Numb Expression Contributes to the Maintenance of an Undifferentiated State in Human Epidermis.

The epidermis is a stratified epithelium with a stem cell subpopulation in the basal layer that constantly replicates and periodically detaches from the base, undergoing a differentiation process that involves various developmental signals and regulatory pathways. During the last 10 years, a number of studies tried to elucidate the intricate scenario that maintains the epithelial shield during the entire life span. In our study, we investigated the role of Numb in the skin compartment and, in particular, its involvement in stem cell maintenance. Numb expression in the skin compartment was assessed by immunofluorescence and immunohistochemistry analysis. We evaluated Numb expression in primary epithelial cells at various differentiative stages. Moreover, we overexpressed Numb in the isolated population enriched for undifferentiated progenitors to establish its involvement in in vitro differentiation. We demonstrated that Numb in high-proliferating epithelial undifferentiated progenitors contributes to the maintenance of an undifferentiated state. This regulation involves the E3 ligases Itch binding. Moreover, the analysis of a cohort of cutaneous carcinomas showed that Numb is highly expressed in squamous cell carcinoma (SCC), where we observed a direct correlation between the expression of Numb and Ki-67. Our data indicate for the first time that Numb is involved in the maintenance of the undifferentiated proliferating stem cell pool in the epithelial basal layer and its expression could become a new marker in skin cancer

Integrin α7 Is a Functional Marker and Potential Therapeutic Target in Glioblastoma

Functionally relevant markers of glioblastoma stem-like cells (GSCs) have potential for therapeutic targeting to treat this aggressive disease. Here we used generation and screening of thousands of monoclonal antibodies to search for receptors and signaling pathways preferentially enriched in GSCs. We identified integrin α7 (ITGA7) as a major laminin receptor in GSCs and in primary high-grade glioma specimens. Analyses of mRNA profiles in comprehensive datasets revealed that high ITGA7 expression negatively correlated with survival of patients with both low- and high-grade glioma. In vitro and in vivo analyses showed that ITGA7 plays a key functional role in growth and invasiveness of GSCs. We also found that targeting of ITGA7 by RNAi or blocking mAbs impaired laminin-induced signaling, and it led to a significant delay in tumor engraftment plus a strong reduction in tumor size and invasion. Our data, therefore, highlight ITGA7 as a glioblastoma biomarker and candidate therapeutic target

New therapeutic and diagnostic strategies in glioblastoma: Cancer Stem Cells targeted by monoclonal antibody

One of the most challenging objective in oncology is the identification of new specific tumor target to design effective therapies. At present it is widely accepted that the tumors are originated from a undifferentiated cell population able to trigger the cancer establishment and responsible for its maintenance. These undifferentiated cells are called Cancer Stem Cells (CSC) or tumor-initiating cells. A direct consequence of the CSC hierarchical model applied to clinic is that is currently taking place the idea that the traditional cancer treatment concern only the bulk tumor and the differentiated population without affecting the stem cell compartment. In that scenario the resistant cancer stem cells can proliferate and diffuse resulting in a temporary success of the treatment followed by a relapse. For this reason an effective therapy should be addressed to the eradication of CSCs that represent a gold target from the clinical treatment. CSCs are relatively slow proliferating in vivo, where they divide asymmetrically and are amplified few time with a massive duplications cycles of division made by progenitors. Therefore CSCs can be resistant to several available therapies which primarily affect actively duplicating cells. Despite to their relative quiescence, several mechanisms could account for this resistance such as the increased expression of drug pumps (Zhou, Schuetz et al. 2001), the expression of anti-apoptotic proteins, the higher efficiency in repairing DNA damage (Bao, Wu et al. 2006). The aim of this work was to specifically target CSCs of one of the most important causes of death for cancer, glioblastoma multiforme (GBM) by generating monoclonal antibodies (mAbs). Glioblastoma multiforme is the most common and most aggressive malignant primary brain tumor in humans. Even after treatments based on multiple approaches, as radiotherapy, surgical resection and chemotherapy, the prognosis of GBM patients remains unfavorable. The generation of mAbs against new surface markers against glioblastoma stem cells could have an impact as for the diagnostic as for therapeutic application. New mAbs could help to set up new diagnostic tests or to follow the effect of therapies. The mAbs generated could be employed as new therapeutic tools with selective toxicity against tumor cells, add efficacy to present therapies and represent an additional line of treatment for patients in advanced stages with multiresistant diseases. Moreover the discovery of a common stemness marker as antigen on glioblastoma stem cells may allow to select for cancer stem cells and this help to isolate and characterize them and possibly may be useful for defining novel diagnostic techniques and targeted therapies. The glioblastoma stem cells maintain the characteristics of tumor initiating cells after prolonged culture in vitro. We used these cells as immunogens in immunocompetent BALB/c mice to develop a hybridoma library. After the initial screening of the library was identified the 1.4A12 antibody that identified integriná7 (ITGA7) as antigen mostly expressed on brain tumor stem cells. The biological function of the integriná7 was evaluated the vitro and in vivo. ITGA7 silencing impairs the growth rate proliferation, clonogenicity, invasion in vitro and the tumor growth reduction in vivo. We analyzed the effect of 1.4A12 antibody and its role in counteracting the ITGA7 pathway. The 1.4 anti-integrin alpha7 antibody interferes with the integrin alpha 7 signaling and suppressed tumor invasion in vitro and tumour growth in vivo. All our data indicated that integrin alpha7 is involved in GBM pathogenesis and that 1.4A12 antibody could be of great help in defining new therapeutic options to increase glioblastoma eradication interfering with the tumor growth and with the invasion of cancer cells

Numb Expression Contributes to the Maintenance of an Undifferentiated State in Human Epidermis

The epidermis is a stratified epithelium with a stem cell subpopulation in the basal layer that constantly replicates and periodically detaches from the base, undergoing a differentiation process that involves various developmental signals and regulatory pathways. During the last 10 years, a number of studies tried to elucidate the intricate scenario that maintains the epithelial shield during the entire life span. In our study, we investigated the role of Numb in the skin compartment and, in particular, its involvement in stem cell maintenance. Numb expression in the skin compartment was assessed by immunofluorescence and immunohistochemistry analysis. We evaluated Numb expression in primary epithelial cells at various differentiative stages. Moreover, we overexpressed Numb in the isolated population enriched for undifferentiated progenitors to establish its involvement in in vitro differentiation. We demonstrated that Numb in high-proliferating epithelial undifferentiated progenitors contributes to the maintenance of an undifferentiated state. This regulation involves the E3 ligases Itch binding. Moreover, the analysis of a cohort of cutaneous carcinomas showed that Numb is highly expressed in squamous cell carcinoma (SCC), where we observed a direct correlation between the expression of Numb and Ki-67. Our data indicate for the first time that Numb is involved in the maintenance of the undifferentiated proliferating stem cell pool in the epithelial basal layer and its expression could become a new marker in skin cancer

Innovative oral spray-dried Idebenone systems to improve patient compliance

Idebenone is a high permeable drug with very slight water solubility that affects the dissolution rate in the biological fluids, causing an irregular and limited in vivo absorption after oral administration. Moreover, it is marketed in Europe as tablets equivalent to 150 mg, with the consequent administration of multiple dose of solid unit to obtain the correct dose, a deterrent for the patients’ compliance. According to these considerations, our goal was to develop spray-dried microparticles using a soluble b-cyclodextrin (CD) polymer and an enhancer of dissolution rate, such as carboxymethyl cellulose, to obtain a formulation easily dosable and soluble in water. The complex in solution was evaluated by phase solubility studies and the Idebenone/CD molar ratio selected was 1:1. According to Higuchi and Connors, adding carboxymethyl cellulose, a Bs-type profile was obtained. This result was due to the presence of carboxymethyl cellulose that competes with the CD in forming Idebenone microsystems, reducing of 10-fold the formulation bulk. UV–Vis absorption, 1H nuclear magnetic resonance and circular dichroism showed the formation of the CD/Idebenone inclusion complex conErmed also by differential scanning calorimetry, Fourier transform infrared spectroscopy and fluorescence microscope (FM). The water solubility data and the in vitro dissolution tests performed in simulated gastric fluid, showed an increase of the drug water interaction due to the presence of the CD and carboxymethyl cellulose, both able to improve drug wettability, water solubility and dissolution rate. This approach seems to be suitable to produce microsystems which are able to enhance the in vivo absorption of Idebenone after oral administration and to increase the patient compliance

Numb expression contributes to the maintenance of an undifferentiated state in human epidermis

The epidermis is a stratified epithelium with a stem cell subpopulation in the basal layer that constantly replicates and periodically detaches from the base, undergoing a differentiation process that involves various developmental signals and regulatory pathways. During the last 10 years, a number of studies tried to elucidate the intricate scenario that maintains the epithelial shield during the entire life span. In our study, we investigated the role of Numb in the skin compartment and, in particular, its involvement in stem cell maintenance. Numb expression in the skin compartment was assessed by immunofluorescence and immunohistochemistry analysis. We evaluated Numb expression in primary epithelial cells at various differentiative stages. Moreover, we overexpressed Numb in the isolated population enriched for undifferentiated progenitors to establish its involvement in in vitro differentiation. We demonstrated that Numb in high-proliferating epithelial undifferentiated progenitors contributes to the maintenance of an undifferentiated state. This regulation involves the E3 ligases Itch binding. Moreover, the analysis of a cohort of cutaneous carcinomas showed that Numb is highly expressed in squamous cell carcinoma (SCC), where we observed a direct correlation between the expression of Numb and Ki-67. Our data indicate for the first time that Numb is involved in the maintenance of the undifferentiated proliferating stem cell pool in the epithelial basal layer and its expression could become a new marker in skin cancer

Two-Step Coimmunoprecipitation (TIP) enables efficient and highly selective isolation of native protein complexes

Coimmunoprecipitation (co-IP) is one of the most frequently used techniques to study protein-protein (PPIs) or protein-nucleic acid interactions (PNIs). However, the presence of coprecipitated contaminants is a well-recognized issue associated with single-step co-IPs. To overcome this limitation, we developed the two-step co-IP (TIP) strategy that enables sequential coimmunoprecipitations of endogenous protein complexes. TIP can be performed with a broad range of mono- and polyclonal antibodies targeting a single protein or different components of a given complex. TIP results in a highly selective enrichment of protein complexes and thus outperforms single-step co-IPs for downstream applications such as mass spectrometry for the identification of PPIs and quantitative PCR for the analysis of PNIs. We bench-marked TIP for the identification of CD95/FAS-interact-ing proteins in primary human CD4 T cells, which recapitulated all major known interactors, but also enabled the proteomics discovery of PPM1G and IPO7 as new interaction partners. For its feasibility and high performance, we propose TIP as an advanced tool for the isolation of highly purified protein-protein and protein-nucleic acid complexes under native expression conditions

Decrease of survivin, p53 and Bcl-2 expression in chemorefractory colorectal liver metastases may be predictive of radiosensivity after radioembolization with yttrium-90 resin microspheres

In a prospective multicenter phase II trial of radioembolization with yttrium-90 (90Y-RE) in chemorefractory liver-dominant metastatic colorectal cancer (mCRC), we showed that median survival was 12.6 months (95% CI 7.0-18.3) with 48% of 50 patients achieving disease control. In this extension retrospective study, we analyzed whether a panel of biomarkers, known to be associated to an adverse clinical outcome, underwent variations in CRC liver metastases pre and post 90Y-RE.Of the 50 patients included in the study, 29 pre-90Y-RE therapy and 15 post-90Y-RE had liver biopsy specimens available. In these series we investigated survivin, p53, Bcl-2 and Ki-67 expression pre- and post-90Y-RE by immuhistochemistry (IHC). Our findings evidenced a decrease of survivin (77% vs 33%), p53 (93% vs 73%), Bcl-2 (37% vs 26%) expression as well as of Ki-67 proliferation index (62.5% vs 40%) on liver biopsies collected post-90Y-RE as compared to pre-90Y-RE. In the subset of 13 matched liver metastases we further confirmed the reduction of survivin (92.3% vs 53.8%; p = 0.06), p53 (100% vs 69.2%; p = 0.05) and Bcl-2 (69.2% vs 53.8%; p = 0.05) expression post- 90Y-RE. This biomarker modulation was accompanied by morphological changes as steatohepatitis, hepatocyte necrosis, collagen deposition, proliferating and/or bile duct ectasia, focal sinusoidal dilatation and fibrosis.Although our analysis was conducted in a very limited number cases, these changes appear strictly related to the response to 90Y-RE therapy and may deserve further investigation on a larger series of patients. \ua9 2013 Melucci et al; licensee BioMed Central Ltd