Synaptosomes: new vesicles for neuronal mitochondrial transplantation.

BACKGROUND: Mitochondrial dysfunction is a critical factor in the onset and progression of neurodegenerative diseases. Recently, mitochondrial transplantation has been advised as an innovative and attractive strategy to transfer and replace damaged mitochondria. Here we propose, for the first time, to use rat brain extracted synaptosomes, a subcellular fraction of isolated synaptic terminal that contains mitochondria, as mitochondrial delivery systems. RESULTS: Synaptosome preparation was validated by the presence of Synaptophysin and PSD95. Synaptosomes were characterized in terms of dimension, zeta potential, polydispersity index and number of particles/ml. Nile Red or CTX-FITCH labeled synaptosomes were internalized in LAN5 recipient cells by a mechanism involving specific protein-protein interaction, as demonstrated by loss of fusion ability after trypsin treatment and using different cell lines. The loading and release ability of the synaptosomes was proved by the presence of curcumin both into synaptosomes and LAN5 cells. The vitality of mitochondria transferred by Synaptosomes was demonstrated by the presence of Opa1, Fis1 and TOM40 mitochondrial proteins and JC-1 measurements. Further, synaptosomes deliver vital mitochondria into the cytoplasm of neuronal cells as demonstrated by microscopic images, increase of TOM 40, cytochrome c, Hexokinase II mitochondrial proteins, and presence of rat mitochondrial DNA. Finally, by using synaptosomes as a vehicle, healthy mitochondria restored mitochondrial function in cells containing rotenone or CCCp damaged mitochondria. CONCLUSIONS: Taken together these results suggest that synaptosomes can be a natural vehicle for the delivery of molecules and organelles to neuronal cells. Further, the replacement of affected mitochondria with healthy ones could be a potential therapy for treating neuronal mitochondrial dysfunction-related diseases.

EBF1, MYO6 and CALR expression levels predict therapeutic response in diffuse large B-cell lymphomas.

Background: Diffuse large B-cell lymphoma (DLBCL) is a hematological malignancy representing one-third of non-Hodgkin's lymphoma cases. Notwithstanding immunotherapy in combination with chemotherapy (R-CHOP) is an effective therapeutic approach for DLBCL, a subset of patients encounters treatment resistance, leading to low survival rates. Thus, there is an urgent need to identify predictive biomarkers for DLBCL including the elderly population, which represents the fastest-growing segment of the population in Western countries. Methods: Gene expression profiles of n=414 DLBCL biopsies were retrieved from the public dataset GSE10846. Differentially expressed genes (DEGs) (fold change >1.4, p-value <0.05, n=387) have been clustered in responder and non-responder patient cohorts. An enrichment analysis has been performed on the top 30 up-regulated genes of responder and non-responder patients to identify the signatures involved in gene ontology (MSigDB). The more significantly up-regulated DEGs have been validated in our independent collection of formalin-fixed paraffin-embedded (FFPE) biopsy samples of elderly DLBCL patients, treated with R-CHOP as first-line therapy. Results: From the analysis of two independent cohorts of DLBCL patients emerged a gene signature able to predict the response to R-CHOP therapy. In detail, expression levels of EBF1, MYO6, CALR are associated with a significant worse overall survival. Conclusions: These results pave the way for a novel characterization of DLBCL biomarkers, aiding the stratification of responder versus non-responder patients.

Recapitulating thyroid cancer histotypes through engineering embryonic stem cells.

Thyroid carcinoma (TC) is the most common malignancy of endocrine organs. The cell subpopulation in the lineage hierarchy that serves as cell of origin for the different TC histotypes is unknown. Human embryonic stem cells (hESCs) with appropriate in vitro stimulation undergo sequential differentiation into thyroid progenitor cells (TPCs-day 22), which maturate into thyrocytes (day 30). Here, we create follicular cell-derived TCs of all the different histotypes based on specific genomic alterations delivered by CRISPR-Cas9 in hESC-derived TPCs. Specifically, TPCs harboring BRAFV600E or NRASQ61R mutations generate papillary or follicular TC, respectively, whereas addition of TP53R248Q generate undifferentiated TCs. Of note, TCs arise by engineering TPCs, whereas mature thyrocytes have a very limited tumorigenic capacity. The same mutations result in teratocarcinomas when delivered in early differentiating hESCs. Tissue Inhibitor of Metalloproteinase 1 (TIMP1)/Matrix metallopeptidase 9 (MMP9)/Cluster of differentiation 44 (CD44) ternary complex, in cooperation with Kisspeptin receptor (KISS1R), is involved in TC initiation and progression. Increasing radioiodine uptake, KISS1R and TIMP1 targeting may represent a therapeutic adjuvant option for undifferentiated TCs.

Targeting of the Peritumoral Adipose Tissue Microenvironment as an Innovative Antitumor Therapeutic Strategy.

The tumor microenvironment (TME) plays a key role in promoting and sustaining cancer growth. Adipose tissue (AT), due to its anatomical distribution, is a prevalent component of TME, and contributes to cancer development and progression. Cancer-associated adipocytes (CAAs), reprogrammed by cancer stem cells (CSCs), drive cancer progression by releasing metabolites and inflammatory adipokines. In this review, we highlight the mechanisms underlying the bidirectional crosstalk among CAAs, CSCs, and stromal cells. Moreover, we focus on the recent advances in the therapeutic targeting of adipocyte-released factors as an innovative strategy to counteract cancer progression.

Dual Inhibition of Myc Transcription and PI3K Activity Effectively Targets Colorectal Cancer Stem Cells.

Despite advances in the curative approach, the survival rate of advanced colorectal cancer (CRC) patients is still poor, which is likely due to the emergence of cancer cell clones resistant to the available therapeutic options. We have already shown that CD44v6-positive CRC stem cells (CR-CSCs) are refractory toward standard anti-tumor therapeutic agents due to the activation of the PI3K pathway together with high HER2 expression levels. Tumor microenvironmental cytokines confer resistance to CR-CSCs against HER2/PI3K targeting by enhancing activation of the MAPK pathway. Here, we show that the CSC compartment, spared by BRAF inhibitor-based targeted therapy, is associated with increased expression levels of CD44v6 and Myc and retains boosted clonogenic activity along with residual tumorigenic potential. Inhibition of Myc transcription, downstream of the MAPK cascade components, and PI3K pathway activity was able to overcome the protective effects of microenvironmental cytokines, affecting the survival and the clonogenic activity of CR-CSCs, regardless of their mutational background. Likewise, the double targeting induced stabilization of mouse tumor avatars. Altogether, these data outline the rationale for dual kinase targeting of CR-CSCs to prevent their adaptive response, which would lead to disease progression.

Effective targeting of breast cancer stem cells by combined inhibition of Sam68 and Rad51.

Breast cancer (BC) is the second cause of cancer-related deceases in the worldwide female population. Despite the successful treatment advances, 25% of BC develops resistance to current therapeutic regimens, thereby remaining a major hurdle for patient management. Current therapies, targeting the molecular events underpinning the adaptive resistance, still require effort to improve BC treatment. Using BC sphere cells (BCSphCs) as a model, here we showed that BC stem-like cells express high levels of Myc, which requires the presence of the multifunctional DNA/RNA binding protein Sam68 for the DNA-damage repair. Analysis of a cohort of BC patients displayed that Sam68 is an independent negative factor correlated with the progression of the disease. Genetic inhibition of Sam68 caused a defect in PARP-induced PAR chain synthesis upon DNA-damaging insults, resulting in cell death of TNBC cells. In contrast, BC stem-like cells were able to survive due to an upregulation of Rad51. Importantly, the inhibition of Rad51 showed synthetic lethal effect with the silencing of Sam68, hampering the cell viability of patient-derived BCSphCs and stabilizing the growth of tumor xenografts, including those TNBC carrying BRCA mutation. Moreover, the analysis of Myc, Sam68 and Rad51 expression demarcated a signature of a poor outcome in a large cohort of BC patients. Thus, our findings suggest the importance of targeting Sam68-PARP1 axis and Rad51 as potential therapeutic candidates to counteract the expansion of BC cells with an aggressive phenotype.

Targeting Phosphatases and Kinases: How to Checkmate Cancer.

Metastatic disease represents the major cause of death in oncologic patients worldwide. Accumulating evidence have highlighted the relevance of a small population of cancer cells, named cancer stem cells (CSCs), in the resistance to therapies, as well as cancer recurrence and metastasis. Standard anti-cancer treatments are not always conclusively curative, posing an urgent need to discover new targets for an effective therapy. Kinases and phosphatases are implicated in many cellular processes, such as proliferation, differentiation and oncogenic transformation. These proteins are crucial regulators of intracellular signaling pathways mediating multiple cellular activities. Therefore, alterations in kinases and phosphatases functionality is a hallmark of cancer. Notwithstanding the role of kinases and phosphatases in cancer has been widely investigated, their aberrant activation in the compartment of CSCs is nowadays being explored as new potential Achille's heel to strike. Here, we provide a comprehensive overview of the major protein kinases and phosphatases pathways by which CSCs can evade normal physiological constraints on survival, growth, and invasion. Moreover, we discuss the potential of inhibitors of these proteins in counteracting CSCs expansion during cancer development and progression.

Messing Up the Cancer Stem Cell Chemoresistance Mechanisms Supported by Tumor Microenvironment.

Despite the recent advances in cancer patient management and in the development of targeted therapies, systemic chemotherapy is currently used as a first-line treatment for many cancer types. After an initial partial response, patients become refractory to standard therapy fostering rapid tumor progression. Compelling evidence highlights that the resistance to chemotherapeutic regimens is a peculiarity of a subpopulation of cancer cells within tumor mass, known as cancer stem cells (CSCs). This cellular compartment is endowed with tumor-initiating and metastasis formation capabilities. CSC chemoresistance is sustained by a plethora of grow factors and cytokines released by neighboring tumor microenvironment (TME), which is mainly composed by adipocytes, cancer-associated fibroblasts (CAFs), immune and endothelial cells. TME strengthens CSC refractoriness to standard and targeted therapies by enhancing survival signaling pathways, DNA repair machinery, expression of drug efflux transporters and anti-apoptotic proteins. In the last years many efforts have been made to understand CSC-TME crosstalk and develop therapeutic strategy halting this interplay. Here, we report the combinatorial approaches, which perturb the interaction network between CSCs and the different component of TME.

Cancer Stem Cells in Thyroid Tumors: From the Origin to Metastasis.

Thyroid tumors are extremely heterogeneous varying from almost benign tumors with good prognosis as papillary or follicular tumors, to the undifferentiated ones with severe prognosis. Recently, several models of thyroid carcinogenesis have been described, mostly hypothesizing a major role of the thyroid cancer stem cell (TCSC) population in both cancer initiation and metastasis formation. However, the cellular origin of TCSC is still incompletely understood. Here, we review the principal epigenetic mechanisms relevant to TCSC origin and maintenance in both well-differentiated and anaplastic thyroid tumors. Specifically, we describe the alterations in DNA methylation, histone modifiers, and microRNAs (miRNAs) involved in TCSC survival, focusing on the potential of targeting aberrant epigenetic modifications for developing novel therapeutic approaches. Moreover, we discuss the bidirectional relationship between TCSCs and immune cells. The cells of innate and adaptive response can promote the TCSC-driven tumorigenesis, and conversely, TCSCs may favor the expansion of immune cells with protumorigenic functions. Finally, we evaluate the role of the tumor microenvironment and the complex cross-talk of chemokines, hormones, and cytokines in regulating thyroid tumor initiation, progression, and therapy refractoriness. The re-education of the stromal cells can be an effective strategy to fight thyroid cancer. Dissecting the genetic and epigenetic landscape of TCSCs and their interactions with tumor microenvironment cells is urgently needed to select more appropriate treatment and improve the outcome of patients affected by advanced differentiated and undifferentiated thyroid cancers.

Metabolic Escape Routes of Cancer Stem Cells and Therapeutic Opportunities.

Although improvement in early diagnosis and treatment ameliorated life expectancy of cancer patients, metastatic disease still lacks effective therapeutic approaches. Resistance to anticancer therapies stems from the refractoriness of a subpopulation of cancer cells-termed cancer stem cells (CSCs)-which is endowed with tumor initiation and metastasis formation potential. CSCs are heterogeneous and diverge by phenotypic, functional and metabolic perspectives. Intrinsic as well as extrinsic stimuli dictated by the tumor microenvironment (TME)have critical roles in determining cell metabolic reprogramming from glycolytic toward an oxidative phenotype and vice versa, allowing cancer cells to thrive in adverse milieus. Crosstalk between cancer cells and the surrounding microenvironment occurs through the interchange of metabolites, miRNAs and exosomes that drive cancer cells metabolic adaptation. Herein, we identify the metabolic nodes of CSCs and discuss the latest advances in targeting metabolic demands of both CSCs and stromal cells with the scope of improving current therapies and preventing cancer progression.