Silvano Piazza

Effects of Age and Heart Failure on Human Cardiac Stem Cell Function

Currently, it is unknown whether defects in stem cell growth and differentiation contribute to myocardial aging and chronic heart failure (CHF), and whether a compartment of functional human cardiac stem cells (hCSCs) persists in the decompensated heart. To determine whether aging and CHF are critical determinants of the loss in growth reserve of the heart, the properties of hCSCs were evaluated in 18 control and 23 explanted hearts. Age and CHF showed a progressive decrease in functionally competent hCSCs. Chronological age was a major predictor of five biomarkers of hCSC senescence: telomeric shortening, attenuated telomerase activity, telomere dysfunction-induced foci, and p21(Cip1) and p16(INK4a) expression. CHF had similar consequences for hCSCs, suggesting that defects in the balance between cardiomyocyte mass and the pool of nonsenescent hCSCs may condition the evolution of the decompensated myopathy. A correlation was found previously between telomere length in circulating bone marrow cells and cardiovascular diseases, but that analysis was restricted to average telomere length in a cell population, neglecting the fact that telomere attrition does not occur uniformly in all cells. The present study provides the first demonstration that dysfunctional telomeres in hCSCs are biomarkers of aging and heart failure. The biomarkers of cellular senescence identified here can be used to define the birth date of hCSCs and to sort young cells with potential therapeutic efficacy.

A Pin1/Mutant p53 Axis Promotes Aggressiveness in Breast Cancer

TP53 missense mutations dramatically influence tumor progression, however, their mechanism of action is still poorly understood. Here we demonstrate the fundamental role of the prolyl isomerase Pin1 in mutant p53 oncogenic functions. Pin1 enhances tumorigenesis in a Li-Fraumeni mouse model and cooperates with mutant p53 in Ras-dependent transformation. In breast cancer cells, Pin1 promotes mutant p53 dependent inhibition of the antimetastatic factor p63 and induction of a mutant p53 transcriptional program to increase aggressiveness. Furthermore, we identified a transcriptional signature associated with poor prognosis in breast cancer and, in a cohort of patients, Pin1 overexpression influenced the prognostic value of p53 mutation. These results define a Pin1/mutant p53 axis that conveys oncogenic signals to promote aggressiveness in human cancers.

Multipotent Progenitor Cells Are Present in Human Peripheral Blood

To determine whether the peripheral blood in humans contains a population of multipotent progenitor cells (MPCs), products of leukapheresis were obtained from healthy donor volunteers following the administration of granulocyte colony-stimulating factor. Small clusters of adherent proliferating cells were collected, and these cells continued to divide up to 40 population doublings without reaching replicative senescence and growth arrest. MPCs were positive for the transcription factors Nanog, Oct3/4, Sox2, c-Myc, and Klf4 and expressed several antigens characteristic of mesenchymal stem cells. However, they were negative for markers of hematopoietic stem/progenitor cells and bone marrow cell lineages. MPCs had a cloning efficiency of ≈3%, and following their expansion, retained a highly immature phenotype. Under permissive culture conditions, MPCs differentiated into neurons, glial cells, hepatocytes, cardiomyocytes, endothelial cells, and osteoblasts. Moreover, the gene expression profile of MPCs partially overlapped with that of neural and embryonic stem cells, further demonstrating their primitive, uncommitted phenotype. Following subcutaneous transplantation in nonimmunosuppressed mice, MPCs migrated to distant organs and integrated structurally and functionally within the new tissue, acquiring the identity of resident parenchymal cells. In conclusion, undifferentiated cells with properties of embryonic stem cells can be isolated and expanded from human peripheral blood after granulocyte colony-stimulating factor administration. This cell pool may constitute a unique source of autologous cells with critical clinical import.

Oncogenic miR-181a/b affect the DNA damage response in aggressive breast cancer

Breast cancer is a heterogeneous tumor type characterized by a complex spectrum of molecular aberrations, resulting in a diverse array of malignant features and clinical outcomes. Deciphering the molecular mechanisms that fuel breast cancer development and act as determinants of aggressiveness is a primary need to improve patient management. Among other alterations, aberrant expression of microRNAs has been found in breast cancer and other human tumors, where they act as either oncogenes or tumor suppressors by virtue of their ability to finely modulate gene expression at the post-transcriptional level. In this study, we describe a new role for miR-181a/b as negative regulators of the DNA damage response in breast cancer, impacting on the expression and activity of the stress-sensor kinase ataxia telangiectasia mutated (ATM). We report that miR-181a and miR-181b were overexpressed in more aggressive breast cancers, and their expression correlates inversely with ATM levels. Moreover we demonstrate that deregulated expression of miR-181a/b determines the sensitivity of triple-negative breast cancer cells to the poly-ADP-ribose-polymerase1 (PARP1) inhibition. These evidences suggest that monitoring the expression of miR-181a/b could be helpful in tailoring more effective treatments based on inhibition of PARP1 in breast and other tumor types.

Mutant p53 Reprograms TNF Signaling in Cancer Cells through Interaction with the Tumor Suppressor DAB2IP

Inflammation is a significant factor in cancer development, and a molecular understanding of the parameters dictating the impact of inflammation on cancers could significantly improve treatment. The tumor suppressor p53 is frequently mutated in cancer, and p53 missense mutants (mutp53) can acquire oncogenic properties. We report that cancer cells with mutp53 respond to inflammatory cytokines increasing their invasive behavior. Notably, this action is coupled to expression of chemokines that can expose the tumor to host immunity, potentially affecting response to therapy. Mechanistically, mutp53 fuels NF-κB activation while it dampens activation of ASK1/JNK by TNFα, and this action depends on mutp53 binding and inhibiting the tumor suppressor DAB2IP in the cytoplasm. Interfering with such interaction reduced aggressiveness of cancer cells in xenografts. This interaction is an unexplored mechanism by which mutant p53 can influence tumor evolution, with implications for our understanding of the complex role of inflammation in cancer.

YAP enhances the pro-proliferative transcriptional activity of mutant p53 proteins

Mutant p53 proteins are present in more than half of human cancers. Yes‐associated protein (YAP) is a key transcriptional regulator controlling organ growth, tissue homeostasis, and cancer. Here, we report that these two determinants of human malignancy share common transcriptional signatures. YAP physically interacts with mutant p53 proteins in breast cancer cells and potentiates their pro‐proliferative transcriptional activity. We found YAP as well as mutant p53 and the transcription factor NF‐Y onto the regulatory regions of cyclin A, cyclin B, and CDK1 genes. Either mutant p53 or YAP depletion down‐regulates cyclin A, cyclin B, and CDK1 gene expression and markedly slows the growth of diverse breast cancer cell lines. Pharmacologically induced cytoplasmic re‐localization of YAP reduces the expression levels of cyclin A, cyclin B, and CDK1 genes both in vitro and in vivo. Interestingly, primary breast cancers carrying p53 mutations and displaying high YAP activity exhibit higher expression levels of cyclin A, cyclin B, and CDK1 genes when compared to wt‐p53 tumors.

GTSE1 is a microtubule plus-end tracking protein that regulates EB1-dependent cell migration.

The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is necessary for cell migration, but how microtubules and microtubule-associated proteins regulate multiple pathways promoting cell migration remains unclear. Microtubule plus-end binding proteins (+TIPs) are emerging as important players in many cellular functions, including cell migration. Here we identify a +TIP, GTSE1, that promotes cell migration. GTSE1 accumulates at growing microtubule plus ends through interaction with the EB1+TIP. The EB1-dependent +TIP activity of GTSE1 is required for cell migration, as well as for microtubule-dependent disassembly of focal adhesions. GTSE1 protein levels determine the migratory capacity of both nontransformed and breast cancer cell lines. In breast cancers, increased GTSE1 expression correlates with invasive potential, tumor stage, and time to distant metastasis, suggesting that misregulation of GTSE1 expression could be associated with increased invasive potential.

A gene expression signature of retinoblastoma loss-of-function is a predictive biomarker of resistance to palbociclib in breast cancer cell lines and is prognostic in patients with ER positive early breast cancer

Palbociclib is a CDK4/6 inhibitor that received FDA approval for treatment of hormone receptor positive (HR+) HER2 negative (HER2neg) advanced breast cancer. To better personalize patients treatment it is critical to identify subgroups that would mostly benefit from it. We hypothesize that complex alterations of the Retinoblastoma (Rb) pathway might be implicated in resistance to CDK4/6 inhibitors and aim to investigate whether signatures of Rb loss-of-function would identify breast cancer cell lines resistant to palbociclib. We established a gene expression signature of Rb loss-of-function (RBsig) by identifying genes correlated with E2F1 and E2F2 expression in breast cancers within The Cancer Genome Atlas. We assessed the RBsig prognostic role in the METABRIC and in a comprehensive breast cancer meta-dataset. Finally, we analyzed whether RBsig would discriminate palbociclib-sensitive and -resistant breast cancer cells in a large RNA sequencing-based dataset. The RBsig was associated with RB1 genetic status in all tumors (p <7e-32) and in luminal or basal subtypes (p < 7e-11 and p < 0.002, respectively). The RBsig was prognostic in the METABRIC dataset (discovery: HR = 1.93 [1.5-2.4] p = 1.4e-08; validation: HR = 2.01 [1.6-2.5] p = 1.3e-09). Untreated and endocrine treated patients with estrogen receptor positive breast cancer expressing high RBsig had significantly worse recurrence free survival compared to those with low RBsig (HR = 2.37 [1.8 − 3.2] p = 1.87e−08 and HR = 2.62 [1.9− 3.5] p = 8.6e−11, respectively). The RBsig was able to identify palbociclib resistant and sensitive breast cancer cells (ROC AUC = 0,7778). Signatures of RB loss might be helpful in personalizing treatment of patients with HR+/HER2neg breast cancer. Further validation in patients receiving palbociclib is warranted.

Translating Proteomic Into Functional Data: An High Mobility Group A1 (HMGA1) Proteomic Signature Has Prognostic Value in Breast Cancer

Cancer is a very heterogeneous disease, and biological variability adds a further level of complexity, thus limiting the ability to identify new genes involved in cancer development. Oncogenes whose expression levels control cell aggressiveness are very useful for developing cellular models that permit differential expression screenings in isogenic contexts. HMGA1 protein has this unique property because it is a master regulator in breast cancer cells that control the transition from a nontumorigenic epithelial-like phenotype toward a highly aggressive mesenchymal-like one. The proteins extracted from HMGA1-silenced and control MDA-MB-231 cells were analyzed using label-free shotgun mass spectrometry. The differentially expressed proteins were cross-referenced with DNA microarray data obtained using the same cellular model and the overlapping genes were filtered for factors linked to poor prognosis in breast cancer gene expression meta-data sets, resulting in an HMGA1 protein signature composed of 21 members (HRS, HMGA1 reduced signature). This signature had a prognostic value (overall survival, relapse-free survival, and distant metastasis-free survival) in breast cancer. qRT-PCR, Western blot, and immunohistochemistry analyses validated the link of three members of this signature (KIFC1, LRRC59, and TRIP13) with HMGA1 expression levels both in vitro and in vivo and wound healing assays demonstrated that these three proteins are involved in modulating tumor cell motility. Combining proteomic and genomic data with the aid of bioinformatic tools, our results highlight the potential involvement in neoplastic transformation of a restricted list of factors with an as-yet-unexplored role in cancer. These factors are druggable targets that could be exploited for the development of new, targeted therapeutic approaches in triple-negative breast cancer.

Critical role of lysosomes in the dysfunction of human Cardiac Stem Cells obtained from failing hearts

UNLABELLED The in vivo reparative potential of Cardiac Stem Cells (CSC), cultured from explanted failing hearts (E-), is impaired by cellular senescence. Moreover, E-CSC are characterized, with respect to CSC obtained from healthy donors (D-), by an arrest in the autophagic degradation. Although the lysosome plays a pivotal role in cellular homeostasis and defects of this organelle may be associated with aging and heart failure, the lysosomal function of CSC has never been investigated. The aim of this work was to focus on the Lysosomal Compartment (LC) of E-CSC, evaluating elements that could jeopardize lysosome functionality. METHODS AND RESULTS Bioinformatics analysis conducted on genes differentially expressed between D- and E-CSC identified lysosomal-related gene sets as significantly enriched. Moreover, 29 differentially expressed genes were part of CLEAR (Coordinated Lysosomal Expression and Regulation) gene network, by which Transcription Factor EB (TFEB) regulates cellular clearance. Consistently, live cell imaging and flow cytometry analyses showed that the lysosomes of E-CSC are less acidic than the D-CSC ones. Furthermore, confocal microscopy showed in E-CSC: an accumulation of intralysosomal lipofuscins, a reduction of cathepsin B activity, evidence of lysosome membrane permeabilization, and the reduction of the nuclear active TFEB. The use of Rapamycin (TORC1 inhibitor) was able on one hand to increase TFEB activation and, on the other hand, to reduce lipofuscin mass, potentiating the lysosomal functionality. CONCLUSIONS This study demonstrated for the first time that E-CSC are characterized by a blunted activation of TFEB and an altered proteostasis. TORC1 hyperactivation plays a central role in this phenomenon.