Small Nucleolar RNAs Determine Resistance to Doxorubicin in Human Osteosarcoma.

Doxorubicin (Dox) is one of the most important first-line drugs used in osteosarcoma therapy. Multiple and not fully clarified mechanisms, however, determine resistance to Dox. With the aim of identifying new markers associated with Dox-resistance, we found a global up-regulation of small nucleolar RNAs (snoRNAs) in human Dox-resistant osteosarcoma cells. We investigated if and how snoRNAs are linked to resistance. After RT-PCR validation of snoRNAs up-regulated in osteosarcoma cells with different degrees of resistance to Dox, we overexpressed them in Dox-sensitive cells. We then evaluated Dox cytotoxicity and changes in genes relevant for osteosarcoma pathogenesis by PCR arrays. SNORD3A, SNORA13 and SNORA28 reduced Dox-cytotoxicity when over-expressed in Dox-sensitive cells. In these cells, GADD45A and MYC were up-regulated, TOP2A was down-regulated. The same profile was detected in cells with acquired resistance to Dox. GADD45A/MYC-silencing and TOP2A-over-expression counteracted the resistance to Dox induced by snoRNAs. We reported for the first time that snoRNAs induce resistance to Dox in human osteosarcoma, by modulating the expression of genes involved in DNA damaging sensing, DNA repair, ribosome biogenesis, and proliferation. Targeting snoRNAs or down-stream genes may open new treatment perspectives in chemoresistant osteosarcomas.

Pharmacogenomics of second-line drugs used for treatment of unresponsive or relapsed osteosarcoma patients.

Second-line treatment of high-grade osteosarcoma (HGOS) patients is based on different approaches and chemotherapy protocols, which are not yet standardized. Although several drugs have been used in HGOS second-line protocols, none of them has provided fully satisfactory results and the role of rescue chemotherapy is not well defined yet. This article focuses on the drugs that have most frequently been used for second-line treatment of HGOS, highlighting the present knowledge on their mechanisms of action and resistance and on gene polymorphisms with possible impact on treatment sensitivity or toxicity. In the near future, validation of the so far identified candidate genetic biomarkers may constitute the basis for tailoring treatment by taking the patients' genetic background into account.

Candidate germline polymorphisms of genes belonging to the pathways of four drugs used in osteosarcoma standard chemotherapy associated with risk, survival and toxicity in non-metastatic high-grade osteosarcoma.

This study aimed to identify associations between germline polymorphisms and risk of high-grade osteosarcoma (HGOS) development, event-free survival (EFS) and toxicity in HGOS patients treated with neo-adjuvant chemotherapy and surgery.Germline polymorphisms of 31 genes known to be relevant for transport or metabolism of all four drugs used in HGOS chemotherapy (methotrexate, doxorubicin, cisplatin and ifosfamide) were genotyped in 196 patients with HGOS and in 470 healthy age and gender-matched controls. Of these 196 HGOS patients, a homogeneously treated group of 126 patients was considered for survival analyses (survival cohort). For 57 of these, treatment-related toxicity data were available (toxicity cohort).Eleven polymorphisms were associated with increased risk of developing HGOS (p < 0.05). The distribution of polymorphisms in patients was characterized by a higher Shannon entropy. In the survival cohort (n = 126, median follow-up = 126 months), genotypes of ABCC2_1249A/G, GGH_452T/C, TP53_IVS2+38G/C and CYP2B6*6 were associated with EFS (p < 0.05). In the toxicity cohort (n = 57), genotypes of ABCB1_1236T/C, ABCC2_1249A/G, ABCC2_3972A/G, ERCC1_8092T/G, XPD_23591A/G, XRCC3_18067T/C, MTHFR_1298A/C and GGH_16T/C were associated with elevated risk for toxicity development (p < 0.05).The results obtained in this retrospective study indicate that the aforementioned germline polymorphisms significantly impact on the risk of HGOS development, EFS and the occurrence of chemotherapy-related toxicity. These findings should be prospectively validated with the aim of optimizing and tailoring HGOS treatment in the near future.

Mitochondria-Targeted Doxorubicin: A New Therapeutic Strategy against Doxorubicin-Resistant Osteosarcoma.

Doxorubicin is one of the leading drugs for osteosarcoma standard chemotherapy. A total of 40% to 45% of high-grade osteosarcoma patients are unresponsive, or only partially responsive, to doxorubicin (Dox), due to the overexpression of the drug efflux transporter ABCB1/P-glycoprotein (Pgp). The aim of this work is to improve Dox-based regimens in resistant osteosarcomas. We used a chemically modified mitochondria-targeted Dox (mtDox) against Pgp-overexpressing osteosarcomas with increased resistance to Dox. Unlike Dox, mtDox accumulated at significant levels intracellularly, exerted cytotoxic activity, and induced necrotic and immunogenic cell death in Dox-resistant/Pgp-overexpressing cells, fully reproducing the activities exerted by anthracyclines in drug-sensitive tumors. mtDox reduced tumor growth and cell proliferation, increased apoptosis, primed tumor cells for recognition by the host immune system, and was less cardiotoxic than Dox in preclinical models of drug-resistant osteosarcoma. The increase in Dox resistance was paralleled by a progressive upregulation of mitochondrial metabolism. By widely modulating the expression of mitochondria-related genes, mtDox decreased mitochondrial biogenesis, the import of proteins and metabolites within mitochondria, mitochondrial metabolism, and the synthesis of ATP. These events were paralleled by increased reactive oxygen species production, mitochondrial depolarization, and mitochondria-dependent apoptosis in resistant osteosarcoma cells, where Dox was completely ineffective. We propose mtDox as a new effective agent with a safer toxicity profile compared with Dox that may be effective for the treatment of Dox-resistant/Pgp-positive osteosarcoma patients, who strongly need alternative and innovative treatment strategies. Mol Cancer Ther; 15(11); 2640-52. ©2016 AACR.

H2S-Donating Doxorubicins May Overcome Cardiotoxicity and Multidrug Resistance.

Doxorubicin (DOXO) is one of the most effective antineoplastic agents in clinical practice. Its use is limited by acute and chronic side effects, in particular by its cardiotoxicity and by the rapid development of resistance to it. As part of a program aimed at developing new DOXO derivatives endowed with reduced cardiotoxicity, and active against DOXO-resistant tumor cells, a series of H2S-releasing DOXOs (H2S-DOXOs) were obtained by combining DOXO with appropriate H2S donor substructures. The resulting compounds were studied on H9c2 cardiomyocytes and in DOXO-sensitive U-2OS osteosarcoma cells, as well as in related cell variants with increasing degrees of DOXO-resistance. Differently from DOXO, most of the products were not toxic at 5 µM concentration on H9c2 cells. A few of them triggered high activity on the cancer cells. H2S-DOXOs 10 and 11 emerged as the most interesting members of the series. The capacity of 10 to impair Pgp transporter is also discussed.

Mechanisms of gene amplification and evidence of coamplification in drug-resistant human osteosarcoma cell lines.

Gene amplification and copy number changes play a pivotal role in malignant transformation and progression of human tumor cells by mediating the activation of genes and oncogenes, which are involved in many different cellular processes including development of drug resistance. Since doxorubicin (DX) and methotrexate (MTX) are the two most important drugs for high-grade osteosarcoma (OS) treatment, the aim of this study was to identify genes gained or amplified in six DX- and eight MTX-resistant variants of the human OS cell lines U-2OS and Saos-2, and to get insights into the mechanisms underlying the amplification processes. Comparative genomic hybridization techniques identified amplification of MDR1 in all six DX-resistant and of DHFR in three MTX-resistant U-2OS variants. In addition, progressive gain of MLL was detected in the four U-2OS variants with higher resistance levels either to DX or MTX, whereas gain of MYC was found in all Saos-2 MTX-resistant variants and the U-2OS variant with the highest resistance level to DX. Fluorescent in situ hybridization revealed that MDR1 was amplified in U-2OS and Saos-2/DX-resistant variants manifested as homogeneously staining regions and double minutes, respectively. In U-2OS/MTX-resistant variants, DHFR was amplified in homogeneously staining regions, and was coamplified with MLL in relation to the increase of resistance to MTX. Gene amplification was associated with gene overexpression, whereas gene gain resulted in up-regulated gene expression. These results indicate that resistance to DX and MTX in human OS cell lines is a multigenic process involving gene copy number and expression changes.

Malignant fibrous histiocytoma of bone: analysis of genomic imbalances by comparative genomic hybridisation and C-MYC expression by immunohistochemistry.

Malignant fibrous histiocytoma (MFH) of bone is a rare, highly malignant tumour. As very little is known about its genetic alterations, 26 bone MFHs were analysed by comparative genomic hybridisation (CGH). Twenty-three tumours (89%) had DNA sequence copy number changes (mean, 7.2 changes per sample). Gains were more frequent than losses (gains:losses=2.5:1). Minimal common regions for the most frequent gains were 8q21.3-qter (35%), 9q32-qter (35%), 7q22-q31 (35%), 1q21-q23 (31%), 7p12-pter (31%), 7cen-q11.2 (31%) and 15q21 (31%). Minimal common regions for the most frequent losses were 13q21-q22 (42%) and 18q12-q22 (27%). High-level amplifications were detected in 8 out of the 26 tumours (31%). The only recurrent amplifications, 1q21-q23 and 8q21.2-q22, were present in two samples (8%). As copy number increase at 8q24 (the locus of C-MYC) was frequent, the expression of C-MYC was studied by immunohistochemistry. Increased levels of c-myc protein were detected in 7 out of 21 tumours studied (33%). 81% of the samples studied both by CGH and immunohistochemistry showed concordant results. Furthermore, the findings of the present study were compared to previous publications on osteosarcoma, soft tissue MFH and fibrosarcoma of bone. Clear differences were detected in CGH aberration patterns, further supporting the concept of bone MFH as an individual bone tumour entity. Finally, the findings of the present study reflect well the high malignancy and aggressive nature of bone MFH.

Prognostic and therapeutic relevance of HER2 expression in osteosarcoma and Ewing's sarcoma.

Expression of HER2 was evaluated by immunohistochemical techniques in 84 osteosarcoma (OS) and 113 Ewing's sarcoma (ES) paraffin-embedded tumour biopsies. HER2 gene status was also assessed in a panel of cell lines as well as in vitro efficacy of trastuzumab (a humanised antibody directed against HER2) as single agent or in combination with the insulin-like growth factor I receptor (IGF-IR) IR3 antibody. Overexpression of HER2 was present in 32% of OS and 16% of ES and was significantly associated with the increased expression of P-glycoprotein, a surface molecule responsible for multidrug resistance. Event-free survival analyses revealed a prognostic value for HER2 and/or P-glycoprotein expression in OS, but not in ES. However, despite its prognostic relevance, no therapeutic effectiveness was observed pre-clinically for trastuzumab-driven therapy, in both OS or ES cell lines, unless the antibody was associated with anti-IGF-IR targeting strategies. Therefore, the therapeutic potential of trastuzumab in these neoplasms may be better exploited in combined treatments with anti-IGF-IR approaches.

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques.

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.

Induction of p21 mRNA synthesis after short-wavelength UV light visualized in individual cells by RNA FISH.

Expression of the cyclin-dependent kinase inhibitor gene p21 is induced after DNA damage and plays a role in cell survival. The exact mechanism of induction is not known, but enhancement of mRNA stability has recently been implicated as an important factor. To obtain further insight into the dynamics of p21 gene expression at the individual cell level, normal fibroblasts, GM1492 fibroblasts from a Bloom's syndrome patient, and U2OS osteosarcoma cells were UVC irradiated, fixed at different time points, and subjected to mRNA fluorescence in situ hybridization (FISH) and immunocytochemical staining. In mock-irradiated normal fibroblasts, a subfraction of cells revealed low levels of p21 mRNA synthesis. After UVC treatment, p21 transcripts accumulated over time in nuclear locations other than transcription foci. At 6 hr after irradiation, almost 50% of the cells displayed p21 mRNA in three different distribution patterns within the nuclei. The highest frequency of cells with cytoplasmic accumulation of p21 mRNA was seen at 17 hr after UVC treatment. We conclude that increased p21 gene transcription and possibly stabilization of newly synthesized p21 mRNA contribute to elevated levels of p21 protein after UVC irradiation. (J Histochem Cytochem 50:81-89, 2002)