quantification-of-self-renewal-in-murine-mammosphere-cultures

Quantification of Self-renewal in Murine Mammosphere Cultures

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...

European Institute of Oncology IRCCS

University of Milan

Loss of p53 function is invariably associated with cancer. Its role in tumor growth was recently linked to its effects on cancer stem cells (CSCs), although the underlying molecular mechanisms remain unknown. Here, we show that c-myc is a transcriptional target of p53 in mammary stem cells (MaSCs) and is activated in breast tumors as a consequence of p53 loss. Constitutive Myc expression in normal mammary cells leads to increased frequency of MaSC symmetric divisions, extended MaSC replicative-potential, and MaSC-reprogramming of progenitors, whereas Myc activation in breast cancer is necessary and sufficient to maintain the expanding pool of CSCs. Concomitant p53 loss and Myc activation trigger the expression of 189 mitotic genes, which identify patients at high risk of mortality and relapse, independently of other risk factors. Altogether, deregulation of the p53:Myc axis in mammary tumors increases CSC content and plasticity and is a critical determinant of tumor growth and clinical aggressiveness.

p53 Loss in Breast Cancer Leads to Myc Activation, Increased Cell Plasticity, and Expression of a Mitotic Signature with Prognostic Value.

Acute myeloid leukemia (AML) is one of the most common hematological malignancies, characterized by high relapse and mortality rates. The inherent intra-tumor heterogeneity in AML is thought to play an important role in disease recurrence and resistance to chemotherapy. Although experimental protocols for cell proliferation studies are well established and widespread, they are not easily applicable to in vivo contexts, and the analysis of related time-series data is often complex to achieve. To overcome these limitations, model-driven approaches can be exploited to investigate different aspects of cell population dynamics.

Modeling cell proliferation in human acute myeloid leukemia xenografts.

Tumor cells may adapt to metabolic challenges by alternating between glycolysis and oxidative phosphorylation (OXPHOS). To target this metabolic plasticity, we combined intermittent fasting, a clinically feasible approach to reduce glucose availability, with the OXPHOS inhibitor metformin. In mice exposed to 24-h feeding/fasting cycles, metformin impaired tumor growth only when administered during fasting-induced hypoglycemia. Synergistic anti-neoplastic effects of the metformin/hypoglycemia combination were mediated by glycogen synthase kinase 3β (GSK3β) activation downstream of PP2A, leading to a decline in the pro-survival protein MCL-1, and cell death. Mechanistically, specific activation of the PP2A-GSK3β axis was the sum of metformin-induced inhibition of CIP2A, a PP2A suppressor, and of upregulation of the PP2A regulatory subunit B56δ by low glucose, leading to an active PP2A-B56δ complex with high affinity toward GSK3β.

Combination of Hypoglycemia and Metformin Impairs Tumor Metabolic Plasticity and Growth by Modulating the PP2A-GSK3β-MCL-1 Axis.

It is not clear how spontaneous DNA double-strand breaks (DSBs) form and are processed in normal cells, and whether they predispose to cancer-associated translocations. We show that DSBs in normal mammary cells form upon release of paused RNA polymerase II (Pol II) at promoters, 5' splice sites and active enhancers, and are processed by end-joining in the absence of a canonical DNA-damage response. Logistic and causal-association models showed that Pol II pausing at long genes is the main predictor and determinant of DSBs. Damaged introns with paused Pol II-pS5, TOP2B and XRCC4 are enriched in translocation breakpoints, and map at topologically associating domain boundary-flanking regions showing high interaction frequencies with distal loci. Thus, in unperturbed growth conditions, release of paused Pol II at specific loci and chromatin territories favors DSB formation, leading to chromosomal translocations.

Release of paused RNA polymerase II at specific loci favors DNA double-strand-break formation and promotes cancer translocations.

Human breast cancer is characterized by a high degree of inter-patients heterogeneity in terms of histology, genomic alterations, gene expression patterns, and metastatic behavior, which deeply influences individual prognosis and treatment response. The main cause of mortality in breast cancer is the therapy-resistant metastatic disease, which sets the priority for novel treatment strategies for these patients. In the present study, we demonstrate that Patient Derived Xenografts (PDXs) that were obtained from metastatic and therapy-resistant breast cancer samples recapitulate the wide spectrum of the disease in terms of histologic subtypes and mutational profiles, as evaluated by whole exome sequencing. We have integrated genomic and transcriptomic data to identify oncogenic and actionable pathways in each PDX. By taking advantage of primary short-term in vitro cultures from PDX tumors, we showed their resistance to standard chemotherapy (Paclitaxel), as seen in the patients. Moreover, we selected targeting drugs and analyzed PDX sensitivity to single agents or to combination of targeted and standard therapy on the basis of PDX-specific genomic or transcriptomic alterations. Our data demonstrate that PDXs represent a suitable model to test new targeting drugs or drug combinations and to prioritize personalized therapeutic regimens for pre-clinal and clinical tests.

Development of Personalized Therapeutic Strategies by Targeting Actionable Vulnerabilities in Metastatic and Chemotherapy-Resistant Breast Cancer PDXs.

Tumor mutational burden (TMB), the total number of somatic coding mutations in a tumor, is emerging as a promising biomarker for immunotherapy response in cancer patients. TMB can be quantitated by a number of NGS-based sequencing technologies. Whole Exome Sequencing (WES) allows comprehensive measurement of TMB and is considered the gold standard. However, to date WES remains confined to research settings, due to high cost of the large genomic space sequenced. In the clinical setting, instead, targeted enrichment panels (gene panels) of various genomic sizes are emerging as the routine technology for TMB assessment. This stimulated the development of various methods for panel-based TMB quantification, and prompted the multiplication of studies assessing whether TMB can be confidently estimated from the smaller genomic space sampled by gene panels. In this review, we inventory the collection of available gene panels tested for this purpose, illustrating their technical specifications and describing their accuracy and clinical value in TMB assessment. Moreover, we highlight how various experimental, platform-related or methodological variables, as well as bioinformatic pipelines, influence panel-based TMB quantification. The lack of harmonization in panel-based TMB quantification, of adequate methods to convert TMB estimates across different panels and of robust predictive cutoffs, currently represents one of the main limitations to adopt TMB as a biomarker in clinical practice. This overview on the heterogeneous landscape of panel-based TMB quantification aims at providing a context to discuss common standards and illustrates the strong need of further validation and consolidation studies for the clinical interpretation of panel-based TMB values.

Tumor mutational burden quantification from targeted gene panels: major advancements and challenges.

Autophagy is a conserved self-eating process that delivers cytoplasmic material to the lysosome to allow degradation of intracellular components, including soluble, unfolded and aggregated proteins, damaged organelles, and invading microorganisms. Autophagy provides a homeostatic control mechanism and is essential for balancing sources of energy in response to nutrient stress. Autophagic dysfunction or dysregulation has been implicated in several human pathologies, including cancer and neurodegeneration, and its modulation has substantial potential as a therapeutic strategy. Given the relevant clinical and therapeutic implications of autophagy, there is emerging intense interest in the identification of the key factors regulating the components of the autophagic machinery. Various post-translational modifications, including ubiquitylation, have been implicated in autophagy control. The list of the E3 ubiquitin protein ligases involved in the regulation of several steps of the autophagic process is continuously growing. In this review, we will focus on recent advances in the understanding of the role of the homologous to the E6AP carboxyl terminus-type E3 ubiquitin ligases in autophagy control.

Emerging roles of HECT-type E3 ubiquitin ligases in autophagy regulation.

Obesity correlates with hematological malignances including leukemias, but risk of specific leukemia subtypes like Acute Promyelocytic Leukemia and underlying molecular mechanisms are poorly understood. We explored multiple datasets for correlation between leukemia, Body Mass Index and molecular features. In a population-based study (n=5.2 million), we correlated Body Mass Index with promyelocytic, other acute myeloid, lymphoid or other leukemias. In cross-sectional studies, we tested body mass index deviation in promyelocytic leukemia trial cohorts from what expected based on national surveys. We interrogated The Cancer Genome Atlas for transcriptional signatures and mutations enriched in promyelocytic leukemia and/or obesity and confirmed correlation between body mass and FLT3 mutations in promyelocytic leukemia cohorts by logistic regression. In the population-based study, Hazard Ratio per 5 kg/m2 increase was: promyelocytic leukemia 1.44 (95% CI 1.0-2.08); non-promyelocytic acute myeloid leukemias 1.17 (1.10-1.26); lymphoid leukemias 1.04 (1.0-1.09); other 1.10 (1.04-1.15). In cross-sectional studies, body mass deviated significantly from expected (Italy p<0.001, Spain p=0.011, USA p<0.001). Promyelocytic leukemia showed upregulation of polyunsaturated fatty acid metabolism genes. Oddds of FLT3 mutations were higher in obese acute myeloid leukemias (Odds Ratio=2.4, p=0.007), whether promyelocytic or not, a correlation confirmed in the pooled promyelocytic leukemia cohorts (OR 1.22, 1.05-1.43 per 5 kg/m2). These results strengthen the evidence for obesity as a bona fide risk factor for myeloid leukemias and in particular APL. FLT3 mutations and polyunsaturated fatty acid metabolism may play a previously underappreciated role in obesity-associated leukemogenesis.

Obesity is a risk factor for acute promyelocytic leukemia: evidence from population and cross-sectional studies studies and correlation with flt3 mutations and polyunsaturated fatty acid metabolism.

Since their appearance, humanized mice carrying human immune system seemed promising tools to study the crosstalk between cancer and immunity. The NOD-scidIL2Rgammanull (NSG) mice engrafted with human cord blood (hCB)-CD34+ cells have been proposed to be a valuable tool to reproduce human immune system in mouse. However, the lack of solid evidences on the functionality of their human immune components limits their usage in immune-oncology. We report that (hCB)-CD34+ cells lose their ability to propagate and originate bone marrow-derived human immune cells after two serial transplantations in NSG mice. We demonstrate that transplants of bone marrow patient-derived acute myeloid leukemias (hAMLs) grow very similarly in the humanized (hCB)-CD34+ NSG and parental NSG mice. The similar extent of engraftment and development of leukemias in (hCB)-CD34+ NSG and controls suggests a poor human immune response against not compatible hAMLs. Our findings suggest that (hCB)-CD34+ NSG mice are transient and/or incomplete carriers of the human immune system and, therefore, represent a suboptimal tool to study the interaction between tumor and immune cells.

Human cord blood (hCB)-CD34+ humanized mice fail to reject human acute myeloid leukemia cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Dual role for miR-34a in the control of early progenitor proliferation and commitment in the mammary gland and in breast cancer.

Development of metastases and drug resistance are still a challenge for a successful systemic treatment in breast cancer (BC) patients. One of the mechanisms that confer metastatic properties to the cell relies in the epithelial-to-mesenchymal transition (EMT). Moreover, both EMT and metastasis are partly modulated through epigenetic mechanisms, by repression or induction of specific related genes.

Pier Giuseppe Pelicci

Department of Experimental Oncology,
IEO,
Department of Experimental Oncology, IEO

European Institute of Oncology IRCCS

Quantification of Self-renewal in Murine Mammosphere Cultures

Pier Giuseppe Pelicci

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Experimental Oncology,IEO,Department of Experimental Oncology, IEO

European Institute of Oncology IRCCS

Quantification of Self-renewal in Murine Mammosphere Cultures

Department of Experimental Oncology,
IEO,
Department of Experimental Oncology, IEO