Synergic activity of FGFR2 and MEK inhibitors in the treatment of FGFR2-amplified cancers of unknown primary.

Patients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96) and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. The tissue-of-origin prediction was obtained from the tumor microRNA expression profile and confirmed by single-cell transcriptomics. Genomic testing and fluorescence in situ hybridization analysis identified FGFR2 gene amplification in both models, in the form of homogeneously staining region (HSR) in CUP#55 and double minutes in CUP#96. FGFR2 was recognized as the main oncogenic driver and therapeutic target. FGFR2-targeting drug BGJ398 (infigratinib) in combination with the MEK inhibitor trametinib proved to be synergic and exceptionally active, both in vitro and in vivo. The effects of the combined treatment by single-cell gene expression analysis revealed a remarkable plasticity of tumor cells and the greater sensitivity of cells with epithelial phenotype. This study brings personalized therapy closer to CUP patients and provides the rationale for FGFR2 and MEK targeting in metastatic tumors with FGFR2 pathway activation.

Epigenetic age acceleration in hematopoietic stem cell transplantation.

Not available.

Old age: the crown of life, our play's last act. Question and answers on older patients undergoing allogeneic hematopoietic cell transplantation.

PURPOSE OF REVIEW: Several studies showed that age alone should not be used as an arbitrary parameter to exclude patients from allogeneic hematopoietic cell transplantation (HCT). The accessibility to allogeneic HCT programs for older patients with hematological diseases is growing up constantly. The Center for International Blood and Marrow Transplant Research has recently shown that over 30% of allogeneic HCT recipients are at least 60 years old and that nearly 4% are aged 70 or more. Historically, the use of allogeneic HCT among elderly patients has been limited by age restrictions, reflecting physicians' concerns regarding prohibitive transplant-related mortality and HCT-associated morbidity. RECENT FINDINGS: The introduction of reduced intensity/toxicity conditioning regimens has allowed transplant Centers to carry out allogeneic HCT on patients previously considered not ideal candidates. The integration of specific risk scores could lead to better capture mental and physical frailties of older patients. Older adults less frequently have available medically fit siblings, able to donate, so, unrelated donors, familial haploidentical donors or umbilical cord blood grafts could potentially abrogate such a difficulty, allowing the curative potential of allogeneic HCT. SUMMARY: The appropriate assessing of allogeneic HCT feasibility for elderly patients should be the resonate application of different clinical and biological principles.

Identification of a serum and urine extracellular vesicle signature predicting renal outcome after kidney transplant.

BACKGROUND: A long-standing effort is dedicated towards the identification of biomarkers allowing the prediction of graft outcome after kidney transplant. Extracellular vesicles (EVs) circulating in body fluids represent an attractive candidate, as their cargo mirrors the originating cell and its pathophysiological status. The aim of the study was to investigate EV surface antigens as potential predictors of renal outcome after kidney transplant. METHODS: We characterized 37 surface antigens by flow cytometry, in serum and urine EVs from 58 patients who were evaluated before, and at 10-14 days, 3 months and 1 year after transplant, for a total of 426 analyzed samples. The outcome was defined according to estimated glomerular filtration rate (eGFR) at 1 year. RESULTS: Endothelial cells and platelets markers (CD31, CD41b, CD42a and CD62P) in serum EVs were higher at baseline in patients with persistent kidney dysfunction at 1 year, and progressively decreased after kidney transplant. Conversely, mesenchymal progenitor cell marker (CD1c, CD105, CD133, SSEEA-4) in urine EVs progressively increased after transplant in patients displaying renal recovery at follow-up. These markers correlated with eGFR, creatinine and proteinuria, associated with patient outcome at univariate analysis and were able to predict patient outcome at receiver operating characteristics curves analysis. A specific EV molecular signature obtained by supervised learning correctly classified patients according to 1-year renal outcome. CONCLUSIONS: An EV-based signature, reflecting the cardiovascular profile of the recipient, and the repairing/regenerative features of the graft, could be introduced as a non-invasive tool for a tailored management of follow-up of patients undergoing kidney transplant.

Changes in the biochemical taste of cytoplasmic and cell-free DNA are major fuels for inflamm-aging.

Inflamm-aging depicts the progressive activation of the innate immune system that accompanies human aging. Its role as a disease-predisposing condition has been proposed, but its molecular basis is still poorly understood. A wealth of literature conveys that, particularly upon stress, nuclear and mitochondrial genomes are released into the cytoplasmic and extracellular compartments. Cytoplasmic (cy) and cell-free (cf) DNA pools trigger inflammation and innate immunity at local and systemic level. In particular, cyDNA plays a crucial role in the phenomenon of cell senescence and in the cognate pro-inflammatory secretome. Here we propose that changes in a variety of biochemical characteristics "tastes" of cy- and cf-DNA (e.g. the amount of 8-oxo-deoxy-guanosine and 5-methyl-deoxy-cytosine, the proportion of DNA hybridized with RNA) potentially affect the capability of these DNA pools to ignite the innate immune system. We also underpin that telomeric sequences are major components of the cy/cfDNA payload. Telomere shortening, a hallmark of aging, causes the depletion of telomeric sequences in cy/cfDNA pool, thus unleashing their potential to exert an age-related activation of the innate immune system. Finally, we posit that various sources of DNA (extracellular vesicles, the commensal metagenome and food) contribute to the cy/cfDNA payloads. We speculate that changes in the biochemical "taste" of cy/cfDNA are major modifiers of inflamm-aging.

Androgen receptor in breast cancer: A wolf in sheep's clothing? A lesson from prostate cancer.

The possibility that a receptor for androgen is expressed in Breast Cancer (BC) is fascinating given that the tumor is predominantly estrogen-dependent. The androgen receptor (AR) is emerging as a new marker and a potential new therapeutic target in the treatment of BC patients. The recent availability of selective AR inhibitors (e.g. bicalutamide, enzalutamide, apalutamide) approved for the treatment of prostate cancer has opened up the possibility to use them in BC patients whose tumors express AR. However, AR appears to have various functions according to the BC subtype, e.g. ER-positive or triple negative BC and the patient prognosis is different on the basis of the presence or absence of estrogen and progesterone receptors. Moreover, a different AR expression was seen according to the various ethnicities. Of note, in population at low economical income, the availability of anti-AR compounds at low cost could open the possibility to treat AR-positive triple negative BC that are highly present in these populations. Up to now, AR detection is not routinely performed in BC. The standardization of AR detection methods could render AR an easily detectable marker in primary BC and metastatic samples. Nevertheless, the overall concordance of 60% of AR expression in primary tumor and metastasis implies that a clinician who need the AR value to give anti-AR therapy should have the data on both the tumor materials. Following the comprehensive studies on prostate cancer the possibility to test AR on liquid biopsies suggest the use of this biomarker for a real-time disease monitoring. Finally, considering the possibility to treat patients with immune checkpoint inhibitors there is the need to know the relation between microenvironment and AR in BC.

Packaging and transfer of mitochondrial DNA via exosomes regulate escape from dormancy in hormonal therapy-resistant breast cancer.

The horizontal transfer of mtDNA and its role in mediating resistance to therapy and an exit from dormancy have never been investigated. Here we identified the full mitochondrial genome in circulating extracellular vesicles (EVs) from patients with hormonal therapy-resistant (HTR) metastatic breast cancer. We generated xenograft models of HTR metastatic disease characterized by EVs in the peripheral circulation containing mtDNA. Moreover, these human HTR cells had acquired host-derived (murine) mtDNA promoting estrogen receptor-independent oxidative phosphorylation (OXPHOS). Functional studies identified cancer-associated fibroblast (CAF)-derived EVs (from patients and xenograft models) laden with whole genomic mtDNA as a mediator of this phenotype. Specifically, the treatment of hormone therapy (HT)-naive cells or HT-treated metabolically dormant populations with CAF-derived mtDNAhi EVs promoted an escape from metabolic quiescence and HTR disease both in vitro and in vivo. Moreover, this phenotype was associated with the acquisition of EV mtDNA, especially in cancer stem-like cells, expression of EV mtRNA, and restoration of OXPHOS. In summary, we have demonstrated that the horizontal transfer of mtDNA from EVs acts as an oncogenic signal promoting an exit from dormancy of therapy-induced cancer stem-like cells and leading to endocrine therapy resistance in OXPHOS-dependent breast cancer.

Immunosuppressive Treg cells acquire the phenotype of effector-T cells in chronic lymphocytic leukemia patients.

BACKGROUND: In chronic lymphocytic leukemia (CLL) disease onset and progression are influenced by the behavior of specific CD4+ T cell subsets, such as T regulatory cells (Tregs). Here, we focused on the phenotypic and functional characterization of Tregs in CLL patients to improve our understanding of the putative mechanism by which these cells combine immunosuppressive and effector-like properties. METHODS: Peripheral blood mononuclear cells were isolated from newly diagnosed CLL patients (n = 25) and healthy volunteers (n = 25). The phenotypic and functional characterization of Tregs and their subsets was assessed by flow cytometry. In vitro analysis of TH1, TH2, TH17 and Tregs cytokines was evaluated by IFN-γ, IL-4, IL-17A and IL-10 secretion assays. The transcriptional profiling of 84 genes panel was evaluated by RT2 Profiler PCR Array. Statistical analysis was carried out using exact non parametric Mann-Whitney U test. RESULTS: In all CLL samples, we found a significant increase in the frequency of IL-10-secreting Tregs and Tregs subsets, a significant rise of TH2 IL-4+ and TH17 IL-17A+ cells, and a higher percentage of IFN-γ/IL-10 and IL-4/IL-10 double-releasing CD4+ T cells. In addition, we also observed the up-regulation of innate immunity genes and the down-regulation of adaptive immunity ones. CONCLUSIONS: Our data show that Tregs switch towards an effector-like phenotype in CLL patients. This multifaceted behavior is accompanied by an altered cytokine profiling and transcriptional program of immune genes, leading to a dysfunction in immune response in the peripheral blood environment of CLL patients.

Convergent adaptation of cellular machineries in the evolution of large body masses and long life spans.

In evolutionary terms, life on the planet has taken the form of independently living cells for the majority of time. In comparison, the mammalian radiation is a relatively recent event. The common mammalian ancestor was probably small and short-lived. The "recent" acquisition of an extended longevity and large body mass of some species of mammals present on the earth today suggests the possibility that similar cellular mechanisms have been influenced by the forces of natural selection to create a convergent evolution of longevity. Many cellular mechanisms are potentially relevant for extending longevity; in this assay, we review the literature focusing primarily on two cellular features: (1) the capacity for extensive cellular proliferation of differentiated cells, while maintaining genome stability; and (2) the capacity to detect DNA damage. We have observed that longevity and body mass are both positively linked to these cellular mechanisms and then used statistical tools to evaluate their relative importance. Our analysis suggest that the capacity for extensive cellular proliferation while maintaining sufficient genome stability, correlates to species body mass while the capacity to correctly identify the presence of DNA damage seems more an attribute of long-lived species. Finally, our data are in support of the idea that a slower development, allowing for better DNA damage detection and handling, should associate with longer life span.

Carbonic Anhydrase 9 mRNA/microRNA34a Interplay in Hypoxic Human Mammospheres.

The hypoxic environment is a crucial component of the cancer stem cell niche and it is capable of eliciting stem cell features in cancer cells. We previously reported that SNAI2 up-regulates the expression of Carbonic Anhydrase iso-enzyme 9 (CA9) in hypoxic MCF7 cells. Here we show that SNAI2 down-regulates miR34a expression in hypoxic MCF7 cell-derived mammospheres. Next, we report on the capability of miR34a to decrease CA9 mRNA stability and CA9 protein expression. We also convey that the over-expression of cloned CA9-mRNA-3'UTR increases the mRNA half-life and protein levels of two miR34a targets JAGGED1 and NOTCH3. The data here reported shows that the SNAI2-dependent down-regulation of miR34a substantially contributes to the post-transcriptional up-regulation of CA9, and that CA9-mRNA-3'UTR acts as an endogenous microRNA sponge. We conclude that CA9/miR34 interplay shares in the hypoxic regulation of mammospheres and therefore, may play a relevant role in the hypoxic breast cancer stem cell niche.

Lamins are rapamycin targets that impact human longevity: a study in centenarians.

The dynamic organisation of the cell nucleus is profoundly modified during growth, development and senescence as a result of changes in chromatin arrangement and gene transcription. A plethora of data suggests that the nuclear lamina is a key player in chromatin dynamics and argues in favour of a major involvement of prelamin A in fundamental mechanisms regulating cellular senescence and organism ageing. As the best model to analyse the role of prelamin A in normal ageing, we used cells from centenarian subjects. We show that prelamin A is accumulated in fibroblasts from centenarians owing to downregulation of its specific endoprotease ZMPSTE24, whereas other nuclear envelope constituents are mostly unaffected and cells do not enter senescence. Accumulation of prelamin A in nuclei of cells from centenarians elicits loss of heterochromatin, as well as recruitment of the inactive form of 53BP1, associated with rapid response to oxidative stress. These effects, including the prelamin-A-mediated increase of nuclear 53BP1, can be reproduced by rapamycin treatment of cells from younger individuals. These data identify prelamin A and 53BP1 as new targets of rapamycin that are associated with human longevity. We propose that the reported mechanisms safeguard healthy ageing in humans through adaptation of the nuclear environment to stress stimuli.

Interleukin-6 and pro inflammatory status in the breast tumor microenvironment.

BACKGROUND: Greater than 50,000 new cases of breast cancer cases were diagnosed in Italy during 2013, with nearly 15,000 women succumbing to the disease. These epidemiological statistics highlight the overwhelming clinical dilemma of breast cancer and emphasize the need for novel therapeutic targets and prevention strategies. Countless studies in the fields of mammary gland development and breast cancer have led to an appreciation of a breast tumor microenvironment that actively contributes to the heterogeneous nature of breast cancer. METHODS: The current review will focus on the impact of IL-6 and in the breast tumor microenvironment. Excessive IL-6 has been demonstrated in primary breast tumors and breast cancer patient sera and is associated with poor clinical outcomes in breast cancer. These clinical associations are corroborated by emerging preclinical data revealing that IL-6 is a potent growth factor and promotes an epithelial-mesenchyme (EMT) phenotype in breast cancer cells to indicate that IL-6 in the breast tumor microenvironment is clinically relevant. RESULTS: High serum levels of interleukin-6 correlate with poor outcome in breast cancer patients. However, few data are yet available on the relationship between IL-6 and stem/progenitor cells, which may fuel the genesis of breast cancer in vivo. Mammospheres (MS) from node invasive breast carcinoma tissues express IL-6 mRNA at higher levels than MS from matched non-neoplastic mammary glands. IL-6 mRNA is detectable only in basal-like breast carcinoma tissues; our results reveal that IL-6 triggers a Notch-3-dependent upregulation of the Notch ligand Jagged-1, whose interaction with Notch-3 promotes the growth of MS and Michigan Cancer Foundation-7 (MCF-7)-derived spheroids. IL-6 induces a Notch-3-dependent upregulation of the carbonic anhydrase IX gene and promotes a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. CONCLUSIONS: In conclusion, our data support the hypothesis that IL-6 induces malignant features in Notch-3-expressing, stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

High-throughput genotyping of high-risk Human Papillomavirus by MALDI-TOF Mass Spectrometry-based method.

A high-throughput matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS)-based method was here developed to genotype 16 high-risk human papillomavirus (HPV) types in cervical cytology specimens. This method was compared to a commercial kit, the Inno-LiPA HPV genotyping assay, which detects a broad spectrum of HPV types. HPV DNA was assessed by the two methods in a total of 325 cervical cytology specimens collected in PreservCyt® solution. The overall agreement was almost perfect (Cohen's k=0.86) in term of positive and negative cases. Indeed, HPV types 16, 35, 56 and 66 showed the highest agreement values (>0.80). The highest agreement values (K >0.80) were found for all 16 HPV types in single infections, but only for HPV 16, 35, 45 and 56 in multiple infections. In conclusion, the high-throughput MS-based method developed here is well-suited for broad spectrum HPV genotyping in large-scale epidemiological studies.

PPARγ and RXR ligands disrupt the inflammatory cross-talk in the hypoxic breast cancer stem cells niche.

Cancer stem cells (CSCs) are affected by the local micro-environment, the niche, in which inflammatory stimuli and hypoxia act as steering factors. Here, two nuclear receptors (NRs) agonists, i.e. pioglitazone (PGZ), a ligand of peroxisome proliferator activated receptor-γ, and 6-OH-11-O-hydroxyphenanthrene (IIF), a ligand of retinoid X receptors, were investigated for their capability to interference with the cross-talk between breast CSCs and the niche compartment. We found that IIF potentiates the ability of PGZ to hamper the mammospheres-forming capability of human breast tumours and MCF7 cancer cells, reducing the expression of CSCs regulatory genes (Notch3, Jagged1, SLUG, Interleukin-6, Apolipoprotein E, Hypoxia inducible factor-1α and Carbonic anhydrase IX). Notably, these effects are not observed in normal-MS obtained from human breast tissue. Importantly, NRs agonists abolish the capability of hypoxic MCF7 derived exosomes to induce a pro-inflammatory phenotype in mammary glands fibroblasts. Moreover, NRs agonist also directly acts on breast tumour associated fibroblasts to downregulate nuclear factor-κB pathway and metalloproteinases (MMP2 and MMP9) expression and activity. In conclusion, NRs agonists disrupt the inflammatory cross-talk of the hypoxic breast CSCs niche.

Slug/ß-catenin-dependent proinflammatory phenotype in hypoxic breast cancer stem cells.

Cancer stem cell survival relies on the activation of inflammatory pathways, which is speculatively triggered by cell autonomous mechanisms or by microenvironmental stimuli. Here, we observed that hypoxic bone marrow stroma-derived transforming growth factor-ß 1 promotes the growth of human breast cancer stem cells as mammospheres. The ensuing Slug-dependent serine 139 phosphorylation of the DNA damage sensor H2AX in breast cancer stem cells induces tumor necrosis factor-α and IL-8 mRNAs, whose stability is enhanced by cytoplasmic ß-catenin. ß-Catenin also up-regulates and binds miR-221, reducing the stability of the miR-221 targets Rad51 and ERα mRNAs. Our data show that the Slug/ß-catenin-dependent activation of DNA damage signaling triggered by the hypoxic microenvironment sustains the proinflammatory phenotype of breast cancer stem cells.

Glutathione transferase-A2 S112T polymorphism predicts survival, transplant-related mortality, busulfan and bilirubin blood levels after allogeneic stem cell transplantation.

Busulfan liver metabolism depends on glutathione, a crucial mediator of cellular and systemic stress. Here we investigated 40 polymorphisms at 27 loci involved in hepatic glutathione homeostasis, with the aim of testing their impact on the clinical outcome of 185 busulfan-conditioned allogeneic transplants. GSTA2 S112T serine allele homozygosity is an independent prognostic factor for poorer survival (RR=2.388), for increased any time- and 100-day transplant-related mortality (RR=4.912 and RR=5.185, respectively). The genotype also predicts a wider busulfan area under the concentration-time curve (1214.36 ± 570.06 vs. 838.10 ± 282.40 mMol*min) and higher post-transplant bilirubin serum levels (3.280 ± 0.422 vs. 1.874+0.197 mg/dL). In vitro, busulfan elicits pro-inflammatory activation (increased NF-KappaB activity and interleukin-8 expression) in human hepatoma cells. At the same time, the drug down-regulates a variety of genes involved in bilirubin liver clearance: constitutive androstane receptor, multidrug resistance-associated protein, solute carrier organic anion transporters, and even GSTA2. It is worthy of note that GSTA2 also acts as an intra-hepatic bilirubin binding protein. These data underline the prognostic value of GSTA2 genetic variability in busulfan-conditioned allotransplants and suggest a patho-physiological model in which busulfan-induced inflammation leads to the impairment of post-transplant bilirubin metabolism.

Inflamm-aging of the stem cell niche: breast cancer as a paradigmatic example: breakdown of the multi-shell cytokine network fuels cancer in aged people.

Inflamm-aging is a relatively new terminology used to describe the age-related increase in the systemic pro-inflammatory status of humans. Here, we represent inflamm-aging as a breakdown in the multi-shell cytokine network, in which stem cells and stromal fibroblasts (referred to as the stem cell niche) become pro-inflammatory cytokine over-expressing cells due to the accumulation of DNA damage. Inflamm-aging self-propagates owing to the capability of pro-inflammatory cytokines to ignite the DNA-damage response in other cells surrounding DNA-damaged cells. Macrophages, the major cellular player in inflamm-aging, amplify the phenomenon, by broadcasting pro-inflammatory signals at both local and systemic levels. On the basis of this, we propose that inflamm-aging is a major contributor to the increase in cancer incidence and progression in aged people. Breast cancer will be presented as a paradigmatic example for this relationship.

Replicative senescence and high glucose induce the accrual of self-derived cytosolic nucleic acids in human endothelial cells.

Recent literature shows that loss of replicative ability and acquisition of a proinflammatory secretory phenotype in senescent cells is coupled with the build-in of nucleic acids in the cytoplasm. Its implication in human age-related diseases is under scrutiny. In human endothelial cells (ECs), we assessed the accumulation of intracellular nucleic acids during in vitro replicative senescence and after exposure to high glucose concentrations, which mimic an in vivo condition of hyperglycemia. We showed that exposure to high glucose induces senescent-like features in ECs, including telomere shortening and proinflammatory cytokine release, coupled with the accrual in the cytoplasm of telomeres, double-stranded DNA and RNA (dsDNA, dsRNA), as well as RNA:DNA hybrid molecules. Senescent ECs showed an activation of the dsRNA sensors RIG-I and MDA5 and of the DNA sensor TLR9, which was not paralleled by the involvement of the canonical (cGAS) and non-canonical (IFI16) activation of the STING pathway. Under high glucose conditions, only a sustained activation of TLR9 was observed. Notably, senescent cells exhibit increased proinflammatory cytokine (IL-1ß, IL-6, IL-8) production without a detectable secretion of type I interferon (IFN), a phenomenon that can be explained, at least in part, by the accumulation of methyl-adenosine containing RNAs. At variance, exposure to exogenous nucleic acids enhances both IL-6 and IFN-ß1 expression in senescent cells. This study highlights the accrual of cytoplasmic nucleic acids as a marker of senescence-related endothelial dysfunction, that may play a role in dysmetabolic age-related diseases.