología tumoral...

La metástasis cerebral se ha descrito hasta en un 40% de los pacientes con tumores diseminados. Esta complicación se asocia con una supervivencia global de menos del 10% de los pacientes a los 2 años desde el diagnóstico y un deterioro significativo en la calidad de vida. La inducción de un estado de inmunosupresión es una de las señas de identidad en el desarrollo de las metástasis cerebrales. Durante este proceso se produce normalmente la modulación de la actividad de STAT3 y de la expresión de PD-L1 tanto en células tumorales como inmunes, entre otras moléculas. La identificación de nuevos biomarcadores de biopsia líquida para el diagnóstico de metástasis cerebrales ofrece diversas ventajas, como su fácil accesibilidad y la posibilidad de una monitorización dinámica no invasiva, y podría permitir una mejor comprensión de los mecanismos moleculares de la diseminación al sistema nervioso central (SNC), con impacto positivo en el pronóstico de los pacientes. Entre otros biomarcadores útiles en biopsia líquida, las vesículas extracelulares se han señalado como especialmente útiles debido a su secreción en fluidos corporales y a su papel en la comunicación intercelular implicada en procesos claves de la biología tumoral..., Around 40% of the cancer patients with metastatic tumors will present brain metastases during the course of their disease. Once diagnosed, brain metastases are associated with a decreased overall survival; less than 10% of the patients survive more than 2 years, with a significant impairment in their quality of life.Brain metastases are often associated with immune system impairment. Among other molecules, both STAT3 activity and PD-L1 levels in tumor and immune cells are modulated. The identification of new biomarkers useful in liquid biopsy offers several advantages in brain metastases diagnosis and prognosis, such as their easy accessibility and the possibility of non-invasive dynamic monitoring. Extracellular vesicles 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...

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.

The truncated somatostatin receptor sst5TMD4 is associated with poor prognosis in breast cancer and increases breast cancer cell malignancy. Here, we examined the cellular/molecular mechanisms underlying this association, aiming to identify new molecular tools to improve diagnosis, prognosis or therapy. A gene expression array comparing sst5TMD4 stably-transfected MCF-7 cells and their controls (empty-plasmid) revealed the existence of profound alterations in the expression of genes involved in key tumoral processes, such as cell survival or angiogenesis. Moreover, sst5TMD4- overexpressing MCF-7 and MDA-MB-231 cells demonstrated increased expression/ production of pro-angiogenic factors and enhanced capacity to form mammospheres. Consistently, sst5TMD4-expressing MCF-7 cells induced xenografted tumors with higher VEGF levels and elevated number of blood vessels. Importantly, sst5TMD4 was expressed in a subset of breast cancers, where it correlated with angiogenic markers, lymphatic metastasis, and reduced disease-free survival. These results, coupled to our previous data, support a relevant role of sst5TMD4 in the angiogenic process and reinforce the role of sst5TMD4 in breast cancer malignancy and metastatic potential, supporting its possible utility to develop new molecular biomarkers and drug therapies for these tumors.

Around, 30-40% of HER2-positive breast cancers do not show substantial clinical benefit from the targeted therapy and, thus, the mechanisms underlying resistance remain partially unknown. Interestingly, ERBB2 is frequently co-amplified and co-expressed with neighbour genes that may play a relevant role in this cancer subtype. Here, using an in silico analysis of data from 2,096 breast tumours, we reveal a significant correlation between Gasdermin B (GSDMB) gene (located 175 kilo bases distal from ERBB2) expression and the pathological and clinical parameters of poor prognosis in HER2-positive breast cancer. Next, the analysis of three independent cohorts (totalizing 286 tumours) showed that approximately 65% of the HER2-positive cases have GSDMB gene amplification and protein over-expression. Moreover, GSDMB expression was also linked to poor therapeutic responses in terms of lower relapse free survival and pathologic complete response as well as positive lymph node status and the development of distant metastasis under neoadjuvant and adjuvant treatment settings, respectively. Importantly, GSDMB expression promotes survival to trastuzumab in different HER2-positive breast carcinoma cells, and is associated with trastuzumab resistance phenotype in vivo in Patient Derived Xenografts. In summary, our data identifies the ERBB2 co-amplified and co-expressed gene GSDMB as a critical determinant of poor prognosis and therapeutic response in HER2-positive breast cancer.

Gasdermin B (GSDMB) belongs to the Gasdermin protein family that comprises four members (GSDMA-D). Gasdermin B expression has been detected in some tumor types such as hepatocarcinomas, gastric and cervix cancers; and its overexpression has been related to tumor progression. At least four splicing isoforms of GSDMB have been identified, which may play differential roles in cancer. However, the implication of GSDMB in carcinogenesis and tumor progression is not well understood. Here, we uncover for the first time the functional implication of GSDMB in breast cancer. Our data shows that high levels of GSDMB expression is correlated with reduced survival and increased metastasis in breast cancer patients included in an expression dataset (>1,000 cases). We demonstrate that GSDMB is upregulated in breast carcinomas compared to normal breast tissue, being the isoform 2 (GSDMB-2) the most differentially expressed. In order to evaluate the functional role of GSDMB in breast cancer two GSDMB isoforms were studied (GSDMB-1 and GSDMB-2). The overexpression of both isoforms in the MCF7 breast carcinoma cell line promotes cell motility and invasion, while its silencing in HCC1954 breast carcinoma cells decreases the migratory and invasive phenotype. Importantly, we demonstrate that both isoforms have a differential role on the activation of Rac-1 and Cdc-42 Rho-GTPases. Moreover, our data support that GSMDB-2 induces a pro-tumorigenic and pro-metastatic behavior in mouse xenograft models as compared to GSDMB-1. Finally, we observed that although both GSDMB isoforms interact in vitro with the chaperone Hsp90, only the GSDMB-2 isoform relies on this chaperone for its stability. Taken together, our results provide for the first time evidences that GSDMB-2 induces invasion, tumor progression and metastasis in MCF7 cells and that GSDMB can be considered as a new potential prognostic marker in breast cancer.

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR- 422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

Somatostatin receptors (sst1-5) are present in different types of tumors, where they inhibit key cellular processes such as proliferation and invasion. Although ssts are densely expressed in breast cancer, especially sst2, their role and therapeutic potential remain uncertain. Recently, we identified a new truncated sst5 variant, sst5TMD4, which is related to the abnormal response of certain pituitary tumors to treatment with somatostatin analogs. Here, we investigated the possible role of sst5TMD4 in breast cancer. This study revealed that sst5TMD4 is absent in normal mammary gland, but is abundant in a subset of poorly differentiated human breast tumors, where its expression correlated to that of sst2. Moreover, in the MCF-7 breast cancer model cell, sst5TMD4 expression increased malignancy features such as invasion and proliferation abilities (both in cell cultures and nude mice). This was likely mediated by sst5TMD4-induced increase in phosphorylated extracellular signal-regulated kinases 1 and 2 and p-Akt levels, and cyclin D3 and Arp2/3 complex expression, which also led to mesenchymal-like phenotype. Interestingly, sst5TMD4 interacts physically with sst2 and thereby alters its signaling, enabling disruption of sst2 inhibitory feedback and providing a plausible basis for our findings. These results suggest that sst5TMD4 could be involved in the pathophysiology of certain types of breast tumors.

The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex), with Ex1-Ex4 encoding a 117 amino-acid (aa) preproprotein that is known to be processed to yield a 28-aa (ghrelin) and/or a 23-aa (obestatin) mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant), which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In) 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site) but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p = 0.0001) but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p = 0.0001), ghrelin receptor-type 1b (GHSR1b; p = 0.049) and cyclin-D3 (a cell-cycle inducer/proliferation marker; p = 0.009), but not with native-ghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cancer.

[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.