Silvana Libertini

Bevacizumab Increases Viral Distribution in Human Anaplastic Thyroid Carcinoma Xenografts and Enhances the Effects of E1A-Defective Adenovirus dl922-947

Purpose: Anaplastic thyroid carcinoma is a prime target for innovative therapy because it represents one of the most lethal human neoplasms and is refractory to conventional treatments such as chemotherapy and radiotherapy. We have evaluated a novel therapeutic approach based on the oncolytic replication-selective adenovirus dl922-947. Experimental Design: The antitumor efficacies of the E1AΔCR2 (dl922-947) and ΔE1B55K (dl1520) mutants were compared in human thyroid anaplastic carcinoma cells in culture and in xenografts in vivo. To enhance the effects of dl922-947, anaplastic thyroid carcinoma tumor xenografts were treated with dl922-947 in combination with bevacizumab. Results: We showed that the efficacy of dl922-947 exceeded that of dl1520 in all tested anaplastic thyroid carcinoma cells in vitro and in vivo. Furthermore, bevacizumab in combination with dl922-947 significantly reduced tumor growth compared with single treatments alone. Bevacizumab treatment significantly improved viral distribution in neoplastic tissues. Conclusions: Our data showed that dl922-947 had a higher oncolytic activity compared with dl1520 in anaplastic thyroid carcinoma cell lines and might represent a better option for virotherapy of anaplastic thyroid carcinoma. Moreover, bevacizumab increased the oncolytic effects of dl922-947 by enhancing viral distribution in tumors. The results described herein encourage the use of the dl922-947 virus in combination with bevacizumab.

Aurora B Expression in Post-Puberal Testicular Germ Cell Tumours

Aurora/Ipl1‐related kinases are a conserved family of proteins that are essential for the regulation of chromosome segregation and cytokinesis during mitosis. Aberrant expression and activity of these kinases occur in a wide range of human tumours and have been implicated in mechanisms leading to mitotic spindle aberrations, aneuploidy, and genomic instability. Previous studies of our group have shown that Aurora B expression is restricted to specific germinal cells. In this study, we have evaluated by immunohistochemical analysis Aurora B expression in post‐puberal testicular germ cell tumours (22 seminomas, 2 teratomas, 15 embryonal carcinomas, 5 mixed germinal tumours with a prominent yolk sac tumour component and 1 choriocarcinoma). The Aurora B protein expression was detected in all intratubular germ cell tumours, seminomas and embryonal carcinomas analysed but not in teratomas and yolk sac carcinomas. The immunohistochemical data were further confirmed by Western blot analysis. In addition, the kinase Aurora B was vigorously expressed in GC‐1 cells line derived from murine spermatogonia. The block of Aurora B function induced by a pharmacological inhibitor significantly reduced the growth of GC‐1 cells suggesting that Aurora B is a potential therapeutic target. J. Cell. Physiol. 221: 435–439, 2009.

Raised expression of the antiapoptotic protein ped/pea-15 increases susceptibility to chemically induced skin tumor development

ped/pea-15 is a cytosolic protein performing a broad antiapoptotic function. We show that, upon DMBA/TPA-induced skin carcinogenesis, transgenic mice overexpressing ped/pea-15 (Tgped/pea-15) display early development of papillomas and a four-fold increase in papilloma number compared to the nontransgenic littermates (P<0.001). The malignant conversion frequency was 24% for the Tgped/pea-15 mice and only 5% in controls (P<0.01). The isolated application of TPA, but not that of DMBA, was sufficient to reversibly upregulate ped/pea-15 in both untransformed skin and cultured keratinocytes. ped/pea-15 protein levels were also increased in DMBA/TPA-induced papillomas of both Tgped/pea-15 and control mice. Isolated TPA applications induced Caspase-3 activation and apoptosis in nontransformed mouse epidermal tissues. The induction of both Caspase-3 and apoptosis by TPA were four-fold inhibited in the skin of the Tgped/pea-15 compared to the nontransgenic mice, accompanied by a similarly sized reduction in TPA-induced JNK and p38 stimulation and by constitutive induction of cytoplasmic ERK activity in the transgenics. ped/pea-15 expression was stably increased in cell lines from DMBA/TPA-induced skin papillomas and carcinomas, paralleled by protection from TPA apoptosis. In the A5 spindle carcinoma cell line, antisense inhibition of ped/pea-15 expression simultaneously rescued sensitivity to TPA-induced Caspase-3 function and apoptosis. The antisense also reduced A5 cell ability to grow in semisolid media by 65% (P<0.001) and increased by three-fold tumor latency time (P<0.01). Thus, the expression levels of ped/pea-15 control Caspase-3 function and epidermal cell apoptosis in vivo and determine susceptibility to skin tumor development.

Chk1 is essential for chemical carcinogen-induced mouse skin tumorigenesis.

Chk1 is a key regulator of DNA damage checkpoint responses and genome stability in eukaryotes. To better understand how checkpoint proficiency relates to cancer development, we investigated the effects of genetic ablation of Chk1 in the mouse skin on tumors induced by chemical carcinogens. We found that homozygous deletion of Chk1 immediately before carcinogen exposure strongly suppressed benign tumor (papilloma) formation, and that the few, small lesions that formed in the ablated skin always retained Chk1 expression. Remarkably, Chk1 deletion rapidly triggered spontaneous cell proliferation, γ-H2AX staining and apoptosis within the hair follicle, a principal site of origin for carcinogen-induced tumors. At later times, the ablated skin was progressively repopulated by non-recombined Chk1-expressing cells and ultimately normal sensitivity to tumor induction was restored when carcinogen treatment was delayed. In marked contrast, papillomas formed normally in Chk1 hemizygous skin but showed an increased propensity to progress to carcinoma. Thus, complete loss of Chk1 is incompatible with epithelial tumorigenesis, whereas partial loss of function (haploinsufficiency) fosters benign malignant tumor progression.

AZD1152 negatively affects the growth of anaplastic thyroid carcinoma cells and enhances the effects of oncolytic virus dl922-947.

Novel therapeutic approaches are required for the treatment of anaplastic thyroid carcinoma (ATC), an incurable disease resistant to current available therapies. Aurora B is an important mitotic kinase involved in chromosome segregation and cytokinesis. It is overexpressed in many cancers including ATC and represents a potential target for chemotherapy. The effects of AZD1152, a specific Aurora B kinase inhibitor, have been evaluated against ATC, showing G(2)/M accumulation, polyploidy and subsequent cell death by mitotic catastrophe upon drug treatment. Only three administrations of AZD1152 significantly reduced the growth of ATC tumour xenogratfs. Oncolytic viruses in association with other forms of treatment have proven highly promising in preclinical and clinical reports. The oncolytic adenovirus dl922-947 is active against ATC cells, and we have evaluated the effects of the association between AZD1152 and dl922-947. In cells treated with virus and drug, we report additive/synergistic killing effects. Interestingly, the phosphorylation of histone H3 (Ser10), the main Aurora B substrate, is inhibited by dl922-947 in a dose-dependent manner, and completely abolished in association with AZD1152. The combined treatment significantly inhibited the growth of ATC tumour xenografts with respect to single treatments. Our data demonstrate that the Aurora B inhibitor AZD1152, alone or in combination with oncolytic virus dl922-947, could represent a novel therapeutic option for the treatment of ATC.

Fascin 1 is transiently expressed in mouse melanoblasts during development and promotes migration and proliferation

Fascins, a family of actin-bundling proteins, are expressed in a spatially and temporally restricted manner during development and often in cancer. Fascin 1 has a clear role in cell migration in vitro, but its role in vivo in mammals is not well understood. Here, we investigate the role of fascin 1 in the melanocyte lineage and in melanoma cells. Fascin 1 knockout causes hypopigmentation in adult mice owing to migration and cell cycle progression defects in melanoblasts, the melanocyte precursor cell. Study of live embryo skin explants reveals that E14.5 fascin 1-null melanoblasts migrate slower, and generate fewer and thinner pseudopods. By contrast, fascin 1 expression drives faster migration and lamellipodia protrusion in melanocytes in vitro. In addition, fascin 1 depletion retards melanoblast proliferation in vivo and melanoma cell growth in vitro. These data indicate that fascin 1 not only promotes cell migration in mouse melanocytes but it also has a role in growth and cell cycle progression.

Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma

Melanoma patients treated with oncogenic BRAF inhibitors can develop cutaneous squamous cell carcinoma (cSCC) within weeks of treatment, driven by paradoxical RAS/RAF/MAPK pathway activation. Here we identify frequent TGFBR1 and TGFBR2 mutations in human vemurafenib-induced skin lesions and in sporadic cSCC. Functional analysis reveals these mutations ablate canonical TGFβ Smad signalling, which is localized to bulge stem cells in both normal human and murine skin. MAPK pathway hyperactivation (through BrafV600E or KrasG12D knockin) and TGFβ signalling ablation (through Tgfbr1 deletion) in LGR5+ve stem cells enables rapid cSCC development in the mouse. Mutation of Tp53 (which is commonly mutated in sporadic cSCC) coupled with Tgfbr1 deletion in LGR5+ve cells also results in cSCC development. These findings indicate that LGR5+ve stem cells may act as cells of origin for cSCC, and that RAS/RAF/MAPK pathway hyperactivation or Tp53 mutation, coupled with loss of TGFβ signalling, are driving events of skin tumorigenesis.

Inhibition of Autophagy Enhances the Effects of E1A-Defective Oncolytic Adenovirus dl922–947 Against Glioma Cells In Vitro and In Vivo

Oncolytic viruses represent a novel therapeutic approach for aggressive tumors, such as glioblastoma multiforme, which are resistant to available treatments. Autophagy has been observed in cells infected with oncolytic viruses; however, its role in cell death/survival is unclear. To elucidate the potential therapeutic use of autophagy modulators in association with viral therapy, we analyzed autophagy induction in human glioma cell lines U373MG and U87MG infected with the oncolytic adenovirus dl922-947. dl922-947 infection triggered an autophagic cellular response, as shown by the development of acidic vesicular organelles, LC3-I→LC3-II conversion, and reduction of p62 levels. However, on infection, the Akt/mTOR/p70s6k pathway, which negatively regulates autophagy, was activated, whereas the ERK1/2 pathway, a positive regulator of autophagy, was inhibited. Accordingly, MEK inhibition by PD98059 sensitized glioma cells to dl922-947 effects, whereas autophagy induction by rapamycin protected cells from dl922-947-induced death. Treatment with two inhibitors of autophagy, chloroquine and 3-methyladenine, increased the cytotoxic effects of dl922-947 in vitro. In vivo, the growth of U87MG-induced xenografts was further reduced by adding chloroquine to the dl922-947 treatment. In conclusion, autophagy acts as a survival response in glioma cells infected with dl922-947, thus suggesting autophagy inhibitors as adjuvant/neoadjuvant drugs in oncolytic virus-based treatments.

PED/PEA-15 Modulates Coxsackievirus–Adenovirus Receptor Expression and Adenoviral Infectivity via ERK-Mediated Signals in Glioma Cells

Glioblastoma multiforme (GBM) is the most aggressive human brain tumor, and is highly resistant to chemo- and radiotherapy. Selectively replicating oncolytic viruses represent a novel approach for the treatment of neoplastic diseases. Coxsackievirus-adenovirus receptor (CAR) is the primary receptor for adenoviruses, and loss or reduction of CAR greatly decreases adenoviral entry. Understanding the mechanisms regulating CAR expression and localization will contribute to increase the efficacy of oncolytic adenoviruses. Two glioma cell lines (U343MG and U373MG) were infected with the oncolytic adenovirus dl922-947. U373MG cells were more susceptible to cell death after viral infection, compared with U343MG cells. The enhanced sensitivity was paralleled by increased adenoviral entry and CAR mRNA and protein levels in U373MG cells. In addition, U373MG cells displayed a decreased ERK1/2 (extracellular signal-regulated kinase-1/2) nuclear-to-cytosolic ratio, compared with U343MG cells. Intracellular content of PED/PEA-15, an ERK1/2-interacting protein, was also augmented in these cells. Both ERK2 overexpression and genetic silencing of PED/PEA-15 by antisense oligonucleotides increased ERK nuclear accumulation and reduced CAR expression and adenoviral entry. Our data indicate that dl922-947 could represent an useful tool for the treatment of GBM and that PED/PEA-15 modulates CAR expression and adenoviral entry, by sequestering ERK1/2.

Aurora A and B Kinases - Targets of Novel Anticancer Drugs

The Aurora Kinases are highly related serine-threonine kinases, essential for accurate and equal segregation of genomic material during mitosis. A large number of studies have linked the aberrant expression of Aurora kinases to cancer, leading to the development of specific Aurora kinases inhibitors. Several small molecules inhibit with a similar efficacy both Aurora A and Aurora B, however, in most cases the effects resemble Aurora B disruption by genetic methods, indicating that Aurora B represents an effective therapeutic target. These drugs are currently under preclinical or clinical evaluation and are reviewed in this article. The relevant patents are discussed.