Cellular and molecular components of the atherosclerotic process: Primary colture and characterization of human vascular smooth muscle cells from carotid artery

Introduction The last years were characterized by several studies proceeding to understand SMC heterogeneity. Animal models of rat or rabbit IT after endothelial lesion are presently the most studied models for atherosclerotic plaque formation and have been useful in order to understand several biological features of SMCs. The disadvantages of animal models are that there is no possibility to reproduce the same history of pathogenesis in animals as that in man. There is no perfect animal model that completely replicates all stages of human atherosclerosis. Howevere, identification of particular SMCs subpopulation in human arteries represents a difficult challenge for understandable reason, such us material availability and experiment standardization. The relevance of SMC heterogenity to human disease still remains to be demonstrated. Aim Aim of this study is to obtain primary culture of human vascular smooth muscle cells (SMCs) from both atherosclerotic and no-atherosclerotic carotid artery, to investigare human SMC heterogeneity. Moreover, the possibility to isolate o SMC subpopulations, from a single human artery, could be usefull to verify the hypothesis that distinct SMC subtypes are involved in atherosclerosis pathogenesis and progression. Materials, Methods and Results Thanks to a collaboration between our laboratory and the Vascular Surgery’s group of Sant’Anna Hospital(University of Ferrara) leading by Francesco Mascoli, we had the possibility to obtain samples of atherosclerotic and noatherosclerotic human carotid artery from patients undergone to carotid endarterectomy. From these specimens we proceeded with tissue explantation in order to obtain primary colture of vascular smooth muscle cells both from no-atherosclerotic and atherosclerotic carotid artery. Cells obtained by tissue explantation were tested by immunoflurescences staining in order to verify the presence of alfa smooth muscle actin, a specific marker of vascular smooth muscle cells. Once we verified the nature of our cells through the presence of alfa smooth muscle actin we have tried to divide the atherosclerotic artery in media and plaque and than proceeding tissue explatation only with media or only with plaque or with complete lesion. We have found out that only from the complete lesion (media +plaque) we have obtained cellular outgrowth, while media and plaque alone has not gave cells at all. From these results we were started to understand which tissue, between media and plaque was the proliferative ones. In order to obtain this data we have proceeded with an experiment of cocolture using transwell insert, wich allows media and plaque to stay phisically separeted but to share the same medium. After these experiments, we have found out that the proliferative tissue was the media but plaque presence was a “condicio sine qua non” for cellular outgrowth in atherosclerotic carotid artery. We have compared cells coming from non atherosclerotic media with the ones coming from atherosclerotic artery: early, we have found out the morphological differences: cells from non atherosclerotic media was large and grew up in a monolayer even at confluence, while cells from atherosclerotic media was small and elongated and, at confluence, grew up forming a multilayer net. We have analyzed the cells also using immunofluorescences finding out that large cells(coming from no-atherosclerotic artery), had much alfa smooth muscle actin and did not shown Desmin, or S100A4(a marker of atheroma prone cells in porcine model of atherosclerosis), but show SMMHC. Small cells (coming from atherosclerotic artery), instead, had less alfa smooth muscle actin compared to large cells, and did not shown desmin and SMMHC but show an high production of S100A4. We also have tried to add plaque in tissue explantation of no-atherosclerotique media and the outgrownig from these experiments was small and by immunofluorescence shown the presences of S100A4. These are only preliminary data. The presence of the mRNA of these markers was analyzed by using RT-PCR. For further information about the differences between these two celluar populations we led a microarray experiment using biological material coming from three patiences. We discovered that there are 375 genes modulated in the two populations; these genes are involved in several biological pathways, in particular inflammation and cellular development. In conclusion we found that there are two kind of cellular populations in human artery with atherosclerotic lesion, they have some important differences that could explain why cells change phenotype and migrate in to the intima. Cells from atherosclerotic artery are strictly dependent of plaque presences that act on on them as a magnet. We identify a group of genes that can help us to understand what are the steps of phenotypical modulation. Presence of plaque nearby no-atherosclerotic artery can switch the cells from a large phenotype to a Small Phenotype (this preliminary data is confirmed by the presence in cells, outgrowed from no-atherosclerotic artery in presece of plaque, of S100A4

miRNA transported by exosomes: a key machinery in tumor microenvironment mediated chemoresistance

It is well established that the tumor microenvironment (TME) actively participates in fundamental processes such as tumor initiation, invasion and response to therapies, apart from its documented scaffold role. Ultimately, the TME can be defined as a tri-dimensional network structured by the tumor-associated extra-cellular matrix (ECM) in which tumor cells interact with various types of stromal cells in an attempt to hijack the normal organization of the host tissue and support cancer growth

EGF and IGF1 affect Sunitinib activity in BP-NEN: new putative targets beyond VEGFR?

Broncho-Pulmonary Neuroendocrine Neoplasms (BP-NENs) are neoplasms orphan of an efficient therapy. Available medical treatments derived from clinical trials are not specific for the management of this malignancy. Sunitinib is a multi-receptor tyrosine-kinases (RTKs) inhibitor that has already shown its efficacy in NENs but there are not available data about its action in BP-NENs. Therefore, our aim was to understand the effects of RTKs inhibition promoted by Sunitinib in order to evaluate new putative targets useful in malignancy treatment. Since our results underlined a role for EGFR and IGF1R in modulating Sunitinib antiproliferative action, we investigated the effects of Erlotinib, an EGFR inhibitor, and Linsitinib, an IGF1R inhibitor, in order to understand their function in regulating cells behaviour. Cell viability and caspase activation were evaluated on two immortalized human BP-NEN cell lines and primary cultures. Our results showed that after treatment with Sunitinib and/or IGF1, EGF and VEGF, the antiproliferative effect of Sunitinib was counteracted by EGF and IGF1 but not by VEGF. Therefore, we evaluated with alpha-screen technology the phosphorylated EGFR and IGF1R levels in primary cultures treated with Sunitinib and/or EGF and IGF1. Results showed a decrease of p-IGF1R after treatment with Sunitinib and an increase after co-treatment with IGF1. Then, we assessed cell viability and caspase activation on BP-NEN cell lines after treatment with Linsitinib and/or Erlotinib. Results demonstrate that these two agents have a stronger antiproliferative effect compared to Sunitinib. In conclusion, our results suggest that IGF1R and EGF1R could represent putative molecular targets in BP-NENs treatment

The multifaceted role of lemur tyrosine kinase 3 in health and disease

In the last decade, LMTK3 (lemur tyrosine kinase 3) has emerged as an important player in breast cancer, contributing to the advancement of disease and the acquisition of resistance to therapy through a strikingly complex set of mechanisms. Although the knowledge of its physiological function is largely limited to receptor trafficking in neurons, there is mounting evidence that LMTK3 promotes oncogenesis in a wide variety of cancers. Recent studies have broadened our understanding of LMTK3 and demonstrated its importance in numerous signalling pathways, culminating in the identification of a potent and selective LMTK3 inhibitor. Here, we review the roles of LMTK3 in health and disease and discuss how this research may be used to develop novel therapeutics to advance cancer treatment

Correction to: LMTK3 inhibition affects microtubule stability

Correction to: Mol Cancer 20, 53 (2021) https://doi.org/10.1186/s12943-021-01345-3 Following the publication of the original article [1], the authors noticed errors on the figures introduced during the production step. Below are the errors: Fig. 2a: The labels for the y- and x-axes have been translocated upwards and need to be realigned with the axes. The x-axis should read: -Log2 (fold change). Fig. 2d: the quantification values in the NUSAP1 blots, for T47D and MDA-MB-231 cells, are no longer visible. The original article has been corrected

The PTTG1-binding factor (PBF/PTTG1IP) regulates p53 activity in thyroid cells

The PTTG1-Binding Factor (PBF/PTTG1IP) has an emerging repertoire of roles, especially in thyroid biology, and functions as a proto-oncogene. High PBF expression is independently associated with poor prognosis and lower disease-specific survival in human thyroid cancer. However, the precise role of PBF in thyroid tumorigenesis is unclear. Here, we present extensive evidence demonstrating that PBF is a novel regulator of p53, a tumor suppressor protein with a key role in maintaining genetic stability, which is infrequently mutated in differentiated thyroid cancer. By coimmunoprecipitation and proximity ligation assays, we show that PBF binds specifically to p53 in thyroid cells, and significantly represses transactivation of responsive promoters. Further, we identify that PBF decreases p53 stability by enhancing ubiquitination, which appears dependent on the E3 ligase activity of Mdm2. Impaired p53 function was evident in a transgenic mouse model with thyroid-specific PBF over-expression (PBF-Tg), which had significantly increased genetic instability as indicated by FISSR-PCR analysis. Consistent with this, ~40% of all DNA repair genes examined were repressed in PBF-Tg primary cultures, including genes with critical roles in maintaining genomic integrity such as Mgmt, Rad51 and Xrcc3. Our data also revealed that PBF induction resulted in upregulation of the E2 enzyme Rad6 in murine thyrocytes, and was associated with Rad6 expression in human thyroid tumors. Overall, this work provides novel insights into the role of the proto-oncogene PBF as a negative regulator of p53 function in thyroid tumorigenesis, where PBF is generally over-expressed and p53 mutations are rare compared to other tumor types

Androgen receptor signaling regulates the transcriptome of prostate cancer cells by modulating global alternative splicing

Androgen receptor (AR), is a transcription factor and a member of a hormone receptor superfamily. AR plays a vital role in the progression of prostate cancer and is a crucial target for therapeutic interventions. While the majority of advanced-stage prostate cancer patients will initially respond to the androgen deprivation, the disease often progresses to castrate-resistant prostate cancer (CRPC). Interestingly, CRPC tumors continue to depend on hyperactive AR signaling and will respond to potent second-line antiandrogen therapies, including bicalutamide (CASODEX®) and enzalutamide (XTANDI®). However, the progression-free survival rate for the CRPC patients on antiandrogen therapies is only 8–19 months. Hence, there is a need to understand the mechanisms underlying CRPC progression and eventual treatment resistance. Here, we have leveraged next-generation sequencing and newly developed analytical methodologies to evaluate the role of AR signaling in regulating the transcriptome of prostate cancer cells. The genomic and pharmacologic stimulation and inhibition of AR activity demonstrates that AR regulates alternative splicing within cancer-relevant genes. Furthermore, by integrating transcriptomic data from in vitro experiments and in prostate cancer patients, we found that a significant number of AR-regulated splicing events are associated with tumor progression. For example, we found evidence for an inadvertent AR-antagonist-mediated switch in IDH1 and PL2G2A isoform expression, which is associated with a decrease in overall survival of patients. Mechanistically, we discovered that the epithelial-specific splicing regulators (ESRP1 and ESRP2), flank many AR-regulated alternatively spliced exons. And, using 2D invasion assays, we show that the inhibition of ESRPs can suppress AR-antagonist-driven tumor invasion. Our work provides evidence for a new mechanism by which AR alters the transcriptome of prostate cancer cells by modulating alternative splicing. As such, our work has important implications for CRPC progression and development of resistance to treatment with bicalutamide and enzalutamide

LMTK3 confers chemo-resistance in breast cancer

Lemur tyrosine kinase 3 (LMTK3) is an oncogenic kinase that is involved in different types of cancer (breast, lung, gastric, colorectal) and biological processes including proliferation, invasion, migration, chromatin remodeling as well as innate and acquired endocrine resistance. However, the role of LMTK3 in response to cytotoxic chemotherapy has not been investigated thus far. Using both 2D and 3D tissue culture models, we found that overexpression of LMTK3 decreased the sensitivity of breast cancer cell lines to cytotoxic (doxorubicin) treatment. In a mouse model we showed that ectopic overexpression of LMTK3 decreases the efficacy of doxorubicin in reducing tumor growth. Interestingly, breast cancer cells overexpressing LMTK3 delayed the generation of double strand breaks (DSBs) after exposure to doxorubicin, as measured by the formation of γH2AX foci. This effect was at least partly mediated by decreased activity of ataxia-telangiectasia mutated kinase (ATM) as indicated by its reduced phosphorylation levels. In addition, our RNA-seq analyses showed that doxorubicin differentially regulated the expression of over 700 genes depending on LMTK3 protein expression levels. Furthermore, these genes were found to promote DNA repair, cell viability and tumorigenesis processes / pathways in LMTK3-overexpressing MCF7 cells. In human cancers, immunohistochemistry staining of LMTK3 in pre- and postchemotherapy breast tumor pairs from four separate clinical cohorts revealed a significant increase of LMTK3 following both doxorubicin and docetaxel based chemotherapy. In aggregate, our findings show for the first time a contribution of LMTK3 in cytotoxic drug resistance in breast cancer