Ilaria Chiodi

Telomere-independent functions of telomerase in nuclei, cytoplasm, and mitochondria.

Telomerase canonical activity at telomeres prevents telomere shortening, allowing chromosome stability and cellular proliferation. To perform this task, the catalytic subunit (telomerase reverse transcriptase, TERT) of the enzyme works as a reverse transcriptase together with the telomerase RNA component (TERC), adding telomeric repeats to DNA molecule ends. Growing evidence indicates that, besides the telomeric-DNA synthesis activity, TERT has additional functions in tumor development and is involved in many different biological processes, among which cellular proliferation, gene expression regulation, and mitochondrial functionality. TERT has been shown to act independently of TERC in the Wnt-β-catenin signaling pathway, regulating the expression of Wnt target genes, which play a role in development and tumorigenesis. Moreover, TERT RNA-dependent RNA polymerase activity has been found, leading to the genesis of double-stranded RNAs that act as precursor of silencing RNAs. In mitochondria, a TERT TERC-independent reverse transcriptase activity has been described that could play a role in the protection of mitochondrial integrity. In this review, we will discuss some of the extra-telomeric functions of telomerase.

Reduced Expression of the ROCK Inhibitor Rnd3 Is Associated with Increased Invasiveness and Metastatic Potential in Mesenchymal Tumor Cells

Background Mesenchymal and amoeboid movements are two important mechanisms adopted by cancer cells to invade the surrounding environment. Mesenchymal movement depends on extracellular matrix protease activity, amoeboid movement on the RhoA-dependent kinase ROCK. Cancer cells can switch from one mechanism to the other in response to different stimuli, limiting the efficacy of antimetastatic therapies. Methodology and Principal Findings We investigated the acquisition and molecular regulation of the invasion capacity of neoplastically transformed human fibroblasts, which were able to induce sarcomas and metastases when injected into immunocompromised mice. We found that neoplastic transformation was associated with a change in cell morphology (from fibroblastic to polygonal), a reorganization of the actin cytoskeleton, a decrease in the expression of several matrix metalloproteases and increases in cell motility and invasiveness. In a three-dimensional environment, sarcomagenic cells showed a spherical morphology with cortical actin rings, suggesting a switch from mesenchymal to amoeboid movement. Accordingly, cell invasion decreased after treatment with the ROCK inhibitor Y27632, but not with the matrix protease inhibitor Ro 28-2653. The increased invasiveness of tumorigenic cells was associated with reduced expression of Rnd3 (also known as RhoE), a cellular inhibitor of ROCK. Indeed, ectopic Rnd3 expression reduced their invasive ability in vitro and their metastatic potential in vivo. Conclusions These results indicate that, during neoplastic transformation, cells of mesenchymal origin can switch from a mesenchymal mode of movement to an amoeboid one. In addition, they point to Rnd3 as a possible regulator of mesenchymal tumor cell invasion and to ROCK as a potential therapeutic target for sarcomas.

Telomerase: cellular immortalization and neoplastic transformation. Multiple functions of a multifaceted complex

The telomerase complex allows telomere length maintenance, which is required for an unlimited cellular proliferation. Telomerase is virtually absent in normal human somatic cells, which are characterized by a definite proliferation potential, while it is present in the vast majority of tumors (around 90%). Restoring telomerase activity in normal somatic cells can indefinitely prolong cellular life span. However, evidence has been reported that this event can be associated with the acquisition of characteristics typical of cellular transformation. Moreover, analysis of telomerase immortalized cells, as well as of tumor cells in which telomerase is inactivated, has highlighted multiple functions of telomerase in tumorigenesis, besides telomere lengthening. In this paper, we will review telomerase immortalization of somatic cells, together with its possible consequences, and we will examine the complex role of telomerase in tumorigenesis.

A comprehensive strategy for the analysis of acoustic compressibility and optical deformability on single cells

We realized an integrated microfluidic chip that allows measuring both optical deformability and acoustic compressibility on single cells, by optical stretching and acoustophoresis experiments respectively. Additionally, we propose a measurement protocol that allows evaluating the experimental apparatus parameters before performing the cell-characterization experiments, including a non-destructive method to characterize the optical force distribution inside the microchannel. The chip was used to study important cell-mechanics parameters in two human breast cancer cell lines, MCF7 and MDA-MB231. Results indicate that MDA-MB231 has both higher acoustic compressibility and higher optical deformability than MCF7, but statistical analysis shows that optical deformability and acoustic compressibility are not correlated parameters. This result suggests the possibility to use them to analyze the response of different cellular structures. We also demonstrate that it is possible to perform both measurements on a single cell, and that the order of the two experiments does not affect the retrieved values.

Cross-Analysis of Gene and miRNA Genome-Wide Expression Profiles in Human Fibroblasts at Different Stages of Transformation

We have developed a cellular system constituted of human telomerase immortalized fibroblasts that gradually underwent neoplastic transformation during propagation in culture. We exploited this cellular system to investigate gene and miRNA transcriptional programs in cells at different stages of propagation, representing five different phases along the road to transformation, from non-transformed cells up to tumorigenic and metastatic ones. Here we show that gene and miRNA expression profiles were both able to divide cells according to their transformation phase. We identified more than 1,700 genes whose expression was highly modulated in cells at at least one propagation stage and we found that the number of modulated genes progressively increased at successive stages of transformation. These genes identified processes significantly deregulated in tumorigenic cells, such as cell differentiation, cell movement and extracellular matrix remodeling, cell cycle and apoptosis, together with upregulation of several cancer testis antigens. Alterations in cell cycle, apoptosis, and cancer testis antigen expression were particular hallmarks of metastatic cells. A parallel deregulation of a panel of 43 miRNAs strictly connected to the p53 and c-Myc pathways and with oncogenic/oncosuppressive functions was also found. Our results indicate that cen3tel cells can be a useful model for human fibroblast neoplastic transformation, which appears characterized by complex and peculiar alterations involving both genetic and epigenetic reprogramming, whose elucidation could provide useful insights into regulatory networks underlying cancerogenesis.

Poly(ADP-ribosylation) and Neoplastic Transformation: Effect of PARP Inhibitors

Poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribosylation) play essential roles in several biological processes, among which neoplastic transformation and telomere maintenance. In this paper, we review the poly(ADP-ribosylation) process together with the highly appealing use of PARP inhibitors for the treatment of cancer. In addition, we report our results concerning poly(ADP-ribosylation) in a cellular model system for neoplastic transformation developed in our laboratory. Here we show that PARP-1 and PARP-2 expression increases during neoplastic transformation, together with the basal levels of poly(ADP-ribosylation). Furthermore, we demonstrate a greater effect of the PARP inhibitor 3-aminobenzamide (3AB) on cellular viability in neoplastically transformed cells compared to normal fibroblasts and we show that prolonged 3AB administration to tumorigenic cells causes a decrease in telomere length. Taken together, our data support an active involvement of poly(ADP-ribosylation) in neoplastic transformation and telomere length maintenance and confirm the relevant role of poly(ADP-ribosylation) inhibition for the treatment of cancer.

Relocalization of cell adhesion molecules during neoplastic transformation of human fibroblasts

Studying neoplastic transformation of telomerase immortalized human fibroblasts (cen3tel), we found that the transition from normal to tumorigenic cells was associated with the loss of growth contact inhibition, the acquisition of an epithelial-like morphology and a change in actin organization, from stress fibers to cortical bundles. We show here that these variations were paralleled by an increase in N-cadherin expression and relocalization of different adhesion molecules, such as N-cadherin, α-catenin, p-120 and β-catenin. These proteins presented a clear membrane localization in tumorigenic cells compared to a more diffuse, cytoplasmic distribution in primary fibroblasts and non-tumorigenic immortalized cells, suggesting that tumorigenic cells could form strong cell-cell contacts and cell contacts did not induce growth inhibition. The epithelial-like appearance of tumorigenic cells did not reflect a mesenchymal-epithelial transition; in fact, cen3tel cells expressed vimentin and did not express cytokeratins at all transformation stages. Moreover, they did not express epithelial proteins such as occluding and claudin-1. In contrast, ZO-1 showed higher levels and a more defined membrane localization in tumorigenic cells compared to non-tumorigenic cells; this confirms its role in adherens junction formation in mesenchymal cells and is in agreement with the strong cell-cell contact formation by neoplastically transformed cells. Finally, we found α-catenin and ZO-1 nuclear localization in non-transformed cells, suggestive of possible additional roles of these proteins besides cell junction formation.

Drug Treatment of Cancer Cell Lines: A Way to Select for Cancer Stem Cells?

Tumors are generally composed of different cell types. In recent years, it has been shown that in many types of cancers a subset of cells show peculiar characteristics, such as the ability to induce tumors when engrafted into host animals, self-renew and being immortal, and give rise to a differentiated progeny. These cells have been defined as cancer stem cells (CSCs) or tumor initiating cells. CSCs can be isolated both from tumor specimens and established cancer cell lines on the basis of their ability to exclude fluorescent dyes, express specific cell surface markers or grow in particular culture conditions. A key feature of CSCs is their resistance to chemotherapeutic agents, which could contribute to the remaining of residual cancer cells after therapeutic treatments. It has been shown that CSC-like cells can be isolated after drug treatment of cancer cell lines; in this review, we will describe the strategies so far applied to identify and isolate CSCs. Furthermore, we will discuss the possible use of these selected populations to investigate CSC biology and develop new anticancer drugs.

Cellular immortalization and neoplastic transformation: Simultaneous, sequential or independent? Telomeres, telomerase or karyotypic variations?

A major difference between normal somatic cells and cancer cells is their proliferation potential. Normal somatic cells in culture are subjected to the well-known Hayflick limit; that is they stop proliferating after a limited number of divisions, while cancer cells can proliferate indefinitely. A large body of evidence has shown that telomeres, the terminal parts of the eukaryotic chromosomes, play a key role in defining cells’ proliferation potential. In fact, in the absence of telomerase, as in somatic cells, they shorten at each cell division and trigger cellular senescence when they reach a length below a threshold level.1 In contrast, in tumors, induction of telomere maintenance mechanisms, prevalently telomerase activity, is a general finding, suggesting that progression of malignancy requires telomere stabilization.2 The seminal paper by Bodnar et al.3 demonstrated that restoration of telomerase activity allows several types of somatic cells to stabilize telomeres, bypass senescence and become immortal, thus defining a causal relationship between telomere maintenance, telomerase activity and cellular immortalization. Cellular immortalization per se, that is the increase of cells’ proliferative potential beyond the Hayflick limit, does not necessarily imply neoplastic transformation. Some authors have shown that telomerase immortalized cells can divide for many passages maintaining a normal phenotype, while others, our group among them, has demonstrated that the artificial extension of somatic cells proliferative capacity can be associated with the development of the neoplastic phenotype (reviewed in ref. 4). In this second case, and in particular in the cellular system set up in our laboratory, named cen3tel, neoplastic transformation followed the lifespan extension achieved through telomerase expression and telomere stabilization. We could identify several phases along the road to transformation of cen3tel cells, each characterized by specific cellular and molecular features.4-7 In a recent paper published in Cell Cycle, Duesberg and McCormack8 analyzed the karyotype of cen3tel cells at different propagation stages and concluded that immortality and tumorigenesis originated simultaneously in cells propagated for about 150 population doublings (PD) after exogenous telomerase expression, together with the acquisition of a “clonal and flexible karyotype” and independently of telomerase activity and telomere stabilization. Analyzing other cell lines expressing exogenous telomerase alone or telomerase and activated oncogenes, these authors reached the same conclusions; that is “clonal and flexible karyotypes” are at the basis of immortalization and transformation, while telomerase and oncogenes are not sufficient to trigger these processes. Without questioning the role of karyotypic variations in tumorigenesis and the acquisition of a clonal and flexible karyotype by tumorigenic cen3tel cells, as described by Duesberg and McCormack,8 we would like to draw the attention on some published characteristics of the cen3tel cellular systems that undermine Duesberg and McCormack’s conclusions. (1) The telomerase-negative cen3 primary fibroblasts, used as recipient to generate cen3tel cells, entered senescence around PD 40, stopped dividing and never became tumorigenic.5 Only cen3 cells expressing exogenous telomerase and proliferating beyond the Hayflick limit underwent neoplastic transformation. Thus, in this cellular system, telomerase expression was the prerequisite for the extension of the cells’ proliferative capacity and the development of the genomic variations driving tumorigenesis. (2) In cen3tel cells, neoplastic transformation was a stepwise process; in fact, cells first became able to grow in agar and showed CDKN2A downregulation (around PD 100) then became tumorigenic.6 Cells around PD 100 had a transformed, even if not yet neoplastic, phenotype. Upon acquisition of the neoplastic phenotype, mutations in the tumor suppressor gene TP53 and overexpression of the c-myc oncogene were found in cen3tel cells. The same genes were found to be impaired in the independent cen3telS2 tumorigenic cells,4,6 confirming the role of oncogenes and tumor suppressor genes in tumorigenesis. Moreover, genome-wide transcription analysis of cen3tel cells at different stages of transformation revealed that there was a sequential evolution of the transcriptome of these cells with the progressive modulation of more and more cancer-related genes, leading to the development of a more and more aggressive phenotype.7 Thus, it is hard to conclude that immortality and tumorigenicity originated simultaneously in cen3tel cells together with the clonal and flexible karyotype. (3) Duesberg and McCormick8 stated that our results themselves indicate that immortalization and neoplastic transformation are independent of telomerase function. They report that according to our results, in cen3tel cells at PD 166, telomere length decreased and stabilized around values lower than in senescent cells. In Mondello et al.,5 we showed that cells around PD 100, and not around PD 166, had a mean telomere length lower than senescent cells; in addition, and more importantly, we pointed out that, despite the reduced length, cen3tel cell telomeres were functional. In fact, we did not detect telomeric fusions, and cells did not stop dividing, indicating that in the presence of telomerase, short telomeres can be stabilized and support cell growth. In a recent paper (Chiodi et al. Biochim Biophys Acta 2013; 1885-93), we have shown that in cen3tel cells just after PD 100, telomeres started to be elongated because of a change in telomere metabolism. On the basis of the observations reported above, we think that cen3tel cells can be considered immortal all along their lifespan, since their generation through the introduction of the hTERT cDNA in cen3 primary fibroblasts. We do not see any experimental evidence that can postpone immortalization to the time of acquisition of the neoplastic phenotype and the occurrence of the “clonal and flexible karyotype.” Cellular immortalization can be viewed as a process that allows cells to keep on dividing; the selection of cells with karyotypic variations, a greater fitness and a tumorigenic phenotype can eventually occur, but this doesn’t necessarily imply that only these cells are immortal. In conclusion, studies on the cen3tel cellular system and on other telomerase immortalized cells strongly support the hypothesis that exogenous telomerase expression stabilizes somatic cell telomeres, allowing cellular immortalization. In the absence of telomerase, as in telomerase-deficient mouse cells, other telomere maintenance mechanisms can stabilize telomeres, allowing immortalization and cellular transformation.9 We believe that the cytogenetic results reported by Duesberg and McCormack8 are very interesting, giving a picture of the evolution of the karyotype during cellular transformation and highlightening chromosome rearrangements that could possibly have a role in tumorigenesis. However, we do not think that these data disprove the link between telomerase, telomere stabilization and cellular immortality.

Cells with stemness features are generated from in vitro transformed human fibroblasts

Cancer stem cells (CSCs) have been involved in the maintenance, progression and relapse of several tumors, but their origin is still elusive. Here, in vitro transformed human fibroblasts (cen3tel cells) and the tumorsphere assay were used to search for and possibly characterize CSCs in transformed somatic cells. Cen3tel cells formed spheres showing self-renewal capacity and Sox2 overexpression, suggesting that they contained a subset of cells with CSC-like features. Sphere cells displayed deregulation of a c-MYC/miR-34a circuitry, likely associated with cell protection from apoptosis. Gene expression profiles of sphere cells revealed an extensive transcriptional reprogramming. Genes up-regulated in tumorspheres identified processes related to tumorigenesis and stemness, as cholesterol biosynthesis, apoptosis suppression, interferon and cytokine mediated signalling pathways. Sphere cells engrafted into NSG mice more rapidly than adherent cells, but both cell populations were tumorigenic. These results indicate that, during transformation, human somatic cells can acquire CSC properties, confirming the high plasticity of tumor cells. However, CSC-like cells are not the only tumorigenic population in transformed cells, indicating that the CSC phenotype and tumorigenicity can be uncoupled.