A tripartite organelle platform links growth factor receptor signaling to mitochondrial metabolism.

One open question in the biology of growth factor receptors is how a quantitative input (i.e., ligand concentration) is decoded by the cell to produce specific response(s). Here, we show that an EGFR endocytic mechanism, non-clathrin endocytosis (NCE), which is activated only at high ligand concentrations and targets receptor to degradation, requires a tripartite organelle platform involving the plasma membrane (PM), endoplasmic reticulum (ER) and mitochondria. At these contact sites, EGFR-dependent, ER-generated Ca2+ oscillations are sensed by mitochondria, leading to increased metabolism and ATP production. Locally released ATP is required for cortical actin remodeling and EGFR-NCE vesicle fission. The same biochemical circuitry is also needed for an effector function of EGFR, i.e., collective motility. The multiorganelle signaling platform herein described mediates direct communication between EGFR signaling and mitochondrial metabolism, and is predicted to have a broad impact on cell physiology as it is activated by another growth factor receptor, HGFR/MET.

Microbial biofilms on macroalgae harbour diverse integron gene cassettes.

Integrons are genetic platforms that capture, rearrange and express mobile modules called gene cassettes. The best characterized gene cassettes encode antibiotic resistance, but the function of most integron gene cassettes remains unknown. Functional predictions suggest that many gene cassettes could encode proteins that facilitate interactions with other cells and with the extracellular environment. Because cell interactions are essential for biofilm stability, we sequenced gene cassettes from biofilms growing on the surface of the marine macroalgae Ulva australis and Sargassum linearifolium. Algal samples were obtained from coastal rock platforms around Sydney, Australia, using seawater as a control. We demonstrated that integrons in microbial biofilms did not sample genes randomly from the surrounding seawater, but harboured specific functions that potentially provided an adaptive advantage to both the bacterial cells in biofilm communities and their macroalgal host. Further, integron gene cassettes had a well-defined spatial distribution, suggesting that each bacterial biofilm acquired these genetic elements via sampling from a large but localized pool of gene cassettes. These findings suggest two forms of filtering: a selective acquisition of different integron-containing bacterial species into the distinct biofilms on Ulva and Sargassum surfaces, and a selective retention of unique populations of gene cassettes at each sampling location.

p140Cap inhibits ß-Catenin in the breast cancer stem cell compartment instructing a protective anti-tumor immune response.

The p140Cap adaptor protein is a tumor suppressor in breast cancer associated with a favorable prognosis. Here we highlight a function of p140Cap in orchestrating local and systemic tumor-extrinsic events that eventually result in inhibition of the polymorphonuclear myeloid-derived suppressor cell function in creating an immunosuppressive tumor-promoting environment in the primary tumor, and premetastatic niches at distant sites. Integrative transcriptomic and preclinical studies unravel that p140Cap controls an epistatic axis where, through the upstream inhibition of ß-Catenin, it restricts tumorigenicity and self-renewal of tumor-initiating cells limiting the release of the inflammatory cytokine G-CSF, required for polymorphonuclear myeloid-derived suppressor cells to exert their local and systemic tumor conducive function. Mechanistically, p140Cap inhibition of ß-Catenin depends on its ability to localize in and stabilize the ß-Catenin destruction complex, promoting enhanced ß-Catenin inactivation. Clinical studies in women show that low p140Cap expression correlates with reduced presence of tumor-infiltrating lymphocytes and more aggressive tumor types in a large cohort of real-life female breast cancer patients, highlighting the potential of p140Cap as a biomarker for therapeutic intervention targeting the ß-Catenin/ Tumor-initiating cells /G-CSF/ polymorphonuclear myeloid-derived suppressor cell axis to restore an efficient anti-tumor immune response.

Aberrant phosphorylation inactivates Numb in breast cancer causing expansion of the stem cell pool.

Asymmetric cell division is a key tumor suppressor mechanism that prevents the uncontrolled expansion of the stem cell (SC) compartment by generating daughter cells with alternative fates: one retains SC identity and enters quiescence and the other becomes a rapidly proliferating and differentiating progenitor. A critical player in this process is Numb, which partitions asymmetrically at SC mitosis and inflicts different proliferative and differentiative fates in the two daughters. Here, we show that asymmetric Numb partitioning per se is insufficient for the proper control of mammary SC dynamics, with differential phosphorylation and functional inactivation of Numb in the two progeny also required. The asymmetric phosphorylation/inactivation of Numb in the progenitor is mediated by the atypical PKCζ isoform. This mechanism is subverted in breast cancer via aberrant activation of PKCs that phosphorylate Numb in both progenies, leading to symmetric division and expansion of the cancer SC compartment, associated with aggressive disease. Thus, Numb phosphorylation represents a target for breast cancer therapy.

Comparison of StemPrintER with Oncotype DX Recurrence Score for predicting risk of breast cancer distant recurrence after endocrine therapy.

OBJECTIVE: Molecular tests predicting the risk of distant recurrence (DR) can be used to assist therapy decision-making in oestrogen receptor-positive (ER+) and human epidermal growth factor receptor 2-negative (HER2-) breast cancer patients after considerations of standard clinical markers. The Oncotype DX Recurrence Score (RS) is a widespread tool used for this purpose. Here, we compared the RS with the StemPrintER Risk Score (SPRS), a novel genomic predictor with a unique biological basis in its ability to measure the expression of cancer stemness genes. MATERIALS AND METHODS: We benchmarked the SPRS vs. RS, alone or in combination with clinicopathological variables expressed by the Clinical Treatment Score (CTS), for the prognostication of DR in a retrospective cohort of 776 postmenopausal patients with ER+/HER2-breast cancer enrolled in the translational arm of the randomised Arimidex, Tamoxifen, Alone or in Combination (ATAC) trial. Likelihood ratio (LR) with χ2 test and C-index were used to assess prognostic performance for the entire ten-year follow-up period and in early (0-5 years) and late (5-10 years) intervals. RESULTS: In all patients, the SPRS provided significantly more prognostic information than the RS for ten-year DR prognostication (C-index = 0.688, LR-χ2 = 33.4 vs. C-index = 0.641, LR-χ2 = 22.1) and for late (5-10 years) DR prognostication (C-index = 0.689, LR-χ2 = 18.8 vs. C-index = 0.571, LR-χ2 = 4.7). The SPRS also provided more prognostic information than the RS when added to the CTS in all patients (CTS + SPRS: LR-Δχ2 = 14.9; CTS + RS: LR-Δχ2 = 9.7) and in node-negative patients (CTS + SPRS: LR-Δχ2 = 11.7; CTS + RS: LR-Δχ2 = 6.6). CONCLUSIONS: In postmenopausal ER+/HER2- breast cancer patients, SPRS provided more prognostic information than RS for DR when used alone or in combination with the CTS. The SPRS could therefore potentially identify high-risk patients, who might benefit from aggressive treatments, from low-risk patients who might safely avoid adjuvant chemotherapy or prolongation of endocrine therapy.

L1CAM promotes ovarian cancer stemness and tumor initiation via FGFR1/SRC/STAT3 signaling.

BACKGROUND: Cancer stem cells (CSC) have been implicated in tumor progression. In ovarian carcinoma (OC), CSC drive tumor formation, dissemination and recurrence, as well as drug resistance, thus contributing to the high death-to-incidence ratio of this disease. However, the molecular basis of such a pathogenic role of ovarian CSC (OCSC) has been elucidated only to a limited extent. In this context, the functional contribution of the L1 cell adhesion molecule (L1CAM) to OC stemness remains elusive. METHODS: The expression of L1CAM was investigated in patient-derived OCSC. The genetic manipulation of L1CAM in OC cells provided gain and loss-of-function models that were then employed in cell biological assays as well as in vivo tumorigenesis experiments to assess the role of L1CAM in OC cell stemness and in OCSC-driven tumor initiation. We applied antibody-mediated neutralization to investigate L1CAM druggability. Biochemical approaches were then combined with functional in vitro assays to study the molecular mechanisms underlying the functional role of L1CAM in OCSC. RESULTS: We report that L1CAM is upregulated in patient-derived OCSC. Functional studies showed that L1CAM promotes several stemness-related properties in OC cells, including sphere formation, tumor initiation and chemoresistance. These activities were repressed by an L1CAM-neutralizing antibody, pointing to L1CAM as a druggable target. Mechanistically, L1CAM interacted with and activated fibroblast growth factor receptor-1 (FGFR1), which in turn induced the SRC-mediated activation of STAT3. The inhibition of STAT3 prevented L1CAM-dependent OC stemness and tumor initiation. CONCLUSIONS: Our study implicate L1CAM in the tumorigenic function of OCSC and point to the L1CAM/FGFR1/SRC/STAT3 signaling pathway as a novel driver of OC stemness. We also provide evidence that targeting this pathway can contribute to OC eradication.

miR-146 connects stem cell identity with metabolism and pharmacological resistance in breast cancer.

Although ectopic overexpression of miRNAs can influence mammary normal and cancer stem cells (SCs/CSCs), their physiological relevance remains uncertain. Here, we show that miR-146 is relevant for SC/CSC activity. MiR-146a/b expression is high in SCs/CSCs from human/mouse primary mammary tissues, correlates with the basal-like breast cancer subtype, which typically has a high CSC content, and specifically distinguishes cells with SC/CSC identity. Loss of miR-146 reduces SC/CSC self-renewal in vitro and compromises patient-derived xenograft tumor growth in vivo, decreasing the number of tumor-initiating cells, thus supporting its pro-oncogenic function. Transcriptional analysis in mammary SC-like cells revealed that miR-146 has pleiotropic effects, reducing adaptive response mechanisms and activating the exit from quiescent state, through a complex network of finely regulated miRNA targets related to quiescence, transcription, and one-carbon pool metabolism. Consistent with these findings, SCs/CSCs display innate resistance to anti-folate chemotherapies either in vitro or in vivo that can be reversed by miR-146 depletion, unmasking a "hidden vulnerability" exploitable for the development of anti-CSC therapies.

Unconventional endocytosis and trafficking of transferrin receptor induced by iron.

The posttranslational regulation of transferrin receptor (TfR1) is largely unknown. We investigated whether iron availability affects TfR1 endocytic cycle and protein stability in HepG2 hepatoma cells exposed to ferric ammonium citrate (FAC). NH4Cl and bafilomycin A1, but not the proteasomal inhibitor MG132, prevented the FAC-mediated decrease in TfR1 protein levels, thus indicating lysosomal involvement. Knockdown experiments showed that TfR1 lysosomal degradation is independent of 1) endocytosis mediated by the clathrin adaptor AP2; 2) Tf, which was suggested to facilitate TfR1 internalization; 3) H-ferritin; and 4) MARCH8, previously implicated in TfR1 degradation. Notably, FAC decreased the number of TfR1 molecules at the cell surface and increased the Tf endocytic rate. Colocalization experiments confirmed that, upon FAC treatment, TfR1 was endocytosed in an AP2- and Tf-independent pathway and trafficked to the lysosome for degradation. This unconventional endocytic regulatory mechanism aimed at reducing surface TfR1 may represent an additional posttranslational control to prevent iron overload. Our results show that iron is a key regulator of the trafficking of TfR1, which has been widely used to study endocytosis, often not considering its function in iron homeostasis.

A self-sustaining endocytic-based loop promotes breast cancer plasticity leading to aggressiveness and pro-metastatic behavior.

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 ß-catenin/TCF4-dependent partial epithelial-to-mesenchymal transition (EMT), followed by the establishment of a TGFß-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 TGFß-dependent regulatory loops conferring cellular plasticity and invasive behavior.

Molecularly Distinct Clathrin-Coated Pits Differentially Impact EGFR Fate and Signaling.

Adaptor protein 2 (AP2) is a major constituent of clathrin-coated pits (CCPs). Whether it is essential for all forms of clathrin-mediated endocytosis (CME) in mammalian cells is an open issue. Here, we demonstrate, by live TIRF microscopy, the existence of a subclass of relatively short-lived CCPs lacking AP2 under physiological, unperturbed conditions. This subclass is retained in AP2-knockout cells and is able to support the internalization of epidermal growth factor receptor (EGFR) but not of transferrin receptor (TfR). The AP2-independent internalization mechanism relies on the endocytic adaptors eps15, eps15L1, and epsin1. The absence of AP2 impairs the recycling of the EGFR to the cell surface, thereby augmenting its degradation. Accordingly, under conditions of AP2 ablation, we detected dampening of EGFR-dependent AKT signaling and cell migration, arguing that distinct classes of CCPs could provide specialized functions in regulating EGFR recycling and signaling.

A novel L1CAM isoform with angiogenic activity generated by NOVA2-mediated alternative splicing.

The biological players involved in angiogenesis are only partially defined. Here, we report that endothelial cells (ECs) express a novel isoform of the cell-surface adhesion molecule L1CAM, termed L1-ΔTM. The splicing factor NOVA2, which binds directly to L1CAM pre-mRNA, is necessary and sufficient for the skipping of L1CAM transmembrane domain in ECs, leading to the release of soluble L1-ΔTM. The latter exerts high angiogenic function through both autocrine and paracrine activities. Mechanistically, L1-ΔTM-induced angiogenesis requires fibroblast growth factor receptor-1 signaling, implying a crosstalk between the two molecules. NOVA2 and L1-ΔTM are overexpressed in the vasculature of ovarian cancer, where L1-ΔTM levels correlate with tumor vascularization, supporting the involvement of NOVA2-mediated L1-ΔTM production in tumor angiogenesis. Finally, high NOVA2 expression is associated with poor outcome in ovarian cancer patients. Our results point to L1-ΔTM as a novel, EC-derived angiogenic factor which may represent a target for innovative antiangiogenic therapies.

CD73 Regulates Stemness and Epithelial-Mesenchymal Transition in Ovarian Cancer-Initiating Cells.

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.

Evidence and uptake routes for Zinc oxide nanoparticles through the gastrointestinal barrier in Xenopus laevis.

The developmental toxicity of nanostructured materials, as well as their impact on the biological barriers, represents a crucial aspect to be assessed in a nanosafety policy framework. Nanosized metal oxides have been demonstrated to affect Xenopus laevis embryonic development, with nZnO specifically targeting the digestive system. To study the mechanisms of the nZnO-induced intestinal lesions, we tested two different nominally sized ZnO nanoparticles (NPs) at effective concentrations. Advanced microscopy techniques and molecular marker analyses were applied in order to describe the NP-epithelial cell interactions and the mechanisms driving NP toxicity and translocation through the intestinal barrier. We attributed the toxicity to NP-induced cell oxidative damage, the small-sized NPs being the more effective. This outcome is sustained by a marked increase in anti-oxidant genes' expression and high lipid peroxidation level in the enterocytes, where disarrangement of the cytoskeleton and cell junctions' integrity were evidenced. These events led to diffuse necrotic changes in the intestinal barrier, and trans- and paracellular NP permeation through the mucosa. The uptake routes, leading NPs to cross the intestinal barrier and reach secondary target tissues, have been documented. nZnOs embryotoxicity was confirmed to be crucially mediated by the NPs' reactivity rather than their dissolved ions. The ZnO NPs' ability to overwhelm the intestinal barrier must be taken into high consideration for a future design of safer ZnO NPs.

A molecular thermometer for nanoparticles for optical hyperthermia.

We developed an all-optical method to measure the temperature on gold (nanorods and nanostars) and magnetite nanoparticles under near-infrared and radiofrequency excitation by monitoring the excited state lifetime of Rhodamine B that lies within =/~20 nm from the nanoparticle surface. We reached high temperature sensitivity (0.029 ± 0.001 ns/°C) and low uncertainty (±0.3 °C). Gold nanostars are =/~3 and =/~100 times more efficient than gold nanorods and magnetite nanoparticles in inducing localized hyperthermia.

Nano-sized CuO, TiO2 and ZnO affect Xenopus laevis development.

The teratogenic potential of commercially available copper oxide (CuO), titanium dioxide (TiO2) and zinc oxide (ZnO) nanoparticles (NPs) was evaluated using the standardized FETAX test. After characterization of NP suspensions by TEM, DLS and AAS, histopathological screening and advanced confocal and energy-filtered electron microscopy techniques were used to characterize the induced lesions and to track NPs in tissues. Except for nCuO, which was found to be weakly embryolethal only at the highest concentration tested, the NPs did not cause mortality at concentrations up to 500 mg/L. However, they induced significant malformation rates, and the gut was observed to be the main target organ. CuO NPs exhibited the highest teratogenic potential, although no specific terata were observed. ZnO NPs caused the most severe lesions to the intestinal barrier, allowing NPs to reach the underlying tissues. TiO2 NPs showed mild embryotoxicity, and it is possible that this substance could be associated with hidden biological effects. Ions from dissolved nCuO contributed greatly to the observed embryotoxic effects, but those from nZnO did not, suggesting that their mechanisms of action may be different.

P53 detection by fluorescence lifetime on a hybrid fluorescein isothiocyanate gold nanosensor.

p53 is a tumor suppressor whose detection in the body is highly valuable as marker for early cancer diagnosis and prognosis. In this report we have studied constructs based on gold nanoparticles (NPs) functionalized with specific anti-p53 antibodies and with a fluoresceine derivative, FITC. The interaction of surface plasmons on gold NPs few nm in size, with fluorophores bound within few nanometers from the surface, induces changes in the fluorophore excited state lifetime. This parameter follows linearly the p53 concentration in solutions up to 200-400 pM, depending on the size of the NP, with an uncertainty approximatley =25 pM. We have evaluated the specificity of the nanosensor for p53 by testing it against bovine serum albumin, beta-lactoglobulin and lysozyme solutions. The titration of total cell extracts from p53 positive or p53-null cells with the anti-p53 antibody decorated gold NPs indicates that this construct is promising for possible applications to in vivo screening.