Alfonso Colombatti

Long non-coding RNAs and cancer: a new frontier of translational research?

Tiling array and novel sequencing technologies have made available the transcription profile of the entire human genome. However, the extent of transcription and the function of genetic elements that occur outside of protein-coding genes, particularly those involved in disease, are still a matter of debate. In this review, we focus on long non-coding RNAs (lncRNAs) that are involved in cancer. We define lncRNAs and present a cancer-oriented list of lncRNAs, list some tools (for example, public databases) that classify lncRNAs or that scan genome spans of interest to find whether known lncRNAs reside there, and describe some of the functions of lncRNAs and the possible genetic mechanisms that underlie lncRNA expression changes in cancer, as well as current and potential future applications of lncRNA research in the treatment of cancer.

Type A modules: interacting domains found in several non-fibrillar collagens and in other extracellular matrix proteins.

A 200-amino acid long motif first recognized in von Willebrand Factor (type A module) has been found in components of the extracellular matrix, hemostasis, cellular adhesion, and immune defense mechanisms. At present the extracellular matrix is the predominant site of expression of type A modules since at least four non-fibrillar collagens and two non-collagenous proteins contain a variable number of modules ranging from one to twelve. The modules conform to a consensus motif made of short conserved subregions separated by stretches of variable length. The proteins that incorporate type A modules participate in numerous biological events such as cell adhesion, migration, homing, pattern formation, and signal transduction after interaction with a large array of ligands.

Targeted intraoperative radiotherapy impairs the stimulation of breast cancer cell proliferation and invasion caused by surgical wounding

Purpose: After apparently successful excision of breast cancer, risk of local recurrence remains high mainly in the area surrounding the original tumor, indicating that wound healing processes may be implicated. The proportional reduction of this risk by radiotherapy does not depend on the extent of surgery, suggesting that radiotherapy, in addition to killing tumor cells, may influence the tumor microenvironment. Experimental Design: We studied how normal and mammary carcinoma cell growth and motility are affected by surgical wound fluids (WF), collected over 24 h following breast-conserving surgery in 45 patients, 20 of whom had received additional TARGeted Intraoperative radioTherapy (TARGIT), immediately after the surgical excision. The proteomic profile of the WF and their effects on the activation of intracellular signal transduction pathways of breast cancer cells were also analyzed. Results: WF stimulated proliferation, migration, and invasion of breast cancer cell lines. The stimulatory effect was almost completely abrogated when fluids from TARGIT-treated patients were used. These fluids displayed altered expression of several cytokines and failed to properly stimulate the activation of some intracellular signal transduction pathways, when compared with fluids harvested from untreated patients. Conclusions: Delivery of TARGIT to the tumor bed alters the molecular composition and biological activity of surgical WF. This novel antitumoral effect could, at least partially, explain the very low recurrence rates found in a large pilot study using TARGIT. It also opens a novel avenue for identifying new molecular targets and testing novel therapeutic agents.

EMILIN-1 deficiency induces elastogenesis and vascular cell defects

ABSTRACT EMILINs constitute a family of genes of the extracellular matrix with high structural similarity. Four genes have been identified so far in human and mouse. To gain insight into the function of this gene family, EMILIN-1 has been inactivated in the mouse by gene targeting. The homozygous animals were fertile and did not show obvious abnormalities. However, histological and ultrastructural examination revealed alterations of elastic fibers in aorta and skin. Formation of elastic fibers by mutant embryonic fibroblasts in culture was also abnormal. Additional alterations were observed in cell morphology and anchorage of endothelial and smooth muscle cells to elastic lamellae. Considering that EMILIN-1 is adhesive for cells and that the protein binds to elastin and fibulin-5, EMILIN-1 may regulate elastogenesis and vascular cell maintenance by stabilizing molecular interactions between elastic fiber components and by endowing elastic fibers with specific cell adhesion properties.

Extracellular Matrix: A Matter of Life and Death

Extracellular matrix (ECM) is an essential component of the stromal microenvironment both from a structural and a functional point of view. The ECM functions as a scaffold for tissue organization and regulates growth factors and chemokines availability thus contributing to maintain tissue homeostasis. Attachment of cells to ECM is essential to support cell survival, growth, and proliferation, and the lack of these interactions can trigger a type of cell death named anoikis. Several studies point out that alterations of ECM composition are often responsible of many pathological conditions such as cancer, of which it has been demonstrated to be occasionally the main promoter. ECM does not always represent a prosurvival stimulus; among the different array of ECM molecules a set of proteins can negatively affect cell viability and are thought to play an important role in tumor progression. For this reason attention has been focused on these molecules as potential tools or targets for therapy.

Stathmin activity influences sarcoma cell shape, motility, and metastatic potential

The balanced activity of microtubule-stabilizing and -destabilizing proteins determines the extent of microtubule dynamics, which is implicated in many cellular processes, including adhesion, migration, and morphology. Among the destabilizing proteins, stathmin is overexpressed in different human malignancies and has been recently linked to the regulation of cell motility. The observation that stathmin was overexpressed in human recurrent and metastatic sarcomas prompted us to investigate stathmin contribution to tumor local invasiveness and distant dissemination. We found that stathmin stimulated cell motility in and through the extracellular matrix (ECM) in vitro and increased the metastatic potential of sarcoma cells in vivo. On contact with the ECM, stathmin was negatively regulated by phosphorylation. Accordingly, a less phosphorylable stathmin point mutant impaired ECM-induced microtubule stabilization and conferred a higher invasive potential, inducing a rounded cell shape coupled with amoeboid-like motility in three-dimensional matrices. Our results indicate that stathmin plays a significant role in tumor metastasis formation, a finding that could lead to exploitation of stathmin as a target of new antimetastatic drugs.

Expression of CCR5 receptors on Reed–Sternberg cells and Hodgkin lymphoma cell lines: Involvement of CCL5/Rantes in tumor cell growth and microenvironmental interactions

The expression of CCL5/Rantes by Hodgkin (H) and Reed‐Sternberg (RS) cells has been recently documented. In the present study we demonstrated that the CCL5 receptor (CCR5) is constitutively expressed by Hodgkin Lymphoma (HL)‐derived cell lines (i.e. L‐428, KM‐H2, L‐1236 and L‐540) as shown by immunohistochemistry, flow cytometry and western blotting and also detected by immunohistochemistry on primary H‐RS cells from lymph node tissues. sCD40L never significantly affected CCR5 expression, whereas a short exposure to doxorubicin down regulated its expression. CCR5 receptors on HL cell lines were functionally active, since neutralizing anti‐CCL5 monoclonal antibodies inhibited basal proliferation of HL‐derived cell lines and recombinant CCR5 ligands (CCL3/Mip‐1α, CCL4/Mip1β and CCL5/Rantes) increased their clonogenic growth. CCL5 secretion by L‐1236, L‐428 and KM‐H2 cells was stimulated by CD40 engagement and also by coculturing L‐1236 cells on primary stromal fibroblasts from HL‐involved lymph nodes (HLF). Coculture experiments indicated that a direct contact of H‐RS cells induces HLF cells to produce CCL5. Supernatants from L‐1236, L‐428 and KM‐H2 cells stimulated migration of purified CD4+ T‐cells and eosinophils in vitro. The migratory response to HL‐cell lines supernatants was only partially neutralized (CD4+ cells: 70%; esinophils: 36%) by anti‐CCL5 antibodies, reinforcing the notion that multiple chemokines are involved in the recruitment of nonmalignant reactive cells in HL tissues. Taken together, our results indicate a possible involvement of the CCR5/CCR5‐ligands signaling in the regulation of H‐RS cells growth and in the formation/maintenance of the typical tissue microenvironment of HL.

The Inflammatory Chemokine CCL5 and Cancer Progression

Until recently, inflammatory chemokines were viewed mainly as indispensable “gate keepers” of immunity and inflammation. However, updated research indicates that cancer cells subvert the normal chemokine system and these molecules and their receptors become important constituents of the tumor microenvironment with very different ways to exert tumor-promoting roles. The CCR5 and the CCL5 ligand have been detected in some hematological malignancies, lymphomas, and a great number of solid tumors, but extensive studies on the role of the CCL5/CCR axis were performed only in a limited number of cancers. This review summarizes updated information on the role of CCL5 and its receptor CCR5 in cancer cell proliferation, metastasis, and the formation of an immunosuppressive microenvironment and highlights the development of newer therapeutic strategies aimed to inhibit the binding of CCL5 to CCR5, to inhibit CCL5 secretion, or to inhibit the interactions among tumor cells and the microenvironment leading to CCL5 secretion.

The EMILIN protein family

The EMILINs are a new family of glycoproteins of the extracellular matrix. The prototype of this family is the chicken EMILIN that was originally identified in extracts of aortas; it was then found to be widely distributed in several tissues associated with elastin and localized at the interface between amorphous elastin and microfibrils. Based on peptide sequences, chicken and human cDNAs coding for EMILIN were isolated by RT/PCR by screening kidney and heart cDNA libraries. By using a C-terminal fragment of human EMILIN-1 as a bait in the yeast two-hybrid system, a second family member, EMILIN-2, has also been isolated. EMILINs are characterized by a C-terminal gC1q globular domain, a short collagenous sequence, a long coiled-coil region and a new cysteine-rich N-terminal domain that can be considered a hallmark of the family being present also in multimerin. The gene for EMILIN-1 was mapped on chromosome 2p23 overlapping with the promoter region of the ketohexokinase gene. The gC1q domain of EMILIN-1 can form relatively stable and compact homotrimers and this association is then followed by a multimeric assembly of disulfide-bonded protomers. Recombinant EMILIN-1 purified from the supernatant of 293 cells represents a very efficient ligand for cell adhesion of several cell types.

EMI, a novel cysteine-rich domain of EMILINs and other extracellular proteins, interacts with the gC1q domains and participates in multimerization.

The N‐terminal cysteine‐rich domain (EMI domain) of EMILIN‐1 is a new protein domain that is shared with two proteins (multimerin and EMILIN‐2) and with four additional database entries. The EMI domains are always located at the N‐terminus, have a common gene organization, and belong to proteins that are forming or are compatible with multimer formation. The potential role of the EMI domain in the assembly of EMILIN‐1 was investigated by the two‐hybrid system. No reporter gene activity was detected when EMI‐1 was co‐transformed with the C‐terminal gC1q‐1 domain excluding a head‐to‐tail multimerization; conversely, a strong interaction was detected when the EMI‐1 domain was co‐transformed with the gC1q‐2 domain of EMILIN‐2.