Search

The mechanistic target of rapamycin complex 1 (mTORC1) is a key metabolic hub that controls the cellular response to environmental cues by exerting its kinase activity on multiple substrates1–3. However, whether mTORC1 responds to diverse stimuli by differentially phosphorylating specific substrates is poorly understood. Here we show that transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy4,5, is phosphorylated by mTORC1 via a substrate-specific mechanism that is mediated by Rag GTPases. Owing to this mechanism, the phosphorylation of TFEB—unlike other substrates of mTORC1, such as S6K and 4E-BP1— is strictly dependent on the amino-acid-mediated activation of RagC and RagD GTPases, but is insensitive to RHEB activity induced by growth factors. This mechanism has a crucial role in Birt–Hogg–Dube syndrome, a disorder that is caused by mutations in the RagC and RagD activator folliculin (FLCN) and is characterized by benign skin tumours, lung and kidney cysts and renal cell carcinoma6,7. We found that constitutive activation of TFEB is the main driver of the kidney abnormalities and mTORC1 hyperactivity in a mouse model of Birt–Hogg–Dube syndrome. Accordingly, depletion of TFEB in kidneys of these mice fully rescued the disease phenotype and associated lethality, and normalized mTORC1 activity. Our findings identify a mechanism that enables differential phosphorylation of mTORC1 substrates, the dysregulation of which leads to kidney cysts and cancer. Dysregulation of an mTORC1 substrate-specific mechanism leads to constitutive activation of TFEB, and promotes kidney cystogenesis and tumorigenesis in a mouse model of Birt–Hogg–Dube syndrome.

Background Increasing evidence linking epigenetic mechanisms and different diseases, including cancer, has prompted in the last 15 years the investigation of histone post-translational modifications (PTMs) in clinical samples. Methods allowing the isolation of histones from patient samples followed by the accurate and comprehensive quantification of their PTMs by mass spectrometry (MS) have been developed. However, the applicability of these methods is limited by the requirement for substantial amounts of material. Results To address this issue, in this study we streamlined the protein extraction procedure from low-amount clinical samples and tested and implemented different in-gel digestion strategies, obtaining a protocol that allows the MS-based analysis of the most common histone PTMs from laser microdissected tissue areas containing as low as 1000 cells, an amount approximately 500 times lower than what is required by available methods. We then applied this protocol to breast cancer patient laser microdissected tissues in two proof-of-concept experiments, identifying differences in histone marks in heterogeneous regions selected by either morphological evaluation or MALDI MS imaging. Conclusions These results demonstrate that analyzing histone PTMs from very small tissue areas and detecting differences from adjacent tumor regions is technically feasible. Our method opens the way for spatial epi-proteomics, namely the investigation of epigenetic features in the context of tissue and tumor heterogeneity, which will be instrumental for the identification of novel epigenetic biomarkers and aberrant epigenetic mechanisms.

Epigenetic aberrations have been recognized as important contributors to cancer onset and development, and increasing evidence suggests that linker histone H1 variants may serve as biomarkers useful for patient stratification, as well as play an important role as drivers in cancer. Although traditionally histone H1 levels have been studied using antibody-based methods and RNA expression, these approaches suffer from limitations. Mass spectrometry (MS)-based proteomics represents the ideal tool to accurately quantify relative changes in protein abundance within complex samples. In this study, we used a label-free quantification approach to simultaneously analyze all somatic histone H1 variants in clinical samples and verified its applicability to laser micro-dissected tissue areas containing as low as 1000 cells. We then applied it to breast cancer patient samples, identifying differences in linker histone variants patters in primary triple-negative breast tumors with and without relapse after chemotherapy. This study highlights how label-free quantitation by MS is a valuable option to accurately quantitate histone H1 levels in different types of clinical samples, including very low-abundance patient tissues.

Establishment of apical–basal cell polarity is necessary for generation of luminal and tubular structures during epithelial morphogenesis. Molecules acting at the membrane/ actin interface are expected to be crucial in governing these processes. Here, we show that the I-BAR-containing IRSp53 protein is restricted to the luminal side of epithelial cells of various glandular organs, and is specifically enriched in renal tubules in human, mice, and zebrafish. Using three-dimensional cultures of renal MDCK and intestinal Caco-2 cysts, we show that IRSp53 is recruited early after the first cell division along the forming apical lumen, and is essential for formation of a single lumen and for positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 directly binds to and controls localization of the inactive form of the small GTPase RAB35, a tethering factor for apical determinants. The interaction of IRSp53 with the actin capping protein EPS8 is critical for restricting IRSp53 localization. Correlative light and electron microscopy shows that IRSp53 loss perturbs the shape and continuity of the opposing apical membrane during the initial phase of lumenogenesis, which leads to preservation of multiple cytoplasmic bridges that interrupt the continuity of the nascent lumen. At the organism level, genetic removal of IRSp53 results in abnormal renal tubulogenesis, with defects in tubular polarity and architectural organization in both IRSp53 zebrafish mutant lines and IRSp53-KO murine models. Thus, IRSp53 acts as a platform for spatiotemporal regulation of assembly of the multi-protein complexes that shape the luminal membrane during the early steps of epithelial lumen morphogenesis.

Summary Cancer-initiating cells (CICs) have been implicated in tumor development and aggressiveness. In ovarian carcinoma (OC), CICs drive tumor formation, dissemination, and recurrence, as well as drug resistance, thus accounting for the high death-to-incidence ratio of this neoplasm. However, the molecular mechanisms that underlie such a pathogenic role of ovarian CICs (OCICs) remain elusive. Here, we have capitalized on primary cells either from OC or from its tissues of origin to obtain the transcriptomic profile associated with OCICs. Among the genes differentially expressed in OCICs, we focused on CD73, which encodes the membrane-associated 5′-ectonucleotidase. The genetic inactivation of CD73 in OC cells revealed that this molecule is causally involved in sphere formation and tumor initiation, thus emerging as a driver of OCIC function. Furthermore, functional inhibition of CD73 via either a chemical compound or a neutralizing antibody reduced sphere formation and tumorigenesis, highlighting the druggability of CD73 in the context of OCIC-directed therapies. The biological function of CD73 in OCICs required its enzymatic activity and involved adenosine signaling. Mechanistically, CD73 promotes the expression of stemness and epithelial-mesenchymal transition-associated genes, implying a regulation of OCIC function at the transcriptional level. CD73, therefore, is involved in OCIC biology and may represent a therapeutic target for innovative treatments aimed at OC eradication., Graphical Abstract, Highlights • CD73 is enriched in ovarian cancer-initiating cells (OCICs) • CD73 orchestrates OCIC stemness and EMT • OC initiation and growth require CD73 activity • OCIC-associated CD73 is a therapeutic target useful for OC eradication, Cavallaro et al. characterized the transcriptome of OCIC-enriched primary cultures and found CD73 as an upregulated gene. CD73 was then shown to regulate the expression of stemness and EMT-associated genes. The expression and function of CD73 in OCICs is required for tumor initiation, and CD73-targeted drugs decrease the rate of tumor take and inhibit cancer growth.

HOXB7 is a homeodomain (HOX) transcription factor involved in regional body patterning of invertebrates and vertebrates. We previously identified HOXB7 within a ten-gene prognostic signature for lung adenocarcinoma, where increased expression of HOXB7 was associated with poor prognosis. This raises the question of how HOXB7 overexpression can influence the metastatic behavior of lung adenocarcinoma. Here, we analyzed publicly available microarray and RNA-seq lung cancer expression datasets and found that HOXB7-overexpressing tumors are enriched in gene signatures characterizing adult and embryonic stem cells (SC), and induced pluripotent stem cells (iPSC). Experimentally, we found that HOXB7 upregulates several canonical SC/iPSC markers and sustains the expansion of a subpopulation of cells with SC characteristics, through modulation of LIN28B, an emerging cancer gene and pluripotency factor, which we discovered to be a direct target of HOXB7. We validated this new circuit by showing that HOXB7 enhances reprogramming to iPSC with comparable efficiency to LIN28B or its target c-MYC, which is a canonical reprogramming factor.

Metastasis is facilitated by the formation of a "premetastatic niche," which is fostered by primary tumor-derived factors. Colorectal cancer (CRC) metastasizes mainly to the liver. We show that the premetastatic niche in the liver is induced by bacteria dissemination from primary CRC. We report that tumor-resident bacteria Escherichia coli disrupt the gut vascular barrier (GVB), an anatomical structure controlling bacterial dissemination along the gut-liver axis, depending on the virulence regulator VirF. Upon GVB impairment, bacteria disseminate to the liver, boost the formation of a premetastatic niche, and favor the recruitment of metastatic cells. In training and validation cohorts of CRC patients, we find that the increased levels of PV-1, a marker of impaired GVB, is associated with liver bacteria dissemination and metachronous distant metastases. Thus, PV-1 is a prognostic marker for CRC distant recurrence and vascular impairment, leading to liver metastases. Copyright © 2021 Elsevier Inc. All rights reserved.

Summary Our understanding of the molecular determinants of cancer is still inadequate because of cancer heterogeneity. Here, using epithelial ovarian cancer (EOC) as a model system, we analyzed a minute amount of patient-derived epithelial cells from either healthy or cancerous tissues by single-shot mass-spectrometry-based phosphoproteomics. Using a multi-disciplinary approach, we demonstrated that primary cells recapitulate tissue complexity and represent a valuable source of differentially expressed proteins and phosphorylation sites that discriminate cancer from healthy cells. Furthermore, we uncovered kinase signatures associated with EOC. In particular, CDK7 targets were characterized in both EOC primary cells and ovarian cancer cell lines. We showed that CDK7 controls cell proliferation and that pharmacological inhibition of CDK7 selectively represses EOC cell proliferation. Our approach defines the molecular landscape of EOC, paving the way for efficient therapeutic approaches for patients. Finally, we highlight the potential of phosphoproteomics to identify clinically relevant and druggable pathways in cancer., Graphical Abstract, Highlights • We analyze ex-vivo-cultured primary cells using phosphoproteomics • We investigate epithelial ovarian cancer (EOC) and healthy tissue • We uncover expression of cancer-specific proteins and kinase signatures • The kinase CDK7 phosphorylates POLR2A and regulates EOC cell proliferation, Francavilla et al. use mass-spectrometry-based phosphoproteomics as a powerful tool to reveal cancer signatures. They analyze changes in the proteome and phosphoproteome of primary cells derived from epithelial ovarian cancer (EOC) compared to healthy tissues and reveal a role for the kinase CDK7 in EOC cell proliferation.

The subversion of endocytic routes leads to malignant transformation and has been implicated in human cancers. However, there is scarce evidence for genetic alterations of endocytic proteins as causative in high incidence human cancers. Here, we report that Epsin 3 (EPN3) is an oncogene with prognostic and therapeutic relevance in breast cancer. Mechanistically, EPN3 drives breast tumorigenesis by increasing E-cadherin endocytosis, followed by the activation of a β-catenin/TCF4-dependent partial epithelial-to-mesenchymal transition (EMT), followed by the establishment of a TGFβ-dependent autocrine loop that sustains EMT. EPN3-induced partial EMT is instrumental for the transition from in situ to invasive breast carcinoma, and, accordingly, high EPN3 levels are detected at the invasive front of human breast cancers and independently predict metastatic rather than loco-regional recurrence. Thus, we uncover an endocytic-based mechanism able to generate TGFβ-dependent regulatory loops conferring cellular plasticity and invasive behavior., It is unclear if genetic alterations in endocytic proteins play a causal role in high incidence human cancers. Here, the authors report the oncogenic role of Epsin3 (EPN3) in breast cancer, and show EPN3 to drive tumorigenesis through induction of a partial epithelial mesenchymal transition state and a TGFβ-dependent regulatory loop that promotes cellular plasticity and invasive behaviour.

Background: Breast cancers show variations in the number and biological aggressiveness of cancer stem cells that correlate with their clinico-prognostic and molecular heterogeneity. Thus, prognostic stratification of breast cancers based on cancer stem cells might help guide patient management. Methods: We derived a 20-gene stem cell signature from the transcriptional profile of normal mammary stem cells, capable of identifying breast cancers with a homogeneous profile and poor prognosis in in silico analyses. The clinical value of this signature was assessed in a prospective-retrospective cohort of 2, 453 breast cancer patients. Models for predicting individual risk of metastasis were developed from expression data of the 20 genes in patients randomly assigned to a training set, using the ridge-penalized Cox regression, and tested in an independent validation set. Findings: Analyses revealed that the 20-gene stem cell signature provided prognostic information in Triple-Negative and Luminal breast cancer patients, independently of standard clinicopathological parameters. Through functional studies in individual tumours, we correlated the risk score assigned by the signature with the proliferative and self-renewal potential of the cancer stem cell population. By retraining the 20-gene signature in Luminal patients, we derived the risk model, StemPrintER, which predicted early and late recurrence independently of standard prognostic factors. Interpretation: Our findings indicate that the 20-gene stem cell signature, by its unique ability to interrogate the biology of cancer stem cells of the primary tumour, provides a reliable estimate of metastatic risk in Triple-Negative and Luminal breast cancer patients independently of standard clinicopathological parameters. Keywords: Breast cancer, Cancer stem cells, Prognostic signatures, Biomarkers, Metastasis

This study unveils a redundant function for the endocytic proteins Eps15 and Eps15L1 in mouse embryo development and erythropoiesis, and a unique nonredundant role for Eps15L1 in the nervous system., EPS15 and its homologous EPS15L1 are endocytic accessory proteins. Studies in mammalian cell lines suggested that EPS15 and EPS15L1 regulate endocytosis in a redundant manner. However, at the organismal level, it is not known to which extent the functions of the two proteins overlap. Here, by exploiting various constitutive and conditional null mice, we report redundant and nonredundant functions of the two proteins. EPS15L1 displays a unique nonredundant role in the nervous system, whereas both proteins are fundamental during embryo development as shown by the embryonic lethality of -Eps15/Eps15L1-double KO mice. At the cellular level, the major process redundantly regulated by EPS15 and EPS15L1 is the endocytosis of the transferrin receptor, a pathway that sustains the development of red blood cells and controls iron homeostasis. Consequently, hematopoietic-specific conditional Eps15/Eps15L1-double KO mice display traits of microcytic hypochromic anemia, due to a cell-autonomous defect in iron internalization.

Aberrations in histone post-translational modifications (PTMs), as well as in the histone modifying enzymes (HMEs) that catalyze their deposition and removal, have been reported in many tumors and many epigenetic inhibitors are currently under investigation for cancer treatment. Therefore, profiling epigenetic features in cancer could have important implications for the discovery of both biomarkers for patient stratification and novel epigenetic targets. In this study, we employed mass spectrometry-based approaches to comprehensively profile histone H3 PTMs in a panel of normal and tumoral tissues for different cancer types, identifying various changes, some of which appear to be a consequence of the increased proliferation rate of tumors, while others are cell-cycle independent. Histone PTM changes found in tumors partially correlate with alterations of the gene expression profiles of HMEs obtained from publicly available data and are generally lost in culture conditions. Through this analysis, we identified tumor- and subtype-specific histone PTM changes, but also widespread changes in the levels of histone H3 K9me3 and K14ac marks. In particular, H3K14ac showed a cell-cycle independent decrease in all the seven tumor/tumor subtype models tested and could represent a novel epigenetic hallmark of cancer.

The mechanisms of tumor cell dissemination and the contribution of membrane trafficking in this process are poorly understood. Through a functional siRNA screening of human RAB GTPases, we found that RAB2A, a protein essential for ER‐to‐Golgi transport, is critical in promoting proteolytic activity and 3D invasiveness of breast cancer (BC) cell lines. Remarkably, RAB2A is amplified and elevated in human BC and is a powerful and independent predictor of disease recurrence in BC patients. Mechanistically, RAB2A acts at two independent trafficking steps. Firstly, by interacting with VPS39, a key component of the late endosomal HOPS complex, it controls post‐endocytic trafficking of membrane‐bound MT1‐MMP, an essential metalloprotease for matrix remodeling and invasion. Secondly, it further regulates Golgi transport of E‐cadherin, ultimately controlling junctional stability, cell compaction, and tumor invasiveness. Thus, RAB2A is a novel trafficking determinant essential for regulation of a mesenchymal invasive program of BC dissemination.

USP6NL, also named RN-tre, is a GTPase-activating protein involved in control of endocytosis and signal transduction. Here we report that USP6NL is overexpressed in breast cancer, mainly of the basal-like/integrative cluster 10 subtype. Increased USP6NL levels were accompanied by gene amplification and were associated with worse prognosis in the METABRIC dataset, retaining prognostic value in multivariable analysis. High levels of USP6NL in breast cancer cells delayed endocytosis and degradation of the EGFR, causing chronic AKT (protein kinase B) activation. In turn, AKT stabilized the glucose transporter GLUT1 at the plasma membrane, increasing aerobic glycolysis. In agreement, elevated USP6NL sensitized breast cancer cells to glucose deprivation, indicating that their glycolytic capacity relies on this protein. Depletion of USP6NL accelerated EGFR/AKT downregulation and GLUT1 degradation, impairing cell proliferation exclusively in breast cancer cells that harbored increased levels of USP6NL. Overall, these findings argue that USP6NL overexpression generates a metabolic rewiring that is essential to foster the glycolytic demand of breast cancer cells and promote their proliferation. Significance: USP6NL overexpression leads to glycolysis addiction of breast cancer cells and presents a point of metabolic vulnerability for therapeutic targeting in a subset of aggressive basal-like breast tumors. Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3432/F1.large.jpg. Cancer Res; 78(13); 3432–44. ©2018 AACR.

Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.

The cell fate determinant Numb is frequently downregulated in human breast cancers (BCs), resulting in p53 inactivation and an aggressive disease course. In the mouse mammary gland, Numb/p53 downregulation leads to aberrant tissue morphogenesis, expansion of the stem cell compartment, and emergence of cancer stem cells (CSCs). Strikingly, CSC phenotypes in a Numb‐knockout mouse model can be reverted by Numb/p53 restoration. Thus, targeting Numb/p53 dysfunction in Numb‐deficient human BCs could represent a novel anti‐CSC therapy. Here, using patient‐derived xenografts, we show that expansion of the CSC pool, due to altered self‐renewing divisions, is also a feature of Numb‐deficient human BCs. In these cancers, using the inhibitor Nutlin‐3 to restore p53, we corrected the defective self‐renewal properties of Numb‐deficient CSCs and inhibited CSC expansion, with a marked effect on tumorigenicity and metastasis. Remarkably, a regimen combining Nutlin‐3 and chemotherapy induced persistent tumor growth inhibition, or even regression, and prevented CSC‐driven tumor relapse after removal of chemotherapy. Our data provide a pre‐clinical proof‐of‐concept that targeting Numb/p53 results in a specific anti‐CSC therapy in human BCs.

RAB5A and RAB4 promote breast tumor cell dissemination by controlling the trafficking of proteins necessary for localized invadosome formation., The mechanisms by which tumor cells metastasize and the role of endocytic proteins in this process are not well understood. We report that overexpression of the GTPase RAB5A, a master regulator of endocytosis, is predictive of aggressive behavior and metastatic ability in human breast cancers. RAB5A is necessary and sufficient to promote local invasion and distant dissemination of various mammary and nonmammary tumor cell lines, and this prometastatic behavior is associated with increased intratumoral cell motility. Specifically, RAB5A is necessary for the formation of invadosomes, membrane protrusions specialized in extracellular matrix (ECM) degradation. RAB5A promotes RAB4- and RABENOSYN-5–dependent endo/exocytic cycles (EECs) of critical cargos (membrane-type 1 matrix metalloprotease [MT1-MMP] and β3 integrin) required for invadosome formation in response to motogenic stimuli. This trafficking circuitry is necessary for spatially localized hepatocyte growth factor (HGF)/MET signaling that drives invasive, proteolysis-dependent chemotaxis in vitro and for conversion of ductal carcinoma in situ to invasive ductal carcinoma in vivo. Thus, RAB5A/RAB4 EECs promote tumor dissemination by controlling a proteolytic, mesenchymal invasive program.

MicroRNAs (miRNAs) are a class of small RNAs (18–22 nt) that post transcriptionally regulate gene expression by binding to complementary sequences on target mRNAs, resulting in translational repression or target degradation and gene silencing. As aberrant expression of miRNAs is implicated in important diseases including cancer miRNA-based therapies are under intensive investigation. We optimized strategies to stably or conditionally generate miRNA inhibitors for a continuous block of miRNA activity that allows for probing miRNA function in long-term cell culture experiments, cancer xenografts, 3D tissue models and for in vivo studies with transgenic organisms.

Recent findings suggest the possibility that tumors originate from cancer cells with stem cell properties. The cancer stem cell (CSC) hypothesis provides an explanation for why existing cancer therapies often fail in eradicating highly malignant tumors and end with tumor recurrence. Although normal stem cells and CSCs both share the capacity for self-renewal and multi-lineage differentiation, suggesting that CSC may be derived from normal SCs, the cellular origin of transformation of CSCs is debatable. Research suggests that the tightly controlled balance of self-renewal and differentiation that characterizes normal stem cell function is dis-regulated in cancer. Additionally, recent evidence has linked an embryonic stem cell (ESC)-like gene signature with poorly differentiated high-grade tumors, suggesting that regulatory pathways controlling pluripotency may in part contribute to the somatic CSC phenotype. Here, we introduce expression profile bioinformatic analyses of mouse breast cells with CSC properties, mouse embryonic stem (mES) and induced pluripotent stem (iPS) cells with an emphasis on how study of pluripotent stem cells may contribute to the identification of genes and pathways that facilitate events associated with oncogenesis. Global gene expression analysis from CSCs and induced pluripotent stem cell lines represent an ideal model to study cancer initiation and progression and provide insight into the origin cancer stem cells. Additionally, insight into the genetic and epigenomic mechanisms regulating the balance between self-renewal and differentiation of somatic stem cells and cancer may help to determine whether different strategies used to generate iPSCs are potentially safe for therapeutic use.

A role for bone-marrow-derived cells (BMDCs) in tissue repair and malignancy onset has been proposed, but their contribution is still debated. We tested the ability of BMDCs containing the inducible krasV12 oncogene to initiate lung adenocarcinoma. For our experimental strategy, we reconstituted lethally irradiated wild type mice with BMDCs carrying inducible krasV12 and subsequently induced oncogene expression by 4-OHT administration. Epithelial lung lesions, from adenoma to adenocarcinomas, appeared at successive time points. These results show that lung tumors were derived from donor BMDCs and indicate a direct involvement of bone marrow cells in the development of epithelial cancers.

Vascular lumen formation is a fundamental step during angiogenesis; yet, the molecular mechanisms underlying this process are poorly understood. Recent studies have shown that neural and vascular systems share common anatomical, functional and molecular similarities. Here we show that the organization of endothelial lumen is controlled at the post-transcriptional level by the alternative splicing (AS) regulator Nova2, which was previously considered to be neural cell-specific. Nova2 is expressed during angiogenesis and its depletion disrupts vascular lumen formation in vivo. Similarly, Nova2 depletion in cultured endothelial cells (ECs) impairs the apical distribution and the downstream signalling of the Par polarity complex, resulting in altered EC polarity, a process required for vascular lumen formation. These defects are linked to AS changes of Nova2 target exons affecting the Par complex and its regulators. Collectively, our results reveal that Nova2 functions as an AS regulator in angiogenesis and is a novel member of the ‘angioneurins' family., The alternative splicing factor Nova2 is best known for its pivotal function in the brain. Giampietro et al. reveal an important role for Nova2 in the regulation of alternative splicing of transcripts in the vascular endothelium that are crucial for the maintenance of endothelial cell polarity and vessel lumen formation in zebrafish.

Mammospheres (MMs) are a model for culturing and maintaining mammary gland stem cells (SCs) or cancer stem cells (CSCs) ex situ. As MMs recapitulate the micro-niche of the mammary gland or a tumor, MMs are a model for studying the properties of SCs or CSCs, and for mapping, isolating, and characterizing the SC/CSC generated lineages. Cancer stem cells share with normal SCs the properties of self-renewal and the capacity to generate all cell types and organ structures of the mammary gland. Analysis of human tumor samples suggests that CSCs are heterogeneous in terms of proliferation and differentiation potential. Mammospheres from CSCs likewise display heterogeneity. This heterogeneity makes analysis of CSC generated MMs challenging. To identify the unique and diverse properties of MM derived CSCs, comparative analysis with MMs obtained from normal SCs is required. Here we present protocols for identifying and enriching cells with SC features from a cancer cell line using the LA7CSCs as a model. A comprehensive and comparative approach for identifying, isolating, and characterizing MMs from SCs and CSCs from human breast is also introduced. In addition, we describe detailed procedures for identifying, isolating, and characterizing mammary gland specific cell types, generated during MM formation.

PMCID: PMC4449154.-- et al., The small GTPase Cdc42 is a key regulator of polarity, but little is known in mammals about its spatial regulation and the relevance of spatial Cdc42 pools for polarity. Here, we report the identification of a GM130-RasGRF complex as a regulator of Cdc42 at the Golgi. Silencing GM130 results in RasGRF-dependent inhibition of the Golgi pool of Cdc42, but does not affect Cdc42 at the cell surface. Furthermore, active Cdc42 at the Golgi is important to sustain asymmetric front-rear Cdc42-GTP distribution in directionally-migrating cells. Concurrently to Cdc42 inhibition, silencing GM130 also results in RasGRF-dependent Ras-ERK pathway activation. Moreover, depletion of GM130 is sufficient to induce E-cadherin down-regulation, indicative of a loss in cell polarity and epithelial identity. Accordingly, GM130 expression is frequently lost in colorectal and breast cancer patients. These findings establish a previously unrecognized role for the GM130-RasGRF-Cdc42 connection in regulating polarity and tumourigenesis., HF is supported by the German Science Foundation (DFG), by the Biotechnology Institute Thurgau, and by the Young Scholar Fund of the University of Konstanz. FB acknowledges support by a fellowship from the Company of Biologists. We thank Beate Reiler for excellent technical assistance on staining the tissue samples. We thank Elisabeth Genot and Paolo Ciufici (University of Bordeaux) for help with the GTP-Cdc42 staining. PPDF acknowledges support by grants from Associazione Italiana per la Ricerca sul Cancro (AIRC IG 14404), MIUR (the Italian Ministry of University and Scientific Research), the Italian Ministry of Health, the European Research Council (Mammastem Project) and The Monzino Foundation.

Epstein-Barr virus (EBV) is a human herpes virus with oncogenic potential, associated with several malignancies. The EBV-encoded latent membrane protein 1 (LMP1) is one of nine proteins regularly expressed in virally infected and immortalised B lymphocytes. We now document the consistent immunoreactivity for LMP1 in 90% of 65 nevi and melanomas, using the monoclonal antibody cocktail CS1-4. The immunocytochemical findings, however, were not confirmed using reverse-transcription polymerase chain reaction (RT-PCR) experiments, which failed to demonstrate any actual expression of LMP1 mRNA. In situ hybridisation for EBV-encoded RNAs (EBERs 1 and 2) and PCR amplification of EBV genomic sequences also failed to document any viral infection. Several normal and neoplastic human tissues have also been immunostained for LMP1, without any positive staining, with the exception of a minor percentage of skin melanocytes and of normal blasts of the myeloid and erythroid lineages. We conclude that the vast majority of nevi and melanomas express a still uncharacterised molecule, cross-reacting with anti-LMP1 (CS1-4) antibodies, which may be considered a consistent marker of melanocytic proliferations. The immunoreactivity of normal and neoplastic human tissues for the anti-LMP1 reagent should not be taken as evidence of EBV infection.

Recent data suggest that drug resistance and/or disease recurrence in ovarian cancer (OC) are driven by a subpopulation of cells in human tumors with stem-like characteristics (cancer stem cells, CSCs). CSCs are defined as a small subpopulation of cells within the tumor bulk that possess the capacity, on one hand, to self-renew and, on the other hand, to give rise to all heterogeneous cancer cell lineages that compose the tumor of origin. The CSC hypothesis provides an attractive cellular mechanism to explain the therapeutic refractoriness, dormant behavior, and relapse of OC. Our study aims at assessing ovarian cancer stem cells (OCSC) as causal players in OC etiology and progression and at defining their molecular and functional traits. Specifically, we are pursuing these objectives through the accomplishment of the following milestones: 1) collection of normal and pathological samples; 2) identification of OCSC based on functional properties; 3) comparison of gene expression profiles between cancer stem cells and their normal counterpart; 4) characterization of novel genes/pathways involved in OCSC function (clonogenicity, tumorigenicity, quiescence, chemoresistance, etc.). This workflow has been applied to surgical samples of OC as well as to normal ovarian surface epithelium (OSE) and fallopian tube epithelium (FTE), namely the tissues of origin of OC. OCSC have been derived from primary cultures exploiting their ability to resist anoikis and form monoclonal spheroids when cultured under nonadherent conditions. The transcriptome of OC-derived spheroids was compared with that of their normal counterparts, yielding a set of differentially expressed genes. Among these, for the initial characterization we have selected two cell surface markers CD73, upregulated in spheroids, and CD24, downregulated in spheroids. The CD73+/CD24- subpopulation was enriched in sphere-forming cells and, most importantly, exhibited higher tumorigenic capacity when xenografted in immunocompromised mice. Thus, by utilizing clinically relevant samples we have characterized the transcriptome of OCSC and of their normal counterparts. Furthermore, we found that the CD73+/CD24- phenotype in ovarian cancer is associated to CSC-like traits. On one hand, our approach revealed novel OCSC biomarkers that can be exploited for imaging and purification purposes. On the other hand, we have identified potential therapeutic targets, such as CD73, that might set the stage for innovative treatments aimed at the eradication of OC through the elimination of OCSC. Citation Format: Michela Lupia, Giovanni Bertalot, Pier Paolo Di Fiore, Nicoletta Colombo, Fabrizio Bianchi, Ugo Cavallaro. The CD73+/CD24- subpopulation of ovarian cancer cells is enriched in cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A66.

TFE-fusion renal cell carcinomas (TFE-fusion RCCs) are caused by chromosomal translocations that lead to overexpression of the TFEB and TFE3 genes (Kauffman et al., 2014). The mechanisms leading to kidney tumor development remain uncharacterized and effective therapies are yet to be identified. Hence, the need to model these diseases in an experimental animal system (Kauffman et al., 2014). Here, we show that kidney-specific TFEB overexpression in transgenic mice, resulted in renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate those observed in both TFEB- and TFE3-mediated human kidney tumors. Analysis of kidney samples revealed transcriptional induction and enhanced signaling of the WNT β-catenin pathway. WNT signaling inhibitors normalized the proliferation rate of primary kidney cells and significantly rescued the disease phenotype in vivo. These data shed new light on the mechanisms underlying TFE-fusion RCCs and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway.

Background The cancer transcriptome is difficult to explore due to the heterogeneity of quantitative and qualitative changes in gene expression linked to the disease status. An increasing number of "unconventional" transcripts, such as novel isoforms, non-coding RNAs, somatic gene fusions and deletions have been associated with the tumoral state. Massively parallel sequencing techniques provide a framework for exploring the transcriptional complexity inherent to cancer with a limited laboratory and financial effort. We developed a deep sequencing and bioinformatics analysis protocol to investigate the molecular composition of a breast cancer poly(A)+ transcriptome. This method utilizes a cDNA library normalization step to diminish the representation of highly expressed transcripts and biology-oriented bioinformatic analyses to facilitate detection of rare and novel transcripts. Results We analyzed over 132,000 Roche 454 high-confidence deep sequencing reads from a primary human lobular breast cancer tissue specimen, and detected a range of unusual transcriptional events that were subsequently validated by RT-PCR in additional eight primary human breast cancer samples. We identified and validated one deletion, two novel ncRNAs (one intergenic and one intragenic), ten previously unknown or rare transcript isoforms and a novel gene fusion specific to a single primary tissue sample. We also explored the non-protein-coding portion of the breast cancer transcriptome, identifying thousands of novel non-coding transcripts and more than three hundred reads corresponding to the non-coding RNA MALAT1, which is highly expressed in many human carcinomas. Conclusion Our results demonstrate that combining 454 deep sequencing with a normalization step and careful bioinformatic analysis facilitates the discovery and quantification of rare transcripts or ncRNAs, and can be used as a qualitative tool to characterize transcriptome complexity, revealing many hitherto unknown transcripts, splice isoforms, gene fusion events and ncRNAs, even at a relatively low sequence sampling.

Tumors derived from rat LA7 cancer stem cells (CSCs) contain a hierarchy of cells with different capacities to generate self-renewing spheres and tubules serially ex vivo and to evoke tumors in vivo . We isolated two morphologically distinct cell types with distinct tumorigenic potential from LA7-evoked tumors: cells with polygonal morphology that are characterized by expression of p21/ WAF1 and p63 and display hallmarks of CSCs and elongated epithelial cells, which generate tumors with far less heterogeneity than LA7 CSCs. Serial transplantation of elongated epithelial cells results in progressive loss of tumorigenic potential; tumor heterogeneity; CD44, E-cadherin, and epithelial cytokeratin expression and increased α-smooth muscle actin I and vimentin expression. In contrast, serial transplantation of LA7 CSCs can be performed indefinitely and results in tumors that maintain their heterogeneity, consistent with self-renewal and multilineage differentiation potential. Collectively, our data show that polygonal cells are CSCs, whereas epithelial elongated cells are lineage-committed progenitors with tumorigenic potential, and suggest that tumor progenitors, although lacking indefinite self-renewal potential, nevertheless may make a substantial contribution to tumor development. Because LA7 cells can switch between conditions that favor maintenance of pure CSCs vs. differentiation into other tumor cell types, this cell system provides the opportunity to study factors that influence CSC self-renewal and differentiation. One factor, p63 , was identified as a key gene regulating the transition between CSCs and early progenitor cells.

Epithelial Ovarian Cancer (OC) is one of the leading causes of death for women, and no significant therapeutic progress has been made in the last decades. Recent data suggest that one of the mechanisms accounting for resistant and/or relapsing disease is a subpopulation of cells in human tumors with stem-like characteristics (cancer stem cells, CSCs). CSCs are defined as a small subpopulation of cells within the tumor bulk that possess the capacity, on one hand, to self-renew and, on the other hand, to give rise to all heterogeneous cancer cell lineages that compose the tumor of origin. The CSC hypothesis provides an attractive cellular mechanism to explain the therapeutic refractoriness, dormant behavior, and relapse of the disease. Our study aims at assessing ovarian cancer stem cells (OCSC) as causal players in OC etiology and progression and at defining their molecular and functional profile. Specifically, we are pursuing the following objectives through the accomplishment of these milestones: 1) collection of normal and pathological samples; 2) identification of OCSC based on functional properties; 3) comparison of gene expression profiles between cancer stem cells and their normal counterpart; 4) characterization of novel genes/pathways involved in OCSC function (clonogenicity, tumorigenicity, quiescence, chemoresistance, etc.). This workflow is being applied to a series of fresh surgical samples of OC as well as to normal ovarian surface epithelium (OSE) and fallopian tube epithelium (FTE), namely the tissues of origin of OC. Thus, we obtained a collection of primary cells that recapitulate many traits of the original tissue both in vitro and in vivo. The gene expression profiles of sphere-forming SC has been compared to that of adherent, parental cells, aimed at identifying stemness-associated genes. This screening has been extended to OC, OSE, and FTE, resulting in a set of genes that are differentially expressed in OCSC as compared to their normal counterparts. A subset of such genes has been selected for the subsequent validation, both as OCSC biomarkers that could have clinical applications and as drivers in the biological and pathogenic function of OCSC. Our study might set the stage for innovative therapeutic approaches aimed at the selective elimination of OCSC, thus preventing tumor recurrence and chemoresistance, namely the two main causes of ovarian cancer lethality. Citation Format: Michela Lupia, Giovanni Bertalot, Nicoletta Colombo, Stefano Confalonieri, Pier Paolo Di Fiore, Ugo Cavallaro. Molecular and functional characterization of ovarian cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1414. doi:10.1158/1538-7445.AM2015-1414

In recent years, enormous effort has been dedicated to transcriptomic profiling of various physiological and pathological conditions, in particular in cancer biology field. Microarray gene expression (mRNA) datasets of thousands of human tumors are now publicly available and can be used to get insights of cancer processes by systems-based analysis and to identify biomarkers for improvement of cancer therapy. However, the high complexity of human transcriptome may be difficult to handle. In this regard, shifting the attention to the miRNome could be an advantage, since its complexity is at least 20-fold lower than that of a reference transcriptome (∼1000 miRNAs vs. ∼20,000 genes). Importantly, patterns of distinct miRNA expression profiles were observed in tumors and there is a growing interest in miRNAs, behaving as potential cancer determinants and biomarkers. Knowing that almost 50% of human miRNA genes are located within introns of coding genes and that they usually share transcriptional regulation, those publicly available datasets are likely to contain “latent” information on intronic-miRNA expression. In other words, it could be possible to predict the regulation of intronic-miRNA expression by simply analyzing their host genes profile (miR-HG). The aim of our work is to take advantage of cancer datasets, focusing mainly on breast cancer datasets, to provide proof of principle evidences that meta-analysis of miR-HG expression profiles can pinpoint intronic-miRNAs with a role in breast cancer cell biology, and a potential utility as cancer biomarkers. Using this approach, we successfully discovered a diagnostic microRNA signature enabling the identification of breast cancer molecular subtypes. Importantly, qRT-PCR analysis of just three intronic-miRNAs, using FFPE samples, was sufficient to identify more aggressive breast tumor subtypes (i.e. basal, HER2 and luminal B subtypes), with a ∼80% of accuracy, in patients with moderately differentiated breast cancer. Unexpectedly, in a number of cases, the regulation of expression of intronic-miRNAs was more relevant to cancer phenotypes than the expression of their host genes. In line with these encouraging results, we propose our data mining strategy as a valid tool for cancer research and other biomedical fields. Citation Format: Simona Monterisi, Giovanni D'Ario, Elisa Dama, Nicole Rotmensz, Stefano Confalonieri, Chiara Tordonato, Flavia Troglio, Giovanni Bertalot, Patrick Maisonneuve, Giuseppe Viale, Francesco Nicassio, Pier Paolo Di Fiore, Fabrizio Bianchi. Mining cancer gene expression databases for latent information on intronic microRNAs. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 233. doi:10.1158/1538-7445.AM2015-233

The potential of recruited bone-marrow-derived cells (BMDCs) to act as a source of malignancy was disputed for a long time. A role for BMDCs in tissue repair has also been proposed, since these cells are capable of homing to the lung and differentiating into multiple epithelial cell lineages so thus contributing in lung tissue repair and regeneration. We tested the ability of bone marrow cells containing an inducible oncogene to initiate lung adenocarcinoma. Since the role of the K-Ras gene in lung cancer is well known, for our experimental strategy, we used a mouse model expressing oncogenic K-RasV12 (K-Ras+/V12RERTert/ert). This model generates a specific neoplastic phenotype induced by the K-RasV12 mutation. Mice usually develop lung tumors with complete penetrance evolving from hyperplasia, through adenoma, to adenocarcinoma, when the oncogene expression is induced by 4-hydroxitamoxifen (4-OHT) administration. Following lethal irradiation, wild-type mice were transplanted with donor bone marrow cells derived from K-RasV12 mice and, once the bone marrow was reconstituted, treated with 4-OHT. Mice were monitored and sacrificed at subsequent post-induction time points (0, 3.5, 5.5, 8.5, 11, 13.5, 15, and 16 months) and lungs and peripheral blood were collected. Analysis of the H&E-stained lung sections from both reconstituted K-RasV12 and control mice revealed diffuse hyperplasia, possibly due to irradiation. Epithelial lesions, adenomas and/or adenocarcinomas, were detected in 4 out of 8 K-RasV12-reconstituted animals at 3.5, 5.5, 13.5 and 15 months post oncogene induction while no lesions were detected in the control group (transplanted but not induced mice). In addition, the K-Ras implication on the origin of the tumor was confirmed by X-Gal staining of lung lesions in reconstituted mice. In our model, -geo is indeed a marker protein coexpressed along with the same K-RasV12 transcript by a bicistronic strategy. Our results suggest a direct implication of BMDCs in lung epithelial tumor development and emphasize the need of further studies to dissect the mechanisms regulating BMDCs’ recruitment to injured tissues and their role in tumor initiation and/or progression. Citation Format: Elena Belloni, Ines Martin-Padura, Elvira Gerbino, Stefania Orecchioni, Fulvia Fusar-Imperatore, Paola Marighetti, Giovanni Bertalot, Pier Giuseppe Pelicci, Francesco Bertolini. K-RasV12 bone marrow cell reconstitution causes lung cancer in wild type mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2282. doi:10.1158/1538-7445.AM2015-2282