Papers by Alessandro Zannini
Nature Communications
In this article, the author name Ilenia Segatto was incorrectly written as Ilaria Segatto. The or... more In this article, the author name Ilenia Segatto was incorrectly written as Ilaria Segatto. The original article has been corrected.
Cell Death & Differentiation, Nov 11, 2016
Biochimica Et Biophysica Acta - General Subjects, Oct 1, 2015
The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome i... more The p53 protein family, comprising p53, p63 and p73, is primarily involved in preserving genome integrity and preventing tumor onset, and also affects a range of physiological processes. Signal-dependent modifications of its members and of other pathway components provide cells with a sophisticated code to transduce a variety of stress signaling into appropriate responses. TP53 mutations are highly frequent in cancer and lead to the expression of mutant p53 proteins that are endowed with oncogenic activities and sensitive to stress signaling. p53 family proteins have unique structural and functional plasticity, and here we discuss the relevance of prolyl-isomerization to actively shape these features. The anti-proliferative functions of the p53 family are carefully activated upon severe stress and this involves the interaction with prolyl-isomerases. In particular, stress-induced stabilization of p53, activation of its transcriptional control over arrest- and cell death-related target genes and of its mitochondrial apoptotic function, as well as certain p63 and p73 functions, all require phosphorylation of specific S/T-P motifs and their subsequent isomerization by the prolyl-isomerase Pin1. While these functions of p53 counteract tumorigenesis, under some circumstances their activation by prolyl-isomerases may have negative repercussions (e.g. tissue damage induced by anticancer therapies and ischemia-reperfusion, neurodegeneration). Moreover, elevated Pin1 levels in tumor cells may transduce deregulated phosphorylation signaling into activation of mutant p53 oncogenic functions. The complex repertoire of biological outcomes induced by p53 finds mechanistic explanations, at least in part, in the association between prolyl-isomerases and the p53 pathway. This article is part of a Special Issue entitled Proline-directed foldases: Cell signaling catalysts and drug targets.
Nature Communications
Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer c... more Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent ...
Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer c... more Reprogramming of amino acid metabolism, sustained by oncogenic signaling, is crucial for cancer cell survival under nutrient limitation. Here we discovered that missense mutant p53 oncoproteins stimulate de novo serine/glycine synthesis and essential amino acids intake, promoting breast cancer growth. Mechanistically, mutant p53, unlike the wild-type counterpart, induces the expression of serine-synthesis-pathway enzymes and L-type amino acid transporter 1 (LAT1)/CD98 heavy chain heterodimer. This effect is exacerbated by amino acid shortage, representing a mutant p53-dependent metabolic adaptive response. When cells suffer amino acids scarcity, mutant p53 protein is stabilized and induces metabolic alterations and an amino acid transcriptional program that sustain cancer cell proliferation. In patient-derived tumor organoids, pharmacological targeting of either serine-synthesis-pathway and LAT1-mediated transport synergizes with amino acid shortage in blunting mutant p53-dependent growth. These findings reveal vulnerabilities potentially exploitable for tackling breast tumors bearing missense TP53 mutations.
Cancer stem cells (CSCs) are proposed to be responsible for breast cancer heterogeneity, chemothe... more Cancer stem cells (CSCs) are proposed to be responsible for breast cancer heterogeneity, chemotherapeutic treatment failure, metastatic spread and disease recurrence. The precise identification of the molecular bases that govern the induction and maintenance of CSCs and their aggressive phenotypes is of utmost importance, since it may provide the rational to develop effective therapeutic strategies. In particular there is a considerable effort in finding common pathways, mutations or histological features that might be targeted for therapy, overcoming breast cancer heterogeneity. Here we now demonstrate that CSC self-renewal, chemoresistance, tumour growth and metastases formation capabilities' are under direct control of Pin1's enzymatic activity on the Notch signalling pathway. In particular Pin1 protects the nuclear activated forms of Notch1 and Notch4 (N1/4-ICD) from their E3-ubiquitin-ligase Fbxw7α, thereby boosting their protein levels and transcriptional activity. Fbxw7α acts as a potent inhibitor of CSCs maintenance by promoting protein degradation of N1-and N4-ICD, and, as a consequence, this ubiquitin-ligase strongly decreased tumour growth and metastases dissemination in vivo. Interestingly, concomitant over-expression of Pin1 almost completely recovered all these aggressive breast cancer traits. In tissues from breast cancer patients, we observed Notch signalling over-activation despite presence of the negative regulator Fbxw7α, which relied on high Pin1 protein levels. Notably, activation of the Notch-Pin1 axis correlated with poor prognosis in these patients. As a consequence of our findings, suppression of Pin1 holds promise in reverting aggressive phenotypes in breast cancer through shrinkage of CSCs number and a concomitant gain in chemosensitivity, carrying important implications for breast cancers therapy. However, the cellular hierarchy of the mammary gland and the cell of origin of different classes of breast tumours are still open questions that await elucidation. 3.1.1 Classification of breast cancer subtypes: advances and pitfalls Gene expression profiles and histological staging offered the opportunity to classify human breast cancers in 5 different major subtypes: normal-like, luminal A, luminal B, HER2+, basal-the great proportion of breast tumours, namely ER, PR and HER2. For this reason, a large percentage of Basal-like breast tumours are also called Triple Negative breast cancers, or TNBC. Moreover, since there is no specific molecular target to date, the therapeutic regime for these patients still relies on chemotherapy. The Claudin-low subtype represents 12-15% of breast tumours and it is defined by low expression of genes involved in the regulation of tight junctions, inter-cellular adhesion (claudin-3,-4,-7) and epithelial markers (E-Cadherin). They are similar to basal-like tumours, but they differ by the fact that the claudin-low subtype is strongly infiltrated by immune system cells and moreover they are characterized by overexpressions of genes linked to mesenchymal differentiation and epithelial-to-mesenchymal transition (EMT). This subtype classification is not only relevant to define the most fitting therapeutic approach, but it is also useful to predict the clinical outcome and to provide, at times, possible explanation Figure 1. Stemness as a guiding principle that governs therapeutic response Three fields in biology-cancer genetics, epigenetics, and microenvironment-are coming together to provide increasing clarity to the processes that determine stemness and in turn influence clinical outcome. These three factors can influence stemness simultaneously, but they can also act independently over time. Through evolutionary time, different forces can impact a cell's stemness properties and thereby shape tumor progression and therapeutic response (Kreso and Dick, 2014). Seminal work performed by Shackleton and Stingl has provided proper means to isolate and characterize a stem cell population capable of reconstituting a functional mammary gland in mice, called mammary repopulating units (MRUs) (Shackleton et al., 2006; Stingl et al., 2006). MRU cells are purified from the mammary gland after digestion and sorted by both excluding cells expressing hematopoietic and endothelial markers and including the expression of CD24, α6-integrin (CD49f), Sca-1 and β1-integrin (CD29). In particular, cells with a CD24 med /CD49f high profile characterize the MRU population and it is present in a proportion of 1 MRU out of 1400 total cells in mice. In summary, a huge number of data allowed identifying a stem cell population that is able to generate, in vitro, mammospheres and colonies composed of both lineages and, more importantly, an entire functional mammary gland in vivo. Notably the isolation and characterisation of human mammary stem cells lacks a real in vivo validation, but the in vitro assays are well established and reliable. 3.1.3 Breast cancer stem cells Breast cancer stem cell (CSCs) research is one of the most investigated fields in breast cancer research in the last years. Like normal stem cells that are able to regenerate an organ, also CSCs are considered to be responsible for re-generation of the tumour, as demonstrated by sequential transplantation assays. From a clinical point of view breast CSCs are associated to aggressiveness in breast cancer since they are chemoresistant, they have been linked to metastases formation and associated to the undifferentiated, poor outcome breast cancer subtypes
Frontiers in Oncology, Feb 25, 2019
Cellular choices are determined by developmental and environmental stimuli through integrated sig... more Cellular choices are determined by developmental and environmental stimuli through integrated signal transduction pathways. These critically depend on attainment of proper activation levels that in turn rely on post-translational modifications (PTMs) of single pathway members. Among these PTMs, post-phosphorylation prolyl-isomerization mediated by PIN1 represents a unique mechanism of spatial, temporal and quantitative control of signal transduction. Indeed PIN1 was shown to be crucial for determining activation levels of several pathways and biological outcomes downstream to a plethora of stimuli. Of note, studies performed in different model organisms and humans have shown that hormonal, nutrient, and oncogenic stimuli simultaneously affect both PIN1 activity and the pathways that depend on PIN1-mediated prolyl-isomerization, suggesting the existence of evolutionarily conserved molecular circuitries centered on this isomerase. This review focuses on molecular mechanisms and cellular processes like proliferation, metabolism, and stem cell fate, that are regulated by PIN1 in physiological conditions, discussing how these are subverted in and hijacked by cancer cells. Current status and open questions regarding the use of PIN1 as biomarker and target for cancer therapy as well as clinical development of PIN1 inhibitors are also addressed.
Cancers, Dec 21, 2020
The possibility to generate in the laboratory faithful models of patients' tumors is of primary i... more The possibility to generate in the laboratory faithful models of patients' tumors is of primary importance to capture cancer complexity and study therapy response in a personalized setting. Tumor organoids are 3D cell cultures, obtained from patients' tumor tissues, that recapitulate several characteristics of the original tumor, thus representing a clinically relevant patient avatar. This study reports the generation and the molecular characterization of patient-derived organoids from invasive breast carcinomas. Our results proved the usefulness of these cancer models for designing patient-specific therapeutic approaches to treat highly aggressive cancers, but also highlighted the need to further improve this methodology to overcome its current limitations.
Embo Molecular Medicine, Dec 16, 2013
Nature Cell Biology, Dec 18, 2017
Tumour-associated p53 missense mutants act as driver oncogenes impacting cancer progression, meta... more Tumour-associated p53 missense mutants act as driver oncogenes impacting cancer progression, metastatic potential and drug resistance (Gain-of-Function, GOF)1. Mutant p53 protein stabilization is a prerequisite for GOF manifestation, however it does not represent an intrinsic property of p53 mutants, but rather requires secondary events2. Moreover, mutant p53 protein levels are often heterogeneous even within the same tumour, raising questions on the mechanisms that control local mutant p53 accumulation in some tumour cells but not in their neighbours 2,3. By investigating the cellular pathways that induce protection of mutant p53 from ubiquitin-*
Nature Communications, Jun 9, 2017
The prolyl isomerase PIN1, a critical modifier of multiple signalling pathways, is overexpressed ... more The prolyl isomerase PIN1, a critical modifier of multiple signalling pathways, is overexpressed in the majority of cancers and its activity strongly contributes to tumour initiation and progression. Inactivation of PIN1 function conversely curbs tumour growth and cancer stem cell expansion, restores chemosensitivity and blocks metastatic spread, thus providing the rationale for a therapeutic strategy based on PIN1 inhibition. Notwithstanding, potent PIN1 inhibitors are still missing from the arsenal of anti-cancer drugs. By a mechanism-based screening, we have identified a novel covalent PIN1 inhibitor, KPT-6566, able to selectively inhibit PIN1 and target it for degradation. We demonstrate that KPT-6566 covalently binds to the catalytic site of PIN1. This interaction results in the release of a quinone-mimicking drug that generates reactive oxygen species and DNA damage, inducing cell death specifically in cancer cells. Accordingly, KPT-6566 treatment impairs PIN1-dependent cancer phenotypes in vitro and growth of lung metastasis in vivo.
Nature Communications, Jan 19, 2017
The Hippo pathway is an oncosuppressor signalling cascade that plays a major role in the control ... more The Hippo pathway is an oncosuppressor signalling cascade that plays a major role in the control of cell growth, tissue homoeostasis and organ size. Dysregulation of the Hippo pathway leads to aberrant activation of the transcription co-activator YAP (Yes-associated protein) that contributes to tumorigenesis in several tissues. Here we identify glucocorticoids (GCs) as hormonal activators of YAP. Stimulation of glucocorticoid receptor (GR) leads to increase of YAP protein levels, nuclear accumulation and transcriptional activity in vitro and in vivo. Mechanistically, we find that GCs increase expression and deposition of fibronectin leading to the focal adhesion-Src pathway stimulation, cytoskeleton-dependent YAP activation and expansion of chemoresistant cancer stem cells. GR activation correlates with YAP activity in human breast cancer and predicts bad prognosis in the basal-like subtype. Our results unveil a novel mechanism of YAP activation in cancer and open the possibility to target GR to prevent cancer stem cells self-renewal and chemoresistance.
Oncotarget, Oct 15, 2015
Targeted anticancer therapies represent the most effective pharmacological strategies in terms of... more Targeted anticancer therapies represent the most effective pharmacological strategies in terms of clinical responses. In this context, genetic alteration of several oncogenes represents an optimal predictor of response to targeted therapy. Integration of large-scale molecular and pharmacological data from cancer cell lines promises to be effective in the discovery of new genetic markers of drug sensitivity and of clinically relevant anticancer compounds. To define novel pharmacogenomic dependencies in cancer, we created the Mutations and Drugs Portal (MDP, http:// mdp.unimore.it), a web accessible database that combines the cell-based NCI60 screening of more than 50,000 compounds with genomic data extracted from the Cancer Cell Line Encyclopedia and the NCI60 DTP projects. MDP can be queried for drugs active in cancer cell lines carrying mutations in specific cancer genes or for genetic markers associated to sensitivity or resistance to a given compound. As proof of performance, we interrogated MDP to identify both known and novel pharmacogenomics associations and unveiled an unpredicted combination of two FDA-approved compounds, namely statins and Dasatinib, as an effective strategy to potently inhibit YAP/TAZ in cancer cells.
Nature Communications, Mar 22, 2019
Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that re... more Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate lipid biosynthesis and adipogenesis by controlling the expression of several enzymes required for cholesterol, fatty acid, triacylglycerol and phospholipid synthesis. In vertebrates, SREBP activation is mainly controlled by a complex and well-characterized feedback mechanism mediated by cholesterol, a crucial bio-product of the SREBP-activated mevalonate pathway. In this work, we identified acto-myosin contractility and mechanical forces imposed by the extracellular matrix (ECM) as SREBP1 regulators. SREBP1 control by mechanical cues depends on geranylgeranyl pyrophosphate, another key bio-product of the mevalonate pathway, and impacts on stem cell fate in mouse and on fat storage in Drosophila. Mechanistically, we show that activation of AMP-activated protein kinase (AMPK) by ECM stiffening and geranylgeranylated RhoA-dependent acto-myosin contraction inhibits SREBP1 activation. Our results unveil an unpredicted and evolutionary conserved role of SREBP1 in rewiring cell metabolism in response to mechanical cues.
Aging cell, Jun 3, 2017
Caloric restriction (CR) can delay onset of several age-related pathophysiologies and extend life... more Caloric restriction (CR) can delay onset of several age-related pathophysiologies and extend lifespan in various species, including rodents. CR also induces metabolic remodeling involved in activation of lipid metabolism, enhancement of mitochondrial biogenesis, and reduction of oxidative stress in white adipose tissue (WAT). In studies using genetically modified mice with extended lifespans, WAT characteristics influenced mammalian lifespans. However, molecular mechanisms underlying CR-associated metabolic remodeling of WAT remain unclear. Sterol regulatory element-binding protein-1c (Srebp-1c), a master transcription factor of fatty acid (FA) biosynthesis, is responsible for the pathogenesis of fatty liver (steatosis). Our study showed that, under CR conditions, Srebp-1c enhanced mitochondrial biogenesis via increased expression of peroxisome proliferator-activated receptor gamma coactivator-1α (Pgc-1α) and upregulated expression of proteins involved in FA biosynthesis within WAT....
Cancers, 2020
Tumor organoids are tridimensional cell culture systems that are generated in vitro from surgical... more Tumor organoids are tridimensional cell culture systems that are generated in vitro from surgically resected patients’ tumors. They can be propagated in culture maintaining several features of the tumor of origin, including cellular and genetic heterogeneity, thus representing a promising tool for precision cancer medicine. Here, we established patient-derived tumor organoids (PDOs) from different breast cancer subtypes (luminal A, luminal B, human epidermal growth factor receptor 2 (HER2)-enriched, and triple negative). The established model systems showed histological and genomic concordance with parental tumors. However, in PDOs, the ratio of diverse cell populations was frequently different from that originally observed in parental tumors. We showed that tumor organoids represent a valuable system to test the efficacy of standard therapeutic treatments and to identify drug resistant populations within tumors. We also report that inhibitors of mechanosignaling and of Yes-associat...
Frontiers in Oncology, 2019
Cellular choices are determined by developmental and environmental stimuli through integrated sig... more Cellular choices are determined by developmental and environmental stimuli through integrated signal transduction pathways. These critically depend on attainment of proper activation levels that in turn rely on post-translational modifications (PTMs) of single pathway members. Among these PTMs, post-phosphorylation prolyl-isomerization mediated by PIN1 represents a unique mechanism of spatial, temporal and quantitative control of signal transduction. Indeed PIN1 was shown to be crucial for determining activation levels of several pathways and biological outcomes downstream to a plethora of stimuli. Of note, studies performed in different model organisms and humans have shown that hormonal, nutrient, and oncogenic stimuli simultaneously affect both PIN1 activity and the pathways that depend on PIN1-mediated prolyl-isomerization, suggesting the existence of evolutionarily conserved molecular circuitries centered on this isomerase. This review focuses on molecular mechanisms and cellular processes like proliferation, metabolism, and stem cell fate, that are regulated by PIN1 in physiological conditions, discussing how these are subverted in and hijacked by cancer cells. Current status and open questions regarding the use of PIN1 as biomarker and target for cancer therapy as well as clinical development of PIN1 inhibitors are also addressed.
Nature cell biology, 2018
Tumour-associated p53 missense mutants act as driver oncogenes affecting cancer progression, meta... more Tumour-associated p53 missense mutants act as driver oncogenes affecting cancer progression, metastatic potential and drug resistance (gain-of-function) . Mutant p53 protein stabilization is a prerequisite for gain-of-function manifestation; however, it does not represent an intrinsic property of p53 mutants, but rather requires secondary events . Moreover, mutant p53 protein levels are often heterogeneous even within the same tumour, raising questions on the mechanisms that control local mutant p53 accumulation in some tumour cells but not in their neighbours . By investigating the cellular pathways that induce protection of mutant p53 from ubiquitin-mediated proteolysis, we found that HDAC6/Hsp90-dependent mutant p53 accumulation is sustained by RhoA geranylgeranylation downstream of the mevalonate pathway, as well as by RhoA- and actin-dependent transduction of mechanical inputs, such as the stiffness of the extracellular environment. Our results provide evidence for an unpredict...
Nature communications, Jun 9, 2017
The prolyl isomerase PIN1, a critical modifier of multiple signalling pathways, is overexpressed ... more The prolyl isomerase PIN1, a critical modifier of multiple signalling pathways, is overexpressed in the majority of cancers and its activity strongly contributes to tumour initiation and progression. Inactivation of PIN1 function conversely curbs tumour growth and cancer stem cell expansion, restores chemosensitivity and blocks metastatic spread, thus providing the rationale for a therapeutic strategy based on PIN1 inhibition. Notwithstanding, potent PIN1 inhibitors are still missing from the arsenal of anti-cancer drugs. By a mechanism-based screening, we have identified a novel covalent PIN1 inhibitor, KPT-6566, able to selectively inhibit PIN1 and target it for degradation. We demonstrate that KPT-6566 covalently binds to the catalytic site of PIN1. This interaction results in the release of a quinone-mimicking drug that generates reactive oxygen species and DNA damage, inducing cell death specifically in cancer cells. Accordingly, KPT-6566 treatment impairs PIN1-dependent cancer...
Nature communications, Jan 19, 2017
The Hippo pathway is an oncosuppressor signalling cascade that plays a major role in the control ... more The Hippo pathway is an oncosuppressor signalling cascade that plays a major role in the control of cell growth, tissue homoeostasis and organ size. Dysregulation of the Hippo pathway leads to aberrant activation of the transcription co-activator YAP (Yes-associated protein) that contributes to tumorigenesis in several tissues. Here we identify glucocorticoids (GCs) as hormonal activators of YAP. Stimulation of glucocorticoid receptor (GR) leads to increase of YAP protein levels, nuclear accumulation and transcriptional activity in vitro and in vivo. Mechanistically, we find that GCs increase expression and deposition of fibronectin leading to the focal adhesion-Src pathway stimulation, cytoskeleton-dependent YAP activation and expansion of chemoresistant cancer stem cells. GR activation correlates with YAP activity in human breast cancer and predicts bad prognosis in the basal-like subtype. Our results unveil a novel mechanism of YAP activation in cancer and open the possibility to...
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Papers by Alessandro Zannini