Giovanni Bertalot

A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination

RAB5A and RAB4 promote breast tumor cell dissemination by controlling the trafficking of proteins necessary for localized invadosome formation.

Spatial control of Cdc42 signalling by a GM130-RasGRF complex regulates polarity and tumorigenesis

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. Concurrent to Cdc42 inhibition, silencing GM130 also results in RasGRF-dependent Ras-ERK pathway activation. Moreover, depletion of GM130 is sufficient to induce E-cadherin downregulation, 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 a GM130-RasGRF-Cdc42 connection in regulating polarity and tumorigenesis.

RAB2A controls MT1‐MMP endocytic and E‐cadherin polarized Golgi trafficking to promote invasive breast cancer programs

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.

Phosphoproteomics of Primary Cells Reveals Druggable Kinase Signatures in Ovarian Cancer

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.

Pre-clinical validation of a selective anti-cancer stem cell therapy for Numb-deficient human breast cancers

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.

Modelling TFE renal cell carcinoma in mice reveals a critical role of WNT signaling

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. DOI: http://dx.doi.org/10.7554/eLife.17047.001

Overlapping genes may control reprogramming of mouse somatic cells into induced pluripotent stem cells (iPSCs) and breast cancer stem cells

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.

Culture and Characterization of Mammary Cancer Stem Cells in Mammospheres

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.

Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function

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.

Circulating and tissue biomarkers in early-stage non-small cell lung cancer

Objective We sought to characterise circulating and tissue tumour biomarkers of patients who developed early-stage non-small cell lung cancer (NSCLC) during long-term follow-up of a chemoprevention trial (NCT00321893). Materials and Methods Blood and sputum samples were collected from 202 high-risk asymptomatic individuals with CT-detected stable lung nodules. Real-time PCR was performed on plasma to quantify free circulating DNA. Baseline serum was investigated with a previously validated test based on 13 circulating miRNAs (miR-Test). Promoter methylation status of p16, RASSF1a and RARβ2 and telomerase activity were assessed in sputum samples. DNA was extracted from each tumour developed during follow-up and subjected to a mutation survey using the LungCarta panel on the Sequenom MassARRAY platform. Results During follow-up (9 years) six individuals underwent surgery for stage I NSCLC with a median time of disease onset of 20.5 months. MiR-Test scores were positive (range: 0.14–7.24) in four out of six baseline pre-disease onset sera. No association was identified between free circulating DNA or sputum biomarkers and disease onset. All tumours harboured at least one somatic mutation in well-known cancer genes, including KRAS (n = 4), BRAF (n = 1), and TP53 (n = 3). Conclusion Circulating miRNA tests may represent valuable tools to detect clinically-silent tumours. Early-stage lung adenocarcinomas harbour recurrent genetic events similar to those described in advanced-stage NSCLCs.